Abstract 8: Differential roles of OCT3/4, SOX2 and NANOG for constitutive high NOXA expression levels in embryonal carcinoma (EC) cells

Abstract

Abstract Recently we found that hypersensitivity of embryonal carcinoma (EC) to chemotherapy is mediated by high constitutive levels of NOXA protein. This pro-apoptotic BH3-only protein primes EC cells to undergo rapid and massive apoptosis in response to p53 activation. Both hypersensitivity as well as high NOXA protein levels were lost upon differentiation in these cells. We here investigated the role of three key regulators of pluripotency, namely OCT3/4, SOX2 and NANOG for NOXA protein and transcript (PMAIP1) expression in two EC cell lines, the pluripotent NTERA-2D1 and the nullipotent 2102EP. We found that siRNA-mediated silencing of POU5F1 (OCT3/4) led to a down-regulation of PMAIP1 mRNA by ∼80% and to an almost complete loss of NOXA protein (by >90%). On the other hand, silencing of SOX2 or NANOG only slightly reduced transcript levels (by ∼20% and ∼30%, respectively). At the same time, a distinct down-regulation of NOXA protein levels (by ∼75%) was observed in SOX2- and NANOG-deprived cells, respectively. These data indicate that the high constitutive levels of NOXA in EC cells depend on two independent mechanisms, (1) transcriptional regulation predominantly mediated by OCT3/4, and (2) post-transcriptional regulation mediated by either one of the stem cell factors. Indeed, we found that siRNA-mediated loss of one stem cell factor led to a ∼2fold reduction of NOXA protein stability in both cell lines reaching a NOXA half-life of approximately 2 hours, which was comparable to that observed in PHA stimulated lymphocytes from normal donors. These data demonstrate that the high constitutive levels of NOXA protein in EC cells are due to OCT3/4 dependent induction of the PMAIP1 transcript and a prolonged NOXA protein stability mediated by either one of the stem cell factors. Citation Format: Christine Bayha, Matthias Gutekunst, Walter E. Aulitzky, Heiko van der Kuip. Differential roles of OCT3/4, SOX2 and NANOG for constitutive high NOXA expression levels in embryonal carcinoma (EC) cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 8. doi:10.1158/1538-7445.AM2015-8