Hepsin regulates TGFβ signaling via fibronectin proteolysis
2021; Springer Nature; Volume: 22; Issue: 11 Linguagem: Inglês
10.15252/embr.202152532
ISSN1469-3178
AutoresDenis Belitškin, Shishir M. Pant, Pauliina Munne, Ilida Suleymanova, Kati Belitškina, Hanna‐Ala Hongisto, Johanna I. Englund, Tiina Raatikainen, Olga Klezovitch, Valeri Vasioukhin, Shuo Li, Qingyu Wu, Outi Monni, Satu Kuure, Pirjo Laakkonen, Jeroen Pouwels, Topi A. Tervonen, Juha Klefström,
Tópico(s)Protease and Inhibitor Mechanisms
ResumoArticle13 September 2021Open Access Source DataTransparent process Hepsin regulates TGFβ signaling via fibronectin proteolysis Denis Belitškin Denis Belitškin orcid.org/0000-0002-0032-2769 Research Programs Unit/Translational Cancer Medicine Research Program and Medicum, Faculty of Medicine, Biomedicum Helsinki, University of Helsinki, Helsinki, Finland Search for more papers by this author Shishir M Pant Shishir M Pant orcid.org/0000-0002-2686-7898 Research Programs Unit/Translational Cancer Medicine Research Program and Medicum, Faculty of Medicine, Biomedicum Helsinki, University of Helsinki, Helsinki, Finland Search for more papers by this author Pauliina Munne Pauliina Munne orcid.org/0000-0002-9720-9964 Research Programs Unit/Translational Cancer Medicine Research Program and Medicum, Faculty of Medicine, Biomedicum Helsinki, University of Helsinki, Helsinki, Finland Search for more papers by this author Ilida Suleymanova Ilida Suleymanova Research Programs Unit/Translational Cancer Medicine Research Program and Medicum, Faculty of Medicine, Biomedicum Helsinki, University of Helsinki, Helsinki, Finland Search for more papers by this author Kati Belitškina Kati Belitškina orcid.org/0000-0002-0902-1014 Pathology Department, North Estonia Medical Centre, Tallinn, Estonia Search for more papers by this author Hanna-Ala Hongisto Hanna-Ala Hongisto Research Programs Unit/Translational Cancer Medicine Research Program and Medicum, Faculty of Medicine, Biomedicum Helsinki, University of Helsinki, Helsinki, Finland Search for more papers by this author Johanna Englund Johanna Englund orcid.org/0000-0001-9637-6843 Research Programs Unit/Translational Cancer Medicine Research Program and Medicum, Faculty of Medicine, Biomedicum Helsinki, University of Helsinki, Helsinki, Finland Search for more papers by this author Tiina Raatikainen Tiina Raatikainen Research Programs Unit/Translational Cancer Medicine Research Program and Medicum, Faculty of Medicine, Biomedicum Helsinki, University of Helsinki, Helsinki, Finland Search for more papers by this author Olga Klezovitch Olga Klezovitch Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA, USA Search for more papers by this author Valeri Vasioukhin Valeri Vasioukhin Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA, USA Search for more papers by this author Shuo Li Shuo Li Department of Cardiovascular and Metabolic Sciences, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA Search for more papers by this author Qingyu Wu Qingyu Wu orcid.org/0000-0003-0561-9315 Department of Cardiovascular and Metabolic Sciences, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA Search for more papers by this author Outi Monni Outi Monni orcid.org/0000-0002-2319-8799 Research Programs Unit/Applied Tumor Genomics Research Program, Faculty of Medicine, Biomedicum Helsinki, University of Helsinki, Helsinki, Finland Search for more papers by this author Satu Kuure Satu Kuure orcid.org/0000-0002-9076-0622 GM-Unit, Laboratory Animal Centre, Helsinki Institute of Life Science (HiLIFE), University of Helsinki, Helsinki, Finland Search for more papers by this author Pirjo Laakkonen Pirjo Laakkonen orcid.org/0000-0002-9620-095X Laboratory Animal Center, Helsinki Institute of Life Science (HiLIFE), University of Helsinki, Helsinki, Finland Search for more papers by this author Jeroen Pouwels Jeroen Pouwels Research Programs Unit/Translational Cancer Medicine Research Program and Medicum, Faculty of Medicine, Biomedicum Helsinki, University of Helsinki, Helsinki, Finland Search for more papers by this author Topi A Tervonen Topi A Tervonen orcid.org/0000-0001-6577-9607 Research Programs Unit/Translational Cancer Medicine Research Program and