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Stromelysin 3, Ets-1, and Vascular Endothelial Growth Factor Expression in Oral Precancerous and Cancerous Lesions: Correlation with Microvessel Density, Progression, and Prognosis

Departments of ¹ Biochemistry, ² Pathology, ³ ENT and ⁴ Surgical Oncology, Institute Rotary Cancer Hospital, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, India

Requests for reprints: Ranju Ralhan, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, 110029, India. Phone: 91-11-26593478; Fax: 91-11-26588663; E-mail: ralhanr{at}hotmail.com.

	ABSTRACT

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Purpose: Identification of molecular changes characteristic of development and progression of oral cancer are of paramount importance for effective intervention. Stromelysin 3 (MMP11) is a unique matrix metalloproteinase shown to have dual function during cancer progression. The transcription factor Ets-1 and vascular endothelial growth factor (VEGF) are important proangiogenic factors in cancer. This study was designed to test the hypothesis that concomitant expression of stromelysin 3, Ets-1, and/or VEGF affects the development, progression, and prognosis of oral cancer.

Patients and Methods: Immunohistochemical analysis of stromelysin 3, Ets-1, VEGF, and platelet/endothelial cell adhesion molecule 1 (a marker for intratumoral microvessel density) was carried out in serial paraffin embedded tissue sections of 220 oral squamous cell carcinomas (OSCC), 90 precancerous lesions (59 hyperplasias and 31 dysplasias), and 81 matched histologically normal oral tissues.

Results: Ets-1, VEGF, and stromelysin 3 expression independently correlated with increased intratumoral microvessel density in precancerous lesions (P = 0.05, 0.001, and 0.026, respectively) as well as in SCCs (P = 0.005, 0.01, and 0.031, respectively). Logistic regression analysis revealed that concomitant expression of stromelysin 3 and Ets-1 (stromelysin 3⁺/ Ets-1⁺ phenotype; odds ratio, 3.7; P = 0.001) was the most significant predictor for transition to precancerous stage, whereas dual expression of stromelysin 3 and VEGF (stromelysin 3⁺/ VEGF⁺ phenotype; odds ratio, 2.07; P = 0.004) was the most important predictor for progression from precancerous stage to frank malignancy. Intriguingly, Ets-1 expression was significantly associated with VEGF expression and stromelysin 3 expression in precancerous tissues as well as OSCCs. Follow-up data for 144 patients for a maximum period of 115 months showed that VEGF [hazards ratio (HR), 4.532; P = 0.004] and Ets-1 (HR = 2.182; P = 0.049) expression significantly correlated with reduced disease-free survival in univariate analysis. In bivariate analysis, patients harboring Ets-1⁺/VEGF⁺ phenotype had the worst survival (median disease-free survival, 50 months; HR, 2.943; P = 0.003). Multivariate analysis using Cox's proportional hazards model showed that increased VEGF expression was the most significant adverse prognosticator in OSCC patients (HR, 4.470; P = 0.004).

Conclusions: In conclusion, this study provides the first evidence of concomitant expression of stromelysin 3, VEGF, and Ets-1 in clinical specimens in different stages of development of oral cancer. In early stages, concomitant expression of stromelysin 3 and Ets-1 favors the development of a precancerous state, whereas dual expression of stromelysin 3 and VEGF is associated with progression from precancerous to cancerous state. VEGF expression is an adverse prognosticator for disease-free survival.

Key Words: angiogenesis • oral cancer • prognosis • progression • transition

	INTRODUCTION

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Head and neck tumorigenesis is a multistep process. Squamous cell carcinomas (SCC) of the oral cavity, a subgroup of head and cancer, are clinically preceded by precancerous lesions, often leukoplakia, with histologic evidence of hyperplasia or dysplasia. Only a small proportion (5-10%) of the early dysplastic lesions progress to SCC over a period of 10 years (1). Despite rapid advances in diagnosis and treatment, the prognosis of patients with oral SCC (OSCC) has remained unchanged over the last two decades (2). At present, positive pathologic neck nodes have the most decisive influence on the prognosis of oral cancer patients (3). Identification of molecular markers that can predict patients with precancerous lesions at high risk of transition to malignancy, or identify patients with biologically aggressive tumors will provide a much needed opportunity to target forceful therapy for effective disease management. Oral SCCs (OSCC) have a high propensity for invasiveness leading to regional lymph node involvement and loco-regional spread, causing severe morbidity and adversely affecting the quality of life of these patients. Elucidation of molecular alterations influencing invasion and loco-regional spread will help to understand the biology of the disease as well as aid in identifying biomarkers to predict malignant potential. Major thrust has been laid on determination of molecular events in OSCCs. However, knowledge of molecular alterations in oral precancerous lesions is meager. Hence, the present study focuses on molecular alterations in early stages of development and progression of oral cancer.

