Agrin Mediates Angiogenesis in the Tumor Microenvironment

Abstract

Angiogenesis represents a hallmark of cancer. Several proteoglycans associate with cell surface receptors and regulate angiogenesis within the tumor microenvironment (TME). We highlight the recent discovery that the proteoglycan Agrin cross talks between the tumor and the endothelium to promote an angiogenesis privileged niche during cancer progression. Angiogenesis represents a hallmark of cancer. Several proteoglycans associate with cell surface receptors and regulate angiogenesis within the tumor microenvironment (TME). We highlight the recent discovery that the proteoglycan Agrin cross talks between the tumor and the endothelium to promote an angiogenesis privileged niche during cancer progression. The TME consists of cancer cells and noncancerous cells such as cancer-associated fibroblasts, macrophages, endothelial cells, and lymphocytes. These cells are surrounded by extracellular matrix (ECM) and require dedicated blood capillaries for their metabolism and survival [1.Carmeliet P. Jain R.K. Angiogenesis in cancer and other diseases.Nature. 2000; 407: 249-257Crossref PubMed Scopus (7443) Google Scholar]. During cancer progression, tumors initiate new capillary formation by angiogenesis. Localized benign tumors are surrounded by a well-developed basal lamina that limits the tumor mass from accessing the blood vessels from the surrounding tissue. However, during malignancy, tumors trigger an ‘angiogenic switch’ and tilt the balance between pro- and antiangiogenic factors towards vascularization [2.Hanahan D. Folkman J. Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis.Cell. 1996; 86: 353-364Abstract Full Text Full Text PDF PubMed Scopus (6068) Google Scholar]. A plethora of secreted and transmembrane proteins including the vascular endothelial growth factor (VEGF)–VEGF receptor 2 (VEGFR2), delta-notch, Tie-angiopoietin pathways regulate angiogenesis that comprises a corrupt TME. Therefore, identification of new proteins that favor a proangiogenic phenotype during tumorigenesis provides a more insightful understanding of the mechanism of tumor angiogenesis, and offers new opportunities to enhance clinical outcomes of antiangiogenic therapies. Proteoglycans are present in the ECM, cell surface, and pericellular and intracellular regions. Different protein modules within proteoglycans and their localization regulate diverse cellular functions including angiogenesis and cancer progression [3.Iozzo R.V. Schaefer L. Proteoglycan form and function: a comprehensive nomenclature of proteoglycans.Matrix Biol. 2015; 42: 11-55Crossref PubMed Scopus (677) Google Scholar]. Although several proteoglycans regulate angiogenesis, the molecular mechanisms as to how they recruit blood vessels within the tumor niche remain poorly understood. Agrin, a heparan sulfate proteoglycan, is a key component in the basal lamina that mediates acetylcholine receptor clustering required for the function of neuromuscular junctions (NMJs) [4.Ruegg M.A. Bixby J.L. Agrin orchestrates synaptic differentiation at the vertebrate neuromuscular junction.Trends Neurosci. 1998; 21: 22-27Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar]. Agrin has a multimodal structural organization with several splice variants. The secretory isoform containing the signal sequence and the N-terminal Agrin binds laminin and serves as a functional link between the cell surface and basement membrane, particularly relevant in the NMJs. The alternatively spliced type II transmembrane isoform is restricted to localizations that are devoid of basal lamina (Figure 1A ). The N-terminal half of Agrin bears serine/threonine glycosylation and glycosaminoglycan attachment sites. The C terminus of Agrin bears three laminin-G (LG)-like domains. The terminal LG3 domain is regulated by alternative splicing with inserts of 8, 11, and 19 amino acids, respectively (Figure 1A). This alternatively spliced variant termed as the Agrin z+ isoform binds to its receptors lipoprotein-related receptor (Lrp)4 and muscle-specific tyrosine kinase (MuSK), which are required for sustaining NMJ functions and oncogenic properties in hepatocellular carcinoma (HCC) [5.Chakraborty S. et al.An oncogenic role of Agrin in regulating focal adhesion integrity in hepatocellular carcinoma.Nat. Commun. 