MicroRNA-31 functions as an oncogenic microRNA in mouse and human lung cancer cells by repressing specific tumor suppressors

MicroRNAs (miRNAs) regulate gene expression. It has been suggested that obtaining miRNA expression profiles can improve classification, diagnostic, and prognostic information in oncology. Here, we sought to comprehensively identify the miRNAs that are overexpressed in lung cancer by conducting miRNA microarray expression profiling on normal lung versus adjacent lung cancers from transgenic mice. We found that miR-136, miR-376a, and miR-31 were each prominently overexpressed in murine lung cancers. Real-time RT-PCR and in situ hybridization (ISH) assays confirmed these miRNA expression profiles in paired normal-malignant lung tissues from mice and humans. Engineered knockdown of miR-31, but not other highlighted miRNAs, substantially repressed lung cancer cell growth and tumorigenicity in a dose-dependent manner. Using a bioinformatics approach, we identified miR-31 target mRNAs and independently confirmed them as direct targets in human and mouse lung cancer cell lines. These targets included the tumor-suppressive genes large tumor suppressor 2 (LATS2) and PP2A regulatory subunit B alpha isoform (PPP2R2A), and expression of each was augmented by miR-31 knockdown. Their engineered repression antagonized miR-31–mediated growth inhibition. Notably, miR-31 and these target mRNAs were inversely expressed in mouse and human lung cancers, underscoring their biologic relevance. The clinical relevance of miR-31 expression was further independently and comprehensively validated using an array containing normal and malignant human lung tissues. Together, these findings revealed that miR-31 acts as an oncogenic miRNA (oncomir) in lung cancer by targeting specific tumor suppressors for repression.     

Identification of suitable reference genes for gene expression measurement in uterine sarcoma and carcinosarcoma tumors

ObjectiveTo date no suitable reference genes have been identified in carcinosarcomas and non-epithelial malignant tumors of corpus uteri for normalizing real-time RT-PCR (qRT-PCR) assays. The purpose of this study was to select appropriate references for gene expression studies in these tumors.Materials and methodsWe used RNA extracts from 75 tissue samples, representing 50 tumors and 25 fragments of normal uterine tissues obtained from 50 patients treated for mixed tumors, smooth muscle sarcoma and stromal sarcoma of the uterus. qRT-PCR for five potential reference (housekeeping) genes, namely B2M, HMBS, HPRT1, TBP and UBC, was performed. The expression stability of these genes was assayed using geNorm software application.ResultsThe analysis of gene expression data with geNorm identified HPRT1 as the most stable reference gene, followed by UBC and HMBS, for all the investigated tissues. When stratified by disease, the results still pointed at HPRT1 as the gene that retained the greatest robustness in mixed tumors as well as in smooth muscle and stromal sarcomas.ConclusionsOur work is the first report on reference gene selection for qRT-PCR applications in mixed tumors, smooth muscle sarcoma and stromal sarcoma of the uterus. A ranking of candidate genes’ stability values for the three types of tumors is provided and might serve as a valuable guide for future gene expression studies of these rare entities.     

Integrated Functional, Gene Expression and Genomic Analysis for the Identification of Cancer Targets

The majority of new drug approvals for cancer are based on existing therapeutic targets. One approach to the identification of novel targets is to perform high-throughput RNA interference (RNAi) cellular viability screens. We describe a novel approach combining RNAi screening in multiple cell lines with gene expression and genomic profiling to identify novel cancer targets. We performed parallel RNAi screens in multiple cancer cell lines to identify genes that are essential for viability in some cell lines but not others, suggesting that these genes constitute key drivers of cellular survival in specific cancer cells. This approach was verified by the identification of PIK3CA, silencing of which was selectively lethal to the MCF7 cell line, which harbours an activating oncogenic PIK3CA mutation. We combined our functional RNAi approach with gene expression and genomic analysis, allowing the identification of several novel kinases, including WEE1, that are essential for viability only in cell lines that have an elevated level of expression of this kinase. Furthermore, we identified a subset of breast tumours that highly express WEE1 suggesting that WEE1 could be a novel therapeutic target in breast cancer. In conclusion, this strategy represents a novel and effective strategy for the identification of functionally important therapeutic targets in cancer.     

The clear cell sarcoma functional genomic landscape

Clear cell sarcoma (CCS) is a deadly malignancy affecting adolescents and young adults. It is characterized by reciprocal translocations resulting in expression of the chimeric EWSR1-ATF1 or EWSR1-CREB1 fusion proteins, driving sarcomagenesis. Besides these characteristics, CCS has remained genomically uncharacterized. Copy number analysis of human CCSs showed frequent amplifications of the MITF locus and chromosomes 7 and 8. Few alterations were shared with Ewing sarcoma or desmoplastic, small round cell tumors, which are other EWSR1-rearranged tumors. Exome sequencing in mouse tumors generated by expression of EWSR1-ATF1 from the Rosa26 locus demonstrated no other repeated pathogenic variants. Additionally, we generated a new CCS mouse by Cre-loxP–induced chromosomal translocation between Ewsr1 and Atf1, resulting in copy number loss of chromosome 6 and chromosome 15 instability, including amplification of a portion syntenic to human chromosome 8, surrounding Myc. Additional experiments in the Rosa26 conditional model demonstrated that Mitf or Myc can contribute to sarcomagenesis. Copy number observations in human tumors and genetic experiments in mice rendered, for the first time to our knowledge, a functional landscape of the CCS genome. These data advance efforts to understand the biology of CCS using innovative models that will eventually allow us to validate preclinical therapies necessary to achieve longer and better survival for young patients with this disease.     

