Methylation of TMEM176A is an independent prognostic marker and is involved in human colorectal cancer development.
ABSTRACT: Colorectal cancer (CRC) is the third most common malignancy and the fourth most common cause of cancer related death worldwide. This study was designed to find tumor suppressors involved in CRC development by performing RNA-seq. Eight CRC cell lines and 130 cases of primary CRC samples were used. RNA-seq, methylation-specific PCR (MSP), flow cytometry, transwell assays, and a xenograft mouse model were used. Reduction of TMEM176A expression was confirmed in human CRC cells by RNA-seq. TMEM176A was expressed in LS180 and SW620 cells, loss of TMEM176A expression was observed in LOVO, HCT116, RKO, and DLD1 cells, and reduced TMEM176A expression was found in HT29 and SW480 cells. Unmethylation of the TMEM176A promoter was found in LS180 and SW620 cells, whereas complete methylation was found in LOVO, HCT116, RKO, and DLD1 cells, and partial methylation was found in HT29 and SW480 cells. Promoter region methylation correlated with loss of/reduced expression of TMEM176A. Re-expression of TMEM176A was induced by 5-aza-2'-deoxycytidine. TMEM176A was methylated in 50.77% of primary colorectal cancers. Methylation of TMEM176A was associated with tumor metastasis (P<0.05) and was an independent prognostic factor for 5-year overall survival (OS) according to Cox proportional hazards model analysis (P<0.05). TMEM176A induced apoptosis and inhibited cell migration and invasion in CRC cells. TMEM176A suppressed CRC cell growth both in vitro and in vivo. Our results suggest that expression of TMEM176A is regulated by promoter region methylation. TMEM176A methylation is an independent prognostic marker for 5-year OS in CRC, and may act as a tumor suppressor in CRC.
Project description:DIRAS1 is a new member of the Ras gene family. It was described as a potential tumor suppressor in human glioblastomas and esophageal cancer. The role of DIRAS1 in colorectal cancer remains unclear.To explore the epigenetic changes and function of DIRAS1 in human colorectal cancer, we studied ten colorectal cancer cell lines and 146 primary colorectal cancer samples and 50 matched adjacent samples using semi-quantitative reverse transcription PCR, immunohistochemistry, methylation-specific PCR and bisulfite sequencing, western blot, flow cytometry, and transwell assays.DIRAS1 expression was found in DKO and HCT116 cells, while reduced expression was detected in LoVo, SW48, LS180, and SW620 cells, and there was no expression detected in DLD1, HT29, RKO, and SW480 cells. Complete methylation was found in the promoter region of DLD1, HT29, RKO, and SW480 cells. Partial methylation was detected in LoVo, LS180, SW48, and SW620 cells, and unmethylation was found in DKO and HCT116 cells. These results indicate that promoter region methylation correlated with loss of/reduced expression of DIRAS1. Re-expression of DIRAS1 was induced by 5-aza-2'-deoxycytidine, suggesting that the expression of DIRAS1 is regulated by promoter region methylation. DIRAS1 was methylated in 47.3% (69/146) of primary colorectal cancer samples, no methylation was found in non-cancerous colonic tissue samples. Methylation of DIRAS1 was significantly associated with TNM stage (P?<?0.05) and short survival time (P?=?0.0121). DIRAS1 induced apoptosis and inhibited cell proliferation, migration, and invasion in colorectal cancer. Finally, DIRAS1 suppressed colorectal cancer cell xenograft growth in nude mice.DIRAS1 is frequently methylated in human colorectal cancer and the expression of DIRAS1 is regulated by promoter region methylation. Methylation of DIRAS1 is a marker of poor prognosis in human colorectal cancer.
