Tumor Suppressor In Colorectal Cancer Research Articles

Identification and Validation of SLC9A2 as A Potential Tumor Suppressor in Colorectal Cancer: Integrating Bioinformatics Analysis with Experimental Confirmation.

To uncover the mechanisms underlying the development of colorectal cancer (CRC), we applied bioinformatic analyses to identify key genes and experimentally validated their possible roles in CRC onset and progression. We performed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis on differentially expressed genes (DEGs), constructed a protein-protein interaction (PPI) network to find the top 10 hub genes, and analyzed their expression in colon adenocarcinoma (COAD) and rectum adenocarcinoma (READ). We also studied the correlation between these genes and immune cell infiltration and prognosis and validated the expression of SLC9A2 in CRC tissues and cell lines using qRT-PCR and Western blotting. Functional experiments were conducted in vitro to investigate the effects of SLC9A2 on tumor growth and metastasis. We found 130 DEGs, with 45 up-regulated and 85 down-regulated in CRC. GO analysis indicated that these DEGs were primarily enriched in functions related to the regulation of cellular pH, zymogen granules, and transmembrane transporter activity. KEGG pathway analysis revealed that the DEGs played pivotal roles in pancreatic secretion, rheumatoid arthritis, and the IL-17 signaling pathway. We identified 10 hub genes: CXCL1, SLC26A3, CXCL2, MMP7, MMP1, SLC9A2, SLC4A4, CLCA1, CLCA4, and ZG16. GO enrichment analysis showed that these hub genes were predominantly involved in the positive regulation of transcription. Gene expression analysis revealed that CXCL1, CXCL2, MMP1, and MMP7 were highly expressed in CRC, whereas CLCA1, CLCA4, SLC4A4, SLC9A2, SLC26A3, and ZG16 were expressed at lower levels. Survival analysis revealed that 5 key genes were significantly associated with the prognosis of CRC. Both mRNA and protein expression levels of SLC9A2 were markedly reduced in CRC tissues and cell lines. Importantly, SLC9A2 overexpression in SW480 cells led to a notable inhibition of cell proliferation, migration, and invasion. Western blotting analysis revealed that the expression levels of phosphorylated ERK (p-ERK) and phosphorylated JNK (p-JNK) proteins were significantly increased, whereas there were no significant changes in the expression levels of ERK and JNK following SLC9A2 overexpression. Correlation analysis indicated a potential link between SLC9A2 expression and the MAPK signaling pathway. Our study suggests that SLC9A2 acts as a tumor suppressor through the MAPK pathway and could be a potential target for CRC diagnosis and therapy.

Abstract 5420: NLRX1 involves in prostate cancer cell growth, survival and migration

Abstract Prostate cancer (PCa) is one of the most common male cancer and is the second leading cause of death in men. The basic working components about its cause and progression are not surely known in the research as well as the practitioner community. The current curing therapies such as surgery, radiation therapy, cryotherapy, and hormone therapy are not very encouraging. Subsequently, there is a need to investigate new potential targets for focused treatment. NLRX1 (NOD9 or NLR family member X1) is one member among the NLRs and is a non-inflammasome forming NLR sub-group localized in mitochondria. Previous studies showed that NLRX1 is involved as a tumor suppressor in colorectal cancer and hepatocellular carcinoma. However, NLRX1 serves as tumor promoter in breast cancer and head and neck cancer sarcoma. The present study focuses on the role of NLRX1 in prostate cancer cells under stress conditions including serum-free and hydrogen peroxide (H2O2) to understand cell regulation and functions of NLRX1. We used human prostate cancer PC3 and LNCaP cell lines as the models and the following methods, including Annexin V/PI staining for cell viability, BrdU incorporation for cell proliferation, mitoSOX staining for mitochondrial ROS, wound healing assay for cell migration and Boyden chamber for cell invasion. First, we assessed the TCGA database via UCSC Xena and found that prostate cancer tumors have high NLRX1 expression than normal tissues. Next, we knock down the NLRX1 through lentivirus transduction. We found that silencing NLRX1 expression can decrease cell proliferation in PC3 cells. Furthermore, silencing NLRX1 can significantly decrease cell viability under H2O2 in PC3 cells and serum-free condition in both cancer cell lines. In addition, we found that H2O2-induced cell death is reversed by necroptosis inhibitor Nec-1. Moreover, NLRX1 silencing reduces cell migration in both PC3 and LNCaP cells and can inhibit cell invasion in both cell lines at 24 h. These results suggest that NLRX1 is a positive regulator of cell growth, migration and invasion in prostate cancer cells, and NLRX1 provides a cell-protective role in response to H2O2 and serum-free stress. Thus, this study will provide a potential target for the development of a therapeutic strategy for prostate cancer. Citation Format: Wan-Wan Lin, Varsha Rathore. NLRX1 involves in prostate cancer cell growth, survival and migration [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 5420.

