Inhibition of Hedgehog signaling sensitizes NSCLC cells to standard therapies through modulation of EMT-regulating miRNAs

Abstract

BackgroundEpidermal growth factor receptor- tyrosine kinase inhibitors (EGFR-TKIs) benefit Non-small cell lung cancer (NSCLC) patients, and an EGFR-TKIi erlotinib, is approved for patients with recurrent NSCLC. However, resistance to erlotinib is a major clinical problem. Earlier we have demonstrated the role of Hedgehog (Hh) signaling in Epithelial-to-Mesenchymal transition (EMT) of NSCLC cells, leading to increased proliferation and invasion. Here, we investigated the role of Hh signaling in erlotinib resistance of TGF-β1-induced NSCLC cells that are reminiscent of EMT cells.MethodsHh signaling was inhibited by specific siRNA and by GDC-0449, a small molecule antagonist of G protein coupled receptor smoothened in the Hh pathway. Not all NSCLC patients are likely to benefit from EGFR-TKIs and, therefore, cisplatin was used to further demonstrate a role of inhibition of Hh signaling in sensitization of resistant EMT cells. Specific pre- and anti-miRNA preparations were used to study the mechanistic involvement of miRNAs in drug resistance mechanism.ResultssiRNA-mediated inhibition as well as pharmacological inhibition of Hh signaling abrogated resistance of NSCLC cells to erlotinib and cisplatin. It also resulted in re-sensitization of TGF-β1-induced A549 (A549M) cells as well the mesenchymal phenotypic H1299 cells to erlotinib and cisplatin treatment with concomitant up-regulation of cancer stem cell (CSC) markers (Sox2, Nanog and EpCAM) and down-regulation of miR-200 and let-7 family miRNAs. Ectopic up-regulation of miRNAs, especially miR-200b and let-7c, significantly diminished the erlotinib resistance of A549M cells. Inhibition of Hh signaling by GDC-0449 in EMT cells resulted in the attenuation of CSC markers and up-regulation of miR-200b and let-7c, leading to sensitization of EMT cells to drug treatment, thus, confirming a connection between Hh signaling, miRNAs and drug resistance.ConclusionsWe demonstrate that Hh pathway, through EMT-induction, leads to reduced sensitivity to EGFR-TKIs in NSCLCs. Therefore, targeting Hh pathway may lead to the reversal of EMT phenotype and improve the therapeutic efficacy of EGFR-TKIs in NSCLC patients.

Sonic hedgehog-glioma associated oncogene homolog 1 signaling enhances drug resistance in CD44+/Musashi-1+ gastric cancer stem cells

“…However, whether other ABC family genes such as ABCCs, ABCDs, ABCFs, ABCEs, ABCAs, and ABCGs members play any role in drug resistance of CD44 + /Musashi-1 + gastric cancer cells still could not be excluded [29,47,48]. Moreover, except modulating the ATP-binding cassette (ABC) family, SHH-GLI1 signaling may affect drug resistance through multiple mechanisms, such as EMT regulating miRNA, glucuronidation, and autophagy [21,49,50]. These respects should be considered and investigated in the future studies.…”

“…In the presence of HH ligands, SMO is activated transducing the signal to the cytoplasm via canonical signaling of GLI proteins [20]. Aberrant activation of SHH signaling has been shown to play crucial roles in the development of drug resistance to chemotherapy in many cancers [21][22][23]. It has recently been shown that SHH signaling is also involved in the resistance of CD44 + gastric adenocarcinoma to chemotherapy [24].…”

Clinical implications of miRNAs in the pathogenesis, diagnosis and therapy of pancreatic cancer

“…Specific to PC, synthetic miR-3548 that targets GLI1 transcription factor resulted in effective inhibition of cell division and cell proliferation [168]. Not only do miRNA regulate SHH signaling, SHH signaling also effects miRNA expression, as evidenced by the fact that use of pharmacological hedgehog inhibitor vismodegib (GDC-0449) upregulates the expression of miR-200b and let-7c that results in reversal of EMT phenotype and attenuation of drug resistance in NSCLC cells [169]; whereas cyclopamine, a SHH inhibitor in PC cells significantly inhibited expression of let7, miR-34, miR-107, miR-125, miR-128, miR-130, miR-132, and miR-141 [170]. Therefore, identification of a complete miRNA profile that exist to regulate various components of SHH signaling, will be helpful in identifying the central mechanism of sustained SHH activation in cancer cells.…”

SMO Gene Amplification and Activation of the Hedgehog Pathway as Novel Mechanisms of Resistance to Anti-Epidermal Growth Factor Receptor Drugs in Human Lung Cancer

“…Inhibition of the Hh pathway can reverse EMT and is associated with enhanced tumor sensitivity to cytotoxic agents (16). Recently, upregulation of the Hh pathway has been demonstrated in the NSCLC cell line A549, concomitantly with the acquisition of a TGFb1-induced EMT phenotype with increased cell motility and invasion (17).…”