deciphering the secret(ome)s of cell communication along ...€¦ · madrid . hanahan &...

Deciphering the SECRET(ome)S of cell

communication along cancer progression.

Verónica Torrano, PhD

[email protected]

Verónica Torrano, PhD

[email protected]

October 18th 2019

Madrid

Hanahan & Weinberg. Cell 2011

Balanced integration of intracellular circuits

Adapted from Lewis N.E. Frontiers in Phisiology 2013

Hallmarks of cancer: GE deregulation at the core of

cancer research field

The “OMICS”: Useful tool for precision medicine

OME layers

Martin-Martin, Carracedo and Torrano, Frontiers 2018

Genomics

Epigenomics

Transcriptomics

Proteomics

Metabolomics

Treatment

decision

Molecular characterisation Molecular stratification

Gehlenborg N. SlideShare 2017

Are we able to exploit all the generated data?

Limitations…

Problem Alternative

CIBERONC Collab

XR Bustelo

RR Gomis

S Vincent

V Quesada

CIBEREHD Collab

AM Aransay

Cortazar et al., Cancer Res 2018

Valcarcel & Macchia et al., Cell Death Dis 2018 http://web.bioinformatics.cicbiogune.es/CANCERTOOL/index.html

Exploiting publicly available datasets for the identification of

new drivers of tumor progression

2016

2016 2018

2018

Productivity of CANCERTOOL

Cancertool serve as a discovery platform

for cancer-related genes

Hallmarks of cancer: GE deregulation at the core of

cancer research field

2016

Oct. 2019

Cancer cell/highly proliferating

NADPH NADPH

Proteins

Lipids (PL)

Nucleic acids

Nutrients

Nutrients

Nutrients

Nutrients

Nutrients Cell Growth/

proliferation

Oxidative

stress

Energetic

homeostasis

Normal cell (non-proliferating)

Energetic

homeostasis Nutrients

Cancer cell/highly proliferating

NADPH NADPH

Proteins

Lipids (PL)

Nucleic acids

Nutrients

Nutrients

Nutrients

Nutrients

Nutrients Cell Growth/

proliferation

Oxidative

stress

Energetic

homeostasis

Normal cell (non-proliferating)

Energetic

homeostasis Nutrients

METABOLIC SWITCH

CATABOLIC PROGRAM

ANABOLIC PROGRAM

HOW?

A2 A1

A3

A4

A2 A1 A3 A4 = + + + + An Metabolic switch

How the metabolic switch is triggered in cancer?

A2 A1

A3

A4

A2 A1 A3 A4 = + + + + An Metabolic switch

How the metabolic switch is triggered in cancer?

Is it regulated?

A1

A2 A3

A4

???

METABOLIC

SWITCH

Hypothesis: There must be a parsimonious process that allows a major

metabolic reprogramming with a limited number of genetic alterations that

trigger the metabolic swith

A1

A2 A3

A4

MASTER

TRANSCRIPTIONAL

REGULATORS OF

METABOLISM

METABOLIC

SWITCH

Hypothesis: There must be a parsimonious process that allows a major

metabolic reprogramming with a limited number of genetic alterations that

trigger the metabolic swith

Lorea Valcarcel, PhD

Ana Rosa Cortazar Verónica Torrano, PhD

Arkaitz Carracedo, PhD

Approach to study deregulation of metabolism in PCa

Cell signaling & Metabolism in PCa

Transcription

Bioinformatics

Metabolism

PCa: prostate cancer

Data mining

23 major transcriptional co-regulators of metabolism

selected

p=1.19e-17 L

og

2 m

RN

A P

GC

1A

Taylor et al.

DFS: Disease free survival

PGC1α is dowregulated in PCa

Low

0 2 5 5 0 7 5 1 0 0 1 2 5

0

5 0

1 0 0

DF

S (

%)

L o w

H ig h

p= 0.0479

Time (months)

Lapointe et al.

Varambally et al.

Tomlins et al.

Grasso et al.

23

Taylor et al.

(TCGA)

PGC1A

Torrano et al. Nature Cell Biology 2016

What is PGC1α?

