A large scale expression study associates uc.283-plus lncRNA with pluripotent stem cells and human glioma.

BACKGROUND: There are 481 ultra-conserved regions (UCRs) longer than 200 bases in the genomes of human, mouse and rat. These DNA sequences are absolutely conserved and show 100% identity with no insertions or deletions. About half of these UCRs are reported as transcribed and many correspond to long non-coding RNAs (lncRNAs). METHODS: We used custom microarrays with 962 probes representing sense and antisense sequences for the 481 UCRs to examine their expression across 374 normal samples from 46 different tissues and 510 samples representing 10 different types of cancer. The expression in embryonic stem cells of selected UCRs was validated by real time PCR. RESULTS: We identified tissue selective UCRs and studied UCRs in embryonic and induced pluripotent stem cells. Among the normal tissues, the uc.283 lncRNA was highly specific for pluripotent stem cells. Intriguingly, the uc.283-plus lncRNA was highly expressed in some solid cancers, particularly in one of the most untreatable types, glioma. CONCLUSION: Our results suggest that uc.283-plus lncRNA might have a role in pluripotency of stem cells and in the biology of glioma.

Pluripotent stem cell miRNAs and metastasis in invasive breast cancer.

BACKGROUND: The purpose of this study is to determine whether microRNA for pluripotent stem cells are also expressed in breast cancer and are associated with metastasis and outcome. METHODS: We studied global microRNA profiles during differentiation of human embryonic stem cells (n =26) and in breast cancer patients (n = 33) and human cell lines (n = 35). Using in situ hybridization, we then investigated MIR302 expression in 318 untreated breast cancer patients (test cohort, n = 22 and validation cohort, n = 296). In parallel, using next-generation sequencing data from breast cancer patients (n = 684), we assessed microRNA association with stem cell markers. All statistical tests were two-sided. RESULTS: In healthy tissues, the MIR302 (high)/MIR203 (low) asymmetry was exclusive for pluripotent stem cells. MIR302 was expressed in a small population of cancer cells within invasive ductal carcinoma, but not in normal breast (P < .001). Furthermore, MIR302 was expressed in the tumor cells together with stem cell markers, such as CD44 and BMI1. Conversely, MIR203 expression in 684 breast tumors negatively correlated with CD44 (Spearman correlation, Rho = -0.08, P = .04) and BMI1 (Rho = -0.11, P = .004), but positively correlated with differentiation marker CD24 (Rho = 0.15, P < .001). Primary tumors with lymph node metastasis had cancer cells showing scattered expression of MIR302 and widespread repression of MIR203. Finally, overall survival was statistically significantly shorter in patients with MIR302-positive cancer cells (P = .03). CONCLUSIONS: In healthy tissues the MIR302(high)/MIR203(low) asymmetry was characteristic of embryonic and induced pluripotency. In invasive ductal carcinoma, the MIR302/MIR203 asymmetry was associated with stem cell markers, metastasis, and shorter survival.

Next generation analysis of breast cancer genomes for precision medicine.

For many years breast cancer classification has been based on histology and immune-histochemistry. New techniques, more strictly related to cancer biology, partially succeeded in fractionating patients, correlated to survival and better predicted the patient response to therapy. Nowadays, great expectations arise from massive parallel or high throughput next generation sequencing. Cancer genomics has already revolutionized our knowledge of breast cancer molecular pathology, paving the way to the development of new and more effective clinical protocols. This review is focused on the most recent advances in the field of cancer genomics and epigenomics, including DNA alterations and driver gene mutations, gene fusions, DNA methylation and miRNA expression.

MicroRNA Expression Profiling in the Histological Subtypes of Barrett's Metaplasia.

