Estradiol increases extracellular levels of vascular endothelial growth factor in vivo in murine mammary cancer

Angiogenesis is essential for tumor growth and metastasis and an important prognostic factor in breast cancer. VEGF, a key factor for angiogenesis, has been correlated with tumor vessel density in breast cancer. Estrogen, another crucial factor in breast cancer, stimulates VEGF; and an ERE in the VEGF gene has been defined. VEGF is bioactive in the extracellular fluid, where it becomes available to endothelial cells. Whether E(2) affects VEGF levels in the extracellular fluid is not known. We show, using intratumoral microdialysis in vivo, that E(2) treatment increased tumor extracellular levels of VEGF in an estrogen-dependent breast cancer model. Moreover, extracellular levels of VEGF in the tumor showed a strong correlation with total tumor VEGF, contrary to plasma levels of VEGF. Ninety-three percent of measured VEGF in the extracellular fluid in the tumor was tumor-derived, while only 45% of VEGF in circulating plasma originated from the tumor. We conclude that E(2) increases extracellular VEGF and that microdialysis is a sensitive method for measurement of local VEGF production in vivo. Our results have potential application to the assessment of tumor characteristics in vivo in human tumors for individualized cancer therapy.     

Tamoxifen inhibits secretion of vascular endothelial growth factor in breast cancer in vivo

Vascular endothelial growth factor (VEGF) is considered a key mediator of tumor angiogenesis, including neovascularization in human breast cancer. High tissue VEGF levels appear to correlate with poor prognosis and decreased overall survival in node-positive and node-negative breast cancer patients. Hormonal regulation of VEGF expression has been demonstrated, and some reports indicate that tamoxifen, a partial estrogen receptor agonist, increases VEGF mRNA in breast cancer cells. These results appear to contradict the efficacy of tamoxifen as an adjuvant for estrogen-dependent breast cancer, yet clinical data show that tamoxifen prevents metastasis and increases overall survival. In this study, we confirmed previous studies showing that intracellular levels of VEGF in vitro increased in response to tamoxifen to levels similar to those observed after estrogen treatment. To further study hormonal effects on the release of VEGF, we used microdialysis to sample the extracellular space, where VEGF is biologically active, in solid tumors in situ. We show for the first time that tamoxifen decreased extracellular VEGF in vivo in solid MCF-7 tumors in nude mice. These in vivo findings were confirmed in vitro where extracellular VEGF in the cell culture medium was decreased significantly by tamoxifen treatment. Furthermore, we illustrate that microdialysis is a viable method that may be applied in human breast tissue to detect soluble VEGF in situ released by the tumor.     

Combination of anti‐angiogenic therapies reduces osteolysis and tumor burden in experimental breast cancer bone metastasis

The clinical efficacy of anti‐angiogenic monotherapies in metastatic breast cancer is less than originally anticipated, and it is not clear what the response of bone metastasis to anti‐angiogenic therapies is. Here, we examined the impact of neutralizing tumor‐derived vascular endothelial growth factor (VEGF) in animal models of subcutaneous tumor growth and bone metastasis formation. Silencing of VEGF expression (Sh‐VEGF) in osteotropic human MDA‐MB‐231/B02 breast cancer cells led to a substantial growth inhibition of subcutaneous Sh‐VEGF B02 tumor xenografts, as a result of reduced angiogenesis, when compared to that observed with animals bearing mock‐transfected (Sc‐VEGF) B02 tumors. However, there was scant evidence that either the silencing of tumor‐derived VEGF or the use of a VEGF‐neutralizing antibody (bevacizumab) affected B02 breast cancer bone metastasis progression in animals. We also examined the effect of vatalanib (a VEGF receptor tyrosine kinase inhibitor) in this mouse model of bone metastasis. However, vatalanib failed to inhibit bone metastasis caused by B02 breast cancer cells. In sharp contrast, vatalanib in combination with bevacizumab reduced not only bone destruction but also skeletal tumor growth in animals bearing breast cancer bone metastases, when compared with either agent alone. Thus, our study highlights the importance of targeting both the tumor compartment and the host tissue (i.e., skeleton) to efficiently block the development of bone metastasis. We believe this is a crucially important observation as the clinical benefit of anti‐angiogenic monotherapies in metastatic breast cancer is relatively modest.     

