Angiogenic Switch Research Articles

Targeting Metabolism to Counteract Tumor Angiogenesis: A Review of Patent Literature.

Massive vessel recruitment is required to sustain rapid tumor growth by delivering oxygen and nutrients. Current strategies to counteract angiogenesis are mostly aimed at reducing tumor vessel density. However, many of these drugs have been shown to trigger hypoxia, thus exacerbating tumor aggressiveness. Promising results come from a completely different approach based on the "normalization" of the endothelial layer and the consequent improvement of the vascular function. This new strategy would ameliorate drug delivery to the tumor meanwhile reducing invasiveness and metastatisation. Since endothelial metabolism has proved essential in the regulation of the angiogenic switch, many recent patents focus on agents able to inhibit specific metabolic pathways in Tumor- Associated Endothelial Cells (TECs) in order to provide vessel normalization. Here, we provide a review of the recent advances in the development of patents on agents targeting endothelial metabolism that have proved effective in several vascular disorders. Results of genetic and pharmacologic studies that brought to the development of patents for methods to counteract aberrant angiogenesis were analysed and sub-divided according to the specific metabolic pathway targeted. Growing evidences indicate that targeting specific molecular players involved in the endothelial metabolic remodelling required to sustain aberrant angiogenesis, is a valuable therapeutic strategy that can be exploited in vascular disorders as well as in tumor angiogenesis. These findings might have important implications in clinics and could be particularly relevant to patients developing resistance to traditional anti-angiogenic drugs.

MT4-MMP Modulates the Expression of miRNAs in Breast Cancer Cells

MT4-MMP is a member of the metalloproteinases family, although with a controversial role in the extracellular matrix remodelation. Overexpression of this metalloproteinase has been observed in breast cancer and it has been suggested that it can regulate tumor growth and cancer progression. The mechanisms by which MT4-MMP participates in breast cancer includes tumor blood vessels desestabilization, the activation of an angiogenic switch, and increase of EGFR signaling. However, all the mechanisms by which MT4-MMP participates in breast cancer are still unknowns. To study if MT4-MMP could modulate the expression of microRNAs (miRNAs) related to biological processes associated with tumor formation and progression. MT4-MMP was ectopically overexpressed in MDA-MB-231cells and the miRNAs expression profile modulated by the metalloproteinase was studied by using miRNAs microarrays. Microarray data were analyzed with different tools to find the molecular and cellular functions related to the differentially expressed miRNAs. The clinical relevance of some miRNAs was analyzed using a public database. MT4-MMP overexpression in breast cancer cells induced the modulation of 65 miRNAs, which were related to the alteration of pathways dependent of p53, TGF-β, MAPK, ErbB, and Wnt, as well as processes such as cell cycle, adherens junctions, apoptosis, and focal adhesion. Several of the upregulated miRNAs were associated to a worse prognosis in breast cancer patients. In breast cancer cells, the overexpression of MT4-MMP modulates the expression of miRNAs involved in several biological processes associated with tumor formation and progression and with clinical relevance.

The Warburg Effect in Endothelial Cells and its Potential as an Anti-angiogenic Target in Cancer.

Endothelial cells (ECs) make up the lining of our blood vessels and they ensure optimal nutrient and oxygen delivery to the parenchymal tissue. In response to oxygen and/or nutrient deprivation, ECs become activated and sprout into hypo-vascularized tissues forming new vascular networks in a process termed angiogenesis. New sprouts are led by migratory tip cells and extended through the proliferation of trailing stalk cells. Activated ECs rewire their metabolism to cope with the increased energetic and biosynthetic demands associated with migration and proliferation. Moreover, metabolic signaling pathways interact and integrate with angiogenic signaling events. These metabolic adaptations play essential roles in determining EC fate and function, and are perturbed during pathological angiogenesis, as occurs in cancer. The angiogenic switch, or the growth of new blood vessels into an expanding tumor, increases tumor growth and malignancy. Limiting tumor angiogenesis has therefore long been a goal for anticancer therapy but the traditional growth factor targeted anti-angiogenic treatments have met with limited success. In recent years however, it has become increasingly recognized that focusing on altered tumor EC metabolism provides an attractive alternative anti-angiogenic strategy. In this review, we will describe the EC metabolic signature and how changes in EC metabolism affect EC fate during physiological sprouting, as well as in the cancer setting. Then, we will discuss the potential of targeting EC metabolism as a promising approach to develop new anti-cancer therapies.

