CRC Tumors Research Articles

Risk SNP-induced lncRNA-SLCC1 drives colorectal cancer through activating glycolysis signaling

Long non-coding RNAs (lncRNAs) play key roles in colorectal carcinogenesis. Here, we aimed to identify the risk SNP-induced lncRNAs and to investigate their roles in colorectal carcinogenesis. First, we identified rs6695584 as the causative SNP in 1q41 locus. The A>G mutation of rs6695584 created a protein-binding motif of BATF, altered the enhancer activity, and subsequently activated lncSLCC1 expression. Further validation in two independent CRC cohorts confirmed the upregulation of lncSLCC1 in CRC tissues, and revealed that increased lncSLCC1 expression was associated with poor survival in CRC patients. Mechanistically, lncRNA-SLCC1 interacted with AHR and transcriptionally activated HK2 expression, the crucial enzyme in glucose metabolism, thereby driving the glycolysis pathway and accelerating CRC tumor growth. The functional assays revealed that lncSLCC1 induced glycolysis activation and tumor growth in CRC mediated by HK2. In addition, HK2 was upregulated in colorectal cancer tissues and positively correlated with lncSLCC1 expression and patient survival. Taken together, our findings reveal a risk SNP-mediated oncogene lncRNA-SLCC1 promotes CRC through activating the glycolysis pathway.

ACBP suppresses the proliferation, migration, and invasion of colorectal cancer via targeting Wnt/beta-catenin signaling pathway

Anticancer bioactive peptide (ACBP), a novel bioactive peptide isolated from spleens of goats immunized with tumor extracts in our lab, can inhibit the proliferation of CRC in vitro and vivo. However, it remains unclear how the proliferation of CRC is inhibited by ACBP at the molecular level. Here, we provide evidences showing that ACBP significantly inhibits the expression of Wnt/β-catenin related genes (cyclin D1, met and c-myc) through pharmacotranscriptomic and qRT-PCR analysis in CRCs. Active β-catenin, a key protein within Wnt pathway, was compromised remarkably by ACBP in three CRCs, including HCT116, RKO and HT29. Thus nuclear accumulation of active β-catenin was retarded and finally lead to the decreased expression of oncogenes cyclin D1, met, and c-myc. In addition, we proved that active β-catenin reduction was mainly due to the inhibition of phospho-LRP6 and stimulation of phospho-β-catenin by ACBP. Based on the detection of Met and C-Myc in CRC tumor tissue without prior radiotherapy or chemotherapy, our results demonstrated that ACBP can act as a promising anticancer agent for CRC by targeting Wnt/β-catenin pathway, especially active β-catenin.

LINC01116 facilitates colorectal cancer cell proliferation and angiogenesis through targeting EZH2-regulated TPM1

BackgroundColorectal cancer (CRC) is a common malignant tumor globally. Meanwhile, LINC01116 has been proposed as risk factor for various tumors, including CRC. But the regulation of LINC01116 in CRC required more validated data. This study aimed to elucidate the potential function of LINC01116 in regulating cell proliferation and angiogenesis of CRC.MethodsLINC01116 expression in 80 pairs of CRC tumor and adjacent non-tumor tissues was determined by qRT-PCR. After transfection of pcDNA3.1-LINC01116, sh-LINC01116, sh-TPM1, pcDNA3.1-EZH2 or sh-EZH2 in SW480 and HCT116 cells, the levels of LINC01116, TPM1 and EZH2 were measured by qRT-PCR or Western blot. The cell biological function of CRC cell lines was determined by CCK-8, colony formation assays, tube formation and scratch assays. RNA pull-down and RIP assays were applied to detect the binding of LINC01116 with EZH2 and H3K27me3. Binding of EZH2 to the TPM1 promoter was assessed by ChIP assay. Finally, xenograft models in nude mice were established to validate the results of in vitro experiments.ResultsLINC01116 was overexpressed in CRC tissues and high expression of LINC01116 was negatively correlated with postoperative survival. In vitro study showed LINC01116 expression could significantly enhance CRC progression, including increasing cell proliferation, migration and angiogenesis. Besides, investigations into the mechanism disclosed that LINC01116 could regulate EZH2 to inactivate TPM1 promoter, thus promoting CRC cell proliferation and angiogenesis. Moreover, consistent results of in vivo experiments were conformed in vitro experiments.ConclusionLINC01116 promotes the proliferation and angiogenesis of CRC cells by recruiting EZH2 to potentiate methylation in the TPM1 promoter region to inhibit the transcription of TPM1.

