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K.H. Sarun
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MA 19 - Mesothelioma: Bench to Bedside (ID 680)
- Event: WCLC 2017
- Type: Mini Oral
- Track: Mesothelioma
- Presentations: 1
- Moderators:Dean A Fennell, Hedy Lee Kindler
- Coordinates: 10/18/2017, 11:00 - 12:30, Room 315
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MA 19.06 - Multiple Mechanisms Contribute to Downregulation of Tumour Suppressor microRNAs in Malignant Pleural Mesothelioma (ID 9745)
11:00 - 12:30 | Author(s): K.H. Sarun
- Abstract
- Presentation
Background:
Malignant pleural mesothelioma (MPM) is a disease with an almost invariably fatal diagnosis with limited therapeutic options. Characteristic patterns of deregulated microRNA expression have been demonstrated in MPM, and many downregulated microRNAs have been shown to have tumour suppressor activity. However, apart from silencing of miR-34b/c by promoter hypermethylation and co-deletion of miR-31 with the CDKN2A locus, the mechanisms responsible for downregulation of other tumour suppressor miRNAs such as miR-16 are yet to be elucidated.
Method:
Tumour samples (n=60) were from MPM patients undergoing extrapleural pneumonectomy, and samples of pleura (n=23) collected from patients undergoing cardiac surgery were used as normal controls. MPM cells lines were obtained from the ATCC. Expression levels of mature microRNAs in MPM tumour samples and cell lines, and pri-miRs and miRNA host genes in cell lines, were determined by RT-qPCR. Copy number variation (CNV) was analysed by droplet digital PCR (ddPCR), and methylation was inferred by miRNA expression following decitabine treatment. MYC was analysed by Western blot, and expression modulated by siRNAs.
Result:
Analysis of microRNA expression in tumour samples revealed a consistent and significant downregulation of miR-15a (4-fold, P<0.01), 15b (10-fold, P<0.01), 16 (22-fold, P<0.05), 34a (1.6-fold, P<0.05), 34b (1.8-fold, P<0.01), 34c (2.3-fold, P<0.0001) and 193a (3.1-fold, P<0.001) compared with normal pleura. Copy number variation analysis showed evidence of heterozygous loss for miR-193a (4 of 5 cell lines) and miR-15a/16-1 (2 of 5), but no change in miR-15b/16-2. Treating cell lines with the demethylating agent decitabine resulted in dramatic upregulation only in the case of miR-34c. RNAi-mediated knockdown of c-MYC led to upregulation of miR-15b and 16, and to a lesser extent miR-15a, as well as a consistent increase in the miR-15b/16-2 host gene SMC4 and the miR-15a/16-1 host gene DLEU2. Analysing the expression of these microRNAs in the tumour samples revealed a strong correlation between miR-15b and 16 (R[2]=0.793) and miR-34b and 34c (R[2]=0.753), but not between others.
Conclusion:
Our data suggest that a combination of deletion, hypermethylation and transcriptional regulation contribute to the downregulation of miR-15a/b, 16, 34a/b/c and 193a. In MPM, unlike other cancers, the downregulation of miR-15a/16-1, miR-15b/16-2 appears to be due to transcriptional changes rather than deletion or promoter hypermethylation. MYC appears to contribute to miR-16 downregulation primarily via control of SMC4 and the miR-15b/16-2 locus, suggesting that the transcriptional control of miR-16 expression by c-Myc contributes to the malignant phenotype of MPM.
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P1.09 - Mesothelioma (ID 695)
- Event: WCLC 2017
- Type: Poster Session with Presenters Present
- Track: Mesothelioma
- Presentations: 2
- Moderators:
- Coordinates: 10/16/2017, 09:30 - 16:00, Exhibit Hall (Hall B + C)
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P1.09-004 - YB-1 Suppresses miR-137 via a Feed Forward Loop, Increasing YB-1 Levels, Migration and Invasion in Malignant Mesothelioma (ID 9681)
09:30 - 16:00 | Author(s): K.H. 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. Besides numerous other functions, YB-1 has been described to stimulate migration and invasion via regulation of EMT-related factors such as Snail and Twist. The microRNA miR-137 is a small, non-coding RNA, which has been shown to have tumour-suppressor functions by targeting multiple oncogenes including YB-1. In this study we characterised the relationship between miR-137 and YB-1 expression in MPM and investigate their roles in regulating malignant behaviour such as migration and invasion.
Method:
Expression levels of miR-137 and YB-1 were determined by RT-qPCR and immunoblot. Synthetic mimics were used to overexpress miR-137. For YB-1 knockdown and overexpression, siRNAs or expression plasmids were used, respectively. Cell migration was measured by live cell videomicroscopy followed by manual single cell tracking. Invasion was assessed by an agarose spot invasion assay.
Result:
While miR-137 expression varied among our panel of MPM cell lines, YB-1 was consistently overexpressed in tumour cells compared to controls. We observed a trend towards an inverse correlation between YB-1 and miR-137 levels. Transfection with a miR-137 mimic resulted in significantly decreased levels of YB-1 and a direct interaction was confirmed by luciferase reporter assays. Interestingly, modulation of YB-1 expression led to inversely correlated changes in miR-137 levels, strongly suggesting that elevated YB-1 levels suppress miR-137. Thus, increases in YB-1 expression reduce expression of the YB-1 regulator miR-137, which in turn leads to further elevation in YB-1 via a feed-forward loop. In terms of functional effects, both miR-137 mimics and YB-1 knockdown significantly inhibited MPM cell migration and invasion. YB-1 overexpression, in contrast, stimulated cell motility and invasive growth.
