Search

DNA methylation was shown previously to be a crucial mechanism responsible for transcriptional deregulation in the pathogenesis of classical Hodgkin lymphoma (cHL). To identify epigenetically inactivated miRNAs in cHL, we have analyzed the set of miRNAs downregulated in cHL cell lines using bisulfite pyrosequencing. We focused on miRNAs with promoter regions located within or &lt, 1000 bp from a CpG island. Most promising candidate miRNAs were further studied in primary Hodgkin and Reed-Sternberg (HRS) cells obtained by laser capture microdissection. Last, to evaluate the function of identified miRNAs, we performed a luciferase reporter assay to confirm miRNA: mRNA interactions and therefore established cHL cell lines with stable overexpression of selected miRNAs for proliferation tests. We found a significant reverse correlation between DNA methylation and expression levels of mir-339-3p, mir-148a-3p, mir-148a-5p and mir-193a-5 demonstrating epigenetic regulation of these miRNAs in cHL cell lines. Moreover, we demonstrated direct interaction between miR-148a-3p and IL15 and HOMER1 transcripts as well as between mir-148a-5p and SUB1 and SERPINH1 transcripts. Furthermore, mir-148a overexpression resulted in reduced cell proliferation in the KM-H2 cell line. In summary, we report that mir-148a is a novel tumor suppressor inactivated in cHL and that epigenetic silencing of miRNAs is a common phenomenon in cHL.

Laryngeal squamous cell carcinoma is a major medical problem worldwide. Although our understanding of genetic changes and their consequences in laryngeal cancer has opened new therapeutic pathways over the years, the diagnostic as well as treatment options still need to be improved. In our previous study, we identified CRKL (22q11) as a novel putative oncogene overexpressed and amplified in a subset of LSCC tumors and cell lines. Here we analyze to what extent CRKL DNA copy number gains correlate with the higher expression of CRKL protein by performing IHC staining of the respective protein in LSCC cell lines (n = 3) and primary tumors (n = 40). Moreover, the importance of CRKL gene in regard to proliferation and motility of LSCC cells was analyzed with the application of RNA interference (siRNA). Beside the physiological cytoplasmic expression, the analysis of LSCC tumor samples revealed also nuclear expression of CRKL protein in 10/40 (25%) cases, of which three (7.5%), presented moderate or strong nuclear expression. Similarly, we observed a shift towards aberrantly strong nuclear abundance of the CRKL protein in LSCC cell lines with gene copy number amplifications. Moreover, siRNA mediated silencing of CRKL gene in the cell lines showing its overexpression, significantly reduced proliferation (p CRKL expression.

In this study, we analyzed the expression profile of four genes (CCNA2, CCNB1, CCNB2, and CDK1) in laryngeal squamous cell carcinoma (LSCC) cell lines and tumor samples. With the application of microarray platform, we have shown the overexpression of these genes in all analyzed LSCC samples in comparison to non-cancer controls from head and neck region. We have selected CDK1 for further analysis, due to its leading role in cell cycle regulation. It is a member of the Ser/Thr protein kinase family of proven oncogenic properties. The results obtained for CDK1 were further confirmed with the application of reverse transcription quantitative polymerase chain reaction (RT-qPCR) technique, Western blot, and immunohistochemistry (IHC). The observed upregulation of CDK1 in laryngeal squamous cell carcinoma has encouraged us to analyze for genetic mechanisms that can be responsible this phenomenon. Therefore, with the application of array-CGH, sequencing analysis and two methods for epigenetic regulation analysis (DNA methylation and miRNA expression), we tried to identify such potential mechanisms. Our attempts to identify the molecular mechanisms responsible for observed changes failed as we did not observe significant alterations neither in the DNA sequence nor in the gene copy number that could underline CDK1 upregulation. Similarly, the pyrosequencing and miRNA expression analyses did not reveal any differences in methylation level and miRNA expression, respectively; thus, these mechanisms probably do not contribute to elevation of CDK1 expression in LSCC. However, our results suggest that alteration of CDK1 expression on both mRNA and protein level probably appears on the very early step of carcinogenesis. Electronic supplementary material The online version of this article (doi:10.1007/s13277-016-4991-4) contains supplementary material, which is available to authorized users.

