Towards effectiveness of cell free DNA based liquid biopsy in head and neck squamous cell carcinoma.

Liquid biopsy is a minimally invasive procedure, that uses body fluids sampling to detect and characterize cancer fingerprints. It is of great potential in oncology, however there are challenges associated with the proper handling of liquid biopsy samples that need to be addressed to implement such analysis in patients' care. Therefore, in this study we performed optimization of pre-analytical conditions and detailed characterization of cfDNA fraction (concentration, length, integrity score) in surgically treated HNSCC patients (n = 152) and healthy volunteers (n = 56). We observed significantly higher cfDNA concentration in patients compared to healthy controls (p < 0.0001) and a time dependent decrease of cfDNA concentration after tumor resection. Our results also revealed a significant increase of cfDNA concentration with age in both, healthy volunteers (p = 0.04) and HNSCC patients (p = 0.000002). Moreover, considering the multitude of HNSCC locations, we showed the lack of difference in cfDNA concentration depending on the anatomical location. Furthermore, we demonstrated a trend toward higher cfDNA length (range 35-10380 and 500-10380 bp) in the group of patients with recurrence during follow-up. In conclusion, our study provide a broad characterization of cfDNA fractions in HNSCC patients and healthy controls. These findings point to several aspects necessary to consider when implementing liquid biopsy in clinical practice including: (I) time required for epithelial regeneration to avoid falsely elevated levels of cfDNA not resulting from active cancer, (II) age-related accumulation of nucleic acids accompanied by less efficient elimination of cfDNA and (III) higher cfDNA length in patients with recurrence during follow-up, reflecting predominance of tumor necrosis.

Defining the mutational profile of lower-risk myelodysplastic neoplasm patients with respect to disease progression using next-generation sequencing and pyrosequencing.

Introduction: Lower-risk myelodysplastic neoplasms (LR-MDS) comprise the majority of MDS. Despite favourable prognoses, some patients remain at risk of rapid progression. We aimed to define the mutational profile of LR-MDS using next-generation sequencing (NGS), Sanger Sequencing (SSeq), and pyrosequencing. Material and methods: Samples from 5 primary LR-MDS (67 exons of SF3B1, U2AF1, SRSF2, ZRSR2, TET2, ASXL1, DNMT3A, TP53, and RUNX1 genes) were subjected to NGS. Next, a genomic study was performed to test for the presence of identified DNA sequence variants on a larger group of LR-MDS patients (25 bone marrow [BM], 3 saliva [SAL], and one peripheral blood [PB] sample/s). Both SSeq (all selected DNA sequence variants) and pyrosequencing (9 selected DNA sequence variants) were performed. Results: Next-generation sequencing results identified 13 DNA sequence variants in 7 genes, comprising 8 mutations in 6 genes (ASXL1, DNMT3A, RUNX1, SF3B1, TET2, ZRSR2) in LR-MDS. The presence of 8 DNA variants was detected in the expanded LR-MDS group using SSeq and pyrosequencing. Mutation acquisition was observed during LR-MDS progression. Four LR-MDS and one acute myeloid leukaemia myelodysplasia-related patient exhibited the presence of at least one mutation. ASXL1 and SF3B1 alterations were most commonly observed (2 patients). Five DNA sequence variants detected in BM (patients: 9, 13) were also present in SAL. Conclusions: We suggest using NGS to determine the LR-MDS mutational profile at diagnosis and suspicion of disease progression. Moreover, PB and SAL molecular testing represent useful tools for monitoring LR-MDS at higher risk of progression. However, the results need to be confirmed in a larger group.

Global DNA Methylation Profiling Reveals Differentially Methylated CpGs between Salivary Gland Pleomorphic Adenomas with Distinct Clinical Course.