Phenotypic miRNA Screen Identifies miR-26b to Promote the Growth and Survival of Endothelial Cells

Endothelial cell (EC) proliferation is a crucial event in physiological and pathological angiogenesis. MicroRNAs (miRNAs) have emerged as important modulators of the angiogenic switch. Here we conducted high-content screening of a human miRNA mimic library to identify novel regulators of EC growth systematically. Several miRNAs were nominated that enhanced or inhibited EC growth. Of these, we focused on miR-26b, which is a conserved candidate and expressed in multiple human EC types. miR-26b overexpression enhanced EC proliferation, migration, and tube formation, while inhibition of miR-26b suppressed the proliferative and angiogenic capacity of ECs. A combinatory functional small interfering RNA (siRNA) screening of 48 predicted gene targets revealed that miR-26b enhanced EC growth and survival through inhibiting PTEN expression. Local administration of miR-26b mimics promoted the growth of new microvessels in the Matrigel plug model. In the mouse model of hindlimb ischemia, miR-26b was found to be downregulated in endothelium in the first week following ischemia, and local overexpression of miR-26b improved the survival of capillaries and muscle fibers in ischemic muscles. Our findings suggest that miR-26b enhances EC proliferation, survival, and angiogenesis. miR-26b is a potential target for developing novel pro-angiogenic therapeutics in ischemic disease.

Expression of ID4 protein in breast cancer cells induces reprogramming of tumour-associated macrophages

BackgroundAs crucial regulators of the immune response against pathogens, macrophages have been extensively shown also to be important players in several diseases, including cancer. Specifically, breast cancer macrophages tightly control the angiogenic switch and progression to malignancy. ID4, a member of the ID (inhibitors of differentiation) family of proteins, is associated with a stem-like phenotype and poor prognosis in basal-like breast cancer. Moreover, ID4 favours angiogenesis by enhancing the expression of pro-angiogenic cytokines interleukin-8, CXCL1 and vascular endothelial growth factor. In the present study, we investigated whether ID4 protein exerts its pro-angiogenic function while also modulating the activity of tumour-associated macrophages in breast cancer.MethodsWe performed IHC analysis of ID4 protein and macrophage marker CD68 in a triple-negative breast cancer series. Next, we used cell migration assays to evaluate the effect of ID4 expression modulation in breast cancer cells on the motility of co-cultured macrophages. The analysis of breast cancer gene expression data repositories allowed us to evaluate the ability of ID4 to predict survival in subsets of tumours showing high or low macrophage infiltration. By culturing macrophages in conditioned media obtained from breast cancer cells in which ID4 expression was modulated by overexpression or depletion, we identified changes in the expression of ID4-dependent angiogenesis-related transcripts and microRNAs (miRNAs, miRs) in macrophages by RT-qPCR.ResultsWe determined that ID4 and macrophage marker CD68 protein expression were significantly associated in a series of triple-negative breast tumours. Interestingly, ID4 messenger RNA (mRNA) levels robustly predicted survival, specifically in the subset of tumours showing high macrophage infiltration. In vitro and in vivo migration assays demonstrated that expression of ID4 in breast cancer cells stimulates macrophage motility. At the molecular level, ID4 protein expression in breast cancer cells controls, through paracrine signalling, the activation of an angiogenic programme in macrophages. This programme includes both the increase of angiogenesis-related mRNAs and the decrease of members of the anti-angiogenic miR-15b/107 group. Intriguingly, these miRNAs control the expression of the cytokine granulin, whose enhanced expression in macrophages confers increased angiogenic potential.ConclusionsThese results uncover a key role for ID4 in dictating the behaviour of tumour-associated macrophages in breast cancer.

Does metronomic chemotherapy induce tumor angiogenic dormancy? A review of available preclinical and clinical data

