Upload
estella-cameron
View
220
Download
3
Tags:
Embed Size (px)
Citation preview
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?????!!!!!!