Abstract 2320: A multi-domain fusion molecule inhibits tumor growth affecting in-vivo angiogenesis

Background: Angiogenesis is essential for the growth and metastasis of solid tumors, including breast cancer. Although the zinc finger-homeodomain transcription factor ZEB1 is implied as a key regulatory factor in multistep carcinogenesis of breast cancer, its direct role in the regulation of tumor angiogenesis has not been well elucidated. In this report, we extended our study to examine the potential effect of ZEB1 in breast cancer angiogenesis and its molecular mechanism using the in vitro and in vivo models. Materials and Methods: Quantitative RT-PCR, immunoblotting, and ELISA were used to detect ZEB1-induced expression and/or secretion of VEGF in the absence or presence of the indicated signaling pathway inhibitors (the PI3K, NF-κB, and MAPKs pathways) in MDA-MB-231 breast cancer cells. Tube formation assay was performed to detect ZEB1-driven angiogenesis using human umbilical vein endothelial cells (HUVEC). Luciferase and CHIP assays were used to determine the transcriptional activity of the wild-type and mutant VEGF promoters affected by ZEB1. A nude mouse tumor xenograft model was used to examine ZEB1-driven angiogenesis in vivo. The relationship between the expression of ZEB1 and VEGF protein in human breast cancer specimens was assessed by immunohistochemistry. Results: We found that ectopic expression of ZEB1 promotes the expression of VEGF at both the mRNA and the protein levels in MDA-MB-231 cells, which effect is specifically mediated through the p38 and PI3K pathways. The conditioned medium derived from ZEB1-overexpressing MDA-MB-231 cells significantly induces the tube formation of HUVEC compared with that from control-overexpressing cells. Importantly, elevated expression of ZEB1 resulted in an increased angiogenesis in a nude mouse tumor xenograft model. We further moved on to detect whether ZEB1 is a bona-fide transcriptional regulator of VEGF. The results of luciferase and CHIP assays showed that ZEB1 transcriptionally induces the promoter activity of human VEGF by recruiting Sp1 to its biding sites on the VEGF promoter, thus up-regulating VEGF expression in MDA-MB-231 cells. Through the study of breast cancer specimens, we found a strong positive correlation between ZEB1 and VEGF protein expression, further supporting the contribution of ZEB1/VEGF cascade to tumor angiogenesis in breast cancer. Conclusions: Our data demonstrated that ZEB1 up-regulates VEGF expression and thus induces tumor angiogenesis in breast cancer. Thus, inhibition of VEGF expression by down-regulating ZEB1 provides a potential new strategy for overcoming tumor angiogenesis. Citation Format: Qi Tong, Lingjia Liu, Lin Liang, Yang Gao, Shuang Yang. Elevated expression of ZEB1 upregulates VEGF expression and induces tumor angiogenesis in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1599. doi:10.1158/1538-7445.AM2014-1599

PDGF signaling system contributes to tumor angiogenesis and vascular remodeling. Here, we show PDGF-BB markedly induces erythropoietin (EPO) mRNA and protein expression by targeting the PDGFR-β+ stromal and perivascular compartments. In mouse tumor models, PDGF-BB-induced EPO promotes tumor growth via two mechanisms: 1) paracrine stimulation of tumor angiogenesis by directly inducing endothelial cell proliferation, migration, sprouting and tube formation; and 2) endocrine stimulation of extramedullary hematopoiesis leading to increased oxygen perfusion and protection against tumor-associated anemia. Similarly, delivery of an adenovirus-PDGF-BB to tumor-free mice markedly increases EPO production and hematopoietic parameters. An EPO-specific antibody attenuates PDGF-BB-induced tumor growth, angiogenesis and hematopoiesis. At the molecular level, we show that the PDGF-BB-PDGFR-β signaling system activates EPO promoter via in part transcriptional regulation of ATF3 by possible association with c-Jun and SP1. These findings uncover a novel mechanism of PDGF-BB-induced tumor growth, angiogenesis and hematopoiesis. Citation Format: Yuan Xue, Sharon Lim, Yunlong Yang, Zongwei Wang, Lasse Dahl Ejby Jensen, Eva–Maria Hedlund, Patrik Andersson, Masakiyo Sasahara, Ola Larsson, Dagmar Galter, Renhai Cao, Kayoko Hosaka, Yihai Cao. PDGF-BB modulates hematopoiesis and tumor angiogenesis by inducing erythropoietin production in stromal cells. [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 1623. doi:10.1158/1538-7445.AM2013-1623

