Validation and genomic interrogation of the MET variant rs11762213 as a predictor of adverse outcomes in clear cell renal cell carcinoma.

BACKGROUND: The exonic single-nucleotide variant rs11762213 located in the MET oncogene has recently been identified as a prognostic marker in clear cell renal cell carcinoma (ccRCC). This finding was validated with The Cancer Genome Atlas (TCGA) cohort, and the biologic implications were explored. METHODS: The genotype status for rs11762213 was available for 272 patients. Paired tumor-normal data, genomic data, and clinical information were acquired from ccRCC TCGA data sets. Cancer-specific survival (CSS) was analyzed with the competing risk method, and Cox proportional hazards regression was used for the analysis of the time to recurrence (TTR). Multivariate competing risk models were fitted to adjust for the validated Mayo Clinic Stage, Size, Grade, and Necrosis (SSIGN) score. RESULTS: The variant allele of rs11762213 was detected in 10.3% of the cohort. After adjustments for the SSIGN score, the risk allele remained a significant predictor for adverse CSS (hazard ratio [HR], 3.88; 95% confidence interval [CI], 1.99-7.56; P < .0001) and for TTR (OR, 2.97; 95% CI, 1.43-6.2; P = .003). The mapping of rs11762213 to regulatory regions within the genome suggested that it might affect a DNA enhancer region. RNA and protein sequencing data for MET did not reveal differences in steady-state expression with stratification by risk allele. CONCLUSIONS: The exonic MET variant rs11762213 is an independent predictor of adverse CSS and TTR in ccRCC and should be integrated into clinical practice for prognostic stratification. Genomic analysis suggests that the single-nucleotide polymorphism may affect an enhancer region located in the coding region of MET. Further biological mechanistic interrogation is currently underway.

The lncRNA MIR31HG regulates p16(INK4A) expression to modulate senescence.

Oncogene-induced senescence (OIS) can occur in response to oncogenic insults and is considered an important tumour suppressor mechanism. Here we identify the lncRNA MIR31HG as upregulated in OIS and find that knockdown of MIR31HG promotes a strong p16(INK4A)-dependent senescence phenotype. Under normal conditions, MIR31HG is found in both nucleus and cytoplasm, but following B-RAF expression MIR31HG is located mainly in the cytoplasm. We show that MIR31HG interacts with both INK4A and MIR31HG genomic regions and with Polycomb group (PcG) proteins, and that MIR31HG is required for PcG-mediated repression of the INK4A locus. We further identify a functional enhancer, located between MIR31HG and INK4A, which becomes activated during OIS and interacts with the MIR31HG promoter. Data from melanoma patients show a negative correlation between MIR31HG and p16(INK4A) expression levels, suggesting a role for this transcript in cancer. Hence, our data provide a new lncRNA-mediated regulatory mechanism for the tumour suppressor p16(INK4A).

Molecular subtypes of uterine leiomyosarcoma and correlation with clinical outcome.

The molecular etiology of uterine leiomyosarcoma (ULMS) is poorly understood, which accounts for the wide disparity in outcomes among women with this disease. We examined and compared the molecular profiles of ULMS and normal myometrium (NL) to identify clinically relevant molecular subtypes. Discovery cases included 29 NL and 23 ULMS specimens. RNA was hybridized to Affymetrix U133A 2.0 transcription microarrays. Differentially expressed genes and pathways were identified using standard methods. Fourteen NL and 44 ULMS independent archival samples were used for external validation. Molecular subgroups were correlated with clinical outcome. Pathway analyses of differentially expressed genes between ULMS and NL samples identified overrepresentation of cell cycle regulation, DNA repair, and genomic integrity. External validation confirmed differential expression in 31 genes (P < 4.4 × 10(-4), Bonferroni corrected), with 84% of the overexpressed genes, including CDC7, CDC20, GTSE1, CCNA2, CCNB1, and CCNB2, participating in cell cycle regulation. Unsupervised clustering of ULMS identified two clades that were reproducibly associated with progression-free (median, 4.0 vs 26.0 months; P = .02; HR, 0.33) and overall (median, 18.2 vs 77.2 months; P = .04; HR, 0.33) survival. Cell cycle genes play a key role in ULMS sarcomagenesis, providing opportunities for therapeutic targeting. Reproducible molecular subtypes associated with clinical outcome may permit individualized adjuvant treatment after clinical trial validation.

