Expression of human protein S100A7 (psoriasin), preparation of antibody and application to human larynx squamous cell carcinoma

Background Up-regulation of S100A7 (Psoriasin), a small calcium-binding protein, is associated with the development of several types of carcinomas, but its function and possibility to serve as a diagnostic or prognostic marker have not been fully defined. In order to prepare antibodies to the protein for immunohistochemical studies we produced the recombinant S100A7 protein in E. coli. mRNA extracted from human tracheal tumor tissue which was amplified by RT-PCR to provide the region coding for the S100A7 gene. The amplified fragment was cloned in the vector pCR2.1-TOPO and sub-cloned in the expression vector pAE. The protein rS100A7 (His-tag) was expressed in E. coli BL21::DE3, purified by affinity chromatography on an Ni-NTA column, recovered in the 2.0 to 3.5 mg/mL range in culture medium, and used to produce a rabbit polyclonal antibody anti-rS100A7 protein. The profile of this polyclonal antibody was evaluated in a tissue microarray. Results The rS100A7 (His-tag) protein was homogeneous by SDS-PAGE and mass spectrometry and was used to produce an anti-recombinant S100A7 (His-tag) rabbit serum (polyclonal antibody anti-rS100A7). The molecular weight of rS100A7 (His-tag) protein determined by linear MALDI-TOF-MS was 12,655.91 Da. The theoretical mass calculated for the nonapeptide attached to the amino terminus is 12,653.26 Da (delta 2.65 Da). Immunostaining with the polyclonal anti-rS100A7 protein generated showed reactivity with little or no background staining in head and neck squamous cell carcinoma cells, detecting S100A7 both in nucleus and cytoplasm. Lower levels of S100A7 were detected in non-neoplastic tissue. Conclusions The polyclonal anti-rS100A7 antibody generated here yielded a good signal-to-noise contrast and should be useful for immunohistochemical detection of S100A7 protein. Its potential use for other epithelial lesions besides human larynx squamous cell carcinoma and non-neoplastic larynx should be explored in future.

Homeobox gene expression profile indicates HOXA5 as a candidate prognostic marker in oral squamous cell carcinoma

The search for molecular markers to improve diagnosis, individualize treatment and predict behavior of tumors has been the focus of several studies. This study aimed to analyze homeobox gene expression profile in oral squamous cell carcinoma (OSCC) as well as to investigate whether some of these genes are relevant molecular markers of prognosis and/or tumor aggressiveness. Homeobox gene expression levels were assessed by microarrays and qRT-PCR in OSCC tissues and adjacent non-cancerous matched tissues (margin), as well as in OSCC cell lines. Analysis of microarray data revealed the expression of 147 homeobox genes, including one set of six at least 2-fold up-regulated, and another set of 34 at least 2-fold down-regulated homeobox genes in OSCC. After qRT-PCR assays, the three most up-regulated homeobox genes (HOXA5, HOXD10 and HOXD11) revealed higher and statistically significant expression levels in OSCC samples when compared to margins. Patients presenting lower expression of HOXA5 had poorer prognosis compared to those with higher expression (P=0.03). Additionally, the status of HOXA5, HOXD10 and HOXD11 expression levels in OSCC cell lines also showed a significant up-regulation when compared to normal oral keratinocytes. Results confirm the presence of three significantly upregulated (>4-fold) homeobox genes (HOXA5, HOXD10 and HOXD11) in OSCC that may play a significant role in the pathogenesis of these tumors. Moreover, since lower levels of HOXA5 predict poor prognosis, this gene may be a novel candidate for development of therapeutic strategies in OSCC.

An in vitro study showing the three-dimensional microenvironment influence over the behavior of head and neck squamous cell carcinoma

