**4.1 The role of VEGF in invasion**

Angiogenesis, the formation of new blood vessels, is not only a normal physiological process, but it is closely linked to both tumor growth and metastasis, providing the principal pathway through tumor cells exit the primary tumor site and enter the circulation. A multiplicity of molecular determinants is involved in these different mechanisms of vascular growth, and VEGFs play a significant role in pathological angiogenesis. In particular, the role of VEGF-A in tumor angiogenesis are clarified by several studies [23, 55], but the results concerning VEGF-expression in normal and dysplastic oral epithelium, as well as about the potential role of VEGF in oral squamous cell carcinoma progression shown contradictions. VEGF-A overexpression has been reported in most types of cancer, including oral squamous cell carcinoma, and it is thought to be a prognostic factor for survival. Some studies revealed no altered VEGF expression in the normal and mildly dysplastic oral epithelium, or the expression was significantly lower than neoplastic epithelium [16–18]. A considerable upregulation of VEGF expression during the transition from normal oral epithelium through dysplasia to OSCC, but no correlation was found between VEGF expression and the grade of dysplasia. Many researchers demonstrated an upregulation of VEGF expression in cancerous tissues in comparison to the normal oral mucosa. These results suggest that VEGF could be implicated in tumor progression by expanding the vascularity during the process of transition from the normal oral mucosa to invasive carcinoma [18, 51–56].

**125**

*Vascular Endothelial Growth Factor Expression in the Pathological Angiogenesis in Oral…*

Immunohistochemical study of VEGF expression in oral squamous cell carcinomas clarify the relationship between vascular endothelial growth factor (VEGF) expression and clinicopathological factors in oral squamous cell carcinoma (OSCC), showing that serum VEGF levels were significantly higher in oral cancer patients as compared to normal controls that further showed an increasing trend with clinical stage and lymph node involvement, suggesting that VEGF level may be a reliable biomarker and may be a potential target for development of chemotherapeutic

Lymphangiogenesis plays a vital role in tumor growth and systemic dissemination of different carcinomas [2]. Although vascular endothelial growth factor C (VEGF-C) is well known to be associated with lymph node metastasis in patients with OSCC, inducing the production of urokinase by cancer and playing a crucial role in the extracellular matrix remodeling and tumor cell invasion, the specific mechanisms of lymphangiogenesis in OSCC are largely unknown. Further, it has been implicated in inducing the growth of both blood and lymphatic vessels and in regulation of proliferation, differentiation and migration of lymphatic endothelial cells in many physiological and pathological conditions [19, 20]. Overexpression of VEGF-C has been detected in various malignancies and is frequently associated with lymphatic invasion, nodal and distant metastasis and consequently poor survival [23, 33]. VEGF-C signaling is involved in the progression of several malignancies that put forward VEGF-C as a potential target for the development of new anticancer therapies to prevent local invasion and metastatic spread of disease. The progression of several malignancies involves VEGF-C signaling. The immunohistochemical analysis and real-time quantitative RT-PCR studies showed a significant correlation between the levels of VEGF-C mRNA expression in a human OSCC cell line, through the phosphoinositide 3-kinase-Akt pathway. The angiogenic action of this growth factor is achievable by binding to two receptor tyrosine kinases (RTK), VEGFR-1 (Flt-1) and VEGFR-2 (KDR, Flk-1) [17, 22]. VEGFR-2 is the major mediator of the mitogenic, angiogenic and permeability-enhancing effects of VEGF-A, while the role of VEGFR-1 in the regulation of angiogenesis is controversial. It is implicated in a paracrine release of growth factors, but the central role of this receptor is to prevent the interaction of VEGF with VEGFR-2. Also, it may promote angiogenesis by recruitment in tumor vasculature of monocytes and other bone marrow-derived cells [48, 52, 57]. Moreover, VEGFR-1 is involved in the induction of matrix metalloproteinases [49] that retrovirus-mediated expression of a dominant negative VEGFR-2, because of inhibition of signal transduction through wild-type VEGFR-2, suppression of the growth of glioblastoma multiforme and other tumor cell lines in vivo. Correlation of VEGF expression with clinical stage of OSCC is controversial. Clinicopathologic findings suggest that evaluation of VEGF expression is of prognostic value in patients with OSCC. VEGF expression was found to increase significantly with advancing stage of the tumor. Elevated expression of VEGF was associated with aggressive phenotype and advanced stage of the tumor [58], as well as demonstrated by Yu Hong Li et al. which found a higher VEGF expression in stage 3 and 4 tumors as compared to stage 1 and 2 [59]. An explanation could be attributed to the correlation with the intensity of VEGF expression and the degree of differentiation or invasiveness of carcinoma. The results support the findings of previous *results* on the average density of immunohistochemistry (IHC), indicating that in normal oral squamous cells, VEGF expression was detected only in the vascular endothelial cells in the mesenchymal tissues [60, 61]. According to these results, to Kim's study on the up-regulation of vascular

