tumor suppressor gene - niu.edu.twtlrc.niu.edu.tw/downloadfiles/a/a21/a21.pdf · i. history of...

Tumor suppressor gene

I. History of tumor suppressor gene1. Cell hybridization2. Human genetics

II. Identify of suppressor genesIII. Suppressor genes:

1 Retinoblastoma (Rb)2. P533. APC4. Deleted in colon carcinoma (DCC)5. Neurofibromatosis tumor suppressor


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.


Cells have brakes, called tumour-suppressor genes.These keep cell numbers down, either by inhibitingprogress through the cell cycle and thereby preventingcell birth, or by promoting programmed cell death(also called apoptosis).

Tumour-suppressor genes.

Tumor suppressor genes

•Genes whose loss contributes to cancer areclassified as tumor suppressor genes becausewhen they function normally they help tosuppress tumors

•Normal functions of tumor suppressor genes isto suppress cellular growth , promote cellulardeath

•Also known as anti-oncogenes

Cell hybridization

Mucoepidermoid

Lung carcinoma cell

•Tumorigenic

•Immortal

Normal bronchial cell•Finite lifespan•Non-tumorigenic

•Finite lifespan

•Non-tumorigenic

CA NCA/N

Human Genetics

LOH:

6Q21, gastric cancer

7Q31, prostate cancer

17Q, ovarian cancer

Human GeneticsCurrent 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 (appearedin both eyes), average 3 independent tumors have familyhistory, prezygotic mutation--95% tumor within 7 yearperiod.

2. Sporadic: no family history, unilateral (one eye). meanage 30 months, only one tumor. Solely in postzygotic 2independent events must have occurred in the Rb gene.One mutation-- growth selection advantage to induce 2ndmutation.

•White glow orglint in one orboth eyes•Have whitepupils inpictures•Has crossed ormisaligned eyes

pRb and retinoblastoma

The disease; retinoblastoma

A B

A/B pocket

NLSLXCXE proteinsE2F

SSSSS TTC-term

TSSTTT SSS

(HDAC, E7)(no LXCXE motif)

The protein; pRb

cyclin docking


3. 180 kb DNA, 4.7 kb mRNA, 928 amino acid, - 105 KD protein(MW 110-114 KD), different degrees of phosphorylation located onchromosome 13.

4. Phosphonuclear protein, has DNA binding ability suggest a role intranscriptional regulation

5. DNA tumor virus association. Adenovirus E1A, SV40 large Tantigen, human papilloma virus (Hpv) E7 oncoproteins formcomplex. Inactive Rb gene by these oncoproteins to transform cells.

6. Mutant Rb protein fail to bind large T antigen.


7. E7 protein of the non oncogenic human papilloma viruses (HPV6B,HPV11) bind p105-Rb with lower affinity than the oncogenic HPV16, 18

E2F homo-or hetero-dimer

Rb/E2F

C-myc, myb,cdc2

TTTCGCGC

pRb

Stephen Taylor4/10/02

BS324: Molecular Biology of Cancer.Lecture 3: The Cell Cycle, Slide: 15

Rb and cell cycle entry

E2FRb

RbPP

PE2F+

Mitogens

CDK 4/6Cyclin D

E2F

Cyclin E gene

Cyclin E

CDK 2Cyclin E

S phaseentry

Sherr (2000)Cancer Research60:3689-3695

8. One of the most well-characterized proteins that interacts with Rbis the transcription factor E2F. E2F binds to unphosphorylated Rb.Upon phosphorylation of Rb, E2F is no longer able to form acomplex with Rb and thus dissociates. Free E2F is then able toactivate a number of target genes. E2F recognizes and binds to aconsensus sequence of TTTCGCGC, which is found in thepromoters of a number of genes that are involved in regulating cellgrowth, including c-myc, myb, cdc2, dihydrofolate reductase,thymidine kinase, and the E2F promoter itself.


