Head And Neck Squamous Cell Carcinoma Tumor Growth Research Articles

Targeting STAT3 inhibits growth and enhances radiosensitivity in head and neck squamous cell carcinoma

Signal transducer and activator of transcription 3 (STAT3) has been implicated in the development and progression of various solid tumors. We examined the efficacy of STAT3 inhibition as a novel therapeutic option for head and neck squamous cell carcinoma (HNSCC). Activation and expression of STAT3 and hypoxia-inducible factor-1α (HIF-1α) in HNSCC cell lines were assessed by immunoblots. The small molecule inhibitor, Stattic, was used to target STAT3 in HNSCC cell lines. MTT assays were performed to determine the effect of STAT3 inhibition on HNSCC cell viability, while clonogenic survival assays were used to assess the ability of Stattic to sensitize HNSCC cells to radiation therapy. We also examined the effect of Stattic on tumor growth and radiosensitivity in vivo using an orthotopic xenograft model of HNSCC. Stattic effectively inhibited STAT3 activation and expression, resulting in decreased cell survival and proliferation and increased radiosensitivity. STAT3-mediated HIF-1α expression was also reduced in response to Stattic treatment. Oral administration of Stattic significantly reduced the growth of HNSCC tumors in a murine orthotopic xenograft, and analysis of tumor lysates confirmed decreased STAT3 phosphorylation. STAT3 inhibition modulates HIF-1α expression, resulting in decreased tumor growth and possible enhanced radiosensitivity in HNSCC. Our results provide support for further exploration of STAT3 as a novel molecular therapeutic target in HNSCC.

Antitumor Mechanisms of Targeting the PDK1 Pathway in Head and Neck Cancer

G-protein-coupled receptors (GPCR) activate the epidermal growth factor receptor (EGFR) and mediate EGFR-independent signaling pathways to promote the growth of a variety of cancers, including head and neck squamous cell carcinoma (HNSCC). Identification of the common signaling mechanisms involved in GPCR-induced EGFR-dependent and EGFR-independent processes will facilitate the development of more therapeutic strategies. In this study, we hypothesized that phosphoinositide-dependent kinase 1 (PDK1) contributes to GPCR-EGFR cross-talk and signaling in the absence of EGFR and suggests that inhibition of the PDK1 pathway may be effective in the treatment of HNSCC. The contribution of PDK1 to the EGFR-dependent and EGFR-independent signaling in HNSCC was determined using RNA interference, a kinase-dead mutant, and pharmacologic inhibition. In vivo xenografts studies were also carried out to determine the efficacy of targeting PDK1 alone or in combination with the U.S. Food and Drug Administration-approved EGFR inhibitor cetuximab. PDK1 contributed to both GPCR-induced EGFR activation and cell growth. PDK1 also mediated activation of p70S6K in the absence of EGFR. Blockade of PDK1 with a small molecule inhibitor (AR-12) abrogated HNSCC growth, induced apoptosis, and enhanced the antiproliferative effects of EGFR tyrosine kinase inhibitors in vitro. HNSCC xenografts expressing kinase-dead PDK1 showed increased sensitivity to cetuximab compared with vector-transfected controls. Administration of AR-12 substantially decreased HNSCC tumor growth in vivo. These cumulative results show that PDK1 is a common signaling intermediate in GPCR-EGFR cross-talk and EGFR-independent signaling, and in which targeting the PDK1 pathway may represent a rational therapeutic strategy to enhance clinical responses to EGFR inhibitors in HNSCC.

Abstract 2721: MEHD7945A, dual specific antibody against EGFR and HER3, augments radiation response in head and neck squamous cell carcinoma

