Energy Balance and Gastrointestinal Cancer

Energy Balance and Cancer

Series Editor: Nathan A. Berger, Case Western Reserve University, Cleveland, OH, USA

For further volumes: http://www.springer.com/series/8282

Sanford D. Markowitz

●

Nathan A. Berger

Editors

Energy Balance and Gastrointestinal Cancer

Editors Sanford D. Markowitz Case Western Reserve University Cleveland, OH, USA

Nathan A. Berger Case Western Reserve University Cleveland, OH, USA

ISBN 978-1-4614-2366-9 e-ISBN 978-1-4614-2367-6 DOI 10.1007/978-1-4614-2367-6 Springer New York Dordrecht Heidelberg London Library of Congress Control Number: 2012932611 © Springer Science+Business Media, LLC 2012 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com)

Preface

As the worldwide pandemic of overweight and obesity continues to expand, with over 1 billion overweight and 315 million obese adults estimated worldwide [1–3], obesity is increasingly recognized as a significant risk factor for cancer. In the USA, approximately 20% of all cancer deaths in women and 14% in men have been attributed to overweight and obesity [4]. Epidemiological studies indicate an association between obesity and specific malignancies in multiple organ systems including colon, postmenopausal breast, endometrial, esophageal adenocarcinoma and renal cell cancer [5–7]. In addition to colon and esophageal adenocarcinoma, more recent evidence supports an association of obesity with other gastrointestinal malignancies including pancreatic, gallbladder, and hepatocellular cancer [4, 7, 8]. In 2009, the American Cancer Society estimated that the combined deaths in the USA from gastrointestinal cancers, including pancreas, colon, and rectum, esophageal, liver, and bile ducts exceeded 135,000 [9]. During the same year, there were 100,000 new cases and 50,000 deaths from colon cancer [9]. The relative risk of mortality from colon cancer according to body mass index in a prospective population study of more than 900,000 US adults [4] was found to be 1.20 for overweight and 1.47–1.84 for obese men, and 1.1 for overweight and 1.3–1.46 for obese women. In contrast, a recent study showed that in men with nonmetastatic colorectal cancer at diagnosis, increased physical activity was associated with improved colorectal cancer mortality and overall mortality [10]. Thus, gastrointestinal cancer in general and colorectal cancer more specifically causes an enormous burden of morbidity and mortality in the USA with significant impact from obesity and benefit associated with physical activity. Persistence of this vexing problem both at the personal and public health levels can be attributed, in part, to the multicomponent and complex nature of the relation between energy balance and cancer in which many of the cytokines, hormones, and other obesity-associated factors may act in combination with environmental and lifestyle factors as both mutagens and cancer promoters. Persistence of the problem is associated also with the difficulty in implementing effective and sustainable biobehavioral interventions to control obesity and associated mediators and comorbidities. v

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This volume assembles a panel of leading investigators to present a transdisciplinary view of the state of the science regarding the linkages between gastrointestinal malignancy and energy balance. In addition to analyzing the extensive epidemiologic data associating energy balance with gastrointestinal malignancies, this volume details the significant progress being made to increase mechanistic understandings and to identify possible mediators of these relationships and the potential interventions that are just beginning to be identified and tested. Chapter 1 presents a comprehensive evaluation by Dr. Nora Nock of the epidemiologic association of obesity with major gastrointestinal malignancies. In Chap. 2, Dr. Graham Casey reviews both the common and the rare genetic changes associated with colon cancer susceptibility. In Chap. 3, Dr. Leonard Augenlicht and associates provide an overview of their ongoing research on the effects of dietary modulation, especially of vitamin D, calcium, and fat, on the incidence of colon cancer in the mouse and new insights on potential mechanisms influenced by diet focused on cellular interactions between macrophages and intestinal epithelial cells. In Chap. 4, Dr. Nathan Berger provides an overview of mouse model studies investigating the effect of exercise on gastrointestinal malignancies. In Chaps. 5 and 6, Drs. Rom Leidner, Amitabh Chak, and Donghui Li review recent studies investigating the relation between obesity, Barrett’s esophagus, esophageal cancer, and pancreatic cancer. In Chaps. 7 and 8, Dr. Li Li explains the important contribution of insulin resistance pathways to the relation of obesity and colon cancer and Drs. Fred Bunz and Nickolas Papadopoulos explain the relation of Ras/Raf mutation to the process. Dr. Jeffrey Meyerhardt, in Chap. 9, evaluates studies on the effect of energy balance on colorectal cancer recurrence and survival in clinical situations and Dr. Monica Bertagnolli describes the role of chronic inflammation in colorectal malignancies. Overall, this volume should provide an important basis for studies to advance the state of the science linking energy balance to gastrointestinal malignancy and serve, also, as a background to develop new therapeutic interventions for prevention and control. Cleveland, OH, USA

Sanford D. Markowitz Nathan A. Berger

References 1. Caterson ID, Gill TP (2002) Obesity: epidemiology and possible prevention. Best Pract Res Clin Endocrinol Metab 16:595–610 2. Ogden CL, Carroll MD, Curtin LR et al (2008) Prevalence of overweight and obesity in the United States, 1999-2004. JAMA 295:1549–1555 3. World Health Organization (2003) World cancer report. IARC Nonserial Publication, Geneva 4. Calle EE, Rodriguez C, Walker-Thurmond K et al (2003) Overweight, obesity, and mortality from cancer in a prospectively studied cohort of U.S. adults. N Engl J Med 348(17): 1625–1638

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5. World Health Organization (2000) Obesity: preventing and managing the global epidemic. Report of a WHO consultation. Rep. 894, World Health Organization 6. World Cancer Research Fund and American Institute for Cancer Research (2007) Food, nutrition, physical activity, and the prevention of cancer: a global perspective. AICR, Washington, DC 7. Renehan AG, Tyson M, Egger M et al (2008) Body-mass index and incidence of cancer: a systematic review and meta-analysis of prospective observational studies. Lancet 371: 569–578 8. Li D, Morris JS, Liu J et al (2009) Body mass index and risk, age of onset, and survival in patients with pancreatic cancer. JAMA 301:2553–2562 9. American Cancer Society (2009) Cancer facts & figures 2009. American Cancer Society, Atlanta. http://www.cancer.org/downloads/STT/500809web.pdf 10. Meyerhardt JA, Giovannucci EL, Ogino S, Kirkner GS, Chan AT, Willett W, Fuch CS (2009) Physical activity and male colorectal cancer survival. Arch Intern Med 169:2102–2108

Contents

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Obesity and Gastrointestinal Cancers: Epidemiology........................ Nora L. Nock

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Genetics of Colon Cancer Susceptibility .............................................. Graham Casey

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Dietary Modulation of Colon Cancer: Effects on Intermediary Metabolism, Mucosal Cell Differentiation, and Inflammation ................................................................................... Lidija Klampfer, Barbara G. Heerdt, Anna Velcich, Erin Gaffney-Stomberg, Donghai Wang, Elaine Lin, and Leonard H. Augenlicht

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The ApcMin/+ Mouse Model to Study the Effects of Exercise on Gastrointestinal Malignancy............................................................ Nathan A. Berger

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Obesity and the Pathogenesis of Barrett’s Esophagus........................ Rom Leidner and Amitabh Chak

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Obesity and Pancreatic Cancer ............................................................ Donghui Li

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Obesity, Insulin Resistance Pathway Factors, and Colon Cancer .................................................................................. Li Li

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Ras/Raf and Their Influence in Glycolysis in Colon Cancer ............. Fred Bunz and Nickolas Papadopoulos

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Energy Balance and Other Modifiable Host Factors on Colorectal Cancer Prognosis............................................................ Jeffrey Meyerhardt

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Cyclooxygenase-2 and Chronic Inflammation: Drivers of Colorectal Tumorigenesis .................................................... Monica M. Bertagnolli

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Index ................................................................................................................

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Contributors

Leonard H. Augenlicht Montefiore Medical Center and Albert Einstein Cancer Center, Albert Einstein College of Medicine, Bronx, NY, USA Nathan A. Berger Center for Science, Health and Society, Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH, USA Monica M. Bertagnolli Division of Surgical Oncology, Dana Farber Cancer Institute, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA Fred Bunz Department of Radiation Oncology and Molecular Radiation Sciences, The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA Graham Casey Department of Preventive Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA Amitabh Chak Department of Medicine, Case Western Reserve University, Cleveland, OH, USA Erin Gaffney-Stomberg Montefiore Medical Center, Albert Einstein College of Medicine, Bronx, NY, USA Barbara G. Heerdt Montefiore Medical Center, Albert Einstein College of Medicine, Bronx, NY, USA Lidija Klampfer Montefiore Medical Center, Albert Einstein College of Medicine, Bronx, NY, USA Rom Leidner Department of Medicine, Case Western Reserve University, Cleveland, OH, USA Donghui Li Department of Gastrointestinal Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

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Li Li Department of Family Medicine—Research Division, Case Western Reserve University, Cleveland, OH, USA Elaine Lin Montefiore Medical Center, Albert Einstein College of Medicine, Bronx, NY, USA Jeffrey Meyerhardt Division of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA Nora L. Nock Department of Epidemiology and Biostatistics, Case Western Reserve University, Cleveland, OH, USA Nickolas Papadopoulos The Ludwig Center for Cancer Genetics and Therapeutics, The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA Anna Velcich Montefiore Medical Center, Albert Einstein College of Medicine, Bronx, NY, USA Donghai Wang Department of Hematology, Peking University First Hospital, Beijing, China

Chapter 1

Obesity and Gastrointestinal Cancers: Epidemiology Nora L. Nock

Abstract There is strong and consistent evidence for associations between obesity and multiple gastrointestinal (GI) cancers. The most consistent and compelling evidence exists for the association between obesity and colon cancer; however, there is emerging evidence for associations between obesity and esophageal adenocarcinoma and, pancreatic and liver cancers. The number of studies evaluating obesity and gastric and gallbladder cancers is limited and results, thus far, have been inconsistent. In this chapter, the epidemiological evidence linking obesity to the development and survival of GI cancers is reviewed. Because obesity plays an integral role in manifestation of the Metabolic Syndrome (MetSyn), epidemiological evidence for associations between MetSyn and GI cancers is also summarized. Other environmental and genetic risk factors for GI cancers and the putative mechanisms linking obesity, MetSyn, and GI cancers are discussed in subsequent chapters of this book.

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Obesity: Definitions

Obesity is often defined using surrogates of body size, most commonly, body mass index (BMI), which is expressed as weight adjusted for height in units of kilograms (kg) per meter squared (m2). The most widely adopted definitions of adult overweight and obesity are those established by the World Health Organization (WHO) [1] as follows:

N.L. Nock, Ph.D. (*) Department of Epidemiology and Biostatistics, Case Western Reserve University, 2103 Cornell Road, Cleveland, OH 44106-7281, USA e-mail: [email protected] S.D. Markowitz and N.A. Berger (eds.), Energy Balance and Gastrointestinal Cancer, Energy Balance and Cancer 4, DOI 10.1007/978-1-4614-2367-6_1, © Springer Science+Business Media, LLC 2012

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205. Spencer M, Yao-Borengasser A, Unal R et al (2010) Adipose tissue macrophages in insulinresistant subjects are associated with collagen VI and fibrosis and demonstrate alternative activation. Am J Physiol Endocrinol Metab 299:E1016–E1027 206. Alessi MC, Bastelica D, Morange P et al (2000) Plasminogen activator inhibitor 1, transforming growth factor-beta1, and BMI are closely associated in human adipose tissue during morbid obesity. Diabetes 49:1374–1380 207. Porreca E, Di Febbo C, Vitacolonna E et al (2002) Transforming growth factor-beta1 levels in hypertensive patients: association with body mass index and leptin. Am J Hypertens 15:759–765 208. Herder C, Zierer A, Koenig W, Roden M, Meisinger C, Thorand B (2009) Transforming growth factor-beta1 and incident type 2 diabetes: results from the MONICA/KORA casecohort study, 1984–2002. Diabetes Care 32:1921–1923 209. Samad F, Pandey M, Loskutoff DJ (1998) Tissue factor gene expression in the adipose tissues of obese mice. Proc Natl Acad Sci USA 95:7591–7596 210. Tseng YH, Kokkotou E, Schulz TJ et al (2008) New role of bone morphogenetic protein 7 in brown adipogenesis and energy expenditure. Nature 454:1000–1004 211. Taha MF, Valojerdi MR, Mowla SJ (2006) Effect of bone morphogenetic protein-4 (BMP-4) on adipocyte differentiation from mouse embryonic stem cells. Anat Histol Embryol 35:271–278 212. Bowers RR, Kim JW, Otto TC, Lane MD (2006) Stable stem cell commitment to the adipocyte lineage by inhibition of DNA methylation: role of the BMP-4 gene. Proc Natl Acad Sci USA 103:13022–13027 213. Dani C, Smith AG, Dessolin S et al (1997) Differentiation of embryonic stem cells into adipocytes in vitro. J Cell Sci 110(Pt 11):1279–1285 214. Tang QQ, Otto TC, Lane MD (2004) Commitment of C3H10T1/2 pluripotent stem cells to the adipocyte lineage. Proc Natl Acad Sci USA 101:9607–9611 215. Matsuzawa Y, Funahashi T, Nakamura T (1999) Molecular mechanism of metabolic syndrome X: contribution of adipocytokines adipocyte-derived bioactive substances. Ann N Y Acad Sci 892:146–154 216. Son JW, Kim MK, Park YM et al (2011) Association of serum bone morphogenetic protein 4 levels with obesity and metabolic syndrome in non-diabetic individuals. Endocr J 58(1):39–46 217. Huang H, Song TJ, Li X et al (2009) BMP signaling pathway is required for commitment of C3H10T1/2 pluripotent stem cells to the adipocyte lineage. Proc Natl Acad Sci USA 106:12670–12675 218. Mohamed-Ali V, Pinkney JH, Coppack SW (1998) Adipose tissue as an endocrine and paracrine organ. Int J Obes Relat Metab Disord 22:1145–1158 219. Bowers RR, Lane MD (2007) A role for bone morphogenetic protein-4 in adipocyte development. Cell Cycle 6:385–389 220. Skillington J, Choy L, Derynck R (2002) Bone morphogenetic protein and retinoic acid signaling cooperate to induce osteoblast differentiation of preadipocytes. J Cell Biol 159:135–146 221. Chen D, Ji X, Harris MA et al (1998) Differential roles for bone morphogenetic protein (BMP) receptor type IB and IA in differentiation and specification of mesenchymal precursor cells to osteoblast and adipocyte lineages. J Cell Biol 142:295–305 222. Sottile V, Seuwen K (2000) Bone morphogenetic protein-2 stimulates adipogenic differentiation of mesenchymal precursor cells in synergy with BRL 49653 (rosiglitazone). FEBS Lett 475:201–204 223. Hata K, Nishimura R, Ikeda F et al (2003) Differential roles of Smad1 and p38 kinase in regulation of peroxisome proliferator-activating receptor gamma during bone morphogenetic protein 2-induced adipogenesis. Mol Biol Cell 14:545–555 224. ten Dijke P, Yamashita H, Sampath TK et al (1994) Identification of type I receptors for osteogenic protein-1 and bone morphogenetic protein-4. J Biol Chem 269:16985–16988 225. Bottcher Y, Unbehauen H, Kloting N et al (2009) Adipose tissue expression and genetic variants of the bone morphogenetic protein receptor 1A gene (BMPR1A) are associated with human obesity. Diabetes 58:2119–2128

Chapter 3

Dietary Modulation of Colon Cancer: Effects on Intermediary Metabolism, Mucosal Cell Differentiation, and Inflammation Lidija Klampfer, Barbara G. Heerdt, Anna Velcich, Erin Gaffney-Stomberg, Donghai Wang, Elaine Lin, and Leonard H. Augenlicht

Abstract We review the profound effects that components of diets commonly consumed in western societies and linked through population studies to risk for colon cancer have on the development of intestinal cancer in humans and in mouse models. Focus is particularly on levels of vitamin D, interactive with calcium and fat, in establishing probability of tumor development even in mouse genetic models in which there is high penetrance of the disease. These dietary factors have also been used to develop a mouse model of dietary-induced sporadic colon cancer

L. Klampfer, Ph.D. • B.G. Heerdt, Ph.D. • A. Velcich • E. Gaffney-Stomberg, Ph.D. • E. Lin, Ph.D. Albert Einstein Cancer Center, Albert Einstein College of Medicine, Bronx, NY 10467, USA Montefiore Medical Center, Albert Einstein College of Medicine, 111 East 210th Street, Bronx, NY 10467, USA e-mail: [email protected]; [email protected]; [email protected]; [email protected]; [email protected] D. Wang, M.D. Albert Einstein Cancer Center, Albert Einstein College of Medicine, Bronx, NY 10467, USA Montefiore Medical Center, Albert Einstein College of Medicine, 111 East 210th Street, Bronx, NY 10467, USA Department of Hematology, Peking University First Hospital, Beijing 100034, China e-mail: [email protected] L.H. Augenlicht, Ph.D. (*) Montefiore Medical Center and Albert Einstein Cancer Center, Albert Einstein College of Medicine, Bronx, NY 10467, USA e-mail: [email protected] S.D. Markowitz and N.A. Berger (eds.), Energy Balance and Gastrointestinal Cancer, Energy Balance and Cancer 4, DOI 10.1007/978-1-4614-2367-6_3, © Springer Science+Business Media, LLC 2012

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which exhibits similar lag, incidence, and frequency of tumor development, and relative incidence of carcinomas and adenomas, as seen for >90% of colon tumors that arise in the general population later in life. Potential mechanisms influenced by diet that alter probability of tumor development are outlined, including altered patterns of intermediary metabolism, differentiation, and inflammation in the intestinal mucosa, all apparent in the histopathologically normal intestinal mucosa well before neoplastic changes become detectable. This includes pathways by which macrophages signal to intestinal epithelial cells, revealing a new paradigm for how vitamin D may influence tumor development.

