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Tumor suppressor gene
Tumor suppressor gene
I. History of tumor suppressor gene
1. Cell hybridization
2. Human genetics
II. Identify of suppressor genes
III. Suppressor genes:
1 Retinoblastoma (Rb)
2. P53
3. APC
4. Deleted in colon carcinoma (DCC)
5. Neurofibromatosis tumor suppressor
Tumor suppressor gene
gene (NF-1)
6. Wilms tumor (WT)
7. Others (KLF6, PTEN)
IV. tumor metastasis suppressor gene--Nm23,
CRMP-1
V. tumor metastasis promotion gene—RhoC,
chemokine, CXCR4, CCR7, PRL-3.
Tumour-suppressor genes.
Cells have brakes, called tumour-suppressor genes.
These keep cell numbers down, either by inhibiting
progress through the cell cycle and thereby preventing
cell birth, or by promoting programmed cell death
(also called apoptosis).
Tumor suppressor genes
•
Genes whose loss contributes to cancer are
classified as tumor suppressor genes because
when they function normally they help to
suppress tumors
•
Normal functions of tumor suppressor genes is
to suppress cellular growth , promote cellular
death
•
Also known as anti-oncogenes
Cell hybridization
CA
N
Normal bronchial cell
Mucoepidermoid
•
Finite lifespan
Lung carcinoma cell •
Non-tumorigenic
•
Tumorigenic
•
Immortal
CA/N
•
Finite lifespan
•
Non-tumorigenic
Human Genetics
LOH:
6Q21, gastric cancer
7Q31, prostate cancer
17Q, ovarian cancer
Human Genetics
Current opinion in genetics & develop. 10:81-93, 2000.
Identify of suppressor genes
1. Construction of subtractive library
2. Differential display
3. c-DNA microarray
4. Proteomic approach
Retinoblastoma (Rb) gene
1. Familial (40%): mean age 14 months, bilateral (appeared
in both eyes), average 3 independent tumors have family
history, prezygotic mutation--95% tumor within 7 year
period.
2. Sporadic: no family history, unilateral (one eye). mean
age 30 months, only one tumor. Solely in postzygotic 2
independent events must have occurred in the Rb gene.
One mutation-- growth selection advantage to induce 2nd
mutation.
•
White glow or
glint in one or
both eyes
•
Have white
pupils in
pictures
•
Has crossed or
misaligned eyes
pRb and retinoblastoma
The disease; retinoblastoma
The protein; pRb
TSSTTT
LXCXE proteins (HDAC, E7)
E2F (no LXCXE motif)
A/B pocket
SSS
A
B
SSSSS TT
C-term
NLS
cyclin docking
Retinoblastoma (Rb) gene
3. 180 kb DNA, 4.7 kb mRNA, 928 amino acid, - 105 KD protein
(MW 110-114 KD), different degrees of phosphorylation located on
chromosome 13.
4. Phosphonuclear protein, has DNA binding ability suggest a role in
transcriptional regulation
5. DNA tumor virus association. Adenovirus E1A, SV40 large T
antigen, human papilloma virus (Hpv) E7 oncoproteins form
complex. Inactive Rb gene by these oncoproteins to transform cells.
6. Mutant Rb protein fail to bind large T antigen.
Retinoblastoma (Rb) gene
7. E7 protein of the non oncogenic human papilloma viruses (HPV6B,
HPV11) bind p105-Rb with lower affinity than the oncogenic HPV
16, 18
p
E2F homo-or hetero-dimer
C-myc, myb,cdc2
Rb
TTTCGCGC
Rb/E2F
Rb and cell cycle entry
Mitogens
Sherr (2000)
Cancer Research
60:3689-3695
CDK 4/6
Cyclin D
P
E2F
Rb P
P
Rb
S phase
entry
+
E2F
CDK 2
Cyclin E
Cyclin E
E2F
Cyclin E gene
Stephen Taylor
4/10/02
BS324: Molecular Biology of Cancer.
Lecture 3: The Cell Cycle, Slide: 15
Retinoblastoma (Rb) gene
8. One of the most well-characterized proteins that interacts with Rb
is the transcription factor E2F. E2F binds to unphosphorylated Rb.
Upon phosphorylation of Rb, E2F is no longer able to form a
complex with Rb and thus dissociates. Free E2F is then able to
activate a number of target genes. E2F recognizes and binds to a
consensus sequence of TTTCGCGC, which is found in the
promoters of a number of genes that are involved in regulating cell
growth, including c-myc, myb, cdc2, dihydrofolate reductase,
thymidine kinase, and the E2F promoter itself.
Retinoblastoma (Rb) gene
9. underphosphorylated in G1, becomes heavily
phosphorylated just prior to the G1 to S transition, remains
phosphorylated in S, G2, and most of M, and reverts to an
under phosphorylated state at or before the M-G1 transition
G1/G0
S
Active Rb
Unphospho.
cdc2kinase
G2
Rb-p,
inactive
M
Retinoblastoma (Rb) gene
10. All 3 viral oncoproteins (T-Ag, E1A, E7) bind to the
underphosphorylated (active form) form of Rb protein
11. Rb is active in growth suppression only in G0 and G1 (under
phosphorylated), and is phosphorylated and rendered inactive for
the remainder of the cell cycle.
Retinoblastoma (Rb) gene
12. Inactivation of Rb (or P53), 3 mechanisms:
(i) mutation gene itself
(ii) phosphorylation
(iii) by binding to the viral oncoprotein (T-Ag, E1A, E7)
13. Introduce Rb gene into Rb cell line (no Rb protein)
(i) inability to form colonies in softagar
(ii) inability to form tumors in nude mice
Deacetylation removes acetyl groups from histone tails, causing the
histones to wrap more tightly around the DNA and interfering with
the transcription of genes by blocking access by transcription factors.
Treatments
Current:
-Emulation (removal of eye)
-Radiation
Future:
-to shrink the retinoblastoma so it can be
treated with a laser freezing and local plague.
Prognosis
•
Emulation, lost of the eye. 40% chances of
loosing both eyes
•
Untreated, retinoblastoma is almost always
fatal
Rb

