The story of Retinoblastoma

Tumor Suppressor Genes

Retinoblastoma is a cancerous disease

1/20,000 children; 300 per yearAverage age is 18 months

Treatment: enucleation = eye removal

Prognosis is good after enucleationover 90% survival with early detection and treatment

Leukocoria or “white pupil”

- Sporadic cancer in 55-65% of all cases- Sporadic cancers are unilateral

Rb is either sporadic or familial

Hereditary childhood cancer: - bilateral tumors in ~75% of cases- unilateral tumors in ~25% of cases

Children with bilateral (familial) Rb have a high risk of developing non-retinal tumors

Germ-line mutations in the Rb gene lead to predisposition to cancer

Sporadic

Familial

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

Rb tumors are associated with a deleted region in chromosome 13

Deletion = loss-of-functionprobably a recessive mutation in the Rb

gene

The Knudson’s “Two Hit” Hypothesis for the Generation of RB

Alfred Knudson, PNAS 68:820 (1971)

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

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

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

Loss of heterozygosity (LOH)

Normal paternal

Mutated maternal

Mutated maternal

-/+

-/-

-/-

-/-

Mutated paternal

The mutated maternal chromosome was duplicated

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

Rb is just one example

Inheritance of brca1(lf) mutation results in predisposition for breast cancer

Rb = A Tumor Suppressor Gene

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

Predisposition is inherited dominantly, but cancer is not inherited

The offspring CANNOT inherit two mutated genes

How can we clone a tumor-recessive gene?

Oncogenes transform cells into cancerous cells

But TSGs are recessive

How do we test candidate genes?

Rb tumors are associated with a deleted region in chromosome 13

Deletion = loss-of-functionprobably a recessive mutation in the Rb

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)

Testing a candidate gene

More on this- Angier book, starting p. 334

Rb gene expression is absent or altered in retinoblastoma tumors

Friend et al. Nature (86) Lee et al. Science (87)

Northern blots (mRNA expression)

Rb tumors WT Other tumors

We have correlation

What about causation?

The RB gene is finally cloned

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.”

Bold Predictions, Further Work

pRb: What does it do?

pRb is a nuclear protein that undergoes phosphorylation and dephospharylation in concert with the cell cycle

Hypo-phosphorylated or un-phosphorylated pRb inhibits the cell from entering a new cell cycle

The guardian of the cell at early-mid G1

Upon further phosphorylation at the R point, hyper-phosphorylated pRb becomes inert and the

cell cycle can proceed

Hypo-phosphorylated Rb inhibits activity of the E2F family of transcription factors

Hyper-phosphorylation of Rb sequesters Rb, and releases E2Fs

E2Fs are needed for transcription of genes that are essential for the cell to enter the cell cycle

Hypo-phosphorylated Rb binds to E2Fs and:

- Inhibits their transcription activation sites

- Recruits proteins that will “close” the chromatin down

Releasing Rb from the E2Fs leads to:

- Release of their transcription activation sites

- Recruitment of proteins that will “open up” the chromatin

Rb, the retinoblastoma protein regulates the cell cycle

Cell cycle = OFFRb binds to E2F: no transcription, no entry into S phase

Cell cycle = ONRb does not bind to E2F: transcription and entry into S phase

w/o 2 copies of Rb: no cell cycle arrest

pRb: What does it do?

pRb is a nuclear protein that undergoes phosphorylation and dephospharylation in concert with the cell cycle

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

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

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

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

Have I grown enough?

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

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

E2Fs

Rb gene alteration is involved many tumorsIn the majority of tumors you will find mutation

involved in the R site

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

What to focus on

Cell cycle controlRegulation of CDKs

Mitogens and the cell cycle

Rb: genetics

The restriction point: cycD, cycE, E2Fs, p16 and Rb(read the textbook – chapter 8)

Details of the cell cycle (e.g. what happens in prometaphase)

What not to focus on

Molecular details of ubiqu. pre-replicative complex, etc.

Her-2