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Introduction

The probability for development and progression of colon cancer is profoundly influenced by both patterns of dietary exposure and by inflammation. The themes of this chapter are: how do nutrients modulate inflammation and how does altered inflammation contribute to tumorigenesis? In a bias dictated by our own research, we focus on the effects of dietary levels of vitamin D and calcium on the intestinal mucosa in the context of higher dietary fat. An important area that will not be addressed is the complexity of genetic background that may influence how the host responds to altered levels of these nutrients.

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Links of Intestinal Tumor Models to Inflammation

In humans, chronic inflammation predisposes for development of intestinal tumors, a striking example being inflammatory bowel disease (IBD), in which elevated risk for colon cancer is a function of duration and severity of inflammation [1]. Moreover, sporadic colon cancers that do not develop as a complication of IBD are also driven by inflammation, established by the fact that regular use of nonsteroidal antiinflammatory drugs (NSAIDs) lowers mortality from sporadic colon cancers and causes regression of adenomas in familial adenomatous polyposis (FAP) patients [2]. Most experimental models of intestinal tumorigenesis in rodents have also been linked to inflammation. For example, in the classic model of AOM (azoxymethane)induced colon tumors in the mouse and rat, tumors and early aberrant crypt foci can be decreased by NSAIDs such as celocoxib [3, 4]. Furthermore, the tumor phenotype is accelerated and increased by challenging the mice with dodecyl sodium sulfate (DSS), which induces damage and dramatic inflammation in the colon [5–8]. Finally, TLR4 signaling, which mediates host intestinal response to commensal bacteria in the gut, can be inhibited by vitamin D3, which reduces colon cancer induced by the combination of AOM and DSS [9]. Genetic models of intestinal tumors confirm the linkage of tumorigenesis to inflammation. In the ApcMin/+ model of intestinal cancer, NSAIDs inhibit tumor

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development [10, 11], and depletion of mast cells, macrophages, or anti-TNFa treatment significantly suppressed polyposis in mice that inherit a mutant Apc allele [12, 13], confirming the role of macrophage-derived factors in the progression of intestinal tumors. Moreover, inactivation of glutathionine S transferase pi in ApcMin/+ mice induces the expression of proinflammatory genes in the colonic mucosa and colon tumor formation [14]. This is similar to the compromise of detoxification systems and induction of chronic low-level inflammation seen in the Muc2−/− mouse. In this mouse, the genetic inactivation of the Muc2 gene, which encodes the principal colonic mucin, eliminates the physical and chemical mucus barrier in the GI tract, and gives rise to tumors in the small and large intestine and in the rectum [15, 16]. While deficiency of the Muc2 protein does not lead to overt inflammation, analysis of mucosal cell expression profiles was consistent with a low-level, chronic inflammatory response of the mucosa [17]. A direct effect of inflammation on tumor development is seen in a new Stat3-IKO model of colon cancer [18]. Here, inactivation of Stat3 is directly targeted to macrophages, although there is also inactivation in some T- and B cells. This causes colitis of the colonic epithelium, and the development of tumors in the regions of inflammation, due to an mTOR–Stat3-mediated response of the epithelial cells [18]. Remarkably, both the inflammation and the tumors are resolved by aggressive antibiotic treatment, pointing to an important role of the intestinal microbiota in inflammatory-mediated effects on tumorigenesis [18]. Finally, a dramatic example of the association of inflammation with intestinal tumorigenesis, and its modification by diet, comes from a mouse in which the gene that encodes the fucosyltransferase Pofut1 is inactivated specifically in the intestinal tract. This enzyme (and its homologue Ofut1 in Drosophila) catalyzes the fucosylation of Notch receptors, a modification that is necessary for the efficient interaction of these receptors with Delta and Jagged ligands presented by neighboring cells. Thus, inactivation of Pofut1 leads to a very efficient elimination of signaling through all Notch receptors [19]. When targeted to the intestine, this results in a massive secretory cell metaplasia, including enormous expansion of goblet cells and the elaboration of a very thick mucus gel [20]. After about 9 months, these mice exhibit extensive inflammation throughout the large and small intestine. While its cause is unknown, this may be due to trapping of intestinal microbes in the mucus gel, thus again implicating the intestinal microbiota. In initial reported experiments, the mice were maintained on standard chow diet; under these conditions, while all mice developed inflammation, only 1 of 11 mice at 9 months exhibited a single flat adenoma [20]. However, it is now clear that maintenance of these mice on a defined AIN76A diet, or a western-style diet that is higher in fat and lower in calcium and vitamin D, causes very rapid development of inflammation, with 100% of the mice also exhibiting extensive dysplasia, adenomas, and invasive carcinomas by 3 months (Peregrina and Augenlicht, unpublished). Wild-type mice fed a high-fat diet (HFD—59% fat) exhibit elevated levels of proinflamatory cytokines, including IL6, TNF, and IL1, both in tumors and in the circulation, and enhanced IL6 production and TNR1 signaling have been shown to be required for HFD-induced tumor promotion in a mouse model of hepatocelullar carcinomas [21], consistent with the hypothesis that an HFD promotes tumorigenesis

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through its proinflammatory effect. We found that mice fed a western-style diet have increased levels of circulating IL1b, CCL2 (MCP1), and CCL5 (Rantes), but when the diet was supplemented with higher levels of vitamin D3 and calcium, the plasma levels of IL1, CCL2, and CCL5 were reduced concomitant with the elimination of dietary-induced tumor formation [22].

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The Importance of Nutrients, in Particular Vitamin D and Calcium, in Human Colon Cancer

It is clear from epidemiological data that patterns of nutrient intake have a dramatic effect on colon tumor incidence. There are numerous studies that demonstrate a relationship of dietary patterns and colon tumor incidence in different countries, with colon cancer much more frequent in developed countries in which individuals consume a “western-style” diet than in undeveloped countries [23, 24]. Furthermore, as dietary patterns change, for example, as the Japanese and the Chinese develop a more western-style culture and dietary habits, the incidence of colon cancer has increased in these populations [24]. Finally, and perhaps most impressive, is the fact that migratory populations rapidly develop the cancer profile of their new locale. Again, for example, the Japanese historically exhibited a low incidence of colon cancer and high incidence of gastric cancer. However, upon moving to the Hawaiian Islands, the incidence of colon cancer begins to rise within one generation concomitant with adoption of a western life-style, including patterns of food consumption, and within two generations the incidence of colon cancer becomes equivalent to that in US males [24]. It is difficult to attribute this rapid rise to a shift in population genetics, emphasizing the dramatic role of environmental factors in modulating tumor development. Moreover, the US population had about a tenfold higher incidence of colon cancer than did the indigenous Japanese, demonstrating that it is theoretically possible to reduce colon cancer incidence—and attendant morbidity and mortality—by 90% if it was understood what needed to be done in terms of altering nutritional patterns. However, it is also important to recognize that implementing such changes in dietary habits would require both societal and personal commitments. Interest in the impact of vitamin D in colon cancer was greatly stimulated by reports of a clear association of colon cancer incidence with latitude throughout the USA and other western societies, providing intriguing evidence linking sunlight exposure, and as a consequence, low endogenous vitamin D levels to increased risk for colon cancer [25]. There have been a great many epidemiological studies supporting this, and data in human populations that suggest low dietary vitamin D and calcium levels, which are linked physiologically, to higher incidence of colon cancer have been reviewed [26]. In this regard, it is difficult to separate the role of vitamin D from that of calcium: a principle effect of vitamin D—but certainly not the only, or even, necessarily, the most important in terms of cancer risk—is the stimulation of intestinal uptake of calcium via activation of the calcium transport

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proteins (TRPV 5/6 and calbindin D-9K). Thus, low intake of calcium induces secondary hyperparathyroidism and increases conversion of 25(OH)VitD to its active metabolite, 1,25(OH)2VitD (vitamin D3) to augment intestinal calcium absorption to maintain serum calcium levels. In addition to promoting intestinal calcium absorption, elevated parathyroid hormone increases bone resorption and can lead to decreased bone mineral density (BMD) and osteoporosis. This intimate physiological relationship between calcium and vitamin D3 levels may also be reflected in the results of cancer intervention studies in the human. Thus, it was reported that in a randomized trial of calcium supplementation for prevention of colon tumor recurrence, calcium supplementation reduced tumor recurrence only among subjects who had higher vitamin D levels, leading to the conclusion that the two nutrients together impact on functions that reduce recurrence [27]. There has been a resurgence of interest in the role of vitamin D in colon cancer based on recent observations that >80% of patients presenting with late stage colon cancer exhibit very low, and likely inadequate, levels of serum 25(OH)vitamin D and that the outcome for these patients is much poorer than for those with higher circulating vitamin D levels [28, 29]. This raises the issue of what levels of vitamin D are necessary for reducing colon cancer incidence and recurrence [26]. A recent Institute of Medicine report concluded that current levels of vitamin D and calcium intake are likely adequate for the maintenance of bone density and minimization of risk for osteoporosis and bone fractures, but hedged discussion of levels that might be necessary for other health considerations, including reduction of cancer risk [30]. Indeed, this has been a subject of considerable discussion, with some investigators concluding that levels of up to 4,000 IU of vitamin D3/day—far exceeding the RDA of 600 IU—may be necessary [31]. This is a complex issue for which conclusive data are not yet available and which also involves consideration of toxicities at higher pharmacological doses. However, it seems clear that much higher levels than the RDA are necessary to have an impact on cancer [31], with a recent metaanalysis of data suggesting that a daily intake of 1,000–2,000 IU could reduce colon cancer incidence significantly by raising 25(OH)D levels to >33 ng/mL [26]. Unfortunately, most intervention studies that have thus far tested the effects of vitamin D on polyp recurrence in human populations have been fundamentally flawed in that the population was not monitored for serum 25(OH)D levels and/or baseline levels of vitamin D intake or sun exposure, or the trials were not of sufficient duration to produce a conclusive result. Thus, conclusive evidence for the efficacy of vitamin D and for the necessity of combining it with increased calcium intake in cancer prevention is not yet available. Studies of 25(OH)D levels in the population emphasize the extent of the problem, and hence the potential impact on cancer incidence that could be achieved. According to the most recent NHANES data, only between 31 and 65% of US adults 19 years and older consume adequate calcium from food and supplement sources combined and only 22–59% consume adequate vitamin D [32]. It is of particular interest that deficient levels are seen at high frequency in pediatric populations (NHANES study, 2001–2004 [33]), and may characterize ~40% of young African-American women [34] and ~30% of young white men and women [35]. Moreover, when they give

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birth, a remarkable 50% of black women and 65% of their infants may be vitamin D deficient [36]. Additionally, 80% of nursing home residents not taking supplements are deficient by the end of winter [37], perhaps in part due to the compromised ability of the elderly to generate vitamin D utilizing sunlight. Thus, vitamin D deficiency is wide-spread in the most vulnerable segments of human populations.

4

Insight on Nutrition and Intestinal Cancer from Mouse Models

The inheritance of a mutant allele of the adenomatous polyposis coli gene (Apc) leads to the development of early and multiple intestinal tumors in the human (FAP) and in the mouse, with the number and timing of tumor development dependent upon the particular mutation in the Apc gene. This phenotype is of very high penetrance, affecting 100% of individuals or of mice that inherit the mutant allele. However, the phenotype can be dramatically modulated by both dietary and genetic means. For example, targeted inactivation of the cyclin-dependent kinase inhibitor p21Waf1/cip1 does not produce a tumor phenotype, but it accelerates and amplifies tumor development in Apc+/− mice as a function of p21 gene dosage [38]. The same is true of maintaining Apc+/− mice on a western-style diet (WD) that is higher in fat and lower in calcium and vitamin D3, at levels calculated on the basis of nutrient density to reflect levels commonly consumed by large segments of the US population [39]. Moreover, when the mice are fed the WD, similar dietary-induced increases in tumor development are seen regardless of genotype—that is, in Apc+/− mice that are either p21+/+, p21+/−, or p21−/− [38]. This indicates that the dietary and genetic modulation of intestinal tumorigenesis initiated by inheritance of an Apc mutation are additive regardless of p21 genotype and influence tumor development by distinct mechanisms. Remarkably, the dietary-induced increases were completely prevented by elevating calcium and vitamin D3 in the diet to levels associated with lower risk for colon cancer in humans, thus identifying these nutrients as key modulators of tumor development in the mouse, consistent with the epidemiological data in humans [38, 40]. Essentially the same influence of these dietary factors are seen regardless of genetic mutation that leads to intestinal tumors, including targeted inactivation of mismatch repair genes, another cdki p27Kip1, or Muc2, the gene that encodes the principle intestinal mucin [17, 41]. However, >90% of human colon cancers are not associated with inheritance of mutations in oncogenes or tumor suppressor genes. While such mutations often develop somatically in these sporadic colon tumors, these tumors generally arise after five to six decades of life. Further, during these 50–60 years, individuals adhere to different dietary patterns, involving multiple macro- and micronutrients that epidemiologically are linked to significantly elevated risk for colon cancer later in life. This raises fundamental basic and clinical questions: what is happening during this long period under the influence of different nutrients to increase the probability of a single tumor arising “stochastically” from more than one trillion cell divisions that

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take place in the mucosa over this period? When do key changes take place during this period? Can these changes be useful in evaluating risk for cancer development in individuals and hence be important factors in clinical care? Are the changes reversible, and if so, how late in life can altering dietary patterns for an individual be effective in altering risk for tumor development? Answering these questions can have a major impact on incidence and management of colon cancer. A major obstacle to addressing these issues has been the lack of mouse models that accurately reflect the etiology and development of sporadic intestinal tumors, in contrast to the commonly used genetic models in which inherited gene mutations rapidly lead to tumors. To address this, Newmark and Lipkin tested the effects of long-term feeding of a modification of the western-style diet—the NWD1—in normal C57Bl6 mice, a strain that very rarely develops intestinal tumors over its approximately 2.5 year lifespan. This diet was formulated to provide levels of intake of a number of dietary constituents that were similar, on the basis of nutrient density [42], to the levels of intake in large segments of western populations at higher risk for colon cancer (i.e., higher fat, reduced vitamin D3, calcium and donors to the single carbon pool—folate, methionine and choline). They reported that after about 1 year on the NWD1, there was an increase in the number of colonic and intestinal tumors, and that this was prevented by elevating levels of vitamin D3 and calcium in the diet to levels associated with lower risk in humans [43]. We collaborated with Lipkin and Newmark in repeating this experiment in a larger number of mice, and fundamentally obtained the same results: while mice fed control AIN76A diet rarely developed intestinal tumors, in mice fed the NWD1 tumor incidence and frequency increased by 1.5 years, and at 2 years, 25% of the mice exhibited 1–3 tumors [40] (Fig. 3.1). This incidence and frequency is similar to that seen in the general population when screened by colonoscopy after the age of 50, or about 2/3 of the organism’s life span. Moreover, the approximately 10% incidence of carcinomas among the dietary-induced tumors in the mouse was similar to that seen in the general human population when routinely screened by endoscopy [44]. Thus, based on the similar dietary etiology, tumor incidence, frequency, and histopathology, this is an important model of sporadic intestinal cancer—both of the small and large intestine—the form of the disease responsible for >90% of intestinal cancer in the USA and other western populations. Importantly, elevating calcium and vitamin D3 in the diet from the lower levels equivalent to 220 mg calcium and 200 IU vitD3 in a 2,000 kcal/day human diet, to higher levels equivalent to a human intake of 3,000 mg and 1,000 IU per day, respectively, completely prevented the dietary induction of colon and small intestinal tumors [40, 43] (Fig. 3.1). This observation was confirmed independently by another group [45]. We recently addressed the issue of differences in mechanisms by which Apc or p21 mutation, or the NWD1, affect tumor development that leads to additive effects of these stimuli [38], as we reviewed earlier. Using a well-validated method that permits isolation of cells according to their position along the crypt-luminal axis [46], we investigated the altered expression profiles in the histopathologically normal mucosa of cells isolated from the top of the villus and from the bottom of the crypt in Apc+/− mice, p21−/− mice, and control wild-type mice fed the NWD1.

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Dietary Initiation of Colon Tumors b

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Fig. 3.1 Large intestinal tumors that arise in C57Bl6 mice fed control AIN76A diet, a new westernstyle diet (NWD) or the NWD supplemented with elevated vitamin D and calcium. (a) Gross pathology of two tumors that arose after 1.5 years of feeding the NWD from weaning and histopathology of one of these; (b) tumor frequency; and (c) tumor incidence. (b, c) are data first published by Yang et al. [40]

There were several striking observations [47]. First, the alterations in cell reprogramming in both villus and crypt cells were highly distinct for each stimulus when assayed at the individual gene or functional group level. Second, while the genetic alterations (Apc+/− and p21−/− mice) primarily altered gene expression in crypt cells, feeding the NWD1 altered gene expression primarily in villus cells. These findings reflect distinct perturbations in mucosal homeostasis introduced by each of the stimuli and explain why they are additive in inducing tumors. There are a number of changes in the flat mucosa of the C57Bl6 mice consuming the NWD1, and many of these are prevented by elevating calcium and vitamin D3 in the diet (NWD2) that prevents eventual tumor development. For example, we reported that there was a downregulation of genes in the tricarboxylic acid cycle that was prevented by the elevation of vitamin D3 and calcium [40]. Additionally, we have found decreased expression of the mitochondrial genome accompanied by diminished activity of oxidative phosphorylation enzyme complexes, both of which are also eliminated by elevation of dietary vitamin D3 and calcium (Heerdt and Houston, unpublished).