Diagnostic performances of leucine-rich α-2-glycoprotein 1 and stem cell factor for diagnosis and follow-up of colorectal cancer

BackgroundColorectal cancer (CRC) is one of the most frequently diagnosed tumors worldwide with high mortality and morbidity. There is an urgent need for biomarkers to improve the outcomes and early detection of CRC. The sensitivity of traditional CRC tumor markers (carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA19-9)) is not ideal. The levels of leucine-rich-alpha-2-glycoprotein 1 (LRG1) and stem cell factor (SCF) were evaluated, but the combined value of both markers is unclear. This case-control study included four groups: CRC patients before treatments (n = 22), CRC patients after treatments (n = 26), 20 patients with benign tumor, and 20 healthy subjects. Levels of routine biochemical and hematological markers, traditional tumor markers (CA19.9 and CEA), and candidate markers (LRG1 and SCF) were determined. Univariate and multivariate logistic regression analysis and area receiver-operating characteristic analysis (ROC) were used for evaluation the diagnostic performances of single and combined markers. ResultsNo significance difference in traditional tumor markers CEA, CA 19.9, and neutrophil–lymphocyte ratio (NLR) were found among study groups. SCF, LRG1, and platelet–lymphocyte ratio (PLR) were significantly decreased (p < 0.05) in non-treated CRC patients than after treated CRC. The combination between SCF and LRG1 showed highly significant difference in CRC patients compared with benign, healthy subjects, and among CRC groups (treated and non-treated) (p < 0.0001). The highest areas under curve (AUCs) were observed when LRG1 was used as a single predictor for discriminating CRC from healthy (0.87), benign (0.84), and non-treated CRC vs treated CRC (0.82). AUCs were jumped to 0.90, 0.84, and 0.84 when LRG1 and SCF were combined. ConclusionOur study revealed that LRG1 and SCF were potential diagnostic and follow-up markers for CRC.

Epigenetic regulation of the Wnt-signaling pathway in CIMP-H BRAFV600E mCRC.

110 Background: BRAFV600E mutation identifies mCRC patients with poor prognosis with only little benefit from standard therapy. Analysis from TCGA revealed that 89% of BRAFV600E CRC tumors were associated with a high CpG island methylator phenotype (CIMP-H), which may result in epigenetic silencing of tumor suppressor genes, while only 29% of BRAF wild-type tumors are CIMP-H. In this study, we define key pathways regulated by global DNA hypermethylation in the context of BRAFV600E mutation. Methods: We analyzed the TCGA Illumina 450k array methylation datasets and RNA-sequencing datasets for 97 CIMP-H CRC tumors (27 BRAFV600E; 69 BRAF WT) identified by five universal CIMP annotations ( p14, p16, MLH1, MINT1, MINT2, MINT31). We defined differential methylation profile according to BRAF mutation status and calculated Spearman correlation between methylation and gene expression to identify CIMP-H BRAF-associated genes. Next, pathways enriched with CIMP-H BRAFV600E tumors were defined using PANTHER pathway analysis. Additionally, β-catenin IHC were conducted on 145 MD Anderson CRC patient samples and compared by CIMP and BRAF status. Results: BRAF mutation is associated with lower rates of APC mutation as has previously been shown (32%, 82%). We identified 6,097 differentially methylated probes by BRAF mutation status (FDR = 10-4), and as expected, our data suggests a higher methylation profile in BRAFV600E mutated tumors compared to BRAF WT. Intriguingly, CIMP-H BRAF-associated genes showed enrichment in the Wnt-signaling and cadherin signaling pathways ( p&lt; 0.0001 (FDR &lt; 0.0001)). Despite the epigenetic Wnt-signaling, nuclear β-catenin expression (as a measure of Wnt activity) in CIMP-H and BRAF tumors remains lower than for non-CIMP, and BRAF wild-type ( p= 0.0003 for comparison of CIMP). Conclusions: Genes under methylation regulation in the BRAF-mutant context showed enrichment in Wnt-signaling pathway. Since BRAFV600E CRC tumors have a low association with APC mutation, this data suggests role of epigenetic regulation of the Wnt-pathway activation. However, as measured by nuclear β-catenin, Wnt activation in these tumors is not as high as traditional APC-mutated CRC tumors. CIMP-H tumors with BRAFV600E mutation is a unique subset of CRC tumor that have Wnt-pathway activation regulated by epigenetic modifications more than a β-catenin activation.

GDF15 expression in metastatic colorectal cancer.