OBJECTIVES: The histological definition of Barrett's esophagus (BE) is debated, particularly regarding the phenotype of its metaplastic columnar epithelium. Histologically proven intestinal metaplasia (IM) was the sine qua non condition for a diagnosis of BE but, more recently, non-intestinalized (i.e., cardiac gastric-type; GM) columnar metaplasia has been re-included in the spectrum of Barrett's histology. MicroRNAs modulate cell commitment, and are also reportedly dysregulated in Barrett's carcinogenesis. This study investigates miRNA expression in the histological spectrum of esophageal columnar metaplastic changes, specifically addressing the biological profile of GM vs. IM. METHODS: A study was performed to discover microRNA microarray in 30 matching mucosa samples obtained from 10 consecutive BE patients; for each patient, biopsy tissue samples were obtained from squamous, GM and intestinalized epithelium. Microarray findings were further validated by qRT-PCR analysis in another bioptic series of 75 mucosa samples. RESULTS: MicroRNA profiling consistently disclosed metaplasia-specific microRNA signatures. Six microRNAs were significantly dysregulated across the histological phenotypes considered; five of them (two overexpressed (hsa-miR-192; -miR-215) and three under-expressed (hsa-miR-18a*; -miR-203, and -miR-205)) were progressively dysregulated in the phenotypic sequence from squamous to gastric-type, to intestinal-type mucosa samples. CONCLUSIONS: A consistent microRNA expression signature underlies both gastric- and intestinal-type esophageal metaplasia. The pattern of microRNA dysregulation suggests that GM may further progress to IM. The clinico-pathological implications of these molecular profiles prompt further study on the "personalized" cancer risk associated with each of these metaplastic transformations.

Comparison of microRNA deep sequencing of matched formalin-fixed paraffin-embedded and fresh frozen cancer tissues.

MicroRNAs regulate several aspects of tumorigenesis and cancer progression. Most cancer tissues are archived formalin-fixed and paraffin-embedded (FFPE). While microRNAs are a more stable form of RNA thought to withstand FFPE-processing and degradation there is only limited evidence for the latter assumption. We examined whether microRNA profiling can be successfully conducted on FFPE cancer tissues using SOLiD ligation based sequencing. Tissue storage times (2-9 years) appeared to not affect the number of detected microRNAs in FFPE samples compared to matched frozen samples (paired t-test p>0.7). Correlations of microRNA expression values were very high across microRNAs in a given sample (Pearson's r = 0.71-0.95). Higher variance of expression values among samples was associated with higher correlation coefficients between FFPE and frozen tissues. One of the FFPE samples in this study was degraded for unknown reasons with a peak read length of 17 nucleotides compared to 21 in all other samples. The number of detected microRNAs in this sample was within the range of microRNAs detected in all other samples. Ligation-based microRNA deep sequencing on FFPE cancer tissues is feasible and RNA degradation to the degree observed in our study appears to not affect the number of microRNAs that can be quantified.

Breast cancer signatures for invasiveness and prognosis defined by deep sequencing of microRNA.

The transition from ductal carcinoma in situ to invasive ductal carcinoma is a key event in breast cancer progression that is still not well understood. To discover the microRNAs regulating this critical transition, we used 80 biopsies from invasive ductal carcinoma, 8 from ductal carcinoma in situ, and 6 from normal breast. We selected them from a recently published deep-sequencing dataset [Farazi TA, et al. (2011) Cancer Res 71:4443-4453]. The microRNA profile established for the normal breast to ductal carcinoma in situ transition was largely maintained in the in situ to invasive ductal carcinoma transition. Nevertheless, a nine-microRNA signature was identified that differentiated invasive from in situ carcinoma. Specifically, let-7d, miR-210, and -221 were down-regulated in the in situ and up-regulated in the invasive transition, thus featuring an expression reversal along the cancer progression path. Additionally, we identified microRNAs for overall survival and time to metastasis. Five noncoding genes were associated with both prognostic signatures--miR-210, -21, -106b*, -197, and let-7i, with miR-210 the only one also involved in the invasive transition. To pinpoint critical cellular functions affected in the invasive transition, we identified the protein coding genes with inversely related profiles to miR-210: BRCA1, FANCD, FANCF, PARP1, E-cadherin, and Rb1 were all activated in the in situ and down-regulated in the invasive carcinoma. Additionally, we detected differential splicing isoforms with special features, including a truncated EGFR lacking the kinase domain and overexpressed only in ductal carcinoma in situ.

miRNA signatures associate with pathogenesis and progression of osteosarcoma.