The uPA/uPAR system is known to play a critical role in angiogenesis of glioblastomas. Previously, using protein array antibody analysis, we demonstrated that shRNA against uPA and uPAR (pU2) inhibits angiogenesis in vitro and in vivo by regulating a number of pro-angiogenic, angiostatic and anti-angiogenic molecules. GMCSF, a pleiotropic cytokine that stimulates proliferation, differentiation, and survival of various hematopoietic cells, was significantly inhibited in U87MG and 4910 cells co-cultured with endothelial cells and transfected with pU2. In this study, we evaluated the effect of shRNA against uPA and uPAR on GMCSF, sVEGFr1, GMCSF receptors and GMCSF-induced signaling and its significance on angiogenesis in glioblastoma and endothelial cells. ELISA revealed that simultaneous knockdown of uPA and uPAR in U87MG, 4910 and HMEC cells decreased secretion of GMCSF but enhanced the secretion of sVEGFr1, a known scavenger of VEGF-A. The results of the western blot analysis were consistent with our ELISA results. In endothelial cells treated with shRNA against GMCSF, recombinant uPA (ruPA) resulted in activation of GMCSFR-α and GMCSFR-β at very early time points. Moreover, endothelial tube formation was inhibited with the addition of rsVEGFr1, and antibody to rsVEGFr1 blocked GMCSF-induced tube formation in endothelial cells. The addition of ruPA to endothelial and glioblastoma cells activated phosphorylation of JAK2 and STAT3. p27, an effector of the GMCSF signaling pathway, was downregulated in puPA-, puPAR- and pU2-transfected U87MG and 4910 cells. To determine the significance of these events in vivo, nude mice were checked for levels of GMCSF and sVEGFr1 before and after tumor formation and also after treatment with puPA, puPAR and pU2. Results showed increased levels of sVEGFr1 but decreased levels of GMCSF in shRNA treated mice in comparison to controls. Taken together, these results suggest that knockdown of uPA/uPAR in tandem induces sVEGFr1, which plays a vital role in inhibition of angiogenesis, and its secretion is independent of GMCSF in endothelial cells. 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 3465. doi:10.1158/1538-7445.AM2011-3465

Purpose of Study: To investigate the role of the Neuropilin-1 (NRP-1) in angiogenesis and regulation by Wnt-signaling pathway in osteosarcoma (OS) cell lines. Methods: In vitro tube formation assay using HUVEC cells to examine the effect of Frzb, WIF1, and DKK3 on angiogenesis. Microarray and real-time PCR to assess the mRNA level of NRP-1 in SaOS-2 cells transfected with Dominant Negative LRP5 (DNLRP5) and PcDNA vector control. Western blots were performed to evaluate the expression of NRP-1 in 8 OS cell lines and normal osteoblast (NHOst), as well as the effect of Wnt inhibitors (DNLPR5, Frzb, WIF-1, DKK3) on NRP-1 expression in OS cell lines (LM7, 143B, and 143.98.2) Results: In order to develop a new therapeutic target for OS, the most common primary malignant bone tumor, we examined the expression of NRP-1 and vascular endothelial growth factor (VEGF) in 8 established OS cell lines, as well as NHOst. Both VEGF and NRP-1 (a co-receptor of VEGF) were up-regulated in most OS cell lines compared with normal osteoblast, suggesting that VEGF and NRP-1 may be responsible for the excessive blood vessel formation in OS. Given our previous report that Wnt inhibitors Frzb, WIF1, DKK3, and DNLRP5, inhibited tumor growth and metastasis of OS, we hypothesize that one of the potential mechanisms may be through inhibition of tumor angiogenesis. When HUVEC assay was examined, DKK3, Frzb, WIF-1 condition medium significantly inhibited tube formation compared with control, suggesting that these Wnt inhibitors suppressed angiogenesis. Further microarray assay, real time PCR and western blots indicated that these Wnt inhibitors (DKK3, Frzb, WIF-1 and DNLRP5) dramatically down-regulated the expression of NRP-1, but VEGF level remained unchanged. These results show that inhibition of angiogenesis by Wnt signaling may be mediated by down-regulation of NRP-1 instead of VEGF. Discussion and Conclusions: Our study suggests that the Wnt-signaling reduces tumor growth and metastasis by inhibiting angiogenesis and by down-regulating NRP-1 expression in OS. We are further studying the role of NRP-1 in osteosarcoma by knockdown of NRP1 expression in OS cells to assess tumor proliferation, metastases and angiogenesis. This may represent a novel therapeutic strategy for osteosarcoma. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. 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 3963.

