Describe the mechanism by which Describe the mechanism by which mutations in tumour suppressor genes can mutations in tumour suppressor genes can lead to the development of cancer?? lead to the development of cancer??

Concentrate primarily on the RB and p53 Concentrate primarily on the RB and p53 genes?? genes??

but give examples of other genes if you but give examples of other genes if you come across significant differences in the come across significant differences in the way in which they act??way in which they act??

MRCPath Self Help Course MRCPath Self Help Course

1212thth January 2010 January 2010Mai M Abd El-AzizMai M Abd El-Aziz

Tumour Suppressor genesTumour Suppressor genes (TS)(TS)

One of three major classes of genes One of three major classes of genes tumorigenesis tumorigenesis Inactivation of TSInactivation of TS development of cancer development of cancer Inactivation arise from Inactivation arise from 1.1. Missense mutationsMissense mutations

2.2. Nonesense mutations Nonesense mutations

3.3. Deletions Deletions

4.4. InsertionsInsertions

5.5. Epigenetic silencingEpigenetic silencing

Mutations in M & P alleles are needed Mutations in M & P alleles are needed selective selective advantage to the cell advantage to the cell

Exceptions: inactivation of one allele can also exert Exceptions: inactivation of one allele can also exert the same action on the cell through the same action on the cell through haploinsufficiency haploinsufficiency

How TS genes operate?How TS genes operate?

Mutations in TS genes drive the neoplastic process Mutations in TS genes drive the neoplastic process by increasing tumour cell number through by increasing tumour cell number through

1.1. Stimulation of cell birth Stimulation of cell birth

2.2. Inhibition of cell death/cell cycle arrestInhibition of cell death/cell cycle arrest

Mutations in TS can be either Mutations in TS can be either 1.1. Germline Germline hereditary predisposition to cancerhereditary predisposition to cancer

2.2. Somatic Somatic sporadic tumourssporadic tumours

The first mutationThe first mutation clonal expansion & initiate the clonal expansion & initiate the neoplastic processneoplastic process

Subsequent mutations Subsequent mutations additional rounds of clonal additional rounds of clonal expansion expansion tumour progression tumour progression

TS Genes and PathwaysTS Genes and Pathways

PathwaysPathways GenesGenes Disease Disease

RBRB RB1RB1

CDKN2ACDKN2AHereditary retinoblastomaHereditary retinoblastoma

Familial malignant melanomaFamilial malignant melanoma

P53P53 TP53TP53

WT1WT1Li Fraumeni syndromeLi Fraumeni syndrome

Familial Wilms tumour Familial Wilms tumour

APCAPC APCAPC FAPFAP

HIF1HIF1 VHLVHL Von Hipple-Lindau syndromeVon Hipple-Lindau syndrome

PI3KPI3K TSC1, TSC2TSC1, TSC2 Tuberous sclerosis Tuberous sclerosis

RTKRTK NF1NF1 NeurofibromaNeurofibroma

SMADSMAD MEN1MEN1 Multiple endocrine neoplasia Multiple endocrine neoplasia type Itype I

pRb and p53 pathways

Loss of function of both p53 and the pRB pathways play a significant role in the development of most human cancers

pRb regulates apoptosis during development, and its loss results in deregulated cell proliferation and apoptosis

p53, however guards against genomic instability and oncogene expression by inducing both arrest of the cell cycle and apoptosis hence its loss will desensitise cells to checkpoint signals, including apoptosis

All DNA tumor viruses that cause tumours in All DNA tumor viruses that cause tumours in experimental animals or humans encode proteins experimental animals or humans encode proteins that inactivate both pRb and p53that inactivate both pRb and p53

The retinoblastoma paradigm The retinoblastoma paradigm

Knudson in 1971 proposed that all retinoblastomas Knudson in 1971 proposed that all retinoblastomas involved two hit mechanisms involved two hit mechanisms

In the familial cases one hit was inherited In the familial cases one hit was inherited In the sporadic cases both hits occurred somatically to In the sporadic cases both hits occurred somatically to

inactivate the inactivate the RBRB gene gene

Complications of the RP paradigm Complications of the RP paradigm BRCA1BRCA1 is inactivated in only 10-15%, when is inactivated in only 10-15%, when

inactivation happens is not by chromosomal inactivation happens is not by chromosomal mechanisms but by DNA methylation mechanisms but by DNA methylation

Some genes lose function of one allele in tumours, but Some genes lose function of one allele in tumours, but the retained allele appears fully functional the retained allele appears fully functional

Cases with three hits, eg, APC certain germline Cases with three hits, eg, APC certain germline mutations are weak and hence two somatic mutations mutations are weak and hence two somatic mutations in addition to the inherited one are needed in addition to the inherited one are needed

The RB pathway The RB pathway

pRb functions as a gatekeeperpRb functions as a gatekeeper

-vely regulates progression through -vely regulates progression through

the G1 phase of the cell cyclethe G1 phase of the cell cycleDuring the G1 phaseDuring the G1 phasepRb is hypo(P) (Active)pRb is hypo(P) (Active)binds E2F binds E2F repression of repression of

E2F-mediated transcription E2F-mediated transcription

In late G1 and through the M phase In late G1 and through the M phase pRb is (P) i.e. inactivated by CDK pRb is (P) i.e. inactivated by CDK E2F is released E2F is released

promote the expression of genes required for promote the expression of genes required for cell division. cell division.