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This raised the possibility that the histologically normal mucosa exhibited an early shift toward glycolysis, a metabolic adaptation known to characterize colonic and other tumors. Recent evidence from metabolomic data is consistent with this, demonstrating altered metabolic profiles and an increase in lactate production induced in the colonic mucosa by the NWD1 that can be prevented by elevating vitamin D3 and calcium (Heerdt, Gaffney-Stomberg and Augenlicht, unpublished). Moreover, it is clear that diet can rapidly alter the intestinal microbiome [48] and that when fed the NWD1, the histologically normal mucosa exhibits oxidative stress and an inflammatory response [49]. We investigated the pattern of expression changes in the mucosa induced by the NWD1 in comparison to the alterations induced by targeted inactivation of Apc or p21 [47]. We found that one of the unique characteristics of both the intestinal villi and colonic crypts of mice fed the NWD1 was the ectopic expression of markers of the Paneth cell lineage, a cell type that is normally not expressed in these compartments but only at the bottom of the small intestinal crypt. Moreover, in each case, this was accompanied by elevated expression of the Wnt receptor Fzd5, the receptor reported to be necessary for the differentiation of the Paneth cell lineage, and this was associated with elevated Wnt signaling. Finally, elevating vitamin D3 and calcium in the diet to levels that prevent the eventual development of dietary-induced tumors by the NWD1 also prevented all of these changes [47]. These data are important for a number of fundamental reasons. First, the data suggest that elevated dietary-induced risk for “sporadic colon cancer” may be assessed by expression of these aspects of the Wnt signaling pathway and/or by ectopic expression of markers of the Paneth cell lineage. Second, Lgr5+ intestinal stem cells, which are nestled at the bottom of the crypt in a manner that maximizes their heterotypic contacts with Paneth cells, in fact require signals from Paneth cells for their viability and expression of stem cell functions [50]. Thus, the ectopic expression of Paneth cell markers in the villus and colonic crypt cell compartments may also encompass expression of Paneth cell factors that cause an expansion of cells with stem-like properties in these compartments, leading to an elevated target cell population for tumor initiation and hence elevated probability of tumor development. Finally, Paneth cells are fundamental in mediating the interaction of gut commensal bacteria with the intestinal mucosa, and hence are an important determinant of if and how the mucosa mounts an inflammatory response. Moreover, dietary factors can rapidly change the composition of the gut microflora [48]. However, the extent to which these changes are mechanistically linked either to potential dietary-induced changes in the intestinal microbiome or to the dietaryinduced inflammatory response is not yet known.

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Cellular and Molecular Effects of Diet and Inflammation on Intestinal Tumorigenesis

Preclinical studies have suggested that the chemopreventive properties of vitamin D stem from its ability to inhibit proliferation and angiogenesis and to induce apoptosis, autophagy, and differentiation in tumor cells [51, 52]. Vitamin D3 has been shown

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to downregulate the expression of Toll-like receptors 2 and 4 (TLR2 and TLR4) on human monocytes resulting in hyporesponsiveness to TLR-activating ligands [53], which contributes to anti-inflammatory properties of vitamin D on which we will focus. The complex role of inflammatory cells and molecules in colon cancer has been reviewed [54, 55] and the potential of targeting the tumor microenvironment for chemoprevention has been emphasized [56]. The particular focus here will be on the anti-inflammatory activity of vitamin D in the colon and its potential role in the prevention of colon cancer. It has recently been established that >80% of colon cancer patients at diagnosis have levels of serum 25(OH)D that would be classified as insufficient to deficient and that the low vitamin D levels are associated with poor outcome [28, 29, 57]. This is also true of patients with IBD, a risk factor for colon cancer [58]. Several studies have addressed mechanisms through which vitamin D deficiency may be part of the network linking inflammation and increased risk of colon cancer. The emerging picture suggests that vitamin D effects on both intestinal epithelial and immune cells contribute to inflammatory responses that impact on tumorigenesis. Mice with a targeted inactivation of the vitamin D receptor do not develop overt inflammation, yet the presence of increased proinflammatory cytokines (TNFa and IL1b) in their colon is indicative of an increased inflammatory background in their mucosa [59]. Interestingly, in mice fed the NWD, low in calcium and vitamin D, we detected an increased circulating level of these same cytokines, as reviewed earlier. Moreover, colitis induced by DSS or by IL10 inactivation in the mouse is exacerbated by inactivating the vitamin D receptor [60, 61], and in the IL10−/− colitis model, the inflammation is moderated by elevating dietary levels of vitamin D and is further ameliorated by elevating calcium levels as well [61]. In addition, not only are T cells more readily reactive and display an inflammatory phenotype [59], but also there are fewer intraepithelial T cells with anti-inflammatory activity in VDR knockout mice due to abnormalities in T-cell homing [62]. On the other hand, VDR deficiency can also compromise mucosal barrier function due to an impairment of tight junctions [63], and this may be linked to increased levels of oxidative stress detected in the colon of VDR knockout mice [64]. Interestingly, moderate disfunction of the intestinal barrier has been documented in mice fed a high-fat diet (reviewed in [65]), while VDR polymorphisms have been linked to increased risk of IBD development [66, 67]. Taken together, these data suggest that abnormalities in levels of vitamin D or its utilization have the dual effect of not only weakening the intestinal mucosal barrier resulting in increased exposure to luminal contents and bacteria but also decreasing the level of immune tolerance resulting in increased intestinal inflammation. Among inflammatory cells in tumors and in the mucosa are macrophages, an important cell that can produce a variety of proinflammatory cytokines and that can have pronounced effects on tumor development and phenotype [68, 69]. These cells can stimulate a variety of processes that promote tumor development and progression, including cell migration, invasion, metastasis, and neovascularization [70, 71]. Klampfer and colleagues have recently defined a paracrine loop between macrophages and colon tumor cells [72–74] as a mechanism by which tumor-associated

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AN AMPLIFYING LOOP BETWEEN COLON CANCER CELLS AND MACROPHAGES AND ITS SHORT CIRCUIT BY VITAMIN D3

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macrophages can become educated by intestinal tumor cells to stimulate growth, chemotherapeutic resistance, and metastatic properties of the tumor cells (summarized in Fig. 3.2). The loop is initiated by the release of versican from the tumors which stimulates IL1b secretion from macrophages via the activation of Stat1 by phosphorylation. In turn, macrophages and IL1 stimulate epithelial cell growth by elevating Wnt signaling in tumor cells via an NFkB–AKT-dependent inhibition of GSKb phosphorylation. Similarly, TNFa activation of Wnt signaling by macrophages was identified in gastric cancer [13], and it was reported that PI3K/AKT signaling activates Wnt signaling in intestinal epithelial cells in the IL10−/− mouse model of inflammation-induced intestinal dysplasia [75]. Increased Wnt signaling in tumor cells has multiple consequences (Fig. 3.2). (a) Tumor cell growth is stimulated through the activation of growth promoting genes, such as c-myc. (b) Snail expression is stimulated, potentially mediating an epithelial–mesenchymal transition promoting progression. (c) TRAIL-induced apoptosis of tumor cells is inhibited [72–74]. Importantly, versican and Snail are Wnt target genes, establishing this as a selfamplifying loop between the epithelial cells and the macrophages. Since genetic depletion of macrophages in ApcD716 mice decreases intestinal tumors [13], this role of macrophages may be fundamental in intestinal cancer. This paracrine loop is linked to nutrition by the striking observation that the cross-talk can be short-circuited by the treatment of the macrophages with vitamin

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D3, which, through a VDR-dependent mechanism, interferes with Stat1 activity in the macrophages, and thus prevents synthesis and secretion of IL1b (Fig. 3.2). Consequently, all of the downstream effects of IL1b on the tumor cells are abrogated [72–74]. This is a new paradigm for how vitamin D may act as a chemopreventive molecule for colon cancer.

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Concluding Remarks

Although highly complex, understanding how nutrients modulate probability for development and progression of colon cancer can have profound impact on both understanding mechanisms of tumorigenesis and clinical approaches to the disease. As regards clinical implications, mouse genetic models of intestinal cancer have been extremely valuable in dissecting fundamental molecular and cellular changes necessary for tumor development—e.g., mutations or epigenetic modulation of the Apc gene and its attendant effects on Wnt signaling in colon tumorigenesis in FAP syndrome and most sporadic colon tumors, or of mismatch repair genes in hereditary non-polyposis colon cancer (HNPCC) [76–78]. These are singular accomplishments with potential high-impact for screening and therapeutic approaches. However, as previously discussed, such genetic models generally reflect how tumors arise in rare genetic risk groups with very high incidence and rapidity of disease development, rather than much more common sporadic colon cancer in which one or two tumors develop over lengthy periods (e.g., five to six decades), where environmental factors have a major impact. The early stages in the pathophysiology of the colon cancer may differ markedly in these situations. For example, as illustrated in Fig. 3.3, in mice or in Familial Polyposis patients that inherit a mutant APC allele, the mutation is present from conception, and clearly has a subtle impact on how the mucosa functions [79, 80]. At some point in individual cells, this mutation is reduced to genetic/functional homozygosity, and these focal changes than initiate the development of tumors. However, the vast majority of patients that will develop colon cancer are wild-type at the APC locus from conception, through parturition, and on into adult life. We do not know when the first mutation (or “hit”) in the APC gene happens, presumably randomly, in very few cells during the many months or decades of life in the mouse and human, respectively, nor when this is reduced to homozygosity to initiate tumor formation. The former may be only after 50% or more of the life span, and the latter likely between 1 and 10 years before the tumor becomes detectable. During this lengthy period, the mucosa is exposed continuously to nutrient influences on its function and ability to maintain homeostasis. These stimuli, in particular nutrients, can affect the composition and functioning of the intestinal microbiome, the differentiation and balance of different cell types, and metabolic adaptation. Since there are over 1012 cell divisions in the mucosa during this time, and yet only one or two will give rise to a tumor, it is clear that perturbations in the mucosa that are very subtle can be highly important in the stochastic processes that result in a clinically detectable neoplasm. Thus, the mucosa is likely primed by

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Inherited APC Mutation (Familial Adenomatous Polyposis, ApcMin mice) – 1% of CRC

conception birth 2ndhit tumor APC+/-

APC-/-

0

b

15-20 years (in the mouse, weeks)

Sporadic Colon Cancer – >90% of CRC

conception birth

1sthit

2ndhit tumor APC+/-

WT APC

APC-/-

environmental factors 0

50-60+ years (in the mouse, weeks)

Fig. 3.3 Depiction of the differences in kinetics of tumor development and underlying alterations in the APC gene in human and mice that inherit a mutation of the APC gene compared to the development of sporadic colon cancer

nutrients and other environmental and genetic influences as to the ultimate fate of mutated cells that have the potential to give rise to tumors. This is akin to the seed:soil hypothesis for metastasis, in which it is postulated that sites of cancer metastasis are adapted to provide a favorable environment for the seeding and growth of cells originating from the primary tumor. Thus, changes that are present in the mucosa long before tumors appear can be important markers of relative risk for tumor development, as well as targets for chemo- and nutritional prevention. For example, we [40, 47] and others [81–85] showed that vitamin D modulates the intensity of Wnt signaling, a major oncogenic signaling pathway in colon cancer. Human colon tumors display heterogeneous levels of Wnt activity [86] and it has been shown that only cells with higher levels of Wnt signaling display colon cancer stem cell (CSC) properties [87]. Significantly, myofibroblast-derived factors have been shown to impose the CSC phenotype by promoting Wnt signaling in tumor cells. Because CSCs mediate tumor growth and often elude therapeutic approaches, understanding how dietary factors regulate their “stemness” will help to improve clinical approaches for colorectal cancer patients. In addition to the impact that understanding these preneoplastic events can have clinically, the changes are profoundly important in understanding mechanisms that give rise to tumors. A prime example of just how interesting it can be to consider these issues comes from the study of HNPCC (Lynch syndrome). It is now understood that this syndrome is due to defects in mismatch repair, and in some populations and pedigrees, the particular gene and mutation have been identified [88–92], as well as some of the consequences of this defective repair (generation of mutations in the TGFb receptor, for example) [93]. This syndrome was first recognized in the

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early 1900s by Warthin, in considering a family in which there was a high incidence of gastric and uterine cancer [94]. However, when this family was revisited by Lynch six decades later, members exhibited a high incidence and clear inheritance of colon, not gastric, cancer [95]. Coincidentally, the major intestinal cancer in the general population had shifted over that period from gastric to colon cancer. Thus, risk was established by inherited mutation in a gene involved in mismatch repair, but environment profoundly influenced the tissue-specific penetrance of the tumor phenotype—a profound example of environmental (diet)–gene interaction that it would be fascinating to understand.

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Chapter 4

The ApcMin/+ Mouse Model to Study the Effects of Exercise on Gastrointestinal Malignancy Nathan A. Berger

Abstract Epidemiologic evidence in humans supports a strong relationship between energy balance and colorectal cancer. Obesity is associated with an increased incidence of both colon adenomas and carcinomas; physical activity and exercise are associated with decreased incidence and better prognosis. Animal models to study the association of energy balance with colorectal cancer focus on the ApcMin/+ mouse model, which carries a mutation in the Adenomatous Polyposis Coli gene, which is known to be mutated early in the development of colorectal cancer in the human hereditary disorder, Familial Adenomatous Polyposis, as well as in sporadic colon cancer. The ApcMin/+ mouse, which has provided a robust model for studying factors that affect the development of colorectal cancer, serves as the focus of this review of a mouse model to study the effects of exercise on gastrointestinal malignancy.

Extensive epidemiological evidence indicates that obesity is associated with an increased risk of colon cancer in humans (see Chap. 1) [1], whereas physical activity and exercise are associated with a decreased risk [2]. In fact, in the recent summary by the World Cancer Research Fund/American Institute for Cancer Research, colon cancer was considered the only malignancy for which there was convincing evidence that physical activity was associated with decreased risk of cancer [2]. At the same time, the evidence that physical activity decreases the risk of postmenopausal breast cancer or endometrial cancers was considered probable [2]. Epidemiological evidence also indicates that higher levels, greater frequency, and increased intensity of exercise are more protective, suggesting that physical activity exerts a dose–response effect [2]. Postulated mechanisms include reduction in insulin levels, lowered levels of circulating tumor-promoting cytokines and adipokines, reduced body fatness,

N.A. Berger, M.D. (*) Center for Science, Health and Society, Case Comprehensive Cancer Center, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106-4971, USA e-mail: [email protected] S.D. Markowitz and N.A. Berger (eds.), Energy Balance and Gastrointestinal Cancer, Energy Balance and Cancer 4, DOI 10.1007/978-1-4614-2367-6_4, © Springer Science+Business Media, LLC 2012

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decreased gut transport time, effects on endogenous steroid hormone levels, and others [2]. Overall it is estimated that, in the USA, 45% of colorectal cancers are preventable by appropriate control of food, nutrition, physical activity, and body fatness [3]. Clinical studies also indicate that physical activity affects prognosis, time to recurrence and survival in patients already diagnosed with colorectal cancer (see Chap. 9) [4]. Although epidemiological evidence for prevention is convincing and a limited number of observational studies provide support for the beneficial effects of physical activity in patients with established diagnosis of colorectal cancer, studies to understand mechanisms, dose–response, effect of different types of exercise, impact of gender, potential surrogate endpoints, biomarkers, mediators, and confounding factors are difficult to study in human populations. Moreover, controlled randomized trials on this subject are difficult, if not impossible in humans. Animal models are useful for this purpose, especially if animal tumors are (1) genetically and pathologically similar to the human condition, (2) animals are affordable, readily accessible and subject to interventions such as exercise, and (3) studies are highly reproducible. Additionally, mouse models can be readily utilized for sample collection of body fluids and cell and tissue harvesting and for systematic biochemical, molecular, physiologic and pathologic analysis, and behavioral monitoring, and manipulation. The ApcMin/+ mouse provides a frequently used and effective model for these types of studies that satisfies most of the requirements outlined above. However, although some insights regarding the effects of physical activity and exercise on colorectal cancer have been investigated using this model, studies require improved standardization to make experiments utilizing the ApcMin/+ mouse valuable in understanding the role of physical activity and exercise in disease. The Min mutation was identified in a mouse lineage derived from a mating between an ethylnitrosourea (ENU)-treated C57BL/6J (B6) male with an AKR/J female, which was then backcrossed to the B6 strain for more than ten generations. The resulting phenotype is characterized by progressive adult onset anemia, bloody feces and multiple, variable sized polyps in the small intestine, leading to intestinal obstruction and early death [5]. In this model, polyps occur also at a low frequency in the colon. Histologically these polyps may be polypoid, papillary or sessile adenomas and occasionally may progress to show small areas of adenocarcinoma in older mice; however, metastasis is generally not seen. On a fixed B6 background, heterozygous mice can develop polyps in both the small intestine and colon that can reach anywhere from 100 to 300 total polyps and typically start to appear around 5–10 weeks of age. The life span of mice with these polyps was found to average 119 ± 31 days [5]. In the original description of this chemically induced mutation, the authors noted that since all cells in the animal contained the mutated gene, but only a limited number of tumors appeared, somatic interactions must be required for tumor development [5]. The mouse gene located on chromosome 18 bearing the Min mutation was subsequently shown to be homologous to the human adenomatous polyposis coli (APC) gene, located on human chromosome 5 [6–10]. The human APC gene contains the germ-line mutation responsible for familial adenomatous polyposis

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(FAP), an inherited cancer syndrome in which patients develop multiple intestinal adenomatous polyps, which if not treated, can progress to colon adenocarcinomas [10]. The human APC gene is also mutated early in the course of most cases of sporadic intestinal cancer [11]. These observations suggest that the mouse ApcMin/+ model should be useful to study human colon cancer, especially in its early stages and in prevention studies. The Min mutation in mice results in a nonsense mutation in codon 850 of APC, resulting in a truncated inactive peptide [10, 12] similar to most common human mutations of APC. Phenotypic expression of the genetic ApcMin/+ mutation requires, in most cases, inactivation of the normal Apc allele, which may occur by deletion or mutation [13]. A small percentage of genetic ApcMin/+ mice may develop phenotypic expression without apparent loss of the normal allele, potentially in association with other factors that result in allelic suppression [13]. A series of nonallelic modifiers have been identified, modifiers of Min, (Mom) that may enhance or suppress polyp number or growth [14]. Under normal physiologic conditions, the protein product of the APC gene serves as a tumor suppressor in the Wnt signaling pathway. The APC protein complexes with several cytoplasmic proteins including beta-catenin that target the latter for cytoplasmic degradation in a ubiquitin-dependent process [15]. When APC is inactive as in the case of mutation, beta-catenin accumulates in the cytoplasm and translocates to the nucleus where it can activates transcription factors involved in cell proliferation, growth and initiation of the intestinal polyp process. When the mutated APC gene is present in the heterozygous state, the cell must lose or inactivate the normal APC allele, before its tumor suppressor function is lost [13]. This requirement for loss of heterozygosity contributes to the lag time required for polyp development in ApcMin/+ mice. Other Apc mutant mice have been developed with similar predisposition to developing intestinal malignancies. In addition to the chemically induced mutation resulting in the Apcmin/+ model, genetic knock out approaches have been used to generate the ApcD716 and the Apc1638N models, with truncation mutations at codons 716 and 1638, respectively [15]. Although the mouse strains produced by knockout technology develop intestinal polyps, the ApcD716 develop many more, approximately 300 polyps, and the Apc1638N develops significantly fewer, approximately three polyps, compared to the ApcMin/+, which most commonly develops approximately 100 polyps on the B6 background . While each of these mutant mouse models have been used in different experimental situations, the ApcMin/+ mice have been the most prevalent model used to study exercise and will serve as the focus of this review. The ApcMin/+ mouse is valuable in that the intestinal phenotype recapitulates the progression from early mutation and polyp development to adenocarcinoma of the colon in humans. Heterozygous mice with Min mutation are fertile, have no apparent embryonic defects, and undergo normal development and maturation. Polyp growth in the ApcMin/+ mice can be evaluated for location and size, in terms of length, diameter, area, and volume as an indicator of tumor burden. Because the polyps can be evaluated quantitatively, the ApcMin/+ mouse provides a useful model to study numerous aspects of intestinal neoplasia that otherwise could not be investigated in humans.

