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

MicroRNAs as cancer therapeutics : A step closer to clinical application

During the last decades, basic and translational research has enabled great improvements in the clinical management of cancer. However, scarcity of complete remission and many drug-induced toxicities are still a major problem in the clinics. Recently, microRNAs (miRNAs) have emerged as promising therapeutic targets due to their involvement in cancer development and progression. Their extraordinary regulatory potential, which enables regulation of entire signalling networks within the cells, makes them an interesting tool for the development of cancer therapeutics. In this review we will focus on miRNAs with experimentally proven therapeutic potential, and discuss recent advances in the technical development and clinical evaluation of miRNA-based therapeutic agents

Regulation of cell-cell adhesion in prostate cancer cells by microRNA-96 through upregulation of E-Cadherin and EpCAM

Prostate cancer is one of the most common cancers in men, yet the biology behind lethal disease progression and bone metastasis is poorly understood. In this study, we found elevated levels of microRNA-96 (miR-96) in prostate cancer bone metastasis samples. To determine the molecular mechanisms by which miR-96 deregulation contributes to metastatic progression, we performed an Argonaute2-immunoprecipitation assay, in which mRNAs associated with cell-cell interaction were enriched. The expression of two cell adhesion molecules, E-Cadherin and EpCAM, was upregulated by miR-96, and potential targets sites were identified in the coding sequences of their mRNAs. We further showed that miR-96 enhanced cell-cell adhesion between prostate cancer cells as well as their ability to bind to osteoblasts. Our findings suggest that increased levels of miR-96 give prostate cancer cells an advantage at forming metastases in the bone microenvironment due to increased cell-cell interaction. We propose that miR-96 promotes bone metastasis in prostate cancer patients by facilitating the outgrowth of macroscopic tumours in the bone

Functional In Vivo Screening Identifies microRNAs Regulating Metastatic Dissemination of Prostate Cancer Cells to Bone Marrow

Distant metastasis is the major cause of cancer-related deaths in men with prostate cancer (PCa). An in vivo functional screen was used to identify microRNAs (miRNAs) regulating metastatic dissemination of PCa cells. PC3 cells transduced with pooled miRZiP™ lentivirus library (anti-miRNAs) were injected intraprostatic to 13 NSG mice followed by targeted barcode/anti-miR sequencing. PCa cells in the primary tumours showed a homogenous pattern of anti-miRNAs, but different anti-miRNAs were enriched in liver, lung, and bone marrow, with anti-miR-379 highly enriched in the latter. The bone metastasis-promoting phenotype induced by decreased miR-379 levels was also confirmed in a less metastatic PCa cell line, 22Rv1, where all mice injected intracardially with anti-miR-379-22Rv1 cells developed bone metastases. The levels of miR-379 were found to be lower in bone metastases compared to primary tumours and non-cancerous prostatic tissue in a patient cohort. In vitro functional studies suggested that the mechanism of action was that reduced levels of miR-379 gave an increased colony formation capacity in conditions mimicking the bone microenvironment. In conclusion, our data suggest that specific miRNAs affect the establishment of primary tumours and metastatic dissemination, with a loss of miR-379 promoting metastases in bone

Prevalent and rare variants show distinctive conservation preference.

<p>(<b>A</b>) Distribution of A,T and G,C nucleotides in the UCEs and SNV positions. <b>(B–D</b>) Cumulative distribution plots of phyloP scores of SNVs with different MAFs. Data from three different data sources (<b>B</b>) SG-CHN, (<b>C</b>) ITA and (<b>D</b>) 1 KG are shown. Shaded grey area represents 95% confidence interval (obtained by bootstrapping) of random G/C content corrected UCE positions (blue line). Numbers in the parentheses indicate analyzed positions or SNVs.</p

UCEs are enriched for the TFBS.

<p>(<b>A</b>) Box plots represent results of one hundred sets (each set contains one thousand randomly chosen positions). The y-axis indicates actual ENCODE TFBS overlap per one thousand tested positions. Boxes show IQR, notches indicate 95% confidence intervals of the median, whiskers extend to 1.5 times the IQR and open circles show outliers. *** P<2.2×10⁻¹⁶, two- tailed Mann–Whitney test. (<b>B</b>) Prevalent SNV positions are depleted for TFBS. All rare and prevalent SNV positions from the three different populations were analyzed for the ENCODE TFBS overlap. Random UCE set represents randomly chosen UCE positions (G,C content matched) that had the same number of analyzed positions as the rare and prevalent SNVs. Prevalent and rare SNVs overlap with the TFBS overlap is shown as relative to random UCE positions. For the statistical analysis each set (Pearson's Chi-squared test) was individually tested. * P<0.01.</p

General characterization of SNVs in the UCEs.