Abstract The HER receptor family members (EGFR, HER2, HER3 and HER4) play an important role in tumorigenesis. High EGFR expression levels correlate with poor prognosis and resistance to therapy for many epithelial tumors. Combining EGFR inhibition with radiation has been extensively studied in head and neck squamous cell carcinoma (HNSCC). Increasing evidence implicates HER3 as a key mediator of response and resistance to EGFR targeted therapies. We therefore examined the capacity of a dual specific antibody MEHD7945A, that can simultaneously target EGFR and HER3, to modulate radiation response in HNSCC. We characterized 10 HNSCC cell lines for their EGFR and HER3 expression. Immunoblot analysis confirmed that MEHD7945A significantly inhibited basal and radiation-induced EGFR and HER3 phosphorylation and downstream MAPK and AKT signaling. MEHD7945A consistently inhibited tumor cell growth in culture ranging from 30∼80%. The growth inhibitory effect of MEHD7945A was more potent than that observed with anti-EGFR or anti-HER3 antibody alone. Using clonogenic survival assay, we found that MEHD7945A significantly enhanced radiosensitivity in HNSCC cells. Once again, this effect of MEHD7945A was more potent than that observed with anti-EGFR or anti-HER3 antibodies. Treatment with MEHD7945A and radiation induced cell cycle arrest in G1 and G2 phases. Tumor cells eventually processed to apoptosis without successful DNA damage repair, as reflected by increased apoptotic populations detected by AnnexinV/PI staining. In addition, an increase of nuclear γ-H2AX foci was observed in cells receiving combination MEHD7945A and radiation. We administered MEHD7945A systemically to athymic mice bearing human tumor xenografts. Using single dose and fractionated radiation schedules, we observed that combined MEHD7945A and radiation was significantly more potent than single modality treatment to induce HNSCC tumor growth inhibition. These in vitro and in vivo results indicate that MEHD7945A can sensitize cells to radiation and inhibit HNSCC tumor growth, suggesting the potential of MEHD7945A as a radiation sensitizer for future clinical investigations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2721. doi:1538-7445.AM2012-2721

Abstract 125: MicroRNA-34a dysregulation promotes tumor growth and tumor angiogenesis in head and neck squamous cell carcinoma

Abstract Objectives: Head and Neck squamous cell carcinoma (HNSCC) results from deregulation of multiple signaling pathways leading to tumor cells acquiring proliferative, invasive, angiogenic and metastatic properties. Interactions between neoplastic cells and the surrounding microenvironment also play a major role in tumor progression. Recent studies have demonstrated that microRNA's (miRs) play a critical role in cancer development where they can act as oncogenes or as onco-suppressors. A microRNA array analysis of a panel of ten HNSCC cell lines reveled that miR-34a is significantly downregulated in all these cell lines as compared to normal human oral keratinocyes (HOK). In addition, miR-34a expression was also downregulated in highly angiogenic endothelial cells (endothelial cells overexpressing Bcl-2; EC-Bcl-2) as compared to normal human endothelial cells. However, little is known about the role of miR-34a in HNSCC tumor growth and tumor angiogenesis. In this study, we examined if dysregulation of miR-34a in HNSCC promotes tumor growth and tumor angiogenesis. Methods and Results: miR-34a expression in tumor samples, HNSCC cell lines and endothelial cells was examined by real time PCR. Lipofectamine-2000 was used to transfect miR-34a in HNSCC cell lines and human endothelial cells. Cell-proliferation and migration was examined by Xcelligence system. miR-34a effect on tumor growth and tumor angiogenesis was examined by in vivo SCID mouse xenograft model. Our results demonstrate that miR-34a is significantly downregulated in HNSCC tumors and cell lines. Ectopic expression of miR-34a in HNSCC cell lines significantly inhibited tumor cell proliferation, colony formation and migration. Tumor cells expressing miR-34a also showed marked decrease in the levels of a key transcription factor, E2F3. Rescue experiments using microRNA resistant E2F3 isoforms suggest that miR-34a-mediated inhibition of cell proliferation and colony formation is predominantly mediated by E2F3a isoform. In addition, tumor samples from HNSCC patients showed inverse relationship between miR-34a and survivin expression. Overexpression of E2F3a completely rescued survivin expression in miR-34a expressing cells, thereby suggesting that miR-34a may be regulating survivin expression via E2F3a. Ectopic expression of miR-34a also significantly inhibited tumor growth and tumor angiogenesis in a SCID mouse xenograft model. Interestingly, miR-34a inhibited tumor angiogenesis by blocking VEGF production by tumor cells as well as directly inhibiting endothelial cell functions. Conclusions: Taken together, these findings suggest that dysregulation of miR-34a expression is common in HNSCC and modulation of miR34a activity might represent a novel therapeutic strategy for the treatment of HNSCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 125. doi:1538-7445.AM2012-125

RANKL Expression Specifically Observed in Vivo Promotes Epithelial Mesenchymal Transition and Tumor Progression