Stromelysin 3, a matrix metalloproteinase (MMP11) originally identified by its overexpression in primary breast carcinomas shows some characteristic properties (4). Unlike other MMPs, pro-stromelysin 3 is processed intracellularly by furin dependent proteolytic cleavage and is then secreted in a potentially active form, suggesting that it may have a unique role among the MMPs (5). During malignancy, the cellular function of stromelysin 3 is proposed to favor survival of cancer cells in the stromal microenvironment by decreasing cancer cell death through apoptosis and necrosis (6). Stromelysin 3 expression has been reported in lung, colorectal, ovarian, and head and neck carcinomas (7–11). In some human tumors, stromelysin 3 expression has been observed in stromal fibroblasts surrounding malignant epithelial cells and suggested to promote tumorigenesis in a paracrine manner, by promoting cancer cell implantation in the connective host tissue at the time of local invasion. Recent studies have focused on understanding the effect of stromelysin 3 on tumor progression resulting in dissemination, invasion at distance, and metastasis development. These studies showed that stromelysin 3 has a dual function during cancer progression being a tumor enhancer in early stages and repressor in processes leading to local or distal invasion (12, 13). Furthermore, stromelysin 3 overexpression has been associated with increased aggressiveness of tumors and a poor clinical outcome (14–16). We have recently reported that stromelysin 3 expression is an early event in oral tumorigenesis (17). The expression of stromelysin 3 in oral hyperplastic and dysplastic lesions suggested its association with progression of phenotypic alterations acquired early during the malignant transformation pathway of oral epithelium.

The growth of solid tumors is dependent on the process of angiogenesis (18). Therefore, solid tumors must produce angiogenic factors at an early point in tumor development (19). Vascular endothelial growth factor (VEGF) is an important proangiogenic molecule, which plays a crucial role in tumor angiogenesis, by increasing blood vessel permeability, endothelial cell growth, proliferation, migration, and differentiation (20). It may also facilitate extravasation of tumor cells and thereby the formation of metastases by degrading the tumor marginal extracellular matrix via activation of proteolytic enzymes. High tumor expression of VEGF protein has been linked to poor clinical outcome in several human tumor sites including stomach, ovary, esophagus, breast, colorectum, lung, and bone (21).

The transcription factor Ets-1 is induced in endothelial cells by angiogenic factors, VEGF, and basic fibroblast growth factor. Ets-1 promotes neovascularization by inducing angiogenesis-related genes such as MMPs and Integrin ß3 (22, 23). Ets-1 expression has been reported in a number of human tumors, including oral carcinomas, astrocytomas, gastric carcinomas, and mammary carcinomas (24–27). Strikingly, Ets-1 antisense oligonucleotides abrogate the invading phenotype and VEGF-induced migration of endothelial cells in vitro (28). The gene for the endothelial cell–specific Flt1/VEGFR1 also contains an Ets-1-responsive element in its promoter, and mutational disruption of this Ets-1 binding site results in a decrease of VEGFR1 transcription (29). Ets factors have also been shown to interact and cooperate with other transcription factors in the activation of promoters of genes encoding extracellular matrix proteases (30, 31). Ets-1 overexpresssion promotes endothelial cell invasiveness and expression of matrix proteases as well as integrin ß3; conferring an angiogenic phenotype to these cells (32). Therefore, we hypothesized that stromelysin 3 may exert its dual effects during cancer development and progression by its association with angiogenic factors VEGF and Ets-1. To address this hypothesis, it was important to determine the relationship between stromelysin 3 and angiogenic factors VEGF and Ets-1 transcription factor in oral tumorigenesis. Thus, the aim of the present study was to determine the clinical significance of concomitant expression of stromelysin 3, VEGF, Ets-1, and tumor vascularity (intratumoral microvessel density) in early stages of oral tumor development, disease progression, and prognosis. The availability of expression status of all four markers in the same set of patients provided a unique opportunity to determine whether alterations in stromelysin 3, VEGF, Ets-1, and intratumoral microvessel density exert a cooperative effect on oral cancer development and prognosis.

	MATERIALS AND METHODS

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Tissue Specimens. This study was approved by the Human Ethics Committee of All India Institute of Medical Sciences, New Delhi, India. Surgically resected tissue specimens from oral cancerous lesions and biopsy samples from precancerous tissues and adjacent normal oral tissues were obtained from Institute Rotary Cancer Hospital, All India Institute of Medical Sciences, with prior informed written consent of the patients. Patients recruited for the study were chosen consecutively. The surgically resected tumors were collected, formalin fixed and paraffin embedded for histopathologic confirmation and immunohistochemical analysis. The diagnosis was based on clinical examination and histopathologic analysis of the tissue specimens. Before processing for Ets-1, VEGF, stromelysin 3, and platelet/endothelial cell adhesion molecule 1 (PECAM-1) immunostaining, all tissue blocks were reviewed for the presence of tumor by the pathologist (M.M). The clinical and pathologic data recorded included age, gender, clinical tumor stage, site of the lesion, histopathologic differentiation, and consumption of betel, areca nut, and tobacco.

