CRC Tumorigenesis Research Articles

Colorectal cancer microbiome programs DNA methylation of host cells by affecting methyl donor metabolism

BackgroundColorectal cancer (CRC) arises from complex interactions between host and environment, which include the gut and tissue microbiome. It is hypothesized that epigenetic regulation by gut microbiota is a fundamental interface by which commensal microbes dynamically influence intestinal biology. The aim of this study is to explore the interplay between gut and tissue microbiota and host DNA methylation in CRC.MethodsMetagenomic sequencing of fecal samples was performed on matched CRC patients (n = 18) and healthy controls (n = 18). Additionally, tissue microbiome was profiled with 16S rRNA gene sequencing on tumor (n = 24) and tumor-adjacent normal (n = 24) tissues of CRC patients, while host DNA methylation was assessed through whole-genome bisulfite sequencing (WGBS) in a subset of 13 individuals.ResultsOur analysis revealed substantial alterations in the DNA methylome of CRC tissues compared to adjacent normal tissues. An extensive meta-analysis, incorporating publicly available and in-house data, identified significant shifts in microbial-derived methyl donor-related pathways between tumor and adjacent normal tissues. Of note, we observed a pronounced enrichment of microbial-associated CpGs within the promoter regions of genes in adjacent normal tissues, a phenomenon notably absent in tumor tissues. Furthermore, we established consistent and recurring associations between methylation patterns of tumor-related genes and specific bacterial taxa.ConclusionsThis study emphasizes the pivotal role of the gut microbiota and pathogenic bacteria in dynamically shaping DNA methylation patterns, impacting physiological homeostasis, and contributing to CRC tumorigenesis. These findings provide valuable insights into the intricate host-environment interactions in CRC development and offer potential avenues for therapeutic interventions in this disease.

Abstract 2086: Effect of XPO1 inhibition on colorectal cancer tumorigenesis

Abstract Colorectal Cancer (CRC) is the second leading cause of cancer-related death in the U.S. A subset of individuals faces a notably higher likelihood of developing CRC within their lifetime. Hence, there is a compelling need for innovative chemopreventive treatments aimed at minimizing CRC tumorigenesis. Exportin 1 (XPO1; also referred to as CRM1) plays a pivotal role in transporting proteins from the nucleus to the cytoplasm. The overexpression of XPO1 has been identified across several cancers, including CRC. XPO1 interacts with a diverse spectrum of more than 1000 proteins. The overexpression of XPO1 leads to the excessive removal of tumor suppressors from the nucleus, rendering these proteins functionally inert. To counteract the overexpression of XPO1 in tumors, a novel class of drugs known as Selective Inhibitors of Nuclear Export (SINE) has been developed. Among these, Eltanexor (KPT-8602) has emerged as a promising therapeutic agent, demonstrating fewer side effects compared to its precursors, and is currently under evaluation in Phase 1/2 clinical trials. The treatment effects of SINE compounds on CRC remain largely unknown. We have assessed Eltanexor's ability to limit CRC tumorigenesis using a genetic CRC model in Apcmin/+ mice. Our findings indicate that Eltanexor substantially reduces tumor burden by approximately 3-fold and induces a significant reduction of tumors exceeding 1mm in size. Furthermore, a xenograft experiment using the human CRC cell line, HCT116, reveals nearly a 3-fold reduction in tumor size upon Eltanexor treatment. In both CRC models, Eltanexor exhibits noteworthy tolerability. Additionally, XPO1 protein levels are consistently diminished in both models, aligning with our in vitro observations. To further ascertain Eltanexor's specificity for CRC tumors versus normal tissue, we conducted drug sensitivity assays using organoids derived from Apcmin/+ tumors and wild-type mice small intestine tissue. We found that Apcmin/+ tumoroids have heightened sensitivity to Eltanexor-treatments, when compared to wild-type organoids. In an endeavor to elucidate Eltanexor's potent in vivo tumorigenesis inhibitory effects, we conducted multiple assays which revealed a reduction in COX-2 RNA and protein expression levels in Eltanexor-treated cells and reduced COX-2 protein in the tumors of Eltanexor-treated Apcmin/+ mice. In CRC, COX-2 is implicated in CRC development and is currently the target of multiple CRC chemopreventive agents. Our observations reveal that Eltanexor-treated CRC cells exhibit reduced COX-2 gene expression through both inhibiting COX-2’s promoter and its mRNA post-transcriptional stability. Collectively, our findings underscore XPO1 as a potent target for inhibiting CRC tumorigenesis. Future studies will aim to further assess how Eltanexor is impacting COX-2’s promoter, while additionally testing Eltanexor’s tumorigenesis-inhibiting potential in a colitis-induced CRC mouse model. Citation Format: Andrew E. Evans, Dan A. Dixon. Effect of XPO1 inhibition on colorectal cancer tumorigenesis [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 2086.

