Thesis title: hMENA splicing regulates cancer-specific type-I IFN signaling, pro-tumoral macrophages polarization and resistance to ICB in NSCLC
Introduction Non-small cell lung cancer (NSCLC) is an heterogeneous disease whose evolution depends on the crosstalk between cancer cells and the tumor immune microenvironment (TME) (1) . The introduction of immune checkpoint blockade (ICB) has revolutionized the treatment of NSCLC but the majority of patients do not respond due to innate or acquired resistance (2, 3).The actin cytoskeleton regulatory protein hMENA and its tissue specific splicing-derived isoforms, differently participate to NSCLC progression. The lack of hMENA11a, along with high overall hMENA expression, is a poor prognostic factor and a prerequisite of Epithelial-Mesenchymal Transition (EMT) (4).
Aim To identify molecular mechanisms underlying the role of hMENA11 as prognostic factor in NSCLC and its role in the TME.
Results and Methods RNA-seq on NSCLC cells depleted for total hMENA isoforms or only for hMENA11a revealed that in hMENA11a depleted cells IFN-target genes were up-regulated, including those coding for IFN-β and PDL-1. Biochemical and immunofluorescence (IF) assays revealed that up-regulation occurs through the activation of JAK/STAT1/IRF-1 axis. Bioplex analyses, confirmed by ELISA, demonstrated that hMENA11a loss unleashes the production of inflammatory cytokines and chemokines (i.e. IL-8, IL-6, CXCL-1), through NF-κB activation as evidenced by luciferase assay. ATAC-seq confirmed the role of hMENA11a loss in PDL-1 expression.
Considering the role of cancer-related actin cytoskeleton dynamics in the regulation of Type I IFN-mediated immune response (5), we looked at the actin-linked viral sensor RIG-I. In hMENA11a silenced cells RIG-I is activated and sustains STAT1 activation, IFNβ production and PD-L1 up-regulation.
Based on i) the role of hMENA11a in the regulation of inflammatory pathways, ii) recent data that hMENA status controls the expression of immune response genes (6) iii) breast invasive cancer cells with Mena11a low along with macrophages, form sites of intravasation, in tumor samples of patient with metastatic risk (7) we reasoned whether a paracrine loop exists between hMENA11a loss and pro-tumoral macrophages.
Conditioned medium obtained from hMENA11a silenced cells affects macrophages polarization, up-regulating immune-checkpoint molecules such as PDL-1/2 and VISTA. By blocking interferon-α/β receptor, we found that IFN-β is the major player in hMENA11a-dependent macrophages polarization. Interestingly, these polarized-macrophages secrete factors that up-regulate the expression of EMT markers (i.e. vimentin) in cancer cell lines, as evidenced by confocal IF. By computational analyses we constructed a macrophage signature related to hMENA11a loss, “MENA11a-macro signature” [C10orf54 (VISTA), CD86, CD163 and ITGAM (CD11b)]. By interrogating The Cancer Genome Atlas Lung Adenocarcinoma (TCGA-LUAD) database we found that this signature identifies patients with reduced overall survival.
To assess the clinical relevance of our results we profiled tumor tissues of 10 Fast Progressors (FP) and 10 Good Responders (GR) ICB-treated NSCLC patients, by Nanostring nCounter® PanCancer IO 360™ Panel and custom probes for hMENA splicing variants. By analysing the IFN pathway score for each patient, a higher score in FP vs GR was found (p< 0,05). In GR hMENA11a transcripts were upregulated whereas IFN-I pathway related genes were downregulated. Macrophage score was significantly higher in FP vs GR.
Conclusions Our data identify hMENA splicing and in particular hMENA11a loss as a crucial mechanism activating NF-κB and in turn IFN-β secretion through RIG-I activation. This leads to a paracrine loop able to polarize a pro-tumoral macrophage subtype which contributes to an EMT-related phenotype of tumor cells. In our model IFN-β sustains PDL-1 on both cancer cells and macrophages, impacting survival of LUAD patients.
Moreover, our data on ICB-treated NSCLC patients strongly support the hypothesis that hMENA11a loss may regulate the chronic IFN-β production which ultimately supports rather than inhibit tumor cells, patients survival and response to ICB.