Clinicopathologic Characteristics of Patients. Two hundred and twenty (167 males and 53 females; ages between 20 and 85 years; mean age, 52 years), diagnosed as having primary SCC of the oral cavity (during 1994-2001) were included in this study, prior to treatment. Patients recruited for the study were chosen consecutively. The diagnosis was based on clinical examination and histopathologic analysis of the tissue specimens. The clinical staging was done following the Unio Internationale Contra Cancrum (1988) staging system (33), the tumor-node-metastasis stages of these tumors varied from T₁ to T₄ (T₁, 24 cases; T₂, 81 cases; T₃, 50 cases; and T₄, 65 cases) and N₀ to N₊ (N₀, 117 cases and N₊, 103 cases). No patients with distant organ metastasis were included in the study. The tumors were histologically graded as well (167 cases), moderate (49 cases) or poorly differentiated (four cases) by the pathologist (M.M) by reviewing the H&E-stained sections without prior knowledge of the clinical diagnosis. All the patients included in this study were habitual consumers of betel and tobacco. The various subsites of oral tumors included buccal mucosa (63 cases), alveolus (32 cases), tongue (78 cases), lip (20 cases), and other sites (27 cases). The lip cancer cases have been included because in India lip cancer is etiologically related to the habit of chewing "khaini" (tobacco and slaked lime). Khaini consumers keep it in between lower labia and gingiva, slaked lime present in the mixture due to exothermic reaction and irritation causes the vasodilation thereby making the mucosa more vulnerable to the carcinogens present in tobacco. Most of the habitual tobacco consumers keep betel quid or tobacco in the side of their mouth and few have the habit of sleeping with the quid in mouth thereby constantly exposing buccal mucosa and tongue to a plethora of carcinogens. Hence, these sites are more frequent targets for development of OSCC.

Ninety patients with oral precancerous lesions (59 cases with histologically confirmed hyperplasia and 31 cases with histologic evidence of dysplasia) were enrolled in this study. The various subsites of oral precancerous lesions included: buccal mucosa (65 cases), tongue (13 cases), alveolus (3 cases), lip (5 cases), and other sites (4 cases). The tobacco, betel, and/or areca nut consumption habits of the patients with oral precancerous or cancerous lesions were correlated with molecular analysis.

Eighty-one histologically normal oral tissues were procured form the patients from an area adjacent to the site of cancer or site of precancerous lesions. The subsites analyzed were buccal mucosa (40), tongue (13), lip (10), alveolus (14), and others (retromolar trigone, palate, four cases).

Immunostaining for Angiogenic Proteins in Oral Squamous Cell Carcinomas. Paraffin-embedded tissue blocks were used to cut 4- to 5-µm-thick sections. H&E staining was done and serial sections were used for immunostaining of Ets-1, VEGF, stromelysin 3, and PECAM-1 proteins. Immunohistochemical staining was done using the avidin-biotin method as previously described (17, 34). Briefly, slides were deparaffinized in xylene, hydrated, and incubated with 0.5% (v/v) H₂O₂ in methanol for 20 minutes, to block the endogenous peroxidase activity. Slides were then washed with TBS and heated for 15 minutes at 100°C in 10 mmol/L sodium citrate buffer (pH 6.0) for antigen retrieval. Nonspecific binding was blocked by incubation with 1% bovine serum albumin for 1 hour. The primary antibodies were diluted for Ets-1 (C-20, 200 µg/mL stock; diluted 1:200), VEGF (C-1, 200 µg/mL stock; diluted 1:200), stromelysin 3 (AB-2; stock 200 µg/mL; diluted 1:100) and PECAM-1 (C-20, PECAM-1 200 µg/mL stock; diluted 1:100) in 1% bovine serum albumin and sections were incubated for 16 hours at 4°C. All the antibodies except stromelysin 3, used in this study were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Stromelysin 3 antibody was obtained from Oncogene Research Products (Darmstadt, Germany). The primary antibody was detected using secondary antibody (rabbit anti-mouse IgG for monoclonal antibodies and goat anti-rabbit IgG for polyclonal antibodies) and avidin-biotin complex by avidin-biotin complex method using LSAB+ kit (Dako Co., Glostrup, Denmark). The color was developed using diaminobenzidine as the chromogen. The slides were washed with TBS extensively after each step. Finally, the slides were counter stained with Mayer's hematoxylin and mounted with DPX mountant. Parallel sections incubated with the primary antibody, replaced by an unrelated monoclonal antibody and nonimmune mouse IgG of the same isotype, were examined to ensure specificity and exclude cross reactivities between the antibodies and conjugates used. Human OSCC cell line, HSC-2, expressing Ets-1 and stromelysin 3 and human placental tissue expressing VEGF were used as positive controls with each batch.

Positive Criteria of Immunohistochemical Staining. The intensity of immunohistochemical staining was evaluated by two of us independently (S.A. and M.M.) in five areas of the slide sections for correlation and confirmation of the tissue analysis. Positive cells were stained dark brown. Tumors were regarded as Ets-1 positive if >10% of the tumor cells showed nuclear/cytoplasmic staining (24). Tumors were regarded as VEGF positive if >.10% of the tumor cells showed cytoplasmic staining (35). Tumors were graded as stromelysin 3 immunopositive if >10% of the stromal fibroblasts were stained or >10% of the tumor cells showed perinuclear staining (17, 36). Stromelysin 3 expression in the tissue sections was evaluated using a semiquantitative scoring system: 0 for absence of immunostaining or detectable immunostaining in <10% stromal/tumor cells, +1 for 10% to 30% positive stromal/tumor cells, +2 for 31 to 60% positive, and +3 for >60% positive stromal/tumor cells. The immunostaining was evaluated in five areas of the slide sections for correlation and confirmation of the tissue analysis.

