Colonic Crypt Cells Research Articles

Comparative study of KI-67 and CD44 expression in serrated polyps of the distal colon

Introduction. Serrated polyps are newly distinguished types of colonic polyps. Nowadays lots of questions about serrated neoplastic pathway are still unclear. The purpose of the study was to compare Ki-67 and CD44 immunohistochemical (IHC) expression levels in different histological types of serrated polyps. Materials and methods. Histopathological and IHC studies of 30 serrated polyps were conducted. IHC study was carried out using antibodies against Ki-67 and CD44. Results of IHC reactions with antibodies against Ki-67 were estimated by immunostained nuclei counting and were expressed in proliferation index (PI) while results of IHC reactions with antibodies against CD44 were estimated by photo digital morphometry and were expressed in immunostained cells relative area (%). Furthermore, distribution of Ki-67+ and CD44+ cells in colonic crypts was examined. Results. It was revealed that hyperplastic polyps (HP) were characterized by medium PI and basal-middle pattern of Ki-67+ cells distribution. HP were also characterized by the median of CD44+ cells area equal to 22,36 (13,15;30,41) % and basal-middle pattern of CD44+ cells distribution. Herewith, direct medium strength correlation between Ki-67 and CD44 expression levels in HP was established. Traditional serrated adenomas (TSA) were characterized by medium PI and diffuse pattern of Ki-67+ cells distribution. TSA were also characterized by the median of CD44+ cells area equal to 25,48 (15,19;29,04) % and upper-middle pattern of CD44+ cells distribution. Direct weak strength correlation between Ki-67 and CD44 expression levels in TSA was established. Sessile serrated adenomas (SSA) were characterized by medium PI and basal pattern of Ki-67+ cells distribution. SSA were also characterized by the median of CD44+ cells area equal to 20,54 (11,25;28,15) % and basal-middle pattern of CD44+ cells distribution. Direct medium strength correlation between Ki-67 and CD44 expression levels in SSA was established.

X irradiation induced colonic mucosal injury and the detection of apoptosis through PARP-1/p53 regulatory pathway

This study aimed to explore whether PARP-1 regulatory pathway mediated X irradiation induced cell cycle arrest and apoptosis or not. In this regard, colonic mucosal injury caused by whole-body X-irradiation induced apoptosis through PARP-1, caspase 3 and p53 regulatory pathway were evaluated in experimental rat models. Eighteen Wistar albino rats were divided into three groups. Two radiation groups received 8.3 Gy dose of whole-body X-irradiation as a single dose and the control group received physiological saline intraperitoneally. Radiation groups were sacrificed after 6 h and 4 days of irradiation. PARP-1 and caspase 3 expression in the nuclei of colonic crypt cells significantly increased 6 h after irradiation, and declined 4 days after irradiation. In conflict with other studies that reported p53 as not being expressed widely in colonic mucosa, in our study the expressions of p53 were elevated both in the cytoplasm and in the nucleus of the crypt cells, especially 6 h after irradiation. In the radiation groups, colonic mucosal injury score was significantly elevated compared with that of the control group. Our data demonstrated that PARP-1, caspase-3 and p53 expression increased in colonic mucosa 6 h after irradiation.

NCoR1 Protects Mice From Dextran Sodium Sulfate-Induced Colitis by Guarding Colonic Crypt Cells From Luminal Insult.

