Molecular diagnosis of tumor angiogenesis and anti-angiogenic cancer therapy

Angiogenesis is regulated by the balance of pro-angiogenic factors and angiogenesis inhibitors, and the imbalance of these regulators is the cause of pathological angiogenesis, including tumor angiogenesis. Angiogenesis is required for tumor growth and metastasis, and thus constitutes an important target for the control of tumor progression. While the benefit of anti-angiogenic therapy is potentially profound, limitations have also been recognized by the results obtained thus far by clinical trials. Precise understanding of the process of angiogenesis should lead us to new regimens for more efficient anti-angiogenic therapy. This review focuses on our current understanding of the molecular mechanism of tumor angiogenic and the status of the anti-angiogenesis approach for cancer treatment.     

Biomarkers in the development of anti-angiogenic therapies for ovarian cancer

The treatment of ovarian cancer remains challenging as the majority of patients will relapse and die from their disease despite successful first-line treatment. New treatment strategies are needed and recently there has been an explosion of new agents being tested in ovarian cancer. Most of these are directed against molecularly defined pathways and a significant proportion target angiogenesis, an important process in the growth of ovarian cancer. We review the role of angiogenesis in the pathophysiology of ovarian cancer and discuss the development of the most promising anti-angiogenic drugs in this disease, including the first large phase III trials with bevacizumab which have demonstrated a disease-modifying role in ovarian cancer. Other studies with this drug and other inhibitors of the angiogenic pathways are underway in the first-line and recurrent disease settings. The financial cost of these agents, increased toxicity and requirement for prolonged therapy necessitates the urgent need to identify and validate biomarkers to guide the use of these drugs in the future. There are over 200 candidate biomarkers being studied in ovarian cancer. However, currently there are no validated biomarkers to predict response or progression of disease. In this review we present a selection of biomarkers that are under investigation and discuss their benefits and limitations.     

Targeting angiogenesis in esophagogastric adenocarcinoma

The possibility of targeting tumor angiogenesis was postulated almost 40 years ago. The vascular endothelial growth factor (VEGF) family and its receptors have since been characterized and extensively studied. VEGF overexpression is a common finding in solid tumors, including esophagogastric cancer, and frequently correlates with poor prognosis. Monoclonal antibodies, soluble receptors, and small-molecule tyrosine kinase inhibitors have been developed to inhibit tumor angiogenesis, and antiangiogenic therapy is now a component of standard treatment for advanced renal cell, hepatocellular, colorectal, breast, and non-small cell lung carcinomas. The small-molecule tyrosine kinase inhibitors sunitinib and sorafenib have been evaluated in phase II studies in esophagogastric cancer but appear to have only modest activity. Similarly, despite promising efficacy signals from phase II studies, the addition of the anti-VEGF-A monoclonal antibody bevacizumab to cisplatin plus capecitabine failed to result in a longer overall survival duration than with the chemotherapy doublet plus placebo. The response rate and progression-free survival interval were significantly greater with bevacizumab, confirming some efficacy in advanced gastric cancer, but with inadequate benefit to justify the high cost of treatment. Evaluation of bevacizumab in the neoadjuvant and perioperative settings continues, hypothesizing that a higher response rate will translate into longer survival in patients with operable disease. Despite extensive research, the discovery of a reliable predictive biomarker for antiangiogenic therapy continues to elude the scientific and oncology communities, and mechanisms of primary and acquired resistance are incompletely understood. We are therefore currently unable to personalize antiangiogenic therapy for established indications, or use molecular selection for clinical trials evaluating novel indications.     

Targeting the tumour microenvironment in ovarian cancer

The study of cancer initiation, growth, and metastasis has traditionally been focused on cancer cells, and the view that they proliferate due to uncontrolled growth signalling owing to genetic derangements. However, uncontrolled growth in tumours cannot be explained solely by aberrations in cancer cells themselves. To fully understand the biological behaviour of tumours, it is essential to understand the microenvironment in which cancer cells exist, and how they manipulate the surrounding stroma to promote the malignant phenotype.Ovarian cancer is the leading cause of death from gynaecologic cancer worldwide. The majority of patients will have objective responses to standard tumour debulking surgery and platinum-taxane doublet chemotherapy, but most will experience disease recurrence and chemotherapy resistance. As such, a great deal of effort has been put forth to develop therapies that target the tumour microenvironment in ovarian cancer. Herein, we review the key components of the tumour microenvironment as they pertain to this disease, outline targeting opportunities and supporting evidence thus far, and discuss resistance to therapy.     

