The actin modulator hMENA regulates GAS 6‐ AXL axis and pro‐tumor cancer/stromal cell cooperation
2020; Springer Nature; Volume: 21; Issue: 11 Linguagem: Inglês
10.15252/embr.202050078
ISSN1469-3178
AutoresRoberta Melchionna, Sheila Spada, Francesca Di Modugno, Daniel D’Andrea, Anna Di Carlo, Mariangela Panetta, Anna Maria Mileo, Isabella Sperduti, Barbara Antoniani, Enzo Gallo, Rita T. Lawlor, Lorenzo Piemonti, Paolo Visca, Michèle Milella, Gian Luca Grazi, Francesco Facciolo, Emily Chen, Aldo Scarpa, Paola Nisticò,
Tópico(s)Pancreatic function and diabetes
ResumoArticle10 September 2020Open Access Source DataTransparent process The actin modulator hMENA regulates GAS6-AXL axis and pro-tumor cancer/stromal cell cooperation Roberta Melchionna Tumor Immunology and Immunotherapy Unit, IRCCS Regina Elena National Cancer Institute, Rome, Italy Search for more papers by this author Sheila Spada Tumor Immunology and Immunotherapy Unit, IRCCS Regina Elena National Cancer Institute, Rome, Italy Search for more papers by this author Francesca Di Modugno Tumor Immunology and Immunotherapy Unit, IRCCS Regina Elena National Cancer Institute, Rome, Italy Search for more papers by this author Daniel D'Andrea Department of Medicine, Centre for Cell Signaling and Inflammation, Imperial College London, London, UK Search for more papers by this author Anna Di Carlo Tumor Immunology and Immunotherapy Unit, IRCCS Regina Elena National Cancer Institute, Rome, Italy Search for more papers by this author Mariangela Panetta Tumor Immunology and Immunotherapy Unit, IRCCS Regina Elena National Cancer Institute, Rome, Italy Search for more papers by this author Anna Maria Mileo Tumor Immunology and Immunotherapy Unit, IRCCS Regina Elena National Cancer Institute, Rome, Italy Search for more papers by this author Isabella Sperduti Biostatistics and Scientific Direction, IRCCS Regina Elena National Cancer Institute, Rome, Italy Search for more papers by this author Barbara Antoniani Pathology Unit, IRCCS Regina Elena National Cancer Institute, Rome, Italy Search for more papers by this author Enzo Gallo Pathology Unit, IRCCS Regina Elena National Cancer Institute, Rome, Italy Search for more papers by this author Rita T Lawlor ARC-NET Research Centre, Department of Diagnostics and Public Health, Section of Pathology, University of Verona, Verona, Italy Search for more papers by this author Lorenzo Piemonti Diabetes Research Institute, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Paolo Visca Pathology Unit, IRCCS Regina Elena National Cancer Institute, Rome, Italy Search for more papers by this author Michele Milella Department of Medical Oncology 1, IRCCS Regina Elena National Cancer Institute, Rome, Italy Search for more papers by this author Gian Luca Grazi Hepato-pancreato-biliary Surgery Unit, IRCCS Regina Elena National Cancer Institute, Rome, Italy Search for more papers by this author Francesco Facciolo Thoracic-Surgery Unit, IRCCS Regina Elena National Cancer Institute, Rome, Italy Search for more papers by this author Emily Chen Thermo Fisher Precision Medicine Science Center, Cambridge, MA, USA Search for more papers by this author Aldo Scarpa ARC-NET Research Centre, Department of Diagnostics and Public Health, Section of Pathology, University of Verona, Verona, Italy Search for more papers by this author Paola Nisticò Corresponding Author [email protected] orcid.org/0000-0003-4409-2261 Tumor Immunology and Immunotherapy Unit, IRCCS Regina Elena National Cancer Institute, Rome, Italy Search for more papers by this author Roberta Melchionna Tumor Immunology and Immunotherapy Unit, IRCCS Regina Elena National Cancer Institute, Rome, Italy Search for more papers by this author Sheila Spada Tumor Immunology and Immunotherapy Unit, IRCCS Regina Elena National Cancer Institute, Rome, Italy Search for more papers by this author Francesca Di Modugno Tumor Immunology and Immunotherapy Unit, IRCCS Regina Elena National Cancer Institute, Rome, Italy Search for more papers by this author Daniel D'Andrea Department of Medicine, Centre for Cell