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L. Mac Donagh



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    MA02 - RNA in Lung Cancer (ID 377)

    • Event: WCLC 2016
    • Type: Mini Oral Session
    • Track: Biology/Pathology
    • Presentations: 1
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      MA02.02 - A Novel 5-miR Signature Shows Promise as a Diagnostic Tool and as a Predictor of Cisplatin Response in NSCLC (ID 5948)

      14:20 - 15:50  |  Author(s): L. Mac Donagh

      • Abstract
      • Slides

      Background:
      MicroRNAs are a class of small non-coding RNAs that range in size from 19-25 nucleotides. They have been shown to regulate a number of processes within tumour biology, including metastasis, invasion and angiogenesis. More recently, miRNAs have been linked to chemoresistance in solid tumours, including lung cancer.

      Methods:
      MicroRNA expression within an isogenic panel of age-matched parent (PT) and cisplatin resistant (CisR) NSCLC cell lines was profiled using the 7[th] generation miRCURY LNA arrays (Exiqon). Significantly altered miRNAs within the CisR sublines were manipulated using antagomirs (Exiqon) and Pre-miRs (Ambion) and functional studies were carried out in the presence and absence of cisplatin. To examine the translational relevance of these miRNAs, their expression was examined in a cohort of chemo-naïve patient-matched normal and lung tumour tissue and serum from NSCLC patients of different histologies. To create a xenograft model of cisplatin resistance 1x10[3 ]cells H460 PT or CisR cells were injected into 5-7week old NOD/SCID mice. Tumour volume was measured over time and harvested once the tumour mass measured 500mm[3] and formalin-fixed and paraffin embedded (FFPE). Expression of the 5-miR signature was analysed within FFPE murine tumours and cisplatin resistance was investigated relative to cisplatin sensitive controls.

      Results:
      Profiling and subsequent validation revealed a 5-miR signature associated with our model of cisplatin resistance (miR-30a-3p, miR-30b-5p, miR-30c-5p, miR-34a-5p, miR-4286). Inhibition of the miR-30 family and miR-34a-5p reduced clonogenic survival of CisR cells when treated cisplatin. Expression of the miRNA signature was significantly altered in both adenocarcinoma (AD) and squamous cell carcinoma (SCC) relative to matched normal lung tissue and between SCC and AD tissue. miR-4286 was significantly up-regulated in SCC sera compared to normal control and AD sera. Similarly to the cell line expression of the miRNAs, the miR-30 family members and miR-34a-5p were up-regulated in the CisR xenograft FFPE tissue relative to PT.

      Conclusion:
      A novel miRNA signature associated with cisplatin resistance was identified in vitro, genetic manipulation of which altered clonogenic response to cisplatin. The 5-miR signature shows both diagnostic and prognostic biomarker potential across a number of diagnostically relevant biological mediums.

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    P2.03a - Poster Session with Presenters Present (ID 464)

    • Event: WCLC 2016
    • Type: Poster Presenters Present
    • Track: Advanced NSCLC
    • Presentations: 2
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      P2.03a-063 - Small Molecule Cancer Stemness Inhibitor, BBI608, Restores Cisplatin Sensitivity in Resistant NSCLC (ID 5962)

      14:30 - 15:45  |  Author(s): L. Mac Donagh

      • Abstract
      • Slides

      Background:
      The cancer stem cell (CSC) hypothesis is now a well-established and widely investigated field within oncology. It hypothesizes that there is a robustly resistant stem-like population of cells that survive initial chemotherapeutic treatment. These surviving CSCs contribute to the recapitulation of a heterogeneous tumour via a combination of asymmetric and symmetric cell division, subsequently resulting in relapse and therapeutic resistance. BBI608 is a small molecule inhibitor of cancer stemness; it targets STAT3, leading to the inhibition of critical genes required for the maintenance of cancer stemness. To date, preclinical studies investigating BBI608 in in vitro and in vivo models of pancreatic and prostate cancer have shown promise.