TCEB1-mutated renal cell carcinoma: a distinct genomic and morphological subtype.

Integrated sequencing analysis identified a group of tumors among clear cell renal cell carcinomas characterized by hotspot mutations in TCEB1 (a gene that contributes to the VHL complex to ubiquitinate hypoxia-inducible factor). We analyzed 11 tumors from two distinct cohorts with TCEB1 mutations along with an expanded cohort to assess whether these should be considered an entity distinct from clear cell renal cell carcinoma and clear cell papillary renal cell carcinoma. All tumors were characterized by hotspot mutations in TCEB1 Y79C/S/F/N or A100P. Morphological and immunohistochemical characteristics of the tumors were assessed by two experienced genitourinary pathologists. Clinical and pathological variables, copy number alterations, mutations, and expression signatures were compared with a cohort of TCEB1 wild-type tumors. All TCEB1-mutated tumors were VHL and PBRM1 wild type and contained distinct copy number profiles including loss of heterozygosity of chromosome 8, the location of TCEB1 (8q21.11). All tumors lacked the clear cell renal cell carcinoma signature 3p loss and contained distinct gene expression signatures. None of the clear cell papillary tumors harbored TCEB1 mutations. Pathologically, all TCEB1-mutated tumors shared characteristic features including thick fibromuscular bands transecting the tumor, pure clear cell cytology frequently with cells showing voluminous cytoplasm, and clear cell renal cell carcinoma-like acinar areas associated with infolding tubular and focally papillary architecture. The presence of voluminous cytoplasm, absence of luminal polarization of tumor nuclei, and lack of extensive cup-like distribution of carbonic anhydrase-IX expression distinguish it from clear cell papillary carcinoma. None of the patients developed metastases at last follow-up (median 48 months). In sum, TCEB1-mutated renal cell carcinoma is a distinct entity with recurrent hotspot mutations, specific copy number alterations, pathway activation, and characteristic morphological features. Further clinical follow-up is needed to determine whether these tumors are more indolent compared with the conventional clear cell renal cell carcinoma.

Down-Regulation of miR-129-5p and the let-7 Family in Neuroendocrine Tumors and Metastases Leads to Up-Regulation of Their Targets Egr1, G3bp1, Hmga2 and Bach1.

Expression of miRNAs in Neuroendocrine Neoplasms (NEN) is poorly characterized. We therefore wanted to examine the miRNA expression in Neuroendocrine Tumors (NETs), and identify their targets and importance in NET carcinogenesis. miRNA expression in six NEN primary tumors, six NEN metastases and four normal intestinal tissues was characterized using miRNA arrays, and validated by in-situ hybridization and qPCR. Among the down-regulated miRNAs miR-129-5p and the let-7f/let-7 family, were selected for further characterization. Transfection of miR-129-5p inhibited growth of a pulmonary and an intestinal carcinoid cell line. Analysis of mRNA expression changes identified EGR1 and G3BP1 as miR-129-5p targets. They were validated by luciferase assay and western blotting, and found robustly expressed in NETs by immunohistochemistry. Knockdown of EGR1 and G3BP1 mimicked the growth inhibition induced by miR-129-5p. let-7 overexpression inhibited growth of carcinoid cell lines, and let-7 inhibition increased protein content of the transcription factor BACH1 and its targets MMP1 and HMGA2, all known to promote bone metastases. Immunohistochemistry analysis revealed that let-7 targets are highly expressed in NETs and metastases. We found down-regulation of miR-129-5p and the let-7 family, and identified new neuroendocrine specific targets for these miRNAs, which contributes to the growth and metastatic potential of these tumors.

Genome-wide analysis of noncoding regulatory mutations in cancer.

