Epigenetic Regulation of a Disintegrin and Metalloproteinase (ADAM) Transcription in Colorectal Cancer Cells: Involvement of ?-Catenin, BRG1, and KDM4.
ABSTRACT: A disintegrin and metalloproteinase (ADAM) family of proteins play versatile roles in cancer development and progression. In the present study, we investigated the role of ADAM proteins in colorectal cancer (CRC) cell migration and invasion focusing on the epigenetic mechanism whereby ADAM transcription is regulated. We report that higher levels of ADAM10, ADAM17, and ADAM19 were detected in SW480 cells than in HCT116 cells. Expression levels of the same set of ADAMs were higher in human CRC biopsy specimens of advanced stages than in those of a less aggressive phenotype. Overexpression of ADAM10/17/19 in HCT116 cells enhanced, whereas depletion of ADAM10/17/19 in SW480 cells weakened, migration and invasion. ADAM expression was activated by the Wnt signaling pathway, which could be attributed to direct binding of ?-catenin on the ADAM promoters. Mechanistically, ?-catenin recruited the chromatin remodeling protein BRG1, which in turn enlisted histone demethylase KDM4 to alter the chromatin structure, thereby leading to ADAM transactivation. In conclusion, our data suggest that the Wnt signaling may promote CRC metastasis, at least in part, by recruiting an epigenetic complex to activate ADAM transcription.
Project description:Mutational inactivation of adenomatous polyposis coli (APC) is an early event in colorectal cancer (CRC) progression that affects the stability and increases the activity of ?-catenin, a mediator of Wnt signaling. Progression of CRC also involves inactivation of signaling via transforming growth factor ? and bone morphogenetic protein (BMP), which are tumor suppressors. However, the interactions between these pathways are not clear. We investigated the effects of loss of the transcription factor Smad4 on levels of ?-catenin messenger RNA (mRNA) and Wnt signaling.We used microarray analysis to associate levels of Smad4 and ?-catenin mRNA in colorectal tumor samples from 250 patients. We performed oligonucleotide-mediated knockdown of Smad4 in human embryonic kidney (HEK293T) and in HCT116 colon cancer cells and transgenically expressed Smad4 in SW480 colon cancer cells. We analyzed adenomas from (APC(?1638/+)) and (APC(?1638/+)) × (K19Cre(ERT2)Smad4(lox/lox)) mice by using laser capture microdissection.In human CRC samples, reduced levels of Smad4 correlated with increased levels of ?-catenin mRNA. In Smad4-depleted cell lines, levels of ?-catenin mRNA and Wnt signaling increased. Inhibition of BMP or depletion of Smad4 in HEK293T cells increased binding of RNA polymerase II to the ?-catenin gene. Expression of Smad4 in SW480 cells reduced Wnt signaling and levels of ?-catenin mRNA. In mice with heterozygous disruption of Apc(APC(?1638/+)), Smad4-deficient intestinal adenomas had increased levels of ?-catenin mRNA and expression of Wnt target genes compared with adenomas from APC(?1638/+) mice that expressed Smad4.Transcription of ?-catenin is inhibited by BMP signaling to Smad4. These findings provide important information about the interaction among transforming growth factor ?, BMP, and Wnt signaling pathways in progression of CRC.
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:Colorectal cancer (CRC) is the third most common cancer in the world. Oplopanax elatus is widely used in traditional medicine. However, little is known about its pharmacological effects and bioactive compounds. We evaluated the effects of the polyyne-enriched extract from O. elatus (PEO) on the progression of colon carcinogenesis in ApcMin/+ mice. In addition, these effects were also investigated in HCT116 and SW480 cells. After PEO oral administration (0.2% diet) for 12 weeks, PEO significantly improved body weight changes and reduced the tumor burden and tumor multiplicity compared with the untreated mice. Meanwhile, western blot and immunohistochemistry results showed PEO significantly reduced the expression of ?-catenin and cyclinD1 in both small intestine and the colon tissues compared with the untreated mice. In addition, PEO treatment significant decreased the cell viability in both HCT116 and SW480 cell lines. It also decreased the levels of ?-catenin, cyclinD1, c-myc and p-GSK-3? in HCT116 and SW480 cells at 25 ?M. These results indicate that PEO may have potential value in prevention of colon cancer by down-regulating Wnt-related protein.
Project description:Wnt signaling pathway is aberrantly activated in a variety of cancers, especially in colorectal cancer and small molecule antagonists of Wnt/?-catenin signaling are attractive candidates for developing effective therapeutics. In the present study, we identified Bisleuconothine A, a bisindole alkaloid with an eburnane-aspidosperma type skeleton, as a novel and selective Wnt signaling inhibitor by using a cell-based luciferase assay system. Our study found that Bisleuconothine A down-regulated the endogenous Wnt target gene expression through promoting phosphorylation of ?-catenin and the subsequent inhibition of its nuclear translocation in HCT116 and SW480 colorectal cancer cells. In vitro, Bisleuconothine A inhibited cell proliferation through induction of apoptosis by increasing the cleavage of caspases in HCT116 and SW480 colorectal cancer cells. Moreover, in vivo, Bisleuconothine A dramatically suppressed tumor growth in HCT116 Xenograft. And further analysis showed that Bisleuconothine A suppressed the Wnt target gene expression in HCT116 Xenograft, which was associated with up-regulation of ?-catenin phosphorylation and subsequent Wnt signaling inhibition. Taken together, our study indicated that bisindole alkaloids could be included as a new chemotype of small-molecule Wnt signaling inhibitors, and have great potential to be further developed for anti-tumor agents.
