Transcript Tumor suppressor genes

The Basics of Cancer Biology
• Lucio Miele, M.D., Ph.D.
Part II: “Partners in Crime -1”
Tumor Suppressors, Oncogenes,
Enablers and Turncoats
Cancer is a genetic disease
“Cancer is, in essence, a genetic disease. Although
cancer is complex, and environmental and other
nongenetic factors clearly play a role in many
stages of the neoplastic process, the tremendous
progress made in understanding tumorigenesis in
large part is owing to the discovery of the genes,
that when mutated, lead to cancer.”
Bert Vogelstein (1988)
NEJM 1988; 319:525-532
Abstract
Cancer is a distinct type of genetic disease
in which not one, but several, mutations
are required. Each mutation drives a wave
of cellular multiplication associated with
gradual increases in tumor size,
disorganization and malignancy. Three to
six such mutations appear to be required to
complete this process.
Vogelstein B and Kinzler KW
Trends Genet. 1993
What are Cancer Genes?
• From a genetic standpoint: Any gene
which, when mutated, increases the risk of
cancer is a cancer gene.
• From a phenotypical or functional
standpoint: Any gene which, when altered,
causes the transformation of normal cells
into cancerous cells is a cancer gene.
• Note: a gene that increases cancer risk
when mutated does not NEED to be
transforming! Its effect could be indirect
Traditional Types of Cancer
Genes
Cancer genes include those whose products:
1)
Directly regulate cell proliferation or survival
a. Tumor suppressor genes (cell proliferation inhibitory)
b. Oncogenes (cell proliferation promoting)
2)
Are involved in the repair of damaged DNA
a. DNA repair genes
b. DNA recombination genes (homologous recombination, nonhomologous end-joining)
3)
Regulate TSGs or oncogenes
a. Non-coding RNAs (miRNAs, lnRNAs)
b. Epigenetic modifiers
Oncogenes and Tumor Suppressors
• There are two broad categories of genes to think about when considering
cancer-forming mutations:
1. Oncogene - An oncogene is a gene whose normal activity promotes
cellular proliferation or division.
2. Tumor Suppressor - a gene that inhibits events leading towards
cancer. (This type of gene will be discussed in tomorrow’s lecture.)
Oncogene = gas pedal
Tumor Suppressor gene = brakes
Sporadic
Small % of cases
Relative large % of cases
Oncogenes:
• Mutation in one copy of the gene - Dominant
• promote cell proliferation
Tumor suppressor genes:
• Mutations in both copies of the gene - Recessive
• promote cell proliferation
DNA repair genes:
Usually recessive, loss-of-function mutations
that increase spontaneously and environmentally
induced mutation rates
Cancer Gene Census
•
•
•
•
Almost 2% of protein coding genes in humans are cancer genes
15% of cancer genes bear germline mutations
90% of them have somatic mutations
10% of them show both germline and somatic mutations
Wellcome Trust Sanger Institute at http://cancer.sanger.ac.uk/cancergenome/projects/census
Tumor Suppressor Genes
• Tumor suppressor genes (TSG) are genes that normally slow
down cell division, repair DNA damage, or cause cells to die (by
apoptosis or programmed cell death, by necrosis, by autophagy or
by or by mixed cell death mechanisms).
• When tumor suppressor genes are damaged, deleted or
epigenetically silenced, cells can grow out of control, which can
lead to cancer.
• More than 30 tumor suppressor genes have been identified,
including p53, BRCA1, BRCA2, APC, and RB1.
• An important difference between oncogenes and TSGs is that
oncogenes result from the activation (turning on) of protooncogenes, but TSGs cause cancer when they are inactivated
(turned off).
• Another major difference is that while the oncogenes develop from
mutations in normal genes (proto-oncogenes) during the life of the
individual (acquired mutations), abnormalities of TSGs can be
inherited as well as acquired.
Tumor suppressor gene
A tumor suppressor gene is like the brake pedal on a car. It normally
keeps the cell from dividing too quickly, just as a brake keeps a car
from going too fast. When something goes wrong with the gene, such
as a mutation, cell division can get out of control.