Differentially expressed miR-4310 functions as a tumor suppressor in colorectal cancer.

Colorectal cancer (CRC) is a digestive tract malignancy microRNAs (miRNAs) have attracted much attention as biomarkers in tumor studies. This work focused on the predictive potential and mechanism of miR-4310 in CRC. The miRNA expression profile sets were obtained from the Gene Expression Omnibus (GEO) database, and the appropriate miRNA was screened by GEO2R. The CRC tissues and control tissues of 88 patients with CRC were collected, and the expression of miR-4310 was detected by quantitative real-time PCR, and the efficacy of miR-4310 in diagnosing CRC was evaluated by the receiver operating characteristic curve (ROC). The effects of miR-4310 on the proliferation, migration, and invasion of CRC cells were explored by Cell Counting Kit-8 (CCK-8) and Transwell experiments. Predicting the potential binding sites of miR-4310 and Runt-related transcription factor 1 (RUNX1) by four predictive websites. The relationship between miR-4310 and RUNX1 was confirmed by a double luciferase reporter gene experiment. The bioinformatics analysis found that miR-4310 was differentially expressed in CRC tissues and this finding was certified by the expression of miR-4310 in CRC tissues of collected patients and cultured CRC cell lines. The expression of miR-4310 had a predictive possibility for CRC patients. MiR-4310/RUNX1 pathway had effects on CRC viability, migration, and invasion. MiR-4310 had the potential to be a biomarker for early screening of CRC. MiR-4310 and RUNX1 participated in the regulation of CRC cells.

CBX2 Deletion Suppresses Growth and Metastasis of Colorectal Cancer by Mettl3-p38/ERK MAPK Signalling Pathway.

Colorectal cancer (CRC) seriously endangers human health owing to its high morbidity and mortality. Previous studies have suggested that high expression of CBX2 may be associated with poor prognosis in CRC patients. However, its functional role in CRC remains to be elucidated. Herein, we found that CBX2 overexpression in colorectal cancer tissue compared with adjacent tissues. Additionally, forest maps and the nomogram model indicated that elevated CBX2 expression was an independent prognostic factor in CRC. Moreover, we confirmed that the deletion of CBX2 markedly suppressed the proliferation and migration of CRC cells in vitro and in vivo. Furthermore, downregulation of CBX2 promotes CRC cell apoptosis and hinders the cell cycle. Mechanistically, our data demonstrated that deletion of CBX2 inhibited the MAPK signaling pathway by regulating the protein levels of Mettl3. In conclusion, our study demonstrated that CBX2 is a vital tumor suppressor in CRC and could be a promising anti-cancer therapeutic target.

The ATF2/miR-3913-5p/CREB5 axis is involved in the cell proliferation and metastasis of colorectal cancer

Various miRNAs have been shown to participate in the tumor progression and development of colorectal cancer (CRC). However, the role of miR-3913-5p in CRC are yet to be clearly defined. In the present study, we determine that miR-3913-5p is downregulated in CRC cell lines and CRC tissues. Exogenous miR-3913-5p expression weakens the CRC cells growth, migration and invasion. Mechanistically, miR-3913-5p directly targets the 3’UTR of CREB5. Overexpression of CREB5 reverses the suppression of CRC cells proliferation, migration and invasion induced by miR-3913-5p. Furthermore, ATF2 negatively regulates the transcription of miR-3913-5p by binding to its promoter. CREB5 can cooperate with ATF2. CREB5 is required for ATF2 in regulating miR-3913-5p. Finally, inverse correlations can be found between the expressions of miR-3913-5p and CREB5 or ATF2 in CRC tissues. Thus, a plausible mechanism of ATF2/miR-3913-5p/CREB5 axis regulating CRC progression is elucidated. Our findings suggest that miR-3913-5p functions as a tumor suppressor in CRC. ATF2/miR-3913-5p/CREB5 axis might be a potential therapeutic target against CRC progression.

ELOA promotes tumor growth and metastasis by activating RBP1 in gastric cancer.