Microvessel Counting. Microvessel density was assessed in tumor areas showing the highest density of staining (hotspots) as determined by an initial scan at low magnification (x100) based on the method of Bossi et al., (37). For vessel counting, a x200 field [high-power field (i.e., x20 objective and x10 occular) 0.739 mm² per field] in each of the three vascular areas was counted, and average counts were recorded. The areas with the highest density of staining were seen predominantly at the invading edge of the tumor. Microvessel density was determined by two of us independently (S.A., M.M.) as described (17).

Follow-up Study. One hundred forty-four of the 220 OSCC patients (112 males and 32 females), ranging in age from 30 to 76 years (median age, 54 years), who underwent treatment of primary OSCC between 1995 and 1999 could be followed, whereas 76 patients were lost to follow-up. Most of these patients were referred to All India Institute of Medical Sciences from different parts of the country. After completion of the treatment, some patients returned to their native place and were lost to follow-up. Survival status of patients was verified and regularly updated from Tumor Registry records, Institute Rotary Cancer Hospital, as of August 2004. Patients were monitored for a period of median 16.5 months and a maximum of 115 months. As per our protocol, OSCC patients with T₁ and T₂ tumors were treated with radical radiotherapy or surgery alone, whereas majority of patients with T₃ and T₄ disease were treated using a combination of radical surgery followed by postoperative radical radiotherapy. Of the 144 OSCC patients included in the prognostic factor analysis, 61 (42%) patients were given radical radiotherapy, 26 (18%) patients were treated by surgery alone, 51 (36%) cases were treated using a combination of radical surgery followed by radical radiotherapy (surgery + radical radiotherapy), and patients with locally advanced inoperable disease were taken up for neoadjuvant chemotherapy (computerized tomography) and the good responders were given radical radiotherapy [computerized tomography + radical radiotherapy, 6 (4%)]. All patients undergoing surgery had tumor-free margins (Ro resection). Radical surgery consisted of radical resection of the tumor with 1- to 2-cm three-dimensional tumor-free margins and an appropriate neck dissection based on nodal status. The radical radiotherapy regime comprised of a total dose of 60 to 70 Gy in 25 to 30 fractions over a period of 5 to 6 weeks. The chemotherapy regimen included two cycles of infusion of 500 mg/m² 5-fluorouracil on day 1 and 30 mg/m² cisplatin on days 1 to 4. The cycle was repeated after 3 weeks. The patients were followed up periodically and time to recurrence was recorded. A new cancer developing after >3 years of treatment of the primary tumor in a different anatomic location was considered as a second primary tumor. A local recurrence was defined as any tumor of similar histology appearing within 2 cm or 3 years of the primary tumor. Disease relapsing in the treated neck was taken as a regional relapse. No distant metastasis was observed in this study. If a patient died during the follow-up, patient survival time was censored at the time of death. Medical history, clinical examination, and radiological evaluation were used to determine whether death resulted from recurrent cancer (relapsing patients) or from any other cause. Disease-free survivors were defined as patients free from clinical and radiological evidence of local, regional, or distant relapse at the time of the last follow-up. Loco-regional relapse/death was observed in 51 of 144 (369%) patients monitored in this study. Ninety-three patients who did not show recurrence were alive till the end of the follow-up period. Among the 76 patients that were lost to follow-up, the number of deaths could not be ascertained; therefore, overall survival could not be considered as a separate variable in our study. Only disease-free survival of the patients was studied. Disease-free survival was expressed as the number of months from the date of surgery to the loco-regional relapse. These patients were in regular follow-up and most of them were staying with their families. The family members were counseled along with the patient about the ill effects of tobacco and alcohol by the health workers in Cancer clinics. Ninety-five patients undergoing treatment had stopped use of any of these tobacco products. However, three patients started smoking after the treatment. Because the number in this group is quite small, no statistically significant inference could be drawn. Consecutive tissue sections were used for immunohistochemical analysis of Ets-1, VEGF, stromelysin 3, and PECAM1 to assess the prognostic effect of these proteins. To investigate the prognostic relevance of these potential biomarkers on clinical outcome, univariate and multivariate analyses were carried out.

Statistical Analyses. The immunohistochemical data were subjected to statistical analyses using the SPSS software, version 10 (Chicago, IL). For purpose of analysis, clinicopathologic variables were evaluated as dichotomized variables. The relationships among Ets-1, VEGF, and stromelysin 3 expression and patient variables were tested in univariate analysis by Fisher's exact test, ² test, ² test for trend, and logistic regression analysis. Association between Ets-1, VEGF, and stromelysin 3 expression and intratumoral microvessel density was assessed by independent t test. Follow-up studies were analyzed by Kaplan-Meier, and Cox's proportional hazards test. Disease-free survival was estimated according to Kaplan-Meier method. Only disease-free survival was evaluated in the present study, as the number of deaths due to disease progression did not allow a reliable statistical analysis. The association between patient outcome and variables was assessed by log-rank test. Two-sided Ps were calculated and P 0.05 was considered significant.

