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R. Hasina
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MINI 21 - Novel Targets (ID 133)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:B.P. Levy, D.S. Tan
- Coordinates: 9/08/2015, 16:45 - 18:15, Mile High Ballroom 2a-3b
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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)
17:15 - 17:20 | Author(s): R. Hasina
- Abstract
- Presentation
Background:
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.
Methods:
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.
Results:
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.
Conclusion:
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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P1.04 - Poster Session/ Biology, Pathology, and Molecular Testing (ID 233)
- Event: WCLC 2015
- Type: Poster
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:
- Coordinates: 9/07/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P1.04-001 - MET/RON Inhibition in KRAS Mutated Non Small Cell Lung Cancer (ID 2174)
09:30 - 09:30 | Author(s): R. Hasina
- Abstract
Background:
Molecular genetics have allowed us to categorize non-small cell lung cancer (NSCLC) based on their genetic profile. KRAS mutations occur in 25-30% of NSCLCs. KRAS regulates cellular function in response to growth factors and their receptors. When mutated, KRAS is constitutively active and is responsible for driving tumor oncogenesis. Direct inhibition of KRAS has not been a successful clinical strategy. The strategy of synthetic lethality (targeting a non-lethal defect in cancer cells combined with a second defect, that together make the cancer cell more susceptible to treatment) has gained traction in recent years. Several synthetic lethal targets have been identified with KRAS. We have previously shown that MET plays an important role in the oncogenic addiction observed in KRAS mutated NSCLC and contributes to both tumor growth and metastasis. However, the development of resistance in MET targeting due to upregulation of RON, a related receptor tyrosine kinase, is also evident. Our hypothesis is that dual targeting of MET and RON may be synthetic lethal to KRAS mutated NSCLC and studies to investigate this as a potential therapeutic strategy are warranted.
Methods:
MET- and RON-specific siRNAs (small molecule inhibitors), crizotinib, and the ligand for MET (hepatocyte growth factor), were used in in vitro assays. Immunoblotting, cell viability, and cell migration assays were carried out in a panel of KRAS mutated as well as KRAS wild type NSCLC cells. In addition, human bronchial epithelial cells (HBECs) that were rendered tumorigenic with sequential mutations in CDk4, hTERT, p53, and KRAS genes were also used.
Results:
Our analysis of a panel of NSCLC cells showed that most KRAS mutant cell lines express both MET and RON, and stimulation with HGF activated KRAS effector pathways such as MAPK, AKT and S6RP. When we silenced MET expression with siRNA, it led to upregulation of RON, indicating the interaction between MET and RON. Cell viability assays using crizotinib showed that KRAS mutant cell lines (A549 and H460) are three-fold more sensitive than KRAS wild type cells (H1975 and H1437), and cells with MET amplification (H1993) showed the highest response. Preliminary data with the KRAS-transformed HBECs also showed that they are more sensitive to crizotinib inhibition than the non-transformed control HBECs. Wound healing assays with these same cells showed a similar trend in MET specific inhibition of cell migration in KRAS-mutated cells compared to wild type cells.
Conclusion:
These data highlight the potential therapeutic benefit of targeting MET and RON simultaneously in a subpopulation of KRAS mutated NSCLC patients who may have MET overexpression or amplification. Based on KRAS oncogenic addiction to MET, we propose that NSCLC cells that are MET amplified and KRAS mutated are potentially synthetic lethal and will benefit from dual MET/RON treatment
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P1.08 - Poster Session/ Thymoma, Mesothelioma and Other Thoracic Malignancies (ID 224)
- Event: WCLC 2015
- Type: Poster
- Track: Thymoma, Mesothelioma and Other Thoracic Malignancies
- Presentations: 1
- Moderators:
- Coordinates: 9/07/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P1.08-005 - Met and PI3K/mTOR as a Potential Combinatorial Therapeutic Target in Malignant Pleural Mesothelioma (ID 1700)
09:30 - 09:30 | Author(s): R. Hasina
- Abstract
Background:
There are a number of genetic alterations such as BAP1 and NF2 that can occur in malignant pleural mesothelioma (MPM). Various studies have shown that both MET and its downstream key intracellular signaling partners PI3K and mTOR are known to be overexpressed and frequently mutated in MPM. Here we have examined the therapeutic efficacy of a new generation small molecule inhibitor of MET receptor tyrosine kinase ARQ 197 and phosphatidylinositol 3-kinase and mTOR (PI3K/mTOR) inhibitors BEZ-235 and GDC-0980 in MPM.
Methods:
The mesothelioma cells were treated with ARQ 197, NVP-BEZ235, or GDC-0980 alone or in combination for 72 hours and cell proliferation was measured by using Alamar Blue assay. Synergistic efficacy was determined by isobologram and combination-index methods of Chou and Talalay. Signaling was assessed by immunoblotting. The mechanism of inhibition was further studied by using apoptosis assays and cell cycle analysis. Cell motility was studied by using scratch assays. We also examined efficacy of the combination of ARQ 197 and GDC-0980 on in vivo tumor growth by using mouse xenograft models.
Results:
MPM cell lines over-express MET and its active form p-MET, PI3K, and p-AKT and total AKT. ARQ 197, NVP-BEZ235, and GDC-0980, when used alone, significantly inhibited the cell proliferation of mesothelioma cells in a dose dependent manner. The combination of MET and PI3K/mTOR inhibitors was synergistic in suppressing MPM cell growth as compared to any single drug alone. Treatment of ARQ 197, NVP-BEZ235, and GDC-0980 alone or in combination inhibited the phosphorylation of AKT and S6 kinase in mesothelioma cells. MET and PI3K/mTOR inhibitors affect cell growth of mesothelioma cells by cell cycle inhibition (cyclin D1) and induction of apoptosis (presence of cleaved PARP, by IF/ confocal microscopy). MET inhibitor ARQ 197 alone inhibits the cell motility of mesothelioma cells in scratch assay. The combination of ARQ 197/ GDC-0980 was much more effective than each single agent alone in inhibiting the tumor growth of mesothelioma xenografts in nude mice. Compared to the control mice (2946±403 mm[3]), the tumors of mice treated with ARQ 197(2262±317 mm[3]) and GDC-0980 (1631±229.57mm[3]) alone had a significant decrease in the tumor volume. The tumor volume of mice treated with the combination of ARQ 197 and GDC-0980 further decreased it to six fold (475±97.43 mm[3]) compared to the control mice.
Conclusion:
Our results suggest that the combined use of ARQ 197/NVP-BEZ235 and ARQ 197/GDC-0980 is far more effective than single drug use in suppressing MPM cell motility and growth in vitro and tumor growth in vivo and therefore merits further translational studies.
