Multisite Evaluation of Next-Generation Methods for Small RNA Quantification.

Small RNAs (smRNAs) are important regulators of many biologic processes and are now most frequently characterized using Illumina sequencing. However, although standard RNA sequencing library preparation has become routine in most sequencing facilities, smRNA sequencing library preparation has historically been challenging because of high input requirements, laborious protocols involving gel purifications, inability to automate, and a lack of benchmarking standards. Additionally, studies have suggested that many of these methods are nonlinear and do not accurately reflect the amounts of smRNAs in vivo. Recently, a number of new kits have become available that permit lower input amounts and less laborious, gel-free protocol options. Several of these new kits claim to reduce RNA ligase-dependent sequence bias through novel adapter modifications and to lessen adapter-dimer contamination in the resulting libraries. With the increasing number of smRNA kits available, understanding the relative strengths of each method is crucial for appropriate experimental design. In this study, we systematically compared 9 commercially available smRNA library preparation kits as well as NanoString probe hybridization across multiple study sites. Although several of the new methodologies do reduce the amount of artificially over- and underrepresented microRNAs (miRNAs), we observed that none of the methods was able to remove all of the bias in the library preparation. Identical samples prepared with different methods show highly varied levels of different miRNAs. Even so, many methods excelled in ease of use, lower input requirement, fraction of usable reads, and reproducibility across sites. These differences may help users select the most appropriate methods for their specific question of interest.

ASXL3 Is a Novel Pluripotency Factor in Human Respiratory Epithelial Cells and a Potential Therapeutic Target in Small Cell Lung Cancer.

In this study, we generated induced pluripotent stem cells (iPSC) from normal human small airway epithelial cells (SAEC) to investigate epigenetic mechanisms of stemness and pluripotency in lung cancers. We documented key hallmarks of reprogramming in lung iPSCs (Lu-iPSC) that coincided with modulation of more than 15,000 genes relative to parental SAECs. Of particular novelty, we identified the PRC2-associated protein, ASXL3, which was markedly upregulated in Lu-iPSCs and small cell lung cancer (SCLC) lines and clinical specimens. ASXL3 overexpression correlated with increased genomic copy number in SCLC lines. ASXL3 silencing inhibited proliferation, clonogenicity, and teratoma formation by Lu-iPSCs, and diminished clonogenicity and malignant growth of SCLC cells in vivo Collectively, our studies validate the utility of the Lu-iPSC model for elucidating epigenetic mechanisms contributing to pulmonary carcinogenesis and highlight ASXL3 as a novel candidate target for SCLC therapy. Cancer Res; 77(22); 6267-81. ©2017 AACR.

Common Molecular Subtypes Among Asian Hepatocellular Carcinoma and Cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) and hepatocellular carcinoma (HCC) are clinically disparate primary liver cancers with etiological and biological heterogeneity. We identified common molecular subtypes linked to similar prognosis among 199 Thai ICC and HCC patients through systems integration of genomics, transcriptomics, and metabolomics. While ICC and HCC share recurrently mutated genes, including TP53, ARID1A, and ARID2, mitotic checkpoint anomalies distinguish the C1 subtype with key drivers PLK1 and ECT2, whereas the C2 subtype is linked to obesity, T cell infiltration, and bile acid metabolism. These molecular subtypes are found in 582 Asian, but less so in 265 Caucasian patients. Thus, Asian ICC and HCC, while clinically treated as separate entities, share common molecular subtypes with similar actionable drivers to improve precision therapy.

Distinct methylation profiles characterize fusion-positive and fusion-negative rhabdomyosarcoma.

