MAD2 interacts with DNA repair proteins and negatively regulates DNA damage repair.

MAD2 (mitotic arrest deficient 2) is a key regulator of mitosis. Recently, it had been suggested that MAD2-induced mitotic arrest mediates DNA damage response and that upregulation of MAD2 confers sensitivity to DNA-damaging anticancer drug-induced apoptosis. In this study, we report a potential novel role of MAD2 in mediating DNA nucleotide excision repair through physical interactions with two DNA repair proteins, XPD (xeroderma pigmentosum complementation group D) and ERCC1. First, overexpression of MAD2 resulted in decreased nuclear accumulation of XPD, a crucial step in the initiation of DNA repair. Second, immunoprecipitation experiments showed that MAD2 was able to bind to XPD, which led to competitive suppression of binding activity between XPD and XPA, resulting in the prevention of physical interactions between DNA repair proteins. Third, unlike its role in mitosis, the N-terminus domain seemed to be more important in the binding activity between MAD2 and XPD. Fourth, phosphorylation of H2AX, a process that is important for recruitment of DNA repair factors to DNA double-strand breaks, was suppressed in MAD2-overexpressing cells in response to DNA damage. These results suggest a negative role of MAD2 in DNA damage response, which may be accounted for its previously reported role in promoting sensitivity to DNA-damaging agents in cancer cells. However, the interaction between MAD2 and ERCC1 did not show any effect on the binding activity between ERCC1 and XPA in the presence or absence of DNA damage. Our results suggest a novel function of MAD2 by interfering with DNA repair proteins.

Decreased adhesiveness, resistance to anoikis and suppression of GRP94 are integral to the survival of circulating tumor cells in prostate cancer.

The presence of circulating tumor cells (CTC) is common in prostate cancer patients, however until recently their clinical significance was unknown. The CTC stage is essential for the formation of distant metastases, and their continuing presence after radical prostatectomy has been shown to predict recurrent or latent disease. Despite their mechanistic and prognostic importance, due both to their scarcity and difficulties in their isolation, little is known about the characteristics that enable their production and survival. The aim of this study was to investigate the molecular mechanisms underlying the survival of CTC cells. A novel CTC cell line from the bloodstream of an orthotopic mouse model of castration-resistant prostate cancer was established and compared with the primary tumor using attachment assays, detachment culture, Western blot, flow cytometry and 2D gel electrophoresis. Decreased adhesiveness and expression of adhesion molecules E-cadherin, beta4-integrin and gamma-catenin, together with resistance to detachment and drug-induced apoptosis and upregulation of Bcl-2 were integral to the development of CTC and their survival. Using proteomic studies, we observed that the GRP94 glycoprotein was suppressed in CTC. GRP94 was also shown to be suppressed in a tissue microarray study of 79 prostate cancer patients, indicating its possible role in prostate cancer progression. Overall, this study suggests molecular alterations accounting for the release and survival of CTC, which may be used as drug targets for either anti-metastatic therapy or the suppression of latent disease. We also indicate the novel involvement of GRP94 suppression in prostate cancer metastasis.

Identification of a novel inhibitor of differentiation-1 (ID-1) binding partner, caveolin-1, and its role in epithelial-mesenchymal transition and resistance to apoptosis in prostate cancer cells.

Recently, ID-1 (inhibitor of differentiation/DNA binding) is suggested as an oncogene and is reported to promote cell proliferation, invasion, and survival in several types of human cancer cells through multiple signaling pathways. However, how Id-1 interacts with these pathways and the immediate downstream effectors of the Id-1 protein are not known. In this study, using a yeast two-hybrid screening technique, we identified a novel Id-1-interacting protein, caveolin-1 (Cav-1), a cell membrane protein, and a positive regulator of cell survival and metastasis in prostate cancer. Using an immunoprecipitation method, we found that the helix-loop-helix domain of the Id-1 protein was essential for the physical interaction between Id-1 and Cav-1. In addition, we also demonstrated that the physical interaction between Id-1 and Cav-1 played a key role in the epithelial-mesenchymal transition and increased cell migration rate as well as resistance to taxol-induced apoptosis in prostate cancer cells. Furthermore, our results revealed that this effect was regulated by Id-1-induced Akt activation through promoting the binding activity between Cav-1 and protein phosphatase 2A. Our study demonstrates a novel Id-1 binding partner and suggests a molecular mechanism that mediates the function of Id-1 in promoting prostate cancer progression through activation of the Akt pathway leading to cancer cell invasion and resistance to anticancer drug-induced apoptosis.

Anti-apoptotic role of TWIST and its association with Akt pathway in mediating taxol resistance in nasopharyngeal carcinoma cells.

TWIST, a basic helix-loop-helix transcription factor, has been reported to be associated with development and progression of human cancer. Recently, over expression of TWIST is found in cancer patients with shorter survival and poor response to chemotherapy. Previously, we found that upregulation of TWIST was responsible for the development of acquired resistance to taxol in a nasopharyngeal carcinoma (NPC) cell line, HNE1-T3 (Wang et al., Oncogene, 2004;24:274). In this study, we investigated the underlying molecular mechanisms responsible for the TWIST-mediated taxol resistance. By comparison of the parental HNE1 and its derivative HNE1-T3 cell lines, we found that the resistance to taxol in HNE1-T3 cells was associated with suppression of taxol-induced apoptosis evidenced by decreased expression of Bak and Bax and increased Bcl-2, as well as inhibition of PARP and caspase cleavage and DNA ladder formation. However, there was no correlation between taxol sensitivity and alterations on G2/M cell cycle distribution, suggesting that the TWIST-induced taxol resistance is mediated through protection against apoptosis but not mitotic arrest. Analysis of additional 8 NPC cell lines showed that upregulation of TWIST was associated with resistance to microtubule disrupting agents, especially taxol, and inactivation of TWIST through small RNA interference led to increased sensitivity to taxol-induced cell death. Subsequent studies also demonstrated that the TWIST-mediated taxol resistance may be regulated through its positive involvement with the Akt pathway. Our findings suggest an underlying molecular mechanism responsible for the TWIST-mediated chemodrug resistance and suggest a target for overcoming taxol resistance in cancer cells.

Significance of TWIST expression and its association with E-cadherin in bladder cancer.

Recently, TWIST, a basic helix-loop-helix transcription factor, has been reported to play a key role in the metastatic progression of several types of human cancer. The aim of this study was to investigate the significance of TWIST expression in bladder cancer using tissue microassays generated from 226 bladder tissue specimens. Using immunohistochemical staining, we studied TWIST expression levels in nonmalignant bladder tissues (n = 37), primary bladder cancer tissues (n = 164), and 25 cases of matched lymph node metastatic lesions. The association between TWIST expression levels and tumor staging and grading, as well as metastatic potential, was analyzed by statistical analysis. Our results showed that TWIST protein expression was significantly higher in bladder cancer specimens compared with nonmalignant tissues (P < .001), indicating its positive role in the development of bladder cancer. In addition, increased TWIST expression levels were associated with advanced-stage and high-grade tumors, suggesting its involvement in the progression of this cancer. Furthermore, TWIST expression was much higher in the metastatic lesion compared with its primary site (P < .05). More importantly, the increased TWIST expression in bladder cancer specimens was correlated with decreased membranous expression of E-cadherin, a cell adhesion molecule that plays a key role in the metastatic progression of human cancer. Our results demonstrate TWIST as a novel positive factor in the development and progression of bladder cancer and suggest a marker for advanced bladder cancer.