2015; 66184Crossref PubMed Scopus (104) Google Scholar]. In healthy livers, the expression of Agrin is restricted to areas surrounding blood vessels with minimal expression reported within the hepatocytes. In contrast, Agrin levels are markedly enhanced in hepatocytes during cirrhosis and transition into HCC [6.Tátrai P. et al.Agrin, a novel basement membrane component in human and rat liver, accumulates in cirrhosis and hepatocellular carcinoma.Lab. Investig. 2006; 86: 1149Crossref PubMed Scopus (65) Google Scholar]. Recent studies have revealed the role of Agrin in ECM sensing and mechanotransducing that integrates integrin-focal adhesion and activation of YAP/TAZ to promote liver tumor growth [5.Chakraborty S. et al.An oncogenic role of Agrin in regulating focal adhesion integrity in hepatocellular carcinoma.Nat. Commun. 2015; 66184Crossref PubMed Scopus (104) Google Scholar,7.Chakraborty S. et al.Agrin as a Mechanotransduction signal regulating YAP through the Hippo pathway.Cell Rep. 2017; 18: 2464-2479Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar]. Agrin contributes to ECM remodeling and stiffness during tumorigenesis, via inactivation of the Hippo pathway to enhance YAP/TAZ–TEAD-dependent transcription of gene signatures promoting tumorigenesis [7.Chakraborty S. et al.Agrin as a Mechanotransduction signal regulating YAP through the Hippo pathway.Cell Rep. 2017; 18: 2464-2479Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar]. Thus, the mechanotransducing activity of Agrin has a broad-ranging impact on shaping the TME. Given that proteoglycans influence angiogenesis as one of the key pathways that favor cancer progression, the role of Agrin in this aspect was lacking until recent studies [8.Njah K. et al.A role of Agrin in maintaining the stability of vascular endothelial growth factor receptor-2 during tumor angiogenesis..Cell Rep. 2019; 28 (e7): 949-965Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar]. Here, we highlight that Agrin impacts angiogenesis within the HCC microenvironment and may have clinical implications in targeting the TME. A key question is whether Agrin recruits blood vessels within a growing lesion, which has been addressed using Agrin-depleted liver cancer cells in a Matrigel plug assay that determined in vivo angiogenic potential. Strikingly, depletion of Agrin in cancer cells reduced the blood vessel infiltration within a growing lesion in addition to the suppression of tumor growth and metastasis of HCC cells to mouse lungs. Moreover, the metastatic lesions that could colonize the lungs by Agrin-depleted cells lacked the capacity to attract surrounding pulmonary blood vessels within these lesions [8.Njah K. et al.A role of Agrin in maintaining the stability of vascular endothelial growth factor receptor-2 during tumor angiogenesis..Cell Rep. 2019; 28 (e7): 949-965Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar]. These results are consistent with an in vitro experiment showing reduced adherence of Agrin-depleted cancer cells to endothelial cells (ECs). Presently, it remains unclear as to how the TME sustains and/or enhances Agrin expression. For instance, Agrin secreted by platelet-derived growth factor (PDGF)-activated hepatic stellate cells (HSCs) supports HCC development in a paracrine fashion [9.Lv X. et al.Agrin para-secreted by PDGF-activated human hepatic stellate cells promotes hepatocarcinogenesis in vitro and in vivo.Oncotarget. 2017; 8: 105340-105355Crossref PubMed Scopus (24) Google Scholar]. Therefore, it is likely that Agrin produced cumulatively by hepatocytes, HSCs, and stromal cells affects liver endothelium by creating more vascular infiltration during HCC development (Figure 1B). A plausible mechanism could be contributed by Agrin-enriched cancer cells that invade the basement membrane, allowing vessel infiltration and metastasis (Figure 1B). Also, whether tumor extravasation is affected by Agrin-mediated vessel alterations needs further investigation. Besides HCC, Agrin is also important for oral squamous cell carcinoma (OSCC) progression [10.Rivera C. et al.Agrin has a pathological role in the progression of oral cancer.Br. J. Cancer. 