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K. Schelch



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    OA02 - Novel Targets and Biomarkers in Malignant Pleural Mesothelioma (ID 369)

    • Event: WCLC 2016
    • Type: Oral Session
    • Track: Mesothelioma/Thymic Malignancies/Esophageal Cancer/Other Thoracic Malignancies
    • Presentations: 2
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      OA02.01 - The microRNA-15/16 Family Regulates Tumour Cell Growth via Fibroblast Growth Factor Signals in Malignant Pleural Mesothelioma (ID 5395)

      11:00 - 12:30  |  Author(s): K. Schelch

      • Abstract
      • Presentation
      • Slides

      Background:
      Malignant pleural mesothelioma (MPM) is a highly aggressive, asbestos-related malignancy characterized by poor outcome and limited therapeutic options. Fibroblast growth factor (FGF) signals play important roles in mesothelioma cell growth and malignant behavior and their inhibition leads to reduced tumor growth. MicroRNAs (miRNAs) are conserved noncoding RNAs controlling gene expression via translational repression of target mRNAs. The miR-15/16 family is downregulated in MPM and has tumor suppressor functions. Several FGFs/FGFRs are predicted miR-15/16 targets. The aim of this study was to explore the link between the miR-15/16 and the FGF/R family in MPM.

      Methods:
      Gene and microRNA expression was determined by RT-qPCR or Taqman Low Density Arrays (TLDAs). Mimics were used for restoring microRNA expression. Stimulation or inhibition of FGF signals or bcl-2 was achieved by recombinant FGF2, siRNAs, or small-molecule inhibitors, respectively. A SYBR green-based proliferation assay and colony formation assays were used to monitor effects on cell growth.

      Results:
      Expression analysis showed a consistent downregulation of target FGF/FGFR genes after transfection with miRNA mimics. Restoration of miR-15/16 led to dose-dependent growth inhibition, which significantly correlated with sensitivity to the specific FGFR1 inhibitor PD166866. Re-expression of microRNAs in combination with FGFR knock-down or pharmacological inhibition resulted in reduced activity, indicating target competition. Combined inhibition of the FGF-axis and bcl-2, another established target of miR-15/16, resulted in enhanced activity. Treatment with recombinant FGF2 further reduced mature as well as pri-microRNA levels and also could prevent/reduce growth inhibition by mimics, but only when added within 24 hours after transfection. TLDA screens after stimulation/inhibition of FGF signals identified regulation of several other miRNAs involved in pathways relevant for tumour growth and aggressiveness.

      Conclusion:
      Our data shows that the post-transcriptional repression of FGF-mediated signals contributes to the tumour-suppressor function of the microRNA-15/16 family. Impairing hyperactivated FGF signals as well as the anti-apoptotic protein bcl-2 through the restoration of this miRNA family might serve as a novel therapeutic strategy in mesothelioma.

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      OA02.03 - Circulating Fibroblast Growth Factor 18 is Elevated in Malignant Pleural Mesothelioma Patients - A Multi-Institutional Study (ID 5988)

      11:00 - 12:30  |  Author(s): K. Schelch

      • Abstract
      • Presentation
      • Slides

      Background:
      Malignant pleural mesothelioma (MPM) is a rare but devastating malignancy. Despite the search for new promising treatment approaches, the outcome for most MPM patients remains dismal. Therefore, the identification of novel biomarkers is urgently needed in order to identify patients with a better prognosis and to support personalized therapeutic decisions. In our previously published study, we were able to show that fibroblast growth factor 18 (FGF18) is overexpressed in MPM tissue specimens and cell models. The objective of this study was the evaluation of FGF18 as a circulating biomarker in MPM.

      Methods:
      Plasma was collected from 107 MPM patients at the time of diagnosis or before surgical resection. Samples were included from the Medical University of Vienna, University Hospital Center in Zagreb and from The Concord Repatriation General Hospital and Strathfield Private Hospital in Sydney. Samples from 49 healthy volunteers and from 8 patients with non-malignant pleural diseases served as controls. Circulating FGF18 was measured by enzyme-linked immunosorbent assay and correlated to clinical, pathologic and radiologic parameters.

      Results:
      Plasma FGF18 level was significantly elevated in MPM patients vs. healthy controls (P<0.0001). A slight increase of circulating FGF18 level was also detected in patients with pleuritis or fibrosis (vs. control, P=0.0067). Sarcomatoid (n=7) morphology was associated with high FGF18 levels when compared to the epithelioid (n=77) histology (P=0.0064). Importantly, MPM patients presenting with FGF18 levels below the median had a significantly longer overall survival when compared to those with high FGF18 levels (median survival 625 versus 382 d, P=0.0038). Data on multivariate analysis, disease-free survival, correlation with other biomarkers and tumor volume will be presented at the conference.

      Conclusion:
      Our findings reveal that FGF18 is a promising blood-derived candidate biomarker in MPM. Furthermore FGF18 may support the histological classification of MPM and the identification of MPM patients with poor prognosis. .