116 Background: Cachexia affects many cancer patients. Growth differentiation factor-15 (GDF15) is a protein that regulates weight and the stress response of cells. The GDF15 gene encodes a ligand of TGF-beta that triggers cachexia and modulates the progression from tumorigenesis to metastasis. Inhibition of GDF15 with an antibody restored muscle mass and fat in animal models. Serum levels rise in proportion to the progression of colon cancer, predict outcome, and have been correlated with CEA. Methods: We retrospectively reviewed 7607 CRC tumors profiled by Caris Life Sciences (Phoenix, AZ) from 2019 to 2020. Profiling included whole transcriptome sequencing (RNA-Seq by NovoSeq). Tumor mutational burden, mismatch repair status, and pathway genomic alterations were evaluated. QuantiSEQ was used to assess immune cell infiltration in the tumor microenvironment. Results: GDF15 expression ranged from 0 to 593 transcripts per million (TPM) with median of 30 (IQR = 15.02). There was no association with age, sex, or primary tumor sidedness. MSI-H/dMMR tumors had higher GDF15 expression (median 37 vs 30, p = 0.0004); TMB &gt; = 17 tumors was seen in 5.9% of bottom quartile (Q1) GDF15 expressors and 8.3% of top quartile (Q4). PDL1 IHC positivity was inversely correlated with GDF15 expression (7.1% in Q1 vs. 2.6% in Q4, p &lt; 0.0001). Genomic alterations associated with higher GDF15 expression (Q4 vs Q1) included genes on TGF-B (SMAD2/4), PI3K (PIK3CA, MTOR), chromatin remodeling (ARID1A, KMT2C), DDR (ATM) and Wnt pathway (APC); those inversely associated included MYC CNA and TP53. Q1 tumors had higher CNA of ERBB2 and FGFR1. Relative neutrophils and NK cells in the TME increased from Q1 to Q4 (p &lt; 0.001). There was a decrease in CD8+ T-cells and Treg cells from Q1 to Q4. Conclusions: GDF15 expression correlates with increased dMMR/MSI-H and TMB, but not with PDL1 expression. Mutations and activated pathways associated with GDF15 expression may explain increased cachexia with more aggressive disease. The association with chromatin remodeling may warrant therapies targeting histone modification and epigenetics. The increase in NK cells but decrease in CD8+ T cells in the TME with increasing GDF15 suggests approaches to treatment. Higher CD8+ lymphocyte counts correlate with PFS with immunotherapy. Anti-PD-L1 therapy reinvigorates the killing function of CD8+ T cells. The decrease in CD8+ T cells and PDL1 positivity with rising GDF15 suggests worse outcome and a lack of response to anti-PDL1 therapy. NK cell checkpoint inhibitors, CARs, and an anti-GFRAL antibody are now in clinical trials and might be utilized in high GDF15 cancers. GDF15 is emerging as a target in the treatment of obesity and cachexia and as a prognostic marker in oncology. Understanding its expression in metastatic colon cancer may reveal which patients could benefit from developing anti-GDF15 targeted therapies against cancer progression.

Epithelial-Mesenchymal Transition and MicroRNAs in Colorectal Cancer Chemoresistance to FOLFOX.

The FOLFOX scheme, based on the association of 5-fluorouracil and oxaliplatin, is the most frequently indicated chemotherapy scheme for patients diagnosed with metastatic colorectal cancer. Nevertheless, development of chemoresistance is one of the major challenges associated with this disease. It has been reported that epithelial-mesenchymal transition (EMT) is implicated in microRNA-driven modulation of tumor cells response to 5-fluorouracil and oxaliplatin. Moreover, from pharmacogenomic research, it is known that overexpression of genes encoding dihydropyrimidine dehydrogenase (DPYD), thymidylate synthase (TYMS), methylenetetrahydrofolate reductase (MTHFR), the DNA repair enzymes ERCC1, ERCC2, and XRCC1, and the phase 2 enzyme GSTP1 impair the response to FOLFOX. It has been observed that EMT is associated with overexpression of DPYD, TYMS, ERCC1, and GSTP1. In this review, we investigated the role of miRNAs as EMT promotors in tumor cells, and its potential effect on the upregulation of DPYD, TYMS, MTHFR, ERCC1, ERCC2, XRCC1, and GSTP1 expression, which would lead to resistance of CRC tumor cells to 5-fluorouracil and oxaliplatin. This constitutes a potential mechanism of epigenetic regulation involved in late-onset of acquired resistance in mCRC patients under FOLFOX chemotherapy. Expression of these biomarker microRNAs could serve as tools for personalized medicine, and as potential therapeutic targets in the future.

A 10-gene-methylation-based signature for prognosis prediction of colorectal cancer

BackgroundColorectal cancer (CRC) is a common malignant tumor of digestive tract which has high incidence and mortality rates. Accurate prognosis prediction of CRC patients is pivotal to reduce the mortality and disease burden. MethodsIn this study, we comprehensively analyzed the gene expression and methylation data of CRC samples from The Cancer Genome Atlas (TCGA). Differential expression genes (DEGs) and methylation CpGs (DMCs) in tumor tissues compared with adjacent normal tissues of CRC were first identified. Functional enrichment analysis of DEGs and DMCs was performed by Database for Annotation, Visualization and Integrated Discovery (DAVID). Spearman correlation analysis was used to screen DMCs that negatively correlated with gene expressions which were subsequently applied to sure independence screening (SIS) along with stepwise regression for screening optimal CpGs for CRC prognosis prediction model construction by Cox regression analysis. ResultsWe identified a total of 1774 DEGs (663 upregulated and 1111 downregulated) and 11,975 DMCs (7385 hypermethylated and 4590 hypomethylated) in CRC tumor samples compared with adjacent normal samples. The hypermethylated loci were mainly located on CpG island, while the hypomethylated loci were mainly located on N-shore. Spearman correlation analysis screened 321 DMCs that negatively correlated with expressions of their annotated genes. Cox regression model consist of 10 CpGs was finally established which could effectively stratified CRC patients that exhibited significantly different overall survival probability independent of age, gender, and pathological staging. ConclusionWe established a prognosis prediction model based on 10 methylation sites, which could evaluate the prognosis of CRC patients.

