Abstract LB-11: PTEN modulates EGFRvIII-induced SHP2 phosphorylation and translocation to affect glioblastoma sensitivity to tyrosine kinase inhibitors (TKIs)

Abstract

Abstract Co-expression of EGFRvIII and PTEN in a small subset of recurrent glioblastoma tumors has been shown to increase sensitivity to tyrosine kinase inhibitors (TKIs). However, the exact mechanism of EGFRvIII and PTEN interaction in response to TKIs is still unresolved. Our recent work has shown that SHP2 PTPase is required for EGFRvIII-mediated transformation. The present work was aimed to study the effect of PTEN on EGFRvIII-induced SHP2 phosphorylation and translocation and modulate glioblastoma sensitivity to tarceva treatment. We show that tarceva treatment (10μM/ml) abolished EGFRvIII, EGFR, Gab1, SHP2 and Erk1/2 phosphorylation in LN229.EGFRvIII cells at all time intervals. On the contrary, phosphorylation of EGFRvIII and Erk1/2 in U87MG.EGFRvIII cells was inhibited at early time points, but was restored in 2 to 6 hours. Interestingly, phosphorylation of Akt, Gab1 and SHP2 (Tyr580) was unaffected in U87MG.EGFRvIII cells, but EGFR and SHP2 (Tyr542) phosphorylation were inhibited in a time-dependent manner. MTT proliferation and soft agar transformation assays demonstrated that U87MG.EGFRvIII cells were resistant to tarceva treatment when compared to LN229.EGFRvIII cells. Immunofluorescent labeling of U87MG.EGFRvIII cells with an anti-phospho-SHP2 (Tyr542) antibody showed membrane, cytoplasmic, perinuclear and nuclear localization of SHP2, whereas LN229.EGFRvIII cells exhibited membrane staining of phosphorylated SHP2. However, both the cell lines exhibited strong nuclear staining of phospho-SHP2 (Tyr580). Interestingly, tarceva treatment inhibited membrane localization of phospho-SHP2 (Tyr542) and nuclear localization of phospho-SHP2 (Tyr580) in LN229.EGFRvIII cells after 1 hr, but did not affect SHP2 phosphorylation and localization in U87MG.EGFRvIII cells at all time intervals. Furthermore, stable expression of PTEN in U87MG.EGFRvIII cells conferred relocalization of phospho-SHP2 (Tyr542) to the membrane. Notably, U87MG.EGFRvIII/PTEN clones showed a partially untransformed phenotype. Furthermore, phosphorylation of SHP2 (Tyr580) and Erk1/2 was totally abolished in U87MG.EGFRvIII/PTEN subclones. Our data indicate that the expression of PTEN in U87MG.EGFRvIII cells conferred a phenotype similar to LN229.EGFRvIII cells. Collectively, these observations infer that SHP2 is a downstream effecter of PTEN and that PTEN deficiency may lead to SHP2 activation and nuclear localization by EGFRvIII, which may result in increased resistance to TKIs. 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 LB-11. doi:10.1158/1538-7445.AM2011-LB-11