Signal peptide-complement protein C1r/C1s, Uegf, and Bmp1 (CUB)-epidermal growth factor (EGF) domain-containing protein 2 (SCUBE2) is a peripheral membrane protein expressed in normal tissue and tumor vascular endothelial cells (ECs); however, its role in angiogenesis remains poorly understood. In this study, we show that endothelial SCUBE2 is upregulated by hypoxia and acts as a co-receptor for VEGFR2 to facilitate VEGF binding to VEGFR2 and augment its signals including VEGFR2 phosphorylation and p44/42 MAPKs/Akt activation, thus promotes cell proliferation and tubule formation in ECs. While physiological angiogenesis remained normal in the endothelial ablation of Scube2 in mice, pathological angiogenesis in experimental tumors was altered, resulting in smaller tumors and reduced microvascular density. To simulate the angiogenic environment of the tumor, Scube2-deficient ECs were isolated and propagated in vitro with VEGF. Mutant ECs exhibited marked reduction in binding of VEGF, proliferation and sprouting responses to VEGF as well as downstream signal activation. Our results reveal that SCUBE2 might act as a novel co-receptor for VEGFR2, and point that targeting such SCUBE2 function in human ECs may represent a potential anti-tumor strategy by inhibiting tumor angiogenesis. Citation Format: Yuh-Charn Lin, Ruey-Bing Yang. SCUBE2 is a co-receptor for VEGFR2 in tumor angiogenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3391.

CK2 is a highly conserved protein serine/threonine kinase that is ubiquitously distributed in eukaryotes, constitutively active and has been implicated in multiple cellular functions, as well as in tumorigenesis and transformation. Elevated CK2 activity has been associated with the malignant transformation of several tissues and is associated with aggressive tumor behavior. Previously, we have described a peptide CIGB-300 (also named P15-Tat) targeting the acidic phophorylation domain of the CK2 substrates. Different groups around the world have tried to manipulate this biochemical event by targeting the ATP-binding site of CK2 or its gene transcription using antisense oligonucleotides. Otherwise, CIGB-300 peptide was developed following the innovative approach of targeting the phosphoaceptor site on the CK2 substrates rather than the enzyme per se. We demonstrated that CIGB-300 induced apoptosis on tumor cells and tumor regression when injected directly into solid tumors or by systemic administration in mice. In this work we have evaluated the CIGB-300 peptide on the angiogenic process, both in vitro and in vivo. We have focused in the exploration of CIGB-300 properties to inhibit proliferation, tube formation and RNA pattern expression on endothelial cells (HUVEC). Likewise we used the chicken chorioallatoic membrane (CAM) assay as a model in vivo to asses the CIGB-300 activity on angiogenesis. Others authors have studied the role of different CK2 inhibitors on angiogenesis finding that CK2 is involved in endothelial cell proliferation, survival, migration and tube formation. Intraperitoneally administered CK2 inhibitors significantly reduced preretinal neovascularization in a mouse model of proliferative retinopathy. Solid tumors require the growth of new blood vessels (angiogenesis) to grow. Tumor angiogenesis utilizes at least some of the angiogenic signalling pathways that are required during vascular development. Tumor angiogenesis has become an important target for antitumor therapy, with most current therapies aimed at blocking the VEGF pathway. However, not all tumors are responsive to VEGF blockers, and some of them that are responsive initially may become resistant during the course of treatment, thus there is a need to explore other angiogenesis signalling pathways. In this paper we have examined the antiangiogenic properties of CIGB-300 peptide in vitro and in vivo. The data suggests that CK2 is involved in angiogenic processes and CIGB-300 could act as promising antiangiogenic inhibitor. 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 1376.

