Colon Adenoma Cells Research Articles

Abstract 5276: Immunomodulatory activity of intermittent low-dose erlotinib plus sulindac: Implications for hereditary and sporadic colorectal cancer

Abstract A once-weekly optimized dosing regimen of erlotinib (ERL) in the polyposis in rat colon (Pirc) model [1] was a reliable predictor of antitumor efficacy in familial adenomatous polyposis (FAP) patients [2]. Pirc rats are increasingly used as a translational tool for FAP studies. Based on transcriptomic data from FAP patients receiving ERL plus sulindac (SUL) vs. placebo [3], we determined the timing of changes in interferon-γ signaling in Pirc adenomas, while performing additional dose titration. In rats that were given ERL administered at 5 mg per kilogram of body weight through oral gavage (two times a week) and that received a diet containing 125 parts per million of SUL, a regimen referred to as SUL125+ERL5x2, significant suppression of tumor growth was observed. This combination represented half of the standard of care SUL dose and about one-quarter of the ERL dose in FAP patients. In adenomas collected after one year of treatment from SUL125+ERL5x2 treated rats, the expression of genes related to the major histocompatibility complex (MHC) class I (β2m, Cnx, Psmb8 and Tap1) and MHC class II (RT1-A2, Cd74, RT-1Bb, Cd74, and Ciita) was increased, compared to vehicle controls, excluding a subset of adenomas that remained resistant. Through the use of multiplex mass cytometry (CyTOF), an increase in tumor-infiltrating CD4+ T cells was observed in adenomas from the SUL125+ERL5x2 treatment group. Administration of single or combined agents increased CD68+ cells and reduced the presence of Foxp3+ and Arg1+ cells in Pirc adenomas. The natural history of adenomatous colon polyps indicated an increase in MHC gene expression as tumors increased in size; however, this trend reversed markedly in fully occluding lesions. Additionally, expression of MHC-related factors increased in Pirc colon adenoma and murine colon carcinoma cells treated with ERL±SUL, resulting in enhanced CD8+ T-cell activation and IL-2 secretion. In conclusion, treatment with intermittent low doses of ERL±SUL increased MHC gene expression and induced changes in the immune cell component in the tumor microenvironment of Pirc rat adenomas. A subset of non-responsive adenomas could benefit from additional immunopreventive approaches, with implications for possible clinical applications in both hereditary and sporadic colorectal cancer.

Abstract 4780: TRAIL-inducing imipridone ONC201/TIC10 demonstrates anti-neoplastic effects in colonic adenoma-derived organoids

