Differential expression of a novel serine protease homologue in squamous cell carcinoma of the head and neck

Differential gene expression between squamous cell carcinoma of the head and neck and matched normal tissue was studied by utilizing Representational Difference Analysis. Using this methodology, a novel gene, DESC1 was isolated. DESC1 possesses strong identity to the serine protease super-family. Comparison of DESC1 expression between primary squamous cell carcinoma and matched normal tissue shows that the level of DESC1 expression is reduced or absent in 11/12 SCC tissue specimens when compared to specimens of matched normal tissue. Tissue-specific expression studies further show that DESC1 expression can only be detected in tissues derived from the head and neck, and in skin, prostate and testes. Cell line studies demonstrate that DESC1 expression is epithelial-specific. Chromosomal localization studies indicate that DESC1 is located on the long arm of chromosome 4 at position q12-13.     

Induction of plasminogen activator inhibitor type-1 (PAI-1) by hypoxia and irradiation in human head and neck carcinoma cell lines

Background  

Squamous cell carcinoma of the head and neck (SCCHN) often contain highly radioresistant hypoxic regions, nonetheless, radiotherapy is a common treatment modality for these tumours. Reoxygenation during fractionated radiotherapy is desired to render these hypoxic tumour regions more radiosensitive. Hypoxia additionally leads to up-regulation of PAI-1, a protein involved in tumour progression and an established prognostic marker for poor outcome. However, the impact of reoxygenation and radiation on PAI-1 levels is not yet clear. Therefore, we investigated the kinetics of PAI-1 expression and secretion after hypoxia and reoxygenation, and determined the influence of ionizing radiation on PAI-1 levels in the two human SCCHN cell lines, BHY and FaDu.     

IGF-1R抑制剂AG1024对头颈鳞癌FaDu细胞放射敏感性的影响

背景与目的:胰岛素样生长因子1受体(insulin-like growth factor 1 receptor,IGF-1R)在多数头颈鳞癌中高表达。本研究拟探讨IGF-1R抑制剂AG1024对头颈鳞癌FaDu细胞的放射增敏作用。以此探索IGF-1R联合放射治疗头颈鳞癌的疗效及可能机理,为临床寻找有效的以IGF-1R为靶点的肿瘤治疗方法提供实验依据。方法:以头颈鳞癌FaDu细胞为研究对象,克隆形成实验分析细胞放射敏感性,应用流式细胞术(flow cytometry,FCM)检测AG1024对FaDu细胞早期凋亡及周期的影响。进一步应用γ-H2AX形成实验检测DNA双键断裂(double-strand breaks,DSBs)的修复情况;蛋白质印迹法(Western blot)及免疫沉淀法测定IGF-1R下游蛋白激酶B(protein B,Akt)和细胞外调节蛋白激酶1/2(extracellular regulated protein kinases1/2,Erk1/2)的活化水平,动物实验观察对肿瘤生长的影响。结果:AG1024联合放疗使细胞存活曲线的肩区明显变窄,反映放射敏感性指标的D0降低;FCM检测结果表明,AG1024联合放疗可增加FaDu细胞的早期凋亡水平(P<0.05),使细胞的G0/G1期比例增高(P<0.05),S期比例降低(P<0.05);在放射后24 h进一步的γ-H2AX形成实验显示,Ag1024能抑制DSBs的修复(P<0.05);同时,经AGl024处理后,细胞中磷酸化Akt(p-Akt)和磷酸化Erk1/2(p-Erk1/2)的表达水平明显降低;动物实验的结果显示药物联合照射能明显抑制肿瘤的生长。结论:IGF-1R抑制剂AG1024在体内和体外对头颈鳞癌FaDu细胞均有放射增敏作用,其机制可能与改变细胞周期并诱导细胞凋亡、抑制DSBs的修复、下调P13K/Akt及Ras/Raf/MAPK信号通路有关。     