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    P3.03 - Poster Session with Presenters Present (ID 473)

    • Event: WCLC 2016
    • Type: Poster Presenters Present
    • Track: Mesothelioma/Thymic Malignancies/Esophageal Cancer/Other Thoracic Malignancies
    • Presentations: 2
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      P3.03-002 - Inducible Changes in Cell Morphology and Gene Expression Reflecting the Histological Subtypes of Mesothelioma (ID 5405)

      14:30 - 15:45  |  Author(s): K. Schelch

      • Abstract
      • Slides

      Background:
      Malignant pleural mesothelioma (MPM) represents an aggressive malignancy with dismal prognosis and limited therapeutic options. MPM occurs in three main histological subtypes: epithelioid, sarcomatoid and biphasic, which are characterized by differences in morphological growth pattern, aggressiveness and patient prognosis. However, the mechanisms and causes responsible for the different cell morphologies are poorly understood. Epithelial-mesenchymal transition (EMT) has been implicated in cancer progression and chemoresistance, but its role in MPM is not well understood. Fibroblast growth factor (FGF) signals promote cell growth, survival and aggressiveness in several tumors including mesothelioma. Aim of this study was to characterize growth factor-induced, EMT-like changes with respect to the MPM histological subtypes.

      Methods:
      Morphological and behavioral changes of treated cell models were analyzed by morphometry, immunoblotting and functional assays. Alterations in gene or microRNA expression were evaluated via qPCR and array hybridization. Pathway enrichment analysis was based on KEGG.

      Results:
      In several cell lines established from biphasic MPM, treatment with FGF2 and EGF induced morphological changes reminiscent of EMT and aggressive behavior such as scattering, increased migration, proliferation and invasiveness. Inhibition of the fibroblast growth factor receptors (FGFR) or the MAPK axis via small-molecule inhibitors could prevent these changes and, in cell lines with sarcomatoid-like shape, reverse scattering and induce a more epithelioid morphology. Comparable results were obtained using an engineered FGFR1 enabling contactless activation via blue light. Analyses of genes and microRNAs regulated by FGF2 or EGF showed an overlap with previously established EMT markers but also identified several novel potential markers such as MMP1, ESM1, ETV4, PDL1, ITGA6 or BDKRB2. Blocking the FGFR or MAPK pathways resulted in the opposite regulation of these genes. Inhibition of MMP1 via siRNAs or pharmacological inhibitors prevented FGF2-induced scattering and invasiveness. In unsupervised clustering, the gene expression profiles of solvent- or cytokine-treated cells were associated with those of epithelioid and sarcomatoid MPM, respectively. Immunohistochemistry showed an association of MMP1 as well as phospho-ERK with the sarcomatoid part of tissue specimens from biphasic tumors. Pathway enrichment analysis of differentially expressed genes as well as the targets of altered microRNAs after FGF2 treatment showed that the regulated genes are assigned to categories important for cell growth and aggressive behavior.

      Conclusion:
      Our data characterize FGFR-mediated signals as important players in MPM aggressiveness and the morphological and behavioral plasticity of mesothelioma cells, leading to a better understanding of the link between the MPM histological subtypes and their influence on patient outcome.

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      P3.03-007 - miR-137 Acts as a Tumour Suppressor via the Down-Regulation of YB-1 in Malignant Pleural Mesothelioma (ID 5579)

      14:30 - 15:45  |  Author(s): K. Schelch

      • Abstract
      • Slides

      Background:
      Malignant pleural mesothelioma (MPM) continues to increase in incidence worldwide and has limited therapeutic options. MPM displays characteristic changes in gene expression, including noncoding RNAs such as microRNAs, which have potential therapeutic relevance. One such miRNA is miR-137, a tumour suppressor whose promoter region is frequently methylated in other cancers and lies in in a commonly deleted chromosomal region in MPM (1p21-23). A potential role for miR-137 has yet to be investigated in MPM. One known target of miR-137 is YB-1, a multifunctional protein often up-regulated in other aggressive cancers, where elevated YB-1 levels are linked to poor clinical outcomes. This study investigates the causes of miR-137 suppression, the relationship between miR-137 and YB-1, one of its targets, as well as their roles in MPM cell growth and malignant behaviour.

      Methods:
      Basal expression of miR-137 and YB-1 was determined in 13 MPM cell lines by RT-qPCR and immunoblotting. Cells were treated with 5’Aza-cytidine and RT-qPCR was conducted to link methylation with miR-137 suppression. Copy number variation (CNV) was investigated by ddPCR. Cells were transfected with miR-137 mimic and subsequent YB-1 expression was investigated using RT-qPCR. Proliferation, colony formation and wound-healing assays were conducted after transfection with miR-137 mimics or YB-1-specific siRNAs.

      Results:
      miR-137 was absent in 4 MPM cell lines (p<0.01) and was up-regulated in response to 5’Aza-cytidine treatment in these lines, as well as other lines with low basal expression. Copy-number loss was evident in 5 cell lines and gain was present in 2. Increasing levels of miR-137 generally inhibited MPM cell migration, proliferation and colony formation. miR-137 mimics significantly down-regulated YB-1 expression, while YB-1 protein was overexpressed in the majority of MPM cell lines, compared to MeT-5A. YB-1 knock-down resulted in dose-dependent growth inhibition over 120 hours, reduced colony formation and also decreased cell migration. Effects were more pronounced in those cell lines showing high YB-1 protein levels.

      Conclusion:
      Our results show that methylation and CNV are likely to play a role in miR-137 down-regulation in MPM and that miR-137 acts as a tumour suppressor in MPM through at least in part the down-regulation of YB-1. We also demonstrated that YB-1 is commonly overexpressed and plays a role in proliferation and migration. These results imply a direct relationship between miR-137 and YB-1 expression, a biological interaction that may prove a useful target in developing future therapeutic approaches in MPM.

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