Caveolin-1 mediates tumor cell migration and invasion and its regulation by miR-133a in head and neck squamous cell carcinoma

MicroRNAs (miRNAs) are small non-coding RNAs of approximately 22 nucleotides that can function as oncogenes or tumor suppressors in human cancer. Down-regulation of the miRNA miR-133a in many type of cancers, and a reduction of cell proliferation, migration, and invasion upon over-expression, suggests that miR-133a is a tumor suppressor. In this study, genome-wide gene expression analysis of HNSCC cells that over-express miR-133a showed that caveolin-1 (CAV1), a multifunctional scaffolding protein, is down-regulated, a result that was confirmed by real-time PCR and Western blot analysis. A luciferase reporter assay revealed that miR-133a is directly bound to CAV1 mRNA. Cancer cell migration and invasion were significantly inhibited in HNSCC cells transfected with si-CAV1. Therefore, CAV1 functions as an oncogene in HNSCC. The identification of tumor suppressive miRNAs and their target genes could provide new insights into potential mechanism of HNSCC carcinogenesis.     

Tumor suppressive microRNA-218 inhibits cancer cell migration and invasion through targeting laminin-332 in head and neck squamous cell carcinoma

Recent our microRNA (miRNA) expression signature revealed that expression of microRNA-218 (miR-218) was reduced in cancer tissues, suggesting a candidate of tumor suppressor in head and neck squamous cell carcinoma (HNSCC). The aim of this study was to investigate the functional significance of miR-218 and its mediated moleculer pathways in HNSCC. Restoration of miR-218 in cancer cells led to significant inhibition of cell migration and invasion activities in HNSCC cell lines (FaDu and SAS). Genome-wide gene expression analysis of miR-218 transfectants and in silico database analysis showed that focal adhesion pathway was a promising candidate of miR-218 target pathways. The laminins are an important and biologically active part of the basal lamina, the function of that are various such as influencing cell differentiation, migration and adhesion as well as proliferation and cell survival. Interestingly, all components of laminin-332 (LAMA3, LAMB3 and LAMC2) are listed on the candidate genes in focal adhesion pathway. Furthermore, we focused on LAMB3 which has a miR-218 target site and gene expression studies and luciferase reporter assays showed that LAMB3 was directly regulated by miR-218. Silencing study of LAMB3 demonstrated significant inhibition of cell migration and invasion. In clinical specimens with HNSCC, the expression levels of laminin-332 were significantly upregulated in cancer tissues compared to adjacent non-cancerous tissues. Our analysis data showed that tumor suppressive miR-218 contributes to cancer cell migration and invasion through regulating focal adhesion pathway, especially laminin-332. Tumor suppressive miRNA-mediated novel cancer pathways provide new insights into the potential mechanisms of HNSCC oncogenesis.     

Insulin-like growth factor-1 receptor and ligand targeting in head and neck squamous cell carcinoma

IGF-1 receptor (IGF-1R) signaling is associated with increased tumorigenesis of epithelial cancers. In light of recent epidemiological studies correlating high circulating levels of IGF-1 with increased risk of second primary tumors (SPTs) of the head and neck, we examined IGF system and epidermal growth factor receptor (EGFR) expression in human head and neck squamous cell carcinoma (HNSCC) matched pairs and a cross-section of HNSCC cell lines. Employing the latter, we demonstrated that IGF-1 stimulated S-phase transition in a PI 3-K/Akt and Erk-dependent manner in 5 of 8 cell lines, with Erk activation being dependent upon EGFR kinase activity. IGF-1 stimulated thymidine incorporation was inhibited by treatment with IGFBP-3, the IGF-1R tyrosine kinase inhibitor NVP-AEW541, or the EGFR tyrosine kinase inhibitor AG1478. Combining IGFBP-3 with NVP-AEW541 or AG1478 abrogated IGF-1 responses at 10-fold lower doses than either compound alone. These results demonstrate the potential for co-targeting the IGF system and EGFR in HNSCC.     

