The Innate Immune Receptor NLRX1 Functions as a Tumor Suppressor by Reducing Colon Tumorigenesis and Key Tumor-Promoting Signals.

NOD-like receptor (NLR) proteins are intracellular innate immune sensors/receptors that regulate immunity. This work shows that NLRX1 serves as a tumor suppressor in colitis-associated cancer (CAC) and sporadic colon cancer by keeping key tumor promoting pathways in check. Nlrx1(-/-) mice were highly susceptible to CAC, showing increases in key cancer-promoting pathways including nuclear factor κB (NF-κB), mitogen-activated protein kinase (MAPK), signal transducer and activator of transcription 3 (STAT3), and interleukin 6 (IL-6). The tumor-suppressive function of NLRX1 originated primarily from the non-hematopoietic compartment. This prompted an analysis of NLRX1 function in the Apc(min/+) genetic model of sporadic gastrointestinal cancer. NLRX1 attenuated Apc(min/+) colon tumorigenesis, cellular proliferation, NF-κB, MAPK, STAT3 activation, and IL-6 levels. Application of anti-interleukin 6 receptor (IL6R) antibody therapy reduced tumor burden, increased survival, and reduced STAT3 activation in Nlrx1(-/-)Apc(min/+) mice. As an important clinical correlate, human colon cancer samples expressed lower levels of NLRX1 than healthy controls in multiple patient cohorts. These data implicate anti-IL6R as a potential personalized therapy for colon cancers with reduced NLRX1.

Localized intestinal radiation and liquid diet enhance survival and permit evaluation of long-term intestinal responses to high dose radiation in mice.

BACKGROUND: In vivo studies of high dose radiation-induced crypt and intestinal stem cell (ISC) loss and subsequent regeneration are typically restricted to 5-8 days after radiation due to high mortality and immune failure. This study aimed to develop murine radiation models of complete crypt loss that permit longer-term studies of ISC and crypt regeneration, repair and normalization of the intestinal epithelium. METHODS: In C57Bl/6J mice, a predetermined small intestinal segment was exteriorized and exposed to 14 Gy-radiation, while a lead shield protected the rest of the body from radiation. Sham controls had segment exteriorization but no radiation. Results were compared to C57Bl/6J mice given 14 Gy-abdominal radiation. Effects of elemental liquid diet feeding from the day prior to radiation until day 7 post-radiation were assessed in both models. Body weight and a custom-developed health score was assessed every day until day 21 post-radiation. Intestine was assessed histologically. RESULTS: At day 3 after segment radiation, complete loss of crypts occurred in the targeted segment, while adjacent and remaining intestine in segment-radiated mice, and entire intestine of sham controls, showed no detectable epithelial damage. Liquid diet feeding was required for survival of mice after segment radiation. Liquid diet significantly improved survival, body weight recovery and normalization of intestinal epithelium after abdominal radiation. Mice given segment radiation combined with liquid diet feeding showed minimal body weight loss, increased food intake and enhanced health score. CONCLUSIONS: The segment radiation method provides a useful model to study ISC/crypt loss and long-term crypt regeneration and epithelial repair, and may be valuable for future application to ISC transplantation or to genetic mutants that would not otherwise survive radiation doses that lead to complete crypt loss. Liquid diet is a simple intervention that improves survival and facilitates long-term studies of intestine in mice after high dose abdominal or segment radiation.

Elevated C-peptide and insulin predict increased risk of colorectal adenomas in normal mucosa.

BACKGROUND: Lower concentrations of the insulin-like growth factor binding protein-1 (IGFBP-1) and elevated concentrations of insulin or C-peptide have been associated with an increase in colorectal cancer risk (CRC). However few studies have evaluated IGFBP-1 and C-peptide in relation to adenomatous polyps, the only known precursor for CRC. METHODS: Between November 2001 and December 2002, we examined associations between circulating concentrations of insulin, C-peptide, IGFBP-1 and apoptosis among 190 individuals with one or more adenomatous polyps and 488 with no adenomatous polyps using logistic regression models. RESULTS: Individuals with the highest concentrations of C-peptide were more likely to have adenomas (OR = 2.2, 95% CI 1.4-4.0) than those with the lowest concentrations; associations that appeared to be stronger in men (OR = 4.4, 95% CI 1.7-10.9) than women. Individuals with high insulin concentrations also had a higher risk of adenomas (OR = 3.5, 95% CI 1.7-7.4), whereas higher levels of IGFBP-1 were associated with a reduced risk of adenomas in men only (OR = 0.3, 95% CI 0.1-0.7). Overweight and obese individuals with higher C-peptide levels (>1(st) Q) were at increased risk for lower apoptosis index (OR = 2.5, 95% CI 0.9-7.1), an association that remained strong in overweight and obese men (OR = 6.3, 95% CI 1.0-36.7). Higher levels of IGFBP-1 in overweight and obese individuals were associated with a reduced risk of low apoptosis (OR = 0.3, 95% CI 0.1-1.0). CONCLUSIONS: Associations between these peptides and the apoptosis index in overweight and obese individuals, suggest that the mechanism by which C-peptide could induce adenomas may include its anti-apoptotic properties. This study suggests that hyperinsulinemia and IGF hormones predict adenoma risk, and that outcomes associated with colorectal carcinogenesis maybe modified by gender.

