Eric A. Murphy

A role for VEGF as a negative regulator of pericyte function and vessel maturation

Angiogenesis does not only depend on endothelial cell invasion and proliferation: it also requires pericyte coverage of vascular sprouts for vessel stabilization. These processes are coordinated by vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) through their cognate receptors on endothelial cells and vascular smooth muscle cells (VSMCs), respectively. PDGF induces neovascularization by priming VSMCs/pericytes to release pro-angiogenic mediators. Although VEGF directly stimulates endothelial cell proliferation and migration, its role in pericyte biology is less clear. Here we define a role for VEGF as an inhibitor of neovascularization on the basis of its capacity to disrupt VSMC function. Specifically, under conditions of PDGF-mediated angiogenesis, VEGF ablates pericyte coverage of nascent vascular sprouts, leading to vessel destabilization. At the molecular level, VEGF-mediated activation of VEGF-R2 suppresses PDGF-Rβ signalling in VSMCs through the assembly of a previously undescribed receptor complex consisting of PDGF-Rβ and VEGF-R2. Inhibition of VEGF-R2 not only prevents assembly of this receptor complex but also restores angiogenesis in tissues exposed to both VEGF and PDGF. Finally, genetic deletion of tumour cell VEGF disrupts PDGF-Rβ/VEGF-R2 complex formation and increases tumour vessel maturation. These findings underscore the importance of VSMCs/pericytes in neovascularization and reveal a dichotomous role for VEGF and VEGF-R2 signalling as both a promoter of endothelial cell function and a negative regulator of VSMCs and vessel maturation.Angiogenesis does not only depend on endothelial cell invasion and proliferation: it also requires pericyte coverage of vascular sprouts for vessel stabilization. These processes are coordinated by vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) through their cognate receptors on endothelial cells and vascular smooth muscle cells (VSMCs), respectively. PDGF induces neovascularization by priming VSMCs/pericytes to release pro-angiogenic mediators. Although VEGF directly stimulates endothelial cell proliferation and migration, its role in pericyte biology is less clear. Here we define a role for VEGF as an inhibitor of neovascularization on the basis of its capacity to disrupt VSMC function. Specifically, under conditions of PDGF-mediated angiogenesis, VEGF ablates pericyte coverage of nascent vascular sprouts, leading to vessel destabilization. At the molecular level, VEGF-mediated activation of VEGF-R2 suppresses PDGF-Rbeta signalling in VSMCs through the assembly of a previously undescribed receptor complex consisting of PDGF-Rbeta and VEGF-R2. Inhibition of VEGF-R2 not only prevents assembly of this receptor complex but also restores angiogenesis in tissues exposed to both VEGF and PDGF. Finally, genetic deletion of tumour cell VEGF disrupts PDGF-Rbeta/VEGF-R2 complex formation and increases tumour vessel maturation. These findings underscore the importance of VSMCs/pericytes in neovascularization and reveal a dichotomous role for VEGF and VEGF-R2 signalling as both a promoter of endothelial cell function and a negative regulator of VSMCs and vessel maturation.

MicroRNA-132–mediated loss of p120RasGAP activates the endothelium to facilitate pathological angiogenesis