Medicum, Faculty of Medicine, Biomedicum Helsinki, University of Helsinki, Helsinki, Finland Search for more papers by this author Juha Klefström Corresponding Author Juha Klefström [email protected] orcid.org/0000-0001-7124-8431 Research Programs Unit/Translational Cancer Medicine Research Program and Medicum, Faculty of Medicine, Biomedicum Helsinki, University of Helsinki, Helsinki, Finland Finnish Cancer Institute & FICAN South, Helsinki University Hospital (HUS), Helsinki, Finland Search for more papers by this author Denis Belitškin Denis Belitškin orcid.org/0000-0002-0032-2769 Research Programs Unit/Translational Cancer Medicine Research Program and Medicum, Faculty of Medicine, Biomedicum Helsinki, University of Helsinki, Helsinki, Finland Search for more papers by this author Shishir M Pant Shishir M Pant orcid.org/0000-0002-2686-7898 Research Programs Unit/Translational Cancer Medicine Research Program and Medicum, Faculty of Medicine, Biomedicum Helsinki, University of Helsinki, Helsinki, Finland Search for more papers by this author Pauliina Munne Pauliina Munne orcid.org/0000-0002-9720-9964 Research Programs Unit/Translational Cancer Medicine Research Program and Medicum, Faculty of Medicine, Biomedicum Helsinki, University of Helsinki, Helsinki, Finland Search for more papers by this author Ilida Suleymanova Ilida Suleymanova Research Programs Unit/Translational Cancer Medicine Research Program and Medicum, Faculty of Medicine, Biomedicum Helsinki, University of Helsinki, Helsinki, Finland Search for more papers by this author Kati Belitškina Kati Belitškina orcid.org/0000-0002-0902-1014 Pathology Department, North Estonia Medical Centre, Tallinn, Estonia Search for more papers by this author Hanna-Ala Hongisto Hanna-Ala Hongisto Research Programs Unit/Translational Cancer Medicine Research Program and Medicum, Faculty of Medicine, Biomedicum Helsinki, University of Helsinki, Helsinki, Finland Search for more papers by this author Johanna Englund Johanna Englund orcid.org/0000-0001-9637-6843 Research Programs Unit/Translational Cancer Medicine Research Program and Medicum, Faculty of Medicine, Biomedicum Helsinki, University of Helsinki, Helsinki, Finland Search for more papers by this author Tiina Raatikainen Tiina Raatikainen Research Programs Unit/Translational Cancer Medicine Research Program and Medicum, Faculty of Medicine, Biomedicum Helsinki, University of Helsinki, Helsinki, Finland Search for more papers by this author Olga Klezovitch Olga Klezovitch Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA, USA Search for more papers by this author Valeri Vasioukhin Valeri Vasioukhin Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA, USA Search for more papers by this author Shuo Li Shuo Li Department of Cardiovascular and Metabolic Sciences, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA Search for more papers by this author Qingyu Wu Qingyu Wu orcid.org/0000-0003-0561-9315 Department of Cardiovascular and Metabolic Sciences, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA Search for more papers by this author Outi Monni Outi Monni orcid.org/0000-0002-2319-8799 Research Programs Unit/Applied Tumor Genomics Research Program, Faculty of Medicine, Biomedicum Helsinki, University of Helsinki, Helsinki, Finland Search for more papers by this author Satu Kuure Satu Kuure orcid.org/0000-0002-9076-0622 GM-Unit, Laboratory Animal Centre, Helsinki Institute of Life Science (HiLIFE), University of Helsinki, Helsinki, Finland Search for more papers by this author Pirjo Laakkonen Pirjo Laakkonen orcid.org/0000-0002-9620-095X Laboratory Animal Center, Helsinki Institute of Life Science (HiLIFE), University of Helsinki, Helsinki, Finland Search for more papers by this author Jeroen Pouwels Jeroen Pouwels Research Programs Unit/Translational Cancer Medicine Research Program and Medicum, Faculty of Medicine, Biomedicum Helsinki, University of Helsinki, Helsinki, Finland Search for more papers by this author Topi A Tervonen Topi A Tervonen orcid.org/0000-0001-6577-9607 Research Programs Unit/Translational Cancer Medicine Research Program and Medicum, Faculty of Medicine, Biomedicum Helsinki, University of Helsinki, Helsinki, Finland Search for more papers by