Immunomodulatory nanomedicine for colorectal cancer treatment: a landscape to be explored?

Colorectal cancer (CRC) is one of the deadliest cancers in the world mainly due to metastasis events. Despite improvements, the available treatment modalities for metastatic cases are limited, being generally associated with poor prognosis. As is well known, the immunosuppressive tumor microenvironment (TME) plays a key role in tumorigenesis, promoting cancer cell immune escape and disease progression. In addition, accumulating evidence indicates that the immunosuppressive microenvironment is a critical barrier for antitumor immunity in CRC, being extremely important to modulate the immune microenvironment to inhibit the tumor-promoting immune response. Therefore, new and effective cancer immunotherapeutic approaches demand a better control over the TME to reverse these immunosuppressive conditions. According to the features of different nanomedicines, nanoparticles can constitute a promising strategy, using different materials with the inherent ability to modulate TME and also with the potential to target immunosuppressive cells, to deliver antigens or immunomodulatory agents to eliminate this tumor. In this review, we summarize the importance of the TME in the progression and treatment response of CRC, exploring the potential of the nanotechnology for the development of immunomodulatory therapeutic strategies.

HIF1α promotes tumor chemoresistance via recruiting GDF15-producing TAMs in colorectal cancer

Chemoresistance is a tremendous challenge to efficacy of systemic chemotherapy which is the preferred treatment for the advanced CRC patients. More tumor-associated macrophages (TAMs) are recruited into the CRC tumor under chemotherapy, which are highly implicated in the chemoresistance development, but the underlying molecular mechanism is unclear. Here, we present that activated HIF1α signaling in CRC cells under chemotherapy drives the expression of HMGB1to promotes macrophage infiltration and in turn chemoresistance development. Chemotherapeutic treatment with 5-FU leads to increased recruitment of macrophages into tumors, which display tumor-protective alternative activation. Mechanistically, tumor HIF1α signaling activated by chemo-induced ROS drives the transcription of HMGB1 to promote more macrophage infiltration into CRC tumor. Furthermore, high levels of GDF15 produced by TAMs impair the chemosensitity of tumor cells via enhancing fatty acids β-oxidation. Together, our current study reveals a new insight into the cross-talking between tumor cells and immune cells, and provides novel drug targets for clinic treatments for CRC.

LncRNA-miRNA-mRNA interaction network for colorectal cancer; An in silico analysis

BackgroundColorectal cancer (CRC) is one of the most frequent and diagnosed diseases. Accumulating evidences showed that mRNAs and noncoding RNAs play important regulatory roles in tumorigenesis. Identification and determining the relationship between them can help diagnosis and treatment of cancer. MethodsHere we analyzed three microarray datasets; GSE110715, GSE32323 and GSE21510, to identify differentially expressed lncRNAs and mRNAs in CRC. The adjusted p-value ≤0.05 was considered statistically significant. Gene set enrichment analysis was carried out using DAVID tool. The miRCancer database was searched to obtain differentially expressed miRNAs in colorectal cancer, and the miRDB database was used to attain the targets of the obtained miRNAs. To predict the lncRNA-miRNA interactions we used DIANA-LncBase v2 and RegRNA 2.0. Finally the lncRNA-miRNA-mRNA-signaling pathway network was constructed using Cytoscape v3.1. ResultsBy analyzing the three datasets, a total of 21 mRNAs (15 up- and 6 down-regulated) and 24 lncRNAs (18 up- and 6 down-regulated) were identified as common differentially expressed genes between CRC tumor and marginal tissues. Nevertheless, the constructed lncRNA-miRNA-mRNA-signaling pathway network revealed a convergence on 6 lncRNAs (3 up- and 3 downregulated), 7 mRNAs (2 up- and 5 downregulated) and 6 miRNAs (3 up- and 3 downregulated). We found that dysregulation of lncRNAs such as PCBP1-AS1, UCA1 and SNHG16 could sequester several miRNAs such as hsa-miR-582-5p and hsa-miR-198 and promote the proliferation, invasion and drug resistance of colorectal cancer cells. ConclusionsWe introduced a set of lncRNAs, mRNAs and miRNAs differentially expressed in CRC which might be considered for further experimental research as potential biomarkers of CRC development.

CircTADA2A suppresses the progression of colorectal cancer via miR-374a-3p/KLF14 axis

