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SHINING A LIGHT ON THE GENOME’S ‘DARK MATTER’ NON-CODING RNAs (nc RNAs) : MICRORNAs AND CANCER SHINING A LIGHT ON THE GENOME’S ‘DARK MATTER’ NON-CODING.

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Presentation on theme: "SHINING A LIGHT ON THE GENOME’S ‘DARK MATTER’ NON-CODING RNAs (nc RNAs) : MICRORNAs AND CANCER SHINING A LIGHT ON THE GENOME’S ‘DARK MATTER’ NON-CODING."— Presentation transcript:

1 SHINING A LIGHT ON THE GENOME’S ‘DARK MATTER’ NON-CODING RNAs (nc RNAs) : MICRORNAs AND CANCER SHINING A LIGHT ON THE GENOME’S ‘DARK MATTER’ NON-CODING RNAs (nc RNAs) : MICRORNAs AND CANCER

2 MICRORNAs (MiRs) AND CANCER Outline of Presentation Non-coding RNAs (ncRNAs) and microRNAs (MiRs)-background and functions MiR expression in tumors and cancer cells MiRs as prognostic factors for cancer patients miRs in body fluids and their potential as prognostic and diagnostic biomarkers Examples of miR functions in cancer MiRs as drug targets

3 ncRNAs IN BACTERIAL AND EUKARYOTIC GENOMES % OF ncRNAs* * From Szymanski and Barciszewski Genome Biol.3, 005.1, % 50% 0% 1.4%29% 27%20%70%86%91%92% Homo Sapiens Arabidopsis thaliana Caenorhabditis elegans Drosophila melanogaster Saccharomyces cerevisiae Escheichia coli Mycobacterium tuberculosis Archaeoglobus fulgidus

4 nc RNAs – “Dark Matter” Function nc RNA* long (>200 nt) * Bioessays 32, 599, 2010; Cancer Res 71, 3, 2011 chromatin association and gene modulation/ silencing small ( nt) microRNAs (~23 nt) decrease mRNA stability/ translation Piwi-interacting RNAs (piRNAs, nt) gene silencing/ methylation Promoter- associated RNAs (small/variable) gene silencing..? many more Long intergenic non-coding RNAs (ncRNAs)

5 miRs interact with 3’-UTR of mRNAs Low miR-mRNA base specificity (6-8) Each miR can potentially interact with several hundred mRNAs Function: block gene expression Pre-miRNA MicroRNA REGULATION OF GENE EXPRESSION IN CANCER CELLS/TUMORS nt (single chain) miR-27a RNA miR

6 COMPLEXITIES OF MiR-mRNA INTERACTIONS MULTIPLE MiRs REGULATE A SINGLE mRNA* the p21 3’UTR can potentially be targeted by 266 miRs (p21 – tumor suppressor) 266-miRs luc Transfected 3’UTR p21 HEK293 cells 28 miRs interacted with 3’-UTR; decreased luciferase activity overexpression of miRs decreased p21 protein and mRNA levels “Oncogene 29, 2302, 2010”

7 MicroRNA ACTIVITY IN CANCER: TUMOR SUPPRESSIVE OR ONCOGENIC miR Tumor suppressive Oncogenic miR Suppress expression of oncogenes, growth promoting, survival and angiogenic genes (low in tumors) Suppress expression of tumor suppressor, growth inhibitory, proapoptotic genes (high in tumors)

8 INDIVIDUAL MiRs ASSOCIATED WITH MULTIPLE TUMORS miR TS/OG Tumors Let-7 Family MiR-159/16-1 cluster MiR cluster MiR-26a MiR-34a/b/c MiR-21 TS OG TS/OG TS OG  10  7 7  7 7  4 4  6 6 TS = tumor suppressor; OG = oncogene

9 SPECIFICITY OF MiR EXPRESSION IN TUMORS* miR Tumor Tissue TS/OG MiR-155 MiR-200/141 family MiR-205 MiR-206 MiR-9 Hematopoietic system Epithelial-specific Skeletal and muscle Nervous system OG TS/OG TS TS/OG

10 TUMOR-SPECIFIC PATTERNS OF MiR EXPRESSION LIVER CANCER-CELL GROWTH/APOPTOSIS* DOWNREGULATEDUPREGULATED let7miR-1 c-MycBcl-XL miR-101 c-MetFoxP1McL-1FOS miR-122 cyclin G1 SRF miR-101 CDK-6cyclin D1 miR-124 CDK6 miR-199 mTORc-Met cyclin D2 miR-283 cyclin E2 miR-2a YAP miR-106b-25 p21Bim APL-5p27 miR-124 p57 multiple let7 ER  miR-18a PTEN miR-21miR-101 RASSFLA miR-602 *Br.J.Cancer 104, 235, 2011 (partial list)

