Abstract
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. 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. 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 tumor growth and aggressiveness. Our data shows that the post-transcriptional repression of FGF-mediated signals contributes to the tumor-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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