Pirc Rats Research Articles

A flavonoid-rich extract from bergamot juice prevents carcinogenesis in a genetic model of colorectal cancer, the Pirc rat (F344/NTac-Apcam1137).

To determine the potential of a flavonoid-rich extract from bergamot juice (BJe) to prevent colorectal carcinogenesis (CRC) in vivo. Pirc rats (F344/NTac-Apcam1137), mutated in Apc, the key gene in CRC, were treated with two different doses of BJe (35mg/kg or 70mg/kg body weight, respectively) mixed in the diet for 12weeks. Then, the entire intestine was surgically removed and dissected for histological, immunohistochemical and molecular analyses. Rats treated with BJe showed a significant dose-related reduction in the colon preneoplastic lesions mucin-depleted foci (MDF). Colon and small intestinal tumours were also significantly reduced in rats supplemented with 70mg/kg of BJe. To elucidate the involved mechanisms, markers of inflammation and apoptosis were determined. Compared to controls, colon tumours from BJe 70mg/kg-supplemented rats showed a significant down-regulation of inflammation-related genes (COX-2, iNOS, IL-1β, IL-6 and IL-10 and Arginase 1). Moreover, in colon tumours from rats fed with 70mg/kg BJe, apoptosis was significantly higher than in controls. Up-regulation of p53 and down-regulation of survivin and p21 genes was also observed. These data indicate a strong chemopreventive activity of BJe that, at least in part, is due to its pro-apoptotic and anti-inflammatory actions. This effect could be exploited as a strategy to prevent CRC in high-risk patients.

Morin-dependent inhibition of low molecular weight protein tyrosine phosphatase (LMW-PTP) restores sensitivity to apoptosis during colon carcinogenesis: Studies in vitro and in vivo, in an Apc-driven model of colon cancer.

LMW-PTP has been associated with the development of colorectal cancer (CRC) and with the resistance to chemotherapy in cancer cells. To clarify its role in vivo, we studied LMW-PTP expression in Pirc rats (F344/NTac-Apc am1137 ), genetically prone to CRC and resistant to apoptosis. In the morphologically normal mucosa (NM) of Pirc rats, a dramatic over-expression of LMW-PTP was found compared to wt rats (about 60 times higher). Moreover, LMW-PTP levels further increase in spontaneously developed Pirc colon tumors. To understand if and how LMW-PTP affects resistance to apoptosis, we studied CRC cell lines, sensitive (HT29 and HCT-116), or resistant (HT29R, HCT116R) to 5-Fluorouracil (5-FU): resistant cells over-express LMW-PTP. When resistant cells were challenged with morin, a polyphenol inhibiting LMW-PTP, a fast and dose-related down-regulation of LMW-PTP was observed. 5-FU and morin co-treatment dramatically decreased cell viability, increased apoptosis, and significantly impaired self-renewal ability of all the cancer cell lines we have studied. Similarly, we observed that, in Pirc rats, one-week morin administration (50 mg/kg) down-regulated LMW-PTP and restored the apoptotic response to 5-FU in the NM. Finally, administration of morin for a longer period led to a significant reduction in colon precancerous lesions, together with a down-regulation of LMW-PTP. Taken together, these results document the involvement of LMW-PTP in the process of CRC in vitro and in vivo. Morin treatment may be envisaged as a system to increase the sensitivity to chemotherapy and to prevent carcinogenesis.

Abstract 2400: Complex gut microbiota modulate rat colon adenoma susceptibility, metabolites, and host gene expression

