Tumor Suppressors Versus Oncogenes

The Cancer Phenotype is Usually Recessive

R. Weinberg, Cancer Biology

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

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

Alfred Knudson, PNAS 68:820 (1971)

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 other cancers later in life.

Retinoblastoma is Associated with Loss of Heterozygosity (LOH) at the RB Locus

~40% of the time thewild type allele is mutated4% of these are deletions

R. Weinberg, Cancer Biology

(LOH)

(LOH)

(nondisjunction)

~30% of the time

Mitotic Nondisjunction Causes (LOH) at the RB Locus

Mitotic Recombination Causes LOH at the RB Locus

~30% of the time

Rb is just one example

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

Rb = A Tumor Suppressor Gene

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

Use a fragment of the candidate gene as a probe for hybridization analysis

Search for absence of the gene in tumors (hoping both mutated copies are deletions)

Testing a candidate gene

More on this in 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

Huang et al Science, 242:1563-6 (1988)

Saos-2 cellsSaos-2 cells infected

with RB retrovirus

Expression of the RB Gene in RBMutant Cells Corrects the Cancer

No virusRB virusControl virus

Parental = WERI-Rb27 cellsLux = control virus infected

Rb = RB virus infected

Tumor formation assayed in “nude” mice

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

Un- and Hypo-phosphorylated pRb inhibits the cell from entering a new cell

cycle

RB regulates progression through G1 phase

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

cell cycle can proceed

Figure 8.23c The Biology of Cancer (© Garland Science 2007)

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

Hyper-phosphorylated Rb cannot bindand inhibit E2F

RB/E2F complexes act as

transcriptional repressors

Releasing Rb from the E2Fs leads to activation of

transcription

E2Fs have 100s of target genes, mostly involved in DNA replication

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

Sites of RB Missense Mutations in Tumors

AB

The A and B domain = pocket = where E2F binds

Figure 8.23b The Biology of Cancer (© Garland Science 2007)

How is Rb activity regulated during the cell cycle?

How is Rb activity regulated during the cell cycle? By Cyclin/CDKs

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

However, E-CDK2 can phosphorylate Rb, only AFTER Rb is phosphorylated by D-CDK4/6

How is Rb activity regulated during the cell cycle? By Cyclin/CDKs

One of the E2F targets: the cycE gene!

Transcription of cycE starts a positive feedback loop

Rb control of E2F IS the restriction point

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

E2Fs

Proliferation Signals

The Canonicalp16/pRB/E2F

“Pathway”

Sherr and McCormick, Cancer Cell ·2:103 (2002)

The RB Pathway in Human Cancer

(p16)

Forward versus reverse genetics

Forward Genetics: isolate mutants with a specific phenotype and then clone the gene

Reverse Genetics: clone the gene and then make a mutant to determine phenotype

How can we study retinoblastoma in the lab?

with GEMs: Genetically Engineered Mice

“Knock Out” your gene in Cultured Pluripotent ES Cells

Formation of ES Cells Carrying a Knockout Mutation

Positive and Negative Selection of Recombinant ES Cells

Making theMutant Mouse

The Phenotype of RB Mutant Mice

Homozygous embryos die at day 13.5massive apoptosis in the CNSanemia caused by lack of blood cells made by the fetal livertherefore RB is not essential for cell division

Heterozygous animals are viablethey develop pituitary tumors with near 100% penetrance

these tumors lose the wild type RB allelealso develop thyroid adenomastherefore the Rb-/+ mouse is not a model for retinoblastoma

but it is a good model for tumor suppressors

And can study it’s mechanism of action genetically

Tumorigenesis in RB Mutant Mice Requires E2F

Genotype Pituitary Tumors Thyroid Tumors

RB (+/-) 19/20 (95%) 10/19 (53%)

RB (+/-); E2F1 (+/-) 36/36 (100%) 2/34 (6%)

RB (+/-); E2F1 (-/-) 16/26 (62%) 0/22 (0%)

Yamasaki et al, Nat. Gen. 18:360 (1998)

The Phenotype of RB Mutant Mice

Homozygous embryos die at day 13.5massive apoptosis in the CNSanemia caused by lack of blood cells made by the fetal livertherefore RB is not essential for cell division

Heterozygous animals are viablethey develop pituitary tumors with near 100% penetrance

these tumors lose the wild type RB allelealso develop thyroid adenomastherefore the Rb-/+ mouse is not a model for retinoblastoma

but it is a good model for tumor suppressors

Why don’t Rb-/+ mice get retinoblastoma?????!!!!!!