Cancer primarily develops because of somatic alterations in the genome. Advances in sequencing have enabled large-scale sequencing studies across many tumor types, emphasizing the discovery of alterations in protein-coding genes. However, the protein-coding exome comprises less than 2% of the human genome. Here we analyze the complete genome sequences of 863 human tumors from The Cancer Genome Atlas and other sources to systematically identify noncoding regions that are recurrently mutated in cancer. We use new frequency- and sequence-based approaches to comprehensively scan the genome for noncoding mutations with potential regulatory impact. These methods identify recurrent mutations in regulatory elements upstream of PLEKHS1, WDR74 and SDHD, as well as previously identified mutations in the TERT promoter. SDHD promoter mutations are frequent in melanoma and are associated with reduced gene expression and poor prognosis. The non-protein-coding cancer genome remains widely unexplored, and our findings represent a step toward targeting the entire genome for clinical purposes.

SQSTM1 is a pathogenic target of 5q copy number gains in kidney cancer.

Clear cell renal cell carcinoma (ccRCC) is the most common form of kidney cancer and is often linked to loss of chromosome 3p, which harbors the VHL tumor suppressor gene, loss of chromosome 14q, which includes HIF1A, and gain of chromosome 5q. The relevant target(s) on chromosome 5q is not known. Here, we show that 5q amplification leads to overexpression of the SQSTM1 oncogene in ccRCC lines and tumors. Overexpression of SQSTM1 in ccRCC lines promoted resistance to redox stress and increased soft agar growth, while downregulation of SQSTM1 decreased resistance to redox stress, impaired cellular fitness, and decreased tumor formation. Therefore, the selection pressure to amplify 5q in ccRCC is driven, at least partly, by SQSTM1.

An epidemiologic and genomic investigation into the obesity paradox in renal cell carcinoma.

BACKGROUND: Obesity increases risk for clear-cell renal cell carcinoma (ccRCC), yet obese patients appear to experience longer survival than nonobese patients. We examined body mass index (BMI) in relation to stage, grade, and cancer-specific mortality (CSM) while considering detection bias, nutritional status, and molecular tumor features. METHODS: Data were available from 2119 ccRCC patients who underwent renal mass surgery at Memorial Sloan-Kettering Cancer Center between 1995 and 2012. Logistic regression models produced associations between BMI and advanced disease. Multivariable competing risks regression models estimated associations between BMI and CSM. Somatic mutation, copy number, methylation, and expression data were examined by BMI among a subset of 126 patients who participated in the Cancer Genome Atlas Project for ccRCC using the Kruskal-Wallis or Fisher exact tests. All statistical tests were two-sided. RESULTS: Obese and overweight patients were less likely to present with advanced-stage disease compared with normal-weight patients (odds ratio [OR] = 0.61, 95% confidence interval [CI] = 0.48 to 0.79 vs OR = 0.65, 95% CI = 0.51 to 0.83, respectively). Higher BMI was associated with reduced CSM in univariable analyses (P < .005). It remained statistically significant after adjustment for comorbidities and albumin level, but it became non-statistically significant after adjusting for stage and grade (P > .10). Genome-wide interrogation by BMI suggested differences in gene expression of metabolic and fatty acid genes, including fatty acid synthase (FASN), consistent with the obesity paradox. CONCLUSIONS: Our findings suggest that although BMI is not an independent prognostic factor for CSM after controlling for stage and grade, tumors developing in an obesogenic environment may be more indolent.

Comprehensive analysis of long non-coding RNAs in ovarian cancer reveals global patterns and targeted DNA amplification.

Long non-coding RNAs (lncRNAs) are emerging as potent regulators of cell physiology, and recent studies highlight their role in tumor development. However, while established protein-coding oncogenes and tumor suppressors often display striking patterns of focal DNA copy-number alteration in tumors, similar evidence is largely lacking for lncRNAs. Here, we report on a genomic analysis of GENCODE lncRNAs in high-grade serous ovarian adenocarcinoma, based on The Cancer Genome Atlas (TCGA) molecular profiles. Using genomic copy-number data and deep coverage transcriptome sequencing, we derived dual copy-number and expression data for 10,419 lncRNAs across 407 primary tumors. We describe global correlations between lncRNA copy-number and expression, and associate established expression subtypes with distinct lncRNA signatures. By examining regions of focal copy-number change that lack protein-coding targets, we identified an intergenic lncRNA on chromosome 1, OVAL, that shows narrow focal genomic amplification in a subset of tumors. While weakly expressed in most tumors, focal amplification coincided with strong OVAL transcriptional activation. Screening of 16 other cancer types revealed similar patterns in serous endometrial carcinomas. This shows that intergenic lncRNAs can be specifically targeted by somatic copy-number amplification, suggestive of functional involvement in tumor initiation or progression. Our analysis provides testable hypotheses and paves the way for further study of lncRNAs based on TCGA and other large-scale cancer genomics datasets.

