Abstract PR06: Molecular and functional interactions between AKT and SOX2 in breast carcinoma
Authors: Schaefer Thorsten , Wang Hui , Mir Perihan , Konantz Martina , Pereboom Tamara C. , Merz Britta , Fehm Tanja , Perner Sven , Rothfuss Oliver K. , Kanz Lothar , Schulze-Osthoff Klaus , Lengerke Claudia 
Published: May 2016

Abstract
The pluripotency-associated transcription factor SOX2 (Sex determining region Y - Box 2) is a key regulator of early organogenesis and embryonic stem cell fate. Supporting the notion that embryonic pathways may reactivate during oncogenesis, SOX2 expression was more recently documented also in several cancers. In breast carcinoma (BC), SOX2 expression is linked to the disease-initiating clonogenic cancer stem cell (CSC) compartment and indicative of disease aggressiveness. However, the molecular regulation of SOX2 in BC remains largely unknown. Here, we describe a functional dependence of SOX2 protein expression on the Ser/Thr-kinase AKT, a central mediator of cell growth, proliferation and metabolism, whose deregulation is a common determinant in BC.
Using qRT-PCR and immunoblot analyses, we confirm SOX2 expression in a series of human BC cell lines and primary patient-derived samples, and more importantly, observe co-induction of SOX2 protein expression and AKT kinase activity in cultivation conditions favoring the stem cell compartment. Suggesting functional coupling, lentiviral-induced knockdown of SOX2 and specific AKT kinase inhibition jointly impair BC cell clonogenicity, as deduced from in vitro tumor spheres assays. Verifying SOX2 as a functional downstream target of AKT, ectopic expression of a mCherry-SOX2 fusion protein rescues spheres formation from AKT-inhibited BC cells in vitro and restores tumorigenicity in xenotransplantation assays in vivo. Moreover, AKT inhibitors effectively impair the growth of putative CSC, as identified by an active SOX2 regulatory region (SRR) reporter construct, while conventional chemotherapeutic drugs (e.g. cisplatin or paclitaxel) suppress growth but allow selective survival and thereby enrichment of this cell population.
On the molecular level, Western analyses indicate a stringent correlation between SOX2 protein expression and AKT kinase activity in all BC cell lines and patient-derived samples tested. Importantly, the inhibition of either AKT itself (MK-2206, Akti1/2) or of its up-stream regulator PI3K (Wortmannin, GDC-0941) impair SOX2 expression, whereas a modulation of de no protein synthesis by either rapamycin or cycloheximide has only minor impact on SOX2 protein levels on the same time-span. Cell fractionation studies further illustrate a preferential cytoplasmic clearance of SOX2 protein in AKT-inhibited cells, which is largely restored by co-treatment with the proteasomal inhibitor bortezomib. Taken together, these data indicate that AKT regulates SOX2 protein expression via post-translational mechanisms in BC.
Supporting this notion, an AKT-phosphorylation motif was identified within the nuclear localization sequence of SOX2 and a direct physical interaction of both proteins verified by co-immunoprecipitation. Confirming a modulation of SOX2 nuclear entry, we observe a temporal cytoplasmic retention of SOX2 in BC cells of stalled AKT-kinase activity. This signal retention is fully restored by co-treatment with leptomycin B, indicating that the observed cytoplasmic accumulation results from nuclear exit of pre-formed SOX2 protein under compromised entry conditions (i.e. persisting AKT kinase inhibition). At extended incubation times, the SOX2 signal gradually disappears in AKT-inhibited cells but can be rescued by concomitant treatment with bortezomib. Importantly, the restored SOX2 protein mislocalizes to the cytosol in anti-AKT and bortezomib co-treated cells, demonstrating that in the absence of AKT activity the rescued SOX2 protein cannot perform nuclear entry.
Taken together, our data indicate an unrecognized coupling of AKT and SOX2 proteins that regulates clonogenicity and in vivo tumorigenicity in human BC. Our investigations reveal important mechanistic details about the regulation of SOX2 subcellular distribution and may provide valuable insights on the potential and limitations of PI3K/AKT/mTOR inhibitors currently used in clinical trials of BC.
Citation Format: Thorsten Schaefer, Hui Wang, Perihan Mir, Martina Konantz, Tamara C. Pereboom, Britta Merz, Tanja Fehm, Sven Perner, Oliver K. Rothfuss, Lothar Kanz, Klaus Schulze-Osthoff, Claudia Lengerke. Molecular and functional interactions between AKT and SOX2 in breast carcinoma. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr PR06.