Elongin A (ELOA), our previous work revealed, serves as a novel tumor suppressor in colorectal cancer. However, the function and mechanism of ELOA in other cancer types, including gastric cancer (GC), remain to be elucidated. The expression of ELOA was measured by quantitative reverse transcription-polymerase chain reaction and western blot. The effects of ELOA on GC growth and metastasis were assessed through a series of in-vitro and in-vivo assays. Furthermore, the potential mechanism of ELOA was revealed by RNA sequencing, dual luciferase reporter assay, chromatin immunoprecipitation, and rescue experiments in GC. We uncovered increased expression of ELOA in GC tissues compared with paired normal tissues via bioinformatic analyses and our sample detection. Enhanced ELOA expression in GC tissues was obviously correlated with poor tumor differentiation, lymph node metastasis, advanced tumor stage, and a poor prognosis. A series of functional experiments showed that ELOA promoted the proliferation and metastasis of GC. Mechanistically, we revealed that the decreased levels of miR-490-3p caused the upregulation of ELOA in GC. Both RNA-seq and ChIP assays revealed that ELOA transcriptionally activated retinol-binding protein 1 (RBP1) by binding to its promotor. Furthermore, specific knockdown of RBP1 reduced the tumor-promoting ability of ELOA in GC cells. In summary, our findings demonstrate that ELOA exerts oncogenic properties by activating RBP1 expression, providing the basis for a promising therapeutic target in GC.

MiR-148a-3p Promotes Colorectal Cancer Cell Ferroptosis by Targeting SLC7A11.

Ferroptosis, an iron-dependent form of cell death, and dysregulated microRNA (miRNA) expression correlate with colorectal cancer (CRC) development and progression. The tumor suppressor ability of miR-148a-3p has been reported for several cancers. Nevertheless, the role of miR-148a-3p in CRC remains largely undetermined. Here, we aim at investigating the molecular mechanisms and regulatory targets of miR-148a-3p in the CRC cell death mechanism(s). To this end, miR-148a-3p expression was evaluated in SW480 and SW620 cells and normal colon epithelial CCD 841 CoN cells with quantitative real-time polymerase chain reaction (qRT-PCR). Data reported a reduction of miR-148a-3p expression in SW480 and SW620 cells compared to non-tumor cells (p < 0.05). Overexpression of miR-148a selectively inhibited CRC cell viability (p < 0.001), while weakly affecting normal CCD 841 CoN cell survival (p < 0.05). At the cellular level, miR-148a-3p mimics promoted apoptotic cell death via caspase-3 activation (p < 0.001), accumulation of mitochondrial reactive oxygen species (ROS) (p < 0.001), and membrane depolarization (p < 0.001). Moreover, miR-148a-3p overexpression induced lipid peroxidation (p < 0.01), GPX4 downregulation (p < 0.01), and ferroptosis (p < 0.01), as revealed by intracellular and mitochondrial iron accumulation and ACSL4/TFRC/Ferritin modulation. In addition, levels of SLC7A11 mRNA and protein, the cellular targets of miR-148a-3p predicted by bioinformatic tools, were suppressed by miR-148a-3p's overexpression. On the contrary, the downregulation of miR-148a-3p boosted SLC7A11 gene expression and suppressed ferroptosis. Together, these in vitro findings reveal that miR-148a-3p can function as a tumor suppressor in CRC by targeting SLC7A11 and activating ferroptosis, opening new perspectives for the rationale of therapeutic strategies through targeting the miR-148a-3p/SLC7A11 pathway.

Loss of Pkd1 limits susceptibility to colitis and colorectal cancer

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. Autosomal dominant polycystic kidney disease (ADPKD), associated with mutations disabling the PKD1 gene, affects as many as 1 in 1000. Intriguingly, some studies have suggested that individuals with germline mutations in PKD1 have reduced incidence of CRC, suggesting a genetic modifier function. Using mouse models, we here establish that loss of Pkd1 greatly reduces CRC incidence and tumor growth induced by loss of the tumor suppressor Apc. Growth of Pkd1−/−;Apc−/− organoids was reduced relative to Apc−/− organoids, indicating a cancer cell-intrinsic activity, even though Pkd1 loss enhanced activity of pro-oncogenic signaling pathways. Notably, Pkd1 loss increased colon barrier function, with Pkd1-deficient animals resistant to DSS-induced colitis, associated with upregulation of claudins that decrease permeability, and reduced T cell infiltration. Notably, Pkd1 loss caused greater sensitivity to activation of CFTR, a tumor suppressor in CRC, paralleling signaling relations in ADPKD. Overall, these data and other data suggest germline and somatic mutations in PKD1 may influence incidence, presentation, and treatment response in human CRC and other pathologies involving the colon.

