Promotion Of Colorectal Cancer Research Articles

The use of individual and multilevel data in the development of a risk prediction model to predict patients’ likelihood of completing colorectal cancer screening

Promotion of colorectal cancer (CRC) screening can be expensive and unnecessary for many patients. The use of predictive analytics promises to help health systems target the right services to the right patients at the right time while improving population health. Multilevel data at the interpersonal, organizational, community, and policy levels, is rarely considered in clinical decision making but may be used to improve CRC screening risk prediction. We compared the effectiveness of a CRC screening risk prediction model that uses multilevel data with a more conventional model that uses only individual patient data.We used a retrospective cohort to ascertain the one-year occurrence of CRC screening. The cohort was determined from a Health Maintenance Organization, in Oregon. Eligible patients were 50–75 years old, health plan members for at least one year before their birthday in 2018 and were due for screening. We created a risk model using logistic regression first with data available in the electronic health record (EHR), and then added multilevel data.In a cohort of 59,249 patients, 36.1% completed CRC screening. The individual level model included 14 demographic, clinical and encounter based characteristics, had a bootstrap-corrected C-statistic of 0.722 and sufficient calibration. The multilevel model added 9 variables from clinical setting and community characteristics, and the bootstrap-corrected C-statistic remained the same with continued sufficient calibration.The predictive power of the CRC screening model did not improve after adding multilevel data. Our findings suggest that multilevel data added no improvement to the prediction of the likelihood of CRC screening.

Promotion of colorectal cancer by transcription factor BHLHE40 involves upregulation of ADAM19 and KLF7.

BHLHE40 is a transcription factor, whose role in colorectal cancer has remained elusive. We demonstrate that the BHLHE40 gene is upregulated in colorectal tumors. Transcription of BHLHE40 was jointly stimulated by the DNA-binding ETV1 protein and two associated histone demethylases, JMJD1A/KDM3A and JMJD2A/KDM4A, which were shown to also form complexes on their own and whose enzymatic activity was required for BHLHE40 upregulation. Chromatin immunoprecipitation assays revealed that ETV1, JMJD1A and JMJD2A interacted with several regions within the BHLHE40 gene promoter, suggesting that these three factors directly control BHLHE40 transcription. BHLHE40 downregulation suppressed both growth and clonogenic activity of human HCT116 colorectal cancer cells, strongly hinting at a pro-tumorigenic role of BHLHE40. Through RNA sequencing, the transcription factor KLF7 and the metalloproteinase ADAM19 were identified as putative BHLHE40 downstream effectors. Bioinformatic analyses showed that both KLF7 and ADAM19 are upregulated in colorectal tumors as well as associated with worse survival and their downregulation impaired HCT116 clonogenic activity. In addition, ADAM19, but not KLF7, downregulation reduced HCT116 cell growth. Overall, these data have revealed a ETV1/JMJD1A/JMJD2A→BHLHE40 axis that may stimulate colorectal tumorigenesis through upregulation of genes such as KLF7 and ADAM19, suggesting that targeting this axis represents a potential novel therapeutic avenue.

LncRNAs, the Molecules Involved in Communications With Colorectal Cancer Stem Cells.

Colorectal cancer stem cells (CRCSCs) can actively self-renew, as well as having multidirectional differentiation and tumor regeneration abilities. Because the high functional activities of CRCSCs are associated with low cure rates in patients with colorectal cancer, efforts have sought to determine the function and regulatory mechanisms of CRCSCs. To date, however, the potential regulatory mechanisms of CRCSCs remain incompletely understood. Many non-coding genes are involved in tumor invasion and spread through their regulation of CRCSCs, with long non-coding RNAs (lncRNAs) being important non-coding RNAs. LncRNAs may be involved in the colorectal cancer development and drug resistance through their regulation of CRCSCs. This review systematically evaluates the latest research on the ability of lncRNAs to regulate CRCSC signaling pathways and the involvement of these lncRNAs in colorectal cancer promotion and suppression. The regulatory network of lncRNAs in the CRCSC signaling pathway has been determined. Further analysis of the potential clinical applications of lncRNAs as novel clinical diagnostic and prognostic biomarkers and therapeutic targets for colorectal cancer may provide new ideas and protocols for the prevention and treatment of colorectal cancer.

Development of a Credible Virtual Clinician Promoting Colorectal Cancer Screening via Telehealth Apps for and by Black Men: Qualitative Study.