Introduction Laryngeal cancer (LSCC) is one of the most common tumours among head and neck squamous cell carcinomas. Recently, analysis of the meaning of epigenetic alterations in LSCC development has gained momentum. In this field we have shown recurrent overexpression of the miR-1290 in LSCC. Therefore, in this study we aim at clarifying the oncomiR functionality of miR-1290 by analysing its interaction with the transcriptional modulator MAF – a putative suppressor gene. Material and methods The identification of miR-1290 overexpression in LSCC was based on global miRNA expression profiles (Agilent) of 16 LSCC cell lines and 3 non-tumour controls which were validated on 50 tumour samples by real-time qPCR (RT-qPCR) with LNA modified primers. Target genes of miR-1290 were selected by in silico analysis using miRDB (TS >90) and miRWALK (gene listed in at least 2 in 4 databases). The influence of miR-1290 on its putative target gene MAF was analysed by transient transfection of 2 LSCC cell lines using miRNA inhibitor and successive expression analysis of MAF in the transfected cells by RT-qPCR and Western blot. To confirm the downregulation of MAF in primary LSCC we conducted RT-qPCR on 22 tumour samples and 5 non-tumour controls. In addition, the nuclear MAF protein abundance was evaluated by immunohistochemistry on tissue microarray containing 128 LSCC tumour samples and 19 tumour free resection margins. Results and discussions Based on the in sillico analysis MAF has been selected as a the putative miR-1290 target gene with high suppressor potential. Importantly, MAF is a transcription factor, involved in regulation of apoptosis and TP53 expression, processes deregulated in LSCC. Functional analyses, showed that inhibition of miR-1290 resulted in 3.47 fold increase in MAF mRNA expression and 50% increase of MAF protein abundance. Moreover, by using RT-qPCR we have shown recurrent MAF downregulation in LSCC tumour samples compared to non-tumour controls. In line with these findings we have indicated by immunohistochemical staining that 74/128 LSCC tumour samples were characterised by lack of nuclear expression of MAF in opposite to nuclear expression observed in 19/19 margins. Conclusion Our results demonstrate MAF as a new potential tumour suppressor gene epigenetically downregulated in LSCC by the overexpression of miR-1290. Further studies are required for fully understanding the role of MAF in LSCC pathogenesis.

The aryl hydrocarbon receptor (AhR) mediates a variety of biological responses to ubiquitous environmental pollutants. In this study the effect of administration of β-naphthoflavone (BNF), potent AhR ligand, on the expression of AhR, AhRR, CYP1A1, CYP1A2, CYP1B1, NQO1, GSTA1, ALDH3A1 and UGT1A genes encoding the enzymes controlled by AhR were examined in thirteen laryngeal tumor cell lines and in HepaRG cell line. The analyzed cell lines were derived from patients with squamous laryngeal cancer, with history of cigarette smoking and without signs of human papillomavirus types 16 and 18 infection in investigated cells. Quantitative real-time RT-PCR analysis revealed huge interindividual differences in expression of genes from AhR regulatory network. Our results strongly suggest predominant effect of DNA methylation on induction of CYP1A1 expression by AhR ligands as well. Our results indicate that differentiated HepaRG cell line appeared to be very good substitute for human liver in studies on xenobiotic metabolism by AhR regulated enzymes.

Relapse and metastasis are the main causes of unfavorable outcome in head and neck cancers. Whereas, understanding of the molecular background of these processes is far from being complete. Therefore, in this study we aimed to identify potential biomarker candidates of relapse and metastasis in laryngeal squamous cell carcinoma (LSCC) by combining the 2D electrophoresis based protein screen and immunohistochemical analysis of candidate proteins. We screened three groups of LSCC cell lines derived from primary tumors, recurrent tumors and metastases and identified seven proteins that differed significantly in relative abundance between the analyzed groups. Among the identified proteins were the heat shock proteins HSP60 and HSP70 that were significantly downregulated both in recurrences- and metastases-derived cell lines but not in primary tumor-derived cell lines. Moreover, we identified significant upregulation of the annexin V, calreticulin and the inorganic pyrophosphatase (PPA1) exclusively in the metastases-derived cell lines. As these upregulated proteins could potentially become novel biomarkers of metastasis, we have compared their abundance in primary tumor LSCC N(0) cases, primary tumor LSCC N(+) cases as well as in LSCC metastases N(+). Our results show an intense increase of cytoplasmic PPA1 abundance in the N(+) (p = 0.000042) compared to the N(0) group. In summary, we show a group of proteins deregulated in recurrences and metastases of LSCC. Moreover, we suggest the PPA1 protein as a potential new biomarker for metastasis in this cancer. Electronic supplementary material The online version of this article (doi:10.1007/s00726-016-2201-8) contains supplementary material, which is available to authorized users.