Abstract 6930: Spatial analysis of multi-ancestral tumors to understand how intercellular signaling impacts tumor invasion

Abstract Colorectal cancer (CRC) persists as a leading cause of cancer-related deaths in the United States. Intratumoral heterogeneity stems from a multi-ancestral origin or clonal divergence and contributes to treatment resistance. Unfortunately, tumors in existing mouse models are largely homogenous, rendering predictions of drug efficacy inaccurate. To investigate whether signaling between distinct neoplastic clones in a tumor and cells in the surrounding microenvironment impacts CRC biology, we performed spatial transcriptomic analysis on samples collected from a transgenic mouse model previously established in the Halberg laboratory. This model (referred to as B6.FCTG3KM) develops three types of tumors: homotypic Adenomatous Polyposis Coli (APC)-deficient tumors that express red fluorescent protein (RFP), homotypic APC-deficient tumors that express constitutively active phosphoinositide 3-kinase (PI3K) and green fluorescent protein (GFP), and heterotypic tumors. Previous characterization of the model clearly demonstrated that heterotypic tumors are significantly more aggressive than either type of homotypic tumor. Analysis was conducted on four serially sectioned slides containing three tumors and adjacent normal tissue obtained from the colon of a male B6.FCTG3KM mouse using the 10x Genomics Visium CytAssist platform with the mouse whole transcriptome probe set. Collected tumors were staged using traditional histopathology techniques by a board-certified pathologist (Matkowskyj). Immunohistochemistry and immunofluorescence were used to assess β-catenin localization and to re-probe for RFP and GFP expression. Sequencing was performed on the Illumina NovaSeqX platform to an average depth of 255,528,294 reads per sample. Data analysis was performed using the 10x Genomics SpaceRanger and Kendziorski laboratory R/SpatialView packages. A fold-change threshold of 1.5 and an adjusted p-value threshold of 0.05 were used when establishing marker genes for each of the 8 clusters identified. One distinct cluster was identified by marker genes including Notum, Wif1, and Dkk2, factors previously implicated in CRC tumorigenesis, and corresponded to the center of each region of surface-level dysplastic epithelia. The remainder of the surface-level dysplastic epithelia corresponded to a second cluster, with marker genes including those coding for TRIM family proteins (Trim5, Trim10, Trim40, Trim12c), and matrix metalloproteinases (Mmp12, Mmp9, Mmp10, Mmp13). A third cluster corresponded to the desmoplastic stroma underlying each neoplasm. Ongoing analyses are underway to identify clonal interactions that alter gene expression and tumor aggressiveness. Citation Format: Mary M. Stangis, Lingxin Cheng, Dawn Albrecht, Santina Snow, Tessa Wirtz, Kristina A. Matkowskyj, Huy Q. Dinh, Christina Kendziorski, Richard B. Halberg. Spatial analysis of multi-ancestral tumors to understand how intercellular signaling impacts tumor invasion [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 6930.

Phosphorylation at tyrosine 317 and 508 are crucial for PIK3CA/p110α to promote CRC tumorigenesis

BackgroundPI3K/AKT signaling pathway plays important role in tumorigenesis of human cancer. Protein phosphorylation is crucial for signaling transduction of this pathway. PIK3CA, encoding the catalytic subunit p110α of PI3K complex, is one of the most frequently mutated oncogenes in human cancers. However, phosphorylation sites of PIK3CA/p110α and their underlying mechanism in tumorigenesis are largely unknown.MethodsTyrosine phosphorylation sites of PIK3CA/p110α are identified with Mass-Spectrum. Crispr/CAS9 strategy is applied to generate Y317F and Y508F mutant knock-in cell clones. The growth and metastasis abilities of cells are evaluated in vitro and in vivo. Phospho-proteomics analysis and Western blots are used to demonstrate downstream signaling pathways of PIK3CA/p110α tyrosine phosphorylation. In vitro kinase assay is applied to identify the kinase of PIK3CA/p110α tyrosine phosphorylation.ResultsTyrosine phosphorylation of PIK3CA/p110α is stimulated by growth factors such as EGF, HGF and PDGF. Two tyrosine residues, Y317 and Y508, are identified on PIK3CA/p110α. Either Y317 or Y508 phosphorylation is essential for tumorigenesis of CRC. Mutation at Y317 of p110α reduces the proliferation, migration, and invasion of cancer cells through Src-MLC2 pathway, while mutation at Y508 of p110α impairs AKT signaling. Moreover, Src interacts with and phosphorylates p110α.ConclusionsPIK3CA/p110α phosphorylation at Y317 and Y508 play important role in tumorigenesis of colorectal cancer through two independent pathways.