Bcl-2 as prognostic factor in head and neck squamous cell carcinoma

A series of 66 cases of oral squamous cell carcinoma (OSCC) was retrospectively analyzed by immunohisto-chemistry for bcl-2 expression to verify its predictive value for clinical outcome in patients with OSCC. After grouping for bcl-2 expression, OSCCs were statistically analyzed for the variables age, gender, histological grading (G), TNM, staging, recurrence, and overall survival rate. Univariate and multivariate (Cox regression) analyses were performed. Thirty-six OSCC (54.5%) showed expression for bcl-2, whereas 30 (44.5%) were negative. No statistical association was found between bcl-2 expression and any variables considered at baseline. Overall disease-specific survival rate at 72 months was 51%, independently from the extent of the tumor. In terms of prognostic significance, the bcl-2-positive group showed more than 60% survival at 72 months whereas the bcl-2-negative group showed none. An independent association of bcl-2 expression was found with an improved overall survival rate (p = 0.048), although grading and staging were established to be the best baseline markers of prognosis. On the basis of these results, it is possible to suggest bcl-2 as an early marker of prognosis: lack of bcl-2 expression could constitute a hallmark of aggressive biological behavior in OSCC.     

MUC1 mucin and carbohydrate associated antigens as tumor markers in head and neck squamous cell carcinoma

An immunological analysis to study MUC1 mucin core protein and carbohydrate associated antigens as tissue tumor markers in head and neck carcinoma was performed. Twenty nine patients with the following tumor localizations were included: tongue (n=10), larynx (n=8), oral cavity (n=4), maxillary sinus (n=3), tonsillar ring (n=3) and pharynx (n=1); seven samples of epithelium obtained from normal organs at the same localizations were studied as controls. Immunohistochemical analysis was performed following standard procedures and reaction was graded according to staining intensity and distribution. From each tissue section, membrane, cytoplasmic and nuclear moieties were obtained by differential centrifugation with subsequent fractionation by density gradient centrifugation (6M guanidium chloride-CsCl); subcellular moieties and CsCl derived fractions were analyzed by immunoblotting. Monoclonal antibodies (MAbs) reacting with the core protein of MUCI (C595) and associated carbohydrate antigens were: Tn, 83D4 MAb; Lewis y antigen (Le y), C14 MAb; Lewis x antigen (Le x), KM380 MAb and sialyl Lewis x (sLe x), KM93 MAb. Statistical analysis was undertaken by Spearman rank correlation. In tumor samples, the immunohistochemical identification of MUCl core protein and associated antigens was extended; differences were found in the pattern and intensity of expression; results were corroborated by immunoblotting although in a few samples there was not coincidence between both methods. Localization, tumor mass or node involvement did not show significant differences for any of the antigens studied. Conclusions: 1) head and neck carcinoma expressed MUCI and associated carbohydrate antigens in high levels; 2) no relationship between antigenic expression and tumor status was found.     

Identification of novel molecular targets regulated by tumor suppressive miR-1/miR-133a in maxillary sinus squamous cell carcinoma

Based on our microRNA (miRNA) expression signature analysis of maxillary sinus squamous cell carcinoma (MSSCC), we found that miR-1 and miR-133a were significantly reduced in tumor tissues. Quantitative real-time RT-PCR revealed that the expression levels of miR-1 and miR-133a were significantly downregulated in clinical MSSCC tumor tissues compared with normal tissues. We focused on the functional significance of miR-1 and miR-133a in cancer cells and identification of the novel cancer networks regulated by these miRNAs in MSSCC. Restoration of downregulated miRNAs (miR-1 or miR-133a) in cancer cells revealed that both miRNAs significantly inhibited cancer cell proliferation and induced cell apoptosis. Molecular target identification of these miRNAs showed that transgelin 2 (TAGLN2) and purine nucleoside phosphorylase (PNP) were regulated by miR-1 and miR-133a. Both TAGLN2 and PNP mRNA expression levels were significantly upregulated in clinical MSSCC tumor tissues. Silencing studies of target genes demonstrated that both genes inhibited cancer cell proliferation. The identification of novel miR-1/miR-133a-regulated cancer pathways could provide new insights into potential molecular mechanisms of MSSCC oncogenesis.     

The tumour-suppressive function of miR-1 and miR-133a targeting TAGLN2 in bladder cancer

Background:

On the base of the microRNA (miRNA) expression signature of bladder cancer (BC), we found that miR-1 and miR-133a were significantly downregulated in BC. In this study, we focussed on the functional significance of miR-1 and miR-133a in BC cell lines and identified a molecular network of these miRNAs.

