Angpt2/Tie2 autostimulatory loop controls tumorigenesis
2022; Springer Nature; Volume: 14; Issue: 5 Linguagem: Inglês
10.15252/emmm.202114364
ISSN1757-4684
AutoresNinelia Minaskan Karabid, Tobias Wiedemann, Sebastian Gulde, Hermine Mohr, Renu Chandra Segaran, Julia Geppert, Maria Rohm, Giovanni Vitale, Germano Gaudenzi, Alessandra Dicitore, Donna P. Ankerst, Yiyao Chen, Rickmer Braren, Georg Kaissis, Franz Schilling, Mathias Schillmaier, Graeme Eisenhofer, Stephan Herzig, Federico Roncaroli, Jürgen Honegger, Natalia S. Pellegata,
Tópico(s)Protein Tyrosine Phosphatases
ResumoArticle10 March 2022Open Access Source DataTransparent process Angpt2/Tie2 autostimulatory loop controls tumorigenesis Ninelia Minaskan Karabid Ninelia Minaskan Karabid Institute for Diabetes and Cancer, Helmholtz Zentrum München, Neuherberg, Germany Joint Heidelberg-IDC Translational Diabetes Program, Heidelberg University Hospital, Heidelberg, Germany Contribution: Conceptualization, Investigation, Writing - original draft Search for more papers by this author Tobias Wiedemann Tobias Wiedemann orcid.org/0000-0002-8101-7407 Institute for Diabetes and Cancer, Helmholtz Zentrum München, Neuherberg, Germany Joint Heidelberg-IDC Translational Diabetes Program, Heidelberg University Hospital, Heidelberg, Germany Contribution: Conceptualization, Formal analysis, Investigation Search for more papers by this author Sebastian Gulde Sebastian Gulde orcid.org/0000-0002-8672-010X Institute for Diabetes and Cancer, Helmholtz Zentrum München, Neuherberg, Germany Joint Heidelberg-IDC Translational Diabetes Program, Heidelberg University Hospital, Heidelberg, Germany Contribution: Formal analysis, Investigation, Writing - review & editing Search for more papers by this author Hermine Mohr Hermine Mohr orcid.org/0000-0002-3061-1166 Institute for Diabetes and Cancer, Helmholtz Zentrum München, Neuherberg, Germany Joint Heidelberg-IDC Translational Diabetes Program, Heidelberg University Hospital, Heidelberg, Germany Contribution: Writing - review & editing Search for more papers by this author Renu Chandra Segaran Renu Chandra Segaran Institute for Diabetes and Cancer, Helmholtz Zentrum München, Neuherberg, Germany Joint Heidelberg-IDC Translational Diabetes Program, Heidelberg University Hospital, Heidelberg, Germany Contribution: Investigation Search for more papers by this author Julia Geppert Julia Geppert Institute for Diabetes and Cancer, Helmholtz Zentrum München, Neuherberg, Germany Joint Heidelberg-IDC Translational Diabetes Program, Heidelberg University Hospital, Heidelberg, Germany Contribution: Investigation Search for more papers by this author Maria Rohm Maria Rohm orcid.org/0000-0003-3926-1534 Institute for Diabetes and Cancer, Helmholtz Zentrum München, Neuherberg, Germany Joint Heidelberg-IDC Translational Diabetes Program, Heidelberg University Hospital, Heidelberg, Germany Contribution: Data curation Search for more papers by this author Giovanni Vitale Giovanni Vitale Istituto Auxologico Italiano IRCCS, Laboratory of Geriatric and Oncologic Neuroendocrinology Research, Cusano Milanino, Milan, Italy Department of Medical Biotechnology and Translational Medicine, University of Milan, Milan, Italy Contribution: Formal analysis, Investigation Search for more papers by this author Germano Gaudenzi Germano Gaudenzi Istituto Auxologico Italiano IRCCS, Laboratory of Geriatric and Oncologic Neuroendocrinology Research, Cusano Milanino, Milan, Italy Contribution: Formal analysis, Investigation Search for more papers by this author Alessandra Dicitore Alessandra Dicitore Department of Medical Biotechnology and Translational Medicine, University of Milan, Milan, Italy Contribution: Investigation Search for more papers by