Loss/Gain of function

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Gli oncogeni specificati da virus a DNA inattivano I soppressori di tumori La loro azione mima una mutazione LOSS OF FUNCTION dei soppressori (gate keeper) Gli oncogeni specificati da virus ad RNA (retrovirus) derivano da geni cellulari (protooncogeni) La loro azione mima una mutazione GAIN OF FUNCTION

Loss/Gain of function

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Come si perde l‘allele wild type di un gene soppressore, durante

la mitosi?

Come si perde un allele?

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Sei differenti meccanismi possono causare la perdita In mitosi della copia buona di un soppressore tumorale

Figure 7.13 The Biology of Cancer (© Garland Science 2007)

RFLP Restriction fragment length polymorphisms to localize tumor suppressors

LOHPerdita dieterozigosità

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Dove cerchereste I tumor suppressor genes???????

All chromosomal regions have some tendencies to undergo LOH

MitoticNon-disjunction

Mitoticrecombination deletion Point mutations

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Mitotic recombination

10exp-5 -10exp-4 per cell , per generation

Gene Conversion (locus-restricted recombination event)

10exp-4/cell/generation

A+ B+ C+

A- B- C-

A+

B+ C-A-

B+ C+SCAMBIO ��NON ��RECIPROCO

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Che tipo di HIT??? First HIT: at tumor suppressor loci is usually a coding TRUNCATING MUTATION (non-sense, frameshift) Second HIT: ALLELIC LOSS/ Loss of heterozygosity (LOH), ALLELIC IMBALANCE

Two hits hypothesis revisited

First Hit: Coding Truncating��Mutation : frameshift, missense,

some nonsenseLarge and small deletions confer a selective disadvantage in all cells and are selected against in gametes. Point mutations are more likely

Second Hit: allelic loss/ LOHMitotic recombination, small interstitial deletions, Promoter methylation or gene conversion are the most attractive mechanisms because they minimise site effects on other loci

Chromosome non-disjunction are not tolerated if hemizigosity of other locus on the same chromosome is disadvantageous or if the mechanism that control genome integrity are still able to cause apoptosis

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Promoter methylation

In normal cells, CpG islands preceding gene promoters are generally unmethylated, and tend to be transcriptionally active, while other individual CpG dinucleotides throughout the genome tend to be methylated. However, in cancer cells, CpG islands preceding tumor suppressor gene promoters are often hypermethylated and transcriptionally inactive, while CpG methylation of oncogene promoter regions and parasitic repeat sequences is often decreased.

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Metilazione e Deacetilazione : 1-Dnmt metilano il DNA = modificazione epigenetica

Il DNA metilato non e’ efficientemente legato da attivatori trascrizionali ma viene legato da proteine che legano metil-citosine 2- (MeCP2, MBD1-4) e repressori trascrizionali Questi a loro volta sono in grado di reclutare diversi HDAC -deacetilasi di istoni > repressione della trascrizione anche alcune Dnmt reclutano HDACs e viceversa

1 2

DNA methyltransferase inhibitors azacitidine and decitabine. These DNA hypomethylating agents are used to treat myelodysplastic syndrome, a blood cancer produced by abnormal bone marrow stem cells. Histone lysine methyltransferases (KMT) and protein arginine methyltransferases (PRMT). Preclinical study has suggested that lunasin (43 aa soy bean peptide) may have potentially beneficial epigenetic effects (PubMed 11606382).

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Lunasin at a concentration of 10 nmol/l was able to decrease the acetylation of histones by between 20 and 25%; by contrast, lunasin at a concentration <1 µmol/l was able to decrease the acetylation of histones H3 and H4 by 80 and 74%, respectivelyOncol Lett. 2017 Jun; 13(6): 3997–4001.

Hypothetical Epigenetic Mechanism: Competitor of HAT

TRICHOSTATIN TSA contro HDAC

Trichostatin A is an organic compound that serves as an antifungal antibiotic (by Streptomyces platensis) and selectively inhibits the mammalian Histone deacetylase family of enzymes: TSA Switches on differentiation or suppressor genes

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Metilazione e regolazione genica: •  Il pattern di metilazione dei geni è

alterato nelle cellule tumorali, e metilazione alle isole CpG di certi geni è associata al loro silenziamento specifico

•  PML-RAR t(15:17) Retinoic Acid Receptor, come proteina chimerica da traslocazione, lega

il promotore del gene Retinoic Acid receptor-β e causa il reclutamento di DNMT -> metilazione -> MBD -> HDAC> deacetilazione> inattivazione trascrizionale gene RARbeta---> APL Acute Promyelocytic leukemia ( 10% )

•  Accumulo Leucociti immaturi •  Diminuzione piastrine e rossi

DifferenziationAzacitidina (ipometilazione)TricostatinAcido retinoico ATRA

Il Controllo del ciclo cellulare Ciclo cellulare

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Checkpoint in G1 Checkpoint in S

L’ATTIVAZIONE DEI CHECKPOINTS DETERMINAUN

RALLENTAMENTO O UN ARRESTO DEL CICLO CELLULARE TALE DA

PERMETTERE DI RIPARARE IL DANNO

CDK e cicline nel cell cycle clock

Tre categorie di cellule:

Cellule molto specializzate: cellule nervose, muscolari, i globuli rossi hanno perso la capacità di dividersi.P27 overexpressed? Senescenti INK4 overexpressed?