BackgroundTraditionally, promotion of colorectal cancer (CRC) screening among Black men was delivered by community health workers, patient navigators, and decision aids (printed text or video media) at clinics and in the community setting. A novel approach to increase CRC screening of Black men includes developing and utilizing a patient-centered, tailored message delivered via virtual human technology in the privacy of one’s home.ObjectiveThe objective of this study was to incorporate the perceptions of Black men in the development of a virtual clinician (VC) designed to deliver precision messages promoting the fecal immunochemical test (FIT) kit for CRC screening among Black men in a future clinical trial.MethodsFocus groups of Black men were recruited to understand their perceptions of a Black male VC. Specifically, these men identified source characteristics that would enhance the credibility of the VC. The modality, agency, interactivity, and navigability (MAIN) model, which examines how interface features affect the user’s psychology through four affordances (modality, agency, interactivity, and navigability), was used to assess the presumed credibility of the VC and likability of the app from the focus group transcripts. Each affordance triggers heuristic cues that stimulate a positive or a negative perception of trustworthiness, believability, and understandability, thereby increasing source credibility.ResultsIn total, 25 Black men were recruited from the community and contributed to the development of 3 iterations of a Black male VC over an 18-month time span. Feedback from the men enhanced the visual appearance of the VC, including its movement, clothing, facial expressions, and environmental surroundings. Heuristics, including social presence, novelty, and authority, were all recognized by the final version of the VC, and creditably was established. The VC was named Agent Leveraging Empathy for eXams (ALEX) and referred to as “brother-doctor,” and participants stated “wanting to interact with ALEX over their regular doctor.”ConclusionsInvolving Black men in the development of a digital health care intervention is critical. This population is burdened by cancer health disparities, and incorporating their perceptions in telehealth interventions will create awareness of the need to develop targeted messages for Black men.

Aberrant expression of lncRNAs SNHG6, TRPM2-AS1, MIR4435-2HG, and hypomethylation of TRPM2-AS1 promoter in colorectal cancer.

Accumulating evidence has indicated that deregulation of lncRNAs plays essential roles in colorectal cancer (CRC) carcinogenesis. The goal of this study was to analyze the expression of lncRNAs in colorectal cancer and their association with clinicopathological variables. Bioinformatics analysis of published CRC microarray data was performed to identify the important lncRNAs. The expression levels of candidate genes were assessed in the human colon cancer/normal cell lines, CRC, adenomatous colorectal polyps, and their marginal tissues by qRT-PCR. Moreover, the methylation status of the TRPM2-AS1 promoter was studied using qMSP assay. Furthermore, we investigated the molecular mechanisms of these lncRNAs in CRC progression using in silico analysis. Microarray analysis revealed that lncRNAs SNHG6, MIR4435-2HG, and TRPM2-AS1 were upregulated in CRC. These results were validated in colon cell lines. Moreover, qRT-PCR showed that the expression levels of SNHG6 and TRPM2-AS1 were upregulated in the colorectal tumor tissues compared with their paired tissues. Nonetheless, there was no significant increase in MIR4435-2HG expression in CRC samples. Furthermore, we observed a significant hypomethylation of TRPM2-AS1 promoter and its activation in CRC tissues. By in silico analysis, we found that the lncRNAs upregulation could promote proliferation and drug resistance of colorectal cancer cells via miRNAs sponging and modulation of their targets expression. In conclusion, based on our results upregulation of SNHG6 and TRPM2-AS1, and hypomethylation of TRPM2-AS1 promoter might be considered as potential diagnostic biomarkers for CRC initiation and development.

