miR-23a impairs bone differentiation in osteosarcoma via down-regulation of GJA1.

Osteosarcoma is the most common type of bone cancer in children and adolescents. Impaired differentiation of osteoblast cells is a distinguishing feature of this aggressive disease. As improvements in survival outcomes have largely plateaued, better understanding of the bone differentiation program may provide new treatment approaches. The miRNA cluster miR-23a~27a~24-2, particularly miR-23a, has been shown to interact with genes important for bone development. However, global changes in gene expression associated with functional gain of this cluster have not been fully explored. To better understand the relationship between miR-23a expression and bone cell differentiation, we carried out a large-scale gene expression analysis in HOS cells. Experimental results demonstrate that over-expression of miR-23a delays differentiation in this system. Downstream bioinformatic analysis identified miR-23a target gene connexin-43 (Cx43/GJA1), a mediator of intercellular signaling critical to osteoblast development, as acutely affected by miR-23a levels. Connexin-43 is up-regulated in the course of HOS cell differentiation and is down-regulated in cells transfected with miR-23a. Analysis of gene expression data, housed at Gene Expression Omnibus, reveals that Cx43 is consistently up-regulated during osteoblast differentiation. Suppression of Cx43 mRNA by miR-23a was confirmed in vitro using a luciferase reporter assay. This work demonstrates novel interactions between microRNA expression, intercellular signaling and bone differentiation in osteosarcoma.

The CDX1-microRNA-215 axis regulates colorectal cancer stem cell differentiation.

The transcription factor caudal-type homeobox 1 (CDX1) is a key regulator of differentiation in the normal colon and in colorectal cancer (CRC). CDX1 activates the expression of enterocyte genes, but it is not clear how the concomitant silencing of stem cell genes is achieved. MicroRNAs (miRNAs) are important mediators of gene repression and have been implicated in tumor suppression and carcinogenesis, but the roles of miRNAs in differentiation, particularly in CRC, remain poorly understood. Here, we identified microRNA-215 (miR-215) as a direct transcriptional target of CDX1 by using high-throughput small RNA sequencing to profile miRNA expression in two pairs of CRC cell lines: CDX1-low HCT116 and HCT116 with stable CDX1 overexpression, and CDX1-high LS174T and LS174T with stable CDX1 knockdown. Validation of candidate miRNAs identified by RNA-seq in a larger cell-line panel revealed miR-215 to be most significantly correlated with CDX1 expression. Quantitative ChIP-PCR and promoter luciferase assays confirmed that CDX1 directly activates miR-215 transcription. miR-215 expression is depleted in FACS-enriched cancer stem cells compared with unsorted samples. Overexpression of miR-215 in poorly differentiated cell lines causes a decrease in clonogenicity, whereas miR-215 knockdown increases clonogenicity and impairs differentiation in CDX1-high cell lines. We identified the genome-wide targets of miR-215 and found that miR-215 mediates the repression of cell cycle and stemness genes downstream of CDX1. In particular, the miR-215 target gene BMI1 has been shown to promote stemness and self-renewal and to vary inversely with CDX1. Our work situates miR-215 as a link between CDX1 expression and BMI1 repression that governs differentiation in CRC.

Whole genome analyses of a well-differentiated liposarcoma reveals novel SYT1 and DDR2 rearrangements.

Liposarcoma is the most common soft tissue sarcoma, but little is known about the genomic basis of this disease. Given the low cell content of this tumor type, we utilized flow cytometry to isolate the diploid normal and aneuploid tumor populations from a well-differentiated liposarcoma prior to array comparative genomic hybridization and whole genome sequencing. This work revealed massive highly focal amplifications throughout the aneuploid tumor genome including MDM2, a gene that has previously been found to be amplified in well-differentiated liposarcoma. Structural analysis revealed massive rearrangement of chromosome 12 and 11 gene fusions, some of which may be part of double minute chromosomes commonly present in well-differentiated liposarcoma. We identified a hotspot of genomic instability localized to a region of chromosome 12 that includes a highly conserved, putative L1 retrotransposon element, LOC100507498 which resides within a gene cluster (NAV3, SYT1, PAWR) where 6 of the 11 fusion events occurred. Interestingly, a potential gene fusion was also identified in amplified DDR2, which is a potential therapeutic target of kinase inhibitors such as dastinib, that are not routinely used in the treatment of patients with liposarcoma. Furthermore, 7 somatic, damaging single nucleotide variants have also been identified, including D125N in the PTPRQ protein. In conclusion, this work is the first to report the entire genome of a well-differentiated liposarcoma with novel chromosomal rearrangements associated with amplification of therapeutically targetable genes such as MDM2 and DDR2.

