James M. Bugni

Mutant N-Ras protects colorectal cancer cells from stress-induced apoptosis and contributes to cancer development and progression

N-RAS is one member of a family of oncoproteins that are commonly mutated in cancer. Activating mutations in NRAS occur in a subset of colorectal cancers, but little is known about how the mutant protein contributes to the onset and progression of the disease. Using genetically engineered mice, we find that mutant N-RAS strongly promotes tumorigenesis in the context of inflammation. The protumorigenic nature of mutant N-RAS is related to its antiapoptotic function, which is mediated by activation of a noncanonical mitogen-activated protein kinase pathway that signals through STAT3. As a result, inhibition of MAP-ERK kinase selectively induces apoptosis in autochthonous colonic tumors expressing mutant N-RAS. The translational significance of this finding is highlighted by our observation that NRAS mutation correlates with a less favorable clinical outcome for patients with colorectal cancer. These data show for the first time the important role that N-RAS plays in colorectal cancer.

Substance P (SP)-Neurokinin-1 Receptor (NK-1R) Alters Adipose Tissue Responses to High-Fat Diet and Insulin Action

Peripheral administration of a specific neurokinin-1 receptor (NK-1R) antagonist to mice leads to reduced weight gain and circulating levels of insulin and leptin after high-fat diet (HFD). Here, we assessed the contribution of substance P (SP) and NK-1R in diet-induced obesity using NK-1R deficient [knockout (KO)] mice and extended our previous findings to show the effects of SP-NK-1R interactions on adipose tissue-associated insulin signaling and glucose metabolic responses. NK-1R KO and wild-type (WT) littermates were fed a HFD for 3 wk, and obesity-associated responses were determined. Compared with WT, NK-1 KO mice show reduced weight gain and circulating levels of leptin and insulin in response to HFD. Adiponectin receptor mRNA levels are higher in mesenteric fat and liver in NK-1 KO animals compared with WT, after HFD. Mesenteric fat from NK-1R KO mice fed with HFD has reduced stress-activated protein kinase/c-Jun N-terminal kinase and protein kinase C activation compared with WT mice. After glucose challenge, NK-1R KO mice remove glucose from the circulation more efficiently than WT and pair-fed controls, suggesting an additional peripheral effect of NK-1R-mediated signaling on glucose metabolism. Glucose uptake experiments in isolated rat adipocytes showed that SP directly inhibits insulin-mediated glucose uptake. Our results further establish a role for SP-NK-1R interactions in adipose tissue responses, specifically as they relate to obesity-associated pathologies such as glucose intolerance and insulin resistance. Our results highlight this pathway as an important therapeutic approach for type 2 diabetes.

Diet-Induced Obesity Promotes Colon Tumor Development in Azoxymethane-Treated Mice

Obesity is an important risk factor for colon cancer in humans, and numerous studies have shown that a high fat diet enhances colon cancer development. As both increased adiposity and high fat diet can promote tumorigenesis, we examined the effect of diet-induced obesity, without ongoing high fat diet, on colon tumor development. C57BL/6J male mice were fed regular chow or high fat diet for 8 weeks. Diets were either maintained or switched resulting in four experimental groups: regular chow (R), high fat diet (H), regular chow switched to high fat diet (RH), and high fat diet switched to regular chow (HR). Mice were then administered azoxymethane to induce colon tumors. Tumor incidence and multiplicity were dramatically smaller in the R group relative to all groups that received high fat diet at any point. The effect of obesity on colon tumors could not be explained by differences in aberrant crypt foci number. Moreover, diet did not alter colonic expression of pro-inflammatory cytokines tumor necrosis factor-α, interleukin-6, interleukin-1β, and interferon-γ, which were measured immediately after azoxymethane treatment. Crypt apoptosis and proliferation, which were measured at the same time, were increased in the HR relative to all other groups. Our results suggest that factors associated with obesity – independently of ongoing high fat diet and obesity – promote tumor development because HR group animals had significantly more tumors than R group, and these mice were fed the same regular chow throughout the entire carcinogenic period. Moreover, there was no difference in the number of aberrant crypt foci between these groups, and thus the effect of obesity appears to be on subsequent stages of tumor development when early preneoplastic lesions transition into adenomas.

The neurotensin receptor-1 promotes tumor development in a sporadic but not an inflammation-associated mouse model of colon cancer.

