The Role of Cyclins and Cyclin-Dependent Kinases in Cell Cycle Regulation

1. Neoplasia 6 DrHeymAwad FRCPath

2. Cyclins and cyclin dependent kinases

3. 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

4. G1 to S transition is critical because if this checkpoint is passed the cells are committed for DNA replication • G1 to S is called the restriction point

5. Progression through cell cycle, especially G1-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

6. The DEAB sequence of cyclins !

7. 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.

9. Checkpoints important to discover DNA damage and prevent cells with DNA damage from continuing cell cycle • G1-S checkpoint monitors integrity of chromosomes before replicating • G2-M checks DNA integrity after replication to decide if cells can safely go into mitosis

10. 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.

11. CDKI • Some inhibit CDK broadly p21, p27, p57 • Others are specific p15,p16,p18,p19 • (you don’t need to remember which is which!!)

12. 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.

13. 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

14. RB gene= retinoblastoma gene = governor of cell cycle

15. 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

16. retinoblastoma

17. Two hit hypothesis • Two mutations (hits) required to develop retinoblastoma • The 2 mutations involve the RB gene on chromosome 13 (13q14)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

18. 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.

19. Two hit hypothesis

20. Two hit hypothesis

21. 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…

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

23. RB gene • RB gene product is a DNA binding protein expressed in all cells • This protein has two forms: active hypophsphorylatedstate and inactive hyperphosphorylated • Bb regulates G1/S checkpoint

24. S phase requires cyclin E/CDK2 complex • Expression of cyclin E needs E2F family of transcription factors • Early in G1 , RB is in its active hypophosphorylated form, it binds and inhibits E2F .. So no cyclin E2 • This is done by two ways: hyposphorylated RB sequesters E2f so it doesn’t interact with transcription factors also RB recruits chromatin remodeling proteins that bind to promoters of E2F, so DNA becomes insensitive to transcription factors

25. 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 E2F • E2F now free and can cause transcription of cyclin E.. • CyclinE 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 .

26. How RB works

27. E2F is not the only transcription factor targeted by RB • Rb stimulates myocyte, adipocyte and other cell specific transcription fatcors… so important for G0-G1 with differentiation

28. 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 cdk4 or overexpression of cyclin D favour cell proliferation by inactivating RB

29. NOTE • Loss of nomal cell cycle control is found in all tumors through mutations of RB, cyclin D, CDK4 or CDKN2A (which is a CDKI)

30. Some virus like HPV have protein E7 which binds to the hypophsphorylated RB so prevents it from inhibiting E2F • So RB is functionally deleted

31. TP53 gene: the guardian of the genome • Tp53 is one of the most commonly mutated genes in cancer • It encodes p53 protein P53 causes growth inhibition by three mechanisms • Temporary cell cycle arrest: quiescence 2. permenant cell cycle arrest: senescence 3. triggers apoptosis

32. Check this pic in the book…

33. P53 monitors internal stress whereas RB senses external signals • P53 is triggered by several stresses: anoxia, inappropriate oncogene activity (MYC or RAS) or DNA damage.

34. In non-stressed healthy cell, p53 is short lived: 20 minutes because it binds MDM2 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 p53 and release it from MDM2 • Now p53 has longer life span and can drive transcription of certain genes.. hundreds of them

35. Genes transcribed by p53 • Suppress growth by three mechanisms • 1. mediating cell cycle arrest. This occurs late in G1. caused by p53 dependent transcription of CDK1 gene= p21= CDKNIA • P21 protein inhibit cyclin/CDK complexes and prevent phosphorylation of RB • So cell is arrested in G1 • Pause to repair any DNA damage

36. P53 also induces expression of DNA damage repair genes • If DNA is repaired successfully ,p53 upregulatestransciption of MDM2.. Destruction of p53.. Removal of the block on cell cycle. • If DNA not repaired p53 makes cells enter apoptosis or senescence

37. Senescence by p53 • Senescence needs activation f p53 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

38. P53 induced apoptosis • Induced by pro-apoptotic genes including BAX and PUMA

39. P53 also represses proliferative and anti-apoptotic genes (bcl2) • ? P53 is a transcriptional activator so how could it repress certain gene expression • Answer by miRNAs

40. More than 70% of human cancers have mutated TP53 • 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)

41. P53 can become nonfunctional by some DNA viruses • HPV, HepB, EBV.. Proteins can bind to p53 and deactivate it • Note p53 activated by phosphorylation