9. underphosphorylated in G1, becomes heavilyphosphorylated just prior to the G1 to S transition, remainsphosphorylated in S, G2, and most of M, and reverts to anunder phosphorylated state at or before the M-G1 transition


G1/G0 S G2 M

Active RbUnphospho.

cdc2kinase

Rb-p,inactive

10. All 3 viral oncoproteins (T-Ag, E1A, E7) bind to theunderphosphorylated (active form) form of Rb protein

11. Rb is active in growth suppression only in G0 and G1 (underphosphorylated), and is phosphorylated and rendered inactive forthe remainder of the cell cycle.


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 thehistones to wrap more tightly around the DNA and interfering withthe transcription of genes by blocking access by transcription factors.

Treatments

Current:-Emulation (removal of eye)-Radiation

Future:-to shrink the retinoblastoma so it can betreated with a laser freezing and local plague.

Prognosis

•Emulation, lost of the eye. 40% chances ofloosing both eyes

•Untreated, retinoblastoma is almost alwaysfatal

Rb

Enhanced Mdm2 activity inhibits pRBfunction via ubiquitin-dependentdegradation

*The EMBO Journal (2005) 24, 160–169

•it is mutated in about half of all human cancers

•it was initially identified as a tumor specific nuclearantigen of molecular weight 53kDa

•isolation of the gene from tumor cells yielded anoncogene: when it was transfected into normal cells itcould transform them together with ras

•when the gene was isolated from normal cells, it wasdiscovered that the tumor derived gene was mutated,and when the wild-type was transfected into tumor cellsit stopped their growth, ie it was a tumor suppressorgene

P53: both oncogene and tumorsuppressor gene

P53 gene

1. P53 (1970) was found initially through its associationwith SV40 large T oncoprotein in virus transformedcells it acts as a negative regulator of cell growth

2. P53 gene 16-20kb of DNA on the small (p) arm ofchromosome 17 in human, 11 of mice

3. 2.2-2.5 kb mRNA producing 53 kd nuclearphosphoprotein

4. primary a.a. sequence (375 a.a.) - 3 distinct domain.

The p53 pathway in response to DNA damage

DNA DamageATM ( DNA-dependentprotein kinase; Rad 3)

No DNA damage

p53Mdm2

P53 is unstable and isdegraded by a ubiquitin-dependent mechanism.

p53Mdm2

PPP

PPP

p53

Phosphorylation in response toDNA damage preventsassociation with Mdm2and stabilizes p53.

Ionizing radiation

Blocks transactivationTargets for degredation

PPP

ATM(DNA-dependent protein kinase)

Ataxia telangiectasia–mutated gene (ATM) isa 350-kDa proteinwhose function isdefective in theautosomal recessivedisorder ataxiatelangiectasia (AT).

Structure of P53

The domain structure of p53 and binding partners

Transactivation Sequence specific DNA bindingOligomerisation/

Nuclear Localisation/Export p53Hsp70May help to

stabilise p53

TBP

E1B55

MDM2

TAF70

TAF31

TFIIH

Theseinteractionspromotetranscription

Theseinteractionsblock p53transcriptionalactivation

SV40 T antigen

Blocks p53 function

95% of mutations in p53 found in cancer cells lie within this domain

•p53 is a tetramer, which does not have an exposednuclear export signal (NES), therefore the NLSpredominates and p53 remains nuclear.

•p53 monomers have an exposed NES which ismore active than the NLS therefore p53 iscytoplasmic where it is targeted for rapiddegradation.

•Mutations which influence oligomerisation of p53influence its subcellular localisation and thereforeactivity.

P53 gene

5. Mouse and human proteins share 80% homology at a.a.level. murine p53 blocked the binding of DNApolymerase to SV40 large T antigen. SV40 DNAreplication was blocked by p53.

6. Detected in all cells, present at low levels, half life 6-20min. Mutations have been shown to increase the stabilityof p53 protein, to alter the conformation of the protein andto result in complex formation with a HSP 70.

p53 tumor suppressor

P53 is a transcription factor which monitors the state ofDNA.

It inhibits cell cycle progression if there is damage.

Following DNA damage (e.g. by radiation), p53 levelsrise, and proliferating cells arrest in G1.

This allows time for DNA repair prior to the next round ofreplication.

