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Thomas George Johnson
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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 - 09:30 | Author(s): Thomas George Johnson
- 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 - 09:30 | Presenting Author(s): Thomas George Johnson
- 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.