Tumor dormancy is the ability of cancer cells to survive in a non-proliferating state. This condition can depend on three main mechanisms: cell cycle arrest (quiescence or cell dormancy), immunosurveillance (immunologic dormancy), or lack of functional blood vessels (angiogenic dormancy). In particular, under angiogenic dormancy, cancer cell proliferation is counterbalanced by apoptosis owing to poor vascularization, impeding tumor mass expansion beyond a microscopic size, with an asymptomatic and non-metastatic state. Tumor vasculogenic or non-angiogenic switch is essential to promote escape from tumor dormancy, leading to tumor mass proliferation and metastasis. In avascular lesions angiogenesis process results blocked from the equilibrium between pro- and anti-angiogenic factors, such as vascular endothelial growth factor (VEGF) and thrombospondin-1 (TSP-1), respectively. The angiogenic switch mainly depends on the disruption of this balance, in favor of pro-angiogenic factors, and on the recruitment of circulating endothelial progenitors (CEPs) that promote the formation of new blood vessels. Metronomic chemotherapy, the regular intake of doses able to sustain low but active concentrations of chemotherapeutic drugs during protracted time periods, is an encouraging therapeutic approach that has shown to upregulate anti-angiogenic factors such as TSP-1 and decline pro-angiogenic factors such as VEGF, suppressing the proangiogenic cells such as CEPs. In this perspective, metronomic chemotherapy may be one of the available therapeutic approaches capable to modulate favorably the angiogenic tumor dormancy, but further research is essential to better define this particular characteristic.

Involvement of NADPH Oxidase 1 in Liver Kinase B1-Mediated Effects on Tumor Angiogenesis and Growth.

The liver kinase B1 (LKB1) gene is a tumor suppressor with an established role in the control of cell metabolism and oxidative stress. However, whether dis-regulated oxidative stress promotes growth of LKB1-deficient tumors remains substantially unknown. Through in vitro studies, we observed that loss of LKB1 perturbed expression of several genes involved in reactive oxygen species (ROS) homeostasis. In particular, this analysis evidenced strongly up-modulated NADPH oxidase 1 (NOX1) transcript levels in tumor cells lacking LKB1. NOX1 accounted in part for enhanced cytotoxic effects of H2O2-induced oxidative stress in A549 LKB1-deficient tumor cells. Notably, genetic and pharmacologic inhibition of NOX1 activity reduced angiogenesis and growth of A549 tumors in mice. These results suggest that NOX1 inhibitors could counteract ROS production and the angiogenic switch in LKB1-deficient tumors.

A putative G-quadruplex structure in the proximal promoter of VEGFR-2 has implications for drug design to inhibit tumor angiogenesis

Tumor angiogenesis is mainly regulated by vascular endothelial growth factor (VEGF) produced by cancer cells. It is active on the endothelium via VEGF receptor 2 (VEGFR-2). G-quadruplexes are DNA secondary structures formed by guanine-rich sequences, for example, within gene promoters where they may contribute to transcriptional activity. The proximal promoter of VEGFR-2 contains a G-quadruplex, which has been suggested to interact with small molecules that inhibit VEGFR-2 expression and thereby tumor angiogenesis. However, its structure is not known. Here, we determined its NMR solution structure, which is composed of three stacked G-tetrads containing three syn guanines. The first guanine (G1) is positioned within the central G-tetrad. We also observed that a noncanonical, V-shaped loop spans three G-tetrad planes, including no bridging nucleotides. A long and diagonal loop, which includes six nucleotides, connects reversal double chains. With a melting temperature of 54.51 °C, the scaffold of this quadruplex is stabilized by one G-tetrad plane stacking with one nonstandard bp, G3-C8, whose bases interact with each other through only one hydrogen bond. In summary, the NMR solution structure of the G-quadruplex in the proximal promoter region of the VEGFR-2 gene reported here has uncovered its key features as a potential anticancer drug target.

P-338 - NADPH oxidase regulates the expression of angiogenic growth factors in Cerebral Cavernous Malformation cellular models

Cerebral Cavernous Malformation (CCM) is a neurovascular disease characterized by abnormal vascular structures associated with defects on vascular lumen formation and vascular fragility, mostly localized in the brain. Lack of KRIT1 protein associated to CCM drives the stable and quiescent endothelium toward an active state characterized by loss of barrier function, increased vascular permeability, loss of adherens junction integrity, increased β-catenin nuclear localization and vascular endothelial growth factor (VEGF) expression and angiogenesis. Multiple pathways are responsible for changes in endothelial phenotype and we contributed to demonstrate that the dysregulation of reactive oxygen species (ROS) production following loss of KRIT1 expression play a key role in increasing vascular permeability and is linked to NADPH oxidase (NOX) up-regulation. By using wt and KRIT1-/- mouse embryonic fibroblasts (MEFs) we showed that loss of KRIT1 induced NOX4 overexpression that is associated with alteration of angiogenic growth factors expression and with the angiogenic switch of endothelial cells. Our data contribute to demonstrate the pivotal role of oxidative stress in the progression of CCM disease, indicating NOX4 and ROS as potential targets for pharmaceutical approaches.

