MA 19.06 Multiple Mechanisms Contribute to Downregulation of Tumor Suppressor microRNAs in Malignant Pleural Mesothelioma M. Williams, 1 M. Kirschner, 2 Y.Y. Cheng, 3 K. Sarun, 3 B. Mccaughan, 4 S. Kao, 5 N. Van Zandwijk, 6 G. Reid 3 1 Asbestos Diseases Research Institute, Sydney/NSW/AU, 2 Divison of Thoracic Surgery, University Hospital Zurich, Zurich/CH, 3 Asbestos Diseases Research Institute, Sydney/AU, 4 Rpah Medical Centre, Sydney/AU, 5 Medical Oncology, Chris O’Brien Lifehouse, Sydney/AU, 6 Asbestos Diseases Research Institute, University of Sydney, Concord/AU 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 tumor 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 tumor suppressor miRNAs such as miR-16 are yet to be elucidated. Method: Tumor samples (n¼60) were from MPM patients undergoing extrapleural pneumonectomy, and samples of pleura (n¼23) 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 tumor samples and cell lines, and pri-miRs and miRNA host genes in cell lines, were determined by RT-qPCR. Copy number variation (CNV) was analyzed by droplet digital PCR (ddPCR), and methylation was inferred by miRNA expression following decitabine treatment. MYC was analyzed by Western blot, and expression modulated by siRNAs. Result: Analysis of microRNA expression in tumor 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 upregu- lation 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. Analyzing the expression of these microRNAs in the tumor samples revealed a strong correlation between miR-15b and 16 (R 2 ¼0.793) and miR-34b and 34c (R 2 ¼0.753), but not between others. Conclusion: Our data suggest that a combination of deletion, hypermethylation and transcriptional regu- lation 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. Keywords: MYC, microRNA, Mesothelioma MA 19.07 Does Loss of Smad7 Lead to Increased Aggressiveness of Malignant Pleural Mesothelioma? J. Gerstmayr, 1 E. Lang, 1 D. Falch, 1 C. Pirker, 1 V. Laszlo, 2 B. Dome, 2 W. Klepetko, 2 M. Hoda, 2 W. Berger, 1 M. Grusch 1 1 Institute of Cancer Research, Medical University Vienna, Vienna/AT, 2 Division of Thoracic Surgery, Medical University Vienna, Vienna/AT Background: Malignant pleural mesothelioma (MPM) is characterized by aggressive growth, limited therapeutic options and rapid recurrence following treatment. A better understanding of biological factors un- derlying MPM aggressiveness offers the chance to improve therapeutic strategies. Growth factors of the TGF-beta superfamily including TGF- beta itself, activins and BMPs have been repeatedly linked to MPM growth. In the current study, we focus on the role of Smad7, a key intracellular antagonist of TGF-beta and activin signaling, in MPM. Method: A panel of 17 human MPM cell lines was screened for tumorigenicity in SCID mice. Comparative genomic hybridization and whole genome gene expression arrays were used for identification of genes correlating with tumorigenicity. Immunoblotting and qPCR were used to detect Smad7 expression levels in cell lines. For ectopic over- expression of Smad7 in MPM cells, a retroviral expression system was used. Various in vitro assays, immunoblotting and reporter gene assays were employed to characterize the effect of Smad7 overexpression on MPM cell proliferation, migration, signal transduction and drug response. Result: When human MPM cell lines were dichotomized into tumorigenic ones and non-tumorigenic ones based on their ability to form tumors in SCID mice, loss of Smad7 was one of the most con- spicuous associations with tumorigenicity identified in CGH arrays. The tumorigenic group also showed a reduced Smad7 transcript expression in gene expression microarrays. Based on these data we screened a larger panel of MPM cell lines for Smad7 mRNA and protein expression and identified cell lines with high, medium and low Smad7 expression. We generated a retroviral expression construct and established an isogenic subline overexpressing Smad7 from the MPM cell line VMC33 that shows low endogenous Smad7 expression. Compared to parental VMC33 cells or VMC33-RFP cells, which overexpress red fluorescent protein and were used as control, VMC33-Smad7 cells showed a reduced growth rate and diminished clone forming capacity in vitro. VMC33-Smad7 also showed reduced cell migration, but no difference in invasion could be detected. Since Smad7 was described as antagonist of TGF-beta, we tested its effect on TGF-beta signaling with a reporter gene assay and indeed found a blunted response to TGF-b in Smad7 overexpressing cells. With respect to sensitivity against kinase in- hibitors targeting TGF-beta receptors, VMC33-Smad7 showed a decreased response to galunisertib and SB-431542 compared to VMC33-RFP. Conclusion: Taken together, these data suggest that Smad7 may have a growth limiting function in MPM, possibly by antagonizing growth-promoting TGF-beta and/or activin signals. Key- words: TGF-beta, Smad7, mesothelioma cell models MA 19.09 The Role of Neoadjuvant Chemotherapy in Patients with Malignant Pleural Mesothelioma S. Voigt, 1 M. Joshi, 2 P. Speicher, 3 B. Tong, 4 M. Onaitis, 5 J. Crawford, 6 T. D’Amico, 4 D. Harpole 4 1 Department of Surgery, Duke University, Durham, NC/US, 2 Duke University, Durham/US, 3 Surgery, Duke University, Durham, NC/US, 4 Department of Thoracic Surgery, Duke Medical Center, Durham, NC/US, 5 Thoracic Surgery, University of California San Diego, La Jolla, CA/US, 6 Medicine, Duke Cancer Institute, Durham, NC/US Background: The treatment of localized malignant pleural mesotheli- oma (MPM) involves multimodality therapy, however, there is no standard of care with respect to operative procedure and timing of chemotherapy. We analyzed data from a single institution to identify whether the use of pemetrexed-platinum neoadjuvant chemotherapy impacts survival. Method: Patients with histologically-proven MPM who had surgery from 1996 to 2016 were identified. Follow-up was complete for a median of 24 months. Survival was calculated from time of diagnosis to last follow up or death. Univariate and multivariate Cox proportional hazards were used. Result: From 1996 to 2016 we identified 376 patients. Mean age was 66+/-8 years and 54 (14%) were female. There was no difference in survival for pleurectomy/decorti- cation or extrapleural pneumonectomy. Neoadjuvant chemotherapy significantly improved survival compared to surgery followed by November 2017 Abstracts S1885