Induction of apoptosis and cell cycle arrest by NS398 in oral squamous cell carcinoma cells via downregulation of E2 promoter-binding factor-1

Overexpression of cyclooxygenase-2 (COX-2) plays an important role in development and progression of different human cancers, but the underlying molecular mechanisms remain to be defined. Tissue specimens of normal oral epithelia (n=9), dysplasia (n=38), and oral squamous cell carcinoma (SCC, n=54) were immunohistochemically analyzed for COX-2 and E2F-1 expression. A human oral SCC cell line, Tca8113, was used to assess NS398 antitumor activity. PGE2 levels were measured by using radioimmunoassay, and COX-2, pRb, and E2F-1 proteins were determined by Western blot assay. We found expression of COX-2 and E2F-1 proteins was significantly increased in both oral dysplasia and SCC compared to the normal epithelium. Increased COX-2 expression was associated with E2F-1 expression in both oral dysplasia and SCC. NS398 treatment reduced viability of Tca8113 cells in a dose- and time-dependent manner. NS398 suppressed PGE2 levels, a product of COX-2 enzyme, in the tumor cells. The reduced cell viability is due to induction of apoptosis by NS398, which activates caspase-3, but does not inhibit bcl-2. NS398 also induced tumor cell arrest at G1 phase of the cell cycle and inhibited expression of COX-2, pRb and E2F-1 proteins. This study provides evidence that E2F-1 and COX-2 are overexpressed in oral cancer, and further supports suppression of COX-2 in control of oral cancer.     

p120(cat) Delocalization in cell lines of oral cancer.

p120(cat) is a novel component of the catenin family, a cytoplasmic molecule closely associated with the cell-cell adhesion molecule E (epithelial)-cadherin, by forming complexes between the cytoplasmic domain of E-cadherin and the cytoskeleton. Recent studies suppose a role for this molecule in human cancers and to date none report its expression in oral squamous cell carcinomas (SCCs). The goal of this study was to evaluate the role of this protein in the oral carcinogenetic process. A linked streptavidin-biotin-alkaline phosphatase technique was used to examine the immunoreactivity and cellular localisation of p120(cat) in five oral epithelial cell lines (NCTC 2544, normal and immortalized keratinocytes; KB, a poorly differentiated SCC cell line; OSC 20, a well differentiated oral SCC cell line; CAL 33 and CAL 27, moderately differentiated oral SCC cell lines) and 10 normal oral epithelium biopsies. RESULTS: As already reported for E-cadherin, beta- and gamma-catenin, p120 expression showed a homogeneous membranous localization in normal oral specimens. The intensity of staining for p120 progressively increased from basal and parabasal layers toward the intermediate spinous layer. No staining for p120 was observed in the upper layer. NCTC showed a membranous positivity. OSC 20, CAL 33 and CAL 27 showed a membranous positivity, even if polarized to cell-cell adhesion sites, in 40-50% of cells. OSC 20, CAL 33 and CAL 27 cells showed also a cytoplasmic delocalization. All positive KB cells showed a prevalent cytoplasmic staining and 10% of these cells showed a nuclear delocalization. In cancer cells, p120 showed an inverse relationship with the degree of differentiation for a progressive displacement of the signal toward the cytoplasm or nucleus in dedifferentiated cells. In conclusions, this nuclear delocalization for p120 could suppose its potential involvement in signalling and cancer transformation.     