TRIM58 functions as a tumor suppressor in colorectal cancer by promoting RECQL4 ubiquitination to inhibit the AKT signaling pathway

BackgroundThis study aimed to investigate the underlying molecular mechanisms of TRIM58 in the development of colorectal cancer (CRC). CRC is one of the most common cancers of the digestive tract worldwide. The ubiquitin–proteasome system regulates many oncogenic or tumor-suppressive proteins. TRIM58, an E3 ubiquitin ligase and a member of the tripartite motif protein family, is a potential prognostic marker that indicates poor prognosis in cancer. Currently, the precise molecular mechanisms for the TRIM58-mediated CRC progression remain unclear.MethodsTo examine the effects of TRIM58 on cell viability, cell cycle progression, and apoptosis in CRC, Cell Counting Kit-8 and flow cytometry assays were employed. The AKT inhibitor LY294002 was used to examine the effects of AKT signaling on TRIM58-mediated cell viability, cell cycle progression, and apoptosis in CRC. Additionally, Co-IP and ubiquitination assays were used to examine the correlation between TRIM58 and RECQL4.ResultsTRIM58 overexpression inhibited CRC cell viability and promoted cell cycle arrest and apoptosis, in which the TRIM58 knockdown demonstrated inversed effects via the AKT signaling pathway. TRIM58 inhibited RECQL4 protein levels through its ubiquitin ligase activity, and RECQL4 overexpression inhibited TRIM58 overexpression-mediated CRC cell viability, cell cycle progression, and apoptosis. The downregulation of TRIM58 and upregulation of RECOL4 were observed in human CRC tissue, and TRIM58 demonstrated antitumor effects in CRC-induced tumor growth in a mouse model.ConclusionsTRIM58 acts as a tumor suppressor in CRC through the promotion of RECQL4 ubiquitination and inhibition of the AKT signaling pathway and may be investigated for the successful treatment of CRC.

Human Neuralized is a novel tumour suppressor targeting Wnt/β-catenin signalling in colon cancer.

While there is growing evidence that many epigenetically silenced genes in cancer are tumour suppressor candidates, their significance in cancer biology remains unclear. Here, we identify human Neuralized (NEURL), which acts as a novel tumour suppressor targeting oncogenic Wnt/β-catenin signalling in human cancers. The expression of NEURL is epigenetically regulated and markedly suppressed in human colorectal cancer. We, therefore, considered NEURL to be a bona fide tumour suppressor in colorectal cancer and demonstrate that this tumour suppressive function depends on NEURL-mediated oncogenic β-catenin degradation. We find that NEURL acts as an E3 ubiquitin ligase, interacting directly with oncogenic β-catenin, and reducing its cytoplasmic levels in a GSK3β- and β-TrCP-independent manner, indicating that NEURL-β-catenin interactions can lead to a disruption of the canonical Wnt/β-catenin pathway. This study suggests that NEURL is a therapeutic target against human cancers and that it acts by regulating oncogenic Wnt/β-catenin signalling.

KLF2 inhibits colorectal cancer progression and metastasis by inducing ferroptosis via the PI3K/AKT signaling pathway.

Krüppel-like factor 2 (KLF2) belongs to the zinc finger family and is thought to be a tumor suppressor gene due to its low expression in various cancer types. However, its functional role and molecular pathway involvement in colorectal cancer (CRC) are not well defined. Herein, we investigated the potential mechanism of KLF2 in CRC cell invasion, migration, and epithelial-mesenchymal transition (EMT). We utilized the TCGA and GEPIA databases to analyze the expression of KLF2 in CRC patients and its correlation with different CRC stages and CRC prognosis. RT-PCR, western blot, and immunohistochemistry assays were used to measure KLF2 expression. Gain-of-function assays were performed to evaluate the role of KLF2 in CRC progression. Moreover, mechanistic experiments were conducted to investigate the molecular mechanism and involved signaling pathways regulated by KLF2. Additionally, we also conducted a xenograft tumor assay to evaluate the role of KLF2 in tumorigenesis. KLF2 expression was low in CRC patient tissues and cell lines, and low expression of KLF2 was associated with poor CRC prognosis. Remarkably, overexpressing KLF2 significantly inhibited the invasion, migration, and EMT capabilities of CRC cells, and tumor growth in xenografts. Mechanistically, KLF2 overexpression induced ferroptosis in CRC cells by regulating glutathione peroxidase 4 expression. Moreover, this KLF2-dependent ferroptosis in CRC cells was mediated by inhibiting the PI3K/AKT signaling pathway that resulted in the suppression of invasion, migration, and EMT of CRC cells. We report for the first time that KLF2 acts as a tumor suppressor in CRC by inducing ferroptosis via inhibiting the PI3K/AKT signaling pathway, thus providing a new direction for CRC prognosis assessment and targeted therapy.