P53 mediated cell cycle arrest is mediated by stimulationof expression of p21CIP1, the cyclin kinase inhibitor.

p53 tumor suppressor

The functional expression of the gene p53 increases thesensitivity of the expressing cells to activation ofprogrammed death induced by a wide variety ofdamaging agents.

Such a universally resistant genotype would provide agreat 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 damageto 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 elevated5-100 fold, half life becomes many hours. Wt p53 assembleinto homotetramers and higher order homo-oligomericstructures.

8. mutant p53 + ras oncogene can transform cells but not wtp53

9. p53 is found in oligomeric complex with oncoproteinSV40 large T antigen, and adenovirus E1b

P53 gene

10. SV40 large T, adenovirus E1b form complex with wt p53and inactive wt p53. Papilloma virus E6 + p53 --- proteolyticpathway

11. p53 is documented as the most frequently mutated gene inhuman 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 theoncoproteins of SV40, adenovirus, and human papillomavirus.

P53 gene

(iii) both proteins must be eliminated from the cell's growth-suppressing pathway in order for full viral transformation tooccur.

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-dependentmanner

2. p14ARF binds to MDM2 and promotes the rapiddegradation of MDM2.

3. ARF-mediated MDM2 degradation is associated withMDM2 modification and concurrent p53 stabilization.

(Cell 92: 725, 1998)

P53 gene

1. Acetylation of lysine or phosphorylation of serine nearthe carboxy terminus of p53 can enhance thebinding of p53 to DNA.

2. Phosphorylation of the amino terminus of p53 doesnot affect its DNA binding, but does affect itsaffinity for MDM2 and subsequently leading todegradation of p53.

3. p14ARF binds to MDM2 and inhibits its activity.Nature 408:306, 2000

The p53 network

p53

p21 gene promoter

p21

1. p21 (waf1/cip1) is a cyclin kinase inhibitor

Cyclin/Cdk complex

p21

2. p53 also induces proteins involved in DNA repair.

Mediators of p53 function

p21 can bind to the cyclin/CDK complex and inhibit its kinase activity

-inactivated-G1 arrest

P53 network-cell cycleinhibition

1. P53 protein stimulates the p21 WAF/CIP1, an inhibitor ofcyclin-dependent kinase (CDKs). CDK are key regulatorof the cell cycle.

2. p21 WAF/CIP1 inhibit both the G1 to S and G2 to mitosis.

3. P53 protein stimulates 14-3-3which sequesterscyclinB1-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 butinhibits Bcl-2.

2. NOXA and p53AIP1 genes are activated by p53.These proteins induce apoptosis when over-expressed.

3. P53 mediators induced apoptosis includeTNT receptor, Fas, PIDD, toxic reactive oxygen etc.

p53 and the DNA Damage ResponseDNA Lesion

x

Stabilisation of p53 which normally has a half life of 20 minutes

p53

BAX

whichpromoteapoptosis

WAF/CIP1 gene - transcriptionalactivation by p53

p21

cyclin

CDK

Kinaseactivity

cyclin

CDKp21

No kinaseactivity

G1 S

G2M

DNA RepairApoptosis

No phosphorylationof Rb therefore norelease of E2F-1therefore cell remains in G1 -allowing time for DNA repair.

Review on p53:Cell 88 p323 (1997)

Go

Putative signal pathway of p53

P53 pathway

1 P53 block RPA(replication protein A)replication

2. P53p21-CIP cdk kinase cell cyclearrest

3. GADD 45 (growth arrest DNA damage)/p21 canbind to PCNA (proliferating cell nuclear antigen)and inhibit DNA replication.

4. Wtp53 bax apoptosis

bcl2

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 p19and p16.

ONCOGENE CHECKPOINT

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 Myc-

induced B-Cell Lymphoma•Melanoma

•Immortal MEFS•RAS-susceptible

•Spontaneous tumors•Acceleration of Myc-

induced B-Cell Lymphoma•No Melanoma

1. P16-INK4a acts as an inhibitor of cyclin D1-dependent kinases 4 or 6 and prevents thephosphorylation of Rb, thereby maintaining anactive Rb and blocking the exit from theG1phase.