血管内皮生长因子D及其受体3在乳腺癌淋巴转移中作用的研究进展

乳腺癌是女性最常见的恶性肿瘤。乳腺癌淋巴转移与患者的预后密切相关。最近研究表明淋巴管生成可能会主动促进淋巴转移的发生,而血管内皮生长因子家族的部分成员在这一过程中发挥了重要作用,如血管内皮生长因子C、血管内皮生长因子D及其受体3等。但是,对乳腺癌中血管内皮生长因子D的作用及其预后价值、血管内皮生长因子受体3与乳腺癌淋巴管生成的关系等问题仍有争议。本文对近年国内外有关血管内皮生长因子D及其受体3在乳腺癌淋巴转移中作用的研究进展作一综述。     

<Emphasis Type="Italic">In vivo</Emphasis> measurement of tumor estradiol and Vascular Endothelial Growth Factor in breast cancer patients

Background  

Angiogenesis, crucial for tumor progression, is a process regulated in the tissue micro-environment. Vascular endothelial growth factor (VEGF) is a potent stimulatory factor of angiogenesis and a negative prognostic indicator of breast cancer. VEGF is biologically active in the extracellular space and hitherto, there has been a lack of techniques enabling sampling of angiogenic molecules such as VEGF in situ. The majority of breast cancers are estrogen-dependent, and estrogen has been shown to regulate VEGF in normal breast tissue and experimental breast cancer. We investigated if microdialysis may be applicable in human breast cancer for sampling of extracellular VEGF in situ and to explore if there is an association with local estradiol and VEGF levels in normal and cancerous breast tissue.     

Effect of the vascular endothelial growth factor receptor-2 antibody DC101 plus gemcitabine on growth,metastasis and angiogenesis of human pancreatic cancer growing orthotopically in nude mice

Vascular endothelial growth factor (VEGF) is the major pro-angiogenic factor for most tumors. VEGF expression has been shown to be associated with a poor prognosis in human pancreatic cancer. The purpose of our study was to determine the effect of blockade of VEGF receptor-2 activity with or without gemcitabine on tumor growth and metastasis in an orthotopic model of human pancreatic cancer in nude mice. Therapy with gemcitabine or DC101, a VEGF receptor-2 antibody, resulted in a significant reduction of primary pancreatic tumor growth compared to untreated controls. The combination of DC101 and gemcitabine inhibited primary pancreatic tumor growth and lymphatic metastasis to a greater degree than either agent alone. Treatment with DC101 decreased vessel counts and increased the area of hypoxic tumor tissue compared to controls. Immunofluorescent double staining for apoptotic endothelial cells demonstrated a significant increase in the number apoptotic endothelial cells 24 days after initiation of therapy with DC101 plus gemcitabine. DC101 plus gemcitabine also increased tumor cell death and decreased tumor cell proliferation in pancreatic tumors. These findings indicate that blockade of VEGF receptor activation interferes with the survival of tumor endothelial cells, resulting in a reduction of primary pancreatic tumor growth in nude mice. Furthermore, the data demonstrate that anti-VEGF receptor-2 therapy potentiates the tumoricidal effect of gemcitabine in this model. Anti-VEGF receptor-2 therapy in combination with gemcitabine may be a novel therapeutic approach for advanced pancreatic cancer.     

