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The ability of focal radiotherapy to promote priming of tumor-specific CD8+ T cells and increase responses to immunotherapy is dependent on infiltration of the tumor by Batf3-dependent conventional dendritic cell type 1 (cDC1) cells. Such infiltration is driven by radiotherapy-induced IFN type I (IFN-I). Other signals may also modulate cDC1 infiltration of irradiated tumors. Here we found increased expression of adenosine-generating enzymes CD38 and CD73 in irradiated mouse and human breast cancer cells and increased adenosine in mouse tumors following radiotherapy. CD73 blockade alone had no effect. CD73 blockade with radiotherapy restored radiotherapy-induced cDC1 infiltration of tumors in settings where radiotherapy induction of IFN-I was suboptimal. In the absence of radiotherapy-induced IFN-I, blockade of CD73 was required for rejection of the irradiated tumor and for systemic tumor control (abscopal effect) in the context of cytotoxic T-lymphocyte–associated protein 4 blockade. These results suggest that CD73 may be a radiation-induced checkpoint, and that CD73 blockade in combination with radiotherapy and immune checkpoint blockade might improve patient response to therapy.

Tumor infiltration of conventional dendritic cells has been shown to be essential for triggering efficient antitumor immune responses. These findings have generated an increasing demand for reliable methods to accurately identify and quantify specific DC-subpopulations, both in immune monitoring of clinical trial samples as well as in preclinical mouse tumor models. Here, we describe a flow cytometric approach to assess percentages and absolute counts of conventional dendritic cells in solid mouse tumors.

Radiotherapy (RT) used at immunogenic doses leads to accumulation of cytosolic double-stranded DNA (dsDNA) in cancer cells, which activates type I IFN (IFN-I) via the cGAS/STING pathway. Cancer cell–derived IFN-I is required to recruit BATF3-dependent dendritic cells (DC) to poorly immunogenic tumors and trigger antitumor T-cell responses in combination with immune checkpoint blockade. We have previously demonstrated that the exonuclease TREX1 regulates radiation immunogenicity by degrading cytosolic dsDNA. Tumor-derived DNA can also activate cGAS/STING-mediated production of IFN-I by DCs infiltrating immunogenic tumors. However, how DNA from cancer cells is transferred to the cytoplasm of DCs remains unclear. Here, we showed that tumor-derived exosomes (TEX) produced by irradiated mouse breast cancer cells (RT-TEX) transfer dsDNA to DCs and stimulate DC upregulation of costimulatory molecules and STING-dependent activation of IFN-I. In vivo, RT-TEX elicited tumor-specific CD8+ T-cell responses and protected mice from tumor development significantly better than TEX from untreated cancer cells in a prophylactic vaccination experiment. We demonstrated that the IFN-stimulatory dsDNA cargo of RT-TEX is regulated by TREX1 expression in the parent cells. Overall, these results identify RT-TEX as a mechanism whereby IFN-stimulatory dsDNA is transferred from irradiated cancer cells to DCs. We have previously shown that the expression of TREX1 is dependent on the RT dose size. Thus, these data have important implications for the use of RT with immunotherapy. Cancer Immunol Res; 6(8); 910–20. ©2018 AACR.

Deciphering the complexity of the tumor microenvironment (TME)is essential to unveil mechanisms of therapy resistance and develop novel microenvironment-related anti-tumor treatment. Actin cytoskeleton dynamics act as platforms for gene regulation and key signaling transduction pathways involved in the cross-talk among tumor cells and cellular and non-cellular components of TME.The actin regulatory protein hMENA undergoes tissue specific splicing, generating two alternatively expressed isoforms hMENA11a and hMENAΔv6 with a crucial role in EMT. We have previously demonstrated that hMENA11a and hMENAΔv6, respectively inhibit or increase cell invasiveness, TGFβ and β1 integrin signaling and the secretion of several key extracellular matrix (ECM) proteins. Early node-negative NSCLC patients show a prolonged disease-free survival (DFS) when expressing high tumor hMENA11a/low stromal FN1. Tertiary Lymphoid Structures (TLS), sites of transient lymphoid neo-genesis and determinants of antitumor immunity, have been associated with a favorable clinical outcome in NSCLC patientsandfound in responding lesions of ICB-treated melanoma patients. The aim of the present study was to analyzethe pattern ofhMENA isoforms as biomarker of EMT signature in the context of ECM composition and TLS presence and localization. We evaluated by gain and loss of function experiments the role of hMENA isoforms in TLS neogenesis. hMENA isoforms expression, TLS presence and stromal fibronectin were evaluated in 110primary tumors of node negative NSCLC patients by immunohistochemical analysis using pan-hMENA, hMENA11a, CD3, CD20 and fibronectin (FN) antibodies. The Chi-Square or Fisher Exact tests were used to estimate associations among categorical variables. We found, by RNA-SEQ analysisand subsequent validation by QRT-PCR, in NSCLC cell lines depleted for the expression of ‘epithelial’ hMENA11a isoform,that hMENA11asustains the expression of lymphotoxin beta receptor (LTBR), a regulator of TLS formation. The evaluation of TLS presence and spatial distribution in the primary tumors indicatedthat the presence of TLS within the tumor core is significantly correlatedwith hMENA11a expression in tumor cells, whereas the presence of TLS at the margin oftumor nests correlates with the absence of hMENA11a. When we evaluated also the fibronectin we found a trend of association between low stromal fibronectin and intratumoral TLS, however a low level of stromal FN in concomitance with the expression of hMENA11ain tumor cells,strongly associated with intra-tumoralTLS presence. Our findings indicate that the alternative splicing of hMENA is crucial in the reciprocal signaling between tumor cells and their immune microenvironment, by participating in tertiary lymphoid structure neo genesis and spatial distribution. Funded by Airc Citation Format: Francesca Di Modugno, Sheila Spada, Anna Di Carlo, Paola Trono, Isabella Sperduti, Barbara Antoniani, Enzo Gallo, Giulia Campo, Francesco Facciolo, Paolo Visca, Paola Nisticò. Tissue specific splicing program of hMENA: impact on tumor immune microenvironment in node-negative NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1482.

