Tumor Angiogenesis In Breast Cancer Research Articles

Breast cancer cell-derived extracellular vesicles transfer miR-182-5p and promote breast carcinogenesis via the CMTM7/EGFR/AKT axis

BackgroundExtracellular vesicles (EVs) derived from tumor cells are implicated in the progression of malignancies through the transfer of molecular cargo microRNAs (miRNAs or miRs). We aimed to explore the role of EVs derived from breast cancer cells carrying miR-182-5p in the occurrence and development of breast cancer.MethodsDifferentially expressed miRNAs and their downstream target genes related to breast cancer were screened through GEO and TCGA databases. miR-182-5p expression was examined in cancer tissues and adjacent normal tissues from patients with breast cancer. EVs were isolated from breast cancer cell line MDA-MB-231 cells and identified. The gain- and loss-of function approaches of miR-182-5p and CKLF-like MARVEL transmembrane domain-containing 7 (CMTM7) were performed in MDA-MB-231 cells and the isolated EVs. Human umbilical vein endothelial cells (HUVECs) were subjected to co-culture with MDA-MB-231 cell-derived EVs and biological behaviors were detected by CCK-8 assay, flow cytometry, immunohistochemical staining, Transwell assay and vessel-like tube formation in vitro. A xenograft mouse model in nude mice was established to observe the tumorigenesis and metastasis of breast cancer cells in vivo.ResultsmiR-182-5p was highly expressed in breast cancer tissues and cells, and this high expression was associated with poor prognosis of breast cancer patients. miR-182-5p overexpression was shown to promote tumor angiogenesis in breast cancer. Moreover, our data indicated that miR-182-5p was highly enriched in EVs from MDA-MD-231 cells and then ultimately enhanced the proliferation, migration, and angiogenesis of HUVECs in vitro and in vivo. Moreover, we found that CMTM7 is a target of miR-182-5p. EVs-miR-182-5p promotes tumorigenesis and metastasis of breast cancer cells by regulating the CMTM7/EGFR/AKT signaling axis.ConclusionsTaken altogether, our findings demonstrates that EVs secreted by breast cancer cells could carry miR-182-5p to aggravate breast cancer through downregulating CMTM7 expression and activating the EGFR/AKT signaling pathway.

O-GlcNAc modification regulates MTA1 transcriptional activity during breast cancer cell genotoxic adaptation

BackgroundChromatin modifier metastasis-associated protein 1 (MTA1), closely associated with tumor angiogenesis in breast cancer, plays an important role in gene expression and cancer cell behavior. Recently, an association between O-GlcNAc transferase (OGT) and MTA1 was identified by mass spectroscopy. However, the potential relationship between MTA1 and O-GlcNAc modification has not yet explored. MethodsIn the current study, the role of MTA1 and its O-GlcNAc modification in breast cancer cell genotoxic adaptation was investigated through quantitative proteomics, chromatin immunoprecipitation followed by sequencing (ChIP-seq), transcriptome analysis, and loss- and gain-of-function experiments. ResultsWe demonstrate that the O-GlcNAc modification promotes MTA1 to interaction with chromatin and thus changes the expression of target genes, contributing to breast cancer cell genotoxic adaptation. MTA1 is modified with O-GlcNAc residues at serine (S) residues S237/S241/S246 in adriamycin-adaptive breast cancer cells, and this modification improves the genome-wide interactions of MTA1 with gene promotor regions by enhancing its association with nucleosome remodeling and histone deacetylation (NuRD) complex. Further, O-GlcNAc modification modulates MTA1 chromatin binding, influencing the specific transcriptional regulation of genes involved in the adaptation of breast cancer cells to genotoxic stress. ConclusionsOur findings reveal a previously unrecognized role for O-GlcNAc-modified MTA1 in transcriptional regulation and suggest that the O-GlcNAc modification is a key to the molecular regulation of chemoresistance in breast cancers.

