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M. Hassanein



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    P2.01 - Poster Session 2 - Cancer Biology (ID 145)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P2.01-020 - Down-regulation of Somatostatin Receptor 2 decreases cell proliferation in Lung Cancer (ID 1889)

      09:30 - 09:30  |  Author(s): M. Hassanein

      • Abstract

      Background
      Somatostatin receptors (SSTRs) have been shown to be overexpressed in 80-90% of all neuroendocrine tumors, including SCLCs. Previous reports have shown that SSTR2 activation modulates cell proliferation by acting through the ERK signaling cascade. We hypothesized that down regulation of SSTR2 will inhibit cell proliferation in lung cancer cell lines that overexpresses this receptor.

      Methods
      SSTR2 expression was assessed by western blot analysis in 28 lung cancer cell lines. H520 (SCC) and H727 (Carcinoid) were used as an in vitro model for studying the effects of targeted down-regulation of SSTR2 by shRNA. Several SSTR2 null-colonies of these cell lines were generated by antibiotic selection. Cell proliferation was measured at four different time points using MTS metabolic assay. In addition, we evaluated the effect of SSTR2 down regulation in colony formation potential of our lung cancer cell lines.

      Results
      Western blot analysis of 28 lung cancer cell lines showed expression of SSTR2 in 17/18 (94%) of SCLCs including carcinoids, 3/6 (50%) of NSCLCs, and 0/4 (0%) of non-immortalized human bronchial cells. Post transfection analysis by western blot revealed a significant decrease in SSTR2 protein expression in multiple SSTR2 null-colonies. We observed a 37% decrease in cell growth in H520 and a 38% decrease in H727 by day 6 with p values of 0.007 and 0.002 respectively. In addition, down regulation of SSTR2 resulted in reduction in the number of colonies formed by both cell lines.

      Conclusion
      These results demonstrate that that knocking down SSTR2 in lung cancer cells induces a marked decrease in cell proliferation. Ongoing studies are focused on understanding the effect of knocking down SSTR2 on oncogenic traits of lung cancer cells including cell survival, apoptosis, migration and invasion and the cellular mechanisms underpinning our original observation.

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    P3.01 - Poster Session 3 - Cancer Biology (ID 147)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P3.01-006 - Discovery of new membrane-associated proteins preferentially expressed in small-cell lung cancer (ID 2306)

      09:30 - 09:30  |  Author(s): M. Hassanein

      • Abstract

      Background
      Small-cell lung cancer (SCLC) is the most aggressive subtype of lung cancers, with no early detection strategy or targeted therapy currently available. We hypothesized that difference gel electrophoresis (DIGE) may allow the identification of membrane-associated proteins specific to SCLC, the advancement of our understanding of SCLC biology, and the discovery of new candidate diagnostic or therapeutic biomarkers.

      Methods
      Membrane-associated protein lysates were prepared in quadruplicate from three SCLC, three non-small-cell lung cancer (NSCLC), and three immortalized normal bronchial epithelial cell lines. The 36 samples were co-analyzed by DIGE and subsequent protein identification was performed by mass spectrometry (MS). Identified proteins were submitted to Ingenuity pathway analysis (IPA). Candidate biomarkers were validated by Western blotting (WB) and immunohistochemistry (IHC), and tested against clinical outcomes.

      Results
      Principle component analysis on the global DIGE dataset demonstrated that the four replicates derived from each of the nine cell lines clustered very closely from one another, as did samples within the same histological group. A total of 135 distinct protein features were differentially expressed in SCLC as compared with NSCLC and bronchial airway epithelial cell lines (P<0.001). Those included 137 different proteins identified by tandem MS. IPA revealed that these proteins were overrepresented in the cellular assembly, organization, morphology, and tissue morphology networks. DPYL2, GNAQ, RSSA, RUVB1, and STMN1 were found to be the most discriminatory candidate biomarkers among the membrane-associated proteins overexpressed in SCLC as compared with NSCLC and normal bronchial airway cell lines. Overexpression of DPYL2, GNAQ, and STMN1 was verified in SCLC cell lines by WB. Intense protein expression of DPYL2, GNAQ, RUVB1, and STMN1 was also confirmed in SCLC by IHC on tissue microarrays (TMA). These proteins’ expression levels measured by IHC were significantly associated with the SCLC subtype and survival in a univariable analysis but could not be verified as independent in a multivariable analysis.

      Conclusion
      Proteomic analysis of membrane-associated proteins in lung cancer and bronchial airway epithelial cell lines revealed 137 proteins differentially expressed in SCLC. These proteins were enriched for cellular assembly, organization, morphology, and tissue morphology networks. Of the five proteins selected for clinical validation, DPYL2, GNAQ, RUVB1, and STMN1 overexpression in SCLC was verified by WB and IHC, suggesting that these results need to be tested for functional implication in SCLC progression. The association with survival requires further validation in a larger clinical dataset. Funding This work was supported by a VA Merit review award 1I01CX000242 to PPM. SO was supported by a Télévie Grant, a Fondation Mont-Godinne Grant and a Clinician-Researcher Mandate from Secteurs des Sciences de la Santé, Université Catholique de Louvain (UCL), Belgium.