Dynamin 2 along with microRNA-199a reciprocally regulate hypoxia-inducible factors and ovarian cancer metastasis.

Hypoxia-driven changes in the tumor microenvironment facilitate cancer metastasis. In the present study, we investigated the regulatory cross talk between endocytic pathway, hypoxia, and tumor metastasis. Dynamin 2 (DNM2), a GTPase, is a critical mediator of endocytosis. Hypoxia decreased the levels of DNM2. DNM2 promoter has multiple hypoxia-inducible factor (HIF)-binding sites and genetic deletion of them relieved hypoxia-induced transcriptional suppression. Interestingly, DNM2 reciprocally regulated HIF. Inhibition of DNM2 GTPase activity and dominant-negative mutant of DNM2 showed a functional role for DNM2 in regulating HIF. Furthermore, the opposite strand of DNM2 gene encodes miR-199a, which is similarly reduced in cancer cells under hypoxia. miR-199a targets the 3'-UTR of HIF-1α and HIF-2α. Decreased miR-199a expression in hypoxia increased HIF levels. Exogenous expression of miR-199a decreased HIF, cell migration, and metastasis of ovarian cancer cells. miR-199a-mediated changes in HIF levels affected expression of the matrix-remodeling enzyme, lysyloxidase (LOX). LOX levels negatively correlated with progression-free survival in ovarian cancer patients. These results demonstrate a regulatory relationship between DNM2, miR-199a, and HIF, with implications in cancer metastasis.

Inhibition of ovarian cancer by RGD-P125A-endostatin-Fc fusion proteins.

Previous studies have shown that a single point mutation in endostatin at position 125 (P125A) can improve the biological activity of endostatin. Addition of an integrin-targeting moiety, R-G-D, resulted in better localization to tumor vasculature and improved the antiangiogenic activity of endostatin. Because endostatin has relatively shorter serum half-life, frequent dosing was required for inhibiting tumor growth. In our study, we have genetically fused RGD-P125A-endostatin to Fc of IgG4 isotype and evaluated its antiangiogenic and antitumor effects in athymic mice. Two genetic constructs were made, RGD-P125A-endostatin-Fc (RE-Fc) and P125A-endostatin-RGD-Fc (ER-Fc). Both constructs were cloned and expressed in mammalian cells. Purified fusion proteins inhibited endothelial cell migration and proliferation better than yeast-derived P125A-endostatin. Both RE-Fc and ER-Fc inhibited ovarian cancer growth and were found to be as effective as Bevacizumab treatment. Fusion protein showed marked increased half-life. Combination treatment with Bevacizumab and ER-Fc showed additive inhibition of ovarian cancer growth. These studies demonstrate that genetic fusion with human IgG4-Fc increases the half-life of P125A-endostatin and can be used along with Bevacizumab to improve antiangiogenic and antitumor activities.

Hypoxia-induced microRNA-424 expression in human endothelial cells regulates HIF-α isoforms and promotes angiogenesis.

Adaptive changes to oxygen availability are critical for cell survival and tissue homeostasis. Prolonged oxygen deprivation due to reduced blood flow to cardiac or peripheral tissues can lead to myocardial infarction and peripheral vascular disease, respectively. Mammalian cells respond to hypoxia by modulating oxygen-sensing transducers that stabilize the transcription factor hypoxia-inducible factor 1α (HIF-1α), which transactivates genes governing angiogenesis and metabolic pathways. Oxygen-dependent changes in HIF-1α levels are regulated by proline hydroxylation and proteasomal degradation. Here we provide evidence for what we believe is a novel mechanism regulating HIF-1α levels in isolated human ECs during hypoxia. Hypoxia differentially increased microRNA-424 (miR-424) levels in ECs. miR-424 targeted cullin 2 (CUL2), a scaffolding protein critical to the assembly of the ubiquitin ligase system, thereby stabilizing HIF-α isoforms. Hypoxia-induced miR-424 was regulated by PU.1-dependent transactivation. PU.1 levels were increased in hypoxic endothelium by RUNX-1 and C/EBPα. Furthermore, miR-424 promoted angiogenesis in vitro and in mice, which was blocked by a specific morpholino. The rodent homolog of human miR-424, mu-miR-322, was significantly upregulated in parallel with HIF-1α in experimental models of ischemia. These results suggest that miR-322/424 plays an important physiological role in post-ischemic vascular remodeling and angiogenesis.