Background & AimsColonic stem cells are essential for producing the mucosal lining, which in turn protects stem cells from insult by luminal factors. Discovery of genetic and biochemical events that control stem cell proliferation and differentiation can be leveraged to decipher the causal factors of ulcerative colitis and aid the development of more effective therapy.MethodsWe performed in vivo and in vitro studies from control (nuclear receptor corepressor 1 [NCoR1F/F]) and intestinal epithelial cell–specific NCoR1-deficient mice (NCoR1ΔIEC). Mice were challenged with dextran sodium sulfate to induce experimental ulcerative colitis, followed by colitis examination, barrier permeability analysis, cell proliferation immunostaining assays, and RNA sequencing analysis. By using crypt cultures, the organoid-forming efficiency, cell proliferation, apoptosis, and histone acetylation were analyzed after butyrate and/or tumor necrosis factor α treatments.ResultsNCoR1ΔIEC mice showed a dramatic increase in disease severity in this colitis model, with suppression of proliferative cells at the crypt base as an early event and a concomitant increase in barrier permeability. Genome expression patterns showed an important role for NCoR1 in colonic stem cell proliferation and secretory cell differentiation. Colonic organoids cultured from NCoR1ΔIEC mice were more sensitive to butyrate-induced cell growth inhibition and apoptosis, which were exaggerated further by tumor necrosis factor α co-treatment, which was accompanied by increased histone acetylation.ConclusionsNCoR1 regulates colonic stem cell proliferation and secretory cell differentiation. When NCoR1 is disrupted, barrier protection is weakened, allowing luminal products such as butyrate to penetrate and synergistically damage the colonic crypt cells. Transcript profiling: RNA sequencing data have been deposited in the GEO database, accession number: GSE136153.

Dietary Fucoxanthin Induces Anoikis in Colorectal Adenocarcinoma by Suppressing Integrin Signaling in a Murine Colorectal Cancer Model.

Fucoxanthin (Fx), abundantly contained in edible brown algae, is a carotenoid with strong anti-cancer potential. Anoikis is an anchor-dependent apoptosis particularly related to integrin signaling, and a target for cancer preventive strategies. We recently demonstrated that Fx prevented colon cancer in azoxymethane-dextrane sodium sulfate (AOM/DSS) carcinogenic model mice, and that it increased anoikis-like integrin β1low/-/cleaved caspase-3high cells in colonic mucosal crypts. However, an induction mechanism of anoikis by Fx in adenocarcinoma tissue remains unresolved. Thus, we investigated anoikis in colonic adenocarcinoma in AOM/DSS mice. Fx administration (30 mg/kg body weight) significantly suppressed the incidence and multiplicity of colonic adenocarcinoma in AOM/DSS mice. A number of anoikis-like integrin β1low/-/cleaved caspase-3high cells in colonic adenocarcinoma and mucosal crypts were significantly increased, 8.3- and 3.5-fold in the Fx group compared with those of the control group, respectively. The results indicated the increase of anoikis-like cells occurred more strongly in colonic adenocarcinoma than in colonic mucosal crypts. In addition, integrin β1 expression, and pFAK (Tyr397) and pPaxillin (Tyr31) activation in mucosal tissue decreased 0.7-, 0.5- and 0.6-fold by Fx administration, respectively. The results suggest that Fx induces anoikis in colonic adenocarcinoma developed by AOM/DSS treatment through attenuation of integrin signaling.

MiR-221 Targets QKI to Enhance the Tumorigenic Capacity of Human Colorectal Cancer Stem Cells.

miRNAs are key players in the integrated regulation of cellular processes and shape many of the functional properties that define the "cancer stem cell" (CSC) phenotype. Little is known, however, about miRNAs that regulate such properties in human colorectal carcinoma. In this study, we compared the expression levels of 754 miRNAs between paired samples of EpCAM+/CD44+ cancer cells (enriched in CSCs) and EpCAM+/CD44neg cancer cells (with CSC depletion) sorted in parallel from human primary colorectal carcinomas and identified miR-221 as the miRNA that displayed the highest level of preferential expression in EpCAM+/CD44+ cancer cells. High levels of miR-221 expression were associated with Lgr5+ cells in mouse colon crypts and reduced survival in patients with colorectal carcinoma. Constitutive overexpression of miR-221 enhanced organoid-forming capacity of both conventional colorectal carcinoma cell lines and patient-derived xenografts (PDX) in vitro. Importantly, constitutive downregulation of miR-221 suppressed organoid-forming capacity in vitro and substantially reduced the tumorigenic capacity of CSC populations from PDX lines in vivo. Finally, the most abundant splicing isoform of the human Quaking (QKI) gene, QKI-5, was identified as a functional target of miR-221; overexpression of miR-221-reduced QKI-5 protein levels in human colorectal carcinoma cells. As expected, overexpression of QKI-5 suppressed organoid-forming capacity in vitro and tumorigenic capacity of colorectal carcinoma PDX cells in vivo. Our study reveals a mechanistic link between miR-221 and QKI and highlights their key role in regulating CSC properties in human colorectal cancer. SIGNIFICANCE: These findings uncover molecular mechanisms underlying the maintenance of cancer stem cell properties in colon cancer.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/20/5151/F1.large.jpg.