BackgroundColorectal cancer (CRC) is one of the causes of cancer-related death worldwide. The aim of our study was to disclose the expression pattern and underlying molecular mechanism of circular RNA TADA2A (circTADA2A) in CRC.MethodsThe levels of circTADA2A, transcriptional adaptor 2A (TADA2A), microRNA-374a-3p (miR-374a-3p) and Kruppel like factor 14 (KLF14) were determined by quantitative real-time polymerase chain reaction (qRT-PCR). Xenograft tumor assay was used to uncover the function of circTADA2A in vivo. The miRNA targets of circTADA2A were searched using circbank and starbase softwares, while DIANA TOOL was used to explore miR-374a-3p-mRNA interactions. Dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were performed to validate the target relationship of circTADA2A/miR-374a-3p/KLF14 axis. Cell cycle and apoptosis were analyzed by flow cytometry. The glycolysis of CRC cells was determined by Seahorse XFe 96 Extracellular Flux Analyzer, Glucose Uptake Colorimetric Assay kit, Lactate Assay Kit II and ATP Colorimetric Assay kit. KLF14 protein level was measured by Western blot assay.ResultsCircTADA2A was abnormally down-regulated in CRC tissues and cell lines. CircTADA2A overexpression impeded CRC tumor growth in vivo. MiR-374a-3p was verified as a target of circTADA2A in CRC cells, and circTADA2A inhibited the malignant potential of CRC cells through targeting miR-374a-3p. MiR-374a-3p interacted with KLF14 messenger RNA (mRNA), and miR-374a-3p deteriorated CRC through down-regulating KLF14. CircTADA2A enhanced the abundance of KLF14 through targeting miR-374a-3p in CRC cells.ConclusionCircTADA2A functioned as a tumor suppressor in CRC to inhibit the glycolysis and cell cycle and potentiate the apoptosis of CRC cells via miR-374a-3p/KLF14 axis.

Abstract 4334: Exploring prognostic and immunological impact of a novel 4 gene signature in colorectal cancer patients

Abstract Colorectal cancer (CRC) affects nearly 1.4 million individuals every year and is the third most common cause of deaths due to cancer. There is a need to identify novel prognostic biomarkers with accurate prognostic and therapeutic implications. In this study, we sought to identify prognostic gene expression signatures and explore their relationship with the tumor microenvironment. Under the IRB approved protocol, FFPE blocks of CRC patients were acquired from the Medical College of Georgia, Augusta, USA. Tumor regions and adjacent normal tissue regions were identified and marked by a board-certified pathologist. RNA isolation was performed from tumor sections (n = 88) and adjacent normal regions (n = 16). A panel of 17 genes with the highest perturbation in TCGA dataset was investigated on the FFPE tissues. The quantification of the mRNA molecules was performed using total RNA (300 ng) on the NanoString platform. It uses a specific molecular barcoding system to tag target nucleic acid molecules with very high sensitivity and specificity. The immunological dynamics of the prognostic genes were analyzed through TIMER (Tumor-Infiltrating Immune Cells) web portal using the TCGA-COAD dataset. In the clinical dataset, MCM4, YWHAB, LRRC59 and DPP7 were highly expressed in tumor tissue compared to adjacent control and were associated with worse prognosis (Log-rank test, p &amp;lt; 0.001*). However, PI4K2B, PCMT1 and PBX1P1 showed higher expression in adjacent normal tissue compared to tumor regions (Student's t-test, p &amp;lt; 0.001*). Further analysis using TIMER portal revealed that copy number alterations in YWHAB were significantly associated with decreased tumor infiltration of B cells, CD8+ T cells, neutrophils and dendritic cells (Wilcoxon test, p &amp;lt; 0.001*). The predominant alterations observed were arm-level gain and high amplification. Further, arm-level gain of MCM4 was associated with lower infiltration of B cells and CD8+ T cells (Wilcoxon test, p &amp;lt; 0.001*) and FBXO46 was associated with lower infiltration of B cells only (Wilcoxon test, p &amp;lt; 0.05*). Additional comparison between CRC tumor and adjacent normal tissue was explored using “DiffExp” module of TIMER web portal. The comparison showed that YWHAB, DPP7 and MCM4 were expressed at higher amounts in tumor tissues compared to adjacent normal tissue. These preliminary analyses, therefore reveal the significance of prognostic genes and their association with the infiltration of immune cells that in turn can be of therapeutic value. A comprehensive network of prognostic gene signatures and its perturbations in immune cell functioning can aid in efficient prognostic classification and design of new therapies. Citation Format: Pankaj Kumar Ahluwalia, Ashis Mondal, Meenakshi Ahluwalia, Nikhil Sahajpal, Kimya Jones, Yasmeen Jilani, Allan Njau, Ravindra Kolhe. Exploring prognostic and immunological impact of a novel 4 gene signature in colorectal cancer patients [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 4334.

Abstract 2830: The impact of gut microbial toxins and PGE2 on IL-23 production and its role in obesity associated colorectal tumorigenesis