Hexafluoro-2-propanol represses colorectal cancer proliferation by regulating transaminases.

Hexafluoro-2-propanol (HFIP) is a metabolite of sevoflurane used as part of the general anaesthesia technique. This study aims to investigate the effect of HFIP in colorectal cancer (CRC) and the regulation of associated genes. The differential expressed genes (DEGs) with HFIP treatment were analysed based on GEO dataset GSE56256. Due to the restricted number of studies performing RNA-Seq in CRC with HFIP treatment, we selected a CRC patient cohort (GSE23878) to obtain DEGs that were compared with DEGs from GSE56256. The DEGs with opposite expression patterns in these two GEO datasets indicate that the genes mediate the function of HFIP, thereby repress the progression of CRC. The DEGs from these two GEO datasets were analysed independently. We obtained 10 up-regulated genes in GSE23878 (CRC patient cohort) were also down-regulated in GSE56256 (HFIP cohort), therefore, these 10 genes may function as tumor suppressors in CRC and may be involved in the function of HFIP. STRING analysis demonstrated an interaction in GPT2, PSAT1 and SLC7A11. The high expression of GPT2, PSAT1 and SLC7A11 were confirmed in TCGA-COAD datasets and in CRC cells. HFIP treatment repressed GPT2, PSAT1 and SLC7A11, which resulted in an inhibited proliferation and colony formation ability of DLD-1 and HCT-116 cells. Silencing GPT2, PSAT1 and SLC7A11 showed similar effect compared to HFIP treatment. Overexpression of GPT2, PSAT1 and SLC7A11 abrogated the effect of HFIP. In conclusion, the sevoflurane metabolite HFIP plays a cancer suppression role depending on cell proliferation in colorectal cancer through the down-regulation of GPT2, PSAT1 and SLC7A11 expression.

Long non-coding RNA CCL14-AS suppresses invasiveness and lymph node metastasis of colorectal cancer cells by regulating MEP1A

BackgroundLong non-coding RNAs (lncRNAs) play important roles in the biology of colorectal cancer (CRC). There are several lncRNAs associated with invasion and metastasis have been characterized in CRC. However, studies focusing on the precise molecular mechanisms by which lncRNAs function in lymph node (LN) metastasis in CRC are still limited.MethodsIn this study, by analyzing TCGA dataset, we identified that AC244100.2 (termed CCL14-AS), a novel lncRNA enriched in the cytoplasm, was negatively correlated with LN metastasis and unfavorable prognosis of CRC. In situ hybridization was used to examine CCL14-AS expression in clinical CRC tissues. Various functional experiments including migration assay and wound-healing assay were used to investigate the effects of CCL14-AS on CRC cells migration. The nude mice popliteal lymph node metastasis model assay further confirmed the effects of CCL14-AS in vivo.ResultsCCL14-AS expression was significantly downregulated in CRC tissues compared to adjacent normal tissues. In addition, low CCL14-AS expression was correlated with advanced T classification, LN metastasis, distant metastasis, and shorter disease-free survival of CRC patients. Functionally, CCL14-AS overexpression inhibited the invasiveness of CRC cells in vitro and LN metastasis in nude mice. On the contrary, knockdown of CCL14-AS promoted the invasiveness and LN metastasis abilities of CRC cells. Mechanistically, CCL14-AS downregulated the expression of MEP1A via interacting with MEP1A mRNA and reduced its stability. Overexpression of MEP1A rescued the invasiveness and LN metastasis abilities in CCL14-AS-overexpressing CRC cells. Moreover, the expression levels of CCL14-AS was negatively correlated with that of MEP1A in CRC tissues.ConclusionsWe identified a novel lncRNA, CCL14-AS, as a potential tumor suppressor in CRC. Our findings supported a model in which the CCL14-AS/MEP1A axis serves as critical regulator in CRC progression, suggesting a novel biomarker and therapeutic target in advanced CRC.

Neogenin suppresses tumor progression and metastasis via inhibiting Merlin/YAP signaling

From in situ growth to invasive dissemination is the most lethal attribute of various tumor types. This transition is majorly mediated by the dynamic interplay between two cancer hallmarks, EMT and cell cycle. In this study, we applied nonlinear association analysis in 33 cancer types and found that most signaling receptors simultaneously associating with EMT and cell cycle are potential tumor suppressors. Here we find that a top co-associated receptor, Neogenin (NEO1), inhibits colorectal cancer (CRC) and Glioma in situ growth and metastasis by forming a complex with Merlin (NF2), and subsequent simultaneous promoting the phosphorylation of YAP. Furthermore, Neogenin protein level is associated with good prognosis and correlates with Merlin status in CRC and Glioma. Collectively, our results define Neogenin as a tumor suppressor in CRC and Glioma that acts by restricting oncogenic signaling by the Merlin-YAP pathway, and suggest Neogenin as a candidate therapeutic agent for CRC and Glioma.

