Advanced adenoid cystic carcinoma (ACC) is featured by SWI/SNF chromatin remodeling complex aberrations.

PURPOSE: Adenoid cystic carcinoma (ACC) is a rare neurotropic cancer with slow progression occurring in salivary glands and less frequently in other body parts. ACC is featured by hyperchromatic nuclei and various mutations in genes encoding chromatin-related machineries. The ACC treatment is mainly limited to the radical surgery and radiotherapy while the chemotherapy remains ineffective. As the knowledge about molecular basis of ACC development is limited, we investigated here the molecular features of this disease. PATIENTS AND METHODS: This study included 50 patients with ACC. Transcript profiling of available ACC samples vs normal salivary gland tissue, quantitative real-time PCR (qRT-PCR) transcript level measurements and the immunohistochemistry (IHC) for SWI/SNF chromatin remodeling complex (CRC) subunits and androgen receptor on surgery-derived paraffin-embedded samples were performed. RESULTS: Transcriptomic study followed by Gene Ontology classification indicated alteration of chromatin-related processes, including downregulated transcript levels of main SWI/SNF CRC subunits and elevated expression of BRM ATPase-coding SMARCA2 gene in ACC. Subsequent IHC indicated broad accumulation of BRM ATPase and several SWI/SNF subunits, suggesting affected control of their protein level in ACC. The IHC revealed ectopic, heterogeneous expression of androgen receptor (AR) in some ACC cells. CONCLUSIONS: Our study indicated that ACC features aberrant expression of genes controlling chromatin status and structure. We found that the balance between SWI/SNF classes is moved towards the BRM ATPase-containing complex in ACC. As BRM is known to be involved in chemoresistance in cancer cells, this observation may be the likely explanation for ACC chemoresistance.

Telomere length and TERT abnormalities in pituitary adenomas.

OBJECTIVES: Pituitary adenomas (PAs) are among the most frequent intracranial tumors in humans. Abnormal telomerase activity and telomere lengthening are features of tumor cells. They may result from mutations in TERT promoter region, gene amplification or aberrant DNA methylation pattern. Such changes were found in variety of tumors including those of brain. Aim of the study was to evaluate the incidence of TERT abnormalities and to assess their role in telomere lengthening in PAs. METHODS: Study involved 101 patients with PA including both nonfunctioning and functioning subtypes. Telomerase length as well as TERT mRNA level and gene amplification were estimated using quantitative PCR (qPCR). Promoter mutations were assessed using Sanger sequencing. The results from genome-wide DNA methylation profiling with HumanMethylation 450K (Illumina) were used for the analysis of TERT locus. RESULTS: Variable telomere length was observed in patients, however no relationship with clinicopathological features was found. We observed a missense variant in TERT promoter in one patient only whereas increased TERT copy number were identified in 6 patients (5.6%). However no relationship between these results and telomere length or TERT expression was found. DNA methylation at TERT locus was not found to be changed when adenoma samples and normal tissue sections were compared. CONCLUSION: The results indicate that telomerase abnormalities do not play a role in pathogenesis of pituitary tumors.

DNA methylation profiling in nonfunctioning pituitary adenomas.

Nonfunctioning pituitary adenomas (NFPAs) are among the most frequent intracranial tumors but their molecular background, including changes in epigenetic regulation, remains poorly understood. We performed genome-wide DNA methylation profiling of 34 NFPAs and normal pituitary samples. Methylation status of the selected genomic regions and expression level of corresponding genes were assessed in a group of 75 patients. NFPAs exhibited distinct global methylation profile as compared to normal pituitary. Aberrant DNA methylation appears to contribute to deregulation of the cancer-related pathways as shown by preliminary functional analysis. Promoter hypermethylation and decreased expression level of SFN, STAT5A, DUSP1, PTPRE and FGFR2 was confirmed in the enlarged group of NFPAs. Difference in the methylation profiles between invasive and non-invasive NFPAs is very slight. Nevertheless, invasiveness-related aberrant epigenetic deregulation of the particular genes was found including upregulation of ITPKB and downregulation CNKSR1 in invasive tumors.

Evaluation of the role of downregulation of SNF5/INI1 core subunit of SWI/SNF complex in clear cell renal cell carcinoma development.

