Development Of CRC Research Articles

DNA Hypomethylation of PDCD1 Gene in Korean Colorectal Cancer Patients

Abstract Colorectal cancer (CRC) is one of the most common and critical cancers, hence considerable effort has been focused on the identification of prognostic, diagnostic markers and therapeutic targets for CRC. Meanwhile, CRC is extensively known to be related to DNA methylation. PD-1, also known as CD279, is a cell surface protein encoded by PDCD1 gene which acts as the immune checkpoint to terminate pro-inflammatory response of T cells at the right time. Recently, PD-1 has been reported to be aberrantly overexpressed in multiple cancers, hampering the anti-tumor function of T cells. Monoclonal antibodies (mAb) which target PD-1 have been used as cancer therapy, but there has been limited success with the use of immune checkpoint inhibitors. We investigated the DNA methylation status of normal and tumor tissues of Korean CRC patients by performing the targeted bisulfite sequencing and identified that the promoter CpG island of PDCD1 is significantly hypomethylated in CRC tissue compared to its paired adjacent normal tissue. We have hypothesized the hypomethylation of promoter CpG island of PDCD1 is associated with aberrant expression of PD-1 and promotes immune evasion, eventually, development of CRC. Additionally, we are currently checking the possibility that hypomethylation of promoter CpG island of PDCD1 hinders the therapeutic effects of PD-1 monoclonal antibodies in CRC patients.

Altered ARID1A expression in colorectal cancer

BackgroundARID1A has been described as a tumor suppressor gene, participating in chromatin re-modeling, epithelial-mesenchymal-transition and many other cellular and molecular processes. It has been cited as a contribute in tumorigenesis. The role of ARID1A in CRC is not yet defined.AimTo investigate the role of ARID1A methylation and CNV in its expression in CRC cell lines and to examine the relationship between ARID1A status with survival and clinicopathologic characteristics in patients with CRC.MethodsWe used RT-PCR to determine both CNV and expression of ARID1A from six CRC cell lines. We used MSP to evaluate methylation of ARID1A. IHC was used to assess ARID1A protein expression. We also evaluated MSI and EMAST status in 18 paired CRC and adjacent normal tissues. 5AzadC was used to assess effect of DNA demethylation on ARID1A expression. Statistical analysis was performed to establish correlations between ARID1A expression and other parameters.ResultsAmong the 18 CRC tumors studied, 7 (38.8%) and 5 tumors (27.7%) showed no or low ARID1A expression, respectively. We observed no significant difference in ARID1A expression for overall patient survival, and no difference between clinicopathological parameters including MSI and EMAST. However, lymphatic invasion was more pronounced in the low/no ARID1A expression group when compared to moderate and high expression group (33% VS. 16.6% respectively. ARID1A promoter methylation was observed in 4/6 (66%) cell lines and correlated with ARID1A mRNA expression level ranging from very low in SW48, to more pronounced in HCT116 and HT-29/219. Treatment with the methyltransferase inhibitor 5-Azacytidine (5-aza) resulted in a 25.4-fold and 6.1-fold increase in ARID1A mRNA expression in SW48 and SW742 cells, respectively, while there was no change in SW480 and LS180 cells. No ARID1A CNV was observed in the CRC cell lines.ConclusionARID1A expression is downregulated in CRC tissues which correlates with it being a tumor suppressor protein. This finding confirms ARID1A loss of expression in CRC development. Our in-vitro results suggest high methylation status associates with reduced ARID1A expression and contributes to CRC tumorigenesis. However, there was no significant association between ARID1A loss of expression and clinicopathological characteristics. Future in-vivo analysis is warranted to further establish ARID1A role in colorectal neoplastic transformation.

