IN VIVO FUNCTIONAL SIGNIFICANCE OF CCAT2 LONG NON-CODING RNA IN MYELODYSPLASTIC SYNDROME

Long non-coding RNAs form the largest part of the mammalian non-coding transcriptome and control gene expression at various levels including chromatin modification, transcriptional and post-transcriptional processing. Although the underlying molecular mechanisms are not yet entirely understood, lncRNAs are implicated in initiation and progression of several cancers. CCAT2 is a lncRNA that spans the highly conserved 8q24 region associated with increased risk for various cancers. CCAT2 has been shown to play an important role in inducing chromosomal instability and supporting cell proliferation and cell cycle arrest. However, a causal role of CCAT2 in initiation of tumorigenesis and the importance of G/T SNP in CCAT2-induced phenotype still remains to be resolved. The purpose of this study was to elucidate the role of CCAT2 and its specific alleles (G/T) in regulation of cellular processes that drive tumorigenesis using a genetically engineered mouse model. We generated transgenic mice for each CCAT2 allele using random integration approach in C57Bl6/N background. CCAT2(G/T) mice displayed spontaneous induction of widespread pancytopenias with splenomegaly and hepatomegaly. CCAT2(G/T) BM biopsies displayed severe myeloid and erythroid hyperplasia with enhanced proliferation and excessive apoptosis, along with extramedullary hematopoiesis in spleen and liver. Percentage of HSPCs was significantly reduced in BM of these mice, with increased presence of immature erythroid blasts and granulocyte-macrophage progenitors suggesting a block in differentiation. HSPCs of CCAT2(G/T) mice showed increased frequency of cytogenetic aberrations, including breaks and chromosomal fusions. However, these mice don’t develop AML, suggesting CCAT2 is critical in initiation of MDS. Microarray expression profiling of CCAT2(G/T) HSPCs revealed enrichment of pathways associated with epigenetic regulation, chromosomal instability and cell cycle regulation. We further identified significantly higher CCAT2 expression in the MDS patients as compared to healthy volunteers. Interestingly, patients with AML had significantly lower expression of CCAT2 as compared to patients with only MDS. Based on these data, we conclude that CCAT2 plays an important role in regulation of normal hematopoiesis, and its deregulation can lead to MDS. CCAT2 lncRNA can be developed into a diagnostic and prognostic marker, as well as a novel intervention target for MDS therapy. CCAT2(G/T) mice can serve as a robust model for studying initiation of de novo MDS and as a pre-clinical model for evaluation of new therapies for low-risk MDS

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

The Long Noncoding RNA CCAT2 Induces Chromosomal Instability Through BOP1-AURKB Signaling

BACKGROUND & AIMS: Chromosomal instability (CIN) is a carcinogenesis event that promotes metastasis and resistance to therapy by unclear mechanisms. Expression of the colon cancer-associated transcript 2 gene (CCAT2), which encodes a long noncoding RNA (lncRNA), associates with CIN, but little is known about how CCAT2 lncRNA regulates this cancer enabling characteristic.METHODS: We performed cytogenetic analysis of colorectal cancer (CRC) cell lines (HCT116, KM12C/SM, and HT29) overexpressing CCAT2 and colon organoids from C57BL/6N mice with the CCAT2 transgene and without (controls). CRC cells were also analyzed by immunofluorescence microscopy, gamma-H2AX, and senescence assays. CCAT2 transgene and control mice were given azoxymethane and dextran sulfate sodium to induce colon tumors. We performed gene expression array and mass spectrometry to detect downstream targets of CCAT2 lncRNA. We characterized interactions between CCAT2 with downstream proteins using MS2 pull-down, RNA immunoprecipitation, and selective 2'-hydroxyl acylation analyzed by primer extension analyses. Downstream proteins were overexpressed in CRC cells and analyzed for CIN. Gene expression levels were measured in CRC and non-tumor tissues from 5 cohorts, comprising more than 900 patients.RESULTS: High expression of CCAT2 induced CIN in CRC cell lines and increased resistance to 5-fluorouracil and oxaliplatin. Mice that expressed the CCAT2 transgene developed chromosome abnormalities, and colon organoids derived from crypt cells of these mice had a higher percentage of chromosome abnormalities compared with organoids from control mice. The transgenic mice given azoxymethane and dextran sulfate sodium developed more and larger colon polyps than control mice given these agents. Microarray analysis and mass spectrometry indicated that expression of CCAT2 increased expression of genes involved in ribosome biogenesis and protein synthesis. CCAT2 lncRNA interacted directly with and stabilized BOP1 ribosomal biogenesis factor (BOP1). CCAT2 also increased expression of MYC, which activated expression of BOP1. Overexpression of BOP1 in CRC cell lines resulted in chromosomal missegregation errors, and increased colony formation, and invasiveness, whereas BOP1 knockdown reduced viability. BOP1 promoted CIN by increasing the active form of aurora kinase B, which regulates chromosomal segregation. BOP1 was overexpressed in polyp tissues from CCAT2 transgenic mice compared with healthy tissue. CCAT2 lncRNA and BOP1 mRNA or protein were all increased in microsatellite stable tumors (characterized by CIN), but not in tumors with microsatellite instability compared with nontumor tissues. Increased levels of CCAT2 lncRNA and BOP1 mRNA correlated with each other and with shorter survival times of patients.CONCLUSIONS: We found that overexpression of CCAT2 in colon cells promotes CIN and carcinogenesis by stabilizing and inducing expression of BOP1 an activator of aurora kinase B. Strategies to target this pathway might be developed for treatment of patients with microsatellite stable colorectal tumors

