The mechanisms by which hypoxic tumours evade immunological pressure and anti-tumour immunity remain elusive. Here, we report that two hypoxia-responsive microRNAs, miR-25 and miR-93, are important for establishing an immunosuppressive tumour microenvironment by downregulating expression of the DNA sensor cGAS. Mechanistically, miR-25/93 targets NCOA3, an epigenetic factor that maintains basal levels of cGAS expression, leading to repression of cGAS during hypoxia. This allows hypoxic tumour cells to escape immunological responses induced by damage-associated molecular pattern molecules, specifically the release of mitochondrial DNA. Moreover, restoring cGAS expression results in an anti-tumour immune response. Clinically, decreased levels of cGAS are associated with poor prognosis for patients with breast cancer harbouring high levels of miR-25/93. Together, these data suggest that inactivation of the cGAS pathway plays a critical role in tumour progression, and reveal a direct link between hypoxia-responsive miRNAs and adaptive immune responses to the hypoxic tumour microenvironment, thus unveiling potential new therapeutic strategies.
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We are grateful to M. C. Ku of the Next Generation Sequencing Core, and C. Benner and M. Shokhirev of the Integrative Genomics and Bioinformatics Core, at Salk Institute for Biological Studies for technical assistance. We would also like to thank D. O’Keefe for his help in preparing the manuscript. This work was supported by The Razavi Newman Integrative Genomics and Bioinformatics Core Facility and the NGS Core Facility of the Salk institute with funding from NIH-NCI CCSG: P30014195, the Chapman Foundation and the Helmsley Charitable Trust. Work in the laboratory of J.C.I.B. was supported by the G. Harold and Leila Y. Mathers Charitable Foundation, The Leona M. and Harry B. Helmsley Charitable Trust (2012-PG-MED002), Fundacion Dr. Pedro Guillen, The Moxie Foundation and UCAM.
The authors declare no competing financial interests.
Integrated supplementary information
(a) FACS analysis for MDSCs showed the effect of HIF-1α signaling on accumulation of CD11b+ Ly6G−Ly6Chigh monocytic MDSCs within tumors. E0771 cells expressing HIF-1α or empty vector were used for tumor formation. (b,c) The population of CD11b+ F4/80+ TAMs or CD11b+ Ly6G+ Ly6Clow granulocytic MDSCs was not affected by ectopic expression of HIF-1α. (d,e) The immune cell profile for tumor infiltrating CD4+ T cells or CD8+ T cells in HIF-1α overexpressing tumors vs. control tumors. For control, n = 5 tumors from 5 different animals. For HIF-1α, n = 7 tumors from 7 different animals. Samples were compared using two-tailed Student’s t test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. ns, not significant. All data shown in this figure represent the mean of the above- indicated sample numbers.
Supplementary Figure 2 Identification of hypoxia responsive miRNAs associated with tumor progression.
(a) Heat map of small RNA sequencing results from MCF7 cells treated with normoxia or hypoxia, MCF7 cells overexpressing HIF-1α, and MCF7-derived BTIC cells. (b) The correlation of selected hypoxia responsive miRNAs with breast tumor progression. Red, molecule is up-regulated. Green, molecule is down-regulated. The intensity of green and red molecule color indicates the degree of down-regulation and up-regulation, respectively. Font bold in red indicates the focus molecule. (c) The correlation of selected hypoxia responsive miRNAs with certain key cancer signaling pathways. (d) The expression level of miR106b-25 cluster in normoxic, hypoxic, HIF-1α overexpressing, and BTIC cells. MCF7 and MDA-MB-231 cells were used as models, as indicated. (e) Knockdown of HIF-1α abolished hypoxia effect on induction of miR25/93 in either hypoxic MCF7 (left upper) or MDA-MB-231 cells (left lower). The protein levels for HIF-1α were presented in right panel. The cells expressing shRNA targeting EGFP were used as control. All the data in the graphs are presented as means ± SD (n = 3 independent experiments). Samples were compared using two-tailed Student’s t test. Asterisk indicates P < 0.05, compared with controls. The immunoblotting shown in panel e are representative of three independent experiments. Unprocessed scans of western blot analysis is available in Supplementary Figure 9. Statistics source data is available in Supplementary Table 4.