Rhabdomyosarcoma comprises two major subtypes, fusion positive (PAX3-FOXO1 or PAX7-FOXO1) and fusion negative. To investigate the significance of DNA methylation in these subtypes, we analyzed methylation profiles of 37 rhabdomyosarcoma tumors and 10 rhabdomyosarcoma cell lines, as well as 8 normal tissues. Unsupervised clustering of DNA methylation clearly distinguished the fusion-positive and fusion-negative subsets. The fusion-positive tumors showed substantially lower overall levels of methylation compared with fusion-negative tumors. Comparison with the methylation pattern of normal skeletal muscle and bone marrow indicates that fusion-negative rhabdomyosarcoma is more similar to these normal tissues compared with fusion-positive rhabdomyosarcoma, and suggests that many of the methylation differences between these subtypes arise from 'aberrant' hyper- and hypomethylation events in fusion-positive rhabdomyosarcoma. Integrative methylation and gene expression analysis revealed that methylation differences between fusion-positive and fusion-negative tumors could either be positively or negatively associated with mRNA expression. There was no significant difference in the distribution of PAX3-FOXO1-binding sites between genes with and without differential methylation. However, the finding that PAX3-FOXO1-binding sites were enriched among genes that were both differentially methylated and differentially expressed suggests that the fusion protein interacts with DNA methylation to regulate target gene expression. An 11-gene DNA methylation signature, classifying the rhabdomyosarcoma tumors into fusion-positive and fusion-negative subsets, was established and validated by pyrosequencing assays. Notably, EMILIN1 (part of the 11-gene signature) showed higher methylation and lower mRNA expression in fusion-positive compared with fusion-negative tumors, and demonstrated demethylation and re-expression in multiple fusion-positive cell lines after treatment with 5-aza-2'-deoxycytidine. In conclusion, our study demonstrates that fusion-positive and fusion-negative rhabdomyosarcoma tumors possess characteristic methylation profiles that contribute to the expression differences between these fusion subtypes. These findings indicate an important relationship between fusion status and epigenetic changes in rhabdomyosarcoma, present a novel approach for ascertaining fusion status, and may identify new therapeutic targets in rhabdomyosarcoma.

An Integrated Prognostic Classifier for Stage I Lung Adenocarcinoma Based on mRNA, microRNA, and DNA Methylation Biomarkers.

INTRODUCTION: Up to 30% stage I lung cancer patients suffer recurrence within 5 years of curative surgery. We sought to improve existing protein-coding gene and microRNA expression prognostic classifiers by incorporating epigenetic biomarkers. METHODS: Genome-wide screening of DNA methylation and pyrosequencing analysis of HOXA9 promoter methylation were performed in two independently collected cohorts of stage I lung adenocarcinoma. The prognostic value of HOXA9 promoter methylation alone and in combination with mRNA and miRNA biomarkers was assessed by Cox regression and Kaplan-Meier survival analysis in both cohorts. RESULTS: Promoters of genes marked by polycomb in embryonic stem cells were methylated de novo in tumors and identified patients with poor prognosis. The HOXA9 locus was methylated de novo in stage I tumors (p < 0.0005). High HOXA9 promoter methylation was associated with worse cancer-specific survival (hazard ratio [HR], 2.6; p = 0.02) and recurrence-free survival (HR, 3.0; p = 0.01), and identified high-risk patients in stratified analysis of stages IA and IB. Four protein-coding gene (XPO1, BRCA1, HIF1α, and DLC1), miR-21 expression, and HOXA9 promoter methylation were each independently associated with outcome (HR, 2.8; p = 0.002; HR, 2.3; p = 0.01; and HR, 2.4; p = 0.005, respectively), and when combined, identified high-risk, therapy naive, stage I patients (HR, 10.2; p = 3 × 10). All associations were confirmed in two independently collected cohorts. CONCLUSION: A prognostic classifier comprising three types of genomic and epigenomic data may help guide the postoperative management of stage I lung cancer patients at high risk of recurrence.

Long-term stability of total RNA in RNAstable® as evaluated by expression microarray.

