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A. Rodriguez

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    MINI 21 - Novel Targets (ID 133)

    • Event: WCLC 2015
    • Type: Mini Oral
    • Track: Biology, Pathology, and Molecular Testing
    • Presentations: 1
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      MINI21.06 - Role of the Focal Adhesion Protein Paxillin in Lung Cancer - From Genetic Alterations to Novel Mitochondrial Functionality (ID 2188)

      16:45 - 18:15  |  Author(s): A. Rodriguez

      • Abstract
      • Presentation
      • Slides

      Cytoskeletal and focal adhesion abnormalities are observed in several types of cancer including lung cancer, which is attributed to a greater number of deaths than prostate, breast and colorectal cancers combined. Paxillin is a 68 kDa protein that is an integral part of the focal adhesion and acts as an adaptor molecule. We initially cloned the gene for paxillin, and localized it to chromosome 12q24. We have previously reported that paxillin can be mutated (approximately 8%), amplified (5-7%), and/or overexpressed in almost 80% of lung cancer patient samples. Paxillin protein is upregulated in more advanced stages of lung cancer compared with earlier stages and is a prognostic factor for non-small cell lung cancer (NSCLC). Paxillin gene is amplified in some pre-neoplastic lung lesions as well as neoplastic lesions. We identified 22 different variants of paxillin mutation in our initial investigation especially between the LD and the LIM domains (Jagadeeswaran et al. 2008). There are mutations that have been validated in the TCGA set. We selected six mutants to perform further studies ((P52L, A127T, P233L, T255I, D399N, and P487L as well as wild-type as control). Our investigations focused on an effort to understand the contribution of molecular abnormalities found in paxillin and their relationship to mitochondrial functionality.

      HEK293 cells as well as a paxillin null NSCLC cell line H522 was used to overexpress the above paxillin mutants and wild-type paxillin. Live cell confocal microscopy was performed to evaluate cell motility, immunoprecipitation to determine interaction with other proteins, and gene expression analysis was performed to evaluate effects on gene expression.

      Among the mutations we investigated, we found that the most common paxillin mutant A127T in lung cancer cells enhanced cell proliferation, focal adhesion formation and co-localized with the anti-apoptotic protein B cell CLL/Lymphoma 2 (BCL-2), which among other sites also localizes to the mitochondria. We further found that when these variant clones of activating mutations were expressed in HEK293 cells, they conferred phenotypic changes resembling neoplastic cells. In gene chip microarrays assay investigating gene expression modulation conferred by these mutations in these same HEK293 cells, we found that P52L, A127T, T255I, P233L and D399N mutations, compared to wild-type paxillin, indeed modulated the expression of a significant number of genes. In particular, there were a number of mitochondrial signature proteins that were altered in the various mutants. Analyzing mitochondrial functions by measuring the interaction of these mutants with mitochondrial proteins MFN2, and DRP1, we identified that they alter mitochondrial dynamics, with significant fission rather than fusion. Paxillin also translocated from the focal adhesion to the mitochondrial membrane. In relationship to cisplatin responsiveness, PXN and mutant overexpression lead to cisplatin resistance.

      These data suggest that wild-type and mutant paxillin variants play a prominent role in neoplastic changes with direct implications in lung cancer progression and hence, its potential as a therapeutic target needs to be explored further.

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