Target For Anti-angiogenic Therapy Research Articles

Sprouting Vessels with Nrf2

Nrf2 enhances neovascularization by suppressing Delta-Notch signaling.

Angiogenic marker associated with resistance to neoadjuvant chemoradiotherapy in rectal cancer.

11030 Background: The ability to achieve pathologic down staging after neoadjuvant chemoradiotherapy (CRT) is correlated with improved survival. However, there is no effective method of predicting which patients will response to neoadjuvant CRT. Neoadjuvant CRT can change the expression of angiogenic factors. However, little is known about its possible changes in response to preoperative CRT. We examined the expression of angiogenic factors in rectal cancer tissues before preoperative CRT and after surgery. Methods: Fifty five patients with locally advanced rectal cancer were studied. All patients were given preoperative CRT of 5040 cGy for 5-6 weeks with concurrent administration of 5-fluorouracil and leucovorin. Surgical resection was performed 6–8 weeks later in all patients. Immunohistochemical staining for angiogenenic markers (vascular endothelial growth factor [VEGF], placenta growth factor [PLGF], hypoxia inducible factor 1α [HIF 1α], stromal cell derived factor [SDF 1α]) were performed on specimens obtained before preoperative CRT and after surgery. A semiquantitative-immunohistochemical score established from the extension and intensity of the angiogenic factors was used for analysis. Results: The positive expression rate of VEGF, PLGF, SDF 1α, and HIF 1α was 56.4% (31/55), 65.5% (36/55), 70.9% (39/55), and 47.3% (26/55), respectively. The expression rate of VEGF, PLGF, SDF 1α, and HIF 1α was increased by 3.6% (2/55), 7.3% (4/55), 30.9% (17/55), and 1.8% (1/55) after neoadjuvant CRT, respectively. Expression of VEGF, PLGF, and HIF 1α protein was downregulated after neoadjuvant CRT in the rectal cancer tissues (P < 0.001, P = 0.001, P = 0.044, respectively). However, SDF 1α was upregulated after neoadjuvant CRT (P < 0.001). And also, upregulated expression of SDF 1α after neoadjuvant CRT was significantly associated with resistance to CRT (P = 0.035). However, SDF 1α showed no correlation with other clinical factors (age, sex, clinical stage). Conclusions: Expression of SDF-1α was increased in the rectal cancer tissue after neoadjuvant CRT, as well as has been associated with CRT resistance. Our data suggests that SDF 1α should be evaluated as new target for antiangiogenic therapy.

Abstract 5475: Endoglin (CD105) is a target for ovarian cancer cell-specific therapy through induction of DNA damage.

Abstract Background: Endoglin (ENG, CD105) is a protein markedly overexpressed in tumor-associated endothelial cells, and a target for anti-angiogenic therapy. Recently we have noted it to be overexpressed in chemoresistant ovarian cancer cells as well. Our objective was to evaluate the effects and mechanisms of targeting endoglin specifically in ovarian cancer cells. Methods: Endoglin expression was assessed in multiple ovarian cancer lines by Western blot, immunohistochemistry, and flow cytometry. Anti-endoglin siRNAs were used to downregulate expression in ES2 and HeyA8MDR. In vitro, the effects of endoglin-knockdown individually and with chemotherapy were evaluated by MTT assay, cell-cycle analysis, alkaline comet assay, γ-H2AX foci formation and qPCR array for mediators of DNA damage and repair. In an orthotopic murine model, mice inoculated with ES2 or HeyA8MDR cell lines were administered chitosan-encapsulated anti-ENG siRNA or control siRNA with and without carboplatin. PCNA, γH2AX, and 53BP1 IHC and the TUNEL assay were performed to examine biologic effects of endoglin knockdown. Results: Endoglin is expressed to varying degrees by multiple ovarian cancer cell lines. Expression was on the cell surface, consistent with its recognized role as a cofactor with TGF-beta receptor, in only 5% of cells, with most other cells having cytoplasmic expression. In ES2 and HeyA8MDR cell lines, endoglin inhibition decreased cell viability, increased apoptosis, induced double-stranded DNA damage, and increased cisplatin sensitivity. Endoglin downregulation led to a decrease in expression of several DNA repair genes, including NBN, NTHL1, BARD1, H2AFX and SIRT1, and an increase in expression of DDIT3 and PPP1R15A. BARD1-specific downregulation, in turn, led to a significant decrease in BRCA1 expression, likely through ubiqutinylation. In vivo, anti-endoglin treatment decreased tumor weight in both ES2 and HeyA8MDR models when compared to control (35-41% reduction, p<0.05). Combination treatment with anti-Endoglin siRNA and carboplatin was associated with even greater inhibitory effect compared to control (58-62% reduction, p<0.001). Conclusions: Targeting endoglin improves platinum sensitivity of ovarian cancer cells both in vivo and in vitro. Endoglin downregulation induces DNA damage at multiple levels, one of which is through decreased expression of BARD1 and BRCA1. Anti-endoglin therapies should be pursued further as their development would allow dual treatment of both tumor angiogenesis and an aggressive subset of chemoresistant tumor cells. Citation Format: Angela J. Ziebarth, Somaria Nowsheen, Adam D. Steg, Monjri M. Shah, Ashwini A. Katre, Zachary C. Dobbin, Anil K. Sood, Michael G. Conner, Eddy S. Yang, Charles N. Landen. Endoglin (CD105) is a target for ovarian cancer cell-specific therapy through induction of DNA damage. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5475. doi:10.1158/1538-7445.AM2013-5475 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Expression of Semaphorin3B protein in human hepatocellular carcinoma and its clinical significance

