title: Inhibition of Hedgehog signaling sensitizes NSCLC cells to standard therapies through modulation of EMT-regulating miRNAs.
abstract: BACKGROUND
  Epidermal 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.
  
  
  METHODS
  Hh 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.
  
  
  RESULTS
  siRNA-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.
  
  
  CONCLUSIONS
  We 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.
authors.forename: Aamir
authors.initials: A
authors.lastname: Ahmad
authors.forename: Ma'in Y
authors.initials: MY
authors.lastname: Maitah
authors.forename: Kevin R
authors.initials: KR
authors.lastname: Ginnebaugh
authors.forename: Yiwei
authors.initials: Y
authors.lastname: Li
authors.forename: Bin
authors.initials: B
authors.lastname: Bao
authors.forename: Shirish M
authors.initials: SM
authors.lastname: Gadgeel
authors.affiliation: Department of Pathology, Wayne State University School of Medicine, Detroit, MI 48201, USA. fsarkar@med.wayne.edu.
authors.forename: Fazlul H
authors.initials: FH
authors.lastname: Sarkar
mesh_terms: D000813:Anilides / Q000008:administration & dosage / Q000494:pharmacology; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology; D002289:Carcinoma, Non-Small-Cell Lung / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D002945:Cisplatin / Q000008:administration & dosage / Q000494:pharmacology; D019008:Drug Resistance, Neoplasm; D058750:Epithelial-Mesenchymal Transition / Q000187:drug effects / Q000235:genetics; D066246:ErbB Receptors / Q000037:antagonists & inhibitors; D000069347:Erlotinib Hydrochloride; D055785:Gene Knockdown Techniques; D053823:Hedgehog Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008175:Lung Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D035683:MicroRNAs / Q000235:genetics*; D011725:Pyridines / Q000008:administration & dosage / Q000494:pharmacology; D011799:Quinazolines / Q000008:administration & dosage / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000235:genetics / Q000494:pharmacology; D015398:Signal Transduction; D014162:Transfection
chemical_list: D000813:Anilides; D053823:Hedgehog Proteins; C538724:HhAntag691; D035683:MicroRNAs; D011725:Pyridines; D011799:Quinazolines; D034741:RNA, Small Interfering; D000069347:Erlotinib Hydrochloride; C512478:EGFR protein, human; D066246:ErbB Receptors; D002945:Cisplatin