3D Cell Culture-Based Global miRNA Expression Analysis Reveals miR-142-5p as a Theranostic Biomarker of Rectal Cancer Following Neoadjuvant Long-Course Treatment

Altered expression of miRNAs in tumor tissue encourages the translation of this specific molecular pattern into clinical practice. However, the establishment of a selective biomarker signature for many tumor types remains an inextricable challenge. For this purpose, a preclinical experimental design, which could maintain a fast and sensitive discovery of potential biomarkers, is in demand. The present study suggests that the approach of 3D cell cultures as a preclinical cancer model that is characterized to mimic a natural tumor environment maintained in solid tumors could successfully be employed for the biomarker discovery and validation. Subsequently, in this study, we investigated an environment-dependent miRNA expression changes in colorectal adenocarcinoma DLD1 and HT29 cell lines using next-generation sequencing (NGS) technology. We detected a subset of 16 miRNAs differentially expressed in both cell lines cultivated in multicellular spheroids compared to expression levels in cells grown in 2D. Furthermore, results of in silico miRNA target analysis showed that miRNAs, which were differentially expressed in both cell lines grown in MCS, are involved in the regulation of molecular mechanisms implicated in cell adhesion, cell-ECM interaction, and gap junction pathways. In addition, integrins and platelet-derived growth factor receptors were determined to be the most significant target genes of deregulated miRNAs, which was concordant with the environment-dependent gene expression changes validated by RT-qPCR. Our results revealed that 3D microenvironment-dependent deregulation of miRNA expression in CRC cells potentially triggers essential molecular mechanisms predominantly including the regulation of cell adhesion, cell–cell, and cell–ECM interactions important in CRC initiation and development. Finally, we demonstrated increased levels of selected miR-142-5p in rectum tumor tissue samples after neoadjuvant long course treatment compared to miR-142-5p expression levels in tumor biopsy samples collected before the therapy. Remarkably, the elevation of miR-142-5p expression remained in tumor samples compared to adjacent normal rectum tissue as well. Therefore, the current study provides valuable insights into the molecular miRNA machinery of CRC and proposes a potential miRNA signature for the assessment of CRC in further clinical research.

Abstract A07: The involvement of a type VII secretion system in the interactions between Streptococcus gallolyticus subspecies gallolyticus and colorectal cancer

Abstract Colorectal cancer (CRC) is the 2nd-3rd most common cancer worldwide and is a leading cause of cancer-related death. Increasing evidence suggests that the gut microbiome plays an important role in the development of CRC. One such organism of the gut microbiome that plays an important role in CRC is Streptococcus gallolyticus subspecies gallolyticus (Sgg). Previously, we reported that Sgg stimulates CRC cell proliferation in a β-catenin dependent manner. Using an azoxymethane-induced mouse model of CRC, we have also shown that Sgg-treated mice had significantly higher tumor burden in the colon compared to mice treated with saline or negative control bacteria, suggesting that Sgg actively promotes the development of CRC. The mechanism underlying Sgg’s role in CRC development, however, has yet to be elucidated. The type VII secretion system (T7SS), also called the Esx secretion system, is a specialized secretion system found primarily in Firmicutes and Actinobacteria and has been shown to play an important role in virulence in Staphylococcus aureus and Mycobacterium tuberculosis. Here, we examined the genome of Sgg and found that it encodes a putative T7SS (SggT7SS) highly similar to the T7SS in S. aureus with respect to sequence identity and gene organization. We further showed that the SggT7SS is functional. To investigate if the SggT7SS is involved in the connection between Sgg and CRC, we generated an Sgg mutant lacking the core components of the T7SS secretion machinery. Functional characterization of the mutant indicates that SggT7SS is intimately involved in the interactions between Sgg and CRC cells. Taken together, our results suggest that a T7SS system may be important in inducing specific host responses to Sgg in the context of CRC. Studies to further elucidate the activities of T7SS and its role in Sgg’s contribution to CRC are currently under way. Citation Format: John Culver Taylor, Jacob Rutherford, Maria Nunez, Yi Xu. The involvement of a type VII secretion system in the interactions between Streptococcus gallolyticus subspecies gallolyticus and colorectal cancer [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 A07.