Recent findings have focused attention on the molecular consequences of the microenvironment in tumor progression, but events occurring in cancer cells themselves in response to their ambient conditions remain obscure. Here, we identify receptor activator of nuclear factor κB ligand (RANKL) as a microenvironment-specific factor essential for tumorigenesis in vivo, using head and neck squamous cell carcinoma (HNSCC) as a model. In human HNSCC tissues, RANKL is abundantly expressed, and its expression level correlates with the histological grade of differentiation. RANKL levels are significantly higher in poorly differentiated SCCs than in well or moderately differentiated SCCs. In contrast, all HNSCC cell lines tested displayed extremely low RANKL expression; however, RANKL is efficiently up-regulated when these cell lines are inoculated in the head and neck region of mice. RANKL expression is restored in a microenvironment-specific manner, and cannot be observed when the cells are inoculated in the hindlimbs. Forced expression of RANKL compensates for tumor growth in the hindlimb milieu, promotes epithelial mesenchymal transition, and induces tumor angiogenesis, in a manner independent of vascular endothelial growth factor (VEGF). These results implicate RANKL expression causatively in tumor growth and progression in HNSCC in vivo. RANKL may provide a novel functional marker for biological malignancy and a therapeutic target based on the specific nature of the microenvironment.

Enhanced local dendritic cell activity and tumor‐specific immunoresponse in combined radiofrequency ablation and interleukin‐2 for the treatment of human head and neck cancer in a murine orthotopic model

Radiofrequency ablation (RFA) is a minimally invasive tumor destruction technique and can provide the antigen source initiating tumor immunity. However, induced immune response is weak and requires additional immunotherapy for optimized RFA treatment against cancer. A murine orthotopic model of head and neck squamous cell carcinoma (HNSCC) was established to investigate the efficacy and mechanism of an RFA + interleukin-2 (IL-2) combination adenoviral gene therapy among 6 groups. Tumor volume change, apoptosis, in situ macrophage recruitment, cytotoxic T lymphocyte (CTL) activity, migration of dendritic cells into the regional lymph nodes, and systemic antitumor immunity were examined. RFA + IL-2 therapy induced the highest levels of macrophage recruitment and dendritic cell migration resulting in enhanced CTL activity, increased tumor apoptosis, enhanced systemic antitumor immunity, and the best inhibition of tumor growth among all groups. RFA + IL-2 combination therapy generates potent tumor immunity to suppress tumor growth in HNSCC.

PS-341 and Histone Deacetylase Inhibitor Synergistically Induce Apoptosis in Head and Neck Squamous Cell Carcinoma Cells

Proteasome inhibitor PS-341 (also known as bortezomib) and histone deacetylase (HDAC) inhibitors have emerged as novel therapeutic agents for a variety of malignancies. In this study, we examined whether PS-341 and the HDAC inhibitor trichostatin A (TSA) induced apoptosis in head and neck squamous cell carcinoma (HNSCC), a common and lethal malignancy. We found that, although TSA treatment alone did not induce apoptosis in HNSCC cells, it significantly enhanced PS-341-induced apoptosis in HNSCC cells in vitro. Consistently, TSA significantly improved PS-341-mediated inhibition of HNSCC tumor growth in nude mice. Mechanistically, we found that TSA increased PS-341-induced Noxa expression and caspase activation in HNSCC cells. The knockdown of Noxa significantly reduced apoptosis induced by cotreatment of PS-341 and TSA. Taken together, our results provide new insight into the mechanisms of synergistic antitumor activity of the PS-341 and HDAC inhibitor regimen, offering a new therapeutic strategy for HNSCC patients.

Abstract LB-354: Dysregulation of microRNA-34a expression in head and neck squamous cell carcinoma promotes tumor growth and tumor angiogenesis

