1

Therapeutic+Target+for+Cancer+
Metastasis+:+Regulation+of+TGF8β+by+Post+Translational+Modification

M2#Fujiwara#2018.11.07#(Wed)Contents
Cancer'metastasis
Cancer'metastasis
Epithelial0to0Mesenchymal'Transfer

Metastasis'inhibition'– Blocking'TGFβ'pathwayTGFβ'(inducing'EMT)'is'promising'target'for'cancer'
metastasis
Control'of'TGFβ'signaling'by'Ubiquitination

Cancer7metastasis

https://www.cancer.gov/types/metastatic5cancer

Cause7of790%7of7deaths7from7solid7tumors
Gene7profiling7for7metastasis7in7primary7tumors7
correlates7strongly7with7the7likelihood7of7metastatic7
recurrence,7and7predicts7prognosis7of7the7patients.

3

Prerequisites$for$Metastasis$
Cancer$cells$must$evade$or$co>opt$mul?ple$rules$and$barriers$that$were$reﬁned$for$long$years$of$organismal$
evolu?on.
Tolerance$for$apoptosis Inappropriate$prolifera?on...
Tolerance$for$tumor$microenvironment$(Hypoxia,$
reac?ve$oxygen,$limited$nutrients)
Loss$of$cellular$adhesion$(E>cadherin),$Increased$mo?lity$
and$invasivenessCollusion$with$Immune$response$(NF>κB,$MMP9,$HGF),$
stromal$cells$(TGF>β,$IL>10,$IL>23...)
Entry$and$survival$in$the$circula?on$(lymph,$vessel)
Angiogenesis,$co>op?ng$blood$platelets.
Exit$into$new$?ssue$and$coloniza?on
Integrins$for$secondary$organs,$Cancer$stem$cells

G.$P.$Gupta$et$al.$Cell$127,%679 (2006)$

4

Cancer$Evolution$for$Metastasis$
Cancer$cells$must$evade$or$co>opt$multiple$rules$and$barriers$that$were$refined$for$long$years$of$organismal$
evolution.
Tolerance$for$apoptosis Inappropriate$proliferation...
Tolerance$for$tumor$microenvironment$(Hypoxia,$reactive$oxygen,$limited$nutrients)
Loss$of$cellular$adhesion$(E>cadherin),$Increased$motility$
and$invasivenessCollusion$with$Immune$response$(NF>κB,$MMP9,$HGF),$
stromal$cells$(TGF>β,$IL>10,$IL>23...)
Entry$and$survival$in$the$circulation$(lymph,$vessel)
Angiogenesis,$co>opting$blood$platelets.
Exit$into$new$tissue$and$colonization
Integrins$for$secondary$organs,$Cancer$stem$cells

G.$P.$Gupta$et$al.$Cell$127,%679 (2006)$

5

Cancer$Evolution$for$Metastasis$
Cancer$cells$must$evade$or$co=opt$mul>ple$rules$and$barriers$that$were$reﬁned$for$long$years$of$organismal$
evolu>on.
Tolerance$for$apoptosis Inappropriate$prolifera>on...
Tolerance$for$tumor$microenvironment$(Hypoxia,$reac>ve$oxygen,$limited$nutrients)
Loss$of$cellular$adhesion$(E=cadherin,$extracellular$matrix),$Increased$mo>lity$and$invasivenessCollusion$with$Immune$response$(NF=κB,$VEGF,$HGF),$
G.$P.$Gupta$et$al.$Cell$127,%679 (2006)$
stromal$cells$(TGF=β,$IL=10,$IL=23...)
Entry$and$survival$in$the$circula>on$(lymph,$vessel)
Angiogenesis,$co=op>ng$blood$platelets.
Exit$into$new$>ssue$and$coloniza>onIntegrins$for$secondary$organs,$Cancer$stem$cells

6Prerequisites*for*Metastasis*
Cancer*cells*must*evade*or*coHopt*multiple*rules*and*barriers*that*were*refined*for*long*years*of*organismal*
evolution.
Tolerance*for*apoptosis Inappropriate*proliferation...
Tolerance*for*tumor*microenvironment*(Hypoxia,*
reactive*oxygen,*limited*nutrients)
Loss*of*cellular*adhesion*(EHcadherin,*extracellular*matrix),*Increased*motility*and*invasivenessCollusion*with*Immune*response*(NFHκB,*VEGF,*HGF),*
stromal*cells*(TGFHβ,*ILH10,*ILH23...)
Carcinoma located within blood vessels.
SquamousHcell carcinoma*() oftongue*().*Fibrin*deposition*(arrows)*and*tissueHlike*architecture.