11 TUMOR-SPECIFIC PATTERNS OF MiR EXPRESSION LIVER CANCER-CELL GROWTH/APOPTOSIS* miRs Corresponding growth promoting/ prosurvival mRNAs- upregulated Corresponding growth inhibitory/anti- survival mRNAs- downregulated miRs

12 CORRELATION OF MIR EXPRESSION WITH PROGRESSION AND PROGNOSIS OF GASTRIC CANCER* PATIENTS: 181 patients from 2 cohorts (Japan) CLASSIFICATION: Stages I-IV Diffuse vs. Intestinal type ANALYSIS: Custom miR microarray chip (Ohio State Univ.) miR expression in 160 paired samples (tumor vs. non-tumor) Correlations of miR expression vs. stage, type and prognosis (survival) * Lancet Oncol. 11,136, 2010

13 GASTRIC CANCER MiR SIGNATURE* UPREGULATED MIRs (22): miR-181 (6), miR-21, miR-25, miR-92 (2), miR-19b (2), miR (7), miR-224, miR-19a miR-345, miR-191, miR-135b, miR-135a (2) DOWNREGULATED MIRs (13): miR-148 (2), miR-375, miR-29b (2), miR-29c, miR-152, miR-218-2, miR-451, miR-30a-d (5), miR-422b Several different miRs in the cluster. Lancet Oncol. 2010

14 MiRs AS PROGNOSTIC FACTORS: GASTRIC CANCER SURVIVAL* Stages I-II Stages III-IV 3.2 high low high low high Let-7g miR-199 miR-495 HAZARD RATIO (disease free survival) HAZARD RATIO (disease free survival) Intestinal-Type Gastric Cancer I-II III-IVI-II III-IV I-II III-IV

15 SERUM AND BODY FLUID MiRs AS BIOMARKERS Multiple miRs have been characterized not only in serum but also tears, urine, breast milk, seminal fluid, saliva, amniotic fluid, bronchial lavage, cerebrospinal fluid, pleural fluid, peritoneal fluid and colostrum (Clin. Chem 56, 1733, 2010) A select number of miRs may serve as diagnostic markers for different tumor types (Mol. Cancer 9:306, 2010)

16 SERUM MiRs AS MARKERS FOR LIVER PATHOLOGIES (Clin. Sci 120, 183, 2011) SERUM MiRs AS MARKERS FOR LIVER PATHOLOGIES (Clin. Sci 120, 183, 2011) Elevated in sera from patients with liver pathologies Primarily expressed in liver Function - not known Rel. miR-885-5p Expr NormalHepato- cellular Carcinoma Liver cirrhosis MiR-885-5p Serum MiR-885-5p Levels

17 MiRs AS PROGNOSTIC FACTORS FOR CANCER: SUMMARY There are unique miR signatures for different cancers Several miRs are up-or downregulated in multiple tumors Tumors and serum miR expression can be prognostic factors for patient survival Since multiple miRs target unique and overlapping mRNAs, are there functional individual miRs and can they be targeted by anticancer agents?

18 EXAMPLES OF FUNCTIONAL MiRs IN TUMORS Highly expressed in multiple tumorsmiR-21 Modulates expression of apoptotic/growth inhibitory mRNAs Highly expressed cluster in multiple tumorsmiR Several paralogs (same seed sequence) modulate expression of anti-carcinogenic mRNAs Low expression in tumorsmiR-335 Expression studies suggest that miR-335 inhibits metastasis

19 MiR-21 IS ONCOGENIC IN VITRO Multiple studies show that knockdown of miR-21 in cancer cells decreases growth and induces apoptosis Overexpression of miR-21 in cancer cells enhances their tumorigenicity miR-21 is a prognostic factor for poor patient survival miR-21 also plays a role in drug-resistance

20 MiR-21 IS A NEGATIVE PROGNOSTIC FACTOR FOR PANCREATIC CANCER PATIENTS* low miR-21 high miR Overall Survival (Mo) % of patients Radically resected metastatic Grade1-23 (1000X higher in tumor vs. non-tumor tissue) *Cancer Res 70, 4528, 2010; PLOS One 5, e10630, 2010 Patients were treated with gemcitabine miR-21 also linked to gemcitabine and drug resistance (5-FU)

