Role and Regulation of Cdk Inhibitors in Development and Cancer Prof. Martine F. Roussel, PhD

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1 Role and Regulation of Cdk Inhibitors in Dept. of Genetics & Tumor Cell Biology St. Jude Children s Research Hospital Memphis, Tennessee USA 1 2 Somatic cell cycles Cdk1/Cyclin B M G2 Cdk1/Cyclin A Quiescence [G 0 ] Under-P RB Cdk4(6)/Cyclin Ds S G1 Mitogen dependent G1 progression Cdk2/Cyclin A Cdk2/Cyclin E RB hyperphosphorylation Restriction point 3 1

2 Negative regulation of G1 progression by Cdk inhibitory proteins p16 Ink4a, p15 Ink4b p18 Ink4c, p19 Ink4d p21 Cip1, p27 Kip1 p57 Kip2 Cyclin D1,2,3 Cdk4,6 Cyclin E/A Cdk2 RB, p107, p130 4 The Rb pathway in human cancer Cancer type Ink4a loss Cyclin D-Cdk4 overexpression Rb loss Small Cell Lung 15% 5% Cyclin D1 80% Non Small Cell Lung 58% 20-30% Pancreatic 80% Breast 31% 50% Cyclin D1 Glioblastoma 60% 40% Cdk4 T-ALL 75% Mantle Cell Lym. 90% Cyclin D1 5 Two groups of Ink4 proteins p16 Ink4a and p15 Ink4b Genes respond to different stress signals, including cell senescence and organismal aging The two genes are closely linked on human Ch. 9p21 and are frequently co-deleted in tumors Not expressed during mouse development; KO strains develop normally, but mice lacking Ink4a are tumor-prone; Loss of Ink4b exacerbates the effects of Ink4a inactivation; Ink4a is lost in gliomas p18 Ink4c and p19 Ink4d Expressed in stereotypic patterns during development; KO strains exhibit focal tissue abnormalities Ink4c (CDKN2C), 1p32 is a haplo-insufficient tumor suppressor gene; Deletions and mutations are rare, but epigenetic silencing is frequent in some tumor types including gliomas and medulloblastoma No evidence that Ink4d, 19p13 is a tumor suppressor gene 6 2

3 Two groups of CIP/KIP proteins p21 Cip1 Gene respond to different stress signals Not expressed during mouse development; KO strains develop normally p27 Kip1, p57 Kip2 Expressed in stereotypic patterns during development, in postmitotic cells, neurons in the CNS Kip1 KO strain exhibit organomegaly, pituitary tumors late in life Kip2 loss induces many developmental anomalies: Beckwith/Wiedemann syndrome Kip1 is a haplo-insufficient tumor suppressor gene; Deletion is rare, but epigenetic silencing is frequent in some tumor types including gliomas and medulloblastoma 7 Expression of INK4 genes during embryogenesis 8 Expression of Ink4 genes in tissues of young mice Role of p18 Ink4c and p19 Ink4d in spermatogenesis 9 3

Testicular atrophy but fertility in Ink4d-null male mice Testicular atrophy is associated with

Tunnel assay +/+ -/- 10 Ink4c/Ink4d double deficient male mice are sterile: absence of

cells p18/p19(-/-)testes Spermatozoa WT epididymis p18/p19(-/-)edpididymis 11 Conclusions p18

4 Testicular atrophy but fertility in Ink4d-null male mice Testicular atrophy is associated with increased apoptosis A. LacZ staining +/+ -/- B. Tunnel assay +/+ -/- 10 Ink4c/Ink4d double deficient male mice are sterile: absence of spermatozoa due to delayed meiosis I and increased apoptosis WT testes Leydig cells Leydig cells p18/p19(-/-)testes Spermatozoa WT epididymis p18/p19(-/-)edpididymis 11 Conclusions p18 Ink4c and p19 Ink4d are essential for male fertility Both Ink4 proteins collaborate to ensure mitotic exit and normal meiotic maturation of spermatocytes 12 4

5 Role of Cdk inhibitors in the CNS 13 Hypothesis Neurons must exit cycle to differentiate and migrate to their right positions in the brain Loss of cell cycle inhibitory proteins that enforce Rb tumor suppressor functions may lead to cancers in the CNS 14 Role of Cdk inhibitors in the CNS (2) In the developing CNS, p18 Ink4c is expressed transiently in proliferating neurons and time their exit from the cell cycle while p19 Ink4d and p27 Kip1 are found mainly in post-mitotic i neurons p19 Ink4d and p27 Kip1 expression is maintained in adult brain and actively keep neurons out of cycle p27 Kip1 -null mice are deaf due to increased proliferation of supporting cells in the organ of Corti 15 5