Transfection. The OSCC cell line AMOS-III (38) was transfected with ets-1 expression vector, psG5-Ets1 (a kind gift from Prof. Bohdan Wasylyk, Institut de Genetioue et de Biologie Moleculaire et Cellulaire, Strasbourg, France) using calcium phosphate method. Briefly, cells were plated 2 hours before transfection. Twenty micrograms of DNA (5 µg of the DNA to be transfected and 15 µg of the control psG5 vector DNA) using transfection buffer (2x BBS: 150 mmol/L BES, 280 mmol/L NaCl, and 1.5 mmol/L Na₂HPO₄) for 14 to 16 hours. Cells were allowed to grow for additional 32 to 34 hours. Thereafter, the Western blot and reverse transcription-PCR analyses were done to determine the expression levels of Ets-1 and VEGF-C in transfectants and control untransfected cells.

Immunoblotting. Cell extracts were prepared by boiling the cell pellets of AMOS-III (control and Ets1-transfectants) in SDS-lysis buffer. Western Blotting was carried out by the method of Towbin et al. (39). Briefly, crude cell extracts (100 µg protein per lane) resolved by SDS-PAGE were transferred onto nitrocellulose membrane using the Bio-Rad mini gel electrophoresis apparatus for 1 hour in transfer buffer [25 mmol/L Tris base, 192 mmol/L glycine, and 20% methanol (pH 8.3)]. The nitrocellulose membranes were treated with blocking solution [5% nonfat milk in TBS: 20 mmol/L Tris (pH 8.0), 500 mmol/L NaCl] o/n at 4°C and probed with primary antibody to VEGF-C and Ets-1 (Santa Cruz Biotechnologies) for 2 hours at 37°C. Thereafter, the membranes were washed with TTBS (TBS containing 0.1% Tween) and probed with the secondary antibody conjugated to horseradish peroxidase for 2 hours at 37°C followed by washing with TTBS thrice. The proteins were detected by enhanced chemiluminescence method (Amersham, Buckinghamshire, United Kingdom) and autoradiographed.

Reverse Transcription-PCR Analysis. Total RNA was isolated from AMOS-III cells (control and Ets-1 transfectants, described above) using standard protocol of Chomczynski and Sacchi (40). Briefly, cells grown in culture were directly lysed in GITC solution [4 mol/L guanidinium thiocyanate, 0.1 mol/L sodium acetate (pH 5.2), and 1% ß-mercaptoethanol] containing 0.5% sarcosyl; 0.1 volume of sodium acetate (3 mol/L, pH 5.2) was added to the cell lysate and RNA was extracted with water saturated phenol. RNA was precipitated by adding equal volume of isopropanol at –20°C o/n. Total RNA was treated with DNase I (Life Technologies, Grand Island, NY) according to manufacturer's instruction. Reverse transcription of RNA from cells was done using 1 µg total RNA (DNA free), 100 pmol/l random hexamer primer, 1 mmol/L DTT, 6 mmol/L MgCl₂, 500 µmol/L of each deoxynucleoside triphosphate, and 20 units Moloney murine leukemia virus reverse transcriptase. The reaction mixture was used directly as a template for PCR at a dilution of 1:20. PCR amplification was carried out in a final volume of 25 µL containing 20 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 1.5 mmol/L MgCl₂, 200 µmol/L deoxynucleotide triphosphates, 0.5 µmol/L of each primer, 1 unit of Taq DNA polymerase (Life Technologies), and 5 µL of radical radiotherapy mix for VEGF amplification. For amplification, the specific 5' and 3' primers complementary to human nucleotide sequences of VEGF-C were used as described by Ohta et al. (41). ß-Actin was used as an internal control to ensure that equal amount of RNA was used in control and Ets-1 transfected cells.

	RESULTS

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Expression of Ets-1, Vascular Endothelial Growth Factor, Stromelysin 3, and Platelet/Endothelial Cell Adhesion Molecule-1 in Human Oral Squamous Cell Carcinomas, Precancerous Lesions, and Normal Tissues. The results of immunohistochemical analyses of Ets-1, VEGF, stromelysin 3, and microvessel density (as assessed by PECAM-1 staining) in 220 cases of OSCCs are summarized in Table 1. Figure 1A-C shows the expression of Ets-1, VEGF, and stromelysin 3 proteins, respectively, in OSCCs. Ets-1 was strongly expressed in 77% of tumors (Fig. 1A). Ets-1 expression was observed heterogeneously in OSCCs and was mainly localized either in the cytoplasm or in both cytoplasm and nuclei of tumor cells. Ets-1 immunostaining was also observed in endothelial cells and the stromal components in the tumor tissues. Ets-1 expression in cases with nodal metastasis was significantly higher than in node negative cases (P = 0.03). VEGF expression was observed in 76% of OSCCs. VEGF expression was predominantly observed in the cytoplasm of tumor cells. The immunostaining pattern of VEGF in tumor cells was heterogenous. In some tumors, the tumor-infiltrating inflammatory cells showed strong VEGF expression. Occasional weak staining for VEGF was also observed in the capillary endothelial cells of tumors. Sixty-seven percent of OSCCs showed stromelysin 3 immunoreactivity. In these tissue sections, the stromelysin 3 immunoreactivity was localized in stromal fibroblasts surrounding the tumor islands. In the stroma near invasive front of the tumor, the cells were intensely stained. Interestingly, stromelysin 3 immunostaining was also observed in the perinuclear region of the epithelial tumor cells, which had attained spindle shaped morphology (Fig. 1C). The fibroblasts in immediate juxtaposition to the tumor cells showed intense stromelysin 3 immunoreactivity. No significant correlation was observed between the expression of these proteins in OSCCs and histologic differentiation, tumor stage, habits of betel and tobacco chewing, or smoking (data not shown).

