Neoplasia 6 Dr Heym Awad FRCPath Cyclins and

Neoplasia 6 Dr Heym Awad FRCPath

Cyclins and cyclin dependent kinases

Normal cell cycle • All the stimuli mentioned in the previous lecture aim for quiescent cells to enter the cell cycle • Replication of cells is stimulated by growth factors or signaling from ECM components through integrins • Each phase in the cell cycle depends on successful completion of the previous one • Cycle stops when essential gene function is lost

• G 1 to S transition is critical because if this checkpoint is passed the cells are committed for DNA replication • G 1 to S is called the restriction point

• Progression through cell cycle, especially G 1 -S is regulated by proteins called cyclins • Cyclins activate kinases CDK (cyclin dependent kinases) • Cyclin and CDK form complexes that phosphorylate target proteins that drive the cell through the cell cycle. • Cyclins : D, E, A , B (they appear in this sequence

The DEAB sequence of cyclins !

• SO: cyclin/CDK complexes cause proliferation. • Theses are inhibited by cyclin dependent kinase inhibitors (CDKI) • CDKI important for enforcing the checkpoints and delaying cell cycle.

• Checkpoints important to discover DNA damage and prevent cells with DNA damage from continuing cell cycle • G 1 -S checkpoint monitors integrity of chromosomes before replicating • G 2 -M checks DNA integrity after replication to decide if cells can safely go into mitosis

• When there is DNA damage the checkpoints are activated. . They delay the cell cycle and trigger DNA repair • If damage is severe: apoptosis or senescence are stimulated.

CDKI • Some inhibit CDK broadly p 21, p 27, p 57 • Others are specific p 15, p 16, p 18, p 19 • (you don’t need to remember which is which!!)

• Mutations causing increased cyclins or CDK cause self sufficiency in growth signals. • Mutations inhibiting CDKI will cause increased growth. • Examples: cyclin D is activated in several tumors mainly lymphomas.

Second hallmark of cancer • 2. insensitivity to growth inhibitors • Growth inhibition is achieved by tumor suppressor genes • Loss or decreased functions of tumor suppressor genes is essential to cause cancer

• RB gene= retinoblastoma gene = governor of cell cycle

retinoblastoma • Rare childhood tumor • 60 % of cases are sporadic, 40% familial • Predisposition to develop the tumor is inherited as autosomal dominant trait • To develop retinoblastoma: two hit hypothesis

retinoblastoma

Two hit hypothesis • Two mutations (hits) required to develop retinoblastoma • The 2 mutations involve the RB gene on chromosome 13 (13 q 14)locus • Both copies of RB gene need to be deactivated to develop retinoblastoma • In familial cases, one hit is inherited (germ line mutation) the other is acquired -In sporadic cases, both mutations are acquired

• In familial cases , one single somatic mutation is needed. . So dominant pattern of inheritance. • NOTE: retinoblastoma disease follows autosomal dominant inheritance. Because the susceptibility of the disease is what is inherited. BUT, RB gene is a recessive gene.

Two hit hypothesis

Two hit hypothesis

• People with inherited RB have increased risk of other cancers. . Mainly osteosarcomas and soft tissue sarcomas • Homozygous loss (both copies lost) of RB gene can be seen in many cancers like breast, bladder…

• A cell heterozygous in RB locus is not neoplastic ( one normal and one abnormal allele) • The two hits are essential for neoplastic transformation

RB gene • RB gene product is a DNA binding protein expressed in all cells • This protein has two forms: active hypophsphorylated state and inactive hyperphosphorylated • Bb regulates G 1/S checkpoint

• S phase requires cyclin E/CDK 2 complex • Expression of cyclin E needs E 2 F family of transcription factors • Early in G 1 , RB is in its active hypophosphorylated form, it binds and inhibits E 2 F. . So no cyclin E 2 • This is done by two ways: hyposphorylated RB sequesters E 2 f so it doesn’t interact with transcription factors also RB recruits chromatin remodeling proteins that bind to promoters of E 2 F, so DNA becomes insensitive to transcription factors