this author Donna Pauler Ankerst Donna Pauler Ankerst Department of Mathematics, Technical University Munich, Garching, Germany Contribution: Data curation, Formal analysis, Writing - original draft, Writing - review & editing Search for more papers by this author Yiyao Chen Yiyao Chen Department of Mathematics, Technical University Munich, Garching, Germany Contribution: Formal analysis Search for more papers by this author Rickmer Braren Rickmer Braren Institute for Diagnostic and Interventional Radiology, Klinikum Rechts der Isar, Technical University Munich, Munich, Germany Contribution: Formal analysis, Investigation Search for more papers by this author Georg Kaissis Georg Kaissis orcid.org/0000-0001-8382-8062 Institute for Diagnostic and Interventional Radiology, Klinikum Rechts der Isar, Technical University Munich, Munich, Germany Contribution: Investigation Search for more papers by this author Franz Schilling Franz Schilling Department of Nuclear Medicine, Klinikum rechts der Isar, Technical University Munich, Munich, Germany Contribution: Formal analysis, Investigation Search for more papers by this author Mathias Schillmaier Mathias Schillmaier Department of Nuclear Medicine, Klinikum rechts der Isar, Technical University Munich, Munich, Germany Contribution: Investigation Search for more papers by this author Graeme Eisenhofer Graeme Eisenhofer orcid.org/0000-0002-8601-9903 Institute of Clinical Chemistry and Laboratory Medicine, University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany Contribution: Resources, Data curation Search for more papers by this author Stephan Herzig Stephan Herzig orcid.org/0000-0003-3950-3652 Institute for Diabetes and Cancer, Helmholtz Zentrum München, Neuherberg, Germany Joint Heidelberg-IDC Translational Diabetes Program, Heidelberg University Hospital, Heidelberg, Germany Contribution: Conceptualization, Data curation Search for more papers by this author Federico Roncaroli Federico Roncaroli Division of Neuroscience and Experimental Psychology, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK Contribution: Resources, Data curation, Writing - original draft Search for more papers by this author Jürgen B Honegger Jürgen B Honegger Department of Neurosurgery, Eberhard Karls University Tübingen, Tübingen, Germany Contribution: Resources, Investigation Search for more papers by this author Natalia S Pellegata Corresponding Author Natalia S Pellegata [email protected] orcid.org/0000-0002-8000-7784 Institute for Diabetes and Cancer, Helmholtz Zentrum München, Neuherberg, Germany Joint Heidelberg-IDC Translational Diabetes Program, Heidelberg University Hospital, Heidelberg, Germany Contribution: Conceptualization, Data curation, Writing - original draft, Writing - review & editing Search for more papers by this author Ninelia Minaskan Karabid Ninelia Minaskan Karabid Institute for Diabetes and Cancer, Helmholtz Zentrum München, Neuherberg, Germany Joint Heidelberg-IDC Translational Diabetes Program, Heidelberg University Hospital, Heidelberg, Germany Contribution: Conceptualization, Investigation, Writing - original draft Search for more papers by this author Tobias Wiedemann Tobias Wiedemann orcid.org/0000-0002-8101-7407 Institute for Diabetes and Cancer, Helmholtz Zentrum München, Neuherberg, Germany Joint Heidelberg-IDC Translational Diabetes Program, Heidelberg University Hospital, Heidelberg, Germany Contribution: Conceptualization, Formal analysis, Investigation Search for more papers by this author Sebastian Gulde Sebastian Gulde orcid.org/0000-0002-8672-010X Institute for Diabetes and Cancer, Helmholtz Zentrum München, Neuherberg, Germany Joint Heidelberg-IDC Translational Diabetes Program, Heidelberg University