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The tumor immune microenvironment is a determinant of response to cancer immunotherapy and, in many cases, is prognostic for patient survival independently of the type of treatment. Radiation therapy is used in most cancer patients for its direct cytotoxic effects on malignant cells but there is increasing evidence that it also reprograms the tumor immune microenvironment. In this review we discuss the main mechanisms whereby the local inflammatory reaction induced by radiation can reset the cross-talk between the tumor and the immune system. The outcome reflects the balance between immunostimulatory signals that lead to increased tumor antigen presentation and effector T cell activation, and immunosuppressive signals that hinder radiation-induced tumor rejection. The emerging role of small extracellular vesicles (exosomes) in this process will be discussed. Overall, preclinical and early clinical findings support the hypothesis that radiation has the potential to generate an immune-permissive tumor microenvironment. An improved understanding of the pathways involved will enable the design of more effective combinations of radiation and immunotherapy, based on a rationale integration of radiation with other interventions.

Supplementary figures and tables

Proteomics profiles of UT-TEX and RT-TEX

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.

Radiation therapy (RT) increases tumor response to CTLA-4 inhibition (CTLA4i) in mice and in some patients, yet deep responses are rare. This raises the possibility that effector T cells are insufficiently activated by the combination. To address this question we use mouse models of triple negative breast cancer highly resistant to immunotherapy. We find that CTLA4i promotes the expansion of CD4+ T helper cells, whereas RT enhances T cell clonality and enriches for CD8+ T cells with an exhausted phenotype. Combination therapy decreases regulatory CD4+ T cells and increases CD8+ T cells with effector memory, early activation and precursor exhausted phenotype. A combined gene signature comprising these three CD8+ T cell clusters is associated with survival in triple negative breast cancer patients. Despite the expression of multiple immune checkpoints on intratumoral T cells, response to RT+CTLA4i is not increased by targeting additional checkpoints, including PD1. In contrast, agonistic CD40 therapy enhances responses in mice, indicating the need to target different immune compartments to increase responses to combinations of RT and CTLA4i.

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.

Most patients with non–small cell lung cancer (NSCLC) do not achieve durable clinical responses from immune checkpoint inhibitors, suggesting the existence of additional resistance mechanisms. Nicotinamide adenine dinucleotide (NAD)–induced cell death (NICD) of P2X7 receptor (P2X7R)–expressing T cells regulates immune homeostasis in inflamed tissues. This process is mediated by mono–adenosine 5′-diphosphate (ADP)–ribosyltransferases (ARTs). We found an association between membranous expression of ART1 on tumor cells and reduced CD8 T cell infiltration. Specifically, we observed a reduction in the P2X7R + CD8 T cell subset in human lung adenocarcinomas. In vitro, P2X7R + CD8 T cells were susceptible to ART1-mediated ADP-ribosylation and NICD, which was exacerbated upon blockade of the NAD + -degrading ADP-ribosyl cyclase CD38. Last, in murine NSCLC and melanoma models, we demonstrate that genetic and antibody-mediated ART1 inhibition slowed tumor growth in a CD8 T cell–dependent manner. This was associated with increased infiltration of activated P2X7R + CD8 T cells into tumors. In conclusion, we describe ART1-mediated NICD as a mechanism of immune resistance in NSCLC and provide preclinical evidence that antibody-mediated targeting of ART1 can improve tumor control, supporting pursuit of this approach in clinical studies.

Here, we developed an unbiased, functional target-discovery platform to identify immunogenic proteins from primary non-small cell lung cancer (NSCLC) cells that had been induced to apoptosis by cisplatin (CDDP) treatment in vitro, as compared with their live counterparts. Among the multitude of proteins identified, some of them were represented as fragmented proteins in apoptotic tumor cells, and acted as non-mutated neoantigens (NM-neoAgs). Indeed, only the fragmented proteins elicited effective multi-specific CD4+ and CD8+ T cell responses, upon a chemotherapy protocol including CDDP. Importantly, these responses further increased upon anti-PD-1 therapy, and correlated with patients’ survival and decreased PD-1 expression. Cross-presentation assays showed that NM-neoAgs were unveiled in apoptotic tumor cells as the result of caspase-dependent proteolytic activity of cellular proteins. Our study demonstrates that apoptotic tumor cells generate a repertoire of immunogenic NM-neoAgs that could be potentially used for developing effective T cell-based immunotherapy across multiple cancer patients., Grimaldi and Cammarata et al. develop a proteomics-based, target discovery platform to identify immunogenic proteins specific to apoptotic tumor cells. This study highlights the importance of protein modifications in apoptotic tumor cells as a mechanism of generating immunogenic neoantigens that can be targeted for T cell-based immunotherapy.

