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
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)
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
Mutated
maternal
Normal
paternal
-/-
-/+
Mutated
paternal
Mutated
maternal
-/-
-/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?
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
Deletion = loss-of-function
probably a recessive mutation in the Rb
gene
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
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)
We have correlation
What about causation?
The RB gene is finally cloned
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
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
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
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 = 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
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
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
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 tumors
In 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 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)