Strand-specific miR-28-5p and miR-28-3p have distinct effects in colorectal cancer cells

The authors thank Sue Moreau from the Department of Scientific Publications at The University of Texas MD Anderson Cancer Center for English language editing of the manuscript. Author contributions: Study concept and design: M.I.A., P.A.Z, G.A.C. Acquisition of data: M.I.A., L.Z., X.Z. Drafting of the manuscript: M.I.A., M.N., R.S., M.F., R.M.R., P.A.Z, G.A.C. Analysis and interpretation of data: M.I.A., M.N., R.S., R.M., P.A.Z, G.A.C. Critical revision of the manuscript for important intellectual content: M.I.A., M.N., R.S., M.F., R.M.R., P.A.Z, G.A.C. Statistical analysis: M.I.A., C.I., L.X. Obtained funding: G.A.C. Administrative, technical, or material support: R.G., I.V., F.F., M.F., G.L. Study supervision: G.A.C. Drs Nicoloso and Spizzo are currently at the Division of Experimental Oncology, CRO, National Cancer Institute, Aviano, ItalyBackground & Aims MicroRNAs (miRNAs) can promote or inhibit tumor growth and are therefore being developed as targets for cancer therapies. They are diverse not only in the messenger RNAs (mRNA) they target, but in their production; the same hairpin RNA structure can generate mature products from each strand, termed 5p and 3p, that can bind different mRNAs. We analyzed the expression, functions, and mechanisms of miR-28-5p and miR-28-3p in colorectal cancer (CRC) cells. Methods We measured levels of miR-28-5p and miR-28-3p expression in 108 CRC and 49 normal colorectal samples (47 paired) by reverse transcription, quantitative real-time polymerase chain reaction. The roles of miR-28 in CRC development were studied using cultured HCT116, RKO, and SW480 cells and tumor xenograft analyses in immunodeficient mice; their mRNA targets were also investigated. Results miR-28-5p and miR-28-3p were down-regulated in CRC samples compared with normal colon samples. Overexpression of miRNAs in CRC cells had different effects and the miRNAs interacted with different mRNAs: miR-28-5p altered expression of CCND1 and HOXB3, whereas miR-28-3p bound NM23-H1. Overexpression of miR-28-5p reduced CRC cell proliferation, migration, and invasion in vitro, whereas miR-28-3p increased CRC cell migration and invasion in vitro. CRC cells overexpressing miR-28 developed tumors more slowly in mice compared with control cells, but miR-28 promoted tumor metastasis in mice. Conclusion miR-28-5p and miR-28-3p are transcribed from the same RNA hairpin and are down-regulated in CRC cells. Overexpression of each has different effects on CRC cell proliferation and migration. Such information has a direct application for the design of miRNA gene therapy trials.M.I.A. is supported by a PhD fellowship (SFRH/BD/47031/2008) from Fundação para a Ciência e Tecnologia, Portugal. G.A.C. is supported as a fellow by The University of Texas MD Anderson Cancer Center Research Trust, The University of Texas System Regents Research Scholar, and the Chronic Lymphocytic Leukemia Global Research Foundation. Work in Dr Calin’s laboratory is supported in part by grants from the National Institutes of Health (CA135444), the US Department of Defense, the Pancreatic Cancer Action Network (2009 Seena Magowitz AACR Pilot Grant), and the US-European Alliance for the Therapy of Chronic Lymphoid Leukemia. STR DNA fingerprinting was done by the Cancer Center Support grant funded Characterized Cell Line core, NCI # CA16672

Modulation of MicroRNA-194 and cell migration by HER2-targeting trastuzumab in breast cancer

