EPIGENETICA E TUMORI

1. EPIGENETICA E TUMORI R. Piazza Dip. Scienze della Salute Università di Milano-Bicocca http://149.132.236.129/lezioni/

2. EPIGENETICA L'epigenetica descrive un insieme di meccanismi di regolazione dell’espressione genica in cui il fenotipo è determinato non dal genotipo in quanto tale, ma dalla sovrapposizione al genotipo stesso di una serie di informazioni aggiuntive che ne influenzano il comportamento.

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11. EPIGENETIC GENE SILENCING EUCHROMATIN GENOMIC DNA METHYLATION CHROMATIN CONDENSATION (HETEROCHROMATIN) RECRUITMENT OF METHYL-CpG BINDING PROTEINS RECRUITMENT OF COREPRESSOR COMPLEXES WITH DEACETYLASE ACTIVITY

12. METILAZIONE DEL DNA LA METILAZIONE DEL DNA AVVIENE IN POSIZIONE 5, ESCLUSIVAMENTE IN CITOSINE SEGUITE DA GUANINE (DINUCLEOTIDI CpG)

13. CpG ?? SEQUENZA PALINDROMICA! m CA ?? GT ?? +

14. DNA methylation 3 human DNA methyltransferases • DNMT1 • DNMT3A • DNMT3B maintenance methyltransferases de novo methyltransferases – highly expressed at embryo implantation when waves of de novo methylation are occurring in the genome ◄daughter strand ◄daughter strand

15. EUCHROMATIN GENOMIC DNA METHYLATION CHROMATIN CONDENSATION (HETEROCHROMATIN) RECRUITMENT OF METHYL-CpG BINDING PROTEINS RECRUITMENT OF COREPRESSOR COMPLEXES WITH DEACETYLASE ACTIVITY

16. Come sono distribuiti i siti CpG nell’ambito di un gene ? Qual è il pattern di metilazione atteso in geni attivamente trascritti ed in geni ‘spenti’ ? Sito CpG non metilato Sito CpG metilato

17. CpG islands CpG islands are typically 300-3,000 base pairs in length. They are in and near approximately 40% of promoters of mammalian genes. The CpG island is a region of at least 200 bp, with a G + C percentage that is greater than 50% and with an observed/expected CpG ratio that is greater than 60%. Probabilità teorica di avere un determinato doppietto (es. CG o AT) ? 1/16 = 6.25% ‘Observed/expected CpG ratio greater than 60%’ si riferisce alla probabilità teorica (6.25%): 60% di 6.25% = ~3.75% A causa del fenomeno noto come mCG suppression la percentuale di CG nel genoma è molto inferiore: 1% Cioè circa 3-4 volte superiore rispetto alla media di doppietti CG dell’intero genoma (1%).

18. mCG SUPPRESSION Timina Metil-Citosina Sito CpG non metilato Sito CpG metilato DEAMINAZIONE OSSIDATIVA ??

19. EUCHROMATIN GENOMIC DNA METHYLATION CHROMATIN CONDENSATION (HETEROCHROMATIN) GENOMIC DNA METHYLATION GENE SILENCING RECRUITMENT OF METHYL-CpG BINDING PROTEINS RECRUITMENT OF COREPRESSOR COMPLEXES WITH DEACETYLASE ACTIVITY

20. Sin3 - NuRD

21. HDACs

24. Histone Methyl-Transferase (HMT)

25. Methylation of K9 of H3 = gene silencing

26. H3 H4 Mechanism: Acetylation of H3 or H4 leads to unfolding and accessibility of chromatin (histone acetyltransferases) Methylation of K4 of H3 = active gene expression Methylation of K9 of H3 = gene silencing

27. Mechanism: • Acetylation of H3 or H4 leads to unfolding and accessibility of chromatin (histone acetyltransferases) • Methylation of K4 of H3 = active gene expression • Methylation of K9 of H3 = gene silencing

28. Historically, Cancer Was Considered to be Driven Mostly by Genetic Changes GENETIC Example: Replication errors X X Altered DNA sequence Oncogenesis Altered DNA/mRNA/proteins Tumor