Retraction: Role of Activated Rac1/Cdc42 in Mediating Endothelial Cell Proliferation and Tumor Angiogenesis in Breast Cancer.

Retraction: Role of Activated Rac1/Cdc42 in Mediating Endothelial Cell Proliferation and Tumor Angiogenesis in Breast Cancer.

Clinicopathological and prognostic significance of nestin expression in patients with breast cancer: a systematic review and meta-analysis

BackgroundNestin has been revealed to promote tumorigenesis, progression, metastasis, and angiogenesis of breast cancer. Although the prognostic and clinicopathological impact of nestin expression on breast cancer patients has been assessed in several independent studies, their results remained conflicting. Therefore, we performed this meta-analysis to elucidate the prognostic and clinicopathological association of nestin expression with breast cancer.MethodsA comprehensive literature search was performed in the electronic databases PubMed, EMBASE, Web of Science, the Cochrane Library, China National Knowledge Infrastructure (CNKI), and the Wangfang Data. The statistical analysis was conducted using Stata 15.0 and Review Manager 5.3.ResultsA total of 15 studies with 6066 breast cancer patients were included in this meta-analysis. Pooled results indicated that positive expression of nestin was significantly associated with reduced breast cancer-specific survival (BCSS, univariate analysis, HR = 2.11, 95% CI [1.79, 2.49], P < 0.00001; multivariate analysis, HR = 1.30, 95% CI [1.06, 1.60], P = 0.01), worse overall survival (OS, univariate analysis, HR = 1.88, 95% CI [1.31, 2.71], P = 0.0007; multivariate analysis, HR = 1.89, 95% CI [1.34, 2.67], P = 0.0003) and poorer recurrence-free survival (univariate analysis, HR = 2.60, 95% CI [1.52, 4.46], P = 0.0005), but not with distant metastasis-free survival in univariate analysis (P > 0.05). In addition, increased nestin expression was correlated with younger age, higher tumor grade, larger tumor size, positive blood vessel invasion and high vascular proliferation index, but not with lymph node metastasis or lymph vessel invasion. Nestin was preferentially expressed in invasive ductal carcinoma, triple-negative breast cancer and basal-like subtypes. Nestin expression was inversely associated with the expression of ER and PR, but not with HER-2. Conversely, nestin expression was positively correlated with the expression of basal-like markers CK5, P-cadherin and EGFR. Moreover, nestin expression was strongly associated with the presence of five basal-like profiles (BLP1-5).ConclusionsThis meta-analysis revealed the prognostic value and clinicopathological significance of nestin expression in breast cancer. Nestin is an independent prognostic factor for worse BCSS and OS of breast cancer patients. Nestin is also a valuable biomarker for unfavorable clinicopathological features and tumor angiogenesis of breast cancer. Therefore, nestin is a promising therapeutic target for malignant breast cancer, especially for TNBC and basal-like phenotype.

San Huang Decoction Targets Aurora Kinase A to Inhibit Tumor Angiogenesis in Breast Cancer.

San Huang Decoction (SHD), a Chinese herb formula, has been popularly prescribed in the clinical treatment of patients suffering from breast cancer. The aim of this study was to explore the anti-angiogenic effects of SHD in breast cancer and explain the underlying mechanism. Transwell and Matrigel assays showed that SHD reduced human umbilical vein endothelial cell migration and tubule formation and ELISA and qRT-PCR assays demonstrated its mediation of vascular endothelial growth factor (VEGF) expression. siRNA silencing of aurora kinase A (AURKA) produced results similar to those obtained by inhibition of AURKA with SHD. In addition, a chorioallantoic membrane assay was carried out to directly examine the effect of SHD on breast cancer anti-angiogenesis and immunofluorescence and immunohistochemical staining analysis showed that SHD reduced the expression of CD31, AURKA, and VEGF in a xenograft model. Furthermore, SHD regulated extracellular signal-regulated kinase expression in breast cancer cells, which was examined by western blotting. In conclusion, our findings indicated that SHD treatment mimicked the decrease in tumor neovascularization in breast cancer cells after the siRNA-mediated knockdown of AURKA. Thus, SHD may inhibit tumor angiogenesis in breast cancer by targeting AURKA and downregulating the ERK signaling pathway.

