Differential expression profiling of head and neck squamous cell carcinoma (HNSCC)

Head and neck squamous cell carcinoma (HNSCC) is the fifth most common cancer in men with an incidence of about 780000 new cases per year worldwide and a poor rate of survival. There is a need for a better understanding of HNSCC, for the development of rational targeted interventions and to define new prognostic or diagnostic markers. To address these needs, we performed a large-scale differential display comparison of hypopharyngeal HNSCCs against histologically normal tissue from the same patients. We have identified 70 genes that exhibit a striking difference in expression between tumours and normal tissues. There is only a limited overlap with other HNSCC gene expression studies that have used other techniques and more heterogeneous tumour samples. Our results provide new insights into the understanding of HNSCC. At the genome level, a series of differentially expressed genes cluster at 12p12-13 and 1q21, two hotspots of genome disruption. The known genes share functional relationships in keratinocyte differentiation, angiogenesis, immunology, detoxification, and cell surface receptors. Of particular interest are the 13 'unknown' genes that exist only in EST, theoretical cDNA and protein databases, or as chromosomal locations. The differentially expressed genes that we have identified are potential new markers and therapeutic targets.     

Calcified nodal metastasis from squamous cell carcinoma of the head and neck

Calcification within cervical lymph nodes is relatively rare, and most commonly ascribed to benign inflammatory or infectious processes. We present a case of a calcified submandibular nodal metastasis from squamous cell carcinoma of the lip and review the current published literature on nodal calcifications in the neck. To the authors’ knowledge, calcification resulting from metastatic squamous cell carcinoma of the head and neck has been described only once previously.     

Role and expression patterns of E-cadherin in head and neck squamous cell carcinoma (HNSCC)

E- cadherin is a member of the cadherin superfamily known as the main mediator of the cell- cell calcium dependent adhesion interactions. Research evidence also yields to this adhesion molecule an important role in carcinogenesis and tumor progression. This review focuses on the differential expression of E- cadherin in the various anatomic sites of the human body where HNSCC arises. Controversies in the results of various studies are discussed and possible prospects for application of all this developing knowledge to prognosis and therapy of the disease are briefly mentioned.     

Role of chemotherapy in nodal metastasis of untreated squamous cell carcinoma of the head and neck

A multiple drug chemotherapeutic regime was used in thirty-two previously untreated patients of advanced squamous cell carcinoma of the head and neck, presenting with nodal metastasis. Each cycle of chemotherapy consisted of methotrexate, 5-flourouracil and cyclophosphamide. An overall response rate of 72% in the involved lymph nodes and 84.4% at the primary site was observed. The nodal metastasis from the tonsil had a major response rate of 75% while those from the larynx-hypo-pharynx responded in only 22.2% of the cases. Fixed lymph nodes displayed the lowest response rate (61.5%). The primary lesion and its nodal metastasis showed an identical response in 78% of the patients.     

MiR-34a suppresses amphiregulin and tumor metastatic potential of head and neck squamous cell carcinoma (HNSCC)

MiR-34a is a well-known tumor metastasis inhibitor, but only a few target genes involved in metastasis have been identified. In HNSCC, the role of miR-34a in metastasis has not been fully elaborated, and the target gene of miR-34a is still blind. Here we addressed that, the relative lower expression of miR-34a is associated with HNSCC lymphatic metastasis. HNSCC metastasis was found to be strongly suppressed in vitro and in vivo by over-expressing miR-34a. In order to screen the possible target genes of miR-34a in HNSCC, a microarray-based differential mRNA profiling mediated by miR-34a over-expression was performed, and AREG was identified as a pivotal target. We demonstrated that the mRNA and protein levels of AREG were greatly reduced when forcing miR-34a expression. The correlation between AREG mRNA levels and HNSCC metastatic phenotype was also significant in HNSCC tissues (p < 0.01). Moreover, the results of luciferase assay provided the further evidence that miR-34a degraded AREG mRNA through targeting the 3′-UTR site. Restoration of AREG expression partially rescued miR-34a-mediated cell invasion defects in vivo and in vitro. Additionally, Over-expressing miR-34a greatly reduced EGFR and uPA, which were reversed by re-expression of AREG. Taken together, these findings indicate that miR-34a targets AREG, and is essential in inhibition of HNSCC metastasis.     