(a) The levels for miR25/93 in either control tumors or miR25/93 overexpressing tumors. (b) FACS analysis showed accumulation of CD11b+ Ly6G− Ly6Chigh monocytic MDSC in miR25/93 overexpressing tumors, as compared with control tumors. E0771 cells were used for generating miR25/93 overexpressing tumors. (c and d) The percentage of CD11b+ F4/80+ TAM or CD11b+ Ly6G+ Ly6Clow granulocytic MDSC inmiR25/93 overexpressing tumors vs. control tumors.(e and f)The immune cell profile for tumor infiltrating CD4+ T cells or CD8+ T cells inmiR25/93 overexpressing tumors vs. control tumors. (g) Gene expression analysis in whole tumor specimen for selected genes responsible for anti-tumor immunity. (h) The elevated levels of tumor promoting factors in miR25/93 overexpressing tumors as compared to control tumors. For panel a, the data are presented as means ± SD (n = 3 independent experiments). Asterisk indicates P < 0.05, compared with controls. For panels b–d, control: n = 4 tumors from 4 different animals, miR25/93: n = 4 tumors from 4 different animals. For panels e,f, control: n = 10 tumors from 10 different animals, miR25/93: n = 13 tumors from 13 different animals. For panels g,h, n = 8 tumors from 8 different animals. Data shown in b–h represent the mean of the above-indicated sample numbers. Samples were compared using two-tailed Student’s t test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ns not significant.
Supplementary Figure 4 The regulation of miR25 and miR93 by TET1 protein-mediated epigenetic change in response to hypoxia.
(a) The binding of HIF-1α to the VEGF gene promoter and miR25/93genome regions. VEGF gene promoter was used a positive control for HIF-1α binding upon hypoxia. Four sets of primers targeting genome regions of miR25/93 were used, as indicated. MCF7 and MDA-MB-231 cells were used, as indicated. (b) Levels of 5hmC were locally increased in miR25/93 genomic regions in response to hypoxia. (c) hMeDIP-qPCR validation of the 5hmC change in the miR25/93 genomic regions upon hypoxia. (d) ChIP analysis showed an increase in binding of TET1, but not TET3, to the miR25/93 genomic regions in response to hypoxia (left). TNF genome region was used as a positive control for either TET1 or TET3 (right). (e) qPCR analysis of TET1 knockdown cells for the expression of miR25-3p, miR93-5p, and miR93-3p upon hypoxia. (f) Western bolt analysis for TET1 level in TET1 knockdown cells. (g) The deficiency of TET3 did not alter the ability of hypoxia to induce miR25 and miR93 expression. (h) Western bolt analysis for TET3 in TET3 knockdown cells (i) In TET1 knockdown cells, hMeDIP-qPCR analysis showed that 5hmC levelson the miR25/93 genomic regions were restored in response to hypoxia by re-expressing wild type TET1, but not a TET1 inactive mutant. As representation, the third primer set out of four primer sets was used in qPCR analysis. The cells expressing shRNA targeting EGFP were used as control in panels d to h. N: normoxia H: hypoxia. All data are presented as means ± SD (n = 3 independent experiments). Samples were compared using two-tailed Student’s t test. Asterisk indicates P < 0.05, compared with controls. The image shown in panel f,h is representative of three independent experiments. Unprocessed scans of western blot analysis is available in Supplementary Figure 9. Statistics source data is available in Supplementary Table 4.
