Attenuated Tumor Angiogenesis Research Articles

Caspase-4 promotes metastasis and interferon-γ-induced pyroptosis in lung adenocarcinoma

Caspase-4 (CASP4) is a member of the inflammatory caspase subfamily and promotes inflammation. Here, we report that CASP4 in lung adenocarcinoma cells contributes to both tumor progression via angiogenesis and tumor hyperkinesis and tumor cell killing in response to high interferon (IFN)-γ levels. We observe that elevated CASP4 expression in the primary tumor is associated with cancer progression in patients with lung adenocarcinoma. Further, CASP4 knockout attenuates tumor angiogenesis and metastasis in subcutaneous tumor mouse models. CASP4 enhances the expression of genes associated with angiogenesis and cell migration in lung adenocarcinoma cell lines through nuclear factor kappa-light chain-enhancer of activated B cell signaling without stimulation by lipopolysaccharide or tumor necrosis factor. CASP4 is induced by endoplasmic reticulum stress or IFN-γ via signal transducer and activator of transcription 1. Most notably, lung adenocarcinoma cells with high CASP4 expression are more prone to IFN-γ-induced pyroptosis than those with low CASP4 expression. Our findings indicate that the CASP4 level in primary lung adenocarcinoma can predict metastasis and responsiveness to high-dose IFN-γ therapy due to cancer cell pyroptosis.

Nuclear PD-L1 promotes EGR1-mediated angiogenesis and accelerates tumorigenesis

Targeting programmed cell death protein ligand 1 (PD-L1) remains one of the most essential immunotherapies in cancer1,2. PD-L1 has been detected in the nucleus in multiple malignancies, playing an oncogenic role independent of immune checkpoint regulation3–5. Howbeit, the regulatory function of nuclear PD-L1 (nPD-L1) remains to be fully understood. Here, we report that nPD-L1 is an endogenous accelerator for cancer angiogenesis. First, we found that an abundant proportion of PD-L1 was distributed within the nucleus of uveal melanoma samples, which is associated with an unfavorable outcome. Moreover, the capacity of promoting angiogenesis was largely attenuated in the nPD-L1-deficient cells both in vivo and in vitro. Mechanistically, nPD-L1 facilitates p-STAT3 binding to the promoter of early growth response-1 (EGR1), resulting in the activation of EGR1-mediated angiogenesis. Therapeutically, the inhibition of histone deacetylase 2 restores the normal acetylation level of PD-L1, blocking its nuclear translocation and thereby attenuating tumor angiogenesis. Conclusively, we reveal that nPD-L1 promotes angiogenesis in malignancies, and provide a novel anti-vascularization strategy through blocking aberrant PD-L1 nuclear translocation for tumor therapy.

Nanosized zingerone-triggered anti-angiogenesis contributes to tumor suppression in human hepatocellular carcinoma

Suppression of tumor angiogenesis has become one of the most important means of antitumorigenesis, tumor development, and metastasis. Therefore, developing novel anti-angiogenic agents for tumor therapeutics is an important issue. Phytochemicals possess numerous effective antitumorigenesis properties and can inhibit cell growth and metastasis and are thus regarded as promising candidates for use as chemopreventive agents and chemotherapeutic adjuvants. In our previous study, we fabricated phytochemical-derived carbon dot-zingerone nanoparticles (zingerone NPs) and demonstrated their potent antitumorigenesis in vivo and in vitro against human hepatocellular carcinoma (HCC). In this study, we further validated the roles of zingerone NPs in antitumor angiogenesis. Here, two human endothelial cell lines, EA.hy293 and HUVECs, and two hepatocellular carcinoma cell lines, SK-Hep-1 and Huh7, were utilized to investigate the effects of zingerone NPs on antitumor angiogenesis. Our data demonstrated that zingerone NPs significantly inhibited the viability of human endothelial EA.hy296 cells and HUVECs. Moreover, zingerone NPs revealed superior anti-angiogenic effects on cell invasiveness, in vitro tube formation, and ex vivo rat aorta ring angiogenesis. By verifying zingerone NP-mediated molecular mechanisms of anti-angiogenesis, we found that zingerone NPs significantly decreased the protein expression of VEGF and VEGFR in endothelial cells. Moreover, zingerone NPs downregulated VEGF expression in hepatoma cell lines. The hepatoma Huh7 xenograft model also showed that zingerone NPs markedly attenuated tumor angiogenesis through inhibition of VEGF and CD31 expression. In addition, we also demonstrated that zingerone NPs significantly suppressed tumorigenesis by inhibiting VEGF-mediated PI3K/Akt/mTOR axis signaling and Akt/eNOS signaling in endothelial and hepatocellular carcinoma cells. These results suggest that the phytochemical-derived carbon dots-zingerone NPs possess superior characteristics in antitumor angiogenesis and antitumorigenesis. Altogether, our study provides recent advances in zingerone NPs as anti-angiogenic agents to enhance chemopreventive and anticancer effects for future tumor therapeutic strategies.