Enhanced Mdm2 activity inhibits pRB
function via ubiquitin-dependent
degradation
*The EMBO Journal (2005) 24, 160–169
P53: both oncogene and tumor
suppressor gene
•
it is mutated in about half of all human cancers
•
it was initially identified as a tumor specific nuclear
antigen of molecular weight 53kDa
•
isolation of the gene from tumor cells yielded an
oncogene: when it was transfected into normal cells it
could transform them together with ras
•
when the gene was isolated from normal cells, it was
discovered that the tumor derived gene was mutated,
and when the wild-type was transfected into tumor cells
it stopped their growth, ie it was a tumor suppressor
gene
P53 gene
1. P53 (1970) was found initially through its association
with SV40 large T oncoprotein in virus transformed
cells it acts as a negative regulator of cell growth
2. P53 gene 16-20kb of DNA on the small (p) arm of
chromosome 17 in human, 11 of mice
3. 2.2-2.5 kb mRNA producing 53 kd nuclear
phosphoprotein
4. primary a.a. sequence (375 a.a.) - 3 distinct domain.
The p53 pathway in response to DNA damage
ATM(DNA-dependent protein kinase)
No DNA damage
DNA Damage Ionizing radiation
ATM ( DNA-dependent
protein kinase; Rad 3)
PPP
p53
Mdm2
Blocks transactivation
Targets for degredation
p53
Mdm2
PPP
PPP
p53
P53 is unstable and is
degraded by a ubiquitindependent mechanism.
Phosphorylation in response to
DNA damage prevents
association with Mdm2
and stabilizes p53.
Ataxia telangiectasia–
mutated gene (ATM) is
a 350-kDa protein
whose function is
defective in the
autosomal recessive
disorder ataxia
telangiectasia (AT).
Structure of P53
The domain structure of p53 and binding partners
Transactivation
May help to
stabilise p53
Hsp70
TBP
These
interactions
promote
transcription
Oligomerisation/
Nuclear Localisation/Export
p53
SV40 T antigen
Blocks p53 function
95% of mutations in p53 found in cancer cells lie within this domain
TAF70
TAF31
TFIIH
These
interactions
block p53
transcriptional
activation
Sequence specific DNA binding
MDM2
E1B55
•p53 is a tetramer, which does not have an exposed
nuclear export signal (NES), therefore the NLS
predominates and p53 remains nuclear.
•p53 monomers have an exposed NES which is
more active than the NLS therefore p53 is
cytoplasmic where it is targeted for rapid
degradation.
•Mutations which influence oligomerisation of p53
influence its subcellular localisation and therefore
activity.
P53 gene
5. Mouse and human proteins share 80% homology at a.a.
level. murine p53 blocked the binding of DNA
polymerase to SV40 large T antigen. SV40 DNA
replication was blocked by p53.
6. Detected in all cells, present at low levels, half life 6-20
min. Mutations have been shown to increase the stability
of p53 protein, to alter the conformation of the protein and
to result in complex formation with a HSP 70.
p53 tumor suppressor

P53 is a transcription factor which monitors the state of
DNA.

It inhibits cell cycle progression if there is damage.

Following DNA damage (e.g. by radiation), p53 levels
rise, and proliferating cells arrest in G1.

This allows time for DNA repair prior to the next round of
replication.

P53 mediated cell cycle arrest is mediated by stimulation
of expression of p21CIP1, the cyclin kinase inhibitor.
p53 tumor suppressor

The functional expression of the gene p53 increases the
sensitivity of the expressing cells to activation of
programmed death induced by a wide variety of
damaging agents.

Such a universally resistant genotype would provide a
great selective advantage to any cell so affected.

