PTEN reconstitution alters glioma responses to c-Met pathway inhibition.

Mutations/deletions of the tumor-suppressor phosphatase and tensin homolog PTEN result in PI3K/Akt pathway hyperactivation and potentially alter oncogenic responses to targeted receptor tyrosine kinase inhibitors. We previously showed that hepatocyte growth factor (HGF):c-Met pathway inhibition decreases tumor growth and oncogenic signaling responses in PTEN-null/Met+ gliomas. Here, we use two tet-on PTENwt-inducible glioma cell lines and xenograft models to examine the influence of PTEN on oncogenic signaling responses to HGF:c-Met pathway inhibitors. Reconstitution of PTEN inhibited Akt by more than 80% and inhibited cell growth by approximately 70-75% in both cell lines in vitro. C-Met inhibition alone inhibited in-vitro cell growth by approximately 80-85% and the magnitude of growth inhibition was not altered by combining PTEN reconstitution with c-Met inhibition. Combining PTEN reconstitution with Met inhibition arrested a higher percentage of cells in G(1)/G(0) phase of the cell cycle when compared with either PTEN reconstitution or c-Met inhibition alone. Both PTEN reconstitution alone and inhibiting autocrine HGF:c-Met signaling alone, using anti-HGF mAb, robustly inhibited the growth of subcutaneous and intracranial glioma xenografts. Combining anti-HGF therapy with PTEN reconstitution did not significantly alter the magnitude of xenograft growth inhibition. Semiquantitative immunohistopathological analyses revealed that the inhibition of glioma xenograft angiogenesis and cell proliferation by anti-HGF mAb was greatest in conjunction with PTEN reconstitution. In contrast, xenograft cell apoptosis was greatest in response to anti-HGF therapy alone and PTEN reconstitution abrogated the apoptotic response to anti-HGF therapy. These results provide new insights into how PTEN modulates glioma responses to the inhibition of HGF:c-Met signaling and possibly other receptor tyrosine kinase pathways.

Cyr61 mediates hepatocyte growth factor-dependent tumor cell growth, migration, and Akt activation.

Certain tumor cell responses to the growth factor-inducible early response gene product CCN1/Cyr61 overlap with those induced by the hepatocyte growth factor (HGF)/c-Met signaling pathway. In this study, we investigate if Cyr61 is a downstream effector of HGF/c-Met pathway activation in human glioma cells. A semiquantitative immunohistochemical analysis of 112 human glioma and normal brain specimens showed that levels of tumor-associated Cyr61 protein correlate with tumor grade (P < 0.001) and with c-Met protein expression (r(2) = 0.4791, P < 0.0001). Purified HGF rapidly upregulated Cyr61 mRNA (peak at 30 minutes) and protein expression (peak at 2 hours) in HGF(-)/c-Met(+) human glioma cell lines via a transcription- and translation-dependent mechanism. Conversely, HGF/c-Met pathway inhibitors reduced Cyr61 expression in HGF(+)/c-Met(+) human glioma cell lines in vitro and in HGF(+)/c-Met(+) glioma xenografts. Targeting Cyr61 expression with small interfering RNA (siRNA) inhibited HGF-induced cell migration (P < 0.01) and cell growth (P < 0.001) in vitro. The effect of Cyr61 on HGF-induced Akt pathway activation was also examined. Cyr61 siRNA had no effect on the early phase of HGF-induced Akt phosphorylation (Ser(473)) 30 minutes after stimulation with HGF. Cyr61 siRNA inhibited a second phase of Akt phosphorylation measured 12 hours after cell stimulation with HGF and also inhibited HGF-induced phosphorylation of the Akt target glycogen synthase kinase 3alpha. We treated preestablished subcutaneous glioma xenografts with Cyr61 siRNA or control siRNA by direct intratumoral delivery. Cyr61 siRNA inhibited Cyr61 expression and glioma xenograft growth by up to 40% in a dose-dependent manner (P < 0.05). These results identify a Cyr61-dependent pathway by which c-Met activation mediates cell growth, cell migration, and long-lasting signaling events in glioma cell lines and possibly astroglial malignancies.