Clear cell renal cell carcinoma (ccRCC) is characterized by stabilization of hypoxia-inducible factor (HIF1), and mutations in von Hippel-Lindau (VHL) gene. Additionally, in about 40% of ccRCC cases the mutation in PBRM1 (POLYBROMO1) gene coding for a non-core subunit of SWI/SNF chromatin remodeling complex was found suggesting potential impairment of this complex function in ccRCC. In this study we assessed the extent to which the core SWI/SNF complex subunit - INI1 (hSNF5/SMARCB1) is affected in ccRCC and whether it has any consequences on the development of this type of cancer. The evaluation of INI1 protein level in samples from 50 patients with diagnosed ccRCC, including three displaying rhabdoid features, showed the INI1 positive staining in rhabdoid cells while the conventional ccRCC cells exhibited reduced INI1 level. This indicated the rhabdoid component of ccRCC as distinct from other known rhabdoid tumors. The reduced INI1 protein level observed in all conventional ccRCC cases used in this study correlated with decreased SMARCB1 gene expression at the transcript level. Consistently, the overexpression of INI1 protein in A498 ccRCC cell line resulted in the elevation of endogenous SMARCB1 transcript level indicating that the INI1-dependent regulatory feedback loop controlling expression of this gene is affected in ccRCC Moreover, the set of INI1 target genes including i.e. CXCL12/CXCR7/CXCR4 chemokine axis was identified to be affected in ccRCC. In summary, we demonstrated that the inactivation of INI1 may be of high importance for ccRCC development and aggressiveness.

Downregulation of PTPRH (Sap-1) in colorectal tumors.

Tyrosine phosphorylation is one of the basic mechanisms for signal transduction in the cell. Receptors exhibiting tyrosine kinase activity are widely involved in carcinogenesis and are negatively regulated by receptor protein tyrosine phosphatases (RPTP). Genes encoding different RPTPs are affected by aberrant epigenetic regulation in cancer. PTPRH (SAP-1) has been previously described to be overexpressed in colorectal cancer (CRC) and classified as an oncogenic factor. Previous microarray-based mRNA expression comparison of colorectal adenomas (AD), CRC and normal mucosa samples (NM) demonstrated that PTPRH tumor expression is the most reduced of all RPTP genes. qRT-PCR validation revealed gene downregulation for CRC (7.6-fold-change; P<0.0001) and AD (3.4-fold-change; P<0.0001) compared to NM. This was confirmed by immunohistochemical staining of tumor and NM sections as pronounced decrease of protein expression was observed in CRCs compared to the corresponding normal tissue. DNA methylation of two PTPRH promoter fragments was analyzed by pyrosequencing in a group of CRC, and AD patients as well as NM samples and CRC cell lines. The mean DNA methylation levels of these two regions were significantly higher in CRC than in NM. Both regions were highly methylated in SW480 and HCT116 cell lines contrary to unmethylated HT29 and COLO205. Cell lines with highly methylated promoters notably showed lower PTPRH expression levels, lower RNA II polymerase concentrations and higher levels of H3K27 trimethylation in the promoter and gene body, measured by chromatin immunoprecipitation. Cells were cultured with 5-aza-deoxycitidine and an increase in PTPRH expression was observed in SW480 and HCT116, whereas this was unchanged in the unmethylated cell lines. The results indicate that PTPRH is downregulated in colorectal tumors and its expression is epigenetically regulated via DNA methylation and chromatin modifications.

Mutational Analysis of Recurrent Meningioma Progressing From Atypical to Rhabdoid Subtype.

BACKGROUND: Rhabdoid meningioma is rare aggressive meningioma histological subtype that develops predominantly through progression from less malignant tumors. Owing to its low incidence, this tumor's biological background is unknown. The aim of this study was to profile somatic mutations in 4 meningioma samples from the same patient, derived previously from 4 subsequent tumor resections. CASE DESCRIPTION: A 58-year-old woman presented with recurrent meningioma progressing from atypical to rhabdoid subtype. Four tumor samples that represent a primary tumor (atypical GII) and 3 recurrent tumors that were subsequently removed (anaplastic GIII, rhabdoid GIII, and anaplastic/rhabdoid GIII) from this patient were subjected to mutational analysis of coding sequences of 952 tumor-related genes. Three mutations were identified in all tumor samples exhibiting a high allelic frequency: ARID1A frameshift deletion, NF2 in-frame deletion, and missense variant of SRSF2. The predicted inactivating effect of ARID1A deletion was confirmed by immunohistochemical staining of tumor sections in which a high proportion of cells lacked protein expression. Additional low-allelic-fraction mutations were observed in all tumor samples, likely representing "passenger," low-effect mutations that reflect a clonal selection of tumor cells through malignant progression of the meningioma. CONCLUSION: The mutation of ARID1A that encodes the subunit of the SWI/SNF complex represents the most likely driver of the tumor's malignant potential. It also may be involved in the acquisition of the rhabdoid phenotype, given that mutations in chromatin remodeling proteins are the hallmark of atypical teratoid/rhabdoid tumors.