Methods and results:

We investigated the miRNA expression signature of BC clinical specimens and identified several downregulated miRNAs (miR-133a, miR-204, miR-1, miR-139-5p, and miR-370). MiR-1 and miR-133a showed potential role of tumour suppressors by functional analyses of BC cells such as cell proliferation, apoptosis, migration, and invasion assays. Molecular target searches of these miRNAs showed that transgelin 2 (TAGLN2) was directly regulated by both miR-1 and miR-133a. Silencing of TAGLN2 study demonstrated significant inhibitions of cell proliferation and increase of apoptosis in BC cell lines. The immunohistochemistry showed a positive correlation between TAGLN2 expression and tumour grade in clinical BC specimens.

Conclusions:

The downregulation of miR-1 and miR-133a was a frequent event in BC, and these miRNAs were recognised as tumour suppressive. TAGLN2 may be a target of both miRNAs and had a potential oncogenic function. Therefore, novel molecular networks provided by miRNAs may provide new insights into the underlying molecular mechanisms of BC.     

HSulf-1 modulates HGF-mediated tumor cell invasion and signaling in head and neck squamous carcinoma

Recently, we cloned a novel sulfatase domain-containing downregulated gene, HSulf-1, which modulates heparin-binding growth factor signaling in ovarian cancer. Based on the pilot data showing the loss of HSulf-1 in head and neck squamous cell carcinoma cell lines (SCCHN), we sought to employ SCCHN as a model to define the role of HSulf-1 in the molecular regulation of tumorigenicity. Three SCCHN lines (012SCC, WMMSCC, and 015SCC) had no detectable HSulf-1 mRNA. Clonal lines of HSulf-1-expressing 012SCC attenuated the activation of ERK/mitogen-activated protein kinase (MAPK) signaling mediated by fibroblast growth factor (FGF-2) and both ERK/MAPK and Akt signaling mediated by hepatocyte growth factor (HGF). Consistent with this downregulation, phosphorylation of HGF receptor, c-Met, which is frequently overexpressed in SCCHN, was also attenuated in HSulf-1 clonal 012SCC cell lines. HGF markedly enhanced the motility and migration of vector-transfected cells in a transwell invasion chamber. However, HGF-mediated motility and invasion was attenuated in HSulf-1 clonal 012SCC cell lines. In addition, transfected cells displayed significant growth inhibition concomitant with a decrease in mitogenicity, as measured by thymidine incorporation and increased sensitivity to staurosporine- and cisplatin-induced apoptosis. These data suggest that HSulf-1 normally functions as a negative regulator in cell growth and loss of HSulf-1 in SCCHN potentiates growth factor signaling, enhances motility, invasiveness and inhibits stress-induced apoptosis, with a resulting increase in tumorigenicity.     

Serum hepatocyte growth factor as a marker of tumor activity in head and neck squamous cell carcinoma

Hepatocyte growth factor (HGF) has previously been reported to be elevated in the serum of patients with malignancy, including breast, colorectal and gastric cancers. Here, we evaluated the correlation between serum HGF and the progression of head and neck squamous cell carcinoma (HNSCC). The mean serum HGF levels in 71 healthy control subjects, 78 patients with primary HNSCC, and eight patients with recurrent HNSCC were 0.538 ± 0.163, 0.701 ± 0.252, and 0.925 ± 0.349 ng/ml, respectively. The increase in the HGF level was significantly correlated with tumor stage progression. The HGF level had decreased to normal at 1 month after curative resection of the tumors. During follow-up for several months, the HGF level significantly increased in recurrent HNSCC patients, whereas there was no increase in nonrecurrent patients. Our data suggest that serum HGF is significantly corrected with tumor progression and may be a strong predictor of recurrence in HNSCC.     

Frequently methylated tumor suppressor genes in head and neck squamous cell carcinoma

