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Glen Reid
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MA 19 - Mesothelioma: Bench to Bedside (ID 680)
- Event: WCLC 2017
- Type: Mini Oral
- Track: Mesothelioma
- Presentations: 2
- Moderators:Dean A Fennell, Hedy Lee Kindler
- Coordinates: 10/18/2017, 11:00 - 12:30, Room 315
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MA 19.04 - Discussant - MA 19.01, MA 19.02, MA 19.03 (ID 10829)
11:15 - 11:30 | Presenting Author(s): Glen Reid
- Abstract
- Presentation
Abstract not provided
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MA 19.06 - Multiple Mechanisms Contribute to Downregulation of Tumour Suppressor microRNAs in Malignant Pleural Mesothelioma (ID 9745)
11:35 - 11:40 | Author(s): Glen Reid
- 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: 5
- 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): Glen Reid
- 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 | Author(s): Glen Reid
- 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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P1.09-006 - JMJ and BRD Domain Family Members in Malignant Pleural Mesothelioma: Potential Therapeutic Targets or Not? (ID 9919)
09:30 - 09:30 | Author(s): Glen Reid
- Abstract
Background:
Malignant pleural mesothelioma (MPM) is an aggressive rare cancer affecting the pleura and is predominantly associated with prior exposure to asbestos. Treatment options are limited, and most patients die within 24 months of diagnosis. There is an urgent unmet need to identify new therapeutic options for the treatment of MPM. Asbestos fibres contain transition metals such as iron, and may cause an alteration of iron homeostasis in the tissue. In addition, asbestos fibres have also been shown to have high affinity for histones, and therefore may result in high accumulation of iron around chromatin. Lysine Demethylases (KDMs) containing a JmjC domain require both Fe2+ and 2-oxoglutarate as co-factors to regulate gene expression. Bromodomain containing proteins a family of chromatin reader proteins, have potential therapeutic efficacy against various malignancies. Long non-coding RNAs (lncRNAs) have also been shown to play a role as oncogenic molecules in different cancers. Several such lncRNAs have now been shown to locate to the same chromosomal region as various KDMs. We therefore examined the expression of various JmjC and Brd members (along with any associated lncRNAs) in MPM and assessed some for their clinical potential using existing small molecule inhibitors.
Method:
A panel of MPM cell lines and a cohort of snap-frozen patient samples isolated at surgery comprising benign, epithelial, biphasic, and sarcomatoid histologies were screened for expression of various BRD and JmjC members and associated lncRNAs by RT-PCR. IHC for KDM4A was performed on a cohort of FFPE specimens. The effects of treatments with small molecule inhibitors targeting these proteins on both cellular health and gene expression were assessed.
Result:
The expression of the various KDMs was detectable across our panel of cell lines. In primary tumours the expression of many of these genes were significantly elevated in malignant MPM compared to benign pleura (p<0.05), and significant differences were also observed when samples were analysed across different histological subtypes. Treatment of mesothelioma cell lines with various small molecule inhibitors caused significant effects on cellular health and on the expression of a panel of genes.
Conclusion:
The expression of various KDMs, BRD genes and associated lncRNAs are significantly altered in MPM. Small molecule inhibitors directed against these show potential therapeutic efficacy with significant anti-proliferative effects. We continue to assess the effects of these compounds on gene expression and cellular health to confirm their potential utility as novel therapies for the treatment of MPM.
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P1.09-008 - A 4-microRNA Signature in Serum Can Discriminate Between Non-Small-Cell Lung Cancer and Malignant Pleural Mesothelioma (ID 9956)
09:30 - 09:30 | Author(s): Glen Reid
- Abstract
Background:
Differential diagnosis of malignant pleural mesothelioma (MPM) and non-small cell lung cancer (NSCLC) can be difficult. For both malignancies various studies have in recent years investigated circulating cell-free microRNAs (miRs) as potential diagnostic markers, with most of these focusing on NSCLC. One of the most recent studies has suggested a 4-miR signature consisting of miR-141, miR-200b, miR-193b and miR-301 in serum to be specific for NSCLC patients. Here, we investigated the value of this 4-miR signature in discriminating serum samples from NSCLC and MPM patients.
