ABSTRACT
Dual specificity phosphatase-3 (Dusp3/Vhr) regulates cell cycle progression by counteracting the effects of mitogen-activated protein kinases (Mapk) Erk1/2 and Jnk. Despite the known upregulation of Dusp3 at M phase in mammalian cells, its mitotic functions are poorly characterized. Here, we report that loss of Dusp3 by RNAi leads to the formation of multipolar spindles in human mitotic cancer cells in an Erk1/2-dependent manner. In the phosphatase-silenced cells, the normal bipolar spindle structure was restored by chemical inhibition of Erk1/2 and ectopic overexpression of Dusp3. We propose that at M phase Dusp3 keeps Erk1/2 activity in check to facilitate normal mitosis.
Subject(s)
Cell Polarity/genetics , Dual Specificity Phosphatase 3/genetics , Mitosis/genetics , Spindle Apparatus/genetics , Cell Cycle/genetics , Dual Specificity Phosphatase 3/biosynthesis , Gene Expression Regulation, Neoplastic , HeLa Cells , Humans , MAP Kinase Kinase 4/genetics , MAP Kinase Signaling System/genetics , Phosphorylation , RNA Interference , TransfectionABSTRACT
Epigenetic dysregulation has emerged as a major contributor to tumorigenesis. Histone methylation is a well-established mechanism of epigenetic regulation that is dynamically modulated by histone methyltransferases and demethylases. The pathogenic role of histone methylation modifiers in non-small cell lung cancer (NSCLC), which is the leading cause of cancer deaths worldwide, remains largely unknown. Here, we found that the histone H3 lysine 36 (H3K36) demethylase KDM2A (also called FBXL11 and JHDM1A) is frequently overexpressed in NSCLC tumors and cell lines. KDM2A and its catalytic activity were required for in vitro proliferation and invasion of KDM2A-overexpressing NSCLC cells. KDM2A overexpression in NSCLC cells with low KDM2A levels increased cell proliferation and invasiveness. KDM2A knockdown abrogated tumor growth and invasive abilities of NSCLC cells in mouse xenograft models. We identified dual-specificity phosphatase 3 (DUSP3) as a key KDM2A target gene and found that DUSP3 dephosphorylates ERK1/2 in NSCLC cells. KDM2A activated ERK1/2 through epigenetic repression of DUSP3 expression via demethylation of dimethylated H3K36 at the DUSP3 locus. High KDM2A levels correlated with poor prognosis in NSCLC patients. These findings uncover an unexpected role for a histone methylation modifier in activating ERK1/2 in lung tumorigenesis and metastasis, suggesting that KDM2A may be a promising therapeutic target in NSCLC.
Subject(s)
Carcinoma, Non-Small-Cell Lung/pathology , Epigenesis, Genetic/physiology , F-Box Proteins/physiology , Gene Expression Regulation, Neoplastic/physiology , Histones/metabolism , Jumonji Domain-Containing Histone Demethylases/physiology , Lung Neoplasms/pathology , MAP Kinase Signaling System , Neoplasm Proteins/physiology , Protein Processing, Post-Translational/physiology , Animals , Carcinoma, Non-Small-Cell Lung/genetics , Cell Division , Cell Line, Tumor , Dual Specificity Phosphatase 3/biosynthesis , Dual Specificity Phosphatase 3/genetics , Dual Specificity Phosphatase 3/physiology , Epigenesis, Genetic/genetics , F-Box Proteins/antagonists & inhibitors , F-Box Proteins/biosynthesis , F-Box Proteins/genetics , Female , Gene Expression Regulation, Neoplastic/genetics , Heterografts , Humans , Jumonji Domain-Containing Histone Demethylases/antagonists & inhibitors , Jumonji Domain-Containing Histone Demethylases/biosynthesis , Jumonji Domain-Containing Histone Demethylases/genetics , Lung Neoplasms/genetics , Male , Methylation , Mice , Mice, Nude , Neoplasm Invasiveness , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/biosynthesis , Neoplasm Proteins/genetics , Prognosis , Promoter Regions, Genetic , Protein Processing, Post-Translational/genetics , RNA Interference , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , RNA, Neoplasm/biosynthesis , RNA, Neoplasm/geneticsABSTRACT
BACKGROUND: The 21-kDa Vaccinia virus VH1-related (VHR) dual-specific protein phosphatase (encoded by the DUSP3 gene) plays a critical role in cell cycle progression and is itself regulated during the cell cycle. We have previously demonstrated using RNA interference that cells lacking VHR arrest in the G1 and G2 phases of the cell cycle and show signs of beginning of cell senescence. METHODS: In this report, we evaluated successfully the expression levels of VHR protein in 62 hysterectomy or conization specimens showing the various (pre) neoplastic cervical epithelial lesions and 35 additional cases of hysterectomy performed for non-cervical pathologies, from patients under 50 years of age. We used a tissue microarray and IHC technique to evaluate the expression of the VHR phosphatase. Immunofluorescence staining under confocal microscopy, Western blotting and RT-PCR methods were used to investigate the localization and expression levels of VHR. RESULTS: We report that VHR is upregulated in (pre) neoplastic lesions (squamous intraepithelial lesions; SILs) of the uterine cervix mainly in high grade SIL (H-SIL) compared to normal exocervix. In the invasive cancer, VHR is also highly expressed with nuclear localization in the majority of cells compared to normal tissue where VHR is always in the cytoplasm. We also report that this phosphatase is highly expressed in several cervix cancer cell lines such as HeLa, SiHa, CaSki, C33 and HT3 compared to primary keratinocytes. The immunofluorescence technique under confocal microscopy shows that VHR has a cytoplasmic localization in primary keratinocytes, while it localizes in both cytoplasm and nucleus of the cancer cell lines investigated. We report that the up-regulation of this phosphatase is mainly due to its post-translational stabilization in the cancer cell lines compared to primary keratinocytes rather than increases in the transcription of DUSP3 locus. CONCLUSION: These results together suggest that VHR can be considered as a new marker for cancer progression in cervix carcinoma and potential new target for anticancer therapy.