Cellule differenziate che normalmente non si dividono, ma lo possono fare in risposta ad uno stimolo appropriato, per esempio le cellule del fegato.

Cellule in G0 ma capaci di proliferare: cellule staminali nei tessuti epiteliali e nel midollo osseo, cellule germinali, satelliti del muscolo.

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Tempi e ploidia nelle quattro fasi del ciclo cellulare eucariotico

Cellula umana in rapida proliferazione:Ciclo di 24 ore

G1= 11-15 ore, accrescimento- minimo, 3hrS= 8 ore replicazioneG2= 4-5 ore M= 1 ora, mitosi e citodieresi

2n

4n

4n

Diversità nel ciclo cellulare tra cellule dell’organismo adulto e cellule embrionali

Early embrionic need LIF Leukemia Inducing Factor, E-RASBut they do not use p53 or pRB control. The only wild type cells able to make a tumor (TERATOMA)

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Punti di controllo del ciclo cellulare

Check points

Fattori di crescita

Oociti aspettanoFattori ormonali in G2

Altri Soppressori Coinvolti nel Ciclo Cellulare

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DNA damage CheckpointIn G1

DNA damage checkpoint in S:Tutti I cromosomi replicati

Anaphase blockEntrance into M

pS20

T68

pS395

P53 stable pS20, active pS15 nuclear

S15

BASC (BRCA1- Associated genome Surveillance Complex

NO ChromosomeSegregation

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La durata del ciclo varia molto, soprattutto nella lunghezza della fase G1. In una cellula adulta può durare da 12 a 36 ore, la differenza dipende dalla fase G1.G1 minima è di 3 ore.

La fase G1 può durare ore, giorni, settimane o più a lungo a seconda del tipo cellulare.Dipende dalla quantità di p27-p21 (p27 Aploinsufficient for tumor protection!!!).

Quando la fase G1 si protrae a lungo, si parla di Go, cioè una fase stazionaria di attesa (QUIESCENT CELLS).

L’ingresso da Go a G1 ed il passaggio da G1 a S richiede l’intervento di messaggi ambientali, chiamati mitogeni o fattori di crescita.

E

A B

D1D2D3

cdk4cdk6

E2FRb

SWI/SNF

cdk2

E2F

DNA synthesis genes

EA

AssemblySeuestration

G1 S G2 MMitogen

p27KIP1P21CIP1

INK4

cdk2

Il controllo del ciclo cellulare Le cicline

p27KIP1

D

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Meccanismo di base del sistema di controllo del ciclo cellulare

Cdk si associa con una ciclina attivando un processo a valle. L’attività di Cdk è interrotta dalla degradazione della ciclina

Quindi il ciclo cellulare non può andare indietro

Le cicline attivano le chinasi

I due componenti principali del sistema di controllo del ciclo cellulare

Il complesso ciclina/Cdk ha una attività kinasica che innesca i processi a valle;

in assenza di ciclina, Cdk è inattiva

Serine/threonineKINASEs

40% aa identity

Quando la cyclinA lega CDK2, laAttività enzimaticaAumenta 400.000Volte!!

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Accumulo e degradazione di “cicline” in embrioni di riccio di mare

Cyclin B (G2-M)

Il citoplasma in fase M è dominante: iniettando in oociti di ranaIl citoplasma da ovocellula in M, si induce mitosi

La degradazione veloce delle cicline rende impossibile tornare indietro

Cyclin D is also exported from nucleus at G1/S

Cyclin E rises after Restriction pointA rises when cell enter SAll cyclins are rapidly degraded by Ubiquitin Ligase

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15hrs

8hrs

5hrs

1hr

6x10exp9bp.