Method:
RNA was extracted from a series of serum samples from 98 NSCLC, 98 MPM and 96 healthy controls collected at the Netherlands Cancer Institute between 1995 and 2011. MicroRNA-specific TaqMan assays were used to quantify serum microRNA levels in the t groups which after initial quality control consisted of 65, 68 and 58 samples in the NSCLC, MPM and control groups, respectively. Expression levels of individual microRNAs between the different groups were analysed using one-way ANOVA with Tukey-Kramer Posthoc Test. Binary logistic regression modelling was used to generate the 4-miR-signature, for which accuracy in discriminating NSCLC from MPM or healthy was analysed by ROC curve analysis.
Result:
Analysis of the signature microRNAs showed for all 4 miRs trends towards higher abundance in serum from NSCLC patients. Statistical significance was however only reached for miR-141, which was found to be increased by 4.4-fold in NSCLC compared to healthy controls (p=0.014) and by 5.6-fold in NSCLC vs MPM (p=0.004). Although we did not observe significant differences in abundance for all microRNAs, ROC curve analysis of the 4-miR signature confirmed the discriminatory potential with an AUC 0.73 (95% CI: 0.62-0.85) for NSCLC vs healthy controls. When applying the best achievable Youden Index as cut-off point, the signature showed a sensitivity of 91.4% and a specificity of 44.4%. In addition to being able to discriminate NSCLC from healthy controls, the 4-miR-signature also proved to be valuable for discriminating NSCLC from MPM, where an AUC of 0.77 (95% CI: 0.66-0.89), a sensitivity of 74.3% and a specificity of 80.4% could be observed.
Conclusion:
Initial analyses have confirmed the diagnostic potential of the previously described NSCLC-specific serum-based microRNA signature for distinguishing NSCLC from healthy controls. In addition, we have shown that the same signature can also discriminate between NSCLC and MPM.
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P1.09-009 - Evaluation of a Combined MicroRNA-Clinical Score as Prognostic Factor for Malignant Pleural Mesothelioma (ID 9245)
09:30 - 09:30 | Author(s): Glen Reid
- Abstract
Background:
In 2015, a 6-microRNA signature (miR-Score, Kirschner et al 2015) was demonstrated to show high prognostic accuracy in a series of surgical specimens (with and without induction chemotherapy) from patients with malignant pleural mesothelioma. In-depth analysis of matching pre- and post-chemotherapy tissue specimens has recently shown that a refined 2-miR-Score appears more suitable for use in diagnostic chemo-naïve specimens (Kirschner et al, WCLC 2016). Here, in addition to continued validation, we also aimed to further improve the prognostic accuracy by combining the 2-miR-Score with known clinical prognostic factors.
Method:
Binary logistic regression modelling was used to build a combined score consisting of the 2-miR-Score and the clinical prognostic factors age (<60 years vs >60 years at diagnosis), gender and histological subtype (epithelioid vs non-epithelioid). In addition, microRNA analysis (RT-qPCR) was performed in an additional 33 pairs of chemo-naïve (diagnostic biopsy) and chemo-treated (EPP) specimens. Accuracy of the investigated scores in predicting a good prognosis (>20 months survival post-surgery) was evaluated by ROC curve analysis.
