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GENReports: Market & Tech Analysis, Produced by Enal Razvi, Ph.D. © 2013GENengnews.com

> Enal Razvi, Ph.D.Biotechnology Analyst,Managing DirectorSELECTBIO [email protected]

> Clare Stirzaker, Ph.D.Group Leader & Senior LecturerGarvan Institute of Medical ResearchUniversity of New South Wales, Sydney, Australia

GENReports: Market & Tech Analysis

Regional Epigenetic Down- and Up-Regulation in Cancer & lncRNAs with Potential Functionality


• The focus of this GEN Market & Tech Analysis Report is to present asnapshot of Regional Epigenetic Down‐ and Up‐regulation inCancer with a focus towards Prostate Cancer

• We present here Long‐Range Epigenetic Silencing (LRES) as ameans for down‐regulation of stretches of the genome as well asLong‐Range Epigenetic Activation (LREA)

– These two opposing effects appear to be mediated by an interplay betweenDNA methylation and chromatin modification

– We’ve focused this analysis on an interrogation of prostate cancer

• Also presented herein are potential associations of lncRNAs withdistinct cancer classes

– The potential utility of lncRNAs in finely‐modulating the expression of distinctregions of the genome makes this class of molecules attractive potentialbiomarkers and perhaps even therapeutic targets in the future

Topic Introduction and Scope


Genes

CpG islands4 Mb

LRES

DNA Methylation Histone H3K9 Methylation

2q14.2

LRES is Associated with DNA methylation and Chromatin Remodelling

Long Range Epigenetic Silencing (LRES)

Source: Frigola et al. Nature Genetics 38: 540‐549 (2006)


LRES regions are scattered across the genome: Potential Therapeutic Targets?

LRES features:‐ 47 novel regions (18 chromosomes)‐ total size = 88.5 Mb

(~2.9% of genome) 547 genes‐ average size = 2Mb‐ average number of genes = 12‐ 23% of regions contain gene clusters, HOXA, KRT, MT‐ 32 of 47 show LOH in prostate cancer‐ 34% tumor suppressor /cancer assoc

47 LRES Regions in Prostate Cancer

Source: Coolen et al., Nat Cell Biol. 2010


Long Range Epigenetic Activation (LREA)“Genetic regions associated with long‐range epigenetic activation can beassociated with an increase in DNA methylation, leading to alternativepromoter usage and ectopic gene activation.”

“These regions are associated with a gain of active chromatin marks,demonstrating complexity in the epigenetic modulation mediated bychromatin marks and DNA methylation.”

Clare Stirzaker, Ph.D.Group Leader & Senior LecturerGarvan Institute of Medical ResearchUniversity of New South Wales, Sydney, AustraliaSource: Bert et al., Cancer Cell 2013


• LncRNA‐PRNCR1 bind androgen receptor (AR) and promotes prostate cancer growth

• PRNCR1 binds to androgen receptor in the enhancer region• PRNCR1 is methylated• The enhancer loops to the target gene promoter Gene

Expression Activated• Source: Yang et al., Nature 2013

lncRNAs Bind the Epigenetic Machinery in vivo and Modulate It


CCAT1 colorectal Recently, colon cancer associated transcript 1 (CCAT1) lncRNA was found to be expressedCRNDE colorectal An uncharacterized gene locus (Chr16:hCG_1815491), now named colorectal neoplasia diH19 colorectal Control of imprinting. Containing miRNA miR‐675.=Oncofetal H19‐derived miR‐675 regulaHOTAIR colorectal Overexpression of the HOTAIR transcript, a cis‐lncRNA associated with the HOXD gene clKCNQ1OT1 colorectal epigenetic disruptionMALAT1 colorectal The 3' end of MALAT‐1 is an important biological motif in the invasion and metastasis of MEG3 colorectal MEG3 expression is lost.NPTN‐IT1 colorectal In this study, we found that the lncRNA Low Expression in Tumor (lncRNA‐LET) was generHULC colorectal HULC expression is not confined to HCC, but also to those colorectal carcinomas that met