Hospital, Heidelberg, Germany Contribution: Formal analysis, Investigation, Writing - review & editing Search for more papers by this author Hermine Mohr Hermine Mohr orcid.org/0000-0002-3061-1166 Institute for Diabetes and Cancer, Helmholtz Zentrum München, Neuherberg, Germany Joint Heidelberg-IDC Translational Diabetes Program, Heidelberg University Hospital, Heidelberg, Germany Contribution: Writing - review & editing Search for more papers by this author Renu Chandra Segaran Renu Chandra Segaran Institute for Diabetes and Cancer, Helmholtz Zentrum München, Neuherberg, Germany Joint Heidelberg-IDC Translational Diabetes Program, Heidelberg University Hospital, Heidelberg, Germany Contribution: Investigation Search for more papers by this author Julia Geppert Julia Geppert Institute for Diabetes and Cancer, Helmholtz Zentrum München, Neuherberg, Germany Joint Heidelberg-IDC Translational Diabetes Program, Heidelberg University Hospital, Heidelberg, Germany Contribution: Investigation Search for more papers by this author Maria Rohm Maria Rohm orcid.org/0000-0003-3926-1534 Institute for Diabetes and Cancer, Helmholtz Zentrum München, Neuherberg, Germany Joint Heidelberg-IDC Translational Diabetes Program, Heidelberg University Hospital, Heidelberg, Germany Contribution: Data curation Search for more papers by this author Giovanni Vitale Giovanni Vitale Istituto Auxologico Italiano IRCCS, Laboratory of Geriatric and Oncologic Neuroendocrinology Research, Cusano Milanino, Milan, Italy Department of Medical Biotechnology and Translational Medicine, University of Milan, Milan, Italy Contribution: Formal analysis, Investigation Search for more papers by this author Germano Gaudenzi Germano Gaudenzi Istituto Auxologico Italiano IRCCS, Laboratory of Geriatric and Oncologic Neuroendocrinology Research, Cusano Milanino, Milan, Italy Contribution: Formal analysis, Investigation Search for more papers by this author Alessandra Dicitore Alessandra Dicitore Department of Medical Biotechnology and Translational Medicine, University of Milan, Milan, Italy Contribution: Investigation Search for more papers by this author Donna Pauler Ankerst Donna Pauler Ankerst Department of Mathematics, Technical University Munich, Garching, Germany Contribution: Data curation, Formal analysis, Writing - original draft, Writing - review & editing Search for more papers by this author Yiyao Chen Yiyao Chen Department of Mathematics, Technical University Munich, Garching, Germany Contribution: Formal analysis Search for more papers by this author Rickmer Braren Rickmer Braren Institute for Diagnostic and Interventional Radiology, Klinikum Rechts der Isar, Technical University Munich, Munich, Germany Contribution: Formal analysis, Investigation Search for more papers by this author Georg Kaissis Georg Kaissis orcid.org/0000-0001-8382-8062 Institute for Diagnostic and Interventional Radiology, Klinikum Rechts der Isar, Technical University Munich, Munich, Germany Contribution: Investigation Search for more papers by this author Franz Schilling Franz Schilling Department of Nuclear Medicine, Klinikum rechts der Isar, Technical University Munich, Munich, Germany Contribution: Formal analysis, Investigation Search for more papers by this author Mathias Schillmaier Mathias Schillmaier Department of Nuclear Medicine, Klinikum rechts der Isar, Technical University Munich, Munich, Germany Contribution: Investigation Search for more papers by this author Graeme Eisenhofer Graeme Eisenhofer orcid.org/0000-0002-8601-9903 Institute of Clinical Chemistry and Laboratory Medicine, University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany Contribution: Resources, Data curation Search for more papers by this author Stephan Herzig