2. P19ARF inhibits cell growth by interactingwith MDM2, thereby blocking MDM2-mediated degradation of the p53 protein andneutralizing MDM2’s inhibition of p53activity.

P16/p19

p53Ub Ub Ub

p53 Pathway

MDM2

P19 ARF

From Sharpless and DePinho (1999)Current Opinions in Genetics and Dev. 9:22

BAXp21mdm2

DEGRADATION

p53

ATMDNA-PK

???

DNA Damage

Tumor Suppressor Genesp53, p19

OncogenesMDM2

Oncogenic Stimuli(e.g.E1A, MYC)

Intersection of p53 and RB Pathways

Dimerization and processing of procaspase-9 by redoxstress in mitochondria.J Biol Chem. 2004 Jan 27

EMBO J. 2004 Jan 28;23(2):460-72. Epub 2004 Jan08. Coupling of caspase-9 to Apaf1 in response to lossof pRb or cytotoxic drugs is cell-type-specific.

p53

Caspases

Apoptosis

Bax

E2F1

BCL2

mdm2 RB Cyclin DCDK4

p16P19-ARF

E2F1

S-Phase Apoptosis

Ink 4

P53 and RB Crosstalk

P19 ARF

p73The human p73 promoter: characterization and identificationof functional E2F binding sites. Neoplasia. 2002 May-Jun;4(3):195-203.

1. E2F1 induces phosphorylation of p53 that iscoincident with p53 accumulation and apoptosis.Mol Cell Biol. 2002 Aug;22(15):5308-18.

MDM2/RbEnhanced Mdm2 activity inhibits pRB function

via ubiquitin-dependent degradationThe EMBO Journal (2005) 24, 160–169.

The central acidic domain of MDM2 is criticalin inhibition of retinoblastoma-mediatedsuppression 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 regionon the long arm of chromosome 5. The mutation ofthese genes is associated with familial adenomatouspolyposis (apc stands for adenomatous polyposiscoli) and gardner's syndrome, as well as somesporadic colourectal cancers.

MSHMultigene family coding for proteins involved inmismatch 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 signalingmolecules important in morphogenesis. First member wasDrosophila wingless , but many vertebrate homologues arenow known. Wnt -1 induces accumulation of -catenin andplakoglobin and affects the association of APC tumoursuppressor protein with catenin.

plakoglobinPolypeptide (83 kD) present at cell-cell but not cell-substratum contacts. Associated with desmosomes and withadherens junctions: soluble 7S form present in cytoplasm.

•An autosomal dominant polyposis syndrome in which thecolon contains few to thousands of adenomatous polyps,often occurring by age 15 to 25.

•Protein in cytoplasm degrades B-catenin, a signal-transduction protein that can enter the nucleus and activatetranscription

•B-catenin is also important in cell-cell adhesion - thereforerole in invasion as well (Proteins associated with thecytoplasmic domain of uvomorulin and presumablyinvolved in linking to the cytoskeleton. ( -catenin 102 kD;-catenin 88 kD; -catenin 80 kD))

APC (Adenomatosis Polyposis Coli)

Uvomorulin

Glycoprotein (120 kD) originally defined as theantigen responsible for eliciting antibodies capableof blocking compaction in early mouse embryos (atthe morula stage), and inhibiting calcium-dependent aggregation of mouse teratocarcinomacells. May be the mouse equivalent of LCAM, thechick cell adhesion molecule.

Polyps in familial polyposis coli

Colon Cancer-common

FAP(FamilialAdenomatousPolyposis)-rare-inherited-associated withbenignadenomatous polypsin early adulthood-some polyps progressto invasivenesspolyps

•SIGNAL TRANSDUCTION INHIBITORS

•named for rare familial condition

- hundreds to thousands of precancerous colonicpolyps, at least one of which will progress to cancerby young adulthood (one allele inherited, oneacquired)

- but also seen in most sporadic colon carcinoma.

APC

The APC tumor suppressor is encoded by theFAP locus

Bert Vogelstein

The tumor suppressor APC

In 1991, Kinzler and Groden identified thetumor suppressor APC encoded by the FAPlocus.