Objectives: The Head and Neck Squamous Cell Carcinoma (HNSCC) ranks sixth worldwide. The mechanisms of growth, invasion and metastasis of this pathology are extensively studied and generally related to specific variations in signaling pathways like the PI3K-Akt; however most of these competent studies have been performed bidimensionally, which may hide important questions. This study sought to analyze the influence of the microenvironment upon the behavior of HNSCC. Study Design: The status of pAkt, NF-kappa B and Cyclin D1 proteins was accessed through immunofluorescence and western blot methods in HNSCC cell lines originating from tongue, pharynx and metastatic lymph node when submitted to a three-dimensional culture model utilizing a matrix system. A bidimensional culture model (monolayer) was used as control. Results: The HNSCC cell lines cultured three-dimensionally exhibited a growth pattern characterized by small isolated islands, different from the control group. When the three-dimensional model was applied, two of the studied cell lines showed the same expression pattern as the bidimensional model regarding nuclear or cytoplasmatic localization, as well as reduction of all protein levels; however, the cell line originated from tongue, which specially has the epidermal growth factor receptor constitutively activated, demonstrated nuclear translocation of pAkt and also an increase in the levels of Cyclin D1. Conclusions: The results suggest the influence of the microenvironment upon the behavior of HNSCC cells due to the changed expression of proteins related to tumor growth and cellular invasion. Furthermore, intrinsically genetic conditions also played important roles over the cells, despite the culture model employed.

Four-gene expression model predictive of lymph node metastases in oral squamous cell carcinoma

Background. Previous knowledge of cervical lymph node compromise may be crucial to choose the best treatment strategy in oral squamous cell carcinoma (OSCC). Here we propose a set four genes, whose mRNA expression in the primary tumor predicts nodal status in OSCC, excluding tongue. Material and methods. We identified differentially expressed genes in OSCC with and without compromised lymph nodes using Differential Display RT-PCR. Known genes were chosen to be validated by means of Northern blotting or real time RT-PCR (qRT-PCR). Thereafter we constructed a Nodal Index (NI) using discriminant analysis in a learning set of 35 patients, which was further validated in a second independent group of 20 patients. Results. Of the 63 differentially expressed known genes identified comparing three lymph node positive (pN+) and three negative (pN0) primary tumors, 23 were analyzed by Northern analysis or RT-PCR in 49 primary tumors. Six genes confirmed as differentially expressed were used to construct a NI, as the best set predictive of lymph nodal status, with the final result including four genes. The NI was able to correctly classify 32 of 35 patients comprising the learning group (88.6%; p = 0.009). Casein kinase 1alpha1 and scavenger receptor class B, member 2 were found to be up regulated in pN + group in contrast to small proline-rich protein 2B and Ras-GTPase activating protein SH3 domain-binding protein 2 which were upregulated in the pN0 group. We validated further our NI in an independent set of 20 primary tumors, 11 of them pN0 and nine pN+ with an accuracy of 80.0% (p = 0.012). Conclusions. The NI was an independent predictor of compromised lymph nodes, taking into the consideration tumor size and histological grade. The genes identified here that integrate our "Nodal Index" model are predictive of lymph node metastasis in OSCC.

Oral candidiasis mimicking an oral squamous cell carcinoma: report of a case

Oral candidiasis is a significant problem in immune-compromised patients. The most common forms of mucosal candidiasis are oropharyngeal, oesophageal and vaginal, and more than 90% of HIV positive persons will manifest at least one episode of oropharyngeal candidiasis. Local and systemic factors such as uninterrupted daily use of a prosthesis by patients, smoking habit, as well as high glucose intake may contribute to the development of the lesion. The aim of this article is to report an uncommon case of oral candidiasis presenting an aggressive clinical behaviour in a 64-year-old male patient, with a significant smoking habit and a medical history of non-controlled diabetes. The lesion affected the hard and soft palate of the right side, revealing erythematous and ulcerated areas, elevated borders and central portions resembling necrosis, mimicking the clinical features of oral squamous cell carcinoma. However, the correct diagnosis of oral candidiasis was obtained after histopathological and cytological examinations and the patient was easily treated with traditional antifungal drugs and correction of his glucose levels.

