Amir C. Patel

Dysregulation of microRNAs in colonic field carcinogenesis: implications for screening.

Colorectal cancer (CRC) screening tests often have a trade-off between efficacy and patient acceptability/cost. Fecal tests (occult blood, methylation) engender excellent patient compliance but lack requisite performance underscoring the need for better population screening tests. We assessed the utility of microRNAs (miRNAs) as markers of field carcinogenesis and their potential role for CRC screening using the azoxymethane (AOM)-treated rat model. We found that 63 miRNAs were upregulated and miR-122, miR-296-5p and miR-503# were downregulated in the uninvolved colonic mucosa of AOM rats. We monitored the expression of selected miRNAs in colonic biopsies of AOM rats at 16 weeks and correlated it with tumor development. We noted that the tumor bearing rats had significantly greater miRNA modulation compared to those without tumors. The miRNAs showed good diagnostic performance with an area under the receiver operator curve (AUROC) of >0.7. We also noted that the miRNA induction in the colonic mucosa was mirrorred in the mucus layer fecal colonocytes isolated from AOM rat stool and the degree of miRNA induction was greater in the tumor bearing rats compared to those without tumors. Lastly, we also noted significant miRNA modulation in the Pirc rats- the genetic model of colon carcinogenesis, both in the uninvolved colonic mucosa and the fecal colonocytes. We thus demonstrate that miRNAs are excellent markers of field carcinogenesis and could accurately predict future neoplasia. Based on our results, we propose an accurate, inexpensive, non-invasive miRNA test for CRC risk stratification based on rectal brushings or from abraded fecal colonocytes.

Topical polyethylene glycol as a novel chemopreventive agent for oral cancer via targeting of epidermal growth factor response.

Head and neck squamous cell carcinoma (HNSCC) is a major cause of morbidity and mortality underscoring the need for safe and effective chemopreventive strategies. Targeting epidermal growth factor receptor (EGFR) is attractive in that it is an early critical event in HNSCC pathogenesis. However, current agents lack efficacy or have unacceptable toxicity. Several groups have demonstrated that the over-the-counter medication, polyethylene glycol (PEG) has remarkable chemopreventive efficacy against colon carcinogenesis. Importantly, we reported that this effect is mediated through EGFR internalization/degradation. In the current study, we investigated the chemopreventive efficacy of this agent against HNSCC, using both the well validated animal model 4-NQO (4-nitroquinoline 1-oxide) rat model and cell culture with the human HNSCC cell line SCC-25. We demonstrated that daily topical application of 10% PEG-8000 in the oral cavity (tongue and cavity wall) post 4NQO initiation resulted in a significant reduction in tumor burden (both, tumor size and tumors/tumor bearing rat) without any evidence of toxicity. Immunohistochemical studies depicted decreased proliferation (number of Ki67-positive cells) and reduced expression of EGFR and its downstream effectors cyclin D1 in the tongue mucosa of 4NQO-rats treated with PEG. We showed that EGFR was also markedly downregulated in SCC-25 cells by PEG-8000 with a concomitant induction of G1-S phase cell-cycle arrest, which was potentially mediated through upregulated p21cip1/waf1. In conclusion, we demonstrate, for the first time, that PEG has promising efficacy and safety as a chemopreventive efficacy against oral carcinogenesis.

134 Brahma-Related Gene 1 (BRG1) as a Novel Epigenetic Modulator of Gene Dysregulation in Early Colorectal Carcinogenesis: Implications for Chemoprevention

Understanding the early molecular events in colorectal carcinogenesis is critical for designing novel diagnostic and chemopreventive strategies. One of the key early events is the diffuse dysregulation of gene expression prior to morphological lesions (field carcinogenesis). The mechanisms are believed to be largely epigenetic with methylation and microRNA being well explored. Recently, interest has focused on the SWI/SNF complex, chromatin remodeling proteins that have been implicated in carcinogenesis. Indeed, the complex member Brahmarelated gene 1 (BRG-1) has been implicated in lung and pancreatic cancer. However, colorectal carcinogenesis is largely unexplored. We therefore wanted to explore the role of BRG-1 in colon carcinogenesis and reversal during chemoprevention. Methods: To study the expression of BRG-1, immunohistochemistry studies were performed using different rat colorectal cancer models: the well-established 40-week azoxymethane treated (AOM) model and polyposis in rat colon (Pirc) model. We used the Pirc rat that harbor germline mutations in the APC mutation, the initiating genetic events in most sporadic colorectal cancer. These animals spontaneously develop colonic adenomas at 10 weeks. We utilized sulindac as a chemopreventive agent that was started at 5-6 weeks of age. Furthermore, BRG-1 expression at a message level was studied using human colon cancer cell line HCT116 with and without celecoxib treatment. Results: Immunohistochemistry revealed significantly reduced nuclear expression of BRG-1 in AOM treated colonic mucosa (50% compared to control). Immunohistochemistry of our Pirc rat model revealed reduced nuclear expression of BRG-1 in colonic mucosa (80% compared to wildtype). (Figure 1). Furthermore, Pirc rats treated with sulindac revealed an increase in BRG-1 expression (139% compared to untreated Pirc). (Figure 1) Finally, PCR data revealed that celecoxib treated HCT 116 cells expressed higher message levels of BRG-1 (137% compared to untreated). (Figure 2) Conclusions: We demonstrate, herein, for the first time that BRG-1 is suppressed early during colorectal carcinogenesis. This occurred both in a novel animal model and humans implicating its role as an important epigenetic regulator of early gene expression alterations in the premalignant mucosa. This suggests a role as a biomarker for risk stratification. Furthermore, treatment with an established chemopreventive agent reversed this process supporting the role that BRG-1 may represent a novel therapeutic target.