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?

1. The conventional form of the APC gene contains 15 exons, withthe most 3’exon containing over three-quarters of the 8,535 bpof coding sequence.

2. Most APC mutations occur within the central third of the gene,designated the mutation cluster region. This region contains twoof the most commonly found mutations, 5-bp deletions creatingstop codons at positions 1061 and 1309, and a nucleotidepolymorphism (ASH) in the Ashkenazi Jewish population .

3. Regions of the gene where mutations are associated withcongenital hypertrophy of the retinal pigmented epithelium(CHRPE) and desmoid tumors (aggressive fibromatosis) areshown.

The APC gene, mutational spectrum, clinicalcorrelates, and APC protein structure

The WNT signaling pathway; absence of Wntsignal

Free -catenin is destabilized bybinding to the destruction complex-glycogen synthase kinase 3-axin/conductin-APC

APC binds to -catenin and GSK3GSK3phosphorylates -cateninP--catenin is ubiquinated and destroyedby the proteosome

Wnt receptor: Frizzled;7-pass transmembrane

cell

WNT signaling pathway; presence of Wnt signal

Presence of Wnt:GSK3is inactivated-catenin is stabilized-catenin shuttles to the nucleus-catenin binds to the TCF-familyof transcription factors and serves asan essential co-activator

Wnt/-catenin signaling: target genes

Gene Organism/system Direct/Indirect up/down Ref.c-myc human colon cancer yes up He 1998Cyclin D human colon cancer yes up Tetsu 1999

Shtutman 1999Tcf-1 human colon cancer yes up Roose 1999PPARdelta human colon cancer yes up He TC, et al 1999c-jun human colon cancer yes up Mann B, 1999fra-1 human colon cancer yes up Mann B, 1999uPAR human colon cancer ? up 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 ofhuman chromosome 18. The absence of these genes isassociated with the formation of colourectal cancer (dccstands for deleted in colourectal cancer). The products ofthese genes show significant homology to neural celladhesion molecules and other related cell surfaceglycoproteins.

Deleted in Colon Carcinoma (DCC)Gene:

1. Allele losses on chromosome 18q21 in colorectalcarcinoma not adenomas

2. 70% colon cancer, DCC reduced expression, pointmutation, insertion, deletion.

3. Genomic DNA--1000 kb, amino acid--190 Kdtransmembrane phosphoprotein cell surface receptor.

4 Ligand for DCC is Netrin (Cell 87:175, 1996). Netrinsare chemoattractants for commissural axons in thevertebrate spinal cord, but the mechanisms through whichthey produce their effects are unknown.

5. DCC mRNA is present in a variety of normal tissueincluding colonic mucosa, but undetectable in 90% ofcolon tumor cell lines.


Netrin

Genes identified in studies of vertebrate neuronaldevelopment. Netrins are chemotropic for embryoniccommisural neurones: netrin 1 is secreted by thefloorplate, whereas netrin 2 is distributed ventrallyexcept for the floorplate. The netrins are homologousto the product of unc 6, a gene identified in studies ofneuronal development of the worm, Caenorhabditiselegans.

6. Gene encodes a member of the immunoglobulinsuperfamily with homology to neural cell adhesionmolecules that presumably regulates cell behaviorthrough interation with other cells


•Kruppel-like factor 6 (KLF6) is a zinc fingertranscription factor of unknown function. Here, we showthat the KLF6 gene is mutated in a subset of humanprostate cancer.

•Loss-of-heterozygosity analysis revealed that one KLF6allele is deleted in 77% (17 of 22) of primary prostatetumors.

•Sequence analysis of the retained KLF6 allele revealedmutations in 71% of these tumors.

Functional studies confirm that whereas wild-type KLF6 up-regulates p21 (WAF1/CIP1) in ap53-independent manner and significantlyreduces cell proliferation, tumor-derived KLF6mutants do not. Our data suggest that KLF6 isa tumor suppressor gene involved in humanprostate cancer.