Abstract IA07: The plasticity of the intestinal microbiota in colorectal cancer

Abstract Gene-environment interaction plays a key role in disease susceptibility, including colorectal cancer. Microbiota, particularly the intestinal bacteriome, plays a central role in host physiology, and the composition and activity of this consortium of microorganisms is directly influenced by known cancer risk factors such as lifestyle, diet, and inflammation. Accumulating evidence points to a role of microbiota in carcinogenesis. The mechanism by which microbiota impacts on cancer development is still unclear, but alteration in genomic stability (genetics and epigenetics) is considered a converging point. Inflammation represents a powerful condition by which microbial composition and biologic activities are altered. Using gnotobiotic technology, microbial genetic manipulation, genetically engineered mice (Apcmin/+; Il10-/-), and microbial genomics, we investigated the role of specific bacterial or consortium of bacteria in the development of colitis-associated colorectal cancer. In this lecture, I will provide evidence that specific microbial genotoxic activities originating from various strains such as Escherichia coli and Campylobacter jejuni promote development of CRC. For example, the presence of DNA-damaging toxins such as colibactin from adherent invasive Escherichia coli or cytolethal distending toxins (cdt) from Campylobacter jejuni is critical for development of colorectal cancer. I will address the relationship between inflammation and bacteria-induced carcinogenesis by studying the effect of neutralizing TNFa antibody, a biologic used to manage intestinal bowel disease, on microbial community activity. These studies represent the first step toward understanding mechanisms by which microbiota influences development of colorectal cancer, and shed light on how alteration of environmental factors modulates microbial carcinogenic potential. Citation Format: Ye Yang, Raad Gharaibeh, Christian Jobin. The plasticity of the intestinal microbiota in colorectal cancer [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 IA07.

Increased Expression of Caspase Genes in Colorectal Cancer Cell Line by Nisin

Background: Nisin is a potent bacteriocin produced by Lactococcus lactis subsp. lactis and anti bacterial peptide. Colorectal cancer (CRC) is considered as one of the most prevalent and mortal cancers throughout the world. Genetic variations and environmental factors are responsible for initiation and development of cancer. Regulation of the apoptosis process plays an important role in the development of crc. bax, bcl2, caspase 3, 6, 8 and 9 genes are involved in induction of apoptosis. Objectives: The present study describes cytotoxic effects of nisin and bax, bcl2, caspase 3, 6, 8 and 9 gene expression levels in SW480 cells incubated with nisin at different doses and different incubation times. Methods: The SW480 cells were treated with concentrations of nisin at different doses and different incubation times. The cytotoxic effects of nisin were studied using MTT assay. Expression levels of bax, bcl2, caspase 3, 6, 8 and 9 genes were determined by real-time PCR technique. Results: Our results showed that treatment with 800,1000,1200 and 1500 μg/mL nisin after 24 hour incubation at 37°C significantly reduced cell viability (P 0.05). Conclusions: Our results confirm that nisin induces mitochondrial dependent apoptosis by up regulation of caspase genes expressions. The results of this study contribute to the use of nisin as a suggested therapeutic factor to reduce the progression of colon cancer.

Fecal Metabolomic Signatures in Colorectal Adenoma Patients Are Associated with Gut Microbiota and Early Events of Colorectal Cancer Pathogenesis.

Colorectal adenomas are precancerous lesions of colorectal cancer (CRC) that offer a means of viewing the events key to early CRC development. A number of studies have investigated the changes and roles of gut microbiota in adenoma and carcinoma development, highlighting its impact on carcinogenesis. However, there has been less of a focus on the gut metabolome, which mediates interactions between the host and gut microbes. Here, we investigated metabolomic profiles of stool samples from patients with advanced adenoma (n = 102), matched controls (n = 102), and patients with CRC (n = 36). We found that several classes of bioactive lipids, including polyunsaturated fatty acids, secondary bile acids, and sphingolipids, were elevated in the adenoma patients compared to the controls. Most such metabolites showed directionally consistent changes in the CRC patients, suggesting that those changes may represent early events of carcinogenesis. We also examined gut microbiome-metabolome associations using gut microbiota profiles in these patients. We found remarkably strong overall associations between the microbiome and metabolome data and catalogued a list of robustly correlated pairs of bacterial taxa and metabolomic features which included signatures of adenoma. Our findings highlight the importance of gut metabolites, and potentially their interplay with gut microbes, in the early events of CRC pathogenesis.IMPORTANCE Colorectal adenomas are precursors of CRC. Recently, the gut microbiota, i.e., the collection of microbes residing in our gut, has been recognized as a key player in CRC development. There have been a number of gut microbiota profiling studies for colorectal adenoma and CRC; however, fewer studies have considered the gut metabolome, which serves as the chemical interface between the host and gut microbiota. Here, we conducted a gut metabolome profiling study of colorectal adenoma and CRC and analyzed the metabolomic profiles together with paired microbiota composition profiles. We found several chemical signatures of colorectal adenoma that were associated with some gut microbes and potentially indicative of future CRC. This study highlights potential early-driver metabolites in CRC pathogenesis and guides further targeted experiments and thus provides an important stepping stone toward developing better CRC prevention strategies.