Pleomorphic adenomas (PAs) are the most frequently diagnosed benign salivary gland tumors. Although the majority of PAs are characterized by slow growth, some develop very fast and are more prone to recur. The reason for such differences remains unidentified. In this study, we performed global DNA methylation profiling using the Infinium Human Methylation EPIC 850k BeadChip Array (Illumina) to search for epigenetic biomarkers that could distinguish both groups of tumors. The analysis was performed in four fast-growing tumors (FGTs) and four slow-growing tumors (SGTs). In all, 85 CpG dinucleotides differentiating both groups were identified. Six CpG tags (cg06748470, cg18413218, cg10121788, cg08249296, cg18455472, and cg19930657) were selected for bisulfite pyrosequencing in the extended group of samples. We confirmed differences in DNA methylation between both groups of samples. To evaluate the potential diagnostic accuracy of the selected markers, ROC curves were constructed. We indicated that CpGs included in two assays showed an area under the curve with an acceptable prognostic value (AUC > 0.7). However, logistic regression analysis allowed us to indicate a more optimal model consisting of five CpGs ((1) cg06748470, (2) cg00600454, (3) CpG located in chr14: 77,371,501−77,371,502 (not annotated in GRCh37/hg19), (4) CpG2 located in chr16: 77,469,589−77,469,590 (not annotated GRCh37/hg19), and (5) cg19930657) with AUC > 0.8. This set of epigenetic biomarkers may be considered as differentiating factors between FGT and SGT during salivary gland tumor diagnosis. However, this data should be confirmed in a larger cohort of samples.

Loss of the MAF Transcription Factor in Laryngeal Squamous Cell Carcinoma.

MAF is a transcription factor that may act either as a tumor suppressor or as an oncogene, depending on cell type. We have shown previously that the overexpressed miR-1290 influences MAF protein levels in LSCC (laryngeal squamous cell carcinoma) cell lines. In this study, we shed further light on the interaction between miR-1290 and MAF, as well as on cellular MAF protein localization in LSCC. We confirmed the direct interaction between miR-1290 and MAF 3'UTR by a dual-luciferase reporter assay. In addition, we used immunohistochemistry staining to analyze MAF protein distribution and observed loss of MAF nuclear expression in 58% LSCC samples, of which 10% showed complete absence of MAF, compared to nuclear and cytoplasmatic expression in 100% normal mucosa. Using TCGA data, bisulfite pyrosequencing and CNV analysis, we excluded the possibility that loss-of-function mutations, promoter region DNA methylation or CNV are responsible for MAF loss in LSCC. Finally, we identified genes involved in the regulation of apoptosis harboring the MAF binding motif in their promoter region by applied FIMO and DAVID GO analysis. Our results highlight the role of miR-1290 in suppressing MAF expression in LSCC. Furthermore, MAF loss or mislocalization in FFPE LSCC tumor samples might suggest that MAF acts as a LSCC tumor suppressor by regulating apoptosis.

Laryngeal squamous cell carcinoma cell lines show high tolerance for siRNA-mediated CDK1 knockdown.

Alterations of the cell cycle checkpoints lead to uncontrolled cell growth and result in tumorigenesis. One of the genes essential for cell proliferation and cell cycle regulation is CDK1. This makes it a potential target in cancer therapy. In our previous study we have shown upregulation of this gene in laryngeal squamous cell carcinoma (LSCC). Here we analyze the impact of siRNA-mediated CDK1 knockdown on cell proliferation and viability, measured with cell growth monitoring and colorimetric test (CCK8 assay), respectively. We proved that a reduction of CDK1 expression by more than 50% has no effect on these cellular processes in LSCC cell lines (n=2). Moreover, using microarrays, we analyzed global gene expression deregulation in these cell lines after CDK1 knockdown. We searched for enriched ontologies in the group of identified 137 differentially expressed genes (>2-fold change). Within this group we found 3 enriched pathways: protein binding (GO:0005515), mitotic nuclear division (GO:0007067) and transmembrane receptor protein tyrosine kinase signaling pathway (GO:0007169) and a group of 11 genes encoding proteins for which interaction with CDK1 was indicated with the use of bioinformatic tools. Among these genes we propose three: CDK6, CALD1 and FYN as potentially dependent on CDK1.

New genetic variants of TET2 and ASXL1 identified by next generation sequencing and pyrosequencing in a patient with MDS-RS-MLD and secondary acute myeloid leukemia.