EPLIN, a Putative Tumour Suppressor in Colorectal Cancer, Implications in Drug Resistance

Colorectal cancer is a serious threat to human health. Poor prognosis and frequently reported drug resistance urges research into novel biomarkers and mechanisms to aid in the understanding of the development and progression of colorectal cancer and to optimise therapeutic strategies. In the current study, we investigated the roles of a putative tumour suppressor, EPLIN, in colorectal cancer. Our clinical colorectal cancer cohort and online databases revealed a downregulation of EPLIN in colorectal cancer tissues compared with normal tissues. The reduced expression of EPLIN was associated with poor clinical outcomes of patients. In vitro cellular function assays showed that EPLIN elicited an inhibitory effect on cellular growth, adhesion, migration and invasion. Utilising a protein microarray on protein samples from normal and tumour patient tissues suggested HSP60, Her2 and other signalling events were novel potential interacting partners of EPLIN. It was further revealed that EPLIN and HSP60 were negative regulators of Her2 in colorectal cancer cells. The clinical cohort also demonstrated that expression of HSP60 and Her2 affected clinical outcomes, but most interestingly the combination of EPLIN, HSP60 and Her2 was able to identify patients with the most unfavourable clinical outcome by independently predicting patient overall survival and disease free survival. Furthermore, EPLIN and HSP60 exhibited potential to regulate cellular response to chemotherapeutic and EGFR/Her2 targeted therapeutic agents. In conclusion, EPLIN is an important prognostic factor for patients with colon cancer and reduced EPLIN in CRC contributes to aggressive traits of CRC cells and their responses to chemotherapeutic drugs. Collectively, EPLIN is a pivotal factor for the development and progression of colorectal cancer and has important clinical and therapeutic values in this cancer type.

Abstract A027: The oncogenic mutation BRAF-V600E is associated with colorectal cancers expressing low levels of CFTR mRNA

Abstract Background and purpose: The cystic fibrosis transmembrane conductance regulator (CFTR) gene is a tumor suppressor in colorectal cancer (CRC). People with cystic fibrosis, caused by biallelic germline mutations in CFTR, have a higher risk developing CRC. We also found that loss of CFTR is implicated in sporadic CRC. In a study of 90 persons diagnosed with stage II CRC, the 23% of patients with lowest CFTR expression had 30% lower disease-free survival at 3 years. The cause of decreased CFTR expression in this group is not known. We analyzed the TCGA COADREAD study to identify associations between low expression of CFTR and cancer-causing genetic alterations. Of these alterations, only BRAF-V600E mutation correlated with low expression of CFTR. The BRAF-V600E mutation is found in ~10% of CRC and is associated with global promoter hypermethylation leading to down regulation of tumor suppressor genes. Accordingly, our hypothesis was that BRAF-V600E mutation contributes to low expression of CFTR via CFTR promoter DNA methylation. Methods: In the TCGA COADREAD database 283 primary colorectal cancers diagnosed at stages II, III and IV had full information needed for this study: CFTR mRNA expression, somatic mutation, overall survival (OS), and DNA methylation status. OS was visualized using Kaplan-Meier analysis. Association between CFTR expression and methylation was determined by Pearson correlation coefficient (PCC) analysis. Associations between mutations and CFTR expression or methylation were determined by Point Biserial correlation coefficient (PBS) analysis. Results: Kaplan-Meier analysis of the 283 cases showed that overall survival was worse in the 25% of cases with lowest CFTR expression (p=0.035). The BRAF-V600E mutation was significantly associated with lower CFTR expression (PBS r=-0.38, p&amp;lt;0.0001) and 81% of BRAF V600E mutations were found in the 25% of cases with lowest CFTR expression. In contrast mutations in APC and KRAS showed a positive association with CFTR expression (PBS r=0.25, p&amp;lt;0.0001; PBS r=0.13, p=0.028 respectively). The CFTR promoter is hypermethylated in several cancers and BRAF-V600E mutation is associated with aberrant promoter DNA methylation. We examined DNA methylation at 9 CpG sites in the CFTR promoter and found that low CFTR expression significantly associated with increased DNA methylation at these 9 sites (PCC for average of 9 sites r= -0.56, p=&amp;lt;0.001). The association between BRAF-V600E mutation and CFTR methylation was modest but significant (PBS r=0.16, p=0.0063). Conclusions: BRAF-V600E mutation was significantly associated with CRC cases expressing low levels of CFTR, a subset with poor overall survival, as was CFTR promoter methylation. BRAF-V600E showed a modest association with CFTR promoter methylation and so may play a role in down-regulation of CFTR expression. However, association of BRAF-V600E with CFTR promoter methylation was modest compared to its association with CFTR expression suggesting that BRAF-V600E mutation may play additional roles in CRC tumors expressing low levels of CFTR. Citation Format: Patricia Scott, Anna Prizment, Rahul Bhattacharya, Zachary Blankenheim, Nathan Pankratz, Timothy Starr, Robert Cormier. The oncogenic mutation BRAF-V600E is associated with colorectal cancers expressing low levels of CFTR mRNA [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr A027.