Analysis of microRNA-target interactions across diverse cancer types.

Little is known about the extent to which individual microRNAs (miRNAs) regulate common processes of tumor biology across diverse cancer types. Using molecular profiles of >3,000 tumors from 11 human cancer types in The Cancer Genome Atlas, we systematically analyzed expression of miRNAs and mRNAs across cancer types to infer recurrent cancer-associated miRNA-target relationships. As we expected, the inferred relationships were consistent with sequence-based predictions and published data from miRNA perturbation experiments. Notably, miRNAs with recurrent target relationships were frequently regulated by genetic and epigenetic alterations across the studied cancer types. We also identify new examples of miRNAs that coordinately regulate cancer pathways, including the miR-29 family, which recurrently regulates active DNA demethylation pathway members TET1 and TDG. The online resource http://cancerminer.org allows exploration and prioritization of miRNA-target interactions that potentially regulate tumorigenesis.

The molecular diversity of Luminal A breast tumors.

Breast cancer is a collection of diseases with distinct molecular traits, prognosis, and therapeutic options. Luminal A breast cancer is the most heterogeneous, both molecularly and clinically. Using genomic data from over 1,000 Luminal A tumors from multiple studies, we analyzed the copy number and mutational landscape of this tumor subtype. This integrated analysis revealed four major subtypes defined by distinct copy-number and mutation profiles. We identified an atypical Luminal A subtype characterized by high genomic instability, TP53 mutations, and increased Aurora kinase signaling; these genomic alterations lead to a worse clinical prognosis. Aberrations of chromosomes 1, 8, and 16, together with PIK3CA, GATA3, AKT1, and MAP3K1 mutations drive the other subtypes. Finally, an unbiased pathway analysis revealed multiple rare, but mutually exclusive, alterations linked to loss of activity of co-repressor complexes N-Cor and SMRT. These rare alterations were the most prevalent in Luminal A tumors and may predict resistance to endocrine therapy. Our work provides for a further molecular stratification of Luminal A breast tumors, with potential direct clinical implications.

cWords - systematic microRNA regulatory motif discovery from mRNA expression data.

BACKGROUND: Post-transcriptional regulation of gene expression by small RNAs and RNA binding proteins is of fundamental importance in development of complex organisms, and dysregulation of regulatory RNAs can influence onset, progression and potentially be target for treatment of many diseases. Post-transcriptional regulation by small RNAs is mediated through partial complementary binding to messenger RNAs leaving nucleotide signatures or motifs throughout the entire transcriptome. Computational methods for discovery and analysis of sequence motifs in high-throughput mRNA expression profiling experiments are becoming increasingly important tools for the identification of post-transcriptional regulatory motifs and the inference of the regulators and their targets. RESULTS: cWords is a method designed for regulatory motif discovery in differential case-control mRNA expression datasets. We have improved the algorithms and statistical methods of cWords, resulting in at least a factor 100 speed gain over the previous implementation. On a benchmark dataset of 19 microRNA (miRNA) perturbation experiments cWords showed equal or better performance than two comparable methods, miReduce and Sylamer. We have developed rigorous motif clustering and visualization that accompany the cWords analysis for more intuitive and effective data interpretation. To demonstrate the versatility of cWords we show that it can also be used for identification of potential siRNA off-target binding. Moreover, cWords analysis of an experiment profiling mRNAs bound by Argonaute ribonucleoprotein particles discovered endogenous miRNA binding motifs. CONCLUSIONS: cWords is an unbiased, flexible and easy-to-use tool designed for regulatory motif discovery in differential case-control mRNA expression datasets. cWords is based on rigorous statistical methods that demonstrate comparable or better performance than other existing methods. Rich visualization of results promotes intuitive and efficient interpretation of data. cWords is available as a stand-alone Open Source program at Github https://github.com/simras/cWords and as a web-service at: http://servers.binf.ku.dk/cwords/.

Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal.

The cBioPortal for Cancer Genomics (http://cbioportal.org) provides a Web resource for exploring, visualizing, and analyzing multidimensional cancer genomics data. The portal reduces molecular profiling data from cancer tissues and cell lines into readily understandable genetic, epigenetic, gene expression, and proteomic events. The query interface combined with customized data storage enables researchers to interactively explore genetic alterations across samples, genes, and pathways and, when available in the underlying data, to link these to clinical outcomes. The portal provides graphical summaries of gene-level data from multiple platforms, network visualization and analysis, survival analysis, patient-centric queries, and software programmatic access. The intuitive Web interface of the portal makes complex cancer genomics profiles accessible to researchers and clinicians without requiring bioinformatics expertise, thus facilitating biological discoveries. Here, we provide a practical guide to the analysis and visualization features of the cBioPortal for Cancer Genomics.

microRNA-146a inhibits G protein-coupled receptor-mediated activation of NF-κB by targeting CARD10 and COPS8 in gastric cancer.

BACKGROUND: Gastric cancer is the second most common cause of cancer-related death in the world. Inflammatory signals originating from gastric cancer cells are important for recruiting inflammatory cells and regulation of metastasis of gastric cancer. Several microRNAs (miRNA) have been shown to be involved in development and progression of gastric cancer. miRNA-146a (miR-146a) is a modulator of inflammatory signals, but little is known about its importance in gastric cancer. We therefore wanted to identify targets of miR-146a in gastric cancer and examine its biological roles. RESULTS: The expression of miR-146a was evaluated by quantitative PCR (qPCR) and found up-regulated in the gastrin knockout mice, a mouse model of gastric cancer, and in 73% of investigated human gastric adenocarcinomas. Expression of miR-146a by gastric cancer cells was confirmed by in situ hybridization. Global analysis of changes in mRNA levels after miR-146a transfection identified two transcripts, caspase recruitment domain-containing protein 10 (CARD10) and COP9 signalosome complex subunit 8 (COPS8), as new miR-146a targets. qPCR, Western blotting and luciferase assays confirmed these transcripts as direct miR-146a targets. CARD10 and COPS8 were shown to be part of the G protein-coupled receptor (GPCR) pathway of nuclear factor-kappaB (NF-kappaB) activation. Lysophosphatidic acid (LPA) induces NF-kappaB activation via this pathway and over-expression of miR-146a inhibited LPA-induced NF-kappaB activation, reduced LPA-induced expression of tumor-promoting cytokines and growth factors and inhibited monocyte attraction. CONCLUSIONS: miR-146a expression is up-regulated in a majority of gastric cancers where it targets CARD10 and COPS8, inhibiting GPCR-mediated activation of NF-kappaB, thus reducing expression of NF-kappaB-regulated tumor-promoting cytokines and growth factors. By targeting components of several NF-kappaB-activating pathways, miR-146a is a key component in the regulation of NF-kappaB activity.

MicroRNA-143 down-regulates Hexokinase 2 in colon cancer cells.

BACKGROUND: MicroRNAs (miRNAs) are well recognized as gene regulators and have been implicated in the regulation of development as well as human diseases. miR-143 is located at a fragile site on chromosome 5 frequently deleted in cancer, and has been reported to be down-regulated in several cancers including colon cancer. METHODS: To gain insight into the role of miR-143 in colon cancer, we used a microarray-based approach in combination with seed site enrichment analysis to identify miR-143 targets. RESULTS: As expected, transcripts down-regulated upon miR-143 overexpression had a significant enrichment of miR-143 seed sites in their 3'UTRs. Here we report the identification of Hexokinase 2 (HK2) as a direct target of miR-143. We show that re-introduction of miR-143 in the colon cancer cell line DLD-1 results in a decreased lactate secretion. CONCLUSION: We have identified and validated HK2 as a miR-143 target. Furthermore, our results indicate that miR-143 mediated down-regulation of HK2 affects glucose metabolism in colon cancer cells. We hypothesize that loss of miR-143-mediated repression of HK2 can promote glucose metabolism in cancer cells, contributing to the shift towards aerobic glycolysis observed in many tumors.