Myelodysplastic syndromes (MDS) are a heterogeneous group of myeloid neoplasms characterized by the presence of cytopenias, ineffective hematopoiesis and frequent transformation into secondary acute myeloid leukemia (secAML). Recent genomic studies provide unprecedented insight into the molecular landscape of clonal proliferation in MDS. Genetic diversity of both MDS and secAML subclones cannot be defined by a single somatic mutation. Mutations of the founding clone may survive over implemented chemotherapy and allogenic hematopoietic cell transplantation (alloHCT), but new subclonal mutations may also appear. Next generation sequencing (NGS) makes it possible to define the mutational profile of disease subclones during the treatment course and has a potential in pre- and post-alloHCT monitoring. Understanding the molecular pathophysiology of MDS may soon allow for monitoring the course of disease and personalized treatment depending on the mutational landscape. In the present paper we report, for the first time in MDS, ASXL1 c.1945G>T, TET2 c.4044+2dupT and c.4076G>T sequence variants. Moreover, we detected RUNX1 c.509-2A>C and SF3B1 c.1874G>T sequence variants. Furthermore, we verify the clinical utility of NGS and pyrosequencing in MDS and secAML.

Copy number gains of the putative CRKL oncogene in laryngeal squamous cell carcinoma result in strong nuclear expression of the protein and influence cell proliferation and migration.

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 < 0.01) as well as cell migration (p < 0.05) rates. Altogether, these results show that the aberrantly strong nuclear localization of CRKL is a seldom but recurrent phenomenon in LSCC resulting from the increased DNA copy number and overexpression of the gene. Moreover, functional analyses suggest that proliferation and migration of the tumor cells depend on CRKL expression.

Justification of direct Sanger sequencing application for detection of KIT and PDGFRα gene mutations in formalin-fixed, paraffin-embedded samples from gastrointestinal stromal tumours.

AIMS: The knowledge concerning genetic background of gastrointestinal stromal tumours (GISTs) is well recognised, and the accurate detection of KIT and PDGFRα mutations is of great importance for the process of disease diagnosis and patient's treatment. In this study, we compare the usefulness of real-time PCR-based techniques and Sanger sequencing to detect mutations of both genes in 41 formalin-fixed, paraffin-embedded GIST samples. METHODS: The analysis encompassed most frequently mutated coding regions of KIT (exons 9, 11, 13 and 17) and PDGFRα (exons 12, 14 and 18) genes. The GIST Mutation Detection Kit (EntroGen), direct Sanger sequencing and high-resolution melting (HRM) analysis were applied to conduct the study. RESULTS: With the application of EntroGen kit, we found alterations in 22/38 samples, with Sanger sequencing variants were found in 36/41 samples. The concordant results for both methods were observed in 19/38 samples. With subsequently applied HRM analysis, we have confirmed that all samples, except one, harboured alterations in the regions indicated by Sanger sequencing. CONCLUSIONS: Our results show that in GIST samples, carrying a broad spectrum of deletions, Sanger sequencing is a better, more sensitive method for mutational analysis of KIT and PDGFRα genes.

DNA repair in cancer initiation, progression, and therapy-a double-edged sword.

Genomic and mitochondrial DNA molecules are exposed continuously for a damaging activity of chemical, physical, and internal genotoxicants. When DNA repair machinery is not working efficiently, the generation of DNA lesions and mutations leads to carcinogenic transformation. The high number of mutation going up to 105 per cell was recognized as a driving force of oncogenesis. Moreover, a high activity of DNA repair genes was hypothesized as a predisposition to metastasis. DNA repair potential has to be taken into account attempting to chemo- and/or radiotherapy. A low activity of DNA repair genes makes tumor cells more sensitive to therapy, but on the other hand, non-tumor cells getting lesions could form second primary cancer. Contrary, high activity of DNA repair genes counteracts attempted therapy. It means an individualized therapy based on recognition of DNA repair potential is recommended.

Expression of Serpin Peptidase Inhibitor B2 (SERPINB2) is regulated by Aryl hydrocarbon receptor (AhR).

