Tumor Vascularization

Tumor vasculature arises through a process known as angiogenesis in which existing blood vessels are recruited [72], remodeled [73], or co-opted [74] to provide a blood supply to avascular tumor tissues.  During angiogenesis, endothelial cells proliferate, produce proteolytic enzymes that degrade the extracellular matrix, change their adhesive properties, migrate, and differentiate into new blood vessels.  This process of tumor vascularization is directed mainly by paracrine angioproliferative tumor factors and extracellular matrix components that bind to specialized receptors such as G-protein-coupled receptors or receptor tyrosine kinases and integrins on endothelial cells [75,76,77].  Ligand activation of these receptors triggers signaling pathways leading to altered protein activity and gene transcription that modulate the cellular behavior. 

One such angioproliferative factor, vascular endothelial growth factor A (hereafter referred to as VEGF), plays a crucial role in regulating tumor angiogenesis.  In vitro, VEGF is a potent mitogen of endothelial cells [78,79].  In vivo, intradermal injection of VEGF causes increased vascular permeability [80].  Inhibition of VEGF release from tumors leads to decreased tumor vascularization and growth [81], and embryonic stem cells that are homozygous null for VEGF are unable to form tumors in nude mice [82].  VEGF also plays an important role in embryonic development.  Mice lacking a single copy of VEGF die at between E11 and E12 as a result of extensive vascular defects in both the embryo and placenta [82,83].  Moreover, whereas homozygous gene targeting of the VEGF receptor VEGFR2 (Flk-1) causes embryonic lethality at E8.5-9.5 that is characterized by the absence of organized blood vessels in the embryo and yolk sac [84], embryos that are homozygous null for the alternate VEGF receptor VEGFR1 (Flt-1) die at E8.5 with abnormally large blood vessels [85,86].

A substantial body of evidence indicates that MKK signaling pathways play essential roles in modulating the release of, and the response to, VEGF.  MKK activity regulates VEGF expression at the transcriptional and post-transcriptional levels [87].  Expression of constitutive MEK1 in fibroblasts elevates expression of VEGF mRNA through binding of the transcription factors Sp1 and AP-2 to its promoter region [88].  Also, VEGF mRNA half-life is increased in cells that over-express p38 MAPK and JNK [87].  MKK signaling pathways are also activated in response to VEGF: treatment of endothelial cells with VEGF has been observed to cause activation of both ERK 1 and 2 [89], as well as p38 MAPK [90].  Moreover, this increase in MKK activity is required for endothelial response to VEGF.  Whereas MEK1/2 inhibitors prevent VEGF-induced cell proliferation [90,91], p38 MAPK inhibitors reportedly prevent VEGF-induced cytoskeletal re-organization and cell migration [90].  The JNK pathway has also been linked to in vitro endothelial cell motility [92,93] and proliferation [92].

Despite evidence of a strong link between MKK signaling, endothelial cell function, and the release of angioproliferative factors in vitro, the role MKK plays in promoting tumor vascularization in vivo is less clear.  Insight into their in vivo functions may be inferred from the effects of MKK inhibitors upon tumor vascularization.  For example, anthrax lethal factor, a protease that inactivates MEK1 and 2 [60] as well as MKK 3, 4, 6, and 7 [62], substantially inhibits vascularization in mouse xenograft studies [63].  Its anti-endothelial effects result not only from its ability to inhibit MKK signaling but also from the up-regulation of anthrax toxin receptors in tumor-associated endothelium [94,95].  Also, expression of an inactive Raf-1 mutant to endothelial cells blocks growth and vascularization of melanomas in mice [96].  BAY 43-9006 (Sorafenib), a compound that inhibits B-Raf and c-Raf, both of which are MKKKs that activate MEK 1 and 2, also reduces tumor vascularization in vivo [97].  However, whether this is due directly to inhibition of MKK signaling is unclear since 1) an anti-angiogenic effect has not been reported for other MEK 1 and 2 inhibitors such as CI1040 (PD184352) and 2) BAY 43-9006 can also inhibit the activity of the VEGF receptor, VEGFR2 [97].  Additional clues come from knock-out studies with mice.  Tumors growing in host mice that are deficient in ERK5 expression are not only reduced in size but also have a reduced vasculature [98].  Although these limited studies indicate that MKK signaling likely plays a role in tumor vascularization, they do not discriminate as to whether all pathways are indispensable or whether particular pathways are more important than others.