ANRIL breast A locus associated with breast cancer.=A genetic susceptibility locus.=More recently, addBCAR4 breast BCAR4 is expressed in 27% of primary breast tumors. Forced expression of BCAR4 in humBCYRN1 breast BC200 RNA was expressed in carcinomas of the breast, cervix, oesophagus, lung, ovary, pDSCAM‐AS1 breast M41 (DSCAM‐AS1) mRNA is expressed at a statistically significantly higher level in humaGAS5 breast GAS5, a non‐protein‐coding RNA, controls apoptosis and is downregulated in breast canceH19 breast Control of imprinting. Containing miRNA miR‐675.=the transcript is stabilized in breast caHOTAIR breast HOTAIR expression level in primary tumors is a powerful predictor of eventual metastasisLSINCT5 breast LSINCT5 is overexpressed in breast and ovarian cancer cell lines and tumor tissues.=OvarMALAT1 breast Sequesters SR splicing factors to regulate alternative splicing.=On a more mechanistic levMEG3 breast MEG3 expression is lost.MIR31HG breast miR‐31 and its host gene lncRNA LOC554202 (MIR31HG) are regulated by promoter hypePINC breast In a finding of relevance to breast cancer pathogenesis, the mammary gland lncRNA PINCPVT1 breast Amplification of PVT1 contributes to the pathophysiology of ovarian and breast cancer.SRA1 breast We have previously found that both fully‐spliced SRAP‐coding and intron‐1‐containing nonXIST breast The intratumoral and intertumoral variability in XIST RNA domain number in BRCA1 tumorZNFX1‐AS1 breast SNORD‐host RNA Zfas1 is a regulator of mammary development and a potential marker fo


BCYRN1 lung BC200 RNA was expressed in carcinomas of the breast, cervix, oesophagus, lung, ovary, pH19 lung Down‐regulation of H19 significantly decreases breast and lung cancer cell clonogenicitylincRNAp21 lung lincRNAp21 is required for the global repression of genes that interfere with p53 functionMALAT1 lung In NSCLC metastasizing tumors, MALAT‐1 expression is three‐fold higher than in non‐metMEG3 lung MEG3 expression is lost.NPTN‐IT1 lung In this study, we found that the lncRNA Low Expression in Tumor (lncRNA‐LET) was gener

DAPK1 pancreatic Differential expressionMALAT1 pancreatic Sequesters SR splicing factors to regulate alternative splicing.MAP3K14 pancreatic Differential expressionPPP3CB pancreatic Differential expressionPVT1 pancreatic PVT1 gene as a regulator of Gemcitabine sensitivity and showed that functional inactivatUCA1 pancreatic Gene expression profiling reveals upregulated UCA1 and BMF in gallbladder epithelia of c

ANRIL prostate Gene silencing of INK4b‐ARF‐INK4a and p15/CDKN2B by recruitment of PRC1 and PRC2.=AC1QTNF9B‐AS1 prostate PCOTH, a novel gene overexpressed in prostate cancers, promotes prostate cancer cell gCBR3‐AS1 prostate The prostate cancer‐up‐regulated long noncoding RNA PlncRNA‐1 (CBR3‐AS1) modulates GAS5 prostate Chromosomal translocations affecting the 1q25 locus containing the Gas5 gene have beeIGF2‐AS prostate Association identified by GWAS.MALAT1 prostate Sequesters SR splicing factors to regulate alternative splicing.MEG3 prostate MEG3 expression is lost.PCA3 prostate Measurement of lncRNA PCA3 in patient urine samples has been shown to allow more sePCAT1 prostate PCAT‐1 is markedly overexpressed in a subset of prostate cancers, particularly metastasePCGEM1 prostate We found a significantly decreased risk of PCa for rs6434568 AC and AC/AA genotype, asPCNCR1 prostate Accumulating evidence indicated that prostate cancer non‐coding RNA 1 (PCNCR1) lncRNPVT1 prostate The risk allele (G) of rs378854 (A>G) reduces binding of the transcription factor YY1 in vit