Signaling and Inflammation, Imperial College London, London, UK Search for more papers by this author Anna Di Carlo Tumor Immunology and Immunotherapy Unit, IRCCS Regina Elena National Cancer Institute, Rome, Italy Search for more papers by this author Mariangela Panetta Tumor Immunology and Immunotherapy Unit, IRCCS Regina Elena National Cancer Institute, Rome, Italy Search for more papers by this author Anna Maria Mileo Tumor Immunology and Immunotherapy Unit, IRCCS Regina Elena National Cancer Institute, Rome, Italy Search for more papers by this author Isabella Sperduti Biostatistics and Scientific Direction, IRCCS Regina Elena National Cancer Institute, Rome, Italy Search for more papers by this author Barbara Antoniani Pathology Unit, IRCCS Regina Elena National Cancer Institute, Rome, Italy Search for more papers by this author Enzo Gallo Pathology Unit, IRCCS Regina Elena National Cancer Institute, Rome, Italy Search for more papers by this author Rita T Lawlor ARC-NET Research Centre, Department of Diagnostics and Public Health, Section of Pathology, University of Verona, Verona, Italy Search for more papers by this author Lorenzo Piemonti Diabetes Research Institute, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Paolo Visca Pathology Unit, IRCCS Regina Elena National Cancer Institute, Rome, Italy Search for more papers by this author Michele Milella Department of Medical Oncology 1, IRCCS Regina Elena National Cancer Institute, Rome, Italy Search for more papers by this author Gian Luca Grazi Hepato-pancreato-biliary Surgery Unit, IRCCS Regina Elena National Cancer Institute, Rome, Italy Search for more papers by this author Francesco Facciolo Thoracic-Surgery Unit, IRCCS Regina Elena National Cancer Institute, Rome, Italy Search for more papers by this author Emily Chen Thermo Fisher Precision Medicine Science Center, Cambridge, MA, USA Search for more papers by this author Aldo Scarpa ARC-NET Research Centre, Department of Diagnostics and Public Health, Section of Pathology, University of Verona, Verona, Italy Search for more papers by this author Paola Nisticò Corresponding Author [email protected] orcid.org/0000-0003-4409-2261 Tumor Immunology and Immunotherapy Unit, IRCCS Regina Elena National Cancer Institute, Rome, Italy Search for more papers by this author Author Information Roberta Melchionna1, Sheila Spada1,†, Francesca Di Modugno1, Daniel D'Andrea2,†, Anna Di Carlo1, Mariangela Panetta1, Anna Maria Mileo1, Isabella Sperduti3, Barbara Antoniani4, Enzo Gallo4, Rita T Lawlor5, Lorenzo Piemonti6, Paolo Visca4, Michele Milella7, Gian Luca Grazi8, Francesco Facciolo9, Emily Chen10, Aldo Scarpa5 and Paola Nisticò *,1 1Tumor Immunology and Immunotherapy Unit, IRCCS Regina Elena National Cancer Institute, Rome, Italy 2Department of Medicine, Centre for Cell Signaling and Inflammation, Imperial College London, London, UK 3Biostatistics and Scientific Direction, IRCCS Regina Elena National Cancer Institute, Rome, Italy 4Pathology Unit, IRCCS Regina Elena National Cancer Institute, Rome, Italy 5ARC-NET Research Centre, Department of Diagnostics and Public Health, Section of Pathology, University of Verona, Verona, Italy 6Diabetes Research Institute, IRCCS San Raffaele Scientific Institute, Milan, Italy 7Department of Medical Oncology 1, IRCCS Regina Elena National Cancer Institute, Rome, Italy 8Hepato-pancreato-biliary Surgery Unit, IRCCS Regina Elena National Cancer Institute, Rome, Italy 9Thoracic-Surgery Unit, IRCCS Regina Elena National Cancer Institute, Rome, Italy 10Thermo Fisher Precision Medicine Science Center, Cambridge, MA, USA †Present address: Department of Radiation Oncology, Weill Cornell Medicine, New York, NY, USA †Present address: MRC Centre for Neuropsychiatric Genetics and Genomics, Cardiff University, Cardiff, UK *Corresponding author. Tel: +39 0652662539; Fax: +39 0652662600; E-mail: [email protected] EMBO Rep (2020)21:e50078https://doi.org/10.15252/embr.202050078 The copyright line for this article was changed on 16 January 2021 after original online publication. PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract 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. Synopsis This study reveals that inhibition of hMENA/hMENADv6 expression reduces pro-tumor CAF-cancer cell crosstalk and inhibits cancer cell invasiveness. CAFs with a pro-tumor activated state express higher levels of hMENA/hMENADv6 compared to normal fibroblasts. CAFs over-expressing hMENADv6 secrete GAS6 and favor the invasiveness of AXL- expressing PDAC and NSCLC cells. Reciprocally in tumor cells hMENA/hMENADv6 regulate AXL expression, and sustain GAS6-AXL paracrine axis. A high hMENA/GAS6/AXL gene expression signature identifies PDAC and NSCLC patients with a poor prognosis. Introduction The tumor microenvironment (TME) is increasingly recognized as a source of novel therapeutic targets (Binnewies et al, 2018), and the identification of paracrine communication between tumor cells and cancer-associated fibroblasts (CAFs) is of great clinical relevance (Carr & Fernandez-Zapico, 2016; Gascard & Tlsty, 2016). This general concept is of significance in non-small cell lung cancer (NSCLC), that despite the therapeutic efficacy of immune checkpoint blockade (ICB; Rizvi et al, 2015) still remains a tumor in which the stroma may hamper treatment efficacy. This scenario is even more detrimental in pancreatic ductal adenocarcinoma (PDAC; Kraman et al, 2010; Provenzano et al, 2012; Shi et al, 2019), a tumor which still lacks effective therapeutic options although estimated to become the second leading cause of cancer-related deaths by 2030 (Hoos et al, 2013; Rahib et al, 2014). CAFs are the main components of tumor stroma and exert tumor-promoting activities by modulating extracellular matrix (ECM), interacting with cancer cells (Olumi et al, 1999; Allinen et al, 2004; Toullec et al, 2010; Jacob et al, 2012; Gascard & Tlsty, 2016; Hammer et al, 2017), and through regulation of inflammation and anti-tumor immunity (Costa et al, 2018; Lakins et al, 2018; Elyada et al, 2019). The recent identification of different CAF subtypes (Costa et al, 2018; Cremasco et al, 2018; Su et al, 2018) calls for the identification of the main players able to convert normal fibroblasts into pro-tumor CAFs and of the molecules used by CAFs to communicate with tumor cells promoting tumor growth and invasiveness. Actin cytoskeleton dynamics and organization regulate cell–ECM and cell–cell contacts and have been also correlated with genome activity (Olson & Nordheim, 2010). We have recently demonstrated that the actin regulatory protein hMENA controls the expression level of β1 integrin by affecting G-ACTIN/F-ACTIN, critical for the nuclear localization of the SRF co-factor myocardin-related transcription factor A (Di Modugno et al, 2018b). hMENA (ENAH) belongs to the Ena/VASP family of actin regulatory proteins, which modulate cell–cell adhesion and cell migration (Bear & Gertler, 2009). The ENAH gene undergoes a splicing process generating multiple tissue-specific isoforms (Di Modugno et al, 2012). We have identified two alternatively expressed isoforms, the epithelial-specific/anti-apoptotic hMENA11a (Di Modugno et al, 2012; Trono et al, 2015) and the mesenchymal-specific/pro-invasive hMENAΔv6 (Di Modugno et al, 2012). hMENA/hMENAΔv6 regulate tumor growth factor TGFβ signaling and are crucial in TGFβ-mediated epithelial mesenchymal transition (EMT) (Melchionna et al, 2016). Clearly, hMENA and its isoforms play a central role in supporting malignant transformation and progression as demonstrated in different tumors (Di Modugno et al, 2006, 2018a,b; Gertler & Condeelis, 2011; Bria et al, 2014; Melchionna et al, 2016; Wang et al, 2017). We have proposed that hMENA isoform expression pattern is a powerful prognostic factor in NSCLC and pancreatic cancer, with a high overall hMENA (including hMENAΔv6) and low hMENA11a expression identifying patients with poor prognosis (Bria et al, 2014; Melchionna et al, 2016). Here we asked whether hMENA may exert its role in cancer progression also by regulating CAF activation and their bi-directional communication with tumor cells. We