Nintedanib and Dasatinib Treatments Induce Protective Autophagy as a Potential Resistance Mechanism in MPM Cells

Abstract

Malignant pleural mesothelioma (MPM) is a rare type of cancer with a grim prognosis. So far, no targetable oncogenic mutation was identified in MPM and biomarkers with predictive value toward drug sensitivity or resistance are also lacking. Nintedanib (BIBF1120) is a small-molecule tyrosine kinase inhibitor that showed promising efficacy preclinically and in phase II trial in MPM as an angiogenesis inhibitor combined with chemotherapy. However, the extended phase III trial failed. In this study, we investigated the effect of nintedanib on one of its targets, the SRC kinase, in two commercial and six novel MPM cell lines. Surprisingly, nintedanib treatment did not inhibit SRC activation in MPM cells and even increased phosphorylation of SRC in several cell lines. Combination treatment with the SRC inhibitor dasatinib could reverse this effect in all cell lines, however, the cellular response was dependent on the drug sensitivity of the cells. In 2 cell lines, with high sensitivity to both nintedanib and dasatinib, the drug combination had no synergistic effect but cell death was initiated. In 2 cell lines insensitive to nintedanib combination treatment reduced cell viability synergisticaly without cell death. In contrast, in these cells both treatments increased the autophagic flux assessed by degradation of the autophagy substrate p62 and increased presence of LC3B-II, increased number of GFP-LC3 puncta and decreased readings of the HiBiT-LC3 reporter. Additionaly, autophagy was synergistically promoted by the combined treatment. At the transcriptional level, analysis of lysosomal biogenesis regulator Transcription Factor EB (TFEB) showed that in all cell lines treated with nintedanib and to a lesser extent, with dasatinib, it became dephosphorylated and accumulated in the nucleus. Interestingly, the expression of certain known TFEB target genes implicated in autophagy or lysosomal biogenesis were significantly modified only in 1 cell line. Finally, we showed that autophagy induction in our MPM cell lines panel by nintedanib and dasatinib is independent of the AKT/mTOR and the ERK pathways. Our study reveals that autophagy can serve as a cytoprotective mechanism following nintedanib or dasatinib treatments in MPM cells.

Keywords: TFEB; autophagy; dasatinib; malignant pleural mesothelioma; nintedanib.

FIGURE 1

Nintedanib does not inhibit SRC in MPM cell lines (A) Cells were treated with 1 μM nintedanib for 24 h and protein lysates were analyzed by western blot (B) Cells were treated with nintedanib for 72 h. Cell viability was analyzed with SRB assay. All experiments were performed in triplicates. Bars represent means ± SEM from three independent experiments.

FIGURE 2

Dasatinib reversed the nintedanib-induced SRC activation (A) Cells were treated with dasatinib for 72 h and cell viability was analyzed with SRB assay (B) Cells were treated with 50 nM or 300 nM dasatinib, alone or in combination with 1 μM nintedanib for 24 h, and protein samples were analyzed by western blot. All experiments were performed in triplicates. Bars represent means ± SEM from three independent experiments.

FIGURE 3

The effect of combination treatment of dasatinib and nintedanib on cell viability and cell cycle (A) Cells were treated with 1 μM, 3 μM, 5 μM nintedanib and 25 nM, 50 nM, 0.1 μM, 0.4 μM dasatinib in all combinations for 72 h and cell viability was analyzed with SRB assay. Combination index (CI) was calculated for each combination and that indicated synergism (CI < 0.9), additive effect (CI is between 0.9 and 1.1) or antagonism (CI > 1.1). Dotted lines indicate these cut-offs (B) Cell cycle analysis was performed after 50 nM or 300 nM dasatinib or 1 μM nintedanib treatment alone or in combinations for 72 h and the percentage of the cells in the subG1 phase was analyzed. Bars represent means ± SEM from three independent experiments.

FIGURE 4

Autophagic response to dasatinib and nintedanib differs across different MPM cell lines. Autophagic flux of the indicated MPM cell lines was analyzed after treatment (Das: dasatinib 50 nM; Nin: nintedanib 1 μM; 24 h) with or without Bafilomycin A1 (Baf A1, 100 nM) by western-blot (A,C,E,G,I), and HiBiT-LC3 luminescence (B,D,F,H). Bars are average ±SEM of three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001, n. s., non-significant, t-test. Note that PF434 cells did not express the LC3-HiBiT after G418 selection despite multiple attempts.

FIGURE 5

Autophagosome formation in response to dasatinib and nintedanib is dependent on the cell line (A–E) GFP-LC3 puncta formation was quantified after treatment (Das: dasatinib 50 nM; Nin: nintedanib 1 μM; 24 h; N + D, combination of nintedanib and dasatinib) with or without Bafilomycin A1 (Baf A1, 100 nM). Representative pictures are shown in (F). Bars are average ±SEM of three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001, n. s., non-significant, t-test.

FIGURE 6

TFEB is de-phosphorylated and translocates to the nucleus upon dasatinib and nintedanib treatments. TFEB mobility shifting was analyzed by western-blot (A,C,E,G,I), and its localization was determined by nuclear fractionation (B,D,F,H,J) upon treatment with the indicated treatments (UT, untreated DMSO control; Das: dasatinib 50 nM; Nin: nintedanib 1 μM; 24 h; N + D, combination of nintedanib and dasatinib). Menin and Tubulin were used as markers for purity of the nuclear and cytosolic fractions, respectively.

FIGURE 7

TFEB target genes are only induced in some MPM cell lines upon dasatinib and nintedanib treatments. Expression of the indicated TFEB target genes was analyzed by qRT-PCR after treatment for 24 h of 50 nM dasatinib and 1 μM nintedanib in (A) SPC111, (B) PF626, (C) SPC212, (D) PF531, and (E) PF434 cell lines. UT, untreated (DMSO) control; Nin, nintedanib; Das, dasatinib; N + D, combination of nintedanib and dasatinib. Bars are average ±SEM. *, p < 0.05; **, p < 0.01; ***, p < 0.001, t-test.

FIGURE 8

Nintedanib treatment does not affect the AKT-mTORC1 or the ERK pathways while dasatinib decreases AKT and pS6 activation in SPC111, PF434 and PF626 cell lines (A) but not in PF531 and SPC212 cells (B).

FIGURE 9

Combination treatment with nintedanib and autophagy inhibitor 3-MA has a synergistic effect in SPC111 cells. Cells were treated with 0.3, 1, 3, 5 μM nintedanib and 1 mM, 2.5 mM, 5 mM, 7.5 mM 3-MA in all combinations for 72 h and cell viability was analyzed with SRB assay. Combination index (CI) was calculated for each combination and that indicated synergism (CI < 0.9), additive effect (CI is between 0.9 and 1.1) or antagonism (CI > 1.1). Dotted lines indicate these cut-offs.