STAT3/miR-130b-3p/MBNL1 feedback loop regulated by mTORC1 signaling promotes angiogenesis and tumor growth

BackgroundAberrantly activated mammalian target of rapamycin complex 1 (mTORC1) plays a vital role in tumor angiogenesis, but its precise mechanisms are still unclear.MethodsMicro-RNA-130b-3p (miR-130b-3p) expression in mTORC1-activated and control cells was examined by quantitative real-time PCR (qRT-PCR). MiR-130b-3p levels and their correlation with mTORC1 activity were evaluated by analyzing publicly available databases and in-house head and neck squamous cell carcinoma (HNSCC) tissues. The role of miR-130b-3p in mTORC1-mediated angiogenesis and tumor growth was examined using tube formation assay, chicken chorioallantoic membrane assay, cell line − derived xenograft models, and an HNSCC patient-derived xenograft (PDX) model. The regulatory mechanisms among signal transducer and activator of transcription 3 (STAT3), miR-130b-3p, and muscleblind-like protein 1 (MBNL1) were investigated via bioinformatics analyses, qRT-PCR, western blot, RNA immunoprecipitation, immunofluorescence, luciferase reporter assay, and chromatin immunoprecipitation assay.ResultsElevated miR-130b-3p enhanced the angiogenic and tumorigenic abilities of mTORC1-activated cells both in vitro and in vivo. STAT3, a downstream effector of mTORC1, transactivated miR-130b-3p by direct binding promoter of the miR-130b gene. MBNL1 was identified as a direct target of miR-130b-3p. MBNL1 depletion rescued the compromised angiogenesis and tumor growth caused by miR-130b-3p inhibition. MiR-130b-3p levels were significantly upregulated and positively correlated with mTORC1 signaling in multiple cancers. MiR-130b-3p inhibition attenuated tumor angiogenesis and growth in an HNSCC PDX model. MBNL1 feedback inhibited STAT3 activation in mTORC1-activated cells.ConclusionsThe STAT3/miR-130b-3p/MBNL1 feedback loop plays a vital role in mTORC1-mediated angiogenesis and tumor progression. This pathway could be targeted for therapeutic intervention of mTORC1-related cancers.

Aiphanol, a multi-targeting stilbenolignan, potently suppresses mouse lymphangiogenesis and lymphatic metastasis.

The high incidence of lymphatic metastasis is closely related to poor prognosis and mortality in cancers. Potent inhibitors to prevent pathological lymphangiogenesis and lymphatic spread are urgently needed. The VEGF-C-VEGFR3 pathway plays a vital role in driving lymphangiogenesis and lymph node metastasis. In addition, COX2 in tumor cells and tumor-associated macrophages (TAMs) facilitates lymphangiogenesis. We recently reported that aiphanol, a natural stilbenolignan, attenuates tumor angiogenesis by repressing VEGFR2 and COX2. In this study, we evaluated the antilymphangiogenic and antimetastatic potency of aiphanol using in vitro, ex vivo and in vivo systems. We first demonstrated that aiphanol directly bound to VEGFR3 and blocked its kinase activity with an half-maximal inhibitory concentration (IC50) value of 0.29 μM in an in vitro ADP-GloTM kinase assay. Furthermore, we showed that aiphanol (7.5-30 μM) dose-dependently counteracted VEGF-C-induced proliferation, migration and tubular formation of lymphatic endothelial cells (LECs), which was further verified in vivo. VEGFR3 knockdown markedly mitigated the inhibitory potency of aiphanol on lymphangiogenesis. In 4T1-luc breast tumor-bearing mice, oral administration of aiphanol (5 and 30 mg· kg-1 ·d-1) dose-dependently decreased lymphatic metastasis and prolonged survival time, which was associated with impaired lymphangiogenesis, angiogenesis and, interestingly, macrophage infiltration. In addition, we found that aiphanol decreased the COX2-dependent secretion of PGE2 and VEGF-C from tumor cells and macrophages. These results demonstrate that aiphanol is an appealing agent for preventing lymphangiogenesis and lymphatic dissemination by synergistically targeting VEGFR3 and inhibiting the COX2-PGE2-VEGF-C signaling axis.