Abstract 2880: CD36, a fatty acid translocase, promotes metastasis in CRC

Abstract Altered fatty acid metabolism, a hallmark of cancer, is an attractive target for therapeutic intervention. Fatty acid translocase (CD36), a multifunctional glycoprotein, has an important role in fatty acid metabolism: it serves as a fatty acid transporter. We have previously shown the importance of CD36 in promotion of colorectal cancer (CRC) cell proliferation and tumorigenesis and its role in compensation of de novo fatty acid synthesis inhibition. CD36 also plays important roles in migration, invasion and metastasis in gastric, ovarian and oral carcinomas. Cell lines with higher metastatic potential express higher levels of CD36; however, the role of CD36 in CRC metastasis has not been previously studied. The purpose of our study is (i) to determine the functional effect of CD36 on CRC metastasis, and (ii) to delineate the mechanistic pathways in which CD36 may regulate CRC metastasis. Methods: In this study we utilized HCT116 and HT29 CRC cell lines. The HT29 LuM3-GFP-Luc cell line was established from HT29 cells and has an increased propensity to metastasize to mouse lungs. The increase in metastatic capacity of HT29 LuM3 is associated with an increase in the expression of CD36. We utilized parental HT29-GFP-Luc and HT29 LuM3-GFP-Luc to overexpress and knockdown CD36, respectively, and we used a CD36 neutralizing antibody to block the activity of CD36 in HCT116 and HT29 LuM3 cells. To measure colonization, migration and invasion in vitro, we used soft agar colony formation assays, a wound healing assay, and spheroid and trans-well invasion assays. In vivo, we utilized the tail-vein injection model to study tumor cell colonization in mouse lungs, and the cecum injection model to study liver metastasis from the primary tumor. We analyzed samples with qRT-PCR, western blot and immunohistochemistry. Results: Knockdown of CD36 significantly reduced CRC cell invasion and colony formation in HCT116 and HT29 LuM3 in vitro. CRC cell invasion and colony formation increased with overexpression of CD36 in HCT116 cells. Overexpression of CD36 in established CRC cell lines increased markers of survival and invasion, such as phospho-Akt, P-Met, and MMP28. Most importantly, knockdown of CD36 in HT29 LuM3-GFP-Luc cells significantly reduced lung metastasis in vivo. Conclusions: CRC cells that have a higher propensity to initiate metastasis in vivo express higher levels of CD36. We showed that this upregulation of CD36 represents a unique advantage for CRC cells to initiate tumor colonies, migrate and invade both in vitro and in vivo. Thus, our study highlights the potential of CD36 as a viable target for metastatic disease in CRC patients. Citation Format: James Michael Drury, Piotr Rychahou, Heidi L. Weiss, Yekaterina Y. Zaytseva. CD36, a fatty acid translocase, promotes metastasis in CRC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2880.

Estrogen Receptor Beta Influences the Inflammatory p65 Cistrome in Colon Cancer Cells.

Inflammation is a primary component of both initiation and promotion of colorectal cancer (CRC). Cytokines secreted by macrophages, including tumor necrosis factor alpha (TNFα), activates the pro-survival transcription factor complex NFκB. The precise mechanism of NFκB in CRC is not well studied, but we recently reported the genome-wide transcriptional impact of TNFα in two CRC cell lines. Further, estrogen signaling influences inflammation in a complex manner and suppresses CRC development. CRC protective effects of estrogen have been shown to be mediated by estrogen receptor beta (ERβ, ESR2), which also impacts inflammatory signaling of the colon. However, whether ERβ impacts the chromatin interaction (cistrome) of the main NFκB subunit p65 (RELA) is not known. We used p65 chromatin immunoprecipitation followed by sequencing (ChIP-Seq) in two different CRC cell lines, HT29 and SW480, with and without expression of ERβ. We here present the p65 colon cistrome of these two CRC cell lines. We identify that RELA and AP1 motifs are predominant in both cell lines, and additionally describe both common and cell line-specific p65 binding sites and correlate these to transcriptional changes related to inflammation, migration, apoptosis and circadian rhythm. Further, we determine that ERβ opposes a major fraction of p65 chromatin binding in HT29 cells, but enhances p65 binding in SW480 cells, thereby impacting the p65 cistrome differently in the two cell lines. However, the biological functions of the regulated genes appear to have similar roles in both cell lines. To our knowledge, this is the first time the p65 CRC cistrome is compared between different cell lines and the first time an influence by ERβ on the p65 cistrome is investigated. Our work provides a mechanistic foundation for a better understanding of how estrogen influences inflammatory signaling through NFκB in CRC cells.

Exosomal Long Non-coding RNA HOTTIP Increases Resistance of Colorectal Cancer Cells to Mitomycin via Impairing MiR-214-Mediated Degradation of KPNA3.