Objective To investigate the expression of Semaphorin3B (SEMA3B) in hepatocellular carcinoma (HCC) and reveal its clinical significance.Methods Immunohistochemistry and Western blotting were performed to examine the expression of SEMA3B protein in 56 hepatocellular carcinoma samples,matched perineoplastic tissues and 14 normal liver samples.Microvessel density (MVD) was determined by counting CD34-positive endothelial cells.Statistical methods were performed to analyse the relationships between the expression of SEMA3B and clinicopathologic factors.Results The rate of the SEMA3B-positive in HCC tissues was 42.9％ and significantly lower than that in normal liver tissues and non-cancerous adjacent liver tissues(78.6％ and 85.7％,respectively)(P＜0.05).MVD in SEMA3B-positive HCC tissus was lower than that in SEMA3B-negative HCC tissus (P＜0.05).The expression of SEMA3B was closely related to tumor nodular number,tumor size,tumor capsulation and CLIP score (P＜0.05 for all).The recurrence and metastasis rate in SEMA3B-positive group was lower than that in the negative group(P＜0.05),and the survival rate in positive group was higher than that in the negative(P＜0.05).Conclusion The expression of SE-MA3B was decreased in HCC tissues,and its expression was closely associated with tumor progression,angiogenesis and prognosis,indicating that it might seve as a predictor of prognosis and a possible novel target of antiangiogenic therapy for patients with HCC. Key words: Hepatocellular carcinoma; Semaphorin3B; Microvessel density; Recurrence; Prognosis

Endocan Is Upregulated on Tumor Vessels in Invasive Bladder Cancer Where It Mediates VEGF-A–Induced Angiogenesis

Tumor-associated blood vessels differ from normal vessels and proteins present only on tumor vessels may serve as biomarkers or targets for antiangiogenic therapy in cancer. Comparing the transcriptional profiles of blood vascular endothelium from human invasive bladder cancer with normal bladder tissue, we found that the endothelial cell-specific molecule endocan (ESM1) was highly elevated on tumor vessels. Endocan was associated with filopodia of angiogenic endothelial tip cells in invasive bladder cancer. Notably, endocan expression on tumor vessels correlated strongly with staging and invasiveness, predicting a shorter recurrence-free survival time in noninvasive bladder cancers. Both endocan and VEGF-A levels were higher in plasma of patients with invasive bladder cancer than healthy individuals. Mechanistic investigations in cultured blood vascular endothelial cells or transgenic mice revealed that endocan expression was stimulated by VEGF-A through the phosphorylation and activation of VEGFR-2, which was required to promote cell migration and tube formation by VEGF-A. Taken together, our findings suggest that disrupting endocan interaction with VEGFR-2 or VEGF-A could offer a novel rational strategy to inhibit tumor angiogenesis. Furthermore, they suggest that endocan might serve as a useful biomarker to monitor disease progression and the efficacy of VEGF-A-targeting therapies in patients with bladder cancer.

PECAM1+/Sca1+/CD38+ Vascular Cells Transform into Myofibroblast-Like Cells in Skin Wound Repair

Skin injury induces the formation of new blood vessels by activating the vasculature in order to restore tissue homeostasis. Vascular cells may also differentiate into matrix-secreting contractile myofibroblasts to promote wound closure. Here, we characterize a PECAM1+/Sca1+ vascular cell population in mouse skin, which is highly enriched in wounds at the peak of neoangiogenesis and myofibroblast formation. These cells express endothelial and perivascular markers and present the receptor CD38 on their surface. PECAM1+/Sca1+/CD38+ cells proliferate upon wounding and could give rise to α-SMA+ myofibroblast-like cells. CD38 stimulation in immunodeficient mice reduced the wound size at the peak of neoangiogenesis and myofibroblast formation. In humans a corresponding cell population was identified, which was enriched in sprouting vessels of basal cell carcinoma biopsies. The results indicate that PECAM1+/Sca1+/CD38+ vascular cells could proliferate and differentiate into myofibroblast-like cells in wound repair. Moreover, CD38 signaling modulates PECAM1+/Sca1+/CD38+ cell activation in the healing process implying CD38 as a target for anti-angiogenic therapies in human basal cell carcinoma.