(a) Hypoxia increased the level of cytosolic mtDNA. Upper: Western blot analysis for specific markers in cytosolic (β-actin), nucleus (Histone H3), and mitochondria (cytochrome c) fractions. Nucleus is referred to the fraction containing both nucleus and mitochondria fraction. Lower: qPCR analysis of cytosolic mtDNA in either hypoxic MCF7 or MDA-MB-231 cells using three sets of primers targeting different mitochondria DNA regions, as indicated. (b) Hypoxia induced mitophagy, as shown by accumulated level of LC-II and decreased level of p62. (c) Hypoxia suppressed mtDNA-induced immune response in either MCF7 (upper) or MDA-MB-231 cells (lower). The IFN-β expression was used as an indicator for mtDNA-induced immune response. (d,e) Loss of function of miR25/93 restored immune response in the presence of cytosolic mtDNA upon hypoxia. (f,g) The analysis of IFN-β expression in cGAS deficient cells indicated cGAS as a major mediator in mtDNA-induced immune response. The cells expressing shRNA targeting EGFP were used as control. N: normoxia H: hypoxia. All the data in graphs are presented as means ± SD (n = 3 independent experiments). Samples were compared using two-tailed Student’s t test. Asterisk indicates P < 0.05. The image shown in panel a,b is representative of three independent experiments. Unprocessed scans of western blot analysis is available in Supplementary Figure 9. Statistics source data is available in Supplementary Table 4.
Supplementary Figure 6 Inhibition of miR25/93 or cGAS is unable to affect cytoplasmic level of mtDNA as a response to hypoxia.
(a) qPCR analysis for cytosolic mtDNA level in MCF7 or MDA-MB-231stable transfectants expressing shRNA against miR25/93 vs. control cells. (b) Knockdown of cGAS was unable to affect the hypoxic effect on increasing the level of cytosolic mtDNA. The cells expressing shRNA targeting EGFP were used as control. N: normoxia H: hypoxia. All the data are presented as means ± SD (n = 3 independent experiments). Samples were compared using two-tailed Student’s t test. Asterisk indicates P < 0.05, compared with controls.
Supplementary Figure 7 Down-regulation of cGAS leads to establishment of immunosuppressive phenotypes.
(a) Tumor growth analysis showed increased proliferation rate of cGAS deficient tumors compared to control tumors. E0771 cells were used for generating tumors with cGAS deficiency. (b–d) FACS analysis for CD11b+ Ly6G− Ly6Chigh monocytic MDSC, CD11b+ F4/80+ TAM, and CD11b+ Ly6G+ Ly6Clow granulocytic MDSC in cGAS down-regulated tumors vs. control tumors. (e,f) The immune cell profile for tumor infiltrating CD4+ T cells or CD8+ T cells in cGAS deficient tumors vs. control tumors. (g) Knockdown of cGAS decreased the percentage of effector CD8+ cytotoxic T cells. Changes in effector CD8+ cytotoxic T cells normalized to total CD8 + T cell proportions. (h) Increased level of Treg cells in cGAS deficient tumors, as compared to control tumors. (i) Gene expression analysis of cGAS deficient tumors showed significantly lower level of anti-tumor immunity factors than control tumors. (j) Increased level of tumor promoting factors in cGAS down-regulated tumors vs. control tumors. (k) cGAS expression in tumor biopsies derived from cGAS deficient cells or control cells. The cells expressing shRNA targeting EGFP were used as control in this figure. For panels b–d, n = 4 tumors from 4 different animals. For panels e–h, control: n = 7 tumors from 7 different animals, cGAS-si: n = 10 tumors from 10 different animals. For panels i–k, n = 8 tumors from 8 different animals. Data shown in panel a are means ± SD of n = 8 tumors from 8 different animals mice for each group. For panel g, data is presented as mean ± SD of indicated sample numbers. For panels b–f and h–k, data represent the mean of the above-indicated sample numbers. Samples were compared using two-tailed Student’s t test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. ns, not significant.
Supplementary Figure 8 TCGA analysis for Bim and PTEN in breast cancer patients harboring high level of miR93.
(a,b) The levels of Bim and PTEN were not correlated with overall survival of breast cancer patients bearing high level of miR93. Sample number and p value were indicated in the panels. The prognostic significance was assessed by Kaplan–Meier analysis. (c) The expression of MCM7 gene was down-regulated by hypoxia. Knocking down HIF-1α partially rescued MCM7 level in hypoxia. The data is presented as means ± SD (n = 3 independent experiments). Samples were compared using two-tailed Student’s t test. Asterisk indicates P < 0.05.
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Wu, MZ., Cheng, WC., Chen, SF. et al. miR-25/93 mediates hypoxia-induced immunosuppression by repressing cGAS. Nat Cell Biol 19, 1286–1296 (2017). https://doi.org/10.1038/ncb3615
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