For example,

Mice with both copies of p53 knocked out develop
multiple malignancies,

Mutation in p53 is associated with many human
cancers.
P53 arrests cells in the G1 stage of the cell cycle if there is damage
to the DNA; this arrest is to allow repair. If repair doesn’
t occur,
the cell undergoes apoptosis.
P53 gene
7. Mutant p53 protein levels in transformed cells are elevated
5-100 fold, half life becomes many hours. Wt p53 assemble
into homotetramers and higher order homo-oligomeric
structures.
8. mutant p53 + ras oncogene can transform cells but not wt
p53
9. p53 is found in oligomeric complex with oncoprotein
SV40 large T antigen, and adenovirus E1b
P53 gene
10. SV40 large T, adenovirus E1b form complex with wt p53
and inactive wt p53. Papilloma virus E6 + p53 --- proteolytic
pathway
11. p53 is documented as the most frequently mutated gene in
human cancer (e.g. bladder, liver, brain, breast, lung, colon)
12. p53 and Rb show several parallels:
(i) growth suppressing proteins found in the nucleus
(ii) both are targeted for sequestration (destruction) by the
oncoproteins of SV40, adenovirus, and human papillomavirus.
P53 gene
(iii) both proteins must be eliminated from the cell's growthsuppressing pathway in order for full viral transformation to
occur.
13. Point mutations create carcinogenic p53.
Viral oncogenes and p53 network
(Nature 408:306, 2000)
P14 ARF
1. p14ARF arrests the cell cycle in a p53-dependent
manner
2. p14ARF binds to MDM2 and promotes the rapid
degradation of MDM2.
3. ARF-mediated MDM2 degradation is associated with
MDM2 modification and concurrent p53 stabilization.
(Cell 92: 725, 1998)
P53 gene
1. Acetylation of lysine or phosphorylation of serine near
the carboxy terminus of p53 can enhance the
binding of p53 to DNA.
2. Phosphorylation of the amino terminus of p53 does
not affect its DNA binding, but does affect its
affinity for MDM2 and subsequently leading to
degradation of p53.
3. p14ARF binds to MDM2 and inhibits its activity.
Nature 408:306, 2000
The p53 network
Mediators of p53 function
1. p21 (waf1/cip1) is a cyclin kinase inhibitor
p21 can bind to the cyclin/CDK complex and inhibit its kinase activity
p21
p53
p21 gene promoter
p21
Cyclin/Cdk complex
-inactivated
-G1 arrest
2. p53 also induces proteins involved in DNA repair.
P53 network-cell cycle
inhibition
1. P53 protein stimulates the p21 WAF/CIP1, an inhibitor of
cyclin-dependent kinase (CDKs). CDK are key regulator
of the cell cycle.
2. p21 WAF/CIP1 inhibit both the G1 to S and G2 to mitosis.
3. P53 protein stimulates 14-3-3which sequesters
cyclinB1-CDK1 complexes, help to maintain a G2 block.
4. Reprimo can also arrest cells in G2 phase.
P53 network-Apoptosis
1. Bax apoptosis-inducing protein. P53 stimulates Bax but
inhibits Bcl-2.
2. NOXA and p53AIP1 genes are activated by p53.
These proteins induce apoptosis when over-expressed.
3. P53 mediators induced apoptosis include
TNT receptor, Fas, PIDD, toxic reactive oxygen etc.
p53 and the DNA Damage Response
DNA Lesion
x
Stabilisation of p53 which normally has a half life of 20 minutes
p53
WAF/CIP1 gene - transcriptional
activation by p53
p21
cyclin
CDK
Kinase
activity
Review on p53:
Cell 88 p323 (1997)
Apoptosis
DNA Repair
cyclin
p21
CDK
No kinase
activity
No phosphorylation
of Rb therefore no
release of E2F-1
therefore cell remains in G1 allowing time for DNA repair.
Go
G1
M
S
G2
BAX
which
promote
apoptosis
Putative signal pathway of p53
P53 pathway
1 P53 block RPA(replication protein A)replication
2. P53p21-CIP
arrest
cdk kinase
 cell cycle
3. GADD 45 (growth arrest DNA damage)/p21 can
bind to PCNA (proliferating cell nuclear antigen)
and inhibit DNA replication.
4. Wtp53 bax apoptosis
bcl2
ONCOGENE CHECKPOINT
P19 ARF and p53
ARF = Alternative Reading Frame.
Both p16 and p19 are in the Ink 4 locus.
--Ink 4 locus mice—tumor prone but viable.
Many tumors have large deletions and would delete p19
and p16.
INK 4 Locus: p16 and p19
Sherr (2001)
Cell:106:531)
•
Immortal MEFS
•
RAS-susceptible
•
Spontaneous tumors
•
Acceleration of Myc
induced B-Cell Lymphoma
•
No melanoma
•
Normal MEFS
•
RAS-resistant
•
?Spontaneous tumors
•No acceleration of Mycinduced B-Cell Lymphoma
•
Melanoma
•
Immortal MEFS
•
RAS-susceptible
•
Spontaneous tumors
•
Acceleration of Mycinduced B-Cell Lymphoma
•
No Melanoma
P16/p19
1. P16-INK4a acts as an inhibitor of cyclin D1dependent kinases 4 or 6 and prevents the
phosphorylation of Rb, thereby maintaining an
active Rb and blocking the exit from the
G1phase.
2. P19ARF inhibits cell growth by interacting
with MDM2, thereby blocking MDM2mediated degradation of the p53 protein and
neutralizing MDM2’
s inhibition of p53
activity.
p53 Pathway
DNA Damage
ATM
DNA-PK
???
Oncogenic Stimuli
(e.g.E1A, MYC)
p53
BAX
p21
mdm2
P19 ARF
MD
M2
Ub Ub Ub
p53
Tumor Suppressor Genes
p53, p19
From Sharpless and DePinho (1999)
Current Opinions in Genetics and Dev. 9:22
Oncogenes
MDM2
DEGRADATION
Intersection of p53 and RB Pathways
Dimerization and processing of procaspase-9 by redox
stress in mitochondria.J Biol Chem. 2004 Jan 27
EMBO J. 2004 Jan 28;23(2):460-72. Epub 2004 Jan
08. Coupling of caspase-9 to Apaf1 in response to loss
of pRb or cytotoxic drugs is cell-type-specific.
P53 and RB Crosstalk
p53
Ink 4
P19-ARF
p16
Bax
E2F1
mdm2
RB
Cyclin D
CDK4
BCL2
Caspases
E2F1
Apoptosis
S-Phase
The human p73 promoter: characterization and identification
of functional E2F binding sites. Neoplasia. 2002 MayJun;4(3):195-203.
Apoptosis
P19 ARF
p73
1. E2F1 induces phosphorylation of p53 that is
coincident with p53 accumulation and apoptosis.
Mol Cell Biol. 2002 Aug;22(15):5308-18.
MDM2/Rb