this author Juha Klefström Corresponding Author Juha Klefström [email protected] orcid.org/0000-0001-7124-8431 Research Programs Unit/Translational Cancer Medicine Research Program and Medicum, Faculty of Medicine, Biomedicum Helsinki, University of Helsinki, Helsinki, Finland Finnish Cancer Institute & FICAN South, Helsinki University Hospital (HUS), Helsinki, Finland Search for more papers by this author Author Information Denis Belitškin1, Shishir M Pant1, Pauliina Munne1, Ilida Suleymanova1, Kati Belitškina2, Hanna-Ala Hongisto1, Johanna Englund1, Tiina Raatikainen1, Olga Klezovitch3, Valeri Vasioukhin3, Shuo Li4, Qingyu Wu4, Outi Monni5, Satu Kuure6, Pirjo Laakkonen7, Jeroen Pouwels1, Topi A Tervonen1,† and Juha Klefström *,1,8,† 1Research Programs Unit/Translational Cancer Medicine Research Program and Medicum, Faculty of Medicine, Biomedicum Helsinki, University of Helsinki, Helsinki, Finland 2Pathology Department, North Estonia Medical Centre, Tallinn, Estonia 3Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA, USA 4Department of Cardiovascular and Metabolic Sciences, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA 5Research Programs Unit/Applied Tumor Genomics Research Program, Faculty of Medicine, Biomedicum Helsinki, University of Helsinki, Helsinki, Finland 6GM-Unit, Laboratory Animal Centre, Helsinki Institute of Life Science (HiLIFE), University of Helsinki, Helsinki, Finland 7Laboratory Animal Center, Helsinki Institute of Life Science (HiLIFE), University of Helsinki, Helsinki, Finland 8Finnish Cancer Institute & FICAN South, Helsinki University Hospital (HUS), Helsinki, Finland † These authors contributed equally to this work as senior authors *Corresponding author. Tel.: +358294125493; E-mail: [email protected] EMBO Reports (2021)22:e52532https://doi.org/10.15252/embr.202152532 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 Transforming growth factor-beta (TGFβ) is a multifunctional cytokine with a well-established role in mammary gland development and both oncogenic and tumor-suppressive functions. The extracellular matrix (ECM) indirectly regulates TGFβ activity by acting as a storage compartment of latent-TGFβ, but how TGFβ is released from the ECM via proteolytic mechanisms remains largely unknown. In this study, we demonstrate that hepsin, a type II transmembrane protease overexpressed in 70% of breast tumors, promotes canonical TGFβ signaling through the release of latent-TGFβ from the ECM storage compartment. Mammary glands in hepsin CRISPR knockout mice showed reduced TGFβ signaling and increased epithelial branching, accompanied by increased levels of fibronectin and latent-TGFβ1, while overexpression of hepsin in mammary tumors increased TGFβ signaling. Cell-free and cell-based experiments showed that hepsin is capable of direct proteolytic cleavage of fibronectin but not latent-TGFβ and, importantly, that the ability of hepsin to activate TGFβ signaling is dependent on fibronectin. Altogether, this study demonstrates a role for hepsin as a regulator of the TGFβ pathway in the mammary gland via a novel mechanism involving proteolytic downmodulation of fibronectin. Synopsis TGFβ is released from the ECM compartments of the mammary glands by hepsin mediated proteolytic cleavage of the ECM component fibronectin. Hepsin depletion in mice leads to impaired TGFβ signaling and increased mammary duct branching. Hepsin overexpression in an in vivo model of breast cancer activates TGFβ signaling. The extracellular matrix protein fibronectin is a target of proteolytic hepsin activity in vitro and in vivo. The regulation of TGFβ by hepsin requires proteolytic cleavage of fibronectin. Introduction The human genome encodes 566 proteases, and of these, 273 have been found in extracellular compartments or the lumen of secretory compartments, 277 in intracellular compartments, and a small fraction at the plasma membrane (Overall & Blobel, 2007). Extracellular proteolysis plays a key role in regulating the physical properties of the extracellular matrix (ECM) through turnover, which, for example, affects branching morphogenesis in the mammary gland (Wiseman et al, 2003; Green & Lund, 2005; Lu et al, 2011). Another critical role of extracellular