Close Association of Metastasis‐Associated Protein 1 Overexpression with Increased Angiogenesis and Poor Survival in Patients with Histologically Node‐Negative Gastric Cancer

Although metastasis-associated protein 1 (MTA1) has been recently demonstrated as a potent angiogenesis-promoting factor in various malignant tumors, its angiogenic property in gastric cancer (GC) remains unclear. This study has detected the expression of MTA1 protein in surgically resected tissues of pathologic N0 (pN0) GC and further investigated its relation with other clinicopathologic factors and tumor angiogenesis and prognosis. MTA1 protein expression was detected immunohistochemically in 111 pN0 GC specimens. Its correlations with clinicopathologic factors and tumor prognosis were evaluated. The intratumoral microvessel density (MVD) was assessed based on CD105 antigen immunoreactivity and analyzed for correlation with MTA1 protein expression. Overexpression of MTA1 was detected in 36.04 % of patients and exhibited a significant association with tumor size and MVD. Survival analysis demonstrated that both overall (OS) and disease-free (DFS) survivals in patients overexpressing MTA1 were significantly poorer than those without MTA1 overexpression 5 years after the operation (both p < 0.001). Multivariate survival analysis demonstrated that MTA1 overexpression was an independent prognosticator for unfavorable OS and DFS (p < 0.001, respectively). MTA1 overexpression is frequently observed in pN0 GC patients and is significantly associated with increased angiogenesis and poor prognosis. Detection of MTA1 protein expression may help predict the relapse and prognosis of pN0 GC. Also, MTA1 protein may form a novel target for antiangiogenic therapy.

Angiopoietins as biomarker of disease activity and response to therapy in multiple myeloma

Tumor angiogenesis is a complex process involving interplay of several angiogenic regulators. In the present study, mRNA expression and circulating levels of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), hypoxia inducible factor (HiF)-1α, circulating endothelial progenitor cells (cEPCs) and bone marrow microvessel density (MVD) were evaluated in multiple myeloma (MM). Compared to healthy controls, the levels of VEGF, bFGF, Ang-2, HiF-1α and cEPCs were significantly higher and Ang-1 and Ang-1/Ang-2 were lower in MM (p < 0.01). cEPC numbers correlated with Ang-1 (p = 0.03), Ang-2 (p = 0.01) and VEGF (p = 0.002). On multivariate analysis, reduced Ang-1/Ang-2 ratio (p = 0.005) at baseline was an independent predictor for response to therapy. After therapy, a decrease in Ang-2 (p < 0.001) and an increase in Ang-1/Ang-2 ratio (p = 0.003) were observed in responders. This study highlights the role of angiopoietins in MM which may, thus, be evaluated as potential targets for anti-angiogenic therapy in future.

A Novel Role for Mesenchymal Stem/Progenitor Cells As Hypoxia Sensors During Initiation of Neo-Vasculogenesis in Vivo

