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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 • 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 • 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 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 • 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 • 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 p21, p27, p57 • Others are specific p15,p16,p18,p19 • (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 (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 • 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 G1/S checkpoint • 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 • 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.. • 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 • 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 • 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 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) • Some virus like HPV have protein E7 which binds to the hypophsphorylated RB so prevents it from inhibiting E2F • So RB is functionally deleted 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 1. Temporary cell cycle arrest: quiescence 2. permenant cell cycle arrest: senescence 3. triggers apoptosis Check this pic in the book… • 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. • 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 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 • P53 also induces expression of DNA damage repair genes • If DNA is repaired successfully ,p53 upregulates transciption of MDM2.. Destruction of p53.. Removal of the block on cell cycle. • If DNA not repaired p53 makes cells enter apoptosis or senescence 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 P53 induced apoptosis • Induced by pro-apoptotic genes including BAX and PUMA • 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 • 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) • P53 can become nonfunctional by some DNA viruses • HPV, Hep B, EBV.. Proteins can bind to p53 and deactivate it • Note p53 activated by phosphorylation