Rocaglamide promotes the infiltration and antitumor immunity of NK cells by activating cGAS-STING signaling in non-small cell lung cancer

Abstract

Background: Natural killer (NK) cell-based immunotherapy is clinically limited due to insufficient tumor infiltration in solid tumors. We have previously found that the natural product rocaglamide (RocA) can enhance NK cell-mediated killing of non-small cell lung cancer (NSCLC) cells by inhibiting autophagy, and autophagic inhibition has been shown to increase NK cell tumor infiltration in melanoma. Therefore, we hypothesized that RocA could increase NK cell infiltration in NSCLC by autophagy inhibition. Methods: Flow cytometry, RNA-sequencing, real-time PCR, Western blotting analysis, and xenograft tumor model were utilized to assess the infiltration of NK cells and the underlying mechanism. Results: RocA significantly increased the infiltration of NK cells and the expressions of CCL5 and CXCL10 in NSCLC cells, which could not be reversed by the inhibitions of autophagy/ULK1, JNK and NF-κB. However, such up-regulation could be suppressed by the inhibitions of TKB1 and STING. Furthermore, RocA dramatically activated the cGAS (cyclic GMP-AMP synthase)-STING (stimulator of interferon genes) signaling pathway, and the inhibition/depletion of STING ablated the up-regulation of CCL5 and CXCL10, NK cell infiltration, and tumor regression induced by RocA. Besides, RocA damaged mitochondrial DNA (mtDNA) and promoted the cytoplasmic release of mtDNA. The mPTP inhibitor cyclosporin A could reverse RocA-induced cytoplasmic release of mtDNA. Conclusions: RocA could promote NK cell infiltration by activating cGAS-STING signaling via targeting mtDNA, but not by inhibiting autophagy. Taken together, our current findings suggested that RocA was a potent cGAS-STING agonist and had a promising potential in cancer immunotherapy, especially in NK cell-based immunotherapy.

Keywords: Mitochondrial DNA; NK cells; Non-small cell lung cancer; Rocaglamide; STING.

Figure 1

RocA increases NK cell infiltration in NSCLC. LLC cells were subcutaneously inoculated onto the upper back of C57BL6 mice on day 0, and 1 mg/kg of RocA was administered by i.p. injection every 2 days from day 3. Mice were sacrificed on day 21, and tumors were excised. The proportion of NK cells in tumors were analyzed by flow-cytometry (A-B). Tumor tissues were stained with anti-NKp46 antibody and DAPI (C), and the NKp46⁺ NK cells were quantified in tumor tissues (D). The proportion of NK cells in spleens in LLC tumor-bearing mice (E) and normal mice without LLC tumor (F) were analyzed by flow-cytometry.

Figure 2

RocA increases the expression of CCL5 in NSCLC cells independent of autophagy inhibition. A549, H1299, and H1975 cells were exposed to different concentrations (0, 12.5, and 25 nM) of RocA for 24 h, and then the expression of CCL5 at the RNA (A-C) and protein (D-F) level was analyzed by real-time PCR and ELISA, respectively. G-I, A549, H1299, and H1975 cells were exposed to different concentrations (0, 25.0, and 50.0 µM) of CQ for 24 h, and then the expression of CCL5 was analyzed by real-time PCR. J, A549, H1299, and H1975 cells were treated with or without 25 nM RocA in the presence or absence of 10µM SP600125 for 24 h, and then the expression of CCL5 was analyzed by real-time PCR. Data were pooled from three independent experiments. K, The expressions of ULK1, p62, and LC3 in ULK1 wild-type (WT) and knockout (KO) H1299 cells were detected by Western blotting analysis. Data represented three independent experiments. L, ULK1^WT, and ULK1^KO H1299 cells were exposed to different concentrations (0, 12.5, and 25 nM) of RocA for 24 h, and then the expression of CCL5 was analyzed by real-time PCR. Data were pooled from three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001; NS, non-statistical significance.