Although it is well established that tumors initiate an angiogenic switch, the molecular basis of this process remains incompletely understood. Here we show that the miRNA miR-132 acts as an angiogenic switch by targeting p120RasGAP in the endothelium and thereby inducing neovascularization. We identified miR-132 as a highly upregulated miRNA in a human embryonic stem cell model of vasculogenesis and found that miR-132 was highly expressed in the endothelium of human tumors and hemangiomas but was undetectable in normal endothelium. Ectopic expression of miR-132 in endothelial cells in vitro increased their proliferation and tube-forming capacity, whereas intraocular injection of an antagomir targeting miR-132, anti–miR-132, reduced postnatal retinal vascular development in mice. Among the top-ranking predicted targets of miR-132 was p120RasGAP, which we found to be expressed in normal but not tumor endothelium. Endothelial expression of miR-132 suppressed p120RasGAP expression and increased Ras activity, whereas a miRNA-resistant version of p120RasGAP reversed the vascular response induced by miR-132. Notably, administration of anti–miR-132 inhibited angiogenesis in wild-type mice but not in mice with an inducible deletion of Rasa1 (encoding p120RasGAP). Finally, vessel-targeted nanoparticle delivery of anti–miR-132 restored p120RasGAP expression in the tumor endothelium, suppressed angiogenesis and decreased tumor burden in an orthotopic xenograft mouse model of human breast carcinoma. We conclude that miR-132 acts as an angiogenic switch by suppressing endothelial p120RasGAP expression, leading to Ras activation and the induction of neovascularization, whereas the application of anti–miR-132 inhibits neovascularization by maintaining vessels in the resting state.

Nanoparticle-mediated drug delivery to tumor vasculature suppresses metastasis

Integrin ανβ3 is found on a subset of tumor blood vessels where it is associated with angiogenesis and malignant tumor growth. We designed an ανβ3-targeted nanoparticle (NP) encapsulating the cytotoxic drug doxorubicin (Dox) for targeted drug delivery to the ανβ3-expressing tumor vasculature. We observed real-time targeting of this NP to tumor vessels and noted selective apoptosis in regions of the ανβ3-expressing tumor vasculature. In clinically relevant pancreatic and renal cell orthotopic models of spontaneous metastasis, targeted delivery of Dox produced an antimetastatic effect. In fact, ανβ3-mediated delivery of this drug to the tumor vasculature resulted in a 15-fold increase in antimetastatic activity without producing drug-associated weight loss as observed with systemic administration of the free drug. These findings reveal that NP-based delivery of cytotoxic drugs to the ανβ3-positive tumor vasculature represents an approach for treating metastatic disease.

Formation of endothelial lumens requires a coordinated PKCε-, Src-, Pak- and Raf-kinase- dependent signaling cascade downstream of Cdc42 activation

In this study, we present data showing that Cdc42-dependent lumen formation by endothelial cells (ECs) in three-dimensional (3D) collagen matrices involves coordinated signaling by PKCϵ in conjunction with the Src-family kinases (SFKs) Src and Yes. Activated SFKs interact with Cdc42 in multiprotein signaling complexes that require PKCϵ during this process. Src and Yes are differentially expressed during EC lumen formation and siRNA suppression of either kinase, but not Fyn or Lyn, results in significant inhibition of EC lumen formation. Concurrent with Cdc42 activation, PKCϵ- and SFK-dependent signaling converge to activate p21-activated kinase (Pak)2 and Pak4 in steps that are also required for EC lumen formation. Pak2 and Pak4 further activate two Raf kinases, B-Raf and C-Raf, leading to ERK1 and ERK2 (ERK1/2) activation, which all seem to be necessary for EC lumen formation. This work reveals a multicomponent kinase signaling pathway downstream of integrin-matrix interactions and Cdc42 activation involving PKCϵ, Src, Yes, Pak2, Pak4, B-Raf, C-Raf and ERK1/2 to control EC lumen formation in 3D collagen matrices.

Notch promotes vascular maturation by inducing integrin-mediated smooth muscle cell adhesion to the endothelial basement membrane

Vascular development and angiogenesis initially depend on endothelial tip cell invasion, which is followed by a series of maturation steps, including lumen formation and recruitment of perivascular cells. Notch ligands expressed on the endothelium and their cognate receptors expressed on perivascular cells are involved in blood vessel maturation, though little is known regarding the Notch-dependent effectors that facilitate perivascular coverage of nascent vessels. Here, we report that vascular smooth muscle cell (VSMC) recognition of the Notch ligand Jagged1 on endothelial cells leads to expression of integrin αvβ3 on VSMCs. Once expressed, integrin αvβ3 facilitates VSMC adhesion to VWF in the endothelial basement membrane of developing retinal arteries, leading to vessel maturation. Genetic or pharmacologic disruption of Jagged1, Notch, αvβ3, or VWF suppresses VSMC coverage of nascent vessels and arterial maturation during vascular development. Therefore, we define a Notch-mediated interaction between the developing endothelium and VSMCs leading to adhesion of VSMCs to the endothelial basement membrane and arterial maturation.