Result:
Combining the refined 2-miR-Score with the clinical prognostic factors histological subtype and age at diagnosis, increased the overall accuracy of the 2-miR-Score in both chemo-naïve diagnostic (AUC=0.80; 95% CI: 0.65-0.95) and post-chemotherapy (AUC=0.86; 95% CI: 0.73-0.98) specimens. Addition of gender as clinical prognostic factor, did not result in further increases, hence this factor was not included in the combined score. Investigation of an additional set of 33 matched pairs of chemo-naïve and post-chemotherapy tissue samples, confirmed the improved prognostic accuracy of the combined score, with AUCs of 0.76 (95% CI: 0.59-0.92) and 0.79 (95% CI: 0.64-0.95) for chemo-naïve and post-Chemotherapy specimens, respectively. Furthermore, addition of the clinical factors resulted in an increase in specificity of the prognostic score from previously 55-65% to now 65-75%, while keeping sensitivities at the previous levels of 75-85%. Importantly, the combined microRNA-clinical Score did not only outperform the 2-miR-Score, but also the clinical factors alone.
Conclusion:
This validation has confirmed the prognostic potential of the novel 2-miR-Score. Furthermore, addition of known clinical prognostic factors was shown to result in a combined Score with increased prognostic accuracy. In addition to continued validation, in currently ongoing analyses we are also investigating combining the 2-miR-Score with our previously proposed multimodality prognostic score (MMPS; Opitz et al 2015).
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P3.02 - Biology/Pathology (ID 620)
- Event: WCLC 2017
- Type: Poster Session with Presenters Present
- Track: Biology/Pathology
- Presentations: 2
- 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 - 09:30 | Author(s): Glen Reid
- 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
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P3.02-079 - A 3D Spheroid Culture Represents an Improved in Vitro Model of Malignant Plural Mesothelioma (MPM) (ID 10472)
09:30 - 09:30 | Author(s): Glen Reid
- Abstract
Background:
Most biologists rely on cell culture in the two-dimensional (2D) format for studying tumour context which does not accurately reflect the in vivo state. 3D cell culture techniques provide cell-to-cell interactions that better mimic pathological conditions such as cancer. Malignant pleural mesothelioma (MPM) is a deadly cancer with no effective treatment and is highly drug resistance. Our study has addressed this problem by growing cells in 3D thus creating an environment that is more closely mimics the realistic tumour state for molecular and cell effect studies.
Method:
MPM cell lines were grown in conventional 2D, our newly optimised 3D format, hanging drop and poly-HEMA methods. Cells were analysed their structure by light, scanning electron (SEM) and transmission electron (TEM) microscopy. Drug infiltration was confirmed by intravital-microscopy. Cell proliferationswith with different dose of drugs (Cisplatin and Gemcitabine) were analysed in 2D and 3D cells. Cells grown as truly spherical spheroids and their 2D counterparts were harvested for tumour suppressor analysis. 16 previously reported tumour suppressors (ANK1, MIB1, RGS22, TNIL, GMC, SVIL, ATG4D, HOXB4, SCLC25A13, CHST11, ATG4D, GTF2A1, KIAA1361, PDZD2, WDR1 and TMSB15B) and 3 oncogenes (YAP1, ABCG2 and YB1) were analysed.
Result:
The 3D spheroids represent an improved 'mini-tumour' model as indicated by the visualization of cell junctions under TEM. The 3D spheroids (11 MPM lines) formed using our method also provided perfect spherical shape and revealed healthy and surface morphology by SEM analysis. We showed in our model drugs was able to penetrate from outside to centre of the spheroids. However, in our hands, the hanging drop and the poly-HEMA versions did not always produce spherical 3D cells. Cells grown in our 3D model display greater drug resistance when compared with 2D cells. Most tumour suppressor biomarkers we analysed showed down-regulation of mRNA expression level compared with cells in 2D. These tumour suppressor genes were host genes of microRNAs (e.g. MIB1 for miR-1). Most of them are frequently down-regulated in MPM. Our 3D model also showed up-regulation of genes that contribute to drug resistance such as Hif1a, YAP1, ABCG2 and YB1.
Conclusion:
3D cells grown with the newly optimised method provide a better mimic of more realistic MPM evidence by the more resistance of MPM cells to cisplatin and gemcitabine when compared to cells grown in 2D. MPM cells grown in 3D also down-regulated of tumour suppressors and up-regulation of drug resistance genes when compared to 2D cells.
