Deregolazione dei sistemi di controllo nella cellula neoplastica

Transcription

1 1 Lezioni Dipartimento di Oncologia Farmacologia Molecolare Deregolazione dei sistemi di controllo nella cellula neoplastica Massimo Broggini 20 febbraio 2006,

2 2 Unlimited replicative potential Defective apoptotic response Insensitivity to inhibitory signals Growth factors independency

3 3 Cell cycle Apoptosis Repair Senescence Alterations in the levels Alterations in function Loss of function Mutations/ampl/deletions Transcription Translation

4 4 Mutations/ampl/deletions Eventi stabili nel genoma relativamente rari importanti quando si verificano in particolari sequenze frameshift mutations/ MI

5 5 transcription Epigenetic effects transcription factor alterations altered degradation chromatin/histones

6 6 transcription Methylation of CpG islands cancer genome is globally hypomethylated gene-specific hypermethylation link between hereditary tumors and somatic

7 7 transcription Methylation of CpG islands

8 8 transcription Methylation of CpG islands Epigenetically silenced genes do not contain mutations Cooperation between genetic and epigenetic changes Epigenetic silencing highly frequent in early stages of cancer progression.

9 9 transcription Histones modification Acetylation, methylation phosphorylation Changes in chromatin structure Cooperation between histone modification and CpG methylation

10 10 transduction Protein degradation ribosomal machinery altered microrna expression

11 11 transduction mirna short RNAs encoded by their own set of genes non coding regions of genome/introns approximately 1000 mirnas each mirna can regulate target genes predictions: 1/3 of the coding genes regulated by mirna

12 BIOGENESIS OF microrna 12

13 13

14 14 mirna expression - stage-specific expression in development - tissue specific - the relative abundance can vary among the different microrna

15 15 mirna and cancer EXPRESSION PROFILES IN HUMAN TUMORS INVOLVED IN THE REGULATION OF KEY REGULATORY GENES ANTISENSE AGAINST mirna NEW POTENTIAL ANTITUMOR STRATEGY

16 mirna and cancer EXPRESSION PROFILES IN HUMAN TUMORS Nature :834 16

17 mirna and cancer EXPRESSION PROFILES IN MURINE TUMORS Nature :834 17

18 18 mirna and cancer THE 3 UTR OF THE RAS GENES CONTAINS MUYLTIPLE LET-7 SITES, ALLOWING LET-7 TO REGULATE RAS EXPRESSION. LET-7 EXPRESSION IS LOWER IN LUNG CANCER THAN IN NORMAL TISSUES, WHILE ON THE CONTRARY RAS PROTEIN IS SIGNIFICANTLY HIGHER IN TUMORS mir-17-5p AND mir-20a NEGATIVELY REGULATE E2F1 EXPRESSION

19 19 mirna and cancer Chk1 3 UTR 425 bp 1 sito potenziale E2F1 3 UTR 1400 bp 2 siti potenziali DRAGO 3 UTR 4100 bp 5 siti potenziali

20 20 Chk1 3 UTR tgaatatagtgctgctatgttgacattattcttcctagagaagattatcctgtcctgcaaactgcaaatag tagttcctgaagtgttcacttccctgtttatccaaacatcttccaatttattttgtttgttcggcatacaaata atacctatatcttaattgtaagcaaaactttggggaaaggatgaatagaattcatttgattatttcttcatg tgtgtttagtatctgaatttgaaactcatctggtggaaaccaagtttcaggggacatgagttttccagctt ttatacacacgtatctcatttttatcaaaacattttgtttaattcaaaaagtacatatttcttccatgttgattt aattctaagatgaaccaataaagacataattcttgcaaaaaaaaaaaaaaaaaaaaaaaaaa UAGCAGCA CAGAAAUAUU GGC mir-195 UAGCAGCA C U AUAUU CACCAGGA UAGCAGCA CGUAA AUAUU GGCG mir-16