Project description:Aberrant activation of the canonical WNT signaling is a feature of colorectal cancer (CRC). Van-Gogh-like 2 (VANGL2) belongs to the non-canonical WNT pathway whose activation inhibits canonical WNT signaling. In this study, we investigated the role of VANGL2 and its epigenetic regulation in CRC.Van-Gogh-like 2 expression and promoter methylation after 5-aza-2'-deoxycytidine (5-aza) treatment were evaluated in CRC cells. DNA samples from 418 sporadic CRCs were tested for VANGL2 promoter methylation and microsatellite instability (MSI). Proliferation, colony formation and activation of the WNT pathway were tested in cells after VANGL2 overexpression.Van-Gogh-like 2 mRNA was significantly higher in 5-aza-treated RKO, LOVO and SW48, whereas no differences were found in SW480. Van-Gogh-like 2 was fully methylated in RKO, SW48, HCT116, DLD1 and Caco2; partially methylated in LOVO, LS174T and SW837; and unmethylated in SW480, SW620 and HT29. Higher expression of VANGL2 mRNA was found in the unmethylated cell lines. In CRC specimens (8.93% MSI), methylated VANGL2 was associated with MSI, higher grade, proximal colon location and BRAF mutation. Van-Gogh-like 2 overexpression in SW480 significantly decreased proliferation, colony formation and ?-catenin levels.Van-Gogh-like 2 is frequently methylated in MSI-CRCs with BRAF mutation and may act as a tumour suppressor gene, counteracting WNT/?-catenin signaling.
Project description:MicroRNAs (miRNAs/miRs) are small non-coding RNAs that serve a post-transcriptional regulatory role in eukaryotes. Previous studies have demonstrated that the expression of miR-34a in colorectal cancer (CRC) tissues is decreased compared with that in normal colorectal tissues. However, the role of miR-34a in the invasion and metastasis of CRC remains unclear. In the present study, the levels of miR-34a expression were measured in various CRC cell lines. The cells were transfected with miR-34a mimics or inhibitors in order to assess the proliferation rate, and the colony forming, invasive and migratory abilities. Furthermore, the protein expression levels of vimentin and early growth response protein 1 (EGR1) were examined by western blot analysis. The results revealed that the expression of miR-34a was low in SW620, RKO, LoVo and Caco-2 cell lines and high in the SW480 and SW1116 cell lines. The migration, invasion and proliferation levels of SW480 cells were facilitated by decreasing the expression of miR-34a. Transient transfection with miR-34a mimics in SW620 cells caused a notable decrease in cell migration, invasion and proliferation levels compared with the control group, and a downregulation of vimentin and upregulation of EGR1 protein expression. The present study demonstrated that miR-34a was deregulated in a highly invasive CRC cell lines, and that it may attenuate the migratory, invasive and proliferative capabilities of CRC cells by enhancing the expression of EGR1 and inhibiting that of vimentin. The results of the present study represent important progress towards understanding the mechanisms of CRC recurrence and metastasis.
Project description:Human colon cancer cell lines (CRCs) RKO, SW480 and SW620 were investigated for TG2 involvement in tumour advancement and aggression. TG2 expression correlated with tumour advancement and expression of markers of epithelial-mesenchymal transition (EMT). The metastatic cell line SW620 showed high TG2 expression compared to the primary tumour cell lines SW480 and RKO and could form tumour spheroids under non- adherent conditions. TG2 manipulation in the CRCs by shRNA or TG2 transduction confirmed the relationship between TG2 and EMT. TGF?1 expression in CRC cells, and its level in the cell medium and extracellular matrix was increased in primary tumour CRCs overexpressing TG2 and could regulate TG2 expression and EMT by both canonical (RKO) and non-canonical (RKO and SW480) signalling. TGF?1 regulation was not observed in the metastatic SW620 cell line, but TG2 knockdown or inhibition in SW620 reversed EMT. In SW620, TG2 expression and EMT was associated with increased presence of nuclear ?-catenin which could be mediated by association of TG2 with the Wnt signalling co-receptor LRP5. TG2 inhibition/knockdown increased interaction between ?-catenin and ubiquitin shown by co-immunoprecipitation, suggesting that TG2 could be important in ?-catenin regulation. ?-Catenin and TG2 was also upregulated in SW620 spheroid cells enriched with cancer stem cell marker CD44 and TG2 inhibition/knockdown reduced the spheroid forming potential of SW620 cells. Our data suggests that TG2 could hold both prognostic and therapeutic significance in colon cancer.