Mechanisms of angiogenesis and their use in the inhibition of tumor growth and metastasis

There is a constant requirement for vascular supply in solid tumors. Tumor-associated neovascularization allows the tumor cells to express their critical growth advantage. Axillary lymph node status is the most important prognostic factor in operable breast cancer, and experimental and clinical evidence suggests that the process of metastasis is also angiogenesis-dependent. Various angiogenic growth factors and cytokines induce neovascularization in tumors, namely members of the vascular endothelial growth factor (VEGF) and angiopoietin (Ang) gene families. A strong correlation has been found between VEGF expression and increased tumor microvasculature, malignancy, and metastasis in breast cancer. Anti-angiogenic therapy approaches offer a new promising anti-cancer strategy and a remarkably diverse group of over 20 such drugs is currently undergoing evaluation in clinical trials.     

Vascular endothelial growth factor reduces tamoxifen efficacy and promotes metastatic colonization and desmoplasia in breast tumors

Clinical studies have shown that decreased tamoxifen effectiveness correlates with elevated levels of vascular endothelial growth factor (VEGF)-A(165) in biopsy samples of breast cancers. To investigate the mechanisms underlying tamoxifen resistance and metastasis, we engineered the estrogen receptor (ER)-positive MCF-7 human breast cancer cell line to express VEGF to clinically relevant levels in a doxycycline-regulated manner. Induction of VEGF expression in orthotopically implanted xenografts that were initially tamoxifen responsive and noninvasive resulted in tamoxifen-resistant tumor growth and metastasis to the lungs. Lung metastases were also observed in a VEGF-dependent manner following tail vein injection of tumor cells. At both primary and metastatic sites, VEGF-overexpressing tumors exhibited extensive fibroblastic stromal content, a clinical feature called desmoplasia. VEGF-induced metastatic colonies were surrounded by densely packed stromal cells before detectable angiogenesis, suggesting that VEGF is involved in the initiation of desmoplasia. Because expression of VEGF receptors R1 and R2 was undetectable in these tumor cells, the observed VEGF effects on reduction of tamoxifen efficacy and metastatic colonization are most likely mediated by paracrine signaling that enhances tumor/stromal cell interactions and increases the level of desmoplasia. This study reveals new roles for VEGF in breast cancer progression and suggests that combination of antiestrogens and VEGF inhibitors may prolong tamoxifen sensitivity and prevent metastasis in patients with ER-positive tumors.     

Human breast tumors override the antiangiogenic effect of stromal thrombospondin-1 in vivo

The antiangiogenic extracellular matrix protein thrombospondin-1 (TSP-1) inhibits tumor growth and metastasis in animals. However, the clinical relevance of such findings are equivocal as increased stromal TSP-1 expression has been associated with either good or poor prognosis. In an effort to obtain a more integrated understanding of the role of TSP-1 in breast cancer, we first used a breast tumorigenesis model in which tumor-associated stromal fibroblasts were engineered to produce high levels of TSP-1. We demonstrate here that stromal TSP-1 delayed human MDA-MB-231/B02 breast tumor growth. However, this delay in MDA-MB-231/B02 tumor growth upon exposure to TSP-1 was associated with an increased vascular endothelial growth factor (VEGF) expression in tumor cells themselves, leading to a tumor growth rate comparable to that of tumors whose fibroblasts did not overproduce TSP-1. Clinical evidence also suggested that primary breast carcinomas have adapted to escape the effects of stromal TSP-1. TSP-1 was found to be expressed in the stroma of human breast carcinomas where, although its level correlated with decreased vascularization, it was unexpectedly associated with a reduction of relapse-free survival. In metastatic axillary lymph nodes, tumor cells expressed high levels of VEGF and TSP-1 expression were no longer associated with a decreased vascularization. Overall, these results suggest that a resistance may develop early in human breast cancers as a result of high in situ exposure to stromal TSP-1, leading to disease progression.     