Loss of S6K1 But Not S6K2 in the Tumor Microenvironment Suppresses Tumor Growth by Attenuating Tumor Angiogenesis

Two isoforms of the 70-kDa ribosomal protein S6 kinase, S6K1 and S6K2, have been identified and are considered key downstream effectors of the mTOR signaling pathway, which is involved in tumor growth and progression. However, their biological roles in the tumor microenvironment are poorly understood. In this study, utilizing tumor xenograft models in S6k1−/− and S6k2−/− mice, we show that loss of S6K1 but not S6K2 in the tumor stroma suppresses tumor growth, accompanied by attenuated tumor angiogenesis. We found that while S6K1 depletion had no effect on the proangiogenic phenotype of endothelial cells, the growth and angiogenesis of tumor xenografts were significantly reduced in wild-type mice upon reconstitution with S6K1-deficient bone marrow cells. Furthermore, upon S6K1 loss, induction of both mRNA and protein levels of Hif-1α and those of the downstream target, Vegf, was compromised in bone marrow–derived macrophages stimulated with lactate. These findings indicate that S6K1 but not S6K2 contributes to establishing a microenvironment that favors tumor growth through mediating angiogenesis, and suggest that attenuated tumor angiogenesis upon loss of S6K1 in the tumor stroma is, at least in part, attributable to impaired upregulation of Vegf in tumor-associated macrophages.

Tumor Targeted Curcumin Delivery by Folate-Modified MPEG-PCL Self-Assembly Micelles for Colorectal Cancer Therapy.

IntroductionCurcumin (Cur) is a natural extract of Asian spice Curcumin longa, showing multi-targeting capability and low toxicity in anti-tumor activities. The low bioavailability restricts its application as a therapeutic agent. Folate (FA) receptors are highly expressed in many malignant tumors while low expressed in normal tissue. Herein, we developed a self-assembled FA modified MPEG-PCL micelle to incorporate Cur (FA/Nano-Cur) and applied it for colorectal cancer therapy.MethodsWe prepared FA/Nano-Cur micelles and identified their characteristics. The drug release behavior, pharmacokinetics and in vitro anti-tumor activities of FA/Nano-Cur were studied. Furthermore, the in vivo anti-tumor ability assessment and anti-tumor mechanisms investigation were carried out in murine colorectal cancer model.ResultsFA/Nano-Cur micelles had an average particle size of 30.47 nm. Elongated T1/2 and larger AUC were found in FA/Nano-Cur group than that in the Free Cur group. MTT assay and apoptotic study indicated the growth inhibitory effect and pro-apoptotic effect of FA/Nano-Cur were the most significant among all treatments. Moreover, the in vivo study demonstrated that FA/Nano-Cur micelles exhibited a much stronger effect to suppress tumor growth, promote tumor apoptosis and attenuate tumor angiogenesis than Free Cur and Nano-Cur micelles.ConclusionThe present study demonstrated FA/Nano-Cur micelles might be a promising therapeutic agent in colorectal cancer treatment with distinctive advantages of improved bioavailability, sustained drug release, tumor-targeted delivery and low toxicity.

NAMPT signaling is critical for the proangiogenic activity of tumor-associated neutrophils.

Tumor-associated neutrophils (TANs) regulate many processes associated with tumor progression, and depending on the microenvironment, they can exhibit pro- or antitumor functions. However, the molecular mechanisms regulating their tumorigenicity are not clear. Using transplantable tumor models, we showed here that nicotinamide phosphoribosyltransferase (NAMPT), a molecule involved in CSF3R downstream signaling, is essential for tumorigenic conversion of TANs and their pro-angiogenic switch. As a result tumor vascularization and growth are strongly supported by these cells. Inhibition of NAMPT in TANs leads to their antitumor conversion. Adoptive transfer of such TANs into B16F10-tumor bearing mice attenuates tumor angiogenesis and growth. Of note, we observe that the regulation of NAMPT signaling in TANs, and its effect on the neutrophil tumorigenicity, are analogous in mice and human. NAMPT is up-regulated in TANs from melanoma and head-and-neck tumor patients, and its expression positively correlates with tumor stage. Mechanistically, we found that targeting of NAMPT suppresses neutrophil tumorigenicity by inhibiting SIRT1 signaling, thereby blocking transcription of pro-angiogenic genes. Based on these results, we propose that NAMPT regulatory axis is important for neutrophils to activate angiogenic switch during early stages of tumorigenesis. Thus, identification of NAMPT as the critical molecule priming protumor functions of neutrophils provides not only mechanistic insight into the regulation of neutrophil tumorigenicity, but also identifies a potential pathway that may be targeted therapeutically in neutrophils. This, in turn, may be utilized as a novel mode of cancer immunotherapy.

