Oral Oncology (2006) 42, 337–342

http://intl.elsevierhealth.com/journals/oron/

REVIEW

The role of vascular endothelial growth factor(VEGF) in oral dysplasia and oral squamous cellcarcinoma

Shane Johnstone *, Richard M. Logan

Department of Oral Pathology, Dental School, The University of Adelaide, Adelaide, South Australia 5005,Australia

Received 3 June 2005; accepted 29 June 2005

Summary A better understanding of oral cancer pathogenesis is essential toimproving patient prognosis and treatment modalities. Research has shown a signif-icant increase in vascularity during the transition from normal oral mucosa, throughdiffering degrees of dysplasia, to invasive carcinoma. A close association betweentumour angiogenesis and tumour progression to late oral squamous cell carcinomahas also been reported. Vascular endothelial growth factor (VEGF) acts to induceendothelial proliferation, migration and specialisation in new and developing vascu-lar beds. VEGF is also a promoter of angiogenesis in many tumour types, and hastherefore been subject to numerous studies in oral dysplasia and squamous cell car-cinomas. The contribution of VEGF to the development of oral dysplasia and invasivecarcinomas is currently disputed due to conflicting results within the literature.More research is required before VEGF technology can be used to improve the diag-nosis, prognosis and treatment of sufferers.

�c 2005 Elsevier Ltd. All rights reserved.

KEYWORDSOral dysplasia;Vascular endothelialgrowth factor;Oral squamous cellcarcinoma;Oral cancer

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Introduction

A good understanding of the mechanisms underly-ing oral cancer pathogenesis is essential to improvetreatment modalities and long-term patient out-

368-8375/$ - see front matter �c 2005 Elsevier Ltd. All rights reseoi:10.1016/j.oraloncology.2005.06.020

* Corresponding author. Tel.: +61 8 8303 4123; fax: +61 8 8303838.E-mail address: shane.johnstone@adelaide.edu.au (S. John-

tone).

comes. Current treatment regimes are guided bytraditional clinicopathologic factors such as TNMstage, histological grade and patient age. An exten-sive amount of research into the development andtreatment of oral cancer over the last 25 years hasgiven little improvement in the 5-year survival rateof sufferers.1,2

For solid tumours to grow and metastasise, anadequate blood supply is essential.3 Vasculardevelopment and factors that regulate it have

rved.

338 S. Johnstone, R.M. Logan

therefore been extensively studied in many tumourtypes. Vascular endothelial growth factor (VEGF)has been shown to be a critical angiogenic cytokineinvolved in the development of a blood supply inseveral different tumours. VEGF is a highly potentangiogenic agent that acts to increase vessel per-meability and enhance endothelial cell growth,proliferation, migration and differentiation.4

A significant increase in vascularity occurs dur-ing the transition from normal oral mucosa,through differing degrees of dysplasia, to invasivesquamous cell carcinoma.5–7 Tumour progressionfrom early to late carcinomas is also closely associ-ated to vascularity.7,8 Since VEGF induces angio-genesis in many tumour types, it has thereforebeen subject to numerous studies in oral dysplasiaand oral cancers. In particular, the role of VEGF inangiogenesis, disease progression and patient prog-nosis has been extensively investigated in oral dys-plasia and squamous cell carcinoma. In addition,the use of VEGF expression as a prognostic indica-tor in oral cancer patients has also been studied.

The contribution of VEGF to the development oforal dysplasia and cancerous lesions is currentlydisputed due to conflicting results within the liter-ature. A greater understanding of the role of angi-ogenesis and VEGF in oral cancer is required beforeVEGF technology can be utilised to improve thediagnosis, prognosis and treatment of patients.

The biology of vascular endothelialgrowth factor

The biological activities of VEGF

The development of a vascular supply is a funda-mental requirement for organ development anddifferentiation during embryogenesis. Vasculardevelopment occurs in several stages, beginningwith the assembly of a vessel plexus from singlecell precursors (vasculogenesis).9 The vascularplexus then undergoes modification by sproutinggrowth and remodelling (angiogenesis), followedby recruitment of vessels into target tissues. Final-ly, new vessels differentiate according to the spe-cific needs of the tissue.9

Vascular endothelial growth factor (VEGF), alsoknown as VEGF-A, is a potent angiogenic cytokineinvolved in every stage of vascular development.9

The multiple roles of VEGF are based on its abilityto induce various responses by endothelial cellsduring vascular development, including cell prolif-eration, migration, specialisation and survival.4,9