ORAOV1 is amplified in oral squamous cell carcinoma

BACKGROUND: Oral cancer overexpressed 1 (ORAOV1) was found as a candidate oncogene in the 11q13 chromosomal region, based on its amplification and overexpression in oral cancer cell lines. Because gene amplification often leads to increased levels of gene expression, we aimed to verify the relationship between ORAOV1 gene status and mRNA expression primarily in oral squamous cell carcinoma (OSCC) by quantitative assay, correlating with clinical and pathological characteristics in patients. METHODS: Levels of ORAOV1 amplification and expression were evaluated by qPCR and RT-qPCR in OSCC cell lines and in tumor and non-tumoral surgical margins from 33 patients with OSCC. All subjects were smokers and habitual alcohol drinkers, mostly men above 40 years of age and with a single primary tumor. RESULTS: ORAOV1 exhibited increased gene expression levels as well as higher copy number in three OSCC cell lines with 11q13 amplified chromosomal region when compared with the OSCC cell line without the amplification (one-way ANOVA, P < 0.05). Weak correlation between ORAOV1 mRNA levels and DNA copy number was seen in tumor samples (Spearman, P = 0.07). Although ORAOV1 was amplified in tumor (Wilcoxon, P < 0.01), high levels of transcripts in margin did not reveal differences in comparison with tumor (Wilcoxon, P = 0.85). Aggressiveness and survival rate did not demonstrate statistical difference for both events in OSCC. CONCLUSION: The overexpression of ORAOV1 in non-tumoral margin samples can occur in the absence of amplification. The weak correlation between ORAOV1 amplification and expression in OSSC suggests that ORAOV1 expression can be regulated by mechanisms other than gene amplification. J Oral Pathol Med (2012) 41: 5460

Diagnostic utility of immunohistochemistry in distinguishing trichoepithelioma and basal cell carcinoma: evaluation using tissue microarray samples

Trichoepithelioma is a benign neoplasm that shares both clinical and histological features with basal cell carcinoma. It is important to distinguish these neoplasms because they require different clinical behavior and therapeutic planning. Many studies have addressed the use of immunohistochemistry to improve the differential diagnosis of these tumors. These studies present conflicting results when addressing the same markers, probably owing to the small number of basaloid tumors that comprised their studies, which generally did not exceed 50 cases. We built a tissue microarray with 162 trichoepithelioma and 328 basal cell carcinoma biopsies and tested a panel of immune markers composed of CD34, CD10, epithelial membrane antigen, Bcl-2, cytokeratins 15 and 20 and D2-40. The results were analyzed using multiple linear and logistic regression models. This analysis revealed a model that could differentiate trichoepithelioma from basal cell carcinoma in 36% of the cases. The panel of immunohistochemical markers required to differentiate between these tumors was composed of CD10, cytokeratin 15, cytokeratin 20 and D2-40. The results obtained in this work were generated from a large number of biopsies and resulted in the confirmation of overlapping epithelial and stromal immunohistochemical profiles from these basaloid tumors. The results also corroborate the point of view that trichoepithelioma and basal cell carcinoma tumors represent two different points in the differentiation of a single cell type. Despite the use of panels of immune markers, histopathological criteria associated with clinical data certainly remain the best guideline for the differential diagnosis of trichoepithelioma and basal cell carcinoma. Modern Pathology (2012) 25, 1345-1353; doi: 10.1038/modpathol.2012.96; published online 8 June 2012

DNA repair gene excision repair cross complementing-group 1 (ERCC1) in head and neck squamous cell carcinoma: analysis of methylation and polymorphism (G19007A), protein expression and association with epidemiological and clinicopathological factors

Aims: To evaluate the associations of excision repair cross complementing-group 1 (ERCC1) (DNA repair protein) (G19007A) polymorphism, methylation and immunohistochemical expression with epidemiological and clinicopathological factors and with overall survival in head and neck squamous cell carcinoma (HNSCC) patients. Methods and results: The study group comprised 84 patients with HNSCC who underwent surgery and adjuvant radiotherapy without chemotherapy. Bivariate and multivariate analyses were used. The allele A genotype variant was observed in 79.8% of the samples, GG in 20.2%, GA in 28.6% and AA in 51.2%. Individuals aged more than 45 years had a higher prevalence of the allelic A variant and a high (83.3%) immunohistochemical expression of ERCC1 protein [odds ratio (OR) = 4.86, 95% confidence interval (CI): 1.2-19.7, P = 0.027], which was also high in patients with advanced stage (OR= 5.04, 95% CI: 1.07-23.7, P = 0.041). Methylated status was found in 51.2% of the samples, and was higher in patients who did not present distant metastasis (OR = 6.67, 95% CI: 1.40-33.33, P = 0.019) and in patients with advanced stage (OR = 5.04, 95% CI: 1.07-23.7, P = 0.041). At 2 and 5 years, overall survival was 55% and 36%, respectively (median = 30 months). Conclusion: Our findings may reflect a high rate of DNA repair due to frequent tissue injury during the lifetime of these individuals, and also more advanced disease presentation in this population with worse prognosis.