2018; 118: 1628-1638Crossref PubMed Scopus (22) Google Scholar]. Evaluating the angiogenic microenvironment contributed by Agrin in OSCC or other cancers would also be of interest. Agrin crosstalk between cancer cells and ECs is underlined by the observation that high Agrin expression correlates with poor prognosis [7.Chakraborty S. et al.Agrin as a Mechanotransduction signal regulating YAP through the Hippo pathway.Cell Rep. 2017; 18: 2464-2479Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar]. The role of endothelial Agrin in this angiogenic crosstalk cannot be devalued in comparison with cancer-cell-derived Agrin. Similar to HCC cells, Agrin is expressed and secreted in macro- and microvascular ECs, albeit at lower levels than in cancer cells [8.Njah K. et al.A role of Agrin in maintaining the stability of vascular endothelial growth factor receptor-2 during tumor angiogenesis..Cell Rep. 2019; 28 (e7): 949-965Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar]. Depletion of EC Agrin hampers in vitro (2D) and sprouting (3D) angiogenesis in several types of ECs. These defects are readily rescued by Agrin from cancer cells, suggesting a unified and collective action of cancer-derived and endothelial Agrin on tumor angiogenesis. Importantly, the angiogenic role of Agrin relies on its receptor complex consisting of Integrin β1, Lrp4, and MuSK, which are also expressed in these ECs. Similar to HCC cells, focal adhesion kinase (FAK) is also critical as part of downstream effectors for Agrin-mediated angiogenesis in ECs (Figure 2). Agrin and its receptors also mediate a strong adhesion of infiltrating ECs to tumor cells. A C-terminal fragment of Agrin promoted an angiogenic phenotype in several ECs and an ex vivo mouse metatarsal sprouting assay. This fragment of Agrin effectively recruited blood vessels in cell-free subcutaneous Matrigel plugs, implying a promising avenue for Agrin as an in vivo mediator of angiogenesis. VEGFR1–3 are well studied in the context of developmental and tumor angiogenesis. VEGFR2, a widely expressed tyrosine kinase receptor in ECs, binds to VEGF-A to initiate a myriad of downstream signaling events including the mitogen-activated protein kinase (MAPK), endothelial NO synthase (eNOS), and Akt pathways [11.Fukumura D. et al.The role of nitric oxide in tumour progression.Nat. Rev. Cancer. 2006; 6: 521-534Crossref PubMed Scopus (999) Google Scholar]. Several proteoglycans regulate the VEGFR family, including VEGFR2 and downstream signaling events to control angiogenesis [3.Iozzo R.V. Schaefer L. Proteoglycan form and function: a comprehensive nomenclature of proteoglycans.Matrix Biol. 2015; 42: 11-55Crossref PubMed Scopus (677) Google Scholar]. As HCC represents a highly vascularized solid tumor, both VEGF and VEGFR2 are highly expressed in the liver endothelium and tumors [12.Semela D. Dufour J.-F. Angiogenesis and hepatocellular carcinoma.J. Hepatol. 2004; 41: 864-880Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar]. Therefore, the stability of VEGFR2 is central for sustaining angiogenesis in HCC. One striking observation is the loss of VEGFR2 stability in the ECs upon Agrin depletion, that also hampers the downstream e-NOS signaling pathway. These findings illustrate the presence of important molecular players within the ECM that may promote angiogenesis by sustaining VEGFR2 signaling. Consistent with the notion that a stiff ECM supports abnormal vasculature, Agrin further contributes to ECM stiffness and remodeling via YAP activation [7.Chakraborty S. et al.Agrin as a Mechanotransduction signal regulating YAP through the Hippo pathway.Cell Rep. 2017; 18: 2464-2479Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar,13.Bordeleau F. et al.Matrix stiffening promotes a tumor vasculature phenotype.Proc. Natl. Acad. Sci. U. S. A. 2017; 114: 492Crossref PubMed Scopus (206) Google Scholar]. The evidence that secreted Agrin from cancer tissues and ECs cooperates with stiffened ECM to stabilize endothelial VEGFR2 via a multiprotein