21 ONCOMIR-21: IN VIVO TRANSGENIC MICE OVEREXPRESSING MIR-21 (Nes Cre 8, miR-21 LSL-Tetoff ) (DOXYCYCLINE ) ONCOMIR-21: IN VIVO TRANSGENIC MICE OVEREXPRESSING MIR-21 (Nes Cre 8, miR-21 LSL-Tetoff ) (DOXYCYCLINE ) Overexpression of miR-21 in mice results in pre-B malignant lymphoid tumors Doxycycline- induced downregulation of miR-21 inhibits oncogenesis Mice with lymphomas (+) Doxycycline (-) Doxycycline Age (days) 100 Survival (%)

22 ONCOMIR-21: IN VIVO KNOCK OUT STUDIES *Cancer Cell 18, 282, 2010 PNAS 108, 10144, 2011 No obvious phenotype in miR- 21 -/- mice Decreased DMBA- induced skin cancer (miR-21 -/- ) miR-21 -/- suppresses K-ras induced lung cancer Lung Tumors 0 3 Lesions/Lung Rel. Tumor Areas Adenoma K-Ras LA2 miR-21 -/- K-Ras LA2 miR-21 -/-

23 REGULATION OF SPECIFICITY PROTEIN (Sp) TRANSCRIPTION FACTORS BY MiRs (ONCOMIRS) Low expression in non tumor tissue High expression in tumor tissue

24 Mechanisms for Sp overexpression Epigenetic effects (hypo/hypermethylation) -no evidence Enhanced expression of genes that regulate Sp TFs -they are self regulatory Inhibition of “Sp repressors”……..by miRNAs? REGULATION OF SPECIFICITY PROTEIN (Sp) TRANSCRIPTION FACTORS BY MiRs (ONCOMIRS)

25 5 microRNAs (miRs) as-MiR-27a ZBTB10 (1.4 Fold) SKBR3 CELLS LAQ824 (HDACi) 5 hr + 22 miRs (including miR-27a) - + Note: miR-27a ZBTB10 mRNA One of several hundred potential miR-27a target Cancer Res. 66, 1277,2006 REGULATION OF SPECIFICITY PROTEIN (Sp) TRANSCRIPTION FACTORS BY MiRs (ONCOMIRS) MiR-27a : ZBTB10?

26 ZBTB10 competitively binds (and displaces Sp) GC rich sequences (JBC 274, 8123, 1999) ZBTB10 is a member of the BTB/POZ family of transcription repressors Does miR-27a repress ZBTB10 and thereby allow for overexpression of Sp1, Sp3 and Sp4? REGULATION OF SPECIFICITY PROTEIN (Sp) TRANSCRIPTION FACTORS BY MiRs (ONCOMIRS) ZBTB10 – an Sp repressor

27 High Basal Sp Expression in Tumors miR-27a ZBTB10 LOW ZBTB10 low HIGH GC EGFR, CD1, c-Met (growth) bcl-2, survivin (survival) VEGF, VEGFR1 VEGFR2 (angiogenesis) NF  B, p65 (inflammation) REGULATION OF SPECIFICITY PROTEIN (Sp) TRANSCRIPTION FACTORS BY MiRs (ONCOMIRS) (a)Cancer Res , 2007 miR-17-5p miR-20a ZBTB4 (a)Oncogene 2012 ZBTB4

28 mitochondria ROS miR-20a miR-27a Sp-repressor (mRNA) EGFR, CD1, c-Met (growth) bcl-2, survivin (survival) VEGF, VEGFR1/VEGFR2 (angiogenesis) NF  B/p65 (inflammation Mechanisms of drug-induced repression of Sp1, Sp3, Sp4 and Sp-regulated genes in cancer cell lines (curcumin, celastrol, aspirin, betulinic acid….) ZBTB10 ZBTB4 SUMMARY OF ONCOMIRS THAT SUPRESS Sp TRANSCRIPTION FACTORS phosphatases proteasomes caspases (active) Sp- regulated genes

29 Several anticancer drugs downregulate miRs and induce Sp repressors which downregulate Sp1, Sp3 and Sp4 genes. Can anticancer drugs induce tumor suppressor-like miRs ? DEVELOPMENT OF ANTICANCER DRUGS THAT TARGET MiRS

