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T. Sasaki



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    P3.04 - Poster Session/ Biology, Pathology, and Molecular Testing (ID 235)

    • Event: WCLC 2015
    • Type: Poster
    • Track: Biology, Pathology, and Molecular Testing
    • Presentations: 1
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      P3.04-012 - Transition of HSP90-Dependent Client Proteins in ALK Rearranged Non-Small Cell Lung Cancer (ID 337)

      09:30 - 09:30  |  Author(s): T. Sasaki

      • Abstract

      Background:
      Heat Shock Protein (HSP) 90 is one of the major intracellular molecular chaperones, the protein expression is increased in the cell stress conditions, is one of the proteins most present in the cytoplasm in normal condition. HSP90 plays a role for the correct folding of proteins in the correct conformation, and function by interacting with various intracellular proteins. The client proteins that interact with HSP90 contains many important role signaling molecules for cell proliferation and differentiation, such as protein kinase and steroid hormone receptors. Luminespib (AUY922) is a specific HSP90 inhibitor bound to the ATP-binding pocket, leading inactivation, destabilization and degradation of HSP90-dependent client protein. In Non-small cell lung cancer, ALK rearranged cancers are reported as highly sensitive to luminespib, which supposed to ALK fusion proteins are the client proteins of HSP90. By comparing the expression changes in intracellular proteins after treatment of luminespib, we determined the significant pathways in the ALK rearranged cancer cells both sensitive to ALK tyrosine kinase inhibitors (TKIs) and resistant to ALK-TKIs.

      Methods:
      HSP90 inhibitor luminespib (AUY922, Novartis Pharm.) and ALK-TKIs, alectinib (CH5424802, Chugai Pharm.) and were used in this study. Alectinib resistant cell lines were established by exposing increased dose of alectinib to ALK rearranged H3122 cell line (AFR). To study the protein expression screening after treatment of luminespib, protein lysate from H3122 and AFR with or without luminespib were collected, and submitted for mass-spectrometry using iTRAQ. We also determined the protein expression of HSP90-dependent client protein and signaling proteins by Western Blotting.

      Results:
      The iTRAQ data analysis and protein identifications were done with ProteinPilot version 4.5. We observed 1118 proteins/peptides in each cell lines. At first we focused on changes in the protein expression of HSP family after treatment of luminespib. The HSP90A after treatment of luminespib was increased in H3122 and the HSP90B after treatment of luminespib was decreased in AFR. The protein expression of HSP70 after treatment of luminespib was increased in H3122 and in AFR. These results suggested that the role of HSP90-dependent client proteins have changed in ALK-TKIs acquired resistant cells. Secondly, we studied the HSP90-dependent client proteins by intracellular pathway analysis. We suggested that the focal adhesion pathway such as paxilin/crkâ…¡revealed significant HSP90-dependent client proteins.

      Conclusion:
      The focal adhesion molecules are one of the significant signaling pathways in the ALK rearranged NSCLC both sensitive and resistant to ALK-TKIs, and that proteins in the focal adhesion pathway could be a potential target of a ALK-rearranged NSCLC.