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.

Microarray-Based Phospho-Proteomic Profiling of Complex Biological Systems.

Protein microarray technology has been successfully used for identifying substrates of purified activated kinases. We used protein microarrays to globally interrogate the effects of PTEN and Akt activity on the phospho-kinome of in vitro and in vivo glioma models and validated results in clinical pathological specimens. Whole cell lysates extracted from tumor samples can be applied to human kinome chip microarrays to profile the global kinase phosphorylation patterns in a high-throughput manner and identify novel substrates inherent to the tumor cell and the interactions with tumor microenvironment. Our findings identify a novel microarray-based method for assessing intracellular signaling events applicable to human oncogenesis and other pathophysiologic states.

Profiling the dynamics of a human phosphorylome reveals new components in HGF/c-Met signaling.

Protein phosphorylation is a dynamic and reversible event that greatly influences cellular function. Identifying the key regulatory elements that determine cellular phenotypes during development and oncogenesis requires the ability to dynamically monitor proteome-wide events. Here, we report the development of a new strategy to monitor dynamic changes of protein phosphorylation in cells and tissues using functional protein microarrays as the readout. To demonstrate this technology's ability to identify condition-dependent phosphorylation events, human protein microarrays were incubated with lysates from cells or tissues under activation or inhibition of c-Met, a receptor tyrosine kinase involved in tissue morphogenesis and malignancy. By comparing the differences between the protein phosphorylation profiles obtained using the protein microarrays, we were able to recover many of the proteins that are known to be specifically activated (i.e., phosphorylated) upon c-Met activation by the hepatocyte growth factor (HGF). Most importantly, we discovered many proteins that were differentially phosphorylated by lysates from cells or tissues when the c-Met pathway was active. Using phosphorylation-specific antibodies, we were able to validate several candidate proteins as new downstream components of the c-Met signaling pathway in cells. We envision that this new approach, like its DNA microarray counterpart, can be further extended toward profiling dynamics of global protein phosphorylation under many different physiological conditions both in cellulo and in vivo in a high-throughput and cost-effective fashion.