Abstract A031: Heterozygote carriers of cystic fibrosis mutations have increased risk of colorectal cancer

Abstract Background and purpose: Colorectal cancer (CRC) is the third leading cause of cancer deaths in the U.S. Establishing new genetic variants associated with CRC risk may lead to early CRC detection and novel treatment options. Recently, mouse genetic studies identified the cystic fibrosis transmembrane conductance regulator (CFTR) gene as a tumor suppressor in CRC. In line with this, a human study using MarketScan claims data showed that cystic fibrosis (CF) carriers – individuals with one copy of mutant CFTR – are at increased CRC risk. Although there are more than 10 million CF carriers in the U.S., to our knowledge, no studies comprehensively examined CFTR mutations in relation to CRC risk. Our hypothesis is that CFTR mutations are associated with an increased risk of CRC. Methods: We used whole exome sequencing (WES) data in 454,333 individuals (age 44-82 years), including 4,975 cases of CRC, in the UK Biobank study. We utilized risk set sampling to select up to four controls per CRC case based on age of diagnosis, gender, race, and year of joining the study. To examine associations between CFTR mutations and CRC risk, logistic regression (odds ratio and p-value) was used after adjusting for age, gender and 10 principal components capturing global ancestry. An aggregate burden score was constructed that included all variants listed as CF-causing in the CFTR2 online database. Results: Of the 465 CFTR variants in the CFTR2 database, 378 are classified as CF-causing, and of those 153 were observed in the UK Biobank WES data set. Individuals with two CF-causing alleles were excluded from further analysis (8 cases and 35 controls), since the association between CF and CRC is already well established. The frequency of the F508del variant, the most common CF-causing mutation, was 3.77% in cases versus 3.11% in controls, while the frequency of any other CF-causing mutation was 1.07% in cases versus 0.80% in controls. Having any CF-causing mutation was associated with a significant 24% increased CRC risk (one-sided p=0.002). Having one copy of the F508del mutation, was associated with a significant 21% increased risk of CRC (one-sided p=0.012), while having one copy of any other CF-causing mutation was associated with a slightly larger 34% increased risk of CRC (one-sided p=0.036). Conclusions: CF-causing mutations in the heterozygous state lead to a significantly increased risk of CRC. Individuals with multiple genetic risk factors, such as in CFTR, may require earlier screening for CRC. Citation Format: Anna E. Prizment, Abigail Standafer, Shuo Wang, Conghui Qu, Ulrike Peters, Patricia Scott, Timothy Starr, Nathan Pankratz. Heterozygote carriers of cystic fibrosis mutations have increased risk of colorectal cancer [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr A031.

M6A-modified circFNDC3B inhibits colorectal cancer stemness and metastasis via RNF41-dependent ASB6 degradation