VEGF在乳腺癌中的表达及其与病理指标的相关性

目的探讨VEGF在乳腺癌中的表达及其与病理指标的相关性。方法随机选取30例乳腺癌患者作为恶性肿瘤组,另随机选取同期乳腺纤维瘤患者30例为良性肿瘤组,选取同期健康体检女性30例作为健康对照组。运用酶联免疫吸附法(ELISA)测定3组女性的血清VEGF表达,然后统计分析3组妇女的血清VEGF表达、阳性情况,并分析恶性肿瘤组患者的血清VEGF表达与其病理指标的相关性。结果恶性肿瘤组患者的血清VEFG表达显著高于良性肿瘤组、健康对照组(P<0.05),阳性率93.3%(28/30)显著高于良性肿瘤组、健康对照组的13.3%(4/30)、10.0%(3/30)(P<0.05),但良性肿瘤组、健康对照组患者的血清VEGF表达、阳性率之间的差异均无统计学意义(P>0.05)。恶性肿瘤组肿块直径<2.0 cm、2.0~5.0 cm、>5.0 cm患者的血清VEGF表达逐渐升高(P<0.05),组织类型乳腺导管癌、乳腺乳头状癌、乳腺单纯癌、乳腺髓样癌患者的血清VEGF表达逐渐升高(P<0.05),病理分期Ⅰ期、Ⅱ期、Ⅲ期、Ⅳ期患者的血清VEGF表达逐渐升高(P<0.05),有腋窝淋巴结转移患者的血清VEGF表达显著高于无腋窝淋巴结转移患者(P<0.05),阳性表达级别1级、2级、3级患者的血清VEGF表达逐渐升高(P<0.05)。结论VEGF在乳腺癌中的表达升高,与病理指标显著相关。     

Endothelial Robo4 suppresses breast cancer growth and metastasis through regulation of tumor angiogenesis

Targeting tumor angiogenesis is a promising alternative strategy for improvement of breast cancer therapy. Robo4 (roundabout homolog 4) signaling has been shown to protect endothelial integrity during sepsis shock and arthritis, and inhibit Vascular Endothelial Growth Factor (VEGF) signaling during pathological angiogenesis of retinopathy, which indicates that Robo4 might be a potential target for angiogenesis in breast cancer. In this study, we used immune competent Robo4 knockout mouse model to show that endothelial Robo4 is important for suppressing breast cancer growth and metastasis. And this effect does not involve the function of Robo4 on hematopoietic stem cells. Robo4 inhibits breast cancer growth and metastasis by regulating tumor angiogenesis, endothelial leakage and tight junction protein zonula occludens protein‐1 (ZO‐1) downregulation. Treatment with SecinH3, a small molecule drug which deactivates ARF6 downstream of Robo4, can enhance Robo4 signaling and thus inhibit breast cancer growth and metastasis. SecinH3 mediated its effect by reducing tumor angiogenesis rather than directly affecting cancer cell proliferation. In conclusion, endothelial Robo4 signaling is important for suppressing breast cancer growth and metastasis, and it can be targeted (enhanced) by administrating a small molecular drug.     

Cathepsin G-mediated enhanced TGF-β signaling promotes angiogenesis via upregulation of VEGF and MCP-1

Transforming growth factor (TGF)-β signaling makes a significant contribution to the pathogenesis of breast cancer bone metastasis. In other tumor types, TGF-β has been shown to promote tumor vascularity. Here, we report that inhibition of TGF-β significantly reduces microvessel density in mammary tumor-induced bone lesions, mediated by decreased expression of both vascular endothelial growth factor (VEGF) and monocyte chemotactic protein (MCP)-1, both known angiogenic factors. Cathepsin G upregulation at the tumor–bone interface has been linked to increased TGF-β signaling, and we also report that inhibition of Cathepsin G reduced tumor vascularity, as well as VEGF and MCP-1 expression.     

Positive correlation between estradiol and vascular endothelial growth factor but not fibroblast growth factor-2 in normal human breast tissue in vivo.