Angiogenesis, or the development of new blood vessels from existing blood vessels, is a process essential to wound healing, reproduction, and tumor growth. Tumor angiogenesis is an important factor in the growth and spread of cancer. In order for tumors to grow beyond 2mm^3, vasculature is necessary to provide oxygen and nutrients and remove waste products. The imperfect blood vessels built within tumors also allow tumor cells to travel through the bloodstream and metastasize. Endogenous inhibitors of angiogenesis are known to form from plasma proteins. For example, angiostatin is derived from plasmin. A newly described angiogenesis inhibitor derived from high molecular weight kininogen (HK) is domain 5 of HK (HK5). HK5 has been shown to inhibit endothelial cell proliferation, migration and tube formation. HK5 binds to the endothelial cell surface receptors uPAR, cytokeratin-1, gC1qR and tropomyosin to disrupt angiogenic signaling. Our laboratory has shown that the iron storage protein ferritin binds to HK5, inhibiting its antiangiogenic effects and thereby allowing endothelial cells to migrate and proliferate. We have begun to investigate the physical interaction of ferritin with the HK5 protein with the long-term goal of developing inhibitors of the HK5-ferritin interaction. Human recombinant HK5 and ferritin H were purified and their interaction studied using fluorescence anisotropy. The two proteins were found to bind with a Kd of 1.36μM. The influence of metals on the interaction was also evaluated. We found that in the presence of iron the interaction between ferritin and HK5 was maximal, while in the presence of zinc, the interaction was minimal. EDTA pretreatment of the two proteins further improved binding. The results suggest that iron is essential to the optimal binding interaction of HK5 and ferritin. Future studies will be directed at the identification of specific amino acid residues involved in this interaction. (supported by NIH R01 DK071892) 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 1285.

The final stage of lung development in humans and rodents occurs principally after birth and involves the partitioning of the large primary saccules into smaller air spaces by the inward protrusion of septae derived from the walls of the saccules. Several observations in animal models implicate angiogenesis as critical to this process of alveolarization, but all anti-angiogenic treatments examined to date have resulted in endothelial cell (EC) death. We therefore targeted the function of platelet endothelial cell adhesion molecule, (PECAM-1), an EC surface molecule that promotes EC migration and has been implicated in in vivo angiogenesis. Administration of an anti-PECAM-1 antibody that inhibits EC migration, but not proliferation or survival in vitro, disrupted normal alveolar septation in neonatal rat pups without reducing EC content. Three-dimensional reconstruction of lungs showed that pups treated with a blocking PECAM-1 antibody had remodeling of more proximal branches resulting in large tubular airways. Subsequent studies in PECAM-1-null mice confirmed that the absence of PECAM-1 impaired murine alveolarization, without affecting EC content, proliferation, or survival. Further, cell migration was reduced in lung endothelial cells isolated from these mice. These data suggest that the loss of PECAM-1 function compromises postnatal lung development and provide evidence that inhibition of EC function, in contrast to a loss of viable EC, inhibits alveolarization.