The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data.

The cBio Cancer Genomics Portal (http://cbioportal.org) is an open-access resource for interactive exploration of multidimensional cancer genomics data sets, currently providing access to data from more than 5,000 tumor samples from 20 cancer studies. The cBio Cancer Genomics Portal significantly lowers the barriers between complex genomic data and cancer researchers who want rapid, intuitive, and high-quality access to molecular profiles and clinical attributes from large-scale cancer genomics projects and empowers researchers to translate these rich data sets into biologic insights and clinical applications.

Integrated analyses of microRNAs demonstrate their widespread influence on gene expression in high-grade serous ovarian carcinoma.

BACKGROUND: The Cancer Genome Atlas (TCGA) Network recently comprehensively catalogued the molecular aberrations in 487 high-grade serous ovarian cancers, with much remaining to be elucidated regarding the microRNAs (miRNAs). Here, using TCGA ovarian data, we surveyed the miRNAs, in the context of their predicted gene targets. METHODS AND RESULTS: Integration of miRNA and gene patterns yielded evidence that proximal pairs of miRNAs are processed from polycistronic primary transcripts, and that intronic miRNAs and their host gene mRNAs derive from common transcripts. Patterns of miRNA expression revealed multiple tumor subtypes and a set of 34 miRNAs predictive of overall patient survival. In a global analysis, miRNA:mRNA pairs anti-correlated in expression across tumors showed a higher frequency of in silico predicted target sites in the mRNA 3'-untranslated region (with less frequency observed for coding sequence and 5'-untranslated regions). The miR-29 family and predicted target genes were among the most strongly anti-correlated miRNA:mRNA pairs; over-expression of miR-29a in vitro repressed several anti-correlated genes (including DNMT3A and DNMT3B) and substantially decreased ovarian cancer cell viability. CONCLUSIONS: This study establishes miRNAs as having a widespread impact on gene expression programs in ovarian cancer, further strengthening our understanding of miRNA biology as it applies to human cancer. As with gene transcripts, miRNAs exhibit high diversity reflecting the genomic heterogeneity within a clinically homogeneous disease population. Putative miRNA:mRNA interactions, as identified using integrative analysis, can be validated. TCGA data are a valuable resource for the identification of novel tumor suppressive miRNAs in ovarian as well as other cancers.

miR-34a repression in proneural malignant gliomas upregulates expression of its target PDGFRA and promotes tumorigenesis.

Glioblastoma (GBM) and other malignant gliomas are aggressive primary neoplasms of the brain that exhibit notable refractivity to standard treatment regimens. Recent large-scale molecular profiling has revealed distinct disease subclasses within malignant gliomas whose defining genomic features highlight dysregulated molecular networks as potential targets for therapeutic development. The "proneural" designation represents the largest and most heterogeneous of these subclasses, and includes both a large fraction of GBMs along with most of their lower-grade astrocytic and oligodendroglial counterparts. The pathogenesis of proneural gliomas has been repeatedly associated with dysregulated PDGF signaling. Nevertheless, genomic amplification or activating mutations involving the PDGF receptor (PDGFRA) characterize only a subset of proneural GBMs, while the mechanisms driving dysregulated PDGF signaling and downstream oncogenic networks in remaining tumors are unclear. MicroRNAs (miRNAs) are a class of small, noncoding RNAs that regulate gene expression by binding loosely complimentary sequences in target mRNAs. The role of miRNA biology in numerous cancer variants is well established. In an analysis of miRNA involvement in the phenotypic expression and regulation of oncogenic PDGF signaling, we found that miR-34a is downregulated by PDGF pathway activation in vitro. Similarly, analysis of data from the Cancer Genome Atlas (TCGA) revealed that miR-34a expression is significantly lower in proneural gliomas compared to other tumor subtypes. Using primary GBM cells maintained under neurosphere conditions, we then demonstrated that miR-34a specifically affects growth of proneural glioma cells in vitro and in vivo. Further bioinformatic analysis identified PDGFRA as a direct target of miR-34a and this interaction was experimentally validated. Finally, we found that PDGF-driven miR-34a repression is unlikely to operate solely through a p53-dependent mechanism. Taken together, our data support the existence of reciprocal negative feedback regulation involving miR-34 and PDGFRA expression in proneural gliomas and, as such, identify a subtype specific therapeutic potential for miR-34a.