Conclusion:
Our data highlight a crucial role of YB-1 in the regulation of migration and invasion, which are key characteristics of MPM. Additionally, we identified a regulatory circle between YB-1 and its targeting microRNA miR-137. Targeting this loop, by both miR-137 overexpression and YB-1 inhibition, could serve as a potential therapeutic strategy in MPM.
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P1.09-005 - Targeting YB-1 Induces Either Drug Sensitisation or Resistance via Distinct Mechanisms in Malignant Pleural Mesothelioma (ID 9809)
09:30 - 16:00 | Author(s): K.H. Sarun
- Abstract
Background:
Malignant pleural mesothelioma (MPM) is an aggressive malignancy and current therapy is essentially palliative. YB-1 is a multifunctional oncoprotein associated with poor patient outcome in tumours including NSCLC and is related to increased chemoresistance. It is widely accepted that YB-1 plays a role in the cell growth of many tumours. YB-1 has been implicated in suppressing apoptotic pathways such as the mTOR/STAT3 pathway and disrupting the cell cycle via transcriptionally regulating cyclins A, B1 and D1 in multiple cancers. We recently found YB-1 to be overexpressed in MPM cells and that siRNA-mediated knockdown inhibited growth. Here we investigate the mechanisms behind YB-1’s role in MPM cell growth and subsequent effects on drug resistance.
Method:
YB-1 expression and YBX1 mRNA was determined by Western blot and RT-qPCR, respectively, in MPM cell lines and their drug resistant sublines. Growth assays and colony formation assays with or without siRNA transfection elucidated the role of YB-1 in MPM growth. These were also conducted in combination with cisplatin, gemcitabine and vinorelbine treatment. TALI apoptosis assays were conducted to investigate the effect of YB‑1 silencing in MPM cells.
Result:
YB-1 siRNA significantly inhibited the growth of MSTO, VMC23 and MM05 cells (P<0.05) and was overexpressed compared to the immortalised mesothelial cell line MeT-5A in MSTO and VMC23. TALI apoptosis assays revealed that growth inhibition was due to apoptosis and necrosis in MSTO cells but not in VMC23, suggesting cell cycle arrest to be the cause of growth inhibition in this cell line. Interestingly, YB-1 knockdown in MSTO cells resulted in a sensitisation to cisplatin, gemcitabine and vinorelbine, but increased resistance to these drugs in VMC23 and MM05, suggesting a link between the mode of growth regulation YB-1 plays and the effect of its silencing on innate drug resistance in MPM cells. Additionally, YB‑1 levels were upregulated in MSTO and MM05 cells with acquired drug resistance, compared to parental cells.
Conclusion:
YB-1 plays different roles in MPM cell growth which are cell type dependent. When acting upon apoptotic pathways, YB-1 knockdown sensitised MPM cells to chemotherapy. In other cases, YB-1-mediated cell cycle arrest resulted in heightened resistance. Finally, YB-1 is upregulated in cells with acquired drug resistance, indicating that it plays an important role in the acquired resistance to cisplatin, gemcitabine and vinorelbine in MPM.
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P3.02 - Biology/Pathology (ID 620)
- Event: WCLC 2017
- Type: Poster Session with Presenters Present
- Track: Biology/Pathology
- Presentations: 1
- Moderators:
- Coordinates: 10/18/2017, 09:30 - 16:00, Exhibit Hall (Hall B + C)
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P3.02-078 - Establishing Malignant Pleural Mesothelioma Primary Cell Lines Using the 3D Spheroid Method Produces a Model with Better Tumour Architecture (ID 10456)
09:30 - 16:00 | Author(s): K.H. Sarun
- Abstract
Background:
Malignant pleural mesothelioma (MPM) is an aggressive malignancy with no effective treatment options. Poor prognosis and drug resistance are the main challenges of this deadly disease. There is also no simple distinctive diagnosis tool for identification of MPM. Better diagnostic markers may also provide better biological information for newer treatment option development. In this study we have established primary MPM cell lines and characterised them with current biomarkers. Our ultimately goal is to use these cell lines for better identification of diagnostic biomarkers.
Method:
MPM cell lines were either established from tissue specimens or pleural effusion from patients with pathologically confirmed MPM. Cells were cultured in standard (2D) and spheroid (3D) versions for characterisation. Cells prepared in 2D and 3D were stained with H&E and analysed 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). Scoring and comments were provided by pathologists experienced in MPM diagnosis (KL, SK). Established cell lines were also analysed for ploidy (flow cytometry) and interphase (fluorescent microscope) for chromosome number. PBMC from healthy donor was used as a control diploid.
Result:
We successfully established nine cell lines from MPM patient specimens. The original tumour histological sub-types were: three epithelioid, four biphasic, one desmoplastic and one not otherwise specified. Cells grown in 3D with H&E staining revealed better tumour architecture, cell-cell contacts and morphology when compared to cells grown in standard 2D culture. Mesothelioma positive markers were more distinctive and intense in biphasic cell lines grown 3D culture. Other sub-types showed similar staining when grown in both formats. Results from ploidy showed no distinctive difference between sub-types, however, 5 out of 9 cell lines established had tetraploid chromosome content.
Conclusion:
Cells grown in 3D provide more tumour architecture when compared with 2D cells. 3D cells also provide more intensity and greater percentage of positive MPM markers