Correction to: The 3'UTR of the pseudogene CYP4Z2P promotes tumor angiogenesis in breast cancer by acting as a ceRNA for CYP4Z1.

After publication of this article [1], it came to our attention that there was an error in Fig. 4b and 4c. In Fig.4b, the migration images of vector groups were incorrect based on this error, the authors re-constructed the migration experiments of Fig. 4b, and the consistent results were obtained. In Fig. 4c, the tube formation image of the Z2P-UTR-siRNA&Z1-UTR group (MCF-7) of Fig. 7d was accidentally misused in control group (MCF-7) and vector group (MCF-7) of Fig. 4c. The corrected Fig. 4 is provided below. This error did not impact the conclusions of the article. The authors apologize for this error.

Aspirin: A Regulator of Tumor Angiogenesis in Breast Cancer

Background and ObjectiveTumor neovascularization / angiogenesisis a pathophysiological process in which new blood vessels are formed in the primary tumor site or distant organs to supply adequate nutrition to cancer cells for metastatic growth. Therefore, controlling tumor angiogenesis is an attractive target for cancer therapy. Structural abnormalities of vasculature (i.e. leakiness due to abnormal lining of pericytes on the microvessels) is one of the critical features of tumor angiogenesis that sensitizes vascular cells to cytokines and helps circulating tumor cells to metastasize to distant organs. Our long term goal is to repurpose the drugs that may prevent tumor angiogenesis or normalize the vessels by repairing leakiness via recruiting pericytes or both. Our recent studies found that Aspirin (ASA)has the potency to inhibit breast cancer growth and metastasis, as well as reprogram the mesenchymal to epithelial transition (MET). Given the importance of ASA, we tested whether ASA may be able to regulate tumor angiogenesis.MethodsWe investigated the effect of low dose ASA (1mM, which is equivalent to 80mg human dose), ASA‐treated(2.5mM) conditioned media (231‐CMASA) or vehicle‐treated conditioned media (231‐CMVT) of MDA‐MB‐231 cells on different endothelial cell physiology. These include endothelial cells' migration towards serum using modified Boyden chamber assay, in vitro capillary‐like structure formation on Matrigel, cell permeability using in vitro endothelial permeability assay and interactions of pericytes‐endothelial cells. We also determined the effect of ASA on various angiogenic factors associated with tumor angiogenesis. Finally, we determined the effect of ASA on invivo tumor angiogenesis using in vivo Angiogenesis Assay.Results and ConclusionsWe found that 231‐CMASA significantly blocks in vitro migration, the formation of in vitro capillary‐like structures parallel with leakiness via incomplete interaction of pericytes and endothelial cells as compared to 231‐CMASA. The antiangiogenic effect of ASA was also documented in in vivo assays. Mechanistically, ASA treatment blocks several angiogenic factors including VEGF‐A that are associated with these three events, implicating a low dose of Aspirin is potentially therapeutic for breast cancer via blocking and normalizing tumor angiogenesis.Support or Funding InformationThis work was supported by VA Merit Review Grants (S. Banerjee and S.K. Banerjee).

