10/31/2017. micrornas and cancer. From the one gene-one enzyme hypothesis to. microrna DNA RNA. Transcription factors.

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1 micrornas and cancer Cellular and Molecular Biology of Cancer (PATH G ) November 1 st, Katia Basso- Columbia University Katia Basso, PhD Office: ICRC RM506 kb451@cumc.columbia.edu From the one gene-one enzyme hypothesis to DNA Transcription factors RNA microrna PROTEIN microrna 5 -UGUGCAAAUCUAUGCAAAACUGA-3 Seed (position 2 to 8) micrornas (mirnas) represent a class of small RNAs characterized by: short length: about 22 nucleotide-long RNAse III endonuclease-mediated processing association with Argonaute proteins 1

2 microrna discovery lin-4 discovery in C. elegans (Lee et al. Cell 1993; Wightman et al., Cell 1993) let-7 discovery in H. sapiens (Pasquinelli et al. Nature 2000) Identification of a large class of small-rna called micrornas (Lagos-Quintana et al.; Lau et al.; Lee and Ambros Science 2001) microrna biogenesis (Hutvagner et al. Science 2001; Lee et al. Nature 2003) mirbase database (Griffiths-Jones, NAR 2004; Griffiths- Jones, et al. NAR 2006) The discovery of micrornas -milestonesmicrorna biogenesis and function 2

3 nucleus 10/31/2017 Canonical microrna biogenesis POLII Transcription CROPPING HSC70-HSP90 cytoplasm ORF RISC Target repression AAAA Adapted from: Ha, M. and Kim, N.V.: Nature Rev Mol. Cell Biol. (2014) Substrate recognition by Rnase III endonucleases Microprocessor complex (Drosha and DGCR8) Dicer tion on Adapted from: Ha, M. and Kim, N.V.: Nature Rev Mol. Cell Biol. (2014) Multifunctional microrna precursors Maute R.L. et al.: Wiley Interdiscip Rev RNA (2014) 3

4 Non-canonical microrna biogenesis Drosha and DGCR8-independent Canonical pathway TUTase-dependent Dicer-independent Ha, M. and Kim, N.V.: Nature Rev Mol. Cell Biol. (2014) Possible mechanisms of microrna-mediated repression Filipowicz W. et al.: Nature Rev Genetics (2008) microrna-mediated pathways Maute R.L. et al.: Wiley Interdiscip Rev RNA (2014) 4

5 microrna nomenclature mirna precursor 5 3 mirna duplex 3 5 Precursor nomenclature: hsa-mir-19a hsa-mir-19b-1 hsa-mir-19b-2 organism family genomic precursor family location member mature mirnas 5 3 hsa-mir-19a-5p hsa-mir-19a-3p Mature nomenclature: hsa-mir-19a-5p hsa-mir-19a-3p hsa-mir-19b-3p hsa-mir-19b-1-5p hsa-mir-19b-2-5p mature 5 arm or 3 arm microrna targets microrna-mediated target repression Mature RISC ORF Poly(A) mrna How do the mature RISC complexes recognize and bind their specific mrna targets? 5

6 Target Repression 10/31/2017 microrna-target recognition - site position- Binding in the 3 UTR is effective for repression Effective site position in the target 3 UTR Effective sites in the 3 UTR are preferentially located in proximity of the termination codon (except the first 15nt) or of the poly-a tail. Adapted from: Grimson A. et al.: Mol. Cell (2007) microrna-target recognition -seed- Canonical mirna complementary sites mrna changes according to type and number of sites in the target 3 UTR Adapted from: Grimson A. et al.: Mol. Cell (2007) microrna-target recognition -3 pairing- Pairing in position of the mirna promotes target regulation Adapted from: Grimson A. et al.: Mol. Cell (2007) 6