KLF6 (Kruppel-like factor)

Frequent inactivation of the tumor suppressorKruppel-like factor 6 (KLF6) in hepatocellularcarcinoma.

Hepatology. 2004 Nov;40(5):1047-52.

A new role for the Kruppel-like transcriptionfactor KLF6 as an inhibitor of c-Jun proto-oncoprotein function.

Oncogene. 2004 Oct 28;23(50):8196-205

PTEN:phosphatase and tensin homolog deleted onchromosome ten; MMAC1/PTEN; TEP-1)

TEP1:TGFa-regulated and epithelial cell-enrichedphosphatase).Also termed PTEN or MMAC1 (mutated inmultiple advanced cancers 1).A protein tyrosine phosphatase with homology to tensin, is atumour-suppressor gene on chromosome 10q23. Somaticmutations in PTEN occur in multiple tumours, mostmarkedly glioblastomas. Germ-line mutations in PTEN areresponsible for Cowden disease (CD), a rare autosomaldominant multiple-hamartoma syndrome. Mutated inMMAC1/PTEN (multiple advanced cancers 1/phosphataseand tensin) homologue.

Cowden disease

(Cowden is the family name of the first reportedcase) (also called multiple hamartoma syndrome(MHS)), autosomal dominant inherited conditioncharacterized by multiple hamartomas and neoplasmsof ectodermal, mesodermal and endodermal origin. Itoccurs mainly in white persons and the mean age atthe time of the diagnosis is 41 years.

Tensin

Actin-binding component of focal adhesions andsubmembranous cytoskeleton. Has SH2 domain andcan be tyrosine phosphorylated; speculated that itmay link signalling systems with the cytoskeleton.

The mechanisms controlling neural stem cell proliferationare poorly understood. Here we demonstrate that the PTENtumor suppressor plays an important role in regulatingneural stem/progenitor cells in vivo and in vitro.

Pten encodes phosphatase.

Mice lacking PTEN exhibited enlarged, histoarchitecturallyabnormal brains, which resulted from increased cellproliferation, decreased cell death, and enlarged cell size.

•Neurosphere cultures revealed a greater proliferationcapacity for tripotent Pten -/- central nervous systemstem/progenitor cells, which can be attributed, at least inpart, to a shortened cell cycle.

•However, cell fate commitments of the progenitors werelargely undisturbed. Our results suggest that PTENnegatively regulates neural stem cell proliferation.

•PTEN phosphatase promotes apoptosis by preventingAkt activation, antagonises the anti-apoptotic effect of PI3K.

•The N-terminal-phosphatase domain (amino acids 1-185) is shown withthe 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

Structure of PTEN

PTEN negatively regulates PKB/Akt-mediatedcell survival

PTEN/Mdm2/P53

summary

Oncogenes and tumor suppressors work together to achievethe full malignant phenotype

Tumor suppressors are proteins that play a role incontroling cell cycle, in check-point control and inmaintenace of a differentiated phenotype

Loss of tumor suppressor genes lead to cell cycle defectssuch as loss of check point control

Inherited mutations in tumor suppressor genes predisposesthe individual to cancer

Tumor cell invasion andmetastasis

A. Invasion-suppressor genes

•E-cadherin

•Tissue inhibitors of metalloproteinases (TIMPS)

•plasminogen activator inhibitors (PAIs)

•Anti-metastatic gene nm23

Molecular aspects of invasion

1. Association of nm23-H1 allelic deletions withdistant metastases in colorectal carcinoma(Lancet 1991; 338: 722-24. )

2. A low nm23H-1 gene expression identifyinghigh malignancy human melanomas(Melanoma Research 1994, 4, p. 179-184 )

3. Levels of nm23 Messenger RNA in metastaticmalignant Melanomas: Inverse Correlation toDisease Progressions (Cancer Res. 52, 6088-6091, 1992 )

Summary of literatures

B. Invasion-Promoter Genes

•Lytic enzymes ( Collagenase)

•Motility factors (epithelial mesenchymal transitionEMT) , extracellular matrix(ECM)remodeling

•Interleukin-1(IL-1)