Tumor Angiogenesis in Breast Cancer: Pericytes and Maturation Does Not Correlate With Lymph Node Metastasis and Molecular Subtypes

BackgroundAngiogenesis, traditionally assessed by microvessel density (MVD), does not give an indication of the functional status of the tumor neovasculature. The structural and functional stability of the tumor vasculature and its potential clinical relevance in breast cancer was evaluated. Materials and MethodsIn invasive breast cancer, immunostaining of endothelial cells and pericytes was performed using anti-CD34 and anti–platelet-derived growth factor receptor-β antibody, respectively. Double immunostaining for the proliferating capillary index (PCI) and microvessel pericyte coverage index (MPI) was performed with CD34/Ki-67 and CD34/smooth muscle actin. ResultsThe mean MVD of 145 vessels/mm2 was significantly greater in grade 3 tumors (P = .018) and in necrotic tumors (P = .022). The PCI ranged from 0% to 17.14% (mean, 4.37%) and was associated with a high proliferative index in tumor tissue (P = .044). The MPI ranged from 13.09% to 88.18% (mean, 41.35%), indicating the stability of the tumor vasculature. However, it was not significantly associated with the tumor size, tumor grade, lymph node metastasis, proliferative index, or molecular subtypes. ConclusionMVD remains the angiogenesis-related parameter associated with tumor grade and necrosis. The PCI was the only functional parameter of angiogenesis associated with a poor prognostic indicator. The MPI did not show any correlation with the known prognostic and predictive factors.

The 3'UTR of the pseudogene CYP4Z2P promotes tumor angiogenesis in breast cancer by acting as a ceRNA for CYP4Z1.

Pseudogenes are now known to regulate their protein-coding counterparts. Additionally, disturbances of 3'UTRs could increase the risk of cancer susceptibility by acting as modulators of gene expression. The aim of this study was to investigate the roles of the pseudogene CYP4Z2P-3'UTR and functional gene CYP4Z1-3'UTR in breast cancer angiogenesis process. The levels of CYP4Z2P- and CYP4Z1-3'UTR and miRNA of interests were measured in 22 cancerous tissues paired with non-cancerous samples by qRT-PCR. The effects of CYP4Z2P- and CYP4Z1-3'UTR were studied by overexpression and RNA interference approaches in vitro and ex vivo. Insights of the mechanism of competitive endogenous RNAs were gained from bioinformatic analysis, luciferase assays, and western blot. The positive CYP4Z2P/CYP4Z1 interaction and negative interaction between predicted miRNAs and CYP4Z2P or CYP4Z1 were identified via qRT-PCR assay and bivariate correlation analysis. CYP4Z2P- and CYP4Z1-3'UTR share several miRNA-binding sites, including miR-211, miR-125a-3p, miR-197, miR-1226, and miR-204. The CYP4Z2P- and CYP4Z1-3'UTRs arrest the interference caused by of these miRNAs, resulting in increased translation of CYP4Z1. Moreover, ectopic expression of the CYP4Z2P- and CYP4Z1-3'UTRs exhibit tumor angiogenesis-promoting properties in breast cancer collectively by inducing the phosphorylation of ERK1/2 and PI3K/Akt. Co-transfection with Dicer siRNA reversed the CYP4Z2P 3'UTR-mediated changes. Additionally, PI3K or ERK inhibitors reversed CYP4Z2P- and CYP4Z1-3'UTR-mediated changes in VEGF-A expression. Increased CYP4Z2P- and CYP4Z1-3'UTR expression promotes tumor angiogenesis in breast cancer partly via miRNA-dependent activation of PI3K/Akt and ERK1/2. The CYP4Z2P- and CYP4Z1-3'UTRs could thus be used as combinatorial miRNA inhibitors.

Abstract 1599: Elevated expression of ZEB1 upregulates VEGF expression and induces tumor angiogenesis in breast cancer

Abstract 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

The oncoprotein HBXIP enhances angiogenesis and growth of breast cancer through modulating FGF8 and VEGF

