Prudence A. E. Scott

The angiogenic switch for vascular endothelial growth factor (VEGF)-A, VEGF-B, VEGF-C, and VEGF-D in the adenoma–carcinoma sequence during colorectal cancer progression

Angiogenesis is essential for tumour growth and metastasis. It is controlled by angiogenic factors, one of the most important being vascular endothelial growth factor (VEGF)‐A. Although its role has been demonstrated in many tumour types including colorectal carcinoma (CRC), the importance of the newer family members in adenoma, invasive tumour growth, and progression to a metastatic phenotype has been poorly characterized in CRC. The aim of this study was to determine the role and timing of the VEGF angiogenic switch during CRC progression. We measured the gene expression of VEGF ligands (VEGF‐A, VEGF‐B, VEGF‐C, and VEGF‐D) and their receptors (VEGFR‐1, VEGFR‐2, and VEGFR‐3), in normal colorectal tissues (n = 20), adenomas (n = 10), and in CRC (n = 71) representing different Dukes stages using ribonuclease protection assay, semi‐quantitative relative reverse transcriptase polymerase chain reaction, together with the pattern of their expression by immunohistochemistry. VEGF‐A mRNA was the most abundant in colorectal tissue, followed by VEGF‐B, VEGF‐C, and VEGF‐D. VEGF‐A and VEGF‐B mRNAs were significantly more abundant in adenomas (p = 0.0003 and p = 0.04 respectively) compared with normal tissues, while VEGF‐A and VEGF‐C were significantly increased in carcinomas compared with normal tissues (p = 0.0006 and p = 0.0009 respectively). A significantly greater amount of VEGF‐C mRNA was present in carcinomas compared with adenomas (p = 0.03), whereas there was a significant reduction of VEGF‐B in carcinomas compared with adenomas (p = 0.0002). VEGF‐D mRNA was significantly more abundant in normal tissues than in adenomas (p = 0.0001) and carcinomas (p < 0.0001). In normal tissues distant from the primary tumour, there was a significantly greater amount of VEGF‐A and VEGF‐D mRNA in patients with Dukes B and Dukes C respectively, compared with Dukes A stage tumours (p = 0.04 and p = 0.01 respectively). Immunohistochemistry showed low basal levels of all ligands in histologically normal tissues and their expression in the epithelium of tumours reflected the levels of mRNA expression identified. VEGF‐A and VEGF‐C mRNA levels correlated significantly with tumour grade (p = 0.01 and p = 0.01 respectively) and tumour size (p = 0.001 and p = 0.01 respectively), but not with patient age, sex, presence of infiltrative margin, lymphocytic response, vascular invasion, Dukes stage, or lymph node involvement (p > 0.05). VEGF‐B mRNA correlated with an infiltrative margin (p = 0.04) but no other clinicopathological variable, and expression of VEGF‐D demonstrated no association with any parameter examined. VEGFR‐1 was significantly correlated with tumour grade (p = 0.02), Dukes stage (p < 0.001), and lymph node involvement (p = 0.004), VEGFR‐2 with lymph node involvement (p = 0.02), and VEGFR‐3 did not correlate with any of the clinicopathological variables tested. These results suggest that VEGF‐A and VEGF‐B play a role early in tumour development at the stage of adenoma formation and that VEGF‐C plays a role in advanced disease when there is more likelihood of metastatic spread. The finding of increased levels of VEGF‐A and VEGF‐D expression in normal tissues collected from a site distant from the primary tumour indicates changes in the surrounding tumour environment that may enhance the subsequent spread of tumour cells. Copyright

The 121 amino acid isoform of vascular endothelial growth factor is more strongly tumorigenic than other splice variants in vivo

Vascular endothelial growth factor (VEGF) is known to occur as at least six differentially spliced variants, giving rise to mature isoforms containing 121, 145, 165, 183, 189 and 206 amino acids. However, little is yet known concerning the in vivo activities of this differential splicing. Stably transfected MCF-7 breast carcinoma cells were constructed that secreted comparable amounts of the 121, 165 or 189 isoforms. Rabbit corneal angiogenesis assays showed the VEGF121 transfectant to have much greater angiogenic activity than the 165 or 189 expressing MCF-7 cells. While the VEGF121-expressing MCF-7 cells were reproducibly more tumorigenic than the control transfectants, this was not the case with the VEGF165- or VEGF189-expressing cells. More surprising was the observation that VEGF189 located to the nucleus, consistent with the presence of a highly conserved nuclear localization sequence in exon 6a that is expressed in VEGF189 but not 121 or 165. It was concluded that the VEGF121 isoform is both more angiogenic and tumorigenic than are the 165 and 189 isoforms. This is probably due to the ability of the 121 isoform, unlike the 165 and 189 isoforms, to freely diffuse from the cells producing it.