*DOI: http://dx.doi.org/10.5772/intechopen.90924*

strategies for patients with oral carcinoma.

**4.2 The role of VEGF in metastasis**

Immunohistochemical study of VEGF expression in oral squamous cell carcinomas clarify the relationship between vascular endothelial growth factor (VEGF) expression and clinicopathological factors in oral squamous cell carcinoma (OSCC), showing that serum VEGF levels were significantly higher in oral cancer patients as compared to normal controls that further showed an increasing trend with clinical stage and lymph node involvement, suggesting that VEGF level may be a reliable biomarker and may be a potential target for development of chemotherapeutic strategies for patients with oral carcinoma.

## **4.2 The role of VEGF in metastasis**

*Oral Diseases*

the growth of new lymphatic vessels [41–45]. The expression of VEGFR-3 (or Flt-4) is relevant in lymphatic vessels [44] and in hematopoietic cells of monocytic lineage [6, 42], and is also expressed in a subset of capillary endothelia [19]. Studies on animal models showed that VEGF-C/VEGFR-3 axis plays a crucial role in cancer metastasis by inducing lymphangiogenesis [46–48], but further investigations would be necessary. Furthermore, it has been documented the link between mutations in VEGFR-3 with hereditary lymphedema, an autosomal dominant disorder of the lymphatic system that can lead to lymphangiosarcomas [13, 49]. Neuropilins-1 and -2 are more important in immunology and neuronal development, but they are also involved in angiogenesis [19, 44]. Neuropilins, bind especially class 3 semaphorins but the Neuropilin-1 also binds VEGF, VEGF-B and PlGF, while Neuropilin-2 binds VEGF, VEGF-C and PlGF [50]. When is coexpressed in cells together with VEGFR-2, Neuropilin-1 enhances the binding of VEGF165 to VEGFR-2 and augments tumor angiogenesis in vivo [51, 52]. Nrp-2 is expressed also on lymphatic ECs, and mutated Nrp-2 forms induce abnormalities in the formation of small lymphatic vessels and lymphatic capillaries in mice [53]. In addition, some isoforms bind to known as non-tyrosine kinase receptors, known as neuropilins (NRPs) (neuropilin-1 and neuropilin-2 (22–44)). Neuropilins-1 and -2 are also involved in immunology and neuronal development [50, 54]. Neuropilins bind, especially class 3 semaphorins but the Neuropilin-1 also binds VEGF, VEGF-B and PlGF, while Neuropilin-2 binds VEGF, VEGF-C and PlGF [50]. Neuropilin-1 is capable to improve the binding of VEGF165 to VEGFR-2 and increase tumor angiogenesis in vivo [51, 53]. Nrp-2 is expressed also on lymphatic ECs, and it has been shown in vivo that mutated Nrp-2 form alters the formation of small lymphatic vessels and lymphatic capillaries in mice [54]. The binding results in stimulation of cell-signaling pathways that act to increase cell nucleus division, and contributing to angiogenesis

through extracellular matrix dissolution, and endothelial cell movement.

Angiogenesis, the formation of new blood vessels, is not only a normal physiological process, but it is closely linked to both tumor growth and metastasis, providing the principal pathway through tumor cells exit the primary tumor site and enter the circulation. A multiplicity of molecular determinants is involved in these different mechanisms of vascular growth, and VEGFs play a significant role in pathological angiogenesis. In particular, the role of VEGF-A in tumor angiogenesis are clarified by several studies [23, 55], but the results concerning VEGF-expression in normal and dysplastic oral epithelium, as well as about the potential role of VEGF in oral squamous cell carcinoma progression shown contradictions. VEGF-A overexpression has been reported in most types of cancer, including oral squamous cell carcinoma, and it is thought to be a prognostic factor for survival. Some studies revealed no altered VEGF expression in the normal and mildly dysplastic oral epithelium, or the expression was significantly lower than neoplastic epithelium [16–18]. A considerable upregulation of VEGF expression during the transition from normal oral epithelium through dysplasia to OSCC, but no correlation was found between VEGF expression and the grade of dysplasia. Many researchers demonstrated an upregulation of VEGF expression in cancerous tissues in comparison to the normal oral mucosa. These results suggest that VEGF could be implicated in tumor progression by expanding the vascularity during the process of transition from the normal oral mucosa to invasive carcinoma [18, 51–56].

