International Journal of Molecular Sciences Article The Role of Methionine Aminopeptidase 2 in Lymphangiogenesis 1, 1, 1, 1 2 Rawnaq Esa y, Eliana Steinberg y, Dvir Dror y, Ouri Schwob , Mehrdad Khajavi , Miriam Maoz 3, Yael Kinarty 4, Adi Inbal 4 , Aviad Zick 3 and Ofra Benny 1,* 1 The Institute for Drug Research, The School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem 91120, Israel; [email protected] (R.E.); [email protected] (E.S.); [email protected] (D.D.); [email protected] (O.S.) 2 Boston Children’s Hospital, Harvard Medical School, Boston, MA 02115, USA; [email protected] 3 Sharett Institute of Oncology, Hebrew University-Hadassah Medical Center, Jerusalem 91120, Israel; [email protected] (M.M.); [email protected] (A.Z.) 4 Department of Medical Neurobiology, Institute for Medical Research—Israel-Canada, The Hebrew University of Jerusalem, Hadassah Medical School, Jerusalem 9112002, Israel; [email protected] (Y.K.); [email protected] (A.I.) * Correspondence: [email protected]; Tel.: +972-2-6757268 These authors contributed equally to this work. y Received: 18 June 2020; Accepted: 16 July 2020; Published: 21 July 2020 Abstract: During the metastasis process, tumor cells invade the blood circulatory system directly from venous capillaries or indirectly via lymphatic vessels. Understanding the relative contribution of each pathway and identifying the molecular targets that affect both processes is critical for reducing cancer spread. Methionine aminopeptidase 2 (MetAp2) is an intracellular enzyme known to modulate angiogenesis. In this study, we investigated the additional role of MetAp2 in lymphangiogenesis. A histological staining of tumors from human breast-cancer donors was performed in order to detect the level and the localization of MetAp2 and lymphatic capillaries. The basal enzymatic level and activity in vascular and lymphatic endothelial cells were compared, followed by loss of function studies determining the role of MetAp2 in lymphangiogenesis in vitro and in vivo. The results from the histological analyses of the tumor tissues revealed a high MetAp2 expression, with detectable sites of co-localization with lymphatic capillaries. We showed slightly reduced levels of the MetAp2 enzyme and MetAp2 mRNA expression and activity in primary lymphatic cells when compared to the vascular endothelial cells. The genetic and biochemical manipulation of MetAp2 confirmed the dual activity of the enzyme in both vascular and lymphatic remodulation in cell function assays and in a zebrafish model. We found that cancer-related lymphangiogenesis is inhibited in murine models following MetAp2 inhibition treatment. Taken together, our study provides an indication that MetAp2 is a significant contributor to lymphangiogenesis and carries a dual role in both vascular and lymphatic capillary formation. Our data suggests that MetAp2 inhibitors can be effectively used as anti-metastatic broad-spectrum drugs. Keywords: MetAp2; angiogenesis; lymphangiogenesis; metastasis; cancer 1. Introduction Cancer metastasis is associated with a high mortality rate and is the primary cause of cancer morbidity. The spread of tumor cells from their primary origin occurs mainly via blood and lymphatic vessels [1]. The 3-dimensional (3D) fast growth of tumors creates a dynamic microenvironment with Int. J. Mol. Sci. 2020, 21, 5148; doi:10.3390/ijms21145148 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 5148 2 of 15 elevated demands for nutrients and oxygen exchange as well as metabolic waste disposal which are accommodated by the expedited angiogenesis process [2,3]. Angiogenesis is regulated by a dynamic balance between inducer and inhibitor molecules. Tumor angiogenesis is a multistep process triggered by diverse factors secreted by the hypoxic tumor as well as by the host cells [4]. This process is characterized by a formation of new blood vessels from pre-existing vessels [2,3]. Similar to angiogenesis, the growth and formation of new lymphatic vessels, known as lymphangiogenesis, occurs in response to multiple triggers [5]. Lymphangiogenesis was shown to play a critical role in tumor progression and metastasis [6]. Unlike hematologic vessels, lymphatic vessels are thin and consist of discontinuous membranes that enable the penetration of tumor cells into the lymphatic circulation and the adjacent capillary lumens. In response to secreted signals, both lymphatic and vascular endothelial cells proliferate and migrate toward the stimulus, assembling into tube-like structures [1]. Several pathways were identified as promoting angiogenesis and lymphangiogenesis, the most studied and dominant one being the vascular endothelial growth factor (VEGF) pathway. VEGF, which is overexpressed