Expression of RARalpha and RARbeta in human oral potentially malignant and neoplastic lesions

Retinoids reverse potentially malignant lesions and inhibit the development of second primary cancers in patients with head-and-neck cancer. Many of the effects of retinoids result from modulation of gene expression by 2 distinct classes of nuclear receptor, RARs and RXRs; alterations in their expression can lead to tumorigenesis. To determine whether aberrations in expression of the receptors are related to the development of betel- and tobacco-related oral cancer, we used specific monoclonal antibodies against RARalpha and RARbeta to detect expression of these proteins in 30 histopathologically normal tissues, 45 potentially malignant lesions (leukoplakia) with histological evidence of either hyperplasia (31 cases) or dysplasia (14 cases) and 64 oral squamous-cell carcinomas (SCCs) by immunohistochemistry. Of the 30 normal oral tissues analysed, 8 cases showed detectable levels of RARalpha protein, while 10 cases did not show detectable RARbeta immunoreactivity. Immunostaining for RARalpha protein was observed in 12/31 (39%) hyperplastic lesions, 6/14 (43%) dysplastic lesions and 43/64 (67%) oral SCCs. Expression of RARalpha in oral SCC was significantly associated with the histological differentiation status of tumours (p = 0.016). In contrast, lack of detectable immunoreactivity was observed in 19/31 (61%) hyperplastic lesions, 8/14 (57%) dysplastic lesions and 21/64 (33%) oral SCCs. The hallmark of the study was the significant increase in RARalpha immunopositivity in oral SCCs compared to normal tissue (p = 0.0005) and hyperplastic lesions (p = 0.016). One intriguing feature was the significant decrease in RARbeta immunopositivity in hyperplastic lesions compared with normal oral mucosa (p = 0.05) as well as in oral SCCs compared with normal tissues (p = 0.0008).     

5-Fluorouracil-induced apoptosis in cultured oral cancer cells

Chemotherapy is commonly used to treat advanced oral squamous cell carcinoma (SCC) and is known to kill cancer cells through apoptosis. Our hypothesis states that 5-fluorouracil (5FU) also kills cultured oral epithelial cells through programmed cell death or apoptosis. Cultured oral cancer cells were exposed to an optimum dose of 20 mg/ml of 5FU. Cells were analyzed for changes in cell cycle distribution and induction of cell death including apoptosis. Normal control, human papilloma virus-immortalized (PP), ATCC SCC cell line (CA1) and two primary oral SCC cell lines (CA3 and -4) were studied. Inhibition of apoptosis by a pan-caspase inhibitor was used. SYTO 11 flow cytometry showed increased apoptosis in all 5FU-treated cell cultures compared to untreated controls. The results show biological variation in apoptotic response. CA1 had the lowest apoptotic rate of the cancer cell lines at 1.5%. Next lowest was CA3, followed by CA4 and PP. In addition, alteration in the G1 and S phase fractions were found. Untreated CA1 showed 28% G1, 53% S compared to 43% G1, and 40% S of treated. We investigated the pathway of apoptosis using the pan-caspase inhibitor IDN-1529 by methylthiazolyl diphenyl tetrazolium bromide (MTT) colorimetric analysis. Results showed mild inhibition of cell death when cells were incubated with 50 microM IDN-1529 for 24 h. This suggests a probable caspase-dependent apoptotic pathway. In conclusion, our data suggest that 5FU induces oral cancer cell death through apoptosis and that biological variation exists between normal and cancer cells and between different types of cancer cells themselves. Our data indicate that cultures of a useful in vitro model for chemosensitivity assays are possible. Our results also suggest a caspase-dependent pathway for chemocytotoxicity in oral SCC.     

Allelic losses in OraTest-directed biopsies of patients with prior upper aerodigestive tract malignancy.