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Fig. 4.1 Schematic illustration of theoretical possibilities for alteration of intestinal polyp numbers as a function of time by factors affecting polyp development and numbers in the ApcMin/+ mouse model. No specific polyp numbers or times are indicated since these are theoretical constructs

In addition to enumerating polyp number, the mice can be used to study genetics, biochemistry, molecular biology, histology, immunology, metabolic pathways and alterations of polyp development as well as factors affecting progression and prevention of adenocarcinoma as well as survival. As indicated above, polyp number, size, and location in the ApcMin/+ mouse can be used to evaluate factors affecting colon cancer growth. Figure 4.1 illustrates some of the ways that interventions can potentially affect polyp growth. Curve 1 illustrates the normal course of polyp development in the ApcMin/+ mouse model. Polyps are usually detected after a lag period of 30–60 days from birth. Polyp numbers increase over 30 days to a relatively characteristic number which remain constant in number or increase slowly while they undergo progressive growth leading to death of the animal. Thus, the ApcMin/+ mouse model can be used to study factors affecting tumor initiation as reflected by polyp numbers or tumor progression as reflected by polyp size. Factors affecting polyp development could function in several different ways, each of which may generate different growth curves and have different mechanistic implications. As shown in curve 2, a factor could suppress the total number of polyps that occur without affecting lag time or initial rate of appearance. Curve 3 shows a situation where the rate of polyp appearance is slower but the total number of polyps unaffected. Curve 4 illustrates a condition where polyps develop more slowly and fewer polyps occur. Curve 5 shows a condition where polyp development undergoes a prolonged lag time but then reaches the same characteristic level as in Curve 1. Examination of the theoretical curves presented in Fig. 4.1 demonstrates that different conclusions can be drawn if observations are performed at different times.

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Thus, polyp count at an early time point would indicate significant differences between curve 1, 3, and 5. In contrast, evaluation at an early time point would suggest no difference between curves 1 and 2; however, later examination would show significant differences between these two curves. Similar differences could occur with curves 3 and 4. The potential variation in shape, slope, and plateau in the polyp growth curve indicates that comparative studies should consist of multiple time points, including at least one that reflects rate of appearance and another that reflects steady state or plateau level. This is significant because it suggests differences in mechanism, i.e., the differences between polyp initiation and progression, decreased polyp development, delayed polyp growth, etc. The ApcMin/+ mouse model has been useful to identify and investigate the role of beta-catenin [15], p53 [16], Smads [17], prostaglandins [18], and prostaglandin dehydrogenase pathways [19], the tumor-promoting effects of cytokines, hormones, and growth factors such as insulin, leptin, and IGFs as well as the effects of different dietary components [20–25]. In addition, this mouse model system has also been useful to investigate the chemopreventative effects of agents such as soybeanderived Bowman-Birk Protease Inhibitor [26], Silibinin [27], and NSAIDS including aspirin [28], sulindac [29], and celecoxib [30]. These studies with ApcMin/+ mice have provided important preclinical evidence for the colorectal cancer preventative efficacy of multiple agents. While epidemiologic evidence supports a beneficial effect of physical activity and exercise on colorectal cancer, the availability of the ApcMin/+ mouse model provided an important tool to directly test the benefits and potential mechanisms of action by which exercise may affect colorectal cancer prevention or development. In one of the first applications of the ApcMin/+ mouse to study the effects of exercise in colorectal cancer, Colbert et al. [31] conducted a 7-week exercise regimen, starting 21 days after birth, and administered as treadmill running for 1 h/day, 5 days/week at speeds of 18–21 m/min, for a total of 1.0–1.36 km/day on a 5% grade. After the exercise program was completed, there was no observed exercise associated decrease in polyp number in female mice (38.0 ± 6.3 vs 40.0 ± 6.3). However, exercise in male mice resulted in a trend, although not significant, toward decreased polyps in the exercised compared to control mice (24.8 ± 3.6 vs. 36.8 ± 4.9). The exercise resulted in no effect on body weight or food intake; however, soleus muscle in the exercised mice showed an increased oxidative capacity as reflected by a 1.64-fold increase in citrate synthetase [31]. Thus although the increased physical activity was shown to have a significant effect on skeletal muscle, the effect on polyp formation did not reach statistical significance. Colbert et al. [32] subsequently showed that greater levels of exercise, approximately 3.8 km/day, achieved by providing mice 24 h access to a running wheel along with caloric restriction, the latter achieved by adjusting food intake to equal that consumed by control mice during the previous week, resulted in lower weight in the exercise plus caloric restriction vs. control mice and fewer polyps, 16.9 ± 2.6 in the experimental group vs. 21.6 ± 1.5 in the control mice. Moreover, for individual mice, polyp number was inversely correlated with total running/day. Thus the combination of exercise plus caloric restriction appeared to decrease polyp development

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and showed dose dependency relative to duration of exercise. However, other studies have shown that caloric restriction alone reduces polyp number in the ApcMin/+ mouse model and no internal controls were performed in these experiments to quantitate the effect of caloric restriction alone [33], thus those experiments did not distinguish between an effect of exercise vs. caloric restriction in reducing polyp number [32]. In studies of potential mechanisms, the authors showed that exercised mice had greater serum levels of IGF-1 levels compared to control mice, but there was no correlation with polyp number [32]. However, no assays were performed to quantitate IGF-1-binding protein levels to more appropriately estimate biologically active, unbound or free IGF-1. Notably, even the control mice in this experiment [32] had fewer polyps than the controls in the previous experiment (21.6 ± 1.5 vs. 36.8 ± 4.9) [31]. These differences may be associated with differences in experimental design including different diets, Tekland, in the earlier experiments vs. AIN 76A in the latter, different duration, 10 weeks in the former vs. 17 weeks in the latter. In addition, mice used in the latter experiment were maintained under quarantine conditions for at least 3 weeks with unspecified diet and unknown treatment [32]. Meanwhile, Mehl et al. [34] studied the effect of different exercise regimens in the ApcMin/+ mouse model by comparing treadmill running and voluntary wheel running to non-exercised controls starting at 3.5 weeks. Polyps were evaluated after a 9-week exercise period during which animals were provided with free access to running wheels vs. 1 h of treadmill running at 18 m/min on a 5% grade for 6 days weekly and ad libitum access to the Harland Tekland Diet #8604, 4.7% fat. Despite more exercise by the mice with voluntary access to the running wheel, 4.7 km/day for males and 3.5 km/day for females compared to 1.1 km/day for treadmill runners, there was no difference in body weight among males or females. In these experiments [34], male treadmill runners showed a 29% decrease in intestinal polyp count, whereas voluntary wheel runners, with greater exercise, showed no significant decrease in polyps. Female mice showed no significant difference in polyp count relative to controls with either mode of exercise. Both males and females showed significant reduction in circulating IL6 and wheel runners showed a greater increase in muscle citrate synthetase than treadmill runners compared to controls. Thus this set of experiments did show a protective effect of exercise in males but did not show the expected dose-dependent protection. Rather, they suggest that lesser amounts of exercise in the form of treadmill running are more effective at preventing polyp development than the fourfold exercise increase associated with voluntary wheel running. Moreover, this experiment suggests that exercise is effective for polyp prevention in male but not female mice. Interestingly, control mice in these experiments showed between 90 and 100 polyps compared to the 21.6 in the previously reported experiments. However, as noted in the previous experiment, wheel running was accompanied by caloric restriction which may have contributed to the reduced polyp number [32]. Moreover, control animals in the present experiment were fed the Harland Tekland Diet #8604, whereas in the latter experiment they were fed the AIN 76 A which, although similar in fat content (4.7% vs. 5%) may still contribute to the difference between polyp counts in control animals from the two different experiments.

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While the discrepancy between the mice subjected to treadmill running vs. voluntary wheel running was not resolved, Baltgalvis et al. [35] conducted studies of inflammation, apoptosis, and b-catenin signaling in polyps derived from the experiments reported by Mehl, which showed 29% decrease in polyp counts in males associated with treadmill running [34]. These experiments showed decreased macrophages, decreased TUNEL positive cells, and decreased Bax staining in polyps derived from exercise mice. Exercise had no effect on the number of COX-2 or b-catenin positive cells; however, there was an increase noted in phosphorylation of b-catenin [35]. These observations suggest that exercise impacts multiple pathways affecting polyp growth including reduced immune cell infiltration, apoptosis, and Wnt signaling. Of concern in this report is the fact that the investigators added an additional series of animals including seven controls and eight exercise mice to provide fresh tissue for protein assays; however, there were no reports of the effect of exercise in these additional animals [35]. To further define the combined effects of diet and exercise on polyp growth, Baltgalvis et al. [34] compared treadmill running exercise to control sedentary behavior in ApcMin/+ mice that were fed either the AIN 76A diet containing 5% fat vs. a Western Diet containing 21% fat. The treadmill exercise regimen, 18 m/min, 60 min/day, 6 days/week was the same as noted above [35] and both diet and exercise were started at 4 weeks of age and continued until 10 weeks of age at which time all mice were sacrificed and studied. Control mice fed the western diet tended to be 5% heavier than those fed the AIN 76A diet and showed greater weight of the epididymal fat pad. Neither weight nor epididymal fat pad mass were affected by exercise. Exercise reduced leptin levels in the high-fat Western diet fed mice but not in those fed AIN 76A. The control sedentary mice, on the high-fat Western diet showed 67% increase in intestinal polyps compared to control mice fed AIN 76A (approximately 35 vs. 20). Interestingly, exercise did not decrease polyp number in mice fed the Western diet. In fact, exercise plus high-fat diet actually increased polyp number (approximately 42 compared to 35 in high-fat diet non-exercise mice). The same exercise regimen decreased polyp number by 50% (10 vs. 20) in the mice fed the AIN 76A diet. This decrease was mostly associated with small polyps (less than 1 mm). These studies confirm that moderate intensity treadmill exercise can reduce polyp numbers in ApcMin/+ mice, especially those on a low-fat diet. In contrast, in these experiments, high-fat diets (21%) both increase polyp count in sedentary animals and overcome the beneficial effects of exercise in animals subjected to a moderate intensity exercise regimen. It is noteworthy that in the present study, polyps were counted at 10 weeks of age compared to 13 weeks of age in previous experiments. These differences in experimental duration result in evaluations being performed at different locations along the horizontal axis in Fig. 4.1 and may contribute to differences in experimental results and interpretation. In a series of experiments with female ApcMin/+ mice, Ju et al. [35] evaluated the effect of voluntary wheel running, approximately 2.8–3.9 km/mouse/day on mice fed a normal AIN 93G or high-fat diet, AIN 76A supplemented with 20% fat. Groups of mice on the normal diet (AIN 93G) exposed to the exercise wheel for 6 weeks showed a decrease in omental fat pad mass relative to control mice and

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showed 29–31% decrease in small intestinal polyps. When mice on the high-fat diet and a 9-week exercise program were sacrificed at 14 weeks of age, they showed decreased circulating IGF-1 and increased IGFBP-3 resulting in lower levels of bioactive IGF-1 and decreased polyps associated with exercise vs. control 30.8 ± 3.4 vs. 22.0 ± 3.8. Thus, these experiments indicate that voluntary wheel running reduced polyp development in female ApcMin/+ mice on both normal fat (5%) and high-fat diets (20%) [37]. In additional experiments, these investigators showed that voluntary wheel running in male CF-1 mice maintained on a high-fat diet and treated with a chemical carcinogen, azoxymethane (AOM) plus dextran sulfate sodium (DSS) protocol, resulted in decreased intestinal tumor formation relative to nonexercised controls [37]. In another comparison of a fixed exercise regimen, using treadmill running vs. free access to a running wheel, Basterfield and Mathers [38] found that intestinal polyp numbers showed no significant difference between stationary, treadmill and wheel running mice, although treadmill running mice showed a tendency to lower numbers of larger polyps (>2 mm). In these experiments, female wheel running mice were reported to run approximately twice as far as males; however, both genders were combined for analysis. In addition, mice were analyzed together on an “intention to treat” approach, independent of actual exercise activity, thereby potentially nullifying any exercise associated events by combining animals with variable exercise intensities. Moreover, in these experiments, any potential effects of exercise may have been counteracted by the high-fat western style diet. In addition, since polyps were enumerated at 17 weeks of age (regimens started 5 weeks of age and continued for 12 weeks before sacrifice), polyp growth may have proceeded far enough along the horizontal axis in Fig. 4.1 to a point where polyp numbers equalize and plateau. A further problem with this report relates to the low mean numbers of polyps in all mice ranging from 11.0 in treadmill runners to 14.1 in controls. This is significantly lower than the 30–100 polyps usually found in ApcMin/+ mice on the C57BL/6 background and suggests the presence of polyp suppressors in the diet. Overall, these experiments demonstrate that exercise can reduce the development of intestinal polyps in the ApcMin/+ mouse model. Results, however, remain inconsistent. Different experiments suggest that both treadmill running [31, 33, 36, 37] and voluntary wheel running [32, 37] are effective at preventing polyp development, while other experiments suggest that they are not. Some experiments, but not all, indicate that exercise protects against polyp development in both male and female mice [37], whereas others discriminate between genders [31, 33]. Caloric restriction together with exercise was shown to reduce polyp development, although these experiments did not distinguish the independent contribution of each component [32]. In contrast, high-fat diets were shown to increase polyp count in one experiment and to interfere with exercise prevention of polyp development in male mice [35]. Another report demonstrated that exercise does prevent polyp formation in both female and male mice fed a high-fat diet [37]. At least, one experiment suggested dose dependency of the polyp preventative effects of exercise, polyp count was inversely related to total running/day [32], whereas another experiment suggested the absence of dose dependence, treadmill running mice that ran ~1 km/day

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showed reduced polyp numbers compared to wheel running mice at ~3.8 km/day which showed no reduction in polyp number [37]. At least one report suggested that exercise affects polyp progression but not total number [37]. The ApcMin/+ mouse continues to provide an interesting model system to study the impact of exercise on intestinal neoplasia; however, experimental conditions need to be refined to assure reproducibility and remove inconsistencies. Clarification is needed of the variable response to different types of exercise, treadmill running vs. voluntary wheel running, dose dependency, apparent gender differences, and effects of differential dietary components including quantity and quality of fat and other components such as antioxidants, anti-inflammatory agents and inhibitors of prostaglandin synthesis. Each of the latter have been shown to have profound effects on polyp initiation and/or progression. In addition, time of intervention and evaluation in the animal’s lifespan is important since as suggested in Fig. 4.1, variable timing may have differential effects on polyp latency, development, or plateau. Starting experiments 3, 4, or 5 weeks after birth will result in different stages and numbers of polyps developed before experimental interventions are ever initiated. Moreover, in the experiments reported above, polyp evaluations were carried out at different times including 10, 11, 13, 14, and 17 weeks of age. Since as indicated in Fig. 4.1, these variable ages at time of evaluation occur at different points along the horizontal axis, they may mask some of the differences that would be observed if all evaluations were performed at a uniform time point. Thus, the experimental approach would be improved by sampling both at an early time point to demonstrate differences in polyp development and at a later point to demonstrate differences in plateau levels. Finally, analyzing the effect of the exercise intervention on survival would provide useful information about the effects of exercise on prevention and progression of established tumors. Acknowledgment Support for this work was derived in part from NIH Grants U54 CA116867 and P30 CA043703 to Nathan A. Berger. We thank Stephanie Doerner for assistance with the figure.