•hepatocyte growth factor (HGF), Scatter factor(SF)- receptor, c-Met

Molecular aspects of invasion

Epithelial cells (left) are tightly interconnected in sheets bynumerous cell-cell interactions; their organization is determined bythe basement membrane (lower left). During epithelial-mesenchymaltransition (EMT), epithelial cells become independent of theirneighbors and can move freely (center). In this way, tumors derivedfrom epithelial cells become motile and may invade lymph or bloodvessels (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 Down-regulates the Expression of Activating TranscriptionFactor 3 in Prostate Cancer

The expression of the KAI1 gene, a tumor metastasissuppressor, is directly activated by p53

[Expression of tumor metastasis-suppressor geneKiSS-1 and matrix metalloproteinase-9 in portalvein tumor thrombus of hepatocellular carcinoma]

TSLC1 is a tumor suppressor gene associated withmetastasis in nasopharyngeal carcinoma.

KAI1/CD82, a tumor metastasis suppressor

Tumor metastasis suppressorgene---nm23

1. nm23 is a tumor metastasis associated gene.

2. nm23 RNA levels were highest in cells and tumors ofrelatively low metastatic potential in the followingsystems.

(i) murine k-1735 metanoma cell lines

(ii) N-nitrosomethylurea - induced rat mammary carcinomas

(iii) human breast carcinoma

(iv) human hepatoma


(v) human melanoma

3. nm23 is homologous to NDP kinase. Nucleosidediphosphate kinase catalyzes a transfer of the terminalphosphate of 5' - triphosphate nucleotides to 5' diphosphatenucleotides (RNA or DNA).

4. Encode a 17 kd nuclear and cytoplasmic protein.


5. Human nm23-H1---chromosome 17.

6. Amino acid sequence contain leucine repeats,basic amino acid region, suggestive of a leucinezipper involved in transcriptional regulation.

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nucleosidestriphosphate。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nm23-H2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 suppressorgene。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-bindingprotein，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 inhighly invasive CL 1 –5 cells,the cells were assessedmorphologically and with an in vitro invasion assay.

•CRMP-1 expression in 80 lung cancer specimens wasdetermined by real-time quantitative RT-PCR.

•Expression of CRMP-1 was inversely associated withinvasive activity in the cell

J Natl. Cancer Inst;93: 1392. 2001

•CRMP-1-transfected CL 1 –5 cells became roundedand lower in vitro invasive activity than untransfectedcells.

•Real-time RT-PCR of lung cancer specimens showedthat reduced expression of CRMP-1 was associatedwith advanced disease, lymph node metastasis, earlypostoperative relapse, and shorter survival.

•Conclusions: CRMP-1 appears to be involved incancer invasion and metastasis and may be aninvasion-suppressor gene.

•PRL-3

•Chemokine, CXCR4, CCR7.

•RhoC

Tumor metastasis promotiongene

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, BertVogelstein 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 ofmetastatic colorectal cancer with that of primarycancers, benign colorectal tumors, and normalcolorectal epithelium.

Among the genes identified, the PRL-3 proteintyrosine phosphatase gene was of particular interest.

•It was expressed at high levels in each of 18 cancermetastases studied but at lower levels in nonmetastatictumors and normal colorectal epithelium.

•In 3 of 12 metastases examined, multiple copies of thePRL-3 gene were found within a small amplicon locatedat chromosome 8q24.3.

•These data suggest that the PRL(phosphatase ofregenerating liver)-3 gene is important for colorectalcancer metastasis and provide a new therapeutic target forthese 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 inhuman gastric carcinomas: closecorrelation with invasion and metastasis.Pathobiology. 2004;71(4):176-84

Chemokines

Chemokines, grouped into CXC and CC subfamiliesbased on the arrangement of the two NH2-terminalcysteine residues, are small secreted proteins thatregulate the chemotactic response for a variety of cells .These ligands and receptors have been predominantlyinvestigated on lymphoid cells.

receptors, cxcr4•Seven-transmembrane G-protein-coupled receptors for -chemokines. They also function as fusion cofactors for T-cell-tropicHIV-1 strains.