The pretubulysin-induced exposure of collagen is caused by endothelial cell retraction that results in an increased adhesion and decreased transmigration of tumor cells.

Microtubule-targeting agents (MTAs) are the most widely used chemotherapeutic drugs. Pretubulysin (PT), a biosynthetic precursor of the myxobacterial tubulysins, was recently identified as a novel MTA. Besides its strong anti-tumoral activities, PT attenuates tumor angiogenesis, exerts anti-vascular actions on tumor vessels and decreases cancer metastasis formation in vivo. The aim of the present study was to analyze the impact of PT on the interaction of endothelial and tumor cells in vitro to gain insights into the mechanism underlying its anti-metastatic effect. The influence of PT on tumor cell adhesion and transmigration onto/through the endothelium as well as its influence on cell adhesion molecules and the chemokine system CXCL12/CXCR4 was investigated. Treatment of human endothelial cells with PT increased the adhesion of breast cancer cells to the endothelial monolayer, whereas their transmigration through the endothelium was strongly reduced. Interestingly, the PT-induced upregulation of ICAM-1, VCAM-1 and CXCL12 were dispensable for the PT-evoked tumor cell adhesion. Tumor cells preferred to adhere to collagen exposed within PT-triggered endothelial gaps via β1-integrins on the tumor cell surface. Taken together, our study provides, at least in part, an explanation for the anti-metastatic potential of PT.

Notch3 signaling-mediated melanoma–endothelial crosstalk regulates melanoma stem-like cell homeostasis and niche morphogenesis

Melanoma is among the most virulent cancers, owing to its propensity to metastasize and its resistance to current therapies. The treatment failure is largely attributed to tumor heterogeneity, particularly subpopulations possessing stem cell-like properties, ie, melanoma stem-like cells (MSLCs). Evidence indicates that the MSLC phenotype is malleable and may be acquired by non-MSLCs through phenotypic switching upon appropriate stimuli, the so–called ‘dynamic stemness'. Since the phenotypic characteristics and functional integrity of MSLCs depend on their vascular niche, using a two-dimensional (2D) melanoma–endothelium co-culture model, where the MSLC niche is recapitulated in vitro, we identified Notch3 signaling pathway as a micro-environmental cue governing MSLC phenotypic plasticity via pathway-specific gene expression arrays. Accordingly, lentiviral shRNA-mediated Notch3 knockdown (KD) in melanoma cell lines exhibiting high levels of endogenous Notch3 led to retarded/abolished tumorigenicity in vivo through both depleting MSLC fractions, evinced by MSLC marker downregulation (eg, CD133 and CD271); and impeding the MSLC niche, corroborated by the attenuated tumor angiogenesis as well as vasculogenic mimicry. In contrast, Notch3 KD affected neither tumor growth nor MSLC subsets in a melanoma cell line with relatively low endogenous Notch3 expression. Thus, Notch3 signaling may facilitate MSLC plasticity and niche morphogenesis in a cell context-dependent manner. Our findings illustrate Notch3 as a molecular switch driving melanoma heterogeneity, and provide the biological rationale for Notch inhibition as a promising therapeutic option.

Dose dependent effects of cadmium on tumor angiogenesis

Angiogenesis is crucial for tumor growth and metastasis. Cadmium (Cd) exposure is associated with elevated cancer risk and mortality. Such association is, at least in part, attributable to Cd-induced tumor angiogenesis. Nevertheless, the reported effects of Cd on tumor angiogenesis appear to be either stimulatory or inhibitory, depending on the concentrations. Ultra-low concentrations of Cd (<0.5 μM) inhibit endothelial nitric oxide synthase activation, leading to reduced endothelial nitric oxide production and attenuated tumor angiogenesis. In contrast, low-lose Cd (1-10 μM) up-regulates vascular endothelial growth factor (VEGF)-mediated tumor angiogenesis by exerting sub-apoptotic levels of oxidative stress on both tumor cells and endothelial cells (ECs). The consequent activation of protein kinase B/Akt, nuclear factor-κB, and mitogen-activated protein kinase signaling cascades mediate the increased secretion of VEGF by tumor cells and the up-regulated VEGF receptor-2 expression in ECs. Furthermore, Cd in high concentrations (>10 μM) induces EC apoptosis via the activation of caspase-3, resulting in destruction of tumor vasculature. In this review, we summarize the current knowledge concerning the roles of Cd in tumor angiogenesis, with a focus on molecular mechanisms underlying the dose dependent effects of Cd on various EC phenotypes.

