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Kadir Harun Sarun
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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: 3
- Moderators:
- Coordinates: 9/25/2018, 16:45 - 18:00, Exhibit Hall
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P2.06-07 - Genomic Deletion of BAP1 and CDKN2A are Better MM Diagnostic Biomarkers. (ID 13623)
16:45 - 18:00 | Author(s): Kadir Harun Sarun
- Abstract
Background
Malignant mesothelioma (MM) is deadly cancer caused by asbestos exposure and has limited treatment options. More reliable MM biomarkers and more accurate detection methods are urgent requirements for early identification of this cancer. MM cell cultures are essential in vitro models for potential treatment options analysis. In this study, we established primary MM cell lines and characterised them with current biomarkers as well as utilising the latest digital droplet PCR instrument for genomic deletion analysis. We aim to discover better diagnostic biomarkers for detection of MM.
a9ded1e5ce5d75814730bb4caaf49419 Method
Pathologically confirmed MM samples from patients were used to establish MM cell lines. MM cells were grown over 20 passages to ensure homogenous MM cell populations. Original MM tissue samples and established MM cells were stained with H&E and verified with a diagnostic biomarker panel (CK-8/18, Calretinin, CK5/6, CD141, WT-1, D2-40, EMA, CEA, Tag-72, BG8, CD15, TTF-1 and BAP1). MM cells were grown in 2D and 3D conformations for biomarker staining and compared to stained tissue biomarkers. Scores and comments were provided by pathologists experienced in MM diagnosis. Laser Capture micro-dissected (LCM) MM samples and established cells were used for DNA isolation. Genomic deletions were analysed by digital droplet PCR for copy number variations of BAP1 and CDKN2A.
4c3880bb027f159e801041b1021e88e8 Result
15 MM cell lines established from original tissue were grown over 20 passages which produced homogeneous cell populations under normal cell culture conditions. Cells grown in 3D with H&E staining exhibited better tumour architecture, cell-to-cell contacts and tumour cell-like morphology when compared to cells grown in standard 2D culture. The percentage of MM biomarker levels showed variation between original tissue and cells established in culture. Genomic deletions of BAP1 and CDKN2A, however, showed consistent levels between the two.
8eea62084ca7e541d918e823422bd82e Conclusion
Conventional immunohistochemistry MM biomarkers did not show sustained correlation between tissues of origin to established cell culture, whereas genomic deletion analysis did. Genomic deletion analysis has the potential to be used as more accurate biomarkers for MM diagnosis.
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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): Kadir Harun Sarun
- Abstract
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.
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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): Kadir Harun Sarun
- Abstract
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.
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