Su1871 Lactate Dehydrogenase-a Induction as an Early Metabolomic Marker of Colon Carcinogenesis: Potential Target for Chemoprevention

Our current study investigated the effect of PHLPP expression on the efficacy of chemotherapeutic drugs in colon cancer cells. Both PHLPP isoforms, PHLPP1 and PHLPP2, were either knocked down or overexpressed in SW480 and HT29 human colon cancer cells, and the rate of cell proliferation and apoptosis were measured upon drug treatment. Specifically, the cells were treated with different concentrations of oxaliplatin, PI3K inhibitor (LY294002), or rapamycin for 48 hours and cell proliferation was determined using MTS assays. Our results showed that knockdown of both PHLPP isoforms resulted in a decrease in the sensitivity to all drugs tested, whereas overexpression of PHLPP enhanced the anti-proliferation effect of these drugs. Moreover, we assessed the effect of the chemotherapy drugs in inducing cell death by monitoring the appearance of apoptotic markers including cleaved PARP and Caspase-3. Similarly, knockdown of both PHLPP isoforms rendered the cells resistant to apoptosis upon drug treatment. In summary, we have identified PHLPP as a critical factor in determining the drug sensitivity in the chemotherapeutic treatment of colon cancer. Adding yet another novel function of PHLPP and reasoning for the possible use of PHLPP as a therapeutic target in colon cancer.

885 Polyethylene Glycol (PEG) Suppresses Adenomas and Aberrant Crypt Foci (ACF) in the Polyposis in Rat Colon Model (Pirc): Implications for Colorectal Cancer Prevention

G A A b st ra ct s with regular aspirin use was 0.60 (95% CI, 0.47-0.76) among those with GT genotypes and 0.55 (95% CI, 0.38-0.81) with the TT genotypes. In contrast, regular aspirin use was not associated with lower risk among individuals with GG genotypes (multivariate OR, 0.99; 95% CI, 0.72-1.38). Among those with GT/TT genotypes, regular aspirin use appeared more strongly associated with risk of colorectal cancer with positive nuclear CTNNB1 expression (multivariate OR, 0.44; 95% CI, 0.26-0.75), but not negative nuclear CTNNB1 expression (multivariate OR, 0.86; 95% CI, 0.57-1.31). Conclusions: This molecular pathological epidemiology (MPE) study suggests that aspirin reduces risk of colorectal cancer, particularly tumors with activated CTNNB1, among individuals with rs6983267 GT/TT genotypes, but not among individuals with GG genotypes. Our results support an influence of aspirin on WNT signaling and suggest that aspirin chemoprevention may be tailored according to rs6983267 genotype.

886 Polyposis in Rat Colon (Pirc): A Robust Preclinical Model for Chemoprevention of Colorectal Cancer

G A A b st ra ct s with regular aspirin use was 0.60 (95% CI, 0.47-0.76) among those with GT genotypes and 0.55 (95% CI, 0.38-0.81) with the TT genotypes. In contrast, regular aspirin use was not associated with lower risk among individuals with GG genotypes (multivariate OR, 0.99; 95% CI, 0.72-1.38). Among those with GT/TT genotypes, regular aspirin use appeared more strongly associated with risk of colorectal cancer with positive nuclear CTNNB1 expression (multivariate OR, 0.44; 95% CI, 0.26-0.75), but not negative nuclear CTNNB1 expression (multivariate OR, 0.86; 95% CI, 0.57-1.31). Conclusions: This molecular pathological epidemiology (MPE) study suggests that aspirin reduces risk of colorectal cancer, particularly tumors with activated CTNNB1, among individuals with rs6983267 GT/TT genotypes, but not among individuals with GG genotypes. Our results support an influence of aspirin on WNT signaling and suggest that aspirin chemoprevention may be tailored according to rs6983267 genotype.