Fibroblast-derived Gremlin1 localises to epithelial cells at the base of the intestinal crypt

Gremlin1 (GREM1) is a secreted glycoprotein member of the differential screening-selected gene in aberrant neuroblastoma (DAN) family of bone morphogenetic protein (BMP) antagonists, which binds to BMPs preventing their receptor engagement. Previous studies have identified that stage II colorectal cancer (CRC) patients with high levels of GREM1 gene expression in their tumour tissue have a poorer prognosis. Using a series of in silico and in situ methodologies, we demonstrate that GREM1 gene expression is significantly higher (p < 0.0001) in CRC consensus molecular subtype 4 (CMS4), compared to the other CMS subtypes and correlates (p < 0.0001) with levels of cancer-associated fibroblasts (CAFs) within the CRC tumour microenvironment (TME). Our optimised immunohistochemistry protocol identified endogenous GREM1 protein expression in both the muscularis mucosa and adjacent colonic crypt bases in mouse intestine, in contrast to RNA expression which was shown to localise specifically to the muscularis mucosa, as determined by in situ hybridisation. Importantly, we demonstrate that cells with high levels of GREM1 expression display low levels of phospho-Smad1/5, consistent with reduced BMP signalling. Taken together, these data highlight a novel paracrine signalling circuit, which involves uptake of mature GREM1 protein by colonic crypt cells following secretion from neighbouring fibroblasts in the TME.

Effects of Nut Consumption on Colon Cell Proliferation and Apoptosis (P06-058-19)

ObjectivesAn imbalance between colon cell proliferation and apoptosis increases one’s risk of colon cancer. Some studies have shown an inverse relationship between nut consumption and colon cancer; however, the underlying mechanism is still not elucidated. This study aimed to determine if mixed nut or single nut (pistachio) consumption is able to decrease colon cancer risk by modulating colonic cell proliferation and apoptosis. It was hypothesized that rats consuming nut diets exhibit fewer colonic proliferating cells and greater apoptotic cells compared to a control. MethodsThirty 21-day-old Sprague Dawley male rats were assigned to one of three isocaloric diets: control diet containing no nuts, pistachio diet (8.1% w/w), or mixed nut diet (7.5% w/w) for 8 weeks. The mixed nut diet contained cashews, almonds, Brazil nuts, pecans, macadamia nuts, walnuts, pistachios, and peanuts. Proliferating colonic cells were marked using Ki-67 quantitative immunostaining and apoptotic cells with Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Cells clearly stained as proliferative and apoptotic were divided by the total cells in each crypt column, multiplied by 100 to determine proliferation and apoptosis indices. The proliferative zone was determined as 100 times the location of the highest labeled proliferating cell, divided by the total number of cells per crypt. ResultsThe majority of proliferating cells were located near the bottom of the colonic crypt and apoptotic cells were found in the upper part of the crypt (P < 0.05). No significant differences were found for proliferating index, proliferative zone and apoptotic index among all three experimental diet groups. ConclusionsSome clinical and experimental studies show the protective effects of nut consumption against colon cancer. Our results indicate that the beneficial effects of nut consumption in colon cancer prevention may be explained by a different mechanism rather than modification of cell proliferation or apoptosis. Additional markers for cell proliferation and apoptosis may need to be used to confirm our results. Other potential mechanisms such as oxidative stress, DNA damage and repair enzymes should be further investigated. Funding SourcesAmerican Heart Association (16GRNT31360007).

Interleukin-1 Signaling Mediates Obesity-Promoted Elevations in Inflammatory Cytokines, Wnt Activation, and Epithelial Proliferation in the Mouse Colon.

