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Cytoplasmic p21 expression levels determine cisplatin resistance in human testicular cancer.

ABSTRACT: Platinum-based chemotherapies such as cisplatin are used as first-line treatment for many cancers. Although there is often a high initial responsiveness, the majority of patients eventually relapse with platinum-resistant disease. For example, a subset of testicular cancer patients still die even though testicular cancer is considered a paradigm of cisplatin-sensitive solid tumors, but the mechanisms of chemoresistance remain elusive. Here, we have shown that one key determinant of cisplatin-resistance in testicular embryonal carcinoma (EC) is high cytoplasmic expression of the cyclin-dependent kinase (CDK) inhibitor p21. The EC component of the majority of refractory testicular cancer patients exhibited high cytoplasmic p21 expression, which protected EC cell lines against cisplatin-induced apoptosis via CDK2 inhibition. Localization of p21 in the cytoplasm was critical for cisplatin resistance, since relocalization of p21 to the nucleus by Akt inhibition sensitized EC cell lines to cisplatin. We also demonstrated in EC cell lines and human tumor tissue that high cytoplasmic p21 expression and cisplatin resistance of EC were inversely associated with the expression of Oct4 and miR-106b seed family members. Thus, targeting cytoplasmic p21, including by modulation of the Oct4/miR-106b/p21 pathway, may offer new strategies for the treatment of chemoresistant testicular and other types of cancer.

SUBMITTER: Koster R

PROVIDER: S-EPMC2947220 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

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Cytoplasmic p21 expression levels determine cisplatin resistance in human testicular cancer.

Koster Roelof R di Pietro Alessandra A Timmer-Bosscha Hetty H Gibcus Johan H JH van den Berg Anke A Suurmeijer Albert J AJ Bischoff Rainer R Gietema Jourik A JA de Jong Steven S

The Journal of clinical investigation 20100901 10

Platinum-based chemotherapies such as cisplatin are used as first-line treatment for many cancers. Although there is often a high initial responsiveness, the majority of patients eventually relapse with platinum-resistant disease. For example, a subset of testicular cancer patients still die even though testicular cancer is considered a paradigm of cisplatin-sensitive solid tumors, but the mechanisms of chemoresistance remain elusive. Here, we have shown that one key determinant of cisplatin-res ...[more]

PMID: 20811155

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Project description:BACKGROUND: P21(WAF1/Cip1) binds to cyclin-dependent kinase complexes and inhibits their activities. It was originally described as an inhibitor of cancer cell proliferation. However, many recent studies have shown that p21 promotes tumor progression when accumulated in the cell cytoplasm. So far, little is known about the correlation between cytoplasmic p21 and drug resistance. This study was aimed to investigate the role of p21 in the cisplatin resistance of ovarian cancer. METHODS: RT-PCR, western blot and immunofluorescence were used to detect p21 expression and location in cisplatin-resistant ovarian cancer cell line C13* and its parental line OV2008. Regulation of cytoplasmic p21 was performed through transfection of p21 siRNA, Akt2 shRNA and Akt2 constitutively active vector in the two cell lines; their effects on cisplatin-induced apoptosis were evaluated by flow cytometry. Tumor tissue sections of clinical samples were analyzed by immunohistochemistry. RESULTS: p21 predominantly localizes to the cytoplasm in C13* compared to OV2008. Persistent exposure to low dose cisplatin in OV2008 leads to p21 translocation from nuclear to cytoplasm, while it had not impact on p21 localization in C13*. Knockdown of cytoplasmic p21 by p21 siRNA transfection in C13* notably increased cisplatin-induced apoptosis through activation of caspase 3. Inhibition of p21 translocation into the cytoplasm by transfection of Akt2 shRNA into C13* cells significantly increased cisplatin-induced apoptosis, while induction of p21 translocation into the cytoplasm by transfection of constitutively active Akt2 in OV2008 enhanced the resistance to cisplatin. Immunohistochemical analysis of clinical ovarian tumor tissues demonstrated that cytoplasmic p21 was negatively correlated with the response to cisplatin based treatment. CONCLUSIONS: Cytoplasmic p21 is a novel biomarker of cisplatin resistance and it may represent a potential therapeutic target for ovarian tumors that are refractory to conventional treatment.

Project description:Ovarian cancer (OC) is a major contributor to cancer?related mortality in women. Despite numerous drugs being available for the treatment and improving the prognosis of OC, resistance to clinical chemotherapy remains a major obstacle for the treatment of advanced OC. Therefore, determining how to reverse the chemoresistance of OC has become a research hotspot in recent years. The present study aimed to reveal the potential mechanism of OC chemoresistance. Reverse transcription?quantitative PCR and western blot analysis were performed to detect the expression levels of Ubiquitin?specific peptidase 46 (USP46) and Pumilio 2 (PUM2) in OC. Cell viability and apoptosis were evaluated by Cell Counting Kit?8 assay and flow cytometry, respectively. The association between USP46 and PUM2 was assessed by RNA immunoprecipitation. The results of the present study revealed that the expression levels of USP46 which is associated with tumor progression, was downregulated, while PUM2 expression levels were upregulated in cisplatin (DDP)?resistant OC cells and patient tissues. The downregulation of USP46 expression levels in SKOV3 cells significantly inhibited cell apoptosis and increased cell viability. In SKOV3/DDP cells, the upregulation of USP46 expression levels notably suppressed cell viability and increased cell apoptosis. The results of the RNA immunoprecipitation chip assay demonstrated that PUM2 bound to USP46 and regulated its expression. Furthermore, following the knockdown of USP46 expression, the mRNA and protein expression levels of the cell apoptosis?related protein, Bcl?2, were upregulated, whereas the expression levels of caspase?3, caspase?9 and Bax were significantly downregulated. In addition, phosphorylated AKT expression levels were notably upregulated. Following the overexpression of USP46 in SKOV3/DDP cells, the opposite trends were observed. In SKOV3 cells, the knockdown of PUM2 could reverse the DDP resistance induced by small interfering RNA?USP46 as the expression levels of Bcl?2 were downregulated whereas those of caspase?3, caspase?9 and Bax were upregulated compared with the small interfering?USP46 group. Similarly, in SKOV3/DDP cells, the overexpression of PUM2 could reverse DDP sensitivity induced by the overexpression of USP46. In conclusion, the findings of the present study suggested that the downregulation of USP46 expression levels may promote DDP resistance in OC, which may be regulated by PUM2. Therefore, targeting PUM2/USP46 may be an effective way to reverse DDP resistance in OC.

Dataset Information

Cytoplasmic p21 expression levels determine cisplatin resistance in human testicular cancer.

Publications

Cytoplasmic p21 expression levels determine cisplatin resistance in human testicular cancer.

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