demonstrate that hMENA/hMENAΔv6 expression play a crucial role in the activation of CAFs derived from both NSCLC and PDAC patients and in their reciprocal interaction with cancer cells. Mechanistically, we identified that this hMENA-mediated pro-tumor function is attributable to its ability to regulate growth arrest-specific 6 (GAS6) in CAFs and AXL in tumor cells, sustaining the pro-tumoral paracrine GAS6-AXL axis, described as crucial in EMT, drug resistance, and immune evasion (Gjerdrum et al, 2010; Jokela et al, 2018; Ludwig et al, 2018). PDAC and NSCLC patients show a worse prognosis when expressing high AXL-GAS6-ENAH gene expression compared with the combined expression of AXL and GAS6 and indicate the relevance of hMENA as both a prognostic marker and a potential therapeutic target. Results hMENA/hMENAΔv6 define a pro-tumor CAF activation state Starting from the observation that stromal compartment of PDAC and NSCLC primary tumors showed in a number of cases a strong immunoreactivity for the Pan-hMENA antibody, compatible with CAF morphology, we evaluated whether hMENA and its isoform expression exert a role not only in tumor cells, but also in pro-tumor CAF biology. We isolated CAFs from resected primary PDACs (P-CAFs) and NSCLCs (L-CAFs) (patient characteristics are shown in Appendix Table S1 and S2, respectively). The isolated CAFs exhibited typical feature of spindle-like mesenchymal cells and lacked the mutations found within primary tumors as revealed by NGS analysis for a panel of 22 genes in all CAFs used for functional studies (Appendix Table S1 and S2). Furthermore, CAF IF analysis and qRT–PCR confirmed that these cells express CAF markers (i.e., FAP, PDGFRB) and are negative for EPCAM (Appendix Fig S1A–C). Characterization of hMENA isoforms clearly showed that CAFs, as expected, were negative for the epithelial hMENA11a isoform (Fig 1A and Appendix Fig S2B and C) and for pan cytokeratin and E-CADHERIN (Fig 1), which are expressed in cancer cells (Ep-PDAC), and were immunostained by α-SMA and Pan-hMENA mAbs (the representative case PDAC#36 in Fig 1A). Figure 1. hMENA isoform expression in CAFs Representative images of immunofluorescence of α-smooth muscle actin (α-SMA), pan cytokeratin, Pan-hMENA, hMENA11a, and E-cadherin expression in CAFs and autologous cancer cells (Ep-PDAC) obtained from enzymatically digested primary PDAC tissue of patient #36. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bar: 20 μm. Representative immunoblot (top) of hMENA/hMENAΔv6 expression levels (detected by Pan-hMENA mAb and by the specific anti-hMENAΔv6 antibody) in normal fibroblasts derived from transplant donor (P-NF), pancreatic serous cystadenoma #71, and cancer-associated fibroblasts (n = 10) obtained from primary PDAC tissues. Densitometry quantified data (bottom) of hMENAΔv6 expression. Quantification of P#110 is relative to the sample shown in the WB on the left. Representative immunoblot (top) of hMENA/hMENAΔv6 expression levels (detected by Pan-hMENA mAb and by the specific anti-hMENAΔv6 antibody) in normal lung fibroblasts (L-NF), cancer-associated fibroblasts obtained from NSCLC tissues (n = 4), and normal dermal fibroblasts (HNF). Densitometry quantified data (bottom) of hMENAΔv6 expression. Data information: Quantified data, are represented as fold change of hMENAΔv6/TUBULIN ratio with respect to control P-NF and L-NF (set as 1). Source data are available online for this figure. Source Data for Figure 1 [embr202050078-sup-0002-SDataFig1.pptx] Download figure Download PowerPoint The tissue specificity of hMENA splicing was confirmed by RT–PCR and WB analysis (Appendix Fig S2B and C) showing that CAFs expressed the hMENA (88 KDa), along with the mesenchymal-specific hMENAΔv6 isoform (80 KDa), but not the hMENA11a (90 KDa). We then compared the expression level of hMENA/hMENAΔv6 between fibroblasts isolated from normal pancreatic tissues derived from transplant donors (P-NFs) and P-CAFs. WB analysis showed that both isoforms were expressed in P-CAF at higher level than in P-NF in the majority of cases evaluated (Fig 