IL-18 is an inflammasome-related cytokine, member of the IL-1 family, produced by a wide range of cells in response to signals by several pathogen- or damage-associated molecular patterns. It can be highly represented in tumor patients, but its relevance in human cancer development is not clear. In this study, we provide evidence that IL-18 is principally expressed in tumor cells and, in concert with other conventional Th1 cell-driven cytokines, has a pivotal role in establishing a pro-inflammatory milieu in the tumor microenvironment of human non-small cell lung cancer (NSCLC). Interestingly, the analysis of tumor-infiltrating CD8+ T cell populations showed that (i) the relative IL-18 receptor (IL-18R) is significantly more expressed by the minority of cells with a functional phenotype (T-bet+Eomes+), than by the majority of those with the dysfunctional phenotype T-bet−Eomes+ generally resident within tumors; (ii) as a consequence, the former are significantly more responsive than the latter to IL-18 stimulus in terms of IFNγ production ex vivo; (iii) PD-1 expression does not discriminate these two populations. These data indicate that IL-18R may represent a biomarker of the minority of functional tumor-infiltrating CD8+ T cells in adenocarcinoma NSCLC patients. In addition, our results lead to envisage the possible therapeutic usage of IL-18 in NSCLC, even in combination with other checkpoint inhibitor approaches.

The splicing of the actin regulator hMENA generates different isoforms and we have demonstrated that the two alternatively expressed isoforms, hMENA11a and hMENAΔv6, have opposite functions in cell invasiveness. This general mechanism is of great clinical relevance in early NSCLC patients, where the pattern of hMENA isoform expression is a powerful prognostic factor. However the mechanism of action of the two isoforms have remained unclear. Herein, we evaluated whether hMENA and its isoforms influence β1 integrin expression and signaling considering the role of this integrin in cancer cell invasiveness and tumor progression. We performed hMENA silencing by siRNA and shRNA, to evaluate by QRT-PCR and biochemical approaches the expression of β1 integrin; by immunofluorescence the MRTF1 localization, by in vivo assay G-Actin/F-Actin ratio and by luciferase reporter assay the SRF activity. β1 integrin activation and signaling was evaluated by flow cytometry using an antibody specific for the β1 active conformation and by biochemical analysis of the phosphorylation of FAK, SRC and Paxillin. The secretoma of hMENA11a transfected cancer cell lines was analyzed by LC-MS/MS. Immunohistochemical analysis was performed using pan-hMENA, hMENA11a, and fibronectin antibodies in primary cancer tissues from node negative NSCLC patients. The Chi-Square or Fisher Exact tests were used to estimate associations among categorical variables and disease-free survival was calculated by the Kaplan-Meier product limit method. We show that the depletion of all hMENA isoforms inhibits the Serum Response Factor (SRF) activity, and the expression of its target gene β1 Integrin, by affecting G-Actin/F-Actin ratio, critical for the nuclear localization of the SRF co-factor myocardin related transcription factor 1 (MRTF1). Furthermore, we provide new insights into the mechanisms involved in the opposite functions of hMENA11a and hMENAΔv6 in cell invasiveness and we identify a new role of these isoforms in the β1 integrin-ECM signalling axis. Indeed, hMENAΔv6-drives cancer cell invasion by increasing β1 integrin activation and signalling, which is reduced by the anti-invasive hMENA11a isoform. Moreover, exogenous expression of hMENA11a in hMENAΔv6 positive cancer cells dramatically reduces secretion of extracellular matrix (ECM) components, including β1 integrin ligands and metalloproteinases. On the other hand overexpression of the pro-invasive hMENAΔv6 increases fibronectin production. In primary tumors high hMENA11a correlates with low stromal fibronectin and favorable clinical outcome of early node-negative non-small cell lung cancer patients. This newly discovered signature, which pays attention to the alternative splicing of hMENA and ECM components such as fibronectin in the stroma, might help fill in the gap in the still controversial clinical management of early node-negative NSCLC patients. Citation Format: Francesca Di Modugno, Sheila Spada, Belinda Palermo, Paolo Visca, Pierluigi Iapicca, Anna Di Carlo, Barbara Antoniani, Isabella Sperduti, Anna Di Benedetto, Irene Terrenato, Marcella Mottolese, Francesco Gandolfi, Francesco Facciolo, Emily Chen, Martin A. Schwartz, Angela Santoni, Mina J. Bissell, Paola Nisticò. hMENA isoforms impact NSCLC patient outcome through fibronectin/β1 integrin axis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5224.

