Tumorsuppressor Gene

09/10/2014
Sinyal Genetik dalam Perkembangan Kanker
Enzim perbaikan DNA
Mutasi induksi
instabilitas genetik
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Negative balance: no proliferation
Balance positive: aberrant growth
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
Gen Tumor suppressor :
› pengkode protein-protein yang berperan dalam
penghambatan pembentukan tumor  mengendalikan
siklus sel (cell cycle guardian)
› Mutasi pada gen-gen ini disebut : mutasi loss-of-function
mutations.
 sel mengandung 1 kopi gen
tumor suppressor yang fungsional
 pembentukan tumor dihambat
 Inaktivasi kedua kopi tumor
suppressor gen 
terjadi pembentukan tumor 
Mutasi pada gen tumor
suppressor : resesif
 (efek virus tumor & onkogen
 pengaruhi pembentukan
tumor saat diekspresikan 
dominan)
Tumor Suppressor Genes
Normal
genes
prevent
cancer
Normal cell
Remove or inactivate
tumor suppressor genes
Cancer cell
Damage to
both genes
leads to
cancer
Mutated/inactivated
tumor suppressor genes
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Tumor Suppressor Genes
Seperti rem
Tumor Suppressor
Gene Proteins
Growth factor
Receptor
Signaling
enzymes
Transcription
factors
DNA
Cell nucleus
Cell proliferation
p53 Tumor Suppressor Protein
Triggers Cell Suicide
p53 protein
Normal cell
Excessive DNA damage
Cell suicide
(Apoptosis)
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Table 7.1 part 1 of 2 The Biology of Cancer (© Garland Science 2007)
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Table 7.1 part 2 of 2 The Biology of Cancer (© Garland Science 2007)
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

selama G1, Rb berikatan dengan E2F  hambat
transkripsi gen untuk fase S
Stimulasi sel untuk membelah  akumulasi G1-Cdk
actif & fosforilasi Rb  afinitas pengikatan pada
E2F turun  aktivasi ekspresi gen untuk fase S

Timbul pada precursor sel fotoreseptor

Secara normal ditemukan pada 1 dari 20.000
anak
Dua kemungkinan :

Timbul secara sporadik  tumor tunggal pada 1 mata
 dibuang/operasi  sembuh
› Timbul karena keturunan  biasanya multiple foci
tumor pada kedua mata terapi/diobati  anak
tersebut tidak dapat terbebas dari kanker
osteosarcoma pada masa dewasanya & adanya
kemungkinan terkena jenis kanker yang lain
›
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Familial retinoblastoma
http://www.djo.harvard.edu/meei/OA



retinoblastoma (RB):
In hereditary cases :
› one mutant allele is inherited and the
other is generated somatically during
growth of the developing eye.
› somatic mitotic recombination
Sporadic retinoblastoma, both the first
and second hits occur in somatic tissue.
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Loss of Heterozygosity (LOH) of Tumor Suppressor Genes
Why do patients who inherit one mutant allele of RB have a high
probability of developing retinal tumors in childhood?
This is characteristic of cancers related to the loss of tumor suppressor
genes and a likely explanation is the predisposition of such genes to a
“lose of heterozygosity” or (LOH).