Stephan Herzig orcid.org/0000-0003-3950-3652 Institute for Diabetes and Cancer, Helmholtz Zentrum München, Neuherberg, Germany Joint Heidelberg-IDC Translational Diabetes Program, Heidelberg University Hospital, Heidelberg, Germany Contribution: Conceptualization, Data curation Search for more papers by this author Federico Roncaroli Federico Roncaroli Division of Neuroscience and Experimental Psychology, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK Contribution: Resources, Data curation, Writing - original draft Search for more papers by this author Jürgen B Honegger Jürgen B Honegger Department of Neurosurgery, Eberhard Karls University Tübingen, Tübingen, Germany Contribution: Resources, Investigation Search for more papers by this author Natalia S Pellegata Corresponding Author Natalia S Pellegata [email protected] orcid.org/0000-0002-8000-7784 Institute for Diabetes and Cancer, Helmholtz Zentrum München, Neuherberg, Germany Joint Heidelberg-IDC Translational Diabetes Program, Heidelberg University Hospital, Heidelberg, Germany Contribution: Conceptualization, Data curation, Writing - original draft, Writing - review & editing Search for more papers by this author Author Information Ninelia Minaskan Karabid1,2,†, Tobias Wiedemann1,2,†, Sebastian Gulde1,2, Hermine Mohr1,2, Renu Chandra Segaran1,2, Julia Geppert1,2, Maria Rohm1,2, Giovanni Vitale3,4, Germano Gaudenzi3, Alessandra Dicitore4, Donna Pauler Ankerst5, Yiyao Chen5, Rickmer Braren6, Georg Kaissis6, Franz Schilling7, Mathias Schillmaier7, Graeme Eisenhofer8, Stephan Herzig1,2, Federico Roncaroli9, Jürgen B Honegger10 and Natalia S Pellegata *,1,2 1Institute for Diabetes and Cancer, Helmholtz Zentrum München, Neuherberg, Germany 2Joint Heidelberg-IDC Translational Diabetes Program, Heidelberg University Hospital, Heidelberg, Germany 3Istituto Auxologico Italiano IRCCS, Laboratory of Geriatric and Oncologic Neuroendocrinology Research, Cusano Milanino, Milan, Italy 4Department of Medical Biotechnology and Translational Medicine, University of Milan, Milan, Italy 5Department of Mathematics, Technical University Munich, Garching, Germany 6Institute for Diagnostic and Interventional Radiology, Klinikum Rechts der Isar, Technical University Munich, Munich, Germany 7Department of Nuclear Medicine, Klinikum rechts der Isar, Technical University Munich, Munich, Germany 8Institute of Clinical Chemistry and Laboratory Medicine, University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany 9Division of Neuroscience and Experimental Psychology, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK 10Department of Neurosurgery, Eberhard Karls University Tübingen, Tübingen, Germany † These authors contributed equally to this work *Corresponding author. Tel: +49 089 31872633; Fax: +49 089 31873360; E-mail: [email protected] EMBO Mol Med (2022)e14364https://doi.org/10.15252/emmm.202114364 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 Invasive nonfunctioning (NF) pituitary neuroendocrine tumors (PitNETs) are non-resectable neoplasms associated with frequent relapses and significant comorbidities. As the current therapies of NF-PitNETs often fail, new therapeutic targets are needed. The observation that circulating angiopoietin-2 (ANGPT2) is elevated in patients with NF-PitNET and correlates with tumor aggressiveness prompted us to investigate the ANGPT2/TIE2 axis in NF-PitNETs in the GH3 PitNET cell line, primary human NF-PitNET cells, xenografts in zebrafish and mice, and in MENX rats, the only autochthonous NF-PitNET model. We show that PitNET cells express a functional TIE2 receptor and secrete bioactive ANGPT2, which promotes, besides angiogenesis, tumor cell growth in an autocrine and paracrine fashion. ANGPT2 