Targeting angiogenesis in metastatic breast cancer

Angiogenesis has become an important target in the treatment of several solid tumors, including breast cancer. As monotherapy, antiangiogenic agents have demonstrated limited activity in metastatic breast cancer (MBC); therefore, they have generally been developed for use in combination with chemotherapies. Thus far, the experience with antiangiogenic agents for MBC has been mixed. The results from one study assessing addition of the monoclonal antibody bevacizumab to paclitaxel led to approval of bevacizumab for MBC. However, the modest improvement of progression-free survival rates in subsequent MBC studies has led to reappraisal of bevacizumab. Phase III studies have not produced evidence supporting use of the multikinase inhibitor sunitinib alone or in combination with MBC chemotherapy. Experience with sorafenib in a phase IIb program indicates potential when used in select combinations, particularly with capecitabine; however, phase III confirmatory data are needed. Although antiangiogenic therapies combined with chemotherapy have increased progression-free survival rates for patients with MBC, increases in overall survival times have not been observed. Some studies have tried to combine antiangiogenic agents such as bevacizumab and sunitinib or sorafenib, but that approach has been limited because of toxicity concerns. Sequential use of antiangiogenic agents with differing mechanisms of action may be an effective approach. Despite setbacks, angiogenesis will likely remain an important target of treatment for selected patients with MBC.     

The importance of angiogenesis in ovarian cancer

Advanced stage ovarian cancer has a high rate of recurrence even after surgery followed by chemotherapy combining carboplatin and a taxane. New strategies are currently under way to combat this situation and one of the most promising ones is based on the knowledge that angiogenesis, the mechanism of formation of new blood vessels coupled with the degradation of the extracellular matrix for metalloproteinases, could be crucial in the development of this tumor. The principal molecule implicated in angiogenesis process of ovarian cancer is the vascular endothelial growth factor (VEGF). Several studies are now in progress to clarify its role as a diagnostic tool or its therapeutic implication. Presently, there is no indication for the use of VEGF in a preliminary diagnosis seeing that an increase in levels can be seen in both benign and malignant ovarian conditions. VEGF is also responsible for an increase in vascular permeability and is directly related to symptoms such as ascites and pleural effusion, both of which are frequent in ovarian cancer. Several papers have analised the role of VEGF as a prognostic factor and some of them do confirm VEGF as an independent prognostic factor in ovarian cancer. VEGF and the metalloproteinase system coupled with angiogenesis are currently being evaluated as therapeutic targets but no positive results have yet to be seen in this field.      

Fifth Ovarian Cancer Consensus Conference of the Gynecologic Cancer InterGroup: Recommendations on incorporating patient-reported outcomes in clinical trials in epithelial ovarian cancer

Despite the support for including patient-reported outcomes (PROs) and health-related quality of life in clinical trials, there have been deficiencies in how these have been assessed and reported in epithelial ovarian cancer (EOC) clinical trials. To redress this, the 5th Ovarian Cancer Consensus Conference, included a plenary session entitled ‘How to include PROs in clinical trials’. The perspective is a summary of the recommendations made by the Gynecologic Cancer InterGroup unanimously agreed on the importance of PROs and PRO end-points in EOC clinical trials. They recognised that effort must be made to ensure the integrity of collection of PRO data and to avoid missing data. PRO end-points should be based on the PRO hypotheses, be context specific and reflect the patient population and the objectives of treatment (e.g. first line, maintenance therapy, early or late relapse). The PRO end-points inform the choice of PRO measures used in the trial and how the results are analysed and reported. There was agreement that progression-free survival should be supported by PROs among patients with late relapse (platinum sensitive) and that progression-free survival alone was not sufficient as the primary end-point of clinical trials in patients with platinum resistant/refractory EOC and PROs should be included as either the primary/co-primary end-point in this subset of patients. Novel approaches to measure the benefit of palliative chemotherapy such as time until definitive deterioration of Health-Related Quality of Life were recommended. There was consensus to endorse the ISOQOL and CONSORT-PRO guidelines on the inclusion and reporting of PRO endpoints in protocols and that all future EOC Gynecologic Cancer InterGroup trials should adhere to these.     