Project description:This investigation was aimed at working out the combined role of lncRNA H19, miR-29b and Wnt signaling in the development of colorectal cancer (CRC). In the aggregate, 185 CRC tissues and corresponding para-carcinoma tissues were gathered. The human CRC cell lines (i.e. HT29, HCT116, SW480 and SW620) and normal colorectal mucosa cell line (NCM460) were also purchased. Si-H19, si-NC, miR-29b-3p mimics, miR-29b-3p inhibitor, si-PGRN and negative control (NC) were, respectively, transfected into the CRC cells. Lucif-erase reporter plasmids were prepared to evaluate the transduction activity of Wnt/?-catenin signaling pathway, and dual-luciferase reporter gene assay was arranged to confirm the targeted relationship between H19 and miR-29b-3p, as well as between miR-29b-3p and PGRN. Finally, the proliferative and invasive capacities of CRC cells were appraised through transwell, MTT and scratch assays. As a result, over-expressed H19 and down-expressed miR-29b-3p displayed close associations with the CRC patients' poor prognosis (P < 0.05). Besides, transfection with si-H19, miR-29b-3p mimic or si-PGRN were correlated with elevated E-cadherin expression, decreased snail and vimentin expressions, as well as less-motivated cell proliferation and cell metastasis (P < 0.05). Moreover, H19 was verified to directly target miR-29b-3p based on the luciferase reporter gene assay (P < 0.05), and miR-29b-3p also bound to PGRN in a direct manner (P < 0.05). Finally, addition of LiCl (Wnt/?-catenin pathway activator) or XAV93920 (Wnt/?-catenin pathway inhibitor) would cause remarkably altered E-cadherin, c-Myc, vimentin and snail expressions, as well as significantly changed transcriptional activity of ?-catenin/Tcf reporter plasmid (P < 0.05). In conclusion, the lncRNA H19/miR-29b-3p/PGRN/Wnt axis counted a great deal for seeking appropriate diagnostic biomarkers and treatment targets for CRC.
Project description:In a high percentage (?85%) of both sporadic and familial adenomatous polyposis forms of colorectal cancer (CRC), the inactivation of the APC tumor suppressor gene initiates tumor formation and modulates the Wnt/?-Catenin transduction pathways involved in the control of cell proliferation, adhesion and metastasis. Increasing evidence showed that the endocannabinoids control tumor growth and progression, both in vitro and in vivo. We evaluated the effect of Rimonabant, a Cannabinoid Receptor 1 (CB1) inverse agonist, on the Wnt/?-Catenin pathway in HCT116 and SW48 cell lines carrying the genetic profile of metastatic CRC poorly responsive to chemotherapies. In these models, Rimonabant inhibited the Wnt/?-Catenin canonical pathway and increased ?-Catenin phosphorylation; in HCT116 cells, but not in SW48, the compound also triggered the Wnt/?-Catenin non canonical pathway activation through induction of Wnt5A and activation of CaMKII. The Rimonabant-induced downregulation of Wnt/?-Catenin target genes was partially ascribable to a direct inhibition of p300/KAT3B histone acetyltransferase, a coactivator of ?-Catenin dependent gene regulation. Finally, in HCT116 xenografts, Rimonabant significantly reduced tumor growth and destabilized the nuclear localization of ?-Catenin. Obtained data heavily supported the rationale for the use of cannabinoids in combined therapies for metastatic CRC harbouring activating mutations of ?-Catenin.
Project description:Metastases cause recurrence and mortality for patients with colorectal carcinomas (CRC). In present study, we evaluated heterogeneity on drug resistance and its underlying mechanism between metastatic and primary CRC. Immunohistochemical results from clinical tissue microarray (TMA) suggested that the expression concordance rates of cancer stem cells (CSCs) and drug resistance relative proteins between lymph-node metastatic and primary CRC foci were low. The apoptotic and proliferation indexes in metastasis CRC specimens were decreased compared with primary. In vitro experimental results indicated that the migration and invasion abilities were upregulated in metastatic cells SW620 compared with primary cells SW480, the cellular efflux ability and WNT/?-catenin activity were also upregulated in SW620 cells. After 5-fluorouracil (5-Fu) treatment, the reduction in the proportion of cell apoptosis, CD133 and TERT expression levels in SW620 were lower than that in SW480 cells. Bioinformatics analysis in whole-genome transcriptional profiling results between metastatic and primary CRC cells suggested that differentially expressed genes were mainly centered on well-characterized signaling pathways including WNT/?-catenin, cell cycle and cell junction. Collectively, heterogeneity of drug resistant was present between metastatic and primary CRC specimens and cell lines, the abnormal activation of WNT/?-catenin signaling pathway could be a potential molecular leading to drug resistant ability enhancing in metastatic CRC cells.