21 3 UTR drago GTTCTCTCAGCCCTTCCTCCCTCTCCCTGTGGGATTGAGCACCCTGTACTCTCCAGCCACCTTACCTGGATACCTGAGCTGCCACCTGTGTATCTGTGTAT CTCTGAGGGCCCTATAGGCCCACCTTGCTGGAAACTCAAGGAAGATTCTCGCCATCTGCCTGTTGGACAGCTGGAGGAGCTGGCTCTTTGCCTGGCCCC GCCTTCCCATCTGTCAGAGACATATTTGAATGTGCTGGATCAAACCCTCCCTTTTCCTAAGCCTCTGGGTCCCCTCCAGCCAGCTCTTTGGCGGCAGCCC CCACCAGCTCCTGTGGGCCTGAGTGCTGCTGTGTTTACTTGTGCCTTTCCCCCACCCTGTCCAGTTTCCCTGTCATGCAGACTTGTTGCTGTCCACAAGCC TTAGTGGCTGCACTGCTGCCCCCTGCCACACAGGGGGCCGGGCCTGGGTCTGTCCTGTTTCCTTTGAGGGTTGCCCCTACTGCCCTTTGCAGGAACAGAT CCAGGTGTGAGAGCTCTTGAGTCAAGAGTGGCAGAAGTGGCTCTAATTGGGGTGAGAGTGTAGTCCCTGGGCTTGCCCTGGGTTGACCCTGGTGGCATA TTTCCTTGGCCGAGGATGGAAGATTTGGAGAATCATGTCCATGCTGGCCCAGGACCCAGCCATCTGGCCCAAAGGCACAAGCTCCTGGCCCTGTTGAGT TGAGAGTTTCCAAGAAGCATCCAGAAGATCCCAAGGGAGAGAAGGAAAATGGCTGATAATGATTGTCTTCCTAATATGCAAGTTCTCACTTCCTACTTC CAGCATCGGCCTTCCTGGCCTTGTCTTTTTTTTGTTTCCCTGGAGTATAATGGGAAGTTGCATGCTGCCTCCTGGGTTTTATCCCAGATAGCTCTGGCTTT CTTGCTGCCCACAGGGGCCTGGGGCAGGAAGGAGACTTGCTGAGATGCCATGGAGTGCCCATCTGGTCACTGGCAGTCTGGGCAGGTTGCCCCTTTCTG GGTTTGTGGTGACGGAGGGGAGGCCGAGAGGCACAGACCAAGTCCCCGGGTGGCTGCAGGCAGCTCCAGCCCGGTCCTGAGGATCCTCCTCACCATGG TCACGTGCCTTAGTAACTGTGCCCAGGAAGTGGCCTGCTGCTTGCTGTGCTGCTGCTTTTCCTACTTCTGCCCTTCCCTGCCACCCCTCGCATGTCACAG CTGACAAGCAATTCCTTGTCTTCCCTGGCCCCCTGGGGGAAGGGCTGAGAAACAGTCCATGTGCACCCCAACCTTAATGGCCTGAGGTGGGCAGAGGG GTGTGGAGCAGCCTGGAGTACAGGGCCCTGGGGGAGGAGCCCACTGATGAGGGGCGCTCTCCCATAGCCATGTGTTGAATGCTAACTAGGCTGGGGTG GACGAACTCTGCCAACTGCTGTCATCTTAGAAGATAGATGCAGCAGTAAGGAATGTTTGTTTTGCTTTTTTCTGAAATTTTCTGAAGCACTGTGGCTGGG AAACTTCGAAGCGGACCCTGTGCTGCATGTCTGCTCCTCCCCTGAGCCTGTCTGCTTGGGGGTGGTAAAAATAAAAATCCCAGTTTATTTTCAGTACCTT ACCTAACAGGGTTGGCTCCAGGCGTGGGTGGCCTAGAAGATGAGGGGAGTGGTCTTCTCCCAGCCTTTTACCCTCTTGCCTCCTGCCTCCGCGCTTACAC ACGCACTTTACCACCCGGTCATTCCCTGGCCTCTTGCTGCCACTTGTAGTCTTCCTTCCTTCCTCTCAGGGTAAGGGCAGTGCCTGCTGTGCCTGTTGGCC ACTCCCACACTTCCCCTCCCCCAGGAGCCCTCATCTGCTGTGCTGAGTCCAGGAAAGCATAGTTAGGTAGGGAGCTGGTTGGAGAAGGTGCTAGAACTA GAAGGCAGATGAGACTAGCATGGGCCCACCTGGAGGGCTGTCCCTAATGGCCCCAGTCGCCTTACCTCACCCACAGCAGTGCCCTTGTCTTCCTCCAAA ACAGAAAGCAGTGACAAAAGGGGGAGGGGTGGTAATCTGAAGTCTCACTGCTGAGCCTTCAGCTTTTATTTTTCACTGTTTCAAAACCCGCATTCTATT