Tumor Suppressors Versus Oncogenes
The Cancer Phenotype is Usually Recessive
R. Weinberg, Cancer Biology
Tumor Suppressor Genes
The story of Retinoblastoma
Retinoblastoma is a cancerous disease
1/20,000 children; 300 per year
Average age is 18 months
Leukocoria or “white pupil”
Treatment:
enucleation = eye removal
Prognosis is good after enucleation
over 90% survival with early
detection and treatment
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
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
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-function
probably 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
(LOH)
(LOH)
~40% of the time the
wild type allele is mutated
4% of these are deletions
R. Weinberg, Cancer Biology
(nondisjunction)
Mitotic Nondisjunction Causes
(LOH) at the RB Locus
~30% of the time
Mitotic Recombination
Causes LOH at the RB Locus
~30% of the time
Rb = A Tumor Suppressor Gene
Predisposition is inherited dominantly,
but cancer is not inherited
The offspring CANNOT inherit two
mutated genes
Rb is just one example
Inheritance of brca1(lf) mutation results
in predisposition for breast cancer
How can we clone a tumor-recessive gene?
How do we test candidate genes?
Oncogenes transform cells into cancerous cells
But TSGs are recessive
Rb tumors are associated with a deleted
region in chromosome 13
Testing a candidate gene
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)
More on this in Angier book, starting p. 334
Rb gene expression is absent or
altered in retinoblastoma tumors
Rb tumors WT
Friend et al. Nature (86)
Other tumors
Lee et al. Science (87)
Northern blots
(mRNA expression)
Expression of the RB Gene in RB
Mutant Cells Corrects the Cancer
No virus
RB virus
Control virus
Parental = WERI-Rb27 cells
Lux = control virus infected
Rb = RB virus infected
Saos-2 cells
Saos-2 cells infected
with RB retrovirus
Tumor formation
assayed in “nude” mice
Huang et al Science, 242:1563-6 (1988)
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.”
pRb: What does it do?
pRb is a nuclear protein that undergoes phosphorylation and
dephospharylation in concert with the cell cycle
RB regulates progression through G1 phase
Un- and Hypophosphorylated 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
Un- and Hypo-phosphorylated Rb inhibits activity
of the E2F family of transcription factors
Figure 8.23c The Biology of Cancer (© Garland Science 2007)
Hyper-phosphorylated Rb cannot bind
and 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 = 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
Sites of RB Missense Mutations in Tumors
B
A
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
How is Rb activity regulated during the cell
cycle? By Cyclin/CDKs
However, E-CDK2 can phosphorylate Rb, only
AFTER Rb is phosphorylated by D-CDK4/6
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 Canonical
p16/pRB/E2F
“Pathway”
The RB Pathway in Human Cancer
(p16)
Sherr and McCormick, Cancer Cell ·2:103 (2002)
How can we study retinoblastoma in the lab?
with GEMs: Genetically Engineered Mice
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
“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 the
Mutant Mouse
The Phenotype of RB Mutant Mice
Homozygous embryos die at day 13.5
massive apoptosis in the CNS
anemia caused by lack of blood cells made by the fetal liver
therefore RB is not essential for cell division
Heterozygous animals are viable
they develop pituitary tumors with near 100% penetrance
these tumors lose the wild type RB allele
also develop thyroid adenomas
therefore 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 13.5
massive apoptosis in the CNS
anemia caused by lack of blood cells made by the fetal liver
therefore RB is not essential for cell division
Heterozygous animals are viable
they develop pituitary tumors with near 100% penetrance
these tumors lose the wild type RB allele
also develop thyroid adenomas
therefore 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?????!!!!!!