Abstract Colorectal cancer (CRC) is the second leading cause of cancer deaths in the United States. Studies indicate that excessive intake of high-fat diet (HFD) promotes CRC by modulating gut microbiome-associated inflammatory mediators. Recent evidence suggests that interleukin-23 (IL-23) plays an important role in the impact of a HFD on obesity, the gut microbiome and colon carcinogenesis. Our study aimed to elucidate the role of IL-23 in obesity associated CRC and explore an anti-IL-23 approach for prevention and treatment. First we profiled human plasma (n=15/arm) for circulating IL-23 levels and found significantly higher levels (p&amp;lt;0.0001) in obese individuals (BMI&amp;gt;30). In addition, IL-23 expression was evaluated in colonic tumors from both humans and preclinical rodent models. Data from the Cancer Genome Atlas showed significant over expression (p&amp;lt;0.0001) of IL-23A in CRC tumors (n=262) compared to normal tissue (n=41). This overexpression correlated with reduced disease free survival. Levels of both IL-23 and its receptor (IL23R) were evaluated in matched human CRC tissues by western blot and IHC and confirmed overexpression in CRC. IL-23 was also upregulated in colonic tumors from both sporadic (AOM-induced rat CRC) and FAP (Apcmin/+ mouse) rodent models when compared to matched normal mucosa. These data reveal a clear association of IL-23 overexpression with both obesity and colonic tumorigenesis. To understand IL-23 production in the context of gut microbial toxins and prostaglandin (PG)E2 levels, we performed several mechanistic studies to mimic the tumor microenvironment. To elucidate PGE2-mediated regulation of IL-23 in colon tumorigenesis we studied both macrophages and dendritic cell lines in in vitro and ex vivo conditions. The immune cells treated with either PGE2 or bacterial toxins (LPA/LTA) showed a significant increase in IL-23 expression which associated with macrophage polarization and dendritic cell phenotypic changes. Interestingly, when CaCo2 cancer cells were co-cultured with PGE2/LTA-LPA-educated macrophages or dendritic cells, the cancer cells displayed enhanced IL-23 expression along with increased tumor cell proliferation and self-renewal. Upon addition of human recombinant IL-23 directly to cancer cells (CaCo2 and HCT116), the cells exhibited increased IL-23R expression along with enhanced self-renewal, cell migration and invasive properties as well as loss of epithelial barrier permeability. In ex-vivo experiments we found that treatment of rat colon tumors with PGE2 increased the expression of IL-23 compared to normal mucosa. Overall our study results demonstrate that IL-23 is associated with both obesity and poor survival of CRC patients and that PGE2 and gut microbe toxins play an important role in IL-23-associated colonic tumor progression. This newly identified nexus represents a potentially important target for prevention of obesity associated colorectal cancer. Citation Format: Janani Panneerselvam, Venkateshwar Madka, Rajani Rai, Parthasarathy Chandrakesan, Courtney W. Houchen, Dharambir K. Sanghera, Katherine T. Morris, Chinthalapally V. Rao. The impact of gut microbial toxins and PGE2 on IL-23 production and its role in obesity associated colorectal tumorigenesis [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 2830.

Abstract LB-063: ARID1A mutations induce an EGFR-like gene expression signature and confer intrinsic and acquired resistance to cetuximab treatment in first line metastatic CRC

Abstract Extensive biological and clinical evidence supports the notion many CRC tumors are addicted to EGFR/KRAS/MAPK signaling. For example, mutations in KRAS, BRAF and other MAPK pathway components confer intrinsic or acquired resistance to anti-EGFR therapy. However, a significant proportion of acquired resistance cases have remained unexplained. ARID1A mutations are found in approximately 11% of CRC tumors, but a connection between ARID1A mutations and EGFR/MAPK signaling activation in CRC has not been previously suspected. By leveraging the genomic biomarker data collected from 333 patients at baseline and at progression who participated in the CALGB/SWOG 80405 trial, we found that mutations in ARID1A, a key component of SWI/SNF complex, were enriched in the cell free DNA of 6/16 of the patients whose tumors had become resistant to anti-EGFR therapy with ARID1A mutations detected at either timepoint (adj. p = 0.03, OR = 0.09, two-tailed fisher's exact test). In contrast, there was no evidence of enriched ARID1A mutations in patients treated with bevacizumab. To investigate the potential role of a broader ARID1A deficiency phenotype in resistance to cetuximab therapy, we developed an ARID1A mutant-like signature from TCGA that captures the transcriptional profile characteristic of ARID1A mutants. Using this signature to stratify CALGB patients based on gene expression data collected on tissue at diagnosis, we also found patients with an ARID1A mutant-like gene expression profile had worst outcome in patients treated with cetuximab than bevacizumab with adjustment for established clinical variables for both OS [p = 0.0002, hazard ratio (HR) 6.2, 95% confidence interval (CI) 2.4-16], and PFS [p = 0.0008, HR 4.7 (1.9 -12)], suggesting that ARID1A mutations may be also a mechanism of intrinsic resistance to anti-EGFR therapies. Gene expression analysis demonstrated that ARID1A deficiency leads to re-activation of an EGFR-like signature, suggesting reactivation of this pathway as a possible mechanism of resistance. Furthermore, CRISPR knockout of ARID1A in the cetuximab-sensitive CRC cell line NCI-H508 conferred elevated MAPK signaling relative to parental line and resistance to cetuximab in culture. Consistent with these findings, we also observed strong mutual exclusivity between ARID1A mutations and mutations in the EGFR/MAPK signaling pathway in more than 40,000 lung and colorectal cancer patients profiled in the FoundationCore® database. Strikingly, in lung cancers where EGFR mutations structural variants (SV) are more prevalent, EGFR SV was the top mutually exclusive alteration with ARID1A mutations in lung cancers. Of 5980 lung cancer patients with ARID1A and/or EGFR SVs, only 100 patients (1.7%) had both SVs. Taken together, our data suggest that ARID1A loss-of-function mutations may promote resistance to cetuximab by driving an EGFR-like transcription program in the absence of ligand-dependent activation of EGFR. Our results suggest that ARID1A defects could be potentially used as an exclusion biomarker for cetuximab treatment decisions, and they provide a rationale for exploring therapeutic MAPK inhibition in ARID1A mutant CRC patients. Citation Format: Radia Marie Johnson, Xueping Qu, Joshua Thomas, Yvonne Kschonsak, Ling-Yuh Huw, Fang-Shu Ou, Ethan Sokol, Nnamdi Ihuegbu, Oliver Zill, Omar Kabbarah, Anneleen Daemen, Richard Bourgon, Alan Venook, Federico Innocenti, Heinz-Josef Lenz, Felipe de Sousa e Melo, Carlos Bais. ARID1A mutations induce an EGFR-like gene expression signature and confer intrinsic and acquired resistance to cetuximab treatment in first line metastatic CRC [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 LB-063.