PURPOSE: Angiogenesis is crucial in tumor development and progression. Ovarian hormones regulate angiogenesis in the reproductive tract but very little is known about its regulation in the normal breast. Sex steroids play an important role in breast cancer development by poorly understood mechanisms. Vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2) are potent stimulators of angiogenesis. Both VEGF and FGF-2 function in autocrine/paracrine pathways and there is a major contribution of bioactive proteins by a posttranslational activation of sequestered molecules in the extracellular space. A direct measurement of these molecules in the extracellular compartment is, therefore, needed. EXPERIMENTAL DESIGN: In this study, microdialysis was used to measure extracellular VEGF and FGF-2 in normal human breast tissue in situ in 11 premenopausal and 5 postmenopausal women. RESULTS: Significantly higher level of VEGF in breast tissue of premenopausal women was found. Plasma as well as local estradiol and breast tissue VEGF exhibited significant correlations, whereas progesterone had no correlation with breast VEGF. FGF-2 did not correlate with either estradiol or progesterone. CONCLUSION: The result suggests that estradiol is a more potent regulator of free VEGF levels than progesterone in the normal breast. The control of free FGF-2 seems to be independent of sex steroids in the breast. Estrogen induction of free extracellular VEGF may be one mechanism involved in sex steroid-dependent breast carcinogenesis.     

乳腺癌组织中VEGF的表达及临床意义

目的 研究血管内皮生长因子(vascular endothelial growth factor,VEGF)在乳腺癌中的表达及临床意义.方法 应用免疫组织化学S-P法检测52例乳腺癌组织和18例乳腺良性病变中VEGF的表达情况,结合临床及病理形态学资料进行统计分析.结果 VEGF正常乳腺组织中阳性表达率低于乳腺癌组织的阳性表达率(P＜0.05).VEGF在腋淋巴结阳性组和复发/远处转移组的阳性率明显高于腋淋巴结阴性组和未发生远处转移/复发组(P＜0.05);乳腺癌VEGF表达与ER(P＜0.05)、PR(P＜0.05)、生存期(P＜0.05)呈负相关;与CerbB-2(P＜0.05)、临床分期(P＜0.05),呈正相关.VEGF与患者年龄、肿块大小无显著性差异(P＞0.05).结论 VEGF可能与乳腺癌的进展、转移及预后有关,作为独立的指标对判定预后和制定治疗方案具有参考意义.     

Vascular endothelial growth factor polymorphisms in relation to breast cancer development and prognosis.

PURPOSE: Angiogenesis is a necessary step in tumor growth and metastasis. Vascular endothelial growth factor (VEGF) is a major mediator of breast cancer angiogenesis. Therefore, we investigated the association of polymorphisms in the VEGF gene with breast cancer risk and prognostic characteristics of the tumors in a large case-control study. EXPERIMENTAL DESIGN: We examined three polymorphisms in the VEGF gene (-2578C/A, -1154G/A, and +936C/T) in 571 familial breast cancer cases from Poland and Germany and -2578C/A, -634G/C, and +936C/T polymorphisms in 974 unselected breast cancer cases from Sweden together with ethnically and geographically selected controls. RESULTS: None of the polymorphisms or any haplotype was significantly associated with either familial or unselected breast cancers. Our study suggests that the +936C/T polymorphism is unlikely to be associated with breast cancer. We also analyzed the unselected cases for genotypes or haplotypes that associated with tumor characteristics. The -634CC genotype and the -2578/-634 CC haplotype were significantly associated with high tumor aggressiveness (large tumor size and high histologic grade, P < 0.01) and the -2578AA genotype and the -2578/-634 AG haplotype with low histologic grade tumors (P = 0.04). The genotypes and haplotypes were not related with other tumor characteristics such as regional or distant metastasis, stage at diagnosis, or estrogen or progesterone receptor status. CONCLUSIONS: Although none of the polymorphisms studied in the VEGF gene was found to influence susceptibility to breast cancer significantly, some of the VEGF genotypes and haplotypes may influence tumor growth through an altered expression of VEGF and tumor angiogenesis.     

