Endothelin-1 receptor drives invadopodia: Exploiting how β-arrestin-1 guides the way

ABSTRACT

Metastatization is a complex multistep process requiring fine-tuned regulated cytoskeleton re-modeling, mediated by the cross-talk of actin with interacting partners, such as the Rho GTPases. Our expanding knowledge of invadopodia, small invasive membrane protrusions composed of a core of F-actin, actin regulators and actin-binding proteins, and hotspots for secretion of extracellular matrix (ECM) proteinases, contributes to clarify critical steps of the metastatic program. Growth factor receptors and their intermediate signaling molecules, along with matrix adhesion and rigidity, pH and hypoxia, act as drivers of cytoskeleton changes and invadopodia formation. We recently pro-posed a novel route map by which cancer cells regulates invadopodia dynamics supporting metastasis as response to the endothelin A receptor (ET_AR), among the highly druggable G-protein coupled receptors in cancer. The metastatic behavior exhibited by ovarian cancer cells overe-xpressing ET_AR is now explained by the interplay with β-arrestin1 (β-arr1), a scaffold protein acting as signal-integrating module of RhoC and cofilin signaling for specific invadopodia formation, accomplished by its interaction with a Rho guanine nucleotide exchange factor (GEF), PDZ-RhoGEF, in a G-protein independent manner. Here, we summarize this novel activation of the RhoC pathway from ET_AR/β-arr1 signaling that may be exploited therapeutically and discuss new perspectives for future directions of investigations.

KEYWORDS:

• cancer
• endothelin receptors
• endothelin
• G-protein coupled receptors
• invadopodia
• PDZ-RHOGEF
• RhoC
• β-arrestin-1