Project description:Adenomatous polyposis coli (APC) and Axin interactions serve an important role in colorectal cancer (CRC) pathogenesis. The aim of the present study was to assess the combined effects of Axin and APC co?expression in CRC cells, and to determine the underlying mechanisms involved. SW480 cells were divided into the following groups: Untransfected (SW480 group), transfected with pEGFP?N3plus pCS2?MT (SW480/vector?vector), transfected with pEGFP?N3?APC5 (SW480/APC5), and transfected with pEGFP?N3?APC5 pluspCS2?MT?Axin (SW480/APC5?Axin). APC5 and Axin mRNA levels were determined by reverse transcription?polymerase chain reaction. MTT assays and flow cytometry analysis were performed to assess cell growth and cell cycle distribution, respectively. Quantitative PCR and western blot analyses were conducted to evaluate the mRNA and protein levels, respectively, of Wnt signaling effectors, including ??catenin, c?myc and survivin. Successful transfection of SW480 cells was determined with APC and APC?Axin plasmids as indicated by the green fluorescence signals. Notably, SW480/APC5 cell growth was inhibited by 40.33%, and cells co?expressing APC5 and Axin demonstrated 61.27% inhibition of cell growth compared with SW480 control cells. The results demonstrate that APC5 may induce G1/S arrest in SW480 cells, and Axin may enhance cell growth arrest induced by APC5. The mRNA and protein levels of ??catenin, c?myc and survivin were significantly reduced in SW480/APC?Axin cells when compared with the SW480/APC group. In conclusion, co?expression of APC5 and Axin genes significantly downregulated Wnt signaling in human SW480 CRC cells and inhibited cell growth, when compared with cells transfected with APC5 alone. These results may provide experimental evidence to support combined gene therapy in CRC.
Project description:Misregulation of the canonical Wnt/?-catenin pathway and aberrant activation of Wnt signaling target genes are common in colorectal cancer (CRC) and contribute to cancer progression. Altered expression of human enhancer of filamentation 1 (HEF1; also known as NEDD9 or Cas-L) has been implicated in progression of melanoma, breast, and CRC. However, the regulation of HEF1 and the role of HEF1 in CRC tumorigenesis are not fully understood. We here identify HEF1 as a novel Wnt signaling target. The expression of HEF1 was upregulated by Wnt-3a, ?-catenin, and Dvl2 in a dose-dependent manner, and was suppressed following ?-catenin downregulation by shRNA. In addition, elevated HEF1 mRNA and protein levels were observed in CRC cell lines and primary tumor tissues, as well as in the colon and adenoma polyps of Apc(Min/+) mice. Moreover, HEF1 levels in human colorectal tumor tissues increased with the tumor grade. Chromatin immunoprecipitation (ChIP) assays and promoter analyses revealed three functional T-cell factor (TCF)-binding sites in the promoter of HEF1 responsible for HEF1 induction by Wnt signaling. Ectopic expression of HEF1 increased cell proliferation and colony formation, while downregulation of HEF1 in SW480 cells by shRNA had the opposite effects and inhibited the xenograft tumor growth. Furthermore, overexpression of HEF1 in SW480 cells promoted cell migration and invasion. Together, our results determined a novel role of HEF1 as a mediator of the canonical Wnt/?-catenin signaling pathway for cell proliferation, migration, and tumorigenesis, as well as an important player in colorectal tumorigenesis and progression. HEF1 may represent an attractive candidate for drug targeting in CRC.
Project description:More than 94% of colorectal cancer cases have mutations in one or more Wnt/?-catenin signaling pathway components. Inactivating mutations in APC or activating mutations in ?-catenin (CTNNB1) lead to signaling overactivation and subsequent intestinal hyperplasia. Numerous classes of medicines derived from synthetic or natural small molecules, including alkaloids, have benefited the treatment of different diseases, including cancer, Piperine is a true alkaloid, derived from lysine, responsible for the spicy taste of black pepper (Piper nigrum) and long pepper (Piper longum). Studies have shown that piperine has a wide range of pharmacological properties; however, piperine molecular mechanisms of action are still not fully understood. By using Wnt/?-catenin pathway epistasis experiment we show that piperine inhibits the canonical Wnt pathway induced by overexpression of ?-catenin, ?-catenin S33A or dnTCF4 VP16, while also suppressing ?-catenin nuclear localization in HCT116 cell line. Additionally, piperine impairs cell proliferation and migration in HCT116, SW480 and DLD-1 colorectal tumor cell lines, while not affecting the non-tumoral cell line IEC-6. In summary, piperine inhibits the canonical Wnt signaling pathway and displays anti-cancer effects on colorectal cancer cell lines.