Enhanced Mdm2 activity inhibits pRB function
via ubiquitin-dependent degradation
The EMBO Journal (2005) 24, 160–
169.

The central acidic domain of MDM2 is critical
in inhibition of retinoblastoma-mediated
suppression of E2F and cell growth.
J Biol Chem. 2004 Dec 17;279(51):53317-22.
APC adenomatous polyposis coli
Tumour suppressor genes located in the 5q21 region
on the long arm of chromosome 5. The mutation of
these genes is associated with familial adenomatous
polyposis (apc stands for adenomatous polyposis
coli) and gardner's syndrome, as well as some
sporadic colourectal cancers.
MSH
Multigene family coding for proteins involved in
mismatch repair. Homologous to S. cerevisiae MutS .
Included in family are MSH1, MSH2, hMSH2,
hMLH1, hPMS1, hPMS2 and probably GTBP.
Wnt
Multigene family encoding various secreted signaling
molecules important in morphogenesis. First member was
Drosophila wingless , but many vertebrate homologues are
now known. Wnt -1 induces accumulation of -catenin and
plakoglobin and affects the association of APC tumour
suppressor protein with catenin.
plakoglobin
Polypeptide (83 kD) present at cell-cell but not cellsubstratum contacts. Associated with desmosomes and with
adherens junctions: soluble 7S form present in cytoplasm.
APC (Adenomatosis Polyposis Coli)
•
An autosomal dominant polyposis syndrome in which the
colon contains few to thousands of adenomatous polyps,
often occurring by age 15 to 25.
•Protein in cytoplasm degrades B-catenin, a signaltransduction protein that can enter the nucleus and activate
transcription
•B-catenin is also important in cell-cell adhesion - therefore
role in invasion as well (Proteins associated with the
cytoplasmic domain of uvomorulin and presumably
involved in linking to the cytoskeleton. ( -catenin 102 kD;
-catenin 88 kD; -catenin 80 kD))
Uvomorulin
Glycoprotein (120 kD) originally defined as the
antigen responsible for eliciting antibodies capable
of blocking compaction in early mouse embryos (at
the morula stage), and inhibiting calciumdependent aggregation of mouse teratocarcinoma
cells. May be the mouse equivalent of LCAM, the
chick cell adhesion molecule.
Colon Cancer
-common
Polyps in familial polyposis coli
polyps
FAP
(Familial
Adenomatous
Polyposis)
-rare
-inherited
-associated with
benign
adenomatous polyps
in early adulthood
-some polyps progress
to invasiveness
APC
•SIGNAL TRANSDUCTION INHIBITORS
•named for rare familial condition
- hundreds to thousands of precancerous colonic
polyps, at least one of which will progress to cancer
by young adulthood (one allele inherited, one
acquired)
- but also seen in most sporadic colon carcinoma.
APC
The APC tumor suppressor is encoded by the
FAP locus
In 1991, Kinzler and Groden identified the
tumor suppressor APC encoded by the FAP
locus.
Bert Vogelstein
The tumor suppressor APC
What does APC do?
-Interacts with -catenin (cadherin-based cellular adhesion).
-Then the Wnt signaling pathway was discovered in Drosophila
-is loss of APC sufficient to cause polyp formation?
APC
The APC gene, mutational spectrum, clinical
correlates, and APC protein structure
1. The conventional form of the APC gene contains 15 exons, with
the most 3’exon containing over three-quarters of the 8,535 bp
of coding sequence.
2. Most APC mutations occur within the central third of the gene,
designated the mutation cluster region. This region contains two
of the most commonly found mutations, 5-bp deletions creating
stop codons at positions 1061 and 1309, and a nucleotide
polymorphism (ASH) in the Ashkenazi Jewish population .
3.
Regions of the gene where mutations are associated with