Human Mena (hMENA), an actin regulatory protein of the ENA/VASP family, cooperates with ErbB receptor family signaling in breast cancer. It is overexpressed in high-risk preneoplastic lesions and in primary breast tumors where it correlates with HER2 overexpression and an activated status of AKT and MAPK. The concomitant overexpression of hMENA and HER2 in breast cancer patients is indicative of a worse prognosis. hMENA is expressed along with alternatively expressed isoforms, hMENA(11a) and hMENAΔv6 with opposite functions. A novel role for the epithelial-associated hMENA(11a) isoform in sustaining HER3 activation and pro-survival pathways in HER2-overexpressing breast cancer cells has been identified by reverse phase protein array and validated in vivo in a series of breast cancer tissues. As HER3 activation is crucial in mechanisms of cell resistance to PI3K inhibitors, we explored whether hMENA(11a) is involved in these resistance mechanisms. The specific hMENA(11a) depletion switched off the HER3-related pathway activated by PI3K inhibitors and impaired the nuclear accumulation of HER3 transcription factor FOXO3a induced by PI3K inhibitors, whereas PI3K inhibitors activated hMENA(11a) phosphorylation and affected its localization. At the functional level, we found that hMENA(11a) sustains cell proliferation and survival in response to PI3K inhibitor treatment, whereas hMENA(11a) silencing increases molecules involved in cancer cell apoptosis. As shown in three-dimensional cultures, hMENA(11a) contributes to resistance to PI3K inhibition because its depletion drastically reduced cell viability upon treatment with PI3K inhibitor BEZ235. Altogether, these results indicate that hMENA(11a) in HER2-overexpressing breast cancer cells sustains HER3/AKT axis activation and contributes to HER3-mediated resistance mechanisms to PI3K inhibitors. Thus, hMENA(11a) expression can be proposed as a marker of HER3 activation and resistance to PI3K inhibition therapies, to select patients who may benefit from these combined targeted treatments. hMENA(11a) activity could represent a new target for antiproliferative therapies in breast cancer.

Background In lung cancer, reliable prognostic indicators of the risk of recurrence are still not available. Alternative splicing represents a potential biomarker of diagnosis, prognosis, invasiveness, and response to therapy in different tumors [1], including lung cancer [2]. Human MENA (hMENA) is an actin regulatory protein that modulates cell adhesion and migration [3]. We have isolated three hMENA splice variants, namely hMENA, hMENA and hMENAΔv6, impacting differently cell shape and function. hMENA expression ensures the integrity of cell-cell adhesion and is associated with an epithelial phenotype, whereas hMENAΔv6 is related to a mesenchymal invasive phenotype. The splicing of hMENA, relevant to epithelial mesenchymal transition, is also regulated by microenvironmental cues [4]. The dynamic reciprocity between tumor and stroma influences the tumor tissue architecture including the T cell localization. This, proposed as a prognostic marker [5], is a prerequisite for antitumor immune surveillance and recently the antibody blockade of immune checkpoints is a new reality in lung cancer treatment [6,7].