Entry*and*survival*in*the*circulation*(lymph,*vessel)
Angiogenesis,*coHopting*blood*platelets.
Exit*into*new*tissue*and*colonization
Integrins*for*secondary*organs,*Cancer*stem*cells

D.*J.*Ruiter,*et*al.*THE*LANCET*Oncology*2,*2001,*1097

Prerequisites$for$Metastasis$
Cancer$cells$must$evade$or$co>opt$multiple$rules$and$barriers$that$were$refined$for$long$years$of$organismal$
evolution.
Tolerance$for$apoptosis Inappropriate$proliferation...
Tolerance$for$tumor$microenvironment$(Hypoxia,$reactive$oxygen,$limited$nutrients)
Loss$of$cellular$adhesion$(E>cadherin,$extracellular$
matrix),$Increased$motility$and$invasivenessCollusion$with$Immune$response$(NF>κB,$VEGF,$HGF),$
stromal$cells$(TGF>β,$IL>10,$IL>23...)
Entry$and$survival$in$the$circulation$(lymph,$vessel)
Angiogenesis,$co>opting$blood$platelets.
Exit$into$new$tissue$and$colonization
Integrins$for$secondary$organs,$Cancer$stem$cells

G.$P.$Gupta$et$al.$Cell$127,%679 (2006)$

8

Contents
Cancer'metastasis
Cancer'metastasis
Epithelial ()2to2Mesenchymal () Transfer

Metastasis'inhibition'– Blocking'TGFβ'pathwayTGFβ'(inducing'EMT)'is'promising'target'for'cancer'
metastasis
Control'of'TGFβ'signaling'by'Ubiquitination

Cancer$cells$escape$from$carcinomas$have$characteristics$of$“EMT”$

(EMTDinduced$cells$gain$property$of$stem$cells)$

M$Angela$Nieto,$Science&342,$1234850$(2013)$
10

Epithelial*to*Mesenchymal2Transition2(EMT)2()
()

()

()

(()

(()

()
((()*mesoderm2** >2muscle,2body2cavity( )...
*endoderm2** >2digestive2tract,2liver,2...2)

)

EMT
Embryonic2cell2migration) Wound2healingCancer2cell2dissemination
Fibroblast2formation
)

METOrgan differentiation
Epithelial2homeostasis
Metastatic2colonization

Features of EMT
N*cadherin,2vimentin...
(mesenchymal2marker)() EMT*TFs2(transcription2factor),2such2as2ZEB1,2Twist,2Snail...
Features2of2MET
E*cadherin,2α*catenin...
(Epithelial2marker)

Embryonic2development2undergo222or232rounds2of2EMT2and2MET2to2form2each2organs. 11

EMT$may$occur$in$cancer cells...
Pancreas cancer

IntesHnal$cancer$
invasive$edge

E6cadherin
β6catenin
Decrease$of$Epithelial$marker$is$found$in$tumor$cells.
And the$many$features$of$EMT$match$the$requirement$
of$cancer$metastasis,$so$it$is$thought$that$EMT$should$
occur$in$cancer$metastasis.

Intestinal$cancer$
invasive$edge

Features of EMT
N6cadherin,$vimentin...
(mesenchymal$marker)
EMT6TFs$(transcription$factor),$such$as$ZEB1,$Twist,$
Snail...
Features$of$MET
E6cadherin,$α6catenin...
(Epithelial$marker)

In%model%mice,%EMT%occur%in%metastasis.

ECcadHI ...%Epithelial%expression
ECcadLO ...%Mesenchymal%expression
Yellow ...%tumor%cells

Motile%tumor%cells%have%undergone%EMT%without%artificially%modifying%EMT%
regulators.
E.%Beerling et%al.%Cell%Reports%
14,%2281%(2016)13

Assumed.role.of.EMT.and.MET.in.cancer.metastasis

J.#P.#Thiery Nature#Reviews#Cancer 2, 442 (2002)
14

Short&summary
Cancer'metastasis'is critical'problem'for'patients,'because'
it causes'death'or'poor'prognosis.
In'metastasis'step,'tumor'cells'should'clear'many'hurdles'
from'various'environmental'pressures.
Cancer'cells'evolve'through'their'own'epi/genomc
instability'or'interaction'with'environment,'and'then'gain'
mesenchymal'features'to'survive harsh'conditions.
EMT'program,'which'is'primary'known'in'embryonic'cells,'
is'now'thought'to'be'the'important'step'in'tumor'metastasis.