Targeted Nanogels: A Versatile Platform for Drug Delivery to Tumors

Although nanoparticle-based drug delivery formulations can improve the effectiveness and safety of certain anticancer drugs, many drugs, due to their chemical composition, are unsuitable for nanoparticle loading. Here, we describe a targeted nanogel drug delivery platform that can (i) encapsulate a wide range of drug chemotypes, including biological, small molecule, and cytotoxic agents; (ii) display targeting ligands and polymeric coatings on the surface; (iii) enhance drug retention within the nanogel core after photo-cross-linking; and (iv) retain therapeutic activity after lyophilization allowing for long-term storage. For therapeutic studies, we used integrin αvβ3–targeted lipid-coated nanogels with cross-linked human serum albumin in the core for carrying therapeutic cargoes. These particles exhibited potent activity in tumor cell viability assays with drugs of distinct chemotype, including paclitaxel, docetaxel, bortezomib, 17-AAG, sorafenib, sunitinib, bosutinib, and dasatinib. Treatment of orthotopic breast and pancreas tumors in mice with taxane-loaded nanogels produced a 15-fold improvement in antitumor activity relative to Abraxane by blocking both primary tumor growth and spontaneous metastasis. With a modifiable surface and core, the lipid-coated nanogel represents a platform technology that can be easily adapted for specific drug delivery applications to treat a wide range of malignant diseases. Mol Cancer Ther; 10(6); 972–82. ©2011 AACR.

RBBP9: A tumor-associated serine hydrolase activity required for pancreatic neoplasia

Pancreatic cancer is one of the most lethal malignancies. To discover functionally relevant modulators of pancreatic neoplasia, we performed activity-based proteomic profiling on primary human ductal adenocarcinomas. Here, we identify retinoblastoma-binding protein 9 (RBBP9) as a tumor-associated serine hydrolase that displays elevated activity in pancreatic carcinomas. Whereas RBBP9 is expressed in normal and malignant tissues at similar levels, its elevated activity in tumor cells promotes anchorage-independent growth in vitro as well as pancreatic carcinogenesis in vivo. At the molecular level, RBBP9 activity overcomes TGF-β-mediated antiproliferative signaling by reducing Smad2/3 phosphorylation, a previously unknown role for a serine hydrolase in cancer biology. Conversely, loss of endogenous RBBP9 or expression of mutationally inactive RBBP9 leads to elevated Smad2/3 phosphorylation, implicating this serine hydrolase as an essential suppressor of TGF-β signaling. Finally, RBBP9-mediated suppression of TGF-β signaling is required for E-cadherin expression as loss of the serine hydrolase activity leads to a reduction in E-cadherin levels and a concomitant decrease in the integrity of tumor cell–cell junctions. These data not only define a previously uncharacterized serine hydrolase activity associated with epithelial neoplasia, but also demonstrate the potential benefit of functional proteomics in the identification of new therapeutic targets.