ROS-induced ROS release orchestrated by Nox4, Nox2, and mitochondria in VEGF signaling driving endothelial metabolism and angiogenesis

Reactive oxygen species (ROS) derived from NADPH oxidase (NOX) as well as mitochondria play a critical role in promoting angiogenic switch from a quiescent endothelial cells (ECs). However, the mechanism by which ROS produced from different sources can organize to enhance VEGF signaling and angiogenic process remains unknown. Using the cytosol- and mitochondria-targed redox-sensitive RoGFP biosensors with real-time imaging, we found that VEGF stimulation in human ECs rapidly increases Nox4-derived H2O2 that activates Nox2 to produce ROS in the cytosol, which in turn induce mitochondrial ROS (mtROS) production via phosphorylation of p66Shc at Ser36. This ROS-induced ROS release orchestrated by the Nox4/Nox2/pS36-p66Shc/mtROS axis promotes activation and sustained angiogenesis signaling program. Moreover, emerging evidence suggest that EC metabolism and mitochondria dynamics regulated by fission and fusion also regulate angiogenesis; however, their relationships with redox signaling remains unclear. Here we provide the novel mechanism for redox regulation of mitochondria fission protein Drp1 and for how VEGF-induced ROS are sensed via Cysteine oxidation to promote glycolysis driving formation of new blood vessels.

Inhibition of mTOR complex 2 restrains tumor angiogenesis in multiple myeloma.

The mammalian Target of Rapamycin (mTOR) is an intracellular serine/threonine kinase that mediates intracellular metabolism, cell survival and actin rearrangement. mTOR is made of two independent complexes, mTORC1 and mTORC2, activated by the scaffold proteins RAPTOR and RICTOR, respectively. The activation of mTORC1 triggers protein synthesis and autophagy inhibition, while mTORC2 activation promotes progression, survival, actin reorganization, and drug resistance through AKT hyper-phosphorylation on Ser473. Due to the mTOR pivotal role in the survival of tumor cells, we evaluated its activation in endothelial cells (ECs) from 20 patients with monoclonal gammopathy of undetermined significance (MGUS) and 47 patients with multiple myeloma (MM), and its involvement in angiogenesis. MM-ECs showed a significantly higher expression of mTOR and RICTOR than MGUS-ECs. These data were supported by the higher activation of mTORC2 downstream effectors, suggesting a major role of mTORC2 in the angiogenic switch to MM. Specific inhibition of mTOR activity through siRNA targeting RICTOR and dual mTOR inhibitor PP242 reduced the MM-ECs angiogenic functions, including cell migration, chemotaxis, adhesion, invasion, in vitro angiogenesis on Matrigel®, and cytoskeleton reorganization. In addition, PP242 treatment showed anti-angiogenic effects in vivo in the Chick Chorioallantoic Membrane (CAM) and Matrigel® plug assays. PP242 exhibited a synergistic effect with lenalidomide and bortezomib, suggesting that mTOR inhibition can enhance the anti-angiogenic effect of these drugs. Data to be shown indicate that mTORC2 is involved in MM angiogenesis, and suggest that the dual mTOR inhibitor PP242 may be useful for the anti-angiogenic management of MM patients.

Shengui Sansheng San extraction is an angiogenic switch via regulations of AKT/mTOR, ERK1/2 and Notch1 signal pathways after ischemic stroke