Obesity is a prominent risk factor for colorectal cancer (CRC). One mechanism by which obesity promotes the development of CRC is by generating a chronic, low-grade state of colonic inflammation. Interleukin-1β (IL-1β), a proinflammatory cytokine often elevated in obesity, is known to activate several procarcinogenic signaling pathways that are implicated in colonic carcinogenesis. We therefore sought to define the role of IL-1β in mediating some of the early biochemical and molecular events leading up to obesity-promoted CRC. Twenty-five wild-type (WT) C57BL/6J mice and 24 lacking a functional IL-1 receptor (IL1R-/-) were each randomized to either low-fat or high-fat diets, resulting in lean and obese mice. Compared to WT lean controls, WT obese mice displayed 30%-80% greater concentrations of IL-1β and tumor necrosis factor-α (TNF-α) in the colonic mucosa (IL-1β: P = 0.04; TNF-α: P < 0.05), activation of the Wnt signaling cascade [evidenced by a 2-fold increase in colonic crypt cells displaying intranuclear β-catenin (P < 0.03)], and a significant expansion of the proliferation zone of the colonic crypt (P < 0.04). These obesity-induced alterations in colonic cytokines, Wnt signaling, and proliferation were absent in the obese IL1R-/- mice. In the absence of IL-1 signaling, obesity-induced elevations of colonic IL-1β, TNF-α, Wnt activation, and enhanced epithelial proliferation no longer occur. These observations underscore the important mechanistic roles that IL-1 signaling appears to play in mediating the procancerous effects of obesity in the colon, thereby identifying a potential target for future strategies aimed at chemoprevention.

Abstract 1136: Decrease in maturation of stem cells along the neuroendocrine lineage contributes to stem cell overpopulation that drives human CRC development

Abstract Stem cell (SC) overpopulation is thought to drive tumor initiation and progression during colon cancer development, but it is unclear what causes the SC overpopulation. Because most neuroendocrine (NE) cells (NECs) in normal colonic crypts reside within the SC niche at the crypt bottom, we thought that aberrant NECs might be linked to the SC overpopulation. We hypothesized that (i) in normal crypts, SC and NEC populations are anatomically and functionally related, and (ii) during tumorigenesis, changes in these relationships quantitatively correlate with increased SC numbers. We tested this hypothesis - using quantitative immunohistochemical mapping - as a first step towards understanding how interactions between SC and NEC might become altered during colon tumorigenesis. In normal human colonic crypts, most (&amp;gt;80%) cells staining for ALDH1, a colonic SC marker, co-stained for chromogranin-A (CGA) and several other NE markers. Neither ALDH1+ nor CGA+ cells co-stained for MCM2, a marker for proliferating cells. These findings indicate that, in normal colonic crypts, many ALDH1+ cells have NE characteristics. In contrast, the proportion of ALDH1+ cells that co-stained for NE markers progressively decreased (to &amp;lt;20%) during the stepwise progression to cancer (from normal crypts to normal-appearing FAP crypts to adenomatous crypts to colon carcinomas). However, the absolute number of ALDH+ cells progressively increased during this progression, reflecting the extent of the SC overpopulation. These findings suggest that (i) NEC maturation is integral to regulation of SC population size and (ii) a decreased rate of NEC maturation contributes to SC overpopulation during colon tumorigenesis. Citation Format: Bruce M. Boman, Tao Zhang, Shirin Modarai, Lynn Opdenacker, Fields Jeremy. Decrease in maturation of stem cells along the neuroendocrine lineage contributes to stem cell overpopulation that drives human CRC development [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 1136.

Abstract 1147: Developing an individualized human colonic crypt cell culture as an ex vivo model for immunotherapy