It has been reported that long non-coding RNA HOXA distal transcript antisense RNA (lncRNA HOTTIP) functions as a tumor promoter in colorectal cancer (CRC). Hence, we paid attention to exploring whether exosomes could carry lncRNA HOTTIP to affect the mitomycin resistance in CRC and to identify the underlying mechanisms. High expression of HOTTIP was detected in mitomycin-resistant CRC cells. Inhibition of HOTTIP reduced the mitomycin resistance. In the co-culture system of mitomycin-resistant cells or their derived exosomes with CRC cells, the HOTTIP was found to be transferred into the parental cells via extracellular vesicles (EVs) secreted from mitomycin-resistant cells and to contribute to the mitomycin resistance. Based on the bioinformatics databases, possible interaction network of HOTTIP, microRNA-214 (miR-214) and Karyopherin subunit alpha 3 (KPNA3) in CRC was predicted, which was further analyzed by dual-luciferase reporter, RNA binding protein immunoprecipitation and RNA pull-down assays. As HOTTIP down-regulated miR-214 to elevate the KPNA3 expression, HOTTIP enhanced the mitomycin resistance through impairing miR-214-dependent inhibition of KPNA3. Finally, HOTTIP was suggested as an independent factor predicting mitomycin response in patients with CRC. Those data together confirmed the promotive effects of EV-carried HOTTIP on the mitomycin resistance, while targeting HOTTIP might be a promising strategy overcoming drug resistance in CRC.

Organoid modelling identifies that DACH1 functions as a tumour promoter in colorectal cancer by modulating BMP signalling.

BackgroundDachshund homologue 1 (DACH1) is highly expressed in LGR5+ intestinal stem cells and colorectal tumours. However, the roles of DACH1 in intestinal cell stemness and colorectal tumorigenesis remain largely undefined.MethodsWe used immunohistochemistry, western blotting and quantitative real-time PCR to analyse DACH1 expression in colorectal cancer (CRC) samples. CRISPR/Cas9 gene editing and lentiviral vector-mediated overexpression and shRNA-mediated knockdown of DACH1 were utilized to modulate DACH1 expression in cell lines and organoids. An intestinal organoid-based functional model was analysed, and cancer cell colony formation, sphere formation assays and murine xenotransplants were performed to reveal the role of DACH1 in CRC cell proliferation, stemness and tumorigenesis. Immunofluorescence, co-immunoprecipitation, RNA interference and microarray data analyses were conducted to demonstrate the association between DACH1 and the bone morphogenetic protein (BMP) signalling pathway.FindingsDACH1 is specifically expressed in discrete crypt base cells, and increased DACH1 expression was found in all stages of CRC. Moreover, the high expression of DACH1 independently predicted poor prognosis. In colon cancer cells, shRNA-mediated suppression of DACH1 inhibited cell growth in vitro and in vivo. By studying the intestinal organoid-based functional model, we found that depletion of DACH1 reduced the organoid formation efficiency and tumour organoid size. DACH1 overexpression stimulated both colonsphere formation and tumour organoid formation in the context of dysregulated BMP signalling. Mechanistic characterizations indicated that overexpression of DACH1 affects a subset of stem cell signature genes implicated in stem cell proliferation and maintenance through the suppression of BMP signalling via SMAD4.InterpretationTogether, our study highlights DACH1 as an integral regulator of BMP signalling during intestinal tumorigenesis, and DACH1 could be a potential prognostic marker and therapeutic target for colorectal cancer patients.

Abstract A041: Community engagement in the development of an m-health app utilizing a black male virtual health assistant (VHA) to promote colon cancer screening: An iterative study of credibility and likeability

Abstract Traditionally, promotion of colorectal cancer (CRC) screening among minority and rural populations including African American men was delivered by Community Health Workers (CHW), Patient Navigators, and decision aids (printed text or video media) at clinics. A novel approach to increase CRC screening of black men includes developing and utilizing a patient-centered, tailored message delivered via mobile-health (m-health) technology. In this study, a Community Based Participatory Research (CBPR) approach provided feedback on a Virtual Health Assistant (VHA) designed to deliver precision and personalized messages promoting the Fecal Immunochemical Test (FIT) for CRC screening. Focus groups of Black men were recruited to understand and employ their perceptions of a Black-male VHA. Specifically, these men identified source characteristics that would enhance the credibility of the VHA and likeability of an m-health app. The MAIN Model which examines how an interface features affect the user’s psychology through four Affordances: Modality, Agency, Interactivity, and Navigability was used to assess the presumed credibility of the VHA and likability of the app from the focus group transcripts. Each affordance triggers heuristic cues that stimulate a positive or negative perception of trustworthiness, believability, and understandability thereby increasing source credibility and likability. Data collected in the focus group included feedback on the different content modalities, both text and visual: size and font of the text, VHA appearance, voice, movement, diction, the environment as well as perceptions of the trustworthiness and expertise of the interface. Data related to interactivity and ease of navigation through the app was also collected and analyzed. All in all, 25 Black men from the community contributed to the development of three iterations of a Black male VHA over an eighteen-month time span. The final version of the Black male-VHA, ALEX is now part of an intervention to increase CRC screening among minority and rural populations. Citation Format: Danyell Wilson-Howard, Melissa Vilaro, Lauren Griffin, Fatemeh Travassoli, Mohan Zalake, Benjamin Lok, Francois Modave, Peter Carek, Thomas George, Janice Krieger. Community engagement in the development of an m-health app utilizing a black male virtual health assistant (VHA) to promote colon cancer screening: An iterative study of credibility and likeability [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr A041.