BackgroundAs a traditional Chinese herbal formula, Shengui Sansheng San (SSS) has been employed for stroke treatment more than 300 years. PurposeWe hypothesize that SSS extraction is an angiogenic switch in penumbra post-stroke, and corresponding mechanisms are investigated. MethodsIn present study, rats were subjected to permanent middle cerebral artery occlusion model (MCAo) and were treated with low, middle and high doses of SSS extraction. We assessed neurological function and survival rate, and measured infarct volume by 2,3,5-triphenyltetrazolium chloride staining on day 7 after ischemia. von Willebrand factor (vWF), stromal cell-derived factor-1 alpha (SDF-1α) /chemokine (C-X-C motif) receptor 4 (CXCR4) axis, vascular endothelial growth factor (VEGF)/VEGF receptor 2 (VEGFR2) as well as protein kinase B (AKT)/mammalian target of rapamycin (mTOR) /hypoxia-inducible factor-1 alpha (HIF-1α), extracellular signal-regulated kinase 1/2 (ERK1/2) and Notch1 signaling pathways were respectively investigated by immunofluorescence assay or western blotting in vivo and oxygen–glucose-deprived (OGD) brain microvascular endothelial cells (BMECs); simultaneously, wound healing of BMECs and tube formation assay were administrated. ResultsCompared to MCAo group, SSS extraction could significantly improve neurological functional scores, survival rate and cerebral infarct volume, enhance vWF+ vascular density and perimeter, SDF-1α/CXCR4 axis, VEGF expression, as well as activate AKT/mTOR/HIF-1α and ERK1/2 and inhibit Notch1 pathways in penumbra. In vitro, containing SSS extraction serum increased BMEC migration, capillary formation and VEGF expression via up-regulations of AKT/mTOR and ERK1/2 pathways in OGD BMECs, but ERK inhibitor (U0126) reversed the result of VEGF expression in high dose of SSS group. Additionally, VEGFR2 and Notch1 expressions were suppressed by containing SSS extraction serum. All results were in dose dependent manner. ConclusionOur study firstly demonstrates that SSS extraction is an angiogenic switch. Due to suppressed VEGFR2/Notch1 cascades and activated AKT/mTOR and ERK1/2 signals in BMECs, a feedback loop of angiogenic homeostasis is established. Furthermore, the comprehensive mediations of SDF-1α/CXCR4 axis, AKT/mTOR/HIF-α, ERK1/2 and Notch1 pathways in penumbra contribute to the improvements of neurological function, survival rate and infarct volume post-stroke.

\u0394Np73 enhances HIF-1\u03b1 protein stability through repression of the ECV complex

Cellular responses to low oxygen conditions are mainly regulated by the Hypoxia-inducible factors (HIFs). Induction of HIF-1α in tumor cells activates the angiogenic switch and allows for metabolic adaptations. HIF-1α protein levels are tightly regulated through ubiquitin-mediated proteosomal degradation; however, high levels of HIF-1α is a common feature in many solid tumors and is thought to enhance cancer cell proliferation, migration, and survival. Here, we report that the oncogenic p73 isoform, ∆Np73, increases HIF-1α protein stability. We found that ∆Np73 represses expression of genes encoding subunits of the ECV complex, in particular Elongin C, Elongin B, Cullin 2, and Rbx1. The ECV complex is an E3 ligase complex responsible for polyubiquitinating HIF-1α. Loss of ∆Np73 increases ubiquitination of HIF-1α, leading to its degradation via the proteosomal pathway, and subsequent decrease of HIF-1α target genes. Taken together, our data demonstrates that high levels of ∆Np73 stabilize HIF-1α protein, allowing for it to accumulate and further potentiating its transcriptional activity and supporting tumor progression.

Endothelial progenitor cells contribute to neovascularization of non-small cell lung cancer via histone deacetylase 7-mediated cytoskeleton regulation and angiogenic genes transcription.

To supply tumor tissues with nutrients and oxygen, endothelial progenitor cells (EPCs) home to tumor sites and contribute to neovascularization. Although the precise mechanism of EPCs-induced neovascularization remains poorly understood in non-small cell lung cancer (NSCLC), histone deacetylase 7 (HDAC7) is considered as a critical regulator. To explore the function of HDAC7 in neovascularization induced by EPCs, tube formation assay, immunofluorescence, microarray, Western blot analysis and animal models were performed. In vitro, HDAC7 abrogation led to the activation of Rho-associated coiled-coil containing protein kinase/myosin light chain 2 pathway concomitant with ERK dephosphorylation, causing the instability of cytoskeleton and collapse of tube formation. In vivo, absence of HDAC7 impaired the vascular lumen integrity and decreased the functional blood perfusion, inhibiting the growth of tumor. At the level of transcription, HDAC7 silencing upregulated antiangiogenic genes and suppressed proangiogenic genes collectively, turning off the angiogenic switch during vessel formation. Taken together, HDAC7 plays a dual role in maintaining the structural and nonstructural functions of EPCs. Our work demonstrates the molecular mechanism by which HDAC7 contributes to the angiogenic property of EPCs and provides a rational basis for specific targeting of antiangiogenic strategies in lung cancer.

MicroRNA Key to Angiogenesis Regulation: MiRNA Biology and Therapy.