Head and neck squamous cell carcinoma (HNSCC) is a very aggressive cancer. In advanced stages, the patient has poor chances of receiving effective treatment, and survival rates are low. To facilitate timely diagnosis and improve treatment, elucidation of early detection markers is crucial. DNA methylation markers are particularly advantageous because DNA methylation is an early event in tumorigenesis, and the epigenetic modification, 5-methylcytosine, is a stable mark. A genome-wide screen using Restriction Landmark Genomic Scanning found a set of genes that are most commonly methylated in head and neck cancers. Five candidate genes: septin 9 (SEPT9), sodium-coupled monocarboxylate transporter 1 (SLC5A8), functional smad-suppressing element on chromosome 18 (FUSSEL18), early B-cell factor 3 (EBF3), and iroquois homeobox 1 (IRX1) were methylated in 27% to 67% of the HNSCC patient samples tested. Furthermore, approximately 50% of the methylated tumor samples shared methylation between two of the five genes (most commonly between EBF3 and IRX1), and 15% shared methylation between three of the five genes. Expression analysis revealed candidate gene down-regulation in 25% to 93% of the HNSCC samples, and 5-aza-2'-deoxycytidine treatment was able to restore expression in at least 2 of 5 HNSCC cell lines for all of the genes tested. Overexpression of the three most frequently down-regulated candidates, SLC5A8, IRX1, and EBF3, validated their tumor suppressor potential by growth curve analysis and colony formation assay. Interestingly, all of the candidates identified may be involved in the transforming growth factor beta signaling pathway, which is often disrupted in HNSCC.     

Serum ferritin as a tumor marker in patients with squamous cell carcinoma of the head and neck

A double antibody enzyme immunoassay was used to measure serum ferritin levels in several different control and tumor-bearing populations collected from two institutions. The control groups consisted of normal volunteers, smokers, and Latter Day Saints. No statistically significant differences were noted in ferritin levels between pairs of these groups. Differences were noted among the normal groups when separated on the basis of age and sex, with higher ferritin levels in individuals older than 32 years of age and in men. By one-way analysis of variance, most control groups and subgroups were shown to have significantly lower levels (P less than 0.05) than the head and neck cancer group, with the exception of male smokers, who had levels comparable to male head and neck cancer patients. Serum ferritin levels were higher in head and neck cancer patients than in controls; however, there was no difference when compared with patients with other types of solid malignancies or when considering the anatomic site of the head and neck lesion. Ferritin levels were significantly (P less than 0.05) higher in patients with advanced (Stages III and IV) cancer than in those individuals with Stage I or II cancer. In patients with no evidence of clinical disease 5 years after treatment, the ferritin level had essentially returned to normal. In a group of head and neck cancer patients followed longitudinally, a significant decline in ferritin levels (P less than 0.05) was seen by 5 months after the completion of successful treatment. Furthermore, ferritin levels showed a tendency to increase or remain at high levels in patients with a poor prognosis and to decrease in those patients with a favorable prognosis, giving support to the contention that ferritin may prove to be a valuable tumor marker in head and neck cancer.     

CD44 as a stem cell marker in head and neck squamous cell carcinoma

In the recent past, evidence is increasing indicating the existence of a subpopulation of resistant tumor cells in head and neck squamous cell carcinoma (HNSCC) that cannot be eradicated by established antineoplastic treatments. These cancer stem cells (CSCs) have features of somatic stem cells such as selfrenewal, proliferation and differentiation. CD44+ cells in tumors of the head and neck are referred to as CSCs of HNSCC. Expression profiling of CD44 in 29 HNSCC tumors was performed by fluorescence microscopy. ELISA analysis was performed to detect concentration of soluble CD44 in the peripheral blood of 29 HNSCC patients and 11 healthy controls. Expression of CD44 was determined in all HNSCC tissue samples (n=29). In all samples a surface staining pattern was found. The concentration of CD44 in the peripheral blood of HNSCC patients was significantly higher compared to a healthy control group (mHNSCC =13.5 ± 0.5 ng/ ml; mCont = 9.3 ± 0.6 ng/ml; P=0.6 x 10(-12)). The role of CD44 as a marker for CSCs in HNSCC remains to be ascertained. Further experiments might reveal its role as a diagnostic and prognostic factor, and possibly as a therapeutic target.     

Macrophage colony-stimulating factor as a tumor marker for squamous cell carcinoma of the head and neck.

It has been demonstrated that in patients with epithelial ovarian cancer and malignant germ cell tumors of the ovary, macrophage colony-stimulating factor (M-CSF) is significantly elevated in the serum compared to healthy individuals. Therefore, M-CSF has been suggested as a tumor marker in these malignancies. In the present study, the tumor marker potential of the serum M-CSF concentration in patients with squamous cell carcinomas of the head and neck (SCCHN) was investigated. The serum M-CSF concentration was determined by a quantitative sandwich enzyme immunoassay in 59 patients suffering from SCCHN and 59 healthy controls. A significant difference in the mean serum concentration of M-CSF between the patients with SCCHN and the control group was found (p = 0.002). The M-CSF serum concentration correlated neither with the stage of disease nor with histopathological grading, and no correlation with serum C-reactive protein was found. The serum M-CSF concentration could be of interest as a tumor marker in SCCHN.     

