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Glen Reid



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    OA08 - Mesothelioma: Immunotherapy and microRNA for Diagnosis and Treatment (ID 907)

    • Event: WCLC 2018
    • Type: Oral Abstract Session
    • Track: Mesothelioma
    • Presentations: 1
    • Moderators:
    • Coordinates: 9/24/2018, 15:15 - 16:45, Room 201 BD
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      OA08.06 - Tumour Suppressor MicroRNAs Modulate Drug Resistance by Targeting Anti-Apoptotic Pathways in Malignant Pleural Mesothelioma (MPM) (ID 13539)

      16:10 - 16:20  |  Author(s): Glen Reid

      • Abstract
      • Presentation
      • Slides

      Background

      Malignant Pleural mesothelioma (MPM) is an aggressive thoracic malignancy with limited treatment options. MPM has a poor prognosis, predominately due to its inherent drug resistance and its limited response to current therapies. Aberrant microRNA expression is a common event in neoplasms with many implicated in chemo-resistance, however their role in MPM drug resistance is largely unexplored.

      a9ded1e5ce5d75814730bb4caaf49419 Method

      To investigate the role of microRNAs in MPM drug resistance, we generated MPM cell lines with acquired drug resistance to cisplatin, gemcitabine and vinorelbine by periodic treatment with the IC50 of each chemotherapeutic agent. Expression levels of mature microRNAs were compared between parental MPM cell lines and cell lines with acquired drug resistance using RT-qPCR. BCL2 is an anti-apoptotic gene and a known target of miR-15a/16-1 and miR-34a. To determine if microRNAs potentiate drug sensitization via a Bcl-2 mediated anti-apoptotic pathway, drug sensitivity assays were carried out following reverse-transfection with microRNA mimics and Bcl-2 siRNAs combined with cisplatin, gemcitabine and vinorelbine treatment. Following microRNA mimic transfection in 6-well plates, levels of apoptosis and necrosis were determined by PI and annexin V staining while Bcl-2 mRNA and protein expression was determined by RT-qPCR and Western blotting respectively.

      4c3880bb027f159e801041b1021e88e8 Result

      Expression of miR-15a/16-1 and miR-34a was downregulated in MPM cells with acquired resistance to cisplatin, gemcitabine and vinorelbine, compared to the parental counterpart. Transfection with mimics corresponding to miR-15a/16-1 were most effective in improving sensitivity to all chemotherapeutics tested in drug resistant cell lines. In parental cell lines, miR-15a/16-1 mimic induced sensitization was also observed but restoration of miR-34a and miR-34b was also capable of improving response to cisplatin and vinorelbine. Forced miR-15/16 and miR-34a expression also sensitized both parental and resistant cell lines to cisplatin, gemcitabine and vinorelbine via induction of apoptosis; their ability to increase levels of drug-induced apoptosis suggest they may sensitize cells to chemotherapeutics via an anti-apoptotic mechanism involving Bcl-2. miR-15a/16-1 and miR-34a transfection caused Bcl-2 mRNA and protein reduction, confirming their regulation of Bcl-2 in MPM. Furthermore, siRNA induced knockdown of Bcl-2 also induced a modest improvement in drug sensitivity.

      8eea62084ca7e541d918e823422bd82e Conclusion

      Restoration of microRNA expression sensitized both drug resistant and parental cell lines to chemotherapeutic agents and increased levels of drug-induced apoptosis. Taken together, this data suggests that miR-15a/16-1 and miR-34a are involved in the acquired and intrinsic drug resistance phenotype of MPM cells in part by modulation of apoptotic mechanisms via targeting Bcl-2.

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    P2.06 - Mesothelioma (Not CME Accredited Session) (ID 955)

    • Event: WCLC 2018
    • Type: Poster Viewing in the Exhibit Hall
    • Track:
    • Presentations: 2
    • Moderators:
    • Coordinates: 9/25/2018, 16:45 - 18:00, Exhibit Hall
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      P2.06-16 - YB-1: An Important Driver of Mesothelioma Drug Resistance and a Potential Novel Therapeutic Target (ID 13653)

      16:45 - 18:00  |  Author(s): Glen Reid

      • Abstract
      • Slides

      Background

      Malignant pleural mesothelioma (MPM) is an aggressive malignancy and current therapy is essentially palliative. Novel therapy targets are urgently needed. YB-1 is a multifunctional oncoprotein associated with poor patient outcome and is related to increased chemoresistance in tumours including NSCLC. It is widely accepted that YB-1 plays a role in the cell growth of many cancers, and we recently confirmed this in MPM cells. Here, we begin to evaluate YB-1 as a therapeutic target in this disease.

      a9ded1e5ce5d75814730bb4caaf49419 Method

      YB-1 expression was determined by Western blot in MPM cell lines and their drug resistant sublines. Growth and colony formation assays were conducted after transfection with YB-1- or control-siRNA. These were also carried out in combination with cisplatin, gemcitabine or vinorelbine treatment. Apoptosis was assessed by PI and annexin V staining in YB‑1 knockdown MPM cells. Migration of MPM cells was measured using videomicroscopy and manual cell tracking. Luciferase-expressing MPM cells transfected with YB-1- or control-siRNA were injected into female SCID mice (n=10, intra-peritoneal injection). Tumour growth was monitored via luminescence by injecting luciferin (intra-peritoneal injection) and measuring bioluminescence on an In Vitro Imaging System (IVIS) once a week for 4 weeks. Tumour weight determined after humane euthanasia of animals at the termination of the experiment.