•CXCR4 is a chemokine receptor that, together with its naturalligand stromal-derived factor-1 (SDF-1), is involved in theregulation of leukocyte migration as well as other immunologicaland developmental processes. In addition, CXCR4 has beenidentified 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) initiallyharbor macrophage-tropic, non-syncytium-inducing (M-tropic, NSI) viruses that mayevolve 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 betweenthe viral envelope and host cell membrane. Fusion occurs only

when both CD4 and an appropriate coreceptor are expressedon the host cell surface; CD4 alone is not sufficient for viralinfection. Recently, members of the chemokine receptorfamily have been shown to serve as HIV-1 coreceptors.Macrophage tropic (M-tropic) strains of HIV-1 use thechemokine receptor CCR5, and T cell tropic (T-tropic) strainsrequire the expression of CXCR4 in conjunction with CD4 formembrane fusion and infection to occur. In addition, otherchemokine receptors such as CCR2b and CCR3 can functionas coreceptors for some viruses, and some viruses can usemore than one chemokine receptor .

1. Breast cancer is characterized by a distinctmetastatic pattern involving the regional lymphnodes, bone marrow, lung and liver.

2. Tumour cell migration and metastasis share manysimilarities with leukocyte trafficking, which iscritically regulated by chemokines and theirreceptors.

3. The chemokine receptors CXCR4 and CCR7 arehighly expressed in human breast cancer cells,malignant breast tumors and metastases.

4. Their respective ligands CXCL12/SDF-1a andCCL21/6Ckine exhibit peak levels of expression inorgans representing the first destinations of breastcancer metastasis.

5. In vivo, neutralizing the interactions of CXCL12/CXCR4significantly impairs metastasis of breast cancer cells to regionallymph nodes and lung.

6. Malignant melanoma, which has a similar metastatic pattern asbreast cancer but also a high incidence of skin metastases, showshigh expression levels of CCR10 in addition to CXCR4 andCCR7.

7. Our findings indicate that chemokines and their receptorshave a critical role in determining the metastatic destinationof tumor cells.

CXCR4 Expression of CXCR4 predicts poor prognosis

in patients with malignant melanomaClin Cancer Res. 2005 Mar 1;11(5):1835-41.

Silencing of CXCR4 blocks breast cancermetastasis

*Cancer Res. 2005 Feb 1;65(3):967-71.

RhoC

•RhoC GTPase is a member of the Ras-homologyfamily of small GTP-binding proteins and is responsiblefor cytoskeletal reorganization during cellular motility .

•RhoC belongs to a highly homologous subfamilycomprised of RhoA, RhoB, and RhoC . Although thesefamily members have >90% sequence homology to oneanother, their roles in the cell are distinct.

•They use an in vivo selection scheme to select highlymetastatic melanoma cells. By analysing these cells onoligo-DNA arrays,

•They define a pattern of gene expression that correlateswith progression to a metastatic phenotype.

•They show enhanced expression of several genes involvedin extracellular matrix assembly and of a second set ofgenes that regulate, either directly or indirectly, the actin-based cytoskeleton.

•One of these, the small GTPase RhoC, enhancesmetastasis when overexpressed, whereas a dominant-negative Rho inhibits metastasis.

•Analysis of the phenotype of cells expressing dominant-negative Rho or RhoC indicates that RhoC is important intumour cell invasion.

RhoC Genomic analysis reveals RhoC as a potential

marker in hepatocellular carcinoma with poorprognosis.Br J Cancer. 2004 Jun 14;90(12):2349-55.

RhoC induces differential expression of genesinvolved in invasion and metastasis in MCF10Abreast cells.Breast Cancer Res Treat. 2004 Mar;84(1):3-12

•RhoC regulates many cellular function, such ascytoskeletal organization, cell motility.

•The genomic approach allows us to identifyfamilies of genes involved in a process, not justsingle genes, and can indicate which molecular andcellular events might be important in complexbiological processes such as metastasis.

tumor suppressor gene - niu.edu.twtlrc.niu.edu.tw/downloadfiles/a/a21/a21.pdf · i. history of...

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