Abstract
Background: There is a great need to understand the molecular aberrations that drive cellular transformation, to enable selective targeting of tumor-promoting molecules and aid in the development of more effective anti-cancer treatments. Hsp90 is a chaperone protein with key roles in maintaining the transformed phenotype. Upon mutation or deregulation many oncogenic proteins display unusually stable association with Hsp90-containing chaperone complexes. Hypothesis: Small molecules that target tumor specific Hsp90 and efficiently lock the chaperone in a client-binding conformation may be employed in the isolation and identification of cancer-specific activating proteins and signaling pathways. Study design: The purine-scaffold (PU) class of Hsp90 inhibitors exert their biological effects via specific interference with the tumor specific Hsp90. In this study, we have immobilized PU-H71 on solid support, and used it as a chemical tool to \#8220;fish out\#8221; proteins tightly bound to tumor-specific Hsp90. To validate the ability of PU-H71 to identify the key subset of malignancy driving proteins, we have investigated the chronic myeloid leukemia cell K562. The major transforming molecule in K562 cells is Bcr-Abl, a constitutively activated kinase that stimulates several signaling pathways, such as JAK/STAT, Raf/MEK/ERK and PI3K/Akt, leading to deregulated cell cycle progression and proliferation, and defective apoptosis. To identify the Hsp90-onco clients in K562 cells, we subjected the protein cargo isolated by the Hsp90 inhibitor-beads to proteomic analysis, and validated it by follow-up western blot analyses. Results: We found that Hsp90 interacts in K562 cells with both Bcr-Abl and its normal counterpart, c-Abl. In contrast, PU-H71 beads selectively isolated the Bcr-Abl bound Hsp90 species. CrkL and Grb2, key proteins streamlining Bcr-Abl activated signaling through the Ras/Raf-1/ERK, Akt/mTOR and STAT5 mediated pathways, were also identified. We found key nodal proteins of the three major activated pathways to be in the PU-H71/Hsp90 complex. Among them is STAT5, whose activation is necessary for Bcr-Abl to confer cytokine independence and protection against apoptosis in CML. mTOR, which is constitutively activated in Bcr-Abl-transformed cells and is a downstream effector of the PI3K/Akt pathway, is also found in the PU-H71 pull down. Similarly is ERK and p90RSK, key effectors of the MAPK pathway. In addition to signaling proteins, PU-H71 pulldowns implicate for the first time Hsp90 in regulating specific pathogenic changes related to CML, such as increased gene methylation by regulating the histone-arginine methyltransferase CARM1. Conclusion: Our method looks at a unique subpopulation of proteins that are clients of the activated tumor Hsp90 protein, and thus, is restricted at identifying abnormal proteins found in and contributing to the oncogenic state.
Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr LB-86.
100th AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO
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