P16 inhibits CDK4/6 kinase P16 inhibits CDK4/6 kinase Loss of P16 function Loss of P16 function loss of pRb function loss of pRb function

inappropriate cell cycling inappropriate cell cycling

The RB pathway cont. The RB pathway cont.

• pRb repression of E2F is believed to be mediated by recruiting chromatin remodelling complexes to the E2F promoter during the resting phase (G0/G1)

• SWI/SNF complex, histone deacteylases, polycomb proteins and methylases are examples of chromatin remodelling complexes that interacts with pRb

• pRb is a member of the pocket proteins which includes p107& p130

• pRb interacts with E2F1-4 whereas p107 interacts with E2F4, p130 interacts with E2F5

Potential functions of RBPotential functions of RB

Besides cell cycle regulation Besides cell cycle regulation DNA damage responses DNA damage responses DNA repair DNA repair DNA replicationDNA replication Protection against apoptosis Protection against apoptosis Differentiation Differentiation

• Rb-/- mice die between days 13-15 gest.

• P170 or p130-null mice develop normally

• Rb+/- predisposition to pitut & thyroid tumours

• Rb+/-&p53 delretinal dysplasia

TP53

The TP53 gene encodes a 53 kDa phosphoprotein which belongs to small family of related proteins (p63 & p73)

95% of the mutations were found to occur in the central DNA binding core of p53

Nearly 28% of mutations affect only six residues (shown) Over 90% of the mutations lead to single amino acid

substitutions

p53 pathway

This is by covalent modification involving phosphorylation of the transactivation domain, and acetylation and phosphorylation of the basic allosteric control region

Normally, levels of p53 are low due to the short half-life of the protein

Stress signals (DNA-damaging agents, such as ultraviolet or γ irradiation and chemotherapeutic drugs) can induce the stabilisation and activation of p53

p53 activation

(UV) (IR) Phosphorylate p53

ATM kinaseATR kinase

A) Stress

Activation of p53

Can be activated by both external and internal aberrations

B) Deregulation of cellular oncogenes

Expression of P14 (ARF)

Deactivation of MDM2

Blocking the degradation of P53

Activation of P53

How P53 prevents tumour formation ??

First, it arrests the G1/S transition to allow DNA repair to happen

Second, it can transcriptionally activate DNA repair proteins

Last, if DNA repair fails then it will induce apoptosis

Arresting the G1/S transition phase

By activating the transcription of the gene encoding P21

Inhibition of CDK4/cyclinD1 & CDK2/cyclin E complexes

Blocking of the G1/S transition phase

P53 apoptosis pathway

APC pathway & FAP Absence of WNT signal

phosphorylation of -catenin at multiple residues

This is dependent on a multiprotein complex made of APC, Axin and GSK3

(P)-catenin will be degraded by ubiquitin and proteosome pathway

WNT sig. stabiliz. of -catenin & its accumul. in the cytoplasm move to the nucleus where it leads to transcription of target genes

Mut. Of APC -catenin transcrip. Of genes & TCF

APC downregulate the WNT pathway by acting as a shuttle for -catenin

HIF1 Pathway & VHLHIF1 Pathway & VHL

VHL in presence of VHL in presence of oxygen oxygen degrade HIF1

In absence of O2 or if VHL is mutated HIF1 will be stabilized

Expression of VEGF stim. of angiogenesis

Implication of oncogenes and TS Implication of oncogenes and TS genes in the process of genes in the process of

angiogenesis angiogenesis

References

Retinoblastoma: revisiting the model prototype of inherited cancer. Lohmann DR and Gallie BL (2004) Am J Med Genet, 129C 23-28

The retinoblastoma tumour suppressor in development and cancer. Classon M and Harlow E (2002) Nat Rev Cancer, 2, 910-7

The p53 tumour suppressor gene. Steele RJ at al (1998) Br J Surg, 85, 1460-67

Live or let die: the cell's response to p53. Vousden KH and Liu X (2002) Nat Rev Cancer,2, 594-604

The role of p53 and pRB in apoptosis and cancer. Hickman ES, Moroni MC, Helin K. Curr Opin Genet Dev. 2002 Feb;12(1):60-6. Review

Apoptosis - the p53 network. Haupt S, Berger M, Goldberg Z, Haupt Y. J Cell Sci. 2003 Oct 15;116(Pt 20):4077-85.

Tumour suppressor genes: Pathways and isolate strategies by Wafik S. El-Deiry

Vogelstein B and Kinzler KW (2004) Nat Med, 10, 789-799