DLEU2 leukemia & lymphoma The microRNAs miR‐15a and miR‐16‐1 are downregulated in multiple tumor types and areANRIL leukemia & lymphoma Gene silencing of INK4b‐ARF‐INK4a and p15/CDKN2B by recruitment of PRC1 and PRC2.=HDLEU1 leukemia & lymphoma Leu1 and Leu2 genes are strong candidates as tumor suppressor gene(s) involved in B‐CLMEG3 leukemia & lymphoma MEG3 hypermethylation occurred in 15 MDS patients (34.9%), and in 20 AML patients (47MIR155HG leukemia & lymphoma MYB transcriptionally regulates the miR‐155 host gene in chronic lymphocytic leukemia.TCL6 leukemia & lymphoma T‐cell leukemia/lymphoma 6WT1‐AS leukemia & lymphoma In AML, there is often abnormal splicing of WT1‐AS, which may play a role in the develop

AIR liver Up regulation in liver by DDC (Diethyl 1,4‐dihydro‐2,4,6,‐trimethyl‐3,5‐pyridinedicarboxylaH19 liver Epigenetic activation of the MiR‐200 family contributes to H19‐mediated metastasis suppHOTAIR liver Clinical significance of the expression of long non‐coding RNA HOTAIR in primary hepatocHULC liver The lncRNA HULC is one of the most upregulated of all genes in hepatocellular carcinomaIGF2‐AS liver IGF‐IR and IGF‐IIR antisense genes could significantly restrain the malignant behavior of hMALAT1 liver Long non‐coding RNA MALAT‐1 overexpression predicts tumor recurrence of hepatocellulaMEG3 liver Enforced expression of MEG3 in HCC cells significantly decreased both anchorage‐depenNPTN‐IT1 liver In this study, we found that the lncRNA Low Expression in Tumor (lncRNA‐LET) was generVL30 LTRs liver Trim24 (Tif1α) and Trim33 (Tif1γ) interact to form a co‐repressor complex that suppresses

BCYRN1 ovarian BC200 RNA was expressed in carcinomas of the breast, cervix, oesophagus, lung, ovary, pDNM3OS ovarian pri‐miR‐199a2 within the human Dnm3os gene. The regulation of MIR199A2/214 expressiH19 ovarian H19 RNA was detected in 90% of patients with OCAF (Ovarian cancer ascites fluid ) as deLSINCT5 ovarian LSINCT5 is overexpressed in breast and ovarian cancer cell lines and tumor tissues.=OvarPVT1 ovarian Amplification of PVT1 contributes to the pathophysiology of ovarian and breast cancer.

BCYRN1 esophageal BC200 RNA was expressed in carcinomas of the breast, cervix, oesophagus, lung, ovary, p7SK gastric Transfection of an siRNA directed against LARP7 (anti‐LARP7 siRNA) into non‐neoplastic gCCAT1 gastric Long noncoding RNA CCAT1, which could be activated by c‐Myc, promotes the progressioHOTAIR gastrointestinal Overexpression of HOTAIR was also strongly associated with high‐risk grade and metastaANRIL nasopharyngeal A genetic susceptibility locus.=A SNP rs1412829 (C49137T) at CDKN2A‐CDKN2B gene clusLINC00312 nasopharyngeal NAG‐7 is a novel gene downregulated in human nasopharyngeal carcinoma.=NAG7 (LINC0


ANRIL skin Risk SNPs (rs3217992, A>G;rs1063192, C>T) for coronary disease, stroke, diabetes, melanBANCR skin BRAF‐regulated lncRNA 1 (BANCR) was identified as a recurrently overexpressed, previouGAS5 skin Chromosomal translocations affecting the 1q25 locus containing the Gas5 gene have beeH19 skin H19 RNA downregulation stimulated melanogenesis in melasma.=Although the IGF2 and LINC00032 skin An analysis of genome‐wide DNA copy number alterations in melanoma tumors revealed PAN skin KSHV infected cells express a highly abundant long noncoding transcript referred to as poSPRY4‐IT1 skin The melanoma‐upregulated long noncoding RNA SPRY4‐IT1 modulates apoptosis and invaUCA1 skin Cancer up‐regulated drug resistant (CUDR) gene, was found to be overexpressed in a dox

H19 bladder Control of imprinting. Containing miRNA miR‐675.=Upregulated H19 contributes to bladdeMEG3 bladder Downregulated MEG3 activates autophagy and increases cell proliferation in bladder canTUG1 bladder Long intergenic non‐coding RNA TUG1 is overexpressed in urothelial carcinoma of the blaUCA1 bladder In adult human tissues, UCA1 gene was not expressed except in the heart and spleen. Th