complex involving Agrin, Lrp4, integrin β1, and FAK in ECs emphasizes the mechanical regulation of angiogenesis within the TME (Figure 2). By contrast, the oncogenic functions of Agrin in YAP/TAZ activation, endothelial VEGFR2 stability, and angiogenesis are moderated by ECM compliance. As induction of angiogenesis with abnormal blood vessels leads to tumor dissemination and poor clinical outcome, understanding the role of Agrin in EC junctional protein stability and vascular leakiness, possibly by enhancing ECM stiffness, sheds light on the long-term corruption of TM’ and the fate of cancer. Agrin may also sequester VEGF, a key proangiogenic ligand in the ECM to facilitate VEGF–VEGFR2 binding, e-NOS activity, and angiogenesis of ECs (Figure 2). Agrin-related proteoglycans perlecan and biglycan also activate the VEGF–VEGFR2 pathway to stimulate angiogenesis [3.Iozzo R.V. Schaefer L. Proteoglycan form and function: a comprehensive nomenclature of proteoglycans.Matrix Biol. 2015; 42: 11-55Crossref PubMed Scopus (677) Google Scholar]. Perlecan also promotes remodeling of ECM that facilitates metastasis of pancreatic cancer cells harboring mutations in the p53 gene, as well as resistance to chemotherapy [14.Vennin C. et al.CAF hierarchy driven by pancreatic cancer cell p53-status creates a pro-metastatic and chemoresistant environment via perlecan.Nat. Comm. 2019; 10: 3637Crossref PubMed Scopus (115) Google Scholar]. Although biglycan promotes angiogenesis, soluble decorin, a closely related counterpart of biglycan suppresses VEGFR2 and angiogenesis by evoking autophagy [15.Buraschi S. et al.Decorin causes autophagy in endothelial cells via Peg3.Proc. Natl. Acad. Sci. U. S. A. 2013; 110: E2582-E2591Crossref PubMed Scopus (149) Google Scholar]. A similar opposing functional interplay is also observed between Agrin and endorepellin, a soluble protein fragment derived from the C terminus of perlecan. Despite high homology in their C-terminal domains, soluble Agrin is angiogenic while endorepellin inhibits angiogenesis via inducing autophagy in ECs [3.Iozzo R.V. Schaefer L. Proteoglycan form and function: a comprehensive nomenclature of proteoglycans.Matrix Biol. 2015; 42: 11-55Crossref PubMed Scopus (677) Google Scholar]. Therefore, understanding the contrasting functions of Agrin, perlecan, and biglycan with endorepellin and decorin that balance ECM rigidity, angiogenesis, and crosstalk with other signaling pathways in the TME would form exciting future studies. From a therapeutic viewpoint, the angiogenic role of Agrin in the TME could be a new target in HCC and other cancers for novel treatments (either alone or in combination with the targeting of other cancer hallmarks). Although elementary at this stage, increasing evidence encourages the use of novel combinations of antibodies scavenging Agrin, and inhibitors against VEGFR2 and FAK for limiting cancer angiogenesis. In accordance, both sorafenib, which inhibits VEGFR2 signaling, and the FAK-PyK2 inhibitor PF562271, suppress Agrin-mediated angiogenesis and tumorigenesis [8.Njah K. et al.A role of Agrin in maintaining the stability of vascular endothelial growth factor receptor-2 during tumor angiogenesis..Cell Rep. 2019; 28 (e7): 949-965Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar,9.Lv X. et al.Agrin para-secreted by PDGF-activated human hepatic stellate cells promotes hepatocarcinogenesis in vitro and in vivo.Oncotarget. 2017; 8: 105340-105355Crossref PubMed Scopus (24) Google Scholar]. Hence, recognizing the role of Agrin as an extrinsic regulator of angiogenesis and the cumulative role of Agrin and perlecan in the tumor ECM, that control tumor growth and metastasis, may offer fresh perspectives to restrain tumor vascularization. This work is supported in part by the Open-Fund Young Investigator grant (NMRC/OFYIRG/068/2018-00), National Medical Research Council (NMRC), Singapore, to S.C., and core funds provided by the Institute of Molecular and Cell Biology. The figures were created using BioRender. We are thankful to many scientists in the field whose seminal works are not cited here due to space constraints.

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