Abstract Objectives of the study: Head and Neck squamous cell carcinoma (HNSCC) results from deregulation of multiple signaling pathways leading to tumor cells acquiring proliferative, invasive, angiogenic and metastatic properties. Interactions between neoplastic cells and the surrounding microenvironment also play a major role in tumor progression. Recent studies have demonstrated that microRNA's (miRs) play a critical role in cancer development where they can act as oncogenes or as onco-suppressors. A microRNA array analysis of a panel often HNSCC cell lines reveled that miR-34a is significantly downregulated in all these cell lines as compared to normal human oral keratinocyes (HOK). In addition, miR-34a expression was also downregulated in highly angiogenic endothelial cells (endothelial cells overexpressing Bcl-2; EC-Bcl-2) as compared to normal human endothelial cells. However, little is known about the role of miR-34a in HNSCC tumor growth and tumor angiogenesis. In this study, we examined if dysregulation of miR-34a in HNSCC promotes tumor growth and tumor angiogenesis. Methodology: The study was performed using a panel of established human head & neck cancer cell lines. miRs expression in tumor and endothelial cells was examined by miR-arrays and further validated by real time PCR. Lipofectamine-2000 was used to transfect miR-34a in 2 HNSCC cell lines (UM-SCC-74A and UM-SCC-47) and human endothelial cells. Cell-proliferation assay was performed by MTT and colony formation assay. Tumor cell and endothelial cell motility was assessed by scratch assay. Expression of miR-34a downstream mediators was measured by Western blot. Results: Ectopic expression of miR34a significantly inhibited cell proliferation of UM-SCC-74A cells (52% and 58% on days 2 and 4) and UM-SCC-47 cells (43% and 47% on days 2 and 4). In addition, enhanced expression of miR34a in UM-SCC-74A and UM-SCC-47 cells significantly inhibited tumor cell colony formation and tumor cell migration. Similar to tumor cells, miR-34a significantly inhibited EC-Bcl-2 proliferation and migration by down regulating c-met, Bcl-2 expression and upregulating phospho p38 MAPK expression. Conclusion: These findings indicate that dysregulation of miR-34a expression is common in HNSCC and modulation of miR34a activity might represent a novel therapeutic strategy for the treatment of head and neck cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-354.

Saracatinib Impairs Head and Neck Squamous Cell Carcinoma Invasion by Disrupting Invadopodia Function

Elevated Src kinase activity is linked to the progression of solid tumors, including head and neck squamous cell carcinoma (HNSCC). Src regulates HNSCC proliferation and tumor invasion, with the Src-targeted small molecule inhibitor saracatinib displaying potent anti-invasive effects in preclinical studies. However, the pro-invasive cellular mechanism(s) perturbed by saracatinib are unclear. The anti-proliferative and anti-invasive effects of saracatinib on HNSCC cell lines were therefore investigated in pre-clinical cell and mouse model systems. Saracatinib treatment inhibited growth, cell cycle progression and transwell Matrigel invasion in HNSCC cell lines. Dose-dependent decreases in Src activation and phosphorylation of the invasion-associated substrates focal adhesion kinase, p130 CAS and cortactin were also observed. While saracatinib did not significantly impact HNSCC tumor growth in a mouse orthotopic model of tongue squamous cell carcinoma, impaired perineural invasion and cervical lymph node metastasis was observed. Accordingly, saracatinib treatment displayed a dose-dependent inhibitory effect on invadopodia formation, extracellular matrix degradation and matrix metalloprotease 9 activation. These results suggest that inhibition of Src kinase by saracatinib impairs the pro-invasive activity of HNSCC by inhibiting Src substrate phosphorylation important for invadopodia formation and associated matrix metalloprotease activity.

Identification of Insulin-Like Growth Factor Binding Protein-3 as a Farnesyl Transferase Inhibitor SCH66336-Induced Negative Regulator of Angiogenesis in Head and Neck Squamous Cell Carcinoma

The farnesyl transferase inhibitor (FTI) SCH66336 has been shown to have antitumor activities in head and neck squamous cell carcinoma (HNSCC) in vitro and in vivo. However, its mechanism of action has not been well defined. Here, we report that the insulin-like growth factor (IGF) binding protein (IGFBP)-3 mediates antitumor activities of SCH66336 in HNSCC by inhibiting angiogenesis. SCH66336 significantly suppressed HNSCC tumor growth and angiogenesis via mechanisms that are independent of H-Ras and RhoB. By inducing IGFBP-3 secretion from HNSCC cells, this compound suppresses angiogenic activities of endothelial cells, including vessel formation in chorioallantoic membranes of chick, endothelial cell sprouting from chick aorta, and capillary tube formation of human umbilical vascular endothelial cells (HUVEC). Knockdown of IGFBP-3 expression in HNSCC cells by RNA interference or depletion of IGFBP-3 in HUVECs by neutralizing antibody effectively blocked the effects of IGFBP-3 secreted from SCH66336-treated HNSCC cells on HUVECs. These findings suggest that IGFBP-3 could be a primary target for antitumor activities of FTIs and that IGFBP-3 is an effective therapeutic approach against angiogenesis in HNSCC.

