ABSTRACT
Gankyrin is a subunit of the 26S proteasome, and has been known to degrade p53 and retinoblastoma protein and promote the tumorigenicity and metastasis in some malignancies. However, the role of gankyrin in breast cancer has not been explored. In this study, we investigated the expression of gankyrin in breast cancer and evaluated its effect on breast cancer. Representative cancer tissues including normal breasts from 60 patients with breast cancer were stained immunohistochemically for gankyrin, estrogen receptor, progesterone receptor, and ErbB2. We evaluated the relationship between gankyrin expression and clinicopathologic parameters or prognostic markers. We also attempted to clarify the mechanism of gankyrin involved in breast carcinogenesis by using MCF7 breast cancer cells. Gankyrin was weakly expressed in normal breast epithelial cells, however, tumor regions of 37/60 (61.7%) cases showed an overexpression of gankyrin. Gankyrin overexpression was associated with extensive intraductal carcinoma (p=0.014) and ErbB2 positivity (p=0.031) in invasive ductal carcinoma. In MCF7 breast cancer cells, downregulation of gankyrin was associated with a reduction of cell proliferation and tumorigenicity. In conclusion, gankyrin was identified in normal breasts and overexpressed in invasive breast cancers. The overexpression of gankyrin was associated with extensive intraductal carcinoma and ErbB2 expression in breast cancer.
Subject(s)
Breast Neoplasms/metabolism , Breast/metabolism , Carcinoma, Intraductal, Noninfiltrating/metabolism , Proteasome Endopeptidase Complex/metabolism , Proto-Oncogene Proteins/metabolism , Receptor, ErbB-2/metabolism , Adult , Aged , Aged, 80 and over , Biomarkers, Tumor/metabolism , Cell Line, Tumor , Cell Proliferation , Female , Gene Expression Regulation, Neoplastic , Humans , MCF-7 Cells , Middle Aged , Proteasome Endopeptidase Complex/genetics , Proto-Oncogene Proteins/genetics , Receptors, Estrogen/metabolism , Receptors, Progesterone/metabolism , Up-Regulation , Young AdultABSTRACT
DNA double strand breaks (DSBs) occur more frequently in TIS21(-/-) mouse embryo fibroblasts than that in wild type MEFs (wt-MEFs). Therefore, the role TIS21 plays in the DNA damage response was investigated. Adenoviral transduction of Huh7 tumor cells with the TIS21 gene accelerated the repair of DSBs induced by etoposide treatment as evaluated by clearance of γH2AX foci and the Comet assay. TIS21 increased methylation of Mre11 and protein arginine methyltransferase 1 (PRMT1) activity, leading to Mre11 activation in vitro and in vivo, as determined by immunoprecipitation and radiolabeling analyses. When downstream DNA damage response mediators were evaluated in various human cancer cells lines, TIS21 was found to strongly inhibit Chk2(T68) and p53(S20) phosphorylation by p-ATM(S1981) but not p53(S15). The loss of Chk2 activation after etoposide treatment reduced apoptosis in the cells by downregulating the expression of E2F1 and Bax. These data suggest that TIS21 regulates DSB repair and apoptosis. Expression of TIS21 promoted the repair of DSBs and reduced apoptosis by blocking the damage signal from p-ATM(S1981) to Chk2(T68)-p53(S20)via the activation of Mre11 and PRMT1.
Subject(s)
DNA Breaks, Double-Stranded , DNA Repair , DNA-Binding Proteins/metabolism , Immediate-Early Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Tumor Suppressor Proteins/metabolism , Adenoviridae/genetics , Adenoviridae/metabolism , Apoptosis , Checkpoint Kinase 2 , Cloning, Molecular , Comet Assay , DNA Methylation , DNA-Binding Proteins/genetics , E2F1 Transcription Factor/genetics , E2F1 Transcription Factor/metabolism , Enzyme Activation , Etoposide/adverse effects , Genetic Vectors/genetics , Genetic Vectors/metabolism , HEK293 Cells , HeLa Cells , Histones/genetics , Histones/metabolism , Humans , Immediate-Early Proteins/genetics , MRE11 Homologue Protein , Phosphorylation , Protein Serine-Threonine Kinases/genetics , Protein-Arginine N-Methyltransferases/genetics , Protein-Arginine N-Methyltransferases/metabolism , Repressor Proteins/genetics , Repressor Proteins/metabolism , Signal Transduction , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Tumor Suppressor Proteins/genetics , bcl-2-Associated X Protein/genetics , bcl-2-Associated X Protein/metabolismABSTRACT
BTG2 is a tumor suppressor gene. It is frequently downregulated in human cancer tissues, and its loss is associated with cancer cell metastasis, suggesting that the suppression of BTG2 plays a critical role in cancer cell migration and invasion. Here, we report that re-expression of BTG2 decreased cell migration and invasion in A549 and PC3 cancer cells. Furthermore, BTG2 expression was correlated with downregulation of focal adhesion kinase (FAK) Tyr576 and Tyr925 residues phosphorylation, while Tyr397 which is the autophosphorylation site was not influenced by BTG2 expression. c-Src phosphorylation which is the upstream of FAK was not influenced, whereas c-Src kinase activity was significantly decreased by BTG2 expression. BTG2 overexpression increased Src reduction state and inhibited reactive oxygen species (ROS) generation by being localized in mitochondria. Mitochondria-target BTG2 also inhibited cell migration via downregulation of Src-FAK signaling. In conclusion, our study reveals that BTG2 negatively regulated cancer cell migration by inhibiting Src activity through downregulation of ROS generation in mitochondria.