Abstract We used a multi-omics approach to identify signatures of differential susceptibility in the F344/NTac-Apc+/Pirc (Pirc) rat model of familial adenomatous polyposis. We assessed how different gut microbiomes (GMs) effect the metabolome and host transcriptome to reveal mechanisms of susceptibility. We previously found that differences in the GM are causative determinants of cancer susceptibility using complex microbiota targeted rederivation (CMTR), in which isogenic Pirc rat embryos are transferred into surrogate dams each harboring distinct specific pathogen free gut microbiota: GM-1 and GM-2. Fecal samples collected from CMTR Pirc rats at 1 month of age were analyzed using ultra-high performance liquid chromatography mass spectrometry (UHPLC/MS) to assess the impact of bacterial metabolites on disease susceptibility. At 6 months of age animals were sacrificed, tumor burden assessed, and normal colonic epithelium (NE) and tumor (T) tissues were collected for RNASeq analysis. Metabolomic analysis identified several features that were unique to either GM-1 or GM-2, and principal component analysis revealed a significant separation of the groups. Based on mass-charge ratios, 117 features were significantly different between the groups, of which 28 were putatively identifiable metabolites through the HMDB (Human Metabolite Database) and METLIN databases. Hierarchical cluster analysis of the 1 month metabolome data based on Euclidean distance measurements (Ward's algorithm) showed separation of samples based on tumor burden at sacrifice. Metabolites present prior to observable disease onset at 1 month were correlated with adenoma burden 5 months later. RNAseq between GM-1 and GM-2 tissues identified 2173 differentially expressed genes (DEGs) in the normal epithelium, and 3406 DEGs between tumor groups (FDR < 0.05). Ordinate analysis confirmed both NE and T samples were significantly distinct between rats harboring the two GM profiles. Integrated pathway (IP) analysis of metabolome and RNASeq data based on detected features showed that bile acid biosynthesis was enriched in GM-1, identifying 14 target genes affecting this pathway. Metabolome data from serum samples from an independent group of 1 month old Pirc and wildtype rats with GM-1 also identified metabolites from bile acid biosynthesis elevated in Pirc serum. Six out of the 14 target genes from the IP analysis showed significant differences (FDR < 0.05) between GM-1 and GM-2 in the NE and T tissues indicating a potential role of metabolites modulating both NE and T gene expression. DEG pathway analysis indicated increased fatty acid metabolism and mucin biosynthesis in the high susceptibility group, and apoptosis and calcium signaling pathways increased in the resistant group. These data show the utility of using metabolomics for early detection of adenomas and provide insights into the mechanism of how the GM modulates colon cancer. Citation Format: Susheel Bhanu Busi, Zhentian Lei, Lloyd Sumner, James Amos-Landgraf. Complex gut microbiota modulate rat colon adenoma susceptibility, metabolites, and host gene expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2400.

Abstract 4987: Biofilm-producing sulfate-reducing bacteria suppress tumor burden in a rat model of colon cancer

Abstract Human epidemiological and animal model studies have shown that the presence of colon cancer is associated with certain microbiota. Previously, we re-derived genetically identical embryos of the Pirc (F344/NTac-Apc+/Pirc) rat model of familial adenomatous polyposis using three strains of surrogate dams (F344/NHsd, LEW/SsNHsd, and Crl:SD), each harboring distinct gut microbiota (GM) that modulated adenoma susceptibility. Bacterial relative abundances as determined by 16S rDNA sequencing showed several taxa correlating with suppression of both tumor growth and phenotype penetrance as early as 1 month of age. One taxon associated with reduced adenoma burden was a sulfate-reducing bacterium, viz. Desulfovibrio spp. To determine if Desulfovibrio was responsible for differences in tumor burden, Pirc rats harboring complex GM were gavaged at 14 and 15 days of age with two genetic isolates of Desulfovibrio (~10^8 colony forming units, CFU): biofilm-forming DvH-MT and biofilm-deficient DvH-MO. We found that the rats stably colonized with the biofilm-forming DvH-MT strain, with the bacteria present at 4 months of age. Adenoma burden at sacrifice showed that the biofilm-competent strain significantly reduced colonic adenoma size (t-test, p<0.05), compared to the biofilm-deficient, DvH-MO-treated rats. To understand how the biofilm-forming capacity of the DvH-MT strain modulated adenoma burden, the genomes of the DvH-MT and DvH-MO strains were sequenced and 12 variants were identified between the two strains. In the DvH-MO strain one variant in the DVU1017 type-1 secretion system ABC transporter binding protein gene was shown to be required for biofilm-formation by Desulfovibrio in vitro. This gene was inactivated in the parent DvH-MT strain and the new construct was designated JWT716. To determine the presence and proximity of Desulfovibrio to adenomas in the colon, we modified the parental DvH-MT to express the fluorescent protein, dTomato and designated the fluorescent strain as JWT733. Pirc rats were treated with the biofilm-forming, JWT733 and the biofilm-deficient, JWT716. 3-months post-treatment we confirmed via qRT-PCR the presence of the biofilm-forming, JWT733 in fecal samples. JWT716 was undetectable in the fecal samples from most rats. Using a narrow-band filter to detect dTomato, we confirmed JWT733 colonization in the treated rats by colonoscopy. At 4 months of age, the rats were sacrificed and adenoma burden was estimated. We found that the biofilm-forming Desulfovibrio suppressed tumor burden in the colon compared to rats treated with the biofilm-deficient strain (t-test, p<0.05). This implicates the role of biofilm formation and potentially sulfate-reduction by this bacterium in the suppression of tumor multiplicity. In this model of colon cancer, Desulfovibrio alone or in concert may be residing in a niche in the intestinal mucus layer creating a beneficial or protective biofilm. Citation Format: Susheel Bhanu Busi, Kara B. De Leon, Judy D. Wall, James M. Amos-Landgraf. Biofilm-producing sulfate-reducing bacteria suppress tumor burden in a rat model of colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4987.