Wnt/β-catenin signaling inhibits death receptor-mediated apoptosis and promotes invasive growth of HNSCC

The Wnt/β-catenin signaling pathway plays a critical role in cell proliferation and oncogenesis. It has been found to be chronically activated in a variety of human cancers, including head and neck squamous cell carcinoma (HNSCC). Previously, we have found that the activation of the Wnt/β-catenin signaling pathway inhibits mitochondria-mediated apoptosis. In this study, we extended our studies to determine whether the Wnt/β-catenin signaling pathway inhibited death receptor-mediated apoptosis in HNSCC cells. We found that Wnt/β-catenin inhibited not only tumor necrosis factor (TNF)/c-Myc-mediated apoptosis, but also cell detachment-mediated apoptosis (anoikis) which is dependent on the death receptor signaling pathway. Interestingly, we also observed that the Wnt/β-catenin signaling pathway induced HNSCC cell scattering and promoted cell invasion in the Matrigel, both of which are hallmarks for the invasive growth of HNSCC. Consistently, the over-expression of β-catenin promoted HNSCC tumor growth in nude mice. Taken together, our results suggest that the Wnt/β-catenin signaling pathway plays dual functions in HNSCC development: promoting both cell survival and invasive growth of HNSCC cells.

Tissue Inhibitor of Matrix Metalloproteinase-1 Is Prognostic in Head and Neck Squamous Cell Carcinoma: Comparison of the Circulating and Tissue Immunoreactive Protein

Tissue inhibitors of metalloproteinases (TIMP) are capable of inhibiting the matrix metalloproteinases, but they also possess other biological functions. Little is known about the role of TIMP-1 in the progression and spreading of cancer cells among patients with head and neck squamous cell carcinoma (HNSCC). In this study, the pretreatment serum levels of TIMP-1 or the overexpression of TIMP-1 immunoreactive protein in the primary tumor was correlated to the clinical course in patients with HNSCC. The TIMP-1 immunoreactive protein was studied in 74 cases representing HNSCC. The tissue immunoreactive protein was evaluated from paraffin-embedded tumor sections in 68 cases using immunohistologic staining with a specific antibody, and in 68 cases the pretreatment serum levels of TIMP-1 were quantitatively measured by ELISA assay. The results were compared with the clinicopathologic factors of the disease and the patients' outcome. A positive correlation was found between the size of the primary tumor (T) and the circulating TIMP-1 level (P = 0.021) or the positive immunoreaction of TIMP-1 in tumor (P = 0.039). The 5-year cause-specific survival was significantly lower in patients presenting with a high serum TIMP-1 level than in those with a low level of TIMP-1 (38% versus 64%, P = 0.034). They also had an unfavorable 5-year relapse-free survival rate (37% versus 56%, respectively). Similarly, the expression of TIMP-1 in tumor was prognostic for shortened survival, the 5-year cumulative relapse-free survival being 42% in patients with a TIMP-1-positive tumor versus 75% in cases with a negative tumor (P = 0.035). Tissue TIMP-1 positivity also seemed associated to the cause-specific survival (P = 0.075) and to be connected with later lymph node or hematogenic relapses. This study shows for the first time that both circulating and tissue TIMP-1 immunoreactive protein predicts the clinical course and dissemination in HNSCC, suggesting that TIMP-1 might be related to both tumor growth and metastasis in HNSCC.

Third place--Resident Research Competition, AAO-2000. Antisense cyclin D1 inhibits growth of head and neck cancer xenografts in nude mice.

Cyclin D1 is a regulatory factor essential in the progression of the cell cycle from G1 through S phase. Amplification and overexpression of cyclin D1 have been observed in many human cancers including head and neck squamous cell carcinoma (HNSCC). We have previously transfected a HNSCC control cell line (CCL23) with an antisense cyclin D1 plasmid and demonstrated inhibition of cell proliferation in vitro. In this study, we examine whether antisense cyclin D1 could inhibit tumor growth in vivo. Methods/measures: The CCL23 and its antisense cyclin D1 transfected clone (CCL23 AS) were injected into the flanks of nude mice. Tumor growth was monitored weekly. After 5 weeks, tumors were removed and studied for tumor size, cyclin D1 expression, cyclin D1-dependent kinase activity, and retinoblastoma (Rb) phosphorylation. Compared with the control tumors, 11 of 19 antisense tumors were smaller, 7 tumors were of equal size, and 1 tumor was larger. Immunohistochemical analysis with an anti-cyclin D1 antibody demonstrated decreased cyclin D1 expression in CCL23 AS and the smaller antisense tumors. Cyclin D1-dependent kinase activity was reduced in CCL23 AS and the smaller antisense tumors, and this was accompanied by a relative decrease in phosphorylated Rb in these samples. Antisense cyclin D1 inhibits growth of HNSCC tumors. Cyclin D1 expression, cyclin D1-dependent kinase activity, and Rb phosphorylation are decreased in these tumors. These findings lend support for the potential use of antisense cyclin D1 as gene therapy for HNSCC.