View larger version (129K): [in this window] [in a new window]   Fig. 1 Representative immunostaining for Ets-1, VEGF, stromelysin 3 (ST3), and PECAM-1 proteins. A, well-differentiated OSCC showing Ets-1 staining in the nuclei and cytoplasm of the tumor cells; stroma around the tumor tissue was also stained. B, VEGF immunostaining in well-differentiated OSCC tissue section showing VEGF immunostaining in the cytoplasm of the tumor cells. C, stromelysin 3 staining in SCC showing epithelial staining; stromal cells are also positively stained. D, hyperplastic epithelium showing nuclear and cytoplasmic Ets-1 immunoreactivity predominantly in the suprabasal layer. E, VEGF expression in dysplastic epithelium showing cytoplasmic staining of the epithelial cells; endothelial cells are also stained. F, precancerous lesion showing epithelial and stromal stromelysin 3 immunostaining. G, Ets-1 immunostaining in histologically normal oral tissue showing no detectable immunoreactivity. H, histologically normal oral tissue showing no detectable immunoreactivity for VEGF protein. I, histologically normal oral tissue showing lack of stromelysin 3 expression. Original magnification, x200.

Association of Angiogenic Factors with Microvessel Density. The expression of Ets-1, VEGF, and stromelysin 3 in OSCCs as well as precancerous lesions correlated independently with increased intratumoral microvessel density as shown in Table 3.

Association of Alterations in Stromelysin 3, Vascular Endothelial Growth Factor, and Ets-1 Expression with Transition from Normal to Precancerous Stage. To determine the independent predictors for transition from normal to precancerous phenotype, logistic regression analysis was carried out in a stepwise manner for all the variables (i.e., age, gender, site, habits, Ets-1, VEGF, stromelysin 3) and their interaction in 81 normal and 90 precancerous lesions (59 cases of hyperplasia and 31 cases of dysplasia). Of these variables, the variables, which emerged significant in univariate and multivariate analysis, are summarized in Table 4. Interaction between Ets-1 and stromelysin 3 [Ets-1⁺/stromelysin 3⁺ phenotype; odds ratio (OR), 3.701; P = 0.001] was the most significant predictor for transition of normal oral epithelium to the precancerous state followed by VEGF expression (OR, 2.970; P = 0.002).

Of the potential biomarkers analyzed, Cox's proportional univariate analysis revealed that Ets-l-positive tumors (n = 112 cases) had a shorter disease-free survival (median disease-free survival, 60 months) than Ets-1-negative tumors (n = 32, median disease-free survival, >86 months; hazards ratio [HR], 2.182; P = 0.049; Fig. 2A and Table 6). Similarly, VEGF overexpression in tumors (n = 111) was associated with poorer disease-free survival (median disease-free survival, 50 months) than VEGF negative tumors (n = 33, median disease-free survival, >98 months; HR, 4.532; P = 0.004; Fig. 2B). When analyzed in combination, the disease-free survival time was further reduced in VEGF overexpressing patients with either loss of stromelysin 3 expression (HR, 1.792; P = 0.061; Fig. 2C) or concomitant increase in Ets-1 protein expression (HR, 2.943; P = 0.003; Fig. 2D). Overexpression of VEGF and Ets-1 proteins together with reduced stromelysin 3 expression was a significant adverse prognosticator for OSCCs (HR, 2.031, P = 0.035, Fig. 2E). However, when analyzed by Cox proportion hazards model for the possible risk attributed by these markers individually or in combination, VEGF expression emerged as the most significant variables to assess the risk of early recurrence in patients harboring these alterations (HR, 4.470; P = 0.004). Tumor stage, nodal metastasis, and mode of treatment were not significant prognostic factors in this cohort of OSCC patients. Interestingly, in node-positive OSCCs, Ets-1 positivity was a significant prognostic factor compared with other OSCC patients who were either node negative or Ets-1 negative (OR, 2.134, P = 0.049). Similarly in node-positive OSCCs, VEGF positivity was a significant prognostic factor compared with other OSCC patients who were either node negative or VEGF negative (OR, 4.561, P = 0.004).

View larger version (21K): [in this window] [in a new window]   Fig. 2 Kaplan-Meier estimation of cumulative proportion of no tumor recurrence or metastasis. A, Ets-1 protein expression. The median time for disease-free survival (no recurrence/metastasis) in Ets-1-positive tumors was 60 months, whereas in Ets-1-negative cases it was >86 months (P = 0.049). B, VEGF protein expression. The median time for disease-free survival (no recurrence/metastasis) in VEGF-positive tumors was 50 months, whereas in VEGF-negative cases it was >98 months (P = 0.004). C, VEGF+/stromelysin 3 (ST3)– proteins expression. The median time for disease-free survival was 24 months. D, VEGF+/Ets-1+ proteins expression. The median time for disease-free survival was 50 months. E, VEGF+/Ets-1+/stromelysin 3– proteins expression. The median time for disease-free survival was 16 months.