Abstract A21: Promotion of colorectal cancer by Streptococcus gallolyticus—a novel mechanism

Abstract Streptococcus gallolyticus subsp. gallolyticus (Sgg) is an opportunistic gut pathobiont. It has a longstanding clinical association with colorectal cancer (CRC). We previously demonstrated that Sgg stimulates CRC cell proliferation and promotes tumor growth in mouse models of CRC (Kumar et al., 2017). Recent studies also indicated that Sgg induced an immune-tolerant environment in the mouse colon conductive for tumor development (Zhang et al., 2018). These results provide strong experimental support for Sgg being an active promoter of tumor development. The molecular mechanism underlying Sgg’s tumor promoting activity was unknown. We investigated the effect of Sgg on CRC cells by analyzing mass spectrometry proteomics data. We found that several types of collagen were upregulated in Sgg-treated cells compared to cells cultured in media only. The upregulation was further confirmed by RT-qPCR and Western blot. Transforming growth factor beta (TGFbeta) is a master regulator of the extracellular matrix. Therefore, we examined the effect of Sgg on the TGFbeta signaling pathway. The results showed that Sgg-treated cells had significantly higher level of phosphorylated Smad2. Sgg treatment also induced the expression of TGFbeta target genes. The effects of Sgg on CRC cells were significantly decreased in the presence of inhibitors for TGFbeta signaling. In addition, we observed that Sgg induced similar effects in mouse colon. Together, these results demonstrate that Sgg activates the TGFbeta signaling pathway in vitro and in vivo. Furthermore, we found that activation of TGFbeta by Sgg is mediated by specific Sgg genes. Deletion of these genes abolished the effect of Sgg on CRC cells in vitro and its ability to promote tumor growth in a mouse model of CRC. These results suggest that TGFbeta activation is a mechanism underlying Sgg’s contribution to tumor growth. Next, we tried to determine if these findings are relevant to CRC patients. Examination of tumor tissues from CRC patients revealed that Sgg-positive tumors exhibit significantly enhanced TGFbeta activity compared to Sgg-negative tumors, suggesting that Sgg-positivity correlates with stronger TGFbeta signaling activities in CRC patients. Taken together, our results highlight a previously unrecognized mechanism in which a colonic microbe activates the TGFbeta signaling pathway to promote the development of CRC. Studies to further delineate this novel mechanism are currently under way. Citation Format: John Taylor, Ritesh Kumar, Amir Sarshekeh, Jennifer Davies, Scott Kopetz, Juan Xu, Yi Xu. Promotion of colorectal cancer by Streptococcus gallolyticus—a novel mechanism [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr A21.

Obesity is an initiator of colon adenomas but not a promoter of colorectal cancer in the Black Women's Health Study.

Evidence for the association of anthropometrics with colorectal neoplasms is limited for African Americans. We examined anthropometric measures with both colorectal adenoma and colorectal cancer (CRC) risk in the ongoing Black Women's Health Study. In a nested case-control analysis, 954 cases of colorectal adenoma were compared with 3,816 polyp-free controls, matched on age and follow-up time. For the CRC analyses, 413 incident CRC cases were identified over a 16-year follow-up (802,783 person-years). Adenoma cases and CRC were verified by medical record review. We used multivariable conditional logistic regression analyses (for adenoma) and Cox proportional hazards analyses (for CRC) that included anthropometric exposures and selected confounders. Overall body mass index (BMI) and other anthropometric factors were not associated with colorectal adenoma or cancer risk in Black women. However, increased risk of adenoma (but not CRC) was observed among especially related to adenomas in the proximal colon. Among women ≥ 50years of age, risk of proximal adenoma increased 14% (95% CI 1.00, 1.31), 35% (95% CI 1.12, 1.63), and 25% (0.93, 1.68) with each standard deviation increase in BMI, waist circumference, and waist-to-hip ratio, respectively. None of the anthropometric factors were associated with young onset CRC or adenoma risk. Our results suggest that obesity might be an initiator for colon adenomas but not a promoter for colorectal cancer among Black women.