Evaluation of a Tumor-Targeting, Near-Infrared Fluorescent Peptide for Early Detection and Endoscopic Resection of Polyps in a Rat Model of Colorectal Cancer.

The goal of these studies was to use a tumor-targeting, near-infrared (NIR) fluorescent peptide to evaluate early detection and to guide surgical removal of polyps in a genetically engineered rat model of spontaneous colorectal cancer. This peptide, LS301, was conjugated to Cy7.5 and applied topically to the colon of adenoma-bearing Pirc rats. Ten minutes after administration, rats underwent targeted NIR laser colonoscopy. Rats were also evaluated by white light colonoscopy and narrow-band imaging, for comparison to the NIR technique. Unlike white light and narrow-band colonoscopy, NIR imaging detected unexpected flat lesions in young Pirc rats. NIR imaging was also used to assess resection margins after electrocauterization of polyps. Tumor margins remained negative at 5 weeks postsurgery, demonstrating successful polypectomy. The present studies show that NIR-targeted colonoscopy is an attractive strategy to improve screening for and resection of colorectal neoplasia.

Pomegranate By-Products in Colorectal Cancer Chemoprevention: Effects in Apc-Mutated Pirc Rats and Mechanistic Studies In Vitro and Ex Vivo.

To investigate the effect of pomegranate mesocarp, a polyphenol-rich by-product of juice production, in colorectal cancer (CRC) chemoprevention. A mesocarp decoction (PMD) is administered for 6 weeks in the diet to Pirc rats, mutated in Apc, a key-gene in CRC. Mucin-depleted foci (MDFs), as CRC biomarkers, are reduced in PMD-fed rats compared to controls (MDF/colon: 34±4 versus 47±3, p = 0.02). There is an increase in apoptosis in MDFs from PMD-treated rats compared to controls (2.5±0.2 versus 1.6±0.2, p<0.01). To elucidate the involved mechanisms, two colon-relevant metabolites of the polyphenolic and fiber PMD components, urolithin-A (u-A) and sodium butyrate (SB), are tested alone or in combination in vitro (colon cancer cells), and ex vivo in adenoma (AD) and normal mucosa (NM) from Pirc rats. u-A 25 μm plus SB 2.5 mm (USB) causes a significant reduction in COX-2 protein expression compared to untreated controls (about -70% in cancer cell cultures, AD, and NM), and a strong increase in C-CASP-3 expression in cells (about ten times), in AD and NM (+74 and +69%). These data indicate a chemopreventive activity of PMD due, at least in part, to pro-apoptotic and anti-inflammatory action of its metabolites that could be exploited in high-risk patients.

Metabolic reprogramming of the premalignant colonic mucosa is an early event in carcinogenesis.