Abstract Colorectal Cancer (CRC) is the third leading cause of cancer death worldwide. Majority of mortality is attributable to metastasis to distal organs. Current treatment targeting the ERK/MAPK pathway used monoclonal anti-EGFR antibody after the first line of chemotherapy fails. However these treatments are only effective for a small percentage of patients with wild-type KRAS, and these same patients eventually develop resistance. Small molecule drugs are toxic and have limited success. A combination of antibodies is an attractive alternative. This study aims to investigate whether anti-KRAS antibody can enter live cells in an individualized ex vivo culture system and whether it can bind and inactivate KRAS. The transient culture has advantages over cell line study as it better mimics the human colonic environment and tumors can be compared to their matched normals to partially address the problem of heterogeneity. Samples were obtained from patients' resected tumour and matched mucosa, cultured ex vivo, and immunostaining performed. We show that anti-KRAS antibody is internalized in both the cultured mucosa and tumor crypt cells prior to fixation. Subcellular localization of the internalized antibody in the mucosa cells is similar in the pre-fixed and post-fixed cells. In contrast, the internal cell membrane staining observed in post-fixed tumor cells appear more cytoplasmic and 'punctated' in the pre-fixed tumors, mimicking that of the mucosa cells suggesting that the internalized anti-KRAS may have altered the sub-cellular localization and hence cycling of KRAS. Furthermore, staining with anti-keratin antibody indicates that live tumor cells treated with anti-KRAS has partial reversal of the phenotype to become more like the regular striated pattern of the matched mucosa. Preliminary double labelling of cultured tumours with monoclonal anti-KRAS prior to fixation and polyclonal anti-KRAS post-fixation revealed punctate staining of KRAS in the cytoplasm that were mainly non-overlapping suggesting that the punctate structures are in different compartments. Citation Format: Peh Yean Cheah, Yee Syuen Low, Michelle Lo, See Aik Tang, Alphaeus Ng, Choong Leong Tang, Siew Heng Wong. Developing an individualized human colonic crypt cell culture as an ex vivo model for immunotherapy [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 1147.

Effect of Spirogyra neglecta on the early stages of 1, 2-dimethylhydrazine-induced colon carcinogenesis in rats.

This study focused on the chemopreventive effects of Spirogyra neglecta extract (SNE) and dried S. neglecta mixed diet on the early stages of 1,2-dimethylhydrazine (DMH)-induced colon carcinogenesis in rats. Male Wistar rats were injected with DMH to initiate aberrant crypt foci (ACF) formation. In the initiation stage, SNE significantly decreased the number of ACF in the colon of DMH-treated rats. Rats that received a low dose of SNE showed enhanced activity of several detoxifying and antioxidant enzymes. In the postinitiation stage, a low dose of SNE significantly decreased the number of ACF in the colon of DMH-treated rats. It significantly reduced the number of proliferating cell nuclear antigen-positive cells and increased the number of apoptotic cells in colonic crypts. S. neglecta thus inhibited the development of the early stages of DMH-induced colon carcinogenesis in rats by modulation of xenobiotic metabolizing enzymes and inhibition of cell proliferation as well as induction of apoptosis.

CPT-11-Induced Delayed Diarrhea Develops via Reduced Aquaporin-3 Expression in the Colon.

While irinotecan (CPT-11) has a potent anti-cancer effect, it also causes serious diarrhea as an adverse reaction. In this study, we analyzed the pathogenic mechanism of CPT-11-induced delayed diarrhea by focusing on water channel aquaporin-3 (AQP3) in the colon. When rats received CPT-11, the expression level of AQP3 was reduced during severe diarrhea. It was found that the expression levels of inflammatory cytokines and the loss of crypt cells were increased in the colon when CPT-11 was administered. When celecoxib, an anti-inflammatory drug, was concomitantly administered, both the diarrhea and the reduced expression of AQP3 induced by CPT-11 were suppressed. The inflammation in the rat colon during diarrhea was caused via activated macrophage by CPT-11. These results showed that when CPT-11 is administered, the expression level of AQP3 in the colon is reduced, resulting in delayed diarrhea by preventing water transport from the intestinal tract. It was also suggested that the reduced expression of AQP3 might be due to the inflammation that occurs following the loss of colonic crypt cells and to the damage caused by the direct activation of macrophages by CPT-11. Therefore, it was considered that anti-inflammatory drugs that suppress the reduction of AQP3 expression could prevent CPT-11-induced delayed diarrhea.