Kallistatin is an endogenous protein that exerts pleiotropic effects, including vasodilation and inhibition of angiogenesis, inflammation, oxidative stress, apoptosis, fibrosis, and tumor progression. Through its two functional domains – an active site and a heparin-binding site – kallistatin regulates differential signaling pathways and a wide spectrum of biological functions. Kallistatin's active site is key for inhibiting tissue kallikrein activity, and stimulating the expression of endothelial nitric oxide synthase (eNOS), sirtuin 1 (SIRT1) and suppressor of cytokine signaling 3 (SOCS3). Kallistatin via its heparin-binding site blocks signaling pathways mediated by growth factors and cytokines, such as vascular endothelial growth factor (VEGF), tumor necrosis factor-α (TNF-α), high mobility group box-1 (HMGB1), Wnt, transforming growth factor-β (TGF-β), and epidermal growth factor (EGF). Kallistatin gene or protein delivery protects against the pathogenesis of hypertension, heart and kidney damage, arthritis, sepsis, influenza virus infection, tumor growth and metastasis in animal models. Conversely, depletion of endogenous kallistatin by neutralizing antibody injection exacerbates cardiovascular and renal injury in hypertensive rats. Kallistatin levels are markedly reduced in rodents with hypertension, sepsis, streptozotocin-induced diabetes, and cardiac and renal injury. Kallistatin levels are also diminished in patients with liver disease, septic syndrome, diabetic retinopathy, severe pneumonia, inflammatory bowel disease, and obesity, prostate and colon cancer. Therefore, circulating kallistatin levels may serve as a new biomarker for human diseases. This review summarizes kallistatin's protective roles and mechanisms in vascular and organ injury, and highlights the therapeutic potential of kallistatin for multiple disease states.

The Semaphorins and their receptors, Neuropilins and Plexins, were originally implicated as guidance signals in the development of the central nervous system. However, a growing body of evidence indicates that semaphorins may regulate tumor progression, by promoting or inhibiting tumor angiogenesis, tumor cell survival and metastatic spreading, in fact mediating a crosstalk between tumor cells and multiple stromal cell types in the surrounding microenvironment. Here, we provide two examples of the multifaceted activity of semaphorins during tumor progression. We found that Semaphorin 3A (SEMA3A) inhibits primary tumor growth and its metastatic dissemination by employing three different experimental approaches in mouse tumor models: (i) over-expression of SEMA3A in tumor cells; (ii) systemic expression of SEMA3A following gene transfer in mice; (iii) tumor-targeted release of SEMA3A using gene-modified Tie2-expressing monocytes (TEMs) as delivery vehicles. In each of these experimental settings, SEMA3A efficiently inhibited tumor growth by disrupting vessel function and inducing tumor hypoxia and necrosis, without promoting metastatic dissemination. Moreover, we have recently shown that another secreted semaphorin known to regulate angiogenesis, Semaphorin 3E (SEMA3E), is abundantly expressed in metastatic human tumors, where it promotes cancer cell invasiveness and metastatic spreading. Notably, this biological activity of SEMA3E depends on a proteolytic conversion by furin-like convertases, generating the active fragment “p61”-SEMA3E. In cancer cells, p61-SEMA3E induces the association of the receptor PlexinD1 with the oncogenic tyrosine-kinase receptor ErbB2, leading to ErbB2 activation and enhanced tumor invasion and metastasis. Here we show that a mutated uncleavable variant of SEMA3E (Uncl-SEMA3E) binds the receptor PlexinD1 but it is unable to trigger ErbB2 signaling in cancer cells and lacks any pro-metastatic activity. On the other hand, Uncl-SEMA3E functions as a potent anti-angiogenic factor and displays striking tumor-suppressor properties in vivo. In summary, SEMA3A and Uncl-SEMA3E are potent inhibitors of tumor angiogenesis and tumor growth which concomitantly block the metastatic spreading of tumor cells. These data provide evidence that support interfering with semaphorin-mediated signals as a promising anti-cancer strategy. 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 3268. doi:10.1158/1538-7445.AM2011-3268