Sa1462 Obesity in Colorectal Adenomas May Have a Gender Specific Relationship

Background/Aims: Stress is well known to suppress appetite, ultimately leading to chronic eating disorders that may require medical treatment. However, no treatment exists for stressinduced loss of appetite, because its detailed mechanism remains unknown. Serotonin regulates emotions and food intake, and serotonin 2c receptor (5-HT2cR) is known to control feeding behavior. In this study, we used an urocortin 1 (UCN)-induced stress model to evaluate the effects of central 5-HT2cR and changes in the expression of c-fos mRNA. 5-HT2bR, another serotonin receptor that controls food intake, was also evaluated, and the effects of both receptor antagonists on loss of appetite were compared to investigate the potential of 5-HT2cR antagonist drugs for anorexia treatment. Methods: Intraventricular (ICV) injection of UCN or phosphate-buffered saline was administered to Sprague-Dawley rats, and the brains were collected after perfusion fixation, and fixed whole brains were cut in the coronal plane. The mRNA expression of 5-HT2cR and c-fos was evaluated by in situ hybridization. In addition, double staining of 5-HT2cR was performed for a sample section in which an increased level of c-fos mRNA expression was observed. The selective 5-HT2cR antagonist SB242084, the selective 5-HT2bR antagonist SB215505 (intraperitoneal), or rikkunshito (oral), which shows properties of both 5-HT2cR and 5-HT2bR antagonism, was administered and their respective effects on food intake decreases were evaluated in order to clarify the role of 5-HT2cR activation on stress-induced anorexia. Results: The 5-HT2cR mRNA expression in rats under UCN-induced stress was increased primarily in the solitary tract nucleus (NTS) of the medulla oblongata and in the paraventricular nucleus (PVN) of the hypothalamus. Concurrent enhancement of c-fos mRNA expression was observed. Expression of 5-HT2cR mRNA in the arcuate nucleus was approximately the same in both stress-induced and non-stress-induced groups. Double staining revealed that 5-HT2cR and c-fos were expressed in the same cells in the solitary tract nucleus and PVN. Intraperitoneal administration of 5-HT2cR antagonist SB242084 and oral administration of rikkunshito significantly inhibited food intake decreases in rats exposed to UCN-induced stress; in contrast, administration of the 5-HT2bR antagonist SB215505 did not affect food intake. Conclusion: 5-HT2cR antagonism is a potential candidate for the treatment of stressrelated anorexia. When the corticotrophin-releasing factor (CRF) receptor is activated, stress responses may be enhanced by increased responsiveness of 5-HT2cR on CRF neurons in the PVN. Signals from the vagus nerve may increase responsiveness of 5-HT2cR in the NTS, leading to further negative regulation of food intake.

M1179 Targeting Epidermal Growth Factor Receptor With Polyethylene Glycol: Applications for Digestive Cancer Prevention

Introduction: H. pylori transactivates the epidermal growth factor (EGFR) and induces extracellular-signal related kinase (ERK) phosphorylation in gastric epithelial cells which may be important in the pathway to gastric carcinogenesis. The green tea catechin epigallocatechin-3-gallate (EGCG) has been reported in some studies to have chemoprotective properties against development of gastric cancer. The aim of this study was to investigate if H. pyloriinduced ERK phosphorylation in A431 epithelial cells was inhibited by EGCG.Methods: H. pylori (NCTC 11637, cag PAI+) were co-cultured with A431 epithelial cells for 2.5 hrs. EGF (100 ng/ml) was used as a positive control. “In-Cell Western” (ICW) assay, which is a rapid quantitative method for determining the inhibitory effects of chemicals of interest on H. pylori epithelial cell signalling responses (REF Du Y et al., 2007), was used to examine the effects of EGCG on ERK1/2 phosphorylation (pERK) in A431 epithelial cells. EGCG (1-200 μM) was pre-incubated with A431 cells for 1 hr prior to the co-culture experiments. Results: EGF significantly (p<0.001, n=4) increased pERK in A431 cells compared to unstimulated controls. EGCG at concentrations greater than 50 μM caused a small but significant (p< 0.01) increase in pERK in A431 cells. EGCG dose-dependently significantly inhibited EGF (100 ng/ml)-induced pERK at a concentration of 10 μM and above (p<0.01, n=4). H. pylori strain NCTC 11637 significantly (p<0.01, n=4) increased pERK in A431 cells compared to unstimulated controls. EGCG dose-dependently inhibited pERK induced by H. pylori. EGCG at 100 μM significantly inhibited (p<0.05, n=4) H. pylori-induced pERK 2.5 hrs postculture. Conclusions: H. pylori-induced ERK phosphorylation in A431 epithelial cells was significantly inhibited by green tea catechin EGCG in a dose-dependent manner. The use of EGCG as dietary phytochemical for H. pylori-induced gastric cancer chemoprevention is plausible, at least in a specific group of patients with higher risk of gastric cancer development but failure for H. pylori eradication.