Inhibition of gastric cancer cells associated angiogenesis by 15d-prostaglandin J2 through the downregulation of angiopoietin-1

Peroxisome proliferator-activated receptor gamma (PPARgamma) ligands have been shown to inhibit angiogenesis. We showed that treatment with 15d-PGJ(2), a PPARgamma ligand, downregulate the expressions of angiopoietin-1 (Ang-1) in gastric cancer cells MKN45. The medium of MKN45 cells treated with 15d-PGJ(2) significantly inhibited the migration and tube formation of human umbilical vein endothelial cells (HUVECs). Moreover, Matrigel plug assay revealed that 15d-PGJ(2) reduced in vivo angiogenesis induced by MKN45 cells. These modulations were restored by the addition of recombinant Ang-1. Our findings supported that 15d-PGJ(2) suppressed angiogenesis of gastric cancer cells by downregulation of Ang-1.     

Knockdown of RAGE expression inhibits colorectal cancer cell invasion and suppresses angiogenesis in vitro and in vivo

The receptor for advanced glycation end-products (RAGE) is a transmembrane receptor in cells, and the interaction of RAGE with ligands results in pro-inflammatory gene activation. Aberrant RAGE activation was reported to promote the pathogenesis of colorectal cancer. This study aimed to investigate the effects of RAGE on the regulation of cell viability, invasion, and angiogenesis, as well as the underlying molecular mechanisms regulating these interactions in colorectal cancer cells.The RAGE mRNA and protein were evaluated in five colorectal cancer cell lines and in 45 cases of colorectal cancer tissue specimens (using immuohistochemistry). RAGE expression was then knockdown using RAGE shRNA for assessing cell viability and invasion assays as well as for tube formation and CAM assays in human umbilical vein endothelial cells and chick embryos, respectively.RAGE was highly expressed in colorectal cancer tissues, and was associated with increased microvessel density. Two of the four RAGE shRNA constructs were able to significantly knockdown RAGE expression in SW480 cells. RAGE knockdown inhibited invasion capacity of SW480 cells, but did not significantly affect cell viability. Furthermore, the conditioned growth medium from stable RAGE shRNA-transfected cells suppressed tube formation of human umbilical vein endothelial cells and angiogenesis of chicken embryos. Knockdown of RAGE inhibited expression of VEGF and SP1 protein in colorectal cancer cells.In summary, these data suggest that silence of RAGE expression could effectively inhibit colorectal cancer angiogenesis in vitro and in vivo.     

Effect of paclitaxel on vascular endothelial growth factor (VEGF) and interleukin (IL)-8 in serum of patients with recurrent ovarian cancer: a comparison of weekly vs triweekly regimens

Antiangiogenic scheduling of cancer chemotherapeutics has been increasingly recognized to be a potential application of the paclitaxel in cancer therapy. The purpose of this study is to confirm antiangiogenic effects of weekly low-dose paclitaxel in recurrent ovarian carcinoma. Measurements of serum vascular endothelial cell growth factor (VEGF) and interleukin (IL)-8 were performed using enzyme-linked immunosorbent assay (ELISA) kits. Serum was collected with written informed consents. Serum levels of VEGF and IL-8 were measured in patients with benign ovarian tumors, low malignant potential ovarian tumors (LMP), and ovarian carcinomas. Among 20 patients with pretreated recurrent ovarian carcinoma, 10 patients receiving treatment with paclitaxel (180 mg/m2) given once every 3 weeks (triweekly paclitaxel) and the other 10 patients receiving treatment with weekly paclitaxel (80 mg/m2) were randomly allocated. Sera from these patients were collected before treatment and 3 weeks after initiation of treatment to determine changes of VEGF and IL-8 levels. Although VEGF levels were highest in patients with ovarian carcinoma, there was no significant difference among benign, LMP, and carcinoma. Among patients with detectable levels of IL-8, ovarian carcinoma showed significantly higher IL-8 levels than benign tumors followed by LMP. VEGF levels in patients with treatment by triweekly paclitaxel did not show any significant change before and after treatment, while those in patients with treatment by weekly paclitaxel decreased significantly after treatment. Similarly, in patients with detectable IL-8 levels, weekly paclitaxel resulted in a significant decrease of IL-8 levels after treatment, while triweekly paclitaxel did not result in any significant change of IL-8 levels. The present study demonstrated that weekly but not triweekly paclitaxel had significant antiangiogenic effects.     