Genetic alterations at critical chromosome loci have been shown to be predictors of the progression of oral premalignancy-to-invasive cancer. We obtained a unique group of oral biopsies, initially collected during a prospective study designed to test the ability of OraTest (toluidine blue), to identify recurrent oral neoplastic lesions in patients with definite therapy for head and neck or upper aerodigestive tract (UADT) cancer. A total of 46 cases, including 13 squamous cell carcinoma (SCC), 11 carcinoma-in situ or dysplasia, and 22 morphologically normal oral biopsies, were analyzed for loss of heterozygosity (LOH) at 9p21, 3p21, and 17p13(TP53) by microsatellite analysis. LOH at one or more tested markers in at least one biopsy was detected in 76% (35 of 46) cases. All of the SCC and carcinoma-in situ cases showed LOH, and, strikingly, more than one-half (69%, 13 of 22) of morphologically normal epithelia also harbored LOH in at least one tested marker. The most frequent LOH was found on chromosome 9p21 (69%, 31 of 45). LOH was observed at 3p21, 17p13(TP53), or in multiple chromosomal arms significantly more often in SCC than in normal epithelia. In the majority of cases, two oral biopsies, one from an OraTest-staining positive area and another from a negative area adjacent to the stain, were collected. Among 25 LOH positive cases with two biopsies, identical allelic losses were confirmed between stained and nonstained biopsies in 16 cases. In the remaining nine cases with discordant LOH patterns between two biopsies, eight cases showed LOH at more genetic loci in OraTest-stained areas. Our data confirm that clonal genetic alterations, especially 9p21 deletion, are often present in the oral epithelia of patients with previous UADT malignancy and, combined with previous studies, suggest that genetic analysis will help stratify patients at risk of developing a secondary oral cancer. In addition to detecting cancer, our study suggests that OraTest can detect clinically occult lesions in the progression pathway to oral cancer.     

Histone deacetylase 2 expression predicts poorer prognosis in oral cancer patients

Histone deacetylase 2 (HDAC2) has been implicated in the development and progression of several human tumors. We immunohistochemically examined the expression of HDAC2 protein in 20 cases of oral epithelial dysplasia (OED) and 93 cases of oral squamous cell carcinoma (OSCC). Positive HDAC2 nuclear staining was observed in 80 of the 93 (86.02%) cases of SCC and 11 of the 20 (55%) cases of ED. The labeling index (LI) for HDAC2 nuclear staining increased significantly from ED (25.8+/-26.5%) to SCCs (59.8+/-28.5%) (p<0.001). No significant correlation was found between the HDAC2 expression level and patient's age, sex, oral habits in oral SCC patients. However, cancer with advanced stage, larger tumor size, or positive lymph node metastasis had higher level of HDAC2 protein expression. Kaplan-Meier curves showed oral SCC patients with high HDAC2 expression (LI>50%), advanced stage, larger tumor size, or positive lymph node metastasis had significantly shorter overall survival (p=0.0158, 0.0267, 0.0029 and 0.02514, respectively by log-rank test) than others. The results of this study show for the first time that overexpression of the HDAC protein is a frequent event in oral cancer and could be used as a prognostic factor in oral SCC.     

Survivin expression predicts poorer prognosis in patients with areca quid chewing-related oral squamous cell carcinoma in Taiwan

Survivin, a recently characterized novel member of the inhibitor of apoptosis (IAP) family, is not detectable in most differentiated normal adult tissues but is expressed in a wide range of cancer tissues. Its expression in cancer has been correlated with poor prognosis, cancer progression and drug resistance. We immunohistochemically examined the expression of survivin in 62 cases of oral epithelial dysplasia (ED) and 96 cases of oral squamous cell carcinoma (SCC). Cytoplasmic survivin staining was detected in 60 of the 62 (97%) ED specimens and 94 of the 96 (98%) SCC specimens but not in adjacent normal oral mucosal tissues. The labeling index (LI) for survivin protein significantly increased from ED (32.3+/-16.3%) to SCC samples (49.4+/-28.5%) (p<0.001). In addition, the mean LI for ED cases with further malignant transformation into SCC (45.6+/-8.8%) was higher than those without malignant transformation (30.1+/-16.3%) (p=0.008). No significant correlation was found between the survivin expression and the patients' age, sex, oral habit, cancer location, or STNM status in SCC cases. Kaplan-Meier curves showed oral SCC patients with high survivin expression (LI>25%), advanced stage, larger tumor size, or positive lymph node metastasis had significantly shorter overall survival (p=0.014, 0.012, 0.005 and 0.011, respectively by log-rank test) than others. The associations remained significant after adjusting for age. These results indicate that survivin protein expression may be an important early event in oral carcinogenesis and predicts unfavorable prognosis for oral SCC. Furthermore, the unique expression of survivin in cancer cells but not in most normal adult tissues suggests that modulation of survivin protein expression may provide a novel strategy for the therapy of oral SCC.     