During vascular morphogenesis VEGF promotes

the differentiation of cell precursors into endothe-lial cells and their early assembly into a primaryvascular plexus. It then induces and guides angio-genic sprouting to expand the primary plexus andvascularise growing tissues. VEGF then aids inendothelial cell proliferation, pericyte coverageand the distribution of arteries and veins.9 In addi-tion, VEGF induces vasodilation and increases per-meability of vascular beds.4

The VEGF gene and its isoforms

VEGF, also known as VEGF-A, belongs to a genefamily that includes placental growth factor(PlGF), VEGF-B, VEGF-C, VEGF-D and VEGF-E.4,10

VEGF gene splicing generates four main VEGFisoforms that differ in their molecular mass andbiological activities.4,11,12 In humans, this corre-sponds to isoforms VEGF121, VEGF165, VEGF189 andVEGF206,

4,12 although other less frequent variationshave been reported.4,13 The most important biolog-ical property that distinguishes the different VEGFisoforms is their ability to bind heparin and theextracellular matrix.12,13

VEGF121 is secreted and is freely diffusiblewithin the extracellular matrix and does not bindheparin or the extracellular matrix.14 VEGF165 isalso secreted, however a portion remains boundto cell surface heparin and the extracellular ma-trix.13 VEGF189 and VEGF206 have a higher affinityto heparin and heparin sulphate than VEGF165.

13

The secreted forms of VEGF induce proliferationof endothelial cells and in vivo angiogenesis.13

The heparin binding forms of VEGF can bind to cellsurface and extracellular matrix proteoglycans andfacilitate the release of other angiogenic factors,such as basic fibroblast growth factor, which arestored on heparin sulphates of the extracellularmatrix.13 In addition, extracellular bound isoformscan be cleaved by plasmin to release an active sol-uble VEGF fragment.4,13 It has been suggested thatVEGF isoforms perform unique functions and act ina co-ordinated fashion to optimise the formationand development of new vessel networks.15

VEGF receptors and co-receptors

VEGF binds to two receptors, VEGF receptor-1(VEGFR-1) and VEGF receptor-2 (VEGFR-2).13 AllVEGF isoforms bind to VEGFR-1 and VEGFR-2 as wellas interacting with a family of co-receptors, theneutropilins.4,13 Activation of VEGFR-1 promotesendothelial cell migration but does not induce cellproliferation.9,13 VEGFR-1 may also act as a decoyreceptor that regulates overall VEGF concentration

VEGF in oral dysplasia and oral squamous cell carcinoma 339

by binding VEGF and preventing its association withVEGFR-2.9 This may help reduce endothelial cellproliferation and prevent overcrowding and vascu-lar disorganisation.16 VEGFR-1 also exists as a solu-ble form, sVEGFR-1, which binds VEGF with highaffinity, preventing binding to functional receptors,and is therefore thought to only play an inhibitoryrole by sequestering free VEGF.12

VEGFR-2 is the major mediator of endothelialcell mitogenesis, proliferation and survival. It isalso essential for the differentiation of endothelialcells and the induction of microvessel permeabil-ity.4,10,12 VEGFR-2’s function is regulated by co-receptors such neutropilin-1, which interact withVEGF isoforms and their binding to VEGFR-2.12,17

Regulation of VEGF gene expression

VEGF is crucial to vascular development, both inthe setting of physiological vascular developmentas well as the development of vasculature in path-ological settings. VEGF gene transcription is in-duced by exposure to low oxygen tension in avariety of circumstances such as embryonic organformation or tumour growth. Hypoxia also pro-motes the stabilisation of VEGF mRNA by proteinsthat bind VEGF mRNA.18

Cytokines and growth factors that do not stimu-late angiogenesis directly can regulate angiogenesisby modulating VEGF expression. Currently, a vari-ety of growth factors, cytokines and nitric oxidehave been found to potentiate or inhibit VEGFexpression.4,13 Specific transformation events suchas oncogenic mutation or inactivation of tumoursuppressor genes can also result in the inductionof VEGF gene expression.13,19

The role of VEGF in angiogenesis

VEGF is crucial for angiogenesis during a number ofphysiological circumstances in embryonic and post-natal development. The development of the car-diovascular system depends on the generation ofa precise VEGF concentration gradient within theembryo.13 Inhibition of VEGF results in an increasedmortality, impaired organ development and im-paired bone cartilage vascularisation.4

VEGF induced angiogenesis has been found toplay an important role in the aetiology of severaldiseases associated with abnormal angiogenesis.For example VEGF has a role in intraocular neovas-cular syndromes, psoriasis,20,21 bullous pemphigoidand erythema multiforme.22