30 inhibition ER ERE Estrogenic Responses Inhibited by AhR-ER Crosstalk Mammary tumor formation and growth (rodent & human) DRE TCDD ( ) 2,3,7,8-TCDD AS AN ANTIESTROGEN Uterine and endometrial responses (rodents) Breast cancer cell responses AhR arnt ER AhR arnt O OCl

31 Ah RECEPTOR AS A DRUG TARGET DRE Drug *Toxic responses (chloracne, wasting…) *Biochemical responses (CYPlA, UGT/GST…) *Pharmacolologic responses (antiestrogenicity, anticancer activity, autoimmune diseases) *Age, sex, species, strain and tissue-dependent AhR arnt

32 1,3,6,8- Alternate-substituted Alkyl PCDFs (synthetic) 2,4,6,8- Indole-3-carbinol (I3C) N H 2 CH 2 Diindolylmethane (DIM) DEVELOPMENT OF NON-TOXIC AhR-BASED ANTIESTROGENS

33 moderate AhR binding affinity low toxicity and poor induction of CYP1A1 exhibits partial AhR antagonist activity (for toxic responses) but elicits high antiestrogenic activity in MCF-7 cells/rat uterus (agonist activity) 6-MCDF PROPERTIES OF ALKYL PCDFs - 6-MCDF* * Selective AhR modulator (SAhRM)

34 SAhRMs FOR BREAST CANCER THERAPY MCDF alone or plus tamoxifen are highly effective against ER+ breast cancer (Cancer Res 61, 3902, 2001) MCDF inhibits ER- breast cancer cell and tumor growth (Endocr Rel. Cancer 16, 835, 2009) Inhibition by MCDF not related to altered kinases, apoptosis or cell cycle genes Do SAhRMs such as MCDF work through miRs ?

35 INDUCTION/REPRESSION OF MiRs BY TCDD/MCDF Repressed MiRsInduced MiRs Let-7d miR-134 miR-198 miR-373 miR-126 miR-205 miR-335

36 Low expression of miR-335 in breast cancer predicts poor metastasis-free survival. Knockdown of miR-335 enhances MDA- MB-231 metastasis whereas overexpression of miR-335 blocks metastasis miR-335 suppresses expression of “prometastatic” genes such as SOX-4 INDUCTION OF POTENTIAL TUMOR SUPPRESSOR MiRs BY TCDD/MCDF –MiR-335* * Massague et al Nature. 451, 177, 2007

37 TCDD/MCDF INDUCE MiR-335 IN MDA-MB-231 CELLS - AHR-DEPENDENT miR-335 AhR Actin iCT iAhR MDA-MB-231 miR-335 MDA-MB-231      

38 TCDD AND MCDF INHIBIT BREAST CANCER CELL MIGRATION AND INVASION-BOYDEN CHAMBER ASSAY Upper Chamber Lower Chamber Membrane Pores Migrated Cells Cells % Control CTL MCDF(uM) 510 MDA-MB-231/24hr * *

39 MIR-335 ALSO INDUCES CELL INVASION BUT THIS RESPONSE IS AHR-INDEPENDENT iCTL+CTLiCTL+miR335 iAhR+miR335iAhR+CTL % control Invasion Assay/MDA-MB-231 * *

40 TCDD/MCDF DECREASE MIR-335-REGULATED SOX4 IN MDA-MB-231 CELLS * iCTLiAhR * SOX4 mRNA (Rel. DMSO)

41 TCDD/MCDF DECREASE SOX4 PROTEIN IN MDA-MBA-231 CELLS SOX4 MDA-MB-231 DMSO TCDD 10 nM MCDF 5 nM iAhR β-Actin

42 MCDF INHIBITS MDA-MB-231 CELL LUNG METASTASIS IN MICE Corn Oil (CTL) MCDF (40 mg/kg/d) No. of colonies No cells CO MCDF +T+T +T+T ND

43 ANTIMETASTATIC ACTIVITY OF AHR AGONISTS IN ER BREAST CANCER Normal cells Preneoplastic cells Cancer cells (Invasive carcinoma) Metastasis SOX4 SOX4 and other miR-335 regulated proteins miR-335 SOX4 SOX4 and other miR-335 regulated metastatic mRNAs arnt AhR Ligand activated Ahr