In vitro and in vivo study of hydralazine, a potential anti-angiogenic agent

Hydralazine (HYD), an old routine clinical anti-hypertension drug, is rarely used in clinic nowadays. Since the strategy of repositioning old drugs was put forward, HYD has been reported to possess various biological activities, including antitumor efficacy and reducing intra-tumor microvessel. Here, we investigated that whether HYD had the ability of anti-angiogeneis and its underlying mechanism. Cells proliferation, wound-healing, Transwell migration and invasion, tube formation and rat aortic ring assays in vitro and chicken chorioallantoic membrane (CAM) model in vivo were designed to investigated HYD's anti-angiogenic effect. Levels of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) were assessed by enzyme-linked immune sorbent assay (ELISA). Hepatocellular carcinoma (HCC) mice model was used to evaluate HYD's effect on tumor growth and microvessel density. Our results showed that HYD not only inhibited human umbilical vascular endothelial cell (HUVEC) proliferation, wound-healing, Transwell migration and invasion and tube formation, but also suppressed the microvessel outgrowth of rat aortic ring in vitro and the neovascularzation of CAM in vivo. Furthermore, we demonstrated that HYD attenuated tumor angiogenesis and tumor growth. In the co-culture system of Transwell migration, the secretion of VEGF and bFGF was reduced by HYD respectively. In sum, our data indicate that HYD has the pharmacological effect of ant-angiogenesis by interference with VEGF and bFGF signaling pathways in endothelial cells. These findings suggest that HYD might be a promising angiogenesis inhibitor and a potential effective therapeutic agent for cancer therapy.

Abstract 2825: Prevention of breast tumor growth by an extract from the muscadine grape

Abstract Grape extracts have garnered attention as chemopreventive agents due their anti-proliferative, anti-inflammatory and anti-oxidant properties. The muscadine grape (Vitis rotundifolia) has a distinct phytochemical composition compared to other grape varieties. The current study examines the preventive effects of a proprietary muscadine grape extract (MGE) on breast cancer formation. The proliferation of actively growing human ER+ or triple negative breast cancer cells was significantly reduced by MGE from grape seeds or skins with an associated decrease in phospho-ERK, suggesting that MGE inhibits breast cancer cell proliferation through a reduction in activation of growth-promoting kinases. Female FVB-Tg(MMTV)NKAMul/J transgenic mice with an activated rat Erbb2 oncogene expressed specifically in the mammary gland (c-neu mice) form mammary tumors by 6-8 months of age. c-Neu mice were treated with MGE in the drinking water (approximately 1.0 mg phenolics/25 g mouse) beginning at weaning and were sacrificed at 7½ months of age. No changes were observed in eating or drinking habits or in gross body, heart or kidney weight, suggesting that the MGE was well tolerated. Although all of the mice developed breast tumors, MGE significantly reduce tumor burden compared to the tumor tissue in mice drinking regular water (9.2±1.2 g versus 3.6±1.4 g, p = 0.001, n = 8-10). A group of c-Neu mice were also sacrificed at 4 months of age, once tumors were palpable, to assess tumor multiplicity. Both tumor burden (1.4±.3 g versus 0.3±0.1 g, p = 0.007) and tumor multiplicity (11.8±2.1 versus 5.1±0.9, p = 0.013, n = 6-11) were reduced in mice treated with MGE. Tumor tissue sections from mice administered MGE had a significant decrease in the proliferation marker Ki67 compared to tumors from control animals [310±92 versus 166±70 immunoreactive cells/field, p = 0.002], in agreement with the reduction in proliferation observed in vitro. Tumor sections from c-neu mice were incubated with an antibody to the endothelial cell marker, CD34, and vessels were identified by a combination of morphology and positive CD34 immunoreactivity; MGE significantly reduced blood vessel density in breast tumor tissue compared to tumors from mice drinking regular water [5.7±0.6 versus 3.6±0.9, p = 0.022], suggesting that the grape extract attenuates tumor angiogenesis. Interstitial tumoral fibrosis was quantified in breast tumor tissue sections using picrosirius red, a nonspecific collagen stain. MGE treatment markedly decreased interstitial fibrosis as compared to the tumors from control animals [2.79±0.70% versus 0.14±0.04%, p = 0.0005], indicating that the extract reduces cancer-associated fibrosis in breast tumors. Taken together, these studies suggest that MGE reduces both breast tumor burden and multiplicity, through decreasing proliferation, angiogenesis and fibrosis, suggesting that extracts from muscadine grapes may represent a novel nutraceutical for the prevention of breast cancer. Citation Format: Patricia E. Gallagher, E. Ann Tallant. Prevention of breast tumor growth by an extract from the muscadine grape. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2825. doi:10.1158/1538-7445.AM2015-2825