Exosomes are extracellular vesicles secreted by cells and involved in intercellular communications among close and distant cells. Exosomes encapsulate and carry biomolecules as cargo to the recipient cells. They contain nucleic acids (DNA, RNA, microRNA) proteins and lipids. Each exosomal components may be isolated and be studied by specific techniques. In this chapter, different methods will be described to isolate DNA from exosomes, since it is important in shaping the response of the recipient cells following the exosome uptake in multiple scenarios, including physiological and pathological conditions. Moreover, the exosomal DNA may be a novel biomarker for diagnosis, disease progression and patient's treatment response.

Exosomes are bi-layered vesicles secreted by the cells in physiological and pathological conditions. They are involved in cell-cell communication facilitating the transfer of functional macromolecules, including DNA, RNA, proteins and lipids. In this chapter, we will focus on specific class of RNA, the microRNAs, that are shuttled from the exosome-producing cells to the recipient cells where they affect biological processes. We will describe the recent methodologies developed to detect and isolate exosomal microRNAs providing a suitable workflow that contributes to quickly expand the field of exosomes-derived microRNAs and their potential use as biomarkers.

The dynamic interplay between cancer cells and cancer‐associated fibroblasts (CAFs) is regulated by multiple signaling pathways, which can lead to cancer progression and therapy resistance. We have previously demonstrated that hMENA, a member of the actin regulatory protein of Ena/VASP family, and its tissue‐specific isoforms influence a number of intracellular signaling pathways related to cancer progression. Here, we report a novel function of hMENA/hMENAΔv6 isoforms in tumor‐promoting CAFs and in the modulation of pro‐tumoral cancer cell/CAF crosstalk via GAS6/AXL axis regulation. LC‐MS/MS proteomic analysis reveals that CAFs that overexpress hMENAΔv6 secrete the AXL ligand GAS6, favoring the invasiveness of AXL‐expressing pancreatic ductal adenocarcinoma (PDAC) and non‐small cell lung cancer (NSCLC) cells. Reciprocally, hMENA/hMENAΔv6 regulates AXL expression in tumor cells, thus sustaining GAS6‐AXL axis, reported as crucial in EMT, immune evasion, and drug resistance. Clinically, we found that a high hMENA/GAS6/AXL gene expression signature is associated with a poor prognosis in PDAC and NSCLC. We propose that hMENA contributes to cancer progression through paracrine tumor–stroma crosstalk, with far‐reaching prognostic and therapeutic implications for NSCLC and PDAC., This study reveals that inhibition of hMENA/hMENADv6 expression reduces pro‐tumor CAF‐cancer cell crosstalk and inhibits cancer cell invasiveness.

Exosomes are small extracellular vesicles released by prokaryotic and eukaryotic cells with a crucial role in cell-to-cell communication in both physiological and pathological conditions. Exosomes contain and transfer active biomolecules, including nucleic acids, proteins and lipids to target recipient cells. In the last decade, many methodologies have been developed for isolating specific exosomal components. In this chapter, we will detail methods to isolate exosomal DNA, considering the crucial role of exosomal DNA in regulating the behavior of recipient cells in multiple settings, including the response of malignant cells to chemo-, radio- and immunotherapy.

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.

Exosomes are extracellular vesicles secreted by cells and involved in intercellular communications among close and distant cells. Exosomes encapsulate and carry biomolecules as cargo to the recipient cells. They contain nucleic acids (DNA, RNA, microRNA) proteins and lipids. Each exosomal components may be isolated and be studied by specific techniques. In this chapter, different methods will be described to isolate DNA from exosomes, since it is important in shaping the response of the recipient cells following the exosome uptake in multiple scenarios, including physiological and pathological conditions. Moreover, the exosomal DNA may be a novel biomarker for diagnosis, disease progression and patient's treatment response.

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.

Exosomes are small extracellular vesicles released by prokaryotic and eukaryotic cells with a crucial role in cell-to-cell communication in both physiological and pathological conditions. Exosomes contain and transfer active biomolecules, including nucleic acids, proteins and lipids to target recipient cells. In the last decade, many methodologies have been developed for isolating specific exosomal components. In this chapter, we will detail methods to isolate exosomal DNA, considering the crucial role of exosomal DNA in regulating the behavior of recipient cells in multiple settings, including the response of malignant cells to chemo-, radio- and immunotherapy.