View larger version (16K): [in this window] [in a new window]   Fig. 3 Kaplan-Meier estimation of cumulative proportion of no tumor recurrence or metastasis in relation to the concomitant expression of Ets-1 and VEGF proteins. Disease-free survival for Ets-1–/VEGF– phenotype was greater than Ets-1–/VEGF+ phenotype, which was in turn greater than Ets-1+/VEGF– phenotype, whereas Ets-1+/VEGF+ phenotype had the worst prognosis with a median disease-free survival of 50 months.

View larger version (17K): [in this window] [in a new window]   Fig. 4 AMOS-III cells were transfected with psG5-Ets-1 expression vector and the levels of Ets-1 and VEGF-C were determined in transfected and untransfected control cells after 48 hours by Western blot analysis. Cell extracts from Ets-1 transfectants and control untransfected cells were prepared in SDS-lysis buffer. Cells extract and concentrated culture supernatant were resolved on a 10% SDS-PAGE gel and transferred onto nitrocellulose membrane. These membranes were then probed with respective antibodies to Ets-1 or VEGF-C as described in MATERIALS AND METHODS. A, representative immunoblot for Ets-1 protein. A 45-kDa band of Ets-1 was observed in control (lane C) and in Ets-1 transfected cells (lane T). B, immunoblot for VEGF-C protein in Ets-1 transfected and control AMOS-III cells. A 52-kDa band was observed in cell extracts from Ets-1 transfected cells (lane 1) and a 45-kDa band for VEGF-C was detected in the culture supernatant of Ets-1 transfected AMOS-III cells (lane 3). No detectable protein was observed in untransfected control cells (lane 2) and their culture supernatant (lane 4).

View larger version (17K): [in this window] [in a new window]   Fig. 5 Reverse transcription-PCR analysis of VEGF-C in Ets-1 transfectants and untransfected control AMOS-III cells. RNA was isolated using GITC and the level of VEGF-C transcripts was determined by reverse transcription-PCR in transfectants and untransfected control cells after 48 hours. Lane1, 100-bp DNA ladder. Lane 2, control cells showing an amplicon of 408 bp. Lane 3, Ets-1-transfectants in addition to the 408-bp amplicon, a 541-bp amplicon corresponding to VEGF 165. Lane 4, negative control.

	DISCUSSION

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

View larger version (10K): [in this window] [in a new window]   Fig. 6 The association of alterations in expression of stromelysin 3 (ST3), VEGF, and Ets-1 proteins in different stages in oral cancer development and progression in the multistep progression model for head and neck cancer proposed by Califano et al. (42).

Although numerous angiogenic factors have been described, the ones responsible for angiogenesis in oral cancer remain to be identified. In our study, stromelysin 3, VEGF, and Ets-1 independently correlated with intratumoral microvessel density in oral precancerous lesions and SCCs, suggesting that these phenotypic alterations though acquired early during the malignant transformation pathway of oral epithelium are maintained after invasion also. An intriguing feature of this study was that stromelysin 3⁺/VEGF⁺ phenotype emerged as the most significant predictor for transition from precancerous stage to frank malignancy.

In support of these clinical findings, we observed that primary cell cultures from precancerous lesion (leukoplakia) transfected with stromelysin 3 showed increased expression of VEGF, suggesting that increase in the expression of stromelysin 3 up-regulates VEGF. We hypothesize that these factors may be associated with the switching on/off a phenotype of tumor angiogenesis in the early stage, prior to the onset of tumor formation.⁵ Based on data from experimental animal models involving chemically induced carcinogenesis and transgenic mice, tumor progression has been suggested to be dependent upon a switch from a prevascular to an angiogenic phase (19). Our data support the previous report of Folkman et al. (45), which showed that a phenotype of tumor angiogenesis was switched on in the early stage of tumor progression, suggesting that angiogenic activity first seemed in a subset of hyperplastic islets before the onset of tumor formation.

Herein we have also shown that Ets-1 transfected oral cancer cells showed increased levels of VEGF and also increased secretion of VEGF in culture supernatants, suggesting that Ets-1 up-regulates VEGF expression in oral cancer cells and its secretion into the extracellular milieu. Our immunohistochemical studies showed expression of Ets-1 in tumor cells, stroma, and endothelial cells indicating an overall increase in the Ets-1 expression in OSCCs. Taken together, the date suggest a role for Ets-1 in the up-regulation of VEGF and increased angiogenesis (intratumoral microvessel density) in oral cancer. Several in vitro studies have shown that Ets-1 is transiently expressed in endothelial cells during angiogenesis or reendothelialization (46, 47). VEGF has been shown to regulate the expression of Ets-1, which in turn regulates the expression of VEGF receptors and MMPs, thus acting at various steps in the process of angiogenic cascade (22, 48, 49). Ets-1 has been shown to induce transcription from the VEGFR-2 gene (flk-1) promoter by cooperating with hypoxia-inducible factor HIF-2 (50). A significant increase in intratumoral microvessel density was observed in VEGF-positive OSCCs compared with the tumors that did not show the expression of VEGF protein. This observation has also been shown in a recent study in HNSCCs, thereby pointing to the undisputed role of VEGF in angiogenesis (51). Thus, an increasing amount of evidence suggests that Ets-1 plays an important role in the angiogenic cascade. In support of these reports, the present study shows that the expression of VEGF and Ets-1 is correlated and together these two proteins regulate the process of angiogenesis.