The effect ofPerilla frutescensleaf on 1, 2-dimethylhydrazine-induced initiation of colon carcinogenesis in rats

In this study, the chemopreventive properties of Perilla frutescens leaf extract (PLE) on rat colon carcinogenesis were investigated during initiation and postinitiation stages. Rats were fed orally by 50 and 500 mg/kg bw of PLE continuously from 1 week before subcutaneously injection of 1,2-dimethylhydrazine (DMH) until the end of experiment. PLE administration reduced the aberrant crypt foci (ACF) number in DMH-treated rats through the inhibition of hepatic cytochrome P4502E1 (CYP2E1) and bacterial β-glucuronidase activity in the colonic lumen. These events led to the reduction of DNA methylation in colonic epithelial cells. For the postinitiation model, rats were injected (s.c.) with DMH, followed by PLE administration until the end of experiment. PLE administration also reduced the ACF number in DMH-induced rats, resulting from apoptosis induction and reduction of proliferation in colon crypt cells. These studies demonstrated that perilla leaf could inhibit ACF formation and progression in DMH-initiated colon carcinogenesis in rats. Practical applications This study revealed the inhibitory effect of PLE on chemical-induced colon carcinogenesis in rats. Altogether, our data supported that PLE acts as a chemopreventive agent for colon cancer. Thus, PLE might be developed as food supplement or functional tea for colon cancer prevention.

Preliminary Report: Multiple Clusters of Proliferating Cells in Non-dysplastic Corrupted Colonic Crypts Underneath Conventional Adenomas.

The finding was recently reported of clusters of colonic crypts lined with indigenous normal epithelium displaying irregular shapes underneath the adenomatous glands of conventional (tubular or villous) adenomas. These abnormal crypts were named non-dysplastic corrupted colonic crypts (NDCs). This study explored the characteristics of cell proliferation in NDCs present in a cohort of conventional adenomas. Sections from six conventional adenomas were challenged with the proliferation marker Ki-67 (MIB1). MIB+ proliferating clusters were regarded as those exhibiting two or more adjoining MIB+ cells. A total of 46 (range=1-18) NDCs were found underneath the six conventional adenomas. Out of the 46 NDCs, two exhibited only one proliferative cluster/crypt, 14 NDCs two clusters/crypt, 14 three clusters/crypt and the remaining 16 NDCs more than four distinct clusters/crypt. This preliminary study showed, evidently for the first time, that multiple, apparently haphazardly distributed clusters of proliferating cells are present in NDCs. Since the Ki-67 proliferation marker only labels progenitor daughter cells generated by stem cells, each MIB+ cluster in each NDC must have been produced by a single stem cell. Consequently, individual NDCs may harbor several stem cells, a deduction that is in concert with recent studies showing that in the normal human colon, the number of stem cells per crypt is of the order of five to six, or about 5% of the cell population of a single crypt.

Orientin, a flavanoid, mitigates 1, 2 dimethylhydrazine-induced colorectal lesions in Wistar rats fed a high-fat diet.

Orientin, a c- glycosyl flavonoid found copiously in roobios tea and various medicinal plants is well known for its antioxidant, anti-inflammatory, and antitumor effects. The present study aims to investigate the anti-cancer efficacy of orientin on 1,2 dimethyl hydrazine induced colonic aberrant crypt foci (ACF) and cell proliferation in Wistar rats. Rats were randomly divided into six groups and fed with high fat diet. Group 1 left as untreated control. Group 2 administered with DMH (20 mg/kg body weight) for initial 4 weeks and left untreated. Group 3 received orientin (10 mg/kg body weight) alone for the entire period. Group 4 received orientin along with DMH for initial 4 weeks and left untreated; Group 5 administered DMH for initial 4 weeks and treated with orientin for remaining 12 weeks; Group 6 administered DMH and treated with orientin throughout the entire period. Our preclinical findings suggest that the administration of orientin decreases the occurrence of DMH induced colonic polyps and aberrant crypt foci, augments antioxidant defense and altered the activities of drug metabolizing phase I and phase II enzymes in colonic and hepatic tissues and thereby ensuring the detoxification of carcinogen. Furthermore, orientin attenuates the aberrant crypt foci formation and reinstates the DMH induced cell proliferation, as evident from the AgNORs staining of colonic tissues of experimental rats. Thus, our study emphasizes that orientin may prevent DMH induced precancerous lesions and proven to be a potent antioxidant and antiproliferative agent.