PGC1α

X Target Genes

Oxidative metabolism Mitochondrial biogenesis

Rowe et al., Circ. Res. 2010

Cystic

lesions

Model human disease using mouse models

Focal

invasion

3m 12m 6m Pten lox/lox ; Pb-Cre

Two copy lost

Cancer lesions

No signs of metastasis

Aim: Evaluate the causal contribution of PGC1α in PCa

DKO PTEN KO

Signs of metastasis

PGC1α is a PCa metastasis suppressor

PanCK

PanCK

DK

O

PT

EN

KO

Dissemination to the bone

DKO DKO


Primary Tumor

Metastatic cell

PT cells

Dissemination

Fibroblast

Migration/Invasion

Adhesion

Blood vessel v

Extravasation

The process of metastasis

1-Tumor initiation:

proliferation and

survival

2- Metastasis initiation:

local invasion and

intravasation 3- Metastasis progression:

survival, arrest at distant organ and

extravasation

4- Metastasis virulence:

micrometastasis formation and

metastatic colonization

v Bone

Lymph node

Lung

Migration

Colonization of secondary organ

(Metastasis)

Intravasation

PGC1A

Primary Tumor

v

1-Tumor initiation:

proliferation and

survival

v

PGC1α expression suppresses cell and tumor growth

ug/ul

2D growth (in vitro) In vivo growth


v

Primary Tumor

v

1-Tumor initiation:

proliferation and

survival

v

PGC1α expression suppresses cell and tumor growth

ug/ul

2D growth (in vitro) In vivo growth


v

In vivo growth

Primary Tumor

Migration/Invasion


local invasion 0.0

0.5

1.0

1.5

50 100 µm

Fo

ld c

ha

ng

e r

ela

tive

to

No

Do

x

** **

3D invasion PC3

Fo

ld c

ha

ng

e r

ela

tive

to

No

Do

x

0.0

0.5

1.0

1.5

** 100µm

No

Do

x

Do

x

Invasive growth PC3

Dox

PGC1α expression suppresses migration and invasion of

PCa cells

Valcarcel et al. Cancer Research 2019

PGC1α expression suppresses PCa metastasis

In vivo metastasis

Dissemination

Adhesion

Extravasation

Bone

Lymph node

Lung

Migration

Colonization of

secondary organ

3- Metastasis

progression

4- Metastasis virulence


0

5

10

15

0 . 0

0 . 5

1 . 0

1 . 5

2 . 0

ERRα drives the tumor and metastasis suppressor activity

of PGC1α

PGC1α

ERRα Transcriptional program

PGC1α

Transcriptional program ERRα

0

5 0

1 0 0

Hin

d l

imb

le

sio

n i

nc

ide

nc

e (

%)

n .s . p = 0 .0 2 5

PGC1α - - + +

+ - + - ERRα

In vivo metastasis

Re

lative

gro

wth

to

da

y 0

**

$

$$$

Fo

ld c

ha

ng

e r

ela

tive

to

No

Do

x

***

$$$

$$

Invasive growth 2D growth

PGC1α - + - + - +

+ + - - - - ERRα

PGC1α + + +

+ - - ERRα

0 5 10 15

Pyruvate metabolism Sirtuin6 regulation and functions

Propionate metabolism p.2 Cytoskeleton remodeling

Leucine, isoleucine and valine metabolism.p.2 Peroxisomal branched chain fatty acid oxidation

PPAR regulation of lipid metabolism Huntington's Disease Tricarbonic acid cycle

Ubiquinone metabolism

-Log(p-val)

PGC1α

ERRα Transcriptional

metabolic program

PGC1α

Up


Functional enrichment (Metacore)

…beyond their canonical function on activation of oxidative

metabolism

…beyond their canonical function on activation of oxidative

metabolism

0 2 4 6 8 10

Development_Slit-Robo signaling Integrin-mediated cell adhesion and migration

Neuroprotective action of lithium Fibronectin-binding integrins in cell motility

Chemotaxis Cell adhesion

Cytoskeleton remodeling Receptor-mediated axon growth repulsion

TGF-beta 1-induced transactivation

-Log(p-val)

0 5 10 15

Pyruvate metabolism Sirtuin6 regulation and functions

Propionate metabolism p.2 Cytoskeleton remodeling

Leucine, isoleucine and valine metabolism.p.2 Peroxisomal branched chain fatty acid oxidation