References 1. Nock NL (2012) Obesity and gastrointestinal cancers: Epidemiology. In: Markowitz SD, Berger NA (eds) Energy balance and gastrointestinal cancer. Springer, New York, pp 1–22 2. World Cancer Research Fund (2007) Physical activity. In: World Cancer Research Fund (ed) Food, nutrition, physical activity, and the prevention of cancer: a global perspective. WCRF/ AICR, Washington, DC, pp 198–209 3. World Cancer Research Fund (2007) The case for action. In: World Cancer Research Fund (ed) Policy and action for cancer prevention: food, nutrition, and physical activity: a global perspective. American Institute for Cancer Research, Washington, DC, pp 12–28 4. Meyerhardt J (2012) Effects of physical activity and other modifying host factors on colon cancer. In: Markowitz SD, Berger NA (eds) Energy balance and gastrointestinal cancer. Springer, New York, pp 141–156 5. Moser AR, Pitot HC, Dove WF (1990) A dominant mutation that predisposes to multiple intestinal neoplasia in the mouse. Science 247(4940):322–324

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6. Cormier RT, Dove WF (2000) Dnmt1N/+ reduces the net growth rate and multiplicity of intestinal adenomas in C57BL/6-multiple intestinal neoplasia (Min)/+ mice independently of p53 but demonstrates strong synergy with the modifier of Min 1(AKR) resistance allele. Cancer Res 60(14):3965–3970 7. Halberg RB, Chen X, Amos-Landgraf JM, White A, Rasmussen K, Clipson L, Pasch C, Sullivan R, Pitot HC, Dove WF (2008) The pleiotropic phenotype of Apc mutations in the mouse: allele specificity and effects of the genetic background. Genetics 180(1):601–609 8. Luongo C, Gould KA, Su LK, Kinzler KW, Vogelstein B, Dietrich W, Lander ES, Moser AR (1993) Mapping of multiple intestinal neoplasia (Min) to proximal chromosome 18 of the mouse. Genomics 15(1):3–8 9. Groden J, Thliveris A, Samowitz W, Carlson M, Gelbert L, Albertsen H, Joslyn G, Stevens J, Spirio L, Robertson M et al (1991) Identification and characterization of the familial adenomatous polyposis coli gene. Cell 66(3):589–600 10. Su LK, Kinzler KW, Vogelstein B, Preisinger AC, Moser AR, Luongo C, Gould KA, Dove WF (1992) Multiple intestinal neoplasia caused by a mutation in the murine homolog of the APC gene. Science 256(5057):668–670 11. Fodde R (2002) The APC gene in colorectal cancer. Eur J Cancer 38(7):867–871 12. Su LK, Johnson KA, Smith KJ, Hill DE, Vogelstein B, Kinzler KW (1993) Association between wild type and mutant APC gene products. Cancer Res 53(12):2728–2731 13. Shoemaker AR, Luongo C, Moser AR, Marton LJ, Dove WF (1997) Somatic mutational mechanisms involved in intestinal tumor formation in Min mice. Cancer Res 57(10):1999–2006 14. McCart AE, Vickaryous NK, Silver A (2008) Apc mice: models, modifiers and mutants. Pathol Res Pract 204(7):479–490 15. Taketo MM (2006) Wnt signaling and gastrointestinal tumorigenesis in mouse models. Oncogene 25(57):7522–7530 16. Patel AC, Nunez NP, Perkins SN, Barrett JC, Hursting SD (2004) Effects of energy balance on cancer in genetically altered mice. J Nutr 134(12 Suppl):3394S–3398S 17. Sodir NM, Chen X, Park R, Nickel AE, Conti PS, Moats R, Bading JR, Shibata D, Laird PW (2006) Smad3 deficiency promotes tumorigenesis in the distal colon of ApcMin/+ mice. Cancer Res 66(17):8430–8438 18. Wang D, Wang H, Shi Q, Katkuri S, Walhi W, Desvergne B, Das SK, Dey SK, DuBois RN (2004) Prostaglandin E(2) promotes colorectal adenoma growth via transactivation of the nuclear peroxisome proliferator-activated receptor delta. Cancer Cell 3:285–295 19. Myung SJ, Rerko RM, Yan M, Platzer P, Guda K, Dotson A, Lawrence E, Dannenberg AJ, Lovgren AK, Luo G, Pretlow TP, Newman RA, Willis J, Dawson D, Markowitz SD (2006) 15-Hydroxyprostaglandin dehydrogenase is an in vivo suppressor of colon tumorigenesis. Proc Natl Acad Sci USA 103(32):12098–12102 20. Mai V, Colbert LH, Perkins SN, Schatzkin A, Hursting SD (2007) Intestinal microbiota: a potential diet-responsive prevention target in ApcMin mice. Mol Carcinog 46(1):42–48 21. Wang B, Bobe G, LaPres JJ, Bourquin LD (2009) High sucrose diets promote intestinal epithelial cell proliferation and tumorigenesis in APC(Min) mice by increasing insulin and IGF-I levels. Nutr Cancer 61(1):81–93 22. Hassan AB, Howell JA (2000) Insulin-like growth factor II supply modifies growth of intestinal adenoma in Apc(Min/+) mice. Cancer Res 60(4):1070–1076 23. Fenton JI, Hursting SD, Perkins SN, Hord NG (2006) Interleukin-6 production induced by leptin treatment promotes cell proliferation in an Apc (Min/+) colon epithelial cell line. Carcinogenesis 27(7):1507–1515 24. Mutanen M, Pajari AM, Oikarinen SI (2000) Beef induces and rye bran prevents the formation of intestinal polyps in Apc(Min) mice: relation to beta-catenin and PKC isozymes. Carcinogenesis 21(6):1167–1173 25. Tammariello AE, Milner JA (2010) Mouse models for unraveling the importance of diet in colon cancer prevention. J Nutr Biochem 21(2):77–88 26. Kennedy AR, Beazer-Barclay Y, Kinzler KW, Newberne PM (1996) Suppression of carcinogenesis in the intestines of Min mice by the soybean-derived Bowman-Birk inhibitor. Cancer Res 56(4):679–682

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27. Rajamanickam S, Kaur M, Velmurugan B, Singh RP, Agarwal R (2009) Silibinin suppresses spontaneous tumorigenesis in APC Min/+ mouse model by modulating beta-catenin pathway. Pharm Res 26(12):2558–2567 28. Barnes CJ, Lee M (1998) Chemoprevention of spontaneous intestinal adenomas in the adenomatous polyposis coli Min mouse model with aspirin. Gastroenterology 114(5):873–877 29. Beazer-Barclay Y, Levy DB, Moser AR, Dove WF, Hamilton SR, Vogelstein B, Kinzler KW (1996) Sulindac suppresses tumorigenesis in the Min mouse. Carcinogenesis 8:1757–1760 30. Jacoby RF, Seibert K, Cole CE, Kelloff G, Lubet RA (2000) The cyclooxygenase-2 inhibitor celecoxib is a potent preventive and therapeutic agent in the Min mouse model of adenomatous polyposis. Cancer Res 60(18):5040–5044 31. Colbert LH, Davis JM, Essig DA, Ghaffar A, Mayer EP (2000) Exercise and tumor development in a mouse predisposed to multiple intestinal adenomas. Med Sci Sports Exerc 32(10):1704–1708 32. Colbert LH, Mai V, Tooze JA, Perkins SN, Berrigan D, Hursting SD (2000) Negative energy balance induced by voluntary wheel running inhibits polyp development in APCMin mice. Carcinogenesis 27(10):2103–2107 33. Mai V, Colbert LH, Berrigan D, Perkins SN, Pfeiffer R, Lavigne JA, Lanza E, Haines DC, Schatzkin A, Hursting SD (2003) Calorie restriction and diet composition modulate spontaneous intestinal tumorigenesis in Apc(Min) mice through different mechanisms. Cancer Res 63(8):1752–1755 34. Mehl KA, Davis JM, Clements JM, Berger FG, Pena MM, Carson JA (2005) Decreased intestinal polyp multiplicity is related to exercise mode and gender in ApcMin/+ mice. J Appl Physiol 98(6):2219–2225 35. Baltgalvis KA, Berger FG, Pena MM, Davis JM, Carson JA (2008) Effect of exercise on biological pathways in ApcMin/+ mouse intestinal polyps. J Appl Physiol 104(4):1137–1143 36. Baltgalvis KA, Berger FG, Pena MM, Davis JM, Carson JA (2009) The interaction of a highfat diet and regular moderate intensity exercise on intestinal polyp development in Apc Min/+ mice. Cancer Prev Res (Phila) 2(7):641–649 37. Ju J, Nolan B, Cheh M, Bose M, Lin Y, Wagner GC, Yang CS (2008) Voluntary exercise inhibits intestinal tumorigenesis in Apc(Min/+) mice and azoxymethane/dextran sulfate sodium-treated mice. BMC Cancer 8:316 38. Basterfield L, Mathers JC (2010) Intestinal tumours, colonic butyrate and sleep in exercised Min mice. Br J Nutr 104(3):355–363

Chapter 5

Obesity and the Pathogenesis of Barrett’s Esophagus Rom Leidner and Amitabh Chak

Abstract Barrett’s esophagus (BE), an intestinal-type metaplasia of normal esophageal squamous epithelium, is the only known precursor of esophageal adenocarcinoma (EAC). This is the protean reason for interest in BE as a clinical entity, as 95% of BE patients die of other causes. EAC has risen at a rate of >350% since the mid1970s, faster than any other cancer in the USA. This has been termed the “EAC epidemic” and has tracked in parallel with rising rates of obesity. Gastroesophageal reflux disease (GERD) is widely accepted as the principle cause of BE. Obesity is the leading modifiable risk factor for BE and GERD. Obesity is also the leading modifiable risk factor for EAC, associated with a threefold increased risk of cancer. Central obesity has consistently correlated with BE and BMI has not. This may explain a significant skew toward white males, but trends are actually rising in all demographic groups.

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Introduction

1. Barrett’s esophagus (BE), an intestinal-type metaplasia of normal esophageal squamous epithelium, is the only known precursor of esophageal adenocarcinoma (EAC). This is the protean reason for interest in BE as a clinical entity. 95% of BE patients die of other causes. 2. Gastroesophageal reflux disease (GERD) is the leading risk factor for BE. Obesity is the leading modifiable risk factor for BE and is independent of GERD. Obesity is also the leading modifiable risk factor for GERD and EAC, associated with a threefold increased risk of EAC. 3. Central obesity, most often measured by waist-to-hip ratio (WHR), has consistently correlated with BE and body mass index (BMI) has not. This may explain skew in incidence toward white males, but trends are actually on the rise in all groups. R. Leidner , M.D. (*) • A. Chak, M.D. (*) Department of Medicine, Case Western Reserve University, Cleveland, OH, USA e-mail: [email protected]; [email protected] S.D. Markowitz and N.A. Berger (eds.), Energy Balance and Gastrointestinal Cancer, Energy Balance and Cancer 4, DOI 10.1007/978-1-4614-2367-6_5, © Springer Science+Business Media, LLC 2012

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4. EAC has risen at a rate of >350% since the mid-1970s, faster than any other cancer in the USA. This has been termed the “EAC epidemic.” 5. Parallel increases in incidence of EAC and obesity have been observed since the 1980s, and this is also the case for GERD. It is impossible to know, however, if BE is also tracking with obesity because population screening for BE does not exist. 6. The true incidence of BE is unknown, but is believed to be increasing at a rate which might, if known, account for the “EAC epidemic.” 7. A well-recognized cancer progression model exists for EAC which is known as the M-D-A sequence. Unlike progression models in nearly all other organs, the EAC sequence does not begin with hyperplasia. The initial and hallmark event is metaplasia (BE), which subsequently proceeds through grades of dysplasia and then adenocarcinoma. The genetic, epigenetic, and molecular basis of this progression is an area of intense research focus and must be rooted in an understanding of the metaplastic initiating event. 8. Unlike preneoplastic precursors of other cancers (polyps and nevi), BE cannot be readily excised and therefore must be monitored clinically for evidence of progression by interval endoscopy and biopsy. As such, this an ideal in vivo model for the study of tumorigenesis. 9. Roughly 40% of patients with BE or EAC do not report reflux symptoms prior to diagnosis. There is no evidence that endoscopic screening improves early detection of BE prior to EAC diagnosis. Most EAC patients survive less than 1 year after diagnosis. 10. Prevention strategies will be most effective at the earliest stage of this progression e.g., preventing emergence of metaplasia, rather than preventing progression of metaplasia once established.

2

Definition: Barrett’s Esophagus

Barrett’s esophagus (BE) is an acquired metaplastic transformation of the mucosal lining of the distal esophagus from a normal stratified (multilayered) squamous epithelium to a single-layered intestinal-type columnar epithelium (intestinal metaplasia). The microscopic hallmark of BE is the presence of single-layered acid mucin-containing goblet cells (Fig. 5.1), which stain intensely blue with Alcian blue pH 2.5 stain, proximal to the normal gastroesophageal junction [1, 2].

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GERD and BE

The strongest risk factor for BE is chronic GERD, conferring a fivefold increased risk [3, 4]. GERD is estimated to affect roughly one in five individuals in the Western World [5]. Inflammation resulting from chronic acid reflux in GERD is widely accepted to be the principal cause of BE [6]. A community-based endoscopic study

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Fig. 5.1 Metaplastic intestinal type epithelium with characteristic goblet cells seen in Barrett’s esophagus

from the Portland, Oregon VA observed a dose–response relationship between duration of GERD symptoms and development of BE. In individuals with 1–5 years of symptoms, BE was threefold more common compared with individuals with less than 1 year of symptoms. BE was fivefold more common in individuals with 6–10 years of symptoms and 6.4-fold more common in individuals with >10 years of symptoms (p < 0.001) [7]. A recent meta-analysis of 26 studies concluded that overall, symptomatic GERD is associated with a threefold increase in BE (p = 0.001, 95% CI 1.9–4.5), and a fivefold increase for long-segment BE (LSBE) in particular [3]. However, only 10% of patients with long-standing GERD symptoms will ultimately develop BE, suggesting that cofactors beyond acid reflux are also involved in the pathogenesis of BE [8]. A Swedish study in a random sample of 1,000 individuals who consented to undergo screening endoscopy and biopsy, found 16 cases of incident BE. Seven of these 16 subjects (44%) reported no history of reflux symptoms in a pre-procedure questionnaire [9]. Of the five individuals with LSBE (>3 cm), 80% reported reflux symptoms and 60% had evidence of esophagitis, whereas only 46% of the 11 individuals with short-segment BE reported reflux symptoms and esophagitis was seen in only 9% of these cases. These data suggest that a complex set of biologic interactions is likely involved in what might otherwise appear to be, on the surface, a straightforward epidemiologic risk association between GERD and BE.

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4 The Cancer Progression Model of Esophageal Adenocarcinoma A two phase cancer progression model, composed of an initial sequence from GERD to intestinal metaplasia (BE) and a later, somewhat delayed, sequence from BE to dysplasia to EAC is widely accepted as the predominant model of the natural history of EAC. This is based on the following observations. BE is the only known precursor to EAC and individuals with BE have a 30- to 100-fold increased risk of developing EAC as compared to the general population [10]. Symptomatic GERD, which along with obesity, is intimately involved in the initiating metaplastic transformation of stratified squamous esophageal epithelium to a specialized singlelayered intestinal metaplasia characteristic of BE, was definitively correlated to risk of EAC in the landmark 3-year nationwide population-based case–control Swedish study reported by Lagergren et al. in 1999 [11]. In this study, a 7.7-fold increased overall risk of EAC was determined for individuals with GERD (95% CI 5.3–11.4) and a dose–response relationship was demonstrated, such that for individuals with long standing and severe reflux in the highest tertile, based on a composite score of frequency of heartburn and/or regurgitation during the day and at night, a 43.5-fold increased risk of EAC was observed (95% CI 18.3–103.5). Esophagectomy specimens characteristically reveal a field of intestinal metaplasia within which an EAC tumor arises [12]. A stepwise cancer progression model of EAC has long been recognized histologically, with evidence of sequential development from metaplasia (BE) to dysplasia and ultimately adenocarcinoma. This is pathologically termed the “M-D-A” sequence. It is fundamentally important to note that unlike cancer progression models in nearly every other organ, characterized by hyperplasia as the initiating event, this model starts with metaplasia, itself a genetically regulated transformation, from which subsequent genetic changes are believed to accumulate and ultimately progress to cancer. The key crossroads, for cancer prevention, therefore, may ultimately lie much further upstream and depend on understanding the cofactors which modulate an initial metaplastic committing event. This, in the main, accounts for the intense interest in BE as a clinical entity.

5

Prevalence of BE and EAC

The incidence of EAC has dramatically increased in recent decades in the Western World, rising over >350% in the USA since the 1970s, more rapidly than any other cancer [4, 13]. At the same time, the predominant histology has reversed from squamous cell carcinoma, located in the upper two-thirds of the esophagus, to adenocarcinoma, located in the distal third of the esophagus. The causes of this dramatic change in epidemiology, sometimes termed the “EAC epidemic” are unknown, but it is well recognized that this trend tracks directly with rising obesity rates (Fig. 5.2) [14]. The incidence of GERD has also increased in step with rising obesity rates [15].