A growing body of evidence in vitro and in vivo, especially by using intravital imaging to visualize cancer cell movement in vivo, has demonstrated that during metastatization invasive cancerous cells possess the ability to extend membrane protrusions, referred as “invadopodia,” tailored for their penetration into the extracellular matrix (ECM), and acquisition of matrix-degrading capability.^1-4 To make them, cytoskeletal changes are expected to match with phenotypic alterations in cancer cells, thus augmenting the ability of these cells to migrate through the stroma, and to intravasate.^1,4,5 Invadopodia, showing both acto-adhesive and degradative functions, are composed of actin filaments and enriched in actin regulators, such as N-WASP, cortactin, Arp2/3, Tks5 and cofilin, as well as in proteinases, signaling and membrane remodeling proteins.^1,6 Although some cancer cells constitutively form invadopodia, emerging evidences highlight that inputs from growth factors and from the tumor microenvironment, such as stromal-cell interactions in an autocrine or paracrine fashion, ECM stiffness or composition, hypoxia and pH, and subsequently cell metabolism, function as drivers of invadopodia.⁷ Growth factors, like platelet-derived growth factor, transforming growth factor-β and epidermal growth factor, which are produced by cancer cells but also by stromal cells, facilitating an invasive milieu for cancer cells, induce invadopodia formation and activity going through the stages of induction, initiation, assembly, and maturation.^7-9 Each of these steps are characterized by a specific signature pattern of protein activities, such as phosphorylation and/or activation, converging on common signaling hub, especially Src kinase, and Rho family GTPase, as well as fine regulation of cofilin activity.⁸ Studies addressing on key regulators of invadopodia focused much attention around the involvement of Rho-family GTPases, identifying the activation and fine-tuned RhoGTPase activities, in particular that of RhoA, CdC42 and Rac1, as able to produce stable “invasive protrusions.”^10,11 More recently, it has been demonstrated that the contribution of different RhoGTPases in invadopodia formation is more complex and involves also the dynamics activity of RhoC in confining the actin polymerization in invadopodia.^12-15 A seminal work first revealed that, in metastatic mammary adenocarcinoma cells, RhoC activity is spatially confined to adhesion rings, areas surrounding the actin core of invadopodia, which in turn increases cofilin inactivation, and is crucial for the geometric confinement of active cofilin within the core of the invasive protrusion.¹⁰ Accordingly, other studies further demonstrated that critical components of invadopodia, such as podoplanin, might control RhoC activity specifically at adhesion rings, allowing the inactivation of cofilin in this specific compartment of invadopodia.¹⁵ In this context, we have recently proposed a picture at the molecular and cellular levels of the new protein complexes mediating actin and membrane dynamics, in which RhoC activity is dynamically regulated to the membrane of invasive ovarian cancer cells to form invadopodia-dependent ECM degradation.¹⁶ Our study has revealed that endothelin-1 (ET-1) by activating the ET_A receptor (ET_AR), among the cancer-related G-protein coupled receptors (GPCR), is responsible for the cell ability to form active invadopodia, tightly controlling the intricate signaling and dynamic organization of cytoskeleton (Fig. 1). ET-1 activates two receptor subtypes known as ET_AR and ET_B receptor (ET_BR), which are differently expressed on human cancers. Indeed, there are cancers that predominantly express ET_AR, such as ovarian, breast, colon, renal, gastric, pancreatic thyroid, and nasopharyngeal cancers; cancers expressing ET_BR, such as melanoma, glioblastoma and astrocytoma, and those cancers expressing both receptors, such as bladder, lung, vulvar cancers and Kaposi's sarcoma.¹⁷ ET-1 initiates downstream signaling cascades, allowing precise control over multiple signaling pathways, to regulate cell motility and metastatic behavior, controlling also multiple and dynamic transitional states between the epithelial and mesenchymal phenotypes, and intravasation.^17-21 One of the most significant findings of this study is the knowledge that the molecular scaffold protein β-arrestin1 (β-arr1) regulates the spatial distribution of actin and actin regulators leading to invadopodia formation and activity through RhoC activity.¹⁶ Our results revealed a previously unknown role for ET_AR/β-arr1 representing a critical route by which the tumor cells form specialized invasive protrusions and acquire maximally pro-invasive capabilities, thus improving our understanding of the molecular specialized functions of this receptor to drive cancer invasion and metastasis (Fig. 1). Since most Rho GTPases are active and stimulate their downstream targets when bound to GTP, a process operated by guanine nucleotide exchange factors (GEFs),²² we exploited putative GEFs implicated in ET_AR-driven RhoC activation and identified ZO-1 homology (PDZ)-RhoGEF as primary RhoGEF implicated in the activation of RhoC. Interestingly, previously studies performed to coupling signaling of many GPCR to this class of proteins have been discovered that PDZ-RhoGEF represents one of the regulator for a specific control of the spatial distribution of RhoA activity acting downstream of GPCR through association with proteins Gα12 or Gα13 belonging to the G-protein family, implicated in cell migration.^23-27 Interestingly, for the first time we report that the link between GPCR and PDZ-RhoGEF is mediated by β-arr1, further highlighting the crucial importance of a β-arr1-dependent signaling in the tumor-promoting activity of ET-1. β-arrs promote signals through its common feature to form multi-protein signaling complexes, leading to a pleiotropic array of responses than originally thought.²⁸ A number of studies demonstrated that β-arrs can regulate cytoskeleton reorganization and chemotaxis induced by GPCR, and this regulation can occur either by scaffolding signaling proteins regulating GTPAses, such as RhoA or Ral, or by directly binding cytoskeleton proteins as those of cytoskeletal filaments, or cytoskeletal accessories and motor proteins, such as cofilin.^29-39 Recent evidences highlight the role of G-protein-dependent induction of invadopodia in cancer cells. Among the major ligands for GPCR, lysophosphatidic acid is implicated in the formation of invadopodia in various cancer cells, such as fibrosarcoma, melanoma, ovarian and prostate cancer cells in a G-protein-dependent manner, and involving Rac, CdC42 or RhoA.^40-43 Interestingly, in melanoma cells, ET-1 signaling is capable to mediate a rapid remodeling of invadopodia, acting through ET_BR/Gi-dependent activation of Cdc42, accompanied by a rapid decrease in Rac activity.⁴⁰ Our findings fill an important gap in the understanding of how GPCR can dynamically control invadopodia formation and activity and depict a G-protein-independent molecular map in which we identify β-arr1 as platform in mediating through PDZ-RhoGEF/ RhoC/ROCK/LIMK pathway, restriction of cofilin activity and recruitment of matrix metalloproteases in these actin-rich structures, for efficient matrix degradation and thus activation of invadopodia, enabling ovarian cancer cells to move, invade and metastatize.¹⁶ By investigating ET_AR-dependent β-arr1 recruitment in driving the metastasis in vivo, our findings also demonstrate that pharmacological blockade of ET-1 receptors by the FDA approved small molecule macitentan, a dual ET_AR and ET_BR receptor antagonist, impairs β-arr1 ‘interactome’ driven by ET-1 axis in cell motility and metastasis formation. Interestingly, in vivo macitentan showed ability to control ovarian cancer metastasis formation, also through the inhibition of cofilin pathway, thus having important implication for cancer therapy because potential advantage of targeting not only cancer cells (which mainly express ET_AR) but also microenvironment associated elements, such as vascular, lymphatic, and inflammatory cells and fibroblasts, which all express ET_BR, that critically contribute to cancer metastasis. Although we defined a molecular mechanism for how cancer cells focus protrusions with efficient matrix degradation during invasion, highlighting ET-1/β-arr1 as critical node in the signaling network, many open questions remain to be evaluated. Since the ability of β-arr1 to directly interact with well-established components of invadopodia, such as N-WASP, or Arp2/3 complex, future studies should focus on determining whether β-arr1 might be a structural critical component within invasive protrusions. Moreover, because the adhesion rings surrounding the actin core of invadopodia, are composed of proteins known to be regulated by ET-1 signaling, such as β1 integrin, it is essential to understand whether β-arr1 might affect invadodopia formation and maturation by the specific segregation of β1 integrin into the adhesion ring/actin core.⁹ Clearly, more advance in this field is likely to improve the knowledge of the molecular events between ET-1/β-arr1 signaling pathways and invadopodia, disclosing so far unexpected involvement of β-arr capable both of promoting actin assembly to form invadopodia and in facilitating gradient sensing and directional cytoskeletal reorganization, as well as by interacting with other GTPases regulating the traffic of specific proteinases through several subcellular compartments and their release at invadopodia.

