The story of Retinoblastoma

1. Tumor Suppressor Genes The story of Retinoblastoma

2. Retinoblastoma is a cancerous disease 1/20,000 children; 300 per year Average age is 18 months Treatment: enucleation = eye removal Prognosis is good after enucleation over 90% survival with early detection and treatment Leukocoria or “white pupil”

3. Rb is either sporadic or familial - Sporadic cancer in 55-65% of all cases - Sporadic cancers are unilateral Hereditary childhood cancer: - bilateral tumors in ~75% of cases - unilateral tumors in ~25% of cases

4. Children with bilateral (familial) Rb have a high risk of developing non-retinal tumors Familial Sporadic Germ-line mutations in the Rb gene lead to predisposition to cancer

5. In cancer patients with a family history of Retinoblastoma: the inheritance seems to be ?

6. Rb tumors are associated with a deleted region in chromosome 13 Deletion = loss-of-function probably a recessive mutation in the Rb gene

8. The Knudson’s “Two Hit” Hypothesis for the Generation of RB Alfred Knudson, PNAS 68:820 (1971)

9. The Knudson’s “two hit” hypothesis for the generation of RB

10. Retinoblastoma is inherited as a dominant trait, but it is recessive at the cellular level People with familial Retinoblastoma carry one mutated copy in ALL their cells. Cells that would get a second hit will develop Rb or later, other cancers

11. Loss of heterozygosity (LOH) Loss of heterozygosity (LOH) in a cell represents the loss of normal function of one allele of a gene in which the other allele was already inactivated

12. Mutated maternal Normal paternal -/- -/+ -/- Mutated paternal Mutated maternal The mutated maternal chromosome was duplicated -/-

13. The presence of one mutated copy increases the chances of a second mutated copy

15. Rb is just one example Inheritance of brca1(lf) mutation results in predisposition for breast cancer Rb = A Tumor Suppressor Gene

16. Retinoblastoma is inherited as a dominant trait, but it is recessive at the cellular level People with familial Retinoblastoma carry one mutated copy in ALL their cells. Cells that would get a second hit will develop Rb or later, other cancers

17. Predisposition is inherited dominantly, but cancer is not inherited The offspring CANNOT inherit two mutated genes

18. How can we clone a tumor-recessive gene? How do we test candidate genes? Oncogenes transform cells into cancerous cells ButTSGsare recessive

19. Rb tumors are associated with a deleted region in chromosome 13 Deletion = loss-of-function probably a recessive mutation in the Rb gene

20. Testing a candidate gene Use a fragment of the candidate gene as a probe for Southern Blot analysis Search for absence of the gene in tumors (hoping both mutated copies are deletions) More on this- Angier book, starting p. 334

21. Rb gene expression is absent or altered in retinoblastoma tumors Rb tumors WT Other tumors Friend et al. Nature (86) Lee et al. Science (87) Northern blots (mRNA expression)

22. We have correlation What about causation? The RB gene is finally cloned

23. Bold Predictions, Further Work Dr. David Abramson, RB expert at New York Hospital (ca. 1986, According to Natalie Angier) “I believe that in fifteen years, at the outside, we’ll be able to stop retinoblastoma before it begins. I’m so sure that I’ve already given the drug a name. I call it retino-revert, or retino-prevent. The drug will be an analogue of the natural protein that is missing in retinoblastoma cells … We’ll be able to diagnose a child prenatally and start giving this retino-revert to the mother to prevent retinoblastomas from growing as the fetus is developing. I know I’m going out on a limb with this one, but … Come back to me in 2001 and tell me if I wasn’t right.”

24. pRb: What does it do? pRb is a nuclear protein that undergoes phosphorylation and dephospharylationin concertwith the cell cycle

25. The guardian of the cell at early-mid G1 Hypo-phosphorylated or un-phosphorylatedpRb inhibits the cell from entering a new cell cycle Upon further phosphorylation at the R point, hyper-phosphorylatedpRb becomes inert and the cell cycle can proceed

26. Hypo-phosphorylatedRbinhibitsactivity of the E2F family of transcription factors E2Fs are needed for transcription of genes that are essential for the cell to enter the cell cycle Hyper-phosphorylation of Rb sequesters Rb, and releasesE2Fs

27. Hypo-phosphorylatedRb binds toE2Fsand: - Inhibits their transcription activation sites - Recruits proteins that will “close” the chromatin down

28. ReleasingRb from the E2Fs leads to: - Release of their transcription activation sites - Recruitment of proteins that will “open up” the chromatin

29. Rb, the retinoblastoma protein regulates the cell cycle Cell cycle = OFF Rb binds to E2F: no transcription, no entry into S phase Cell cycle = ON Rb does not bind to E2F: transcription and entry into S phase w/o 2 copies of Rb: no cell cycle arrest

30. pRb: What does it do? pRb is a nuclear protein that undergoes phosphorylation and dephospharylationin concertwith the cell cycle

31. Rb activity is tightly regulated by the cell cycle clock Hypo-phosphorylation is catalyzed by cycD-CDK4/6 Hyper-phosphorylation is catalyzed by cycE-CDK2

32. pRb is hyper-phosphorylated and inhibited (and released from its role as a guardian), only upon cycE expression

33. Rb activity is tightly regulated by the cell cycle clock However, E-CDK2 can phosphorylateRb, only AFTER Rbis phosphorylated by cycD-CDK4/6

34. Have I grown enough? Only after we have enough mitogen signaling (and, as a result, enough cycD-CDK4/6 activity), cycE can phosphorylateRb and allow entry to the cell cycle

35. E2Fs have more than 100 target genes, mostly involved in the first steps of DNA replication One of the targets: the cycE gene Transcription of cycEstarts a positive feedback loop

36. As E2Fs are necessary for expression of cycE, think how critical negative regulation by Rbis for cell cycle control E2Fs

37. Rbgene alteration is involved many tumors In the majority of tumors you will find mutation involved in the R site

38. Uncontrolled crossing of the R site can be due to loss of Rb function (e.g. mutation), loss of CKIs or oncogenic activity of cyclins E and D

40. What not to focus on Details of the cell cycle (e.g. what happens in prometaphase) Molecular details of ubiqu. pre-replicative complex, etc. What to focus on Her-2 Cell cycle control Regulation of CDKs Mitogens and the cell cycle Rb: genetics The restriction point: cycD, cycE, E2Fs, p16 and Rb (read the textbook – chapter 8)