1B). Similar results were evidenced for L-CAF compared to lung normal fibroblasts (L-NF) (Fig 1C) and to paired "distal" fibroblasts (L-DFs) derived from non-"tumoral" tissue isolated at least 5 cm away from the tumor core (Appendix Fig S3A). Thus, hMENAΔv6 is expressed, although at heterogeneous level, in all our non-immortalized CAF cultures tested (Fig 1B and C and in Appendix Fig S3B), with the exception of #97 which was derived from a PDAC peritoneal metastasis (Fig 1B). Furthermore, we were able to isolate fibroblasts from a pancreatic serous cystadenoma #71 showing a very low hMENA/hMENAΔv6 expression (Fig 1B). This heterogeneous hMENA expression in the stroma was also evidenced in the IHC analysis of primary NSCLC tissues (Appendix Fig S3C). To confirm that CAF present in primary tumor tissues overexpress hMENA/hMENAΔv6, we performed confocal analysis of NSCLC and PDAC tissues co-stained with Pan-hMENA and α-SMA antibodies, showing that Pan-hMENA decorates α-SMA-positive stromal cells and, as expected, also tumor cells which were α-SMA negative (Fig 2A and B). Figure 2. hMENA expression in tumor and stroma of PDAC and NSCLC tissues Representative images of immunofluorescence of Pan-hMENA (yellow) and α-SMA (red) in the primary PDAC tissue of patient #138 from whom high hMENAΔv6 CAFs were obtained. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bar: 50 μm. The inset of the dashed area is provided on the right as a zoomed-in and cropped fluorescence image. Scale bar: 20 μm. αSMA-positive CAFs are also positive for Pan-hMENA (arrow). Representative images of immunofluorescence of Pan-hMENA (yellow) and α-SMA (red) in NSCLC case #484 from whom high hMENAΔv6 CAFs were obtained. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bar: 50 μm. The inset of the dashed area is provided on the right as a zoomed-in and cropped fluorescence image. Scale bar: 20 μm. As in A, α-SMA signal is evident in stromal cells which are also positive for Pan-hMENA (arrow). Boxplots showing the mean mRNA expression of ENAH gene (hMENA) for 22 patients in the 10 groups of cell types (Peng et al, 2019; total number of cells: 38,487, mean of cells for patient: 1,603). Shown in each boxplot are the median value (horizontal line), 25th–75th percentiles (box outline), and highest and lowest values within 1.5× of the interquartile range (vertical line). Expressions from each cell-type group were compared to all other groups by using Mann–Whitney U-test (two-sided) and P values were adjusted for multiple testing using the Benjamini–Hochberg method. Stromal cell-type groups with significantly up-regulated ENAH expression respect to other stromal groups are: Fibroblast, ***q = 0.0002; Stellate, ***q = 1.5e-07. Non-stromal cell-type groups with significantly up-regulated ENAH expression with respect to other non-stromal groups are: Ductal 1, **q = 0.0013. Boxplots showing the mRNA expression of ENAH gene (hMENA) in the seven groups of cell types from (Lambrechts et al, 2018) (total n = 52). Shown in each boxplot are the median value (horizontal line), 25th–75th percentiles (box outline), and highest and lowest values within 1.5× of the interquartile range (vertical line). Cell expression from each group were compared to all other stromal/not-stromal cells by using Mann–Whitney U-test (two-sided) and P values were adjusted for multiple testing using the Benjamini–Hochberg method (Fibroblast group vs other stromal groups, **q = 0.0022; Other comparisons, q > 0.05). Download figure Download PowerPoint Analysis of the Navab dataset (Navab et al, 2011) confirmed that in primary cultures of CAFs and matched non-malignant distal fibroblasts (NFs) from 15 resected NSCLC, hMENA (ENAH) expression correlated with the expression of α-SMA (ACTA2) and FAP, two of the main CAF markers (Appendix Fig S4). Furthermore, in PDAC single-cell RNA-Seq data (Peng et al, 2019) we evidenced that hMENA expression levels are higher in fibroblasts and stellate cells, but not in immune cells, compared to the other stromal cell types (Benjamini–Hochberg adjusted q-values: Fibroblast, q = 0.0002; Stellate, q = 1.5e-07) (Fig 2C). Yet, hMENA expression levels are higher in Ductal 1 cells compared to the other tumor cells (Benjamini–Hochberg adjusted q = 0.0013). Similarly, single-cell RNA-Seq of the lung tumor microenvironment identified different stromal cell subtypes (Lambrechts et al, 2018). From this analysis we gathered results that hMENA (ENAH) is expressed (although heterogeneously among the clusters) at higher levels in fibroblasts compared to the other stromal cell types (Benjamini–Hochberg adjusted q = 0.0022) (Fig 2D). To further detail the hMENA/hMENAΔv6 functional significance, we analyzed whether hMENA/hMENAΔv6 expression level was correlated to CAF activity by performing functional experiments in a number of P-CAF and L-CAF and in P-NF and L-NF. We found that the higher expression of hMENA/hMENAΔv6 in CAFs with respect to P-NF and L-NFs correlated with a different ability to contract the collagen gel, a measure of their matrix remodeling capacity, and to secrete and activate MMP-2 (Appendix Fig S5B–D). The activated phenotype of CAFs was confirmed by the increased expression of FAP in these cells compared to normal primary fibroblasts NFs (Appendix Fig S5A). This hMENA-related CAF functionality (Fig 3) was evident when we silenced all hMENA isoforms by using a pool of three different siRNAs (sihMENA(t)), in CAFs with high hMENAΔv6 expression (P-CAF#36; P-CAF#138; L-CAF#189; L-CAF#484, Fig 1B and C). SihMENA(t) reduced the ability of CAFs to invade, as measured by Matrigel transwell invasion assay (Fig 3A) and to activate MMP-2 in both L-CAFs and P-CAFs (Appendix Fig S6B). Moreover, we observed a significant decrease of the ability of CAFs to contract collagen gels in hMENA/hMENAΔv6 silenced CAFs compared to control CAFs (Appendix Fig S6A). Figure 3. hMENA/hMENAΔv6 regulate pro-tumor CAF functional activity Quantification of in vitro Matrigel invasion assay (bottom) of P-CAF and L-CAF (P-CAF # 36, 138 and L-CAF #189, 484) transfected with control siRNA (CNT) or hMENA siRNA (hMENA(t)) indicating that the siRNA-mediated knock-down of hMENA/hMENAΔv6 reduces the invasive ability of CAFs with respect to siCNT CAFs. Number of invading cells was measured by counting 6 random fields. Data are presented as the mean ± SD of two biological replicates, performed in triplicates each. Immunoblot showing hMENA/hMENAΔv6 expression (detected by Pan-hMENA mAb and by the specific anti-hMENAΔv6 antibody) of the CAFs employed is reported (top). TUBULIN was used as loading control. P values were calculated by two-sided Student's t-test. *P < 0.05, ***P < 0.001. Quantification of in vitro Matrigel invasion assay (bottom) of P-NF and L-NF and P-CAF#110 and L-CAF#400 transfected with control or hMENAΔv6 expressing vectors, demonstrating that the overexpression of hMENAΔv6 isoform induced the invasiveness of P-NFs and L-NFs and/or P-and L-CAFs. Number of invading cells was measured by counting 6 random fields. Data are presented as the mean ± SD of two biological replicates, performed in triplicates each. Immunoblot of hMENAΔv6 expression (detected by the specific anti-hMENAΔv6 antibody) in fibroblasts employed is reported (top). TUBULIN was used as loading control. P values were calculated by two-sided Student's t-test. *P < 0.05, ***P < 0.001. Source data are available online for this figure. Source Data for Figure 3 [embr202050078-sup-0003-SDataFig3.pptx] Download figure Download PowerPoint Of relevance when we overexpressed the hMENAΔv6 in P-NFs and L-NFs as well in CAF with low hMENAΔv6 expression (P-CAF#110 and L-CAF#400, Fig 1B and C), we found that the non-tumoral fibroblasts and CAFs hMENAΔv6 low increased their functional activities (Fig 3B and Appendix Fig S6C). Collectively, these data point for the first time to our knowledge to the role of hMENA/hMENAΔv6 as marker of pro-tumor CAF activation state. hMENA is crucial in the cooperativity between tumor cells and CAFs It is well established that CAFs promote tumor progression and invasion in various cancers through the activation of paracrine signaling (Gascard & Tlsty, 2016). To identify whether hMENA/hMENAΔv6 expression affects paracrine pro-invasive pathways, we collected conditioned medium (CM) from P-NFs and P-CAFs. According to hMENAΔv6 expression, evaluated in WB analysis, these were classified in P-CAF high (hMENAΔv6 expression greater than 2-fold of the average expression in NFs) and P-CAF low (hMENAΔv6 expression lower than 2-fold; Fig 1B). We evaluated CM effects on PANC-1 cell invasion, and we found that when PANC-1 were treated with the P-CAF-CM for 48 h an increase of cancer cell invasion was associated with high hMENAΔv6 expression. Indeed, as shown in Fig 4A, CM derived from P-CAFs high have a higher pro-invasive effect compared to CM derived from P-CAF low and/or from NFs (Fig 4A) indicating that capability to increase cancer cell invasion of CAF-CM is associated with hMENA/hMENAΔv6 expression. In agreement, when CAFs were silenced for hMENA/hMENAΔv6, their CM fails to induce PANC-1 and KP4 PDAC cell invasion (Fig 4B and Appendix Fig S7A). These data were confirmed in H1975 (Fig 4C and D) and A549 NSCLC cells (Appendix Fig S9C). In addition, the CM of silenced P-CAFs also showed a reduced ability to induce in vitro tumor cell growth (Appendix Fig S8). Figure 4. hMENA/hMENAΔv6 mediates the reciprocal dialogue between tumor cells and CAFs Quantification of in vitro Matrigel invasion assay of PANC-1 cells cultured for 48 h with conditioned media (CM) of NFs (P-NFs-CM), CAF low #44 and #110 and CAFs high #36 and 138. Histograms show the number of invading cells measured by counting 6 random fields. Data are presented as the mean ± SD of three biological replicates, performed at least in duplicate each. Statistical analysis was performed with one-way ANOVA P < 0.0001, followed by Bonferroni's multiple comparison test. *P < 0.05, **P < 0.01, ***P < 0.001. Quantification of in vitro Matrigel invasion assay of PANC-1 cultured for 48 h with CM derived from control P-CAFs#36 (siCNT-P-CAF-CM#36) and hMENA/hMENAΔv6 silenced P-CAFs (sihMENA(t)-P-CAF-CM#36), showing that the siRNA-mediated knock-down of hMENA/hMENAΔv6 affects PANC-1 invasive ability mediated by CAF-CM. Culture medium (DMEM) was used as control. Cells invading Matrigel were counted in 6 random fields. Data are presented as the mean ± SD of three biological replicates. Statistical analysis was performed with one-way ANOVA P = 0.006, followed by Bonferroni's multiple comparison test. *P < 0.05, **P < 0.01. Quantification of in vitro Matrigel invasion assay of H1975 cells cultured for 48 h with control media (culture medium) or conditioned media (CM) of L-CAF low #400 and CAFs high #189, as described above. Data are presented as the mean ± SD of two biological replicates, performed in triplicates each. Statistical analysis was performed with one-way ANOVA P < 0.0001, followed by Bonferroni's multiple comparison test. ***P < 0.001. Quantification of in vitro Matrigel invasion assay of H1975 cultured for 48 h with CM derived from control L-CAFs#484 (siCNT-L-CAF-CM#484) and hMENA/hMENAΔv6 silenced L-CAFs (sihMENA(t)-L-CAF-CM#484), showing that the siRNA-mediated knock-down of hMENA/hMENAΔv6 affects H1975 invasive ability mediated by CAF-CM. Culture medium (DMEM) was used as control. Cells invading Matrigel were counted in 6 random fields. Data are presented as the mean ± SD of six replicates. Statistical analysis was performed with one-way ANOVA P = 0.006, followed by Bonferroni's multiple comparison test. *P < 0.05, **P < 0.01. Representative immunoblot (top) and densitometry quantification (bottom) of hMENAΔv6 expression level in P-NFs grown in RPMI control medium (−) or PANC-1–CM for 24 h (n = 3). Data are represented as fold increase with respect to control medium ± SD (set as 1). Data were analyzed using two-sided Student's t-test. *P < 0.05. Representative images (top) and quantification (bottom) of collagen gel contraction ability of CAFs (monoculture) or CAFs co-cultured with siCNT (co-siCNT) or sihMENA(t) PANC-1 cells (co-sihMENA(t)). Dashed white cir
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