Raymond M. Shaheen

Inhibited growth of colon cancer carcinomatosis by antibodies to vascular endothelial and epidermal growth factor receptors

Vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF) regulate colon cancer growth and metastasis. Previous studies utilizing antibodies against the VEGF receptor (DC101) or EGF receptor (C225) have demonstrated independently that these agents can inhibit tumour growth and induce apoptosis in colon cancer in in vivo and in vitro systems. We hypothesized that simultaneous blockade of the VEGF and EGF receptors would enhance the therapy of colon cancer in a mouse model of peritoneal carcinomatosis. Nude mice were given intraperitoneal injection of KM12L4 human colon cancer cells to generate peritoneal metastases. Mice were then randomized into one of four treatment groups: control, anti-VEGFR (DC101), anti-EGFR (C225), or DC101 and C225. Relative to the control group, treatment with DC101 or with DC101+C225 decreased tumour vascularity, growth, proliferation, formation of ascites and increased apoptosis of both tumour cells and endothelial cells. Although C225 therapy did not change any of the above parameters, C225 combined with DC101 led to a significant decrease in tumour vascularity and increases in tumour cell and endothelial cell apoptosis (vs the DC101 group). These findings suggest that DC101 inhibits angiogenesis, endothelial cell survival, and VEGF-mediated ascites formation in a murine model of colon cancer carcinomatosis. The addition of C225 to DC101 appears to lead to a further decrease in angiogenesis and ascites formation. Combination anti-VEGF and anti-EGFR therapy may represent a novel therapeutic strategy for the management of colon peritoneal carcinomatosis.

Induction of VEGF in perivascular cells defines a potential paracrine mechanism for endothelial cell survival

Small tumor vessels are composed of endothelial cells (ECs) surrounded by pericytes. Pericytes are believed to be an EC survival factor, but their mechanism of action is unknown. One possible mediator, VEGF, promotes angiogenesis, EC proliferation, and EC permeability, and it protects ECs from apoptosis. We hypothesized that PDGF (platelet‐derived growth factor)‐BB, a cytokine released from tumor and ECs, mediates pericyte function by inducing VEGF, which in turn may affect EC survival. Using two pericyte‐like cell lines, 10T1/2 cells (murine pericyte cell line) and human vascular smooth muscle cells (hVSMCs), we showed that PDGF‐BB increased VEGF mRNA transcription. Although PDGF‐BB activated both the mitogen‐activated protein kinase and phosphatidylinositol 3‐kinase (PI3‐K) pathways, activation of the PI3‐K pathway was the most important pathway for VEGF induction. Conditioned medium derived from colon cancer cells also induced VEGF in pericyte‐like cells via the PI3‐K pathway, which was blocked by SU6668, a tyrosine kinase inhibitor that blocks the receptors for PDGF, VEGF, and basic fibroblast growth factor. Conditioned medium from hVSMCs pretreated with PDGF‐BB prevented apoptosis of ECs, and this effect was partially abrogated by neutralizing antibodies to VEGF. These studies suggest that pericytes may protect ECs from apoptosis, in part, by cytokine signaling that increases VEGF.

Endothelial cell survival and apoptosis in the tumor vasculature

Angiogenesis is essential for the growth and metastasis of solid tumors. The balance of endothelial cell (EC) proliferation and apoptosis is a major determinant in tumor angiogenesis. Recently, several studies demonstrated that numerous angiogenic factors not only induce angiogenesis but also function as EC survival factors. Vascular endothelial growth factor (VEGF), a potent angiogenic factor, is also an EC survival factor in embryonic vasculogenesis and tumor angiogenesis. VEGF activates specific intracellular survival pathways in ECs including Bcl-2, A1, IAP, Akt, and Erk. Integrins may function as EC survival factors by preventing anoikis by enhancing binding to the extracellular matrix. In addition, integrins may function in concert with VEGF to promote EC survival. Angiopoietin-1 (Ang-1) has recently been shown to stabilize EC networks by binding to the EC-specific tyrosine kinase receptor Tie-2. Pericytes also function as EC survival factors, by cell-cell contact, secretion of survival factors, or both. Targeting any of the above mechanisms for EC survival may provide novel antineoplastic strategies.

