Crosstalk between proliferation and migration

How alternative splicing converts p53 tumour suppressor into an oncogene

Pierre Roux CRBM/ UMR5237 Montpellier, France

Crosstalk between proliferation and migration!

Dynamics of cell invasion in cancer

Epithelial

Mesenchymal

Amœboid

Invasive modes

Dynamics of cancer cell invasion

Primary tumor

Invasive tumor

Metastasis

Mode of migration

Mesenchymal Amœboid

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EMT(epithelio-mesenchymal transition) - Loss of cell-cell adhesion - Increased motility: Rac1- and metalloprotease dependent EAT (epithelio-amoeboid transition) - Loss of cell-cell and cell-ECM adhesion - Increased motility: RhoA-dependent Independent of Rac1 and metalloproteases

« Guardian of the genome »

•! The most frequent mutated gene in cancer

•! Inactivated function in almost all tumors

•! Activates DNA repair when DNA has sustained damages

•! Arrests growth by holding the cell cycle in G1/S or G2

•! Initiates apoptosis if DNA damage proves to be irreparable

The «!Peto’s paradox!»: no correlation between body size and cancer risk&

•! Larger and longer-lived animals should have a higher risk of cancer than smaller, shorter-lived animals!

•! No link between the size of an animal and its risk of developing cancer. !

•! Elephants: Enhanced DNA-damage response and tumor resistant Why don't elephants get cancer?"

Haupt S., and Haupt, Y. ; F1000Res. 2017 Nov 22;6:2041; Sulak et al.;Elife. 2016 Sep 19;5.

Species Humans Elephants

Average survival ~70 years 60–70 years Number of pairs of p53 copies

1 1

Number of pairs of p53 transgenes

0 19

Cancer incidence 1:4 0

The elephant genome encodes 20 copies of the tumor suppressor gene TP53

Comparison of humans and elephants and parameters of relevance to cancer-free survival

•! The most frequent mutated gene in cancer

•! Inactivated function in almost all tumors

The role of p53 is crucial in cancer: more than 97000 publications

The role of p53 is crucial in cancer

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A novel tumor suppressor activity for p53: inhibition of migration and invasion

p53 Apoptosis Cell cycle arrest

p53: The most frequently mutated gene in cancer Inactivated in almost all cancers

Senescence Adhesion Invasion

Migration Rho GTPases

Mutant p53 controls EMT/EAT and promotes invasion

p53 and EMT: Gadea et al., EMBO J., 2002 Gadea et al., J. Cell Science, 2005 Gadea et al., J. Cell Biol, 2007; Vinot et al., Meth. Enzymol., 2008; Roger et al., J. Cell Sci., 2010.

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Mode of migration


1: Mutant p53 disrupts adherens junctions in inhibiting E-Cadherin expression through Slug and Zeb1 2: Mutant p53 promotes amoeboid-like mode of migration in 3D. 3: Mutant p53 activates the RhoA/ROCK pathway.

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Wt TP53 generates alternative splice isoforms

Biomarker of metastasis risk? Poorly reliable biomarker

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Mutant p53 Alternative Splicing Mutation

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TP53 gene and its products

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Alternative splicing generates modified proteins

Alternative splicing as an unexplored program in oncology

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!133p53" is associated with bad prognosis in breast cancer (A. Thompson, JC Bourdon)

#133p53ß is: -! associated with reduced disease free survival (DFS) and global survival, independently of p53

mutations -! a clinical biomarker to predict high risk of relapse and metastases in advanced breast cancer.

Breast: 276 tumors

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Development of highly metastatic MDA-MB231-luc cell lines by in vivo selection (breast model)

Implantation into immunodeficient mice

In vitro isolation and expansion

immunodeficient

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!133p53 is associated with bad prognosis in CRC

#133p53 is: -! associated with reduced disease free survival (DFS) and global survivall -! a clinical biomarker to predict high risk of relapse and metastases in advanced CRC

Rectum: 60 tumors

P = 0.027

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E-Cadherin

p53 status SW480 CoLo205 HCT116 LoVo SKCo1 SW620

wt mutant mutant wt mutant mutant

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The %133p53ß isoform promotes RhoA/ROCK rounded blebbing invasion&=K0!!E#W#X!<<3?<Y"Z[O#

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•! Mice modelling $133p53 :

•! are highly tumour-prone,

•! display chronic inflammation and autoimmunity.