44 REFERENCES 1.Brase, J. C., Wuttig, D., Kuner, R. and Sultmann, H. Serum microRNAs as non-invasive biomarkers for cancer. Mol Cancer 9:306, Giovannetti, E., Funel, N., Peters, G. J., Del Chiaro, M., Erozenci, L. A., Vasile, E., Leon, L. G., Pollina, L. E., Groen, A., Falcone, A., Danesi, R., Campani, D., Verheul, H. M. and Boggi, U. MicroRNA-21 in pancreatic cancer: correlation with clinical outcome and pharmacologic aspects underlying its role in the modulation of gemcitabine activity. Cancer Res 70: , Gui, J., Tian, Y., Wen, X., Zhang, W., Zhang, P., Gao, J., Run, W., Tian, L., Jia, X. and Gao, Y. Serum microRNA characterization identifies miR-885-5p as a potential marker for detecting liver pathologies. Clin Sci (Lond) 120:183-93, Hatley, M. E., Patrick, D. M., Garcia, M. R., Richardson, J. A., Bassel-Duby, R., van Rooij, E. and Olson, E. N. Modulation of K-Ras-dependent lung tumorigenesis by MicroRNA-21. Cancer Cell 18:282-93, Huang, S. and He, X. The role of microRNAs in liver cancer progression. Br J Cancer 104:235-40, Hwang, J. H., Voortman, J., Giovannetti, E., Steinberg, S. M., Leon, L. G., Kim, Y. T., Funel, N., Park, J. K., Kim, M. A., Kang, G. H., Kim, S. W., Del Chiaro, M., Peters, G. J. and Giaccone, G. Identification of microRNA-21 as a biomarker for chemoresistance and clinical outcome following adjuvant therapy in resectable pancreatic cancer. PLoS One 5:e10630, Kim, K., Chadalapaka, G., Lee, S. O., Yamada, D., Sastre-Garau, X., Defossez, P. A., Park, Y. Y., Lee, J. S. and Safe, S. Identification of oncogenic microRNA-17-92/ZBTB4/specificity protein axis in breast cancer. Oncogene Ma, X., Kumar, M., Choudhury, S. N., Becker Buscaglia, L. E., Barker, J. R., Kanakamedala, K., Liu, M. F. and Li, Y. Loss of the miR-21 allele elevates the expression of its target genes and reduces tumorigenesis. Proc Natl Acad Sci U S A 108: , 2011.

45 REFERENCES 9.McDougal, A., Wormke, M., Calvin, J. and Safe, S. Tamoxifen-induced antitumorigenic/antiestrogenic action synergized by a selective aryl hydrocarbon receptor modulator. Cancer Res 61:3902-7, Medina, P. P., Nolde, M. and Slack, F. J. OncomiR addiction in an in vivo model of microRNA-21-induced pre-B- cell lymphoma. Nature 467:86-90, Mertens-Talcott, S. U., Chintharlapalli, S., Li, X. and Safe, S. The oncogenic microRNA-27a targets genes that regulate specificity protein transcription factors and the G2-M checkpoint in MDA-MB-231 breast cancer cells. Cancer Res 67: , Szymanski, M. and Barciszewski, J. Beyond the proteome: non-coding regulatory RNAs. Genome Biol 3:reviews0005, Ueda, T., Volinia, S., Okumura, H., Shimizu, M., Taccioli, C., Rossi, S., Alder, H., Liu, C. G., Oue, N., Yasui, W., Yoshida, K., Sasaki, H., Nomura, S., Seto, Y., Kaminishi, M., Calin, G. A. and Croce, C. M. Relation between microRNA expression and progression and prognosis of gastric cancer: a microRNA expression analysis. Lancet Oncol 11:136-46, Wu, S., Huang, S., Ding, J., Zhao, Y., Liang, L., Liu, T., Zhan, R. and He, X. Multiple microRNAs modulate p21Cip1/Waf1 expression by directly targeting its 3' untranslated region. Oncogene 29:2302-8, Zhang, S., Kim, K., Jin, U. H., Pfent, C., Cao, H., Amendt, B., Liu, X., Wilson-Robles, H. and Safe, S. Aryl hydrocarbon receptor (AHR) agonists induce microRNA-335 expression and inhibit lung metastasis of estrogen receptor negative breast cancer cells. Mol Cancer Therap. 11, , Zhang, S., Lei, P., Liu, X., Li, X., Walker, K., Kotha, L., Rowlands, C. and Safe, S. The aryl hydrocarbon receptor as a target for estrogen receptor-negative breast cancer chemotherapy. Endocr Relat Cancer 16:835-44, 2009.


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