CTAGAATGGTTTTTAAAATGGAAGATCTTACCTTTTTCTATCTTGTTACTCTGGGGTTTTGTCCCCCTAAGAGATTGCACTTTTTGTTTGGGGTTTATTCA GCTGCATAGATGACCAGCTTGATCCCTGGTGAAATGAAAAGCCTTCCTTCTCCTGAAGCCTCTTTCCGCCCTGCCCTCCACTAACAACACTGAGGAGCAC AAGCCCAGGCTTGCCCACCTGGTAGGAAAGGAAGAAATTAGAACAATGGGAGCCTTGGCTCCCCTCTCGTCTCCTCCCCTCCTTCTTGTCACTGGCTTTG ATGAGGCCCACTTCCCAGAGGCTCCTGGGCCTGTGAGTGCAGGAGCTCATTCTCCCCTCACTGCTGAAGTCTGTGACAGCTTCTTCCTCCAGTTATGTCT TTCTTCCAAAGCAATTTCTTAACCATCAGCCATGTGCTGCTATTTCTAGGGCTTCTGGGCTTTGTCCCTTACTGAGAGATTAGGGACTCCACAGCTGCCT TGAGGTAGGGCCTGGCTGAGAGACAAGGGTAGCAGCAGGTGGCAGGCTGTTAAAAGACAGGCTGCCTGAGGAGCCTGGAGCAGGTGGAAACAGGTGG AAGAAACCGGCCACAGCCCTGCTTTACCGGGCTCACCTCTAGGGCATTCCAGCAAGAGGCTGATGCAGGAGAATGGCCAGCACCAAAGGACATTTAAA AGAGTTTTTGGGTTTTTTTGTTTGTTTGTTGTTGGTGTTTGTTTTTTTTTTTTTTTTTTGGCACACTTGAGCTGACTCAGTGCAGGTTTAATATCCTGGTGAC TTGCAGTCACATTCTAATGACTTTCAAGGGCCAGAATATGGTGAAAATCACTTAAAATATCCGTCCCTTCCATGCCTTAGTTTAGCAGGTAGGCTCTATC TTTTGCCATTTCTGTATTTTATGTGCTGTGTTCCCGTTTCACTGGGTATGAACTGTGAAATGGACTGAATCCTGGCCACTTTATGAGTTTGTTTGGTTTTA TAAGGCATTTCAATGTACATTCTATAAATACAAGCACTCCATTTGCAAACAGATCTTAAGCTAATATTTTCTTTCCCATTCATCTTGCCCTCCCCCTCCTC CCGCCAGCTTTAAAGTTCAGTGGAGAAGCCAGATGGCAATTCAGACAAAGGTATACTCTTCCTGCTTCATGGGTGGTGGCACGGGAATAGATAGCCCT TAGCCCTTTCCCTCCCAGTCCCAGCTGAGCCCTCAGACCACTTGCTTCCCACATAACAATGTCGCCTCCATTTCCGAGGAACATCCTTGCGTAGAGAATG AAATATGCTGCAATCATTTCTGCATCCTTACTCCTCACCCCCAAAGAAAAAAAAAAGGCCTAGCAGGGAAGCAGCATGCAGGCTTCACAGCTTAATGCC AAGGACAGCGAGTGAGGCTGGGAGCTTCTCTTGGGCCTGCTGGGTCTGTCAGCTCTCGGAATAGGGACAGTCCTTACTGGTGCCCCAAGGTGGGACTTG GAGAATATTTTGCTTGGCATATGTTTGGTCTGAATGGTGTAGTTGCTGGTTCCCTAGAGAGGAAAAGGTGGCAGGCCCAGCTTTGCTGGGAAATGGCTC TTAATTTCCAGTTGAAACCCTAGTAGAATTGTGAATGAAAACCTCAAGGTTGAGCCCCTCTGCCAAGCAGCAGAGCTAGTAGAAGGGGATGCAGGGGC AAAGCACTCAGTTGCCAAGCAAGGAGGAGAGATGTACGTGGGCTGTGTGGCAGTCCCCACACCCTGCCCTGGCTTCTTCAGGTTATCGCACCACTATGG AATCCTTTGCAGAATGGTACTCATATAATGGTTTAAAACAACACATTCATAATTGACTCTGTGCAGGATGTCACTCAATCAGTTTGGGTTTGCTTTATTT TATTTTATATATATATTTTTTGGTATCCTGTACATTGCAGTGGGTGTGAAGATAGTATTTTAATATTTGTACAAAGTTTAATTTAATTTTAATTGTTCTATG TATATAACTGCATTTCTAAATAATTAAAAAAAAGTTCTTATGAAGGCAAAAAAAAAAAAAAA 21