BackgroundColorectal cancer (CRC) is the second leading cause of cancer-related mortality in the United States. There is an increasing need for the identification of biomarkers of pre-malignant and early stage CRC to improve risk-stratification and screening recommendations. In this study, we investigated the possibility of metabolic and mitochondrial reprogramming early in the pre-malignant colorectal field.MethodsRectal biopsies were taken from 81 patients undergoing screening colonoscopy, and gene expression of metabolic and mitochondrial markers were assessed using real time quantitative PCR. Validation studies were performed in two different animal models of colon carcinogenesis: Pirc rats and AOM-treated rats.ResultsWe found evidence of a Warburg effect in the normal-appearing rectal mucosa of patients harboring precancerous lesions elsewhere in the colon compared to control patients, with a significant increase in HIF1a, SLC2A1 (referred to as GLUT1), PKM2, and LDHA. We also found evidence of early mitochondrial changes in the colorectal field of patients harboring pre-cancerous lesions, with significantly increased mitochondrial gene expression of DRP1 (fission), OPA1 (fusion), PGC1-a (biogenesis), UCP2 (uncoupling) and mtND1 (copy number). Similar results were observed in the two different animal models.ConclusionsThese results demonstrate for the first time evidence of early Warburg-like metabolic changes as well as changes in mitochondrial function, dynamics and mtDNA copy number in endoscopically normal premalignant colorectal mucosal field. These findings provide an opportunity for the development of metabolic biomarkers that could be used for improving screening recommendations and risk-stratification. This also provides a potential target for novel chemopreventive strategies in the pre-malignant colorectal field.

RETRACTED ARTICLE: Astragali radix: could it be an adjuvant for oxaliplatin-induced neuropathy?

Neurotoxicity is a major side effect of platinum derivatives both during and after treatment. In the absence of effective pharmacological compounds, the opportunity to identify safe adjuvant treatments among medicinal plants seems appropriate. Astragali radix is an adaptogenic herbal product recently analyzed in platinum-treated cancer patients. With the aim of evaluating the anti-neuropathic profile of Astragali radix, a previously characterized aqueous (Aqu) and two hydroalcoholic (20%HA and 50%HA) extracts were tested in a rat model of oxaliplatin-induced neuropathy. Repeated administrations significantly reduced oxaliplatin-dependent hypersensitivity with 50%HA, the most effective, fully preventing mechanical and thermal hypersensitivity. Ex vivo, 50%HA reduced morphometric and molecular alterations induced by oxaliplatin in peripheral nerve and dorsal-root-ganglia. In the spinal cord and in brain areas, 50%HA significantly decreased activation of microglia and astrocytes. Furthermore, 50%HA prevented the nephro- and hepato-toxicity induced by the anticancer drug. The protective effect of 50%HA did not alter oxaliplatin-induced apoptosis in colon tumors of Pirc rats, an Apc-driven model of colon carcinogenesis. The hydroalcoholic extract (50%HA) of Astragali radix relieves pain and promotes the rescue mechanisms that protect nervous tissue from the damages triggering chronic pain. A safe profile strongly suggests the usefulness of this natural product in oxaliplatin-induced neuropathy.

Gene Expression Profile of Colon Mucosa after Cytotoxic Insult in wt and Apc-Mutated Pirc Rats: Possible Relation to Resistance to Apoptosis during Carcinogenesis.

Apc-mutated Pirc rats, spontaneously developing intestinal tumours, are resistant to 1,2-dimethylhydrazine- (DMH-) induced colon apoptosis. To understand this phenomenon, we analyzed the expression of genotoxic stress-related genes Mgmt, Gsta1, and Gstp1 in the colon of wt and Pirc rats in basal conditions and 24 h after DMH; plasmatic oxidant/antioxidant status was also evaluated. After DMH, Mgmt expression was increased in both genotypes but significantly only in wt rats; Gsta1 expression was significantly increased in both genotypes. Gstp1 expression did not vary after DMH but was lower in Pirc rats. Moreover, for each genotype, we studied by microarray technique whole gene expression profile after DMH. By unsupervised cluster analysis, 28 genes were differentially modulated between the two genotypes. Among them were interferon-induced genes Irf7, Oas1a, Oasl2, and Isg15 and the transcription factor Taf6l, overexpressed in DMH-treated wt rats and unchanged in Pirc rats. RT-PCR confirmed their overexpression in DMH-treated wt rats and showed a slighter variation in DMH-treated Pirc rats. Taken together, despite a blunted induction of Irf7, Oas1a, and Mgmt, defective apoptosis in Pirc rats 24 h after DMH is not mirrored by major differences in gene expression compared with wt rats.

Differential susceptibility to colorectal cancer due to naturally occurring gut microbiota.