congenital hypertrophy of the retinal pigmented epithelium
(CHRPE) and desmoid tumors (aggressive fibromatosis) are
shown.
The WNT signaling pathway; absence of Wnt
signal
cell
Wnt receptor: Frizzled;
7-pass transmembrane
Free -catenin is destabilized by
binding to the destruction complex
-glycogen synthase kinase 3
-axin/conductin
-APC
APC binds to -catenin and GSK3
GSK3phosphorylates -catenin
P--catenin is ubiquinated and destroyed
by the proteosome
WNT signaling pathway; presence of Wnt signal
Presence of Wnt:
GSK3is inactivated
-catenin is stabilized
-catenin shuttles to the nucleus
-catenin binds to the TCF-family
of transcription factors and serves as
an essential co-activator
APC
Wnt/-catenin signaling: target genes
Gene
Organism/system Direct/Indirect
c-myc human colon cancer
yes
Cyclin D human colon cancer
yes
Tcf-1
human colon cancer
PPARdelta human colon cancer
c-jun
human colon cancer
fra-1
human colon cancer
uPAR
human colon cancer
yes
yes
yes
yes
?
up/down
up
up
Ref.
He 1998
Tetsu 1999
Shtutman 1999
up
up
up
up
up
Roose 1999
He TC, et al 1999
Mann B, 1999
Mann B, 1999
Mann B, 1999
So, just another signaling pathway that controls cell cycle.
Deleted in Colon Carcinoma
(DCC) Gene:
Tumour suppressor genes located in the 18q21 region of
human chromosome 18. The absence of these genes is
associated with the formation of colourectal cancer (dcc
stands for deleted in colourectal cancer). The products of
these genes show significant homology to neural cell
adhesion molecules and other related cell surface
glycoproteins.
Deleted in Colon Carcinoma (DCC)
Gene:
1. Allele losses on chromosome 18q21 in colorectal
carcinoma not adenomas
2. 70% colon cancer, DCC reduced expression, point
mutation, insertion, deletion.
3. Genomic DNA--1000 kb, amino acid--190 Kd
transmembrane phosphoprotein cell surface receptor.
Deleted in Colon Carcinoma (DCC)
Gene:
4 Ligand for DCC is Netrin (Cell 87:175, 1996). Netrins
are chemoattractants for commissural axons in the
vertebrate spinal cord, but the mechanisms through which
they produce their effects are unknown.
5. DCC mRNA is present in a variety of normal tissue
including colonic mucosa, but undetectable in 90% of
colon tumor cell lines.
Netrin
Genes identified in studies of vertebrate neuronal
development. Netrins are chemotropic for embryonic
commisural neurones: netrin 1 is secreted by the
floorplate, whereas netrin 2 is distributed ventrally
except for the floorplate. The netrins are homologous
to the product of unc 6, a gene identified in studies of
neuronal development of the worm, Caenorhabditis
elegans.
Deleted in Colon Carcinoma (DCC)
Gene:
6. Gene encodes a member of the immunoglobulin
superfamily with homology to neural cell adhesion
molecules that presumably regulates cell behavior
through interation with other cells
•
Kruppel-like factor 6 (KLF6) is a zinc finger
transcription factor of unknown function. Here, we show
that the KLF6 gene is mutated in a subset of human
prostate cancer.
•
Loss-of-heterozygosity analysis revealed that one KLF6
allele is deleted in 77% (17 of 22) of primary prostate
tumors.
•
Sequence analysis of the retained KLF6 allele revealed
mutations in 71% of these tumors.
Functional studies confirm that whereas wildtype KLF6 up-regulates p21 (WAF1/CIP1) in a
p53-independent manner and significantly
reduces cell proliferation, tumor-derived KLF6
mutants do not. Our data suggest that KLF6 is
a tumor suppressor gene involved in human
prostate cancer.
KLF6 (Kruppel-like factor)

Frequent inactivation of the tumor suppressor
Kruppel-like factor 6 (KLF6) in hepatocellular
carcinoma.
Hepatology. 2004 Nov;40(5):1047-52.

