Differential regulation of STING expression and cisplatin sensitivity by autophagy in non-small cell lung cancer cells

Abstract

The cGAS-STING pathway is a central signalling mechanism in inflammatory responses and can be activated by cisplatin. Increased autophagic activity has been linked to cisplatin resistance in non-small cell lung cancer (NSCLC); however, how autophagy-STING interactions influence the cisplatin response remains unclear. This study investigates how autophagy modulation affects STING expression and cisplatin sensitivity in NSCLC cells with different basal STING levels. Autophagy was inhibited using chloroquine and induced by serum starvation in Calu-1 and H2030 cells. In Calu-1 cells, cisplatin treatment increased STING expression, activated the cGAS-STING pathway, and induced interferon responses correlated with cisplatin concentration. Autophagy inhibition reduced STING expression and interferon activation while enhancing cisplatin sensitivity. Conversely, autophagy induction caused fluctuations in STING expression and decreased cisplatin sensitivity, with ISG15 expression being selectively increased under serum starvation. In contrast, H2030 cells exhibited low basal STING expression and showed minimal responses to cisplatin or autophagy modulation. These findings suggest that STING expression levels critically influence autophagy-mediated responses to DNA-damaging chemotherapy in NSCLC.

Keywords: Autophagy; Chloroquine; Cisplatin; NSCLC; Starvation; cGAS-STING.

Fig. 1

Cisplatin treatment induces STING expression in epidermoid carcinoma but not adenocarcinoma NSCLC cells. Cytotoxic effects of cisplatin in A Calu-1 and B H2030 cell lines; C mRNA expression results of STING and IRF3 in Calu-1 cells; D Western blot results for STING, p-STING and IRF3 proteins in Calu-1 cells. Β-actin was used as loading control; E mRNA expression results of STING and IRF3 in H2030 cells; F qRT-PCR results for IFIT2, IFI44, IL6, and ISG15 genes in Calu-1 and H2030 cells. The IC₂₅, IC₅₀ and IC₇₅ treatment doses are; 4.58 µM, 9.16 µM and 13.75 µM for Calu-1 cells and 6.76 µM, 13.52 µM and 20.28 for H2030 cells, respectively. p-values of significant comparisons were indicated on the graphs using ANOVA and Dunnett’s multiple comparisons test

Fig. 2

Cisplatin treatment induces autophagy in Calu-1 epidermoid carcinoma cells. Cisplatin was applied to the cells at IC₂₅, IC_50, and IC₇₅ concentrations for 24–48 h. A Western Blot results for LC3A, LC3B, ATG13, and B-actin proteins in Calu-1 and H2030 cells. B MDC staining of autophagosomes and bar graph of the autophagosome ratios in B Calu-1 and C H2030 cell line (n = 100). Scale bar = 100 µm. The same β-actin loading control is shown in Figs. 1(D) and 2(A) as the membrane was stripped and re-probed for multiple target proteins

Fig. 3

Impact of inhibition of autophagy on cisplatin sensitivity, STING expression and interferon response. A Viability curves of cisplatin (Cis.) and Cis. + Chloroquine (CQ) treated Calu-1 cells. Cisplatin doses: 60-50-40-30-20-10-5 μM and CQ dose: 50 µM; B qRT-PCR results of STING and IRF3 gene expression in Calu-1 cells. Cisplatin concentrations: 4.58, 9.16, 13.75 µM for IC₂₅, IC₅₀ and IC₇₅ groups, respectively. CQ dose: 50 μM. C Protein levels of STING, p-STING and IRF3 in Calu-1 cells. β-actin protein used as a loading control. D Protein levels of autophagy markers (LC3A-II, LC3B-II, and ATG13) analysed by Western Blot. 20 μg total protein was loaded for all Western Blot analysis. E MDC staining of autophagosomes, Scale bar = 50 µm and 100 µm. F Bar graph showing the number of autophagosomes (n = 100 cells). G mRNA expression levels of IFIT2, IFI44, IL6, and ISG15 genes. Significant p values were calculated using ANOVA and Dunnett’s multiple comparisons test

Fig. 4

Impact of induction of autophagy on cisplatin sensitivity and STING expression. Cytotoxic effects of cisplatin in serum-starved A Calu-1, and B H2030 cells. Cisplatin treatments were applied at concentrations of 60-50-40-30-20-10-5 μM for 72 h.; C qRT-PCR results of STING and IRF3 gene expressions. p values for significant comparisons are indicated on the graphs using ANOVA and Dunnett’s multiple comparisons test. (*:p < 0.0001, **:p = 0.0002 for IRF3). The IC₅₀ concentration was used for cisplatin treatments; D Western blot result of LC3A, LC3B, STING, p-STING, IRF3, and ATG13 proteins in Calu-1 and H2030 cell lines; E MDC staining of serum-starved and cisplatin treated cells, Scale bar = 100 µm.; F Bar graphs showing the number of autophagosomes

Fig. 5

Impact of induction of autophagy on interferon response. qRT-PCR results of IFIT2, IFI44, IL-6 and ISG15 gene expressions in serum-starved and cisplatin treated A Calu-1, and B H2030 cells. The IC₅₀ concentration was used for cisplatin treatments. p values for significant comparisons were calculated using ANOVA and Dunnett’s multiple comparisons test