Regulation of TGF-β1-Smad-1-7 signaling to Inhibit Epithelial Mesenchymal Transition by Repurposed Anti-Fibrotic Drug Pirfenidone can Attenuate Lung Cancer Progression

Author(s): Ritu Kulshrestha, Pawan Kumar, Amit Singh, Divya S Nair, Meenu R, Himanshu Dhanda, Apoorva Pandey, Dahiya R, Mishra AK, Dinda AK

Background: Lung cancer cells undergo EMT, and metastasize to preferential sites such as brain, bone and adrenal glands. Pirfenidone is an anti-fibrotic agent used for treatment of pulmonary fibrosis. Its efficacy as therapeutic adjuvant in lung cancer via regulation of TGF-β1-Smad-1-7- EMT signaling is postulated.

Method: Lung adenocarcinoma (A-549) cells were cultured in DMEM at 37°C in 5% CO2. Cells were treated with saline (Control), bleomycin (20 mM) and Bleomycin+Pirfenidone (500μg/ml) and harvested at 4, 6, 8, 24, and 48h. The time-course of bleomycin induced TGF-β-BMPSmad- 1-7 signaling, EMT (E-Cadherin and Vimentin) and their inhibition by pirfenidone was assessed.

Results: Bleomycin caused a bimodal upregulation of TGF-β1 at 6, 48 h and EMT of A-549 cells (progressive E-cadherin downregulation, vimentin upregulation). An associated differential upregulation of (i) receptor Smads-(rSmads-2,3,5): Smad-2,5 (Bimodal upregulation at 4,48 hrs), Smad-3 (persistent upregulation- 4 to 48hrs), (ii) Co-Smad-4-persistent upregulation from 4 to 48 hrs, (iii) iSmad-1, 6,7: Smad-1 (upregulation at 24,48 hrs), Smad-6,7- early up-regulation at 4, 6 hrs respectively, was seen. Pirfenidone significantly reversed EMT by downregulating the TGF-β1/Smad-2-5 signaling, Vimentin expression and upregulating BMPiSmads- 6,7 signaling, E-Cadherin expression. This reduced cell migration.

Conclusion: Pirfenidone attenuates lung cancer cell migration by differentially inhibiting the TGF-β1-Smads-1-7 signaling pathway and EMT. Thus indicating the utility of pirfenidone as an adjuvant therapy to attenuate lung cancer progression and improve the prognosis of lung cancer patients on chemotherapy.