Expression of the angiopoietins and their receptor Tie2 in human renal clear cell carcinomas; regulation by the von Hippel-Lindau gene and hypoxia

Angiogenesis is essential for tumour growth and metastasis, and is co‐ordinated by several classes of angiogenic factors. To determine the significance and regulation of the angiopoietin (Ang) pathway in highly vascular human renal cell carcinomas (RCCs), this study has investigated the expression of the Ang‐1, Ang‐2, Ang‐4, and Tie2 genes in a series of normal (n = 26) and neoplastic (n = 45; clear cell n = 35, papillary n = 10) human kidney tissues, examined the pattern of Ang‐2 and Tie2 protein expression, and correlated expression with clinicopathological variables. The effect of the von Hippel‐Lindau (VHL) gene and hypoxia in the renal cell lines RCC786‐0 and RCC4 has also been investigated. Ang‐1, Ang‐2 and Tie2, but not Ang‐4 mRNA, were detected in normal and tumour samples. A significant increase in Ang‐2 (p < 0.001) and a decrease in Tie2 receptor mRNA (p = 0.001) were observed, but no significant difference was observed in Ang‐1 mRNA abundance between normal kidney and RCC (p = 0.37). Immunohistochemistry for Ang‐2 showed strong expression in vascular endothelium and weak expression in tumour cells, whereas Tie2 was expressed exclusively on endothelium. Tie2 gene expression was positively correlated with Ang‐2 expression in cancers (p = 0.001) and showed a borderline significant association with Ang‐1 (p = 0.06), but there was no significant relationship between Ang‐1 and Ang‐2 (p = 0.69). No significant relationships were observed in clear cell carcinomas between Ang‐1, Ang‐2 and Tie2 mRNA abundance and patient sex, patient age, or tumour size (p > 0.05). However, there was significantly greater Ang‐1 (p = 0.02), Ang‐2 (p = 0.03), and Tie2 (p = 0.04) mRNA abundance in clear cell than in chromophil RCCs. Ang‐2 gene expression was down‐regulated by hypoxia in VHL wild‐type RCC786‐0 and RCC4 transfectants (p = 0.0002 and p = 0.04, respectively), mirroring the low expression in human tumour cells. These data suggest that it is endothelial induction of Ang‐2 in tumours that regulates vessel stability and supports targeting Tie2 as an effective novel anti‐angiogenic therapy in clear cell RCCs. Copyright

Expression of VEGF in routinely fixed material using a new monoclonal

The aim of this study was to raise and characterize a monoclonal antibody reactive with VEGF (vascular endothelial growth factor) in routinely fixed specimens and to use it to investigate its tissue distribution in normal and pathological specimens. Recombinant VEGF 189 protein was used to raise a monoclonal antibody. The specificity of the antibody was confirmed using COS cells transfected with cDNA coding for VEGF 121, 165 and 189 protein and by western blotting studies. The resulting antibody VG1 was shown to react with the 121, 165 and 189 isoforms of VEGF protein in routinely processed material. In normal tissues, there was strong staining of endometrial and salivary glands and of the mucosa of the gastro‐intestinal tract. In tumours, a proportion of the neoplastic cells in lung and breast cancer and in melanoma were labelled. In all tissues, whether normal or malignant, striking VEGF positivity was seen in plasma in vessels and stroma. This study has shown that antibody VG1 detects the 121, 165 and 189 VEGF isoforms in routinely fixed specimens. The results of the normal tissue and tumour labelling are in agreement with other studies using alternative methods of detection. This should be a useful and reliable reagent for studies of VEGF and angiogenesis in human pathological material.Copyright

Breast cancer angiogenesis — new approaches to therapy via antiangiogenesis, hypoxic activated drugs, and vascular targeting

SummarySeveral groups have shown that quantitation of tumor angiogenesis by counting blood vessels in primary breast cancer gives an independent assessment of prognosis. Poor prognosis is associated with high blood vessel counts. We have shown that the rate of cell division in endothelial cells is much higher in breast tumours than in normal breast. Breast cancer cell lines and primary human breast tumours express a wide range of vascular growth factors, including VEGF, placenta growth factor, pleiotrophin, TGFβ1, acidic and basic FGF, and platelet-derived endothelial cell growth factor. Inhibiting angiogenesis by blocking vascular growth factors would be difficult with highly specific agents, but drugs with a broader spectrum of antagonism may be effective. We have developed several suramin analogues which are less toxic than suraminin vivo but more potent in inhibiting angiogenesis, and these have been developed for Phase I. A combination of anti-angiogenesis agents with drugs activated by hypoxia may also be useful, because anti-angiogenesis alone may not kill cells, whereas activation of hypoxic drugs could synergize.New endpoints may be necessary because inhibition of new blood vessel formation may not cause tumour regression. Thus, the endpoint of stable disease and biochemical assessment of inhibition of angiogenesis may be much more important in therapeutic studies and for drug development in the future. The prognostic importance of angiogenesis suggests that this should be a major new therapeutic target.