proteolysis lies in the processing of growth factor pro-forms (e.g., pro-HGF; Herter et al, 2005, pro-MSP Ganesan et al, 2011, and EGF Higashiyama et al, 2011). Therefore, extracellular proteolysis orchestrates the interaction between ECM remodeling and growth factor signaling during development and tissue regeneration (Mohammed & Khokha, 2005; Fukushima et al, 2018). Most studies on proteolytic regulation of the ECM have focused on the diverse class of matrix metalloproteinases (MMPs) (Chang & Werb, 2001). Interestingly, in addition to MMPs, serine proteases have been demonstrated to be involved in pericellular proteolysis of ECM components and plasma membrane proteins, allowing localized ECM remodeling and receptor signaling regulation (Del Rosso et al, 2002). Hepsin is a protease belonging to the type II transmembrane serine protease (TTSP) family, a special group of membrane-anchored proteases whose enzymatic activity is confined to the pericellular space (Hooper et al, 2001), making TTSPs accessible for function-blocking antibodies and small molecule inhibitors (Antalis et al, 2010). Hepsin knockout mice display defects in inner ear development and have altered kidney function (Guipponi et al, 2007; Olinger et al, 2019). A recent study reported reduced liver size and browning of fat tissue in hepsin knockout mice, leading to a reduction of body fat in mice on a high-fat diet (Li et al, 2020). Apart from a role for hepsin-mediated cleavage of pro-HGF in the liver (Hsu et al, 2012) and laminin-332 during invasion (Klezovitch et al, 2004; Tripathi et al, 2008; Pant et al, 2018a), the molecular mechanisms underlying other physiological functions of hepsin remain poorly defined. Hepsin is one of the most frequently overexpressed proteins in prostate cancer (Dhanasekaran et al, 2001; Luo et al, 2001; Magee et al, 2001; Stamey et al, 2001; Welsh et al, 2001; Ernst et al, 2002; Chen et al, 2003; Stephan et al, 2004). Hepsin is also very frequently overexpressed in breast cancer, in up to 70% of breast tumors (Tervonen et al, 2016). Hepsin overexpression is suggested to promote cancer progression and metastasis (Tanimoto et al, 1997; Klezovitch et al, 2004; Tervonen et al, 2016), and several hepsin-regulated oncogenic pathways have been described. For example, overactive hepsin damages epithelial integrity and promotes degradation of the basement membrane (Klezovitch et al, 2004; Tripathi et al, 2008; Partanen et al, 2012; Tervonen et al, 2016), and hepsin directly cleaves oncogenic growth factors pro-HGF and pro-MSP (Herter et al, 2005; Ganesan et al, 2011). However, given that most proteases have a broad substrate range, additional signaling pathways downstream of hepsin probably exist that contribute to its oncogenic properties. Here, we demonstrate that hepsin controls TGFβ signaling via proteolytic regulation of fibronectin. The findings are corroborated in vivo by using a new hepsin CRISPR knockout mouse model, alongside an established model of hepsin overexpression in breast cancer. Results CRISPR/Cas9-generated Hpn knockout mice manifest diminished liver size and partial loss of hearing We generated hepsin knockout mice with CRISPR/Cas9-mediated gene editing using a guide RNA targeting exon 4 of the Hpn gene. Genetic analysis revealed a 50 bp frameshift deletion in the coding region of the Hpn gene (Fig 1A), which resulted in the loss of the hepsin protein in mice homozygous for the frameshift allele (Figs 1A and EV1A). Hpn knockout mice did not display any obvious histopathological changes, gross morphological, or tissue architecture deviations, nor did they show any differences in external appearance, lean mass, free fluid, or body fat (Fig EV1B–D). Consistent with Hpn knockout mice created via traditional homologous recombination methods (Wu et al, 1998; Li et al, 2020), our CRISPR hepsin knockout (Hpn−/−) mice displayed diminished liver size and reduced hearing (Figs 1B and C, and EV1E). Figure 1. The knockout of hepsin with CRISPR/Cas9 inhibits TGFβ signaling in the mouse mammary gland Hepsin knockout mice harbor a 50 bp frameshift deletion in the 4th exon of the Hpn gene (TM = transmembrane domain, SRCR = scavenger receptor cysteine-rich domain, SPD = serine protease