Downregulation of Siah1 promotes colorectal cancer cell proliferation and migration by regulating AKT and YAP ubiquitylation and proteasome degradation

BackgroundColorectal cancer (CRC) is one of the most common malignant tumors in the world. Siah E3 ubiquitin protein ligase 1 (Siah1) has been identified as a tumor suppressor gene and plays an important role in the development of malignant tumors. However, the potential role and molecular mechanism of Siah1 in the development and progression of CRC is still unclear.MethodsTo explore the role and molecular mechanism of Siah1 in the development and progression of CRC, we examined the expression of Siah1 in CRC tissue samples and analyzed its association with progression and prognosis in CRC. In addition, overexpression and knockdown of Siah1 was used to investigate its activity in CRC cells. We also use bioinformatics to analyze and verify the significant roles of Siah1 in critical signaling pathways of CRC.ResultsWe found that the expression of Siah1 was significantly downregulated in CRC tissues, and low expression of Siah1 was associated with aggressive TNM staging and poor survival of CRC patients. Moreover, we revealed that overexpression of Siah1 in CRC cells markedly inhibited CRC cell proliferation and invasion in vitro and in vivo, while knockdown of Siah1 enhanced CRC cell proliferation and invasion. Furthermore, we found that Siah1 prohibited cell proliferation and invasion in CRC partially through promoting AKT (the serine-threonine protein kinase) and YAP (yes associated protein) ubiquitylation and proteasome degradation to regulate the activity of MAPK(mitogen-activated protein kinase 1), PI3K-AKT (phosphatidylinositol 3-kinase-the serine-threonine protein kinase) and Hippo signaling pathways.ConclusionsThese findings suggested that Siah1 is a novel potential prognostic biomarker and plays a tumor suppressor role in the development and progression of CRC.

CircPRMT5 circular RNA promotes proliferation of colorectal cancer through sponging miR-377 to induce E2F3 expression.

CircPRTM5 is associated with cell proliferation and migration in many kinds of malignancies. However, the functions and mechanisms of CircPRTM5 in CRC progression remain unclear. We explored the role and the mechanisms of CircPRTM5 in the development of CRC. Tissues of CRC patients and matched adjacent non‐tumour tissues were collected to evaluate the expression of CircPRTM5. The expression of CircPRTM5 in CRC tissues was significantly higher than that in adjacent tissues. The biological functions of CircPRTM5 in CRC were determined by overexpression and down‐regulation of CircPRTM5 in CRC cells in vitro and in vivo. The results indicate that knockdown of CircPRTM5 can significantly inhibit the proliferation of CRC cells. The potential mechanisms of CircPRTM5 in CRC development were identified by RT‐qPCR, Western blotting analysis and luciferase reporter assay. CircPRTM5 competitively regulates the expression of E2F3 by capillary adsorption of miR‐377. CircPRMT5 regulates CRC proliferation by regulating the expression of E2F3, which affects the expression of the cell cycle‐associated proteins cyclinD1 and CDK2. CircPRTM5 exerts critical regulatory role in CRC progression by sponging miR‐377 to induce E2F3 expression.

Preventive effects of gender and metabolic syndrome in 40s on colorectal cancer by colonoscopy.

