Cytoplasmic p21 expression levels determine cisplatin resistance in human testicular cancer
Roelof Koster, Alessandra di Pietro, Hetty Timmer-Bosscha, Johan H. Gibcus, Anke van den Berg, Albert J. Suurmeijer, Rainer Bischoff, Jourik A. Gietema, Steven de Jong
Roelof Koster, Alessandra di Pietro, Hetty Timmer-Bosscha, Johan H. Gibcus, Anke van den Berg, Albert J. Suurmeijer, Rainer Bischoff, Jourik A. Gietema, Steven de Jong
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Research Article Oncology

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.

Authors

Roelof Koster, Alessandra di Pietro, Hetty Timmer-Bosscha, Johan H. Gibcus, Anke van den Berg, Albert J. Suurmeijer, Rainer Bischoff, Jourik A. Gietema, Steven de Jong

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Figure 6

Positivity for p21 and p-p21 protein is inversely associated with the expression of the miR-106b seed family in ECs.

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Positivity for p21 and p-p21 protein is inversely associated with the ex...
(A) Quantitative RT-PCR on miR-106b seed family expression in EC cell lines. Note that the intrinsic cisplatin-resistant EC cell lines (2102EP and Scha) have lower levels of the miR-106b seed family and lower Oct4 expression compared with cisplatin-sensitive EC cell lines (Tera and 833KE). All reactions were run in triplicate. miRNA expression was normalized to RNU48 expression, resulting in a ΔCt from which the 2–ΔCt value was derived and depicted. (B) Enhanced p21 levels in Tera 24 hours after treatment with synthetic anti–miR-106b seed family members. Synthetic anti–miR-220 was used as negative control, since miR-220 expression was not detectable in TC (32). (C) Differences in p21 enhancement in EC cells 24 hours after synthetic anti miR-17-5p treatment. (D) Levels of cytoplasmic localized p21 were enhanced after treatment with synthetic anti–miR-17-5p (a-miR-17), compared with control (a-miR-220). Treatment with 10 μM LY294002 (LY) decreased phosphorylated levels of p21 (Thr145) and p21 became more localized in the nucleus of synthetic anti-miR-17-5p transfected Tera cells. Scale bar: 30 μm. (E) Synthetic anti–miR-17-5p strongly upregulates p21 and reduces cisplatin-induced apoptosis and PARP cleavage in Tera. Cells were treated with 0, 4, and 8 μM cisplatin. ***P < 0.001 compared with matching anti-miR-220 control. (F) Reduced apoptosis induction and reduced PARP cleavage depend on upregulation of p21 by synthetic anti–miR-17-5p, as shown by cotransfection with siRNA against p21. Cells were treated with 0, 4, and 8 μM cisplatin. ***P < 0.001 compared with matching anti–miR-17-5p plus scrambled siRNA. (G) Lower expression levels of p21 and enhanced PARP cleavage 24 hours after cisplatin treatment in pre–miR-17-5p transfected Scha and 2102EP cells. Data are represented as mean ± SD.