Abstract 4180: TGF-beta promotes angiogenesis in an RB-deficient, Kras-driven mouse model of pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease that is projected to become the 2nd leading cause of cancer deaths by 2030. PDACs are associated with a high frequency of KRAS mutations (95%) and overexpression of many pro-angiogenic cytokines and growth factors. Using a genetically engineered mouse model (GEMM) that we established in which oncogenic Kras is combined with loss of RB (KRC), we have determined that PDACs arising in these mice (mPDACs) harbor many endothelial cells (ECs) and sinusoidal-like vessels that have blood flow, as determined by intravital confocal microscopy. Array analysis of pancreatic cancer cells (PCCs) derived from KRC tumors revealed gene expression profiles that correlate with active TGF-β signaling pathways, as determined by gene set enrichment analysis (GSEA), and include increased expression of multiple pro-angiogenic genes. In silico analysis indicated that many of these pro-angiogenic genes were TGF-β targets, and inhibition of the type I TGF-β receptor (TβRI) with SB505124 confirmed that TGF-βs drive pro-angiogenic gene expression in KRC PCCs. Moreover, TGF-β increased the levels of pro-angiogenic cytokines in conditioned media (CM) prepared from KRC PCCs, which when added to ECs, activated canonical TGF-β signaling pathways and stimulated EC proliferation and migration. Although SB505124 blocked TGF-β pathway activation in ECs, it failed to suppress CM-enhanced EC proliferation and migration. By contrast, SB505124 attenuated tumor angiogenesis, growth and metastasis in a syngeneic orthotopic mouse model using KRC PCCs. Taken together, these results suggest that TGF-β promotes angiogenesis in an indirect manner, by up-regulating pro-angiogenic factors in PCCs that act on ECs in a paracrine manner. Therefore, targeting TGF-β in PDAC could be a beneficial anti-angiogenic strategy. Citation Format: Jesse Gore, Kelly E. Craven, Julie L. Wilson, Murray Korc. TGF-beta promotes angiogenesis in an RB-deficient, Kras-driven mouse model of pancreatic cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4180. doi:10.1158/1538-7445.AM2015-4180

Fully human VEGFR2 monoclonal antibody BC001 attenuates tumor angiogenesis and inhibits tumor growth.

The critical role of VEGFR2 in tumor neovascularization and progression has allowed the design of clinically beneficial therapies based on it. Here we show that BC001, a new fully human anti-VEGFR2 monoclonal antibody, inhibits VEGF-stimulated endothelial cell migration, tube formation, and effectively suppressed the transdifferentiation of cancer stem cells into endothelial cells invitro. Since BC001 exhibited no activity against the mouse VEGFR2 and mouse based study was required to confirm its efficacy invivo, BC101, the mouse analogue of BC001, was developed. BC101 significantly attenuated angiogenesis according to Matrigel plug assay and resulted in ~80% growth inhibition of mouse B16F10 homograft tumors relative to vehicle control. Similarly, human analogue BC001 suppressed the growth of human xenograft tumors HCT116 and BGC823. Furthermore, immunohistochemical results showed reduced expression of CD31, VEGFR2 and Ki-67, as well as increased expression of Caspase3 in BC001-treated tumor, which indicated BC001 was able to significantly decrease microvessel density, suppress proliferation and promote apoptosis. These results demonstrate the fully human VEGFR2 monoclonal antibody BC001 can work as an effective inhibitor of tumor angiogenesis and tumor growth both invitro and invivo.

Xiaotan Sanjie decoction attenuates tumor angiogenesis by manipulating Notch-1-regulated proliferation of gastric cancer stem-like cells.