7 microrna-target recognition -AU content- Nucleotide composition in proximity of effective and conserved sites Score changes based on the presence of AU relatively to the site Effectiveness of sites with different local AU content Adapted from: Grimson A. et al.: Mol. Cell (2007) microrna-target recognition - summary- >15nt ORF NNNNNNNNNN NNNNNNNNA Poly(A) NNNNNNNNNN NNNNNNNNN mirna seed 1 mrna In summary, mirnas bind to the 3 UTR of their mrna targets according to the following rules: site position in proximity of the termination codon (except the first 15nt) or of the poly-a tail perfect and contiguous base pairing of mirna nucleotides 2 to 8 (seed region) an A residue in position 1 of the site and/or 3 complementarity especially at position of the mirna improve the site efficiency bulges or mismatches must be present in the middle of the mirna-mrna duplex, precluding the AGO2-mediated cleavage of mrna AU-rich neighborhoods host more effective sites Identification of microrna targets Target Prediction Algorithms TargetScan PicTar MiRanda PITA many others Cell context specificity Gene Expression Profiles Differential Expression Proteomic Profiles Experimental Validation 3 UTR-reporter assay AGO-IP CLIP (Cross-Link and ImmunoPrecipitation) 7

8 microrna-mediated networks The competing endogenous RNA (cerna) hypothesis All types of RNA transcripts communicate through a new language mediated by mirna response elements A cerna Hypothesis: The Rosetta Stone of a Hidden Language? Salmena, L., Poliseno, L., Tay, Y., Kats, L. and Pandolfi, P.P Cell (2011) Effects of microrna-target perturbation Competing target overexpression Steady-state Competing mirna overexpression Maute R.L. et al.: Wiley Interdiscip Rev RNA (2014) 8

9 microrna-mediated modulation of PTEN Poliseno L. et al.: Nature (2010); Tay Y. et al.: Cell (2011) micrornas and cancer micrornas and cancer research -milestones Discovery of mir-15 and mir-16 as oncosuppressors in Chronic Lymphocytic Leukemia (Calin et al., PNAS 2002) mirna & cancerassociated genomic regions (Calin et al., PNAS 2004) Oncomir-1 (mir cluster) (He et al., Nature 2005; O Donnell et al., Nature 2005) mirna profiling classifies tumors (Lu et al., Nature 2005) microrna & metastasis (Ma et al., Nature 2007; Tavazoie et al., Nature 2008) mirna detection in serum and plasma (Chen et al., Cell Res. 2008; Mitchell et al., PNAS 2008) 9

10 micrornas in cancer no mirna expression impaired target repression increased mirna expression enhanced target repression tumor suppressor oncogene Adapted from: Esquela-Kerscher A. and Slack F.: Nat Rev Cancer (2006) Genetic, transcriptional and post-transcriptional microrna alterations in cancer Genomic Alterations Amplification Deletion Mutation Transcription & Processing Kasinski A. and Slack F. J.: Nat Rev Cancer (2011) microrna deregulated activity induced by target modifications Kasinski A. and Slack F. J.: Nat Rev Cancer (2011) 10

11 Pathways leading to altered microrna expression and/or activity TRANSCRIPTION and PROCESSING mirna TARGETS PATHWAYS Genomic lesions (gains, losses, rearrangements) Deregulated transcription Mutations/SNPs affecting mirna and/or its processing Target abundance Mutations creating/destroying sites Genomic lesions that change the 3 UTR (i.e. translocations) Alternative polyadenylation micrornas as tumor suppressors Chronic Lymphocytic Leukemia (CLL) CLL represents the most frequent B-cell malignancy in the elderly Characteristically expresses the CD5 cell surface antigen It can be divided into cases with somatically mutated (~60%) or unmutated (~40%) immunoglobulin variable genes Most frequent genetic alteration is the deletion of chromosomal region 13q14 11

12 Recurrent genetic aberrations in CLL Trisomy 12 (16%) - is thought to affect gene dosage of unknown genes Deletion of 11q (18%) - deletion of the ATM gene; may predispose to genomic instability and the development of lymphoid malignancy Deletion of 17q (7%) - deletion of the TP53 gene, and in most cases, the remaining TP53 allele is mutated; predisposes to genomic instability and the development of lymphoid malignancy Deletion of 13q14 (55%) -? The 13q14 Minimal Deleted Region (MDR) contains multiple genetic elements Ch13 q14 MDR mir-15a/16-1 DLEU2 DLEU1 mir-15a/16-1 DLEU2 E5 E4 E3 E2 E1a mir-16-1 mir-15a 381bp DLEU2 Ex 4 Mouse model of the 13q14 deletion Human 13q14 KCNRG 13q14 DLEU5 MDR (Minimal Deleted Region) mir-15a/16-1 DLEU2 DLEU1 Mouse 14qC3 14qC3 loxp dleu5 Kcnrg mir-15a/16-1 dleu2 loxp Mouse model #1 MDR conditional knock-out dleu2 loxp loxp 430 bp dleu2 mir-15a/ dleu2 exon 5 mir-16-1 exon 4 Mouse model #2 mir-15a/16-1 conditional knock-out Gapdh RT-PCR for dleu2 Klein, U. et al.: Cancer Cell (2010) 12