Osteosarcoma remains a leading cause of cancer death in adolescents. Treatment paradigms and survival rates have not improved in two decades. Driving the lack of therapeutic inroads, the molecular etiology of osteosarcoma remains elusive. MicroRNAs (miRNAs) have demonstrated far-reaching effects on the cellular biology of development and cancer. Their role in osteosarcomagenesis remains largely unexplored. Here we identify for the first time an miRNA signature reflecting the pathogenesis of osteosarcoma from surgically procured samples from human patients. The signature includes high expression of miR-181a,miR-181b, and miR-181c as well as reduced expression of miR-16, miR-29b, and miR-142-5p. We also demonstrate that miR-181b and miR-29b exhibit restricted expression to distinct cell populations in the tumor tissue. Further, higher expression of miR-27a and miR-181c* in pre-treatment biopsy samples characterized patients who developed clinical metastatic disease. In addition, higher expression of miR-451 and miR-15b in pre-treatment samples correlated with subsequent positive response to chemotherapy. In vitro and in vivo functional validation in osteosarcoma cell lines confirmed the tumor suppressive role of miR-16 and the pro-metastatic role of miR-27a. Furthermore, predicted target genes for miR-16 and miR-27a were confirmed as down-regulated by real-time PCR. Affymetrix array profiling of cDNAs from the osteosarcoma specimens and controls were interrogated according to predicted targets of miR-16, miR142-5p, miR-29b, miR-181a/b, and miR-27a. This analysis revealed positive and negative correlations highlighting pathways of known importance to osteosarcoma, as well as novel genes. Thus, our findings establish a miRNA signature associated with pathogenesis of osteosarcoma as well as critical pre-treatment biomarkers of metastasis and responsiveness to therapy.

p53 regulates epithelial-mesenchymal transition through microRNAs targeting ZEB1 and ZEB2.

p53 suppresses tumor progression and metastasis. Epithelial-mesenchymal transition (EMT) is a key process in tumor progression and metastasis. The transcription factors ZEB1 and ZEB2 promote EMT. Here, we show that p53 suppresses EMT by repressing expression of ZEB1 and ZEB2. By profiling 92 primary hepatocellular carcinomas (HCCs) and 9 HCC cell lines, we found that p53 up-regulates microRNAs (miRNAs), including miR-200 and miR-192 family members. The miR-200 family members transactivated by p53 then repress ZEB1/2 expression. p53-regulated miR-192 family members also repress ZEB2 expression. Inhibition or overexpression of the miRNAs affects p53-regulated EMT by altering ZEB1 and ZEB2 expression. Our findings indicate that p53 can regulate EMT, and that p53-regulated miRNAs are critical mediators of p53-regulated EMT.

MicroRNA expression profiling in human Barrett's carcinogenesis.

Barrett's esophagus (BE) is characterized by the native stratified squamous epithelium (N) lining the esophagus being replaced by a columnar epithelium with intestinal differentiation (Barrett's mucosa; BM). BM is considered as the main risk factor for esophageal adenocarcinoma (Barrett's adenocarcinoma; BAc). MicroRNAs (miRNAs) are a class of small noncoding RNAs that control gene expression by targeting messenger RNAs and they are reportedly dysregulated in BM. To test the hypothesis that a specific miRNA expression signature characterizes BM development and progression, we performed miRNA microarray analysis comparing native esophageal mucosa with all the phenotypic lesions seen in the Barrett's carcinogenic process. Specimens were collected from 14 BE patients who had undergone esophagectomy, including: 14 with N, 14 with BM, 7 with low-grade intraepithelial neoplasia, 5 with high-grade intra-epithelial neoplasia and 11 with BAc. Microarray findings were further validated by quantitive real-time polymerase chain reaction and in situ hybridization analyses using a different series of consecutive cases (162 biopsy samples and 5 esophagectomies) of histologically proven, long-segment BE. We identified a miRNA signature of Barrett's carcinogenesis consisting of an increased expression of 6 miRNAs and a reduced expression of 7 miRNAs. To further support these results, we investigated target gene expression using the Oncomine database and/or immunohistochemical analysis. We found that target gene expression correlated significantly with miRNA dysregulation. Specific miRNAs are directly involved in BE progression to cancer. miRNA profiling significantly expands current knowledge on the molecular history of Barrett's carcinogenesis, also identifying molecular markers of cancer progression.

GAMES identifies and annotates mutations in next-generation sequencing projects.

MOTIVATION: Next-generation sequencing (NGS) methods have the potential for changing the landscape of biomedical science, but at the same time pose several problems in analysis and interpretation. Currently, there are many commercial and public software packages that analyze NGS data. However, the limitations of these applications include output which is insufficiently annotated and of difficult functional comprehension to end users. RESULTS: We developed GAMES (Genomic Analysis of Mutations Extracted by Sequencing), a pipeline aiming to serve as an efficient middleman between data deluge and investigators. GAMES attains multiple levels of filtering and annotation, such as aligning the reads to a reference genome, performing quality control and mutational analysis, integrating results with genome annotations and sorting each mismatch/deletion according to a range of parameters. Variations are matched to known polymorphisms. The prediction of functional mutations is achieved by using different approaches. Overall GAMES enables an effective complexity reduction in large-scale DNA-sequencing projects. AVAILABILITY: GAMES is available free of charge to academic users and may be obtained from http://aqua.unife.it/GAMES.