22 drago 3 UTR 22 UAGCAGCACAU GGCUG AUGGUU A AGA AAUU UAGCAGCACAU A AUGGUU UGUG mir-15a UAGCAGCACAU C AUGGUU U ACA mir-15b UUCACAGU UCA UA CCCAGUGAAACGGGAACACAG UUCACAGU GGC UA AGUUCCGCC mir-27a UUCACAGU GGC UA AGU UCUG mir-27b

23 telomeres TTAGGG repeats (10-15Kb) at the chromosomal ends that recruit proteins and protect ends from degradation. 23

24 telomeres Loss of repeats at cell division leading to telomere shortening. Nature Rev Gen :611 24

25 telomeres 25 Telomerase (RT) / ALT not expressed in normal somatic cells. Cancer cells?

26 SENESCENCE 26 STABLE GROWTH ARREST LIFETIME IMPRISONMENT AS A TEMPTATIVE TO PROTECT THE BODY FROM POTENTIAL CANCER CELLS ONCOGENE INDUCED SENESCENCE ARF-p53 AND p16-rb AS MAJOR PATHWAYS CONTROLLING ONC-INDUCED SENESCENCE

27 SENESCENCE 27 MANY EXAMPLES IN VIVO OF ONCOGENE INDUCING SENESCENCE B-RAF IN NEVI SENESCENCE IN EARLY STAGE PROSTATE ABNORMALITIES INDUCED BY PTEN DELETION SENESCENCE IN LUNG ADENOMAS INDUCED BY K-RAS MUT EXPRESSION

28 SENESCENCE 28 PTEN- p19/ arf p53 SENESCENCE RAS ERK p16/ INK4a Rb RAF

29 SENESCENCE Science :886 29

30 SENESCENCE 30 CHROMATIN REMODELLING / METHYLATION Suv39h1/ H3K9 / HP1 HDAC INHIBITORS / HYPOMETHYLATING AGENTS?

31 p27 A CDK2 S p21 CHECKPOINT p53 p27 E CDK2 G1 p21 TRASCRIZIONE E2F + pprb FATTORI DI CRESCITA p16 D CDK4,6 p15 Rb-E2F p21 M G0 p21 A CDK1 G2 CHECKPOINT CHK1/2 B CDK1 CDC25C 31

