MicroRNA & Carcinogenesis

Transcription

1 2009 June MGEL W/S MicroRNA & Carcinogenesis 2009 June MGEL W/S 성현아 성현아

2 Presentation Summary MicroRNA, Discovery and Biogenesis MicroRNA & Carcinogenesis MiRNA and Breast Cancer

3 How few genes there were! There weren't that many more than a fruit fly or a unicellular organism, yeast. Saccharomyces cerevisiae ~ 6,000 genes Drosophila melanogaster ~ 14,000 genes Homo sapisens ~ 19,000 genes 29 May March April 2001

4 What makes human to be human? "Junk" RNA May Have Played Role in Vertebrate Evolution

5 Right about when vertebrates split off fro m other animals, there was an explosion o f these micrornas in their DNA. The complexity didn't come from the numb er of genes an animal has. The complexity comes from how similar g enes are used. This is where micrornas come in because they affect how an animal (or plant) uses it s genes. MicroRNAs Control How Genes are Used. Evidence that Vertebrate Complexity Com es from non-codingrna

6 Non coding RNA A non-coding RNA (ncrna) is any RNA molecule that is not trans lated into a protein trna / rrna snrna/mirna/grna/pirna/sirna/tmrna small RNA(sRNA), non-messenger RNA (nmrna), small non-mess enger RNA (snmrna), or functional RNA (frna)

7 lin-4 is essential for the normal temporal control of diverse postembryonic developmental events in C. elegans lin-4 downregulates LIN-14 larval stage (L1) lin-4 does not encode a protein Two small lin-4 transcripts of approximately 22 and 61 nt sequences complementary to a repeated sequence element in the 3 UTR of lin-14 mrna lin-4 regulates lin-14 translation via an antisense RNA-RNA interaction.

8 The Biogenesis of mirna & Assembly into mirisc Txn. Of pri-mirna(rna pol II) pri-mirnas pre-mirna(drosha) Transport into cytoplasm small RNA duplexes(dicer) mirisc(rna-induced silencing complex) target mrna sequence target mirna silencing

9 Possible Mechanisms of mirisc-mediated Repression : post-transcriptional gene regulation Usually via either translational repression or direct mrna cleavage

10 MicroRNA(miRNA) Endogenous 22-nt non-coding RNAs (22-24nts) Protein level regulation in a sequence-specific manner Developmental timing in worms, cell death and fat metabolism in flies, and leaf development and floral patterning in plants(evolutionary conserved) In mammals, including cell growth and apoptosis, hematopoietic lineage differentiation, insulin secretion, brain morphogenesis, and muscle cell differentiation and proliferation From 450 to 1000 mirnas in the human genome Most human protein-coding genes influenced A single mirna may bind to as many as 200 target genes Half of the known mirna mapped to fragile sites in the genome, providing the first clue to their role in cancer

11 At least 10% of the protein coding mrnas might be conserved targets of mirna

12 Expression and function of micro RNAs in immune cells during normal or disease state Esmerina Tili 1, Jean-Jacques Michaille 1, 2, George Adrian Calin 3 Int J Med Sci 2008; 5:73-79

13 microrna and Human Disease

14 Potential mechanisms of aberrant mirna regulation mirnas located n ear fragile sites Mutation/SNP in the 3 UTR target mrna (poly-mirts) epigenetic changes Roughly 20,000 poly-mirts catalogued in databases such as PolymiRTS(

15 Method to determine mirna expression levels High-throughput tech. for hundreds of mirnas in a large number of samples(commonly used ) Bead-based flow-cytometric tech. mirage : a genome-wide mirna analysis with serial analysis of gene expression (SAGE) qrt PCR

16 mirna & Carcinogenesis

17 How mirna contribute to oncogenesis? 1> Function either as tumor suppressors or oncogenes 2> The genomic abnormalities found to influence the activity of mirna : frequently located near fragile sites 3> Epigenetic changes such as alterations in CpG methylation patterns 4> SNP and/or mutations in the mirna themselves 5> Differences in the expression levels or affinities of any protein involv ed in mirna Transcription and Processing : Drosha, Dicer, Argonaut etc. 6> the role of polymorphisms in the sites of target mrnas 3 UTR

18 mirnas as cancer players : mirna, frequently located near fragile sites Calin G. A. et.al. PNAS 2004;101: by National Academy of Sciences

19 A model of mirna involvement in cancer by modulation of expression of tumor suppressor genes and oncogenes Carlos Caldas et al. Sizing up mirnas as cancer genes, Nature Medicine 11, (2005)

20 mirna-expression profiles classify human cancers.