stimulation of TIE2 in tumor cells activates downstream cell proliferation signals, as previously demonstrated in endothelial cells (ECs). Tie2 gene deletion blunts PitNETs growth in xenograft models, and pharmacological inhibition of Angpt2/Tie2 signaling antagonizes PitNETs in primary cell cultures, tumor xenografts in mice, and in MENX rats. Thus, the ANGPT2/TIE2 axis provides an exploitable therapeutic target in NF-PitNETs and possibly in other tumors expressing ANGPT2/TIE2. The ability of tumor cells to coopt angiogenic signals classically viewed as EC-specific expands our view on the microenvironmental cues that are essential for tumor progression. Synopsis There is currently no treatment for nonfunctioning pituitary neuroendocrine tumors (NF-PitNETs), which are intracranial tumors associated with frequent relapse and severe comorbidities. This study establishes the ANGPT2/Tie2 signaling axis as a novel therapeutic target for NF-PitNETs. Circulating ANGPT2 levels were elevated in NF-PitNET patients and correlated with tumor proliferation rate, an indicator of tumor aggressiveness. Bioactive ANGPT2 was expressed and secreted by NF-PitNET cells, and promoted angiogenesis and tumor cell growth in an autocrine and paracrine fashion. Functional TIE2 receptor was expressed by NF-PitNET cells, and was directly activated by angiopoietins. PitNET cell growth was suppressed upon TIE2 knockout in mouse xenografts in vivo. ANGPT2/TIE2 pharmacological inhibition reduced the growth of NF-PitNET primary tumor cells in vitro, as well as tumor xenografts and autochthonous NF-PitNETs in MENX rats. The paper explained Problem Nonfunctioning pituitary neuroendocrine tumors (NF-PitNETs) are the second most common type of PitNETs and affect patients' functional status and mortality. Given the lack of symptoms linked to hormone hypersecretion, NF-PitNETs are often diagnosed upon tumor mass detection. At this stage, 50% of the tumors have invaded surrounding structures and cannot be completely removed by surgery. Relapse is thus frequent, causing significant comorbidities. These tumors do not respond to standard-of-care treatments, which makes the identification of novel therapeutic targets mandatory to improve patients' management. Results Circulating levels of the angiopoietin 2 (ANGPT2) cytokine are elevated in NF-PitNET patients and correlate with tumor aggressiveness. NF-PitNET cells express and secrete ANGPT2, which stimulates the proliferation/survival of tumor cells in vitro, and angiogenesis in PitNET cell xenografts in zebrafish embryos in vivo. Noteworthy, NF-PitNET cells possess a functional TIE2 receptor, which is activated by ANGPT2 and further stimulates downstream mitogenic signals. This establishes an autocrine/paracrine stimulatory loop in NF-PitNET cells, as demonstrated in endothelial cells. Deletion of TIE2 in PitNET cells suppresses their growth in mouse xenografts in vivo. Proof-of-principle pharmacological inhibition of ANGPT2/TIE2 signaling antagonizes NF-PitNETs in primary tumor cultures, as well as in mouse xenografts and in MENX rats, the only model of spontaneous NF-PitNETs. Impact Our study identifies an active ANGPT2/TIE2 signaling cascade in NF-PitNET cells. The role of this axis in sustaining tumor cell growth and mediating a crosstalk between tumor and endothelial cells in the tumor microenvironment makes it an attractive therapeutic target for the treatment of NF-PitNETs. Introduction Pituitary adenomas are the third most common intracranial neoplasm and considerably impact morbidity and mortality of affected patients (Ntali et al, 2016; Lobatto et al, 2018). Now renamed pituitary neuroendocrine tumors (PitNETs), these tumors, though usually benign, often invade