microRNA Therapeutics in Cancer — An Emerging Concept

MicroRNAs (miRNAs) are an evolutionarily conserved class of small, regulatory non-coding RNAs that negatively regulate protein coding gene and other non-coding transcripts expression. miRNAs have been established as master regulators of cellular processes, and they play a vital role in tumor initiation, progression and metastasis. Further, widespread deregulation of microRNAs have been reported in several cancers, with several microRNAs playing oncogenic and tumor suppressive roles. Based on these, miRNAs have emerged as promising therapeutic tools for cancer management. In this review, we have focused on the roles of miRNAs in tumorigenesis, the miRNA-based therapeutic strategies currently being evaluated for use in cancer, and the advantages and current challenges to their use in the clinic

OncomiR-10b hijacks the small molecule inhibitor linifanib in human cancers

Abstract The pervasive role of microRNAs (miRNAs) in cancer pathobiology drives the introduction of new drug development approaches such as miRNA inhibition. In order to advance miRNA-therapeutics, meticulous screening strategies addressing specific tumor targets are needed. Small molecule inhibitors represent an attractive goal for these strategies. In this study, we devised a strategy to screen for small molecule inhibitors that specifically inhibit, directly or indirectly, miR-10b (SMIRs) which is overexpressed in metastatic tumors. We found that the multi-tyrosine kinase inhibitor linifanib could significantly inhibit miR-10b and reverse its oncogenic function in breast cancer and liver cancer both in vitro and in vivo. In addition, we showed that the efficacy of linifanib to inhibit tyrosine kinases was reduced by high miR-10b levels. When the level of miR-10b is high, it can “hijack” the linifanib and reduce its kinase inhibitory effects in cancer resulting in reduced anti-tumor efficacy. In conclusion, our study describes an effective strategy to screen for small molecule inhibitors of miRNAs. We further propose that miR-10b expression levels, due to the newly described “hijacking” effect, may be used as a biomarker to select patients for linifanib treatment

Tumour angiogenesis regulation by the miR-200 family

The miR-200 family is well known to inhibit the epithelial-mesenchymal transition, suggesting it may therapeutically inhibit metastatic biology. However, conflicting reports regarding the role of miR-200 in suppressing or promoting metastasis in different cancer types have left unanswered questions. Here we demonstrate a difference in clinical outcome based on miR-200's role in blocking tumour angiogenesis. We demonstrate that miR-200 inhibits angiogenesis through direct and indirect mechanisms by targeting interleukin-8 and CXCL1 secreted by the tumour endothelial and cancer cells. Using several experimental models, we demonstrate the therapeutic potential of miR-200 delivery in ovarian, lung, renal and basal-like breast cancers by inhibiting angiogenesis. Delivery of miR-200 members into the tumour endothelium resulted in marked reductions in metastasis and angiogenesis, and induced vascular normalization. The role of miR-200 in blocking cancer angiogenesis in a cancer-dependent context defines its utility as a potential therapeutic agent

Allele-Specific Reprogramming of Cancer Metabolism by the Long Non-coding RNA CCAT2

textabstractAltered energy metabolism is a cancer hallmark as malignant cells tailor their metabolic pathways to meet their energy requirements. Glucose and glutamine are the major nutrients that fuel cellular metabolism, and the pathways utilizing these nutrients are often altered in cancer. Here, we show that the long ncRNA CCAT2, located at the 8q24 amplicon on cancer risk-associated rs6983267 SNP, regulates cancer metabolism in vitro and in vivo in an allele-specific manner by binding the Cleavage Factor I (CFIm) complex with distinct affinities for the two subunits (CFIm25 and CFIm68). The CCAT2 interaction with the CFIm complex fine-tunes the alternative splicing of Glutaminase (GLS) by selecting the poly(A) site in intron 14 of the precursor mRNA. These findings uncover a complex, allele-specific regulatory mechanism of cancer metabolism orchestrated by the two alleles of a long ncRNA. Redis et al. report that the two alleles of the lncRNA, CCAT2, induce distinct metabolic phenotypes. By interacting with the CFIm complex with allele-specific affinities, CCAT2 regulates the alternative splicing of GLS, resulting in the preferential expression of the more aggressive splice isoform