Abstract 613 Background:Hypoxia is a major stimulus of neo-vasculogenesis. Under hypoxic conditions endothelial colony-forming progenitor cells (ECFCs) arrange tubular structures, which can connect to the pre-existing vasculature forming functional perfused vessels. The current view is that mesenchymal stem and progenitor cells (MSPCs) or their pericyte progeny are recruited subsequently to stabilize vessels. So far, clinical applications of endothelial progenitors to restore tissue oxygenation after ischemia, cardiovascular disease or stroke largely failed to meet medical needs. Based on previous work demonstrating patent vessel formation after MSPC/ECFC co-transplantation in vivo (Blood 2009), we hypothesized that MSPCs have a decisive role in the vasculogenic response to hypoxia. Here we show for the first time that ECFCs in hypoxic conditions in vivo strictly require the presence of functional MSPCs not only to stabilize but primarily to initiate neo-vasculogenesis by a hypoxia-inducible transcription factor (HIF)-dependent mechanism. Methods:Adult human ECFCs were isolated from blood and MSPCs from bone marrow aspirates and expanded under humanized culture conditions. Progenitor cell phenotype, long-term proliferation, HIF stabilization, wound repair as well as migratory and vasculogenic functions were monitored under severe hypoxia (1% O2), venous oxygen (5% O2) and standard ambient air culture conditions (20% O2). ECFC and MSPC crosstalk in vivo was studied in immune-deficient NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) after subcutaneous transplantation in various extracellular matrices (matrigel, collagen/fibronectin, human platelet lysate gel). Cell type-specific chemical and genetic inhibition of HIF (YC-1, shRNA) was used to delineate the role of hypoxia sensing in MSPCs and ECFCs, respectively, during vasculogenesis in vivo. To determine if downstream target proteins of HIF-1α could substitute for MSPC presence during vasculogenesis, selected growth factors and cytokines were tested. Results:Progenitor proliferation and function in vitro were reduced with declining oxygen levels. ECFCs stabilized hypoxia-inducible factor-1α (HIF-1α) only at 1% O2, while MSPCs stabilized HIF-1α already at 5% O2. In an NSG mouse model, ECFCs transplanted into a hypoxic environment did not stabilize HIF-1α, while transplanted sole MSPCs or MSPCs in co-transplants showed strong nuclear HIF-1α stabilization 1 day after transplantation preceding any vessel formation or perfusion. In the absence of MSPCs, the majority of ECFCs underwent apoptosis within 24h in vivo. Inhibition of HIF-1α stabilization in MSPCs but not in ECFCs significantly abrogated vessel formation in vivo. Blocking the prominent HIF-1α down-stream target vascular endothelial growth factor (VEGF) resulted in the expected inhibition of neo-vasculogenesis. Interestingly, substitution of VEGF alone could not restore vessel formation, neither when injected together with sole ECFCs nor in a model where ECFCs were co-transplanted with HIF-depleted MSPCs. Substitution of a complex mixture of platelet-derived factors in vivo partly restored the vasculogenic function of HIF-depleted MSPCs. Conclusions:MSPCs react to a low oxygen environment by stabilizing HIF-1α earlier and more sensitively than ECFCs. MSPCs promote vessel formation at least in part by rescuing ECFCs from hypoxia-induced apoptosis in the initial phase of vasculogenesis by a HIF-dependent trophic mechanism. Surprisingly, therapeutic vasculogenesis can occur independently of endothelial HIF stabilization. These results argue in favor of MSPC/ECFC co-transplantation as a promising strategy for vascular regenerative therapy. The observation that VEGF alone could not compensate for the vasculogenic competence of pericyte precursors in vivo underlines the complexity of the hypoxia-induced cytokine network. The fact that hypoxia sensing in MSPCs but not in ECFCs is crucial to initiate vascular regeneration supports a shift of focus from endothelial cells to perivascular mesenchymal cells as a therapeutic target in anti-angiogenic therapy. Disclosures:No relevant conflicts of interest to declare.

Angiopoietin-2 and tumor angiogenesis

Tumor angiogenesis is an important way of rapid proliferation and metastasis for malignancies,and a variety of cellular and molecular factors involved in the process.Angiopoietin-2(Ang-2) is one of the important vascular endothelial growth factor,which influences tumor angiogenesis mainly through releasing vascular structures and damaging the vessel stability.Recent researches reveal that Ang-2 modulates tumor angiogenesis interacting with other vascular growth factors.With the new research progress,Ang-2 will not only be an important target for antiangiogenic therapy,but also further improve clinical efficacy of antitumor therapy combined with other signal pathways as common targets. Key words: Angiopoietins; Neoplasms; Neovascularization, pathologic

Abstract 5275: Biglycan is a specific marker and an autocrine angiogenic factor of tumor endothelial cells

Abstract Tumor angiogenesis is necessary for solid tumor progression and metastasis. Tumor blood vessels are morphologically different from their normal counterparts. We isolated tumor endothelial cells (TECs), demonstrated their abnormalities, compared gene expression profiles of TECs and normal endothelial cells (NECs) by microarray analysis and identified several genes upregulated in TECs. We focused on the gene encoding biglycan, a small leucine-rich repeat proteoglycan. Biglycan is overexpressed in inflammation and fibrosis. However, there has been no report about the expression or the function of biglycan in TEC. In this study, we investigated the expression and the role of biglycan in TECs. Real-time PCR, western blotting and immunocytochemistry revealed higher biglycan expression levels in TECs than in NECs. Furthermore, we confirmed that biglycan was secreted from TECs. Biglycan knockdown inhibited cell migration and tube formation in TECs. TLR2 and TLR4 are the biglycan receptors. TLR2 and TLR4 blocking antibodies suppressed biglycan mediated cell migration and tube formation. We isolated TECs from human renal cell carcinoma tissue and NECs from normal renal tissue in the same patients. TECs and NECs were obtained from six patients. Real-time RT-PCR revealed that the biglycan expression levels were significantly higher in four of the six TEC samples than in the corresponding NEC samples. Furthermore, immunostaining revealed strong biglycan expression in vivo in several human tumor vessels, as in mouse TECs. Biglycan was detected in the sera of cancer patients but was hardly detected in those of healthy volunteers. Biglycan is an autocrine angiogenic factor stimulating tumor endothelial cell migration and tube formation. These findings suggested that biglycan is a novel TEC marker and a target for anti-angiogenic therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5275. doi:1538-7445.AM2012-5275