High Ets-1 levels have been reported in breast, ovary, and cervix carcinoma and correlated with poor prognosis, whereas in lung, colorectal, and OSCC, its high levels have been associated with lymph node metastasis (reviewed in ref. 52). The role of Ets-1 in acquisition of an invasive behavior, angiogenesis, and tumor progression is under intense investigation. In epithelial cancers, Ets-1 has been proposed to fulfill a dual function, providing the cancer cells with nutrients and oxygen by inducing tumor vascularization and promoting tumor invasion by activating extracellular matrix–degrading proteases in the cancer and/or in stromal cells. Consequently, high levels of Ets-1 in tumors often correlate with poor prognosis (52).

The acquisition of invasive behavior may be dependent on the stimulation of MMPs, MMP-1, MMP-3, and MMP-9 by Ets-1 (52). Among the MMPs, the expression of MMP-2, MMP-9, and membrane type-1-MMP (MT1-MMP) has been widely reported in head and neck cancers, although their correlation with clinical features is still controversial (ref. 53 and references therein). Increasing evidence suggests that MMPs contribute to the formation of a microenvironment that promotes tumor growth during early stages of tumorigenesis (54). Proteolytically mediated extracellular matrix degradation is crucial for angiogenesis as well as for the detachment of malignant cells from the primary tumor and migration through the surrounding stroma (55). Two of the most widely studied MMPs in relation to angiogenesis and cancer are MMP-2 and MMP-9 (gelatinase-A and -B), which among other molecules degrade collagen IV, one of the major components of the basement membrane. MMP-2 and MMP-9 have been shown to play an important role in triggering the angiogenic switch by inducing expression of VEGF (55–57). In addition, enhanced mRNA and protein expression of both enzymes has been reported in head and neck, breast, colon, and pancreatic cancer (58). London et al. (59) showed that the MMP-9 antisense phosphorodiamidate morpholino oligomer inhibited in vitro prostate cancer cell proliferation, invasion, and in vivo angiogenesis.

Multivariate analysis revealed that VEGF was the most significant predictor of reduced disease-free survival in OSCC patients. Expression of VEGF has been correlated with increased vascularization and poor prognosis in a variety of human tumors. VEGF has also been shown an independent predictor of disease-free survival in HNSCCs (60, 61). Most of the prognostic studies in oral cancer have been done on smaller patient subgroups and hence these observations need to be conclusively shown in a large cohort of patients. The only other study which involves a larger patient cohort in HNSCCs is by Salven et al. (62), wherein the authors did not determine the correlation between VEGF immunopositivity and disease-free survival, but VEGF expression was correlated with overall survival of the patients.

Interestingly, we observed that there was a strong additive effect of these angiogenic markers on disease-free survival of OSCC patients. Tumors, which did not show VEGF and Ets-1 expression, had the most favorable disease-free survival, whereas Ets-1⁺/VEGF⁺ phenotype tumors had the shortest disease-free survival suggesting that concomitant expression of these proteins is associated with greater angiogenic and metastatic potential of oral tumors and hence poor prognosis.

Our data support several studies reporting VEGF overexpression and increased microvessel density in HNSCC to be associated with metastasis, recurrence, and poor prognosis (62–69). However, there are conflicting data in HNSCC about the correlation among tumor microvessel density, metastasis, and prognosis. Some studies do not show such correlation with clinical variables. Tae et al. (70) showed that VEGF expression is down-regulated during head and neck tumorigenesis and suggest that VEGF may play an important role in the early stage head and neck tumorigenesis, whereas other genetic factors might play a role in the latter stages. Thus, further studies are required to better understand the mechanism of VEGF regulation in head and neck tumorigenesis.

In conclusion, our results showed that expression of stromelysin 3, Ets-1, and VEGF is altered in the early stages of oral tumorigenisis associated with switching on of the angiogenic phase. Concomitant stromelysin 3 and Ets-1 expression is important in the acquisition of precancerous stage, whereas stromelysin 3 and VEGF expression may be associated with the transition from precancerous stage to frank malignancy. However, in depth studies entailing long-term follow-up of patients with oral precancerous lesions are warranted to determine if stromelysin 3⁺/Ets-1⁺ phenotype can serve as a predictor of risk of malignant transformation. VEGF expression is an adverse prognosticator in oral cancer.

	ACKNOWLEDGMENTS

 
We thank Dr. Rajvir Singh (Department of Biostatistics, All India Institute of Medical Sciences) for helping in the statistical analyses.

	FOOTNOTES

 
Grant support: University Grants Commission (India) senior research fellowship award (S. Arora).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

⁵ Kaur and Ralhan, unpublished data.

Received 3/24/04; revised 11/10/04; accepted 12/21/04.

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