13 CD5 CD5 10/31/2017 Malignancies in mouse models of the 13q14 deletion Lymphoid involvement Clonal Ig WT CD5 + MBL in PB + MDR- and mir-15a/16-1-deleted CLL/SLL + + NHL (DLBCL) + + Spleen (H&E) Spleen (FACS) 1.5% 1.4% 66.2% 1.5% B220 Peripheral Blood (FACS) B % 13.2% 45.0% 1.6% Klein, U. et al.: Cancer Cell (2010) Tumor incidence Event-free survival Klein, U. et al.: Cancer Cell (2010) mir-15a and mir-16 modulate cellular proliferation CLL-I83E95 Mouse B cells WT mir EV mir / a-igm h 4.9 CCNE CHK MCM CCND CDK CDK CCND CDC25A +1.7 b-actin CCND2 CCND3 CDK4 CDK6 G 0/G 1 mir-15a/16-1 Rb E2F CDC25A + : mir targets p-rb E2F CCNE CDK2 S-phase CHK1 MCM5 p27 Malignancies in mouse models of the 13q14 deletion -low penetrance and indolent phenotype- mir- 15a/ 16-1 Klein, U. et al.: Cancer Cell (2010) 13

14 micrornas and host genes: synergistic and antagonistic functions Maute R.L. et al.: Wiley Interdiscip Rev RNA (2014) micrornas as oncogenes 13q31-q32 amplification in B cell lymphoma -searching for candidate oncogene(s)- Ota, A. et al.: Cancer Res (2004) 14

15 mir cluster on chr 13q31-q32 and its paralogues Olive, V. et al.: The International Journal of Biochemistry & Cell Biology (2010) mir cluster: oncomir-1 Acceleration of MYC-induced lymphomagenesis in mice Over-expression in cancer He, L. et al.: Nature (2005) mir cluster: function and regulation Olive, V. et al.: The International Journal of Biochemistry & Cell Biology (2010) 15

16 micrornas in cancer MicroRNA Expression in patients Confirmed targets Experimental data Function mir-15a mir-16-1 downregulated in CLL Bcl-2, Wt-1 induce apoptosis and TS decrease tumorigenicity let-7 (a,-b,-c,-d) downregulated in lung and breast cancer RAS, c-myc, HMGA2 induce apoptosis TS mir-29 (a,-b,-c) downregulated in CLL, AML a (11q23), lung TCL-1, MCL1, DNMT3sinduce apoptosis and and breast cancers, and decrease tumorigenicity cholangiocarcinoma mir-34a-b-c downregulated in pancreatic, colon, and CDK4, CDK6, cycline2, induce apoptosis breast cancers E2F3 mir-155 upregulated in CLL, DLBCL, AML, BL, and c-maf induces lung and breast cancers lymphoproliferation, pre- B lymphoma/leukemia in mice mir cluster upregulated in lymphomas and in breast, E2F1, Bim, PTEN cooperates with c-myc to lung, colon, stomach, and pancreas induce lymphoma in cancers mice, transgenic mir develop lymphoproliferative disorder mir-21 upregulated in breast, colon, pancreas, PTEN, PDCD4, TPM1 induces apoptosis and lung, prostate, liver, and stomach cancer; decreases tumorigenicity AML(11q23); CLL; and glioblastoma mir-372/mir-373 upregulated in testicular tumors LATS2 promote tumorigenesis in cooperation with RAS TS TS OG OG OG OG Garzon R. et al.: Annu Rev Med (2009) microrna-mediated target repression pri-mirna DROSHA DGCR8 5 3 Impaired processing Argonaute XPO5 DICER1 5 3 RNA POLII mirna alterations 5 3 pre-mirna RISC mature mirna 5 target Target mrna alterations? Translational repression mrna degradation 3 the OMICS era DNA (Genome and Epigenome) Transcription factors (Transfactome) RNA (Transcriptome) microrna (mirnome) PROTEIN (Proteome) 16