MicroRNA encapsulated nanocells (EnGeneIC)

Malignant pleural mesothelioma (MPM) is an aggressive malignancy with very limited therapeutic options. Fibroblast growth factor (FGF) signals play important roles in mesothelioma cell growth. Several FGFs and FGF receptors (FGFRs) are predicted targets of the miR‐15/16 family, which is downregulated in MPM. The aim of this study was to explore the link between the miR‐15/16 family and the FGF axis in MPM. Expression analyses via RT‐qPCR showed downregulation of the FGF axis after transfection with miR‐15/16 mimics. Direct interaction was confirmed by luciferase reporter assays. Restoration of miR‐15/16 led to dose‐dependent growth inhibition in MPM cell lines, which significantly correlated with their sensitivity to FGFR inhibition. Treatment with recombinant FGF2 prevented growth inhibition and further reduced the levels of FGF/R‐targeting microRNAs, indicating a vicious cycle between miR‐15/16 down‐ and FGF/FGFR signaling upregulation. Combined inhibition of two independent miR‐15/16 targets, the FGF axis and Bcl‐2, resulted in additive or synergistic activity. Our data indicate that post‐transcriptional repression of FGF‐mediated signals contributes to the tumor suppressor function of the microRNA‐15/16 family. Inhibiting hyperactivated FGF signals and Bcl‐2 might serve as a novel therapeutic combination strategy in MPM.

MicroRNAs are frequently downregulated in cancer, and restoring expression has tumour suppressive activity in tumour cells. Our recent phase I clinical trial investigated microRNA-based therapy in patients with malignant pleural mesothelioma. Treatment with TargomiRs, microRNA mimics with novel sequence packaged in EGFR antibody-targeted bacterial minicells, revealed clear signs of clinical activity. In order to detect delivery of microRNA mimics to tumour cells in future clinical trials, we tested hydrolysis probe-based assays specific for the sequence of the novel mimics in transfected mesothelioma cell lines using RT-qPCR.

The custom assays efficiently and specifically amplified the consensus mimics. However, we found that these assays gave a signal when total RNA from untransfected and control mimic-transfected cells were used as templates. Further investigation revealed that the reverse transcription step using stem-loop primers appeared to introduce substantial non-specific amplification with either total RNA or synthetic RNA templates. This suggests that reverse transcription using stem-loop primers suffers from an intrinsic lack of specificity for the detection of highly similar microRNAs in the same family, especially when analysing total RNA. These results suggest that RT-qPCR is unlikely to be an effective means to detect delivery of microRNA mimic-based drugs to tumour cells in patients.