Figure 1. Endothelin A receptor (ET_AR) regulates formation of invadopodia by β-arrestin-1-dependent in cancer cells. Schematic view of an invadopodium that is actively degrading the extracellular matrix via signals induced by ET-1 and its receptor ET_AR in ovarian cancer cells. The activation of ET-1 receptor initiates a G-protein-independent signaling through the recruitment of the scaffold protein β-arrestin1, which binds PDZ-RhoGEF favoring its tyrosine-phosphorylation, leading to RhoC activation and downstream RHO-associated coiled-coil containing protein kinase 1 (ROCK1), and LIMK activation, to control regulation of cofilin activity by Ser3 phosphorylation at adhesion ring. Indeed, ET-1/β-arrestin1 enhances the ability of tumor cells to form F-actin cores in membrane protrusions, followed by recruitment of invadopodia component proteins, like cortactin and Tks5, before activation of proteases (MT1-MMP, MMP-2, MMP-9) to complete invadopodia maturation, promoting invasion and metastasis.

A better knowledge of the role of ET-1R/β-arr1 in the regulation of the invadopodia may suggest the possibility that targeting ET-1R-driven β-arr1 signaling pathways can represent a possible route of therapeutic intervention against cancer.⁴⁴

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Acknowledgments

We would acknowledge all authors of the original report.¹⁶

Additional information

Funding

This work was supported by Associazione Italiana Ricerca sul Cancro (AIRC) to A.B. (AIRC 14199) and L.R. (AIRC 16965).