Angiogenesis is also essential for the survivalof tumour cells and the development of solid

tumours. Tumour cells and associated stroma arethought to be the major source of VEGF in tumours,whose progression is characterised by excessiveangiogenesis in an uncontrolled or disorganisedmanner.4 As tumour growth develops, cells withinthe mass are deprived of oxygen because of theirdistance from nearest blood vessels. This resultsin the generation of hypoxic regions within tu-mours, which respond by producing VEGF, whichthen triggers angiogenesis. The simplicity of thismechanism may explain why VEGF seems to be in-volved in the induction of tumour angiogenesis inmany solid tumours. In addition to endothelial cellproliferation and migration, VEGF induces perme-abilisation of tumour blood vessels through whichproteins can then extravasate.23,24 This leakage isthen thought to form the basis of a new extracellu-lar matrix which facilitates angiogenesis.

Therapeutic implications

The ability of VEGF to promote angiogenesis andneovascularisation has been exploited for thetreatment of diseases with impaired blood supplysuch as ischaemic heart disease and limb ischae-mia.4,13 These treatments aim to induce collateralblood supply to the area of ischaemia by a localdelivery of VEGF.4

VEGF has also been targeted for use in conditionsthat are associated with pathological angiogenesis.Studies have shown that combining anti-VEGF treat-ment with chemotherapy or radiation therapy forthe treatment of solid tumours resulted in increasedanti-tumour effect than either conventional treat-ment modality alone.4 Clinical trials have shownan increased time to disease progression and sur-vival in some cancer patients,25,26 however in manycases progression of disease usually occurs.

The role of VEGF in oral dysplasia andoral squamous cell carcinoma

Despite extensive research into the pathogenesisand treatment of oral cancer, the 5-year survivalrate for patients has shown little improvement.1,2

Current treatment regimes are guided by clinico-pathologic factors such as TNM stage, histologicalgrade and patient age. A better understanding ofthe underlying mechanism of oral cancer pathogen-esis is therefore essential to improving treatmentmodalities and long term patient outcomes.

Solid tumour growth cannot exceed 1–2 mm3

without an adequate blood supply.3 Angiogenesisis therefore a crucial step in the growth, invasion

340 S. Johnstone, R.M. Logan

and metastasis of a tumour.3,27,28 Several studiessuggest that tumour angiogenesis is associated withtumour progression and aggressiveness in a numberof malignancies,29,30 including head and neckcarcinomas.8

Within the oral cavity, 10–20% of dysplastic le-sions progress to carcinomas.31 Research has showna significant increase in vascularity during the tran-sition from normal oral mucosa, through differingdegrees of dysplasia, to invasive carcinoma.5–7 Sev-eral studies have also reported a close associationbetween tumour angiogenesis and tumour pro-gression from early to late oral squamous cellcarcinomas.5,7,8,32 VEGF is a powerful promoter ofangiogenesis in many tumours types and has there-fore been subject to numerous studies in oral dys-plasia and oral cancers. However the link betweenVEGF expression and its role in angiogenesis, diseaseprogression and long-term prognosis in dysplasia andtumours of the oral cavity is not well understood.

VEGF and oral cancer angiogenesis

Several studies have shown an increase in vascular-ity amongst varying degrees of oral dysplasiaand carcinomas when compared to normaltissue,6–8,32 suggesting that it may represent angi-ogenesis in these tumours.

Researchers have traditionally measured oral tu-mour angiogenesis by means of highest microvesseldensity (hMVD) within a tissue sample,7,33,34 thoughmicrovessel volume has also been used.7 Themajority of research has found no correlation be-tween VEGF expression and angiogenesis in oraldysplasia or carcinoma6,31,35,36 or that tumour angi-ogenesis is mediated by factors in addition toVEGF.37 However, several studies have found thatan increase in microvessel density correlated withan increase in VEGF expression by oral squamouscell carcinomas.38,39 Whether VEGF contributes toangiogenesis within these oral lesions, and to whatextent, therefore requires considerably more re-search. In particular, if VEGF is found to be animportant contributor to tumour angiogenesis,determining the underlying mechanism will beessential to improving our understanding of oraltumour pathogenesis.

VEGF and oral cancer progression

Histological grade (degree of differentiation) hastraditionally been used to measure tumour severityand aggressiveness. Few studies have investigatedwhether VEGF plays a role in the transition fromnormal oral mucosa to squamous cell carcinoma.

