Nuclear localization of p70 S6 kinase promotes epithelial-mesenchymal transition in ovarian cancer

Abstract

The 70 kDa ribosomal S6 kinase (p70S6K) is frequently activated in ovarian and a variety of cancers and plays crucial roles in mediating cancer growth and survival. Although it was originally described as a cytoplasmic protein, accumulating evidence suggests that p70S6K can be located in the nucleus. Our laboratory has demonstrated that p70S6K has also a key role in other aspects of tumor progression such as metastasis, in particular epithelial-to-mesenchymal transition (EMT), a crucial process to render cancer cells with abilities to migrate and invade. Interestingly, this function seems to be associated with the nuclear localization of p70S6K. The objective of this study is to investigate the molecular mechanisms and regulation of nuclear p70S6K in EMT. The data showed that the nuclear localization of p70S6K was positively correlated with the metastatic propensities, especially EMT, in ovarian cancer cells. Moreover, overexpression of nuclear p70S6K could effectively induce EMT, as evidenced by a change of cell morphology and marker expression. The findings also identified the splicing factor heterogeneous nuclear ribonucleoprotein (hnRNPA2/B1) as an important mediator of this process. p70S6K could interact with hnRNPA2/B1 in the nucleus in a phosphorylation-dependent manner. Knockdown of hnRNPA2/B1 effectively abolished the nuclear p70S6K induced EMT. The nuclear p70S6K-hnRNPA2/B1 interaction was further revealed to control the alternative splicing of p70S6K pre-mRNA upregulating the short isoforms of p70S6K, h6a and h6c, to drive the EMT phenotypes. The overexpression of nuclear p70S6K, but not cytoplasmic p70S6K, upregulated the expression of h6a and h6c, confirming that this function is specific to the nuclear localization of p70S6K. The phosphorylation, but not kinase activity, of p70S6K was shown to be required for its nuclear localization. In particular, the phosphorylation of p70S6K at the four autoinhibitory S/T-P (S411, S418, T421, S424) and T389 sites. The nuclear import of p70S6K was shown to be importin β-dependent. By analyzing a series of p70S6K C-terminal deletion mutants, the nuclear localization signal of p70S6K was mapped to the first 300 Nterminal residues. Through site-directed mutagenesis, the sequence was refined to residues 100- 105. Collectively, this study uncovers a novel role for the nuclear localization of p70S6K in EMT through alternative splicing activation via hnRNPA2/B1 and the molecular mechanism underlying its nuclear localization, which enhance our understanding of the oncogenic roles of p70S6K.