•! display elevated serum IL-6

Δ122p53 (a model of Δ133p53) mice show decreased survival and a very aggressive tumor

spectrum"

Antony Braithwaite, University of Otago, NZ

Loss of IL-6 in !122p53 mice reduces tumour incidence"

#122p53 IL6 null

X

Loss of IL-6 reduces cell migration and actin polarisation in !122p53-expressing MEFs."

!122p53-expressing cells have constitutively active JAK-STAT and RhoA-ROCK signalling pathways"

!133p53 isoform promotes tumour invasion and metastasis via interleukin-6 activation of JAK-STAT and RhoA-ROCK signalling

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#133p53

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Tumour cell invasion Immune cell migration

Campbell et al.,"Nat. Commun; 2018 Jan 17;9(1):254. !

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#133p53! reprograms epithelial cells to pro-metastatic cells

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!133p53ß is a transcript produced by the WT TP53 gene.

-! Promotes invasiveness in WT p53 cells -! Induces amoeboid movement -! Promotes cancer stem cell formation

Mode of migration


To conclude

•! p53 isoforms expression is a more important mechanism than TP53 mutations to predict patients outcome

•! This explains why patients with WT TP53 can have metastasis and reciprocally why patients with mutant TP53 gene do not systematically have metastasis and relapse

•! The %133p53s isoforms are reliable targets to reduce metastasis

1. How %133p53 isoforms interfer with p53 tumor suppressor expression and activity ?

2. How %133p53 isoforms interact with oncogenic mutant p53 activity ?

1! 2!

!Cell cycle arrest and senescence"

!

p53"

p21"

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and bypass senescence "!

Inactive accumulated form

"133p53!"133p53!

1. How %133p53 isoforms interfer with p53 tumor suppressor expression and activity ?

Normal cell: induced expression

Cancer cells: constitutive expression

Véronique Gire, Fanny Tomas

2. How %133p53s isoforms interact with mutant p53 ?

HUMAN TP53 GENE

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p53 mutants p53 mutants

Tumor suppressor p53

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Mutant p53

Dominant negative effect!

Nikola Arsic

What make differences between WT $133p53 isoforms and mutant p53?

•! Mutant p53 and %133p53 isoforms share:

#! Similar pro-oncogenic functions opposite to p53 tumour suppression

#! Identical cellular localisation, both present in cytoplasm and nucleus, contrary to WT p53, which is exclusively detected in the nucleus.

#! Unfolded protein conformation

#! Specific proteins partners (Pontin, HSP70 and p63 family members), which do not bind to WT p53

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Mutant p53 and %133p53s isoforms promote cancer aggressiveness

From: Gain of function of mutant p53 by coaggregation with multiple tumor suppressors, Xu et al, 2011; Nature Chemical Biology

Pro-oncogenic functions of mutant p53 is implemented by coaggregation of wild-type p53 that promotes

dominant-negative effect

p53R273H"p53R175H"

WT p53"

Only p53 structural mutant p53R175H creates protein aggregates

p53"amyloid aggregates"

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Control"

3D reconstitution"H1299 cells"

wt $133p53 creates protein aggregates in cells

]N))'()&'()& Nikola Arsic

Δ133p53βR175H"

Control" WT Δ133p53β"

$133p53!R280K

3D reconstitution"

$133p53" creates protein aggregates independently of the mutation status

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Nikola Arsic

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Δ133p53βR175H"


$133p53!R280K

3D reconstitution"

$133p53" creates protein aggregates independently of the mutation status

H1299 cells"

Δ133p53β"

amyloid aggregates"Hoechst "

Nikola Arsic

$133p53" interacts with p63 isoforms

Nikola Arsic

p63 regulates invasion/EMT

!Np63

metastasis

Primary tumor

!"#&

TAp63

p63

Interaction with protein partner p63 reduces aggregation state of $133p53"

!"