Colorectal cancer (CRC) is the third most frequently diagnosed cancer with unfavorable clinical outcomes worldwide. circFNDC3B plays as a tumor suppressor in CRC, however, the mechanism of circFNDC3B in CRC remains ambiguous. The stem-like properties of CRC cells were detected by the evaluation of stemness markers, sphere formation assay and flow cytometry. qRT-PCR, FISH, IHC, and western blotting assessed the expression and localization of circFNDC3B, RNF41, ASB6, and stemness markers in CRC. The metastatic capabilities of CRC cells were examined by wound healing and Transwell assays, as well as in vivo liver metastasis model. Bioinformatics analysis, RNA immunoprecipitation (RIP), RNA pull-down assay and co-IP were used to detect the associations among circFNDC3B, FXR2, RNF41, and ASB6. Downregulated circFNDC3B was associated with unfavorite survival in CRC patients, and circFNDC3B overexpression suppressed CRC stemness and metastasis. Mechanistically, studies revealed that YTHDC1 facilitated cytoplasmic translocation of m6A-modified circFNDC3B, and circFNDC3B enhanced RNF41 mRNA stability and expression via binding to FXR2. circFNDC3B promoted ASB6 degradation through RNF41-mediated ubiquitination. Functional studies showed that silencing of RNF41 counteracted circFNDC3B-suppressed CRC stemness and metastasis, and ASB6 overexpression reversed circFNDC3B- or RNF41-mediated regulation of CRC stemness and metastasis. Elevated ASB6 was positively correlated with unfavorite survival in CRC patients. In vivo experiments further showed that circFNDC3B or RNF41 overexpression repressed tumor growth, stemness and liver metastasis via modulating ASB6. Taken together, m6A-modified circFNDC3B inhibited CRC stemness and metastasis via RNF41-dependent ASB6 degradation. These findings provide novel insights and important clues for targeted therapeutic strategies of CRC.

Long Non-Coding RNA CASC2 Functions as A Tumor Suppressor in Colorectal Cancer via Modulating The miR-18a-5p/BTG3 Pathway.

Reportedly, long non-coding RNA (lncRNA) cancer susceptibility candidate 2 (CASC2) is involved in regulating colorectal cancer (CRC) progression. However, the function and detailed downstream mechanism of CASC2 in CRC progression are not fully elucidated. The aim of the study was to investigate the potential function and molecular mechanism of CASC2 in CRC progression. In this experimental study, quantitative real-time polymerase chain reaction (qRT-PCR) was adopted to probe CASC2, microRNA-18a-5p (miR-18a-5p) and B cell translocation gene 3 (BTG3) mRNA expression in CRC tissues and cell lines. After CASC2 was overexpressed in Colo-678 and HCT116 cell lines, methylthiazol tetrazolium (MTT) and 5-bromo-2'-deoxyuridine (BrdU) assays were employed to examine the proliferation of CRC cells. Transwell migration and invasion assays were executed to evaluate the metastatic potential of CRC cells. The targeting relationships among CASC2, miR-18a-5p and BTG3 were validated by dual luciferase reporter gene assay. Western blot assay was applied to examine the regulatory effects of CASC2 and miR-18a-5p on BTG3 protein expression. CASC2 was decreased in CRC tissues and cell lines, and its low expression in CRC tissues was associated with larger tumor size and lymph node metastasis. CASC2 overexpression restrained proliferative, migrative and invasive capabilities of CRC cells. CASC2 could function as a molecular sponge for miR-18a-5p and repress the expression of miR-18a-5p. Furthermore, the inhibitory effects of CASC2 on the malignant phenotypes of CRC cells was counteracted by miR-18a-5p mimics. Additionally, CASC2 could positively regulate BTG3 expression via suppressing miR-18a-5p. CASC2 inhibits CRC development by suppressing miR-18a-5p and raising BTG3 expression.

Genome-wide screening for differentially methylated long noncoding RNAs identifies LIFR-AS1 as an epigenetically regulated lncRNA that inhibits the progression of colorectal cancer

BackgroundAberrant DNA methylation is an epigenetic marker that has been linked to the pathogenesis of colorectal cancer (CRC). Long noncoding RNAs (lncRNAs) have been increasingly identified to be associated with tumorigenic processes of CRC. Identifying epigenetically dysregulated lncRNAs and characterizing their effects during carcinogenesis are focuses of cancer research.MethodsDifferentially methylated loci and expressed lncRNAs were identified by integrating DNA methylome and transcriptome analyses using The Cancer Genome Atlas database. Bisulfite sequencing PCR (BSP) was performed to analyze LIFR-AS1 promoter methylation status. The functional roles of LIFR-AS1 in CRC were determined by in vitro and in vivo experiments.ResultsWe identified a novel hypermethylated lncRNA, LIFR-AS1, that was downregulated and associated with tumorigenesis, metastasis, and poor prognosis in CRC. High methylation burden of LIFR-AS1 indicated a poor survival of CRC patients. Promoter hypermethylation of LIFR-AS1 in tumor tissues was confirmed by BSP. Functional assays revealed that LIFR-AS1 could competitively bind to hsa-miR-29b-3p, and repressed colon cancer cell proliferation, colony formation and invasion. LIFR-AS1 also inhibited tumor growth in a mouse xenograft model of CRC.ConclusionsOur results showed that the identified DNA methylation-dysregulated lncRNAs may be potential biomarkers and highlighted a role for LIFR-AS1 as a tumor suppressor in CRC.