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. Germ-line mutations in the Rb gene lead to predisposition to cancer
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. The mutated maternal chromosome was duplicated -/-
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 = A Tumor Suppressor Gene
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
But TSGs are 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. More on this- Angier book, starting p. 334
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. The RB gene is finally cloned
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 dephospharylation in concert with the cell cycle

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

26. Hypo-phosphorylated Rb inhibits activity 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 releases E2Fs

27. Hypo-phosphorylated Rb binds to E2Fs and:
- Inhibits their transcription activation sites
- Recruits proteins that will “close” the chromatin down

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

29. w/o 2 copies of Rb: no cell cycle arrest
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 dephospharylation in concert with 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 phosphorylate Rb, only AFTER Rb is 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 phosphorylate Rb 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 cycE starts a positive feedback loop

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

37. Rb gene 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. Mitogens and the cell cycle
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)