surrounding structures and cannot be cured using standard therapies (Asa et al, 2017). Invasive pituitary tumors, including nonfunctioning (NF) adenomas derived from gonadotroph cells (NFPAs or NF-PitNETs), cannot be completely resected thereby leading to frequent relapse and significant comorbidities (Tampourlou et al, 2018). Dopamine agonists, somatostatin analogues, and temozolomide have all yielded disappointing therapeutic results, and radiotherapy is associated with significant side effects (Greenman & Melmed, 1996). Thus, novel treatment strategies are needed to treat invasive/recurrent NF-PitNETs. Tumorigenesis is a highly dynamic process driven by the interplay of cellular and non-cellular elements of the tumor microenvironment (TME). Among the formers are endothelial cells (ECs), the main elements that drive angiogenesis, a hallmark of tumor progression needed critical for supply of oxygen and nutrients and removal of metabolic waste. Angiogenesis is regulated by various pro- and anti-angiogenic factors, including angiopoietins (ANGPT), a family of cytokines that bind the receptor tyrosine kinase TIE2 on ECs and drive vasculogenesis, vessel homeostasis, and vascular remodeling (Eklund & Olsen, 2006). The best-known members are Angpt1 and Angpt2. Angpt1 is a Tie2 agonist that triggers receptor autophosphorylation, internalization, and activation of downstream signaling leading to vessel stabilization (Eklund & Saharinen, 2013). In contrast, Angpt2 usually works as Angpt1/Tie2 antagonist and causes endothelium destabilization (Mandriota & Pepper, 1998; Fiedler et al, 2004). However, Angpt2 at high concentration and/or in the absence of Angpt1 can function as a Tie2 agonist (Kim et al, 2000b; Daly et al, 2013). Active Tie2 is phosphorylated at various tyrosine (Tyr) residues. Phosphorylation at Tyr1102 stimulates PI3K and Grb7, which in turn activate focal adhesion kinase p125 (Fak) (Han & Guan, 1999; Kim et al, 2000a; Brindle et al, 2006). The adaptor Grb2 is also recruited by phosphorylated Tyr1102 and stimulates Erk1/2 (Fukuhara et al, 2008). In tumor tissues, ECs secrete high levels of Angpt2, which works as the main Tie2 ligand and together with VEGFA promotes angiogenesis via both autocrine and paracrine mechanisms (Eklund & Saharinen, 2013). Accordingly, elevated circulating levels of Angpt2 have been reported in patients with various solid tumors, including gastro-enteropancreatic (GEP) NETs (Detjen et al, 2010), ovarian cancer (Sallinen et al, 2010), and malignant melanoma (Helfrich et al, 2009) where they correlate with more advanced disease stage and/or poor prognosis. Interestingly, the biological function and therapeutic potential of angiopoietins in preclinical models of PitNETs or human PitNETs are currently unknown. Results Circulating ANGPT2 levels in NF-PitNET patients correlate with tumor aggressiveness High circulating levels of ANGPT2 have been observed in various human cancers, where they associate with aggressive tumor behaviors and poor prognosis (Helfrich et al, 2009; Detjen et al, 2010; Sallinen et al, 2010). To determine whether a similar situation occurs also in patients with NF-PitNETs, we used a validated ELISA kit to measure ANGPT2 in the pre-operative plasma of 69 patients with NF-PitNETs versus age- and gender-matched healthy controls (n = 69). The clinical parameters of the patients are reported in Appendix Table S1. The mean plasma ANGPT2 concentration was 0.87 ng/ml in healthy individuals, and 1.79 ng/ml in patients (P < 0.0001) (Fig 1A; Appendix Fig S1A). Circulating ANGPT2 in patients correlated with the Ki67 labeling index (LI) of the tumors (P = 0.014; Fig 1B), which represents the most reliable marker of biological behavior in PitNETs (Osamura, 2017), but not with other clinical parameters (Appendix Fig S1B–D). Ki67 immunohistochemistry (IHC) of primary tumors representative of the different labeling index (LI) groups is shown in Appendix Fig S1E. Figure 1. ANGPT2 is elevated in NF-PitNET patients' plasma, and is expressed in human primary NF-PitNETs together with Tie2 and ANGPT1 ANGPT2 plasma concentration in 69 NF-NF-PitNET patients and in 69 age- and gender-matched healthy controls. Each dot represents one individual. Healthy controls: mean ± SEM = 0.8698 ± 0.05757; PitNET patients; mean ± SEM = 1.786 ± 0.1383; Difference between means = 0.9158 ± 0.1498; 95% confidence interval = 0.6181 to 1.213. ***P-value < 0.0001 by unpaired t-test with Welch's correction. Correlation between circulating ANGP T2 levels and Ki67 LI (i.e., % of Ki67-positive cells). All box plots show 25th to 75th percentiles (box) and 5th to 95th percentiles (whiskers). The line in the box represents the median. Results are expressed as mean ± SEM. **P = 0.014 by one-way ANOVA (P = 0.007 by Kruskal–Wallis test). Expression of ANGPT1 and ANGPT2 in human unaffected anterior pituitary (control, n = 3) and in human NF-PitNETs. Thirty-seven human NF-PitNETs were analyzed for ANGPT2 expression and 14 for ANGPT1. NF-PitNET panels show representative cases. Original magnification: 400×; scale bar: 20 µm. Summary of the IHC results for human NF-PitNETs. Expression of Tie2 receptor in human normal pituitary (n = 3) and human NF-PitNET tissues (n = 10). Original magnification: 400×; scale bar: 20 µm. Download figure Download PowerPoint Following the aforementioned novel finding, we investigated the expression of ANGPT2 in NF-PitNET tissues by quantitative (q)RT–PCR. ANGPT2 expression was elevated (> 2-fold) in 5/14 samples, including 4/5 invasive tumors (Appendix Fig S2A; Appendix Table S2). For sake of completeness, ANGPT1 levels were also assessed and found to be significantly reduced in NF-PitNETs (versus normal tissue) in 13/14 samples (Appendix Fig S2A). Increased ANGPT2/ANGPT1 ratio at the mRNA level has been reported to correlate with neo-angiogenesis and poor prognosis in many cancer types (Tait & Jones, 2004). In NF-PitNETs, the ANGPT2 to ANGPT1 ratio was shifted toward ANGPT2 (i.e., > 1) in 13 out of 14 samples (Appendix Fig S2B). To verify whether changes in gene expression translate into altered protein levels and to determine the cell population expressing these molecules, we performed IHC on human NF-PitNETs for ANGPT2 (n = 37) and ANGPT1 (n = 15; Fig 1C and D). In parallel, three unaffected pituitary glands were stained as controls (Appendix Fig S2C). As expected, both angiopoietins were expressed in some endothelial cells (ECs) of both unaffected pituitaries and PitNETs (Fig 1C; Appendix Fig S2C). ANGPT2 was also expressed in the cytoplasm of sparse adenohypophyseal cells in unaffected pituitaries and, more importantly, in tumor cells, where moderate to strong cytoplasmic immunoreactivity was seen in 27/39 cases (Fig 1C and D; Appendix Fig S2C and D). In contrast, cytoplasmic ANGPT1 expression in tumors was weak (n = 8) or moderate (n = 5), whereas higher levels were observed in adenohypophyseal cells in unaffected pituitaries (Fig 1C and D; Appendix Fig S2C). Rarely, co-expression of ANGPT1 and ANGPT2 was observed in few tumor cells (Appendix Fig S2E). In unaffected human pituitary glands, some cells showed positivity for ANGPT2 (Fig 1C). To determine whether they were gonadotroph cells, from which NF-PitNETs derive, we conducted co-immunofluorescence (IF) for ANGPT2 and the alpha subunit of gonadotropin hormones (αGSU), a marker of gonadotroph cells, on control pituitary tissue from unaffected individuals (Appendix Fig S2F). The two proteins