<p>(<b>A</b>) Number of SNVs per mega base (Mb) of UCE sequence per sample. SNVs from three data sources- Singaporean Chinese cohort (SG-CHN), Italian cohort (ITA) and 1000 Genome Project (1 KG) were used. SNVs are discriminated according to their minor allele frequency (MAF). Numbers in the parentheses represent sample size used in this study (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110692#s2" target="_blank">Materials and Methods</a>). Random set represents random genomic regions that have the same total length as the UCEs set. Y-axis represents SNVs per Mb divided by sample count in the analyzed population. (<b>B–D</b>) Shared and distinct SNVs between SG-CHN, ITA and 1 KG populations. Venn diagrams of (<b>B</b>) all, (<b>C</b>) prevalent (MAF>0.5%) and rare (<b>D</b>) (MAF<0.5%) SNVs from three analyzed population. Numbers in the parentheses indicate analyzed SNVs in the corresponding population.</p

UCEs comparison to the less constraint SE.

<p>(<b>A</b>) SE are less constraint compared to UCEs. Cumulative distribution plots of phyloP scores of all SE positions (purple line), all UCE positions (red line), random genomic positions (orange line) and SE rare (MAF<0.5%, green line) and prevalent (MAF>5%, black line) SNVs. Prevalent and rare SNVs are extracted from the 1 KG project using global MAFs. Shaded grey area represents 95% confidence interval (obtained by bootstrapping) of random UCE positions (blue line). The numbers of analyzed SNVs are given in the parentheses. (<b>B</b>) SE have a higher overlapping TFBSs count compared to UCEs. Box plots represent results of one hundred sets (each set contains one thousand randomly chosen positions). The y-axis indicates actual ENCODE TFBS overlap per one thousand tested positions. Boxes show IQR, notches indicate 95% confidence intervals of the median, whiskers extend to 1.5 times the IQR and open circles show outliers. *** P <2.2 ×10⁻¹⁶, two- tailed Mann–Whitney test. (<b>C</b>) Venn diagram showing overlap of ENCODE TF and UCE bound TF described by Viturawong et. al <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110692#pone.0110692-Viturawong1" target="_blank">[12]</a>. (<b>D</b>) Comparison of ENCODE TF and UCE bound TF <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110692#pone.0110692-Viturawong1" target="_blank">[12]</a> protein domains identifies RNA recognition domain,RRM1 (marked with dashed circle), as the most prevalent domain among UCE bound proteins. Protein domain (Pfam) annotations were done by using the Perseus module in the MaxQuant software suite.</p

Therapeutic potential of FLANC, a novel primate-specific long non-coding RNA in colorectal cancer

Objective To investigate the function of a novel primate-specific long non-coding RNA (lncRNA), named FLANC, based on its genomic location (co-localised with a pyknon motif), and to characterise its potential as a biomarker and therapeutic target. Design FLANC expression was analysed in 349 tumours from four cohorts and correlated to clinical data. In a series of multiple in vitro and in vivo models and molecular analyses, we characterised the fundamental biological roles of this lncRNA. We further explored the therapeutic potential of targeting FLANC in a mouse model of colorectal cancer (CRC) metastases. Results FLANC, a primate-specific lncRNA feebly expressed in normal colon cells, was significantly upregulated in cancer cells compared with normal colon samples in two independent cohorts. High levels of FLANC were associated with poor survival in two additional independent CRC patient cohorts. Both in vitro and in vivo experiments demonstrated that the modulation of FLANC expression influenced cellular growth, apoptosis, migration, angiogenesis and metastases formation ability of CRC cells. In vivo pharmacological targeting of FLANC by administration of 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine nanoparticles loaded with a specific small interfering RNA, induced significant decrease in metastases, without evident tissue toxicity or pro-inflammatory effects. Mechanistically, FLANC upregulated and prolonged the half-life of phosphorylated STAT3, inducing the overexpression of VEGFA, a key regulator of angiogenesis. Conclusions Based on our findings, we discovered, FLANC as a novel primate-specific lncRNA that is highly upregulated in CRC cells and regulates metastases formation. Targeting primate-specific transcripts such as FLANC may represent a novel and low toxic therapeutic strategy for the treatment of patients. © 2020 BMJ Publishing Group. All rights reserved