Aryl hydrocarbon receptor (AhR) is a highly conserved ligand-activated transcription factor with high affinity to aromatic planar compounds, such as ß-naphthoflavone (BNF), benzo[a]pyrene (BaP) or dioxin (TCDD). After binding the ligand, AhR triggers induction of the expression of phase I and phase II drug-metabolizing genes, together with numerous other genes that are not directly involved in the metabolism of xenobiotics. Several studies have shown that AhR plays a role in tumor initiation, promotion and progression, but the molecular mechanisms involved in these processes are not fully understood. A previous study from our laboratory indicated that the SERPINB2 gene is presumably regulated by AhR. To prove that such induction is really AhR-dependent, in the present study we knocked down the expression of AhR by stable transfection of a laryngeal squamous cell carcinoma cell line (UT-SCC-34) with shRNA, resulting in 92% reduction of BNF-induced expression of SERPINB2. However, in silico analysis did not reveal AhR-dependent responsive elements in the promoter of the SERPINB2 gene. Therefore, to address this problem, we have used cycloheximide, an inhibitor of translation, and our results clearly indicate that an additional, newly synthesized protein is involved in AhR-dependent induction of SERPINB2 expression by BNF. So, to exclude that AhR binds to the putative xenobiotic-responsive elements (XREs) localized upstream of the SERPINB2 gene, we performed chromatin immunoprecipitation assays. As expected, we found no direct binding of AhR to its responsive elements in the vicinity of the SERPINB2 gene, further demonstrating the indirect SERPINB2 induction by AhR. However, the further analysis demonstrated that the expression of the enhancer RNA encoded by the region of DNA 20 kbp upstream from the SERPINB2 gene was AhR-dependent. Although AhR-mediated SERPINB2 induction clearly requires the synthesis of an additional protein, the kinetics of SERPINB2 induction is as fast as the kinetics of CYP1A1 and CYP1B1 induction (both genes directly regulated by AhR). Therefore, given previous studies regarding the induction of SERPINB2 expression by bacterial lipopolysaccharides (LPS), we think that, similarly, the interaction with pause-release proteins may be responsible for AhR-dependent regulation of SERPINB2 expression.

Oral-genital human papillomavirus infection in Polish couples: frequent detection of HPV 42.

BACKGROUND: Human papillomavirus (HPV) contributes to the development of cervical and oropharyngeal tumors. The increased incidence of HPV associated oropharyngeal tumors is lately being observed also in Polish population. The worldwide distribution of HPV varies and the studies rarely combine analysis of virus genotypes in both: genital and oropharyngeal locations. Therefore, in our study, we investigated HPV distribution in both anatomical sites of females with previous history of cervical cancer or with pre-cancerous lesion and their partners to establish the dominant types in Polish couples in genital and oropharyngeal areas, as they can be easily sexually transmitted. METHODS: The study group consisted of 197 females and their partners. Each female had current or previous cervical pathology and HPV detected in gynecological swab with the use of Anyplex™ II HPV28 Detection system. This system is based on multiplexed real time PCR and enables detection of 19 high-risk and 9 low-risk HPVs. RESULTS: Beside women, the virus was found in 114/197 of men in their foreskin swabs. Additionally, HPV was detected in oropharyngeal swabs of 39/197 female and 56/197 male participants. HPV 16/31/42/39/54 dominated in female and HPV 66/42/16/31/53 in male genital locations. The incidence of HPV in oropharynx was lower, top five genotypes included: HPV 6/39/42/35/16 in women compared to HPV 39/6/42/40/33 in men. HPV16 was the most frequently detected virus type, found in 70/197 examined cervical swabs. It was significantly more prevalent as single infection in females, previously treated for the cervical cancer (p = 0.035). Moreover, regular presence of low risk type 42 was noticeable in both sexes, in both kind of swabs. There was a trend observed towards its prevalence as single infectious agent in women with previous history of cervical cancer (p = 0.069). CONCLUSIONS: Our results showed the distribution of HPV genotypes in Polish couples, in which each woman is HPV positive, indicating a common infection of HPV 42, regardless of sex and anatomical site. These findings shed new light on HPV 42 significance, however they should be verified on a larger group of Polish participants, followed regularly in 6 months intervals, in oral as well as in genital areas.

Expression of ELF1, a lymphoid ETS domain-containing transcription factor, is recurrently lost in classical Hodgkin lymphoma.

Loss of B cell-specific transcription factors (TFs) and the resulting loss of B-cell phenotype of Hodgkin and Reed-Sternberg (HRS) cells is a hallmark of classical Hodgkin lymphoma (cHL). Here we have analysed two members of ETS domain containing TFs, ELF1 and ELF2, regarding (epi)genomic changes as well as gene and protein expression. We observed absence or lower levels of ELF1 protein in HRS cells of 31/35 (89%) cases compared to the bystander cells and significant (P < 0·01) downregulation of the gene on mRNA as well as protein level in cHL compared to non-cHL cell lines. However, no recurrent loss of ELF2 protein was observed. Moreover, ELF1 was targeted by heterozygous deletions combined with hypermethylation of the remaining allele(s) in 4/7 (57%) cell lines. Indeed, DNA hypermethylation (range 95-99%, mean 98%) detected in the vicinity of the ELF1 transcription start site was found in all 7/7 (100%) cHL cell lines. Similarly, 5/18 (28%) analysed primary biopsies carried heterozygous deletions of the gene. We demonstrate that expression of ELF1 is impaired in cHL through genetic and epigenetic alterations, and thus, it may represent an additional member of a TF network whose downregulation contributes to the loss of B-cell phenotype of HRS cells.