Abstract Background: Colorectal cancer (CRC) is the second leading cause of cancer-related death inthe United States and screening is the most effective method to reduce mortality. Chemoprevention, which aims to prevent CRC through eradication or prevention of precursor colonic adenomas, represents an alternative strategy to address this problem. The imipridone dordaviprone (ONC201) has been shown to induce apoptosis in cancer cells through both upregulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and the integrated stress response. More recently, we have shown that ONC201 induces TRAIL and apoptosis in a dose-dependent manner and reduces adenoma formation in the Apcmin/+ CRC mouse model. We hypothesized that ONC201 would similarly enhance human colonic adenoma cell death through both TRAIL-dependent and TRAIL-independent mechanisms. We therefore evaluated the effects of ONC201 in two adenoma-derived organoid lines: one developed from an adenoma obtained from an individual with familial adenomatous polyposis (FAP) and the second from a sessile serrated adenoma (SSA). Methods: Sensitivity of the FAP and SSA organoid lines to ONC201 was assessed using CellTiter-Glo® luminescent cell viability assay. Organoids were plated in triplicates and treated with ONC201 (concentration ranging from 0 uM to 20 uM). Results were analyzed after 72 hours of incubation. Western blotting was performed on lysates generated from organoids treated with either 1.69 uM DMSO, 1.69 uM ONC201, or 3.38 uM ONC201 for FAP and 1.23 uM DMSO, 1.23 uM ONC201, or 2.46 uM ONC201 for SSA for 0, 24, or 48 hours. Co-culture experiments were performed with the adenoma-derived organoids (dyed using blue CMAC dye) and human NK-92MI cells (dyed with green CMFDA dye) treated with either 845 nM DMSO, 845 nM ONC201, or 1.69 uM ONC201 for FAP and 615 nM DMSO, 615 nM ONC201, or 1.23 uM ONC201 for SSA. Fluorescent images following administration of red live/dead dye were performed using DAPI, FITC, and cherry red channels on ImageXpress® at 0, 24, 48, and 72hours. Results: ONC201 demonstrated antineoplastic effects in adenoma-derived FAP and SSA organoids. Half maximal inhibitory concentrations (IC50) of ONC201 in FAP and SSA organoids were 1.69 uM and 1.23 uM, respectively. As was noted in rodent studies, the mechanism of action of ONC201 appears to be mediated through modulation of multiple pathways including the TRAIL pathway (TRAIL and DR5 were both upregulated) and the integrated stress response (ATF4 was upregulated). Co-culture with NK cells revealed an increase in NK-mediated organoid cell death. The ability of ONC201 to enhance NK-mediated killing is still being evaluated. Further analysis of the effects of ONC201 on stemness markers and on T-cell-mediated organoid cell killing are currently underway and may reveal other potential biomarkers and chemopreventive strategies for ONC201. Citation Format: Alexis J. Lannigan, Jasper Chan, Maximilian Schwermann, Lanlan Zhou, Varun V. Prabhu, Michael Dame, Dean Brenner, Wafik S. El-Deiry, Alexander G. Raufi. TRAIL-inducing imipridone ONC201/TIC10 demonstrates anti-neoplastic effects in colonic adenoma-derived organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4780.

Chemopreventive effects of α-tocopherol and its long-chain metabolites α-13'-hydroxy- and α-13'-carboxychromanol in LT97 colon adenoma cells.

Anticancer effects of vitamin E (tocopherols) have been studied extensively. While in vitro and animal studies showed promising results regarding anticancer effects of tocopherols, human intervention studies failed to reproduce these results. In vivo, α-tocopherol (α-TOH) is metabolized to the long-chain metabolites (LCM) 13'-hydroxychromanol (α-13'-OH) and 13'-carboxychromanol (α-13'-COOH), which likely reach the large intestine. The LCM showed antiproliferative effects in different colon cancer cell lines, but the exact mechanism of action remains unclear. To further clarify the chemopreventive action of the LCM, premalignant LT97 colon adenoma cells were treated with α-TOH, α-13'-OH and α-13'-COOH to study their impact on growth, apoptosis, antigenotoxicity, and ROS-scavenging capacity as well as expression of selected genes involved in detoxification and the cell cycle. Growth inhibitory potential was observed for α-13'-OH (IC50: 37.4 μM) and α-13'-COOH (IC50: 5.8 μM) but not for α-TOH in the tested concentrations. Levels of caspase-3 activity and expression of genes regulating the cell cycle and detoxification remained unchanged. However, α-TOH, α-13'-OH and α-13'-COOH exhibited antigenotoxic and partly ROS-scavenging capacity. The results indicate that the LCM exert chemopreventive effects via ROS-scavenging capacity, the protection against DNA damage and the induction of cell death via caspase-independent mechanisms in premalignant colon cells.

Potential Role of ROS in Butyrate- and Dietary Fiber-Mediated Growth Inhibition and Modulation of Cell Cycle-, Apoptosis- and Antioxidant-Relevant Proteins in LT97 Colon Adenoma and HT29 Colon Carcinoma Cells.