Angiogenesis is involved in maintaining normal physiological processes like embryonic development, wound healing, inflammation and reproduction. Pathogenesis of various diseases like diabetic retinopathy, rheumatoid arthritis and cancer are associated with imbalanced angiogenesis. Angiogenic stimulators and inhibitors act together for keeping angiogenic switch in balance. Recently, miRNAs have been found to regulate various stages of angiogenesis. miRNAs are 21-23 nucleotides long, single stranded, noncoding RNA molecules generated endogenously. miRNA's ability to target multiple genes within a signaling pathway makes them promising target for the development of second generation anti-angiogenesis drugs. This review was conceived with the notion of availability of specific and comprehensive knowledge about AngiomiRs at one place. This will facilitate the research in basic understanding and in the development of new drugs. In this review, we have summarized the biology and therapeutic potential of the miRNAs, which are involved in controlling angiogenesis process. In miRNA biology, we have provided the updated summary of miRNAs in the regulation of endothelial cells, showed role of miRNAs in the signaling pathways of angiogenesis and, discussed the gaps in complete knowledge of mechanism. We have also provided exclusive insights regarding therapeutic potential of these miRNAs, in angiogenesis related disorders. Additionally, we have discussed the challenges in miRNA based drug delivery and updated the current efforts in the development of miRNA delivery methods. Though much research is needed to discover the complete miRNA network regulating angiogenesis but once it is done, targeting miRNA may be considered as a potential candidate for therapeutic invention against angiogenesis related disorders.

A Phase I/II Study Targeting Angiogenesis Using Bevacizumab Combined with Chemotherapy and a Histone Deacetylase Inhibitor (Valproic Acid) in Advanced Sarcomas.

Epigenetic events and genetic alterations under the control of the tumor microenvironment potentially mediate tumor induced angiogenesis involved in soft tissue sarcoma (STS) metastasis. Addition of antiangiogenic agent, such as bevacizumab, to standard chemotherapy in treatment of sarcoma has been studied in clinical trials, but most of the findings have not supported its use. We hypothesized the existence of an epigenetically mediated “angiogenic switch”, and the tumor microenvironment, prevents bevacizumab from truly blocking angiogenesis. The addition of valproic acid (VPA), a weak histone deacetylase inhibitor, and bevacizumab, a monoclonal antibody against vascular endothelial growth factor, together with the cytotoxic effects of gemcitabine and docetaxel, may enhance responses and alter chemoresistance. This was designed as a phase I/II trial with primary endpoints including safety of the treatment combination and tumor response. Unresectable or metastatic sarcoma patients >18 years of age, irrespective of number of prior treatments, received VPA 40 mg/kg orally for 5 days prior to day 1, bevacizumab at 15 mg/kg IV on day 1, gemcitabine 900 mg/m2 (day 1, day 8), and docetaxel 75 mg/m2 (day 8). Cycles were of 28 day duration. Bevacizumab and VPA were continued as maintenance after 6 cycles, until disease progression. A standard 3 + 3 phase I dose de-escalation design was utilized to evaluate safety. Gain of function p53 gene mutation testing was performed on available archival tissue specimens. A total of 46 patients (30 female, 16 male) with median age of 60 (range 24–81) years were enrolled; 34 (73.9%) patients received prior chemotherapy, 14 (30%) of which received prior gemcitabine and docetaxel. Patients received a median of 5.5 cycles (range 0–24 of treatment (min 0, one patient died prior to completing the first cycle; max: 24, one patient received 6 cycles and 18 maintenance cycles before progressing). Seventeen patients underwent dose reduction, of which VPA was reduced in 6 patients. Forty-one patients were evaluable for response. There was a confirmed complete response in 1 (epithelioid sarcoma), and a partial response (PR) in 6 (1 carcinosarcoma, 2 extrauterine leiomyosarcoma (LMS), 2 undifferentiated pleomorphic sarcoma, and 1 uterine LMS) patients. Stable disease (SD) was seen in 21 patients for at least 2 months. One subject with prior gemcitabine and docetaxel had PR, and 7 had SD. Median progression-free survival (PFS) was 5.7 months (95% CI: 2.1–8.0), and overall survival (OS) was 12.9 months (95% CI: 8.3–14.5). Three patients died due to tumor progression while on the study. The combination of VPA, bevacizumab, gemcitabine, and docetaxel appears to be moderately safe and well tolerated. Given that there are very limited options for patients with relapsed refractory STS, this drug combination may be an important therapy to consider. This combination treatment deserves further investigation in epithelioid and carcinosarcoma subtypes.