did not co-localize. Instead, ANGPT2 was found to co-localize with the growth hormone (GH), marker of somatotroph cells, another pituitary cell lineage (Appendix Fig S2F). These results indicate that the expression of ANGPT2 is acquired de novo by gonadotroph cells during tumorigenesis. In ECs, angiopoietins exert their function by binding to the TIE2 receptor (Fiedler et al, 2004; Eklund & Olsen, 2006; Eklund & Saharinen, 2013). We assessed receptor expression in unaffected human pituitaries and in PitNET tissues. As expected, a positive signal was seen for ECs in control non-diseased pituitaries and in some tumor-associated ECs (Fig 1E; Appendix Fig S2C and D). Surprisingly, TIE2 positivity was also observed in the cytoplasm and the plasma membrane of human NF-PitNET cells (Fig 1E; Appendix Fig S2D), a pattern compatible with published data on HeLa cells with ectopic TIE2 expression (Leppänen et al, 2017). Pituitary cells in the control tissues showed no Tie2 staining. The specificity of all antibodies used for IHC and IF was validated on known positive control tissues (Appendix Fig S3A) or on control cells (i.e., rat aortic ECs, RAOECs; Appendix Fig S3B and C). In the laboratory, we work with MENX rats, developing spontaneous, autochthonous NF-PitNETs. Rat pituitary tumors (frequency 100%) closely resemble human NF-PitNETs at histo-pathological and molecular levels (Lee et al, 2013; Marinoni et al, 2013). We checked whether the rat tumors express angiopoietins and Tie2 receptor similarly to human tumors. Mining previous transcriptome data of rat NF-PitNETs, we found that Angpt2 was upregulated (+2.4-fold), whereas Angpt1 was downregulated (−3.4-fold) compared to normal rat pituitary tissues (Lee et al, 2013). qRT–PCR confirmed the high levels of Angpt2 and low levels of Angpt1 in rat NF-PitNETs (Fig EV1). We then stained rat pituitaries from wild-type (WT) rats and MENX tumor-bearing rats and confirmed that Angpt1 is downregulated whereas Angpt2 is upregulated in the tumors (Fig EV1). Moreover, we found that not only ECs (in both animal groups) but also NF-PitNET cells express the Tie2 receptor (Fig EV1). IF of consecutive tissue sections of rat PitNET tissues confirmed the expression of both Angpt2 and Tie2 in tumor cells, as well as in CD31-positive ECs used as the positive control (Fig EV1). Co-staining of rat tumor tissues for known markers of NF-PitNETs (Marinoni et al, 2013) (i.e., αGSU and the transcription factor SF-1) and for Tie2 showed that the receptor is co-expressed with both proteins in the tumor cells (Appendix Fig S4A–F). In contrast, co-expression of αGSU or SF1 together with Tie2 was not observed in WT rat pituitaries, nor in adjacent non-tumor areas (Appendix Fig S4C and F), thereby suggesting that Tie2 expression is acquired during tumor progression by the gonadotroph cells. This parallels what we observed in human non-diseased pituitary samples: ANGPT2 is not present in normal gonadotroph cells (Appendix Fig S2F), but its expression is acquired during tumorigenesis. Thus, altogether, rat NF-PitNETs express Angpt2 at high levels and further express Tie2. Click here to expand this figure. Figure EV1. Angpt2 is expressed in rat primary NF-PitNETs together with Tie2 and Angpt1 qRT–PCR for Angpt1 and Angpt2 was performed on pituitary samples from tumor-bearing MENX mutant rats (n = 10, 12, respectively) and wild-type control rats (n = 5, 8). Data are expressed as mean ± SEM. *P = 0.049; **P = 0.008 by t-test. IHC was performed on pituitary tissues from wild-type (n = 5) and MENX mutant rats (n = 10) using antibodies against Angpt1, Angpt2. Representative stainings are shown. Original magnification: 400×; sc
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