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Loss of miR-29a/b1 cluster reprograms the tumor microenvironment and contributes to immunosuppression in lung cancer

ABSTRACT: Immune checkpoint inhibitors (ICI), like those that block PD-1/PD-L1, have revolutionized oncological therapy for patients with non-small cell lung cancer (NSCLC). However, most patients demonstrate no clinical benefit or acquire resistance, even in PD-L1+ populations, highlighting the critical need to dissect tumor survival dependencies to overcome resistance. Using our unique Kras/p53-driven lung cancer models which demonstrate acquired or intrinsic resistance to ICI, we performed single cell RNA-sequencing and focused on predicted upstream regulators of differentially expressed genes (DEGs) in the malignant cell cluster of resistant tumors. We found that the micro-RNA, miR-29, was downregulated in tumors with anti-PD-1 resistance, leading to significant upregulation of a multitude of miR-29 targets. Interestingly, we found that the immunosuppressive molecule Enpp2/ATX was one of these genes modulated due to miR-29 loss, and re-expression of miR-29 in anti-PD-1 resistant models diminished ATX tumor expression, diminished the fibrotic microenvironment, and increased CD8 infiltration. Additionally, analysis of lung adenocarcinoma patients revealed miR-29-high patients had increased CD8A expression and enrichment in immunoregulatory pathways. Together, these data provide evidence that the miR-29 family broadly regulates the tumor microenvironment, including anti-tumor immune-related pathways in lung cancer, through control of ATX among many other target genes, with implications in ICI response.

ORGANISM(S): Mus musculus

PROVIDER: GSE285606 | GEO | 2026/02/19

REPOSITORIES: GEO

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Project description:Background: The tumor cell-intrinsic function of programmed cell death-ligand 1 (PD-L1) is poorly understood. The roles of the intrinsic function of PD-L1 in interleukin (IL)-6-mediated immunosuppression and the response to immune checkpoint inhibitors (ICIs) in non-small cell lung cancer (NSCLC) were investigated. Methods: Cohorts of NSCLC patients treated with ICI and public datasets were analyzed. PD-L1-overexpressing and PD-L1-knockdown NSCLC cells were submitted to RNA-seq, in vitro analyses, ChIP-qPCR, CUT&Tag, and biochemical assays. Human myeloid-derived suppressor cells (MDSCs) sorted from peripheral blood mononuclear cells were co-cultured with NSCLC cells, and then assessed for their immunosuppressive activity on T-cells. Mouse Lewis lung carcinoma (LLC) cells with PD-L1 overexpression/or knockdown were subcutaneously injected into wild-type or PD-1-knockout C57BL/6 mice in the presence of IL-6 and/or PD-1 blockade. Results: In the ICI cohort with RNA-seq data, the IL-6/Jak/Stat3 pathway was enriched and IL-6 expression was higher in patients with no response to ICI in PD-L1-high NSCLCs. IL-6 expression in PD-L1-high NSCLCs correlated positively with MDSCs and Tregs, but negatively with cytotoxic lymphocytes. In another ICI cohort, a higher baseline serum IL-6 level was associated with poor clinical outcome after ICI therapy. PD-L1 activated Jak2/Stat3 signaling by binding to and inhibiting tyrosine phosphatase PTP1B. PD-L1 also bound to p-Stat3 in the nucleus, thus promoting the activity of p-Stat3 on the transcription of several cytokines (IL-6, TGFβ, TNFα, IL-1β) and chemokines (CXCL1, CXCL3, CXCL8). PD-L1-overexpressing NSCLC cells enhanced the migration and immunosuppressive activity of human MDSC in vitro, mediated by IL-6 and CXCL1. In both wild-type and PD-1-knockout mice, PD-L1-overexpressing mouse lung tumors were heavily infiltrated by MDSCs with high immunosuppressive function. Treg numbers were increased while the numbers of granzyme B+ or IFNγ+ CD8 T-cells decreased. These responses were shown to be mediated by IL-6 secreted from PD-L1-overexpressing tumor cells. Combined blockade of PD-1 and IL-6 was effective in tumor control and decreased MDSCs while increasing granzyme B+ or IFNγ+ CD8 T-cells. Conclusions: The tumor-cell-intrinsic function of PD-L1 contributes to immunosuppression and tumor progression by MDSCs through the IL-6/Jak/Stat3 pathway, which may serve as therapeutic target to improve ICI efficacy in patients with PD-L1-high NSCLC.

Project description:PD-L1 Inhibitor Regulates the miR-33a-5p/PTEN Signaling Pathway and Can Be Targeted to Sensitize Glioblastomas to Radiation. Glioblastoma (GBM) is the most common and lethal brain tumor in adults. Ionizing radiation (IR) is a standard treatment for GBM patients and results in DNA damage. However, the clinical efficacy of IR is limited due to therapeutic resistance. The programmed death ligand 1 (PD-L1) blockade has a shown the potential to increase the efficacy of radiotherapy by inhibiting DNA damage and repair responses. The miR-33a-5p is an essential microRNA that promotes GBM growth and self-renewal. In this study, we investigated whether a PD-L1 inhibitor (a small molecule inhibitor) exerted radio-sensitive effects to impart an anti-tumor function in GBM cells by modulating miR-33a-5p. U87 MG cells and U251 cells were pretreated with PD-L1 inhibitor. The PD-L1 inhibitor-induced radio-sensitivity in these cells was assessed by assaying cellular apoptosis, clonogenic survival assays, and migration. TargetScan and luciferase assay showed that miR-33a-5p targeted the phosphatase and tensin homolog (PTEN) 3' untranslated region. The expression level of PTEN was measured by western blotting, and was also silenced using small interfering RNAs. The levels of DNA damage following radiation was measured by the presence of γ-H2AX foci, cell cycle, and the mRNA of the DNA damage-related genes, BRCA1, NBS1, RAD50, and MRE11. Our results demonstrated that the PD-L1 inhibitor significantly decreased the expression of the target gene, miR-33a-5p. In addition, pretreatment of U87 MG and U251 cells with the PD-L1 inhibitor increased radio-sensitivity, as indicated by increased apoptosis, while decreased survival and migration of GBM cells. Mir-33a-5p overexpression or silencing PTEN in U87 MG and U251 cells significantly attenuated PD-L1 radiosensitive effect. Additionally, PD-L1 inhibitor treatment suppressed the expression of the DNA damage response-related genes, BRCA1, NBS1, RAD50, and MRE11. Our results demonstrated a novel role for the PD-L1 inhibitor in inducing radio- sensitivity in GBM cells, where inhibiting miR-33a-5p, leading to PTEN activated, and inducing DNA damage was crucial for antitumor immunotherapies to treat GBM.

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Loss of miR-29a/b1 cluster reprograms the tumor microenvironment and contributes to immunosuppression in lung cancer

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