The aim of the present study was to examine whether reactive oxygen species (ROS) contribute to chemopreventive effects of fermentation supernatants (FS) of different dietary fibers (Synergy1®, oat-, barley-, yeast β-glucan, Curdlan) and butyrate as a fermentation metabolite. LT97 and HT29 cells were treated with butyrate and FS alone or with N-acetyl-cysteine (NAC) and their impact on ROS formation, cell growth, and protein expression (Cyclin D2, p21, PARP, Bid, GPx2) was investigated. Butyrate and FS significantly decreased cell growth. ROS levels were significantly increased, particularly in LT97 cells, while co-treatment with NAC decreased ROS formation and growth inhibitory effects in both cell lines. After treatment with butyrate and FS, Cyclin D2 expression was reduced in LT97 cells and p21 expression was increased in both cell lines. Levels of full-length PARP and Bid were decreased, while levels of cleaved PARP were enhanced. GPx2 expression was significantly reduced by fiber FS in HT29 cells. A notable effect of NAC on butyrate- and FS-modulated protein expression was observed exclusively for PARP and Bid in HT29 cells. From the present results, a contribution of ROS to growth inhibitory and apoptotic effects of butyrate and FS on LT97 and HT29 cells cannot be excluded.

Use of the β-Glucan-Producing Lactic Acid Bacteria Strains Levilactobacillus brevis and Pediococcus claussenii for Sourdough Fermentation-Chemical Characterization and Chemopreventive Potential of In Situ-Enriched Wheat and Rye Sourdoughs and Breads.

The aim of the present study was to examine β-glucan production and the potential prebiotic and chemopreventive effects of wheat and rye sourdoughs and breads generated with wild-type and non-β-glucan-forming isogenic mutant strains of Levilactobacillus brevis and Pediococcus claussenii. Sourdough and bread samples were subjected to in vitro digestion and fermentation. Fermentation supernatants (FS) and pellets (FP) were analyzed (pH values, short-chain fatty acids (SCFA), ammonia, bacterial taxa) and the effects of FS on LT97 colon adenoma cell growth, viability, caspase-2 and -3 activity, genotoxic and antigenotoxic effects and on gene and protein expression of p21, cyclin D2, catalase and superoxide dismutase 2 (SOD2) were examined. Concentrations of SCFA were increased and concentrations of ammonia were partly reduced in the FS. The relative abundance of Bifidobacteriaceae was increased in all FPs. Treatment with FS reduced the growth and viability of LT97 cells and significantly increased caspase-2 and -3 activities without exhibiting genotoxic or antigenotoxic effects. The p21 mRNA and protein levels were increased while that of cyclin D2 was reduced. Catalase and SOD2 mRNA and protein expression were marginally induced. The presented results indicate a comparable chemopreventive potential of wheat and rye sourdoughs and breads without an additional effect of the formed β-glucan.

Combined Treatment with a WNT Inhibitor and the NSAID Sulindac Reduces Colon Adenoma Burden in Mice with Truncated APC.

Colorectal cancer is one of the most common cancers worldwide with limited therapeutic options. APC and other Wnt signaling mutations occur in the majority of colorectal cancers but there are currently no Wnt inhibitors in the clinic. The combination of Wnt pathway inhibition with sulindac provides an opportunity for killing Apc-mutant colon adenoma cells and suggests a strategy for colorectal cancer prevention and new treatments for patients with advanced colorectal cancer.

Colocalization with MMP-7 in the Distal Colon is Crucial for Syndecan-2 Shedding in Dextran Sulfate Sodium-Induced Colitis Mice.

IntroductionSyndecan-2 expression is elevated during chronic inflammation and cancer development, and its shedding is observed in cancer patients. However, it remained unknown whether inflammation triggers syndecan-2 shedding.MethodsThe colitis model was produced in C57BL/6 mice by oral administration of 2–3% dextran sulfate sodium (DSS) in the drinking water. Syndecan-2 and MMP-7 expression levels in tissues and cells were detected by real-time PCR, Western blotting, and immunohistochemistry. Shed syndecan-2 levels were detected by slot blotting. For tissue culture, colon tissues were divided into proximal, transverse, and distal parts, and incubated in culture media.ResultsIn C57BL/6 mice with DSS-induced colitis, syndecan-2 shedding began to increase after week 12 of chronic inflammation and continued to increase at week 15. The level of shed syndecan-2 correlated with the colocalization of syndecan-2 and MMP-7 in distal colon tissues. The mRNA expression of IL-6 was increased specifically in trans-distal colon tissues from weeks 9 to 15. IL-6 induced syndecan-2 expression and shedding and MMP-7 expression in ex vivo-cultured distal colon tissues and adenoma cell lines derived from the distal colon. IL-6 treatment induced STAT3 phosphorylation and MMP-7 expression in DLD-1 cells. The application of MMP-7 to ex vivo-cultured colon tissues increased the shedding of syndecan-2 to the culture medium.ConclusionOur findings suggest that chronic inflammation induces syndecan-2 shedding via the site-specific colocalization of syndecan-2 with MMP-7 in the distal colon.