Sphingosine-1-phosphate (S1P) is a plasma lipid mediator with pleiotropic activities; it is constitutively produced in red blood cells and vascular endothelial cells through phosphorylation of sphingosine by one of two S1P synthesizing enzymes, sphingosine kinase 1 and 2 (SphK 1, 2), and exported into plasma to bind to high density lipoprotein and albumin. Sphingosine-1-phosphate acts through five members of the G protein-coupled S1P receptors (S1PR1-S1PR5) to exert diverse actions, which include vascular maturation in embryonic stage and postnatal angiogenesis, maintenance of functional integrity of vascular endothelium, regulation of vascular tonus, and lymphocyte trafficking. Sphingosine-1-phosphate is unique in its ability to regulate cell migration either positively or negatively by acting through different receptor subtypes. S1PR1 and S1PR3 mediate chemotactic cell migration toward S1P via Gi/Rac pathway, whereas S1PR2 mediates S1P inhibition of chemotaxis via G12/13/Rho-dependent inhibition of Rac. Sphingosine-1-phosphate positively or negatively regulates tumor cell migration, invasion in Matrigel, and hematogenous metastasis in manners strictly dependent on S1P receptor subtypes expressed in tumor cells. S1PR1 (and S1PR3) also mediates activation of Gi/phosphatidylinositol 3-kinase (PI3K)/Akt and stimulation of cell proliferation/survival, whereas S1PR2 could mediate suppression of cell proliferation/survival through G12/13/Rho/Rho kinase/PTEN-dependent Akt inhibition. S1PR1 (and S1PR3) expressed in endothelial cells mediates angiogenic action of S1P by stimulating endothelial cell migration, proliferation and tube formation. In a mouse model of hindlimb ischemia after femoral artery resection, repeated local administration or sustained delivery of S1P, or transgenic overexpression of SphK1, accelerates post-ischemic angiogenesis, through the S1P actions on both endothelial cells and bone marrow-derived myeloid cells (BMDCs). In tumor cells, SphK1 is upregulated especially in advanced stages, through mechanisms involving both activating Ras mutation and hypoxia, which leads to increased S1P production and also decreased cellular content of pro-apoptotic sphingolipid ceramide, a metabolic precursor of S1P. Apoptotic tumor cells also produce S1P through SphK2 activation, thus implicated in tumor angiogenesis by acting on endothelial cells through S1PR1/S1PR3, as well as tumor-infiltrating macrophages and BMDCs. Inhibition of S1PR1 function by either an anti-S1P antibody or FTY720 inhibits tumor angiogenesis and tumor growth. Differently from S1PR1, S1PR2 expressed in host cells mediates inhibition of tumor angiogenesis and tumor growth, through mechanisms involving the suppression of endothelial cell migration, proliferation and tube formation, and inhibition of BMDC recruitment to tumor stroma with suppressed expression of pro-angiogenic factor and matrix metalloprotease 9. These findings provide the molecular basis for S1P receptor subtype-selective targeting strategies aiming at angiogenic therapy for occlusive peripheral arterial diseases, and anti-angiogenic and anti-tumor therapies against cancer. Biomedical Reviews 2011; 22: 15-29.

Background: The tumor suppressor gene p63 and its family members p53/p73 have been described as critical determinants of tumorigenesis. ΔNp63, a splice variant of p63 lacking the N-terminal transactivation domain, is thought to antagonize the transcriptional regulation of the p53, p63 and p73 target genes and blocks their tumor suppressor activity. Overexpression of ΔNp63 has been observed in a number of adult human cancers, suggesting a role of this isoform in tumor formation. While some studies have proposed that ΔNp63 might simply inhibit p53 function within cells, leading to malignant transformation, we found that this model is challenged by the lack of consistent correlation between p53 mutation and ΔNp63 expression suggesting that ΔNp63 might possess a gain of function. Methods: Since expression of ΔNp63 isoforms in primary tumors has been shown to correlate with poor prognosis, we attempted to analyze their relationships with tumor angiogenesis. To test whether overexpression of oncogenic ΔNp63 in HEK-293T cells can stimulate tube formation in endothelial cells using HUVEC tube formation assay. Further, we performed gene affymetrix as well as protein micro array analysis in primary cells compared to cells overexpressed ΔNp63 isoforms. In addition, we used siRNA techniques to verify any potential role of ΔNp63 isoforms in tumor angiogenesis in vitro as well as in vivo experiments. Results: We found that overexpression of the oncogenic isoform of p53 family member ΔNp63 in HEK-293T cells resulted in stimulation of tubular structures in HUVEC cells. As 293T cells lack functional p53, these results strongly suggest a gain of function for ΔNp63. In addition, we found that ΔNp63 is overexpressed in neuroblastoma and ΔNp63 overexpression results in increasing secretion of the inflammatory cytokines interleukin-6 (IL-6) and -8 (IL-8) leading to elevated phosphorylation of STAT-3. Our further analysis showed that elevated phosphorylation of STAT-3 induced stabilization of HIF-1α protein resulting in VEGF secretion. Conclusions: We found that ΔNp63 is overexpressed in neuroblastoma and resulted in elevated secretion of the inflammatory cytokines IL-6 and -8 leading to increased STAT3 activation. Our further studies showed that elevated ΔNp63 induced stabilization of HIF-1α protein resulting in VEGF secretion in a p53 independent manner. Importantly, we show in vitro and in vivo experiments that depletion of ΔNp63 isoform by specific siRNAs significantly reduced angiogenic potential of Neuroblastoma. In summary, our study revealed the underlying molecular mechanism how ΔNp63, as a master transcription factor, modulates tumor angiogenesis in Neuroblastoma. Citation Format: Hemant K. Bid, Maren Cam, Antony Audino, Raushan Kurmasheva, Jiayuh Lin, Peter Houghton, Hakan Cam. ΔNp63 promotes neuroblastoma by regulating tumor angiogenesis. [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 2308. doi:10.1158/1538-7445.AM2013-2308