Radioimmunotherapy of experimental head and neck squamous cell carcinoma (HNSCC) with E6-specific antibody using a novel HPV-16 positive HNSCC cell line

Background

This study aimed to evaluate clinical, laboratory, ultrasound (US) and scintigraphyc parameters in thyroid nodule and to develop an auxiliary model for clinical application in the diagnosis of malignancy.

Methods

We assessed 143 patients who were surgically treated at a single center, 65% (93) benign vs. 35% (50) malignant lesions at final histology (1998-2008). The clinical, laboratory, scintigraphyc and US features were compared and a prediction model was designed after the multivariate analysis.

Results

There were no differences in gender, serum TSH and FT4 levels, thyroid auto-antibodies (TAb), thyroid dysfunction and scintigraphyc results (P = 0.33) between benign and malignant nodule groups. The sonographic study showed differences when the presence of suspected characteristics was found in the nodules of the malignant lesions group, such as: microcalcifications, central flow, border irregularity and hypoechogenicity. After the multivariate analysis the model obtained showed age (>39 years), border irregularity, microcalcifications and nodule size over 2 cm as predictive factors of malignancy, featuring 81.7% of accuracy.

Conclusions

This study confirmed a significant increase of risk for malignancy in patients of over 39 years and with suspicious features at US.     

The roles of HOXD10 in the development and progression of head and neck squamous cell carcinoma (HNSCC)

Background:

HOX gene expression is altered in many cancers; previous microarray revealed changes in HOX gene expression in head and neck squamous cell carcinoma (HNSCC), particularly HOXD10.

Methods:

HOXD10 expression was assessed by qPCR and immunoblotting in vitro and by immunohistochemistry (IHC) in tissues. Low-expressing cells were stably transfected with HOXD10 and the phenotype assessed with MTS, migration and adhesion assays and compared with the effects of siRNA knockdown in high-HOXD10-expressing cells. Novel HOXD10 targets were identified using expression microarrays, confirmed by reporter assay, and validated in tissues using IHC.

Results:

HOXD10 expression was low in NOKs, high in most primary tumour cells, and low in lymph node metastasis cells, a pattern confirmed using IHC in tissues. Overexpression of HOXD10 decreased cell invasion but increased proliferation, adhesion and migration, with knockdown causing reciprocal effects. There was no consistent effect on apoptosis. Microarray analysis identified several putative HOXD10-responsive genes, including angiomotin (AMOT-p80) and miR-146a. These were confirmed as HOXD10 targets by reporter assay. Manipulation of AMOT-p80 expression resulted in phenotypic changes similar to those on manipulation of HOXD10 expression.

Conclusions:

HOXD10 expression varies by stage of disease and produces differential effects: high expression giving cancer cells a proliferative and migratory advantage, and low expression may support invasion/metastasis, in part, by modulating AMOT-p80 levels.     

Expression of E-cadherin and vimentin correlates with metastasis formation in head and neck squamous cell carcinoma patients

Purpose

E-cadherin is a transmembrane glycoprotein, involved in cell-cell adhesion and epithelial-mesenchymal transition (EMT). Vimentin is highly expressed in mesenchymal cells and is positively correlated with increased metastasis. Here we set out to determine the expression of E-cadherin and vimentin in head and neck squamous cell carcinomas (HNSCC).

Patients and methods

Twenty-six patients with primary stage II-IV HNSCC were included. E-cadherin and vimentin were visualised using immunohistochemistry, semi-automatically analysed for expression patterns and correlated with the clinical behaviour of these tumours.

Results

A large variation in E-cadherin and vimentin expression was observed between tumours (median 17% range 0-51% respectively median 0% range 0-20%). Tumours with low E-cadherin expression showed a significantly higher incidence of metastasis formation compared to tumours with high expression (81% versus 19%, p = 0.004). Enhanced expression of vimentin was associated with a trend towards a higher metastatic risk (33% versus 77%) compared to tumours without expression of vimentin. All patients with low E-cadherin and high vimentin expression (an EMT-phenotype) developed distant metastases versus only 44% of the other patients (p = 0.008).

Conclusion

Loss of E-cadherin and gain of vimentin may be associated with enhanced migration of tumour cells, leading to higher metastatic risk of HNSCC patients.     