Abstract 3803: The landscape of circular RNAs in metastatic colorectal cancer

Abstract Colorectal cancer (CRC) is the most common gastrointestinal malignancy and a leading cause of cancer death in the U.S.. Approximately 50% of patients with CRC develop metastatic disease (mCRC) with an average 5-year survival rate of 13%. Circular RNAs (circRNAs), covalently closed RNA molecules produced from pre-mRNAs through backsplicing, have recently emerged as an important class of non-coding RNAs regulating CRC tumorigenesis and progression. In this study we established a comprehensive catalog of circRNAs in mCRC. To evaluate circRNAs in mCRC progression, total RNA-Seq was performed on 30 matched normal, primary, and distant metastatic samples from 14 mCRC patients. Additionally, the transcriptome of five CRC cell lines were sequenced to evaluate circRNAs expressed in cell lines. CircRNAs were identified and quantified using the number of backspliced reads, normalized against the total number of mapped reads. Differential expression (DE) analysis was performed to identify circRNAs deregulated between normal, primary, and metastatic tissues. Our analysis identified approximately 62,000 expressed circRNAs in mCRC including approximately 32,000 expressed in patients and 48,000 expressed in cell lines, originating from approximately 9200 linear genes. Approximately 42,000 circRNAs (67%) were unannotated when compared with the circRNA consortium MiOncoCirc annotation. DE analysis identified 376 circRNAs deregulated in CRC progression from normal to primary, and metastasis. A higher proportion of DE circRNAs (82%) was downregulated in primary or metastasis, consistent with earlier reports of lower circRNA concentration in tumor due to rapid cell cycles. Among DE circRNAs were the downregulation of circRNAs that have been known to suppress tumor proliferation, migration, and invasion in mCRC such as circSMARCA5, circFBXW7, and circSETD3, and the upregulation of circFIRRE, a circRNA known to promote tumor progression in osteosarcoma. A significant portion (37%) of deregulated circRNAs also had their parental linear genes found to be deregulated, suggesting parental gene regulation contributed to circRNA regulation. Interestingly, a large portion of circRNAs (63%) were found to be deregulated while their parental linear gene expression was unchanged, suggesting alternative mechanisms of circRNA deregulation. Gene set enrichment analysis showed that linear genes co-deregulated with circRNAs were enriched in signatures of tumor progression including proliferation, epithelial mesenchymal transition, and metastasis, suggesting potential involvement of deregulated circRNAs in tumor progression. Through RNA sequencing of both patients and cell lines, we established a comprehensive compendium of circRNAs and identified those deregulated in mCRC. This provides a valuable resource for further experimental characterization of circRNAs function in mCRC progression. Citation Format: Sidi Zhao, Amy Ly, Jacqueline L. Mudd, Emily B. Rozycki, Ghofran Othoum, Jingqin Luo, Ha X. Dang, Ryan C. Fields, Christopher A. Maher. The landscape of circular RNAs in metastatic colorectal cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3803.

Single-cell genomic and transcriptomic landscapes of primary and metastatic colorectal cancer tumors

BackgroundColorectal cancer (CRC) ranks as the second-leading cause of cancer-related death worldwide with metastases being the main cause of cancer-related death. Here, we investigated the genomic and transcriptomic alterations in matching adjacent normal tissues, primary tumors, and metastatic tumors of CRC patients.MethodsWe performed whole genome sequencing (WGS), multi-region whole exome sequencing (WES), simultaneous single-cell RNA-Seq, and single-cell targeted cDNA Sanger sequencing on matching adjacent normal tissues, primary tumors, and metastatic tumors from 12 metastatic colorectal cancer patients (n=84 for genomes, n=81 for exomes, n=9120 for single cells). Patient-derived tumor organoids were used to estimate the anti-tumor effects of a PPAR inhibitor, and self-renewal and differentiation ability of stem cell-like tumor cells.ResultsWe found that the PPAR signaling pathway was prevalently and aberrantly activated in CRC tumors. Blocking of PPAR pathway both suppressed the growth and promoted the apoptosis of CRC organoids in vitro, indicating that aberrant activation of the PPAR signaling pathway plays a critical role in CRC tumorigenesis. Using matched samples from the same patient, distinct origins of the metastasized tumors between lymph node and liver were revealed, which was further verified by both copy number variation and mitochondrial mutation profiles at single-cell resolution. By combining single-cell RNA-Seq and single-cell point mutation identification by targeted cDNA Sanger sequencing, we revealed important phenotypic differences between cancer cells with and without critical point mutations (KRAS and TP53) in the same patient in vivo at single-cell resolution.ConclusionsOur data provides deep insights into how driver mutations interfere with the transcriptomic state of cancer cells in vivo at a single-cell resolution. Our findings offer novel knowledge on metastatic mechanisms as well as potential markers and therapeutic targets for CRC diagnosis and therapy. The high-precision single-cell RNA-seq dataset of matched adjacent normal tissues, primary tumors, and metastases from CRCs may serve as a rich resource for further studies.