Thermal Processing has no Impact on Chemopreventive Effects of Oat and Barley Kernels in LT97 Colon Adenoma Cells

The unique dietary fiber composition with high contents of β-glucan contributes to the health-promoting properties of oat and barley and may mediate a reduction of colon cancer risk. In the present study, chemopreventive effects of oat and barley (beta®barley) kernels were investigated. In order to address the impact of thermal processing on these effects, kernels were roasted (150–180 °C, approx. 20 min), digested and fermented using an In Vitro human digestion model. Concentrations of short-chain fatty acids (SCFA) and ammonia were determined in fermentation supernatants (FS). Growth inhibition, apoptosis, DNA integrity and gene expression of catalase were analyzed in LT97 colon adenoma cells. Concentrations of SCFA, particularly butyrate, were higher in oat/barley FS (2.2-fold, on average), while ammonia levels were significantly lower (0.7-fold, on average) than in the fermentation control. Treatment of LT97 cells with FS of oat/barley kernels led to a significant time- and dose-dependent growth reduction, a significant increase in caspase-3 activity and enhanced levels of catalase mRNA, without exhibiting genotoxic effects. In general, the results indicate a chemopreventive potential of In Vitro fermented oat and waxy winter barley mediated mainly by growth inhibitory and apoptotic effects, which are preserved after thermal processing.

Impact of processing degree on fermentation profile and chemopreventive effects of oat and waxy barley in LT97 colon adenoma cells

The chemopreventive effects of β-glucan-rich cereals such as oat and barley (beta®barley) have been examined previously, but studies comparing fermentation characteristics and chemopreventive effects of oat and barley of different processing stages are rare. Therefore, the present study aims at investigating the fermentation end points (pH values, concentrations of short-chain fatty acids (SCFA) and ammonia) in fermentation supernatants (FS) obtained from differently processed oat and barley samples (kernels, thick and thin flakes). Chemopreventive effects of FS, such as growth inhibition, apoptosis, and induction of cell cycle- and redox-relevant genes (p21, SOD2), were analysed in LT97 colon adenoma cells. After fermentation, pH values were reduced (∆ pH − 1.3, on average) and SCFA concentrations were increased (∆ + 59 mmol/L, on average) with a shift towards butyrate formation in FS obtained from oat and barley samples compared to the fermentation negative control (FS blank). Ammonia was reduced more effectively in FS obtained from barley (∆ − 4.6 mmol/L, on average) than from oat samples (∆ − 1.0 mmol/L, on average). Treatment of LT97 cells with FS resulted in a time- and dose-dependent reduction of cell number, an increase in caspase-3 activity (up to 9.0-fold after 24 h, on average) and an induction of p21 (2.1-fold, on average) and SOD2 (2.3-fold, on average) mRNA expression, while no genotoxic effects were observed. In general, the results indicate no concrete effect of the type of cereal or processing stage on fermentation and chemopreventive effects of oat and barley.

