Abstract 703: GSTP1 inhibition of HSP27 phosphorylation contributes to glioma drug resistance

Cell confluence induces resistance to chemotherapeutic agents in solid tumor cells and phosphorylation of heat shock protein 27 (HSP27), a low-weight molecular chaperon and stress response protein. Here, we show that human glutathione S-transferase P1 (GSTP1) inhibits phosphorylation of HSP27 induced by both epidermal growth factor (EGF) stimulation and cell confluence in malignant brain tumor cells, resulting in enhancement of resistance to cisplatin. Using the paired human medulloblastoma cell lines, UW228 (GSTP1-ve) and UW228-GSTP1 (GSTP1+ve), we showed that growth at high cell density (confluence) induced resistance to cisplatin, regardless of GSTP1 status. Western blotting analysis of extracts of GSTP1-ve cells growing at high density or stimulated with EGF stimulation showed significantly higher phosphorylation levels of HSP27, whereas the HSP27 phosphorylation was inhibited in GSTP1+ve cells. Interestingly, the induction of HSP27 phosphorylation in GSTP1-ve cells was independent of HSP27 upstream kinase activation and serum starvation, suggesting that the inhibition in GSTP1+ve cells results from a direct interaction of HSP27 with GSTP1, rather than a blockade of HSP27 upstream signaling. Inhibition of HSP27 phosphorylation by kinase-specific inhibitors and siRNA-mediated knock-down of the MAP kinase-activated protein kinase-2, MK2, both showed significant inhibition of HSP27 phosphorylation and enhancement of cisplatin resistance in UW228 cells. Using a cell-free system, recombinant GSTP1 protein inhibited HSP27 phosphorylation via MK2. Immunoprecipitation with anti-HSP27 and anti-GSTP1 antibodies showed formation of a GSTP1-HSP27 complex in starved cells and that the level of this complex increased significantly following EGF treatment. In in vivo xenografts of U87MG, HSP27 formed a complex, not only, with GSTP1, but also with p38, Akt, and MK2. shRNA-mediated GSTP1 knockdown in MGR3 cells increased HSP27 phosphorylation significantly, compared with shRNA negative control. Similarly, GSTP1-knockout mouse embryonic fibroblasts (MEFs) showed higher phosphorylation levels of HSP27 induced by both confluence and EGF than control human GSTP1 knock-in MEF cells. Using 2-D native/SDS-PAGE, the level of large (>200 kD) oligomeric HSP27 species was increased in GSTP1+ve, relative to GSTP1-ve cells. Taken together, these results indicate that GSTP1 binds directly to and inhibits HSP27 phosphorylation resulting in an enhanced HSP27 large oligomer formation and increased chaperon activity. The inhibition of HSP27 occurs directly, rather than through a blockade of HSP27 upstream signaling. The data define HSP27 as a novel downstream effector of GSTP1-mediated cellular signaling that can impact outcome of treatment. Supported by NIH grants RO1 CA 153050, RO1CA127872, RO1 CA 112519, P50CA108786 and P30-CA14236. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 703. doi:10.1158/1538-7445.AM2011-703