**4. Role of VEGF in oral squamous cell carcinoma**

**4.1 The role of VEGF in invasion**

**124**

Lymphangiogenesis plays a vital role in tumor growth and systemic dissemination of different carcinomas [2]. Although vascular endothelial growth factor C (VEGF-C) is well known to be associated with lymph node metastasis in patients with OSCC, inducing the production of urokinase by cancer and playing a crucial role in the extracellular matrix remodeling and tumor cell invasion, the specific mechanisms of lymphangiogenesis in OSCC are largely unknown. Further, it has been implicated in inducing the growth of both blood and lymphatic vessels and in regulation of proliferation, differentiation and migration of lymphatic endothelial cells in many physiological and pathological conditions [19, 20]. Overexpression of VEGF-C has been detected in various malignancies and is frequently associated with lymphatic invasion, nodal and distant metastasis and consequently poor survival [23, 33]. VEGF-C signaling is involved in the progression of several malignancies that put forward VEGF-C as a potential target for the development of new anticancer therapies to prevent local invasion and metastatic spread of disease. The progression of several malignancies involves VEGF-C signaling. The immunohistochemical analysis and real-time quantitative RT-PCR studies showed a significant correlation between the levels of VEGF-C mRNA expression in a human OSCC cell line, through the phosphoinositide 3-kinase-Akt pathway. The angiogenic action of this growth factor is achievable by binding to two receptor tyrosine kinases (RTK), VEGFR-1 (Flt-1) and VEGFR-2 (KDR, Flk-1) [17, 22]. VEGFR-2 is the major mediator of the mitogenic, angiogenic and permeability-enhancing effects of VEGF-A, while the role of VEGFR-1 in the regulation of angiogenesis is controversial. It is implicated in a paracrine release of growth factors, but the central role of this receptor is to prevent the interaction of VEGF with VEGFR-2. Also, it may promote angiogenesis by recruitment in tumor vasculature of monocytes and other bone marrow-derived cells [48, 52, 57]. Moreover, VEGFR-1 is involved in the induction of matrix metalloproteinases [49] that retrovirus-mediated expression of a dominant negative VEGFR-2, because of inhibition of signal transduction through wild-type VEGFR-2, suppression of the growth of glioblastoma multiforme and other tumor cell lines in vivo. Correlation of VEGF expression with clinical stage of OSCC is controversial. Clinicopathologic findings suggest that evaluation of VEGF expression is of prognostic value in patients with OSCC. VEGF expression was found to increase significantly with advancing stage of the tumor. Elevated expression of VEGF was associated with aggressive phenotype and advanced stage of the tumor [58], as well as demonstrated by Yu Hong Li et al. which found a higher VEGF expression in stage 3 and 4 tumors as compared to stage 1 and 2 [59]. An explanation could be attributed to the correlation with the intensity of VEGF expression and the degree of differentiation or invasiveness of carcinoma. The results support the findings of previous *results* on the average density of immunohistochemistry (IHC), indicating that in normal oral squamous cells, VEGF expression was detected only in the vascular endothelial cells in the mesenchymal tissues [60, 61]. According to these results, to Kim's study on the up-regulation of vascular

endothelial growth factor (VEGF) expression in oral squamous cell carcinoma, immunohistochemical staining with VEGF, found a very little expression of VEGF in normal oral squamous cell tissues and a significant positive relationship between VEGF expression and the degree of differentiation or invasiveness of carcinoma [62]. In addition, histopathological findings showed that the VEGF had *higher expression* levels in less-differentiated invasive oral squamous cell carcinoma, while it was expressed at slightly higher levels in well-differentiated and less-invasive intraepithelial carcinoma tissues or highly differentiated oral squamous cell carcinoma than in normal cells [62]. In contrast, Shang and Li found that VEGF expression was significantly higher in patients in patients with stage 1 and 2 tumors as compared to when there were a stage 3 and 4 tumors [63].