Treatment of Type 1 Myotonic Dystrophy by Engineering Site-specific RNA Endonucleases that Target (CUG)n Repeats

Myotonic dystrophy type 1 (DM1) is caused by the expansion of (CTG)_n in the 3′ untranslated region of the dystrophia myotonica-protein kinase (DMPK) gene, which is transcribed as (CUG)_n repeats that accumulate in the nucleus. The RNA repeats specifically sequester or change the expression levels of several RNA-binding proteins, leading to aberrant splicing of many target genes. In this study, we developed artificial site-specific RNA endonucleases (ASREs) that specifically bind and cleave (CUG)_n repeats RNA. We have generated one ASRE that can target the expanded RNA repeats in DM1 patient cells and specifically degrade the pathogenic DMPK messenger RNAs with minimal effect on wild-type alleles. Such ASRE treatment significantly decreased the number of nuclear foci in DM1 patient cells and can reverse the missplicing of many genes affected in DM1 patients. Taken together, the application of ASRE provides a new route of gene therapy for DM1 treatment.     

Transduction of Fetal Mice With a Feline Lentiviral Vector Induces Liver Tumors Which Exhibit an E2F Activation Signature

Lentiviral vectors are widely used in basic research and clinical applications for gene transfer and long-term expression; however, safety issues have not yet been completely resolved. In this study, we characterized hepatocarcinomas that developed in mice 1 year after in utero administration of a feline-derived lentiviral vector. Mapped viral integration sites differed among tumors and did not coincide with the regions of chromosomal aberrations. Furthermore, gene expression profiling revealed that no known cancer-associated genes were deregulated in the vicinity of viral integrations. Nevertheless, five of the six tumors exhibited highly significant upregulation of E2F target genes, of which a majority are associated with oncogenesis, DNA damage response, and chromosomal instability. We further show in vivo and in vitro that E2F activation occurs early on following transduction of both fetal mice and cultured human hepatocytes. On the basis of the similarities in E2F target gene expression patterns among tumors and the lack of evidence implicating insertional mutagenesis, we propose that transduction of fetal mice with a feline lentiviral vector induces E2F-mediated major cellular processes that drive hepatocytes toward uncontrolled proliferation culminating in tumorigenesis.     

MLL1 and DOT1L cooperate with meningioma-1 to induce acute myeloid leukemia

Meningioma-1 (MN1) overexpression is frequently observed in patients with acute myeloid leukemia (AML) and is predictive of poor prognosis. In murine models, forced expression of MN1 in hematopoietic progenitors induces an aggressive myeloid leukemia that is strictly dependent on a defined gene expression program in the cell of origin, which includes the homeobox genes Hoxa9 and Meis1 as key components. Here, we have shown that this program is controlled by two histone methyltransferases, MLL1 and DOT1L, as deletion of either Mll1 or Dot1l in MN1-expressing cells abrogated the cell of origin–derived gene expression program, including the expression of Hoxa cluster genes. In murine models, genetic inactivation of either Mll1 or Dot1l impaired MN1-mediated leukemogenesis. We determined that HOXA9 and MEIS1 are coexpressed with MN1 in a subset of clinical MN1^hi leukemia, and human MN1^hi/HOXA9^hi leukemias were sensitive to pharmacologic inhibition of DOT1L. Together, these data point to DOT1L as a potential therapeutic target in MN1^hi AML. In addition, our findings suggest that epigenetic modulation of the interplay between an oncogenic lesion and its cooperating developmental program has therapeutic potential in AML.     

Delineating Bacteriostatic and Bactericidal Targets in Mycobacteria Using IPTG Inducible Antisense Expression

In order to identify novel high value antibacterial targets it is desirable to delineate whether the inactivation of the target enzyme will lead to bacterial death or stasis. This knowledge is particularly important in slow growing organisms, like mycobacteria, where most of the viable anti-tubercular agents are bactericidal. A bactericidal target can be identified through the conditional deletion or inactivation of the target gene at a relatively high cell number and subsequently following the time course of survival for the bacteria. A simple protocol to execute conditional inactivation of a gene is by antisense expression. We have developed a mycobacteria specific IPTG inducible vector system and monitored the effect of antisense inhibition of several known essential genes in mycobacteria by following their survival kinetics. By this method, we could differentiate between genes whose down regulation lead to bacteriostatic or bactericidal effect. Targets for standard anti-tubercular drugs like inhA for isoniazid, rpoB and C for rifampicin, and gyr A/B for flouroquinolones were shown to be bactericidal. In contrast targets like FtsZ behaved in a bacteriostatic manner. Induction of antisense expression in embB and ribosomal RNA genes, viz., rplJ and rpsL showed only a marginal growth inhibition. The specificity of the antisense inhibition was conclusively shown in the case of auxotrophic gene ilvB. The bactericidal activity following antisense expression of ilvB was completely reversed when the growth media was supplemented with the isoleucine, leucine, valine and pantothenate. Additionally, under these conditions the expression of several genes in branched chain amino acid pathway was severely suppressed indicating targeted gene inactivation.