Author of

• 18974

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  P3.02 - Biology/Pathology (ID 620)

  • Event: WCLC 2017
  • Type: Poster Session with Presenters Present
  • Track: Biology/Pathology
  • Presentations: 2
  • Moderators:
  • Coordinates: 10/18/2017, 09:30 - 16:00, Exhibit Hall (Hall B + C)

  • 23994

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    P3.02-046 - EGFR-Grb2-GEP100 Complex Promoted Its Invasive and Metastatic Potential via Arf6 Pathway in Lung Adenocarcinoma (ID 9546)

    09:30 - 09:30  |  Author(s): H. Motoyama
    • Abstract

    Background:
    Invasive and metastatic activities are the most challenging hallmark of cancer. We previously showed that GEP100-Arf6 pathway signals from stimulated ErbB family receptors was pivotal for epithelio-mesenchymal transition (EMT) and induced cancer invasive activity leading to nodal and distant metastasis. Here we have examined the enhancing effect of Grb2 on the binding of GEP100 with EGFR leading to Arf6 activation and EMT, and the significance of EGFR-Grb2-GEP100 binding complex on the invasive and metastatic potentials in lung cancer cells as well as in clinical settings.
    
    Method:
    A549 lung cancer cells with overexpressed HA-tagged Grb2 or HA alone were stimulated with EGF, and the lysates were applied for the immunoprecipitation assay against GEP100. Arf6 activities of these cells were examined using the pulldown assay with GST-tagged GGA protein. In vitro invasive activities of those cells were measured by Matrigel invasion assays. To clarify the mutual binding sites between Grb2 and GEP100, GST-tagged proteins including Plekstrin homology (PH) domain of GEP100 or SH2/SH3 domain of Grb2 inserted into pGEX vector were produced in bacteria with glutathione-beads purification. Furthermore, mutant Grb2-R86K which impairs the binding to pEGFR was transfected into A549 cells to examine the changes of the binding affinity between pEGFR and GEP100. Last, we performed immunohistochemistry against Grb2, GEP100, and phosphorylated receptor tyrosine kinases (pRTKs) comprising EGFR, Her2, c-Met, and VEGFR, which have been known to bind GEP100 leading to Arf6 activation, for 239 cases of primary lung adenocarcinoma specimens resected in our hospital. The expression of these molecules integrated with the clinicopathologic data and EMT status information were statistically analyzed.
    
    Result:
    Grb2 overexpression via Grb2-GEP100 interaction promoted EGFR-GEP100 binding leading to Arf6 activation and tumor invasive activity in A549 cells. PH domain of GEP100 and N-terminal SH3/SH2 domain of Grb2 were contributed to this binding. Mutant Grb2-R86K exogenous expression in A549 cells resulted in the decrease of the binding between pEGFR and GEP100. Among 155 cases of positive pRTKs, the combination of Grb2 and GEP100 positivity were significantly associated with activated EMT levels, node metastasis, and poor disease-free survival (p=0.048, p=0.08, and p=0.0075, respectively).
    
    Conclusion:
    EGFR-Grb2-GEP100 complex promoted its invasive and metastatic potential via Arf6 pathway in lung adenocarcinoma. The inhibition of the binding among these molecules may be useful to control lung cancer progression.
    
    

  • 24019

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    P3.02-071 - Statins May Improve the Prognosis of Patients with Lung Adenocarcinoma by Suppressing Mutant p53-Induced EMT (ID 9152)

    09:30 - 09:30  |  Author(s): H. Motoyama
    • Abstract

    Background:
    Epithelial-mesenchymal transition (EMT) is known to be pivotal for driving metastasis and recurrence in lung cancer. Some reports have shown statins suppressed EMT by inactivating mutant p53 functions in vitro. Although several clinical trials regarding conventional treatments with statins have been performed, the effect of statins on the prognosis is still controversial. The purpose of the present study is to clarify the impact of statins on EMT and the prognosis of patients with lung adenocarcinoma harboring p53 mutation.
    
    Method:
    Firstly, we transfected wild type p53 or mutant p53 (R175H, R273H) to H1650 cells and administrated simvastatin. We evaluated morphological changes by microscopic measurement and analyzed EMT markers (E-cadherin, vimentin) through western blotting of whole cell lysate. We also analyzed their invasive ability through Matrigel invasion assay. Secondly, a total of 282 lung adenocarcinoma specimens were collected from patients who underwent surgery in our institute from January 2001 to December 2007. We analyzed EMT markers through immunostaining of tumor specimens and we determined p53 mutation by single stranded conformational polymorphism followed by direct sequencing. The association between EMT, p53 mutation status, and statin use as well as the patients’ clinical information was statistically analyzed. Correlations were compared using Pearson's chi-square test and overall survival were compared using the log-rank test.
    
    Result:
    When mutant p53 (R175H, R273H) were transfected, H1650 cells showed EMT like morphological changes. E-cadherin expression was decreased and vimentin expression was increased in H1650 harboring mutant p53 (H1650[mut.p53]). Additionally, H1650[mut.p53] obtained more aggressive invasiveness compared to H1650 harboring wild type p53. However, simvastatin-treated H1650[mut.p53] lost EMT character changes and aggressive invasiveness. According to the medical records, about 20% of the patients took statins (simvastatin, pravastatin, and so on) as a treatment of hyperlipidemia or coronary artery disease. Consistent with the results of in vitro experiments, IHC showed that statin administration was correlated to fewer EMT only in the patients with mutant p53. Moreover, the statin-administrated group showed significantly better survival compared to the non-statin group (p=0.04), which was observed only in the patients with mutant p53.
    
    Conclusion:
    Statins suppressed EMT and improved the prognosis of patients with lung adenocarcinoma in a p53 mutation-dependent manner.