Small RNA sequencing and functional characterization reveals MicroRNA-143 tumor suppressor activity in liposarcoma.

Liposarcoma remains the most common mesenchymal cancer, with a mortality rate of 60% among patients with this disease. To address the present lack of therapeutic options, we embarked upon a study of microRNA (miRNA) expression alterations associated with liposarcomagenesis with the goal of exploiting differentially expressed miRNAs and the gene products they regulate as potential therapeutic targets. MicroRNA expression was profiled in samples of normal adipose tissue, well-differentiated liposarcoma, and dedifferentiated liposarcoma by both deep sequencing of small RNA libraries and hybridization-based Agilent microarrays. The expression profiles discriminated liposarcoma from normal adipose tissue and well differentiated from dedifferentiated disease. We defined over 40 miRNAs that were dysregulated in dedifferentiated liposarcomas in both the sequencing and the microarray analysis. The upregulated miRNAs included two cancer-associated species (miR-21 and miR-26a), and the downregulated miRNAs included two species that were highly abundant in adipose tissue (miR-143 and miR-145). Restoring miR-143 expression in dedifferentiated liposarcoma cells inhibited proliferation, induced apoptosis, and decreased expression of BCL2, topoisomerase 2A, protein regulator of cytokinesis 1 (PRC1), and polo-like kinase 1 (PLK1). The downregulation of PRC1 and its docking partner PLK1 suggests that miR-143 inhibits cytokinesis in these cells. In support of this idea, treatment with a PLK1 inhibitor potently induced G(2)-M growth arrest and apoptosis in liposarcoma cells. Taken together, our findings suggest that miR-143 re-expression vectors or selective agents directed at miR-143 or its targets may have therapeutic value in dedifferentiated liposarcoma.

Off-target effects dominate a large-scale RNAi screen for modulators of the TGF-ß pathway and reveal microRNA regulation of TGFBR2.

BACKGROUND: RNA interference (RNAi) screens have been used to identify novel components of signal-transduction pathways in a variety of organisms. We performed a small interfering (si)RNA screen for novel members of the transforming growth factor (TGF)-ß pathway in a human keratinocyte cell line. The TGF-ß pathway is integral to mammalian cell proliferation and survival, and aberrant TGF-ß responses have been strongly implicated in cancer. RESULTS: We assayed how strongly single siRNAs targeting each of 6,000 genes affect the nuclear translocation of a green fluorescent protein (GFP)-SMAD2 reporter fusion protein. Surprisingly, we found no novel TGF-ß pathway members, but we did find dominant off-target effects. All siRNA hits, whatever their intended direct target, reduced the mRNA levels of two known upstream pathway components, the TGF-ß receptors 1 and 2 (TGFBR1 and TGFBR2), via micro (mi)RNA-like off-target effects. The scale of these off-target effects was remarkable, with at least 1% of the sequences in the unbiased siRNA library having measurable off-target effects on one of these two genes. It seems that relatively minor reductions of message levels via off-target effects can have dominant effects on an assay, if the pathway output is very dose-sensitive to levels of particular pathway components. In search of mechanistic details, we identified multiple miRNA-like sequence characteristics that correlated with the off-target effects. Based on these results, we identified miR-20a, miR-34a and miR-373 as miRNAs that inhibit TGFBR2 expression. CONCLUSIONS: Our findings point to potential improvements for miRNA/siRNA target prediction methods, and suggest that the type II TGF-ß receptor is regulated by multiple miRNAs. We also conclude that the risk of obtaining misleading results in siRNA screens using large libraries with single-assay readout is substantial. Control and rescue experiments are essential in the interpretation of such screens, and improvements to the methods to reduce or predict RNAi off-target effects would be beneficial.