A p21-ZEB1 complex inhibits epithelial-mesenchymal transition through the microRNA 183-96-182 cluster.

The tumor suppressor p21 acts as a cell cycle inhibitor and has also been shown to regulate gene expression by functioning as a transcription corepressor. Here, we identified p21-regulated microRNAs (miRNAs) by sequencing small RNAs from isogenic p21(+/+) and p21(-/-) cells. Three abundant miRNA clusters, miR-200b-200a-429, miR-200c-141, and miR-183-96-182, were downregulated in p21-deficient cells. Consistent with the known function of the miR-200 family and p21 in inhibition of the epithelial-mesenchymal transition (EMT), we observed EMT upon loss of p21 in multiple model systems. To explore a role of the miR-183-96-182 cluster in EMT, we identified its genome-wide targets and found that miR-183 and miR-96 repressed common targets, including SLUG, ZEB1, ITGB1, and KLF4. Reintroduction of miR-200, miR-183, or miR-96 in p21(-/-) cells inhibited EMT, cell migration, and invasion. Conversely, antagonizing miR-200 and miR-183-96-182 cluster miRNAs in p21(+/+) cells increased invasion and elevated the levels of VIM, ZEB1, and SLUG mRNAs. Furthermore, we found that p21 forms a complex with ZEB1 at the miR-183-96-182 cluster promoter to inhibit transcriptional repression of this cluster by ZEB1, suggesting a reciprocal feedback loop.

Enforced expression of Lin28b leads to impaired T-cell development, release of inflammatory cytokines, and peripheral T-cell lymphoma.

LIN28A and LIN28B, the mammalian homologs of lin-28, are implicated in malignant transformation in part because of their ability to promote degradation of the let-7 family of miRs. In the present study, we show that overexpression of Lin28b in vivo leads to an aggressive peripheral T-cell lymphoma (PTCL) characterized by widespread infiltration of parenchymal organs with malignant CD4(+) cells. Similar to patients with PTCL, Lin28b-transgenic mice show signs of inflammation such as eosinophilia, increased C-reactive protein, release of inflammatory cytokines, and pleural effusion. The PTCLs that develop in Lin28b mice are derived from activated T cells and show decreased let-7 expression, increased Il6 expression, activation of NF-κB, and infiltration of B cells, all resulting in an inflammatory microenvironment. In addition, LIN28B is overexpressed 7.5-fold in PTCL patient samples compared with activated CD4(+) cells. The results of the present study demonstrate for the first time that Lin28b can transform primary cells in vivo, identify a previously unsuspected link between Lin28b and PTCL, and provide a unique animal model for the study of PTCL biology and therapy.

Gene expression identifies heterogeneity of metastatic propensity in high-grade soft tissue sarcomas.

BACKGROUND: Metastatic propensity of soft tissue sarcoma (STS) is heterogeneous and may be determined by gene expression patterns that do not correlate well with morphology. The authors have reported gene expression patterns that distinguish 2 broad classes of clear cell renal carcinoma (ccRCC-gene set), and other patterns that can distinguish heterogeneity of serous ovarian carcinoma (OVCA-gene set) and aggressive fibromatosis (AF-gene set); however, clinical follow-up data were not available for these samples. METHODS: In the current study, gene expression patterns in 73 samples of high-grade STS were examined using spotted cDNA microarray slides that contained ∼16,000 unique UniGene clusters. Approximately 50% of the genes present in the ccRCC-, OVCA-, and AF-gene sets were also represented in the data from this chip set, and these were combined to form a composite gene set of 278 probes. RESULTS: Hierarchical clustering using this composite gene set suggested the existence of subsets of the STS samples. Analysis revealed differences in the time to development of metastatic disease between the clusters defined by the first branch point of the clustering dendrogram (P = .005), and also among the 4 different clusters defined by the second branch points (P = .001). CONCLUSIONS: This approach suggests the existence of >2 subsets of high-grade pleomorphic STS, each with distinct clinical behavior. A composite gene set such as that described here may be useful to stratify STS in clinical trials, and may be of practical utility in patient management.