PPAR regulation of lipid metabolism Huntington's Disease Tricarbonic acid cycle

Ubiquinone metabolism

-Log(p-val)

PGC1α


metabolic program

PGC1α


Cytoskeleton/adhesion

program

PGC1α

Up Down

Valcarcel et al. Cancer Research 2019 Torrano et al. Nature Cell Biology 2016

Functional enrichment (Metacore) Functional enrichment (Metacore)

0

10

20

30

40

pM

LC

/Are

a

***

pMLC2 F-actin pMLC2 / F-actin / Dapi

No

do

x

Do

x

50um

No dox Dox

The transcriptional axis PGC1α/ERRα impacts on integrin

signaling and cytoskeleton organization

0

10

20

30

40

pM

LC

/Are

a

***

pMLC2 F-actin pMLC2 / F-actin / Dapi

No

do

x

Do

x

50um

No dox Dox

140KDa

GAPDH 35 KDa

160KDa

PGC1α

ERRα 45 KDa

80KDa

17 KDa

17 KDa

- + - + - +

Cont sgERRα#1 sgERRα#2

ITGβ1

ITGβ4

70KDa p-Src

70KDa total-Src

total-Cofilin

p-Cofilin

The transcriptional axis PGC1α/ERRα impacts on integrin

signaling and cytoskeleton organization

…and preceded by a transcriptional deregulation of MYC

0 8 16 24

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

Time (h)

Ge

ne

exp

ressio

n (

Fo

ld c

ha

ng

e r

ela

tive

to

No

Do

x)

MYC TCF4

ODC

*

*** ******

p=0.05

ITGB1

ITGA3

qPCR

…and preceded by a transcriptional deregulation of MYC

0 8 16 24

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

Time (h)

Ge

ne

exp

ressio

n (

Fo

ld c

ha

ng

e r

ela

tive

to

No

Do

x)

MYC TCF4

ODC

*

*** ******

p=0.05

ITGB1

ITGA3

qPCR

GAPDH

MYC

ITGβ4

ITGβ1

ITGα3

35KDa

70 KDa

80KDa

140KDa

160KDa

160KDa

- - - + + +

16h 24h 48h 5 days

GAPDH 35KDa

Dox

PGC1α

Metastasis PGC1α-ERRα

Stratification potential

Cytoskeleton-MYC (?)

Torrano* and Valcarcel* et al. Nat Cell Bio 2016

Valcarcel* and Torrano* et al. Cell Cyle 2016

Valcarcel*, Gaude*, Torrano* et al. Trends Endo Metabolism. 2017

Torrano and Carracedo. Cell Metabolism 2017

Martin-Martin, Carracedo, Torrano. Frontiers 2018

Valcarcel et al. Cancer Research 2019

Anabolism Catabolism

Metabolic vulnerability

Primary Tumor

Metastatic cell

PT cells

Dissemination

Fibroblast

Migration/Invasion

Adhesion

Blood vessel v

Extravasation

Cell-Autonomous metastasis suppressor

1-Tumor initiation:

proliferation and

survival


local invasion and

intravasation 3- Metastasis progression:

survival, arrest at distant organ and

extravasation

4- Metastasis virulence:

micrometastasis formation and

metastatic colonization

v Bone

Lymph node

Lung

Migration

Colonization of secondary organ

(Metastasis)

Intravasation

PGC1A

Secreted factors: active mediators of tumorigenesis

Hoshino et al. Nature 2016

Peinado et al. Nature Medicine 2012

Costa-Silva et al. Nature Cell Bio 2015

Brady et al. CancerCell 2016

Olmeda et al. Nature 2017

Secretome/Soluble factors Extracellular vesicles

Kaplan et al. Nature 2005

Obenauf et al. Nature 2013

Primary Tumor

Metastatic cell

PT cells

Metastatic niche

Pre-metastatic niche

Fibroblast

Blood vessel

v

Bone

Lymph node

Dormant cell


v

EV: extracellular vesicles

Ariane Schaub

Metabolic regulation of auto and paracrine

cell-communication

v v

PGC1A negative cells PGC1A positive cells

Stroma cell

EVs

Soluble factors:

