Angiogenesis/tumor hypoxia: new treatment options?

Angiogenesis, the development of a neovascular blood supply, is a critical step in the development and progression of malignant tumors and metastasis. It is well known that without new blood vessels a tumor cannot grow to be larger than about 1–2mm² since blood supplies nutrients such as glucose, oxygen and minerals to the cells.

Angiogenesis in healthy humans is largely restricted to wound healing and reproduction. Therefore, to initiate neovascularization, a tumor must switch to an angiogenic phenotype.

Oncogenes and tumor suppressor genes appear to be associated with the angiogenic switch leading to an angiogenic phenotype are ras, myc, raf, c-erbB2, c-jun and src.

Although in angiogenic tumors there is a high number of newly formed vessels, due to their abnormal and leaky structure with blind sacs and a reversed and intermittent flow oxygen and blood supply is much poorer than in normal tissue. Therefore, hypoxia is a common feature in solid tumors. Tumors respond to low oxygen tension by enhancing the hypoxia-inducible factor (HIF) response which is mediated through the inducible dimer HIF-1α and constitutively expressed HIF-1β. In hypoxic conditions oxygen-dependent degradation of HIF-1α is not available leading to HIF-1α stabilization and translocation to the nucleus where it binds to HIF-1β and hypoxia response elements on gene promoters. Thereby transcriptional activation of different gene pathways involved in angiogenesis occurs. Overexpression of HIF-1α has been identified in breast cancers with high levels being associated with more aggressive tumor types, higher clinical stages and poor prognosis.

There are several mechanisms of tumor neovascularization known. In breast cancer, angiogenesis and vascular remodeling appear to be most important. In inflammatory breast cancer vascular mimicry may play an additional role. Angiogenesis is defined as the generation of new blood vessels from the preexisting vasculature. The different steps of angiogenesis include degradation of the basement membrane, followed by endothelial cell migration, then invasion of the extracellular matrix, followed by endothelial cell proliferation and formation of capillary lumen, and finally stabilization of the new vessel. In contrast, vascular remodeling is understood as the use of existing vasculature by the tumor instead of an angiogenic response. Vascular mimicry is a neovascularization strategy which has been reported in inflammatory breast cancer. It is defined as tumor cells themselves (instead of vascular endothelial cells) forming a complete capillary network. Tumors having mimicry as predominant mechanism may not respond ton conventional anti-angiogenic agents.

Neovascularization of tumors is regulated by different angiogenic promoters and inhibitors secreted by the tumor cells themselves or originating from e.g. inflammatory cells or platelets which are often elevated in tumors or from the extracellular matrix. It is well known that breast carcinomas express various angiogenic factors, including the vascular endothelial growth factor (VEGF) family, fibroblast growth factor (FGF)-1, FGF-2, placenta growth factor (PlGF), and transforming growth factor (TGF)-ß₁ at different stages of tumor development.

VEGF is the central player in tumor-mediated angiogenesis and has emerged as key target for anti-angiogenic treatment strategies in oncology. The VEGF family comprises different glycoproteins, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, and PlGF. VEGF-A (often referred to as VEGF) is best characterized and involved in multiple angiogenic processes. The VEGF-A gene undergoes alternative splicing; VEGF165 is the predominant isoform being overexpressed in many human solid tumors. The angiogenic effects of VEGF are mediated via its tyrosine kinase domain receptors VEGFR-1 (Flt-1) and VEGFR-2 (KDR). VEGFR-2 is thought to play the major role in VEGF-promoted angiogenesis. VEGFR-3 is primarily mediating lymphangiogenesis.

VEGF induces angiogenesis through several effects on endothelial cells including increased permeability, survival, proliferation, migration and invasion.

Anti-angiogenesis inhibits tumor-induced formation of new blood vessels. In contrast to conventional cytotoxic drugs anti-angiogenic compounds do not target the tumor cells directly but they target their blood supply needed for survival and growth. Therefore, these drugs may offer an effective, less toxic way to treat cancer. Most anti-angiogenic therapies directly target the VEGF pathway due to its pivotal role in pathological angiogenesis. Agents that have been developed for inhibition of VEGF-A-mediated angiogenesis comprise antibodies targeting VEGF-A (e.g. bevacizumab), soluble receptor constructs (e.g. VEGF Trap), antibodies targeting VEGF-receptors (e.g. IMCL-1121b, IMC-18F1), and tyrosine kinase inhibitors of VEGF-receptors (e.g. ZD6474/zactima). Thereby different points of the VEGF pathway can be targeted, including direct targeting of ligand (VEGF) or receptor (VEGFR-1, VEGFR-2), the latter at extracellular and intracellular tyrosine kinase domains), targeting of regulatory proteins downstream of the receptor, and inhibition of upstream regulators of VEGF. Another approach is multireceptor targeting; these drugs inhibit tyrosine kinases of a broad panel of pro-angiogenic receptors (e.g. sunitinib, sorafenib, vatalanib). To date, clinical trials evaluating these agents in metastatic breast cancer are ongoing.

In breast cancer, bevacizumab (Avastin®) is the only drug that have been extensively studied in phase III clinical trials and that is now approved in combination with chemotherapy for 1^st line treatment by the U.S. Food and Drug Administration (FDA) and the European Medicine Agency (EMEA). Bevacizumab is a recombinant humanized monoclonal VEGF antibody that recognizes all isoforms of VEGF-A thereby preventing receptor binding. This leads to inhibition of angiogenesis and tumor growth. Preclinical studies showed that bevacizumab significantly inhibited the growth of human tumor xenografts and metastasis. In phase I studies doses of 0.3mg/kg neutralized all free circulating VEGF in patients. The half-life is relatively long (mean half-life 20 days) allowing i.v. administrations up to every 3 weeks. Toxicities observed with bevacizumab were acceptable, including headaches and hypertension. Bevacizumab can readily be delivered with chemotherapeutic agents leading to synergistic effects.

Compared to clinical trials in colorectal cancer and NSCLC development of bevacizumab in metastatic breast cancer has been more complex. In a first phase III clinical trial that combined bevacizumab with capecitabine in patients with metastatic breast cancer pretreated with anthracyclines and taxanes (≥2^nd line) the combination therapy resulted into a significantly improved response rate (19.8% versus 9.1%, p=0.001), but neither progression-free nor overall survival was different between the two arms. In contrast, a subsequent phase III trial (E2100) designed as 1^st line trial comparing paclitaxel alone versus paclitaxel plus bevacizumab demonstrated significantly improved response rates (36.9% versus 21.2%, p<0.001) as well as a significant better progression-free survival (11.8 months versus 5.9 months, p<0.001) in the combination arm. Recently, another phase III clinical trial (AVADO) presented 2008 ASCO Annual Meeting also demonstrated efficacy of combining bevacizumab and docetaxel at two different doses (7,5mg/kg q3w vs. 15mg/kg q3w) in 1^st line treatment of HER2-negative metastatic breast cancer patients.

Over the last years there has been a huge increase in understanding of tumor angiogenesis. However, transferring preclinical data to the clinic has turned out to be not that simple as expected. Nevertheless, several clinical approaches are promising and first anti-angiogenic therapies are now being integrated into the daily routine of breast cancer treatment. But there is still much to learn. Full understanding of the complexity of angiogenesis is required to select appropriate tumors and suitable patients for anti-angiogenic treatment. Especially, surrogate markers predictive of response have to be identified to facilitate an effective targeted and tailored anti-angiogenic treatment.