Genetic profiles of gastroesophageal cancer: combined analysis using expression array and tiling array--comparative genomic hybridization.

We aimed to characterize the genomic profiles of adenocarcinomas in the gastroesophageal junction in relation to cancers in the esophagus and the stomach. Profiles of gains/losses as well as gene expression profiles were obtained from 27 gastroesophageal adenocarcinomas by means of 32k high-resolution array-based comparative genomic hybridization and 27k oligo gene expression arrays, and putative target genes were validated in an extended series. Adenocarcinomas in the distal esophagus and the gastroesophageal junction showed strong similarities with the most common gains at 20q13, 8q24, 1q21-23, 5p15, 13q34, and 12q13, whereas different profiles with gains at 5p15, 7p22, 2q35, and 13q34 characterized gastric cancers. CDK6 and EGFR were identified as putative target genes in cancers of the esophagus and the gastroesophageal junction, with upregulation in one quarter of the tumors. Gains/losses and gene expression profiles show strong similarity between cancers in the distal esophagus and the gastroesophageal junction with frequent upregulation of CDK6 and EGFR, whereas gastric cancer displays distinct genetic changes. These data suggest that molecular diagnostics and targeted therapies can be applied to adenocarcinomas of the distal esophagus and gastroesophageal junction alike.

Indistinguishable genomic profiles and shared prognostic markers in undifferentiated pleomorphic sarcoma and leiomyosarcoma: different sides of a single coin?

Soft tissue sarcoma (STS) diagnostics and prognostics are challenging, particularly in highly malignant and pleomorphic subtypes such as undifferentiated pleomorphic sarcoma (UPS) and leiomyosarcoma (LMS). We applied 32K BAC arrays and gene expression profiling to 18 extremity soft tissue LMS and 31 extremity soft tissue UPS with the aim of identifying molecular subtype signatures and genomic prognostic markers. Both the gains/losses and gene expression signatures revealed striking similarities between UPS and LMS, which were indistinguishable using unsupervised hierarchical cluster analysis and significance analysis for microarrays. Gene expression analysis revealed just nine genes, among them tropomyosin beta, which were differentially expressed. Loss of 4q31 (encompassing the SMAD1 locus), loss of 18q22, and tumor necrosis were identified as independent predictors of metastasis in multivariate stepwise Cox regression analysis. Combined analysis applying loss of 4q31 and 18q22 and the presence of necrosis improved the area under receiver operating characteristic curve for metastasis prediction from 0.64 to 0.86. The extensive genetic similarities between extremity soft tissue UPS and LMS suggest a shared lineage of these STS subtypes and the new and independent genetic prognosticators identified hold promise for refined prognostic determination in high-grade, genetically complex STS.

Diagnostic and prognostic gene expression signatures in 177 soft tissue sarcomas: hypoxia-induced transcription profile signifies metastatic potential.

BACKGROUND: Soft tissue sarcoma (STS) diagnosis is challenging because of a multitude of histopathological subtypes, different genetic characteristics, and frequent intratumoral pleomorphism. One-third of STS metastasize and current risk-stratification is suboptimal, therefore, novel diagnostic and prognostic markers would be clinically valuable. We assessed the diagnostic and prognostic value of array-based gene expression profiles using 27 k cDNA microarrays in 177, mainly high-grade, STS of 13 histopathological subtypes. RESULTS: Unsupervised analysis resulted in two major clusters--one mainly containing STS characterized by type-specific genetic alterations and the other with a predominance of genetically complex and pleomorphic STS. Synovial sarcomas, myxoid/round-cell liposarcomas, and gastrointestinal stromal tumors clustered tightly within the former cluster and discriminatory signatures for these were characterized by developmental genes from the EGFR, FGFR, Wnt, Notch, Hedgehog, RAR and KIT signaling pathways. The more pleomorphic STS subtypes, e.g. leiomyosarcoma, malignant fibrous histiocytoma/undifferentiated pleomorphic sarcoma and dedifferentiated/pleomorphic liposarcoma, were part of the latter cluster and were characterized by relatively heterogeneous profiles, although subclusters herein were identified. A prognostic signature partly characterized by hypoxia-related genes was identified among 89 genetically complex pleomorphic primary STS and could, in a multivariate analysis including established prognostic markers, independently predict the risk of metastasis with a hazard ratio of 2.2 (P = 0.04). CONCLUSION: Diagnostic gene expression profiles linking signaling pathways to the different STS subtypes were demonstrated and a hypoxia-induced metastatic profile was identified in the pleomorphic, high-grade STS. These findings verify diagnostic utility and application of expression data for improved selection of high-risk STS patients.