Role of the hypoxia sensing system, acidity and reproductive hormones in the variability of vascular endothelial growth factor induction in human breast carcinoma cell lines.

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor implicated in many pathological processes. We investigated the regulation of 4 alternatively spliced isoforms (121, 165, 189 and 206 amino acids) by hypoxia, hypoglycemia, acidity, female reproductive hormones and vitamin D in breast carcinoma cell lines representing different tumor phenotypes. There was a 17‐fold difference in total VEGF mRNA expression across the cell lines. The isoform expression, 121 > 165 > 189, was unchanged by different culture conditions. Hypoxia was the most potent stimulus, and the cell lines demonstrated a 1.4‐ to 6.9‐fold range of VEGF induction, maintained when other hypoxically regulated genes (phosphoglycerate kinase 1 and glucose transporter 1) and a HIF‐1‐dependent reporter gene were examined. The relative inducibility of the genes was maintained in each cell line, but basal expression was independent of ‐fold induction. VEGF expression decreased under acidic conditions in 2 cell lines, but the hypoxia stimulus remained effective under acidic conditions. Hypoglycemia, female reproductive hormones and vitamin D exerted no effect on expression, nor did inhibitors of mutant ras. Our results show that VEGF expression varies widely between cell lines and that capacity to respond to hypoxia is also cell specific, relating mostly to the hypoxic sensing of the cell and the signal transduction mechanism. Such characteristics, if maintained in vivo, have implications for the angiogenic potential of different tumor cells under normal and hypoxic conditions. Int. J. Cancer 75:706–712, 1998.© 1998 Wiley‐Liss, Inc.

VEGF-B expression in human primary breast cancers is associated with lymph node metastasis but not angiogenesis.

Angiogenesis is essential for tumour growth and metastasis. It is regulated by numerous angiogenic factors, one of the most important being vascular endothelial growth factor (VEGF). Recently VEGF‐B, a new VEGF family member that binds to the tyrosine kinase receptor flt‐1, has been identified. Although the importance of VEGF has been shown in many human tumour types, the contribution of VEGF‐B to tumour neovascularization is unknown in any tumour type. This study therefore measured the mRNA level of VEGF‐B and its receptor flt‐1 by ribonuclease protection assay and the pattern of VEGF‐B expression by immunohistochemistry in 13 normal breast samples and 68 invasive breast cancers. Flt‐1 expression was significantly higher in tumours than in normal breast (p=0.02) but no significant difference was seen in VEGF‐B between normal and neoplastic breast (p=0.3). There was a significant association between VEGF‐B and node status (p=0.02) and the number of involved nodes (p=0.01), but not with age (p=0.7), size (p=0.6), oestrogen receptor (ER) (p=0.2), grade (p=0.5) or vascular invasion (p=0.16). No significant relationship was present between VEGF‐B and flt‐1 (p=0.2) or tumour vascularity (p=0.4). VEGF‐B was expressed mostly in the cytoplasm of tumour cells, although occasional stromal components including fibroblasts and endothelial cells were also positive. No difference in VEGF‐B expression was observed adjacent to regions of necrosis, in keeping with this VEGF family member not being hypoxically regulated. These findings suggest that VEGF‐B may contribute to tumour progression by a non‐angiogenic mechanism, possibly by increasing plasminogen activators and hence metastasis, as has been described in vitro. Measurement of VEGF‐B together with other angiogenic factors may identify a poor prognostic patient group, which may benefit from anti‐VEGF receptor therapy targeted to flt‐1 (VEGFR1) as well as kdr (VEGFR2). Copyright

Angiogenic Polypeptides in Breast Cancer: Expression of Mrna’s in Primary Human Tumours, MCF-7 Cell Transfection and Xenograft Models

Screening of 84 primary human breast carcinomas for the mRNA expression of seven angiogenic polypeptides showed that the most commonly expressed and/or the most highly expressed when compared to normal breast tissue were vascular endothelial growth factor (VEGF) and platelet-derived endothelial cell growth factor/thymidine Phosphorylase (PDECGF/TP)1. The neurokines midkine (MK) and pleiotrophin were also fairly commonly expressed in tumour but not normal tissue (unpublished data from this group and ref. 1).