Reprogramming of miRNA networks in cancer and leukemia.

We studied miRNA profiles in 4419 human samples (3312 neoplastic, 1107 nonmalignant), corresponding to 50 normal tissues and 51 cancer types. The complexity of our database enabled us to perform a detailed analysis of microRNA (miRNA) activities. We inferred genetic networks from miRNA expression in normal tissues and cancer. We also built, for the first time, specialized miRNA networks for solid tumors and leukemias. Nonmalignant tissues and cancer networks displayed a change in hubs, the most connected miRNAs. hsa-miR-103/106 were downgraded in cancer, whereas hsa-miR-30 became most prominent. Cancer networks appeared as built from disjointed subnetworks, as opposed to normal tissues. A comparison of these nets allowed us to identify key miRNA cliques in cancer. We also investigated miRNA copy number alterations in 744 cancer samples, at a resolution of 150 kb. Members of miRNA families should be similarly deleted or amplified, since they repress the same cellular targets and are thus expected to have similar impacts on oncogenesis. We correctly identified hsa-miR-17/92 family as amplified and the hsa-miR-143/145 cluster as deleted. Other miRNAs, such as hsa-miR-30 and hsa-miR-204, were found to be physically altered at the DNA copy number level as well. By combining differential expression, genetic networks, and DNA copy number alterations, we confirmed, or discovered, miRNAs with comprehensive roles in cancer. Finally, we experimentally validated the miRNA network with acute lymphocytic leukemia originated in Mir155 transgenic mice. Most of miRNAs deregulated in these transgenic mice were located close to hsa-miR-155 in the cancer network.

MicroRNA-451 regulates LKB1/AMPK signaling and allows adaptation to metabolic stress in glioma cells.

To sustain tumor growth, cancer cells must be able to adapt to fluctuations in energy availability. We have identified a single microRNA that controls glioma cell proliferation, migration, and responsiveness to glucose deprivation. Abundant glucose allows relatively high miR-451 expression, promoting cell growth. In low glucose, miR-451 levels decrease, slowing proliferation but enhancing migration and survival. This allows cells to survive metabolic stress and seek out favorable growth conditions. In glioblastoma patients, elevated miR-451 is associated with shorter survival. The effects of miR-451 are mediated by LKB1, which it represses through targeting its binding partner, CAB39 (MO25 alpha). Overexpression of miR-451 sensitized cells to glucose deprivation, suggesting that its downregulation is necessary for robust activation of LKB1 in response to metabolic stress. Thus, miR-451 is a regulator of the LKB1/AMPK pathway, and this may represent a fundamental mechanism that contributes to cellular adaptation in response to altered energy availability.

Karyotype-specific microRNA signature in chronic lymphocytic leukemia.

Chromosomal abnormalities, immunoglobulin heavy chain variable-region (IGHV) gene mutation status, and zeta-associated protein 70 (ZAP-70) expression levels have independent prognostic relevance in chronic lymphocytic leukemia (CLL); however, their concordance is variable. Because deregulation of microRNAs has been linked to disease initiation and progression in CLL, we studied the value of the microRNAs as a signature for CLL patients with specific chromosomal abnormalities. We identified 32 microRNAs able to discriminate the 11q deletion, 17p deletion, trisomy 12, 13q deletion, and normal karyotype cytogenetic subgroups. The expression values of 9 among the 32 microRNAs (miR-151-3p, miR-34a, miR-29c, miR-29b, miR-155, miR-148a, miR-146a, miR-146b5p, and miR-640) were correlated with gene expression data from the same samples to assess their biologic impact on CLL. In this study we also found that IGHV unmutated, high expression of ZAP-70 protein, and low expression of the miR-223, miR-29c, miR-29b, and miR-181 family were strongly associated with disease progression in CLL cases harboring 17p deletion, whereas in those harboring trisomy 12 only high expression of the miR-181a, among the analyzed parameters, suggested more aggressive disease. Thus, the use of the microRNA-based classifications may yield clinically useful biomarkers of tumor behavior in CLL.

Genome wide identification of recessive cancer genes by combinatorial mutation analysis.