Project description:5-Fluorouracil (5-FU)-based chemotherapy is widely used for the treatment of colorectal cancer (CRC). While optimal doses of 5-FU are generally established based on a patient's estimated body surface area, the plasma concentrations of 5-FU vary among patients. In addition, hyperglycemia in patients with CRC has been reported as a risk factor in poor prognosis. The aim of the present study was to investigate whether hyperglycemia affects antiproliferative effect of 5-FU on the human colon cancer cells (SW480, SW620, LoVo, and HCT116). Growth inhibition of 5-FU was accessed by WST-8 assay. The effect of high glucose (HG, 15?mM) and 5-FU on the cellular proliferation was evaluated by flow cytometry analysis using 5-ethynyl-2'-deoxy-uridine (EdU) incorporation plus 7-AAD. Cell death was determined by flow cytometry using Annexin V-FITC and PI. The results showed that HG, compared to physiological normal glucose (NG) concentration (5?mM), leads to increased cell proliferation and increased GI50 of 5-FU in the four colon cancer cell lines. When the cells were pretreated with a low-dose 5-FU in NG condition, subsequent HG treatment eliminated inhibitory effect of 5-FU in cancer cell growth. In the presence of 5-FU (0.5??g/mL for LoVo and HCT116; 1??g/mL for SW480 and SW620), culture with HG for 72?h does not significantly altered cell cycle profile in the four cell lines but significantly increased DNA replication in SW620 (21%) and LoVo (17%). Flow cytometric analysis showed that HG protects cells against 5-FU-induced cell death in SW480. Finally, HG did not alter intracellular level of reactive oxygen species (ROS), although 5-FU indeed induced higher intracellular level of ROS. In conclusion, HG attenuates growth inhibition of 5-FU and our results indicate that decreased cell death and increased DNA replication may account for the attenuating effect of a HG environment on 5-FU-induced tumor growth inhibition.
Project description:A correlation of TCF12 mRNA overexpression with colorectal cancer (CRC) metastasis was suggested by microarray data and validated by the survey of 120 patients. Thirty-three (27.5%) of the 120 patients showed tumor TCF12 mRNA overexpression and had a higher rate of metastatic occurrence (p = 0.020) and a poorer survival outcome (p = 0.014). Abundant TCF12 levels were also observed in human CRC cell lines such as SW620 and LoVo, but a relatively low level was detected in SW480 cells. Knockdown of TCF12 expression in SW620 and LoVo cells drastically reduced their activities of migration, invasion, and metastasis. Tight cell-cell contact and an increase in E-cadherin but a concomitant decrease in fibronectin were observed in TCF12-knockdown cells. Connexin 26, connexin 43, and gap-junction activity were also increased upon TCF12-knockdown. In contrast, ectopic TCF12 overexpression in SW480 cells facilitated fibronectin expression and cell migration and invasion activities but diminished cellular levels of E-cadherin, connexin 26, connexin 43, and gap junction. A physical association of TCF12 with the E-cadherin promoter was evidenced by chromatin immunoprecipitation assay. TCF12 was tightly correlated with cellular expression of Bmi1 and EZH2 and was co-immunoprecipitable with Bmi1 and EZH2, suggesting that TCF12 transcriptionally suppressed E-cadherin expression via polycomb group-repressive complexes. Clinically, TCF12 mRNA overexpression was also correlated with E-cadherin mRNA down-regulation in the tumor tissues of our 120 patients (p = 0.013). These studies suggested that TCF12 functioned as a transcriptional repressor of E-cadherin and its overexpression was significantly correlated with the occurrence of CRC metastasis.
Project description:EGFR-targeted therapy is a key treatment approach in patients with RAS wildtype metastatic colorectal cancers (CRC). Still, also RAS wildtype CRC may be resistant to EGFR-targeted therapy, with few predictive markers available for improved stratification of patients. Here, we investigated response of 7 CRC cell lines (Caco-2, DLD1, HCT116, HT29, LS174T, RKO, SW480) to Cetuximab and correlated this to NGS-based mutation profiles, EGFR promoter methylation and EGFR expression status as well as to E-cadherin expression. Moreover, tissue specimens of primary and/or recurrent tumors as well as liver and/or lung metastases of 25 CRC patients having received Cetuximab and/or Panitumumab were examined for the same molecular markers. In vitro and in situ analyses showed that EGFR promoter methylation and EGFR expression as well as the MSI and or CIMP-type status did not guide treatment responses. In fact, EGFR-targeted treatment responses were also observed in RAS exon 2 p.G13 mutated CRC cell lines or CRC cases and were further linked to PIK3CA exon 9 mutations. In contrast, non-response to EGFR-targeted treatment was associated with ATM mutations and low E-cadherin expression. Moreover, down-regulation of E-cadherin by siRNA in otherwise Cetuximab responding E-cadherin positive cells abrogated their response. Hence, we here identify ATM and E-cadherin expression as potential novel supportive predictive markers for EGFR-targeted therapy.