Hypermethylation of the tumor suppressor gene RASSFIA and frequent concomitant loss of heterozygosity at 3p21 in cervical cancers

Loss of heterozygosity (LOH) at chromosome 3p21 is frequent in cervical cancers. The candidate tumor suppressor gene, RASSF1A located at 3p21.3, is found to be inactivated in several major human cancers, implicating its significance in carcinogenesis. We aimed to investigate the status of RASSF1A in cervical cancers. The mutation and methylation status of RASSF1A were analysed in 4 cervical cancer cell lines, 50 primary cervical cancers including 33 squamous cell carcinoma (SCC), 17 adenocarcinoma (AC) and 11 normal controls. The primary cancer samples were also detected for LOH at 3p21 and human papillomavirus (HPV). Hypermethylation of RASSF1A was detected in 30% of SCC, 12% of AC and in 1 of the 4 cancer cell lines but was absent in all normal cases. Methylation of the cancer cell line was associated with loss of gene expression, which was restored by demethylation. About 67% (8 of 12) of hypermethylated primary cancers showed concomitant LOH at 3p21. No somatic mutation was found in all primary cancer samples or cell lines but 2 cases showed germline polymorphism at codon 133. Oncogenic HPV DNAs were found in most cancer samples. No correlation was detected between RASSF1A-hypermethylation or LOH at 3p21 and age of patient, HPV genotype, tumor grade and stage. Hypermethylation of RASSF1A occurs in a subset of cervical cancers, among which concomitant LOH at 3p21 is common. The results supported that RASSF1A may be one of the cervical cancer-related tumor suppressor genes located at 3p21 regions.     

Simultaneous expression of furin and vascular endothelial growth factor in human oral tongue squamous cell carcinoma progression.

PURPOSE: Squamous cell carcinoma (SCC) of the tongue is a common malignancy of the oral cavity. Furin convertase activates several precursor matrix metalloproteinases involved in the degradation of the extracellular matrix. The pattern of expression of furin and vascular endothelial growth factor-C (VEGF-C), two key molecules in neoplasm development, was examined during the progression from normal epithelium to invasive SCC. EXPERIMENTAL DESIGN: We evaluated furin and VEGF-C expression and microvessel density (MVD) by immunohistochemistry in human tongue sections harboring normal epithelium, dysplastic epithelium, and/or SCC. Sections from 46 glossectomy specimens were assessed for furin expression. A selected group of 15 cases, each containing normal epithelium, precursor lesions, and invasive SCC, were further studied for furin and VEGF-C expression and MVD quantification. We also evaluated the pattern of furin expression and VEGF-C processing by Western blot analysis in three SCC cell lines with different degrees of aggressiveness. RESULTS: Furin and VEGF-C expression was notably higher in most precursor lesions and SCCs than in normal epithelia. Approximately 60% (n = 26) and 100% (n = 15) of the normal epithelia showed low-intensity staining for furin and VEGF-C, respectively. Intense staining for furin and VEGF-C was detected in approximately 80% (n = 34) and 100% (n = 15) of the SCCs, respectively. A significant correlation was seen between the expression of these two markers (Spearman's test, P < 0.00002). We found a statistically significant increase in MVD when either dysplasia (432 +/- 19.06; P < 0.05) or SCC (546 +/- 17.24) was compared with normal epithelium (315 +/- 17.27; P < 0.0001). SCC71, the most aggressive cell line analyzed, was the one with the highest furin expression. This cell line totally processed the VEGF-C proform, whereas the less aggressive line SCC9, exhibiting the least furin expression, did not. SCC15, of intermediate aggressiveness and furin expression, showed intermediate pro-VEGF-C processing. CONCLUSIONS: These findings suggest that furin is a useful marker of tumor progression and is responsible for VEGF-C processing. This in turn would enhance angiogenesis, leading to increased MVD associated with preinvasive and invasive neoplasia.     