• Mitogenic signals. . lead to cyclin d expression and activate cyclin D/CDK 4/6 complexes. . These complexes phosphorylate RB. . • Phosphorylated RB is inactivate … this leads to release of E 2 F • E 2 F now free and can cause transcription of cyclin E. . • Cyclin E stimulates DNA replication • So cells enter S phase • Once in s phase cells are committed to division. . They don’t need additional growth signals • In M phase phosphate removed from RB , so it goes back to its inactive state.

How RB works

• E 2 F is not the only transcription factor targeted by RB • Rb stimulates myocyte, adipocyte and other cell specific transcription fatcors… so important for G 0 -G 1 with differentiation

• Rb is important for tumerogenesis but it is not mutated in all cancers • If it is not mutated, other gene mutations mimicking RB mutation must play a role • Mutations in genes affecting RB phosphorylation: Mutatiomnal activation of cdk 4 or overexpression of cyclin D favour cell proliferation by inactivating RB

NOTE • Loss of nomal cell cycle control is found in all tumors through mutations of RB, cyclin D, CDK 4 or CDKN 2 A (which is a CDKI)

• Some virus like HPV have protein E 7 which binds to the hypophsphorylated RB so prevents it from inhibiting E 2 F • So RB is functionally deleted

TP 53 gene: the guardian of the genome • Tp 53 is one of the most commonly mutated genes in cancer • It encodes p 53 protein P 53 causes growth inhibition by three mechanisms 1. Temporary cell cycle arrest: quiescence 2. permenant cell cycle arrest: senescence 3. triggers apoptosis

Check this pic in the book…

• P 53 monitors internal stress whereas RB senses external signals • P 53 is triggered by several stresses: anoxia, inappropriate oncogene activity (MYC or RAS) or DNA damage.

• In non-stressed healthy cell, p 53 is short lived: 20 minutes because it binds MDM 2 which is a protein that targets it for destruction • When cells are stressed. . sensors that include protein kinases are activated (ATM is one of these kinases) • These activated kinases catalyze post translational modifications of p 53 and release it from MDM 2 • Now p 53 has longer life span and can drive transcription of certain genes. . hundreds of them

Genes transcribed by p 53 • Suppress growth by three mechanisms • 1. mediating cell cycle arrest. This occurs late in G 1. caused by p 53 dependent transcription of CDK 1 gene= p 21= CDKNIA • P 21 protein inhibit cyclin/CDK complexes and prevent phosphorylation of RB • So cell is arrested in G 1 • Pause to repair any DNA damage

• P 53 also induces expression of DNA damage repair genes • If DNA is repaired successfully , p 53 upregulates transciption of MDM 2. . Destruction of p 53. . Removal of the block on cell cycle. • If DNA not repaired p 53 makes cells enter apoptosis or senescence

Senescence by p 53 • Senescence needs activation f p 53 and or RB and expression of their mediators like CDKI • Mechanisms of senescence unclear but seem to involve global chromatin change, which permanent change gene expression

P 53 induced apoptosis • Induced by pro-apoptotic genes including BAX and PUMA

• P 53 also represses proliferative and anti-apoptotic genes (bcl 2) • ? P 53 is a transcriptional activator so how could it repress certain gene expression • Answer by mi. RNAs

• More than 70% of human cancers have mutated TP 53 • Both copies of the gene need to be lost for cancer to develop • Mostly somatic • Rare li Fraumini syndrome: inherit defect in one allele. . More predisposition to cancer (Sarcoma, breast carcinoma , leukemia and brain tumor)

• P 53 can become nonfunctional by some DNA viruses • HPV, Hep B, EBV. . Proteins can bind to p 53 and deactivate it • Note p 53 activated by phosphorylation