domain; red bar indicates gRNA-binding site). Immunoblot from whole mammary lysates against hepsin protein (Hpn+/+ = Wt, Hpn+/− = heterozygous deletion, Hpn−/− = homozygous deletion) (representative of 3 mice per group). Weight of indicated organs isolated from Wt (N = 3) and Hpn−/− (N = 4) mice. Acoustic startle reflex test to compare hearing ability between Wt (N = 7) and Hpn−/− (N = 4) mice. Cytoscape enrichment map of pathways affected in Hpn−/− whole mammary glands compared with Wt controls (N = 3 per group). Node size correlates with the number of genes in the signature; node color correlates with either gene set enrichment (red) or reduction (blue) in Hpn−/− mammary glands. A full list of gene signatures affected in Hpn−/− mammary glands is shown in Table EV1. Gene Set Enrichment Analysis (GSEA) graphs showing enrichment of indicated TGFβ1 signaling gene sets in Hpn−/− mammary glands compared with Wt mammary glands (FDRp—P-value; FWERq—false discovery rate; NES—normalized enrichment score). Immunoblot analysis of phospho-Smad2/3 (TGFβ pathway signaling marker) and total Smad2/3 in lysates from indicated tissues isolated from Wt, Hpn+/−, and Hpn−/− mice. GAPDH was used as the loading control. The histogram depicts quantification of pSmad2/3 compared to Wt, normalized to total Smad2/3. Representative Carmine alum stained mammary gland whole mounts from Wt and Hpn−/− mice. The histogram depicts quantification of duct length normalized to duct length in Wt mammary glands. The scale bar represents 1 mm. Data in (B, C, F, G) are represented as mean ± SD, and Student's t-test was used for statistical analyses. Source data are available online for this figure. Source Data for Figure 1 [embr202152532-sup-0002-SDataFig1.xlsx] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Description of gross morphology and histology of Hpn−/− mice Immunoblot analysis of hepsin in Wt and Hpn−/− tissues. Photographs of representative 6-week-old Wt and Hpn−/− mice. Scale bar is equal to 3 cm. Graph showing body composition and body weight analysis of 6-week-male and female Wt and Hpn−/− mice using the Bruker minispec LF50 NMR Body Composition Analyzer (N = 5 mice each). Representative H&E stainings of paraffin tissue sections from the mammary gland, lung, skin, kidney, spleen, and liver from female Wt and Hpn−/− mice (6-week-old littermates). Scale bar equals to 100 μm. Liver mass (% of whole-body weight) in 6-week-old virgin female Wt (N = 3) and Hpn−/− (N = 4) mice. Data in (C, E) are represented as mean ± SD, and Student's t-test was used for statistical analyses (n.s. = not significant). Download figure Download PowerPoint Hpn−/− mice are deficient in TGFβ signaling in the mammary gland and show increased ductal branching Hepsin is expressed in human and mouse epithelial tissues, where it typically localizes to desmosomal and hemidesmosomal junctions (Miao et al, 2008). Moreover, in the mammary gland, oncogenic deregulation of hepsin has been coupled to loss of epithelial integrity (Partanen et al, 2012; Tervonen et al, 2016). These findings prompted us to examine whether the loss of hepsin might affect signaling pathways relevant to cohesiveness or morphogenesis of the mammary epithelial tissue. Whole mammary glands were isolated from wild-type and Hpn−/− mice and analyzed in parallel by RNAseq and Reverse Phase Protein Array (RPPA). The RPPA analysis revealed Hpn knockout-specific changes in the levels of 12 proteins, including osteopontin, periostin, and endostatin (Fig EV2A and B). RNAseq analysis demonstrated that while the expression levels of mRNAs encoding 2 of these proteins were also altered, expression changes in other 10 proteins were not accompanied by corresponding changes in mRNA expression, suggesting that hepsin regulates proteomes via both transcription-associated and post-translational/proteolytic mechanisms (Fig EV2A–D). Click here to expand this figure. Figure EV2. Molecular profiling of Hpn−/− mammary glands Proteome profiling of whole mammary tissue lysates prepared from 6-week-old virgin female Wt and Hpn−/− littermates. Small rectangles indicate proteins with differential expression (see (B)). Graph showing quantification of signal intensity from proteome profiling