Identification of a DNA methylation signature and distinct microRNA variants in breast cancer

Summary Deregulation of factors involved in the epigenetic regulation of gene expression is a hallmark of cancer. In this dissertation, DNA methylation and microRNA (miRNA) expression, as two components implicated in epigenetic regulation, were studied in breast cancer. In the first study, the establishment of a DNA methylation signature for breast cancer was aimed at, based on the early occurrence and stability of abnormal DNA methylation patterns in tumors. Specialized oligonucleotide microarrays were developed and optimized to screen DNA methylation patterns of the candidate loci associated with breast cancer. The assay was applied to analyze DNA methylation patterns of breast cancer and control samples. The profiling data was subjected to multiple bioinformatic analyses in order to identify a DNA methylation signature with appropriately high potential for diagnosis. Methylation patterns of CpG sites associated with two genes, SFRP2 and GHSR, were identified as promising markers. Up-regulation and activation of estrogen receptor α (ERα) signaling is a common feature of almost 70% of breast cancers. However, our understanding of the molecular mechanisms underlying deregulation of this signaling pathway is scarce. In the second study, based on miRNA profiling data of human mammary cell lines, miR-375 was identified as an overexpressed miRNA in ERα-positive cells. Analysis of the miRNA locus revealed that miR-375 overexpression is mainly caused by the loss of epigenetic marks, such as H3K9me2 and local DNA hypomethylation, which, in turn, triggers the dissociation of the transcriptional repressor CCCTC-binding factor (CTCF) from the promoter and enables interactions of ERα with regulatory regions of miR-375. Inhibition of miR-375 in ERα-positive MCF7 cells resulted in reduced ERα activation and cell proliferation. In addition, RASD1, an estrogen inducible gene, was identified as a miR-375 target mediating miR-375 effect on ERα. These data indicate the existence of a positive feedback regulation between ERα and miR-375. The methylation signature identified shows a promising potential to be applicable for diagnosis/risk assessment of breast cancer. Furthermore, our findings from miRNA study provide significant insight into the deregulation of ERα pathway, which may open new therapeutic strategies for breast cancer

Do we use the appropriate controls for the identification of informative methylation markers for early cancer detection?

It is possible to miss potential DNA methylation markers of tumorigenesis because profiling results are initially filtered on the basis of inappropriate controls

Morphological findings in frozen non-neoplastic kidney tissues of patients with kidney cancer from large-scale multicentric studies on renal cancer.

Funder: FP7 Ideas: European Research Council; doi: http://dx.doi.org/10.13039/100011199; Grant(s): 241669There are unexplained geographical variations in the incidence of kidney cancer with the high rates reported in Baltic countries, as well as eastern and central Europe. Having access to a large and well-annotated collection of "tumor/non-tumor" pairs of kidney cancer patients from the Czech Republic, Romania, Serbia, UK, and Russia, we aimed to analyze the morphology of non-neoplastic renal tissue in nephrectomy specimens. By applying digital pathology, we performed a microscopic examination of 1012 frozen non-neoplastic kidney tissues from patients with renal cell carcinoma. Four components of renal parenchyma were evaluated and scored for the intensity of interstitial inflammation and fibrosis, tubular atrophy, glomerulosclerosis, and arterial wall thickening, globally called chronic renal parenchymal changes. Moderate or severe changes were observed in 54 (5.3%) of patients with predominance of occurrence in Romania (OR = 2.67, CI 1.07-6.67) and Serbia (OR = 4.37, CI 1.20-15.96) in reference to those from Russia. Further adjustment for comorbidities, tumor characteristics, and stage did not change risk estimates. In multinomial regression model, relative probability of non-glomerular changes was 5.22 times higher for Romania and Serbia compared to Russia. Our findings show that the frequency of chronic renal parenchymal changes, with the predominance of chronic interstitial nephritis pattern, in kidney cancer patients varies by country, significantly more frequent in countries located in central and southeastern Europe where the incidence of kidney cancer has been reported to be moderate to high. The observed association between these pathological features and living in certain geographic areas requires a larger population-based study to confirm this association on a large scale

Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples

Funder: NCI U24CA211006Abstract: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts

Epigenome Aberrations: Emerging Driving Factors of the Clear Cell Renal Cell Carcinoma

Clear cell renal cell carcinoma (ccRCC), the most common form of Kidney cancer, is characterized by frequent mutations of the von Hippel-Lindau (VHL) tumor suppressor gene in ~85% of sporadic cases. Loss of pVHL function affects multiple cellular processes, among which the activation of hypoxia inducible factor (HIF) pathway is the best-known function. Constitutive activation of HIF signaling in turn activates hundreds of genes involved in numerous oncogenic pathways, which contribute to the development or progression of ccRCC. Although VHL mutations are considered as drivers of ccRCC, they are not sufficient to cause the disease. Recent genome-wide sequencing studies of ccRCC have revealed that mutations of genes coding for epigenome modifiers and chromatin remodelers, including PBRM1, SETD2 and BAP1, are the most common somatic genetic abnormalities after VHL mutations in these tumors. Moreover, recent research has shed light on the extent of abnormal epigenome alterations in ccRCC tumors, including aberrant DNA methylation patterns, abnormal histone modifications and deregulated expression of non-coding RNAs. In this review, we discuss the epigenetic modifiers that are commonly mutated in ccRCC, and our growing knowledge of the cellular processes that are impacted by them. Furthermore, we explore new avenues for developing therapeutic approaches based on our knowledge of epigenome aberrations of ccRCC

Cell&ndash;Cell Contact Mediates Gene Expression and Fate Choice of Human Neural Stem/Progenitor Cells

Transplantation of Neural Stem/Progenitor Cells (NPCs) is a promising regenerative strategy to promote neural repair following injury and degeneration because of the ability of these cells to proliferate, migrate, and integrate with the host tissue. Precise in vitro control of NPC proliferation without compromising multipotency and differentiation ability is critical in stem cell maintenance. This idea was highlighted in recent clinical trials, where discrepancies in NPC culturing protocols produced inconsistent therapeutic benefits. Of note, cell density plays an important role in regulating the survival, proliferation, differentiation, and fate choice of stem cells. To determine the extent of variability produced by inconsistent culturing densities, the present study cultured human-induced pluripotent NPCs (hiPSC-NPCs) at either a low or high plating density. hiPSC-NPCs were then isolated for transcriptomic analysis or differentiation in vitro. Following sequencing analysis, genes involved in cell&ndash;cell contact-mediated pathways, including Hippo-signaling, NOTCH, and WNT were differentially expressed. Modulation of these pathways was highly associated with the regulation of pro-neuronal transcription factors, which were also upregulated in response to higher-density hiPSC-NPC culture. Moreover, higher plating density translated into a greater neuronal and less astrocytic differentiation in vitro. This study highlights the importance of precisely controlling culture conditions during the development of NPC transplantation therapies