A new role for the Kruppel-like transcription
factor KLF6 as an inhibitor of c-Jun protooncoprotein function.
Oncogene. 2004 Oct 28;23(50):8196-205
PTEN:phosphatase and tensin homolog deleted on
chromosome ten; MMAC1/PTEN; TEP-1)
TEP1:TGFa-regulated and epithelial cell-enriched
phosphatase).Also termed PTEN or MMAC1 (mutated in
multiple advanced cancers 1).
A protein tyrosine phosphatase with homology to tensin, is a
tumour-suppressor gene on chromosome 10q23. Somatic
mutations in PTEN occur in multiple tumours, most
markedly glioblastomas. Germ-line mutations in PTEN are
responsible for Cowden disease (CD), a rare autosomal
dominant multiple-hamartoma syndrome. Mutated in
MMAC1/PTEN (multiple advanced cancers 1/phosphatase
and tensin) homologue.
Cowden disease
(Cowden is the family name of the first reported
case) (also called multiple hamartoma syndrome
(MHS)), autosomal dominant inherited condition
characterized by multiple hamartomas and neoplasms
of ectodermal, mesodermal and endodermal origin. It
occurs mainly in white persons and the mean age at
the time of the diagnosis is 41 years.
Tensin
Actin-binding component of focal adhesions and
submembranous cytoskeleton. Has SH2 domain and
can be tyrosine phosphorylated; speculated that it
may link signalling systems with the cytoskeleton.
The mechanisms controlling neural stem cell proliferation
are poorly understood. Here we demonstrate that the PTEN
tumor suppressor plays an important role in regulating
neural stem/progenitor cells in vivo and in vitro.
Pten encodes phosphatase.
Mice lacking PTEN exhibited enlarged, histoarchitecturally
abnormal brains, which resulted from increased cell
proliferation, decreased cell death, and enlarged cell size.
•
Neurosphere cultures revealed a greater proliferation
capacity for tripotent Pten -/- central nervous system
stem/progenitor cells, which can be attributed, at least in
part, to a shortened cell cycle.
•
However, cell fate commitments of the progenitors were
largely undisturbed. Our results suggest that PTEN
negatively regulates neural stem cell proliferation.
•
PTEN phosphatase promotes apoptosis by preventing
Akt activation, antagonises the anti-apoptotic effect of PI3K.
Structure of PTEN
•
The N-terminal-phosphatase domain (amino acids 1-185) is shown with
the catalytic core.
•
The C-terminal domain (amino acids 186- 403)
C2 domain -lipid-binding
PEST domains-regulate protein stability
PDZ domain-is important in protein-protein interactions.
CK2 phosphorylation sites -are important for stability
PTEN negatively regulates PKB/Akt-mediated
cell survival
PTEN/Mdm2/P53
summary
Oncogenes and tumor suppressors work together to achieve
the full malignant phenotype
Tumor suppressors are proteins that play a role in
controling cell cycle, in check-point control and in
maintenace of a differentiated phenotype
Loss of tumor suppressor genes lead to cell cycle defects
such as loss of check point control
Inherited mutations in tumor suppressor genes predisposes
the individual to cancer
Tumor cell invasion and
metastasis
Molecular aspects of invasion
A. Invasion-suppressor genes
•
E-cadherin
•
Tissue inhibitors of metalloproteinases (TIMPS)
•
plasminogen activator inhibitors (PAIs)
•
Anti-metastatic gene nm23
Summary of literatures
1. Association of nm23-H1 allelic deletions with
distant metastases in colorectal carcinoma
(Lancet 1991; 338: 722-24. )
2. A low nm23H-1 gene expression identifying
high malignancy human melanomas
(Melanoma Research 1994, 4, p. 179-184 )
3. Levels of nm23 Messenger RNA in metastatic
malignant Melanomas: Inverse Correlation to
Disease Progressions (Cancer Res. 52, 60886091, 1992 )
Molecular aspects of invasion
B. Invasion-Promoter Genes
•
Lytic enzymes ( Collagenase)
•
Motility factors (epithelial mesenchymal transition
EMT) , extracellular matrix(ECM)remodeling
•
Interleukin-1(IL-1)
•
hepatocyte growth factor (HGF), Scatter factor
(SF)- receptor, c-Met
Epithelial cells (left) are tightly interconnected in sheets by
numerous cell-cell interactions; their organization is determined by
the basement membrane (lower left). During epithelial-mesenchymal
transition (EMT), epithelial cells become independent of their
