Priority Report Cisplatin Hypersensitivity of Testicular Germ Cell Tumors Is Determined by High Constitutive Noxa Levels Mediated by Oct-4 Matthias Gutekunst 1 , Thomas Mueller 3 , Andrea Weilbacher 1 , Michael A. Dengler 1 , Jens Bedke 4 , Stephan Kruck 4 , Moshe Oren 5 , Walter E. Aulitzky 2 , and Heiko van der Kuip 1 Abstract Testicular germ cell tumors (TGCT) are considered a paradigm of chemosensitive tumors. Embryonal carcinoma cells represent the pluripotent entity of TGCTs and are characterized by expression of Oct-4, a key regulator of pluripotency and a determinant of their inherent hypersensitivity to cisplatin. However, the mechanisms underlying this Oct-4–mediated sensitivity are poorly understood. We previously showed that p53 is a major player in cisplatin hypersensitivity and therefore investigated whether Oct-4 may directly affect p53 activity. Despite a significant decrease in sensitivity, depletion of Oct-4 neither did alter cisplatin-induced transactivation of p53 target genes nor its subcellular localization. These data indicate that, rather than directly modulating p53 activity, Oct-4 provides a cellular context that augments the proapoptotic activity of p53. As mitochondrial priming by the Bcl-2 family is a known determinant of chemosensitivity, we compared the constitutive levels of these proteins in Oct-4–positive and -depleted cells. We identified Noxa as the only Bcl-2 family protein to be highly correlated with Oct-4 status and cisplatin sensitivity. Compared with differentiated cells, constitutive Noxa levels were significantly higher in Oct-4–positive cell lines and cancer patient samples. Furthermore, RNA interference–mediated knockdown of Oct-4 resulted in reduced Noxa transcript, in an almost complete loss of constitutive Noxa protein and decreased cisplatin hypersensitivity to a similar extent as did Noxa depletion. In conclusion, our study indicates that Noxa is a central determinant of hypersensitivity to cisplatin. Oct-4–dependent high constitutive levels of this BH3-only protein prime embryonal carcinoma cells to undergo rapid and massive apoptosis in response to p53 activation. Cancer Res; 73(5); 1460–9. Ó2012 AACR. Introduction Testicular germ cell tumors (TGCT) are the most frequent carcinomas in young male adults with a still increasing inci- dence worldwide. Cisplatin-based chemotherapy cures the majority of patients even in advanced stages due to the extraordinary sensitivity of TGCTs to cisplatin. Thus, TGCTs are considered as the paradigm of a chemosensitive tumor (1). Several mechanisms have been considered to underlie this hypersensitivity, including the high apoptotic propensity of TGCT cells (2, 3). The transcription factor Oct-4, a key regulator of pluripotency, is exclusively expressed in cells of nonmalignant pluripotent nature, that is, embryonic stem cells (ESC) and germ cells, as well as their malignant counterparts embryonal carcinoma and seminoma, where it serves as a specific malignancy marker (4, 5). Loss of Oct-4 causes a significant reduction of cisplatin hypersensitivity and was proposed to account for acquired cisplatin resistance in refractory tumors (6, 7). We and others have shown that cisplatin hypersensitivity is highly dependent on a functional p53 protein (8–10). The central role of this tumor suppressor was also shown by the finding that these cells are sensitive not only to cisplatin but also to DNA damage–inde- pendent p53 activators such as Nutlin-3 (10–12). Together, these results indicate that hypersensitivity to p53 activation requires an Oct-4–mediated cellular context. We now report that the low apoptotic threshold of Oct-4–positive TGCT cells is dictated by the constitutive presence of high Noxa protein levels. Materials and Methods Cell culture Cell lines from non-seminomatous germ cell tumors were analyzed: H12.1 (embryonal carcinoma), H12.5 (embryonal carcinoma), 2102EP (embryonal carcinoma), 1777NRpmet (dif- ferentiated state), 1411HP (differentiated state), NTERA-2D1 Authors' Affiliations: 1 Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology and University of Tuebingen; 2 2 nd Department of Internal Medicine, Oncology and Hematology, Robert Bosch Hospital, Stuttgart; 3 Department of Internal Medicine IV, Oncology and Hematology, Martin- Luther-University of Halle-Wittenberg, Halle; 4 Department of Urology, Eberhard Karls University of Tuebingen, Tuebingen, Germany; and 5 Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel Corresponding Author: Heiko van der Kuip, Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Auerbachstr. 112, Stuttgart 70376, Germany. Phone: 49-711-8101-3730; Fax: 49-711-859295; E-mail: heiko.van-der-kuip@ikp-stuttgart.de doi: 10.1158/0008-5472.CAN-12-2876 Ó2012 American Association for Cancer Research. Cancer Research Cancer Res; 73(5) March 1, 2013 1460 Downloaded from http://aacrjournals.org/cancerres/article-pdf/73/5/1460/2693919/1460.pdf by guest on 21 June 2022