The clinicopathological and prognostic significances of IGF-1R and Livin expression in patients with colorectal cancer

BackgroundColorectal cancer (CRC) is the third most common cancer worldwide. However, limited effective biomarkers are associated with the tumorigenesis and prognosis of CRC.MethodsThe present study identified potential signatures from The Cancer Genome Atlas (TCGA) database and further validated the identified biomarkers in CRC tissues by immunohistochemistry (IHC).ResultsThe expression of insulin-like growth factor 1 receptor (IGF-1R) and Livin gene was significantly upregulated in CRC samples compared to the adjacent normal samples in the TCGA dataset. IHC indicated that IGF-1R and Livin protein levels are increased in CRC and adenoma tissues compared to normal tissues. Notably, the IGF-1R protein levels differed significantly between adenoma and CRC. The elevated IGF-1R and Livin expression was associated with CRC clinicopathological features, including age, gender, histological subtype, individual cancer stages, nodal metastasis, and TP53-mutant in TCGA. Additionally, the IGF-1R promoter methylation level was closely related to CRC. Consistent with the TCGA study, IHC indicated that overexpressed IGF-1R and Livin proteins were independent risk factors for stage and metastasis. A marked correlation was established between IGF-1R and Livin expression in CRC, while the survival map showed no significant correlation with CRC. Kaplan–Meier survival curves showed that CRC patients with high IGF-1R or Livin expression had a prolonged overall disease-free survival than those with low expression in TCGA.ConclusionIGF-1R and Livin are associated with CRC tumorigenesis and might be valuable for novel biomarker identification and targeted therapeutic strategy development.

Abstract 940: Loss of Pkd1 limits colorectal cancer by increasing colon barrier function

Abstract Colorectal cancer (CRC) is one of the most common cancers, with an annual incidence of ~135,000 in the US, associated with ~ 50,000 deaths. CRC typically arises from mutations that inactivate the tumor suppressor APC, which activates the WNT/CTNNB1 pathway to drive cell transformation and tumor growth. Identifying factors that reduce incidence of CRC is of high interest. Intriguingly, a large population study has found a decreased incidence of CRC in patients with autosomal dominant polycystic kidney disease (ADPKD), an inherited disease affecting approximately 1 in 1,000 individuals. ADPKD typically arises from germline mutations in the PKD1 gene, which encodes a transmembrane signaling protein that is ubiquitously expressed. Puzzlingly, activated WNT/CTNNB1 signaling is also found in ADPKD, which would be expected to enhance rather than reduce likelihood of CRC. To investigate the role of PKD1 in modulating CRC tumorigenesis, we established a mouse model with tamoxifen-inducible loss of Apc with or without parallel loss of Pkd1 in the colon. Pkd1 loss vey significantly reduced tumor incidence (~80%) and rate of growth. In addition, growth of Pkd1-/-;Apc-/- organoids was reduced relative to Apc-/-organoids, indicating a cancer cell-intrinsic activity. Analysis of signaling pathway activation by RNA-seq indicated that Pkd1-deficient CRC organoids had enhanced WNT/CTNNB1 signaling, excluding reduced activation of this core driver pathway as a mechanism of action. Considering alternative mechanisms, a prominent feature of ADPKD is reprogramming barrier function in renal cells to reduce permeability, supporting cyst development; notably, compromised integrity of the epithelial barrier is associated with CRC. We tested the hypothesis that loss of Pkd1 increased colon barrier integrity. As a first approach, we compared susceptibility of wt versus Pkd1-/- mice to dextran-sodium sulfate (DSS), which induces colitis by damaging epithelial barriers. Histopathological assessment confirmed significantly less DSS-induced damage of colon tissue in Pkd1-/- mice. Further, treatment of DSS-treated mice with orally gavaged FITC-dextran led to elevated FITC-dextran in the serum of wt versus Pkd1-/- mice, indicating greater passage through a functionally impaired barrier. In ADPKD, decreased barrier permeability is mediated by upregulation of specific claudins (CLDN4, 7); in CRC, these claudins are typically reduced during and promote tumor formation. We detected strong elevation of CLDN4 and CLDN7 in the colonic epithelium of Pkd1-/- versus wt mice, with the expressed proteins having consistently greater localization to cell junctions. Based on these and other data, we identify PKD1 control of barrier function as an important regulatory mechanism in CRC tumorigenesis, which may suggest novel therapeutic strategies based on targeting PKD1-associated effector pathways. Citation Format: Anna S. Nikonova, Alexander Y. Deneka, Flaviane Silva, Rosella Tricarico, Anna Kiseleva, Sergey Grivennikov, Erica A. Golemis. Loss of Pkd1 limits colorectal cancer by increasing colon barrier function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 940.

Ferroptosis-associated molecular classification characterized by distinct tumor microenvironment profiles in colorectal cancer

Ferroptosis is a non-apoptotic form of cell death recognized in recent years. Nonetheless, the potential role of ferroptosis-associated genes in immune regulation and tumor microenvironment formation remains unknown. In this study, we characterized the ferroptosis-associated patterns of colorectal cancer through integrative analyses of multiple datasets with transcriptomics, genomics, and single-cell transcriptome profiling. Three distinct ferroptosis-associated clusters (FAC1, FAC2 and FAC3) were identified from 1251 CRC bulk samples, which were associated with different clinical outcomes and biological pathways. The TME characterization revealed that the three patterns were highly consistent with known immune profiles: immune-desert (FAC1), immune-inflamed (FAC2) and immune-excluded (FAC3), respectively. Ferroptosis-associated immune and stromal-activated genes were obtained and characterized by corresponding function in CRC tumorigenesis. Further single-cell analyses identified the ferroptosis-associated immune responding tumor cells and ferroptosis-associated stromal cells infiltration pattern. Based on the Fersig score, which was extracted from the ferroptosis phenotype-related signature, patients with lower Fersig score were characterized by prolonged survival time and effective immune responses. Collectively, we uncovered the ferroptosis-associated patterns associated with TME diversity and immune response phenotype. The Fersig we constructed could be the potential therapeutic target genes to improve the efficacy of patients' immunotherapy. The Fersig scoring scheme could enhance the understanding of TME infiltration associated with ferroptosis and prediction of immunotherapy efficacy.