Abstract 5292: Identification of antiangiogenic small molecule natural products targeting anthrax toxin receptor 2

Abstract Bacillus anthracis protective antigen (PA), the B-subunit of the binary anthrax toxin, binds to the cellular receptors capillary morphogenesis gene protein 2 (CMG2) and tumor endothelial marker 8 (TEM8) with high affinity. Both CMG2 and TEM8 are expressed on endothelial cells during angiogenesis. We have shown that wild-type PA inhibits both VEGF-induced and bFGF-induced angiogenesis at moderate but statistically significant levels. Structure-activity studies identified a PA mutant that exhibited markedly enhanced inhibition of angiogenesis and also inhibited tumor growth in vivo. This mutant, PASSSR, is unable to undergo normal cellular processing and thus, remains bound to the surface receptor. Further mutation of PASSSR so that it does not bind to these cell surface receptors abolished its ability to inhibit angiogenesis. We conclude that high affinity anthrax receptor ligands, such as PA and PASSSR, are angiogenesis inhibitors, and that anthrax toxin receptors are useful targets for anti-angiogenic therapy. The long-term goal of our research is to determine the extent to which antrax toxin receptor directed therapeutics can control tumor growth and metastasis. Here we show that in addition to inhibition of subcutaneous lung tumors, PASSSR can inhibit the growth of orthotopically implanted breast cancer cells and extend survival in this mouse model. We also sought to identify small molecule natural products that can inhibit the interaction with the anthrax toxin receptor CMG2 and to determine their antiangiogenic and antitumor capacity in vivo. This work was based on the hypothesis that small molecule natural products can inhibit CMG2-ECM interactions required for breast cancer angiogenesis. This work leverages the wide variety of chemical entities generated by endophytic fungi, and the ability of other fungal compounds such as fumagillin to provide interesting insights into angiogenesis inhibitors. Following a high-throughput screen of &amp;gt;10,000 natural product extracts, we identified 48 extracts with significant inhibitory activity, of which ∼0.5% exhibited measurable activity. Of these, 9 exhibited 1:1 binding to CMG2 with IC50 &amp;lt;100 ug/ml total solids. Purification and chemical and in vitro characterization resulting in the identification of six compounds with anti-CMG2 and endothelial cell inhibitory, one of which exhibits antiangiogenic activity. Thus, we have identified several small molecule natural product inhibitors of CMG2 and demonstrated that CMG2 inhibitors can inhibit angiogenesis and breast cancer growth. While none of these molecules is currently suitable as a lead for the development of a breast cancer therapeutic, we believe that these studies confirm that the approach that we have taken is suitable for the development of such a therapeutic and propose to dramatically expand the number of molecules screened for such activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5292. doi:1538-7445.AM2012-5292

Biglycan is a specific marker and an autocrine angiogenic factor of tumour endothelial cells

Background:We isolated tumour endothelial cells (TECs), demonstrated their abnormalities, compared gene expression profiles of TECs and normal endothelial cells (NECs) by microarray analysis and identified several genes upregulated in TECs. We focused on the gene encoding biglycan, a small leucine-rich repeat proteoglycan. No report is available on biglycan expression or function in TECs.Methods:The NEC and TEC were isolated. We investigated the biglycan expression and function in TECs. Western blotting analysis of biglycan was performed on sera from cancer patients.Results:Biglycan expression levels were higher in TECs than in NECs. Biglycan knockdown inhibited cell migration and caused morphological changes in TECs. Furthermore, immunostaining revealed strong biglycan expression in vivo in human tumour vessels, as in mouse TECs. Biglycan was detected in the sera of cancer patients but was hardly detected in those of healthy volunteers.Conclusion:These findings suggested that biglycan is a novel TEC marker and a target for anti-angiogenic therapy.