Effects of hypoxia and reoxygenation on the expression levels of the urokinase-type plasminogen activator, its inhibitor plasminogen activator inhibitor type-1 and the urokinase-type plasminogen activator receptor in human head and neck tumour cells

One aim during oncological radiation therapy is to induce reoxygenation in hypoxic tumours in order to enhance radiosensitivity and ultimately increase cell death. In squamous cell carcinomas of the head and neck (SCCHN), hypoxia is considered a pivotal physiological modulator for malignant progression, whereby the plasminogen activation system is involved in overlapping functions such as the shaping of the extracellular matrix, cell proliferation and signal transduction. Since little is known about reoxygenation and the plasminogen activation system in SCCHN, three human SCCHN cell lines (BHY, FaDu, and CAL27) and a non-transformed control cell line (VH7) were exposed to hypoxic (<0.5% O2) conditions for up to 72 h and subsequently reoxygenated for 24 h at normoxic conditions. The mRNA expression of the urokinase-type plasminogen activator (uPA), the plasminogen activator inhibitor type-1 (PAI-1) and the urokinase-type plasminogen activator receptor (uPAR) was assessed by means of real-time semi-quantitative RT-PCR, and the protein expression was determined by immunoenzymometric quantification (ELISA). Both hypoxia and reoxygenation induced statistically significant changes in uPA, PAI-1 and uPAR mRNA and protein levels in the various cell lines investigated, showing that oxygen tension is a strong modulator of the plasminogen activation system in vitro. However, no uniform correlation pattern was found between the mRNA and protein levels analysed over all three time-points (24, 48, and 72 h) and oxygen treatment variants (N, H, R) nor according to oxygen treatment conditions over all three time-points. Changes in oxygen tension could therefore be modulating the fragile balance between the various components of the plasminogen activation system in SSCHN ultimately leading to an increased tumour matrix disruption, alterations in cell invasiveness, and the dissemination of tumour cells to distant organs.     

Preclinical modeling of EGFR inhibitor resistance in head and neck cancer

The epidermal growth factor receptor (EGFR) is widely expressed in head and neck squamous cell carcinomas (HNSCC) and can activate many growth and survival pathways within tumor cells. Despite ubiquitous EGFR expression, therapies targeting the receptor are only modestly effective in the treatment of HNSCC. A consistent mechanism of resistance to EGFR targeting agents has not yet been identified in HNSCC likely due, in part, to the paucity of preclinical models. We assessed the in vitro and in vivo responses of a panel of 10 genotypically validated HNSCC cell lines to the EGFR inhibitors erlotinib and cetuximab to determine their validity as models of resistance to these agents. We defined a narrow range of response to erlotinib in HNSCC cells in vitro and found a positive correlation between EGFR protein expression and erlotinib response. We observed cross-sensitivity in one HNSCC cell line, 686LN, between erlotinib and cetuximab in vivo. We attempted to generate models of cetuximab resistance in HNSCC cell line-derived xenografts and heterotopic tumorgrafts generated directly from primary patient tumors. While all 10 HNSCC cell line xenografts tested were sensitive to cetuximab in vivo, heterotopic patient tumorgrafts varied in response to cetuximab indicating that these models may be more representative of clinical responses. These studies demonstrate the limitations of using HNSCC cell lines to reflect the heterogeneous clinical responses to erlotinib and cetuximab, and suggest that different approaches including heterotopic tumorgrafts may prove more valuable to elucidate mechanisms of clinical resistance to EGFR inhibitors in HNSCC.     

Immunologic effector cells in head and neck cancer.