Storage of labile RNA in laboratories is accomplished through ultra-low freezing of the nucleic acids. This however requires expensive freezers, convenient storage, reliable electrical power, and increased shipping costs, thereby making it a less viable option. Biomatrica (San Diego, CA) has created RNAstable(®), a stabilization reagent that is used to store RNA in a dehydrated state at room temperature (RT) and protects the RNA from degradation. Our objective was to investigate the sequence integrity and suitability of RNA when stored in RNAstable at extended time periods and at varying temperatures through use of Illumina and Agilent RNA expression microarrays. We observed in Bioanalyzer electropherograms that total RNA extracted from 293 cells stored at RT in RNAstable for 4.5 and 11.5 months is similar in quality to RNA stored at -80°C. Illumina mRNA expression array QC metrics and gene expression patterns from RNAstable-protected RNA, in contrast to RNA stored without RNAstable, correlated well with those of freezer controls. Significantly, when RNA was stored in RNAstable at 45°C for 4.5 months, equivalent to 22 months RT storage, RNA quality, microarray probe signal intensities, probe detection rates, and expression profiles remained similar between RNAstable-protected RNA at RT and the -80°C controls. At 10.5 months, miRNA levels were compared among the storage conditions using miRNA expression arrays. Here too we found strong concordance between miRNA expression patterns when total RNA was stored in RNAstable or at -80°C. Further, Bioanalyzer electrophoresis of RNAstable-protected samples stored at RT for a relative total of 33 months or 50.5 months showed comparable integrity scores to those of -80°C controls. We conclude that use of RNAstable holds promise as an effective stabilization reagent for total RNA and should be useful in situations where shipping and storage options are limited resources.

Genome-wide methylation patterns in papillary thyroid cancer are distinct based on histological subtype and tumor genotype.

CONTEXT: Aberrant DNA methylation is known to be a major factor in oncogenesis and cancer progression, but effects of methylation in papillary thyroid cancer (PTC) are not well defined. OBJECTIVE: The objective of the study was to identify altered methylation patterns, which may be associated with PTC disease behavior. DESIGN: This study was a genome-wide methylation analysis of PTC. SETTING: The study was conducted at the National Institutes of Health Clinical Center. PATIENTS: PTC tissue from 51 patients were analyzed and compared with normal thyroid tissue from seven patients. INTERVENTIONS: CpG methylation status was assessed using advanced genome-wide methylation bead chips. OUTCOME MEASURES: Altered methylation patterns in PTC were analyzed by stage, recurrence, histological subtype of tumor, and tumor genotype. RESULTS: PTC is globally hypomethylated compared with normal thyroid with 2837 differentially methylated CpG sites. The follicular variant of PTC demonstrated less differential methylation with only 569 differentially methylated CpG sites. Tumors with mutations in BRAF, RET/PTC, and RAS demonstrated a 3.6-fold increase in the number of differentially methylated sites compared with wild-type tumors. The differentially methylated genes were associated with oncological pathways including cellular movement, growth, and proliferation. CONCLUSION: PTC is epigenetically distinct from the follicular variant of PTC and by gene mutation status (BRAF, RET/PTC, and RAS).

MYC-driven accumulation of 2-hydroxyglutarate is associated with breast cancer prognosis.

Metabolic profiling of cancer cells has recently been established as a promising tool for the development of therapies and identification of cancer biomarkers. Here we characterized the metabolomic profile of human breast tumors and uncovered intrinsic metabolite signatures in these tumors using an untargeted discovery approach and validation of key metabolites. The oncometabolite 2-hydroxyglutarate (2HG) accumulated at high levels in a subset of tumors and human breast cancer cell lines. We discovered an association between increased 2HG levels and MYC pathway activation in breast cancer, and further corroborated this relationship using MYC overexpression and knockdown in human mammary epithelial and breast cancer cells. Further analyses revealed globally increased DNA methylation in 2HG-high tumors and identified a tumor subtype with high tissue 2HG and a distinct DNA methylation pattern that was associated with poor prognosis and occurred with higher frequency in African-American patients. Tumors of this subtype had a stem cell-like transcriptional signature and tended to overexpress glutaminase, suggestive of a functional relationship between glutamine and 2HG metabolism in breast cancer. Accordingly, 13C-labeled glutamine was incorporated into 2HG in cells with aberrant 2HG accumulation, whereas pharmacologic and siRNA-mediated glutaminase inhibition reduced 2HG levels. Our findings implicate 2HG as a candidate breast cancer oncometabolite associated with MYC activation and poor prognosis.