Neurotensin receptor‐1 (NTR‐1) is overexpressed in colon cancers and colon cancer cell lines. Signaling through this receptor stimulates proliferation of colonocyte‐derived cell lines and promotes inflammation and mucosal healing in animal models of colitis. Given the causal role of this signaling pathway in mediating colitis and the importance of inflammation in cancer development, we tested the effects of NTR‐1 in mouse models of inflammation‐associated and sporadic colon cancer using NTR‐1‐deficient (Ntsr1−/−) and wild‐type (Ntsr1+/+) mice. In mice treated with azoxymethane (AOM) to model sporadic cancer, NTR‐1 had a significant effect on tumor development with Ntsr1+/+ mice developing over twofold more tumors than Ntsr1−/− mice (p = 0.04). There was no effect of NTR‐1 on the number of aberrant crypt foci or tumor size, suggesting that NT/NTR‐1 signaling promotes the conversion of precancerous cells to adenomas. Interestingly, NTR‐1 status did not affect tumor development in an inflammation‐associated cancer model where mice were treated with AOM followed by two cycles of 5% dextran sulfate sodium (DSS). In addition, colonic molecular and histopathologic analyses were performed shortly after a single cycle of DSS. NTR‐1 status did not affect colonic myeloperoxidase activity or histopathologic scores for damage and inflammation. However, Ntsr1−/− mice were more resistant to DSS‐induced mortality (p = 0.01) and had over twofold higher colonic expression levels of Il6 and Cxcl2 (p < 0.04), cytokines known to promote tumor development. These results represent the first direct demonstration that targeted disruption of the Ntsr1 gene reduces susceptibility to colon tumorigenesis.

Targeted Cox2 gene deletion in intestinal epithelial cells decreases tumorigenesis in female, but not male, ApcMin/+ mice

Mice heterozygous for mutations in the adenomatous polyposis coli gene (Apc+/− mice) develop intestinal neoplasia. Apc+/− tumor formation is thought to be dependent on cyclooxygenase 2 (COX2) expression; both pharmacologic COX2 inhibition and global Cox2 gene deletion reduce the number of intestinal tumors in Apc+/− mice. COX2 expression is reported in epithelial cells, fibroblasts, macrophages and endothelial cells of Apc+/− mouse polyps. However, the cell type(s) in which COX2 expression is required for Apc+/− tumor induction is not known. To address this question, we developed ApcMin/+ mice in which the Cox2 gene is specifically deleted either in intestinal epithelial cells or in myeloid cells. There is no significant difference in intestinal polyp number between ApcMin/+ mice with a targeted Cox2 gene deletion in myeloid cells and their control littermate ApcMin/+ mice. In contrast, ApcMin/+ mice with a targeted Cox2 deletion in intestinal epithelial cells have reduced intestinal tumorigenesis when compared to their littermate control ApcMin/+ mice. However, two gender‐specific effects are notable. First, female ApcMin/+ mice developed more intestinal tumors than male ApcMin/+ mice. Second, targeted intestinal epithelial cell Cox2 deletion decreased tumorigenesis in female, but not in male, ApcMin/+ mice. Considered in the light of pharmacologic studies and studies with global Cox2 gene knockout mice, our data suggest that (i) intrinsic COX2 expression in intestinal epithelial cells plays a gender‐specific role in tumor development in ApcMin/+ mice, and (ii) COX2 expression in cell type(s) other than intestinal epithelial cells also modulates intestinal tumorigenesis in ApcMin/+ mice, by a paracrine process.

