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L.A. Mac Donagh

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    P1.02 - Poster Session 1 - Novel Cancer Genes and Pathways (ID 144)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P1.02-007 - Validation and Function of a Novel miRNA signature in Cisplatin Resistant Non-Small Cell Lung Cancer Cells (ID 1410)

      09:30 - 16:30  |  Author(s): L.A. Mac Donagh

      • Abstract

      Lung cancer is the leading cause of cancer-related deaths worldwide, where non-small cell lung cancer (NSCLC) accounts for 85% of cases. While cisplatin-based chemotherapy remains the gold standard treatment for lung cancer, response rates are low due to increasing development of resistance to cisplatin. Circumventing cisplatin resistance, therefore, remains a critical goal for anti-cancer therapy. The aim of this study is to examine the role of miRNA’s in the regulation of cisplatin resistance in NSCLC using a panel of isogenic cisplatin resistant NSCLC cell lines developed in our laboratory, and to examine the putative cancer stem cell markers within this chemoresistant phenotype.

      MicroRNA profiling of a panel of isogenic cisplatin resistant (CisR) NSCLC cell lines, and age-matched parent cells (PT) was carried out using a 749 miRNA in-situ hybridisation array platform (Nanostring Technologies). The miRNA signature obtained was validated by qPCR using miRCURY LNA[TM ]Universal RT miRNA PCR technology (Exiqon). In order to determine the role of these miRNA’s in conferring cisplatin resistance, transfection of cell lines using miR-specific antagomirs and pre-miR’s will be carried out to examine this effect on sensitising lung cancer cells to cisplatin using clonogenic survival assays, apoptosis (APC), proliferation (BrdU) and DNA damage repair (γH2AX) assays. Furthermore, the characterisation and potential role of exosomes and exosomal-derived miRNA in modulating the cellular response to cisplatin chemotherapy will also be examined in this model of resistance. Putative stem cell markers (Oct-4, Sox-2, Nanog, SSEA4, Klf-4 and c-Myc) will be assessed in holoclones derived from resistant sublines. An in vivo model will be used to The tumourigenic potential of putative cancer stem-like cells within the cisplatin resistant population will be investigated in vivo using NOD/SCID mice. In parallel, asymmetric division assays will also be used.

      MicroRNA profiling of MOR, H460, A549, SKMES-1 and H1299 cisplatin resistant cell lines deduced a 3-miR signature across all five chemoresistant cell lines. Validation of this miR signature by qPCR showed differential expression of only one miRNA, miR30-c. miR-30c was significantly up-regulated (15-40 fold) in MOR, H460, SKMES-1 and H1299 CisR NSCLC cell lines and significantly down-regulated (5-fold) in A549 CisR cells relative to PT cells. The effects of miR-30c antagomirs and pre-miR’s in reversing the cisplatin resistant phenotype of NSCLC cells are currently being examined.

      Differential expression of 3 specific miRNA’s was demonstrated in CisR lung cancer cells, relative to their parent counterparts, using an in-situ miRNA profiling hybridisation platform. While validation of this panel of miRNA’s identified only one differentially regulated miRNA species, miR30-c, further studies are warranted to explore the role of miR-30c in the cisplatin resistance phenotype of NSCLC. Exosome and cancer stem cell studies are currently under investigation.