In normal tissues, the balance of pro-angiogenic and anti-angiogenic growth factors and proteins favors inhibition of angiogenesis, so that as new capillaries are needed, the balance can be adjusted to stimulate vascular growth. This corresponds to the angiogenic switch and the balance hypothesis proposed by Hananah and Folkman in 1996 (see Figure 1).1 The critical angiogenic activator is vascular endothelial growth factor (VEGF), but several other growth factors participate in the process. Basic fibroblast growth factor (bFGF), matrix metalloproteinases (MMP), transforming growth factor-α (TGF-α), platelet derived growth factor (PDGF), placenta growth factor (PlGF), angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), and hepatocyte growth factor (HGF) are all pro-angiogenic factors. Endogenous angiogenic inhibitors are proteins including endostatin, angiostatin, thrombospondin-1 (Tsp-1), tumstatin, platelet factor 4, and certain interleukins, including IL-12. Some of these proteins, including endostatin, angiostatin, and Tsp-1, are known to promote apoptosis in addition to preventing angiogenesis.
Hypoxia within the tumor microenvironment upregulates many of the angiogenic growth factors, including VEGF, PDGF, PlGF, and HGF.2 Hypoxia inducible factor 1α (HIF-1α) is considered to be the foundation for the environmental hypoxic-activated response.2-4 Under normal oxygenation (normoxic) conditions, HIF-1α is the target of ubiquitination by the von Hippel-Lindan tumor suppressor (pVHL), and the resulting ubiquitinated HIF-1α undergoes proteosomal degradation, terminating any downstream growth factor expression. Alternatively, under hypoxic conditions HIF-1α is not ubiquitinated and instead binds to p300 and cAMP responsive element binding protein (CREB). The resulting HIF-1α protein complex translocates to the nucleus and heterodimerizes with HIF-β, whereupon the heterodimer can initiate the transcription of target genes.5-7 HIF-1α binds to the hypoxia responsive element (HRE) of VEGFA, PDGF, and TGFA and induces the expression of the protein factors VEGF, PDGF, and TGF-α, respectively.
In addition to HIF-1α activation, two alternative signaling pathways initiated by hypoxia have been identified. The unfolded protein response (UPR) signaling pathway is initiated by stress of the endoplasmic reticulum (ER). The reader is directed to the article by Wouters and Koritzinsky for further discussion of the mechanisms of hypoxia induced ER stress.8 Signaling by the kinase mammalian target of rapamycin (mTOR) is also associated with hypoxia.8.9 Under normal environmental conditions, the mammalian target of rapamycin complex (mTORC1) phosphorylates downstream kinases that affect cell growth and protein synthesis. Signaling by mTOR is inhibited under certain hypoxic conditions and this inhibition occurs through multiple HIF-1α independent pathways. Under these conditions, hypoxia may act to reduce tumor growth in early tumor development, since several of the negative regulators of mTORC1 signaling also function as tumor suppressors. In advanced malignant tumors, hypoxia regulates mRNA translation and increases gene expression of factors that promote tumor growth. In addition, hypoxic conditions have been found to downregulate the expression of the pro-apoptotic proteins BH-interacting domain death agonist (BID), Bcl-2 antagonist of cell death (BAD), and Bcl-2 associated X protein (BAX). The shift in expression of these proteins reduces the potential for apoptosis and increases the likelihood of cell survival. It has been hypothesized in this case hypoxia aids in the selection of more malignant cell mutants, a concept that will be discussed later in this review. As a result of the relationship between these alternative signaling pathways and hypoxia, cancer therapies are in development that target factors involved in mTOR and UPR signaling pathways.
Since tumors with abnormal mTOR signaling are also highly vascularized, mTOR signaling has been shown to have a direct effect on HIF-mediated transcription in addition to alternative signaling. Land and Tee found that activation of mTOR increased the transcriptional activity of HIF-1α and VEGF under hypoxic conditions. Overexpression of the Ras homolog enriched in brain (Rheb) activated mTOR, while the addition of rapamycin reversed the mTOR activation.9
VEGF and its receptors VEGFR-1 and VEGFR-2 are recognized as the primary factors responsible for angiogenesis, and so their mechanisms are of great interest for the development of therapeutic inhibitors. VEGF encourages angiogenesis through multiple functions, including inducing endothelial cells to form capillary-like structures and mediating the secretion of enzymes to degrade extracellular matrix proteins. VEGF enhances cell survival by inducing the expression of anti-apoptotic proteins Bcl-2 and its homolog A1.10 VEGF receptors (VEGFR) are cell surface tyrosine kinases, and VEGFR dimerization activates tyrosine kinase activity, resulting in the phosphorylation of tyrosine residues and downstream activation of signal transduction molecules (see Figure 2).
The concentration of angiogenic factors other than VEGF also increases with malignant progression. Early tumor development may primarily use VEGF to support the angiogenic signaling process, while advanced tumors rely on other growth factors, including fibroblast growth factor-1 (FGF-1), TGF-β1 and PlGF.11 Jain has suggested that inhibitor cocktails used to target multiple pathways may be more efficient than a single VEGF inhibitor to block angiogenesis.12
Other pathways upstream can induce VEGF expression by methods other than through hypoxia and HIF-1α signaling. The oncogene ras is able to upregulate VEGF expression and downregulate the expression of Tsp-1 and other endogenous angiogenic inhibitors. Conversely, activation of the tumor suppressor genes p53, PTEN, and Smad4 increases thrombspondin-1 expression, shifting the balance to prevent angiogenesis. p53 has been reported to block angiogenesis through regulation of other unidentified inhibitors.5
The microtubule cytoskeleton has been cited as another possible target for angiogenesis inhibition. Microtubule targeting agents including 2-methoxyestradiol have been shown to block angiogenesis and inhibit HIF-1α signaling. Albendazole was demonstrated to downregulate VEGF in both in vitro experiments and in mouse models.13
Gene silencing by microRNAs has been associated with the regulation of multiple genes after hypoxic induction. Kulshreshtha, et al., identified approximately thirty hypoxia-regulated microRNAs (HRM) in breast or colon cancer cells that were consistently regulated in response to hypoxic conditions. Using predictive software, the gene targets of those HRM were predicted. As the HRM could theoretically downregulate multiple genes (between 10 and 200), the specific targets have not been identified. The researchers identified a group of HRM that theoretically target VEGF, implying the VEGF signaling pathway may become even more challenging to construct.14
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The “angiogenic switch” is illustrated as a balance between pro-angiogenic factors (represented by red spheres) and angiogenesis inhibitors (represented by gray spheres). When the level of angiogenic inhibitors predominates (left image), the microenvironment remains angiostatic and the tumor is quiescent. When the level of pro-angiogenic factors increases and overcomes the effects of the angiogenic inhibitors (right image), the process of constructing new vasculature is initiated.