#$""

%$$%!$&"$

""

!"

#$""

%$$%!$&"$

'&( )"(

6*+,!

_!

6*+,!

_6%!!+"!

`-./0123!

456.751

6%!!+"!

`-./0123

6*+,!

_!

6*+,!

_6%!!+"!

`-./0123!

456.751

6%!!+"!

`-./0123

89/0123/�6%!!+"!`

89/+,!

89!`�24.:6;<+=762.26.

6%!!+"!`89/0123

3D reconstitution"

H1299 c"


ΔNp63α" WT Δ133p53β+ΔNp63α"

Δ133p53β"ΔNp63α"Hoechst"

Current view: p53 and p63 are independent EMT regulators

metastasis

Primary tumor

!"#&

p63 p53

- p53 and p63 do not interact

metastasis

!"#&

Mode of migration


Δ133p53β and p63 cooperate in disrupting adherens junctions and in promoting invasion

Δ133p

53βE

GFP

ΔNp6

3α

ΔNp6

3γ

ΔNp6

3γ

ΔNp6

3α

control

E-Cadherin

TubulinN-Cadherin

Adhesion

Inva

sion

inde

x

sh6N

p63

shC

ontr

ol

0

2

4

6

8

*

6133p53`

sh6

Np6

3

shC

ontr

ol

*10

Inva

sion

inde

x

613

3p53

`

6N

p63a

6N

p63_

6N

p63a

6N

p63_

6133p53`

Con

trol

0

20

40

60

80

*

MCF7:WTp53

WT ∆133p53ß synergizes with ∆Np63 in promoting adherent junctions disruption and enhanced invasion

Co-expression of $133p53" and $Np63! synergistically induces cell invasion

"Np63!"133p53ß!

"133p53ß Storage of inactive %133p53ß in aggregates

"133p53ß!

"Np63!

CCT! Invasion

Release

Active complexes CCT!

Oncoprotein reversible aggregation mechanism

Nikola Arsic

-! Stored in aggregates in an inactive form -! Aggregation state of %133p53ß inversely correlates to its activity level

-! %133p53ß aggregates are reversible depending on presence or absence of interacting partners

"Np63!"133p53ß!Storage of inactive

%133p53ß in aggregates "133p53ß!

"Np63!

CCT!Invasion

Release

Active complexes CCT!

mutant p53 and WT $133p53" aggregates are distinct

Nikola Arsic

Mutant p53

WT p53

Inhibition of WT p53 function in aggregates

Invasion

%133p53ß

To conclude

•! %133p53 isoform: oncoprotein reversible aggregation mechanism

•! %133p53 oncogenic activity mechanism differ from this of mutant p53

•! p53 and p63 (p73?) isoforms activities are coordinated

p63 and p53 signaling cooperate in regulating EMT

metastasis

Primary tumor

!"#&p63 isoforms

p53 isoforms

- p63 and p53 isoforms interact each other

p53!p53!

- This extends the number of combinaison of transcriptional heterocomplex complex

3?<Y#

X!<<3?<Y#

3?<#

X!<<3?<#

X93E<a#

0R3E<#

TAp63!TAp63TAp63!TAp63

"Np63!

TAp63

Np63"133p53ß!

p53!p53!p53

p53!

p53

"133p53ß!

3?<b#

X!<<3?<b#

X93E<c#

0R3E<c#

TAp63!TAp63TAp63!TAp63

"Np63!

TAp63

Np63"133p53ß!

p53

p53!

p53

"133p53ß!

X93E<b#

0R3E<b#

Etc ………

How can p53 integrate so many signals at once?