DNA methylation profiling identifies global methylation differences and markers of adrenocortical tumors.

CONTEXT: It is not known whether there are any DNA methylation alterations in adrenocortical tumors. OBJECTIVE: The objective of the study was to determine the methylation profile of normal adrenal cortex and benign and malignant adrenocortical tumors. METHODS: Genome-wide methylation status of CpG regions were determined in normal (n = 19), benign (n = 48), primary malignant (n = 8), and metastatic malignant (n = 12) adrenocortical tissue samples. An integrated analysis of genome-wide methylation and mRNA expression in benign vs. malignant adrenocortical tissue samples was also performed. RESULTS: Methylation profiling revealed the following: 1) that methylation patterns were distinctly different and could distinguish normal, benign, primary malignant, and metastatic tissue samples; 2) that malignant samples have global hypomethylation; and 3) that the methylation of CpG regions are different in benign adrenocortical tumors by functional status. Normal compared with benign samples had the least amount of methylation differences, whereas normal compared with primary and metastatic adrenocortical carcinoma samples had the greatest variability in methylation (adjusted P ≤ 0.01). Of 215 down-regulated genes (≥2-fold, adjusted P ≤ 0.05) in malignant primary adrenocortical tumor samples, 52 of these genes were also hypermethylated. CONCLUSIONS: Malignant adrenocortical tumors are globally hypomethylated as compared with normal and benign tumors. Methylation profile differences may accurately distinguish between primary benign and malignant adrenocortical tumors. Several differentially methylated sites are associated with genes known to be dysregulated in malignant adrenocortical tumors.

BP1 transcriptionally activates bcl-2 and inhibits TNFalpha-induced cell death in MCF7 breast cancer cells.

INTRODUCTION: We have previously shown that the Beta Protein 1 (BP1) homeodomain protein is expressed in 81% of invasive ductal breast carcinomas, and that increased BP1 expression correlates with tumor progression. The purpose of our current investigation was to determine whether elevated levels of BP1 in breast cancer cells are associated with increased cell survival. METHODS: Effects on cell viability and apoptosis of MCF7 cells stably overexpressing BP1 were determined using MTT and Annexin V assays, and through examination of caspase activation. TNFalpha was used to induce apoptosis. The potential regulation of apoptosis-associated genes by BP1 was studied using real-time PCR and western blot analyses. Electrophoretic mobility shift assays, site-directed mutagenesis, and transient assays were performed to specifically characterize the interaction of BP1 with the promoter of the bcl-2 gene. RESULTS: Stable overexpression of BP1 led to inhibition of apoptosis in MCF7 breast cancer cells challenged with TNFalpha. Increased BP1 resulted in reduced processing and activation of caspase-7, caspase-8, and caspase-9, and inactivation of the caspase substrate Poly(ADP-Ribose) Polymerase (PARP). Increased levels of full-length PARP and a decrease in procaspase-8 were also associated with BP1 overexpression. The bcl-2 gene is a direct target of BP1 since: (i) BP1 protein bound to a consensus binding sequence upstream of the bcl-2 P1 promoter in vitro. (ii) MCF7 cells overexpressing BP1 showed increased levels of bcl-2 mRNA and protein. (iii) Transient assays indicated that increased bcl-2 promoter activity is due to direct binding and modulation by BP1 protein. BP1 expression also prevented TNFalpha-mediated downregulation of bcl-2 mRNA and protein. CONCLUSION: These findings suggest mechanisms by which increased BP1 may impart a survival advantage to breast cancer cells, which could lead to increased resistance to therapeutic agents in patients.