21 217 mirna, 334 samples including multiple human cancers A general downregulation of mirnas in tumours compared with normal tissues Classifying poorly differentiated tumours using mirna expression profiles A. Most of mirna (129 out of 217, P<0.05) had lower expression levels in human tumour samples B. Normal lungs and lung adenocarcinomas of K-RasLA1 mice C. HL-60 cells, treated with all-trans retinoic acid or vehicle Highlightening the potential of mirna profiling in cancer diagnosis

22 mir15 and mir16 are deleted or down-regulated in the majority ( 68%) of CLL cases. mir15 and mir16 are located at chromosome 13q14, a region deleted in more than half of B-CLL

23 Genome wide expression profiling of mirnas in human B-CLL using microarray 41 samples from 38 pts. with CLL and 6 normal samples ( 1 lymph node, 2 tonsillar CD5+ B cells, 3 blood MNC)

24 Top 25 mirnas differentially expressed in CLL cells vs. CD5+ cells Calin G. A. et.al. PNAS 2004;101:

25 mirnas differentially expressed bet. the two main CLL clusters

26 mirna signatures associaed with prognosis in B-CLL

27 1st Set 94 pts. of CLL cells 2nd Set - 50 pts. of CLL cells to validate the predictive power of the mirna signature Northern blotting mirna-microchip procedures( 368 probes, corresponding to 245 human and mouse mirna genes) Prognostic Factor IgVh status unmutated IgVh status mutated ZAP-70 >20% ZAP-70 <20% Group 1(36pts) Group3(10pts) Group 2(10pts) Group 4(47pts)

28 Group1 Poor prognosis (unmutated IgVh & high ZAP-20) 13 mirna associated with prognostic factors

29 Relationship bet. the level of expression of mirna and the time from diagnosis to initial therapy 94 pts 9 mirnas, all members of the 13-me mber prognostic signature Short interval(40±39 months)/ longer interval (88±42 months) mirna expression can be included in the markers with prognostic signifi cance in CLL Germ-line or somatic mutations were found in 5 of 42 sequenced mirnas in 11 of 75 pts. with CLL, but no such mutations were found in 160 subjects without cancer

30 tool. 104 pairs of primary lung cancer and corresponding normal ti ssue 43 mirnas differentially expressed in lung cancer vs. normal t issue thru microarray 6 mirnas differentially expressed in adenocarcinoma and squ amous cell carcinoma Correlation bet. mirna expression profiles and prognosis of l ung adenocarcinoma pts Validation using an independent set of adenocarcinoma

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32 2008 Jan08 Using real-time RT-PCR, mirna expression profiling in 112 NSCLC pt s. divided into the training and testing sets a five-mirna signature for the prediction of treatment outcome of N SCLC in the training set Validation by the testing set and an independent cohort Patients with high-risk scores in their mirna signatures had poor ove rall and disease-free survivals compared to the low-risk-score pts. This mirna signature is an independent predictor of the cancer relapse and survival of NSCLC patients.

33 mirna-expression profiles classify human cancers. Calin and Croce Nature Reviews Cancer 6, (November 2006)

34 Common mirna genes differentially expressed in various cancers, suggesting common altered regulatory pathways? mir-21 ; directly target the tumor suppressor PTEN in cholangiocarcinoma cells (Meng et al. 2006, Gastroenterology) mrr-21 ; knockdown in cultured glioblastoma cells triggers apoptosis by a caspase-dependent mechanism (Chan, J. A. et al. 2005, Cancer Res) mir-21 as an anti-apoptotic and pro-survival factor Let-7 mirna family ; Ras regulation(voorhoeve, P. M. et al,cell, 2006) mir-372 and mir-373 ; facilitate the proliferation and transformation of cells mir-155 transgenic mouse in B-cells ; polyclonal pre-leukaemic pre- B-cell proliferation followed by B-cell malignancy(early event in oncogenesis that needs additional genetic alterations for the development of the fully malignant phenotype) (Costinean, S. et al. PNAS, 2006)

35 A new form of cancer predisposition? Abnormalities of mirnas in the germline, an inherited predisposing event(blue person) The overexpression of target oncogenes (red protein) in the case of mirna deletion The downregulation of target tumour-suppressor genes in the case of mirna amplificatio n (purple protein)

36 Short Summary mirnas located in genomic regions amplified in cancers function as oncogenes, whereas mirnas located in portions of chromosomes del eted in cancers function as tumour suppressors. Abnormal expression of mirnas has been found in both solid and haematopoietic tumours by various genome-wide techniques. The abnormally expressed mirnas targeting essential protein-coding genes involved in tumorigenesis, such as the Ras by let-7 family mem bers, the BCL2 anti-apoptotic gene by the mir-15a mir-16-1 cluster, mirna expression fingerprints correlate with clinical and biological characteristics of tumors, including tissue type, differentiation, aggres sion and response to therapy. Germline sequence abnormalities in mirna genes and targeted sequ ences in mrnas.