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Fig. 5.2 Ecological study showing the correlation of increasing obesity (yellow curve) with rising incidence of esophageal adenocarcinoma in the USA (purple bars) at a time when the incidence of squamous cell carcinoma of the esophagus (blue bars) is decreasing [14]

It would, therefore, be predicted that the incidence of BE would likewise track with rising rates of obesity. This is generally believed to be the case, but remains elusively difficult to confirm. The true incidence and prevalence of BE remains unknown because this would require random population-based screening endoscopy studies. Such studies are not feasible or justifiable from a medical economics standpoint. There is, therefore, a selection bias in available incidence data aggregated from referral-based endoscopic series. Under current practice, patients are selected for endoscopic evaluation according to severity of reflux symptoms and refractoriness to medical treatment. To our knowledge, the only random population data on the true prevalence of BE comes from a Swedish study involving a random population sample of 1,000 volunteers who consented to endoscopy [9]. Sixteen incident cases of BE were thus identified, representing a population prevalence of 1.6%, with a 95% CI of 0.8–2.4%, leaving the true prevalence a matter of ongoing conjecture. A communitybased study of BE incidence between 1994 and 2006 in the Northern California Kaiser Permanente health system, which included 4,205 eligible new cases in that time period, found an annual incidence of 3.6%, adjusted for the volume of endoscopies performed in 2006, the final year of the study (95% CI 3.3–3.9%) [16]. Temporal trends across the health care organization showed a steady linear increase in the prevalence of BE over the 12-year time course of the study, reaching 131/100,000 member years in the final year of the study. In agreement with other studies, prevalence was significantly higher among men than women, and among whites than, in descending order of prevalence, Hispanics, Asians, and African-Americans.

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Obesity Is the Key Modifiable Risk Factor at Each Stage of Neoplastic Progression

The established risk factors for BE and EAC, such as white race, male gender and advanced age are impossible to modify. Modifiable cofactors represent the logical focus of preventative research efforts. Obesity is far and away the strongest identified modifiable risk factor for each phase of the cancer progression sequence from GERD to BE to EAC. The other identified modifiable risk factors are a diet low in fruits and vegetables and smoking, each of which confers a roughly twofold increased level of independent risk [4, 17].

6.1

GERD, EAC, and Obesity

The National Health and Nutrition Examination Survey (NHANES) data have tracked rising obesity in American adults from 15% in 1980 to 34% in 2008 [18]. Obesity is believed to contribute to GERD through several mechanisms, including reduced lower esophageal sphincter pressure, impaired gastric emptying, increased intragastric pressure, and hiatal hernia [19]. A meta-analysis of eight studies showed a significant and dose–dependent association between obesity and GERD, with odds ratio increasing from 1.5 for BMI 25–30 kg/m2 (95% CI 1.1–2.0) to 2.8 for BMI >30 kg/m2 (95% CI 1.9–4.2) [20]. In the Nurses’ Health Study of 10,545 women, a dose–dependent relationship was observed between increasing BMI and frequent reflux symptoms, which were threefold more common in the upper quartile of BMI (p-trend < 0.001) [21]. In a meta-analysis of 14 studies representing a total of 2,488 EAC cases, Kubo et al. found that EAC was increased 1.9-fold for BMI of 25–30 kg/m2 (95% CI 1.5–2.4) and 2.4-fold for BMI >30 kg/m2 (95% CI 2.0–2.8) [22]. This is consistent with results we have observed associating obesity and earlier age of onset of EAC (Fig. 5.3) [23]. Perhaps most convincing are the findings of Whiteman et al., reporting the combined effects of obesity, GERD, and smoking on EAC risk, in an Australian case– control study which included 367 EAC cases and 1,580 population controls [24]. Obesity, defined as BMI ³30 kg/m2, without reflux symptoms, was associated with a 2.2-fold increased risk of EAC (95% CI 1.1–4.3), while frequent reflux, defined as at least weekly symptoms, without obesity, was associated with a 5.6-fold increased risk of EAC (95% CI 2.8–11.3). The combination of obesity and frequent reflux was associated with a 16.5-fold increased risk of EAC (95% CI 8.9–30.6). These results suggest the interaction of GERD and obesity as comodulators of EAC risk.

7

BE and Obesity

To our knowledge, the first case–control data supporting an association between obesity and BE appeared in a 2002 study of familial aggregation in BE by Chak et al. in Cleveland [25]. This study—which was also the first to systematically demonstrate

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Fig. 5.3 Histograms of age of diagnosis in cancer patients without obesity (left) and those with obesity (right) 1 year before diagnosis. Mean age of cancer diagnosis in those with history of obesity was 58.99 years vs. 63.6 years for those without obesity, p = 0.008 [23]

a significant familial component in BE—collected anthropometric data including BMI at 0, 1, 5, 10, and 20 years prior for the 35 BE cases and 106 matched GERD controls in the report. Obesity was defined as BMI ³27.3 kg/m2 for females and ³27.8 kg/m2 for males. Foreshadowing subsequent reports, obesity, as determined by BMI at time of diagnosis, was not associated with BE. However, duration of obesity was found to be significantly associated with BE with a dose–response relationship, (e.g., for 10 prior years obesity OR = 2.3/p = 0.02 and for 20 prior years obesity OR = 3.2/p = 0.004) (Fig. 5.4). Much of the subsequent data regarding an association between obesity and BE comes from studies conducted through the Veterans Affairs medical system (VA). A cross-sectional study from the Palo Alto VA reported by Gerson et al. in 2002 enrolled 110 asymptomatic primarily white male volunteers over age 50 who were scheduled to undergo routine screening colonoscopy and agreed to also undergo endoscopy with research biopsies [26]. Among 110 participants, 27 incident cases of BE were identified (25%). Obesity, defined as BMI >25 kg/m2, was not significantly associated with BE cases (p = 0.7). However, the 83 participants categorized as non-BE for statistical comparison, included 17/83 (20%) with specialized intestinal metaplasia restricted to the GE junction. Among the 27 incident BE cases, 19/27 were short segment BE (70%) and

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Fig. 5.4 Duration of obesity is associated with Barrett’s esophagus [25]

8/27 (30%) were LSBE. The 7% overall prevalence of LSBE in this asymptomatic group (8/110) was in line with rates previously reported in the symptomatic GERD population, but the prevalence of obesity, given a lenient threshold BMI >25 kg/m2, was surprisingly low at 15 and 12% for BE and non-BE, respectively. A retrospective cross-sectional study from the Southern Arizona VA reported by Stein et al. in 2005 compared 65 BE cases and 385 non-BE among patients undergoing endoscopy over a 6-year period from 1988 to 1994 [27]. BMI of 25–30 kg/m2 was associated with a 2.4-fold higher risk of BE (p = 0.03, 95% CI 1.1–5.3). BMI ³30 kg/m2 was associated with 2.5-fold higher risk of BE (p = 0.03, 95% CI 1.1–5.4). The presence and frequency of reflux symptoms, however, was not included in the analysis. No correlation was seen between length of BE segment (short or long) and BMI at time of diagnosis. A case–control study from Queensland, Australia reported by Smith et al. in 2005, compared 117 BE cases, 50 dysplasia cases, and 261 matched controls enrolled in 2003 [28]. The investigators did not find a significant association between BE and BMI at time of diagnosis. In the unadjusted analysis, a significant association was seen between dysplasia and obesity, defined as BMI ³30 kg/m2, but statistical significance fell out after adjustment for reflux symptoms.

7.1

BMI and BE Meta-analysis

Three meta-analyses involving 10–11 studies each have reached similar conclusions. Between the three meta-analyses, results from 21 separate BE studies are represented which collected sufficient anthropometric data for comparison of the association of BMI to BE cases versus controls. Two of the three meta-analyses found no significant

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association between BMI and BE [29, 30]. The meta-analysis by Kamat el al. in 2009, which included 11 studies, reported a statistically significant 1.4-fold increased risk of BE for BMI ³30 kg/m2 (p < 0.001, 95% CI 1.2–1.6) and 1.5-fold increased risk of BE for BMI ³25 kg/m2 (p < 0.001, 95% CI 1.2–1.8) [31]. However, only 2 of the 11 studies evaluated, included GERD controls, and in a separate analysis of these two studies there was no significant association between BMI and BE.

7.2

Central Obesity and BE

In light of the conflicting data regarding BMI and BE, other studies have undertaken to refine measures of obesity, beyond BMI, in assessing the association between obesity and BE. The first data to support this approach was reported in a Seattle study of NSAIDs, anthropometric parameters and BE genetic biomarkers by Vaughan et al. in 2002 [32]. This cross-sectional study of the Seattle cohort of 429 individuals diagnosed with BE, used a logistic regression model adjusting for age, gender, NSAIDs, smoking and found that increasing WHR was significantly associated with genetic abnormalities previously demonstrated by the same group to increase the risk of neoplastic progression, including aneuploidy on flow cytometry (p trend = 0.01), 9p LOH (p trend = 0.005), and 17p LOH (p trend = 0.007). In an innovative 2005 computed tomography (CT) hospital-based case–control study reported by El-Serag et al., visceral adipose tissue (VAT) surface area at the level of the L4–5 vertebral disk by CT was compared in 36 BE cases and 93 matched controls who underwent upper endoscopy between 2000 and 2003 at the Houston VA [33]. A significant association with BE was noted for the upper tertile of visceral obesity by VAT, with 3.2-fold increased risk (p = 0.03, 95% CI 1.1–9.3). A similar CT-based measure of subcutaneous adipose tissue (SAT) surface area at the same level was not significantly associated with BE. The strongest univariate association with BE was observed for BMI >30 kg/m2 with a fourfold increased risk (p = 0.008, 95% CI 1.4–11.1). However, multivariate logistic regression models suggested that the association of VAT and BE was independent of BMI (p = 0.03), whereas BMI was not independently associated with BE (p = 0.55). A 2007 nested case–control study reported by Corley et al. in San Francisco enrolled 317 randomly selected population controls and 316 GERD controls with negative endoscopy and 320 incident cases of BE diagnosed between 2002 and 2005 through the Northern California Kaiser Permanente health system [34]. Abdominal circumference >80 cm, after adjusting for BMI, was independently associated with a 2.2-fold increased risk of BE compared with population controls (95% CI 1.2–4.2), but this risk was attenuated to 1.5-fold when compared with the GERD control group (95% CI 0.8–2.7). No association was noted between BMI and BE. Probably the most definitive data for this association were reported in landmark study by Edelstein et al. in Seattle [35]. This study identified 193 BE cases diagnosed between 1997 and 2000 by upper endoscopy for refractory GERD with matched population controls. Anthropometric measurements assessed included BMI and three measures of central adiposity: waist circumference (WC), WHR, and waist-to-thigh ratio (WTR). All three measures of central adiposity were significantly associated

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with BE with a 2.3-fold increased risk for elevated WC (p trend = 0.001, 95% CI 1.2–4.4), 2.8-fold for elevated WHR (p trend < 0.001, 95% CI 1.5–5.1), and 4.5-fold for elevated WTR (p trend = 0.002, 95% CI 2.1–9.2). The association with BMI was mixed. BE by standard criteria was not associated with BMI, but a significant association was observed for BMI ³30 kg/m2 and the presence of focal intestinal-type metaplasia on microscopic review with Alcian blue acid stain, with a 2.6-fold increased risk (p = 0.001, 95% CI 1.5–4.4). The frequency of reflux symptoms, as anticipated, was highly associated with BE. Weekly symptoms were associated with an impressive 8.1-fold increased risk (95% CI 4.5–14.5). However, elevated WHR was found to be independently associated with BE in a model which adjusted for reflux symptom frequency, with a 2.9-fold increased risk (95% CI 1.3–6.3). Elevated WHR was also independently associated with BE after adjusting for BMI, with a similar 2.9-fold increased risk (p = 0.002, 95% CI 1.5–5.7). One of the most important conclusions to be drawn from this study was to highlight the significant role of central obesity in BE. Importantly, this suggests a plausible cofactor contributing to the striking discrepancy in BE and EAC incidence between males and females (BE M:F ratio 2.5 to 1 and EAC M:F ratio 7 to 1). An increased visceral distribution of visceral body fat, as opposed to a truncal distribution, may also account for the higher incidence of BE and EAC in whites as compared to Asians and African-Americans. A cross-sectional study of 80,100 patients in the Northern California Kaiser Permanente health systems found that increased abdominal circumference was an independent risk factor for reflux symptoms after adjusting for BMI, with a significant 85% increased risk for whites (95% CI 1.6–2.2) but not for African-Americans (OR 0.95, 95% CI 0.6–1.5) or Asians (OR 0.64, 9% CI 0.2–2.3) [36].

8

Central Obesity and GERD as Comodulators of BE Pathogenesis

It is important to note that in the landmark 2007 study by Edelstein et al., WHR was shown to be independently determinant of BE risk, regardless of reflux symptom frequency [35]. A 2.9-fold increased risk of BE was observed for increased WHR, independent of reflux, which rose to a striking 8.1-fold risk in association with weekly reflux symptoms. The significant conclusion from these findings is that although obesity and GERD cleary overlap in the pathogenesis of BE, the effects of obesity, in particular visceral obesity, are further mediated through factors other than chronic acid reflux, to a significant extent. In the 2005 study by Smith et al., the coexistence of frequent reflux symptoms and obesity was shown to result in a striking combined 30-fold increased risk of BE [28]. The biologic basis of the interaction between these two cofactors is being elucidated by a growing body of research. Adipocytes, in particular visceral adipocytes, produce an excess of bioactive compounds known as adipokines. These include leptin, adiponectin, free fatty acids, and inflammatory cytokines.

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Evidence for Leptin

Serum leptin levels are upregulated in human obesity and associated with several human cancers [37–39]. Keen interest in the action of leptin in esophageal malignancy emerged following in vitro studies demonstrating enhanced proliferation of EAC cell lines through anti-apoptotic (OE33), mitogenic (OE33), and angiogenic mechanisms (OE19 and OE33), mediated through COX-2-dependent prostaglandin E2 activation of EGFR signaling and synergism of leptin and acid to activate AKT signaling through NFkB [40–43]. The truncated leptin receptor was subsequently shown to be expressed on esophageal epithelium in BE [44]. The association of serum leptin levels and BE has been investigated in two epidemiologic studies. Kendall et al. in 2008 reported a case–control study design utilizing a pilot cohort of 51 BE cases and 67 population controls, followed by validation cohort of 306 BE cases and 309 controls [45]. A significant 3.3-fold increased risk of BE was observed for men in the highest quartile of serum leptin levels independent of BMI (95% CI 1.7–6.6). After adjusting for GERD symptoms, the risk association was attenuated but remained significant at 2.4-fold (95% CI 1.1–5.2). A significant association was not observed for women, despite significant inclusion (25 pilot cases and 98 validation cases). Thompson et al. in 2010 reported a study of 177 intestinal metaplasia cases and 173 population controls, in which serum leptin levels in the upper tertile were associated with a twofold increased risk for both genders (95% CI 1.1–3.4), which was no longer significant following adjustment for WHR [46]. Of the 177 intestinal metaplasia cases, 90 had endoscopically visible changes which would qualify for BE diagnosis. A subset analysis using these 90 BE cases did not show a significant association between increased serum leptin and risk of BE, however.

8.2

Evidence for Adiponectin

Adiponectin, unlike leptin, is inversely correlated with obesity and has insulin sensitizing and anti-inflammatory effects [47]. Reduced adiponectin levels are associated with several cancers including breast, endometrial, and colorectal [48]. Adiponectin inhibits humoral production of IL6 and TNFa while stimulating anti-inflammatory IL10 production. Adiponectin binds circulating growth factors involved in proliferation and angiogenesis signaling pathways, such that reduced circulating adiponectin levels are thought to confer a proproliferative milieu [49]. Adiponectin has been shown to activate proapoptotic signaling in vitro through cell surface receptor interactions and inhibit leptin-induced proliferation in EAC cell lines (OE19 and OE33) [43]. Reduced expression at the mRNA level of adiponectin receptors has been demonstrated in mucosal BE biopsies [50]. Rubenstein et al. in 2009 examined the association between reduced serum adiponectin and risk of BE by comparing 112 BE cases and 1999 GERD controls [51]. Reduced levels of low molecular weight adiponectin multimer, in particular, was associated with threefold increased risk of

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BE (95% CI 0.2–0.7). Thompson et al. in 2010 reported a case–control study of 177 BE cases and 173 population controls, finding a twofold increased risk of BE for serum adiponectin in the lower tertile (95% CI 0.3–0.8) [46].

8.3

Evidence for Free Fatty Acids

Excess free fatty acid synthesis by visceral adipocytes contributes to insulin resistance [52, 53]. The resulting hyperinsulinemia is a cardinal manifestation of metabolic syndrome and can promote proliferation and inhibit apoptosis through decreased IGFBP levels and increased bioavailability of IGF1, as well as contributing to increased IL6 and TNFa levels. A cross-sectional study of metabolic syndrome and BE by Ryan et al. in 2008 enrolled 102 BE cases in Ireland [54]. Anthropomorphic measurements were recorded and serum analysis was performed to evaluate levels of various adipocytokines including CRP, leptin, adiponectin, VEGF, IL1-10, and INFg. Significant associations favoring LSBE, over short-segment, included abdominal circumference, hyperinsulinemia, increased IL6 levels, and metabolic syndrome diagnosis.

9

Future Directions: Physical Activity as a Modifiable Risk Factor

Prospective interventions targeting the modifiable factors which modulate the initial metaplastic transformation in the distal esophageal epithelium may prove to be the most effective strategy to attenuate the rising incidence of EAC. This is an active focus of ongoing research. Three studies are briefly reviewed. The NIH-AARP Diet and Health Study is a longitudinal cohort study, which enrolled 487,732 individuals, aged 50–71, from eight sites in the USA who returned a mail-in baseline questionnaire between 1995 and 1996 [55]. Incident cases of esophageal or gastric carcinoma between 1995 and 2003 were ascertained from state cancer registries in the eight states of origin, as well as TX, AZ, and NV, and classified by histology and anatomic site according to ICD codes. The AARP questionnaire included self-reporting of average frequency of physical activity, defined as lasting at least 20 min and resulting in increased heart rate, breathing, or working up a sweat. Participants were divided into five categories according to physical activity frequency per week (0, 50% overall sequence homology and 84% homology in tyrosine kinase domain of the b-subunit, insulin, and IGF1 can interact either with IR or IGF1R. The IR/IGF1R-mediated initiation of signal transduction activates important intracellular signal pathways, including the Ras/Raf/MEK/ERK and phosphoinositide-3 kinase/Akt/mTORE pathways. Genetic variants of the IGF axis genes have been associated with both the risk and survival of pancreatic cancer [91, 92]. The IGF1R-mediated signaling pathway has been recognized as a therapeutic target in the treatment of pancreatic cancer.