We devised a novel procedure to identify human cancer genes acting in a recessive manner. Our strategy was to combine the contributions of the different types of genetic alterations to loss of function: amino-acid substitutions, frame-shifts, gene deletions. We studied over 20,000 genes in 3 Gigabases of coding sequences and 700 array comparative genomic hybridizations. Recessive genes were scored according to nucleotide mismatches under positive selective pressure, frame-shifts and genomic deletions in cancer. Four different tests were combined together yielding a cancer recessive p-value for each studied gene. One hundred and fifty four candidate recessive cancer genes (p-value < 1.5 x 10(-7), FDR = 0.39) were identified. Strikingly, the prototypical cancer recessive genes TP53, PTEN and CDKN2A all ranked in the top 0.5% genes. The functions significantly affected by cancer mutations are exactly overlapping those of known cancer genes, with the critical exception for the absence of tyrosine kinases, as expected for a recessive gene-set.

Fine-needle aspiration biopsy of non-Hodgkin lymphoma for use in expression microarray analysis.

BACKGROUND: The majority of patients with lymphoma undergo a single biopsy for diagnosis, and there are few opportunities to acquire posttreatment material. Fine-needle aspiration (FNA) biopsy is a minimally invasive procedure, and acquiring routine posttreatment material would require minimal effort and provide needed material for gene expression profiling. METHODS: Ex vivo FNA biopsies were performed using a standard clinical technique with 21-gauge, 22-gauge, 23-gauge, and 25-gauge needles for 8 lymph node specimens and were collected in either RNA stabilization reagent (RNAlater) or Trizol. Eight patients with known or suspected Non-Hodgkin lymphoma (NHL) underwent interoperative (in vivo) FNA biopsies based on the best technique derived from the ex vivo aspirates. RNA derived from the in vivo FNA biopsies and the matched, snap-frozen surgical lymph node biopsy remnant was used for gene expression analysis with proprietary U133A chips. RESULTS: The results confirmed the authors' experience, that RNA isolated from FNA biopsies of lymph nodes collected in Trizol is superior both quantitatively and qualitatively to RNA collected in RNAlater. Gene expression profiles of NHL derived from in vivo FNA biopsies and matched, frozen surgical specimens showed good overall correlation. CONCLUSIONS: High-throughput gene expression analysis in patients with NHL derived from material acquired by FNA biopsy is a feasible approach to developing a platform for real-time analysis of treatment responses in this group of patients.

ADOA3R as a therapeutic target in experimental colitis: proof by validated high-density oligonucleotide microarray analysis.

Adenosine A3 receptors (ADOA3Rs) are emerging as novel purinergic targets for treatment of inflammatory diseases. Our goal was to assess the protective effect of the ADOA3R agonist N(6)-(3-iodobenzyl)-adenosine-5-N-methyluronamide (IB-MECA) on gene dysregulation and injury in a rat chronic model of 2,4,6-trinitrobenzene sulfonic acid (TNBS)--induced colitis. It was necessary to develop and validate a microarray technique for testing the protective effects of purine-based drugs in experimental inflammatory bowel disease. High-density oligonucleotide microarray analysis of gene dysregulation was assessed in colons from normal, TNBS-treated (7 days), and oral IB-MECA-treated rats (1.5 mg/kg b.i.d.) using a rat RNU34 neural GeneChip of 724 genes and SYBR green polymerase chain reaction. Analysis included clinical evaluation, weight loss assessment, and electron paramagnetic resonance imaging/spin-trap monitoring of free radicals. Remarkable colitis-induced gene dysregulation occurs in the most exceptional cluster of 5.4% of the gene pool, revealing 2 modes of colitis-related dysregulation. Downregulation occurs in membrane transporter, mitogen-activated protein (MAP) kinase, and channel genes. Upregulation occurs in chemokine, cytokine/inflammatory, stress, growth factor, intracellular signaling, receptor, heat shock protein, retinoid metabolism, neural, remodeling, and redox-sensitive genes. Oral IB-MECA prevented dysregulation in 92% of these genes, histopathology, gut injury, and weight loss. IB-MECA or adenosine suppressed elevated free radicals in ex vivo inflamed gut. Oral IB-MECA blocked the colitis-induced upregulation (90% of genes tested (33 of 37 genes). We conclude that our validated high-density oligonucleotide microarray analysis is a powerful technique for molecular gene dysregulation studies to assess the beneficial effects of purine-based or other drugs in experimental colitis. ADOA3R is new potential therapeutic target for inflammatory bowel disease.