Contents
Cancer'metastasis
Cancer'metastasis
Epithelial0to0Mesenchymal'Transfer
Metastasis'inhibition'– Blocking TGFβ'pathwayTGFβ'(inducing'EMT)'is'promising'target'for'cancer'
metastasis
Inhibiting'TGFβ'pathway'through'Ubiquitination

EMT&is&new&target&for&cancer?
EMT&is&essential&for&progression&and&metastasis&in&the&tumor&metastasis.
EMT;TFs&are&associated&with&chemo;resistance&in&cancer (see&table&below).
EMT;TFs&can&affect...
drug&metabolism&by&changing&the&expression&of&&drug;transportersrender&tumor&cells&less&sensitive&to&therapy&induced&apoptosis
kinase&inhibitor&resistance&(?)
EMT&&can&result&in&extensive&changes&in&the&cytoskeleton&and&the&tumor&cell&surface&repertoire,&which&alters&tumor&immunogenicity&on&the&cell&surfaces.

17

Molecular%mechanism%of%EMT

J.%Staalduinen et%al.%Oncogene%(2018)%

18

EMT<inducible$signal$pathway

HIF<αCellular$crosstalk$from
macrophages,$cancer<associated$fibroblasts
Cellular$environment
PTM
Ub<degradation
miRNA$reguation
Epigenetic$modifications
DNA$methylation
Chromatin$remodeling
EMT−TFs Histone$code
Regulating$alternative$splicing

S.#Imani#et#al.#Scientific#Reports volume6,$Article number: 28587$(2016)

19

Anti'cancer,strategy,toward,EMT
Targe6ng,EMT'TFs,or,EMT'TFs,signaling,pathways(siRNA,,miRNA,,small,inhibitor/ac6vator)
Reduc6on,of,EMT'TFs,results,in,MET,(partly)Combina6on,therapy,with,chemotherapy,,Cancer,metastasis20To+prevent+EMT+of+cancer+cells,+blocking+TGFβ+pathway+seems+to+be+promising.
Combination+of+αTGFβ+++αPD5L1+provoked+tumor+regression.+21

Lack(of(response(to(anti:PD:L1(was(associated(with(a(signature(of(TGF:β(signaling(in(fibroblasts((from(a large cohort of patients of MUC treated with anti:PD:L1((atezolizumab).(Pathways(associated(with(response(to(atezoilzumab and(cancer–immune(phenotypesTGFBR2(gene(expression in(is(signiﬁcantly(associated(with(non:response.

Nature(vol554,(2018(54422

Combination+of+αTGFβ+++αPD5L1+provoked+tumor+regression.+

EMT6+mouse+mammary+carcinoma+model(immune5excluded+phenotype)
Anti5TGFβ+treatment+significantly+reduced+
TGFβ+receptor+signaling+(pSMAD2/3).
Combined+antibody led+increase+in+tumor5
infiltrating+T+cells.

Nature+vol554,+2018+544

23

Contents
Cancer'metastasis
Cancer'metastasis
Epithelial0to0Mesenchymal'Transfer
Metastasis'inhibition'– Blocking TGFβ'pathwayTGFβ'(inducing'EMT)'is'promising'target'for'cancer'
metastasis
Inhibiting'TGFβ'pathway'through'Ubiquitination

TGFHβ'Signal'...'one'of'the'upstream'pathways'of'EMT.
Transforming'growth'factor'(TGF)Hβ

TGFHß

SMAD7

T/Smad2

TCell'growth'inhibitory'function
Induce EMTHTFs'through'SMADs'
signaling

cofactor(ZEB1)EMTHTFs

Moses'H.L.'et'al.'Cold'Spring'Harb.'Perspect.'Biol.'8,'a021865

miRNA200'family inhibitstranscription of EMTHTFs'induced'by'TGFβ.
P.'A.'Gregory'et.'al.'Nature'cell'biology,'10,'593,'2008.

25

TGFHβ'Signal'is'regulated'by'ubiquitin
TGFHβ–SMAD'signal'path'is'maintained'
by'many'ubiquitination.

USP2a Zhao'et'al.'2018,'Cell'Reports'22,'2442.

TTAc

Ub

OTUD1

Nedd4H2Z.'Zhang'et'al.''2017,'8,'2116
T
OTUB1 L.'Herhaus et'al.'Nature'commun.'2013,'4,'2519

miRNA200'family
P.'A.'Gregory'et.'al.'Nature'cell'biology,'10,'593,'2008.

T

X.Tang et'al.'Nat.'commun.

Ub

Ub

T

/Smad2

SMAD7

T

T

Ub

T

T

TGFHß

cofactor(ZEB1)EMTHTFs

Moses'H.L.'et'al.'Cold'Spring'Harb.'Perspect.'Biol.'8,'a021865

26

Role'of'Ubiquitin'in'inhibition'of'the'TGFβ'signaling
Steric'hinderance'(K33Htype)USP2aKO'Hep3B injected mice or
ML364'(inhibitor)'to'injected'mice
Zhao'et'al.'2018,'Cell'Reports'22,'2442.
DegradaRon'(K48Htype)degradation'by'proteasome
TGFβ'signal'is'stopped

siRNA'(OTUB1)'inhibits'cell'migration
Nuclear'importation
EMTHTFs'transcription

L.'Herhaus et'al.'
Nature'commun.'2013,'4,'2519

27

SIRT7(antagonizes(TGF4β(signaling(and(inhibits(breast(cancer(metastasis(de4Ub
acetylation

SMAD4(degradation
44>(inhibit(TGFβ(SMAD2(affinity(↑
44>(promote TGFβ(SIRT7&(E3(Ubligase
EMT4TFs

Resveratrol(activates(SIRT7(and(inhibit(TGFβ,(metastasis(is(inhibited(in(model(mouse.