Freshly isolated tumor-infiltrating lymphocytes (TIL) and lymph node lymphocytes (LNL) in patients with head and neck cancer (HNC) often have low or undetectable functional responses. Because impaired ability of these cells to produce cytokines could be responsible for their functional incompetence, spontaneous and in vitro-induced production of interleukin-2 (IL2), interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), and interferon gamma (IFN-gamma) by TIL, LNL from tumor-free as well as tumor-involved lymph nodes (LN), and peripheral blood lymphocytes (PBL) were measured. Although TIL or PBL of patients with HNC produced IL-1 beta and TNF-alpha spontaneously or after in vitro activation, LNL did not produce measurable levels of these cytokines. LNL also produced lower levels of IFN-gamma than PBL. In situ hybridization for cytokine mRNA performed with tumor tissues, and LN of patients with HNC showed that TIL as well as LNL localized in the immediate proximity of the tumor were activated, as evidenced by the expression of mRNA for IL2, IFN-gamma, IL-1 beta, TNF-alpha, and both alpha- and beta-chains of the IL2 receptor. In addition, many LNL located next to the tumor expressed mRNA for transforming growth factor-beta (TGF-beta). In contrast, LNL not adjacent to the tumor in involved LN, as well as those in tumor-uninvolved LN, did not express mRNA for cytokines or IL2 receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

原癌基因Bmi-1与头颈部恶性肿瘤

Bmi-1基因是多梳基因家族的核心成员之一.作为一种原癌基因,Bmi-1基因在细胞的自我更新、增殖及凋亡方面发挥重要作用.多项研究已证实,Bmi-1基因在鼻咽癌、喉癌等多种头颈部恶性肿瘤中均有异常表达,并与肿瘤的发生、发展、侵袭、预后密切相关.Bmi-1基因有望成为一种新的肿瘤分子标志物,为头颈部恶性肿瘤的诊断、治疗提供新的方向和手段.     

XRCC1 polymorphisms and head and neck cancer

Inter-individual differences in DNA repair capacity have been demonstrated using a variety of phenotypic assays, including reduced repair among patients with squamous cell carcinoma of the head and neck (SCCHN). The XRCC1 DNA repair gene may facilitate DNA strand break and base excision repair. A recent case-control study of SCCHN reported associations with two polymorphisms of the XRCC1 including the exon 6, 194Arg/Arg genotype and the exon 10, 399 Gln/Gln genotype. We conducted an analysis of these two XRCC1 polymorphisms using data from a case-control study of SCCHN. Among white subjects, we found a weak elevation in risk associated with the Arg194Trp polymorphism [odds ratio (OR)=1.3; 95% confidence interval (CI)=0.6-2.9] and a decreased risk for the Arg399Gln polymorphism (OR=0.6; CI=0.4-1.1). We found a markedly decreased odds ratio for the Gln/Gln genotype among whites (OR=0.1; CI=0.04-0.6) and blacks (OR=0.01; CI=0.0004-0.3). We also found a suggestion of an interaction between the Arg194Trp and Arg399Gln polymorphisms and tobacco use. Additional epidemiologic and functional studies are needed to resolve the importance of these XRCC1 polymorphisms in SCCHN.     

Tobacco carcinogen mediated up-regulation of AP-1 dependent pro-angiogenic cytokines in head and neck carcinogenesis

Tobacco is notably genotoxic and associated with head and neck carcinogenesis. Cigarette carcinogens have the capacity to alter early response gene expression in tobacco-related malignancies via genes such as nuclear factor kappa B (NFκB). A number of early response gene activation events are also facilitated by fos/jun activator protein 1 (AP-1) associated pathways. In the present study, we hypothesize that tobacco products may induce microenvironment alterations, promoting angiogenesis and providing a permissive environment for head and neck cancer progression. In an in vitro analysis, we employed immortalized oral keratinocyte (HOK-16B) and laryngeal squamous carcinoma (UM-SCC-11A) cells to investigate interleukin (IL)-8 and vascular endothelial growth factor (VEGF) induction by cigarette smoke condensate (CSC). IL-8 and VEGF expression is based on interactions between NFκB, AP-1, and NF-IL6. We identified at least 1.5-fold dose-dependent induction of AP-1, VEGF, and IL-8 promoter/reporter gene activity after 24 h exposure to CSC. Next, we stably transfected UM-SCC-11A cells with A-Fos, a dominant negative AP-1 protein. Treatment with CSC of the A-Fos cell lines compared to empty vector controls significantly down-regulated AP-1, VEGF, and IL-8 promoter/reporter gene expression. We also performed ELISAs and discovered significant up-regulation of IL-8 and VEGF secretion by UMSCC 11A after treatment with phorbol 12-myristate 13-acetate, tumor necrosis factor alpha, and CSC, which was down-regulated by the A-Fos dominant negative protein. We conclude tobacco carcinogens up-regulate AP-1 activity and AP-1 dependent IL-8 and VEGF gene expression in head and neck cancer. This up-regulation may promote an angiogenic phenotype favoring invasion in both premalignant and squamous cancer cells of the head and neck.     