2 TSG:
› Caretaker
 Caretaker genes are genes responsible for
keeping other genes healthy (i.e. suppressing
mutation)
 P53, mlh1 and mlh2
› Gatekeeper
 ‘gatekeeper’ genes that are responsible for
controlling, or inhibiting, cell growth.
 pRB, BRCA1, BRACA2, APC
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A black gradient represents a continuum of expression that is also related to the number of
alleles present (gray numbering). Left, the two-hit paradigm as exemplified by the tumour
suppressor, RB. Loss of one allele induces cancer susceptibility; loss of two alleles induces
cancer. Middle, classic haploinsufficiency. Loss of one allele is sufficient for induction of
cancer. Right, Quasi-sufficiency and obligate haploinsufficiency. Quasi-sufficiency refers to
the phenomenon whereby tumour suppression is impaired after subtle expression
downregulation without loss of even one allele. Obligate haploinsufficiency occurs when
TSG haploinsufficiency is more tumourigenic than complete loss of the TSG, usually due to
the activation of fail-safe mechanisms following complete loss of TSG expression
Tissue specificity and context dependency of tumour suppression
The phenotypic outcome of a reduction in PTEN expression is differentially manifested
depending upon tissue type and genetic background. WD, well-differentiated. PD,
poorlydifferentiated.
LA, lymphadenopathy. SM, splenomegaly. HSC, hematopoietic stem cell.
BM, bone marrow. The effect of complete loss of PTEN is highly context dependent due to
the obligate haploinsufficiency caused by PTEN loss-induced cellular senescence (PICS).
Data summarized here come from multiple groups and studies of genetically-engineered
mice with differing PTEN alleles and expression
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The continuum model of tumour suppression
The classical discrete, stepwise model of tumour suppression (left) is contrasted with a continuum
model of tumour suppression and oncogenesis (centre and right, respectively). In the discrete model,
tumourigenesis is induced by either complete loss of a TSG (two-hit paradigm, dark blue) or after singlecopy loss of a TSG (haploinsufficiency, light blue). In contrast, we propose a continuum model (centre
and right), in which tumour suppression is related to a continuum of TSG expression, rather than to
discrete changes in DNA copy number. A continuum of increasing TSG expression will generally be
negatively correlated with malignancy (centre, light green) whereas increasing oncogene expression will
generally be positively correlated with malignancy (right, red). A linear relationship is depicted for
schematic purposes, but the dose-response relationship need not be linear. In some cases, fail-safe
mechanisms are induced by complete loss of TSG expression or by massive oncogene overexpression.
In these cases, complete loss of TSG expression (centre, dark green) or massive overexpression of an
oncogene (right, orange) will be negatively correlated with malignancy,
Figure 4. Mechanisms of regulation of TSG dosage
The interaction between coding and non-coding factors determines final TSG dosage. Classic
mechanisms such as DNA copy number (1), transcriptional regulation (2), and epigenetic silencing
can impact expression of TSG mRNA. TSG mRNA level or translation into protein is then regulated
by miRNAs (4). The availability of miRNAs for TSG downregulation is further regulated by ceRNAmediated sponging effects (3). Finally, additional translational regulation (6) or post-translational
modifications contribute to the final protein expression, function and effective dosage. The protein
structure shown is PTEN (PDB ID: 1D5R).
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Hipermetilasi  mekanisme
pengendalian ekspresi gen epigenetic
 nongenetic  silencing gene  enzim
DNA maintenance methylase (DNMT)
 Reversible

Jain PK. Annals of the New York Academy of the Sciences. 2003. 983:71-83.
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ER Methylation and Age in Normal Colon
And colon cancers
ER+ ER- 8yo 60yo
N
T
Breast Normal Colon
Cancer Colon Cancer
% ER Methylation
ER =
Estrogen receptor
25
20
15
10
5
0
0
10
20
30
40
50
Age
60
70
80
90 100
From: J.P. Issa lectures (Texas)
Cell cycle
Signal transduction
Apoptosis
DNA repair
Carcinogen metabolism
RB1, INK 4a, INK4b, p14 ARF
APC, LKB1/STK11, RASSF1
DAPK, caspase-8
MGMT, BRCA1, MLH1
GSTP1
Hormonal response
Metastasis
ER, PR, RAR
E-cadherin, VHL
Hanya metilasi dalam atau
dekat daerah promotor saja
yang menyebabkan gene
silencing
Worm, J. Guildberg P.
Journal of Oral Pathology and Medicine. 2002. 31(8):443-9.
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Metilasi DNA Methylation mempengaruhi proses
perkembangan kanker
Pengaruhi ekspresi berbagai gen
DNA Repair
Hormonal
Regulation
Carcinogen
Metabolism
DNA
Methylation
Differentiation
Cell Cycle
Apoptosis
Antisense DNMT-3b dalam
liposom sudah disuntikkan
intravena pada nude mice
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
P53: protein tumor suppressor
› Diaktivasi oleh sinyal stress seperti kerusakan DNA
› Mutasi p53 dapat ditemukan pada 50% kanker pada
manusia

Transformasi p53
› terhentinya siklus sel - Cell cycle arrest (penuaan sel/
cellular senescence)
› Apoptosis
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p53



Faktor transkripsi
waktu hidupnya pendek – sangat tidak stabil, akan
didegradasi tak lama sesudah disintesis
Mekanisme pengaturan activities TF:
› Modulasi level TF dalam inti
› Pengaturan level TF dalam inti konstan
› Modulasi level protein yang berkolaborasi dengan TF