in most cancers and functions as a crucial regulator in angiogenesis and lymphangiogenesis [3,7–9], has become an important target for inhibiting tumor metastasis. Among the various VEGF family members, VEGF-C and VEGF-D are secreted by the tumor and stromal cells, playing a dominant role in mediating tumor-related lymphangiogenesis [6,10–12]. However, there are many additional mediators that are involved in capillary formation and remodeling. Among these mediators is MetAp2, which belongs to a family of post-translational modification enzymes, acting downstream to VEGF. There are two forms of MetAp in eukaryotes: MetAp1 and MetAp2, the latter having received more interest. This is because the induction of Metap2 was shown to mediate cell proliferation and lead to G1 arrest when inhibited [13–17], since it affects protein synthesis via the post-translation removal of methionine [18–21]. Given MetAp2’s major role in protein synthesis in activated vascular endothelial cells, we hypothesize that MetAp2 also affects lymphangiogenesis by mediating the lymphatic endothelium formation via shared mechanisms. In patient-derived breast cancer tissues, we found that MetAp2 is expressed and co-localized with lymphatic vessels. At the cellular level, we found that the basal expressions of MetAp2 and its activities in human lymphatic endothelial cells (LECs) versus vascular endothelial cells (VECs) were comparable, with slightly higher levels in VECs. In functionality assays including cell proliferation, adhesion, and tube formation, LECs showed suppression upon exposure to TNP-470, a MetAp2 inhibitor. In vivo, in a non-cancerous context using the zebrafish developmental model, the specific inhibition of MetAp2 led to a significant reduction in the development of lymphatic vessels. Parachordal cells (PACs), which make up the lymphatic vessels, were observed in only 0.76 out of 3 segments after treatment with 500 µM of the MetAp2 inhibitor. Moreover, in a subcutaneous (S.C.) murine model using B16/F10 melanoma tumors overexpressing VEGF-C, systemic treatment with 30 mg/kg q.o.d induced over a 50% reduction in the tumors’ volume. A histological examination of the tumors suggested an associated dual reduction of angiogenesis and lymphangiogenesis induced by MetAp2 inhibition. Taken together, our data suggest that MetAp2 is a mediator of both angiogenesis and lymphangiogenesis and that its inhibition may slow cancer progression and metastasis. 2. Results 2.1. MetAp2 Expression and Activity in LECs and Tissues Human tumor tissues were surgically resected and collected from breast cancer patients for the histological examination and detection of lymphatic endothelium (indicated by LYVE1 expression) as well as MetAp2 expression. The immunofluorescent staining of the tissue sections showed both MetAp2 and LYVE1 expression with a few loci sites of co-localization (Figure1A,B). Int. J. Mol. Sci. 2020, 21, 5148 3 of 15 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 15 of 15 Figure 1. Cancer tissue samples retrieved from two breast cancer patients express MetAp2 and LYVE1. (AFigure,B) Immunofluorescence 1. Cancer tissue samples staining ofretrieved MetAp2 from (red), two LYVE1 breast (green), cancer and patients DAPI (blue) express reveal MetAp2 lymphatic and structuresLYVE1. (A co-localized,B) Immunofluorescence with MetAp2 staining expression. of MetAp2 Black and (red), white LYVE1 images (green), represent and DAPI fluorescence (blue) reveal in a singlelymphatic channel. structures Left panel co-localized scale bar = with100 µ m.MetAp2 Right panelexpression. scale bar Black= 20–50 andµ whitem. images represent fluorescence in a single channel. Left panel scale bar = 100 µm. Right panel scale bar = 20-50 µm. Immunoblotting and quantitative real-time PCR (qRT-PCR) were used to measure protein expressionImmunoblotting at the cell-level. andThe quantitative basal cellular real-time protein PC levelsR (qRT-PCR) (baseline expressionwere used in to the measure activated protein state ofexpression cells grown at inthe optimal cell-level. growth The conditions)basal cellular of MetAp2protein levels were comparable(baseline expression in both the in vascular the activated and lymphaticstate of cells endothelial grown in cellsoptimal (VECs growth and LECs,conditions) respectively), of MetAp2 with were slightly comparable higher in levels both ofthe MetAp2 vascular inand the lymphatic vascular endothelium endothelial (Figurecells (VECs2A). Inand addition, LECs, qRT-PCRrespectively), was usedwith toslightly quantify higher the MetAp2levels of mRNAMetAp2 levels in the in human vascular umbilical endothelium vein endothelial (Figure 2A). cells In (HUVECs) addition, andqRT-PCR in human was dermal