4.3

Inflammation

Obesity reflects a state of chronic low-grade inflammation and is associated with an increased number of macrophages in adipose tissue [57]. Accumulation of lipids in the adipocytes and enlarged adipose tissues lead to a relative hypoxia state, which induces hypoxia-inducible factor-1a and IL-6 and decreases the expression of adiponectin [93, 94]. Hypoxia-inducible factor-1a is involved in the overexpression of leptin in tumors and in mediating the attraction of macrophages into adipose tissue, which initiate the inflammatory response [95]. In a well-established animal model of pancreatic cancer, a high-fat diet predisposes mice with oncogenic K-ras activation to enhanced pancreatic intraepithelial neoplasm (PanIN) development [96]. In this model, tumor promotion is closely associated with increased inflammation without the presence of insulin resistance, and abrogation of TNF-a receptor signaling significantly blocks PanIN development, which underlines a central role for TNF-a in obesity-mediated enhancement of PanIN lesions. An increased level of TNF-a stimulates the production of MCP-1 and activates the IKKb and nuclear factor (NF)-kB axis, the master regulator of innate and adaptive immunity. IKKbmediated suppression of tuberous sclerosis (TSC1), a growth inhibitory protein,

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activates the mTOR pathway, enhances angiogenesis, and promotes tumor development [97]. NF-kB is constitutively activated in pancreatic cancer [98], and increased NF-kB activity can inhibit apoptosis and promote growth, tumorigenesis, angiogenesis, invasion, and metastasis in pancreatic cancer [99]. In addition to the IKKb/ NF-kB signaling pathway, other transcription factor-signaling pathways involved in the pro-inflammatory effect of obesity are the c-Jun NH2-terminal kinase (JNK) pathway [100] and the Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) pathway [101], both are critical for cell survival, proliferation, and differentiation. The obesity-related proinflammatory cytokines such as TNF-a, IL-6, resistin, MCP-1, and plasminogen activator inhibitor-1 not only contribute to insulin resistance but also affect the endothelium function leading to upregulated adhesion molecule synthesis and increased vascular permeability. Other obesity-related inflammatory components that could contribute to the development of cancer include the matrix metalloproteinases, which are associated with cancer cell invasion and metastasis, and vascular endothelial growth factor, an angiogenic factor [102]. Non-alcoholic fatty pancreas disease (NAFPD) has been proposed as another link between obesity and pancreatic cancer. NAFPD is a disease with a pancreatic phenotype ranging from deposition of fat in the pancreas through fat deposition with inflammation, resultant pancreatic fibrosis, and possibly pancreatic cancer. This pancreatic phenotype is similar to that of obesity-induced liver disease, non-alcoholic fatty liver disease, which describes a spectrum from hepatic steatosis through steatohepatitis to cirrhosis, and primary hepatocellular cancer [103, 104]. In an animal study, pancreata from leptin-deficient obese mice fed a 15% high-fat diet had greater amounts of triglycerides, free fatty acids, cholesterol, and total fat as well as a higher levels of the cytokines IL-1b and TNF-a compared with those from control lean mice [103]. These data suggest that obesity leads to NAFPD. Another experiment showed that maternal obesity led to significant increases in body weight, pancreatic triglyceride content, TGF-b expression (a marker for the pancreatic stellate cell), and collagen gene expression in offspring, indicating a dysmetabolic and NAFPD phenotype [105]. As discussed above, fat is a dynamic endocrine organ and central adiposity is possibly associated with an increased risk of pancreatic cancer. Central adiposity leads to organ steatosis and altered serum adipokines, including a reduced adiponectin level and markedly elevated levels of leptin and proinflammatory cytokines. This abnormal adipokine milieu results in increased tissue infiltration of the monocytes and macrophages which produce the chronic inflammation state. Over many years, the combination of steatosis and local inflammation leads to fibrosis and eventually to cancer [104]. Pancreatic cancer exhibits extensive proliferation of stromal fibroblasts and deposition of extracellular matrix components (desmoplasia) [106]. The stromal interaction, or tumor microenvironment, plays an important role in tumor growth and invasion as well as in drug resistance [107]. The molecular basis of this phenotype is not well defined. The role of adipose stromal cells and adipose endothelial cells in tumorigenesis is intriguing. A recent animal study [108] demonstrated the recruitment of cells from the white adipose tissue to the tumor and went on to

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demonstrate that adipose stromal and epithelial cells migrate to and become engrafted into tumor compartments. These data support the hypothesis that the expanded white adipose tissue in obesity results in increased levels of adipose stromal cells, which are mobilized from the white adipose tissue by obesity-associated inflammation to contribute to tumor development.

4.4

Genetic Susceptibility

Genetic factors that predispose to obesity might also predispose to the development of pancreatic cancer. In a case–control study, we observed that two FTO (fat mass and obesity associated) gene variants and one adiponectin gene variant were differentially associated with the risk of pancreatic cancer by BMI level (Pinteraction = 0.0001, 0.0015, and 0.03, respectively). For example, the adjusted OR for the FTO IVS12777 AC/AA genotype was 0.72 (95% CI: 0.55–0.96) in participants with a BMI 1 cm) and invasive cancers harbor activated RAS oncogenes, implying that constitutive activation of Ras is important for the outgrowth of malignant cell clones. Raf kinase activity is also directly targeted by driver mutations. Mutations in the RAF kinase family member BRAF occur in about 10% of colorectal cancers, and arise at a similar stage of tumor development as RAS mutations [8]. The most frequent BRAF alteration is a missense mutation (V600E) that increases the basal kinase activity of the encoded protein approximately 500-fold [9]. The BRAF V600E mutation and a mutant RAS gene are never found together in the same tumor, suggesting that both mutations confer a similar selective advantage during tumorigenesis [10]. Interestingly, BRAF mutations occur disproportionately in tumors with deficient mismatch repair [8]. The frequency of genetic alterations that affect the Ras/Raf pathway and the timing of these alterations in developing colorectal neoplasia suggest that dysregulated

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Ras/Raf signaling is required at a critical transition point, at which point tumors acquire the ability to invade neighboring tissues. As large tumors grow beyond the normal confines of their tissue compartment and attendant vasculature, hypoxia, acidosis and hypoglycemia increasingly represent barriers to further growth and invasion. From an evolutionary perspective, constitutive Ras/Raf signaling must provide neoplastic clones with a selective advantage during this transitional interval. Presumably, the unique microenvironment of a large colorectal adenoma creates selective pressure that favors cells harboring oncogenic RAS or RAF genes. Studies in the past several years have begun to reveal how the activation of common oncogenes and frequent losses of tumor suppressor genes can provide selective advantages as tumors outgrow their blood supply. For example, the hypoxic conditions that are found in poorly vascularized cancers have been found to favor the selection of cells that have activated PIK3CA or CMYC oncogenes, or loss of the p53 tumor suppressor [11, 12].

4

Upregulation of GLUT1 in Cells with Constitutive Ras/Raf Signaling

Despite the detailed level of understanding of RAS biochemical interactions and the numerous paths that a RAS-mediated signal can presumably take, it is not yet clear how activated Ras/Raf signaling results in the selective growth of colorectal cancer cells. There is even less information about the precise microenvironmental conditions surrounding colorectal cancer cells that specifically favor the selection and expansion of cells with KRAS or BRAF mutations over those with wild-type KRAS and BRAF. As it is the case with other oncogene- and tumor suppressor gene-encoded proteins, RAS proteins have been implicated in the regulation of metabolism and energy balance in cancer cells. For example, transformation of rodent cells with HRAS can result in the upregulation of the facilitative glucose transporter encoded by GLUT1 (SLC2A) [13, 14]. However, this upregulation of GLUT-1 in rodent cells had also been ascribed to HIF1A and linked to hypoxia [15– 17]. The patterns and timing of KRAS and BRAF mutations during colorectal tumorigenesis indicate that they provide a redundant survival advantage to cancer cells growing in a microenvironment that would otherwise not be conducive to neoplastic growth. Important clues as to how constitutive Ras/Raf signaling confers this survival advantage have been obtained from rigorous studies of human somatic cell knockouts. In isogenic human colorectal cancer cell lines that differ only on the genetic status of KRAS or BRAF, GLUT1 was only one of the three genes consistently upregulated in all cell lines with either a KRAS or BRAF mutation, providing an unbiased link between Ras/Raf activation in colon cancer and increased glucose uptake. Isogenic cell lines with altered PIK3CA and HINF1A, derived from the same maternal cell lines, did not result in the consistent upregulation of GLUT1 under normoxic conditions, suggesting that this effect was highly specific for activated Ras/Raf [4] (Fig. 8.2).

Ras/Raf and Their Influence in Glycolysis in Colon Cancer

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Survival

Fig. 8.2 Mutations in the KRAS–BRAF pathway in colon cancer promote cell survival by increasing glucose uptake. In the low glucose microenvironment of a developing colorectal neoplasm, mutant KRAS promotes activation of downstream Raf–MEK–ERK signaling, which increases the expression of the glucose transporter GLUT-1 (left panel). A similar upregulation of GLUT-1 occurs in cancers harboring a BRAF mutation (right panel), demonstrating the importance of downstream signaling pathways to the control of glucose transport

The growth of cell lines with either a KRAS or a BRAF mutation was equivalent to that of cells with wild-type KRAS/BRAF when all were maintained under numerous microenvironmental conditions, including hypoxia. There was one significant exception; under low glucose conditions, the cell lines with mutant KRAS or BRAF exhibited increased survival compared with the isogenic wild-type controls. Consistent with this phenotype and the upregulation of GLUT1, glucose uptake and lactate production was increased in the Ras/Raf activated cell lines, indicating an increase in glycolysis. When wild-type KRAS cell lines were forced to grow under glucose deprivation most cells died. However, clones resistant to low glucose environment emerged. These clones had GLUT1 upregulated and, a fraction of them, also had KRAS mutations in most cases different than the KRAS mutation of the maternal cell line. These results suggest that the microenvironment facilitates adaptation of the cells to the low glucose state by causing increased expression of GLUT1, and resultant uptake of glucose, through oncogenic mutations in the Ras/ Raf pathway. Furthermore, the adaptation of the colorectal cancer cells to anaerobic glycolysis apparently occurs independently of hypoxia. This finding runs contrary to previous models of clonal selection, in which hypoxia has been proposed to be the primary selective pressure that favors adaptation to glycolysis [18]. Indeed, as Warburg originally observed, cancer cells use glycolytic metabolism even in normoxic environments. For example, leukemic cells are highly glycolytic, yet they are present in an environment with high oxygen, the blood stream [19, 20]. Similarly,

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lung cancers growing in the airways are highly glycolytic, despite the presence of abundant oxygen [21]. Clinical data also provide support for the idea that limiting glucose provides a selective barrier during colorectal tumorigenesis. F-18-Fluoro-deoxyglucose (FDG)—positron emission tomography (PET) scans routinely used to scan cancers allow the visualization in tumors of increased glucose transporter expression and glucose uptake [22]. It is tantalizing that these abnormal FDG-signals are observed in pre-malignant colorectal neoplasms congruent with the stage during tumorigenesis in which KRAS or BRAF mutations appear [23, 24]. Not surprisingly, both KRAS mutations and increased FDG-PET signal are markers for poor prognosis in colon cancer [25, 26].

5

Clinical Implications

The link between KRAS or BRAF mutations and the upregulation of GLUT1 suggests that blocking either, glucose uptake and/or KRAS and BRAF signaling would be effective therapeutic approaches. In a proof of principle experiment, the growth of xenografts with KRAS or BRAF mutations was retarded by 3-bromo-pyruvate, an alkylation agent that blocks glycolysis [4]. This experiment provides new evidence that colorectal cancers with KRAS or BRAF mutations can be treated with agents that block glucose transport or interfere with glycolysis, but the development of such therapeutic agents has not been successful thus far. Unfortunately, the development of direct KRAS inhibitors has fallen short of expectations, too. The development of anticancer agents that target BRAF have been more promising. New strategies in medical chemistry, innovative approaches to drug design and greater selectivity for the mutated protein have contributed to the development of a new generation of BRAF inhibitors [27]. In vitro, PLX4032/RG7204 (Plexxicon/ Roche) selectively inhibits all three RAF kinases at nanomolar concentrations [27–29]. In preclinical studies, PLX4032 inhibits the MAP kinase pathway in cells with BRAF V600E mutations. However, in the cells with wild-type BRAF, including the cells with KRAS mutations, not only it does not block the downstream MAP pathway, it actually stimulates it [30–32]. In cells with KRAS mutations, and therefore increased Kras activity, RAF proteins form dimers; binding of the drug to one Raf molecule in the dimer thereby activates the other. In cells with BRAF mutations and low Kras activity, BRAF kinase is active, dimerization is not promoted and binding of the drug inhibits Braf activity. In clinical trials, metastatic melanomas harboring the BRAF V600E mutation showed remarkable PLX4032 sensitivity resulting in complete or partial regression in the vast majority of the patients [33]. This success has prompted optimism for the treatment of patients with colorectal cancers harboring BRAF mutations; additional trials are under way. As is the case in many targeted therapies, cancers can acquire resistance to PLX4032 treatment. Recently, it was shown that the mechanism of resistance involves Braf bypass and restoration of activity of MEK activation of the MAP

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kinase pathway downstream of BRAF [34, 35]. That the resistance mechanism to BRAF inhibitor involves the reactivation of the MAP kinase pathway underscores the ongoing dependence of cancer cells to this pathway, either because it still provides the necessary survival advantage or because the cells were “hard coded” during their selection to use this pathway even when it does not provide a selective advantage anymore. While the development of BRAF inhibitors provides hope for the patients with colorectal cancer that carry BRAF mutations, such drugs would unfortunately not be useful for patients that have KRAS mutations. This is not the only time that KRAS mutations have been a negative indicator for the administration of a targeted anticancer therapy. KRAS mutations also predict a lack of response in patients with colorectal cancer to EGFR inhibitors cetuximab and panitumumab, either as single agents or in combination with chemotherapy [36– 39]. For this reason, companion diagnostics for the detection of KRAS mutations have become a must for the stratification of colorectal patients for treatment with EGFR inhibitors.

6

Conclusion

Mutations that activate Ras/Raf signaling are pivotal for the development of colon cancer. They appear right at the cusp of the transition to more aggressive disease. KRAS is one of the most studied oncogenes. Nonetheless, our understanding of the signaling pathway has not been sufficient for the development of effective therapeutic interventions. Borrowing from the studies described in this chapter and through the use of appropriate experimental models, advances in pharmacology and genetics, new insights into the mechanisms by which these genes promote cancer will continue to emerge. The hope is that new opportunities for the development of therapies will emerge too.

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8. Rajagopalan H, Bardelli A, Lengauer C, Kinzler KW, Vogelstein B, Velculescu VE (2002) Tumorigenesis: RAF/RAS oncogenes and mismatch-repair status. Nature 418:934 9. Gray-Schopfer V, Wellbrock C, Marais R (2007) Melanoma biology and new targeted therapy. Nature 445:851–857 10. Yuen ST, Davies H, Chan TL, Ho JW, Bignell GR, Cox C, Stephens P, Edkins S, Tsui WW, Chan AS et al (2002) Similarity of the phenotypic patterns associated with BRAF and KRAS mutations in colorectal neoplasia. Cancer Res 62:6451–6455 11. Kim JW, Gao P, Dang CV (2007) Effects of hypoxia on tumor metabolism. Cancer Metastasis Rev 26:291–298 12. Matoba S, Kang JG, Patino WD, Wragg A, Boehm M, Gavrilova O, Hurley PJ, Bunz F, Hwang PM (2006) p53 regulates mitochondrial respiration. Science 312:1650–1653 13. Chen C, Pore N, Behrooz A, Ismail-Beigi F, Maity A (2001) Regulation of glut1 mRNA by hypoxia-inducible factor-1. Interaction between H-ras and hypoxia. J Biol Chem 276:9519–9525 14. Flier JS, Mueckler MM, Usher P, Lodish HF (1987) Elevated levels of glucose transport and transporter messenger RNA are induced by ras or src oncogenes. Science 235:1492–1495 15. Airley R, Loncaster J, Davidson S, Bromley M, Roberts S, Patterson A, Hunter R, Stratford I, West C (2001) Glucose transporter glut-1 expression correlates with tumor hypoxia and predicts metastasis-free survival in advanced carcinoma of the cervix. Clin Cancer Res 7:928–934 16. Blum R, Jacob-Hirsch J, Amariglio N, Rechavi G, Kloog Y (2005) Ras inhibition in glioblastoma down-regulates hypoxia-inducible factor-1alpha, causing glycolysis shutdown and cell death. Cancer Res 65:999–1006 17. Zhang JZ, Behrooz A, Ismail-Beigi F (1999) Regulation of glucose transport by hypoxia. Am J Kidney Dis 34:189–202 18. Gatenby RA, Gillies RJ (2004) Why do cancers have high aerobic glycolysis? Nat Rev Cancer 4:891–899 19. Elstrom RL, Bauer DE, Buzzai M, Karnauskas R, Harris MH, Plas DR, Zhuang H, Cinalli RM, Alavi A, Rudin CM, Thompson CB (2004) Akt stimulates aerobic glycolysis in cancer cells. Cancer Res 64:3892–3899 20. Gottschalk S, Anderson N, Hainz C, Eckhardt SG, Serkova NJ (2004) Imatinib (STI571)mediated changes in glucose metabolism in human leukemia BCR-ABL-positive cells. Clin Cancer Res 10:6661–6668 21. Christofk HR, Vander Heiden MG, Wu N, Asara JM, Cantley LC (2008) Pyruvate kinase M2 is a phosphotyrosine-binding protein. Nature 452:181–186 22. Gu J, Yamamoto H, Fukunaga H, Danno K, Takemasa I, Ikeda M, Tatsumi M, Sekimoto M, Hatazawa J, Nishimura T, Monden M (2006) Correlation of GLUT-1 overexpression, tumor size, and depth of invasion with 18F-2-fluoro-2-deoxy-D-glucose uptake by positron emission tomography in colorectal cancer. Dig Dis Sci 51:2198–2205 23. van Kouwen MC, Nagengast FM, Jansen JB, Oyen WJ, Drenth JP (2005) 2-(18F)-fluoro-2deoxy-D-glucose positron emission tomography detects clinical relevant adenomas of the colon: a prospective study. J Clin Oncol 23:3713–3717 24. Yasuda S, Fujii H, Nakahara T, Nishiumi N, Takahashi W, Ide M, Shohtsu A (2001) 18F-FDG PET detection of colonic adenomas. J Nucl Med 42:989–992 25. Riedl CC, Akhurst T, Larson S, Stanziale SF, Tuorto S, Bhargava A, Hricak H, Klimstra D, Fong Y (2007) 18F-FDG PET scanning correlates with tissue markers of poor prognosis and predicts mortality for patients after liver resection for colorectal metastases. J Nucl Med 48:771–775 26. Samowitz WS, Sweeney C, Herrick J, Albertsen H, Levin TR, Murtaugh MA, Wolff RK, Slattery ML (2005) Poor survival associated with the BRAF V600E mutation in microsatellitestable colon cancers. Cancer Res 65:6063–6069 27. Tsai J, Lee JT, Wang W, Zhang J, Cho H, Mamo S, Bremer R, Gillette S, Kong J, Haass NK et al (2008) Discovery of a selective inhibitor of oncogenic B-Raf kinase with potent antimelanoma activity. Proc Natl Acad Sci USA 105:3041–3046