Proto-oncogene Bmi-1 and head and neck cancer

Bmi-1 is a core member of the polycomb group genes. As a proto-oncogene, Bmi-1 plays an important role in cell self-renewal, proliferation and apoptosis. Several studies have shown that Bmi-1 is highly expressed in some head and neck malignant tumors, such as nasopharyngeal cancer and laryngeal carcinoma. Furthermore, the expression level of Bmi-1 is closely related to the occurrence, development, incursion and prognosis of tumor. Bmi-1 is expected to become a novel tumor molecular marker, and provides a new direction for the treatment of the head and neck malignant tumor.     

The biology of head and neck cancer stem cells

Emerging evidence indicates that a small population of cancer cells is highly tumorigenic, endowed with self-renewal, and has the ability to differentiate into cells that constitute the bulk of tumors. These cells are considered the "drivers" of the tumorigenic process in some tumor types, and have been named cancer stem cells. Epithelial-mesenchymal transition (EMT) appears to be involved in the process leading to the acquisition of stemness by epithelial tumor cells. Through this process, cells acquire an invasive phenotype that may contribute to tumor recurrence and metastasis. Cancer stem cells have been identified in human head and neck squamous cell carcinomas (HNSCC) using markers such as CD133 and CD44 expression, and aldehyde dehydrogenase (ALDH) activity. The head and neck cancer stem cells reside primarily in perivascular niches in the invasive front where endothelial-cell initiated events contribute to their survival and function. In this review, we discuss the state-of-the-knowledge on the pathobiology of cancer stem cells, with a focus on the impact of these cells to head and neck tumor progression.     

Expression of the serine protease inhibitor serpinA3 in human colorectal adenocarcinomas

Proteases facilitate a number of steps in cancer progression. The serine protease inhibitors (serpins) are a protein superfamily with inhibitory activity against proteases. One of these proteases, serpinA3, appears to have a multifaceted role and is associated with inflammatory reactions, Alzheimer's disease, malignant melanoma and gastric cancer. To gain insight into the potential effect of serpinA3 on colorectal cancer (CRC) we determined whether serpinA3 is altered in colorectal tissue or plasma in CRC patients. Collectively, by using ELISA we noted a significantly lower serpinA3 level in cancer tissue compared to paired normal tissue. Moreover, the tumour serpinA3 level tended to be higher in disseminated disease as compared to localised disease. No significant difference in the plasma levels of serpinA3 was noted in the patients when compared to the controls. However, plasma serpinA3 and C-reactive protein (marker of inflammation) in the CRC patients and controls were significantly positively correlated. To confirm and detect localization of serpinA3 expression, immunohistochemistry was performed. Immunohistochemistry showed heterogeneous immunoreactivity in epithelial cells in the cancer and normal tissue and extracellular staining within bands of stroma as well as in some stromal cells. A Taq Man system was used to investigate a single nucleotide polymorphism (rs4934) in the serpinA3 signal sequence gene with supposed effect on serpinA3 secretion and expression. No significant difference was observed between CRC and control subjects regarding genotype and allelic distributions, nor were associations noted between clinical characteristics and serpinA3 levels. In conclusion, an altered serpinA3 concentration in CRC tissue may be a potential biomarker in CRC progression. SerpinA3 concentrations in plasma appear to be correlated with systemic inflammation, but do not appear to be specific to CRC patients. Further studies are warranted to improve our understanding of the role of serpinA3 in CRC.     