Project description:Asporin has been implicated as an oncogene in various types of human cancers; however, the roles of asporin in the development and progression of colorectal cancer (CRC) have not yet been determined. With clinical samples, we found that asporin was highly expressed in CRC tissues compared to adjacent normal tissues and the asporin expression levels were significantly associated with lymph node metastasis status and TNM stage of the patients. Through knockdown of asporin in CRC cell lines RKO and SW620 or overexpression of asporin in cell lines HT-29 and LoVo, we found that asporin could enhance wound healing, migration and invasion abilities of the CRC cells. Further more, with the human umbilical vein endothelial cells (HUVECs) tube formation assays and the xenograft model, we found that asporin promoted the tumor growth through stimulating the VEGF signaling pathway. The portal vein injection models suggested that asporin overexpression stimulated the liver metastasis of HT29 cell line, while asporin knockdown inhibited the liver metastasis of RKO cell line. In addition, asporin was found to augment the phosphorylation of EGFR/src/cortactin signaling pathway, which might be contributed to the biological functions of asporin in CRC metastasis. These results suggested that asporin promoted the tumor growth and metastasis of CRC, and it could be a potential therapeutic target for CRC patients in future.
Project description:Colorectal carcinoma (CRC) recurrence is often accompanied by metastasis. Most metastasis undergo through epithelial-mesenchymal transition (EMT). Studies showed that retinol X receptor alpha (RXR?) and 20(S)-Protopanaxadiol (PPD) have anti-tumour effects. However, the anti-metastasis effect of 20(S)-PPD and the effect of RXR? on EMT-induced metastasis are few studies on. Therefore, the role of RXR? and 20(S)-PPD in CRC cell metastasis remains to be fully elucidated. RXR? with clinicopathological characteristics and EMT-related expression in clinical samples were examined. Then, RXR? and EMT level in SW480 and SW620 cells, overexpressed and silenced RXR? in SW620 cells and SW480 cells, respectively, were evaluated. Finally, 20(S)-PPD effect on SW620 and SW480 cells was evaluated. The results showed that a lower RXR? expression in cancer tissues, and a moderate negative correlation between RXR? and N stage, and tended to higher level of EMT. SW480 and SW620 cells had the highest and lowest RXR? expression among four CRC cell lines. SW480 had lower EMT level than SW620. Furthermore, 20(S)-PPD increased RXR? and inhibited EMT level in SW620 cell. Finally, 20(S)-PPD cannot restore SW480 cells EMT level to normal when RXR? silencing. These findings suggest that 20(S)-PPD may inhibit EMT process in CRC cells by regulating RXR? expression.
Project description:Background:An increasing number of studies demonstrate that long non-coding RNAs (lncRNAs) are regulators in cancer biology. Nevertheless, the expression and mechanism of LINC01089 in colorectal cancer (CRC) remain unclear. Methods:Quantitative real-time polymerase chain reaction (qRT-PCR) was taken to investigate the expression levels of LINC01089 and miR-27b-3p in CRC tissues and cells. MTT method and transwell test were employed to assess the proliferation and invasion of CRC cells, respectively. Dual-luciferase activity reporter assay, RNA immunoprecipitation assay, Pearson's correlation analysis, and Western blot were performed to investigate the regulatory mechanism of LINC01089/miR-27b-3p/HOXA10 axis in CRC. Results:LINC01089 was down-regulated in CRC tissues and cell lines. LINC01089 overexpression impeded the proliferation and invasion of SW620 and LoVo cells, whereas LINC01089 knockdown increased the malignancy of SW480 and HT29 cells. Moreover, LINC01089 directly interacted with miR-27b-3p to repressed its expression and indirectly promoted the expression of HOXA10. Conclusion:LINC01089 impedes the proliferation and invasion of colorectal cancer cells by adsorbing miR-27b-3p and up-regulating the expression of HOXA10.