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Immunoreactivity for bFGF

FIGURE 7.8 bFGF protein expression in HNSCC tumor tissue correlates with the mean number of microvessels per microscopic field. Adapted from Riedel et al, Head Neck 22, 183-189 (2000).

proangiogenic cytokine, with a pivotal role in the regulation of normal and pathological angiogenesis. VEGF was initially characterized for its specific and potent ability to promote endothelial cell proliferation in a paracrine mode of action [89,90,147]. In addition, VEGF can induce permeability and vascular leakage of plasma proteins. In particular, it has been demonstrated that extravasation of plasma fibrinogen alters the fibrin deposition in the extracellular matrix. As a consequence, the extracellular matrix is transformed into the mature stroma characteristic of tumors where increased proliferation of fibroblasts and endothelial cells occurs [28].

Disruption of a single VEGF allele or both VEGF alleles in mice results in embryonic lethality due to severe vasculature abnormalities or almost a complete absence of vasculature, respectively [148,149]. To date, five other members belonging to the VEGF family have been identified based on their homology to VEGF: placenta growth factor (PIGF), VEGF-B, VEGF-C, VEGF-D, and VEGF-E [150-152], The different members of the VEGF family have an overlapping ability to bind to and activate their respective cell surface receptors. Three high-affinity vascular endothelial growth factor receptors (VEGFRs) have been cloned and characterized biochemically: VEGFR-l/Flt-1, VEGFR-2/Flk-l/KDR, and VEGFR-3/Flt-4 [153], Studies have demonstrated that both VEGFR-2 and VEGFR-1 are essential for the normal development of embryonic vasculature. However, their respective roles in endothelial cell proliferation and differentiation appear to be distinct. The major growth and permeability functions of VEGF seem to be mediated exclusively by the engagement of VEGFR-2. In contrast, VEGFR-1 appears to exert an opposite negative role, either by sequestering VEGF and impairing its binding to VEGFR-2 or by directly suppressing VEGFR-2 signaling. In fact, VEGFR-2 knockout mice fail to develop a vasculature and have a limited number of endothelial cells, whereas VEGFR-1-deficient mice are characterized by the presence of disorganized tubules, resulting from the abnormal clustering of an excessive number of endothelial cells [91, 154, 155], VEGFR-3 is mostly expressed in lymphatic vessels, and its contribution to the development of blood vessels is not yet fully defined [156],

VEGFRs are transmembrane receptor tyrosine kinases whose activation occurs on ligand binding, followed by receptor dimerization and activation by autophosphory-lation of tyrosine residues in the cytoplasmic tail [157-159]. The activated receptor is likely to provide docking sites for SH2 domain-containing proteins and, upon the engagement of multiprotein aggregates, initiates a downstream signaling cascade, associated with endothelial cell proliferation and survival. Activated VEGFR-2, but not VEGFR-1, has been demonstrated to bind to She, Grb2, Nek, ras GTPase, P13-K, PLC-y, and PKC, as well as to the protein phosphatases SHP-1 and SHP-2 [90,91,160,161], Furthermore, phosphorylated VEGFR-2 leads to the activation of both p44/42MAPK [162] and AKT/PKB [163] signal transduction pathways. The engagement of VEGFR-2 by its ligand results in diverse cellular biological responses, such as changes in cell morphology, actin reorganization, membrane ruffling, chemotaxis, and mitogenesis [161]. VEGF-dependent endothelial cell migration may occur focal adhesion kinase (FAK) and paxillin tyrosine phosphorylation and consequent activation [162]. Understanding of a VEGFR-1 downstream signal transduction pathway(s) is yet to be fully elucidated. However, its importance in the promotion of normal vascular development in the fetus [164], as well as the lethality of VEGFR-1-deficient mouse embryos [155], strongly suggests that this receptor may signal through novel mechanisms.

Numerous studies have demonstrated that VEGF mRNA is upregulated in the vast majority of human tumors, such as lung [165], thyroid [166], breast [44,167], gastrointestinal tract [168], kidney and bladder [169], ovary [170], and cervix uteri carcinomas [48], as well as angiosarcomas [171] and glioblastomas [172]. VEGF gene expression has been described to be upregulated by different mechanisms, among which oxygen tension seems to play a major role, both in vitro and in vivo [173-176], Hypoxia-dependent VEGF gene transcription has been demonstrated to occur in both normal and tumor tissues [173] and to be mediated by the transcriptional activity of hypoxia-inducible factor-1 (HIF-1) [177], Furthermore, upregulation of VEGF mRNA expression and increased VEGF secretion may be dependent on the effect of various mitogenic growth factors and cytokines, such as TGF-P [178], IL-la, PGE2 [179], IL-6 [180], and IGF-1 [181]. These factors, in addition to their direct mitogenic effect on malignant cells, may facilitate the tumor growth via increased VEGF production and secretion [91],

Several studies reported the elevated expression of VEGF mRNA in HNSCC. In situ hybridization analysis revealed a significantly greater expression of both VEGF and VEGFR-2 in 29 cases of oral cavity and larynx invasive squamous cell carcinoma compared to normal control tissues [182]. Similar results were obtained in an immunohistochemical analysis conducted on 63 specimens of various HNSCCs [183]. In addition, a study conducted on 156 patients revealed that high VEGF expression was detectable not only in the cancer cells, but also in the tumor-infiltrating inflammatory cells, such as plasma cells and macrophages, and in the cells of the tumor bordering tissues [184].

A correlation between VEGF expression with increased microvessel density, early recurrence, and worse prognosis has been observed in primary breast cancer [185, 186], lung [187], and gastric carcinoma [188]. The first direct evidence of a correlation between VEGF expression and increased microvessel density in HNSCC stems from a study conducted on a series of patients affected by different stages of head and neck lesions. In particular, the greatest number of microvessels was detected in premalignant lesions.

Although increased angiogenesis was also evident in early and late stage HNSCC, compared to normal tissues, these findings suggest that VEGF is likely to regulate the early angiogenic steps of the tumor progression toward more aggressive phenotypes [189] (Fig. 7.9). Moreover, in 29 specimens from human nasopharyngeal carcinoma, a significant relationship between microvessel density and high VEGF expression was determined, thus suggesting the importance of VEGF-dependent angiogenesis in the occurrence of lymph node metastasis [190], Likewise, the immunohistochemical analysis of 29 oral SCC specimens revealed a correlation between the intensity of VEGF expression with lymph node metastasis, but in contrast to the study by Wakisaka et al. [190], not with vessel density [191]. In addition a poor prognostic outcome is likely to be a direct consequence of high VEGF expression in HNSCC [192].

Wild-type p53 downregulates VEGF transcription, whereas p53 mutants have no effect on the promoter activity [193]. In this regard, the number of p53 mutations in VEGF-positive HNSCC has been found significantly higher than VEGF-negative tumors, thus suggesting the important role of p53 in the regulation of VEGF-dependent angiogenesis in HNSCC [194]. It has been demonstrated that tumor cells secrete VEGF to the bloodstream [195] and, as a consequence, an elevation in VEGF protein levels has been found in the serum of some cancer patients [187,196,197], as well as in patients affected by HNSCC [198,199] (Fig. 7.10). High serum levels of VEGF

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