37 mirna & Breast Cancer

38 Numbers of publications on the relationship of Breast Cancer & mirnas since Orginal Ariticale Review

39 Numbers of publications according to research objective Human BC Expression Profile 40 mirna/target polymorphism Functional Analysis Genomic Localization Method

40 mirna expression profiles in Breast Cancer Pts. Tissue

41 76 breast cancers and 34 normal samples Deregulated expression of 29 mirnas by microarray analysis A minimum predictive gene set of 15 candidates able to separate normal from cancer tissue The correlations bet. mirna expression and standard pathologic features of breast cancers including lymph node status, vascular invasion, metastasis, ER, PR, and p53 status with the exception of ERBB2 expression Only a small set of 15 mirna correctly predict the nature of the sample analyzed (i.e., tumor or normal breast tissue) with 100% accuracy.

42 Figure 1. Cluster analysis and PAM prediction in breast cancer and normal breast tissues Iorio, M. V. et al. Cancer Res 2005;65: Deregulated expression of 29 mirnas by microarray analysis

43 Deregulated expression of 29 mirnas in tumor breast tissue

44 mirna name Median expression ANOVA * P SVM predic tion strengt h PAM score Cancer Normal Cancer Normal Chromosome map mir q22 mir-010b q31 mir q23.2 mir p36.22 mir-102 (mir-29b) > q mir-123 (mir-126) q34 mir-125a q13.4 mir-125b q24.1 mir-125b q11.2 mir-140-as q22.1 mir q32-33 mir-155 (BIC) q21 mir > q41 mir q21.1 mir q31.3-q32.1

45 Differentially expressed mirnas associated With biopathologic features of breast cancers ER and PR

46 lymph node status, vascular invasion, metastasis, p53 status

47 What mrna could be targeted by these targets? Now, using PITA algorithms mir-10b targets ; FLT1(v-crk homologue), the growth factor BDNF, and the transducing factor SHC1 mir-125b target ; YES, ETS1, TEL, and AKT3; the growth factor receptor FGFR2; mitogen-activated signal transduction pathway VTS58635, MAP3K10, MAP3K11, and MAPK14. mir-145 target ; cell cycle promoters such as cyclins D2 and L1; and MAPK transduction proteins such as MAP3K3 and MAP4K4 mir-21 target ; the TGF-beta gene mir-155 ; tumor suppressor genes SOCS1 and APC, and the kinase WEE1, which blocks the activity of Cdc2 and prevents entry into mitosis. HIF1A.

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49 Correlation analysis between DNA copy number alteration and mirna expression

50 mirna & BC metastasis

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52 mir-10b is highly expressed in metastatic breast cancer cells and positively regulates cell migration and invasion.

53 mir-10b induces tumour invasion. Muscular Invasion Stromal Invasion Vascular Invasion Ki-67 MECA-32 Ki-67 MECA-32

54 mir-10b induces distant metastasis Clusters of metastatic cells

55 mir-10b expression level is associated with the metastasis outcome in breast cancer patients a. mir-10b expression level is associated with the metastasis outcome in breast cancer patients. b. mir-10b suppresses HOXD10, leading to induction of RHOC. HOXD10 : preveously reported to be progressively lost in BC showing increasing degree of malignancy(metastasis supression)

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58 Clinical association of mir-335 and mir-126 with metastasis-free survival 20 primary breast tumor samples mir-335 and mir-126 ; metastasis suppressor mirnas in human breast cancer

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60 mirna signatures classify breast cancer subtype

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62 Expression profiling of 453 mirnas perfomed in 29 early-stage breast cancer specimens NA michip array pl atforms (Exiqon versi on 7, containing 453 mirna sequences)

63 the genome-wide expression profiling of mirnas in primary BC microarray containing 435 mature human mirna oligonucleotide probes 9 mirnas(mir-21, mir-365, mir-181b, let-7f, mir-155, mir-29b, mir-181 d, mir-98, and mir-29c) up-regulated greater than 2 fold in BC compare d with NAT 7 mirnas (hsa-mir-497, hsa-mir-31, hsa-mir-355, hsa-mir-320, rno-mir- 140, hsa-mir-127 and hsa-mir-30a-3p) down-regulated greater than 2 fol d. RNA November; 14(11):