59 Background: This study was aimed at measuring the preventive effect of colonoscopy for CRC development depending on age of index colonoscopy, gender and metabolic syndrome among persons aged 40 to 59 years. Methods: Between January 2005 and December 2006, data for the population aged from 40 to 59 who underwent colonoscopy (CSP cohort) claimed were collected from National Health Insurance Service (NHIS). Non-CSP (N-CSP) subjects were also collected by 1:5 propensity score matching with parameters of age, sex, and metabolic profiles, smoking, alcohol and past history of cancer. After one year of washout period, the risk of developing CRC was estimated by the occurrence of new case from January 2009 to December 2014 using the link of National CRC Registry to NHIS database. Compared with N-CSP cohort, hazard ratios (HRs) were obtained via conditional logistic regression analysis to estimate the risk of CRC in CSP cohort by age groups. Results: A total of 2,339,359 subjects were included (CSP cohort: 395,738 and matched N-CSP cohort: 1,943,621). The HRs for developing CRC by ages of 40-44, 45-49, 50-54, and 55-59 years in a CSP cohort were 0.864 ( P = NS), 0.591 ( P < 0.001), 0.599 ( P < 0.001), and 0.524 ( P < 0.001) in men, and 0.774 ( P = NS), 0.841 ( P = NS), 0.598 ( P < 0.001), and 0.605 ( P < 0.001) in women, respectively. Interestingly, when confined to patients with metabolic syndrome in their 40s, HRs for CRC in the colonoscopy cohort were statistically significantly lowered to 0.372 in early 40’s and 0.386 in men of late 40s, respectively, but not in women of 40s. Conclusions: The CRC prevention effect of colonoscopy is expected from late 40s in men and early 50s in women. Furthermore, in cases with metabolic syndrome, the preventive effect of colonoscopy is expected in men of early forties.

Contribution of ABCG2 gene polymorphisms (G34A and C376T) in the prognosis of colorectal cancer

This study aims to assess the association of two single nucleotide polymorphisms (SNPs), G34A and C376T, in the ABCG2 gene with the risk of developing CRC. To the best of our knowledge, this is the first study that determined the role of genetic variations in the ABCG2 gene with the risk of CRC in Saudi Arabia. The gDNA was extracted from the blood of 58 CRC patients and 48 healthy subjects. The DNA sequencing was used to determine the distribution of genotypes. The results showed that CRC patients carried a heterozygous (GA) genotype for SNP G34A had a low risk of developing CRC (odds ratio=0.015, 95% CI [0.00–0.12]; risk ratio=0.35, 95% CI [0.25–0.12], P <0.0001). On the other hand, patients that carried a heterozygous (CT) genotype for SNP C376T had a high risk of developing CRC (odds ratio=13.83, 95% CI [4.31–44.38]; risk ratio=4.88, 95% CI [1.95–12.24], P <0.0001). In conclusion, the results indicated that a heterozygous (GA) genotype in SNP G34A may decrease the risk of CRC development, whereas, the heterozygous (CT) genotype in SNP C376T may increase the risk of CRC. The results may suggest a protective role of ABCG2 SNP G34A against CRC and a deleterious effect of ABCG2 SNP C376T for increasing the risk of CRC.

TSPAN8 promotes colorectal cancer cell growth and migration in LSD1-dependent manner

AimsColorectal cancer (CRC) is the fourth leading cause of cancer-related mortality worldwide. Over-expression of tetraspanin 8 (TSPAN8) is related to the development and progression of CRC. Whether TSPAN8 plays a role in the growth of colorectal cancer and its epigenetic mechanisms regulated by Lysine Specific Demethylase 1 (LSD1) are still unknown. Main methodsIn this study, RT-PCR and western blotting were used to analyze the mRNA and protein expression, respectively; cell viability was assayed with MTS analysis; cell migration was measured with Trans-well analysis. Key findingsIn the present study, the results indicated that the mRNA levels of LSD1 and TSPAN8 in CRC were significantly higher than that in corresponding adjacent non-tumor tissue. Down-regulation of LSD1 or TSPAN8 as well as LSD1 inhibitor Tranylcypromine hemisulfate inhibited the proliferation and migration of CRC cells, while over-expression of LSD1 exhibited opposite effects. LSD1 up-regulated TSPAN8 expression and reduced H3K9me2 occupancy on the TSPAN8 promoter in CRC cells. TSPAN8 promoted epithelial-mesenchymal transition (EMT) in CRC cells in LSD1-dependent manner. SignificanceTSPAN8 may be considered as a promising biomarker for the diagnosis and prognosis in patients with CRC. Furthermore, TSPAN8 could be a novel therapeutic target and potent LSD1 inhibitors could be designed and developed in the treatment of CRC.