-pH-Glucose-Nutrients

Neighboring cells - cellcontact

- secreAonofcytokines

H2O2

.OH

.O2

-

Protein oxidation - Meta-tyrosine - Amino group - Thiol group

Nucleic Acid oxidation Damages to:

• Genomic DNA • Mitochondrial DNA • Ribosome (rRNA)

• tRNA • mRNA

• microRNA, snoRNA…

Carbohydrate oxidation Extracellular signals

Lipid Peroxidation - Cytoplasmic mb (Na+/K+ )

- Endoplasmic Reticulum mb (Ca2+ ) - Mitochondrial mb (ATP )

- Lysosomal mb (release protease) - Nuclear mb (nuclear structure)

mitochondria

UV

Carcinogenes

proliferation Energy

metabolism

Cell cycle arrest G1/G2 Cell motility,

angiogenesis

Senescence Differentiation Inflammation

Organogenesis

Cell death : -extrinsic apoptosis -intrinsic apoptosis

-necroptosis - Autophagic cell death

Cell Stemness Cell repair

How do p53 choose the target genes to induce in order to trigger a coordinated and defined cellular response adapted to the damages and the tissue type?

EMT

Responses p53

-pH-Glucose-Nutrients

Neighboring cells - cellcontact

- secreAonofcytokines

H2O2

.OH

.O2

-

Protein oxidation - Meta-tyrosine - Amino group - Thiol group

Nucleic Acid oxidation Damages to:

• Genomic DNA • Mitochondrial DNA • Ribosome (rRNA)

• tRNA • mRNA

• microRNA, snoRNA…

Carbohydrate oxidation Extracellular signals

Lipid Peroxidation - Cytoplasmic mb (Na+/K+ )

- Endoplasmic Reticulum mb (Ca2+ ) - Mitochondrial mb (ATP )

- Lysosomal mb (release protease) - Nuclear mb (nuclear structure)

mitochondria

UV

Carcinogenes


metabolism


angiogenesis


Organogenesis




How do p53 choose the target genes to induce in order to trigger a coordinated and defined cellular response adapted to the damages and the tissue type?

EMT

Responses p53/p63/p73familyisoforms

How can p53 integrate so many signals at once?

Conventional view: biological response specificity is driven by p53 levels


metabolism


angiogenesis


Organogenesis




Levelofp53

Target genes: > 8000/10000 genes

p53

- transcripAon- post-translaAonalmodificaAons- degradaAon


metabolism

Cell cycle arrest G1/G2 EMT


Organogenesis




'()$'b)$'a)&@1.;.621&4.2'/*\*1&

Target genes: > 8000/10000 genes

Model: biological response specificity is driven by composition of p53 family members complexes

X!<<3?<#

3?<Y#

X!<<3?<Y#

3?<#

X!<<3?<#

X93E<#

0R3E<#

Invasion Migration

Angiogenesis

TAp63!TAp63!

Target genes: "Np63!

TAp63

Np63 "133p53ß!

p53!p53!

p53!

p53

"133p53ß!

3CK"9&I0H./,&%6104&W0*66*&C.=\&de6.80<=*&V06*&W:0/0''*&P.6+&3/,06*&#./M,&&V0//*1&V,>*,&3:601+*//*&%8?=0//*&&J,=6*8+&f=//0*8&7+e':,80*&d08.+&J,=6e/08*&C.?*6&W*??5&C,58,=>&!22,8=*/&d0?8,/&7,2*6&%->,//,:&P,885&#.2,1&&

7'/04.19&E0>0*6&74:*66*6&g3!L$37Lh&3,61+*8&K6.4H&I,+:,/0*&3,:=M,4&3e40/*&%'./0+&",5/01&36*8&&Pi&",:=+*,=&g3=60*&@81A+=+*h&Ci&I,j2,8&&

3.//,-.6,A.819&f3&K.=6>.8&gE=8>**h&fi&#,M0&g".8+'*//0*6h&Vi&K,6/.B,+M&g!B65h&Vi&P,B6*&g#.=/.=1*h&%i! Q,5,B,&g3CK"h&&&

3.//,-.6,A.81&g4/0804h9&%i! #:.2'1.8gE=8>**h&Ki! "i&E*/&60.&g".8+'*//0*6h&!i&36,'*M&g".8+'*//0*6h&fW&E*/.6>&g#.=/.=1*h&

Crosstalk between proliferation and migration

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