64 mir-21, serve as a molecular prognostic marker for BC and disease progression TaqMan RT PCR in 113 BC tumors High level expression of mir-21 : with advanced clinical stage (P = ), lymph node metastasis (P = 0.007), shortened survival of the patients (HR= 5.476, P < 0.001) Multivariate Cox regression analysis rev ealed this mir-21 (HR=4.133,P = 0.001) to be independent of disease stage (HR =2.226, P = 0.013) and histological gra de (HR=3.681, P = 0.033)

65 Cancer Treatment Reviews 25 (2009)

66 MiRNA Polymorphisms & Breast Cancer Prasun J Mishra et al Pharmacogenomics, Mar 2009

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68 3 important aspects in analyzing mirna target site mutation/polymorpism (i) functional: testing SNP-mediated differential mirna targeting (ii) genetic: testing association of the SNP with a disease or quantitative trait and (iii) mechanistic: testing a mechanism by which the differential mirna activity can lead to disease Recommend for the convincing evidence for all three components by (i) providing experimental (in vivo if possible) validation of SNP-mediated differential mirna targeting (ii) minimizing or eliminating the confounding effects of population stratification (iii) explicitly testing an underlying mechanism by which the poly-mirts could contribute to disease pathogenesis.

69 7 Case-control study using a familial study population Study population : 1223 German breast cancer families(braca1/2 mutation negative) and 1495 unrelated German controls Sample : genomic DNA from peripheral blood lymphocytes collected during the years by 7 centers of the German Consortium for Hereditary Breast and Ovarian Cancer SNP selection : 11 putative functional SNPs in mirna target sites located in genes involved in cancer and breast cancer Genotyping : TaqMan allelic discrimination. Primers and TaqMan probes, 5 ng of genomic DNA per assay Conclusions : a variant in the ESR1 affecting a putative mirna-binding site of mir-453 was significantly associated with familial breast cancer risk, especially in premenopausal and high-risk women

70 11 putative functional SNPs in mirna target sites located in genes involved in cancer and breast cancer

71 Genotype frequencies polymorphisms in breast cancer-related genes in the German study population

72 Genotype frequencies of ESR1 polymorphism rs in the German study population according age stratification and high-risk families From the in silico analysis, C variant allele of mir-453 is stronger than T allele T allele the binding of mir-453 mirna-mediated ESR1-repression ESR1 an increased breast cancer risk : protective effect observed for the C allele is biologically reasonable.

73 42 pts. w/ familial breast cancer 23 of pts were BRCA1 mutation, 8 had BRCA2 mutation, 7 were non carriers of BRCA1/2 17 selected mirna gene predicted to regulate key BC genes 7 novel genetic variants mir-30c-1 and mir-17 vatiants in noncarriers of BRCA1/2

74 7 Novel genetic variants in selected mirna genes in BC Genomic seq. PCR/ Sequencing 17 mirna genes Precursur seq.

75 Sequence variations in the pri-mirna can translate into structural alterations C(-48.1kcal/mol, more stable) T(-43.0kcal/mol, less stable) conformational change in the predicted secondary structure(rnahybrid) altered exp.of mature mirna

76 Effects of mir-17 on the luciferase reporter gene bearing 3 UTR segment from BRCA1

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78 mirna, as a new, novel, CANCER BIOMARKER?

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80 mirna being evaluated as therapeutic targets as well as diagnostic targets Designed to Differentiate bet. pancreatic cancer and pancreatitis

81 Why mirna better than mrna as a diagnostic tool? Remarkably, the number of mirnas analyzed was fa r smaller than the number of mrna transcripts ( 2 00 mirnas vs mrnas). More useful because of their regulatory role and th eir functions in modulating cellular differentiation.

82 Research Fields on mirna, as a Breast Cancer Biomarker SUSCEPTIBILITY MARKER for BC mirna gene variation/genomic alteration of mirna genes polymorphism in mirna target gene/mirna related gene copy number variation in mirna gene in breast cancer DIAGNOSTIC MARKER to BC - Deregulation of mirna biogenesis related genes in breast cancer - Differential mirna expression associated with biopathologic features of breast cancer[receptor status, TNM stages, vascular invasion status etc.] PROGNOSTIC MARKER to BC mirna profiling / response to therapeutics / disease outcome

83 Small RNAs Are Raising Big Expectations Karyn Hede Vol.101 Issue 12 June