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Chapter 9

Energy Balance and Other Modifiable Host Factors on Colorectal Cancer Prognosis Jeffrey Meyerhardt

Abstract Epidemiologic and scientific research indicates that host factors that influence energy balance have a significant influence on the risk of developing colorectal cancer. Obesity and certain diets increase one’s risk of developing colorectal cancer, while physical activity decreases this risk. Until recently, it was largely unknown if any of these modifiable factors influence the outcomes of patients already diagnosed with colorectal cancer. However, data are emerging of factors that may influence disease recurrences and mortality for colorectal cancer survivors. Prospective observational studies have shown that increased exercise after diagnosis and avoidance of a Western pattern diet are associated with reduced risk of cancer recurrence and improved overall survival in early stage colorectal cancer after standard therapy. Patients with class II and III obesity (BMI ³ 35 kg/m2) have a modestly increased risk of recurrence. In contrast, change of weight after diagnosis is not associated with outcomes after diagnosis. The data supporting these observations will be reviewed and potential mechanisms of actions will be discussed.

1

Colorectal Cancer Statistics

Colorectal cancer afflicts 1.2 million people globally each year and results in over 600,000 deaths [1]. The highest incidence rates of colorectal cancer are found in countries associated with unfavorable energy balance host factors, including obesity,

J. Meyerhardt, M.D., MPH. (*) Division of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA Department of Medicine, Harvard Medical School, 450 Brookline Ave, Boston, MA 02215-5450, USA e-mail: [email protected] S.D. Markowitz and N.A. Berger (eds.), Energy Balance and Gastrointestinal Cancer, Energy Balance and Cancer 4, DOI 10.1007/978-1-4614-2367-6_9, © Springer Science+Business Media, LLC 2012

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lack of physical activity, and Western pattern diets. Rates of colorectal cancer are lowest in Africa and South-East Asia, regions with populations that maintain a more favorable energy balance profile. In the USA, an estimated 142,570 individuals will be diagnosed with colorectal cancer and 51,370 will die from the disease in the USA annually [2]. The incidence of colorectal cancer has decreased over the past several decades, despite growing adiposity rates in the USA. Screening and removal of precancerous precursors (adenomas) has been associated with this decline.

2

Colorectal Cancer Staging and Standard Treatments

Upon diagnosis of colorectal cancer, pathologic stage (including depth of invasion in the bowel, involvement of regional lymph nodes, and distant metastasis) is considered critical in determining relative prognosis and in deciding the modalities of therapy [3]. Surgery is the primary modality of management for colorectal cancer, and a “curative intent” resection occurs in 80–85% of patients with non-metastatic disease (stages I–III) [4]. For patients with metastatic (stage IV) cancer, 5-year survival is less than 5% [4]. In contrast, for patients with stage I cancer (no lymph nodes involved, tumor limited to the muscular layer of the bowel wall), 5-year overall survival exceeds 90%, similar to the survival for an otherwise healthy agematched population [3]. Stage I patients do not receive adjuvant therapy and the absolute benefit of any additional intervention will be minimal at best. For patients with stage II cancer (no lymph nodes involved, but extension of the tumor beyond the muscle layer), surgery alone conveys a 5-year survival of 80% [5]. The use of adjuvant therapy after surgery is controversial in stage II disease; an expert panel convened by the American Society of Clinical Oncology determined that the absolute benefit from chemotherapy would not exceed 5% [5]. In contrast, 35–40% of patients with stage III colon cancer (regional lymph nodes involved but no evidence of metastatic spread of disease) will develop cancer recurrence despite “curative” surgery and postoperative adjuvant chemotherapy [6]. Overall, 38% of colorectal cancer patients have stage III disease at diagnosis (~56,500 people in the USA annually). Although many patients with stage III disease may be cured with standard therapy, there remains a critical need to improve the outcome for these patients.

3

Risk Factors for Colorectal Cancer: Energy Balance

As with many cancers, the risk of developing colorectal cancer increases with age [7]. Colorectal cancer has been associated with various genetic syndromes, including adenomatous polyposis coli and hereditary nonpolyposis colorectal cancer syndrome, as well as nonsyndromic family history. In addition, colorectal cancer has been consistently associated with a variety of potentially modifiable host factors, including various energy balance factors.

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Prospective cohort studies support the relationship between adiposity and the risk of developing colon cancer [8–21]. In a report of over 360,000 men and women in Europe, higher body mass index (BMI), waist-to-hip ratio, and waist circumference were associated with increased rates of colon cancer; the estimated absolute risk of developing colon cancer within 5 years of follow-up was 203 and 131 cases per 100,000 men and 129 and 86 cases per 100,000 women in the highest and lowest quintiles of waist-to-hip ratio, respectively [22]. Physically active people have a reduced risk of developing colon cancer [11, 14, 16, 21, 23–29]. A meta-analysis of 52 observational studies found an inverse association between physical activity and colon cancer development, with an overall relative risk of 0.76 (95% confidence interval, 0.72–0.81) [30]. When physical activity and BMI are assessed jointly, the highest risk of colon cancer occurs among those both physically inactive and with high BMI levels [31, 32]. Epidemiologic and scientific research indicates that diet may have a significant influence on the risk of developing colon cancer. Consumption of red meat [17, 33–36], alcohol [37, 38], calcium [5, 13, 39–42], vitamin D [26, 29, 30, 43–45], vitamin E [1, 12, 46], and folic acid [38, 43, 47–49] are among factors that appear to influence the risk of developing the disease. Specific dietary patterns have been associated with the development of colorectal cancer in case–control and cohort studies [27, 50–54]. In two large prospective cohort studies, increasing consumption of a Western diet (characterized by higher intakes of red and processed meats, sweets and desserts, French fries, and refined grains) was associated with a significantly increased risk of colon cancer, whereas a prudent diet (higher intakes of fruits, vegetables, legumes, fish, poultry, and whole grains) was nonsignificantly associated with a reduced risk [27, 54]. Recent hypotheses have linked physical activity, obesity, dietary patterns, and adipose distribution to circulating insulin and free insulin-like growth factor-1 (IGF-1) [55–57], which accounts for the integrated actions of circulating IGF-1 and IGF binding proteins (BPs) [56]. In observational studies, colon cancer risk is elevated in individuals with higher circulating levels of insulin or C-peptide (a marker of insulin secretion) [58–60] and IGF-1 or IGF-1/IGFBP-3 ratio [61–65]. Preclinically, insulin stimulates pathways that increase levels of free IGF-1, and both insulin and IGF-1 promote cell proliferation and inhibit apoptosis in colon cancer cells [9, 66–69]. In the Physicians’ Health Study, obesity and physical inactivity influenced colon cancer risk primarily through the insulin axis, but non-hyperinsulinemic (and nonoverweight, physically active) men were still at elevated risk if they had high-IGF-1 levels. Thus, high circulating IGF-1 (or IGF-1/IGFBP-3 ratio) and hyperinsulinemia may represent two different axes that influence colorectal neoplasia risk [70].

4

Risk Factors Influencing Outcomes

Despite advances in therapy, patients and oncologists continually seek further ways to improve outcomes [71–73]. Patients will utilize complementary therapies, either with advice and support of their oncologist or without consultation with their oncologist.

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Until recently, there was a lack of data for such adjunctive therapies in patients with colorectal cancer. However, in the past decade, there are increasing data that certain modifiable factors related to physical activity, adiposity, and diet may impact on the outcomes of subpopulations of colorectal cancer patients [74–82]. The mechanism that are believed in part to underline how these host factors influence tumorigenesis may be influential in the progression and metastases of established disease.

5

Obesity and Colorectal Cancer Prognosis

Studies on the impact of adiposity on outcomes colorectal cancer have led to mixed results (Table 9.1) [24, 76, 79, 80, 87]. An early analysis by Tartter and colleagues reported a significant difference in recurrence-free rates by Quetelet’s index (defined in the study as weight × 100/height2) in female patients with stage II and III colon cancer but not male patients [88]. However, the study was performed prior to the standard initiation of adjuvant chemotherapy, and residual confounding by other known predictors of cancer recurrence could not be excluded in that analysis. After excluding underweight patients, Slattery and colleagues found that increasing baseline BMI was associated with a nonsignificant increase in overall mortality in a cohort of colorectal cancer patients identified through the Utah Cancer Registry [89]. However, that study was limited by the heterogeneous patient population and stage of disease, and an inability to adjust for cancer treatment and other potential clinical predictors of colorectal cancer outcome. In a large randomized trial of adjuvant chemotherapy for stage II and III colon cancer (INT 0089), obesity (BMI ³ 30 kg/m2) was associated a 24% nonstatistically significant worse disease-free survival compared with normal weight women (BMI 21–24.9 kg/m2) [76]. Among patients enrolled in two National Surgical Adjuvant Breast and Bowel Project (NSABP) adjuvant chemotherapy trials for colon cancer, very obese patients (BMI ³ 35 kg/m2) experienced a 27% statistically significant increase in cancer recurrence or death compared with normal weight participants [24]. Both studies were limited by a single anthropometric data point at the time of initiation of adjuvant therapy. Using data from a randomized phase III study of adjuvant therapy in stage III colon cancer patients (CALGB 89803) in which BMI measurements were collected at the time of initiation of adjuvant therapy and approximately 6 months are the completion of adjuvant therapy (14 months after surgery), Meyerhardt and colleagues reported that patients with class II and III obesity (BMI > 35 mg/m2) had a nonsignificant 20% worse disease-free survival compared with normal weight patients [79]. Only one study has observed the influence of change in weight after diagnosis on cancer recurrences and survival [79]. In breast cancer survivors, gain in weight has been associated with increased risk of cancer in some [16, 90] but not all studies [14, 15]. Increasing weight after diagnosis (between time on adjuvant therapy and 6 months after completion of adjuvant therapy) was not associated with disease-free survival or overall survival in the CALGB 89803 cohort.

1976–1981

1988–1992

1990–1992

1989–1994

1999–2001

1981–2001

Slattery [49]

Meyerhardt [84]

Meyerhardt [85]

Dignam [24]

Meyerhardt [86]

Hines [68]

496

1053

4288

1792

3759

411

Sample size 279

TNM tumor, node, metastases staging, CI confidence interval

Year of diagnosis 1976–1979

Author Tartter [83]

TNM stage I–IV colon cancer

TNM stage III colon cancer

Duke B and C colon cancer

Duke B2, B3, C colon cancer TNM stage II and III rectal cancer

Stages of disease Duke B2, C1, C2 colon cancer All stages

Table 9.1 Summary of studies of body mass index in colorectal cancer survivors

Overall survival

Recurrence-free survival Overall survival

Disease-free survival

Colon cancer events

Disease-free survival Overall survival Disease-free survival Overall survival Local recurrences Disease-free survival

Overall survival

Outcome measure Recurrence rate

p > 0.05 for trend with increasing BMI quintile 1.11 (0.94–1.30) BMI ³ 30 kg/m2 1.11 (0.96–1.29) BMI ³ 30 kg/m 1.10 (0.91–1.32) BMI ³ 30 kg/m2 1.09 (0.90–1.33) BMI ³ 30 kg/m2 1.31 (0.91–1.88) BMI ³ 30 kg/m2 1.06 (0.93–1.21) BMI 30–34.9 kg/m2 1.27 (1.05–1.53) BMI ³ 35 kg/m2 1.04 (0.88–1.24) BMI 30–34.9 kg/m2 1.38 (1.10–1.73) BMI ³ 35 kg/m2 1.00 (0.72–1.40) BMI 30–34.9 kg/m2 1.24 (0.84–1.83) BMI ³ 35 kg/m2 0.97 (0.69–1.37) BMI 30–34.9 kg/m2 1.27 (0.85–1.89) BMI ³ 35 kg/m2 0.90 (0.61–1.34) BMI 30–34.9 kg/m2 0.87 (0.54–1.42) BMI ³ 35 kg/m2 0.77 (0.61–0.97) BMI ³ 25 all stages 0.92 (0.65–1.30) stage I and II 0.92 (0.59–1.45) stage III 0.58 (0.37–0.90) stage IV

Hazard ratio (95% CI) or p value (compared to normal weight) p = 0.003 for above median weight

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Obesity can lead to additional complications in patients with rectal cancer. Obesity has been associated with increased perioperative complications, including anastomotic leakage and blood transfusion requirements from rectal cancer [6, 91]. Using data from a large, adjuvant chemoradiotherapy trial of patients with stage II and stage III rectal cancer (INT 0114), Meyerhardt and colleagues reported that a higher baseline BMI was associated with an increased rate of abdominoperineal resections and, consequently, permanent colostomy [80]. In a study from a specialty cancer center, operative time was longer in obese than nonobese patients (4.3 h vs. 3.7 h, p < 0.01) and length of stay was longer in obese than nonobese patients (8 days vs. 7 days, p < 0.01) [18]. Obesity was also predictive of an increased risk of local recurrence among male, though not female, patients. The influence of adiposity in patients with metastatic colorectal cancer is largely unknown. In one study of 120 patients with metastatic colorectal cancer who received combination chemotherapy, higher BMI and higher visceral fat adiposity (VFA) was associated with shorter time to progression to second-line therapy and higher VFA was associated with shorter survival [44].

6

Physical Activity and Colorectal Cancer Prognosis

Observational studies have shown that higher physical activity levels and/or meeting physical activity guidelines is associated with better patient-reported quality of life, physical functioning, and fatigue [10, 21, 22, 92–94]. Only one randomized trial has investigated the effects of an exercise intervention in colorectal cancer survivors [23]. Conclusions from the study are limited due to greater than anticipated contamination in the comparison group that undermined the intention-to-treat analyses. In a secondary analysis, participants whose fitness increased over the course of the intervention, regardless of group assignment, reported significantly improved quality of life, physical functioning, and psychosocial distress compared with participants whose fitness decreased. Data are emerging for the potential influence of exercise in colorectal cancer survivors with stage I to III disease. Haydon and colleagues identified 526 colorectal cancer survivors from a prospective observational cohort of 41,528 Australians who completed pre-diagnosis questionnaires, including assessment of physical activity [45]. Increased exercise was associated with improved disease-specific survival [adjusted hazard ratio (HR) = 0.73; 95% CI: 0.54–1.00]. In subgroup analyses, the association seemed restricted to stage II and III tumors (HR = 0.49; 95% CI: 0.30– 0.79). In efforts to correlate these findings with molecular markers, the investigators reported that physically active colorectal cancer survivors had higher insulin-like growth factor binding protein-3 (IGFBP-3), which was associated with a significant reduction in disease-specific death (HR = 0.52; 95% CI: 0.33–0.83) [45]. These data suggest that the association between physical activity and disease-specific survival in colorectal cancer survivors may be through the IGF axis particularly IGFBP-3. Three studies have examined the association between post-diagnosis physical activity and disease outcomes in colorectal cancer survivors (Table 9.2) [77, 95, 96].

0.01

0.002

0.01

CALGB Cancer and Leukemia Group B, HR hazard ratio, CRC colorectal cancer

0.0003

Referent 1.00 (0.68–1.48) 1.12 (0.74–1.70) 0.74 (0.46–1.20) 0.59 (0.41–0.86)

P trend

Referent 0.77 (0.48–1.23) 0.50 (0.28–0.90) 0.43 (0.25–0.74) for >18 MET-hours/ week 0.003

Referent 1.06 (0.55–2.08) 1.30 (0.65–2.59) 0.76 (0.33–1.77) 0.47 (0.24–0.92)

Referent 0.92 (0.50–1.69) 0.57 (0.27–1.20) 0.39 (0.18–0.82) for >18 MET-hours/ week 0.008

Referent 0.87 (0.58–1.29) 0.90 (0.57–1.40) 0.51 (0.26–0.97) 0.55 (0.33–0.91)