ABSTRACT
Hepatocellular carcinoma (HCC) is a common liver cancer that accounts for 90% of cases. Doxorubicin exhibits a broad spectrum of antitumor activity and is one of the most active agents in HCC. WW domain-containing protein 2 (WWP2) is highly expressed in HCC tissues and activates protein kinase B (AKT) signaling pathway to enhance tumor metastasis. However, the role of WWP2 in the glycolysis and antitumor effects of doxorubicin and the epigenetic alterations of WWP2 in HCC remain to be elucidated. The levels of WWP2 and N6-methyladenosine methyltransferase-like 3 (METTL3) in clinical samples and cells were investigated. WWP2 were silenced or overexpressed to study the role of WWP2 in regulating cell proliferation, colony formation, and glycolysis. RNA immunoprecipitation was performed to test m6 A levels. Quantitative reverse-transcription polymerase chain reaction (RT-PCR) and Western blot were used to measure mRNA and protein, respectively. WWP2 silencing inhibits cell proliferation, colony formation, and glycolysis, while WWP2 overexpression has the inverse effects via the AKT signaling pathway. Silencing WWP2 enhances doxorubicin's antitumor effect, while WWP2 overexpression suppresses doxorubicin's antitumor effect. Data also support that METTL3 mediates WWP2 m6A modification, and m6A reader, IGF2BP2, binds to the methylated WWP2 to promote the stability of WWP2, leading to upregulation of WWP2. METTL3 mediates WWP2 m6A modification, which can be recognized and bound by IGF2BP2 to increase the stability of WWP2, leading to WWP2 overexpression which inhibits the antitumor effects of doxorubicin through METTL3/WWP2/AKT/glycolysis axis.
Subject(s)
Carcinoma, Hepatocellular , Liver Neoplasms , Ubiquitin-Protein Ligases , Carcinoma, Hepatocellular/drug therapy , Doxorubicin/pharmacology , Doxorubicin/therapeutic use , Humans , Liver Neoplasms/metabolism , Methyltransferases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , RNA-Binding Proteins , Ubiquitin-Protein Ligases/metabolismABSTRACT
Primary liver cancer is one of the most common and aggressive human malignancies worldwide. As numerous studies have revealed that WW domain containing E3 Ubprotein ligase 2 (WWP2) exerts cancerspecific functions, the present study assessed the role of WWP2 in liver cancer. WWP2 was revealed to be significantly overexpressed in liver cancer tissues compared with paired normal tissues at the mRNA as well as at the protein level. Furthermore, small interfering RNA-mediated WWP2 knockdown in liver cancer cell lines was demonstrated to inhibit cell proliferation, cause cell cycle arrested in G1 phase and to induce apoptosis as revealed by a Cell Counting Kit-8 assay and flow cytometric analysis. In addition, western blot analysis revealed that WWP2 knockdown significantly increased the expression of apoptosis-associated markers caspase7, caspase8 and B-cell lymphoma 2 (Bcl-2)-associated X in liver cancer cell lines, while Bcl2 was significantly decreased. In conclusion, the present study suggested that WWP2 may exert important functions in the overproliferation and evasion of apoptosis of liver cancer, likely through regulating the expression of apoptosis-associated markers. Furthermore, WWP2 may represent a novel diagnostic marker and molecular therapeutic target for liver cancer.
Subject(s)
Apoptosis , G1 Phase Cell Cycle Checkpoints , Liver Neoplasms/pathology , Ubiquitin-Protein Ligases/metabolism , Adult , Aged , Aged, 80 and over , Apoptosis/genetics , Biomarkers, Tumor/metabolism , Blotting, Western , Cell Line, Tumor , Cell Proliferation , Cell Survival/genetics , Female , G1 Phase Cell Cycle Checkpoints/genetics , Gene Expression Regulation, Neoplastic , Gene Knockdown Techniques , Humans , Liver Neoplasms/genetics , Male , Middle Aged , RNA, Messenger/genetics , RNA, Messenger/metabolism , Signal Transduction/genetics , Ubiquitin-Protein Ligases/genetics , Up-Regulation/geneticsABSTRACT
The role and clinical implication of the WWP2 E3 ubiquitin ligase in liver cancer are poorly understood. In the current study, we investigated the expression level of WWP2 and its functions in cell adhesion, invasion, and migration in liver cancer. We used real-time PCR to detect the expression of WWP2 in liver cancer and adjacent samples from the People's Hospital of Lishui and also analyzed The Cancer Genome Atlas (TCGA) RNA-seq data by bioinformatics. Migration and invasion were detected by transwell analysis. We detected a strong WWP2 expression in tumor tissues of the People's Hospital of Lishui, and the survival rate was significantly higher in patients with lower WWP2-expressing tumors. WWP2 small hairpin RNA (shRNA) lentivirus stably infected cells (shWWP2), Huh7, showed slower growth speed compared with scramble control-infected cells in a xenograft mouse model. Knockdown of WWP2 Huh7 and BEL-7404 cells demonstrated a reduction in adhesion, invasion, and migration. Gene set enrichment analysis (GSEA) showed that WWP2 is positively correlated to cancer-related pathways including the chemokine signaling pathway. WWP2 also regulated MMP-9, caspase-9, CXCR3, and CCR5 expression in liver cancer cells. In addition, knockdown of CXCR3 and CCR5 significantly inhibited cell proliferation, adhesion, invasion, and migration in Huh7 and BEL-7404 cells. Our data suggest that targeting of WWP2 may be a therapeutic strategy for liver cancer treatment.
Subject(s)
Gene Silencing , Liver Neoplasms/genetics , Ubiquitin-Protein Ligases/genetics , Animals , Apoptosis/genetics , Cell Adhesion/genetics , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation , Cell Survival/genetics , Chemokines/genetics , Chemokines/metabolism , Disease Models, Animal , Gene Expression Regulation, Neoplastic , Heterografts , Humans , Liver Neoplasms/metabolism , Liver Neoplasms/mortality , Liver Neoplasms/pathology , Mice , Prognosis , RNA, Messenger/genetics , RNA, Small Interfering/genetics , Signal TransductionABSTRACT
DAPK1 can induce apoptosis in several cells; to determine the effect of DAPK1 would provide a new potential therapeutic strategy for treating pancreatic cancer. The aim of the present study was to investigate the effect of DAPK1 gene on proliferation, migration, and invasion of carcinoma of pancreas BxPC-3 cell line and explore the possible mechanisms. In our study, DAPK1 over-expressed cells were established by using the lentiviral transfection method, and DAPK1 obviously increased in BxPC-3 cells after transient transfection. Cell Counting Kit-8 (CCK-8) assay was used to determine the BxPC-3 cells proliferation after transfection. Apoptosis of the BxPC-3 cells was determined by using flow cytometry analysis. In addition, cell adhesion assay and in vitro invasion assay were performed. Western blotting was used to determine the protein expressions of caspase-3, DAPK1, VEGF, PEDF, MMP2, AKT, P-AKT, P-ERK, Bcl2, and Bax. Our results demonstrated that DAPK1 gene over-expression can suppress the proliferation, migration, and invasion of carcinoma of pancreas BxPC-3 cell line, and the possible mechanisms may be correlated to induction of mitochondria-mediated apoptosis, down-regulations of MMP-2 and VEGF, up-regulations of PEDF, through the PI3K/Akt and ERK pathways.