ADAMTS12 acts as a cancer promoter in colorectal cancer via activating the Wnt/β-catenin signaling pathway in vitro.

BackgroundADAMTS12, a member of the ADAMTS family, is reported to be associated with the clinic outcome of colorectal cancer (CRC) patients. However, the functions and precise mechanism in CRC progression have yet to be fully understood.MethodsBy analyzing The Cancer Genome Atlas (TCGA) database, we examined the mRNA level of ADAMTS12 and assessed the prognostic value of ADAMTS12 in CRC patients using a tissue microarray containing 41 CRC patient samples. Cell Counting Kit-8 (CCK-8), colony formation, and transwell assays were used to quantify the impact of ADAMTS12 on cell proliferation and migration in ADAMTS12-overexpressing and ADAMTS12-deficient cell lines. The signaling pathways that ADAMTS12 mediated were identified by dual-luciferase reporter assays, and confirmed by western blotting and quantitative teal-time polymerase chain reaction (qRT-PCR).ResultsThe ADAMTS12 mRNA level was upregulated in CRC tissues, and CRC patients with a high level of ADAMTS12 showed worse prognosis when compared with the patients with a low level of ADAMTS12. In vitro functional assays demonstrated that overexpression of ADAMTS12 significantly boosted cell proliferation and migration while ADAMTS12 deficiency remarkably impaired both tumor cell behaviors. Mechanical studies further verified that ADAMTS12 overexpression enhanced the transcriptional activity of β-catenin in the Wnt/β-catenin signaling pathway. In the ADAMTS12-deficient context, the downstream gene expression of myc and cyclin D1 was significantly reduced compared with that in wild-type cancer cells.ConclusionsADAMTS12 fulfills the tumor-promotor role by activating Wnt/β-catenin signaling pathway in colon cells and may represent a new option in CRC target treatment.

Haem iron reshapes colonic luminal environment: impact on mucosal homeostasis and microbiome through aldehyde formation

BackgroundThe World Health Organization classified processed and red meat consumption as “carcinogenic” and “probably carcinogenic”, respectively, to humans. Haem iron from meat plays a role in the promotion of colorectal cancer in rodent models, in association with enhanced luminal lipoperoxidation and subsequent formation of aldehydes. Here, we investigated the short-term effects of this haem-induced lipoperoxidation on mucosal and luminal gut homeostasis including microbiome in F344 male rats fed with a haem-enriched diet (1.5 μmol/g) 14–21 days.ResultsChanges in permeability, inflammation, and genotoxicity observed in the mucosal colonic barrier correlated with luminal haem and lipoperoxidation markers. Trapping of luminal haem-induced aldehydes normalised cellular genotoxicity, permeability, and ROS formation on a colon epithelial cell line. Addition of calcium carbonate (2%) to the haem-enriched diet allowed the luminal haem to be trapped in vivo and counteracted these haem-induced physiological traits. Similar covariations of faecal metabolites and bacterial taxa according to haem-induced lipoperoxidation were identified.ConclusionsThis integrated approach provides an overview of haem-induced modulations of the main actors in the colonic barrier. All alterations were closely linked to haem-induced lipoperoxidation, which is associated with red meat-induced colorectal cancer risk.

Su1976 – Impacts of Additive Food E171 (Titanium Dioxide) on the Gut Microbiota and Colorectal Carcinogenesis in ApcMIN/+ Murine Model

