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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 anti-angiogenic activity of endostatin. Since endostatin has relatively shorter serum half-life, frequent dosing was required for inhibiting tumor growth. In the present study, we have genetically fused RGD-P125A-endostatin to Fc of IgG4 isotype and evaluated its anti-angiogenic and anti-tumor 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 anti-angiogenic and anti-tumor activity.

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.

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 (por= 0.02) and proliferative capacity of implanted cells significantly increased (por= 0.01) with the IGF-1-transduced myoblasts. We conclude that the genetic modification of skeletal myoblasts with the IGF-1 gene offers a potential means for enhanced cell survival following transplantation.

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.

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. (Cancer Res 2006; 66(8): 4319-28)

Vascular Endothelial Growth Factor (VEGF) functions as a key regulator in tumor angiogenesis. In addition, VEGF is an important survival factor for endothelial cells under chemical or physical stress. In our report, we show that treatment of endothelial cells with the chemotherapeutic agent carboplatin significantly increased the expression of VEGF. Furthermore, neutralization of secreted VEGF with specific polyclonal anti-VEGF antibodies or monoclonal antibody sensitized endothelial cells to carboplatin treatment and increased apoptosis several-fold. Interestingly, carboplatin treatment did not alter VEGF expression in tumor cells. Similarly, antibody to VEGF did not change the chemosensitivity of tumor cells to this drug. Most importantly, tumor-bearing animals treated with carboplatin showed an increase in VEGF immunoreactivity in the tumor vasculature, confirming the in vitro studies. Based on these observations, we determined whether neutralization of VEGF could enhance the anti-tumor activity of carboplatin in an in vivo ovarian cancer model system. A combination therapy consisting of a suboptimal dose of carboplatin (32.5 mg/kg/inj., q3d x 5; i.p.) and polyclonal anti-VEGF antibody (2 mg/inj., q3d x 10; i.p.) significantly enhanced solid tumor growth inhibition over individual monotherapies and included multiple complete responses. These findings suggest that VEGF is a critical endothelial cell specific survival factor that is induced by carboplatin and contributes to the protection of tumor vasculature during chemotherapy treatment. In addition, these results provide evidence for a potential mechanism that underlies enhanced anti-tumor activity achieved with chemotherapy and anti-VEGF antibody combination treatment regimens as recently reported in a number of clinical trials. We conclude that a similar type of combination therapy may be applicable to many types of malignancies since VEGF expression was differentially induced in the tumor host environment (i.e., tumor vasculature) and not in the tumor cells themselves; hence, this phenomenon may be independent of the type and origin of the primary cancer.

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.

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 potentiates the antimitotic effects of paclitaxel (taxol) on endothelial cells (ECs). P125A-endostatin and taxol-treated ECs showed multipolar spindles and nuclear lobulation, leading to mitotic catastrophe and cell death. Induction of nuclear abnormalities was found to be dependent on β-catenin levels as wnt-mediated overexpression of β-catenin reversed the changes in nuclear morphology. These results prompted us to investigate whether antiangiogenic gene therapy and paclitaxel chemotherapy can synergistically inhibit angiogenesis and tumor growth. We first determined the effect of combination treatment in a transgenic mouse model of breast cancer. Intramuscular injection of recombinant adeno-associated virus type-2 virus induced sustained expression of P125A-endostatin. In vivo studies showed that combination therapy inhibited mammary cancer growth, delayed the onset of multifocal mammary adenocarcinomas, decreased tumor angiogenesis and increased survival in treated mice. In a second model, female athymic mice were orthotopically transplanted with a metastatic human breast cancer cell line. Antiangiogenic gene therapy in combination with paclitaxel inhibited tumor angiogenesis and lung/lymph-node metastasis in this model. These studies demonstrate cooperation between endostatin gene therapy and chemotherapy to inhibit tumor initiation, growth and metastasis.

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. [Cancer Res 2007;67(8):3683–90]

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.

Earlier studies have shown that a point mutation in human endostatin at position 125 (human endostatin wherein proline 125 was substituted with alanine, P125A-endostatin) improves endothelial cell binding and antiangiogenic activity. In the present study, we investigated the effect of recombinant adeno-associated virus (rAAV)-mediated gene delivery of P125A-endostatin (rAAV-P125Aendo) in a mouse model of ovarian carcinoma. Intramuscular (i.m.) injection of rAAV-P125Aendo resulted in a dose-dependent increase in serum endostatin levels. Consequently, vascular endothelial growth factor- and basic fibroblast growth factor-mediated angiogenesis was significantly inhibited in mice injected with rAAV-P125Aendo as compared to control mice injected with rAAV-LacZ. Furthermore, gene therapy using rAAV-P125Aendo construct showed sustained secretion of P125A-endostatin for up to 9 weeks after a single i.m. administration. Recombinant AAV-P125Aendo injection significantly inhibited the growth of human ovarian cancer cells in athymic nude mice. Immunofluorescence studies of residual tumors surgically removed from the rAAV-P125Aendo-treated animals showed decreased number of vessel ends and vessel length, indicating inhibition of angiogenesis. These studies suggest that recombinant AAV-mediated antiangiogenic gene therapy methods can be used to inhibit ovarian cancer growth.