Abstract 63: Navitoclax (ABT-263) induces apoptosis in colon cancer cells by upregulating caspase 3 through inhibition of Bcl-2 family members

Abstract Colorectal cancer (CRC) is the third most common cancer in the United States. Most CRCs in patients receiving therapeutic agents are resistant to apoptotic stimuli. Two protein families, caspase enzymes and B-cell lymphoma-2 (Bcl-2) family proteins are involved in apoptosis. Caspase enzymes promote apoptosis, functioning in a cascade, of which caspase 3 is the pivotal protein. Members of the Bcl-2 family, Bcl-2, Bcl-xL, and Bax, are functionally opposed: Bcl-2 and Bcl-xL inhibit apoptosis, whereas Bax counteracts this effect. Only Bcl-2 family members that have the BH3-domain trigger apoptosis by binding to the pro-survival proteins, thereby neutralizing their functional activity. The approach of “BH-3 mimetics” was utilized for discovery and development of navitoclax (ABT-263), a small-molecule that mimics BH3-only proteins and thus induces apoptosis. Despite advances made in targeted therapy, the mechanism of action of ABT-263 is less studied, particularly for CRCs, which have over-expression of anti-apoptotic proteins, supporting avoidance of cell death, a hallmark of cancer. To identify the cellular mechanism of ABT-263 on apoptosis, we quantified the expression of anti-apoptotic Bcl-2-family members in CRC tumor samples relative to their corresponding controls and found several-fold higher expression of Bcl2, Bcl-xL, and Mcl-1 in the CRC tumor samples. Since ABT-263 is a classical drug for Bcl-2 inhibition, we studied its effect (2.5 µM for 24 hr) on RKO CRC cells, which have high endogenous expression of Bcl-2, by measuring gene expression profiles of Bcl-2 family members by qPCR, along with other proteins involved in apoptosis. ABT-263 treatment resulted in down-regulation of anti-apoptotic modulators, Bcl-2, Bcl-xL, and Bcl-w, by 27%, 29%, and 13%, respectively; these effects were concomitant with two-fold higher expression of caspase 3. Flow cytometry results with Annexin V/PI staining showed that ABT-263 induced apoptosis in RKO cells with 1.59-, 2.59-, and 2.76-fold elevations of early apoptotic, late apoptotic, and necrotic cells. Further, ABT-263 (2.5 µM) lowered cell proliferation by 15% after 24 hr. Similar lower gene expressions of Bcl-2 family members were evident for LS174T CRC cells, which also exhibit high endogenous Bcl-2 expression, when treated with ABT-263. These results highlight a previously unexplored effect of ABT-263-induced apoptosis in CRC cells, by upregulating caspase 3, through inhibition of Bcl-2 family members. These studies were supported by the Richard Elkus, MD, Eminent Scholars Program in Gastro-Intestinal Cancer Research at University of Alabama at Birmingham. Citation Format: Prachi Bajpai, Amr Elkholy, Sumit Agarwal, Hyung Gyoon Kim, Michael Behring, Upender Manne. Navitoclax (ABT-263) induces apoptosis in colon cancer cells by upregulating caspase 3 through inhibition of Bcl-2 family members [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 63.

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.

Abstract 1251: Epigenetics in early onset colorectal cancer (EOCRC)