p16 (INK4a) has clinicopathological and prognostic impact on oropharynx and larynx squamous cell carcinoma

CDKN2A encodes proteins such as p16 (INK4a), which negatively regulate the cell-cycle. Molecular genetic studies have revealed that deletions in CDKN2A occur frequently in cancer. Although p16 (INK4a) may be involved in tumor progression, the clinical impact and prognostic implications in head and neck squamous cell carcinoma (HNSCC) are controversial. The objective of this study was to evaluate the frequency of the immunohistochemical expression of p16 (INK4a) in 40 oropharynx and 35 larynx from HNSCC patients treated in a single institution and followed-up at least for 10 years in order to explore potential associations with clinicopathological outcomes and prognostic implications. Forty cases (53.3%) were positive for p16 (INK4a) and this expression was more intense in non-smoking patients (P = 0.050), whose tumors showed negative vascular embolization (P = 0.018), negative lymphatic permeation (P = 0.002), and clear surgical margins (P = 0.050). Importantly, on the basis of negative p16 (INK4a) expression, it was possible to predict a probability of lower survival (P = 0.055) as well as tumors presenting lymph node metastasis (P = 0.050) and capsular rupture (P = 0.0010). Furthermore, increased risk of recurrence was observed in tumors presenting capsular rupture (P = 0.0083). Taken together, the alteration in p16 (INK4a) appears to be a common event in patients with oropharynx and larynx squamous cell carcinoma and the negative expression of this protein correlated with poor prognosis.

High-throughput sequencing of small RNA transcriptomes reveals critical biological features targeted by microRNAs in cell models used for squamous cell cancer research

Abstract Background The implication of post-transcriptional regulation by microRNAs in molecular mechanisms underlying cancer disease is well documented. However, their interference at the cellular level is not fully explored. Functional in vitro studies are fundamental for the comprehension of their role; nevertheless results are highly dependable on the adopted cellular model. Next generation small RNA transcriptomic sequencing data of a tumor cell line and keratinocytes derived from primary culture was generated in order to characterize the microRNA content of these systems, thus helping in their understanding. Both constitute cell models for functional studies of microRNAs in head and neck squamous cell carcinoma (HNSCC), a smoking-related cancer. Known microRNAs were quantified and analyzed in the context of gene regulation. New microRNAs were investigated using similarity and structural search, ab initio classification, and prediction of the location of mature microRNAs within would-be precursor sequences. Results were compared with small RNA transcriptomic sequences from HNSCC samples in order to access the applicability of these cell models for cancer phenotype comprehension and for novel molecule discovery. Results Ten miRNAs represented over 70% of the mature molecules present in each of the cell types. The most expressed molecules were miR-21, miR-24 and miR-205, Accordingly; miR-21 and miR-205 have been previously shown to play a role in epithelial cell biology. Although miR-21 has been implicated in cancer development, and evaluated as a biomarker in HNSCC progression, no significant expression differences were seen between cell types. We demonstrate that differentially expressed mature miRNAs target cell differentiation and apoptosis related biological processes, indicating that they might represent, with acceptable accuracy, the genetic context from which they derive. Most miRNAs identified in the cancer cell line and in keratinocytes were present in tumor samples and cancer-free samples, respectively, with miR-21, miR-24 and miR-205 still among the most prevalent molecules at all instances. Thirteen miRNA-like structures, containing reads identified by the deep sequencing, were predicted from putative miRNA precursor sequences. Strong evidences suggest that one of them could be a new miRNA. This molecule was mostly expressed in the tumor cell line and HNSCC samples indicating a possible biological function in cancer. Conclusions Critical biological features of cells must be fully understood before they can be chosen as models for functional studies. Expression levels of miRNAs relate to cell type and tissue context. This study provides insights on miRNA content of two cell models used for cancer research. Pathways commonly deregulated in HNSCC might be targeted by most expressed and also by differentially expressed miRNAs. Results indicate that the use of cell models for cancer research demands careful assessment of underlying molecular characteristics for proper data interpretation. Additionally, one new miRNA-like molecule with a potential role in cancer was identified in the cell lines and clinical samples.