      Methods:
      Aldefluor (Stemcell Technologies) staining and flow cytometry analysis of an isogenic panel of matched parent (PT) and cisplatin resistant (CisR) NSCLC cell lines identified the ALDH1-positive (ALDH1+ve) subpopulation of cells as a key CSC subset across cisplatin resistant NSCLC cell lines. PT and CisR cell lines were treated with BBI608 (1μM) and stemness factors investigated, the presence of the ALDH1+ve CSC population was reassessed by flow cytometry and expression of stemness factors (Nanog, Oct-4, Sox-2, Klf4 and cMyc) were examined by reverse transcriptase PCR. The functional parameters of proliferation, clonogenic survival and apoptosis were investigated with increasing concentrations of cisplatin (0-100μM) in the presence and absence of 1μM BBI608.

      Results:
      The NSCLC CisR sublines showed a significantly greater ALDH1+ve CSC population relative to their PT counterparts. Treatment of the CisR sublines with 1μM BBI608 significantly depleted the ALDH1+ve CSC population and decreased gene expression of stemness markers. BBI608 significantly decreased the proliferative capacity and clonogenic survival of the CisR sublines when in combination with cisplatin relative to cisplatin alone. Cisplatin in combination with BBI608 significantly increased cisplatin-induced apoptosis in the CisR sublines indicating restoration of cisplatin sensitivity.

      Conclusion:
      To date, BBI608 has not been investigated in terms of a cisplatin resistant CSC population in lung cancer. BBI608, via the inhibition of STAT3, pharmacologically depleted the CSC subpopulation and stemness expression while simultaneously restoring cisplatin sensitivity. There are currently a number of clinical trials recruiting patients to further investigate BBI608. These data suggest a promising role for BBI608 in the treatment of non-responsive or recurrent NSCLC.

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      P2.03a-064 - Inhibition and Exploitation of Aldehyde Dehydrogenase 1 as a Cancer Stem Cell Marker to Overcome Cisplatin Resistant NSCLC (ID 5954)

      14:30 - 15:45  |  Author(s): L. Mac Donagh

      • Abstract
      • Slides

      Background:
      The root of therapeutic resistance is hypothesized to be the presence a rare CSC population within the tumour population which survives chemotherapeutic treatment and has the potential to recapitulate a heterogenic tumour. Aldehyde dehydrogenase 1 (ALDH1) is involved the catalytic conversion of vitamin A (retinol) to retinoic acid and has been identified as a CSC marker in a number of solid malignancies.

      Methods:
      FACS analysis of an isogenic panel of matched parent (PT) and cisplatin resistant (CisR) NSCLC cell lines identified ALDH1 as a promising CSC-marker associated with cisplatin resistance across NSCLC histologies. The H460 CisR subline was separated by FACS into ALDH1-positive and negative subpopulations and subcutaneously injected into NOD/SCID mice to assess tumour initiation and growth. ALDH1 was inhibited in vitro within the cell lines using two pharmacological ALDH1 inhibitors, DEAB and disulfiram, alone and in combination with cisplatin. Cell lines were treated in vitro with retinol and all-trans retinoic acid (ATRA) to exploit the vitamin A/retinoic acid axis in which ALDH1 is involved.

      Results:
      The CisR sublines showed significantly greater ALDH1 activity relative to their PT counterparts. In vivo subcutaneous injection of ALDH1-positive and negative subpopulations revealed no significant difference in tumour initiation or growth rate. ALDH1 inhibition in combination with cisplatin significantly decreased clonogenic and proliferative competencies and increased apoptosis cell death compared to cisplatin alone. Vitamin A supplementation and ATRA treatment in combination with cisplatin showed similar re-sensitising effects.

      Conclusion:
      This pharmacological CSC depletion in conjunction with cisplatin treatment resulted in re-sensitisation of cisplatin resistant cells to the cytotoxic effects of cisplatin. These data suggest vitamin A supplementation or the addition of ATRA or an ALDH1 inhibitor to the cisplatin-based chemotherapeutic regimen may be of clinical benefit in overcoming tumour recurrence and cisplatin resistance.

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