depicted in (A). Proteins were considered differently expressed starting from a 20% change in expression (indicated with red x-axis labels). Quantification of mRNA levels (read counts) corresponding to proteins quantified in the RPPA (A–B), normalized to the housekeeping gene Pum1 mRNA levels. Data were derived from bulk RNA sequencing data from whole mammary tissue lysates prepared from 6-week-old virgin female Wt and Hpn−/− mice (see Fig 1D and E). Data are presented as mean ± SD, and Student's t-test was used for statistical analyses (n.s. = not significant, N = 3 indicates the number of biological replicates). Venn diagram showing differential expression of mRNAs (bulk RNA seq; Fig EV2C and D) and proteins (RPPA; Fig EV2A and B) in mammary tissue of 6-week-old virgin female Wt and Hpn−/− littermates. Resistin and IGFBP-5 are differentially expressed both on mRNA and protein levels. Download figure Download PowerPoint Gene Set Enrichment Analysis (GSEA) was performed on the RNAseq data. Functional clustering of gene signatures affected by the absence of hepsin suggests the downregulation of TGFβ1 signaling in Hpn−/− mammary glands (Fig 1D and E, Table EV1). TGFβ signaling regulation occurs via two discrete activation steps: the processing of latent-TGFβ in the extracellular compartment and TGFβ receptor-induced activation of downstream signaling, which includes phosphorylation of receptor-associated Smads (R-Smads), such as Smad2 and Smad3 (Derynck & Budi, 2019). To confirm the downmodulation of TGFβ signaling in Hpn−/− mice, we determined the levels of phospho-Smad2/3 in tissue extracts from mammary, lung, kidney, liver, skin, and spleen. The level of phosphorylated Smad2/3 was significantly decreased in Hpn−/− mammary tissue (Fig 1F), indicating deficient canonical TGFβ signaling in the mammary gland upon loss of hepsin. Reduced phosphorylated Smad2/3 levels were not observed in the other tissues examined (Fig 1F), suggesting that this phenomenon is specific to the mammary gland. TGFβ is a well-established suppressor of mammary duct branching and proliferation (Ewan et al, 2002; Ingman & Robertson, 2008; Moses & Barcellos-Hoff, 2011). Analysis of mammary gland whole mounts from 6-week-old wild-type and Hpn−/− littermates showed that loss of hepsin increased duct branching into the fat pad (Fig 1G). This phenotype resembles the one observed in TGFβ1+/− mice (Ewan et al, 2002; Ingman & Robertson, 2008), thus showing that loss of hepsin affects mammary morphogenesis in a manner consistent with reduced TGFβ signaling. Hepsin overexpression activates TGFβ signaling in a Wap-Myc model of breast cancer The TGFβ pathway is commonly dysregulated in human cancer but its impact on tumorigenesis is highly contextual—while TGFβ has tumor-suppressive effects, it also exerts pro-tumorigenic effects by modulating processes such as cell invasion, production of ECM, and inflammatory immune responses (Yeung et al, 2013; Bellomo et al, 2016; Mariathasan et al, 2018; Tauriello et al, 2018). To test whether ectopic overexpression of hepsin induces TGFβ signaling in the context of tumorigenesis, we made use of a previously published tumor syngraft model of Myc-driven breast cancer (Partanen et al, 2012; Utz et al, 2020). Wap-Myc mammary tumor cells, which express high Myc levels, were isolated from donor mice, transduced with the pIND21-HPN lentiviral construct that allows doxycycline (DOX)-induced hepsin overexpression (Tervonen et al, 2016), and subsequently transplanted into recipient mice (Fig 2A). Consistent with the notion that hepsin promotes TGFβ signaling, Western blot analysis revealed increased phospho-Smad2/3 and upregulation of the TGFβ signaling downstream target SNAIL in Wap-Myc tumors with DOX-induced hepsin overexpression compared with control (DOX−) tumors (Fig 2B). RNAseq analysis of these tumors provided additional evidence for TGFβ pathway upregulation by hepsin as the most significantly upregulated gene signatures corresponded to the TGFβ signaling pathway (Figs 2C, D, and E, and EV3A and B). The largest cluster of gene signatures affected by hepsin overexpression, however, was related to the ECM and integrins (Fig 2C). Figure 2. Overexpression of Hpn in Wap-Myc-driven