Targeting HER 2 and angiogenesis in gastric cancer

Gastric cancer is one of the most commonly diagnosed and the second leading cause of cancer death worldwide. Surgery combined with multimodal therapy remains the only curative therapy. However, local relapse or distant metastases occur in more than 50% of radically resected patients. Due to molecular therapies, targeting HER2 and angiogenesis, major advances in the treatment of gastric cancer could be achieved. Nevertheless, development of resistance to monoclonal antibodies, such as trastuzumab, is arising. Currently a number of promising new therapeutic are under investigation, combining chemotherapy with newly developed agents to overcome cancer resistance. In this review we report current clinical applications of targeted therapies and overview ongoing trials, investigating the use of monoclonal antibodies in (HER2 positive) gastric cancer.     

人血管生成素-1基因转染对胃癌细胞体内致瘤力及血管生成的影响

目的　研究人血管生成素 1 (Ang1 )基因转染人胃癌细胞系SGC790 1后 ,对肿瘤细胞生物学特性以及对小鼠体内致瘤力和血管生成的影响 ,探讨其在胃癌发生及血管生成中的作用。方法构建重组正、反义Ang1真核表达载体 ,采用脂质体转染法 ,将重组载体转染人胃癌细胞系SGC790 1 ,分别得到正、反义载体稳定转染的细胞株 7Ang1 +和 7Ang1 - ,并以半定量PCR及Westernblot方法鉴定转染效果。以MTT比色试验绘制生长曲线 ;以流式细胞仪检测细胞周期分布。通过裸鼠成瘤实验 ,比较转染前后小鼠体内成瘤能力的差别 ,并检测肿瘤组织微血管密度 (MVD) ,判定血管生成程度。结果　 7Ang1 -组细胞Ang1在蛋白及mRNA水平的表达均下调。体内成瘤实验结果显示 ,空载体对照组、7Ang1 +组和 7Ang1 -组瘤体的平均重量 ( x±s)分别为 6 2 4 .0 0± 77.78,6 5 2 .6 7± 1 32 .0 7和2 93.0 0± 95 .5 4 ,7Ang1 -组的细胞成瘤性显著低于 7Ang1 +组和空载体对照组 (P <0 .0 1 )。空载体对照组、7Ang1 +组和 7Ang1 -组肿瘤组织的MVD( x±s)分别为 8.4 4± 1 .33,8.78± 1 .92和 6 .0 0± 1 .73,7Ang1 -组细胞的血管生成显著减少 (P <0 .0 1 )。结论　Ang1在胃癌形成和发展中可能通过诱生血管起促进作用 ,通过反义技术可部分阻断这种作用。     

Current status and perspective of antiangiogenic therapy for cancer: urinary cancer

Angiogenesis is considered a prerequisite for solid tumor growth. Antiangiogenic therapy reduces tumor size and extends host survival in a number of preclinical animal models. However, in humans antiangiogenic therapy is a poor promoter of tumor regression and has shown minimal effect on patient survival. In urinary cancers, such as renal cell cancer, prostate cancer, and bladder cancer, advanced refractory disease is a good candidate for antiangiogenic therapy because of its resistance to ordinary chemotherapy, radiotherapy, and hormonal therapy. Unique characteristics of molecular mechanisms underlie the induction of angiogenesis in urinary cancers. In this review, we summarize these unique mechanisms and review the results of clinical trials of antiangiogenic therapy for these cancers, discussing prospects and problems relating to antiangiogenic therapy.     

Overexpression of macrophage migration inhibitory factor induces angiogenesis in human breast cancer

Macrophage migration inhibitory factor (MIF) is known to be an important contributor to tumor progression. Overexpression of MIF has been reported in different types of tumors. However, the correlation between MIF expression and tumor pathologic features in patients with breast cancer has not been elucidated. In this study, we examined the expression of MIF, vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) in human tissues with or without tumor. In addition, we investigated the expression of MIF in MDA-MB-231, MCF-7 (breast cancer cell lines) and MCF-10A (epithelial cell line) cells, and its effect on VEGF and IL-8. We found that MIF was overexpressed in breast cancer tissues compared with normal ones. The level of MIF showed the positive correlation between the expression of IL-8 and tumor microvessel density (MVD). The patients with positive MIF expression in tumor tissues showed a significantly worse disease-free survival compared with negative ones. Increased MIF serum levels were also found to correlate with higher levels of IL-8 in the sera of the patients with breast cancer. In vitro experiments successfully detected MIF in breast cell lines. However, the expression level of it by normal epithelial cells was much less than that of cancer cells. Exogenous MIF did not cause endothelial tube formation and migration but induced a dose dependent increase in VEGF and IL-8 secretion in breast cancer cell lines. In summary, our studies show that human breast cancer tissue expresses MIF. Its in vitro effect on VEGF and IL-8 indicates that MIF may contribute to tumor in angiogenesis and thus play an important role in the pathogenesis of breast cancer.     