A Chemerin Peptide Analog Stimulates Tumor Growth in Two Xenograft Mouse Models of Human Colorectal Carcinoma.

Simple SummaryThe chemoattractant adipokine chemerin has been found to be elevated in several types of cancer, including colorectal carcinoma. The functional role of chemerin in colorectal carcinoma, however, has not been elucidated to date. This study analyses the impact of the chemerin analog CG34 on proliferation, colony formation, and migration in the human colorectal cancer cell lines HCT116, HT29 and SW620. In addition, the effect of systemic CG34 treatment is investigated in two xenograft mouse models of colorectal cancer (HCT116-luc and HT29-luc). The results of this study suggest there is a stimulatory role of chemerin receptor activation on the growth of colorectal carcinoma.Background: Chemerin plasma concentration has been reported to be positively correlated with the risk of colorectal cancer. However, the potential regulation of CRC tumorigenesis and progression has not yet been investigated in an experimental setting. This study addresses this hypothesis by investigating proliferation, colony formation, and migration of CRC cell lines in vitro as well as in animal models. Methods: In vitro, microscopic assays to study proliferation, as well as a scratch-wound assay for migration monitoring, were applied using the human CRC cell lines HCT116, HT29, and SW620 under the influence of the chemerin analog CG34. The animal study investigated HCT116-luc and HT29-luc subcutaneous tumor size and bioluminescence during treatment with CG34 versus control, followed by an ex-vivo analysis of vessel density and mitotic activity. Results: While the proliferation of the three CRC cell lines in monolayers was not clearly stimulated by CG34, the chemerin analog promoted colony formation in three-dimensional aggregates. An effect on cell migration was not observed. In the treatment study, CG34 significantly stimulated both growth and bioluminescence signals of HCT116-luc and HT29-luc xenografts. Conclusions: The results of this study represent the first indication of a tumor growth-stimulating effect of chemerin signaling in CRC.

Metadherin (MTDH) overexpression significantly correlates with advanced tumor grade and stages among colorectal cancer patients.

Colorectal cancer is the 4th leading cause of cancer related deaths affecting both men and women worldwide. In the present study, any probable role of MTDH mRNA expression in CRC tumorigenesis was explored using both discovery and validation cohorts. After prior ethical and biosafety approvals, tumor tissue samples along with their adjacent controls were collected for this study from Pakistani patients diagnosed with colorectal cancer. RNA was isolated using Trizol reagent, followed by cDNA synthesis. Transcript analysis of MTDH was performed by using qPCR. Moreover, genome-wide expression of MTDH was also determined through micro-array data analysis using BRB-array tools software. MTDH expression was significantly high in tumor tissue samples (p < 0.05) compared to their respective controls. Likewise, results of microarray analysis also revealed overamplification of MTDH in tumor samples as compared to controls. Expression of MTDH was also found to be positively correlated with KI-67 index (p < 0.05) and were observed to be significantly upregulated in advance tumor grade (p < 0.05) and stage (p < 0.05). However, no association of MTDH overexpression with age and gender could be established. Hence, it can be concluded that MTDH is a core element that plays a pivotal role in colorectal tumorigenesis irrespective of patient's age and gender. Molecular insight into the tumor microenvironment revealed MTDH as a niche, representing distinctive framework for cancer progression, thus, making it an innovative target strategy for colorectal cancer treatment.

Autophagy Upregulates miR-449a Expression to Suppress Progression of Colorectal Cancer.

Many studies reported that microRNAs (miRNAs) target autophagy-related genes to affect carcinogenesis, however, autophagy-deficiency-related miRNA dysfunction in cancer development remains poorly explored. During autophagic progression, we identified miR-449a as the most up-regulated miRNA. MiR-449a expression was low in the tumor parts of CRC patient specimens and inversely correlated with tumor stage and metastasis with the AUC (area under the curve) of 0.899 and 0.736 as well as poor overall survival rate, indicating that miR-449a has the potential to be a prognostic biomarker. In the same group of CRC specimens, low autophagic activity (low Beclin 1 expression and high p62 accumulation) was detected, which was significantly associated with miR-449a expression. Mechanistic studies disclosed that autophagy upregulates miR-449a expression through degradation of the coactivator p300 protein which acetylates the transcription factor Forkhead Box O1 (FoxO1). Unacetylated FoxO1 translocated to the nucleus and bound to the miR-449a promoter to drive gene expression. Either activation of autophagy by the inducer or overexpression of exogenous miR-449a decreases the expression of target gene LEF-1 and cyclin D1, which lead to decreased proliferation, colony formation, migration, and invasion of CRC cells. Autophagy-miR-449a-tartet genes mediated suppression of tumor formation was further confirmed in the xenograft mouse model. In conclusion, this study reveals a novel mechanism wherein autophagy utilizes miR-449a-LEF1-cyclin D1 axis to suppress CRC tumorigenesis. Our findings open a new avenue toward prognosis and treatment of CRC patients by manipulating autophagy-miR-449a axis.