Human Mena (hMENA), an actin regulatory protein of the ENA/VASP family, cooperates with ErbB receptor family signaling in breast cancer. It is overexpressed in high-risk preneoplastic lesions and in primary breast tumors where it correlates with HER2 overexpression and an activated status of AKT and MAPK. The concomitant overexpression of hMENA and HER2 identifies breast cancer patients with a worse prognosis. hMENA is expressed along with alternatively expressed isoforms, hMENA11a and hMENAΔv6 with opposite functions. By Reverse Phase Protein Assay, we identified a novel role for the epithelial associated hMENA11a isoform in sustaining HER3 activation and pro-survival pathways in HER2 overexpressing breast luminal cancer cells. Since HER3 activation is crucial in mechanisms of cell resistance to PI3K inhibitors, we explored whether hMENA11a is involved in these resistance mechanisms. The specific hMENA11a depletion switched off the HER3-related pathway activated by PI3K inhibitors and impaired the nuclear accumulation of HER3 transcription factor FOXO3a induced by PI3K inhibitors. On the other hand, PI3K inhibitors activated hMENA11a phosphorylation and affected its localization. At the functional level, we found that hMENA11a sustains cell proliferation and survival in response to PI3K inhibitor treatment whereas hMENA11a silencing increases molecules involved in cancer cell apoptosis. As shown in three-dimensional cultured breast cancer cells hMENA11a contributes to cancer cells resistance to PI3K inhibition since the depletion of hMENA11a drastically reduced cell viability upon treatment with PI3K inhibitor BEZ235. Altogether, these results indicate that hMENA11a in HER2 overexpressing breast cancer cells sustains HER3/AKT axis activation and contributes to HER3-mediated resistance mechanisms to PI3K inhibitors. Thus, hMENA11a expression can be proposed as a marker of HER3 activation and of resistance to PI3K inhibition therapies, to select patients who can benefit from these combined targeted treatments. hMENA11a activity may represent a new target for anti-proliferative therapies in breast cancer. Citation Format: Paola Trono, Francesca Di Modugno, Rita Circo, Sheila Spada, Roberta Melchionna, Belinda Palermo, Mariangela Panetta, Silvia Matteoni, Silvia Soddu, Ruggero De Maria, Paola Nisticò. hMENA11a contributes to HER3-mediated resistance to PI3K inhibitors in HER2 overexpressing breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4316. doi:10.1158/1538-7445.AM2015-4316

Background Epithelial to mesenchymal transition (EMT) is an early event in pancreatic cancer and has been involved in cancer invasiveness. An intense stromal reaction, peculiar to the pancreatic tumor microenvironment, includes cancer-associated fibroblasts(CAFs), abundant cells in the tumor stroma, recently linked to the induction of EMT. On the other hand, the EMT process requires a dynamic remodeling of the actin cytoskeleton, and the splicing program of hMENA, a regulator of actin, has been associated with the EMT process. We have described two alternatively expressed isoforms, hMENA11a and hMENAΔv6, with opposite functions in invasiveness in breast cancer (1). hMENA expression was detected in human pancreatic ductal adenocarcinoma samples (PDAC) (2), but no data are available on the alternative isoform expression in this neoplasia. The aim of this study is to investigate the role of hMENA splicing in TGF-β -mediated EMT in pancreatic cancer, the mechanisms involved in hMENA induction in PDAC and the role of CAFs in this process. Methods hMENA isoform expression was evaluated in PDAC tissues by immunohistochemistry using isoform-specific antibodies. Human PDAC cell lines, untreated or TGF-β treated, were characterized for the expression of hMENA isoforms and markers of EMT by qRT-PCR and WB analysis. The effects of both hMENAΔv6 knockdown or overexpression were also evaluated. Pancreatic cancer associated-fibroblasts were isolated from primary PDAC tissues. To study the role of fibroblast-cancer cell interactions on hMENA expression, a noncontact coculture system was used. Results IHC analysis of PDAC tissues revealed that epithelial hMENA11a is rarely expressed in primary pancreatic tumors that express a high level of hMENA and hMENAΔv6 isoforms. In a panel of pancreatic cancer cell lines, hMENA11a expression correlates with an epithelial phenotype, wherea hMENAΔv6 expression is associated with a mesechymal phenotype. TGF-β treatment specifically upregulated the invasive hMENAΔv6 isoform expression. Knockdown of endogenous hMENA/hMENAΔv6 isoform reduced cell invasiveness, reverted cells to an epithelial -“like” phenotype with an increased E-cadherin expression and impaired the TGF-β-mediated vimentin up-regulation. Conversely, overexpression of hMENAΔv6 increased the expression of the mesenchymal marker vimentin. Freshly explanted CAFs expressed the “mesenchymal” hMENAΔv6, and not hMENA11a and produced paracrine factors involved in the induction of hMENA isoforms in tumor cells. Conclusions These data provide new and critical insights into the role of hMENA splicing in TGF-β mediated EMT and identify the hMENA splicing program as a promising pathway for the development of new diagnostics and therapeutics in PDAC. (1) Di Modugno F. et al PNAS 2012 (2) Pino S. et al Clin Cancer Res 2008 Citation Format: Roberta Melchionna, Pierluigi Iapicca, Francesca Di Modugno, Paola Trono, Novella Gualtieri, Maria Grazia Diodoro, Sheila Spada, Giuliana Falasca, Gian Luca Grazi, Mina J Bissell, Paola Nisticò. hMENA splicing program and TGF-β1-mediated EMT in pancreatic cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1035. doi:10.1158/1538-7445.AM2014-1035