      4c3880bb027f159e801041b1021e88e8 Result

      YB-1-siRNA significantly inhibited the growth of MPM cell lines in vitro and was overexpressed in MPM cells compared to the immortalised mesothelial cell line MeT-5A. Growth of MeT-5A and primary mesothelial cell lines was not affected significantly by YB-1 knockdown. Mice injected with YB-1 knockdown cells displayed significantly lower tumour burden, evidenced by bioluminescence in live mice using IVIS and lower tumour weight after harvest. TALI assays showed an increase in apoptotic cells after YB-1 siRNA transfection in vitro, and cells transfected with siRNA showed sensitisation to cisplatin and vinorelbine. YB-1 was expressed at higher levels, and higher migratory capacity was observed in drug resistant MPM cell lines compared to parental cell lines.

      8eea62084ca7e541d918e823422bd82e Conclusion

      These results highlight the importance of YB-1 in MPM biology both in vitro and in vivo. YB-1 knockdown inhibits growth via apoptosis, sensitises MPM cells to commonly prescribed drugs and contributes to a change in behaviour of drug resistant MPM cells. This project serves as a basis for the further investigation of YB-1 as a novel therapeutic target.

      6f8b794f3246b0c1e1780bb4d4d5dc53

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      P2.06-32 - YB-1 - A Key Factor in Mesothelioma Aggressive Growth and Behaviour (ID 13647)

      16:45 - 18:00  |  Author(s): Glen Reid

      • Abstract
      • Slides

      Background

      Malignant pleural mesothelioma (MPM) is a devastating disease characterized by aggressive growth and local invasion, poor outcome and limited therapeutic options. YB-1 is a multifunctional oncoprotein, which is often up-regulated in cancer and associated with aggressiveness and poor patient outcome. Also, YB-1 is actively secreted by cells upon various stresses. Besides numerous other functions, YB-1 has been described to stimulate cancer cell migration and invasion via regulation of EMT-related factors such as Snail and Twist.

      a9ded1e5ce5d75814730bb4caaf49419 Method

      Cells were treated with LPS (20 ng/ml) or grown under hypoxic conditions (1% O2, N2 balanced) for 24 hours. YB-1 levels in supernatants were quantified via western blot. Changes in EMT markers were measured by qPCR. Cell migration and cell cycle was assessed by live cell videomicroscopy followed by manual single cell tracking and analysis using ImageJ and DiPer software, respectively. YB-1 overexpression was achieved by stable transfection with an expression plasmid and confirmed via qPCR and western blot.

      4c3880bb027f159e801041b1021e88e8 Result

      YB-1 is secreted by MPM cells as well as the immortalised mesothelial cell line Met-5A after exposure to LPS or under hypoxic conditions. When MPM cells as well as primary mesothelial cells derived from pericardial fluid were exposed to soluble YB-1, we observed an upregulation of EMT markers such as SNAIL and TWIST as well as significantly increased migratory capacity. Similar effects were observed in cell lines which overexpress YB-1. YB-1 overexpressing cells migrated at significantly higher speed and covered a larger area. Also, when grown at low density, EMT-like changes in cell morphology as well as scattering was observed. Additionally, while cell divisions occurred at higher frequencies, the duration of the M phase was significantly prolonged.

      8eea62084ca7e541d918e823422bd82e Conclusion

      Our data highlight a crucial role of both intracellular and soluble YB-1 in the regulation of migration and invasion, which are key characteristics of MPM. Additionally, YB-1 also interferes with the cell cycle of MPM cells. These findings contribute to a better understanding of the biology of MPM and highlight YB-1’s potential as a therapeutic target.

      6f8b794f3246b0c1e1780bb4d4d5dc53

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    SH03 - Highlight of the Previous Day Sessions (ID 885)

    • Event: WCLC 2018
    • Type: Highlight of the Day Session
    • Track:
    • Presentations: 1
    • Moderators:
    • Coordinates: 9/26/2018, 07:00 - 08:00, Room 107
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      SH03.03 - Biology (ID 14786)

      07:24 - 07:36  |  Presenting Author(s): Glen Reid

      • Abstract
      • Presentation
      • Slides

      Abstract not provided

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