X.Tang et(al.(Nat.(commun.(8:318((2017)28

SIRT7+is+downregulated+in+breast+cancer+lung+metastasisbrown : IHC staining of SIRT7SIRT7+was+downregulated+in+lung+metastasis+compared+to+in+primary+tumors.
SIRT7+level+is+inversely+correlated+
with+metastatic+capacity+of+various+
breast+cancer+cell+lines.
X.Tang et+al.+Nat.+commun.+8:318+(2017)29

SIRT7+inhibits+breast+cancer+lung+metastasis
4T1-shSirt7 cells
4 weeks

Balb/c mice

SIRT7 KD promotes lung metastasis.

MDA-MB231 cells
over expressing SIRT7
4 weeks

SIRT7 over expressing+prevents
lung+metastasis.
X.Tang et+al.+Nat.+commun.+8:318+(2017)30

SIRT7(expression(is(associated(with(TGF9β(signaling(KEGG*(analysis(in(TCGA**9RNA9seq deta set(obtained(from(1097(individuals*Kyoto(Encyclopedia(of(Genes(and(GenomesDatabase(for(bioinformatics,(based(on(molecular(interaction(network((signal(path,(metabolism...)**Database(of(The(Cancer(Genome(Atlas(National(Cancer Institute(&National Human Genome(Research(Institute)*Extra(Cellular Matrix

SIRT7(is(important(for(TGFβ(signal(??
31

SIRT7(indeed(inhibits(TGF3β(signaling and EMT.(TGF3β(downstream(gene<Trans(well(assay((cell(invasion)>

Pharmaceutics(2011,(3,(1073204(over(expression)(Knock(Down)
Genes(whichmediate(EMT(is(enriched(by(SIRT7(KD.

<Trans(well(assay((cell(migration)>(over(expression)

X.Tang et(al.(Nat.(commun.(8:318((2017)32

SIRT7'interacts'with'SMAD4,'especially'
SMAD4(MH2)>SIRT7(Catalytic'domain).

SMAD4

33

SIRT7(deacetylates(SMAD4(K428)(SMAD4)(catalyJc(dead)SMAD4(KR(mutants(in(MH2SMAD4(K428Ac)(is(important(for(SMAD2@SMAD4(binding,(nuclear(Localization(of(SMAD4localization(by(TGF@β(siganal.

(SMAD4)

SMAD4(≒Ac) (≒ Lys)weak(signalweak localization

34

SMAD4(K428)-is-important-for-its-degradation (Ubiquitination)
SMADK428(TGFβ)
Ac(Q)-... SMAD2-binding-↑ (↑)
Ub degradation-↓ (↑)
K (R)--... SMAD2--binding-↓ (↓)(SIRT7) Ub degradation--↑ (↓)

SMAD4-(Ac)-is-important-for-TGFβDinduced-metastasis

35

Resveratrol,activates,SIRT7,and,inhibits,lung,metastasis.
(*Resveratrol,is,known,as,SIRT1,activator)(in,vitro)

SMAD4,in,BT549,cells
resveratrol

Bulb/c,mice,injected,with,4T1,cells36

Summary''
EMT$is critical step$for$tumor$metastasis,$so$blocking$EMT$
can$be$a$effective$treatment$for$cancer metastasis.
EMT$is$induced$by$cellular$crosstalk$with$extracellular$
environment,$then$intracellular environment$is$maintained$(epigenetically).
TGFβ$signaling$pathway$is$one$of$EMTCinducing$pathways,$
and$blocking$TGFβ$in$combination$with$conventional$antiC
cancer$treatment.
TGFβ$signaling$is$mediated$by$SMADs,$which$are$regulated$by$Ubiquitination.$These$PTMs$control$activity$or$
stability$of$SMADs.

Role'of'Ubiquitin'in'inhibition'of'the'TGFβ'signaling
Steric'hinderance'(K33>type)DegradaCon'(K48>type)

TGFβ'STOPPEDSMAD7'degradation
inhibition'of'
TGFβ'Promoted' SMURF2'bindingTGFBR'degradation
TGFβ'STOPPEDIn'OTUD1'mutant'cells'EMT'are'promoted
injected'mice
39
Z.'Zhang'et'al.'Nat.'commun.'8:2116'(2017)