ILF3 is a substrate of SPOP for regulating serine biosynthesis in colorectal cancer

The Serine–Glycine–One-Carbon (SGOC) pathway is pivotal in multiple anabolic processes. Expression levels of SGOC genes are deregulated under tumorigenic conditions, suggesting participation of oncogenes in deregulating the SGOC biosynthetic pathway. However, the underlying mechanism remains elusive. Here, we identified that Interleukin enhancer-binding factor 3 (ILF3) is overexpressed in primary CRC patient specimens and correlates with poor prognosis. ILF3 is critical in regulating the SGOC pathway by directly regulating the mRNA stability of SGOC genes, thereby increasing SGOC genes expression and facilitating tumor growth. Mechanistic studies showed that the EGF–MEK–ERK pathway mediates ILF3 phosphorylation, which hinders E3 ligase speckle-type POZ protein (SPOP)-mediated poly-ubiquitination and degradation of ILF3. Significantly, combination of SGOC inhibitor and the anti-EGFR monoclonal antibody cetuximab can hinder the growth of patient-derived xenografts that sustain high ERK-ILF3 levels. Taken together, deregulation of ILF3 via the EGF–ERK signaling plays an important role in systemic serine metabolic reprogramming and confers a predilection toward CRC development. Our findings indicate that clinical evaluation of SGOC inhibitor is warranted for CRC patients with ILF3 overexpression.

Plasma circular RNA panel acts as a novel diagnostic biomarker for colorectal cancer

BackgroundColorectal cancer (CRC) is one of the most common cancers worldwide, and emerging lines of evidence have implicated circular RNAs (circRNAs), a novel class of endogenous noncoding RNAs, in CRC development. However, whether plasma circRNAs might be novel diagnostic biomarkers for CRC remains unclear. MethodsWe investigated the plasma levels of selected circRNAs by quantitative real-time PCR (qRT-PCR). The presence of the candidate circRNAs was confirmed through RNase R assays, qRT-PCR and DNA sequencing, and their diagnostic value was evaluated using a receiver operating characteristic (ROC) curve. ResultsThe plasma levels of three circRNAs (circ-CCDC66, circ-ABCC1 and circ-STIL) were significantly decreased in CRC patients (n = 45) compared with healthy controls (n = 61). The ROC curve analysis showed that the area under the ROC curve (AUC) of the three-circRNA panel was 0.780, which is higher than that of traditional protein biomarkers, such as carcinoembryonic antigen (CEA) and carbohydrate antigen 19–9 (CA19-9). Combining the circRNA panel with CEA and CA19-9 might improve the ability to diagnose CRC (AUC = 0.855). In addition, the plasma circ-ABCC1 level was related to tumor growth and progression, and the plasma circ-CCDC66 and circ-ABCC1 levels were decreased in precursor lesions of CRC, including colon adenomas and adenomatous polyps. More importantly, circ-CCDC66 and circ-STIL were found to be useful for diagnosing early-stage CRC, and the three-circRNA panel improved the ability to diagnose CEA-negative and CA19-9-negative CRC. ConclusionOur study provides the first identification of a panel of three plasma circRNAs that could serve as a novel and independent diagnostic biomarker for CRC.

2611. Enterotoxigenic Bacteroides fragilis Alters the Genome of Colon Epithelial Cells

BackgroundIndividuals born in 1990 have twice the risk of developing colon cancer and four times the risk of developing rectal cancer as those born in 1950. The gut microbiome is being proposed as a potential contributor to this difference because of the surge in obesity in the United States, the link between obesity and gut dysbiosis, and the growing number of studies which have associated a dysbiotic gut microbiome with CRC. Enterotoxigenic Bacteroides fragilis (ETBF) is one of the bacteria most studied in relation to CRC development; it is found at a higher frequency in both the stool and mucosa of CRC patients, and it rapidly induces tumor formation in an Apcmin/+ mouse model of CRC. In this model, tumor formation typically occurs via loss of heterozygosity (LOH) of the Apc gene, the genetic mutation found in approximately 80% of sporadic CRC cases. ETBF produces a potent exotoxin (BFT) which induces E-cadherin cleavage, β-catenin nuclear localization and colonic epithelial cell proliferation. But we still do not understand how these downstream effects cause lasting changes in the genome of colon epithelial cells that then initiate tumor formation and growth. As cancer is ultimately a disease that arises and progresses via changes in the genome, understanding these interactions is essential.MethodsWe hypothesize that ETBF induces DNA mutations via BFT that encourage tumor formation, and enhance tumor growth. To test this hypothesis, we performed whole-exome sequencing on tumors and normal tissue isolated from Apcmin/+ mice after ETBF or sham inoculation. Additionally, we isolated colon organoids from Apcmin/+ mouse normal tissue (colonoids) and Apcmin/+ mouse tumors (tumoroids) after ETBF or sham inoculation. We performed in vitro DNA damage assays and qPCR for Apc LOH on these colon organoids.ResultsOur preliminary data indicate that ETBF-induced tumors have lower rates of Apc LOH and that double-stranded DNA breaks are observed as soon as 3-hours after BFT treatment of colonoids and as soon as 72-hours after ETBF inoculation.ConclusionThese data suggest that in vivo, ETBF may induce mutations in cancer-driver genes which cause tumor formation via pathways other than somatic recombination at the Apc locus, a result we are now testing with additional (N = 19) whole-exome tumor sequencing in-progress. Disclosures All authors: No reported disclosures.