Denhart et al.40 found that normal or mildly dys-plastic oral epithelium expressed no VEGF, whilstmoderately dysplastic epithelium had a low levelof VEGF expression. VEGF was also shown to bestrongly expressed in some high grade dysplasiasand squamous cell carcinomas. However statisticalanalysis was lacking in this study and not all carci-nomas or high grade dysplasias expressed VEGF.Carlile et al.6 on the other hand found no differ-ence between VEGF expression in normal oralmucosa when compared to epithelial dysplasia,indicating that any increase in dysplasia is notaccompanied by an increase in VEGF expression.Amongst oral squamous cell carcinomas, themajority of studies have found no significant asso-ciation between differentiation levels and VEGFexpression.36,39,41,42 One study did however find asignificantly more number if VEGF positive tumourswere well differentiated compared to moderatelyor poorly differentiated.38

Clinical staging is commonly used as a determi-nant of tumour progression and is often helpful indefining treatment protocols and management oforal cancer patients. VEGF expression has beenfound to significantly increase between stages Iand II tumours43 and when stage I/II tumours arecompared with stage III/IV.39 However other re-search has found no significant link between VEGFexpression and clinical staging in oral squamouscell carcinoma.38,41,42 The use of VEGF as an indi-cator of oral tumour progression as an adjunct toclinical staging is therefore currently not reliableand further investigation is required.

Tumour metastasis is often an indicator of anadvanced lesion and is therefore associated witha poorer survival rate and more aggressive treat-ment. Metastases are often difficult to diagnoseclinically and a reliable chemical marker of metas-tasis could prove more advantageous. Unfortu-nately the majority of research has found nosignificant difference between VEGF positivity orVEGF expression and metastases.38,41,43–45 On theother hand several studies have found that VEGFexpression was significantly higher in patients withregional lymph node involvement35,36,39 and there-fore may promote oral cancer progression. At thisstage however, the use of VEGF as a predictor ormeasurement of tumour progression remains ele-mentary and further research to clarify its useful-ness is required.

VEGF and oral cancer prognosis

If VEGF is to emerge as an important factor inestablishing patient treatment options, then its

VEGF in oral dysplasia and oral squamous cell carcinoma 341

ability to act as a prognostic indicator needs to bereliable. Only two studies have looked at serumconcentrations of VEGF as an indicator of patientprognosis in oral squamous cell carcinoma.46,47

Both studies found the mean VEGF serum concen-tration significantly higher in patients with oralsquamous cell carcinoma when compared to nor-mal subjects. Higher serum VEGF concentrationsalso correlated with an increase in metastasis andclinical stage. In addition, one study found a de-crease in the serum VEGF concentrations after sur-gical treatment in 77% of patients.46 Unfortunatelyneither study reported the number of normal con-trol patients that exceeded the upper limit ofserum VEGF concentrations, therefore it is difficultto conclude that VEGF levels are consistentlyelevated in patients with oral squamous cellcarcinoma.

VEGF expression by oral squamous cell carcino-mas have also been studied as a prognostic indica-tor. The overall survival rates of VEGF positive oralcarcinomas are significantly poorer,41,42,44,48 andpatients with VEGF positive tumours are threetimes more likely to die when compared to patientswith VEGF negative tumours.41 Several studies havealso found that VEGF expression was a more signif-icant predictor of overall survival outcome thanstandard clinicopathologic predictors such as TNMstage.41,48 In addition, VEGF positive tumours havealso correlated with tumour recurrence,41 althoughothers have found only a borderline significance.48

Conclusion

VEGF status has the potential to emerge as animportant factor in the diagnosis, prognosis andtreatment of oral dysplasia and squamous cell car-cinoma. However the contribution of VEGF to dys-plastic or cancerous lesions is not clear due toconflicting results within the literature. It has beensuggested that the expression of VEGF isoformswithin the tissues, together with cross reactivityof antibodies may account for the varying re-sults.6,36 In addition, the quantification of VEGFexpression methods is often subject to observervariability or experimental procedures used.6 Theoral cavity is also highly vascular and it is thereforedifficult to determine new tumour-dependant angi-ogenesis from vessels that may have formed fromnormal oral endothelial proliferation. Future re-search would therefore benefit from a standardisa-tion of the methodology used by researchers. Inparticular, consistency with the antibody used todetect VEGF and methods used for measuring new

angiogenesis would allow for more comparable re-sults. The determination of VEGF isoform profileswithin oral tissues is also necessary to improveour understanding of the role of VEGF.

Future therapies targeted at the molecularmechanism of angiogenesis, especially those thatinteract with VEGF and its receptors or antibodiesagainst VEGF and its receptors may prove usefulin the management of oral cancers. However agreater understanding of the role of VEGF in oralcancer is required before VEGF technology can beutilised to improve treatment.

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