32 32 S CYC D AMPL CDK4 AMPL RB DELETION p16deletion G1 E2F + pprb FATTORI DI CRESCITA p16 D CDK4,6 p15 Rb-E2F G0 p27 degradation CYC E AMPL p21(p53) CDC25s over chk2 chk1 mut G2 M

33 33 p53 Mutated in more than 50% of human cancer p53 mdm2 Amplification of mdm2 / SNP viral inactivation cytoplasmic sequestration

34 34 Hereditary tumors Li Fraumeni tumor prones BRCA-1 defects in repair HNPCC defects in repair / MI

35 MI 35 Defects in mismatch repair (hmlh1, hmsh2) less efficient repair of pol-induced errors insertion/deletion of 1 base which induces frameshift mutations bax, bcl-10, fas, Apaf-1, caspase-5, CHK1, hmsh6

36 DNA METHYLATION 36 Prostate cancer GST-pi heavily methylated protein not expressed Alteration present in precancerous lesions Re-expression of GST-pi does not reverse the phenotype

37 37 TRASDUZIONE DEL SEGNALE DUE PATHWAYS COME ESEMPIO DI RILEVANZA IN ONCOLOGIA RAS-RAF-MEK-ERK PTEN-PI3K-AKT -mtor

38 RAS-RAF-MEK-ERK 38

39 39 RAS-RAF-MAPK-ERK GTP GDP RAS RAF RAF P MEK MEK P ERK ERK P

40 RAS-RAF-MAPK-ERK 40

41 RAS-RAF-MAPK-ERK 41 3 GENI A-RAF, B-RAF, C-RAF (RAF-1) CONTENONO UN RBD S/T CHINASI CHE FOSFORILANO MEK ALTA PERCENTUALE DI MUTAZIONI DI B- RAF IN TUMORI UMANI. 90% DELLE MUTAZIONI IN UN RESIDUO AA - (V599) ATTIVAZIONE COSTITUTIVA A-RAF E RAF-1 MUTATI RARAMENTE NECESSARIO UN DIFFERENTE LIVELLO DI FOSFORILAZIONE TRA LE DIVERSE PROTEINE - B-RAF VS A- e C-RAF

42 RAS-RAF-MAPK-ERK 42 MUTAZIONI DI B-RAF

43 43 RAS-RAF-MEK-ERK Costitutivamente attivato per mutazione RAS INIBITORI FT B-RAF attivato per mutazione RAF INIBITORI B-RAF MEK INIBITORI MEK1 E 2 ERK INIBITORI ERK

44 PTEN-PI3K-AKT -mtor 44

45 PTEN-PI3K-AKT -mtor PI3K PTEN AKT TSC2 TSC1 mtor 6SK 4EBP1 45

46 46 Inibizione di una fosfatasi? 6SK1 DOWNSTREAM mtor Fosforilazione in T389 mtor Fosforilazione in T37, T46, S65, T70 4EBP1 eif4e P P 6SK1 eif4e 4EBP1 Traduzione di RNA con un tratto 5 oligopirimidinico (TOP) FORMAZIONE DEL COMPLESSO eif4f Componenti dell apparato traslazionale Inizia traslazione di mrna coinvolti nel ciclo cellulare cycd c-myc

47 inibitori amplificato in tumori PTEN-PI3K-AKT -mtor AKT sovraespresso in tumori PI3-KINASE RHEB TSC 1/2 mtor AKT-p amplificato in tumori PTEN inibitori mutate in tumori inibitori deleto in tumori 6SK, 4EBP1 amplificato o sovraespresso in tumori Traduzione di geni coinvolti nella crescita cellulare 47

48 48 COMMENTS DIFFERENT GENETIC AND EPIGENETIC ALTERATIONS COOPERATE IN GENERATING GROWTH ADVANTAGED TUMOR CELLS CRUCIAL POINT TO IDENTIFY THOSE ALTERATIONS RESPONSIBLE FOR THE MALIGNANCY ALTERED PATHWAY RATHER THAN ALTERED GENE THERAPEUTIC STRATEGIES NEW HYPOTHESIS ON THE EXISTANCE OF A CANCER STEM CELL