Expression of bcl-2 in oral squamous cell carcinoma: an immunohistochemical study of 90 cases with clinico-pathological correlations

Apoptosis is a genetically determined process playing an active role in tissue size regulation, morphogenesis and removing damaged cells that could be potentially dangerous for their host. Several agents involved in apoptosis regulation, such as the bcl-2 family components, act as oncogenes and are involved in oral carcinogenesis. Aim of this study is to explore bcl-2 immunoreactivity in oral cancers and to assess its potential clinico-pathological implications. Ninety oral squamous cell carcinoma and 10 normal mucosal formalin-fixed, paraffin-embedded samples were analysed for bcl-2 expression by immunohistochemistry. Normal oral mucosa showed a cytoplasmic pattern of bcl-2 immunoreactivity in the basal cell layers. Seventy-four cases of carcinoma (83%) showed no immunoreactivity, at variance with 16 cases (17%) manifesting consistent cytoplasmic positivity. Overall, the peripheral cells of differentiating epithelial tumour islands were intensely stained, with decreasing immunoreactivity toward the centre of the neoplastic nests. Fully keratinised tumour cells showed inconspicuous or absent bcl-2 immunoreactivity. No statistically significant correlations could be demonstrated between bcl-2 immunoreactivity and the sex of the patients, tumour size and with the occurrence of lymph node metastases. Though a direct correlation was found between bcl-2 immunoreactivity and increasing tumour stage, this did not reach statistical significance. Furthermore, G1 and G3 tumours displayed higher percentages of bcl-2-positive cells in comparison with G2 neoplasms and the different distribution of bcl-2 immunoreactivity in G2 and G3 was statistically significant (p<0.05). Finally, patients with absent or low (scores 0 and 1) bcl-2 immunoreactive tumours manifested poorer overall survival rates in comparison with patients with moderate or high (scores 2 and 3) bcl-2 immunoreactive tumours but the difference was not statistically significant. In normal oral mucosa bcl-2 protein is selectively present in the basal cell layers and possibly participates in the control of the terminal keratinocytes differentiation. The study of bcl-2 immunoreactivity possibly may be useful for better characterising and predicting the prognosis of oral SCC but cooperative studies are needed to assess its applications in the clinical practice.     

Reduction of p12DOC-1 expression is a negative prognostic indicator in patients with surgically resected oral squamous cell carcinoma.

PURPOSE: p12DOC-1 is a growth suppressor that negatively regulates cyclin-dependent kinase 2 (CDK2) activities. Expression of p12DOC-1 is reduced and/or lost in tumor tissues. The purpose of this study is to correlate in vivo the expression of p12DOC-1 in oral cancer tissues by immunohistochemistry with clinical and pathological parameters. EXPERIMENTAL DESIGN: Twenty-five cases of normal oral mucosa and 127 cases of oral squamous cell carcinomas were evaluated. Patients' charts were reviewed for clinical, pathological, and 10-year survival data. Because p12DOC-1 is a growth suppressor and associates with CDK2, parallel immunostaining was done for proliferating cell nuclear antigen and CDK2 to evaluate cell proliferation and potential correlation with CDK2. RESULTS: Our results showed that strong p12DOC-1 staining was uniformly seen in normal oral mucosa. p12DOC-1 staining was reduced or absent in 81 cases (63.8%) of oral squamous cell carcinomas. Decreased p12DOC-1 staining (<25% of cells stained) correlated with tumor mode of invasion (P = 0.001) and higher proliferating cell nuclear antigen (P = 0.0028) and CDK2 (P = 0.0020) expression. Survival analysis showed significant correlation of low p12DOC-1 expression with the risk of cervical lymph node metastasis (P = 0.001) and patients' 10-year survival status (P = 0.0214). CONCLUSIONS: These results allow us to conclude that reduction of p12DOC-1 protein expression is a frequent event in oral cancers. Intratumor immunohistochemical evaluation of p12DOC-1 expression can be an adjunctive prognostic indicator for patients with oral cancer.     