Background and aim: Contaminant molecules could play a key role in the initiation and/or promotion of colorectal cancer (CRC) which is the second leading cause of cancer worldwide. Among the different pollutants, titanium dioxide (TiO2) is a white natural metal oxide which is particularly monitored. It is one of the most widely used engineered nanomaterials in daily consumer products, including food. Long-term inhalation studies with TiO2 nanoparticles (NPs) in rats reported development of lung tumors leading to classify this compound as a possible carcinogen for humans by International Agency for Research on Cancer. The food additive TiO2, referred to as E171 in the European Union, is commonly used as a whitening and brightening agent in sugar confectionary, sauces, ice creams and pastries. The gastro-intestinal tract may be an important absorption route for TiO2 NPs and additive E171 could be involved in the CRC etiology. Thus, this project aimed to assess the impact of chronic exposure of food additive E171 in CRC development using APCmin/+ mouse model which spontaneously develop intestinal tumors. Because intestinal microbiota play a role in colorectal carcinogenesis, carcinogenic properties of TiO2 will be evaluated in association with microbiota dysbiosis. Methods: Additive E171 composition was characterized by X-ray diffraction (XRD) and thermogravimetric analysis (TGA). APCmin/+ mice were chronically exposed (twice per week) to E171 (1 and 10 mg/kg of body weight (BW)) or vehicle (water) for 28 days via intragastric gavage. Micro-inflammation was assessed by the quantification of fecal lipocalin-2 (LCN-2) marker. At the end of the experimental protocol, macroscopic and histologic examinations of colon were performed and colonic mucosa-associated microbiota was analyzed by quantitative PCR. Results: As shown by the TGA analysis, the E171 compound is purely mineral and the size of NPs administrated to mice in this study did not exceed 28 nm (XRD analysis). No significant body weight loss was observed between mice groups suggesting no toxicity in our experimental conditions. However macroscopic and histological observations showed a more advanced tumor development in mice receiving the additive E171 (at a dose of 10 mg/kg of BW) compared to control animals. At the end of the experiment, high level of fecal LCN-2 concentration correlated with increase of tumoral volume. Mice with adenocarcinoma within this group also exhibit an increase of Bacteroides and Akkermansia as well as a higher prevalence of Escherichia coli and a decrease of Bifidobacterium. Conclusion: Additive E171 promotes colonic tumorigenesis and induces change in gut microbiota composition. Underlying carcinogenic mechanisms focusing on microbiota dysbiosis implication are in progress. This study supports the carcinogenic properties of TiO2 in the context of colorectal cancer.

RACK1 Acts as a Potential Tumor Promoter in Colorectal Cancer.

Background The receptor of activated protein kinase C 1 (RACK1) promotes the progression and invasion of several cancers. However, the role of RACK1 in the pathogenesis of colorectal cancer (CRC) has not been clearly defined. Herein, we aimed to investigate the biological role of RACK1 in CRC. Materials and Methods The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) dataset were searched, and the expression of RACK1 in CRC tissues and adjacent normal tissues was evaluated. Immunohistochemical staining was performed to detect the expression of RACK1 in human CRC, adenoma, and normal tissues. Western blotting was used to detect the expression of RACK1 in human CRC cell lines. Functional assays, such as BrdU, colony formation, and wound healing and transwell invasion assays, were used to explore the biological role of RACK1 in CRC. Results RACK1 was upregulated in CRC tissues compared with its expression in adjacent normal tissues in TCGA and the GEO dataset (P < 0.05). Moreover, RACK1 was significantly overexpressed in CRC and adenoma tissues compared with its expression in normal tissues (P < 0.05). Loss-of-function experiments showed that RACK1 promoted cell proliferation, migration, and invasion in vitro. Conclusions Our data indicated that RACK1, as an oncogene, markedly promoted the progression of CRC, which suggested that RACK1 is a potential therapeutic target for CRC management.

Wild-type p53 regulates OTOP2 transcription through DNA loop alteration of the promoter in colorectal cancer.

Colorectal cancer (CRC) is the third most commonly diagnosed malignancy worldwide and remains a major public health issue. Therefore, further investigation is required to delineate the cellular and molecular mechanisms underlying colorectal tumorigenesis. Using CRC data taken from The Cancer Genome Atlas, we determined that the expression of otopetrin 2 (OTOP2) is highly correlated with malignancy grade and rate of patient survival. Here, we report that OTOP2 is down‐regulated in cancerous tissues and that elevated OTOP2 effectively suppresses tumor proliferation in vitro. We demonstrate that wild‐type p53 (wtp53), but not mutant p53 (mtp53), can regulate the transcription of otop2 in CRC cells. Subsequently, we investigate the chromatin architecture of the otop2 promoter, whereby we discover alterations in p53‐dependent DNA loop organization and CCCTC‐binding factor (CTCF) binding between cells with wtp53 and mtp53. In conclusion, our study promotes an in‐depth understanding of tumorigenesis, which may also lead to the development of therapeutic applications targeting human malignancy.

Twist1/2 activates MMP2 expression via binding to its promoter in colorectal cancer.