Downregulation of CEACAM6 gene expression in laryngeal squamous cell carcinoma is an effect of DNA hypermethylation and correlates with disease progression.

We have turned our attention to CEACAM6 gene, already described as deregulated in various types of cancer. By using the expression microarrays performed on the set of 16 laryngeal squamous cell carcinoma (LSCC) samples: 11 cell lines and 5 primary tumors we have shown downregulation of CEACAM6 gene as compared to non cancer controls from head and neck region. CEACAM6 gene downregulation, further confirmed by quantitative PCR on 25 LSCC cell lines, was observed in cell lines derived from recurrent tumors in comparison to controls. A significant gene downregulation was observed in cell lines derived from advanced, high grade tumors in comparison to controls. Intrigued by the recurrent transcriptional loss of CEACAM6 we searched for the mechanism potentially responsible for its downregulation and hence we analyzed DNA copy number changes (a-CGH), promoter DNA methylation status and occurrence of gene mutations (in silico). Neither the analysis of gene copy number, nor the mutation screen has shown recurrent deletions or mutations, that could contribute to the observed downregulation of the gene. However, by using bisulfite pyrosequencing, we have shown DNA hypermethylation (mean DNA methylation > 78%) of CEACAM6 promoter region in 9/25 (36%) LSCC cell lines. Importantly, the 5-aza-2-deoxycytidine-induced inhibition of DNA methylation resulted in restoration of CEACAM6 expression in the two LSCC cell lines on mRNA level. In summary, we have shown that recurrent downregulation of CEACAM6 in LSCC is dependent on the gene's promoter DNA methylation and is observed predominantly in large, poorly differentiated tumors and recurrences.

Single nucleotide polymorphism rs11614913 associated with CC genotype in miR-196a2 is overrepresented in laryngeal squamous cell carcinoma, but not salivary gland tumors in Polish population.

The miRNA-196a2 has shown significance in the development of various neoplasms, including head and neck squamous cell carcinoma (HNSCC). The oncogenic functionality of this miRNA is mediated via its potential to target annexin A1 mRNA, a tumor suppressor gene involved in inhibition of the NF-κB pathway. Interestingly, recent data indicate a susceptibility for aforementioned neoplasms in patients with the CC genotype vs the CT and TT genotypes of the rs11614913 SNP located within the DNA sequence of the miR-196a2 that results in elevated expression of the gene. To further investigate this phenomenon, we genotyped this SNP in 40 patients with laryngeal squamous cell carcinoma (LSCC), the most common tumor of the head and neck region and 60 patients with salivary gland tumors (SGT) that show a yet unexplained incidence increase in the last two decades. In agreement with previous reports, we have identified a statistically significant (p < 0.05) overrepresentation of the CC genotype in LSCC patients and demonstrated in LSCC cell lines that it results in elevated expression of miR-196a2 as compared to cell lines with the TT genotype of the respective SNP. Importantly, none of these correlations was found in patients with SGT. These findings underline the importance of the SNP rs11614913 for LSCC development in the Polish population and moreover highlight the different genetic background of the two studied neoplasms of the head and neck region.

Recurrent transcriptional loss of the PCDH17 tumor suppressor in laryngeal squamous cell carcinoma is partially mediated by aberrant promoter DNA methylation.