HSPA4 Knockdown Retarded Progression and Development of Colorectal Cancer.

PurposeColorectal cancer (CRC) is a common malignancy associated with high morbidity and mortality. Heat shock 70 kDa protein 4 (HSPA4) has been shown to exert regulatory roles during tumor progression in different cancer types. Here, we investigated the expression and cellular functions of HSPA4 in CRC.Materials and MethodsExpression of HSPA4 in CRC tissues and paracancerous tissues was analyzed by RT-qPCR and immunohistochemistry IHC staining. The functional roles of HSPA4 were explored using shRNA-mediated knockdown in HCT116 and RKO CRC cell lines, both in vitro and in tumor xenograft studies.ResultsHSPA4 expression was significantly increased at the RNA and protein levels in CRC tissues compared with noncancerous tissues. Moreover, HSPA4 expression was positively associated with tumor stage and its high expression of HSPA4 indicated poor patient prognosis. In vitro studies established that HSPA4 knockdown inhibited proliferation and migration, causing arrest in the G2-phase of the cell cycle along with increased levels of apoptosis. This phenotype was recapitulated in vivo where HSPA4 knockdown suppressed xenograft growth. Mechanistic investigations showed silencing of HSPA4 reduced activation of the PI3K, Akt signaling axis while also downregulating the cell cycle progression markers, CCND1 and CDK6. Similarly, there was altered expression of apoptosis-related proteins consistent with the increase in apoptosis.ConclusionOur findings demonstrate clinical significance for HSPA4 in CRC, further showing that HSPA4 contributes to CRC tumorigenesis through effects on proliferation, migration and survival. Thus, HSPA4 represents a novel prognostic indicator as well as a promising therapeutic target in CRC.

Correlation of LINE-1 Hypomethylation With Size and Pathologic Extent of Dysplasia in Colorectal Tubular Adenomas.

INTRODUCTION:Conventional adenomas (tubular adenoma [TA] or tubulovillous adenoma) and sessile serrated lesions (SSLs) are neoplastic precancerous lesions frequently detected in patients undergoing average risk screening colonoscopy and polyp surveillance. Metachronous risk stratification of adenomas is currently limited to histologic features and size of polyps. We report long interspersed nucleotide element-1 (LINE-1) methylation levels in SSL in comparison to TA and the impact of TA size and presence of high-grade dysplasia (HGD) on LINE-1 methylation.METHODS:LINE-1 methylation was assessed by pyrosequencing of bisulfite-converted DNA. We compared LINE-1 methylation between TA and SSL, among varying sizes of TA, and between TA with HGD and low-grade dysplasia (LGD).RESULTS:LINE-1 methylation declined with increasing polyp size in TA when comparing those <5 mm (72.31 ± 6.11), 5 to <10 mm (67.50 ± 7.00), and ≥10 mm (66.75 ± 11.89). There were lower LINE-1 methylation levels in TA with LGD (n = 119) compared with SSLs (n = 29) (69.11 ± 8.62 vs 81.41 ± 2.43, P < 0.001). TA containing HGD (n = 26) had lower LINE-1 methylation levels than those with LGD (n = 119) (59.86 ± 7.93 vs 69.11 ± 8.62, P < 0.001).DISCUSSION:HGD and increasing size of TA/tubulovillous adenoma were associated with lower LINE-1 methylation. This supports a hypothesis that LINE-1 hypomethylation in TAs indicates advancement along the CRC tumorigenesis pathway. Lower LINE-1 methylation and greater variance of global DNA methylation was seen in TA compared with SSL. LINE-1 methylation in adenomas correlates with polyp size and degree of dysplasia and deserves further study as a predictor of metachronous colorectal cancer risk.

Activation of the clock gene TIMELESS by H3k27 acetylation promotes colorectal cancer tumorigenesis by binding to Myosin-9

BackgroundColorectal cancer (CRC) is a common tumor characterized by its high mortality. However, the underlying molecular mechanisms that drive CRC tumorigenesis are unclear. Clock genes have important roles in tumor development. In the present study, the expression and functions of clock gene TIMELESS (encoding the Timeless protein) in CRC were investigated.MethodsImmunohistochemistry, cell proliferation, migration, invasion, EMT and xenograft tumor experiments were used to prove the function of Timeless in the tumorigenesis of CRC. Immunoprecipitation, mass spectrometry, Immunofluorescence and Chromatin immunoprecipitation (ChIP) were utilized to clarify the mechanism of Timeless in regulating CRC tumorigenesis.ResultsWe found that Timeless was upregulated in CRC tissues compared with corresponding normal tissues and its expression was closely associated with the TNM stages and overall survival of CRC patients. Functional studies demonstrated that Timeless promoted the proliferation, invasion, and EMT of CRC cells in vitro and in vivo. Mechanistic investigations showed that Timeless activated the β-catenin signal pathway by binding to Myosin-9, which binds to β-catenin to induce its nuclear translocation. The upregulation of Timeless was attributed to CREB-binding protein (CBP)/p300-mediated H3K27 acetylation of the promoter region of Timeless.ConclusionTimeless regulates the tumorigenesis of CRC by binding to and regulating myosin-9, suggesting Timeless might be a potential prognostic biomarker and therapeutic target for CRC.