Disruption of angiogenesis and tumor growth with an orally active drug that stabilizes the inactive state of PDGFRβ/B-RAF

Kinases are known to regulate fundamental processes in cancer including tumor proliferation, metastasis, neovascularization, and chemoresistance. Accordingly, kinase inhibitors have been a major focus of drug development, and several kinase inhibitors are now approved for various cancer indications. Typically, kinase inhibitors are selected via high-throughput screening using catalytic kinase domains at low ATP concentration, and this process often yields ATP mimetics that lack specificity and/or function poorly in cells where ATP levels are high. Molecules targeting the allosteric site in the inactive kinase conformation (type II inhibitors) provide an alternative for developing selective inhibitors that are physiologically active. By applying a rational design approach using a constrained amino-triazole scaffold predicted to stabilize kinases in the inactive state, we generated a series of selective type II inhibitors of PDGFRβ and B-RAF, important targets for pericyte recruitment and endothelial cell survival, respectively. These molecules were designed in silico and screened for antivascular activity in both cell-based models and a Tg(fli1-EGFP) zebrafish embryogenesis model. Dual inhibition of PDGFRβ and B-RAF cellular signaling demonstrated synergistic antiangiogenic activity in both zebrafish and murine models of angiogenesis, and a combination of previously characterized PDGFRβ and RAF inhibitors validated the synergy. Our lead compound was selected as an orally active molecule with favorable pharmacokinetic properties which demonstrated target inhibition in vivo leading to suppression of murine orthotopic tumors in both the kidney and pancreas.

A MEK-independent role for CRAF in mitosis and tumor progression.

RAF kinases regulate cell proliferation and survival and can be dysregulated in tumors. The role of RAF in cell proliferation has been linked to its ability to activate mitogen-activated protein kinase kinase 1 (MEK) and mitogen-activated protein kinase 1 (ERK). Here we identify a MEK-independent role for RAF in tumor growth. Specifically, in mitotic cells, CRAF becomes phosphorylated on Ser338 and localizes to the mitotic spindle of proliferating tumor cells in vitro as well as in murine tumor models and in biopsies from individuals with cancer. Treatment of tumors with allosteric inhibitors, but not ATP-competitive RAF inhibitors, prevents CRAF phosphorylation on Ser338 and localization to the mitotic spindle and causes cell-cycle arrest at prometaphase. Furthermore, we identify phospho-Ser338 CRAF as a potential biomarker for tumor progression and a surrogate marker for allosteric RAF blockade. Mechanistically, CRAF, but not BRAF, associates with Aurora kinase A (Aurora-A) and Polo-like kinase 1 (Plk1) at the centrosomes and spindle poles during G2/M. Indeed, allosteric or genetic inhibition of phospho-Ser338 CRAF impairs Plk1 activation and accumulation at the kinetochores, causing prometaphase arrest, whereas a phospho-mimetic Ser338D CRAF mutant potentiates Plk1 activation, mitosis and tumor progression in mice. These findings show a previously undefined role for RAF in tumor progression beyond the RAF-MEK-ERK paradigm, opening new avenues for targeting RAF in cancer.

Oncogenic Ras/Src cooperativity in pancreatic neoplasia

Pancreas cancer is one of the most lethal malignancies and is characterized by activating mutations of Kras, present in 95% of patients. More than 60% of pancreatic cancers also display increased c-Src activity, which is associated with poor prognosis. Although loss of tumor suppressor function (for example, p16, p53, Smad4) combined with oncogenic Kras signaling has been shown to accelerate pancreatic duct carcinogenesis, it is unclear whether elevated Src activity contributes to Kras-dependent tumorigenesis or is simply a biomarker of disease progression. Here, we demonstrate that in the context of oncogenic Kras, activation of c-Src through deletion of C-terminal Src kinase (CSK) results in the development of invasive pancreatic ductal adenocarcinoma (PDA) by 5–8 weeks. In contrast, deletion of CSK alone fails to induce neoplasia, while oncogenic Kras expression yields PDA at low frequency after a latency of 12 months. Analysis of cell lines derived from Ras/Src-induced PDAs indicates that oncogenic Ras/Src cooperativity may lead to genomic instability, yet Ras/Src-driven tumor cells remain dependent on Src signaling and as such, Src inhibition suppresses growth of Ras/Src-driven tumors. These findings demonstrate that oncogenic Ras/Src cooperate to accelerate PDA onset and support further studies of Src-directed therapies in pancreatic cancer.