Przeciwnowotworowe działanie składników propolisu. Cz. 1. Ester fenyloetylowy kwasu kawowego (CAPE)

The paper presents a review of the publications on the anticancerogenic activity of the biologically active component of propolis – caffeic acid phenethyl ester (CAPE). Literature data indicate numerous biological properties of CAPE, namely: antioxidant, anti-inflammatory, antiviral, immunostimulatory, anti-angiogenic and others. In numerous tests, both in vitro and in vivo, the significant activity of CAPE has been confirmed, including an action against HT-29 human colon adenoma cells, and five: human, murine and other tumor cell cultures. The authors also emphasize that CAPE supports the anticancerogenic effect of drugs, including doxorubicin and cisplatin, due to the reduction of cancer cell survival by 45% and 34%, respectively, compared to the above-mentioned drugs used alone. The conducted research indicates that the induction of apoptosis in cells, i.e. programmed cell death, can be mentioned among the main mechanisms of the anticancerogenic activity of CAPE.

Chemopreventive effects of raw and roasted oat flakes after in vitro fermentation with human faecal microbiota

The aim of the present study was to analyse chemopreventive effects of oat flakes under consideration of processing. Thin and thick flakes were roasted and subjected to an in vitro digestion and fermentation. Fermentation supernatants (FS) were characterised and chemopreventive effects were analysed in LT97 colon adenoma cells. Compared to the fermentation control, pH values were decreased (from pH 6.3 to pH 5.0) and concentrations of SCFA, in particular butyrate, were increased in oat FS (2.6-fold, on average). Ammonia levels were not altered. Oat FS significantly decreased cell growth time- and dose-dependently. Caspase 3 activity was significantly increased (9.7-fold, on average). Oat FS slightly increased the mRNA expression of CAT (2.0-fold), SOD2 (1.7-fold) and GSTP1 (2.8-fold), on average, while GPX1 mRNA (0.3-fold) was decreased. The results indicate a chemopreventive potential of in vitro digested oat flakes regarding colon cancer development mediated mostly by growth inhibition and apoptosis, unaffected by roasting.

Study on chemopreventive effects of raw and roasted β-glucan-rich waxy winter barley using an in vitro human colon digestion model.

Due to their unique dietary fibre composition, in particular β-glucan, the consumption of barley may contribute to a healthy diet and the prevention of colon cancer. The aim of the present study was to analyse chemopreventive effects of barley flakes obtained from a β-glucan-rich barley cultivar. In order to address the impact of heat treatment on potential chemopreventive effects, barley flakes were roasted (160 °C-180 °C, approx. 20 min). The flakes were subjected to in vitro digestion and fermentation. Fermentation supernatants (FS) were analysed for the concentrations of short-chain fatty acids (SCFA) and ammonia. Chemopreventive endpoints (growth inhibition, apoptosis, DNA integrity, gene expression of detoxifying enzymes) were analysed in LT97 colon adenoma cells. Concentrations of SCFA were increased in barley FS (2.5-fold, on average) with a shift of molar ratios towards butyrate production, while ammonia levels were significantly decreased (0.7-fold, on average) compared to the fermentation control. The growth of LT97 cells was significantly reduced by barley FS in a time- and dose-dependent manner, and caspase-3 activity of treated cells was significantly enhanced (up to 6.3-fold, on average). On average, treatment of cells resulted in increased mRNA levels of CAT (2.1-fold), SOD2 (2.2-fold) and GSTP1 (3.9-fold), while expression of GPx1 (0.3-fold) was significantly decreased in some cases. The roasting process did not cause genotoxic effects of barley FS and had no impact on chemopreventive properties. Our results indicate chemopreventive potential of in vitro fermented waxy winter barley, mediated primarily by growth inhibitory and apoptotic effects, which is largely unaffected by roasting.

Postmortem vs. neoplastic gene expression: Clues to cancer development and therapy