To determine the underlying mechanisms of action and influence of Xiaotan Sanjie (XTSJ) decoction on gastric cancer stem-like cells (GCSCs). The gastric cancer cell line MKN-45 line was selected and sorted by FACS using the cancer stem cell marker CD44; the stemness of these cells was checked in our previous study. In an in vitro study, the expression of Notch-1, Hes1, Vascular endothelial growth factor (VEGF), and Ki-67 in both CD44-positive gastric cancer stem-like cells (GCSCs) and CD44-negative cells was measured by Western blot. The effect of XTSJ serum on cell viability and on the above markers was measured by MTT assay and Western blot, respectively. In an in vivo study, the ability to induce angiogenesis and maintenance of GCSCs in CD44-positive-MKN-45- and CD44-negative-engrafted mice were detected by immunohistochemical staining using markers for CD34 and CD44, respectively. The role of XTSJ decoction in regulating the expression of Notch-1, Hes1, VEGF and Ki-67 was measured by Western blot and real-time polymerase chain reaction. CD44(+) GCSCs showed more cell proliferation and VEGF secretion than CD44-negative cells in vitro, which were accompanied by the high expression of Notch-1 and Hes1 and positively associated with tumor growth (GCSCs vs CD44-negative cells, 2.72 ± 0.25 vs 1.46 ± 0.16, P < 0.05) and microvessel density (MVD) (GCSCs vs CD44-negative cells, 8.15 ± 0.42 vs 3.83 ± 0.49, P < 0.001) in vivo. XTSJ decoction inhibited the viability of both cell types in a dose-dependent manner in vitro. Specifically, a significant difference in the medium- (82.87% ± 6.53%) and high-dose XTSJ groups (77.43% ± 7.34%) was detected at 24 h in the CD44(+) GCSCs group compared with the saline group (95.42% ± 5.76%) and the low-dose XTSJ group (90.74% ± 6.57%) (P < 0.05). However, the efficacy of XTSJ decoction was reduced in the CD44(-) groups; significant differences were only detected in the high-dose XTSJ group at 48 h (78.57% ± 6.94%) and 72 h (72.12% ± 7.68%) when compared with the other CD44- groups (P < 0.05). Notably, these differences were highly consistent with the Notch-1, Hes1, VEGF and Ki-67 expression in these cells. Similarly, in vivo, XTSJ decoction inhibited tumor growth in a dose-dependent manner. A significant difference was observed in the medium- (1.76 ± 0.15) and high-dose XTSJ (1.33 ± 0.081) groups compared with the GCSCs control group (2.72 ± 0.25) and the low-dose XTSJ group (2.51 ± 0.25) (P < 0.05). We also detected a remarkable decrease of MVD in the medium- (7.10 ± 0.60) and high-dose XTSJ (5.99 ± 0.47) groups compared with the GCSC control group (8.15 ± 0.42) and the low-dose XTSJ group (8.14 ± 0.46) (P < 0.05). Additionally, CD44 expression was decreased in these groups [medium- (4.43 ± 0.45) and high-dose XTSJ groups (3.56 ± 0.31) vs the GCSC control (5.96 ± 0.46) and low dose XTSJ groups (5.91 ± 0.38)] (P < 0.05). The significant differences in Notch-1, Hes1, VEGF and Ki-67 expression highly mirrored the results of XTSJ decoction in inhibiting tumor growth, MVD and CD44 expression. Notch-1 may play an important role in regulating the proliferation of GCSCs; XTSJ decoction could attenuate tumor angiogenesis, at least partially, by inhibiting Notch-1.

Novel Tubulin Antagonist Pretubulysin Displays Antivascular Properties In Vitro and In Vivo

Pretubulysin (PT) is a novel, synthetically accessible myxobacterial compound that acts as a tubulin-depolymerizing agent and inhibits cancer cell growth in vitro and in vivo. Moreover, PT was found to attenuate tumor angiogenesis. Here, we hypothesized that PT could exert antivascular activities on existing tumor vessels. We aimed to characterize the antivascular effects of PT and to elucidate the underlying mechanisms in endothelial cells. In vitro, PT rapidly induced endothelial hyperpermeability and a concentration-dependent disassembly of established endothelial tubes on Matrigel and in an ex vivo aortic ring model. It disrupted endothelial cell junctions and triggered F-actin stress fiber formation and cell contraction by the RhoA/Rho-associated protein kinase pathway without causing cell death. In vivo, using a hamster dorsal skinfold chamber preparation, PT significantly decreased blood flow and vessel diameter in hamster A-Mel-3 amelanotic melanoma tumors but not in the neighboring healthy tissue. In a second tumor model using mice with subcutaneous murine B16 melanoma tumors, a single dose of PT (10 mg/kg) caused a shut down of tumor blood flow and a strong central tumor cell necrosis within 24 hours. Repeated PT administration significantly decelerates tumor growth and seems to be well tolerated. In summary, we could show for the first time that the antitumor effect of PT is, at least in part, mediated via its antivascular activities on existing tumor vessels.

