BackgroundTET1 is a tumor suppressor gene (TSG) that codes for ten-eleven translocation methyl cytosine dioxygenase1 (TET1) catalyzing the conversion of 5-methylcytosine to 5-hydroxy methyl cytosine as a first step of TSG demethylation. Its hypermethylation has been associated with cancer pathogenesis. However, whether TET1 plays any role in nasopharyngeal carcinoma (NPC) remains unclear. This study investigated the expression and methylation of TET1 in NPC and confirmed its role and mechanism as a TSG.ResultsTET1 expression was downregulated in NPC tissues compared with nasal septum deviation tissues. Demethylation of TET1 in HONE1 and HNE1 cells restored its expression with downregulated methylation, implying that TET1 was silenced by promoter hypermethylation. Ectopic expression of TET1 suppressed the growth of NPC cells, induced apoptosis, arrested cell division in G0/G1 phase, and inhibited cell migration and invasion, confirming TET1 TSG activity. TET1 decreased the expression of nuclear β-catenin and downstream target genes. Furthermore, TET1 could cause Wnt antagonists (DACT2, SFRP2) promoter demethylation and restore its expression in NPC cells.ConclusionsCollectively, we conclude that TET1 exerts its anti-tumor functions in NPC cells by suppressing Wnt/β-catenin signaling via demethylation of Wnt antagonists (DACT2 and SFRP2).Electronic supplementary materialThe online version of this article (10.1186/s13148-018-0535-7) contains supplementary material, which is available to authorized users.
BackgroundNasopharyngeal carcinoma (NPC) is prevalent in South China, including Hong Kong and Southeast Asia, constantly associated with Epstein-Barr virus (EBV) infection. Epigenetic etiology attributed to EBV plays a critical role in NPC pathogenesis. Through previous CpG methylome study, we identified Disheveled-associated binding antagonist of beta-catenin 2 (DACT2) as a methylated target in NPC. Although DACT2 was shown to regulate Wnt signaling in some carcinomas, its functions in NPC pathogenesis remain unclear.MethodsRT-PCR, qPCR, MSP, and BGS were applied to measure expression levels and promoter methylation of DACT2 in NPC. Transwell, flow cytometric analysis, colony formation, and BrdU-ELISA assay were used to assess different biological functions affected by DACT2. Immunofluorescence, Western blot, and dual-luciferase reporter assay were used to explore the mechanisms of DACT2 functions. Chemosensitivity assay was used to measure the impact of DACT2 on chemotherapy drugs.ResultsWe found that DACT2 is readily expressed in multiple normal adult tissues including upper respiratory tissues. However, it is frequently downregulated in NPC and correlated with promoter methylation. DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine restored its expression in NPC cells. DACT2 methylation was further detected in 29/32 (91%) NPC tumors but not in any (0/8) normal nasopharyngeal tissue samples. Ectopic expression of DACT2 in NPC cells suppressed their proliferation, migration, and invasion through downregulating matrix metalloproteinases. DACT2 expression also induced G2/M arrest in NPC cells through directly suppressing β-catenin/Cdc25c signaling, which sensitized NPC cells to paclitaxel and 5-FU, but not cisplatin.ConclusionOur results demonstrate that DACT2 is frequently inactivated epigenetically by CpG methylation in NPC, while it inhibits NPC cell proliferation and metastasis via suppressing β-catenin/Cdc25c signaling. Our study suggests that DACT2 promoter methylation is a potential epigenetic biomarker for the detection and chemotherapy guidance of NPC.