Ink4d, Kip1-null mice p27 Kip1 p19 Ink4d

6 Progressive sensory hair cell loss in Ink4d -/- mice induces progressive deafness 16 Ink4d is required for the active maintenance of the post-mitotic state in sensory hair cells Myosin VIIa Caspase 3 BrdU 17 Postnatal development in Ink4d, Kip1-null mice p27 Kip1 p19 Ink4d Mice die by 18 days after birth with major neurological defects 18 6

Conclusions (2) p19 Ink4d and p27 Kip1 are required to actively keep differentiated neurons out of cycle Loss of either protein induces deafness, loss of both induces neurological defects and death

7 Conclusions (2) p19 Ink4d and p27 Kip1 are required to actively keep differentiated neurons out of cycle Loss of either protein induces deafness, loss of both induces neurological defects and death by day 18 after birth, but no brain tumors 19 Role of the Rb pathway in cerebellar development Cyclins D2 and D1 by binding to Cdk4 regulate cerebellar development (Sicinski s lab) N-Myc dependent suppression of p18 Ink4c and p27 Kip1 expression is required for proper cerebellar development (Knoepfler, Eisenman) Deletion of Rb and p53 in the cerebellum induces 100% medulloblastoma (Marino, Berns) 20 Medulloblastoma Represents the most common pediatric brain tumor Cerebellar neuronal tumor, peak incidence ages 2-7 years Arises from granule neuron precursors (GNPs) that proliferate and differentiate in the postnatal cerebellum ~25% of human primary tumors have mutational defects in sonic hedgehog or Wnt signaling pathways ~10% have p53 mutations 21 7

8 Childhood medulloblastoma D I A G N O S I S Average Risk Surgery Chemotherapy Radiotherapy High risk ~75% long term survival rate but quality of life is severely compromised neurocognitive defects <50% long term survival rate Better therapies are required to improve survivors quality of life 22 Cerebellum = little brain Represents only 10% of the total brain mass, hence its Latin name, but contains >50% of our neurons It coordinates sensory inputs from the periphery p to fine tune our movements and balance Although it is the first brain structure to differentiate, its maturation occurs mainly after birth for 16 months in humans, two weeks in the mouse This protracted developmental process makes the cerebellum an easier brain structure to study, but it also makes it especially vulnerable to developmental anomalies, leading to structural defects and cancer 23 Postnatal cerebellar development P1 P5 P10 P20 EGL Purkinje layer IGL External Granular Layer (EGL) Internal Granular Layer (IGL) Molecular Layer (ML) Adapted from Cynthia Wetmore 24 8

Cerebellar development in the mouse E11 Rhombic Lip E13 E15

Cerebellar area (mm 2 ) Cerebellar volume (mm 2 ) Stage of

SHH/PATCHED signaling pathway SHH Ptc Smo Sufu Gli Gli

Rb-dependent cell cycle exit and differentiation of GNPs p18

9 Cerebellar development in the mouse E11 Rhombic Lip E13 E15 P0 EGL P7 EGL Adult IGL ML Area of cerebellum (mm 2 ) Cerebellar area (mm 2 ) Cerebellar volume (mm 2 ) Stage of development volume of cerebellum (mm 2 ) 25 The (simplified) SHH/PATCHED signaling pathway SHH Ptc Smo Sufu Gli Gli targets N-Myc Gli Cyclin E Cyclin D1 Cyclin D2 FoxM1 Ptc 26 Rb-dependent cell cycle exit and differentiation of GNPs p18 Ink4c p27 Kip1 Cyclin D1,2 Cdk4/Cdk6 Cyclin E/A Cdk2 Frederique Zindy RB, p

10 Ink4c expression in the mouse cerebellum 28 p27 Kip1 is expressed in non-dividing granule cells and its expression is maintained in the IGL throughout adulthood P5 P14 1 Month Wt 29 N-Myc in GNP proliferation Shh N-Myc Cyclin D1 Cyclin D2 p18 Ink4c p27 Kip1 Wei Du, Sicinski, McKinney& Rowitch, Knoepfler & Eisenman 30 10