头颈部鳞癌手术切缘分子标记物

手术是头颈部鳞癌治疗的主要方法之一,阴性切缘对于手术治疗的效果十分关键.研究显示,分子生物学检测较常规病理检查可更早期、更准确地对手术切缘进行评估,为术后治疗策略的制定和预后预测提供更佳的依据.     

Regulation of microRNA expression by hepatocyte growth factor in human head and neck squamous cell carcinoma

Hepatocyte growth factor (HGF) is a multifunctional molecule that acts as mitogen, motogen, and/or morphogen in a variety of cells. MET, a specific receptor tyrosine kinase for HGF, is upregulated in various tumors including squamous cell carcinoma of the human head and neck (HNSCC), but how HGF affects the expression of downstream functional genes has not yet been elucidated in detail. In the present study, we examined the expression of microRNA (miRNA), non-coding small RNA that regulate cell proliferation and functions by interfering with the translation of target mRNA, with or without HGF stimulation in HNSCC cell line HSC3. Among several miRNAs, in which the expression was altered after HGF stimulation, we focused on miR-200c and miR-27b, both of which were drastically downregulated after HGF stimulation. Expression of ZEB1, a target mRNA for miR-200c, was upregulated 3 and 6 h after HGF stimulation, and that of E-cadherin, a downstream molecule of ZEB1, was downregulated 12 h after HGF stimulation. Expression of ST14/matriptase, an enzyme for extracellular matrix (ECM) degradation and HGF activation and a target mRNA for miR-27b, was drastically upregulated in the protein level after HGF stimulation, although it was not statistically altered in the mRNA level. These results suggest that miR-200c and miR-27b downregulated by HGF might play an important role in epithelial-mesenchymal transition mediated by ZEB1/E-cadherin and ECM degradation and HGF autoactivation mediated by ST14/matriptase, respectively. Altered expression of miRNA directly regulated by HGF might contribute enhanced progressive and invasive characteristics of HNSCC by regulating the translation of HGF-induced functional molecules.     

Re-irradiation for head and neck squamous cell carcinoma

Introduction

Local recurrences after curative treatment have a potential for cure with salvage surgery or with re-irradiation.

Tumour-suppressive microRNA-874 contributes to cell proliferation through targeting of histone deacetylase 1 in head and neck squamous cell carcinoma

Background:

Our recent studies of microRNA (miRNA) expression signature demonstrated that microRNA-874 (miR-874) was significantly downregulated in maxillary sinus squamous cell carcinoma (MSSCC), and a putative tumour-suppressive miRNA in human cancers. Our aim of this study was to investigate the functional significance of miR-874 in cancer cells and to identify novel miR-874-mediated cancer pathways and responsible genes in head and neck squamous cell carcinoma (HNSCC).

Methods:

Gain-of-function studies using mature miR-874 were performed to investigate cell proliferation and cell cycle distribution in HNSCC cell lines (SAS and FaDu). To identify miR-874-mediated molecular pathways and targets, we utilised gene expression analysis and in silico database analysis. Loss-of-function assays were performed to investigate the functional significance of miR-874 target genes.

Results:

Expression levels of miR-874 were significantly downregulated in HNSCC tissues (including oral, pharyngeal and laryngeal SCCs) compared with normal counterpart epithelia. Restoration of miR-874 in SAS and FaDu cell lines revealed significant inhibition of cell proliferation and induction of G2/M arrest and cell apoptosis. Our expression data and in silico analysis demonstrated that miR-874 modulated the cell cycle pathway. Moreover, histone deacetylase 1 (HDAC1) was a candidate target of miR-874 regulation. Luciferase reporter assays showed that miR-874 directly regulated HDAC1. Silencing of the HDAC1 gene significantly inhibited cell proliferation and induced G2/M arrest and cell apoptosis in SAS cells.

Conclusions:

Downregulation of miR-874 was a frequent event in HNSCC. miR-874 acted as a tumour suppressor and directly targeted HDAC1. Recognition of tumour-suppressive miRNA-mediated cancer pathways provides new insights into the potential mechanisms of HNSCC oncogenesis and suggests novel therapeutic strategies for the disease.