Dynamic reciprocity between cellular components of the tumor microenvironment and tumor cells occurs primarily through the interaction of soluble signals, i.e., cytokines produced by stromal cells to support cancer initiation and progression by regulating cell survival, differentiation and immune cell functionality, as well as cell migration and death. In the present study, we focused on the analysis of the functional response of non-small cell lung cancer cell lines elicited by the treatment with some crucial stromal factors which, at least in part, mimic the stimulus exerted in vivo on tumor cells by microenvironmental components. Our molecular and functional results highlight the role played by the autophagic machinery in the cellular response in terms of the invasive capacity, stemness and drug resistance of two non-small lung cancer cell lines treated with stromal cytokines, also highlighting the emerging role of the YAP pathway in the mutual and dynamic crosstalk between tumor cells and tumor microenvironment elements. The results of this study provide new insights into the YAP-mediated autophagic mechanism elicited by microenvironmental cytokines on non-small cell lung cancer cell lines and may suggest new potential strategies for future cancer therapeutic interventions.

Background: Understanding how cancer signaling pathways promote an immunosuppressive program which sustains acquired or primary resistance to immune checkpoint blockade (ICB) is a crucial step in improving immunotherapy efficacy. Among the pathways that can affect ICB response is the interferon (IFN) pathway that may be both detrimental and beneficial. The immune sensor retinoic acid-inducible gene I (RIG-I) induces IFN activation and secretion and is activated by actin cytoskeleton disturbance. The actin cytoskeleton regulatory protein hMENA, along with its isoforms, is a key signaling hub in different solid tumors, and recently its role as a regulator of transcription of genes encoding immunomodulatory secretory proteins has been proposed. When hMENA is expressed in tumor cells with low levels of the epithelial specific hMENA^11a isoform, identifies non-small cell lung cancer (NSCLC) patients with poor prognosis. Aim was to identify cancer intrinsic and extrinsic pathways regulated by hMENA^11a downregulation as determinants of ICB response in NSCLC. Here, we present a potential novel mechanism of ICB resistance driven by hMENA^11a downregulation. Methods: Effects of hMENA^11a downregulation were tested by RNA-Seq, ATAC-Seq, flow cytometry and biochemical assays. ICB-treated patient tumor tissues were profiled by Nanostring IO 360 Panel enriched with hMENA custom probes. OAK and POPLAR datasets were used to validate our discovery cohort. Results: Transcriptomic and biochemical analyses demonstrated that the depletion of hMENA^11a induces IFN pathway activation, the production of different inflammatory mediators including IFNβ via RIG-I, sustains the increase of tumor PD-L1 levels and activates a paracrine loop between tumor cells and a unique macrophage subset favoring an epithelial-mesenchymal transition (EMT). Notably, when we translated our results in a clinical setting of NSCLC ICB-treated patients, transcriptomic analysis revealed that low expression of hMENA^11a, high expression of IFN target genes and high macrophage score identify patients resistant to ICB therapy. Conclusions: Collectively, these data establish a new function for the actin cytoskeleton regulator hMENA^11a in modulating cancer cell intrinsic type I IFN signaling and extrinsic mechanisms that promote protumoral macrophages and favor EMT. These data highlight the role of actin cytoskeleton disturbance in activating immune suppressive pathways that may be involved in resistance to ICB in NSCLC.

Background: Cancer-associated fibroblasts (CAFs) significantly impact cancer progression and CAF subtypes are key determinants of response to immune checkpoint therapy (ICT). The transforming growth factor-β (TGF-β) signaling is a main pathway in protumorigenic activity of CAFs and resistance to ICT. The actin cytoskeleton regulator hMENA plays crucial roles in epithelial–mesenchymal transition (EMT) and regulates pathways critical to antitumor immune response, such as interferon-I and Axl-GAS6. Methods: We constructed a single-cell atlas of CAFs using integrated public dataset. Experimental data were obtained by biochemical and molecular approaches in CAFs from freshly explanted non-small cell lung cancer (NSCLC) tissues hMENA silenced and in tumor cell lines or peripheral blood mononuclear cells treated with CAF conditioned medium. Multiparametric flow cytometry was used to characterize T cells. A gene signature indicative of ICT response was developed by a machine learning model. Results: Computational analysis indicates that hMENA is primarily overexpressed in a myofibroblast cluster enriched for a TGF-β-activated CAF signature. Experimentally, we showed that TGF-β1 treatment increases hMENA expression and, reciprocally, hMENA/hMENAΔv6 modulate TGF-β1/2 production and secretion and transforming growth factor-β type II receptor expression in CAFs. Functionally, hMENA contributes to TGF-β1-driven CAF phenotype, programmed death-ligand 1 (PD-L1) upregulation, extracellular matrix remodeling and secretion of immunosuppressive cytokines/chemokines. The hMENA-driven TGF-β secretion in CAFs promotes PD-L1 expression and EMT in cancer cells by activating TGF-β signaling. On the tumor cell side, hMENA expression sustains the TGF-β signaling and EMT mediated by hMENA-driven CAFs secretome. This immunosuppressive secretome favors regulatory T cell (Treg) abundance and reduces CD8+ and CD4+ T cell functionality. Finally, based on the hMENA and TGF-β enriched CAF subtype, we developed a 9-gene signature, which in combination with hMENA/hMENAΔv6 correlates with increased Treg abundance and poor prognosis in the Cancer Genome Atlas NSCLC and associates with ICT resistance in Stand Up To Cancer (SU2C) and in phase III clinical trial (OAK) ( NCT02008227 ) datasets. Conclusions: Our findings indicate that hMENA overexpression in CAFs defines a myofibroblast-like subset predominantly driven by TGF-β signaling, which sustains TGF-β1-mediated crosstalk between cancer cells and CAFs and impairs T-cell functionality. In NSCLC tissues, hMENA ^high CAFs associate with TGF-β and regulatory T-cell signatures and correlate with poor patient prognosis and resistance to immune checkpoint therapies, supporting their role as key contributors to an immunosuppressive, ICT-refractory tumor microenvironment.

Non-small cell lung cancer (NSCLC) remains a leading cause of cancer-related mortality. Smoking is the primary etiological factor, but growing evidence suggests the involvement of estrogen in its development and progression, although its role remains unclear. This study explores: (i) the effects induced by estrogen, namely, 17β-estradiol (E2), alone or in combination with a mixture of inflammatory cytokines (Mix), in two human NSCLC cell lines, A549 and Calu1, and (ii) whether and how tumor cells can modulate the activation of normal lung fibroblasts. We found that E2 significantly enhances migration, invasion, and epithelial–mesenchymal transition in NSCLC cells, as well as their resistance to cisplatin, particularly in combination with Mix. Pharmacological inhibition of ERβ reversed the E2-induced effects, implicating ERβ in E2-mediated signaling. Furthermore, E2 increased autophagic flux and induced a shift toward secretory autophagy and the release of extracellular vesicles, which activated normal lung fibroblasts, as demonstrated by the increased expression of α-SMA, FAP, PDGFR-β, and PDPN. The clinical relevance of these data was supported by computational analyses revealing an elevated expression of the Mix gene signature, including TGF-β, IL-6, IL-8, CCXL-16, and ERβ, which was associated with shorter overall survival in NSCLC patients. This molecular profile was linked to the elevated expression of secretory autophagy genes and cancer-associated fibroblast markers. Validation in three large clinical cohorts (TCGA-LUNG, OAK and POPLAR) strengthens the clinical relevance of this E2-related pro-tumor axis while suggesting a promising therapeutic avenue for NSCLC patients.