Recent studies investigating the human microbiome have identified particular bacterial species that correlate with the presence of colorectal cancer. To evaluate the role of qualitatively different but naturally occurring gut microbiota and the relationship with colorectal cancer development, genetically identical embryos from the Polyposis in Rat Colon (Pirc) rat model of colorectal cancer were transferred into recipients of three different genetic backgrounds (F344/NHsd, LEW/SsNHsd, and Crl:SD). Tumor development in the pups was tracked longitudinally via colonoscopy, and end-stage tumor burden was determined. To confirm vertical transmission and identify associations between the gut microbiota and disease phenotype, the fecal microbiota was characterized in recipient dams 24 hours pre-partum, and in Pirc rat offspring prior to and during disease progression. Our data show that the gut microbiota varies between rat strains, with LEW/SsNHsd having a greater relative abundance of the bacteria Prevotella copri. The mature gut microbiota of pups resembled the profile of their dams, indicating that the dam is the primary determinant of the developing microbiota. Both male and female F344-Pirc rats harboring the Lewis microbiota had decreased tumor burden relative to genetically identical rats harboring F344 or SD microbiota. Significant negative correlations were detected between tumor burden and the relative abundance of specific taxa from samples taken at weaning and shortly thereafter, prior to observable adenoma development. Notably, this naturally occurring variation in the gut microbiota is associated with a significant difference in severity of colorectal cancer, and the abundance of certain taxa is associated with decreased tumor burden.

Sulindac, 3,3'-diindolylmethane and curcumin reduce carcinogenesis in the Pirc rat, an Apc-driven model of colon carcinogenesis.

BackgroundRecently, we showed that Sulindac (SU; 320 ppm) reduces precancerous lesions in the colon of Pirc rats, mutated in the Apc gene. Surprisingly, previous data in Apc-mutated mice showed that SU, with reported efficacy in Familial Adenomatous Polyposis (FAP), increases colon carcinogenesis. Therefore, we assessed the effect of SU 320 ppm in a long-term carcinogenesis experiment in Pirc rats. Moreover, since side effects of SU hamper its chronic use and a combination of drugs could be more effective and less toxic than single agents, we also studied whether two natural compounds, 3,3’-diindolylmethane (DIM; 250 ppm) and curcumin (CUR; 2000 ppm), with or without lower doses of SU could affect carcinogenesisMethodsPirc rats were fed an AIN76 diet containing SU, DIM and CUR and sacrificed at 8 months of age to measure intestinal tumours. Apoptosis and proliferation in the normal colon mucosa, as well as gene expression profile were studiedResultsColon tumours were significantly reduced by SU 320 ppm (62 % reduction over Controls), by DIM and CUR without or with SU 80 and 160 ppm (50, 53 and 58 % reduction, respectively) but not by SU 80 ppm alone. Total tumours (colon and small intestine) were reduced by SU (80 and 320 ppm) and by DIM and CUR. Apoptosis in the normal mucosa was significantly increased by SU 320 ppm, and slightly increased by DIM and CUR with or without SU. A slight reduction in Survivin-Birc5 expression was observed with all the treatments compared to Controls. Proliferative activity was not variedConclusionsThe results on SU reinforce the validity of Pirc rats to identify chemopreventive products. Moreover, the efficacy of the DIM and CUR combination to lower colon tumours, suggests an alternative strategy to be exploited in patients at risk.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1627-9) contains supplementary material, which is available to authorized users.

Apc‐driven colon carcinogenesis in pirc rat is strongly reduced by polyethylene glycol

Polyethylene glycol (PEG) is one of the most powerful agents in reducing chemically induced carcinogenesis in rat colon. However, contrasting results in Min mice dampened the enthusiasm on this potentially strong and virtually safe, cancer chemopreventing agent. Pirc (F344/NTac-Apc (am1137) ) rats carrying a germline heterozygous mutation in the Apc gene, spontaneously develop multiple tumours in the colon thus modelling both familial adenomatous polyposis (FAP) and sporadic colorectal cancer (CRC). Given this similarity, we thought that these rats could be appropriate to test the efficacy of PEG 8000 in reducing carcinogenesis. Pirc male rats aged one month were treated with 5% PEG in drinking water for 2 or 6 months. Precancerous lesions were dramatically reduced after 2 months of PEG treatment (Mucin depleted foci (MDF)/colon were 99 ± 17 and 12 ± 8 in Controls and PEG-treated rats, respectively; p < 0.001; mean ± SD). Similarly, colon tumors were significantly reduced after 6 months of treatment (tumors/rat were 8.1 ± 2.3 and 3.6 ± 2.2 in Controls and PEG-treated rats, respectively; p < 0.05; mean ± SD). Colon proliferation, a parameter correlated to cancer risk, was also significantly lower in PEG-treated rats than in Controls, while apoptosis was not significantly affected. In conclusion, PEG markedly reduces colon carcinogenesis in Pirc rats mutated in Apc; we thus suggest that PEG may be used as chemopreventive agent to reduce cancer risk in FAP and CRC patients.