mammary tumors induces TGFβ signaling Schematic representation of the mouse experiment. Immunoblot analysis of Wap-Myc mammary tumor lysates for the indicated TGFβ signaling markers and hepsin (T# denotes individual tumors). GAPDH was used as the loading control. Lysates were derived from Wap-Myc mammary tumors from six mice with and six mice without DOX-induced hepsin overexpression (see (A)). Gene Set Enrichment Analysis (GSEA) enrichment map of pathways upregulated in hepsin overexpressing Wap-Myc tumors (DOX+) compared with control tumors (DOX−) (N = 5 tumors in each group). Node size correlates with the number of genes in the signature; node color red correlates with enrichment in hepsin overexpressing Wap-Myc tumors. GSEA graph comparing the expression of the HALLMARK_TGF_BETA_SIGNALING gene set in hepsin overexpressing (DOX+) to control (DOX−) Wap-Myc tumors (N = 5 tumors per group; FDRp—P-value; FWERq—false discovery rate; NES—normalized enrichment score). Heatmap showing changes in expression of all genes in the HALLMARK_TGF_BETA_SIGNALING gene set in hepsin overexpressing (DOX+) compared with control (DOX−) Wap-Myc tumors. Red color indicates upregulation, and blue color indicates the downregulation of the indicated genes. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Regulation of TGFβ signaling and levels, fibronectin protein, and mRNA A, B. GSEA enrichment maps from tumors overexpressing hepsin (DOX+) and control tumors (DOX−). C. Graph depicting quantification of Tgfβ1 mRNA levels (mean ± SD mRNA read counts), normalized to the housekeeping gene Pum1, in mammary tissue of 6-week-old virgin female Wt and Hpn−/− mice. Student's t-test was used for statistical analyses (n.s. = not significant, N = 3 indicates the number of biological replicates). D. Immunoblot analysis of PAI-1, hepsin, and β-tubulin (loading control) in MCF10A-pIND20-HPN cells treated with doxycycline, galunisertib, and latent-TGFβ (small latent complex) as indicated. Numbers below the PAI-1 blot indicate PAI-1 levels, normalized to β-tubulin. E. Immunoblot analysis of TGFβ-V5 (antibody detecting V5), pSmad2/3, PAI-1, and GAPDH (loading control) in cell lysates from MCF10A-pIND20-HPN cells with or without TGFβ-V5 overexpression. F–I. Silver-stained protein gels with samples from in vitro protease activity assays with recombinant hepsin and fragments of fibronectin. The respective fragments (30 kDa (F), 45 kDa (G), 40 kDa (H), and 120 kDa (I); marked by arrowheads) were incubated (1 μg/reaction) with increasing concentrations of recombinant hepsin (nM). J. Schematic mapping of the fibronectin fragments used in (G–J) onto full-length fibronectin. RGD indicates the RGD-binding domain in fibronectin. K. Graph depicting quantification of Fn1 mRNA levels (mean ± SD mRNA read counts), normalized to the housekeeping gene Pum1, in mammary tissue of 6-week-old virgin female Wt and Hpn−/− mice. Student's t-test was used for statistical analyses (n.s. = not significant, N = 3 indicates the number of biological replicates). Download figure Download PowerPoint Hepsin regulates the extracellular activation of TGFβ signaling Pro-TGFβ protein, which consists of both the mature TGFβ and latency-associated peptide (LAP), is released from cells in a latent form. This so-called TGFβ small latent complex (SLC) consists of the receptor-binding TGFβ growth factor dimer (12 kDa Western blot band under reducing conditions) and a dimer of the latency-associated peptide (LAP) (40 kDa Western blot band under reducing conditions) (Miyazono et al, 1988), where LAP inhibits the TGFβ growth factor dimer from binding and activating TGFβ receptors. The latent-TGFβ SLC is stored in the ECM through a covalent bond between LAP and latent-TGFβ-binding protein (LTBP), forming the large latent complex (LLC), which binds to fibronectin and fibrillins (Fig 3A) schematically depicts the TGFβ-LAP-LTBP complex). TGFβ can be released from LAP-LTBP, and thus from ECM storage, and activated by multiple types of stimuli, such as reactive oxygen species, proteases, or an acidic environment (Lyons et al, 1988, 1990; Sato & Rifkin, 1989; Yu & Stamenkovic, 2000; Jenkins, 2008; Sounni e
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