Cancer-associated rs6983267 SNP and its accompanying long noncoding RNA CCAT2 induce myeloid malignancies via unique SNP-specific RNA mutations

The cancer-risk-associated rs6983267 single nucleotide polymorphism (SNP) and the accompanying long noncoding RNA CCAT2 in the highly amplified 8q24.21 region have been implicated in cancer predisposition, although causality has not been established. Here, using allele-specific CCAT2 transgenic mice, we demonstrate that CCAT2 overexpression leads to spontaneous myeloid malignancies. We further identified that CCAT2 is overexpressed in bone marrow and peripheral blood of myelodysplastic/myeloproliferative neoplasms (MDS/MPN) patients. CCAT2 induces global deregulation of gene expression by down-regulating EZH2 in vitro and in vivo in an allele-specific manner. We also identified a novel non-APOBEC, non-ADAR, RNA editing at the SNP locus in MDS/MPN patients and CCAT2-transgenic mice. The RNA transcribed from the SNP locus in malignant hematopoietic cells have different allelic composition from the corresponding genomic DNA, a phenomenon rarely observed in normal cells. Our findings provide fundamental insights into the functional role of rs6983267 SNP and CCAT2 in myeloid malignancies

Allele-specific reprogramming of cancer metabolism by the long non-coding RNA CCAT2

Altered energy metabolism is a cancer hallmark as malignant cells tailor their metabolic pathways to meet their energy requirements. Glucose and glutamine are the major nutrients that fuel cellular metabolism, and the pathways utilizing these nutrients are often altered in cancer. Here, we show that the long ncRNA CCAT2, located at the 8q24 amplicon on cancer risk-associated rs6983267 SNP, regulates cancer metabolism in vitro and in vivo in an allele-specific manner by binding the Cleavage Factor I (CFIm) complex with distinct affinities for the two subunits (CFIm25 and CFIm68). The CCAT2 interaction with the CFIm complex fine-tunes the alternative splicing of Glutaminase (GLS) by selecting the poly(A) site in intron 14 of the precursor mRNA. These findings uncover a complex, allele-specific regulatory mechanism of cancer metabolism orchestrated by the two alleles of a long ncRNA.G.A.C. is The Alan M. Gewirtz Leukemia & Lymphoma Society Scholar. Work in G.A.C.’s laboratory is supported in part by the NIH/NCI grants 1UH2TR00943-01 and 1 R01 CA182905-01, the UT MD Anderson Cancer Center SPORE in Melanoma grant from NCI (P50 CA093459), Aim at Melanoma Foundation and the Miriam and Jim Mulva research funds, the Brain SPORE (2P50CA127001), the Center for Radiation Oncology Research Project, the Center for Cancer Epigenetics Pilot project, a 2014 Knowledge GAP MDACC grant, a CLL Moonshot pilot project, the UT MD Anderson Cancer Center Duncan Family Institute for Cancer Prevention and Risk Assessment, a SINF grant in colon cancer, the Laura and John Arnold Foundation, the RGK Foundation, and the Estate of C.G. Johnson, Jr. I.B.-N. was financed by a grant entitled Non-Invasive Intelligent Systems for Colorectal Cancer Diagnosis and Prognosis Based on circulating miRNAs Integrated in the Clinical Workflow – INTELCOR. S.M.G.D., A.L.B.A., and D.A. are supported by the São Paulo Research Foundation FAPESP under grants 2014/15968-3, 2014/20673-2, and 2014/17820-3, respectively. W.L. was partly supported by grants from The University of Texas MD Anderson Cancer Center Sheikh Ahmed Bin Zayed Al Nahyan Center for Pancreatic Cancer Research. J.A.B. was supported by the Cancer Center Support Grant (P30 CA016672), and the HP imaging program of the Small Animal Facility (SAIF) was supported by the Cancer Prevention and Research Institutes of Texas grant RP-101243P5. H.L. was supported by NIH/NCI grant R01CA175486, a grant (RP140462) from the Cancer Prevention and Research Institute of Texas, and the R. Lee Clark Fellow Award from The Jeanne F. Shelby Scholarship Fund. I.B.-N. was financed by a Fulbright fellowship and by a grant entitled Non-Invasive Intelligent Systems for Colorectal Cancer Diagnosis and Prognosis Based on circulating miRNAs Integrated in the Clinical Workflow – INTELCOR.Peer Reviewe