Circ_0082182 promotes oncogenesis and metastasis of colorectal cancer in vitro and in vivo by sponging miR-411 and miR-1205 to activate the Wnt/\u03b2-catenin pathway

BackgroundCircular RNAs (circRNAs) are a class of endogenous single-strand RNA transcripts with crucial regulation in human cancers. The objective of this study is to investigate the role of circ_0082182 in CRC and its specific functional mechanism.MethodsThe quantitative real-time polymerase chain reaction (qRT-PCR) was performed to measure the levels of circ_0082182, microRNA-411 (miR-411) and microRNA-1205 (miR-1205). Cell proliferation was detected by Cell counting Kit-8 (CCK-8) and colony formation assays. Flow cytometry was used for determining cell cycle and cell apoptosis. Cell apoptosis was also assessed by caspase3 and caspase9 activities. Cell migration and invasion were examined using scratch assay and transwell assay. The interaction between circ_0082182 and miRNA was validated by the dual-luciferase reporter and biotinylated RNA pull-down assays. Wnt/β-catenin pathway and epithelial-mesenchymal transition (EMT)-associated proteins were quantified by Western blot. Xenograft model was established for the research of circ_0082182 in vivo.ResultsCirc_0082182 was upregulated in CRC and could predict the poor prognosis of CRC patients. Functionally, circ_0082182 promoted CRC cell proliferation, cell cycle progression, and metastasis while inhibited apoptosis. Subsequently, circ_0082182 was shown to act as the sponges of miR-411 and miR-1205. MiR-411 and miR-1205 were identified as tumor inhibitors in CRC. Furthermore, circ_0082182 promoted the CRC progression via sponging miR-411 and miR-1205. Moreover, circ_0082182 facilitated the Wnt/β-catenin pathway and EMT process by targeting miR-411 and miR-1205. In vivo, circ_0082182 accelerated the CRC tumorigenesis and EMT process by activating the Wnt/β-catenin pathway by downregulating the expression of miR-411 or miR-1205.ConclusionThis study showed that circ_0082182 functioned as an oncogene in the developing process of CRC by sponging miR-411 or miR-1205 to activate the Wnt/β-catenin pathway. Circ_0082182 might be a molecular target in the diagnosis and treatment of CRC.

Abstract 5924: A clock gene of TIM promotes colorectal cancer tumorigenesis through binding with myosin-9

Abstract Colorectal cancer (CRC) is a common tumor that is characterized by its high mortality rate. However, the underlying molecular mechanisms that drive CRC tumorigenesis are still not clear. Clock genes have been recognized as playing key roles in tumor development. In our present study, we found the expression of TIM (encoding protein of Timeless), one of the clock genes was up-regulated in CRC tissues comparing to the corresponding normal tissues by the results of IHC and its expression was closely associated with tumor clinical stage and poor overall survival of CRC patients through analysis of clinical CRC patients. Functional studies demonstrated that TIM promoted CRC cell proliferation, migration and invasion in vitro and in vivo. Mechanistic investigations showed that TIM plays a key role in EMT. Immunoprecipitation followed by mass spectrometry revealed that Timeless bound with Myosin-9. Furthermore, we also found TIM regulated the expression level of Myosin-9. And Myosin-9 knockdown retarded the migration and invasion of TIM-overexpressed CRC cells. Furthermore, we found the expression of TIM was regulated by histone acetylation at its promoter area. Our results show that TIM could regulate tumorigenesis of CRC by binding and regulating Myosin-9. Our results also implicate TIM as a potential prognostic biomarker and therapeutic target for CRC. Citation Format: Xiaoqin Yuan, Yi Wang, Lei Wu. A clock gene of TIM promotes colorectal cancer tumorigenesis through binding with myosin-9 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5924.