Mo1609 Obesity Drives Colon Tumor Development in AOM-Treated Mice

Background/Aims: Obesity is an important risk factor for colon cancer in humans. Numerous studies in experimental animals demonstrated that a high fat diet enhances colon cancer development. A potential mechanism for this increased risk is that adipose tissue, a major source of adipokines, growth factors, and cytokines promotes tumor growth. Alternatively, a high-fat diet causes metabolic changes that can independently enhance tumor growth. Here we determined the independent effects of these factors on colon tumor development. Methods: C57BL/6J male mice were fed regular chow or high fat diet (HFD) for 8 weeks. Half of the animals on high fat diet were switched to regular chow, and half on regular chow were switched to the HFD with the resulting 4 groups being regular (R), HFD (H), regular switched to HFD (RH) and HFD switched to regular (HR). One week after the switch, 5 weekly doses of azoxymethane (AOM) were given to induce colon tumors. Mice were euthanized 1 day after the final dose of AOM to analyze gene expression, proliferation, and apoptosis in the colon or 25 weeks after AOM to analyze numbers of colon tumors and aberrant crypt foci (ACF). Body composition was measured by echo MRI. Results: We previously showed the significantly increased tumor multiplicities in all groups that received HFD (groups H, HR, and RH) relative to animals on regular diet (group R). In this study, an ELISA based array analysis of cytokines and growth factors in serum revealed that obesity was associated with differential expression of molecules involved in insulin-like growth factor (Igf) signaling. Relative to group R mice, group HR mice had ratios 1.5, 0.7, and 0.75 in serum concentrations of Igf-II, Igf binding protein (Igfbp) 5, and Igfbp6, respectively (p = 0.05 for each). These differences were also reflected in mRNA expression patterns in mesenteric adipose tissue, as all obese groups had significantly elevated expression of Igf-I and Igf-II and significantly depressed expression of Igfbp5 and 6. Group HR mice appeared to have a greater toxic response to AOM relative to all other groups as measured by apoptotic index and lengthening of the Ki67-positive proliferative zone in colonic crypts following AOM treatment. Conclusions: We have identified obesity itself as a tumor promoter. A mechanism for the increased tumor responses may involve heightened signaling through the Igf1-receptor due to increased expression of ligands and decreased expression of binding proteins in mesenteric adipocytes. Additionally, the greater tumor response of mice in HFD that were switched to regular diet likely involves a heightened toxic response to AOM. Supported by NIH RO-1 DK60729 (CP); A Pilot and Feasibility Study from CURE: Digestive Diseases Research Center 41301; and the Martin Blinder Fund for IBD Research

Colitis-Associated Adipose Tissue Responses to Corticotropin-Releasing Hormone Family of Neuropeptides

Background and Aims: Numerous studies have indicated that adipose tissue is an active endocrine organ and several adipose-associated molecules have been implicated as potential mediators of intestinal inflammation. Results from our group and others indicate that corticotrophin releasing hormone (CRH or CRF), and its peptide family members urocortins (UCNs) participate in the pathophysiology of intestinal inflammation and inflammatory bowel disease (IBD). Recent evidence also indicates that CRH receptors 1 and 2 are expressed by human adipocytes. Whether these ligands and receptors are expressed in the mesenteric fat and are modulated during colitis has never been studied. Here we compared levels of expression of the CRH peptide family at in the mesenteric fat depots of mice before and after experimental TNBS-induced colitis. We also exposed isolated human mesenteric preadipocytes to CRH and measured changes in cytokine production at the mRNA and protein levels. Methods: C57BL6 mice (n=10 per group) were injected intracolonically with either vehicle (40% ethanol, control) or TNBS (5 mg/20 grams) for 48 hr. Mesenteric adipose tissue was harvested from the mice and mRNA was isolated and converted to cDNA for real time RT PCR. Human mesenteric pre-adipocytes grown in culture were stimulated with 100nM CRH for 8 hours. Supernatants were collected for protein array and ELISA while mRNA was isolated for conversion to cDNA and use in real time RT PCR experiments. Data were analyzed using a two tailedMannWhitney test. Results: CRHmRNAwas significantly increased inmesenteric fat of TNBS-exposed mice compared to controls (by 112.9 fold, p<0.01). UCNs 2 and 3 were also significantly increased in these tissues (by 30.7 and 9.2 fold, p< 0.0016 and p< 0.0052, respectively). Protein arrays on CRH stimulated human mesenteric preadipocytes showed increased secretion of TIMP-2 and Rantes (CCL5 gene product) and decreased MIF, MCP-3 and PAI-1 levels. Changes in released MIF were also confirmed by ELISA in the medium of preadipocytes (p< 0.01). Conclusions: Increased expression of CRH and urocortins in adipose tissue during colitis together with the ability of CRH to induce changes in cytokine release from isolated adipocytes suggest that the CRH family of peptides may mediate inflammatory responses in mesenteric fat and affect the development of intestinal inflammation. These findings may represent a novel signaling pathway in colitis and could have important implications for inflammatory bowel disease treatment targets. Research Support: NIH NIDDK T32 DK07180-36 Gastroenterology Training Grant and P01 DK 33506