1880PD - Genetic variants in the one-carbon metabolism pathway to predict outcome in patients with metastatic colorectal cancer (mCRC): Data from TRIBE and FIRE-3 phase III trials

1880PD - Genetic variants in the one-carbon metabolism pathway to predict outcome in patients with metastatic colorectal cancer (mCRC): Data from TRIBE and FIRE-3 phase III trials

Rectal swab gene expression as a non-invasive biomarker for CRC screening

Abstract BACKGROUND Aberrant gene levels in rectal swab specimens are potential non-invasive biomarkers for the early detection of colorectal cancer. The aim of this study was to investigate the expression level of dysregulated genes in CRC in rectal swab specimens from normal subjects and CRC patients, and evaluate the potential of this approach as a non-invasive biomarker for CRC diagnosis. METHODS We performed RNA extraction and reverse transcription of the rectal swab specimens, then applied quantitative polymerase chain reaction to determine the expression level of 15 genes which have been previously reported to show dysregulation during CRC development. RESULTS Our results showed that CD44, IL8, CXCR2 and c-myc were significantly overexpressed in rectal swab specimens of CRC patients when compared to control subjects in both the training and validation studies, suggesting that gene levels of these four genes were potential biomarker to discriminate CRC from non-CRC subjects. Further evaluation showed that a panel of these 4 genes was able to identify CRC patients with an area under the curve of 0.74 for the training study and 0.79 for the validation study. Finally, we tested the predictive value of the 4-gene signature in a prediction study of 55 subjects (including 25 CRC patients and 30 non-CRC control subjects). The 4-gene signature correctly identified 16 of 20 CRC patients (80% sensitivity) and 29 of 35 non-CRC subjects (82.9% specificity). CONCLUSION Our data demonstrated the promising potential of the gene signature in rectal swab specimens as a noninvasive biomarker for CRC diagnosis.

Upregulated METTL3 promotes metastasis of colorectal Cancer via miR-1246/SPRED2/MAPK signaling pathway

Backgroundm6A modification has been proved to play an important role in many biological processes. METTL3 as the main methyltransferase for methylation process has been found to be upregulated in many cancers, including CRC. Here, we investigate m6A modification and the underlying mechanism of METTL3 in the development of CRC.MethodsThe expression of METTL3 was detected in large clinical patient samples. To evaluate the function of METTL3 in vitro and in vivo, colony formation, CCK-8, cell migration and invasion assays were performed. To find out the downstream target of METTL3, GEO dataset was re-mined. We analyzed expression and metastasis-related miRNA by Pearson correlation, and miR-1246 was selected. Here, to identify the downstream target of miR-1246, Targetscan and miRWalk were used. RIP and luciferase reporter assay further confirmed SPRED2 as the direct target of miR-1246.ResultsWe found that upregulated METTL3 is responsible for abnormal m6A modification in CRC and correlates positively with tumor metastasis. The gain- and loss-of-function indicates that METTL3 promotes cell migration and invasion in vitro and in vivo. Additionally, we confirmed that METTL3 can methylate pri-miR-1246, which further promotes the maturation of pri-miR-1246. By using bioinformatics tools, anti-oncogene SPRED2 was identified as the downstream target of miR-1246, wherein downregulated SPRED2 further reverses the inhibition of the MAPK pathway.ConclusionsThe present study demonstrates that the METTL3/miR-1246/SPRED2 axis plays an important role in tumor metastasis and provides a new m6A modification pattern in CRC development.