Abstract 5142: VEGFA gene amplification and protein expression in breast cancer

Abstract Background: The vascular endothelial growth factor A (VEGFA) protein is a chemical signaling molecule that is known to be a major factor in the induction of angiogenesis during tumor initiation and progression, and is also a target of anti-angiogenic therapies. Recently, we discovered the genomic amplification of the VEGFA gene in a small subset of colorectal cancers. Aim of this study was to investigate the presence of VEGFA gene amplification in breast cancer and to determine its potential impact on VEGFA protein expression. Methods: VEGFA gene amplification was evaluated by fluorescence in situ hybridization (FISH) on a multitumor tissue microarray (MTMA) comprising 3417 tissue samples from more than 100 different tumor types. Further, a small tissue microarray was constructed from breast carcinoma samples whose VEGFA protein concentration had been previously quantified by chemiluminescence immunoassay. In order to interrogate tissue heterogeneity, VEGFA gene amplification was also analyzed on large tissue sections from 70 primary breast cancers. Results: We detected VEGFA gene amplification in 2% of the breast cancer samples from the MTMA and in 5% of the breast cancer samples with known VEGFA protein concentration. In addition, 8% of the samples (5 out of 70) were characterized by a high polysomy. Interestingly, elevated VEGFA gene copy number was strongly correlated with higher VEGFA protein levels (p&amp;lt;0.0001). Conclusions: VEGFA gene amplification defines a small subset of breast carcinomas with elevated VEGFA protein expression. Our data suggest that FISH analysis of VEGFA could represent an additional evaluation system for the identification of breast cancer patients who might benefit from anti-VEGFA therapies. 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 5142. doi:10.1158/1538-7445.AM2011-5142

Abstract 1596: Human choriocarcinomas: Placental growth factor-dependent preclinical tumor models

Abstract Choriocarcinomas are an aggressive form of cancer that develops in the uterus from tissue that would normally become the placenta. Choriocarcinomas are a trophoblastic gestational disease and the tumors are highly angiogenic. Human placental growth factor (hPlGF) is an angiogenic growth factor that promotes the growth of blood vessels in the placenta. hPlGF levels can be higher under pathological conditions such as cancer. Some human tumor specimens have higher PlGF levels than normal tissue, thus hPlGF is a target for anti-angiogenic therapy. The human choriocarcinoma cell lines BeWo, JAr, and JEG-3 were evaluated as preclinical models of hPlGF overexpression. The levels of hVEGF-A and hPlGF secreted by the choriocarcinomas and 11 human and mouse cancer cell lines in culture were measured by ELISA. The choriocarcinomas secreted higher levels of hPlGF and lower levels of hVEGF-A than 10/11 other cancer cell lines. The hPlGF serum levels in mice bearing subcutaneous choriocarcinoma tumors correlated with tumor volume and reached &amp;gt;2,000 pg/ml. By comparison, no hPlGF was detected in the serum of mice bearing H460 non-small lung carcinoma, HT29 colon carcinoma or MOLM-13 acute myeloid leukemia xenograft tumors. Although choriocarcinoma cells secreted both hPlGF and hVEGF-A in culture, serum levels of hVEGF-A in mice bearing choriocarcinoma xenografts were undetectable or &amp;lt;50 pg/ml at the time points serum was collected indicating that angiogenic factor secretion by the cells in culture was not the same in vivo. JEG-3 tumors grew faster than JAr tumors following the subcutaneous implantantion of 1×106 cells into nude mice. BeWo tumors grew the slowest and had the highest serum PlGF levels. Immunohistochemical methods confirmed expression of hPlGF in the choriocarcinoma xenografts. Studies involving double immunofluorescence on the xenograft tumors are ongoing to investigate endothelial cell and pericyte interactions and stromal involvement in the tumors. sFLT01, a novel fusion protein that neutralizes PlGF and VEGF-A, was added to choriocarcinoma cells in culture and administered to mice bearing choriocarcinoma xenografts in efficacy studies. Exposure of JAr and JEG-3 cells in culture to sFLT01 resulted in a phenotypic change whereby the cells produced less hPlGF and no significant hVEGF-A. In vivo, sFLT01 (25 mg/kg, IP injection, 2x per week) slowed tumor growth in JAr and JEG-3 tumors and increased median survival by 5-30 days compared to vehicle controls. These results indicate that human choriocarcinoma xenograft models are a valuable research tool for evaluating anti-angiogenic agents that target hPlGF. 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 1596. doi:10.1158/1538-7445.AM2011-1596

Abstract 5145: Nestin as a novel angiogenic marker and target for anti-angiogenic therapy in human pancreatic cancer