Protocadherins are cell-cell adhesion molecules encoded by a large family of genes. Recent reports demonstrate recurrent silencing of protocadherin genes in tumors and provide strong arguments for their tumor supresor functionality. Loss of protocadherins may contribute to cancer development not only by altering cell-cell adhesion, that is a hallmark of cancer, but also by enhancing proliferation and epithelial mesenchymal transition of cells via deregulation of the WNT signaling pathway. In this study we have further corroborated our previous findings on the involvement of PCDH17 in laryngeal squamous cell carcinoma (LSCC). We used bisulfite pyrosequencing to analyze a cohort of primary LSCC tumors for alterations in PCDH17 promoter DNA methylation as an alternative gene inactivation mechanism to the homozygous deletions reported earlier. Moreover, we analyzed primary LSCC samples by immunohistochemistry for PCDH17 protein loss. We identified recurrent elevation of PCDH17 promoter DNA methylation in 32/81 (40%) primary tumors (P < 0.001) and therein hypermethylation of 12 (15%) cases in contrast to no tumor controls (n = 24) that were all unmethylated. Importantly, DNA demethylation by decitabine has restored low level PCDH17 expression in LSCC cell lines. In conclusion, we provide a mechanistic explanation of recurrently observed PCDH17 silencing in LSCC by demonstrating the role of promoter methylation in this process. In light of these findings and recent reports showing that PCDH17 methylation is detectable in serum of cancer patients we suggest that testing PCDH17 DNA methylation might serve as a potential biomarker in LSCC.

Frequent methylation of DAB2, a Wnt pathway antagonist, in oral and oropharyngeal squamous cell carcinomas.

BACKGROUND: Aberrations in Wnt signaling pathway are related to the pathogenesis of head and neck carcinomas and their activation frequently results from epigenetic alterations. This study aimed to assess the frequency of the methylation of DAB2, which acts as a negative regulator of Wnt signaling, and correlate it with clinicopathological features in a group of oral cancer patients. MATERIAL AND METHODS: Forty nine patients with primary oral squamous cell carcinoma were enrolled in the study. DNA samples were isolated from surgical sections using phenol-chloroform extraction. Methylation-specific PCR was used to detect gene promoter methylation. RESULTS: The analysis of the occurrence of DAB2 promoter methylation in primary oral carcinomas indicated that the gene is methylated in 70% of cases. However, no correlation was found between its methylation and TNM staging or overall survival. CONCLUSIONS: Our findings corroborate that DAB2 is a frequent target of epigenetic silencing in oral carcinomas and may be potentially used for tumor detection.

Combined deletion and DNA methylation result in silencing of FAM107A gene in laryngeal tumors.

Larynx squamous cell carcinoma (LSCC) is characterized by complex genotypes, with numerous abnormalities in various genes. Despite the progress in diagnosis and treatment of this disease, 5-year survival rates remain unsatisfactory. Therefore, the extended studies are conducted, with the aim to find genes, potentially implicated in this cancer. In this study, we focus on the FAM107A (3p14.3) gene, since we found its significantly reduced expression in LSCC by microarray profiling (Affymetrix U133 Plus 2.0 array). By RT-PCR we have confirmed complete FAM107A downregulation in laryngeal cancer cell lines (15/15) and primary tumors (21/21) and this finding was further supported by FAM107A protein immunohistochemistry (15/15). We further demonstrate that a combined two hit mechanism including loss of 3p and hypermethylation of FAM107A promoter region (in 9/15 cell lines (p < 0.0001) and in 15/21 primary tumors (p < 0.0001)) prevails in the gene transcriptional loss. As a proof of principle, we show that Decitabine - a hypomethylating agent - restores FAM107A expression (5 to 6 fold increase) in the UT-SCC-29 cell line, characterized by high DNA methylation. Therefore, we report the recurrent inactivation of FAM107A in LSCC, what may suggest that the gene is a promising tumor suppressor candidate involved in LSCC development.

Recurrent epigenetic silencing of the PTPRD tumor suppressor in laryngeal squamous cell carcinoma.

Cellular processes like differentiation, mitotic cycle, and cell growth are regulated by tyrosine kinases with known oncogenic potential and tyrosine phosphatases that downmodulate the first. Therefore, tyrosine phosphatases are recurrent targets of gene alterations in human carcinomas. We and others suggested recently a tumor suppressor function of the PTPRD tyrosine phosphatase and reported homozygous deletions of the PTPRD locus in laryngeal squamous cell carcinoma. In this study, we investigated other gene-inactivating mechanisms potentially targeting PTPRD, including loss-of-function mutations and also epigenetic alterations like promoter DNA hypermethylation. We sequenced the PTPRD gene in eight laryngeal squamous cell carcinoma cell lines but did not identify any inactivating mutations. In contrast, by bisulfite pyrosequencing of the gene promoter region, we identified significantly higher levels of methylation (p = 0.001 and p = 0.0002, respectively) in 9/14 (64%) laryngeal squamous cell carcinoma cell lines and 37/79 (47%) of primary laryngeal squamous cell carcinoma tumors as compared to normal epithelium of the upper aerodigestive tract. There was also a strong correlation (p = 0.0001) between methylation and transcriptional silencing for the PTPRD gene observed in a cohort of 497 head and neck tumors from The Cancer Genome Atlas dataset suggesting that DNA methylation is the main mechanism of PTPRD silencing in these tumors. In summary, our data provide further evidence of the high incidence of PTPRD inactivation in laryngeal squamous cell carcinoma. We suggest that deletions and loss-of-function mutations are responsible for PTPRD loss only in a fraction of cases, whereas DNA methylation is the dominating mechanism of PTPRD inactivation.

