CLIC1 plasma concentration is associated with lymph node metastases in oral squamous cell carcinoma.

BACKGROUND: Previous studies have shown that the chloride intracellular channel 1 (CLIC1) protein is overexpressed in oral squamous cell carcinoma (OSCC) and nasopharyngeal carcinoma. Patients with these diseases had significantly higher CLIC1 plasma levels than healthy controls. OBJECTIVES: To determine the plasma concentration of CLIC1 in patients with OSCC and laryngeal squamous cell carcinoma (LSCC). MATERIAL AND METHODS: We collected blood samples from patients diagnosed with OSCC (n = 13) and LSCC (n = 7), as well as from healthy controls (n = 8). The blood samples were centrifuged to obtain plasma and stored at -80°C. The CLIC1 plasma concentration was determined using enzyme-linked immunosorbent assay (ELISA). RESULTS: The mean CLIC1 plasma concentration was higher in the OSCC group than in the LSCC and control groups. Patients with OSCC and nodal metastases had significantly higher CLIC1 plasma concentration levels than nonmetastatic patients (p < 0.0001; Tukey's multiple comparisons test) and controls (p = 0.0004). The CLIC1 concentration correlated significantly with the presence of nodal spread (p = 0.0003; Spearman's r = 0.8613) and overall TNM staging (p = 0.0167; Spearman's r = 0.6620). No differences in CLIC1 plasma levels were observed between the LSCC and control groups. The CLIC1 plasma concentration was not associated with age, sex, tumor stage, or tumor grade. CONCLUSIONS: There were no differences in CLIC1 plasma concentration between healthy controls and patients with LSCC. However, our findings suggest that the presence of this protein in plasma may be associated with lymphatic metastasis in patients with OSCC. More research is needed to confirm this possible association.

miRNAs as Biomarkers for Diagnosing and Predicting Survival of Head and Neck Squamous Cell Carcinoma Patients.

Head and Neck Squamous Cell Carcinoma (HNSCC) is the sixth most common cancer worldwide. These tumors originate from epithelial cells of the upper aerodigestive tract. HNSCC tumors in different regions can have significantly different molecular characteristics. While many microRNAs (miRNAs) have been found to be involved in the regulation of the carcinogenesis and pathogenesis of HNSCC, new HNSCC related miRNAs are still being discovered. The aim of this study was to explore potential miRNA biomarkers that can be used to diagnose HNSCC and prognose survival of HNSCC patients. For this purpose, we chose a panel of 12 miRNAs: miR-146a-5p, miR-449a, miR-126-5p, miR-34a-5p, miR-34b-5p, miR-34c-5p, miR-217-5p, miR-378c, miR-6510-3p, miR-96-5p, miR-149-5p, and miR-133a-5p. Expression of these miRNAs was measured in tumor tissue and neighboring healthy tissue collected from patients diagnosed with HNSCC (n = 79) in either the oral cavity, oropharynx, or larynx. We observed a pattern of differentially expressed miRNAs at each of these cancer locations. Our study showed that some of these miRNAs, separately or in combination, could serve as biomarkers distinguishing between healthy and tumor tissue, and their expression correlated with patients' overall survival.

Ionizing radiation exposure of stem cell-derived chondrocytes affects their gene and microRNA expression profiles and cytokine production.

Human induced pluripotent stem cells (hiPSCs) can be differentiated into chondrocyte-like cells. However, implantation of these cells is not without risk given that those transplanted cells may one day undergo ionizing radiation (IR) in patients who develop cancer. We aimed to evaluate the effect of IR on chondrocyte-like cells differentiated from hiPSCs by determining their gene and microRNA expression profile and proteomic analysis. Chondrocyte-like cells differentiated from hiPSCs were placed in a purpose-designed phantom to model laryngeal cancer and irradiated with 1, 2, or 3 Gy. High-throughput analyses were performed to determine the gene and microRNA expression profile based on microarrays. The composition of the medium was also analyzed. The following essential biological processes were activated in these hiPSC-derived chondrocytes after IR: "apoptotic process", "cellular response to DNA damage stimulus", and "regulation of programmed cell death". These findings show the microRNAs that are primarily responsible for controlling the genes of the biological processes described above. We also detected changes in the secretion level of specific cytokines. This study demonstrates that IR activates DNA damage response mechanisms in differentiated cells and that the level of activation is a function of the radiation dose.

Head and Neck Squamous Cell Carcinoma: Epigenetic Landscape.

Head and neck squamous carcinoma (HNSCC) constitutes the sixth most prevalent cancer worldwide. The molecular pathogenesis of HNSCC includes disorders in cell cycle, intercellular signaling, proliferation, squamous cell differentiation and apoptosis. In addition to the genetic mutations, changes in HNSCC are also characterized by the accumulation of epigenetic alterations such as DNA methylation, histone modifications, non-coding RNA activity and RNA methylation. In fact, some of them may promote cancer formation and progression by controlling the gene expression machinery, hence, they could be used as biomarkers in the clinical surveillance of HNSCC or as targets for therapeutic strategies. In this review, we focus on the current knowledge regarding epigenetic modifications observed in HNSCC and its predictive value for cancer development.

hTERT promoter methylation status in peripheral blood leukocytes as a molecular marker of head and neck cancer progression.