Organismal death does not immediately end gene expression. Studies of postmortem gene expression in zebrafish and mice and in the myocardium, liver, prostate, pericardial fluid, and blood of human cadavers have identified genes whose expression is increased after organismal death. Cancer can be considered a form of “un-death” since excessively proliferating cells are typically unusually resistant to apoptosis (programmed cell death), and are subject to strong selective pressure for “uncontrolled life.” The changes in gene expression observed in organismal death, particularly in mammals (mice and humans), can be compared to that observed in human neoplasia, and the comparison of these expression patterns can inform us about human cancer. Here we present a hypothesis based on the following three tenets: (a) there will be distinct and opposing patterns of gene expression between the postmortem state and cancer with respect to key physiological outputs such as growth, apoptosis, invasion, and prognosis; (b) cancer cells considered more aggressive (e.g., derived from a metastasis and/or resistant to agents that suppress growth or induce apoptosis) will exhibit expression of relevant genes more unlike that of the postmortem condition while less aggressive neoplastic cells will exhibit gene expression more similar to the postmortem condition; and (c) targeting gene expression in cancer to produce a more postmortem-like pattern will promote less tumorigenic and less aggressive cell phenotypes. To evaluate components (a) and (b) of our hypothesis, we focus on previously published gene expression data from colorectal cancer (CRC) and colonic adenoma cells and compare that to postmortem expression data. This preliminary analysis in general supports our hypothesis, with more aggressive neoplastic cell types exhibiting gene expression patterns most unlike that found in the postmortem condition; this suggests that cancer and the postmortem condition represent opposing ends of a gene expression spectrum in the balance between life and death. Subsequently, we discuss the possibilities for further testing of the hypothesis, particularly for part (c), and we also discuss the possible implications of the hypothesis for cancer therapeutics.

Senescence-associated tissue microenvironment promotes colon cancer formation through the secretory factor GDF15.

The risk of colorectal cancer (CRC) varies between people, and the cellular mechanisms mediating the differences in risk are largely unknown. Senescence has been implicated as a causative cellular mechanism for many diseases, including cancer, and may affect the risk for CRC. Senescent fibroblasts that accumulate in tissues secondary to aging and oxidative stress have been shown to promote cancer formation via a senescence‐associated secretory phenotype (SASP). In this study, we assessed the role of senescence and the SASP in CRC formation. Using primary human colon tissue, we found an accumulation of senescent fibroblasts in normal tissues from individuals with advanced adenomas or carcinomas in comparison with individuals with no polyps or CRC. In in vitro and ex vivo model systems, we induced senescence using oxidative stress in colon fibroblasts and demonstrated that the senescent fibroblasts secrete GDF15 as an essential SASP factor that promotes cell proliferation, migration, and invasion in colon adenoma and CRC cell lines as well as primary colon organoids via the MAPK and PI3K signaling pathways. In addition, we observed increased mRNA expression of GDF15 in primary normal colon tissue from people at increased risk for CRC in comparison with average risk individuals. These findings implicate the importance of a senescence‐associated tissue microenvironment and the secretory factor GDF15 in promoting CRC formation.

Antioxidative, Anti-Inflammatory, and Anticancer Effects of Purified Flavonol Glycosides and Aglycones in Green Tea.

(1) Background: Extensive research has focused on flavan-3-ols, but information about the bioactivities of green tea flavonols is limited. (2) Methods: In this study, we investigated the antioxidative, anti-inflammatory, and anticancer effects of flavonol glycosides and aglycones from green tea using in vitro cell models. The fractions rich in flavonol glycoside (FLG) and flavonol aglycone (FLA) were obtained from green tea extract after treatment with tannase and cellulase, respectively. (3) Results: FLG and FLA contained 16 and 13 derivatives, respectively, including apigenin, kaempferol, myricetin, and quercetin, determined by mass spectrometry. FLA exhibited higher radical-scavenging activity than that of FLG. FLG and FLA attenuated the levels of intracellular oxidative stress in neuron-like PC-12 cells. The treatment of RAW 264.7 murine macrophages with FLG and FLA significantly reduced the mRNA expression of inflammation-related genes in a dose-dependent manner. Furthermore, FLG and FLA treatments decreased the viability of the colon adenoma cell line DLD-1 and breast cancer cell line E0771. Moreover, the treatment with FLG or FLA combined with paclitaxel had synergistic anticancer effects on the DLD-1 cell line. (4) Conclusions: Flavonols from green tea exerted beneficial effects on health and may be superior to flavan-3-ols.

Up-regulation of syndecan-2 in proximal colon correlates with acute inflammation.