Extracellular Vesicles As miRNA Nano-Shuttles: Dual Role in Tumor Progression.

Tumor-derived extracellular vesicles (EVs) have a pleiotropic role in cancer, interacting with target cells of the tumor microenvironment, such as fibroblasts, immune and endothelial cells. EVs can modulate tumor progression, angiogenic switch, metastasis, and immune escape. These vesicles are nano-shuttles containing a wide spectrum of miRNAs that contribute to tumor progression. MiRNAs contained in extracellular vesicles (EV-miRNAs) are disseminated in the extracellular space and are able to influence theexpression of target genes with either tumor suppressor or oncogenic functions, depending on bothparental and target cells. Metastatic cancer cells can balance their oncogenic potential by expressing miRNAs with oncogenic function, whilst exporting miRNAs with tumor suppressor roles out of the cells. Importantly, treatment of cancer cells with specific natural and chemical compounds could induce the elimination of miRNAs with oncogenic function, thereby reducing their aggressiveness. In this review, we discuss the mechanisms by which EV-miRNAs, acting as miRNAs with oncogenic or tumor suppressor functions, could contribute to cancer progression.

P064 INTERFERON-GAMMA INDUCED VASCULAR IMPAIRMENT CONTRIBUTES TO THE PATHOGENESIS OF INFLAMMATORY BOWEL DISEASES

Inflammatory Bowel Diseases (IBD) are characterized by upregulation of Interferon-gamma (IFN-γ). Recently we showed that IFN-γ blockade by a specific antibody in Dextran Sodium Sulfate (DSS)-colitis mouse model results in increased angiogenesis and reduced vessel permeability(1). The aim of the present project was to validate the specific contribution of vascular effects of IFN-γ to the pathogenesis of IBD. For examination of vascular IFN-γ effects in vivo, we established an endothelial cell-specific Ifngr2-knock-out (Ifngr2ΔEndoC) mouse model. Vessel sprouting after IFN-γ stimulation of metatarsals was used for functional validation of the knock out. In order to analyze the specific contribution of vascular effects of IFN-γ to the pathogenesis of IBD, Ifngr2ΔEndoC and control mice were subjected to DSS-colitis. Angiogenesis and vascular barrier function of the colon were compared. Comparison of metatarsal angiogenic sprouting from Ifngr2ΔEndoC and control mice showed that endothelial-specific knock-out of Ifngr2 abrogates the angiostatic effects of IFN-γ. Induction of DSS-colitis revealed that Ifngr2ΔEndoC mice, compared to control mice, had a reduced inflammation of the colon. Investigation of specific vascular alterations showed an increased angiogenesis rate for Ifngr2ΔEndoC mice accompanied by an improved vascular barrier function, marked by higher vascular pericyte coverage and reduced vessel permeability. Our results indicate for first time an important pathofunction of the blood vessels in IBD. Specifically an IFN-γ-induced disruption of the vascular barrier function exaggerated the course of disease. This may open new therapy options. 1. Haep et al. Interferon Gamma Counteracts the Angiogenic Switch and Induces Vascular Permeability in Dextran Sulfate Sodium Colitis in Mice. Inflamm Bowel Dis. 2015.

Abstract B23: A pilot study targeting angiogenesis using bevacizumab combined with gemcitabine/docetaxel and valproic acid (VPA), a histone deacetylase inhibitor (HDACi), in advanced soft tissue sarcomas (STS)