29. Recent Evidence Shows that Epigenetic Changes are Also Important in Causing Cancer GENETIC EPIGENETIC Example: Chromatin modification errors Example: Replication errors X X Altered chromatin structure Altered DNA sequence Oncogenesis Altered DNA/mRNA/proteins Altered levels ofmRNA/proteins Tumor

30. Epigenetics Can Cooperate With Genetic Mutations to Promote Oncogenesis GENETIC EPIGENETIC  Oncogene levels  Oncogene function ↓ Tumor suppressor function ↓ Tumor suppressor levels Oncogenesis Tumor

31. Epigenetics Play Important Roles in Normal Cellular Development and in Cancer Epigenetic mechanisms can regulate genes involved in differentiation, cell cycle, and cell survival Deregulation of epigenetic mechanisms results in aberrant gene expression, which can lead to cancer Reversal of deregulated epigenetic changes is a rational strategy for targeting cancer EPIGENETICS Deregulated epigenetic mechanisms Normal epigenetic mechanisms Normal differentiated cells, e.g. embryonic cells, hematopoetic cells Malignant progenitor cell Tumor

32. HAT TF TF Ac Ac Ac Ac Ac Ac Ac Ac Ac Balance of Histone Acetylation is a Key Factor in Transcriptional Regulation in Normal Cells HISTONE ACETYLATION Deacetylated Histones Closed chromatin Transcription factors cannot access DNA Acetylated Histones Open chromatin Transcription factors can access DNA Geneexpression Geneexpression

33. Balance of Histone Acetylation is a Key Factor in Transcriptional Regulation in Normal Cells HAT TF TF Ac Ac Ac Ac Ac Ac Ac Ac Ac HDAC HISTONE ACETYLATION HISTONE DEACETYLATION Deacetylated Histones Closed chromatin Transcription factors cannot access DNA Acetylated Histones Open chromatin Transcription factors can access DNA HDAC Geneexpression Geneexpression

34. Imbalanced Levels of Histone Acetylation in Cancer Deregulate Gene Expression Increased HDAC activity or decreased HAT activity may result in aberrant gene expression, contributing to cancer HAT TF Ac Ac Ac Ac Ac Ac Ac Ac Ac HDAC HISTONE ACETYLATION TF HISTONE DEACETYLATION Acetylated Histones Open chromatin Transcription factors can access DNA Deacetylated Histones Closed chromatin Transcription factors cannot access DNA

35. Increased HDAC activity, which has been associated with certain tumors, can alter expression of genes involved in normal cell development, resulting in: Loss of cell-cycle arrest Inhibition of differentiation Cell growth and proliferation Evasion of apoptosis Migration and metastasis Increased HDAC Activity Can Alter Gene Expression and Result in Cancer HDAC TF Gene expression Cell nucleus

36. Evidence of Aberrant Epigenetic States in Cancer Genes involved in DNA methylation and histone modifications are deregulated in multiple tumor types Loss of specific chromatin modifications, such as trimethylated Lys-20 and acetylated Lys-16, is associated with gene silencing Found in lymphomas and colorectal cancer Over 40 genes have been shown to be silenced by DNA hypermethylation in a variety of tumor types p15 (CDKN2B), MLH1, BRCA1, Rb, ER, AR, APC

37. CLASSIFICATION OF EPIGENETIC DRUGS

38. Since DNA methylation and histone deacetylation can co-operate to silence tumor suppressors, inhibition of both DNMT and HDAC activities can synergize to restore expression of silenced genes Therapeutic Targeting of Both Histone and DNA Modifications Can Synergize DNMT Inhibitor DNMT HDAC Ac Ac Ac TF Ac Ac Ac DAC Inhibitor Cell-cycle arrest Differentiation Gene expression Growth control Apoptosis Adhesion Cell nucleus

39. Summary Epigenetic processes regulate gene expression and cell behavior Epigenetic changes that lead to silencing of genes are central to the pathogenesis of hematologic and solid malignancies Histone deacetylation and DNA methylation represent two epigenetic modifications with clinical relevance to oncogenesis Therapeutic targeting of epigenetics can restore expression of genes that are critical for control of normal cell growth HDAC inhibitors display synergy with DNA demethylating agents to inhibit tumor growth

40. Protocols