Sangivamycin, an antibiotic with anti-tumor and anti-herpes virus activities by inhibiting both DNA/RNA synthesis and protein kinase C activity, was reported to suppress selectively DNA synthesis and growth of human umbilical vein endothelial cells and their tube formation in vitro. Here, to address the potential clinical use of sangivamycin in future, we investigated its anti-angiogenic effect in in vivo chicken chorioallantoic membrane (CAM) and mouse dorsal air sac (DAS) assays, and investigated underlying mechanism. The effect of sangivamycin on blood vessel formation in CAM was observed under the microscope after treating for two days. For DAS assays, chambers fulfilled with tumor cells were implanted beneath mouse dorsal skin. After the mice were administered with sangivamycin, tumor-induced angiogenesis was observed under the microscope. The effect of sangivamycin on ATP synthesis on the endothelial cell surface was assayed by measuring ATP production with bioluminescence assay. Sangivamycin suppressed angiogenesis within CAM down to 94-71%, which was partially blocked by simultaneous addition of a 40-fold excess of adenosine. Sangivamycin also inhibited tumor-angiogenesis in the DAS assay by 61%, and suppressed ATP production on the endothelial cell surface by 75%. Sangivamycin inhibits the in vivo angiogenesis within CAM and tumor-induced angiogenesis within mouse dorsal skin, at least in part via inhibiting endothelial cell surface ATP metabolism in addition to inhibition of DNA/RNA synthesis and/or protein kinase C activity, suggesting a potential clinical use of sangivamycin as a novel anti-cancer reagent capable of targeting not only cancer cells but also endothelial cells.