Cyclophilin A-EMMPRIN interaction induces invasion of head and neck squamous cell carcinoma

Extracellular matrix remodeling crucial to tumorigenesis involves proteolytic enzymes, primarily matrix metalloproteinases (MMPs). MMP production is stimulated by multiple factors, including the extracellular matrix metallo-proteinase inducer EMMPRIN/CD147. Overexpression of EMMPRIN, a member of the immunoglobulin superfamily, promotes invasion, metastasis, growth and survival of malignant cells. Cyclophilin A (CypA) is a multifunctional protein that promotes cancer progression in various cancer types. CypA can interact with and activate EMMPRIN; however, the role of CypA-EMMPRIN interaction in oncogenicity is not completely understood. To investigate tumorigenicity induced by the CypA-EMMPRIN interaction, we stimulated EMMPRIN-expressing head and neck squamous cell carcinoma (HNSCC) cells with CypA. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye assay revealed that HNSCC cell proliferation increased upon stimulation of the cells with CypA, whereas cisplatin-induced cell death decreased after stimulation. Gelatin zymography showed that CypA also induced MMP-9 up-regulation. Moreover, HNSCC cell invasion through Matrigel?-coated membranes was increased upon stimulation of cells with CypA. This elevated invasive potential was abrogated by an EMMPRIN function-blocking antibody. These findings suggest that CypA, through its interaction with EMMPRIN, contributes to HNSCC tumorigenesis.     

Differential diagnosis between primary lung squamous cell carcinoma and pulmonary metastasis of head and neck squamous cell carcinoma

Differentiation between lung squamous cell carcinoma and pulmonary metastasis of head and neck squamous cell carcinoma is clinically important because the prognoses and therapeutic options are considerably different. However, the clinical, pathological, and immunohistochemical diagnostic methods have not yet been fully established. Although various molecular methods have been developed, they have not yet been practically applied. A combined approach involving molecular and immunohistochemical analysis, such as one that uses antibodies selected on the basis of comprehensive genetic analysis results, may be effective. We suggest a new diagnostic criteria using the clinical characteristics and the result of immunohistochemical analysis. However, there are two underlying problems in the development of new diagnostic methods: tumor heterogeneity and determination of the diagnostic accuracy.     

Amplification of Cyclin L1 is associated with lymph node metastases in head and neck squamous cell carcinoma (HNSCC)

Overrepresentation of chromosomal bands 3q25-q29 has been associated with shortened disease-specific survival in head and neck squamous cell carcinoma (HNSCC). To assess the prevalence of copy number gains (>4 signals per cell) and high-level amplifications (>8 signals per cell) from putative oncogenes in this chromosomal region (CCNL1, SNO, PIK3CA, TP73L), tissue microarray analysis was applied on 280 HNSCCs by fluorescence in situ hybridization. Overall frequency of additional copy numbers was 34.3% for CCNL1, 31.8% for SNO, 39.0% for PIK3CA and 38.3% for TP73L, respectively. In general, gains were more frequently detected in stage IV compared to stage I-III tumours. Performing multivariate logistic regression analysis, a significant association of CCNL1 gains and the presence of lymph node metastases was found, which was independent of anatomical site and T-stage of the primary tumour (P=0.049). Site-specific subgroup analysis further showed that copy number gains of CCNL1 and SNO occurred more frequently in oral carcinomas in advanced clinical stages as compared to N0 oral lesions (CCNL1: P=0.03; SNO: P=0.03). Finally, Kaplan-Meier analysis revealed that high-level amplifications of CCNL1 correlated with shorter overall survival of the patients. Our results indicate that CCNL1 plays a critical role in the loco-regional progression of HNSCC and may serve as an indicator for occult advanced tumour stages.     

Validity of sentinel lymph node (SLN) detection following adjuvant radiochemotherapy (RCT) in head and neck squamous cell carcinoma (HNSCC)

The effect of preoperative radio chemotherapy on lymphatic drainage and intraoperative gamma probe-guided sentinel lymph node detection has yet not been investigated. In this study, we study 13 patients with SCC. Sentinel lymph node (SLN) imaging of the patients was performed using SPECT-CT. Special care was taken to use identical injection sites for both studies. Imaging comprised planar and SPECT, iterative reconstruction and were viewed with the co-registered CT image. The results were validated by comparison with the histological results of intraoperative gamma probe detection and histology of the completed neck dissection. Identical SLNs were found in 6/13 patients. In 2/13 cases SLN biopsies were false-negative. In 4/13 patients preoperative SLN imaging identified more/additional nodes than the initial imaging, whereas fewer nodes were seen in 3/13 patients. Neither the primary tumor site nor the TNM stage was predictive for changes in the lymphatic drainage pattern. No constant effect of irradiation could be demonstrated. Preoperative radio chemotherapy has an unpredictable influence on the lymphatic drainage pattern in HNSCC. Consequently, the intraoperative gamma probe-guided sentinel lymph node detection after radio chemotherapy does not reveal the SLN of carcinogenesis. Thus, we advise fused functional/anatomical imaging (SPECT-CT) before and after radiochemotherapy if the SLN concept is utilized in HNSCC.     