Tumor suppressors are mutated in both inherited
and non-inherited cancers
Inherited cancer
(Germline mutation)
Abnormal gene
Non-inherited cancers
(Somatic mutations)
Retinoblastoma
RB1
Retinoblastoma, osteosarcoma, lung cancer
Li-Fraumeni Syndrome (sarcomas,
brain tumors, leukemias)
p53
Brain tumors, skin cancers, lung cancer, head
and neck cancers, others
Melanoma
INK4a
Many different cancers
Colorectal cancer (due to familial
polyposis)
APC
Many GI cancers
Colorectal cancer (without polyposis),
Lynch Syndrome
MLH1, MSH2, or
MSH6
Colorectal, gastric, endometrial cancers
Breast and/or ovarian
BRCA1, BRCA2,
PALB2, CHEK2
Breast cancer, ovarian cancers
Wilms Tumor
WT1
Wilms tumors
Nerve tumors, including CNS
NF1, NF2
Small numbers of colon cancers, melanomas,
neuroblastoma
Von Hippel-Lindau Syndrome
VHL
Certain types of kidney cancers
Examples of Tumor Suppressor
Genes
•
BRCA 1, BRCA 2:
–
–
–
•
Wilms’ Tumor Suppressor (WT1):
–
–
•
Both proteins involved in DNA repair pathway (usually double
strand break repair).
Loss of these proteins can increase the acquisition of subsequent
mutations and chromosomal instability.
Mutations greatly increase the susceptibility to develop breast or
ovarian cancer in women.
This gene encodes a transcription factor with multiple functions
Mutation is associated with Wilms’ tumor (a renal cancer called
nephroblastoma).
Von Hippel‐Lindau (VHL):
–
–
VHL protein degrades protein important for promoting
angiogenesis, thereby inhibiting the ability to form new blood
vessels.
Mutation is associated with renal cell carcinoma and other types
of cancers.
Functions of Tumor Suppressor genes
•
1. Regulator of cell cycle:
–
Serve as genes that insure proper progression through the cell cycle
to mediate proper cell division.
–
Rb gene, INK-4 gene, P53
•
2. Inducer of apoptosis:
–
Proteins that promote apoptosis in response to improper signals,
presence of DNA damage, etc.
–
Bim, a pro-apoptotic member of the Bcl-2 family of genes
•
3. Transcription factors:
–
Repressor transcription factors: WT1 is a repressor that appears to
suppress a factor (Insulin like growth factor) that can contribute to the
development of a tumor.
–
Activator transcription factors: Members of the SMAD family are
activated by TGF-β, leading to inhibition of cell proliferation
•
4. Regulator of angiogenesis response (VHL)
Hereditary cancer susceptibility
syndromes
About 10% of the most
common cancers are
due to a hereditary
predisposition
• Examples:
Breast/ovarian cancer
Colorectal
Retinoblastoma
RB - the first tumor suppressor
gene cloned (1986)
Loss of both alleles of RB
leads to the development of
Retinoblastoma in humans
RB is seldom mutated in sporadic human cancers
Inhibits proliferation, promotes differentiation but
also inhibits apoptosis
Children with RB1 mutations will develop retinoblastoma very early
in life because retinoblastoma originates from neuroblasts during retinal
development. Therefore, retinoblastoma is not observed in adults
Burkhart D et al., Nat Rev Can 2008
Current model of Rb in G1 cell cycle regulation
Narasimha AM et al., eLife, 2014
Rb functions as a protein adaptor with at least 4
mechanisms of action: multitasking proteins
Burkhart D et al., Nat Rev Can 2008
Burkhart D et al., Nat Rev Can 2008
Burkhart D et al., Nat Rev Can 2008
Burkhart D et al., Nat Rev Can 2008
Two-Hit Hypothesis
• The relatively high frequency of the
second hit explains the early age of onset
of tumors and that they are often bilateral.
• In sporadic cancers two independent rare
events within the target tissue must occur
to form a tumor; hence, the later age of
onset.
• Loss of Heterozygosity (LOH) analysis can
help identify tumor suppressor genes.
Knudson two-hit model
1st hit
2nd hit
1st hit
LOH
LOH
D10S107
T/T
D17S855
T/C
C/C
Loss of Heterozygosity (LOH)
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Mechanisms leading to LOH
p53: the most commonly mutated
TSG in human cancers
DNA damage
Oncogenic signals
p53
Inactive
GADD45
DNA repair
p53R2
PP
P
p53
p21
Cell cycle arrest
post-translational
modifications Active
Bax
PUMA
Noxa
Pigs
Apoptosis
p53 - a Classic Tumor Suppressor
• Point mutations account for nearly
75% of all of the mutated forms of
p53 found in tumors. (Compare
to the other commonly mutated
genes found in cancer.)