Figure 3

RocA increases the expression of CXCL10 in NSCLC cells independent of autophagy inhibition. A, LLC cells were subcutaneously inoculated onto the upper back of C57BL6 mice on day 0, and 1 mg/kg of RocA was administered by i.p. injection every 2 days from day 3. Mice were sacrificed on day 14, and tumors were isolated and used for RNA-Seq. A, DEGs related to NK activation and traffic were richened. B-D, The expressions of CCL5, CXCL10, and IFN-γ in tumors were detected by real-time PCR. E, The expression of CD107α on the surface of NK cells was analyzed by flow-cytometry. A549 (F), H1299 (G), H1975 (H), and LLC (I) cells were exposed to different concentrations (0, 12.5, and 25 nM) of RocA for 24 h, and then the expression of CXCL10 was analyzed by real-time PCR. J, ULK1^WT, and ULK1^KO H1299 cells were exposed to different concentrations (0, 12.5, and 25 nM) of RocA for 24 h, and then the expression of CXCL10 was analyzed by real-time PCR. K, A549, H1299, and H1975 cells were treated with or without 25 nM RocA in the presence or absence of 10µM SP600125 for 24 h, and then the expression of CXCL10 was analyzed by real-time PCR. Data were pooled from three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001; NS, non-statistical significance.

Figure 4

RocA increases the expressions of CCL5 and CXCL10 depending on TBK1 and STING. A, DEGs related to the cGAS-STING signaling pathway were richened. A549, H1299, H1975, and LLC cells were treated with or without 25 nM of RocA in the presence or absence of 5 µM CYT387 (B), 5 µM H-151 or 5 µM C-176 (C), or 10 µM BAY11 for 24 h, and then the expressions of CCL5 and CXCL10 were analyzed by real-time PCR. Data were pooled from three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 5

RocA activates the cGAS-STING signaling pathway and increases the expressions of CCL5 and CXCL10 depending on such pathway. A, A549, H1299, and H1975 cells were exposed to 25 nM of RocA for different durations (0, 1, 3, 6, 12, and 24 h), and then the expressions of cGAS, STING, pTBK1, TBK1, pIRF3, and IRF3 were detected by Western blotting analysis. B, A549, H1299, and H1975 cells were exposed to different concentrations (0, 12.5, 25, and 50 nM) of RocA for 24 h, and then the expressions of cGAS, pTBK1, TBK1, p65, and pp65 were detected by Western blotting analysis. C, A549, H1299, and H1975 cells were transfected with STING siRNA or negative control (NC) for 24 h and then exposed to 25 nM of RocA for 24 h, followed by the detection of STING, pTBK1, and TBK1 by Western blotting analysis. Data represented three independent experiments. E, A549, H1299, and H1975 cells were transfected with STING siRNA or NC for 24 h and then exposed to different concentrations (0, 12.5, and 25 nM) of RocA for 24 h, followed by the detection of CCL5 and CXCL10 by real-time PCR. F, A549, H1299, and H1975 cells were transfected with STING siRNA or NC for 24 h and then exposed to 25 nM of RocA for 24 h, followed by the analysis of NK cell migration. Data were pooled from three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 6

NK cell infiltration and tumor regression by RocA depend on STING. A, The expressions of STING, pTBK1, and TBK1 in STING^WT and STING^KO LLC cells were detected by Western blotting analysis. Data represented three independent experiments. STING^WT and STING^KO LLC cells were exposed to different concentrations (0, 12.5, and 25 nM) of RocA for 24 h, and then the expressions of CCL5 (B) and CXCL10 (C) were analyzed by real-time PCR. Data were pooled from three independent experiments. STING^WT and STING^KO LLC cells were subcutaneously inoculated onto the upper back of C57BL6 mice on day 0, and 1 mg/kg of RocA was administered by i.p. injection every 2 days from day 3. Tumor size was measured every 2 days (D, E). Mice were sacrificed on day 18, and tumors were excised, photographed (F), weighed (G), and used to detect the proportions of NK cells (H-I). Data represented three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001; NS, non-statistical significance.

Figure 7

RocA damages mtDNA and promotes the cytoplasmic release of mtDNA. A549, H1299, and H1975 cells were exposed to 25 nM of RocA or 5 µM of CPT for 24 h. A, The cells were stained with anti-γH2AX antibody and DAPI, and observed under laser confocal microscopy. B, The proportions of γH2AX⁺ cells in the cytoplasm. C, The proportions of γH2AX⁺ cells in the nucleus. D-F, A549, H1299, and H1975 cells were exposed to different concentrations (0, 12.5, and 25 nM) of RocA for 24 h, and then the expressions of CO1, ND1, and D-Loop in the cytoplasm were detected by real-time PCR. G, A549, H1299, and H1975 cells were exposed to different concentrations (0, 12.5, and 25 nM) of RocA or different concentrations (0, 6.25, and 12.5 µM) of CsA for 24 h, and then the expression of CO1 in the cytoplasm was detected by real-time PCR. Data represented three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001; NS, non-statistical significance.