Sa1911 Mitochondrial Alterations in Early Colon Carcinogenesis: Evidence for Warburg Effect in the Initiation of Colorectal Cancer (CRC) Carcinogenesis

Background: There is emerging realization of the central role of metabolism in carcinogenesis with the Warburg effect (preferential utilization of glycolysis in normoxic environment) (e.g. Weinberg, Cell 2012). We have previously noted an increase in microvascular blood supply in the predysplastic colonic mucosa (field carcinogenesis) (Gut 2005, Gastro 2008, Clin Cancer Res 2009, Cancer Prev Res 2010) which may reflect altered metabolism, however molecular determinants remain unexplored. The role of mitochondria in CRC is well established in frank tumors with dysfunction reflected by increased fission leading to less efficient oxidative phosphorylation, enabling shunting of precursors for cell growth. However, the presence of mitochondrial dysfunction and Warburg effect in early colon carcinogenesis has yet been explored. Therefore, we chose to investigate potential mitochondrial changes in the pre-malignant colon. Methods: To investigate mitochondrial changes we incorporated both human and animal studies: For human studies we obtained biopsies from endoscopically normal rectal muscosa from 80 patients undergoing screening colonoscopies. Samples were processed for gDNA and RNA isolation. Real Time PCRwas conducted to assess mitochondrial mass, OPA-1, UCP2, and PKM2 expression. For animal experiments we utilized the Polypopsis in Rat Colon (Pirc) rat, which possesses an APC truncation, consistent with the initiating factor in ~80% of sporadic CRCs. 7 Pirc rats with age-matched controls were euthanized after 24 wks of age. Colons were excised from these animals to obtain colonic gDNA and RNA. Real Time PCR was conducted to assess mitochondrial mass, OPA-1 and UCP2 expression. Results: In patient biopsies, mitochondrial mass was shown to be upregulated with adenomal presence (~72% increase, p=0.05). Pirc rats were also shown to possess a 63.6% induction of mitochondrial mass (p=0.03). OPA-1 expression was upregulated in both patients with adenomas as well as Pirc rats (49%, p=0.05 and 67%, p<0.03 respectively). Induction of UCP2 was detected in patients with adenomas (99%, p<0.04) and in Pircs (101%, p=0.05). Interestingly, patients with lesions had a 80% increase in PKM2 (p=0.002), suggestive of a Warburg effect in early colon carcinogenesis. Conclusions: We demonstrate for the first time, that in the predysplastic mucosa there is profound metabolic alterations consistent with a Warburg effect. Specifically, we noted increased mitochondria mass (fission) which suppresses oxidative phosphorylation. Aside from structural alterations, electron transport system is further inhibited by overexpression of UCP2. Biologically, this provides insight into early metabolic events in colon carcinogenesis. Clinically, these insights may enable novel biomarkers for early detection and also a myriad of potentially druggable targets for chemoprevention.

The concentrations of EGFR, LRG1, ITIH4, and F5 in serum correlate with the number of colonic adenomas in ApcPirc/+ rats.

The development of noninvasive methods for early detection of colon cancer is critical for the successful management of this disease. Using a targeted quantitative proteomics technique, we assessed the ability of 12 serum proteins to detect the presence of colonic polyps in the Apc(Pirc) (/+) rat model of familial colon cancer. Serum protein candidates were selected from gene transcripts upregulated in colonic tumors of Apc(Pirc) (/+) rats and from a prior study of serum proteins differentially expressed in mice carrying intestinal adenomas. Proteins were quantified at early stages of polyp formation in a rat cohort monitored longitudinally by colonoscopy over a period of 75 days. Of the 12 proteins monitored at three distinct time points, seven showed differential expression in at least one time point in the serum from Apc(Pirc) (/+) rats compared with wild-type rats. Tumor multiplicity correlated with protein expression changes, and most tumors grew during the study. EGFR, LRG1, ITIH4, and F5 displayed the most robust tumor-associated protein expression changes over time. Receiver operator characteristic analysis using these four proteins resulted in a sensitivity of 100%, a specificity of 80%, and an area under the curve of 0.93 at 135 days of age, when the Pirc rats bore an average of 19 tumors in the colon and seven in the small intestine. The results of this study demonstrate that the quantitative analysis of a panel of serum proteins can detect the presence of early intestinal tumors in a rat model, and provides support for future measurements in humans.