### **4.3 The role of VEGF in prognosis**

Interestingly, some studies have focused on the potential prognostic importance of *VEGF* polymorphisms in head and neck cancers [14, 20, 21]. Results showed the association between −1154GG *VEGF* genotype, located in the promoter region of the gene, and higher VEGF production [12]. A study conducted by Ku et al., reported an association of −460C/T polymorphism and VEGF overexpression in oral cancer, showing a higher risk for oral cancer in the patients with a high −460TC ratio [66, 67]. Further investigations showed an association between +960C/T *VEGF* polymorphism and oral cancer [23], indicating that the low production of VEGF by the T allele is correlated with increased risk of oral cancer, and vascular invasion in oral squamous cell carcinoma. In contrast, Vairaktaris et al. [56], did not demonstrate an influence of gene polymorphism on the oral cancer in the logistic regression models. However, the genotype *VEGF* −460CT was associated with early stage tumors. Nasr et al. analyzed the polymorphism *VEGF* −2578C/A and suggested that carriers of *VEGF* −2578C allele may play a role in susceptibility to nasopharyngeal carcinoma [64, 65]. The risk for laryngeal squamous cell carcinoma seems to be linked with the increase of the −1154G/G genotype of the *VEGF* gene, in the −1154G/A polymorphism of the *VEGF* [27]. Moreover, the polymorphism −1154G/A *VEGF* has been shown to be associated with differential expression of VEGF *in vitro*. However, some of the data are contradictory.

### **4.4 Vascular endothelial growth factor and advances in developing novel therapeutic strategies for oral squamous cell carcinoma**

There is a great need for therapies to prevent and/or slow the progression of OSCC. Recent studies focused on potential of drugs that target VEGF or its receptors-signaling system because their angiogenesis-promoting activity at the level of endothelial cells. Therefore, modulation of these factors to inhibit tumor angiogenesis, currently, is a major focus in developing OSCC therapies. The development of angiogenesis inhibitors as anticancer agents have been developed with data showing promising efficacy at reducing OSCC in in vitro models [13, 14]. Recent studies found that administration of angiogenesis inhibitors agents in association with chemotherapy and radiotherapy can improve the efficacy of these treatments. For example, Teicher et al. observed that coadministration of the TNR-470, angiogenesis inhibitor agent, induces a substantial cyclophosphamideinduced tumor cell-killing. Jain's study confirmed these findings suggesting that it was a consequent normalization of the tumor vasculature, as a result of endothelial cell death. Different therapeutic strategies to induce the inhibition of VEGF or its receptor signaling system are being emerging for treatment of OSCC, as VEGFR-1 ribozymes, VEGF toxin conjugates, and soluble VEGF receptors.

**127**

Italy

**Author details**

Biagio Rapone1

\* and Elisabetta Ferrara<sup>2</sup>

\*Address all correspondence to: biagiorapone79@gmail.com

"Aldo Moro" University of Bari, Bari, Italy

provided the original work is properly cited.

1 Department of Basic Medical Sciences, Neurosciences and Sense Organs,

2 Complex Operative Unit of Odontostomatology, Hospital S.S. Annunziata, Chieti,

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

*Vascular Endothelial Growth Factor Expression in the Pathological Angiogenesis in Oral…*

Angiogenesis is *essential* for the growth and metastasis of solid *tumors*, including oral squamous cell *carcinoma*. The main factor responsible for angiogenesis is *VEGF* and its receptors. Specifically, *VEGF*-A is known to be a *key* angiogenic factor, and its overexpression has been reported in most types of *cancer*, including *oral squamous cell carcinoma*, and it is thought to be a prognostic factor for survival. It was pointed out that VEGF as an attractive candidate for therapeutic intervention, but more studies are needed to clarify the real potential of angiogenesis inhibitors agents in OSCC, to determine optimal timing for VEGF, and to search for drug

*DOI: http://dx.doi.org/10.5772/intechopen.90924*

The authors declare no conflict of interest.

**5. Conclusions**

candidates.

**Conflict of interest**

*Vascular Endothelial Growth Factor Expression in the Pathological Angiogenesis in Oral… DOI: http://dx.doi.org/10.5772/intechopen.90924*
