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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 RNAs ( nc RNAs) : MICRORNAs AND CANCER. MICRORNAs ( MiRs ) AND CANCER. Outline of Presentation Non-coding RNAs ( ncRNAs ) and microRNAs ( MiRs )-background and functions MiR expression in tumors and cancer cells

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

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  1. 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* 1.4% 29% 27% 20% 70% 86% 91% 92% 100% 50% 0% 1 2 3 4 5 6 7 8 Mycobacterium tuberculosis Drosophila melanogaster 6 4 1 Homo Sapiens Archaeoglobusfulgidus Arabidopsis thaliana Saccharomycescerevisiae 5 2 7 6 Caenorhabditiselegans 3 Escheichia coli * From Szymanski and Barciszewski Genome Biol.3, 005.1, 2002

  4. nc RNAs – “Dark Matter” Function nc RNA* microRNAs (~23 nt) • decrease mRNA stability/ translation small (20-300 nt) Piwi-interacting RNAs (piRNAs, 26-31 nt) • gene silencing/ methylation Promoter-associated RNAs (small/variable) • gene silencing..? many more • chromatin association and gene modulation/ silencing Long intergenic non-coding RNAs (ncRNAs) long (>200 nt) * Bioessays 32, 599, 2010; Cancer Res 71, 3, 2011

  5. MicroRNA REGULATION OF GENE EXPRESSION IN CANCER CELLS/TUMORS miR-27a miR 20-22 nt (single chain) Pre-miRNA • 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 miR-27a RNA

  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) • 28 miRs interacted with • 3’-UTR; decreased luciferase activity 266-miRs Transfected • overexpression of miRs • decreased p21 protein • and mRNA levels luc 3’UTR p21 HEK293 cells “Oncogene 29, 2302, 2010”

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

  8. INDIVIDUAL MiRs ASSOCIATED WITH MULTIPLE TUMORS miRTS/OGTumors Let-7 Family MiR-159/16-1 cluster MiR-17-92 cluster MiR-26a MiR-34a/b/c MiR-21 TS TS OG TS/OG TS OG 10  7  7  4  6  10 TS = tumor suppressor; OG = oncogene

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

  10. TUMOR-SPECIFIC PATTERNS OF MiR EXPRESSION LIVER CANCER-CELL GROWTH/APOPTOSIS* DOWNREGULATED UPREGULATED let7 miR-1 miR-101 let7 miR-106b-25 miR-18a c-Met FoxP1 McL-1 FOS p21 Bim multiple ERa c-Myc Bcl-XL miR-122 miR-124 miR-101 miR-21 miR-124 miR-101 CDK-6 cyclin D1 p27 APL-5 CDK6 p57 cyclin G1 SRF PTEN miR-199 miR-283 miR-2a miR-602 cyclin D2 YAP cyclin E2 mTOR c-Met RASSFLA *Br.J.Cancer 104, 235, 2011 (partial list)

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

  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-17-92 (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* Intestinal-Type Gastric Cancer miR-495 10 5 miR-199 9 8 4 7 3.2 6 3 5 HAZARD RATIO (disease free survival) HAZARD RATIO (disease free survival) Let-7g 4 2 3 1 2 1 0 0 Stages III-IV low low high Stages I-II high high low I-II III-IV I-II I-II III-IV 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) MiR-885-5p Serum MiR-885-5p Levels 10 • Elevated in sera from patients with liver pathologies • Primarily expressed in liver • Function - not known 8 6 Rel. miR-885-5p Expr 4 2 Normal Hepato- cellular Carcinoma Liver cirrhosis

  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 miRsandcan they be targeted by anticancer agents?