This study aimed to characterize the effect of Twist2 on epithelial-to-mesenchymal transition (EMT) and the invasive potential of colorectal cancer (CRC) cells and to explore the mechanisms underlying the regulative effect of Twist1 and Twist2 on matrix metalloproteinase 2 (MMP2) expression in CRC. Data mining was performed in colorectal cancer cohort (COADREAD) in the Cancer Genome Atlas (TCGA-COADREAD). CRC LoVo and HCT116 cells were used as in vitro cell models. CRC tumors with lymphatic invasion (N = 102) had a significantly higher expression of TWIST1 (p = 0.01) and TWIST2 (p = 0.02) than the lymphatic invasion negative cases (N = 228). TWIST2 overexpression enhanced EMT and the invasive potential of the CRC LoVo and HCT116 cells, while TWIST2 knockdown reversed the EMT process and weakened the invasive potential of the cells. TWIST1 and TWIST2 were co-upregulated with MMP2 and MMP9 in COADREAD cohort. TWIST1 or TWIST2 overexpression significantly elevated nuclear β-catenin accumulation, which is a known signaling pathway elevating MMP2 and MMP9 expression. More importantly, we found that both Twist1 and Twist2 could transcriptionally activate MMP2 via directly binding to its promoter. However, this mechanism was not observed in the MMP9 promoter. TWIST1/2 is associated with lymphatic invasion in CRC. TWIST2 upregulation enhances EMT and the invasive potential of CRC cells. TWIST1/2 can enhance the Wnt/β-catenin signaling pathway in CRC cells. In addition, Twist1/2 can bind to the MMP2 promoter and promote its transcription.

Methylation of CDX2 gene promoter in the prediction of treatment efficacy in colorectal cancer.

The aim of the present study was to examine the diagnosis of methylation of CDX2 gene promoter in colorectal cancer (CRC) and assessed its value in the prediction of treatment efficacy. Sixty patients who were diagnosed as CRCs for the first time, 60 patients with hyperplastic polyps (HPs) and adenomas, and 60 patients with inflammatory lesions or healthy patients (control group) were included in the present study. The methylation levels of CDX2 gene promoter were detected by methylation-specific polymerase chain reaction (MSP), and the expression levels of CDX2 mRNA were detected by fluorescence quantitative PCR. Treatment options, such as surgery, radiotherapy and chemotherapy, were chosen on the basis of TNM staging of CRC patients. The tumor-free survival, relapse rate and mortality were also recorded. The methylation rate was 71.67% (43/60) and significantly higher in the CRC group as compared to the HP/adenoma and control groups, P<0.05. Moreover, they showed further increase with higher degree of TNM staging. The expression levels of CDX2 mRNA was significantly lower in the CRC group in comparison to HP/adenoma and control groups, P<0.05, and showed a further decrease with a higher degree of TNM staging. The tumor-free survival was shorter, and the relapse rate and mortality were higher in patients with positive methylation in the CRC group, P<0.05. Multivariate logistic regression analysis demonstrated that TNM staging and positive methylation were independent risk factors of mortality. In conclusion, higher methylation degree of CDX2 gene promoter resulted in decreased expression of CDX2 gene, and was closely associated with TNM staging and prognosis. TNM staging and positive methylation were independent risk factors of mortality for CRC patients.

Downregulation of SM22α protein by hypermethylation of its promoter in colorectal cancer.

Silencing of tumor suppressor genes by hypermethylation in gene promoter regions is a crucial mechanism in carcinogenesis. Gene methylation may be reversible and evaluated easily, thus providing a promising substrate for the development of biomarkers for the detection and prevention of cancer, including colorectal cancer (CRC). In the present study, the protein expression and methylation status of smooth muscle protein 22α (SM22α) was investigated in 78 cases of CRC and adjacent normal tissue. The aim of the study was to investigate the function of SM22α in the pathogenesis of CRC and to identify a candidate biomarker for the early detection of CRC. The methylation status of promoter of SM22α gene was detected using methylation-specific polymerase chain reaction. The protein expression of SM22α was evaluated using western blot analysis. The results demonstrated a significant decrease of SM22α protein expression in 50 (68.5%) cases of CRC compared with that in adjacent normal tissues (P<0.001). The methylation status of SM22α promoter in CRC was significantly increased compared with that in adjacent normal tissues (P<0.001). Additionally, there was a negative correlation between the expression of SM22α protein and methylation levels of SM22α gene in CRC (P<0.001). Kaplan-Meier curves revealed that patients with CRC with an unmethylated promoter of SM22α gene exhibited an increased survival time (34.8±0.6 vs. 30.9±1.3 months; P=0.025) compared with that in patients with a methylated promoter of SM22α gene. The present study demonstrated that the protein expression of SM22α is downregulated in CRC tissues by hypermethylation of its promoter, and that the methylation of SM22 α promoter may be used as a biomarker for early detection, prognosis and prediction of CRC.