Fish Oil Contaminated with Persistent Organic Pollutants Induces Colonic Aberrant Crypt Foci Formation and Reduces Antioxidant Enzyme Gene Expression in Rats

BackgroundEpidemiologic, clinical, and experimental studies have suggested that fish oil (FO), a rich source of n–3 (ω-3) polyunsaturated fatty acids, protects against colon cancer. However, this message is confounded by the FDA's warning that the consumption of certain types of fish should be restricted because of contamination with persistent organic pollutants (POPs), such as polychlorinated biphenyls (PCBs) and organochlorine pesticides. ObjectiveWe examined FO contaminated with POPs (PCBs, dichlorodiphenyltrichloroethane, and chlordane) compared with unmodified FO on the risk factors of colon cancer development. MethodsMale Sprague-Dawley rats aged 28 d (n = 30) were allocated into 3 groups and fed 15% corn oil (CO), FO, or POP-contaminated FO for 9 wk with a subcutaneous injection of colon carcinogen azoxymethane at weeks 3 and 4. Colonic aberrant crypt foci (ACF) and cell proliferation were enumerated, and the gene expression of inflammation, antioxidant enzymes, and repair enzymes were determined with the use of real-time quantitative polymerase chain reaction analysis. ResultsFO-fed rats had a lower number of ACF (mean ± SE: 29 ± 4.0 for FO compared with 53 ± 8.4 for CO and 44 ± 4.6 for POP FO) and higher-multiplicity ACF than the CO and POP FO groups (4.7 ± 0.9 for FO compared with 11 ± 1.5 for CO and 9.6 ± 1.8 for POP FO) (P < 0.05). FO feeding lowered the proliferation index compared with the CO and POP FO feeding groups (18% ± 1.1% for FO compared with 25% ± 1.6% for CO and 23% ± 0.7% for POP FO) (P = 0.009). Superoxide dismutase [2.4 ± 0.6 relative quantification (RQ) for FO compared with 1.2 ± 0.2 RQ for CO and 1.3 ± 0.3 RQ for POP FO] and catalase gene expression (10 ± 2.0 RQ for FO compared with 5.4 ± 1.1 RQ for CO and 6.6 ± 1.5 RQ for POP FO) were higher in the FO group than in the CO and POP FO groups (P < 0.05). There were no differences between CO and POP FO on the variables. ConclusionThese results indicate that POPs in FO reduce the preventive effects of FO on colon carcinogenesis by increasing preneoplastic lesion formation through the downregulation of antioxidant enzyme expression and increasing cell proliferation in rats.

Abstract 2229: Prevention by tolfenamic acid and characterization of the molecular targets in Pirc and FAP colorectal adenomas

Abstract A critical need exists in sporadic and high-risk colorectal cancer patients for endoscopic screening combined with safe and effective preventive agents. To prioritize molecular targets for prevention studies in vivo, RNA-seq analyses were conducted on colorectal adenomas from familial adenomatous polyposis (FAP) patients, and from the Apc-mutant polyposis in rat colon (Pirc) model. Using a new murine endoscopic polypectomy methodology (F.U. Ertem et al., Gastrointest Endosc 2016;83:1272-6), we evaluated agents that might substitute for the current standard of care, Sulindac, and identified tolfenamic acid (TA) as a promising candidate for further investigation. At clinically-relevant doses in the Pirc model, TA suppressed tumorigenesis significantly both in the small intestine and in the colon. Inhibition by TA of colonic crypt cell proliferation, and the concomitant activation of apoptosis, coincided with reduced expression in target tissues of β-catenin, cyclin D1, Survivin and matrix metalloproteinase 7. Based on the oncogene candidates defined by RNA-seq analysis, endoscopic tracking revealed the time-course of tumor suppression by TA, and the temporal changes in S100a9, Nppb, Aldh1a3 and Mmp7, highlighting their value as potential early biomarkers for prevention in the clinical setting. As an “old drug” repurposed from migraine, TA appears to offer an exciting new therapeutic avenue in FAP and other high-risk colorectal cancer patient populations. This work was supported in part by NIH grants CA090890, CA122959, ES00210, ES023512, the John S. Dunn Foundation, and a Chancellor’s Research Initiative. 1 Citation Format: Furkan U. Ertem, Wenqian Zhang, Kyle Chang, Wan-Mohaiza Dashwood, Praveen Rajendran, Deqiang Sun, Ala Abudayyeh, Maen Abdulrahim, Eduardo Vilar, Roderick H. Dashwood. Prevention by tolfenamic acid and characterization of the molecular targets in Pirc and FAP colorectal adenomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2229. doi:10.1158/1538-7445.AM2017-2229

Acetylcholine-related proteins in non-neoplastic appearing colonic mucosa from patients with colorectal neoplasia.