ANRIL brain Association identified by GWAS.=Risk SNPs (rs3217992, A>G;rs1063192, C>T) for coronaryanti‐NOS2A brain anti‐NOS2A is a lncRNA that is expressed in meningiomas and glioblastomas from a genoCCDC26 brain In the pooled analysis, the odds ratio for low‐grade glioma associated with rs55705857 wESRG brain HESRG was expressed strongly and diffusively in the nuclei of tumor cells in intracranial gH19 brain A study of medulloblastomas and medulloblastoma cell lines showed partial loss of impriMEG3 brain Overexpression of the long non‐coding RNA MEG3 impairs in vitro glioma cell proliferatio

BCYRN1 cervix BC200 RNA was expressed in carcinomas of the breast, cervix, oesophagus, lung, ovary, pH19 cervix High incidence of loss of heterozygosity and abnormal imprinting of H19 and IGF2 genes iMALAT1 cervix MALAT1 was involved in cervical cancer cell growth, cell cycle progression, and invasion CASC2 endometrial CASC2, in a region of common allelic loss at chromosome 10q26 is a novel candidate gen


ANRIL kidney Disruptions to the expression of ANRIL have accordingly been associated with the develoH19 kidney This disease has been associated with structural abnormalities at the 11p15 locus, whichHIF1A‐AS1 kidney The antitumor DNA topoisomerase I (Top1) inhibitor camptothecin (CPT) increases the ceIGF2‐AS kidney Loss of imprinting of IGF2 sense and antisense transcripts in Wilms' tumor.=PEG8/IGF2ASKCNQ1DN kidney A novel imprinted gene, KCNQ1DN, within the WT2 critical region of human chromosome MALAT1 kidney We identified a shorter transcriptional initiation site and found that CREB binds to the deMEG3 kidney MEG3‐DMR is completely methylated in neuroblastoma cell lines.MYCNOS kidney Both DeltaMYCN and MYCNOS are expressed in all NBs examined. In NBs with MYCN‐amncRAN kidney It is now becoming increasingly evident that N‐myc also regulates the expression of long NDM29 kidney The synthesis of a pol III‐transcribed noncoding (nc) RNA (NDM29) strongly restricts NB dT‐UCRs kidney It is now becoming increasingly evident that N‐myc also regulates the expression of long WT1‐AS kidney A CTCF‐binding silencer regulates the imprinted genes AWT1 and WT1‐AS and exhibits se

BC040587 bone These CNAs (copy number alterations) in osteosarcoma often involve the noncoding RNAsLSAMP‐AS3 bone These CNAs (copy number alterations) in osteosarcoma often involve the noncoding RNAsMALAT1 bone Sequesters SR splicing factors to regulate alternative splicing.=Osteosarcoma patients' su

BCYRN1 salivary BC200 RNA was expressed in carcinomas of the breast, cervix, oesophagus, lung, ovary, pBOK‐AS1 testicular A natural antisense transcript, BOKAS, regulates the pro‐apoptotic activity of human Bok.TDRG1 testicular The significantly reduced expression of the TDRG1 in patients with seminoma or teratomaXIST testicular XIST expression was common in seminomatous testicular germ cell tumors but not in nonNAMA thyroid Identification of a novel noncoding RNA gene, NAMA, that is downregulated in papillary tPTCSC thyroid papillary thyroid carcinoma susceptibility candidate=Gene expression analysis indicated t


• Epigenetic Modulations Can Result in Either Target Gene Repression or Activation– There appears to be interplay between DNA methylation which

is believed to be the de facto gene modulatory mechanism and chromatin modifications such as histone post‐translational modifications This interplay most probably determines gene activation or silencing

• We have Sought to Present here the Various lncRNAs and their associations thus far with Different Types of Cancer– We expect this field to expand over the coming years wherein

more lncRNAs will be annotated and will provide the backdrop for determining epigenetic modulations

– Thus specific signatures of lncRNAs may offer value as biomarkers for cancer and perhaps other disease classes

How Do These Fit Together?

lncRNAs May Potentially Have Biomarker Potential by Virtue of their SpecificModulation of Genetic Elements which are Involved in Cancer Initiation and/or

Progression There is Precedent for this today with the PCA3 marker which is an lncRNA‐based test for Prostate Cancer

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