We previously reported that syndecan-2 expression is increased on the colonic epithelium during chronic inflammation. Here, we report that syndecan-2 exhibits a different pattern of site-specific colonic expression during acute inflammation. Syndecan-2 expression was up-regulated predominantly in the proximal colon of dextran sulfate sodium-induced colitis mice. The colitis-associated up-regulation of syndecan-2 was barely detected in Rag-1-/- (recombination activating gene 1 knockout) mice under colitis-inducing conditions. Increased syndecan-2 expression correlated with increased levels of infiltrated CD4+ IL-17A+ T cells in the proximal colon. Serum levels of IL-17A were increased during the acute inflammatory response in normal mice but not Rag-1-/- mice. IL-17A directly induced IL-17 receptor (IL-17RA) and syndecan-2 expression in ex vivo-cultured proximal colon tissues and adenoma cell lines from proximal colon. IL-17RA knockdown reduced the IL-17A-mediated syndecan-2 expression in SNU1235 cells. No elevation of syndecan-2 or IL-17RA was observed in colonic tissues from IL-17A-/- mice during colitis induction. Finally, increased expression of syndecan-2 and IL-17RA was observed in the proximal colons of cecal ligation and puncture-induced sepsis mice and infectious pan colitis patients. Together, these data suggest that acute inflammation induces syndecan-2 expression predominantly in the proximal colon via IL-17A-IL-17RA signaling during the early stage of the inflammatory response and that proximal colonic syndecan-2 might be a biomarker for acute inflammation.-Hong, H., Song, H.-K., Hwang, E. S., Lee, A. R., Han, D. S., Kim, S.-E., Oh, E.-S. Up-regulation of syndecan-2 in proximal colon correlates with acute inflammation.

Hydrogen Sulfide Promotes Proliferation of HT-29 Colon Cancer Cells in a Mitochondria-independent Pathway

Several studies reported the carcinogenic and anticarcinogenic effects of hydrogen sulfide. The present study evaluated the role of mitochondria in mediating the anti/pro-carcinogenic effect of hydrogen sulfide on colon cancer cells as mitochondrial KATP channel and mitochondrial electron transport chain are one of the promising targets for cancer treatment. The colon adenoma cell line and normal small intestinal epithelial cell lines were used to study the antiproliferative effect of hydrogen sulfide in the presence of enzyme inhibitors, mitochondrial KATP channel modulators and in presence of inhibitors of endogenous H2S metabolizing enzymes namely cystathionine-β-synthase and cystathionine-γ-lyase. Antiproliferative effect of hydrogen sulfide was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, crystal violet, sulforhodamine B and lactate dehydrogenase assays with its donor sodium hydrogen sulfide in both HT-29 and IEC-6 cells, where only IEC-6 cells showed significant cytotoxic effect at a concentration of 49.88 µg (IC50) but HT-29 failed to exhibit cytotoxicity with the same H2S concentration. In order to identify the mitochondrial role, several electron transporting chain inhibitors and KATP channel modulators were used, but still H2S could able to enhance the colon adenoma cell line growth indicating mitochondrial in-dependency in the pro-carcinogenic effect. However, anticarcinogenic effect of hydrogen sulfide was observed only when the cells were incubated in the presence of cystathionine-β-synthase and cystathionine-γ-lyase inhibitor, indicating their influential role in determining the exogenous hydrogen sulfide toxicity in colon adenoma cell line cells.

Fascin protein stabilization by miR-146a implicated in the process of a chronic inflammation-related colon carcinogenesis model.