The pathogenesis of colorectal neoplasia (CRN) has been associated with altered non-neuronal acetylcholine (ACh) metabolism. The aim of this study was to characterize expression, function, and cellular location of ACh-related proteins in biopsies obtained from endoscopic normal-appearing sigmoid colon in patients with and without CRN. Messenger-RNA (mRNA) levels of 17 ACh-related proteins were quantified by rt-qPCR. Functional responses to ACh, measured as electrogenic transepithelial short circuit current (SCC), were recorded using the Ussing chamber technique. Finally, cellular localization of choline transporter-like proteins (CTLs) and butyryl-cholinesterase enzyme (BChE) was determined by immunohistochemistry. mRNA expression of CTL1 and CTL4 was increased in patients with CRN (P = 0.002 and P = 0.04, respectively). In functional experiments, baseline SCC was increased in CRN patients. ACh induced rapid biphasic changes in SCC. An initial decreasing phase was observed in the minority of CRN patients versus the majority of controls (25% vs 69%, respectively, P = 0.031). For the second increasing phase of SCC, data indicated ACh-activation of two receptors. For both parts of the biphasic response, the half maximal effective concentration and maximal responses showed no difference between patient groups. Immunohistochemistry demonstrated CTL1, 3 and 4 and BChE to be localized to colonic crypt cells. We conclude that CRN is associated with increased expression of CTL1 and CTL4, augmented basal prostaglandin-dependent secretion, and altered functional channel response to ACh in human endoscopic normal-appearing colonic mucosa. The immunohistochemical findings support CTL1, CTL3, CTL4, and BChE to be involved in non-neuronal mucosal ACh metabolism.

The G-protein coupled receptor 56, expressed in colonic stem and cancer cells, binds progastrin to promote proliferation and carcinogenesis.

Overexpression of human progastrin increases colonic mucosal proliferation and colorectal cancer progression in mice. The G-protein coupled receptor 56 (GPR56) is known to regulate cell adhesion, migration, proliferation and stem cell biology, but its expression in the gut has not been studied. We hypothesized that the promotion of colorectal cancer by progastrin may be mediated in part through GPR56. Here, we found that GPR56 expresses in rare colonic crypt cells that lineage trace colonic glands consistent with GPR56 marking long-lived colonic stem-progenitor cells. GPR56 was upregulated in transgenic mice overexpressing human progastrin. While recombinant human progastrin promoted the growth and survival of wild-type colonic organoids in vitro, colonic organoids cultured from GPR56−/− mice were resistant to progastrin. We found that progastrin directly bound to, and increased the proliferation of, GPR56-expressing colon cancer cells in vitro, and proliferation was increased in cells that expressed both GPR56 and the cholecystokinin-2 receptor (CCK2R). In vivo, deletion of GPR56 in the mouse germline abrogated progastrin-dependent colonic mucosal proliferation and increased apoptosis. Loss of GPR56 also inhibited progastrin-dependent colonic crypt fission and colorectal carcinogenesis in the azoxymethane (AOM) mouse model of colorectal cancer. Overall, we found that progastrin binds to GPR56 expressing colonic stem cells, which in turn promotes their expansion, and that this GPR56-dependent pathway is an important driver and potential new target in colorectal carcinogenesis.

Modelling the dynamics of stem cells in colonic crypts

We present a theoretical and computational framework to model the colonic crypt organisation in the human intestine. We construct a theoretical and computational framework to model the colonic crypt behaviour, using a Voronoi tessellation to represent each cell and elastic forces between them we addressed how their dynamical disfunction can lead to tumour masses and cancer. Our results indicate that for certain parameters the crypt is in a homeostatic state, but slight changes on their values can disrupt this behaviour.