• These mutations almost always
lead to amino acid substitutions.
• A majority of the identified point
mutations (from over 15,000
mutated p53 genes sequenced in
cancers) are present in the DNAbinding domain of the gene.
• Therefore, these mutations inhibit
the ability of p53 to bind to DNA
and therefore inhibit its ability to
activate (or repress) the genes
listed in the previous table.
p53 - a Classic Tumor Suppressor
• In addition to loss of or mutation to
p53, p53 activity can be altered
through deletion or mutations to other
genes.
• MDM2 binds to p53, inhibits the ability
of p53 to act as a transcription factor,
and targets it for ubiquitylation and
subsequent degradation.
• The action of MDM2 insures the 20
minutes half life of p53 in normal cells.
• The amplification of the MDM2 gene
in cancer would therefore interfere
with the normal ability of p53 to
respond to extracellular stimuli and
prevent the required cell cycle arrest
and promotion of apoptosis.
The effects of p53 are DOSE-DEPENDENT
• When activated at low levels, p53 binds differentially to
promoters that encode genes that suppress cell cycle
progression and allow DNA repair (e.g., p21)
• When DNA damage is not promptly repaired and active p53
accumulates, it binds lower affinity chromatin sites that
contain the promoters of genes that cause apoptosis (e.g.,
NOXA, DAXX)
• This mechanism permits DNA repair and survival if the
damage is not irreparable, and causes the cell to commit
suicide if the damage is irreparable
• Hence, p53 has been called “the guardian of the genome”
DNA
Damage
E2F
(1)
(2)
1) Wt-p53 binds to p21 promoter; 2) Elevated p21 inhibits CDK2/4 kinases, reducing
Rb1 phosphorylation; 3) unphosphorylated Rb1 forms complex with E2F; 4) E2F
cannot activate genes important for G1/S transition - no tumor.
2) Mut-p53 is unable to bind to p21 promoter, causing reduced p21 level; 2) CDK2/4
kinases induce Rb1 phosphorylation; 3) more E2F free from Rb1 complex; 4) more E2F
will promote cell cycle progression - Tumor formation.
Dominant-negative
mechanism
WTp53 WTp53
Transcriptional activation
WTp53 WTp53
Tetramer formation
Mutant
p53
WTp53
WTp53
Mutant
p53 WTp53
p53 mutations
• More than 50% of human tumors contain p53 mutations.
• 80% are missense mutations in the DNA binding domain.
• “Hot spot” mutations at pp175, 248, 249, 273, and 281
• Mutations of p53 are more frequent events and show worse
prognosis compared to deletion of p53 gene.
175
248 273
249
N
I
Activation domain
II
III
IV
DNA binding domain
281
V
C
Tetramerization domain
Some mutations turn p53 from a TSG into an
oncogene: a Turncoat!
• Over 50% of human cancers either have lost p53 protein or have mutations in
the gene.
• About 70% of Li-Fraumeni syndrome patients carry p53 alterations
(heterozygous).
• Loss of p53 in mice causes tumors dependent on gene dosage.
• Mutant p53 accumulates as a nuclear phosphoroprotein.
• The mutant p53 functions as dominant-negative that inactivates wild-type p53
function through a tetramer formation.
• However, some forms of mutant p53 show unexpected oncogenic functions
which can not be explained simply by loss of p53. These mutants activate
transcription at some chromatin sites
• Therefore, p53 (mutant p53) was originally believed to be an oncogene.
Identification of TSG
• Before 2007
- Linkage analysis of familial cancer families and
positional cloning of TSG
- Loss of heterozygosity analysis to clone TSG
• After 2007
- Exome sequencing of familial cancer family
members and bioinformatics analysis
- Mutation co-segragates with phenotype in family
Cancer gene (TSG) discovery
by Exome sequencing
Exclude
variants
in control
Exome capture
Exome sequencing
Some tumor suppressors don’t encode proteins
miRNAs and lncRNAs can be
TSGs
• By downregulating the expression of
oncogenes
– mRNA stability
– mRNA translation arrest
• By regulating the expression of cancer risk
genes that are not themselves oncogenes
– E.g., genes in the immune system that control
cancer immune surveillance
• By regulating the splicing of transcripts or
the activity of regulatory elements in DNA