D1,2,3

E1,E2A1,A2

B1, B2Numerosi geniCodificano per lecicline

The level of D-type cyclins is controlled by EXTRACELLULAR SIGNAL

- They are transcribed when growth factors are added- They are destroyed, if growth factor is removed, by SCF Skip Cullin Fbox

Because they inform the cell of the current environmentalcondition

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Cyclins D inform ��cell cycle clock

La cellula controllaL’ambiente che la circonda:Il livello di cycline D Aumenta proporzionalmenteAi fattori di crescita

Ingresso nel ciclo: l’aggiunta di fattori di crescita attiva

il passaggio tra la fase G0 e G1

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Perché tre cicline D? Risposta a differenti recettori modulata da differenti promotori


Cell cycle

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TGFbeta DNA Damage

RB

INK

p53

GSK3-------|

Notare che le cycline-kinasi attivano le seguenti e disattivano le precedenti

Come si attivano???? Due cicline , una chinasi

To be active, the CDK must be phosphorylated on a threonine in activation loop by CAK Cdk Activating Kinase. A CDC25APhosphatase must remove a tyrosine inhibitory phosphorylation

Association of CDK2 with cyclinA= enzymatic activity increased 400,000 fold

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>P24941|CDK2_HUMAN Cell division protein kinase 2 - Homo sapiens (Human).MENFQKVEKIGEGTYGVVYKARNKLTGEVVALKKIRLDTETEGVPSTAIREISLLKELNHPNIVKLLDVIHTENKLYLVFEFLHQDLKKFMDASALTGIPLPLIKSYLFQLLQGLAFCHSHRVLHRDLKPQNLLINTEGAIKLADFGLARAFGVPVRTYTHEVVTLWYRAPEILLGCKYYSTAVDIWSLGCIFAEMVTRRALFPGDSEIDQLFRIFRTLGTPDEVVWPGVTSMPDYKPSFPKWARQDFSKVVPPLDEDGRSLLSQMLHYDPNKRISAKAALAHPFFQDVTKPVPHLRL

T14,Y15 by WEE checkpoint kinase (inhibitory) removed by CDC25AT160 by CAK (activatory)P>L glioblastoma= cyclin independent

Ingresso nel ciclo e transizione G1-S:

il fattore di crescita attiva la via di trasduzione del segnale (Ras/MAPK) che porta alla trascrizione genica. Vengono prodotti fattori trascrizionali (risposta precoce), che a loro volta regolano in cascata la trascrizione di altre molecole (risposta tardiva) che permettono la transizione da G1 ad S. Le chinasi ciclina dipendenti agiscono sulla Fosforilazione di : proteine associate ai centrosomi, istoni, membrana nucleare (lamin , nucleoporine)

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D cyclins have other jobs important for the molecular biology of tumors:

Cyclin D associates with ESTROGEN RECEPTOR ER , mimics its ligand and stimulates transcriptional activity.70% of breast cancer overexpress ER and CyclinD

Cyclin D1 plays an important role in the development of breast cancer and is required for normal breast cell proliferation and differentiation associated with pregnancy. We show that ectopic expression of cyclin D1 can stimulate the transcriptional activity of the estrogen receptor in the absence of estradiol and that this activity can be inhibited by 4-hydroxytamoxifen and ICI 182,780. Cyclin D1 can form a specific complex with the estrogen receptor. Stimulation of the estrogen receptor by cyclin D1 is independent of cyclin-dependent kinase 4 activation. Cyclin D1 may manifest its oncogenic potential in breast cancer through binding to the estrogen receptor and activation of the transcriptional activity of the receptor.

Cyclin D associates with C/EBPbeta transcriptional factor and inhibits it. C/EBPbeta (CCAAT-Enhancer-Binding Protein) is involved in differentiation

Mol Cell Biol. 1997 Sep; 17(9): 5338–5347. PMC232384/

Il Paradosso di p21 e p27

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P21 CIP1 (Cdk Interacting Protein) WAF1(Wild type p53 Associated Fragment) (CDKN1A gene, 6p21) P38936

P27 KIP1 (Kinase Inhibitor)CDKN1B

THE TERNARY INHIBITORS OF THE CELL CYCLE PROGRESSION

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Mediate p53 independent Cell-cycle arrest (G1-S specific)

Inhibit All phases of cell cycle. P53activated

P16B EA

Soppressore p27 KIP1��CDKN1B

Breast CarcinomaMultiple Endocrine Neoplasia 4

Multiple endocrine neoplasia type 4 (MEN4) is a rare autosomal dominant endocrine disorder characterized by the occurrence of parathyroid adenoma/hyperplasia, duodeno–pancreatic neuroendocrine tumors (NETs), and anterior pituitary tumors in the same individual

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The p21Cip1 and p27Kip1 CDK ‘inhibitors’ are essential activators of cyclin D‐dependent kinases in murine fibroblastsMangeng Cheng, Paul Olivier, J.Alan Diehl, Matthew Fero, Martine F. Roussel, James M. Roberts, Charles J. SherrDOI 10.1093/emboj/18.6.1571 The EMBO Journal (1999) 18, 1571-1583

Component of the ternary complex, cyclin D-CDK4-CDKN1A. Interacts (via its N-terminal domain) with CDK4; the interaction promotes the assembly of the cyclin D-CDK4 complex, its nuclear translocation and promotes the cyclin D-dependent enzyme activity of CDK4.