Tumor angiogenesis plays an important role in the development of cancer. Previously, we reported that hepatitis B X-interacting protein (HBXIP) functioned as an oncoprotein in breast cancer. However, the role of HBXIP in angiogenesis in breast cancer remains poorly understood. In the present study, we show that the oncoprotein HBXIP plays crucial roles in the event. We observed that the expression levels of HBXIP were positively correlated with those of fibroblast growth factor 8 (FGF8) or vascular endothelial growth factor (VEGF) in clinical breast cancer tissues. Then, we demonstrated that HBXIP was able to upregulate FGF8 through activation of its promoter involving direct binding to cAMP response element-binding protein (CREB) in breast cancer cells and thereby increased its secretion. Strikingly, we identified another pathway that HBXIP upregulated FGF8 and VEGF through inhibiting miRNA-503, which directly targeted 3' untranslated region of FGF8 or VEGF mRNA in the cells. Moreover, we revealed that HBXIP-induced FGF8 could upregulate VEGF expression through activating phosphoinositide 3-kinase (PI3K)/Akt/hypoxia-inducible factor 1-alpha (HIF1α) signaling and increase its secretion. In function, matrigel angiogenesis assay and hemoglobin content analysis uncovered that HBXIP-enhanced FGF8/VEGF boosted tumor angiogenesis and growth in breast cancer in vitro and in vivo in a paracrine/autocrine manner. Thus, we conclude that HBXIP enhances angiogenesis and growth of breast cancer through modulating FGF8 and VEGF. Our finding provides new insights into the mechanism of tumor angiogenesis in breast cancer. Therapeutically, HBXIP may serve as a novel target of tumor angiogenesis.

Abstract A009: Effect of melatonin on the tumor growth and angiogenesis of breast cancer

Abstract Background: Breast cancer has a high mortality rate, mainly due to tumor progression and metastatic spread. Once tumor lesion exceeds a few millimeters in diameter, hypoxia triggers a cascade of events to allow neovascularization. The major contributors to angiogenesis are the vascular endothelial growth factor (VEGF) and its receptors, which promote cell proliferation, migration, permeability and survival. Thus, controlling tumor-associated angiogenesis is a promising tactic in limiting cancer development. The melatonin administration, a hormone naturally produced and secreted in the pineal gland, appears to play an important oncostatic role in cancer and have been currently proposed different mechanisms of action. In this study we evaluated the benefits of melatonin treatment on tumor growth and angiogenesis in breast cancer. Methods: MDA-MB-231 breast cancer cell line was cultured and cell viability was measured by MTT assay after incubation with different concentrations of melatonin (5 mM, 10 mM, 20 mM). We performed an in vivo study implanting cells in athymic nude mice. Mice were treated with 1mg of melatonin (n = 5) or vehicle (n = 8) daily, starting in the same day as tumor implantation and continued for 21 days. Tumors were measured with a digital caliper on day 7, 14 and 21 and the animals underwent SPECT scanning with Tc-99m tagged VEGF-c to detect in vivo angiogenesis (expression of VEGFR2/3). In addition, the markers of angiogenesis (VEGFR2, VEGFR3 and von Willebrand Factor (vWF)) and cell proliferation (Ki-67) were evaluated by immunohistochemistry in mammary tumor tissues. Results: Melatonin treatment in vitro was able to significantly decrease cell viability, after 4 hours of exposure, maintaining its efficacy at higher doses after 24 hours exposure (p&amp;lt;0.05). The animals treated with melatonin showed reduced tumor size compared to control animals after 21 days of treatment (p&amp;lt;0.05). The mean tumor volume of control and treated animals were 282.00 ± 88.53 mm3 and 144.90 ± 38.38 mm3, respectively. The mean of tumor volume in control animals increased significantly from day 14 (118.90 ± 40.17 mm3) to day 21 (282.00 ± 88.53 mm3) (p&amp;lt;0.05), which was not observed in the treated group. Furthermore, there was tumor regression in an animal treated with melatonin (Day 7 = 27.38 mm3; Day 14 = 8.79 mm3; Day 21 = 4.8 mm3), similar pattern was not seen in any of the control. Animals treated with melatonin showed lower activity of Tc-99-VEGF-C in the tumors compared to that of the animals that were treated with vehicle. The intensity of radioactivity in the control animals was 183.55 ± 20.92%, while the intensity of radioactivity in animals treated with melatonin was 150.46 ± 17.06%. Although there was difference in the radioactivity in the tumors between the two group, statistically significant difference was not achieved (p&amp;gt;0.05). However, decrease of VEGFR2 in melatonin treated mice was confirmed by immunohistochemistry (p&amp;lt;0.05). In addition, there was a decrease of micro-vessel density (vWF) and cell proliferation (Ki-67) in melatonin treated tumors (p&amp;lt;0.05). Conclusion: Taken together, our results showed that melatonin inhibits tumor growth, cell proliferation and blocks tumor angiogenesis in breast cancer, directing the development of a clinical study to determine its therapeutic potential for breast cancer. Citation Format: Bruna Victorasso Jardim-Perassi, Ali S. Arbab, Lívia Carvalho Ferreira, Thaiz Ferraz Borin, Nadimpalli Ravi S. Varma, Adarsh Shankar, Asm Iskander, Meser M. Ali, Debora Aparecida Pires de Campos Zuccari. Effect of melatonin on the tumor growth and angiogenesis of breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A009.