Antisense cyclin D1 inhibits growth of head and neck cancer xenografts in nude mice: Third Place—Resident Research Competition, AAO-2000

Problem: Cyclin D1 is a regulatory factor essential in the progression of the cell cycle from G1 through S phase. Amplification and overexpression of cyclin D1 have been observed in many human cancers including head and neck squamous cell carcinoma (HNSCC). We have previously transfected a HNSCC control cell line (CCL23) with an antisense cyclin D1 plasmid and demonstrated inhibition of cell proliferation in vitro. In this study, we examine whether antisense cyclin D1 could inhibit tumor growth in vivo. Methods/measures: The CCL23 and its antisense cyclin D1 transfected clone (CCL23 AS) were injected into the flanks of nude mice. Tumor growth was monitored weekly. After 5 weeks, tumors were removed and studied for tumor size, cyclin D1 expression, cyclin D1-dependent kinase activity, and retinoblastoma (Rb) phosphorylation. Results: Compared with the control tumors, 11 of 19 antisense tumors were smaller, 7 tumors were of equal size, and 1 tumor was larger. Immunohistochemical analysis with an anti-cyclin D1 antibody demonstrated decreased cyclin D1 expression in CCL23 AS and the smaller antisense tumors. Cyclin D1-dependent kinase activity was reduced in CCL23 AS and the smaller antisense tumors, and this was accompanied by a relative decrease in phosphorylated Rb in these samples. Conclusion: Antisense cyclin D1 inhibits growth of HNSCC tumors. Cyclin D1 expression, cyclin D1-dependent kinase activity, and Rb phosphorylation are decreased in these tumors. Clinical significance: These findings lend support for the potential use of antisense cyclin D1 as gene therapy for HNSCC. (Otolaryngol Head Neck Surg 2001;124:656-62.)

Expression of basic fibroblast growth factor and its receptors by head and neck squamous carcinoma tumor and vascular endothelial cells

Basic fibroblast growth factor (bFGF) is a potent angiogenic factor implicated in tumor growth and metastasis. To determine if bFGF and basic fibroblast growth factor receptor 1 (bFGFR1) and 2 (bFGFR2) are upregulated in head and neck squamous cell carcinoma (HNSCC), we measured the distribution and levels of each in HNSCC specimens and control specimens. Head and neck squamous cell carcinoma and control tissue specimens were analyzed qualitatively (40 patients, 10 controls) using immunohistochemistry, and quantitatively (26 patients, 8 controls) using immunoassays and graded immunohistochemistry. Control tissue consisted of palatal tissue obtained during uvulopalatopharyngoplasty (UPPP). Immunohistochemical analysis revealed that bFGF, bFGFR1, and bFGFR2 antigens were strongly associated with cancer and vascular endothelial cells in HNSCC. Control tissue had moderate staining of vascular endothelium and no stromal staining. Quantitative analysis of bFGF in tissue homogenates indicated that bFGF levels in cancer specimens were significantly elevated compared with control tissues (420.3 +/- 360.9 ng/mg total protein versus 49.2 +/- 48.7, respectively, P < or =0.05). When analyzed by clinical stage, bFGF levels were significantly higher in stage III patients as compared with stage IV patients (P < or =0.01). When immunohistochemistry results were correlated with clinical stage, bFGF (P < or =0.01), bFGFR1 (P < or =0.001), and bFGFR2 (P < or =0.0001) staining was significantly more intense in the cancer cells of stage III versus stage IV patients. Enhanced expression of bFGF and bFGF receptors by cancer and vascular endothelial cells is present in HNSCC, and may contribute to tumor growth and metastasis in HNSCC by mediating angiogenesis. Strategies aimed at decreasing the expression of bFGF and its receptors may be of therapeutic benefit in HNSCC, particularly at an early stage of disease.