Abstract Colorectal cancer (CRC) incidence rates have increased among young adults while the rates have decreased among older adults over 50 years of age. While the etiology of the growing prevalence of young adults with newly diagnosed CRC is unknown, there has been growing interest in the roles implicated for diabetes, obesity, environmental changes, and epigenetics. Among the epigenetic marks implicated in EOCRC, the most widely studied are the global DNA hypomethylation. Using mouse studies, our group has demonstrated that DNA methylation as an epigenetic mechanism to control gene expression is essential to postnatal development. We have discovered a key set of genes that undergo epigenetic changes in colonic epithelial stem cells, and these changes are stable over a lifetime. We have shown that the gut microbiome influences this process. Importantly, perturbation of established intestinal DNA methylation leads to accelerated intestinal tumor development. To translate our mechanistic studies in the animal model to humans, we have confirmed these CpG loci that are concordant between the two species (i.e. homologous regions and/or conserved CpG islands). Here, we hypothesize that the dysregulation of developmentally programmed intestinal DNA methylation is associated with enhanced susceptibility to colon cancer in young adult. We have identified a population of patients within the Harris Health and Baylor College of Medicine system under the age of 50 with mismatch repair stable colorectal adenocarcinoma. Patient characteristics including age, sex, race, and primary tumor profile (stage at diagnosis, histology, and RAS status) have been defined. Using a panel of 6 genes and bisulfite pyrosequencing for quantitative assessment of DNA methylation, we compared the level of methylation in EOCRC tumors to paired normal adjacent tissues as well as normal adult tissue (age 50 or greater) to traditional CRC tumors. Our preliminary data suggest hypermethylation patterns are distinct in patients of EOCRC relative to traditional CRC. Our ultimate goal is to better understand the etiology of EOCRC in order to devise better prevention and treatment strategies for EOCRC. Citation Format: Karen Riggins, Benjamin Musher, Michael Scheurer, Li Yang, Patricia Castro, Neda Zarrin-Khameh, Lanlan Shen. Epigenetics in early onset colorectal cancer (EOCRC) [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 1251.

A five-immune-related genes-based prognostic signature for colorectal cancer

BackgroundColorectal cancer (CRC) is a common malignancy with high morbidity and mortality. Prognosis of CRC is highly heterogeneous which hinders the making of appropriate clinical decision tremendously. AimsWe here propose to comprehensively define prognosis of CRC patients in the context of mRNA levels of immune-related genes (IRGs). MethodsCRC samples were acquired from both the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO). IRGs were obtained from ImmProt database. Univariate Cox-regression analysis was adopted to screen prognostically significant IRGs for CRC patients. LASSO Cox-regression analysis was used to identify the optimal prognostic IRGs and construct the prognostic model. Multivariate Cox-regression analysis was performed to determine the independence of associations of specific factors with CRC’s OS. Nomogram that combines the independent prognostic factors was constructed to predict CRC patients’ 1-year, 3-years, and 5-years OS probability. ResultsThere were a total of 76 differentially expressed IRGs (DEGs) in CRC tumor compared with paracancerous samples. Five out of those DEGs were significantly associated with the CRC patients’ OS probability and used for the construction of prognostic model. CRC samples were divided into high-risk and low-risk group based on prognostic model. Significant differences in OS probability were obtained between high-risk and low-risk CRC patients. Additionally, a nomogram containing risk score, age and stage could effectively predict long-term OS probability of CRC patients. ConclusionIn conclusion, we developed a robust IRGs-based prognostic signature which should be helpful for the understanding of heterogeneity of OS probability and the future clinical research.

Long non-coding RNA KCNQ1OT1 up-regulates CTNND1 by sponging miR-329-3p to induce the proliferation, migration, invasion, and inhibit apoptosis of colorectal cancer cells

BackgroundLong non-coding RNAs (lncRNAs) have been certified to be involved in the occurrence and growth of diverse cancers, including CRC. The purpose of the research was to explore the effects of lncRNA KCNQ1 overlapping transcript 1 (KCNQ1OT1) on proliferation, migration, invasion, and apoptosis in CRC cells and its mechanism.MethodsThe levels of KCNQ1OT1 and miR-329-3p were examined by quantitative real-time polymerase chain reaction (qRT-PCR) in CRC tissues and cells. The mRNA and protein levels of catenin delta-1 (CTNND1) were measured by qRT-PCR and western blot analysis, respectively. The targets of KCNQ1OT1 and miR-329-3p were predicted by online software and confirmed by luciferase reporter assay. The cell proliferation, migration, invasion, and apoptosis were examined using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), transwell, and apoptosis assay. The expression levels of CyclinD1, Bcl-2, MMP9, Cleaved-casp-3, and E-cadherin in SW480 and LS1034 cells were gauged by western blot analysis. Xenograft tumor model was structured to prove the biological role of KCNQ1OT1 of CRC in vivo.ResultsThe levels of KCNQ1OT1 and CTNND1 were significantly increased in CRC tissues and cells. Knockdown of KCNQ1OT1 suppressed proliferation, migration, invasion, and induced apoptosis in CRC cells. Conversely, CTNND1 overexpression reversed the impact of KCNQ1OT1 knockdown on CRC cells. Moreover, CTNND1 was verified as a direct target of miR-329-3p, and miR-329-3p could specially bind to KCNQ1OT1. Also, the down-regulation of KCNQ1OT1 triggered the CRC progress by up-regulating CTNND1 expression in CRC cells. Besides, KCNQ1OT1 knockdown inhibited CRC tumor growth through the miR-329-3p/CTNND1 axis in vivo.ConclusionOur results indicated that KCNQ1OT1 could positively regulate CTNND1 expression by sponging miR-329-3p, thereby boosting the progression of CRC. Our findings provided the underlying therapy targets for CRC.