Effects of MDM2 inhibitors on vascular endothelial growth factor-mediated tumor angiogenesis in human breast cancer

Mouse double minute 2 (MDM2) is overexpressed in many malignant tumors, and MDM2 levels are associated with poor prognosis of several human cancers, including breast cancer. In the present study, we investigated the function of MDM2 in vascular endothelial growth factor (VEGF)-mediated tumor angiogenesis of breast cancer and the potential value of MDM2 as an anti-angiogenic therapy target for cancer therapy by inhibiting MDM2 with antisense oligonucleotides (ASO) or other antagonist nutlin-3. Anti-MDM2 ASO and nutlin-3 were evaluated for their in vitro and in vivo anti-angiogenesis activities in different human breast cancer models with a different p53 status: MCF-7 cell line containing wild-type p53 and MDA-MB-468 cell line containing mutant p53. MCF-7 and MDA-MB-468 cells were incubated with different concentrations of ASO or nutlin-3 for various periods of time. VEGF gene and protein expression in tumor cells was measured by qPCR and Western blot. The level of VEGF protein secreted in the culture supernatant of treated cells was quantified by enzyme-linked immunosorbent assay (ELISA). Nude mouse xenograft models were further established to determine their effects on tumor growth and angiogenesis. Serum levels of VEGF were measured by ELISA. VEGF expression and microvessel density in tumor tissues were studied by immunohistochemistry. Both angiogenesis and tumor growth were digitally quantified. In both MCF-7 and MDA-MB-468 cells, VEGF expression and secretion were reduced, resulting from specific inhibition of MDM2 expression by ASO. In vivo assay, after administration of ASO, VEGF production reduced and anti-angiogenesis activity occurred in nude mice bearing MCF-7 or MDA-MB-468 xenograft. However, in both models treated with nutlin-3, VEGF production was not changed and anti-angiogenesis activity was not observed. In summary, the ASO construct targeting MDM2 specifically suppresses VEGF expression in vitro and VEGF-mediated tumor angiogenesis in vivo in breast cancer. Furthermore, the suppression of VEGF expression subsequent to inhibition of MDM2 in p53 mutant cells suggests that MDM2 has a regulatory role on VEGF expression through a p53-independent mechanism.