Overview of angiogenesis: Biologic implications for antiangiogenic therapy

Tumor angiogenesis is essential for the growth of primary and metastatic tumors. This process requires the coordinated activities of multiple factors and cell types. For tumors to develop a neovascular blood supply, tumor cells and host cells must secrete proangiogenic factors that offset the activities of inhibitory angiogenic factors. In addition, the newly derived tumor endothelium must respond to signals in the microenvironment to survive under conditions such as hypoxia and acidity. Moreover, because the process of angiogenesis is regulated by redundant factors and pathways, inhibition of any single pathway is likely to select for cells whose angiogenesis is driven by other factors. Because antiangiogenic therapy is unlikely to induce tumor regression, the criteria for efficacy must be evaluated by means other than the standard criteria used to evaluate cytotoxic chemotherapy. Understanding the basic principles that drive tumor angiogenesis will lead to the development of therapies that will likely prolong survival without the toxicity associated with standard chemotherapy.

Regulation of vascular endothelial growth factor expression in human colon cancer by interleukin-1β

SummaryExpression of vascular endothelial growth factor (VEGF), an important angiogenic factor in colon cancer, is tightly regulated by factors in the microenvironment. However, specific factors indigenous to the organ microenvironment of colon cancer growth that regulate VEGF expression in human colon cancer are not well defined. We investigated interleukin-1β (IL-1β) induction of VEGF expression in colon cancer cells and the mechanism by which this occurs. HT29 human colon cancer cells were treated with IL-1β for various periods. Induction of VEGF mRNA by IL-1β peaked at 24 h (> fivefold) and returned to baseline by 48 h. SW620 human colon cancer cells also reached a peak induction of VEGF mRNA 24 h after treatment with IL-1β. VEGF was induced at a dose range between 1 and 20 ng ml–1 of IL-1β. IL-1β induction of VEGF was also confirmed at the protein level. To examine the mechanism for VEGF induction by IL-1β, we transiently transfected VEGF promoter-reporter constructs into HT29 cells. IL-1β increased the activity of the VEGF promoter-reporter construct. Pretreatment of HT29 cells with dactinomycin abrogated the induction of VEGF mRNA by IL-1β. The half-life of VEGF mRNA was not prolonged by treatment with IL-1β. These findings suggest that IL-1β regulates VEGF expression in human colon cancer cells by increasing transcription of the VEGF gene.

Role of the Tumor Microenvironment in Mediating Response to Anti-angiogenic Therapy

Despite the development of innovative anti-angiogenic strategies, early clinical trials have not replicated the results observed from preclinical models. One reason for this apparent discrepancy is the fact that tumor endothelium is phenotypically distinct from normal tissue endothelium. Moreover, it has recently become apparent that each individual tumor may display a different angiogenic phenotype. The expression of angiogenic factors in tumors is controlled by both intrinsic factors in the tumor cell and the influence of the host microenvironment. The diversity of angiogenic factor expression in tumors growing at different sites, combined with the fact that endothelial cells in different organs and tumors are phenotypically distinct, constitutes a formidable challenge for the development of effective anti-angiogenic regimens. This review provides an overview of how the microenvironment regulates tumor angiogenesis and affects the efficacy of anti-angiogenic therapy.

Regulators of colon cancer angiogenesis

Folkman’s discovery that the growth and spread of tumours depend on angiogenesis has created new avenues of research designed to better understand cancer biology and facilitate the development of new therapeutic strategies. The survival and metastasis of tumours depend on a shift in the normal balance among myriad endogenous angiogenic and anti-angiogenic factors in favour of increased angiogenesis. Several growth factors that regulate angiogenesis in colon cancer have been identified, including the pro-angiogenic factors vascular endothelial growth factor (VEGF), platelet-derived endothelial cell growth factor (PD-ECGF) and the anti-angiogenic factor, thrombospondin. A thorough understanding of the roles that these factors play in the angiogenic process has led to the development of agents intended to inhibit tumour angiogenesis. However, the complexity and redundancy of the angiogenic process continue to present substantial challenges to the development of anticancer therapies.