Abstract Background: Tumor angiogenesis is an important factor in the proliferation and metastasis of pancreatic ductal adenocarcinoma (PDAC). CD34, CD31 and factor VIII-related antigen are commonly used as endothelial cell markers for tumor vessels. However, these markers identify not only newly formed small tumor vessels, but also pre-existing large blood vessels. Nestin, a class VI intermediate filament protein, has been reported to be up-regulated in endothelial cells accompanying the process of angiogenesis. In an acute pancreatitis murine model, we have previously reported that nestin is strongly expressed in proliferating endothelial cells. Furthermore, we have reported that nestin-positive blood vessels correlated with a poor prognosis in colorectal cancer. In this study, we examined the effectiveness of nestin as an angiogenic marker and potent target for anti-angiogenic therapy in PDAC. Methods: Tissues from 45 patients with PDAC were immunostained with nestin and other common vascular endothelial markers including CD34, CD31 and factor VIII-related antigen. We measured the number and dimension of the nestin- and CD34-positive blood vessels using image analyzing software. In addition, we compared proliferation activity between nestin- and CD34-positive vessels as determined by PCNA-labeling indices. To clarify the roles of nestin in endothelial cells, we transfected siRNA targeting nestin transcripts to mouse endothelial TKD2cells, and performed cell growth and migration assays. Results: Immunohistochemically, CD34, CD31 and factor VIII-related antigen were localized in blood vessels of all sizes, while nestin was localized only in the small blood vessels in PDAC tissues. Nestin was also expressed in myofibroblasts and nerve fibers, but not in lymphatic vessels. In image analyzing software, nestin-positive vessels were small in number, and they formed small lumen compare with CD34-positive blood vessels. Nestin-positive vessels showed higher PCNA-labeling indices than those of CD34-positive vessels. Nestin was expressed in small and proliferating blood vessels in PDAC tissues, suggesting that nestin plays important roles in tumor angiogenesis in cancer. Knock down of nestin in mouse endothelial cells using siRNA inhibited the cell growth, but not cell migration in vitro. Conclusion: Nestin was specifically expressed in small and proliferating blood vessels in pancreatic cancer tissues, indicating that nestin is a useful angiogenic marker in cancer. Furthermore, nestin may be a novel therapeutic target for inhibition of tumor angiogenesis in pancreatic cancer patients. 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 5145. doi:10.1158/1538-7445.AM2011-5145

Abstract 3469: EphrinB2 promotes endothelial cell survival and vascular integrity

Abstract EphrinB transmembrane ligands and their cognate EphB receptors critically contribute to vascular development through cell-to-cell signaling by the EphB receptor (forward signaling) and by the EphrinB ligand (reverse signaling). Previous studies demonstrated that mutant mice lacking ephrinB2 or EphB4 and a proportion of mice lacking EphB2 and EphB3 display severe defects in the embryonic vascular system and die at the embryo stage likely due to these vascular defects. Previously, we showed that EphB2, EphB4 and EphrinB2 are required for mature endothelial cell assembly into capillary structures. In the currently studies, we observed that silencing EphrinB2 expression in different types of primary human endothelial cells causes significant and time-dependent apoptotic cell death, as measured by cleavage of Poly(ADP-ribose) Polymerase (PARP), caspase-3 and caspase-8. VEGF, bFGF and SDF-1 variously modulated apoptosis induced by loss of EphrinB2 expression in endothelial cells. Gene expression profiling revealed that EphrinB2 silencing is associated with reduced expression of genes linked to regulation of the cell cycle progression and cytoskeleton organization, and with increased expression of pro-apoptotic genes. Breast cancer tissue from a patient treated with bevacizumab, a humanized monoclonal antibody that neutralizes VEGF, displayed evidence of endothelial cell apoptosis, which was associated with selectively reduced EphrinB2 expression, suggesting a link between VEGF signaling, EphrinB2 expression and cell death. These studies show that EphrinB2 expression is critical to endothelial cell survival in vitro and suggest that EphrinB2 contributes to blood vessel integrity in vivo. Thus, in addition to its well-characterized function as a regulator of cell-to-cell adhesion and repulsion, EphrinB2 is an endothelial pro-survival molecule, which may serve as a novel target for anti-angiogenic therapies. 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 3469. doi:10.1158/1538-7445.AM2011-3469

Abstract SY16-03: Dose matters and angiogenesis

Abstract Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that plays a fundamental role in integrin and growth factor mediated signaling and is an important player in cell migration and proliferation, processes vital for angiogenesis. However, the role of FAK in adult pathological angiogenesis is unknown. We have generated endothelial-specific tamoxifen-inducible FAK knockout mice by crossing FAK-floxed (FAKfl/fl) mice with the Pdgfb-iCreER mice. Tamoxifen-treatment of Pdgfb-iCreER;FAKfl/fl mice results in FAK deletion in adult endothelial cells (EC) without any adverse effects. Importantly however, endothelial FAK-deletion in adult mice inhibited tumor growth and reduced tumor angiogenesis. Furthermore, in in vivo angiogenic assays FAK-deletion impairs vascular endothelial growth factor (VEGF)-induced neovascularization. In addition, in vitro deletion of FAK in ECs resulted in reduced VEGF-stimulated AKT phosphorylation, and correlating reduced cellular proliferation and increased cell death. Our data suggest that FAK is required for adult pathological angiogenesis and validates FAK as a possible target for antiangiogenic therapies. In a follow up study we are testing the effects of reduced, as apposed to null, levels of FAK on tumor angiogenesis and will be reporting on that at the meeting. 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 SY16-03. doi:10.1158/1538-7445.AM2011-SY16-03