The Expression Analysis of Intestinal Cancer Stem Cell Marker Lgr5 in Colorectal Cancer Patients and the Correlation with Histopathological Markers.

Cancer stem cells (CSCs) have frequently been utilized in the cell characterization and identified responsible for tumor development, metastasis, recurrence, and chemoresistance. CSC surface markers function in cancer cell signaling and are indicated as potential biomarkers for cancer diagnosis and prognosis. As well, dysregulation of cancer-related signaling pathways could promote CSC development and progression. Our aim was to evaluate the expression of colorectal CSC markers and their correlation with cancer proliferation and angiogenesis. In this case-control study, total RNA was extracted from a total of 74 colorectal tumors and 74 adjacent normal tissue biopsies. Then, using a quantitative real-time PCR, the relative expression levels of Lgr5 and Lrig1 were measured in all malignant and healthy samples. Also, immunohistochemical (IHC) staining of tumor tissues was performed for Ki-67 (proliferation) and CD34 (angiogenesis) markers, and the immunoexpression staining scores were obtained. The diagnostic value of the genes was evaluated using receiver operating characteristic (ROC) curve. Possible correlation between CSC markers and immunohistochemical markers in CRC was analyzed by Pearson's correlation test and linear regression. The expression level of Lgr5 in tumor samples showed a significant increase compared with normal samples (p < 0.001) with a fold change of 2.54 (± 0.182). However, there was no significant difference in the relative expression of Lrig1 gene in tissue samples of healthy subjects and patients. The analysis of the ROC showed an AUC of 0.92 for Lgr5 and sensitivity 80% and specificity 96%. Further analysis revealed a significant correlation between mRNA levels of Lgr5 and immunoexpression of Ki-67 (r2 = 0.680, p < 0.001). The high expression levels of Lgr5 found in tumor tissues were correlated with histological parameters, indicating a significant role in CRC development and diagnosis.

ETV/PEA3 Transcription Factor Family Correlated with Immune Infiltrates and Clinical Prognosis in Human Colon Cancer

Background: Numerous studies have recently pointed to the influence of the immune microenvironment on CRC development, suggesting that infiltration of different types of immune cells might be a promising source of novel diagnostic and prognostic biomarkers. Methods: The correlation of ETV1, ETV4 and ETV5 with tumor-infiltrating immune cells (TIICs) in colon cancer tumor microenvironments via TIMER (Tumor Immune Estimation Resource) and GEPIA (Gene Expression Profiling Interactive Analysis), we validate colon cancer clinical data via immunohistochemistry (IHC). Furthermore, Oncomine database, R2 platform analysis and Oncomine database and R2 platform analysis were applied in our research to validate CRC clinical prognosis via PEA3 members expression level. Findings: Our findings indicated that the upregulation of PEA3 members, ETV1 and ETV5 is correlated with poor prognosis, meanwhile ETV1 and ETV5 may play a role in promoting CRC development. Furthermore, PEA3 members, especially ETV1 and ETV5 tend to have positive correlation with T cell exhaustion markers, and associate with Colon cancer immune infiltration. Interpretation: These findings reveal that ETV1 and ETV5 is a new potential diagnostic and prognostic marker in CRC and that targeting signalling pathways controlled by ETV5 may be a promising strategy for CRC therapy. Funding Statement: Nature Science Foundation of China (NSFC: 81772558), Clinical Skill and Innovation 3 year program of Shanghai Hospital Development Center (16CR2064B), Shanghai Municipal Commission of Health and Family Planing (201540026), Indiana Clinical and Translational Sciences Institute (CTSI) KL2 Young Investigator Award ( KL2TR001106 and UL1TR001108). This is an open access article under the CC BY-NC-ND license. This is an open access article under the CC BY-NC-ND. license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Declaration of Interests: No potential conflicts of interest are disclosed. Ethics Approval Statement: All the experiments involving in human specimens and animals were in accordance with the ethical code and recommendation issued by Ethics Committee of Human Experimentation and Chinese Animal Community and with the Helsinki Declaration of 1975, as revised in 2008.