Prognostic significance of the methylation of Wnt pathway antagonists-CXXC4, DACT2, and the inhibitors of sonic hedgehog signaling-ZIC1, ZIC4, and HHIP in head and neck squamous cell carcinomas.

OBJECTIVES: Aberrations in Wnt and Shh signaling pathways are related to the pathogenesis of head and neck carcinomas, and their activation frequently results from epigenetic alterations. This study aimed to assess the frequency of methylation of negative regulators of Wnt signaling: CXXC4, DACT2, HDPR1, and FBXW11 and Shh signaling: HHIP, PTCH1, SUFU, ZIC1, and ZIC4 and correlate it with clinicopathological features in this group of patients. MATERIALS AND METHODS: Methylation-specific PCR was used to detect gene promoter methylation, and real-time PCR was used to assess gene expression level. RESULTS: The analysis of the occurrence of gene promoter methylation in head and neck carcinoma cell lines indicated that CXXC4, DACT2, HHIP, ZIC1, and ZIC4 are methylated in these tumors. These genes were further analyzed in tumor sections from oral and laryngeal cancer patients. Gene methylation rate was higher in laryngeal tumors. The methylation index in tumor samples correlated with the overall survival in a subgroup of oral cancer patients who died of the disease. Moreover, ZIC4 methylation correlated with lymph node involvement in oral cancer patients. CONCLUSIONS: Our findings corroborate that the activation of Wnt signaling in head and neck squamous cell carcinoma (HNSCC) is related to epigenetic silencing of its negative regulators. Moreover, the results indicate that the same mechanism of activation may operate in the case of Shh signaling. CLINICAL RELEVANCE: The methylation of ZIC4 may be considered a new prognostic marker in oral cavity and oropharyngeal tumors. Further investigations should determine the diagnostic significance of methylation of ZIC4, HHIP, and DACT2 in head and neck carcinomas.

Induction of expression of aryl hydrocarbon receptor-dependent genes in human HepaRG cell line modified by shRNA and treated with ß-naphthoflavone.

The aryl hydrocarbon receptor (AhR) mediates a variety of biological responses to ubiquitous environmental pollutants. In this study, the effects of administration of ß-naphthoflavone (BNF), a potent AhR ligand, on the expression of AhR-dependent genes were examined by microarray and qPCR analysis in both, differentiated and undifferentiated HepaRG cell lines. To prove that BNF-induced changes of investigated genes were indeed AhR-dependent, we knock down the expression of AhR by stable transfection of HepaRG cells with shRNA. Regardless of genetical identity, our results clearly demonstrate different expression profiles of AhR-dependent genes between differentiated and undifferentiated HepaRG cells. Genes involved in metabolism of xenobiotics constitute only minute fraction of all genes regulated by AhR in HepaRG cells. Participation of AhR in induction of expression of genes associated with regulation of apoptosis or involved in cell proliferation as well as AhR-dependent inhibition of genes connected to cell adhesion could support suggestion of involvement of AhR not only in initiation but also in progression of carcinogenesis. Among the AhR-dependent genes known to be involved in metabolism of xenobiotics, cytochromes P4501A1 and 1B1 belong to the most inducible by BNF. On the contrary, expression of GSTA1 and GSTA2 was significantly inhibited after BNF treatment of HepaRG cells. Among the AhR-dependent genes that are not involved in metabolism of xenobiotics SERPINB2, STC2, ARL4C, and TIPARP belong to the most inducible by BNF. Our results imply involvement of Ah receptor in regulation of CYP19A1, the gene-encoding aromatase, and an enzyme responsible for a key step in the biosynthesis of estrogens.