P53 disintesis dalam
konsentrasi tinggi dan
didegradasi dengan cepat
pula
Sinyal pengaktifan p53:
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Merupakan gen yang paling banyak
mengalami mutasi pada kanker
 Mutasi pada sel gamet (Li-Fraumeni)
 Mutasi menyebabkan inaktivasi gen
 Punya jalur mutasi komplemen (mdm2)
 Memerlukan 2 perubahan (mutasi +
delesi)
 Spektrum mutasi bervariasi pada kanker
 Seringkali mutasi pada kodon

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Kaplan –Meier Plot untuk tikus mutan dengan genotip p53 berbeda. P53 +/- tidak
terlalu berbeda dengan p53 +/+. P53-/- mengembangkan kanker yang malignan.
P53+/- juga mulai mengembangkan kanker pada umur 250 hari
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

P53 tidak mengikuti aturan Knudson “two hit
elimination of tumor suppresor genes” dan resesif
P53  mutan p53 masih dapat mengubah fenofip
sel, p53 punya fungsi dominan  dominantinterfering atau dominant-negative alleles
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P53  homotetramer
 Dominan negative (dn) alel 
menyebabkan hilangnya fungsi p53
15/16 sedangkan null allel  kehilangan
50% fungsi p53

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P53 diperlukan untuk apoptosis
 P53 merupakan tumor suppressor
 Tidak diekspresikannya p53 berperan
pada perkembangan tumor pada
mencit dan manusia
 Merupakan faktor transkripsi yang atur
Bcl-2, Bax dan kelompok famili Bcl-2
lainnya

P53 memberi respons
terhadap sinyal stress
berupa:
• DNA damage,
• oncogenes activation,
• hypoxia.
Ketiga sinyal tersebut
meningkatkan level p53
Efek aktivasi p53:
• Penghambatan
pertumbuhan sel
melalui:
• cell cycle arrest
(senescence)
• Induksi apoptosis
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Stabilisasi p53 oleh kerusakan DNA &
deregulasi signal pertumbuhan

Double stranded DNA break  induksi
p53  melalui :
› ATM kinase
› Casein kinase II (CKII)
› Deregulasi pengendalian siklus sel pRb-E2F
• ATM dan ATR untuk fosforilasi
komponen yang berperan
dalam checkpoint : p53,
ChK1 dan ChK2.
• Adanya UV/iradiasi  ChK1
atau ChK2 fosforilasi p53 
stabilisasi p53.
• protein p53 induksi transkripsi
p21Waf1 G1 cell-cyclearrest
• protein p53 induksi transkripsi
GADD45, p21 dan14-3-3 dan
represi transkripsi Cyclin B 
G2cell-cycle-arrest
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Mdm2 dan ARF  p53
 Degradasi
p53 direguladi oleh mdm2 (sel mencit) atau
hdm2 (sel manusia)  p53 menjadi target untuk di
ubiquitinasi tak lama sesudah disintesis
 Half life p53 : 20 menit
 Pencegahan kerja mdm2 :
› fosforilasi p53 pada ujung amina  oleh ATM, Chk1&Chk2
(teraktivasi saat adanya kerusakan DNA) gambar 9-11&9-12,
9-13
› Lewat p14ARF (manusia) atau p19ARF (mencit).
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 p53
memberi respons
terhadap sinyal stress
berupa:
• DNA damage,
• oncogenes activation,
• hypoxia

Respon yang timbul :
• Represi transkripsi (e.g.
survivin or Bcl2)
• Transaktivasi target down
stream melalui :
• (mitochondrial pathway
(Bax, Bak, NUMA, NOXA)
• death receptor pathway
(Fas, KILLER/DR5, Bid);
• endoplasmic recticulum
pathway (Scotin).
• Jalur-jalur ini
crosscommunication  cell
death

induksi apoptosis oleh p53:
› Tergantung transkripsi p53
› Tidak tergantung transkripsi p53

Sinyal stress
› stabilisasi protein p53 inti 
transaktivasi atau represi p53  apoptosis
› P53 sitoplasma ditranslokasikan ke daerah mitokondria
dan berinteraksi dengan Bax atau Bak dan menyebabkan
oligomerisasi dari kelompok famili proapoptotik Bcl-2
family members untuk pelepasan protien BH3 yang
berikatan dengan Bcl-XL/Bcl2 pada mitokondria
› Pelepasan protein BH3  aktivasi bax dan menyebabkan
pelepasan sitokrom c  apoptosis
› Interaksi p53 dengan Bak bersamaan dengan hilangnya
interaksi antara Bak dengan anti-apoptotik famili Bcl-2,
Mcl1.
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Beberapa faktor yang berkontribusi untuk
memutuskan hidup atau matinya sel yang
mendapat stimulus stress.
 Aktivasi p53 pada sel normal  aktivasi gen yang
berperan dalam cell cycle arrest (p21Waf1, GADD45,
atau14-3-3)  penghambatan proliferasi sel
 Pada sel tumor, faktor-faktor yang akan berikatan
dengan p53 tissue-specific dan modulasi p53 (ASPP,
JMY, p63 dan p73 ) mengarahkan p53 ke promotor
 apoptosis

