Abstract 2982: Unraveling the antifibrotic mode of action of nintedanib against the TGF-β pathway in tumor-associated fibroblasts in non-small cell lung cancer

Abstract

Abstract Non-small cell lung cancer (NSCLC) is the leading cause of cancer related death, and its major histotypes are lung adenocarcinoma (ADC) and squamous cell carcinoma (SCC). Nintedanib (Vargatef) is a multi-tyrosine kinase inhibitor of VEGF, FGF and PDGF receptors that has been approved as second-line treatment of advanced lung adenocarcinoma patients, owing to the positive therapeutic effects reported selectively on ADC but not SCC patients in the LUME-1 clinical trial. Nintedanib has been also approved to treat idiopathic pulmonary fibrosis due to its antifibrotic effects reported in the INPULSIS trial. Of note, we recently reported that tumor fibrosis is larger in ADC than SCC due to the stronger epigenetic repression of the pro-fibrotic TGF-β transcription factor SMAD3 in tumor-associated fibroblasts (TAFs) in SCC compared to ADC. Moreover, we also showed that nintedanib elicits a larger inhibition of TGF-β-induced fibrosis in ADC-TAFs compared to SCC-TAFs. However, the detailed antifibrotic mechanisms of nintedanib on the TGF-β1 pathway remain poorly understood. TGF-β1 signaling begins with its binding to type II TGF-β receptor, which phosphorylates type I TGF-β receptor ALK5, that subsequently phosphorylates SMAD2 and SMAD3, upon which they form heterotrimeric complexes with the co-factor SMAD4 that translocate to the nucleus to regulate gene expression. We found that nintedanib markedly inhibited both the activation of SMAD2 and SMAD3 as well as the increase in nuclear SMAD4 in response to TGF-β1 in pulmonary fibroblasts, thereby supporting that ALK5 may be an off-target of nintedanib. To examine this possibility, we performed a time-course analysis of phospho-ALK5 (pALK5) and pSMAD3 in fibroblasts in response to TGF-β1 with or without nintedanib. As expected, TGF-β1 elicited a peak in pALK5 before that of pSMAD3. Moreover, nintedanib downregulated pALK5 but not total ALK5, supporting that ALK5 is an unintended target of this drug. In addition, we examined the expression of total and phospho-Erk1/2 in the same samples, since Erk1/2 has been previously reported as a regulator of cellular responses to TGF-β. TGF-β1 elicited a peak in pErk1/2 within the same time-window than pSMAD3, which is consistent with previous observations on non-canonical TGF-β signaling. Interestingly, nintedanib abrogated both total and pErk1/2 expression in a time-dependent fashion. These results support that the antifibrotic effects of nintedanib may be mediated through their inhibition of the pro-fibrotic transcription factor SMAD3 as well as the downregulation of Erk1/2. In addition, our results support that Erk1/2 may be implicated in the limited responses of SCC-TAFs to nintedanib, since we previously showed that SCC-TAFs exhibit an enhanced activity of Erk1/2 compared to ADC-TAFs. Citation Format: Rafael Ikemori, Marta Gabasa, Paula Duch, Frank Hilberg, Noemí Reguart, Jordi Alcaraz. Unraveling the antifibrotic mode of action of nintedanib against the TGF-β pathway in tumor-associated fibroblasts in non-small cell lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2982.