Cytosolic DNA sensing is emerging to be a critical component of the antitumor immune response by jumpstarting innate immune responses subsequent to the stimulation of the cGAS and STING pathway. Investigating the accumulation of DNA species in the cytosol is therefore an essential readout for promising anticancer strategies. In this chapter, we present different techniques that can be utilized to detect and quantify cytosolic DNA accumulation.

Radiation therapy (RT) used at immunogenic doses (8GyX3) leads to cytosolic accumulation of DNA that binds to the DNA sensor cGAS and activates the cGAMP synthesis, resulting in STING activation and downstream interferon type I (IFN-I) production by cancer cells [1-3]. RT-induced IFN-I is critical for effective anti-tumor immune responses in combination with immune-checkpoint blockade therapy [1]. We have recently demonstrated that tumor-derived exosomes (TEX) secreted by irradiated (8GyX3) TSA murine carcinoma cells (RT-TEX) contain more DNA compared to TEX produced by untreated cells (UT-TEX). Interestingly, only the DNA carried by RT-TEX is capable of STING-dependent activation of recipient dendritic cells (DCs) resulting in CD40, CD80 and CD86 upregulation and IFN-I production. Mice vaccination with RT-TEX, but not UT-TEX, elicits tumor-specific CD8+ T-cell responses that protect mice from tumor development [4]. Here, we tested the hypothesis that not only quantitative but also qualitative differences between the DNA cargo of RT-TEX and UT-TEX may explain its differential ability to activate DCs. To this end, internal DNA purified from TEX and from the cytosolic fraction of the parent TSA cells was characterized for length using Agilent Bioanalyzer. Furthermore, DNA TEX was analyzed by whole-genome sequencing (WGS) and by whole-genome bisulfite sequencing for methylation status. In addition, the percentage of global methylation of DNA of the TSA cells was quantified by 5-methyl cytosine DNA Elisa kit. All experiments were performed in biological triplicates. DNA size analysis revealed an enrichment of DNA fragments with size between 60 and 250 bp in RT-TEX compared to UT-TEX, and in the cytosolic fraction of irradiated compared to untreated TSA cells. The WGS showed that the entire genome was represented in DNA contained within TEX, regardless of the treatment of the parent cells. More than 99% of TEX DNA was of nuclear origin, but mitochondrial DNA was increased in RT-TEX (6.2 fold change, p=0.006). Interestingly, the RT treatment increases the DNA abundance of several regions (median p=0.006). Interestingly, we found reduced levels of methylation in exosomal and total DNA purified from irradiated compared to untreated TSA cells. Our data support the hypothesis that there are potentially important qualitative differences in the DNA cargo of TEX derived from irradiated compared to untreated cancer cells that reflect molecular changes occurring in parent cells. For instance, the enrichment in DNA fragments with size between 60-250 bp in RT-TEX is especially intriguing in light of the recent report that cGAS is optimally activated by this DNA length range [5]. The impact of these qualitative differences in the cargo DNA on activation of the IFN-I pathway in innate immune cells that uptake TEX is under investigation. Identification of DNA signature associated with TEX ability to activate the cGAS/STING pathway could provide a blood-based biomarker for the immunogenic tumor response to radiotherapy. Citation Format: Sheila Spada, Paul Zumbo, Doron Betel, Tuo Zhang, Nils-Petter Rudqvist, Sandra Demaria. Characteristics of the interferon-stimulatory DNA cargo of exosomes produced by irradiated breast cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2263.

Cytosolic DNA sensing is emerging to be a critical component of the antitumor immune response by jumpstarting innate immune responses subsequent to the stimulation of the cGAS and STING pathway. Investigating the accumulation of DNA species in the cytosol is therefore an essential readout for promising anticancer strategies. In this chapter, we present different techniques that can be utilized to detect and quantify cytosolic DNA accumulation.

We demonstrated previously that the splicing of the actin regulator, hMENA, generates two alternatively expressed isoforms, hMENA11a and hMENAΔv6, which have opposite functions in cell invasiveness. Their mechanisms of action have remained unclear. Here we report two major findings: (i) hMENA regulates β1 integrin expression. This was shown by depleting total hMENA, which led to loss of nuclear expression of serum response factor (SRF)-coactivator myocardin-related transcription factor 1 (MRTF-A), leading to an increase in the G-actin/F-actin ratio crucial for MRTF-A localization. This in turn inhibited SRF activity and the expression of its target gene β1 integrin. (ii) hMENA11a reduces and hMENAΔv6 increases β1 integrin activation and signaling. 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 a favorable clinical outcome of early node-negative non-small-cell lung cancer patients. These data provide new insights into the roles of hMENA11a and hMENAΔv6 in the druggable β1 integrin-ECM signaling axis and allow stratification of patient risk, guiding their clinical management.

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