11 Partial rescue in adult cerebellum WT Ink4c/Kip1/N-myc Fl/Fl Nestin-Cre+ N-myc Fl/Fl Nestin-Cre+ In coll. with Knoepfler and Eisenman 31 Hypothesis (2) By canceling the growth suppressive functions of Rb-family proteins, loss of Ink4c, Kip1, alone or in combination, may contribute to tumor formation by providing cerebellar granule neuronal precursors (GNPs) with a proliferative advantage Results Mice lacking Kip1, Ink4c, or both exhibit organomegaly, which includes the cerebellum, but there are no overt defects in cerebellar or CNS functions 32 Mice lacking Ink4c and p53 develop medulloblastoma (25%) with anaplastic features at 2-5 months of age Crosses into an Arf-null background did not induce tumors 33 11

rvival Percent su Ink4c loss increases medulloblastoma incidence in Ptch1 +/- mice Medulloblastoma incidence 100 Ptc+/-p18+/+ 90 Ptc+/-p18+/- 80 70 Ptc+/-p18-/- 60 50 40 The wild-type Ptc allele is

12 rvival Percent su Ink4c loss increases medulloblastoma incidence in Ptch1 +/- mice Medulloblastoma incidence 100 Ptc+/-p18+/+ 90 Ptc+/-p18+/ Ptc+/-p18-/ The wild-type Ptc allele is lost in MBs, 30 but there is no LOH at the Ink4c locus; 20 Therefore, p18 is haplo-insufficient for tumor suppression Months Role and Regulation of Cdk Inhibitors in 34 Do p18 INK4C contribute to human medulloblastoma? No loss of copy number of INK4c was detected by array CGH in 46 primary medulloblastomas (SJCRH) 4/23 (17%) human medulloblastomas exhibited partial (3 cases) or complete (1 case) methylation of the CDKN2C/INK4C promoter; No normal human cerebellar tissues (0/9) exhibited CDKN2C/INK4C methylation INK4a promoter was not methylated in 23/23 cases of medulloblastoma 35 p18ink4c/cdkn2c protein expression is lost in primary medulloblastoma grade 2 17/73 (23%) grade 1 42/73 (57%) grade 0 14/73 (20%) Uziel et al., Genes & Development 19: , 2005 Sarah Sherr & Richard Gilbertson 36 12

13 Conclusions (3) Loss of Ink4c collaborates with the lack of p53 or Patched to induce medulloblastoma in mice; Loss of Ink4c is found in ~ 20% of human medulloblastomas Loss of Ink4a/Arf and Ink4c (Solomon et al., April 2008; Wiedemyer et al., April 2008) or Cdk4 overexpression induces glioblastoma when combined with mutations of EGFR or p53 in mouse and man 37 Phenotype of Ink4d or Ink4c deficient mice Ink4d KO Ink4c KO Ink4c KO Testicular atrophy (associated with apoptosis) Fertile Organomegaly (spleen, kidney, heart, adrenal gland, thymus and testis) Testicular tumors (seminomas) Fertile Deafness Pituitary adenoma (intermediate lobe) T-cells hyperplasia after mitogen stimulation (in vitro) Fertile Pituitary adenoma (intermediate lobe) Adrenal gland tumor (pheochromocytoma) Glomerulopathies Atrophyc glomeruli Glomerular cyste Zindy et al., (1999) Franklin et al., (1998) E. Latres et al., (2000) 38 Acknowledgments Charles J. Sherr Olivier Ayrault Tamar Uziel Haotian Zhao Frederique Zindy Suqing Xie Antoine Forget Brain Tumor Program Steven Clifford and Sarah Millner, Univ. New Castle, UK Charles Eberhart, Philip A.Beachy, Johns Hopkins, Baltimore, Md Lee Rubin, Curis Inc., Boston The Children s Brain Tumor Foundation (CBTF), The Pediatric Brain Tumor Foundation (PBTF), The American Brain Tumor Association (ABTA), NCI, SJCRH and ALSAC 39 13

14 40 14

Polycomb protein Ezh2 regulates pancreatic β-cell Ink4a/Arf expression and regeneration in streptozotocin-induced diabetes mellitus

Chen et al. 1 Polycomb protein Ezh2 regulates pancreatic β-cell Ink4a/Arf expression and regeneration in streptozotocin-induced diabetes mellitus Hainan Chen 1, Xueying Gu 1, I-hsin Su 2, Rita Bottino