Deregulation of msh homeobox 1 (MSX1) has been identified to be associated with multiple human malignant neoplasms. However, the association of the expression and biological function of MSX1 with breast tumorigenesis, and the underlying mechanism remain largely unknown. Therefore, the present study examined the expression and promoter methylation of MSX1 in breast tumor cell lines, primary breast tumors and normal breast tissues using semi-quantitative, quantitative and methylation-specific reverse transcription‑polymerase chain reaction. Colony formation assays, flow cytometric analysis, and wound healing and Transwell assays were used to assess various functions of MSX1. Western blot analyses were also conducted to explore the mechanism of MSX1. The results revealed that MSX1 was broadly expressed in normal human tissues, including breast tissues, but was frequently downregulated or silenced in breast cancer cell lines and primary tumors by promoter methylation. Methylation of the MSX1 promoter was observed in 7/9 (77.8%) breast cancer cell lines and 47/99 (47.5%) primary tumors, but not in normal breast tissues or surgical margin tissues, suggesting that tumor-specific methylation of MSX1 occurs in breast cancer. Pharmacological demethylation reduced MSX1 promoter methylation levels and restored the expression of MSX1. The ectopic expression of MSX1, induced by transfection with a lentiviral vector, significantly inhibited the clonogenicity, proliferation, migration and invasion of breast tumor cells by inducing G1/S cell cycle arrest and apoptosis. Ectopic MSX1 expression also inhibited the expression of active β-catenin and its downstream targets c-Myc and cyclin D1, and also increased the cleavage of caspase-3 and poly (ADP-ribose) polymerase. In conclusion, MSX1 exerts tumor-suppressive functions by inducing G1/S cell cycle arrest and apoptosis in breast tumorigenesis. Its methylation may be used as an epigenetic biomarker for the early detection and diagnosis of breast cancer.
Dickkopf-related protein 2 (DKK2) is one of the antagonists of Wnt/β-catenin signaling, with its downregulation reported in multiple cancers. However, how DKK2 contributes to breast tumorigenesis remains unclear. We examined its expression and promoter methylation in 10 breast tumor cell lines, 98 primary tumors, and 21 normal breast tissues. Compared with normal tissues, DKK2 was frequently silenced in breast cell lines (7/8). DKK2 promoter methylation was detected in 77.8% of cell lines and 86.7% of breast tumors; while rarely detected in normal breast tissues (19%), indicating common DKK2 methylation in breast cancer. Ectopic expression of DKK2 changed breast tumor cell morphology, inhibited cell proliferation and colony formation by inducing G0/G1 cell cycle arrest and apoptosis, and suppressed tumor cell migration by reversing epithelial-mesenchymal transition (EMT) and downregulating stem cell markers. Moreover, restored expression of DKK2 in MCF7 cells disrupted the microtube formation of human umbilical vein endothelial cells on Matrigel®. In vivo, the growth of MDA-MB-231 cells in nude mice was markedly decreased after stable expression of DKK2. DKK2 suppressed canonical Wnt/β-catenin signaling by inhibiting β-catenin activity with decreased active β-catenin protein. Thus, our findings demonstrate that DKK2 functions as a tumor suppressor through inhibiting cell proliferation and inducing apoptosis via regulating Wnt signaling during breast tumorigenesis.
Background and AimThe high mortality and poor prognosis of hepatocellular carcinoma (HCC) have raised the public attention. Gene therapy is considered as a promising treatment option for cancer; thus, finding a new therapeutic target for HCC is urgently needed. GATA4 is a tumor suppressor gene in multiple cancers, but its role in HCC is unclear. In this study, we explored the function of GATA4 in HCC.MethodsReverse transcription–polymerase chain reaction and quantitative polymerase chain reaction were used to detect the mRNA expression of GATA4 in HCC cells and tissues. Cell viability, transwell, colony formation, and flow cytometry assays were applied to examine different aspects of biological effects of GATA4 in vitro. Xenografts, immunohistochemistry, and terminal deoxynucleotidyl transferase‐mediated digoxigenin‐dUTP nick‐end labeling assays were performed to evaluate the effect of GATA4 on tumorigenicity in vivo. Western blotting, immunofluorescence, and β‐galactosidase staining were used to investigate the mechanism underlying the function of GATA4.ResultsWe found that GATA4 was silenced in 15/19 (79%) HCC tissues. Restoring the expression of GATA4 induced G0/G1 phase arrest, promoted apoptosis, suppressed HCC proliferation in vitro, and inhibited HCC tumor growth in vivo. Our data further showed that the ectopic expression of GATA4 induced cellular senescence through regulating nuclear factor‐κB and inducing mesenchymal‐to‐epithelial transition.ConclusionsOur data demonstrated that by inducing cellular senescence and mesenchymal‐to‐epithelial transition, GATA4 plays a crucial role as a tumor suppressor in HCC. It may thus be a potential cancer therapeutic target for HCC.
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