Correction: Role of Activated Rac1/Cdc42 in Mediating Endothelial Cell Proliferation and Tumor Angiogenesis in Breast Cancer

Correction: Role of Activated Rac1/Cdc42 in Mediating Endothelial Cell Proliferation and Tumor Angiogenesis in Breast Cancer

Role of Activated Rac1/Cdc42 in Mediating Endothelial Cell Proliferation and Tumor Angiogenesis in Breast Cancer

Angiogenesis is a well-established target in anti-cancer therapy. Although vascular endothelial growth factor (VEGF)-mediated angiogenesis apparently requires the Rho GTPases Rac1 and Cdc42, the relevant mechanisms are unclear. Here, we determined that activated Rac1/Cdc42 in MCF-7 breast cancer cells could decrease p53 protein levels and increase VEGF secretion to promote proliferation and tube formation of human umbilical vein endothelial cells (HUVECs). However, these effects are reversed after ubiquitin-proteasome breakage. In exploring potential mechanisms for this relationship, we confirmed that activated Rac1/Cdc42 could enhance p53 protein ubiquitination and weaken p53 protein stability to increase VEGF expression. Furthermore, in a xenograft model using nude mice that stably express active Rac1/Cdc42 protein, active Rac1/Cdc42 decreased p53 levels and increased VEGF expression. Additionally, tumor angiogenesis was inhibited, and p53 protein levels were augmented, by intratumoral injection of the ubiquitin-proteasome inhibitor MG132. Finally in 339 human breast cancer tissues, our analyses indicated that Rac1/Cdc42 expression was related to advanced TNM staging, high proliferation index, ER status, and positive invasive features. In particular, our data suggests that high Rac1/Cdc42 expression is correlated with low wt-p53 and high VEGF expression. We conclude that activated Rac1/Cdc42 is a vascular regulator of tumor angiogenesis and that it may reduce stability of the p53 protein to promote VEGF expression by enhancing p53 protein ubiquitin.

Angiotensin II type 1 receptor antagonist candesartan is an angiogenic inhibitor in MDA-MB 468 human breast cancer cells

14114 Background: The present study examined the effect of angiotensin II on the regulation of different angiogenesis associated genes in human breast cancer cells and attempted to determine whether administration of At1R blocker candesartan could suppress the angiotensin II dependent gene regulation. Methods: Angiotensin II dependent expression of vascular endothelial growth factor (VEGF), tissue inhibitor of matrix metalloproteinases 1 (TIMP-1), and hypoxia inducible transcription factor 2alpha (HIF-2alpha) as well as administration of angiotensin II and At1R blocker candesartan was analyzed in MDA-MB 468 human breast cancer cells using immunofluorescence and TaqMan-Real-Time PCR analysis. Results: VEGF, TIMP-1, HIF-2alpha and the At1R were expressed in MDA-MB 468 human breast cancer cells. Angiotensin II significantly increased gene expression of VEGF, TIMP-1, and HIF-2alpha in these cells. This effect was completely inhibited by candesartan. Conclusion: It is hypothesized that angiotensin II is involved in regulation of tumor angiogenesis in breast cancer by regulation of angiogenesis associated genes via At1R. These findings are the first evidence for targeting tumor angiogenesis by inhibition of At1R in human breast cancer cells and may lead to new therapeutical anticancer strategies proposing administration of candesartan. No significant financial relationships to disclose.

Up-regulation of cyclooxygenase-2 gene expression correlates with tumor angiogenesis in human breast cancer

Objective: To study the relationship between cyclooxygenase-2 (COX-2) expression and tumor angiogenesis in human breast cancer. Methods: Archival primary breast carcinomas (n=62), adjacent ductal carcinomain situ (DCIS, n=13) and DCIS alone (n=5) were analyzed for COX-2 and VEGF expression by immunohistochemistry using specific monoclonal antibodies. Microvessel density (MVD) was also examined the using CD34 staining. Results: A significant correlation was found between COX-2 and VEGF expression (P<0.01). Both COX-2 and VEGF were significantly correlated with MVD (P<0.05) andP<0.01, respectively). COX-2 and VEGF genes were overexpressed in tumor specimens as compared with normal epithelia. Conclusion: COX-2 is related to tumor angiogenesis in breast cancer. It is likely that VEGF is one of the most important mediators of the COX-2 angiogenic pathway.