Multiple mucin depleted foci, high proliferation and low apoptotic response in the onset of colon carcinogenesis of the PIRC rat, mutated in Apc.

PIRC rats (F344/NTac-Apc (am1137) ) mutated in the Apc gene spontaneously develop colon tumors thus mimicking familial adenomatous polyposis (FAP) and sporadic colorectal cancer (CRC) more closely than Apc-based rodent models developing tumors mostly in the small intestine. To understand whether microscopic dysplastic lesions precede the development of macroscopic tumors, PIRC rat colon was examined for the presence of mucin depleted foci (MDF), microadenomas of the rodent and human colon. Few MDF (about 4/animal) were already present in 1-month-old rats and their number rapidly increases to about 250 in 8-month-old rats. These lesions showed Wnt signaling activation (nuclear β-catenin accumulation) and were dramatically decreased by sulindac (320 ppm), a drug with chemopreventive activity (MDF/rat at 4 months: 156 ± 8 and 38 ± 6 in controls and sulindac-treated rats, respectively, means ± SE, p < 0.001). Since altered proliferation and apoptosis could underlie the early phases of carcinogenesis, we studied these processes in the apparently normal colon mucosa (NM) of 1-month-old PIRC and wt rats. Colon proliferation (PCNA expression) was significantly higher in PIRC rats. Notably, PIRC rat NM showed resistance to apoptosis since it sustained proliferation and had lower apoptosis after a cytotoxic insult with 1,2 dimethylhydrazine. Gene expression of Myc, p21, Birc5, Ogg1, Apex1 and Sod2 were significantly up-regulated in the NM of PIRC rat. The overall results put forward PIRC rat as useful model of colon carcinogenesis, either to study the process itself or to test in vivo chemopreventive agents in both short- and long-term studies.

Nano-Architectural Alterations in Mucus Layer Fecal Colonocytes in Field Carcinogenesis: Potential for Screening

Current fecal tests (occult blood, methylation, DNA mutations) target minute amounts of tumor products among a large amount of fecal material and thus have suboptimal performance. Our group has focused on exploiting field carcinogenesis as a modality to amplify the neoplastic signal. Specifically, we have shown that endoscopically normal rectal brushings have striking nano-architectural alterations which are detectable using a novel optical technique, partial wave spectroscopic microscopy (PWS). We therefore wished to translate this approach to a fecal assay. We examined mucus layer fecal colonocytes (MLFC) at preneoplastic and neoplastic time points (confirmed with rat colonoscopy) in the azoxymethane (AOM)-treated rat model and conducted PWS analysis to derive the nano-architectural parameter, disorder strength (Ld). We confirmed these results with studies in a genetic model (the Pirc rat). We showed that MLFC appeared microscopically normal, consistent with field carcinogenesis. Ld was elevated at an early time point (5 weeks post-AOM injection, effect size = 0.40, P = 0.024) and plateaued before adenoma formation (10 weeks post-AOM, effect size = 0.66, P = 0.001), with no dramatic increase once tumors developed. We replicated these data in the preneoplastic Pirc rat with an effect size in the MLFC that replicated the rectal brushings (increase vs. age-matched controls of 62% vs. 74%, respectively). We provide the first demonstration of a biophotonics approach to fecal assay. Furthermore, targeting the nano-architectural changes of field carcinogenesis rather than the detection of tumor products may provide a novel paradigm for colorectal cancer screening.

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.

135 Discovery and Validation of a Novel DNA Methylation Biomarker for Colorectal Cancer With Application to Blood Testing

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.

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.

136 Live-Cell Imaging of Growing Intestinal Crypts Reveals Dynamic Relationships

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.