Abstract
3530
The protein tyrosine phosphatase, SHP-2, regulates RTK-mediated cell proliferation, differentiation, transformation and cell survival. It also plays a critical role in the regulation of cell spreading, migration, and cytoskeletal organization. Previously, we showed that a phosphatase-inactive form of SHP-2, SHP-2 C459S, and shp-2 short interfering RNA (siRNA) inhibited transformation of PTEN-deficient U87MG.EGFRvIII human glioblastoma (GBM) cells. In order to further evaluate the importance of SHP-2 in GBM transformation, particularly that mediated by the EGFRvIII oncoprotein, we employed a PTEN-positive GBM cell line LN229 expressing the EGFRvIII oncoprotein (LN229.EGFRvIII) to test whether SHP-2 is generally required for GBM transformation. Here, we report that the Shp-2 siRNA inhibited the expression of SHP-2 at the protein level in both cell lines. Both U87MG.EGFRvIII and LN229.EGFRvIII GBM cells with decreased SHP-2 expression grew slower and these cells exhibited a completely nontransformed phenotype. Consistent with this, shp-2 siRNA inhibited U87MG.EGFRvIII and LN229.EGFRvIII cells grow in soft agar. Inhibition of SHP-2 by shp-2 siRNA resulted in dephosphorylation of EGFRvIII, while the total EGFRvIII remained the same in U87MG.EGFRvIII cells. However, inhibition of SHP-2 by shp-2 siRNA resulted in decreased EGFRvIII oncoprotein levels in LN229.EGFRvIII cells. Interestingly, shp-2 siRNA expression arrested U87MG.EGFRvIII cells at G2/M while LN229.EGFRvIII cells were arrested at G0/G1. Moreover, immunofluorescence (IF) techniques showed that EGF treatment induced SHP-2 phosphorylation at Tyr542 and phosphorylated SHP-2 accumulated in the cellular membrane in parental and EGFRvIII-containing U87MG and LN229 cells. However, EGFRvIII expression induced SHP-2 phosphorylation at Tyr542 that localized to the perinuclear region only in U87MG.EGFRvIII cells. Total SHP-2 distributed in both the cytosolic and perinuclear regions in all cell lines and its localization was not changed under these conditions. These data provide further support that SHP-2 may be a downstream factor of PTEN in GBM cells. They also suggest that inhibition of SHP-2 expression inhibits the transformation of both PTEN-deficient and -intact GBMs but via different mechanisms. Therefore, use of shp-2 siRNAs may represent a potential therapeutic approach for the treatment of human GBMs, in particular primary GBMs that express the EGFRvIII oncoprotein.
99th AACR Annual Meeting-- Apr 12-16, 2008; San Diego, CA
