Whole genome DNA methylation and mutational profiles identify novel changes in proliferative verrucous leukoplakia.

OBJECTIVE: Proliferative verrucous leukoplakia (PVL) is a rare form of oral leukoplakia with a relatively high transformation rate resulting in oral squamous cell carcinoma (OSCC). Molecular analysis of PVL at the genome level is limited and has only identified molecular similarities between PVL and OSCC. However, the clinical profile of PVL suggests that molecular differences may be more important. STUDY DESIGN: Whole exome sequencing of 5 PVL-associated OSCC (PVL-OSCC) and paired blood samples was used to identify somatic mutations common to the tumors. Whole methylome analysis of samples from 4 PVL-associated OSCC and 3 OSCC of non-PVL origin samples was conducted to explore differential methylation. RESULTS: In contrast to conventional OSCC, PVL-associated OSCC showed infrequent TP53 mutation and altered spectra of PIK3CA and NOTCH1 mutations. Unsupervised hierarchical clustering identified 63 probes that discriminated between PVL-associated OSCC and OSCC of non-PVL origin. Differences in methylation were most significant for divalent metal ion transport, particularly calcium movement. CONCLUSIONS: Specific differences in mutation and methylation profiles between PVL-derived OSCC and OSCC of non-PVL origin suggest differences in their transformation pathways. Further studies of early PVL lesions may identify markers of transformation that are also applicable to more common oral premalignant disorders such as oral epithelial dysplasia.

Development and validation of molecular methodologies to assess PALB2 expression in sporadic breast cancer.

OBJECTIVES: Recent reports have included PALB2 (Partner and localizer of BRCA2) in the growing list of hereditary cancer genes. PALB2 mutations confer a moderate breast cancer risk in heterozygotes and Fanconi anemia in biallelic mutation carriers. PALB2 protein co-localizes with BRCA2 and BRCA1 in nuclear structures and enables error-free homologous recombination repair of double-stranded DNA breaks. This important contribution could be severely diminished if affected by epigenetic mechanisms such as promoter CpG island methylation. The aim of our study was to develop molecular methodologies in order to assess accurately PALB2 expression in breast cancer tissues. DESIGN AND METHODS: DNA and RNA were extracted from 91 sporadic fresh-frozen breast tissues with known histopathological data. DNA was subjected to sodium bisulfite conversion reaction and the CpG island of the PALB2 promoter was analyzed by pyrosequencing. RNA was converted to cDNA and analyzed by a newly developed and validated RT-qPCR assay based on a hydrolysis probe (TaqMan) in the Light Cycler. RESULTS: PALB2 promoter was not methylated in any of the samples tested. 87 out of 91 (95.6%) primary tumors were positive for PALB2 expression, as checked at the mRNA level. When levels of PALB2 mRNA were compared to histopathological data (tumor size, grade, lymph node involvement, metastasis, hormone receptors and HER2 overexpression), no significant statistical correlation was found. CONCLUSIONS: DNA methylation is an unlike mechanism for PALB2 transcriptional regulation. PALB2 mRNA expression does not seem be a promising prognostic biomarker for sporadic breast cancer.

N-bromotaurine surrogates for loss of antiproliferative response and enhances cisplatin efficacy in cancer cells with impaired glucocorticoid receptor.

Glucocorticoids (GCs) are frequently used in anticancer combination regimens; however, their continuous use adds selective pressure on cancer cells to develop GC-resistance via impairment of the glucocorticoid receptor (GR), therefore creating a need for GC-alternatives. Based on the drug repurposing approach and the commonalities between inflammation and neoplasia, drugs that are either in late-stage clinical trials and/or already marketed for GC-refractory inflammatory diseases could be evaluated as GC-substitutes in the context of cancer. Advantageously, unlike new molecular entities currently being de novo developed to restore GC-responsiveness of cancer cells, such drugs have documented safety and efficacy profile, which overall simplifies their introduction in clinical cancer trials. In this study, we estimated the potential of a well-established, multistage, cell line-based, mouse skin carcinogenesis model to be exploited as an initial screening tool for unveiling covert GC-substitutes. First, we categorized the cell lines of this model to GC-sensitive and GC-resistant, in correlation with their corresponding GR status, localization, and functionality. We found that GC-resistance starts in papilloma stages, due to a dysfunctional GR, which is overexpressed, DNA binding-competent, but transactivation-incompetent in papilloma, squamous, and spindle stages of the model. Then, aided by this tool, we evaluated the ability of N-bromotaurine, a naturally occurring, small-molecule, nonsteroid anti-inflammatory drug which is under consideration for use interchangeably/in replacement to GCs in skin inflammations, to restore antiproliferative response of GC-resistant cancer cells. Unlike GCs, N-bromotaurine inhibited cell-cycle progression in GC-resistant cancer cells and efficiently synergized with cisplatin, thus indicating a potential to be exploited instead of GCs against cancer.

Sp1 binds to the external promoter of the p73 gene and induces the expression of TAp73gamma in lung cancer.

The p73 gene possesses an extrinsic P1 promoter and an intrinsic P2 promoter, resulting in TAp73 and DeltaNup73 isoforms, respectively. The ultimate effect of p73 in oncogenesis is thought to depend on the apoptotic TA to antiapoptotic DeltaN isoforms' ratio. This study was aimed at identifying novel transcription factors that affect TA isoform synthesis. With the use of bioinformatics tools, in vitro binding assays, and chromatin immunoprecipitation analysis, a region extending -233 to -204 bp upstream of the transcription start site of the human p73 P1 promoter, containing conserved Sp1-binding sites, was characterized. Treatment of cells with Sp1 RNAi and Sp1 inhibitor functionally suppress TAp73 expression, indicating positive regulation of P1 by the Sp1 protein. Notably Sp1 inhibition or knockdown also reduces DeltaNup73 protein levels. Therefore, Sp1 directly regulates TAp73 transcription and affects DeltaNup73 levels in lung cancer. TAp73gamma was shown to be the only TA isoform overexpressed in several lung cancer cell lines and in 26 non-small cell lung cancers, consistent with Sp1 overexpression, thereby questioning the apoptotic role of this specific p73 isoform in lung cancer.