Anti-epidermal growth factor receptor monoclonal antibody 225 upregulates p27(KIP1) and p15(INK4B) and induces G1 arrest in oral squamous carcinoma cell lines

Epidermal growth factor receptor (EGFR) regulates the growth and progression of human oral squamous cell carcinoma (SCC). Recently, the link between EGFR signaling and the cell cycle has been identified. Some reports have described that EGFR-blocking monoclonal antibody 225 (mAb225) induced G1 arrest and inhibited the growth of various cancer cells. The purpose of this study was to evaluate the effect of mAb225 on human oral SCC cell lines. Exposure to mAb225 in culture inhibited the growth of oral SCC cell lines in an EGFR number-independent manner, with the percent inhibition ranging from 13.8 to 76.6%. Flow-cytometric analysis demonstrated that treatment with mAb225 induced cell accumulation in G1 phase, accompanied by a decrease in the percentage of cells in the S phase. Apoptosis was not seen in this study. G1 arrest was accompanied by a decrease in CDK2-, CDK4-, and CDK6-associated histone H1 kinase activities, and an increase in the expression levels of cell cycle inhibitors p27(KIP1) and p15(INK4B). These results suggested that the antiproliferative effect of EGFR blockade by mAb225 in oral SCC may be mediated by p27(KIP1) and p15(INK4B).     

Expression of cyclin A is related to progression of oral squamous cell carcinoma in Taiwan

Cyclin A is required for DNA synthesis during the S phase and progression through the G2/M transition. Increased expression of cyclin A protein has been correlated with poor prognosis in a variety of human tumors. To investigate the possible influence(s) of cyclin A protein on the progression and prognosis of oral squamous cell carcinomas (SCCs) in Taiwan. We examined the expression of cyclin A in oral SCC, epithelial dysplasia (ED) and normal oral mucosa (NOM) by immunohistochemistry using antibodies to cyclin A. Results and Conclusions. The mean labeling indices (LI) in NOM, ED and SCCs were 7.0+/-3.1%, 12.1+/-3.9% and 21.3+/-12.3%, respectively. The cyclin A LI for oral SCCs was significantly higher than that for NOM (P=0.002) or ED (P<0.001). In addition, a high LI for cyclin A was found to correlate with advanced stage (P=0.0048), larger tumor size (P=0.0017), lymph node involvement (P=0.0006) and cancer recurrence (P<0.0001). The Kaplan-Meier analysis showed patients with tumors containing more than 15% cyclin A-positive cells had significantly shorter overall survival than those with tumors containing less than 15% cyclin A-positive cells (P<0.00001). These results indicate that overexpression of cyclin A protein is associated with tumor progression and patient prognosis for oral SCC.     

Differential response of normal, premalignant and malignant human oral epithelial cells to growth inhibition by chemopreventive agents