Background: Pancreatic cancer, an aggressive and lethal cancer is the fourth leading cause of cancer-related deaths in the United States. Gemcitabine is currently the standard therapeutic agent for advanced pancreatic cancer, but has limited efficacy due to chemoresistance and dose escalation toxicity. We have shown that natural vitamin E δ-tocotrienol is the most bioactive tocotrienols against pancreatic cancer in vitro using pancreatic cancer cells lines as well as in vivo using xenograft models. In this study we evaluated the combination of δ-tocotrienol and gemcitabine on survival, tumor growth, apoptosis, angiogenesis and oncogenic signaling in a transgenic mouse model of pancreatic cancer Methods: Offspring of LSLKRASG12D x PDX-1-Cre x Trp53R172H intercrosses (triple positive by genotyping) were randomized to 4 groups: 1) Vehicle (olive oil, 1.0 ml/kg x 2/ day, PO and PBS 1.0 ml/kg x 2/week, IP), 2) Gemcitabine (100 mg/kg, x 2/week, IP), 3) δ-tocotrienol (200 mg/kg x 2/day, PO) and 4) Gemcitabine + δ-tocotrienol. The treatment was started at the age of 5 weeks and continued for 12 weeks. The survival of the mice in three groups was plotted by Kaplan-Meir graph. The weights of mice were recorded every week and the tumor weights were recorded at the end of the study. The apoptosis markers in plasma and pancreatic tumor, epithelial to mesenchymal transition (EMT), angiogenesis and kras/p53 signaling markers were determined by ELISA, Western blotting and immunostaining. Results: No significant difference in food intake and body weight gain between drug treatment and control groups was observed. δ-tocotrienol treatment alone significantly increased overall survival compared to vehicle (P<0.01). Gemcitabine slightly increased survival whereas combination of two drugs significantly enhanced survival (P<0.05). The tumor weights were more significantly decreased in the combination group (p<0.001) than in each drug alone groups (p<0.02). Gemcitabine and δ-tocotrienol alone and in combination decreased EMT (E-cadherin to vimentin) in tumor tissues. Gemcitabine and δ-tocotrienol combination profoundly induced apoptosis (increased CK18 in plasma), PARP1 cleavage, and Bax expression in tumor tissues than either drug alone. δ-tocotrienol was more effective than gemcitabine in the induction of cyclin-dependent kinase inhibitors (p27Kip1 and p21Cip1), inhibition of angiogenesis (VEGF), vascularity (CD31) and Kras down stream signaling (pAKT. pMEK and pERK). However the combination exerted synergistic effect on the above parameters. Conclusion: δ-tocotrienol synergizes with gemcitabine to inhibit pancreatic tumor growth through inhibition of angiogenesis, cell cycle, Kras signaling and induction of apoptosis in transgenic mouse model of pancreatic cancer. Citation Format: Kazim Husain, Barbara Centeno, Marta Perez, Chen Dung-Tsa, Said M. Sebti, Mokenge P. Malafa. Natural vitamin E delta-tocotrienol combined with gemcitabine prolongs the survival, induces apoptosis, inhibits tumor growth, angiogenesis and oncogenic signaling in a transgenic mouse model of pancreatic cancer. [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 2070. doi:10.1158/1538-7445.AM2013-2070

Tumor endothelial marker 8 (TEM8) is a gene upregulated on colonic tumor endothelium compared to quiescent normal endothelium. Inhibition of TEM8 using antibodies potently inhibits the growth of a variety of tumor types in mice. This inhibition of tumor growth is accompanied by a reduction in microvessel density within the tumors. Thus, TEM8 appears to represent a tumor specific endothelial marker that has a functional role in tumor angiogenesis. TEM8 is also a receptor for anthrax toxin. In the current study, we used chemical libraries to identify small molecule inhibitors of TEM8 that may have efficacy as inhibitors of tumor angiogenesis and anthrax toxin. We conducted a fluorescence resonance energy transfer (FRET) based screen of over 270,000 small molecules in order to identify molecules that inhibit the interaction of TEM8 with its well-known ligand anthrax protective antigen (PA). We describe here the identification and anti-angiogenic effects of small molecules that bind to TEM8. From the initial screen 280 molecules inhibited TEM8/PA interaction by greater than 50%. A secondary screen involved the elucidation of IC50 values for these 280 compounds as inhibitors of the TEM8/PA interaction. Twelve compounds had measurable IC50s in the micromolar range. The anti-angiogenic effects of these compounds were studied in vitro. Of the 12 compounds tested in a human microvascular endothelial cell (HMVEC) proliferation assay, 3 compounds significantly inhibited endothelial cell proliferation after 24 hours (P <0.05), and 5 compounds significantly inhibited proliferation at 72 hours (P <0.05). None of the compounds exhibited measurable cytotoxicity. One of the five compounds tested for effects on HMVEC migration, significantly inhibited migration on a fibronectin coated-surface by 78% (P<0.01). We have identified a number of small molecule inhibitors of TEM8, some of which display anti-angiogenic effects in vitro. Should these inhibitors also display potent anti-angiogenic effects in vivo, they may serve as lead compounds for the future development of TEM8 therapeutic small molecules. Future studies will focus on determining the effects of these molecules on tumor growth and angiogenesis in mouse models. Citation Format: Lorna Cryan, Marinya Roznik, Aaron McBride, Robert D'Amato, Kenneth Christensen, Michael S. Rogers. Identification and anti-angiogenic effects of small molecule inhibitors of TEM8. [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 5076. doi:10.1158/1538-7445.AM2013-5076