Early cancer in the head and neck

In head and neck tumors, there is no definition of early cancer. If T1N0M0, no lymph node and distant metastasis, will be early cancer, it is difficult to detect the tumor at early phase in head and neck tumor except for tongue and laryngeal cancer. Because it is hard to define it at early stage in the tumor surrounding by bony structure or tumor growing endophytically. Although almost tumor of head and neck were easily diagnosed by inspection and palpation, total imaging examination should be done actively for detection of early stage of cancer.     

Docetaxel suppresses invasiveness of head and neck cancer cells in vitro

The combination of docetaxel, cisplatin, and fluorouracil significantly enhances the survival of head and neck cancer patients compared to cisplatin and fluorouracil. We hypothesized that docetaxel may affect invasiveness of the head and neck cancer cells in addition to its tumor‐killing effect. Two different head and neck cancer cell lines (HEp‐2 and Ca9‐22) were treated with docetaxel at IC₁₀ and IC₅₀ concentrations. Cell migration and invasive growth was evaluated by wound healing assay and three‐dimensional (3D) culture of multicellular tumor spheroids, respectively. Expression levels of possible downstream effectors for cell migration/invasiveness were measured by immunoblotting in conditions with or without docetaxel. Docetaxel, but not cisplatin, suppressed filopodia formation compared with no treatment (control) condition. Consistent with this, docetaxel suppressed two‐dimensional (2D) cell migration and 3D cell invasion compared with control or cisplatin. Only docetaxel treated cells exhibited thick tubulin bundle and had lower activity of Cdc42, a member of the Rho family of small GTPases. In conclusion, Docetaxel treatment suppressed migration and invasiveness of head and neck cancer cells in vitro, which is likely to be mediated by regulating Cdc42 activity. (Cancer Sci 2010; 00: 000–000)     

Controversies in management of the neck in head and neck cancer

Opinion statement As definitive external radiation and multimodality organ preservation strategies (eg, combined chemotherapy and radiation therapy [CCRT]) improve, the role of surgery is being re-examined in the management of locally advanced head and neck cancer. Consensus regarding the use of neck dissections for complete responders and incomplete responders has yet to be achieved and the data are surprisingly controversial. A possible benefit from neck dissection after a complete response of the primary tumor after CCRT or definitive external radiation for advanced squamous cell carcinoma of the head and neck may only be anticipated in patients with persisting subclinical neck disease who have no other sites of disease. Some clinicians have even argued that the salvage rate for clinically detectable residual neck disease does not justify neck dissection. Randomized data addressing these questions and a trial addressing the accuracy of new imaging modalities, such as postchemotherapy and postradiation positron emission tomography scanning, across multiple institutions would be appropriate. As a department, we are aggressive in our treatment of isolated residual neck disease after CCRT or definitive external radiation and for patients initially diagnosed with N3 nodal disease. We are investigating the use of adjuvant neck brachytherapy at the time of neck dissection and we are pleased with our early results.     

Effect of radiation on oropharyngeal flora in patients of head and neck cancer

In the Present study 124 cases of head and heck cancers subjected to radiation therapy were studied and the change in oropharyngeal flora with the radiation therapy noted. 50 cases for comparison with no ENT complaints were taken control. The oropharyngeal flora in the control cases was established as the normal flora. Any other floral constituent was considered to be abnormal oropharyngeal flora. It was found that with radiation there was a statistically significant increase in several constituents of normal as well as abnormal oropharyngeal flora. This increase could be the predisposing factor for post radiation infections especially in post operative patients. Oropharyngeal swabs were sent before, during and after radiation therapy for detailed bacterial and mycotic flora smear and culture examinations.