neighbors and can move freely (center). In this way, tumors derived
from epithelial cells become motile and may invade lymph or blood
vessels (right).
Phenotype of tumor progression
Molecular mechanism of cancer invasion
Tumor metastasis suppressor gene
Nm23(nonmetastatic protein 23)
CRMP-1
The Tumor Metastasis Suppressor Gene Drg-1 Downregulates the Expression of Activating Transcription
Factor 3 in Prostate Cancer
The expression of the KAI1 gene, a tumor metastasis
suppressor, is directly activated by p53
[Expression of tumor metastasis-suppressor gene
KiSS-1 and matrix metalloproteinase-9 in portal
vein tumor thrombus of hepatocellular carcinoma]
TSLC1 is a tumor suppressor gene associated with
metastasis in nasopharyngeal carcinoma.
KAI1/CD82, a tumor metastasis suppressor
Tumor metastasis suppressor
gene---nm23
1. nm23 is a tumor metastasis associated gene.
2. nm23 RNA levels were highest in cells and tumors of
relatively low metastatic potential in the following
systems.
(i) murine k-1735 metanoma cell lines
(ii) N-nitrosomethylurea - induced rat mammary carcinomas
(iii) human breast carcinoma
(iv) human hepatoma
Tumor metastasis suppressor
gene---nm23
(v) human melanoma
3. nm23 is homologous to NDP kinase. Nucleoside
diphosphate kinase catalyzes a transfer of the terminal
phosphate of 5' - triphosphate nucleotides to 5' diphosphate
nucleotides (RNA or DNA).
4. Encode a 17 kd nuclear and cytoplasmic protein.
Tumor metastasis suppressor
gene---nm23
5. Human nm23-H1---chromosome 17.
6. Amino acid sequence contain leucine repeats,
basic amino acid region, suggestive of a leucine
zipper involved in transcriptional regulation.
Tumor metastasis suppressor
gene---nm23
NDPK(Nucleoside diphosphate kinase)是細胞中負責
nucleosides triphosphate生合成(biosynthesis)的酵素，
在in vitro的酵素活性分析，NDPK能將所有
nucleosides diphosphate磷酸化為nucleosides
triphosphate。因此NDPK是DNA生合成途徑中的一個
重要酵素，在細胞中負責製造DNA 合成的原料
NDPK是由nm23基因所合成，除了nm23-H1及nm23H2之外，到目前為止又有DR-nm23、nm23-H4、
nm23-H5、nm23-H6等四個isoforms被發現。研究顯
示nm23 mRNA及protein在一些human tumor cell的表
現都較正常細胞為少；而在會轉移的tumor cell中，
NDPK的活性比在不會轉移的tumor cells為低，所以
nm23 基因被推測為一個tumor metastasis suppressor
gene。在其他研究中也顯示nm23的基因產物可以磷
酸化small GDP-binding protein成為small GTP-binding
protein，此活化過程被認為可能是NDPK在調控細胞
生理現象的一個重要的步驟。
•
After transfecting and over-expressing CRMP-1 in
highly invasive CL 1 –5 cells,the cells were assessed
morphologically and with an in vitro invasion assay.
•
CRMP-1 expression in 80 lung cancer specimens was
determined by real-time quantitative RT-PCR.
•
Expression of CRMP-1 was inversely associated with
invasive activity in the cell
J Natl. Cancer Inst;93: 1392. 2001
•
CRMP-1-transfected CL 1 –5 cells became rounded
and lower in vitro invasive activity than untransfected
cells.
•
Real-time RT-PCR of lung cancer specimens showed
that reduced expression of CRMP-1 was associated
with advanced disease, lymph node metastasis, early
postoperative relapse, and shorter survival.
•
Conclusions: CRMP-1 appears to be involved in
cancer invasion and metastasis and may be an
invasion-suppressor gene.
Tumor metastasis promotion
gene
•
PRL-3
•
Chemokine, CXCR4, CCR7.
•
RhoC
癌症轉移所造成的死亡率約為90％。對於癌症如何開始，
科學界已經有相當的認識；但是對於癌症如何轉移，是否
由於部分基因啟動或失去所造成、是否與細胞內的訊息傳
導有關。翰霍普金斯醫學院的Kenneth Kinzler, Bert
Vogelstein 等幾位科學家們，由1995年開始在『由腸癌轉
移到肝臟的癌細胞』中，廣泛搜尋其基因表現情形，他們
發現，在癌症病程中無論癌細胞或是正常細胞均有部分基
因會被過度表現或是抑制，這情形隨細胞種類不同而異，
相當複雜而很難找到規則性。但是有一個名為PRL-3的酵
素，卻相當的有規則性：該酵素不會在正常細胞中表現，
而普遍在轉移的癌細胞中被大量表現！
To gain insights into the molecular basis for metastasis,
we compared the global gene expression profile of
metastatic colorectal cancer with that of primary
cancers, benign colorectal tumors, and normal
colorectal epithelium.
Among the genes identified, the PRL-3 protein
tyrosine phosphatase gene was of particular interest.
•
It was expressed at high levels in each of 18 cancer
metastases studied but at lower levels in nonmetastatic
tumors and normal colorectal epithelium.
•
In 3 of 12 metastases examined, multiple copies of the
PRL-3 gene were found within a small amplicon located
at chromosome 8q24.3.
•
These data suggest that the PRL(phosphatase of
regenerating liver)-3 gene is important for colorectal
cancer metastasis and provide a new therapeutic target for
these intractable lesions.
PRL-3