Downregulation of IRS-1 promotes metastasis of head and neck squamous cell carcinoma

Lymph node metastasis is responsible for the high morbidity of head and neck squamous cell carcinoma (HNSCC). To date, the role of insulin receptor substrate?1 (IRS-1) in tumorigenesis and metastasis of head and neck cancer has not been elucidated. In this study, we found a negative correlation of IRS-1 expression with tumor metastasis both in human tissue samples and in cell lines. Furthermore, we found that knockdown of IRS-1 expression enhanced cell invasive potency and induced EMT in parallel with upregulation of miR-9 expression. We propose that IRS-1 suppresses metastasis of head and neck cancer possibly through miR-9.     

Lupeol suppresses cisplatin-induced nuclear factor-kappaB activation in head and neck squamous cell carcinoma and inhibits local invasion and nodal metastasis in an orthotopic nude mouse model

A poor prognosis in head and neck squamous cell carcinoma (HNSCC) patients is commonly associated with the presence of regional metastasis. Cisplatin-based chemotherapy concurrent with radiation therapy is commonly used in the treatment of locally advanced HNSCC. However, the result is dismal due to common acquisition of chemoresistance and radioresistance. Epidemiologic studies have shown the importance of dietary substances in the prevention of HNSCC. Here, we found that lupeol, a triterpene found in fruits and vegetables, selectively induced substantial HNSCC cell death but exhibited only a minimal effect on a normal tongue fibroblast cell line in vitro. Down-regulation of NF-kappaB was identified as the major mechanism of the anticancer properties of lupeol against HNSCC. Lupeol alone was not only found to suppress tumor growth but also to impair HNSCC cell invasion by reversal of the NF-kappaB-dependent epithelial-to-mesenchymal transition. Lupeol exerted a synergistic effect with cisplatin, resulting in chemosensitization of HNSCC cell lines with high NF-kappaB activity in vitro. In in vivo studies, using an orthotopic metastatic nude mouse model of oral tongue squamous cell carcinoma, lupeol at a dose of 2 mg/animal dramatically decreased tumor volume and suppressed local metastasis, which was more effective than cisplatin alone. Lupeol exerted a significant synergistic cytotoxic effect when combined with low-dose cisplatin without side effects. Our results suggest that lupeol may be an effective agent either alone or in combination for treatment of advanced tumors.     

Combined cytotoxic action of paclitaxel and ceramide against the human Tu138 head and neck squamous carcinoma cell line

Purpose: Paclitaxel, a chemotherapeutic agent used in the treatment of recalcitrant ovarian and breast as well as other neoplasms, is being investigated for the treatment of squamous cell carcinoma of the head and neck. Our previous studies have demonstrated that exogenously added ceramide enhances apoptosis in paclitaxel-exposed human leukemic cells. In this study, we showed that exogenous ceramide augmented paclitaxel-induced apoptosis in Tu138 cells in vitro when added simultaneously in combination with the paclitaxel. Methods: The combined cytotoxic effects of paclitaxel and ceramide exposure against Tu138 cells were assessed by an MTT dye assay, cell cycle analysis, TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) assay, and isobologram analysis for synergistic activity. Results: The MTT dye assay results indicated augmentation of time- and concentration-dependent paclitaxel-mediated cell cytotoxicity by simultaneous ceramide treatment. Paclitaxel treatment of Tu138 cells also resulted in an accumulation of cells in the G₂-M phase of the cell cycle. This paclitaxel-mediated G₂-M phase accumulation decreased significantly with the addition of ceramide, indicating that combined paclitaxel/ceramide treatment resulted in the elimination of Tu138 cells from the S and/or G₂-M phases of the cell cycle. Furthermore, ceramide enhancement of paclitaxel-mediated apoptosis was also detected by the TUNEL assay. Conclusion: Our results suggest that paclitaxel/ceramide combination therapy may be an attractive alternative to conventional methods of chemotherapy for head and neck cancer, and should be further explored. Received: 22 April 1999 / Accepted: 14 March 2000