In sporadic colon tumors, multistep process of well-known genetic alterations accelerates carcinogenesis; however, this does not appear to be the case in inflammation-related ones. We previously established a model of inflammation-related colon carcinogenesis using human colonic adenoma cells, and identified fascin as a driver gene of this process. We analyzed the microRNAs involved in the stable fascin expression in colon adenocarcinoma cells. miRNA microarray analysis was performed using FPCK-1-1 adenoma cells and its-derived FPCKpP1-4 adenocarcinoma cells through chronic inflammation. To assess the involvement of miRNA in the inflammation-related carcinogenesis, sphere-forming ability, expression of colon cancer stemness markers, and stability of fascin protein via the proteasome using tough decoy RNA technique. We found that 17 miRNAs including miR-146a were upregulated and 16 miRNAs were downregulated in FPCKpP1-4 adenocarcinoma cells. We revealed that miR-146a in the adenocarcinoma cells brought about acquisition of sphere formation, cancer stemness, and inhibition of proteasomal degradation of the fascin protein. We found that stable fascin expression is brought about via the inhibition of proteasome degradation by miR-146a in the process of a chronic inflammation-related colon carcinogenesis.

Identification, isolation and characterization of human LGR5-positive colon adenoma cells.

The intestine is maintained by stem cells located at the base of crypts and distinguished by the expression of LGR5. Genetically engineered mouse models have provided a wealth of information about intestinal stem cells, whereas less is known about human intestinal stem cells owing to difficulty detecting and isolating these cells. We established an organoid repository from patient-derived adenomas, adenocarcinomas and normal colon, which we analyzed for variants in 71 colorectal cancer (CRC)-associated genes. Normal and neoplastic colon tissue organoids were analyzed by immunohistochemistry and fluorescent-activated cell sorting for LGR5. LGR5-positive cells were isolated from four adenoma organoid lines and were subjected to RNA sequencing. We found that LGR5 expression in the epithelium and stroma was associated with tumor stage, and by integrating functional experiments with LGR5-sorted cell RNA sequencing data from adenoma and normal organoids, we found correlations between LGR5 and CRC-specific genes, including dickkopf WNT signaling pathway inhibitor 4 (DKK4) and SPARC-related modular calcium binding 2 (SMOC2). Collectively, this work provides resources, methods and new markers to isolate and study stem cells in human tissue homeostasis and carcinogenesis.

Effect of tumor progression on colon cancer stem cell plasticity.

688 Background: Stage I colon cancer initiation is optimized through the activation of the embryonic stem cell (SC)-like program in colon adenoma cells (Le Rolle AF, et al., 2016). This malignant transformation requires cellular dedifferentiation since the embryonic SC-like program does not exist a priori in colon cells. High initial tumor plasticity provides a competitive advantage as the tumor has more intrinsic phenotypic flexibility to survive environmental challenges. Inhibition of the embryonic SC-like program represents a novel therapeutic strategy. Herein, we examine the evolution this high tumor plasticity in stage I-IV colon cancer. Methods: Human colon cancer tissues were collected prospectively under an MSKCC IRB protocol (Jan 1990-Dec 2000). RNA in situ hybridization of LGR5, a colon SC marker, was carried out on human colon tumors before and after therapeutic interventions. We performed Gene Set Enrichment Analysis (GSEA 2.0 Broad Institute software) using Affymetrix U133A expression profiles from normal colon mucosa (n = 33) and colon cancer epithelia from stage I (n = 17), II (n = 35), III (n= 39) and IV (n= 46) tumors. Statistical analyses were conducted with GraphPad Prism 5 and Microsoft Excel. Results: Putative colon SC markers and differentiation markers are increased and decreased, respectively, in stage I-IV colon cancer in comparison to normal colon. Although colon adenoma originates from LGR5+ colon SC, LGR5+ overexpression per se correlates with good prognosis stage IV colon cancer and is not a colon cancer stem cell biomarker. Maximal embryonic SC-like program enrichment occurs in stage I colon cancer. GSEA comparisons of moderately differentiated stages II-IV versus stage I colon cancer reveal progressive tumor differentiation from the stage I embryonic SC-like program toward the intestinal SC program at more advanced tumor stages. Notably, poorly differentiated stage IV colon cancer retains an embryonic SC-like program. Conclusions: We conclude that except for poorly differentiated tumors, colon cancer progression from stage I to IV involves a heterogeneous tumor differentiation process from an embryonic SC-like program towards the intestinal SC or more differentiated intestinal cell programs.