TARGETS OF CYCLIN D1-CDK

Transcription

1

2 TARGETS OF CYCLIN D1-CDK FIRST TARGET OF THE COMPLEX CYCLIN D-KINASI: prb, IS THE PRODUCT OF THE GENE CONFERRING SUSCEPTIBILITY TO RETINOBLASTOMA - ABSENT OR MUTATED IN SEVERAL HUMAN CANCERS - TRANSCRIPTIONL REPRESSOR: IT CONTROLS THE EXPRESSION OF THE GENES INVOLVED IN CELL CYCLE G0 vs cycle

3 prb CONTROLS TEMPORALLY REGULATED TRANSCRIPTION OF CELL CYCLE GENES Mitogenic signals Phosho-pRb P prb E2F dimers 5..TTTCCGCG 3 cell cycle gene repression cyc D1 cdk4/6 Cyc D/kinase P RNA pol II 5..TTTCCGCG 3 Cell cycle genes on G0 arrest towards S G0 vs cycle

4 Regolatori delle origini di replicazione Enzimi del riparo Avanzamento del ciclo cellulare Regolatori della mitosi Enzimi della replicazione G0 vs cycle

5 Ipotesi di Knudson, un pediatra di Houston (Texas), 1971

6 LOSS OF FUNCTION OF prb TUMOR SUPPRESSOR GENE INHERITANCE OF HETEROZYGOUS STATUS WILD-TYPE STATUS inheritable mutation (deletion) wild type allele INACTIVATION OF WT ALLELE IN SOMATIC CELLS -> MUTANT CELL CLONES INFECTION BY ONCOGENIC VIRUSES prb-inactivating oncoproteins (E7 HPV, E1A AdV) POINT MUTATION: ALTERED GENE PRODUCT prb mutation EPIGENETIC MUTATION (methylation of wt allele) NO GENE PRODUCT INACTIVE GENE PRODUCT IN INFECTED CELLS G0 vs cycle CONTINUOUS CELL-CYCLE RE-ENTRY

7

8 Mutatons in different regulatory PATHWAYS contribute to affect proliferation G0 vs cycle

9 TOO MUCH OF A GOOD THING? Cyclin D1: a proto-oncogene, overexpressed in tumors Normal epithelium Esophageal cancer cells G0 vs cycle

10 The cell cycle is regulated by subsequent activity of digìfferent complexes I (CYC-CDK) and by CHECKPOINTs attacco dei cromosomi al fuso assemblaggio del fuso G 0 attivazione di prb check integrità DNA G1 fosfo-prb check integrità DNA replicazione cell cycle

11 Cyclins are short-life proteins which are degraded through the ubiquitin pthway Glycogen synthase kinase-3beta (GSK-3beta), a serine/threonine protein kinase E3 ligase FBW7

12 Wild-type Dominant negative Proteolisi regolata Associazione con chinasi (cdk) ACTIVE COMPLEX DELETED PEST SEQUENCE: CYCLINS ARE NOT DEGRADED: THE EXPRESSION OF THESE MUTANTS BLOCKS CELL CYCLE PROGRESSION cell cycle catalytic ser/thr kinase inactive without cyclin partner constitutively expressed during cell cycle multiple mammalian CDKs (1-9) CYCLIN-DEPENDENT KINASE (CDK)

13 RB CONTROLS THE G1-S TRANSITION

14 THE RB FAMILY Viral oncoprotein binding Chromatin remodelers E2F binding B-myb Sp1 Smad3 Cdk inhibitor Cyclin binding p107! A B B p130 A B B Rb A B

15 Evaluation of the quiescence, cellular senescence and gene expression in human fibroblasts after knockdown of each pocket protein. Chromatin immunoprecipitation and unbiased sequence for each function (quiscence senescence, gene exp). + oncogenic ras Ridondanza: all three functions immunoprecipitate E2F targets. All 3 proteins must be silenced to bypass quiscence induced by serum starvation or contact inhibition. Senescence instead was bypassed only by RB inhibition. temporary senescence block Tessuto specificità? Da chiarire Burd C et al. Cancer Cell Aprile 2010 p. 313 RB induces cyclin E1 that, alone (no cdk), recruits Minichromosome Maintenance Proteins (MCM). Cyclin E1 is responsible for the aberrant replication.