Mucosal healing and fibrosis after acute or chronic inflammation in wild type FVB-N mice and C57BL6 procollagen α1(I)-promoter-GFP reporter mice.

BACKGROUND: Injury and intestinal inflammation trigger wound healing responses that can restore mucosal architecture but if chronic, can promote intestinal fibrosis. Intestinal fibrosis is a major complication of Crohn's disease. The cellular and molecular basis of mucosal healing and intestinal fibrosis are not well defined and better understanding requires well characterized mouse models. METHODS: FVB-N wild type mice and C57BL6 procollagen α1(I)-GFP reporter mice were given one (DSS1) or two (DSS2) cycles of 3% DSS (5 days/cycle) followed by 7 days recovery. Histological scoring of inflammation and fibrosis were performed at DSS1, DSS1+3, DSS1+7, DSS2, DSS2+3, and DSS2+7. Procollagen α1(I)-GFP activation was assessed in DSS and also TNBS models by whole colon GFP imaging and fluorescence microscopy. Colocalization of GFP with α-smooth muscle actin (α-SMA) or vimentin was examined. GFP mRNA levels were tested for correlation with endogenous collagen α1(I) mRNA. RESULTS: Males were more susceptible to DSS-induced disease and mortality than females. In FVB-N mice one DSS cycle induced transient mucosal inflammation and fibrosis that resolved by 7 days of recovery. Two DSS cycles induced transmural inflammation and fibrosis in a subset of FVB-N mice but overall, did not yield more consistent, severe or sustained fibrosis. In C57BL6 mice, procollagen α1(I)-GFP reporter was activated at the end of DSS1 and through DSS+7 with more dramatic and transmural activation at DSS2 through DSS2+7, and in TNBS treated mice. In DSS and TNBS models GFP reporter expression localized to vimentin(+) cells and much fewer α-SMA(+) cells. GFP mRNA strongly correlated with collagen α1(I) mRNA. CONCLUSIONS: One DSS cycle in FVB-N mice provides a model to study mucosal injury and subsequent mucosal healing. The procollagen α1(I)-GFP transgenic provides a useful model to study activation of a gene encoding a major extracellular matrix protein during acute or chronic experimental intestinal inflammation and fibrosis.

Insulin resistance, apoptosis, and colorectal adenoma risk.

Compelling evidence from epidemiologic studies indicates that elevated circulating insulin-like growth factor (IGF)-I, insulin resistance, and associated complications, such as elevated fasting plasma insulin, glucose and free fatty acids, glucose intolerance, increased body mass index, and visceral adiposity, are linked with increased risk of colorectal cancer. However, the role of insulin and markers of glucose control in the development of adenomas, precursors to colorectal cancer, has not been fully explored. We evaluated the relationship between plasma insulin, glucose, IGF-I, IGF-II, IGF-binding protein-3 (IGFBP-3), apoptosis, and colorectal adenomas in a case-control study. Participants were drawn from consenting patients undergoing colonoscopy at the University of North Carolina hospitals (Chapel Hill, NC). Participants were classified as cases or controls based on whether they had one or more colorectal adenomatous polyps. Fasting plasma insulin, IGF-I, IGF-II, and IGFBP-3 levels were assessed by ELISA. Glucose was measured by glucose hexokinase assay. Apoptosis was assessed by morphology on H&E-stained sections. Dietary and lifestyle information were obtained by telephone interview. Logistic regression was used to examine the association between adenoma status and insulin-IGF markers. Adenoma cases (n = 239) and adenoma-free controls (n = 517) provided rectal biopsies and/or blood samples and interview data. Consistent with prior findings, cases were more likely to be males, older, have higher waist-to-hip ratio, lower calcium intake, lower apoptosis, and less likely to report nonsteroidal anti-inflammatory drug use. Those in the highest quartile of insulin (adjusted odds ratio, 2.2; 95% confidence interval, 1.1-4.2) and glucose (adjusted odds ratio, 1.8; 95% confidence interval, 0.9-3.6) were more likely to have an adenoma compared with the lowest quartile. Similarly, subjects in the highest two quartiles of insulin were more likely to be in the lowest two quartiles of apoptosis. Overall, there were no significant differences between mean circulating levels of glucose, IGF-I, IGF-II, and IGFBP-3 among cases and controls and no association between these variables and apoptosis. The results provide novel evidence that elevated insulin and glucose are associated with increased adenoma risk and decreased apoptosis in normal rectal mucosa. These findings suggest that insulin may act early in the adenoma-carcinoma sequence to promote the development of colorectal adenoma by decreasing apoptosis in the normal mucosa.