Anti-angiogenic effects of purine inhibitors of cyclin dependent kinases

Small molecular inhibitors of Cyclin dependent kinases (Cdks) are currently being developed as anticancer therapeutics due to their antiproliferative properties. The purine Cdk specific inhibitor (R)-roscovitine (seliciclib, CYC202) represents one of the most promising of these compounds. It is currently evaluated in clinical trials concerning cancer therapy. Recently, we have shown that roscovitine exerts potent antiangiogenic effects and elucidated Cdk5 as a new player in angiogenesis. These findings introduce Cdk5 as novel target for antiangiogenic therapy, and Cdk5 inhibitors as an attractive therapeutic approach. Here, we present the antiangiogenic profile of 15 derivatives of roscovitine in vitro and in vivo and provide structure activity relationships of the roscovitine analogs. The (S)-isomer LGR561 and the respective (R)- and (S)-isomers LGR848 and LGR849 strongly inhibited proliferation and cell cycle progression, induced cell death, and reduced migration of endothelial cells in vitro. In comparison to roscovitine, these compounds showed an increased potency to inhibit Cdk2, Cdk5, Cdk7, and Cdk9. By analyzing the effects of LGR561, LGR848, and LGR849 on endothelial cell tube formation, mouse aortic ring sprouting, angiogenesis in the chick chorioallantoic membrane, and neovessel formation in the mouse cornea, we elucidate the two (S)-isomers LGR561 and LGR849 as highly potent inhibitors of angiogenesis. This study provides first information on how to modify roscovitine to develop Cdk inhibitors with increased antiangiogenic activity and suggests the application of existing and the development of new Cdk inhibitors to inhibit both, cancer cell proliferation and angiogenesis.

A target for antiangiogenic therapy: Vascular endothelium derived from glioblastoma

Glioblastoma is the most frequent primary brain tumor in the adult, accounting for 53.8% of all gliomas (http://www.cbtrus.org), and it is one of the most deadly among all human tumors. Despite aggressive treatment at diagnosis, consisting of resection followed by radiation with concurrent and subsequent adjuvant chemotherapy with temozolomide, the tumor almost invariably recurs or progresses, with a patient median survival of 14.6 mo (1). The hallmark of glioblastoma that distinguishes it from all of the other glial tumors is microvascular proliferation in conjunction with necrosis. Therefore, treatment with antiangiogenic agents holds great promise to block the growth of this most vascularized tumor. The best-known antiangiogenic agents are inhibitors of VEGF-A, an indispensable angiogenic factor during developmental organogenesis and growth of numerous tumors. However, treatment with bevacizumab, a neutralizing antibody to VEGF-A, at relapse only confers transient benefit and a marginal increase in survival, indicating at tumor progression a VEGF-independent angiogenic mechanism of glioblastoma resistance (2). Several mechanisms have been implicated in angiogenesis. One is the sprouting of capillaries from preexisting blood vessels by endothelial proliferation (3). Another is the cooption of preexisting blood vessels by tumor cells, leading to expression of angiopoietin-2 by those vessels’ endothelial cells and tumor cell proliferation, followed later by involution of preexisting vessels in the core of the tumor, massive tumor cell apoptosis, organization of remaining tumor cells into pseudopalisading that resides around areas of necrosis, and tumor rescue at the margins by angiogenesis (4, 5). Expression of HIF-1α and up-regulation of VEGF-A have been identified in hypoxic perinecrotic pseudopalisading tumor cells (4–6). Hypoxia induces elevated levels of VEGF-A (6) and VEGF-A receptors that appear up-regulated in tumor endothelial cells but not in normal brain (7). Another mechanism is the release of angiogenic factors by the tumor that recruit bone marrow-derived endothelial progenitors, hematopoietic stem and progenitor cells that participate in vessel formation (8–10). In PNAS, Soda, Verma, and colleagues reveal a new paradigm for glioblastoma angiogenesis whose main contribution is transdifferentiation of glioblastoma cells into endothelial cells (11) (Fig. 1). Notably, these tumor-derived endothelial cells (TDECs) are refractory to inhibition of both VEGF-A and basic fibroblast growth factor (bFGF, FGF-2) pathways. By mapping GFP+ p53-deficient glioblastoma established in glial-specific Cre mice (GFAP-Cre) (12), Soda et al. find that tumor cells can directly transdifferentiate into CD31+CD34+ endothelial cells that lack VEGF-A receptors (VEGFR), constituting over 20% of total CD31+CD34+ tumor endothelial population. These TDECs are capable of forming patent vessels. Moreover, further analysis by hypoxyprobe unraveled their preferential localization in deep hypoxic areas of the tumors. The hypoxic-associated distribution of TDECs and elevated expression of HIF-1α, a hypoxia-induced transcription factor, indicate the role of hypoxia as the key determinant in forcing putative glioma cells to differentiate into endothelial-like cells.