Inibitori ciclo cellulare

Active in earlymid-G1

INhibitors of CDK4

INIBITORI solo degli eterodimeri In S-G2_M

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P27 kip1

Blocks function by obstructing the ATP binding site

Figure 8.14a The Biology of Cancer (© Garland Science 2007)

TGFbeta is a early, mid-G1 growth inhibitor

p53

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TGFbetaAnti-mitosis

Associa'on of MYC with MIZ-‐1 displaces the coac'vators, (e.g., p300 and NPM-‐1), thereby blocking transcrip'on of MIZ-‐1-‐dependent genes.

The model proposes that MIZ-‐1 ac'vates target genes in coopera'on with upstream transcrip'on factors that are regulated by specific an'mitogenic signals (e.g., TGFbeta)

B-‐cell lymphoma 2 interac'ng mediator of cell death (BIM) is a potent pro-‐apopto'c

The Role of MIZ-‐1 in MYC-‐Dependent Tumorigenesis Cold Spring Harb Perspect Med. PMID: 24296348 A hallmark of MYC-‐transformed cells is their aberrant response to an7mitogenic signals. Key examples include the inability of MYC-‐transformed cells to arrest prolifera'on in response to an'mitogenic signals such as TGF-‐β or DNA damage and their inability to differen7ate into adipocytes in response to hormonal s'muli. Given the plethora of an'mitogenic signals to which a tumor cell is exposed, it is likely that the ability to confer resistance to these signals is central to the transforming proper'es of MYC in vivo. At the same 'me, the inability of MYC-‐transformed cells to halt cell-‐cycle progression on stress may establish a dependence on muta'ons that impair or disable apoptosis. We propose that the interac'on of MYC with the zinc finger protein MIZ-‐1 mediates resistance to an'mitogenic signals. In contrast to other interac'ons of MYC, there is currently li\le evidence that MIZ-‐1 associates with MYC in normal, unperturbed cells. The func7onal interac7on of both proteins becomes apparent at oncogenic expression levels of MYC and associa7on with MIZ-‐1 mediates both oncogenic func7ons of MYC as well as tumor-‐suppressive responses to oncogenic levels of MYC.

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IL DOPPIO GIOCO DI P21 P27 Genes Dev 1997 Apr 1;11(7):847-62. New functional activities for the p21 family of CDK inhibitors. LaBaer J, Garrett MD, Stevenson LF, Slingerland JM, Sandhu C, Chou HS, Fattaey A, Harlow E.

Cytoplasmic Cip/Kip proteins as ASSEMBLY FACTORS: Kinetic studies demonstrate that p21 and p27 lead to a 35- and 80-fold increase in K(a) of Cdk4 and their cyclin D1, respectively, mostly because of a decrease in K(off) All three CIP/KIP inhibitors, TARGET cdk4 and cyclin D1 to the NUCLEUS P27 can bind Nuclear Pore Associated Protein mNPAP60 (increased translocation) P21 blocks interaction of cyclin D1 and exportin CRM1 (increased D nuclear)

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The freepool of p21 and p27 dwindles

Mitogen= Cyclin D increase

Mitogens decrease: D decreases

. E - CDK2 phosphorylate p27 that goes to proteasome

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Attivazione di pochi CDK1/2 E/A causa fosforilazione e degradazioneDi p27= irreversibilità del ciclo, RB, p21

pTy15

CDC25 CAK

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DOI: https://doi.org/10.1124/mol.115.099325

Leland H. Hartwell, R. Timothy Hunt, and Paul M. Nurse won the 2001 Nobel Prize in Physiology or Medicine

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Cyclin E - Cdk2 complexes control the transition from G1 into S-phase. In this case, the binding of p21Cip1/Waf1 or p27kip1 is inhibitory. Important substrates for Cyclin E - Cdk2 complexes include proteins involved in the initiation of DNA replication. The two Cyclin E proteins are subjected to ubiquitin-dependent proteolysis, under the control of an E3 ubiquitin ligase known as the SCF. Cyclin A - Cdk2 complexes, which are also regulated by p21Cip1/Waf1 and p27kip1, are likely to be important for continued DNA synthesis, and progression into G2.

An additional level of control of Cdk2 is reversible phosphorylation of Threonine-14 (T14) and Tyrosine-15 (Y15), catalyzed by the Wee1 and Myt1 kinases, and dephosphorylation by the three Cdc25 phosphatases, Cdc25A, B and C.

Loss/Gain of function

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