Abstract 4916: MUC13 enhances anchorage independent survival and cooperates with YAP1 and β-catenin towards colorectal cancer metastasis

Abstract Colorectal Cancer (CRC) is the 3rd most common and deadliest cancers in the United States. About 90% of all cancer related deaths are due to the development of metastatic sites in the body. Metastasis develops slowly over time, due to it being an inefficient process and having to overcome several natural defenses, anchorage independent growth aka Anoikis, being one of them. Understanding the mechanism overcoming Anoikis will help develop new therapeutic options to prevent the metastatic spread of cancer cells. Low adhesion cell culture model was developed and standardized to study the key pathways and proteins responsible for anchorage independent survival and metastasis progression in CRC. We identified mucin MUC13 in correspondence with YAP1 and β-catenin as key upregulated proteins in anchorage independent survival and metastasis progression. In an isogeneic CRC cell line model, overexpression of MUC13 in non-metastatic SW480 cells (originally expressing minimal MUC13) increased anchorage independent survival and enhanced tumorigenesis compared to SW480+Vector cells. Metastatic SW620 CRC cells (highly expressing MUC13) showed a decreased anchorage independent survival and tumorigenesis after knockdown using specific shRNA targeted against MUC13, compared to its vector. When co-cultured with fibroblast cells SW480+MUC13 cells showed an increase in spheroid size and percentage of live cells after 36hrs as compared to control cells. A decrease in spheroid size and live cells was observed with SW620+shMUC13 cells, compared to its control. We also observed an upregulation of key downstream β-catenin target genes c-Myc, Axin2 and an increase in YAP1 expression in MUC13 over-expressing cells. With MUC13 overexpression we observed and increase in the formation of YAP1/β-catenin survival complex within the nucleus at 36hrs compared to SW480+Vector. Enhanced interaction between MUC13/YAP1 and MUC13/β-catenin in the nucleus was also observed. In human CRC tissues, MUC13 and YAP1 expression was high in tumor compared to normal adjacent tumor. MUC13 and YAP1 expression in human CRC tissue was highest at Stage II. However, highest YAP1 expression was observed while MUC13 and/or β-catenin present in the nucleus. This supports the notion that MUC13 is a key factor in enhancing anchorage independence survival and CRC tumorigenesis through cooperation with YAP1 and β-catenin. This study for the first time demonstrates complex formation between MUC13, YAP1 and β-catenin, and define their role in CRC progression and metastasis. Citation Format: Kyle Doxtater, Radhika Sekhri, Utkarsh Mishra, Meena Jaggi, Manish Tripathi, Subhash Chauhan. MUC13 enhances anchorage independent survival and cooperates with YAP1 and β-catenin towards colorectal cancer metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4916.

Abstract 294: Altered ARID1A expression in colorectal cancer

Abstract Background: ARID1A 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. Aim: To investigate the role 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. Methods: We used RT-PCR to determine both CNV and expression of ARID1A from six CRC cell lines. And used MSP to evaluate methylation of ARID1A. We used (IHC) to ARID1A protein expression, and, evaluate both MSI and EMAST status in 18 paired CRC and adjacent normal tissues. Statistical analysis was performed to establish correlations between ARID1A expression and other parameters. Results: Among 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%) of 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. Conclusion: ARID1A expression is downregulated in CRC tissues correlate 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 contribute to CRC tumorigenesis. There was no significant association between the ARID1A loss of expression and clinicopathological characteristics. ARID1A might be a useful biomarker for colorectal cancer. Future in- vivo analysis is warranted to further established this role. Citation Format: Mehran Erfani, Seyed Vahid, Maral Mokhtari, Mozhdeh Zamani, Kamran Tahmasebi, Mahvash Alizadeh, Alireza Taghavi, John Carethers, Minoru Koi, Hassan Brim, Pooneh Mokarram, Hassan Ashktorab. Altered ARID1A expression in colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 294.

RNA-binding protein CELF1 enhances cell migration, invasion, and chemoresistance by targeting ETS2 in colorectal cancer.

Colorectal cancer (CRC) is often diagnosed at later stages after it has metastasized to other organs. The development of chemoresistance also contributes to a poor prognosis. Therefore, an increased understanding of the metastatic properties of CRC and chemoresistance could improve patient survival. CUGBP elav-like family member 1 (CELF1) is an RNA-binding protein, which is overexpressed in many human malignant tumors. However, the influence of CELF1 in CRC is unclear. V-ets erythroblastosis virus E26 oncogene homologue 2 (ETS2) is an evolutionarily conserved proto-oncogene known to be overexpressed in a variety of human cancers including CRC. In thespresent tudy, we investigated the association between CELF1 and ETS2 in CRC tumorigenesis and oxaliplatin (L-OHP) resistance. We found a positive correlation between the elevated expression of CELF1 and ETS2 in human CRC tissues. Overexpression of CELF1 increased CRC cell proliferation, migration, and invasion in vitro and in a xenograft tumor growth model in vivo, and induced resistance to L-OHP. In contrast, CELF1 knockdown improved the response of CRC cells to L-OHP. Overexpression of ETS2 increased the malignant behavior of CRC cells (growth, migration, and invasion) and L-OHP resistance in vitro. Moreover, L-OHP resistance induced by CELF1 overexpression was reversed by ETS2 knockdown. The results of luciferase reporter and ribonucleoprotein immunoprecipitation assays indicated that CELF1 up-regulates ETS2 by binding to its 3'-UTR. Taken together, our findings have identified that CELF1 regulates ETS2 in a mechanism that results in CRC tumorigenesis and L-OHP resistance, and CELF1 may be a promising target for overcoming chemoresistance in CRC.