Gene expression profiles relate to SS18/SSX fusion type in synovial sarcoma.

We applied 27k spotted cDNA microarray slides to assess gene expression profiles in 26 samples from 24 patients with synovial sarcomas (SS). The data were analyzed in relation to histopathologic type, cytogenetic aberrations, gene fusion type and development of distant metastases. Supervised analysis based on gene fusion type in 12 SS with SS18/SSX1 and 9 with SS18/SSX2 revealed significant differences in gene expression profiles. Among the discriminators were several genes that have previously been found to be upregulated in SS, including AXL, ZIC2, SPAG7, AGRN, FOXC1, NCAM1 and multiple metallothioneins. Histopathology and degree of cytogenetic complexity did not significantly influence expression, whereas a genetic signature that related to development of metastases could be discerned, albeit with a high false-positive rate. In conclusion, our findings demonstrate differentially expressed genes for the 2 major gene fusion variants in SS, SS18/SSX1 and SS18/SSX2, and thereby suggest that these result in different downstream effects.

Intratumor versus intertumor heterogeneity in gene expression profiles of soft-tissue sarcomas.

Soft-tissue sarcomas (STSs) constitute more than 30 histologic entities. In addition, within each entity, tumors are often heterogeneous in macroscopic features, genetic alterations, microscopic appearance, and clinical course. Therefore, there has been concern about whether a single tumor sample can provide a gene expression profile representative of the entire tumor. We used 27-k cDNA microarray slides to assess the importance of intratumor versus intertumor heterogeneity of the gene expression profiles of 2 morphologically heterogeneous STSs. Multiple pieces of tumor (8 and 10 pieces) were obtained from a myxoid variant of malignant fibrous histiocytoma (MFH) and a leiomyosarcoma (LMS), respectively, and the expression patterns were compared with single tumor samples from 20 MFHs and 16 LMSs. Hierarchical clustering analysis of the expression profiles showed that samples from the same tumor clustered together. The average intratumor distance was considerably shorter than the average intertumor distance in both LMS and MFH. In addition, tumor subclusters that distinguished different macroscopic parts of the tumor could be discerned. We concluded that intratumor variability exists but that accurate gene expression profiling also could be obtained using single samples from a large STS.

Whole genome expression profiles of yeast RNA polymerase II core subunit, Rpb4, in stress and nonstress conditions.

Organisms respond to environmental stress by adopting changes in gene expression at the transcriptional level. Rpb4, a nonessential subunit of the core RNA polymerase II has been proposed to play a role in non-stress-specific transcription and in the regulation of stress response in yeast. We find that in addition to the temperature sensitivity of the null mutant of Rpb4, diploid null mutants are also compromised in sporulation and show morphological changes associated with nitrogen starvation. Using whole genome expression analysis, we report here the effects of Rpb4 on expression of genes during normal growth and following heat shock and nutritional starvation. Our analysis shows that Rpb4 affects expression of a small yet significant fraction of the genome in both stress and normal conditions. We found that genes involved in galactose metabolism were dependent on the presence of Rpb4 irrespective of the environmental condition. Rpb4 was also found to affect the expression of several other genes specifically in conditions of nutritional starvation. The general defect in the absence of Rpb4 is in the expression of metabolic genes, especially those involved in carbon metabolism and energy generation. We report that various stresses are affected by RPB4 and that on overexpression the stress-specific activators can partially rescue the corresponding defects.