Chronic hypoxia, a hallmark of many tumors, is associated with angiogenesis and tumor progression. Adaptive changes to oxygen availability are critical for cell survival and homeostasis. 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 a novel mechanism regulating HIF-1α and HIF-2α levels in endothelial cells during hypoxia. Hypoxia differentially increased microRNA-424 (miR-424) levels in microvascular EC, HUVEC and EC progenitor cells purified from the blood. 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 in EC was regulated by PU.1-dependent transactivation. PU.1 expression was in turn controlled by RUNX-1 and C/EBPα. Knocking down either C/EBPα or RUNX-1 or PU.1 attenuated hypoxia-driven expression of miR-424 and nuclear translocation of HIF-1α. Furthermore, miR-424 promoted angiogenesis in vitro and in mice, which was blocked by specific morpholinos. These results suggest that miR-424 plays an important physiological role in angiogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3980. doi:10.1158/1538-7445.AM2011-3980

Tumor growth and progression is intricately linked to hypoxia induced adaptive changes. Hypoxia is known to alter chemosensitivity, upregulation of putative cancer initiating stem cells and the microenvironment. The multitude of these changes is brought about by differential gene expression. Hypoxia inducible transcription factor-1 alpha (HIF-α) is a key transducer of altered gene expression under low oxygen or ischemia. In addition to the transcriptional activation of multiple genes, hypoxia also alters miRNA levels in tumor and vascular endothelial cells. miRNAs are small noncoding RNAs ∼22 nucleotides in length representing 1%-2% of the eukaryotic transcriptome. We used microarrays to determine relative changes in 467 miRNAs in endothelial and MA148 ovarian cancer cells exposed to hypoxia. One of the miRNAs, miR-210 was significantly up-regulated under hypoxia by microarray analysis (2-fold, P Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2020.

HIF1 is the main transcriptional regulator of the hypoxic response in mammalian cells and plays an important role in the adaptation of tumor cells to hypoxic microenvironments. While normally degraded under normoxic conditions via oxygen-dependent, ubiquitin-based mechanisms, HIF1α protein, which, with the constitutively expressed HIF1β forms the HIF1 heterodimer, is stabilized in hypoxic tumor cells. Stabilization of HIF1α protein under hypoxia leads to transactivation of a variety of angiogenic and tumorigenic genes including VEGF and matrix metalloproteinase 2. Clinically, high levels of HIF1 in tumors translate to increased patient morbidity and mortality. Our studies show that HIF1α, in addition to being regulated at the protein level, is also directly regulated by microRNAs, small 20-22 base ribonucleotides that specifically bind to the 3’ untranslated regions (UTRs) of target mRNA to inhibit translation or induce degradation. Based on bioinformatics and microRNA-target databases, we determined that HIF1α contains a potential binding site in its 3’ UTR for mir-199a, which, based on our microarray and RT-qPCR data, is significantly downregulated in hypoxic ovarian cancer cells compared to normoxia. Using a luciferase reporter construct in which luciferase expression is under control of the HIF1α 3’ UTR, we showed that overexpression of mir-199a in A2780 or MA148 cells leads to suppression of the luciferase signal. Mutation of the mir-199a binding site in the HIF1α 3’ UTR resulted in no change in luciferase signal with mir-199a overexpression, indicating that mir-199a directly targets the HIF1α 3’ UTR. Furthermore, decreasing mir-199a levels, either by incubating cells in hypoxia or using a morpholino specifically targeting mir-199a, leads to increased levels of HIF1α protein as determined by western blot and increased transcripts of HIF1-activated genes such as VEGF as measured by RT-qPCR. Conversely, overexpression of mir-199a leads to decreased HIF1α protein and VEGF transcript levels. Together, our findings have significant implications in the study of HIF1α regulation in cancers. Mir-199a may have a role in future therapeutic strategies to modulate the tumor microenvironment to limit hypoxia-driven tumor progression and metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2070.

Inhibiting angiogenesis is a promising strategy for cancer therapy. Endostatin, a C-terminal fragment of collagen XV[[Unsupported Character - Ш]], is an endogenous inhibitor of angiogenesis. We have earlier shown that addition of integrin-targeting sequences to a mutant endostain (P125A-endo) improved localization to tumor vasculature. In the present study, we investigated whether genetically fusing endostatin to Fc region of human IgG can prolong circulatory half-life and improve anti-tumor activity. Two types of constructs were made: a) RGD-endostatin-Fc and b) Endostatin-RGD-Fc. The location of the RGD moiety was either placed at the NH2-terminus or between the carboxyl terminus of endostatin and Fc. Both constructs were expressed in HEK-293 cells and purified using affinity chromatography. Our results show the biological activity of endostatin was significantly enhanced by linking to the Fc region of IgG when compared to P125A-endostatin expressed in yeast. Both RGD-Endo-Fc and Endo-RGD-Fc were able to bind endothelial cells very efficiently and inhibited endothelial cells proliferation, migration in vitro and angiogenesis in vivo. We then investigated the ability of the fusion protein to inhibit ovarian cancer growth in a xenograft model. Once a week administration of the fusion protein into mice bearing established ovarian tumors inhibited tumor growth and increased their survival. Anti-tumor activity of the fusion protein was comparable to Bevacizumab-mediated suppression of tumor growth. Furthermore, when mice were treated with a combination of the fusion protein and Bevacizumab, more than additive inhibition of tumor growth was observed. These data suggest that fusion with Fc-fragment can improve the anti-angiogenic and anti-tumor activity of endostatin. Moreover, our results support a strategy to combine two anti-angiogenic reagents such as anti-VEGF antibody and endostatin-Fc fusion protein to additively inhibit ovarian cancer growth. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1385.