PRL-3 and PRL-1 promote cell migration,
invasion, and metastasis.
Cancer Res. 2003 Jun 1;63(11):2716-22

Expression of PRL-3 phosphatase in
human gastric carcinomas: close
correlation with invasion and metastasis.
Pathobiology. 2004;71(4):176-84
Chemokines
Chemokines, grouped into CXC and CC subfamilies
based on the arrangement of the two NH2-terminal
cysteine residues, are small secreted proteins that
regulate the chemotactic response for a variety of cells .
These ligands and receptors have been predominantly
investigated on lymphoid cells.
receptors, cxcr4
•
Seven-transmembrane G-protein-coupled receptors for chemokines. They also function as fusion cofactors for T-cell-tropic
HIV-1 strains.
•
CXCR4 is a chemokine receptor that, together with its natural
ligand stromal-derived factor-1 (SDF-1), is involved in the
regulation of leukocyte migration as well as other immunological
and developmental processes. In addition, CXCR4 has been
identified as a cofactor for HIV entry into T cells .
•
CCR7 is prevalent in various subsets of T cells and DCs.
Most individuals infected with human immunodeficiency virus type 1 (HIV-1) initially
harbor macrophage-tropic, non-syncytium-inducing (M-tropic, NSI) viruses that may
evolve into T-cell-tropic, syncytium-inducing viruses (T-tropic, SI) after several years.
For HIV type 1 (HIV-1) to infect a cell, the viral envelope
(env) protein must bind to CD4 and mediate fusion between
the viral envelope and host cell membrane. Fusion occurs only
when both CD4 and an appropriate coreceptor are expressed
on the host cell surface; CD4 alone is not sufficient for viral
infection. Recently, members of the chemokine receptor
family have been shown to serve as HIV-1 coreceptors.
Macrophage tropic (M-tropic) strains of HIV-1 use the
chemokine receptor CCR5, and T cell tropic (T-tropic) strains
require the expression of CXCR4 in conjunction with CD4 for
membrane fusion and infection to occur. In addition, other
chemokine receptors such as CCR2b and CCR3 can function
as coreceptors for some viruses, and some viruses can use
more than one chemokine receptor .
1. Breast cancer is characterized by a distinct
metastatic pattern involving the regional lymph
nodes, bone marrow, lung and liver.
2. Tumour cell migration and metastasis share many
similarities with leukocyte trafficking, which is
critically regulated by chemokines and their
receptors.
3. The chemokine receptors CXCR4 and CCR7 are
highly expressed in human breast cancer cells,
malignant breast tumors and metastases.
4. Their respective ligands CXCL12/SDF-1a and
CCL21/6Ckine exhibit peak levels of expression in
organs representing the first destinations of breast
cancer metastasis.
5. In vivo, neutralizing the interactions of CXCL12/CXCR4
significantly impairs metastasis of breast cancer cells to regional
lymph nodes and lung.
6. Malignant melanoma, which has a similar metastatic pattern as
breast cancer but also a high incidence of skin metastases, shows
high expression levels of CCR10 in addition to CXCR4 and
CCR7.
7. Our findings indicate that chemokines and their receptors
have a critical role in determining the metastatic destination
of tumor cells.
CXCR4
Expression of CXCR4 predicts poor prognosis
in patients with malignant melanoma
Clin Cancer Res. 2005 Mar 1;11(5):1835-41.

Silencing of CXCR4 blocks breast cancer
metastasis
*Cancer Res. 2005 Feb 1;65(3):967-71.
RhoC
•
RhoC GTPase is a member of the Ras-homology
family of small GTP-binding proteins and is responsible
for cytoskeletal reorganization during cellular motility .
•
RhoC belongs to a highly homologous subfamily
comprised of RhoA, RhoB, and RhoC . Although these
family members have >90% sequence homology to one
another, their roles in the cell are distinct.
•
They use an in vivo selection scheme to select highly
metastatic melanoma cells. By analysing these cells on
oligo-DNA arrays,
•
They define a pattern of gene expression that correlates
with progression to a metastatic phenotype.
•
They show enhanced expression of several genes involved
in extracellular matrix assembly and of a second set of
genes that regulate, either directly or indirectly, the actinbased cytoskeleton.
•
One of these, the small GTPase RhoC, enhances
metastasis when overexpressed, whereas a dominantnegative Rho inhibits metastasis.
•
Analysis of the phenotype of cells expressing dominantnegative Rho or RhoC indicates that RhoC is important in
tumour cell invasion.
RhoC


Genomic analysis reveals RhoC as a potential
marker in hepatocellular carcinoma with poor
prognosis.
Br J Cancer. 2004 Jun 14;90(12):2349-55.
RhoC induces differential expression of genes
involved in invasion and metastasis in MCF10A
breast cells.
Breast Cancer Res Treat. 2004 Mar;84(1):3-12
•
RhoC regulates many cellular function, such as
cytoskeletal organization, cell motility.
•
The genomic approach allows us to identify
families of genes involved in a process, not just
single genes, and can indicate which molecular and
cellular events might be important in complex
biological processes such as metastasis.