Endostatin induces autophagy in endothelial cells by modulating Beclin 1 and beta-catenin levels.

Endostatin is a well-characterized endogenous inhibitor of angiogenesis that affects cell proliferation and migration by inhibiting integrin and Wnt-mediated signalling pathways. Here, we show that endothelial cells treated with native and P125A-endostatin activate autophagy. Because autophagy can either be protective or induce programmed cell death, experiments were carried out to understand the signalling pathways leading to autophagy in endothelial cells. P125A-endostatin treatment increased the levels of Beclin 1, a crucial molecule in vesicle nucleation and autophagy. The treatment also reduced the levels of Bcl-2, Bcl-x(L) and beta-catenin; however, progressively increasing amounts of Bcl-2 and Bcl-x(L) were found to be complexed with Beclin 1. Increased beta-catenin and Wnt-mediated signalling reduced Beclin 1 levels and rescued endothelial cells from endostatin-induced autophagy. Finally, knocking down Beclin 1 levels by RNA interference decreased autophagy and accelerated caspase activation in endostatin-treated cells. These studies suggest that endothelial cells may initiate autophagy as a survival response to limit the effects of angiogenesis inhibitors. Thus, interfering with autophagy can potentiate the effects of endostatin by promoting a switch to apoptosis.

AAV-2-mediated expression of IGF-1 in skeletal myoblasts stimulates angiogenesis and cell survival.

The transplantation of skeletal myoblasts is being tested in various organ systems to facilitate tissue repair and regeneration. Previous studies have indicated that transplanted cells for varied reasons were not surviving in sufficient numbers following transplantation, thus negatively affecting overall therapeutic efficacy of the approach. We hypothesize that the genetic modification of myoblasts to express insulin-like growth factor 1 (IGF-1) locally may enhance the survival of transplanted cells by stimulating neo-vascularization, decreasing apoptosis, and promoting cell proliferation. Using an adeno-associated virus (adeno-associated virus type 2) vector system, the IGF-1 gene was introduced into canine skeletal myoblasts. As a negative control, myoblasts transduced with the green fluorescence protein (GFP) was used. Relative angiogenic response induced by IGF-1 myoblast was compared to VEGF165-induced neo-vascularization using Matrigel plugs under similar conditions. In vitro evaluation and characterization revealed that the secreted IGF-1 protein was biologically and functionally active in promoting endothelial cell proliferation, migration and assembly into vessel-like structures. Matrigel plugs containing the three test groups were implanted subcutaneously in nude mice (n = 5). After 3 weeks, analysis of explanted samples revealed an enhanced neo-vascularization with an average microvessel density per field for IGF-1 at 55.9 versus 33.4 for vascular endothelial growth factor and 24 for GFP. Additionally, apoptosis was significantly reduced (p

Autophagy and angiogenesis inhibition.

Angiogenesis, the process by which new blood vessels are formed is critical for embryonic development and physiological functioning of normal tissues. Angiogenesis also plays a critical role in the pathology of many diseases including cancer, wherein the supply and demand for blood vessels determines the rate of cancer growth. A number of therapeutic strategies are being developed to inhibit pathological angiogenesis. Kringle domains of plasminogen such as kringle 5 (K5) and a proteolytic fragment of collagen type XVIII (endostatin) are well-characterized, potent angiogenesis inhibitors. These inhibitors activate different intracellular signaling pathways to induce apoptosis and inhibit cell proliferation. Recent studies from our group have shown that K5 and endostatin can also induce autophagy in addition to apoptosis in endothelial cells. A common feature of the two treatments was the upregulation of Beclin 1 levels leading to alterations in the Beclin 1-Bcl-2 complex. Angiogenesis inhibitor-induced autophagy in endothelial cells was independent of nutritional or hypoxic stress and initiated even in the presence of endothelial-specific survival factors such as vascular endothelial growth factor (VEGF). Interfering with the autophagic response by knocking down Beclin 1 levels dramatically increased apoptosis of endothelial cells. These findings identify the autophagic response as a novel target for enhancing the therapeutic efficacy of angiogenesis inhibitors.

Modulation of angiogenic phenotype alters tumorigenicity in rat ovarian epithelial cells.