Squamous cell carcinoma (SCC) of the oral cavity is a multistep process, progressing through a series of discrete, irreversible and complementary alterations in genes that control cell growth, death, and differentiation. In the premalignant state, the oral mucosa progresses through various grades of epithelial dysplasia, with the potential to convert to SCC. Chemopreventive strategies are designed to suppress, reverse, or prevent the formation of premalignant lesions and their subsequent progression to SCC. In the present study, we determined the growth inhibitory effect of 21 chemopreventive agents in a cell culture model using normal, premalignant, and malignant human oral mucosal cell lines. There were significant differences in the growth inhibitory responses of these cell lines to selected retinoids and non-retinoid analogs. Among the retinoids tested, the synthetic retinamides, as a class, showed selective growth inhibition of both premalignant and malignant cells compared to normal human oral epithelial cells in culture. Within the retinamide class, 2CPR exhibited the greatest selectivity in the growth inhibition of premalignant and malignant cells. Among the non-retinoids analyzed, DFMO was a moderate to potent inhibitor of malignant and premalignant oral cell growth, respectively, and stimulated normal oral cell growth at low concentrations. Using this in vitro approach, we have identified several potential chemopreventive agents for oral cancer as selective growth inhibitors of premalignant ahd malignant human oral mucosa cells.     

Prognostic role of p27(Kip1) expression in oral squamous cell carcinoma in Taiwan

The cyclin-dependent kinase inhibitor p27(Kip1) can inhibit the G1 to S transition of the cell cycle and is a putative tumor suppressor. Decreased expression of p27(Kip1) protein has been correlated with poor prognosis in a variety of human tumors. We examined the expression of p27(Kip1) in oral squamous cell carcinoma (SCC), epithelial dysplasia (ED) and normal oral mucosa (NOM) using antibodies to p27(Kip1). Positive p27(Kip1) nuclear staining was detected in all the specimems from ED and NOM, whereas positive p27(Kip1) staining was observed in 16 of the 63 (25%) cases of oral SCC. The labeling index for p27(Kip1) protein was significantly reduced from NOM through ED to oral SCCs, indicating that changes of p27(Kip1) protein expression may be an early event in oral carcinogenesis in Taiwan. The Kaplan-Meier analysis showed patients with p27(Kip1)-positive tumors had significantly higher overall survival than those with p27(Kip1)-negative tumors in a total of 63 patients (P=0.015) and 47 patients with areca quid chewing habit (P=0.026). Multivariate analysis showed decreased p27(Kip1) protein expression was an independent significant predictor of poor overall survival in the patients with oral SCCs. These results indicate that p27(Kip1) protein expression may serve as a putative new adjuvant prognostic marker for routine assessment of oral SCC patients.     

Immunohistochemical expression of basic fibroblast growth factor and fibroblast growth factor receptors 1 and 2 in the pathogenesis of lung cancer

PURPOSE: To identify the patterns of protein expression of basic fibroblast growth factor (bFGF) and FGF receptors 1 and 2 in non-small cell lung carcinoma (NSCLC) and their role in the early pathogenesis of squamous cell carcinoma (SCC) of the lung. EXPERIMENTAL DESIGN: Archived tissue from NSCLC (adenocarcinoma and SCC; n = 321) and adjacent bronchial epithelial specimens (n = 426) were analyzed for the immunohistochemical expression of bFGF, FGFR1, and FGFR2, and the findings were correlated with clinicopathologic features of the patients. RESULTS: High expression of bFGF, FGFR1, and FGFR2 was shown in most NSCLC tumors. The pattern of expression for all markers varied according to tumor histologic type and cellular localization. Cytoplasmic expression scores were significantly higher in tumors than in normal epithelia. Nuclear bFGF (P = 0.03) and FGFR1 (P = 0.02) levels were significantly higher in women than in men. Although cytoplasmic FGFR1 expression was significantly higher (P = 0.002) in ever smokers than in never smokers, nuclear FGFR1 (P = 0.0001) and FGFR2 (P = 0.003) expression was significantly higher in never smokers. Different prognostic patterns for the expression of these markers were detected for both NSCLC histologic types. Dysplastic changes showed significantly higher expression of all markers compared with squamous metaplasia. CONCLUSIONS: bFGF, FGFR1, and FGFR2 are frequently overexpressed in SCC and adenocarcinoma of the lung. bFGF signaling pathway activation may be an early phenomenon in the pathogenesis of SCC and thus an attractive novel target for lung cancer chemopreventive and therapeutic strategies.