  18. EXAMPLES OF FUNCTIONAL MiRs IN TUMORS miR-21 • Highly expressed in multiple tumors • Modulates expression of apoptotic/growth inhibitory mRNAs miR-17-92 • Highly expressed cluster in multiple tumors • Several paralogs (same seed sequence) modulate expression of anti-carcinogenic mRNAs miR-335 • Low expression in tumors • 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* • Patients were treated with gemcitabine • miR-21 also linked to gemcitabine and drug resistance (5-FU) low miR-21 Radically resected 100 high miR-21 20 metastatic 50 Overall Survival (Mo) % of patients 0 0 Grade 1-2 3 (1000X higher in tumor vs. non-tumor tissue) *Cancer Res 70, 4528, 2010; PLOS One 5, e10630, 2010

  21. ONCOMIR-21: IN VIVO TRANSGENIC MICE OVEREXPRESSING MIR-21 (NesCre 8, miR-21LSL-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 100 Survival (%) (-) Doxycycline Age (days) 100

  22. ONCOMIR-21: IN VIVO KNOCK OUT STUDIES • No obvious phenotype in miR-21-/- mice • Decreased DMBA-induced skin cancer (miR-21-/-) • miR-21-/- suppresses K-ras induced lung cancer Lung Tumors Adenoma 20 3 Lesions/Lung Rel. Tumor Areas 10 0 0 K-RasLA2 K-RasLA2 miR-21-/- K-RasLA2 K-RasLA2 miR-21-/- *Cancer Cell 18, 282, 2010 PNAS 108, 10144, 2011

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

  24. REGULATION OF SPECIFICITY PROTEIN (Sp) TRANSCRIPTION FACTORS BY MiRs (ONCOMIRS) • 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?

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

  26. REGULATION OF SPECIFICITY PROTEIN (Sp) TRANSCRIPTION FACTORS BY MiRs (ONCOMIRS) ZBTB10 – an Sp repressor • 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?

  27. REGULATION OF SPECIFICITY PROTEIN (Sp) TRANSCRIPTION FACTORS BY MiRs (ONCOMIRS) High Basal Sp Expression in Tumors Cancer Res. 67 11011, 2007 Oncogene 2012 LOW HIGH miR-17-5pmiR-20a miR-27a ZBTB10 ZBTB4 GC Sp Sp Sp low ZBTB4 ZBTB10 bcl-2, survivin (survival) NFkB, p65 (inflammation) EGFR, CD1, c-Met (growth) VEGF, VEGFR1 VEGFR2 (angiogenesis)

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

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

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

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

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

  33. 6-MCDF PROPERTIES OF ALKYL PCDFs - 6-MCDF* • 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) * 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 MiRs Induced MiRs Let-7d miR-134 miR-198 miR-373 miR-126 miR-205 miR-335

  36. INDUCTION OF POTENTIAL TUMOR SUPPRESSOR MiRs BY TCDD/MCDF –MiR-335* • 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 * Massague et al Nature. 451, 177, 2007

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

  38. TCDD AND MCDF INHIBIT BREAST CANCER CELL MIGRATION AND INVASION-BOYDEN CHAMBER ASSAY MDA-MB-231/24hr * Upper Chamber * Lower Chamber % Control Cells Membrane Pores Migrated Cells 5 10 CTL MCDF(uM)

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

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

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

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

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

  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, 2010. 2.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 70:4528-38, 2010. 3.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. ClinSci (Lond) 120:183-93, 2011. 4.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, 2010. 5.Huang, S. and He, X. The role of microRNAs in liver cancer progression. Br J Cancer 104:235-40, 2011. 6.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, 2010. 7.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 2011. 8.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. ProcNatlAcadSci U S A 108:10144-9, 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, 2001. • 10.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, 2010. • 11.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 67:11001-11, 2007. • 12.Szymanski, M. and Barciszewski, J. Beyond the proteome: non-coding regulatory RNAs. Genome Biol 3:reviews0005, 2002. • 13.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, 2010. • 14.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, 2010. • 15.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, 108-118, 2012. • 16.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. EndocrRelat Cancer 16:835-44, 2009.

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