P53 vs apoptosis

Apoptosis akibat p53  ditrigger oleh peningkatan aktivitas
E2F
pRb --┤E2F  ARF --l Mdm2 --l p53 apoptosis
Protein p53 – berikatan dengan promotor (daerah domain
DNA binding) perlu modifikasi protein pada ujung karboksi :
asetilasi, glikosilasi, fosforilasi, ribosilasi, sumoylasi (
pemambahan gugus sumo: ubiquitin like peptida)  bantu
berinteraksi dengan faktor transkripsi lainnya
 mutasi pada p53  protein mdm2 tidak disintesis (p53 :
faktor transkripsi untuk mdm2  jumlah p53 dalam sel
meningkat
 Target p53 lainnya : p21 (inhibitor CDK). Induksi p21 berakibat
pada efek sitotaktik  p21 hambat 2 CDK: CDK2 dan CDC2
yang aktif pada akhir G1, S, G2 dan M dari siklus sel

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P53 tidak terlibat dalam apoptosis rutin
 terdapat mekanisme lain yang terlibat
dalam apoptosis sel rutin untuk
maintenance jaringan
 Aksi biologis p53 :

› P53 berperan untuk sitotaksis untuk hentikan
siklus sel
› Suatu mesin apoptosis laten  apoptosis

Myc dapat trigger apoptosis dependent
p53
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Figure 1. Revised model for p53 activation. The figure
summarizes data discussed in the text concerning
the time course of events occurring during p53
activation. A. Unstressed cells. We suggest that in
unstressed cells, p53 tetramers are inactive, but able
to bind their response elements in chromatin. As
MDM2 and MDMX are both required to inactivate
p53, we show them heterodimerized via their
respective RING domains and bound to the
Nterminal p53 transactivation domain. The MDM2MDMX heterodimer may engage an E2 to enable
poly-ubiquitination of MDM2 and MDMX and the
mono-ubiquitination of p53.
According to current information, p53 degradation
would require poly-ubiquitination, which would imply
the activity of at least one other protein (or protein
complex). In the unstressed state, RNA polymerase is
bound in an inactive state to the promoter, indicated
by the phosphorylation of serine 5 in its C-terminal
tail. B. Early after DNA damage. Soon after DNA
damage, ATM and other DNA damage-activated
kinases phosphorylate both p53 and MDM2.
However, at the early time, even though p53 is
phosphorylated on serine 15 (and presumably other
sites), MDM2 (and MDMX) are still bound, and all are
unstable. p53 is inactive at this early time after
damage induction. C. Peak DNA damage response.
Thepeak of the DNA damage response correlates
with the stabilization of p53 and increased
p53abundance (due to its increased stability). p53 is
stabilized because of the accelerated
autodegradation of MDM2, and the accelerated
MDM2-mediated degradation of MDMX, and
perhaps reduced affinity for the MDM2-MDMX
complex due to p53 N-terminal phosphorylation at
residues ser15, thr18 and ser20. We propose that the
accelerated
Figure 1. p53 stabilisation A central player in p53 stabilisation in response to oncogenic events
is an increase in the p19ARF protein. Several oncogenic events can cascade to increase
p19ARF expression. Immediate to it is the overexpression of myc. This, in turn may b
caused
by loss of repression of the transcription factor E2F. And this E2F de-repression may be caused
by inactivation (e.g. adenovirus E1A protein), phosphorylation (e.g. by cyclindependent
kinases) or loss expression (e.g. retinoblastoma) of the Rb protein. p19ARF inhibits mdm2mediated degradation of p53 probably by sequestering mdm2 in the nucleolus (for reviews
see Sherr and Weber, 2000; Vousden and Lu 2002). While the p19ARF pathway does not
appear to mediate p53 stabilisation in response to genotoxic stresses (Stott et al., 1998), most
genotoxic/cytotoxic stresses will disrupt nucleolar function which, in turn appears to lead to
p53 stabilisation (Rubbi and Milner, 2003c).
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