MicroRNAs Control Vascular Endothelial Growth Factor Signaling

Angiogenesis is a very tightly controlled process, in which endothelial cells need to migrate and proliferate toward ischemic tissue. A long-known factor that provides a gradient for endothelial cells to migrate toward is vascular endothelial growth factor (VEGF).1 Carefully titrated levels of VEGF are crucial for blood vessel development, because even the heterozygous deletion of VEGF is lethal in mice.2 In the past decades, it has become clear that VEGF signaling is very complex, with different VEGF isoforms and VEGF receptors that tightly control endothelial cell behavior. Interestingly, although VEGF receptor 2 (kinase insert domain receptor [KDR]) is one of the main proangiogenic VEGF receptors,3 binding of VEGF to VEGF receptor 1 (fms-related tyrosine kinase 1 [FLT1]) does not result in proangiogenic signaling, which raised the concept that FLT1 acts as a trap or decoy for VEGF. Thus, FLT1 can negatively regulate VEGF signaling, and this is of crucial importance, for instance, to keep the cornea avascular,4 but also aids in controlling the fine balance between proangiogenic and antiangiogenic factors. To make matters more complex, FLT1 mRNA is alternatively spliced, giving rise to a membrane-bound FLT1 and secreted soluble FLT1,5 but they both function as VEGF traps, preventing VEGF from activating KDR. In this issue of Circulation Research , Hassel et al6 …

Ginsenoside Rg3 attenuates tumor angiogenesis via inhibiting bioactivities of endothelial progenitor cells

Accumulating evidence suggests that Ginsenoside Rg3 appears to inhibit tumor growth including Lewis lung carcinoma, intestinal adenocarcinomas or B16 melanoma by inhibiting cell proliferation, tumor cell invasion and metastasis. Endothelial progenitor cells (EPCs) appear to play a key role in the growth of early tumors by intervening with the angiogenic switch promoting tumor neovessel formation by producing angiogenic cytokines during tumor progression. This paper reports a novel mechanism of Ginsenoside Rg3, a candidate anticancer bio-molecule, on tumor angiogenesis by inhibiting the multiple bioactivities of EPCs. When Ginsenoside Rg3 was applied to the ex vivo cultured outgrowth ECs, a type of EPCs, it inhibited the cell proliferation, cell migration and tubular formation of EPCs. Importantly, Ginsenoside Rg3 attenuated the phosphorylation cascade of the VEGF dependent p38/ERK signaling in vitro. The xenograft tumor model clearly showed that Ginsenoside Rg3 suppresses tumor growth and tumor angiogenesis by inhibiting the mobilization of EPCs from the bone marrow microenvironment to the peripheral circulation and modulates VEGF-dependent tumor angiogenesis. In conclusion, this study provides a potential therapeutic molecule, Ginsenoside Rg3, as an anticancer drug by inhibiting the EPC bioactivities.

Abstract LB-3: Evidence that dibenzofuran based anti-angiogenic, anti-tumor peptidomimetics target galectin-1

Abstract Galectins, named for their selective binding to ß-galactoside-containing glycans, play crucial roles in many physiological as well as pathological processes. Galectin-1 (gal-1) in particular, has been shown to be differentially elevated in tumor endothelial cells in several cancers including cancer of the breast, colon, prostate and ovaries. Gal-1 targeting 33mer peptide, Anginex, inhibits tumor endothelial cell proliferation via anoikis and attenuates tumor angiogenesis and tumor growth. Based on structure-activity relationships of anginex, partial-peptide mimetic were designed, using dibenzofuran (DBF) as a ß-turn mimetic to maintain the two key ß-strands and the bioactive amphipathic ß-sheet conformation of the peptide. The best DBF-based compound 6DBF7 (13mer) inhibited tumor angiogenesis and tumor growth in vivo better than parent anginex. Here we report on the optimization of the anti-angiogenic activity of 6DBF7 analogs in vitro, by substitution of natural and non-natural amino acids, and subsequently the antiangiogenic and anti-tumor effects of the most promising analogs in vivo. In addition, by using NMR spectroscopy and flow cytometry, we demonstrate that the lead 6DBF7 analogs interact with galectin-1 at a site near to its carbohydrate binding domain and by doing so can compete off galectin-1 from binding to ß-galactoside-containing glycans on the surface of cells in vitro. Overall, this research contributes to our ability to design pharmaceutical agents that target galectin-1 and thereby inhibit tumor growth and are potentially clinical candidates for use in the management of human cancers. This work was supported by NIH grant CA096090 to KHM 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 LB-3. doi:1538-7445.AM2012-LB-3