Vascular endothelial growth factor (VEGF) expression correlates with microvessel density, stage, malignant ascites, metastasis, and survival in ovarian cancer. By transducing VEGF165 into a nontumorigenic rat ovarian surface epithelial cell line (ROSE199), we investigated the direct effect of an angiogenic phenotype on tumor development. The neu oncogene, which is overexpressed in >30% of ovarian cancers, was used in comparison. Neu-transfected ROSE199 cells showed phenotypic characteristics of transformation in vitro with an abundance of focus-forming units in monolayer cultures and anchorage-independent growth in soft agar. In contrast, VEGF-secreting ROSE199 cells (VR) retained normal morphology and in vitro growth characteristics (e.g., proliferation rate) compared with parental ROSE199 cells. Interestingly, injection of VR cells into athymic mice formed malignant ascites in 100% of the animals when injected into the peritoneum and developed vascularized tumors in 85% of the mice when injected s.c. Furthermore, blocking VEGF-mediated signaling by the Flk-1/KDR receptor kinase inhibitor SU5416 significantly inhibited the growth of VR tumors. To validate that the proangiogenic switch is responsible for tumor development, the angiogenic phenotype was balanced by the inducible coexpression of endostatin under the control of Tet-activated promoter. Coexpression of endostatin along with VEGF reversed the tumorigenic phenotype of VR cells. These studies show that alterations in the angiogenic characteristics of ovarian surface epithelium may play an important role in the etiology of ovarian cancer, and that inhibition of angiogenesis can be effective in the treatment of epithelial ovarian cancer.

Kringle 5 of human plasminogen, an angiogenesis inhibitor, induces both autophagy and apoptotic death in endothelial cells.

Inhibition of endothelial cell proliferation and angiogenesis is emerging as an important strategy in cancer therapeutics. Kringle 5 (K5) of human plasminogen is a potent angiogenesis inhibitor. Previous studies have shown K5 exposure promotes caspase activity and apoptosis in endothelial cells. Here we report that K5 treatment evokes an autophagic response in endothelial cells that is specific and initiated even in the absence of nutritional stress. Endothelial cells exposed to K5 up-regulated Beclin 1 levels within a few hours. Furthermore, progressively increasing amounts of antiapoptotic Bcl-2 were found to be complexed with Beclin 1, although total levels of Bcl-2 remained unchanged. Prolonged exposure to K5 ultimately led to apoptosis via mitochondrial membrane depolarization and caspase activation in endothelial cells. Knocking down Beclin 1 levels by RNA interference decreased K5 induced autophagy but accelerated K5-induced apoptosis. These studies suggest that interfering with the autophagic survival response can potentiate the antiangiogenic effects of Kringle 5 in endothelial cells.

Adeno-associated virus-mediated delivery of a mutant endostatin in combination with carboplatin treatment inhibits orthotopic growth of ovarian cancer and improves long-term survival.

A human ovarian cancer cell line, which migrates to mouse ovaries and establishes peritoneal carcinomatosis, was used to evaluate the cooperative effect of an antiangiogenic gene therapy combined with chemotherapy. The ovarian carcinoma cell line MA148 was genetically modified by "Sleeping Beauty" transposon-mediated delivery of DsRed2 fluorescent protein. Stable, high-level expression of DsRed protein enabled in vivo imaging of peritoneal dissemination of ovarian cancer. Both external and internal imaging, along with histopathology, showed migration of i.p. injected human ovarian cancer cell line to mouse ovaries. Using this model, we evaluated the effect of adeno-associated virus (AAV)-mediated expression of a mutant endostatin either alone or in combination with carboplatin treatment. A single i.m. injection of recombinant AAV (rAAV)-mutant human endostatin with P125A substitution (P125A-endostatin) showed sustained expression of mutant endostatin. Antiangiogenic gene therapy inhibited orthotopic growth of ovarian cancer and resulted in 33% long-term tumor-free survival. A single cycle of carboplatin treatment combined with mutant endostatin gene therapy resulted in 60% of the animals remaining tumor free for >200 days, which was significantly better than rAAV-LacZ and/or carboplatin. Combination treatment delayed tumor appearance in 40% of the animals, wherein the residual tumors were smaller in size with limited or no peritoneal metastasis. These studies suggest that AAV-mediated gene therapy of P125A-endostatin in combination with carboplatin is a useful method to inhibit peritoneal dissemination of ovarian carcinoma.