Abstract Introduction: Many of the signaling pathways involved in angiogenesis also drive STS tumor cell growth. Autocrine and paracrine vascular endothelial growth factor (VEGF) and platelet-derived growth factor mediated growth plays a role in the pathogenesis of several STS subtypes. Relatively few clinical trials have evaluated the role of combining angiogenesis inhibitors, like bevacizumab, with chemotherapy in sarcoma with mostly negative results. We hypothesized that the microenvironment may prevent bevacizumab from truly blocking angiogenesis and that there may exist an “angiogenic switch” that is epigenetically mediated. Epigenetically modifying the tumor microenvironment prior to chemotherapy has been shown to improve cytotoxic cell kill and response rates. Gain-of-function p53 mutant tumors are poor chemotherapy responders and cause genome-wide increase in histone methylation and acetylation. VPA is a known HDACi with antitumor effects and suppresses neovascularization. Herein we combined pulse dose of VPA with bevacizumab and standard chemotherapy (gemcitabine/docetaxel) to better modulate the cytotoxic effects against STS. Materials and Methods: Subjects with locally advanced, unresectable, or metastatic STS of any site and all subtypes with measurable disease were enrolled. GIST, bone, and Kaposi's sarcomas were excluded. VPA at 40-mg/kg dosage was given for 5 days prior to each cycle and was repeated before each cycle. Gemcitabine 900 mg/m2 on days 1 and 8 and docetaxel 75 mg/m2 on day 8 were administered on a 28-day schedule. Bevacizumab 15 mg/kg every 4 weeks was administered on day 1 throughout chemotherapy and after chemotherapy was completed. Subjects were treated for a total of 6 cycles and assessed every 2 cycles for progression. Treatment was terminated on disease progression or with unacceptable toxicity. All pathologic diagnoses were confirmed by a pathologist with subspecialty training in bone and soft-tissue pathology. Results: 46 subjects (30 female, 16 male) with median age of 60 (range 24-81) years and a median of 1 (range 0-6) prior line of chemotherapy were enrolled. Sarcoma subtypes included extrauterine leiomyosarcoma (LMS) (9), uterine LMS (8), undifferentiated pleomorphic (7), angiosarcoma, carcinosarcoma, liposarcoma (4 each), and other types (10). 34 (73.9%) subjects received prior chemotherapy, 14 (30%) of whom received prior gemcitabine and docetaxel. 16 (34.8%) received prior radiation and 44 (95.7%) had prior surgery. 5 (12%) of 41 subjects had gain-of-function p53 mutation. Subjects received a median of 5.5 cycles (0-15) of treatment. 17 subjects underwent dose reduction of which VPA was reduced in 6 subjects. 42 subjects were evaluable for response. There was a complete response in 1 (epithelioid) and a partial response (PR) in 6 (1 carcinosarcoma, 2 extrauterine LMS, 2 undifferentiated pleomorphic, and 1 uterine LMS) subjects. Stable disease (SD) was seen in 21 subjects. One subject with prior gemcitabine and docetaxel had PR and 7 had SD. Median progression-free survival (PFS) was 5.7 months (95% CI: 2.1-8) and overall survival (OS) was 12.9 months (95% CI: 8.3-14.5). In LMS subgroup median PFS was 8.4 months (95% CI: 2-8.6) and median OS was 16.3 months (95% CI: 8.1-26.5). Grade 3/4 bevacizumab and VPA-related hematologic toxicities were observed in 2% and 13% and nonhematological toxicities in 9% and 13% of subjects, respectively. Conclusions: Addition of VPA and bevacizumab to gemcitabine and docetaxel showed a clinical benefit rate of 61.54 % in subjects with prior gemcitabine and docetaxel. This regimen is safe and toxicities are manageable. The overall response rate in the study was 16.67%. The combination deserves further investigation in epithelioid and carcinosarcoma subgroups. Citation Format: Umang Swami, Varun Monga, Munir Tanas, Aaron Bossler, Sarah Mott, Brian Smith, Mohammed Milhem. A pilot study targeting angiogenesis using bevacizumab combined with gemcitabine/docetaxel and valproic acid (VPA), a histone deacetylase inhibitor (HDACi), in advanced soft tissue sarcomas (STS) [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr B23.

Integration of pro- and anti-angiogenic signals by endothelial cells.

Angiogenesis or neovascularization is a complex multi-step physiological process that occurs throughout life both in normal tissues and in disease. It is tightly regulated by the balance between pro-angiogenic and anti-angiogenic factors. The angiogenic switch has been identified as the key step during tumor progression in which the balance between pro-angiogenic and anti-angiogenic factors leans toward pro-angiogenic stimuli promoting the progression of tumors from dormancy to dysplasia and ultimately malignancy. This event can be described as either the outcome of a genetic event occurring in cancer cells themselves, or the positive and negative cross-talk between tumor-associated endothelial cells and other cellular components of the tumor microenvironment. In recent years, the mechanisms underlying the angiogenic switch have been extensively investigated in particular to identify therapeutic targets that can lead to development of effective therapies. In this review, we will discuss the current findings on the regulatory pathways in endothelial cells that are involved in the angiogenic switch with an emphasis on the role of anti-angiogenic protein, thrombospondin-1 (TSP-1) and pro-angiogenic factor, vascular endothelial growth factor (VEGF).