16 THE ORIGIN OF RETINOBLASTOMA Human retinal progenitor cells give rise to seven distinct cell types. Retinoblastoma develops specifically from differentiating cone precursors, owing to the molecular circuitry in these cells, which includes high expression of N-Myc, SKP2 and MDM2 proteins. This expression pattern permits the cells to proliferate and undergo a cancerous transformation when the tumour-suppressor protein RB is lost. In other retinal cell types, loss of RB either has no detectable effect or induces cell death NATURE 24 SEPTEMBER 2014.

17 The signalling in the cone precursor cells can be involved in retinoblastoma

18 SKP2 IS AN ONCOGENE

19 p53 is a Very Important Tumor Suppressor Most commonly mutated gene in human cancer p53 is the Guardian of the Genome -Geoff Wahl, the Salk Institute p53 is Activated in Response to DNA Damage -p53 is a Transcription Factor for: -p21 (CKI) -DNA repair genes -Apoptotic (Cell Death) genes In the absence of p53 cells cannot arrest after DNA damage

20 The chance to get cancer increases with age. So, why elephants do not get cancer? The researchers have found that their DNA contains 40 copies of the gene that encodes the protein p53, one of the most important mechanisms for preventing cancer. If cells have DNA damage that could spawn tumors, p53 prevents them from dividing until they make repairs or spurs them to commit suicide. Asian elephants harbor 30 to 40 copies of the gene.

21 P53 has been firstly identified as a protein that was up-regulated in many types of cancer In the effort to identify the differences between normal and cancer cells, the researchers analyzed the proteins by SDS gels, looking for reproducible differences. A protein of ~53 kd was usually found increased in tumor cells. The protein was purified and antibodies were made that stained tumor cells only.

22 p53 was First Characterized as an Oncogene Disruption of p53 function by Microinjection of p53 antibody causes cell cycle arrest p53 function to promote proliferation? From Thierry Soussi, Institute Curie, France

23 p53 was First Characterized as an Oncogene p53 cdna can immortalize Rat Embryonic Fibroblasts From Thierry Soussi, Institute Curie, France

24 Evidence that p53 is a Tumor Supppressor Polymorphisms in mouse p53 lie in regions that are conserved among all vertebrates, and must therefore be p53 mutations True Wild Type p53 Fails to Immortalize / Transform Cells From Thierry Soussi, Institute Curie, France

25 Evidence that p53 is a Tumor Supppressor Inactivation of p53 causes cancer in mice: Friend Murine Leukemia -Integration of virus into a single p53 allele -Second allele inactivated by LOH From Thierry Soussi, Institute Curie, France

26 p53 is Mutated in Many Human Cancers roughly half of all human tumors contain p53 mutations From Thierry Soussi, Institute Curie, France

27 An hereditable form of p53-dependent tumors does exist: Li-Fraumeni syndrome The families carrying this mutation have a high incidence of various cancers (more often sarcoma); bones, breast, leukaemia. Usually early start: 50% of individuals carrying the mutation, have a cancer when they are 30 years old.

28 P53 ACTIVATION

29

30 MDM2 and MDM4 bind to the transcriptional activation domain therefore inhibiting p53 transactivation functions MDM2 has additional activity as E3-ubiquitin ligase causing proteasome-mediated degradation of p53

31 p53 controls several different pathways

32 P53:structure and function TAD: transcriptional activation domain PRD: prolin rich domain DBD: DNA binding domain TET: tetramerization domain Basic domain

33 How can Mutations in a Tumor Suppressor Gene be Oncogenic? Dominant Negative Mutations can Prevent Wild Type p53 Function Dominant Negative DNA binding mutant Adapted from Goodsell (1999) The Oncologist 4:138

34 Mdm2 is an E3 Ubiquitin Ligase

35

36

37

38 HOW P53 DISCRIMINATES BETWEEN CELL CYCLE ARREST AND APOPTOSIS?

39 p21: multiple acivities Bind to PCNA and other proteins involved in DNA synthesis givnig more time for PCNA-independent DNA repair ABBAS T. p21 in cancer:intricate networks and multiple activity : naure reviews of Cancer 9:400

40 but p21 can also have oncogenic features blocking apoptosis

41 Hemeking: p53 enters the microrna world. Cancer Cell 12:414.

42 Functions of mir-34a

43 Gain of function of p53 mutants

44 SUMMRY OF THE OVERALL EVENTS ASSOCIATED WITH MUTANT P53

45