Tina P. Gibson

Neo-angiogenesis and the premalignant micro-circulatory augmentation of early colon carcinogenesis

Spectroscopic techniques have demonstrated that in the microscopically normal mucosa, there is an increase in mucosal micro-circulation in patients harboring neoplasia elsewhere in the colon (i.e. marker of field carcinogenesis). However, the physiological and molecular basis of this early increase in blood supply (EIBS) has not been elucidated. We, therefore, investigated the microvessel density (MVD) and angiogenic gene expression in the premalignant colonic mucosa from the well-validated azoxymethane (AOM)-treated rat experimental model of colon carcinogenesis. Fisher 344 rats were treated with AOM (15 mg/kg i.p.) or saline and euthanized 14 weeks later (a time-point that precedes carcinoma development). Colon sections were studied for MVD via immunohistochemical assessment for CD31 and location was compared with optical assessment of mucosal hemoglobin with low-coherence enhanced backscattering spectroscopy (LEBS). Finally, we performed a pilot real-time PCR angiogenesis microarray (84 genes) from the microscopically normal colonic mucosa of AOM and age-matched saline treated rats. AOM treatment increased MVD in both the mucosa and submucosa of the rats (125% increase in mucosa; p<0.007, and 96% increase in submucosa; p<0.02) but the increase was most pronounced at the cryptal base consistent with the LEBS data showing maximal hemoglobin augmentation at 200-225 μm depth. Microarray analysis showed striking dysregulation of angiogenic and anti-angiogenic factors. We demonstrate, for the first time, that neo-angiogenesis occurs in the microscopically normal colonic mucosa and was accentuated at the bottom of the crypt. This finding has potential implications as a biomarker for risk-stratification and target for chemoprevention.

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.

Association of stem-like cells in gender-specific chemoprevention against intestinal neoplasia in MIN mouse

This study was undertaken to examine the gender-sensitivity and chemopreventive responsiveness of celecoxib on intestinal stem-like cells as a biomarker of colon carcino-genesis, using the MIN mouse model. Male and female MIN mice (6-7-weeks old) were randomized to either control diet or to a diet supplemented with celecoxib (1,500 ppm). The animals were euthanized ten weeks later and the intestines were flushed and opened longitudinally to assess tumor count. Small intestinal segments were formalin-fixed and tissue sections were subjected to immunohistochemical evaluation of DCAMKL1, a known marker of stem-like cells. We found that in animals receiving control (AIN 76A diet) alone, female MIN mice had a higher polyp count than males (52.32 ± 13.89 vs. 35.43 ± 16.05; p<0.0005). However, compared to control diet groups, celecoxib supplementation caused a larger reduction in the number of polyps in females than their male cohorts (6.38 ± 1.43 vs. 12.83 ± 6.74; a reduction of 88% in females to 64% in males). Significant differences (p=0.013) were observed in the number of DCAMKL1-stained cells in the crypts of the wild-type (WT) (10.01 ± 1.07 stem cells per high powered field; HPF) compared to the MIN mice (24.15 ± 8.08 stem cells per HPF), illustrating increased stem-like cells in animals that are more prone to neoplasia. DCAMKL1 labeled stem-like cells were equal in number in the male and female groups receiving the control AIN 76A diet alone (females, 25.73 stem-like cells/HPF); males, 24.15 stem-like cells/HPF). However, females showed a greater reduction in the number of DCAMKL1-labeled stem-like cells with celecoxib supplementation than the respective males (16.63 ± 4.23 vs. 21.56 ± 9.06; a reduction of 35.4% in females to 10.7% in males). We conclude that a higher number of stem-like cells in the uninvolved mucosa paralleled tumorigenesis and mirrored greater chemopreventive responsiveness of female MIN mice compared to males.

MicroRNAs as Novel Targets for NSAID Chemoprevention of Colon Carcinogenesis

Background & Objective: In the United States, colorectal cancer (CRC) is the third most prevalent and deadly malignancy. Development of novel CRC-specific abnormal DNA biomarkers may advance non-invasive, cost-efficient population-based CRC screening and ultimately reduces CRC death. DNA hypermethylation is a common epigenetic abnormality in CRCs and represents a promising class of cancer biomarkers. The objective of the current study was to develop optimal DNA hypermethylation-based biomarkers for use in fecesor serum-based average-risk CRC screening. Design: We first applied DNAmethylationmicroarray analysis in order to identify novel loci demonstrating neoplasia-specific methylation in the colon. This array analysis allowed us to investigate 55% of CpG islands within the genome and was applied to 17 primary CRCs relative to 8 non-neoplastic colonic tissues (NCs) from neoplasia-free subjects. The detected CRC-associated hypermethylation events were then individually measured in 113 colonic tissues comprising 51 CRCs, 9 adenomas, 19 NCs from CRC patients (CRC-NCs), and 34 NCs from neoplasia-free subjects (control NCs) using highly sensitive and quantitative real-time quantitative methylation-specific PCR (qMSP) assays. Receiver-operator characteristics (ROC) curve analysis was applied to the qMSP data in order to assess each individual event as well as combination of events for their ability to discriminate neoplastic from non-neoplastic cases. Results: Microarray-based global methylation profiles discriminated CRCs from NCs. A bioinformatic filtering of the microarray data identified 169 candidate CRC-associated hypermethylation events, including one previously validated hypermethylation marker for fecal DNA-based CRC detection, SFRP2. Fourteen of these 169 loci were evaluated using qMSP assays. Ten of these 14 methylation events significantly distinguished CRCs from control NCs (p<.01). Of these ten events, methylation of VSX2 achieved the highest discriminative accuracy (83.3% sensitivity and 92.3% specificity; Area under ROC curve, or AUC, 0.93, p<1E-6). Similarly, CRC-NCs were significantly discriminated from control NCs by methylation of ALX3 (AUC 0.78, p<1E-4). The discrimination of CRC-NCs from control NCs was improved by a multi-locus methylation panel (AUC 0.83, p<1E-6) relative to ALX3. Although the sample numbers were small, two methylation events significantly distinguished adenomas from control NCs (p<.01). Conclusions: Systematic methylome analysis has identified 11 novel methylation events in neoplastic and non-neoplastic colonic mucosa from CRC patients that accurately discriminate CRC patients from controls. These markers merit further evaluation as candidate biomarkers for stool and circulating DNA-based CRC detection.