Conceived and designed the experiments: XFL GAC RCB. Performed the experiments: XFL MIA WM RS MSN SZ. Analyzed the data: XFL SR. Contributed reagents/materials/analysis tools: YW GAC. Wrote the paper: XFL RCB.Trastuzumab, a humanized monoclonal antibody directed against the extracellular domain of the HER2 oncoprotein, can effectively target HER2-positive breast cancer through several mechanisms. Although the effects of trastuzumab on cancer cell proliferation, angiogenesis and apoptosis have been investigated in depth, the effect of trastuzumab on microRNA (miRNA) has not been extensively studied. We have performed miRNA microarray profiling before and after trastuzumab treatment in SKBr3 and BT474 human breast cancer cells that overexpress HER2. We found that trastuzumab treatment of SKBr3 cells significantly decreased five miRNAs and increased three others, whereas treatment of BT474 cells significantly decreased two miRNAs and increased nine. The only change in miRNA expression observed in both cell lines following trastuzumab treatment was upregulation of miRNA-194 (miR-194) that was further validated in vitro and in vivo. Forced expression of miR-194 in breast cancer cells that overexpress HER2 produced no effect on apoptosis, modest inhibition of proliferation, significant inhibition of cell migration/invasion in vitro and significant inhibition of xenograft growth in vivo. Conversely, knockdown of miR-194 promoted cell migration. Increased miR-194 expression markedly reduced levels of the cytoskeletal protein talin2 and specifically inhibited luciferase reporter activity of a talin2 wild-type 39-untranslated region, but not that of a mutant reporter, indicating that talin2 is a direct downstream target of miR-194. Trastuzumab treatment inhibited breast cancer cell migration and reduced talin2 expression in vitro and in vivo. Knockdown of talin2 inhibited cell migration/invasion. Knockdown of trastuzumab-induced miR-194 expression with a miR-194 inhibitor compromised trastuzumab-inhibited cell migration in HER2-overexpressing breast cancer cells. Consequently, trastuzumab treatment upregulates miR-194 expression and may exert its cell migration-inhibitory effect through miR-194-mediated downregulation of cytoskeleton protein talin2 in HER2-overexpressing human breast cancer cells.This work was supported by the Anne and Henry Zarrow Foundation, kind gifts from Stuart and Gaye Lynn Zarrow and from Mrs. Delores Wilkenfeld, the Laura and John Arnold Foundation, the RGK Foundation, and the MD Anderson NCI CCSG P30 CA16672. G.A.C. is supported as a Fellow at the University of Texas MD Anderson Research Trust, as a University of Texas System Regents Research Scholar and by the CLL Global Research Foundation

N-BLR, a primate-specific non-coding transcript leads to colorectal cancer invasion and migration

Background: non-coding RNAs have been drawing increasing attention in recent years as functional data suggest that they play important roles in key cellular processes. N-BLR is a primate-specific long non-coding RNA that modulates the epithelial-to-mesenchymal transition, facilitates cell migration, and increases colorectal cancer invasion. Results: we performed multivariate analyses of data from two independent cohorts of colorectal cancer patients and show that the abundance of N-BLR is associated with tumor stage, invasion potential, and overall patient survival. Through in vitro and in vivo experiments we found that N-BLR facilitates migration primarily via crosstalk with E-cadherin and ZEB1. We showed that this crosstalk is mediated by a pyknon, a short ~20 nucleotide-long DNA motif contained in the N-BLR transcript and is targeted by members of the miR-200 family. In light of these findings, we used a microarray to investigate the expression patterns of other pyknon-containing genomic loci. We found multiple such loci that are differentially transcribed between healthy and diseased tissues in colorectal cancer and chronic lymphocytic leukemia. Moreover, we identified several new loci whose expression correlates with the colorectal cancer patients' overall survival. Conclusions: the primate-specific N-BLR is a novel molecular contributor to the complex mechanisms that underlie metastasis in colorectal cancer and a potential novel biomarker for this disease. The presence of a functional pyknon within N-BLR and the related finding that many more pyknon-containing genomic loci in the human genome exhibit tissue-specific and disease-specific expression suggests the possibility of an alternative class of biomarkers and therapeutic targets that are primate-specific

CCAT2, a novel noncoding RNA mapping to 8q24, underlies metastatic progression and chromosomal instability in colon cancer

The functional roles of SNPs within the 8q24 gene desert in the cancer phenotype are not yet well understood. Here, we report that CCAT2, a novel long noncoding RNA transcript (lncRNA) encompassing the rs6983267 SNP, is highly overexpressed in microsatellite-stable colorectal cancer and promotes tumor growth, metastasis, and chromosomal instability. We demonstrate that MYC, miR-17-5p, and miR-20a are up-regulated by CCAT2 through TCF7L2-mediated transcriptional regulation. We further identify the physical interaction between CCAT2 and TCF7L2 resulting in an enhancement of WNT signaling activity. We show that CCAT2 is itself a WNT downstream target, which suggests the existence of a feedback loop. Finally, we demonstrate that the SNP status affects CCAT2 expression and the risk allele G produces more CCAT2 transcript. Our results support a new mechanism of MYC and WNT regulation by the novel lncRNA CCAT2 in colorectal cancer pathogenesis, and provide an alternative explanation of the SNP-conferred cancer risk

Fez1/Lzts1 absence impairs Cdk1/Cdc25C interaction during mitosis and predisposes mice to cancer development

The FEZ1/LZTS1 (LZTS1) protein is frequently downregulated in human cancers of different histo-types. LZTS1 is expressed in normal tissues, and its introduction in cancer cells inhibits cell growth and suppresses tumorigenicity, owing to an accumulation of cells in G2/M. Here, we define its role in cell cycle regulation and tumor progression by generating Lzts1 knockout mice. In Lzts1(-/-) mouse embryo fibroblasts (MEFs), Cdc25C degradation was increased during M phase, resulting in decreased Cdk1 activity. As a consequence, Lzts1(-/-) MEFs showed accelerated mitotic progression, resistance to taxol- and nocodazole-induced M phase arrest, and improper chromosome segregation. Accordingly, Lzts1 deficiency was associated with an increased incidence of both spontaneous and carcinogen-induced cancers in mice