Different features of angiogenesis between ovarian and breast carcinoma

Angiogenesis assessed by immunohistochemical staining for endothelial cells has been widely accepted as an independent prognostic factor in human breast carcinoma. However, the clinicopathologic significance of angiogenesis is still being argued in ovarian carcinoma. Therefore, we retrospectively analyzed the clinicopathologic significance of angiogenesis in ovarian carcinoma compared with that in breast carcinoma. After vessels were stained with CD34-monoclonal antibody, the areas with the highest number of intratumoral microvessels were assessed in a 200× field in 42 ovarian carcinoma and 41 breast carcinoma. Intratumoral microvessel density (IMD) in ovarian carcinoma was significantly lower than that in breast carcinoma. Further, the difference of IMD from tumor to tumor in ovarian carcinoma was smaller than that in breast carcinoma. IMD was correlated with tumor grade, but not with other clinicopathologic variables in ovarian carcinoma. Although the patients with high-IMD tumor revealed a poorer prognosis than those with low-IMD tumor in breast carcinoma, IMD had no influential effects on the survival of the patients with ovarian carcinoma. Our comparative analysis of IMD in ovarian carcinoma with that in breast carcinoma indicates that angiogenesis may play an important role in the transient of ovarian neoplasms, but not in the progression of ovarian carcinomas, and that the biological roles of angiogenesis might be different, depending on histologic subtype.     

mRNA EXPRESSION OF PTEN AND VEGF GENES IN EPITHELIAL OVARIAN CANCER

VEGF gene, also known as vascular permeability factor, has been mapped to chromosome 6p21.3[11]. Biochemically, it is a heparin binding glycoprotein that has in at least four molecular isoforms, among which, VEGF165 occurs most common. VEGF can act as a specific mitogen for a variety of endothelial cells in vitro and as an angiogenic molecule in vivo [11,12]. VEGF is a potent multifunctional cytokine that exerts several potentially independent actions on the vascular endothelium, includin…     

Microvessel density and p53 mutations in advanced-stage epithelial ovarian cancer

We planned to determine the relationship between angiogenesis and p53 mutational status in advanced-stage epithelial ovarian cancer. Using 190 tumor samples from patients with stage III and IV ovarian cancer we performed p53 sequencing, immunohistochemistry, and CD31 microvessel density (MVD) determination. MVD was elevated in tumors with p53 null mutations compared to p53 missense mutation or no mutation. Disease recurrence was increased with higher MVD in both unadjusted and adjusted analyses. In adjusted analysis, p53 null mutation was associated with increased recurrence and worse overall survival. Worse overall survival and increased recurrence risk were also associated with the combination of CD31 MVD values >25 vessels/HPF and any p53 mutation. P53 mutation status and MVD may have prognostic significance in patients with advanced-stage ovarian cancer. Tumors with p53 null mutations are likely to be more vascular, contributing to decreased survival and increased recurrence probability.     

Clinical and biological significance of HLA-G expression in ovarian cancer

Ovarian cancer is the most lethal gynecologic neoplastic disease in which the molecular etiology remains largely unclear. Like other cancer types, evolution of ovarian tumor cell species is accompanied by acquisition of novel gene products and these new tumor-associated antigens elicit a host immune response that creates selection pressure upon the emerging tumor clones. One of the mechanisms that ovarian cancer cells evade immune surveillance is by upregulating human leukocyte antigen-G (HLA-G) expression. HLA-G is a non-classical MHC class I molecule and accumulated evidence has suggested its biological role in inactivating immune response. It has been well known that HLA-G expression is frequently detected in the most aggressive type of ovarian cancer, i.e., high-grade serous carcinoma, and measurement of HLA-G protein levels has shown promise for detection and prognosis prediction in ovarian cancer. This review summarizes those recent studies on HLA-G expression in ovarian cancer with special focus on its clinical and biological significance which is fundamental to elucidate the molecular mechanisms in ovarian cancer development and paves the foundation for future HLA-G-based diagnostics and therapeutics.