Sa1835 Nanocytomics as a Novel Modality for Determining Transcriptional Activity

to the squamocolumnar junction (SCJ) were quantified and compared between Pre-CE-IM and Post-CE-IM groups. RESULTS: 3D-OCT provided 80x 100x larger field-of-view compared to biopsy and reliably (in 100% cases) sufficient imaging depth, into the muscularis mucosa, for detecting SSIM. In comparison, only 40% to 80% biopsies reached the lamina propria as reported in recent studies. SSIM was found in 72% (13/18) patients in the PreCE-IM group, and 63% (10/16) patients in the Post-CE-IM group. The number of SSIM per patient in the Post-CE-IM group [7.1 (9.3)] was significantly lower compared to the Pre-CE-IM group [34.4 (44.6); p = 0.02]. SSIM gland size (p = 0.69) and distribution (p = 0.54) were not significantly different before and after CE-IM. CONCLUSIONS: The prevalence of SSIM in patients who responded to treatment after RFA is higher than previously reported using four-quadrant biopsy. Compared to other advanced imaging modalities only imaging surface [narrow band imaging (NBI), chromoendoscopy] or small focal areas [confocal, elastic scattering spectroscopy (ESS), etc.], 3D-OCT provides both subsurface and broad area of view uniquely suited to potentially evaluate patients before and after ablation treatment. ACKNOWLEDGEMENT: NIH 5R01-CA075289-14, K99-EB010071-01A1, and AFOSR FA9550-10-1-0063 and FA9550-10-1-0551.

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.

Mutual Regulation of Expression Between MUC4 and CDx2 in Colon Cancer Cells

Background:Mucins are a family of large glycoproteins that have been implicated in colorectal carcinogenesis. Our group has explored the role of MUC4, a transmembrane mucin, which is generally upregulated in most cancers (pancreas, lung etc), during colorectal carcinogenesis and noted that MUC4 is paradoxically downregulated in colorectal cancers. The progressive downregulation occurs since early stages of carcinogenesis (premalignant mucosa). However, the mechanism remains unclear. CDX2 is a caudal-related homeobox gene that has been known to be dysregulated in colon carcinogenesis. Intriguingly, a recent report implicated CDX2 in MUC4 regulation in Barretts esophagus. We, therefore, wanted to elucidate the role of CDX2 in the MUC4 loss in colon carcinogenesis Materials and Methods:HT29 cells were incubated in standard conditions and transiently transfected with CDX2 siRNA using Lipofectamine. Separately, HT29 cells were stably transfected withMUC4 shRNA and suitable control vector (CV). MUC4 shRNA transfected HT29 cells and CV transfected HT29 cells were treated with PI3K inhibitor LY294002. Protein lysates and RNA were obtained from the transfected cells and western blotting and qRT-PCR performed to assess the expression of MUC4, CDX2 and pAKT (S473). Results:In HT29 cells, a modest decrease in CDX2 expression by SiRNA (34% decreased CDX2 expression) concomitantly reduced MUC4 expression by 20%. Conversely, MUC4 shRNA transfection of HT29 cells (75% reduced MUC4 expression; p=0.05) resulted in 80% reduction in CDX2 mRNA expression and 60% reduction (p<0.02) in CDX2 protein levels. Since PI3K-AKT signaling cascade is known to modulate CDX2 levels, we assessed this pathway in MUC4 shRNA transfected HT29 cells, and demonstrated 50% increase (p<0.02) in Serine 473 phosphyrated AKT, which was mitigated by PI3K inhibitor, LY294002. However, despite the fact that MUC4 knockdown activated AKT, there was no effect of LY294002 treatment on the expression of CDX2 in MUC4 shRNA transfected HT29 cells, indicating that MUC4 modulates CDX2 expression via PI3K/pAKT independent manner. Conclusions: We report, herein, for the first time, that the dysregulation of two putative tumor suppressor genes, MUC4 and CDX2, is intimately related in colon cancer. Not only do CDX2 and MUC4 mirror each others expression but also mutually regulate each other. Further studies are ongoing to dissect this important novel pathway in colon carcinogenesis.