转录因子 Sp1和 VEGF 在乳腺癌中的表达和意义

目的：检测转录因子 Sp1和 VEGF 在乳腺癌中的表达,探讨两者表达的相关性及临床意义。方法：采用免疫组织化学方法检测68例乳腺癌患者肿瘤组织及18例癌旁组织中 Sp1和 VEGF 的表达。结果：Sp1和 VEGF 在68例乳腺癌中表达的阳性率分别为72．05％（49／68）和64．71％（44／68）,与在18例正常乳腺组织中表达阳性率（分别为33．3％和16．67％）的差异有统计学意义（P 均＜0．01）。Sp1的过表达与乳腺癌的TNM分期、脉管浸润及淋巴结转移相关（P ＜0．05）,而与患者的年龄、肿瘤大小、组织学分级无明显相关性;在乳腺癌组织中 Sp1和 VEGF 的表达之间呈明显正相关（P ＜0．01）。结论：Sp1和 VEGF 的表达与乳腺癌的生物学行为密切相关,且 Sp1高表达与乳腺癌的血管生成及患者的预后相关。     

Inhibiting Colorectal Carcinoma Growth and Metastasis By Blocking the Expression of VEGF Using RNA Interference

Angiogenesis plays an essential role in tumor growth and metastasis and is a promising target for cancer therapy. Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis. The present study was designed to determine the role of VEGF in tumor growth and metastasis using RNA interference (RNAi) technology. Four small interfering RNA (siRNA) sequences for the VEGF gene were cloned into expression plasmids and transfected into human colorectal carcinoma (CRC) SW620 cells. Stable transfection of these plasmids decreased VEGF protein expression, leading to the potent suppression of tumor cell proliferation, migration, invasion, and angiogenesis in vitro. Furthermore, in subcutaneous and intrasplenic/portal injection models involving athymic nude mice, the tumor growth and metastasis of SW620 cells expressing VEGF siRNA were significantly inhibited compared with untransfected cells or cells transfected with control vector alone. Immunohistochemical analyses of tumor sections revealed a decreased vessel density and decreased VEGF expression in the animals where siRNA against VEGF were expressed. These results indicate that RNAi of VEGF can be an effective antiangiogenic strategy for CRC.     

Pigment epithelium-derived factor as an impending therapeutic agent against vascular epithelial growth factor-driven tumor-angiogenesis

In spite of the recent epidemiological study indicating a positive decrease in cancer trends, cancer remains to be one of the major causes of deaths and there is an anticipated increase in the number of new cancer cases to be recorded in the following years to come. It is important for researchers to improve the current therapeutic agents involved against cancer, particularly targeting to inhibit tumor cell growth, survival, and metastasis. Many researchers investigate the crucial role of a proangiogenic factor, vascular endothelial growth factor (VEGF) in the process of tumor angiogenesis, where the formation of new blood vessels carrying essential nutrients to the tumor cell becomes a critical factor for tumor growth. Since the establishment of VEGF's integral role in mediating tumor angiogenesis and tumor cell survival, current efforts are dedicated to developing therapeutic agents against VEGF and one of the emerging candidate under this category is pigment epithelium-derived factor (PEDF).     

Review of bevacizumab in the treatment of metastatic breast cancer

Vascular endothelial growth factor (VEGF) inhibition is a rational therapeutic approach in breast cancer because VEGF plays an important role in tumour blood vessel growth, tumour progression and metastasis. Bevacizumab is a recombinant humanised antibody targeted at VEGF. As VEGF is overexpressed in early tumour development, there is a strong rationale for studies using bevacizumab in a first-line setting. The €2100 trial demonstrated significantly greater progression-free survival and overall response rates with a bevacizumab plus paclitaxel combination compared with paclitaxel alone as first-line therapy in previously untreated metastatic breast cancer patients. Other studies in heavily pretreated patients have shown that bevacizumab in combination with capecitabine confers varying survival benefits, which might be due to the decreased significance of VEGF in advanced stages of tumour development.