Combined biological therapy of breast cancer using monoclonal antibodies directed against HER2/neu protein and vascular endothelial growth factor.

Vascular endothelial growth factor (VEGF) is one of the most important endothelial mitogens involved in the development and differentiation of the vascular system. Vascular endothelial growth factor is a highly conserved, homodimeric glycoprotein with multiple isoforms. The most abundant isoform, VEGF165, binds to vascular endothelial growth factor receptors-1 (Flt-1) and 2 (KDR/Flk-1) with picomolar affinity. Recently, correlation between microvessel density and engineered expression of VEGF in human breast xenografts was observed. A role of VEGF in breast cancer progression is evident from clinical studies showing elevated serum VEGF in invasive breast cancers. Vascular endothelial growth factor in breast tumor cytosols is correlated with microvessel density, and VEGF165 content correlates with disease-free and overall survival in primary breast cancers. Preliminary data indicate a transcriptional upregulation of VEGF in HER2-overexpressing breast cancer cells. We hypothesize that the upregulation of VEGF in HER2-overexpressing breast cancers contributes to the aggressive phenotype observed in HER2-positive cases and that the "angiogenic switch" associated with HER2 can be attenuated by trastuzumab. Although tumor angiogenesis in breast cancer is complex, the VEGF/vascular endothelial growth factor receptor (VEGFR) system provides a useful model for testing new angiogenesis inhibitors that target this pathway. The VEGF/VEGFR system provides a number of opportunities for therapeutic intervention in breast cancer. Understanding the biology of this system is paramount to fully exploiting VEGF as a therapeutic target in breast cancer. We hypothesize that new therapeutic molecules targeting VEGF and/or its receptors, such as recombinant humanized monoclonal anti-VEGF antibody (rhuMAb VEGF), may have unique activity against HER2-overexpressing breast cancers.

Combined biological therapy of breast cancer using monoclonal antibodies directed against HER2/ neu protein and vascular endothelial growth factor

Vascular endothelial growth factor (VEGF) is one of the most important endothelial mitogens involved in the development and differentiation of the vascular system. Vascular endothelial growth factor is a highly conserved, homodimeric glycoprotein with multiple isoforms. The most abundant isoform, VEGF165, binds to vascular endothelial growth factor receptors-1 (Flt-1) and 2 (KDR/Flk-1) with picomolar affinity. Recently, correlation between microvessel density and engineered expression of VEGF in human breast xenografts was observed. A role of VEGF in breast cancer progression is evident from clinical studies showing elevated serum VEGF in invasive breast cancers. Vascular endothelial growth factor in breast tumor cytosols is correlated with microvessel density, and VEGF165 content correlates with disease-free and overall survival in primary breast cancers. Preliminary data indicate a transcriptional upregulation of VEGF in HER2-overexpressing breast cancer cells. We hypothesize that the upregulation of VEGF in HER2-overexpressing breast cancers contributes to the aggressive phenotype observed in HER2-positive cases and that the “angiogenic switch” associated with HER2 can be attenuated by trastuzumab. Although tumor angiogenesis in breast cancer is complex, the VEGF/vascular endothelial growth factor receptor (VEGFR) system provides a useful model for testing new angiogenesis inhibitors that target this pathway. The VEGF/VEGFR system provides a number of opportunities for therapeutic intervention in breast cancer. Understanding the biology of this system is paramount to fully exploiting VEGF as a therapeutic target in breast cancer. We hypothesize that new therapeutic molecules targeting VEGF and/or its receptors, such as recombinant humanized monoclonal anti-VEGF antibody (rhuMAb VEGF), may have unique activity against HER2-overexpressing breast cancers. Semin Oncol 29 (suppl 11):29-37. Copyright 2002, Elsevier Science (USA). All rights reserved.

Prognostic Significance of Tumor Angiogenesis in Breast Cancer

Prognostic Significance of Tumor Angiogenesis in Breast Cancer