Cancer cells, including head and neck cancer cell carcinoma (HNSCC), are characterized by an increased telomerase activity. This enzymatic complex is active in approximately 80-90% of all malignancies, and is regulated by various factors, including methylation status of hTERT gene promoter. hTERT methylation pattern has been thoroughly studied so far. It was proved that hTERT is aberrantly methylated in tumor tissue versus healthy counterparts. However, such effect has not yet been investigated in PBLs (peripheral blood leukocytes) of cancer patients. The aim of this study was to analyze the hTERT gene promoter methylation status in blood leukocytes. DNA was extracted from PBL of 92 patients with histologically diagnosed HNSCC and 53 healthy controls. Methylation status of whole hTERT promoter fragment with independent analysis of each 19 CpG sites was performed using bisulfide conversion technique followed by sequencing of PCR products. Not significant (p = 0.0532) differences in the general frequency of hTERT CpG sites methylation were detected between patients and healthy controls. However, it was discovered that some of analyzed positions (CpG islands: 1 [p = 0.0235], 5 [p = 0.0462], 8 [p = 0.0343]) are significantly more often methylated in HNSCC patients than in controls. The opposite finding was observed in case of CpG position 2 (p = 0.0210). Furthermore, closer analysis of single CpG positions revealed differences in methylation status dependent on anatomical site and TNM classification. To conclude, hTERT promoter methylation status (general or single CpG sites) would be considered as a molecular markers of HNSCC diagnostics.

hTERT gene knockdown enhances response to radio- and chemotherapy in head and neck cancer cell lines through a DNA damage pathway modification.

The aim of the study was to analyze the effect of hTERT gene knockdown in HNSCC cells by using novel in vitro models of head and neck cancer (HNSCC), as well as improving its personalized therapy. To obtain the most efficient knockdown siRNA, shRNA-bearing lentiviral vectors were used. The efficiency of hTERT silencing was verified with qPCR, Western blot, and immunofluorescence staining. Subsequently, the type of cell death and DNA repair mechanism induction after hTERT knockdown was assessed with the same methods, followed by flow cytometry. The effect of a combined treatment with hTERT gene knockdown on Double-Strand Breaks levels was also evaluated by flow cytometry. Results showed that the designed siRNAs and shRNAs were effective in hTERT knockdown in HNSCC cells. Depending on a cell line, hTERT knockdown led to a cell cycle arrest either in phase G1 or phase S/G2. Induction of apoptosis after hTERT downregulation with siRNA was observed. Additionally, hTERT targeting with lentiviruses, followed by cytostatics administration, led to induction of apoptosis. Interestingly, an increase in Double-Strand Breaks accompanied by activation of the main DNA repair mechanism, NER, was also observed. Altogether, we conclude that hTERT knockdown significantly contributes to the efficacy of HNSCC treatment.

Tissue and serum microRNA profile of oral squamous cell carcinoma patients.

Head and neck cancer is characterized by malignant tumors arising from the epithelium covering the upper aerodigestive tract, and the majority of these epithelial malignancies are squamous cell carcinomas (SCCs) of the oral cavity (OSCCs). The aim of the current work was to identify miRNAs regulated in OSCC cancerous tissue when compared to a healthy adjacent tissue and to verify the presence of the same miRNAs in the circulation of these patients. For that serum samples and biopsies of healthy and tumor tissues were collected from five patients diagnosed with OSCC of the oral cavity, RNA was extracted from these samples and microRNAs libraries were prepared and sequenced. A total 255 miRNAs were identified in tissue and 381 different miRNAs were identified in serum samples. When comparing the miRNA expression between tumor and healthy tissue we identified 48 miRNAs (25 down- and 23 up-regulated) that were differentially expressed (FDR < 0.05). From these 48 differentially expressed miRNAs in tissue, 30 miRNAs were also found in the serum of the same patients. hsa-miR-32-5p was up-regulated in tumor compared to healthy tissue in our study, and was previously shown to be up-regulated in the serum of OSCC patients. Therefore, this suggests that miRNAs can be used as potential non-invasive biomarkers of OSCC.

hTERT C250T promoter mutation and telomere length as a molecular markers of cancer progression in patients with head and neck cancer.

Squamous cell carcinoma of the head and neck (HNSCC) is the sixth leading cause of cancer worldwide, representing over half a million incidents every year. Cancer cells, including HNSCC, are characterized by increased telomerase activity. This enzymatic complex is active in ~90% of all cancer types and is responsible for the lengthening of telomeres. Highly recurrent point mutations in the human telomerase reverse transcriptase (hTERT) promoter have recently been reported in a number of human neoplasms. The aim of the present study was to analyze the prevalence of the hTERT promoter C250T mutation and telomere length in the blood leukocytes of 61 patients with HNSCC and 49 healthy individuals. Quantitative polymerase chain reaction identified the hTERT promoter mutation in 36% of patients with HNSCC. To the best of our knowledge this is first report indicating the presence of shorter telomeres in early stage tumors. In addition, the results suggest that the C250T hTERT promoter mutation and telomere length assessment may serve as important molecular markers of HNSCC progression.

RNA interference in head and neck oncology.

Head and neck squamous cell carcinoma (HNSCC) is the sixth leading cause of cancer worldwide. The treatment of choice in case of head and neck cancer is surgery, followed by chemo- or/and radiotherapy. A potentially effective instrument to improve the outcome of numerous diseases, including viral infections, diabetes and cancer, is RNA interference (RNAi). It has been demonstrated that small interfering RNA and microRNA molecules are strongly involved in the regulation of various different pathological processes in cancer development. RNAi has become a valuable research tool allowing a better understanding of the mechanisms regulating cancer pathogenesis. Considering those advantages over other current therapeutics (including specificity and high efficacy), RNAi appears to be a potentially useful tool in cancer treatment. The present review discusses the current knowledge about the possibility of using RNAi in HNSCC therapy.