Citokines/metabolites


Metabolic regulation of auto and paracrine

cell-communication

Lymph node

Prostate PT

IL from prostate

tumors

SN of cells

Metabolic regulation of cell communication in PCa

OXPHOS-PGC1α high

Glycolysis-PGC1α low

High risk Met Low risk Met

Cell autonomous

function

Torrano & Valcarcel.2016

IL from prostate

tumors

SN of cells

OXPHOS-PGC1α high


Functional and Molecular

Characterization

EVs/SF EVs/SF


Cell autonomous

function


Has PGC1α a non-cell autonomous function?

Is the TS of PGC1α “transferable”?

EVs/SF

Metabolic regulation of cell communication in PCa

Functional characterization- Experimental approach

Secretome (No dox, Dox)


Soluble factors (No dox, Dox)

Extracellular vesicles (No dox, Dox)

1/2

1/2

Torrano & Valcarcel et al. 2016

Functional characterization- Experimental approach



Soluble factors (No dox, Dox)

Extracellular vesicles (No dox, Dox)

1/2

1/2

Torrano & Valcarcel et al. 2016

?

PGC1α has a non-cell autonomous anti-proliferative

activity in aggressive PCa cells….

S -

S +

0.0

0.5

1.0

1.5

Fold

change r

ela

tive t

o -

Dox

*

PC3

S - S +

0.0

0.5

1.0

1.5

Fold

change r

ela

tive t

o -

Dox

**

DU145

S - S +

0.0

0.5

1.0

Fold

change r

ela

tive t

o -

Dox

**

22RV1

Unpublished data. Please do not post

…. Dependent on ERRα

sgCtl sg#1 sg#2

0.6

0.7

0.8

0.9

1.0

1.1

1.2

Fold

change r

ela

tive to e

ach N

o d

ox

**

$

$$

(+ Dox)

PC3

S (-) S (+)

sgCtrl

sgERRα#1

sgERRα#2


Which component on the CM mediates the

non-cell autonomous anti-proliferative

effect of PGC1α?

Which component on the CM mediates the

non-cell autonomous anti-proliferative

effect of PGC1α?

Conditioned media = extracelular vesicles + soluble factors

Ultracentrifuge, 45 Ti rotor and tubes used for EVs isolation. (Beckman Coulter).

The PGC1α anti-proliferative effect is only

maintained in the SF fraction

SF- SF+

Dox0.0

0.2

0.4

0.6

0.8

1.0

Fold

change r

ela

tive to N

o D

ox

*

Dox0.0

0.5

1.0

PC3

Fold

change r

ela

tive to N

o D

ox

Exo- Exo+


…. And caused by the “protein” fraction of the

secretome

sgctl+dox sg69+dox sg290+dox

0.8

0.9

1.0

1.1

1.2

Fold

change r

ela

tive to e

ach N

o d

ox

sgctl+dox sg69+dox sg290+dox

0.6

0.8

1.0

1.2

Fold

change r

ela

tive to e

ach N

o d

ox

***

$$

$$$

**

Secretome Metabolites

Protein fraction

Protein fraction Metabolites


Molecular characterization of secretomes: Proteomics

PGC1α

negative

162

differential

proteins

Gluconeogenesis

Glycolysis

TCA cycle

ECM organization/degradation

Collagen binding

Focal adhesión

Senescence (SASP)

Hypoxia

Functional enrichment

PGC1α

positive

80

DOWN

82 UP L

ab

el F

ree

LC

-MS

/MS

Pro

teo

mic

s A

na

lysis


PGC1α

negative

162

differential

proteins 80

DOWN

82

UP

Gluconeogenesis

Glycolysis

TCA cycle


Collagen binding

Focal adhesión

Senescence (SASP)

Hypoxia


Focal adhesion

Pyrimidine biosynthesis

Gluconeogenesis

TCA cycle

Pentose Phosphate Pathway

Cellular respiration

Senescence (SASP)

Hypoxia

PGC1α

positive

La

be

l F

ree

LC

-MS

/MS

Pro

teo

mic

s A

na

lysis



PGC1α

negative

162

differential

proteins 80

DOWN

82

UP

Gluconeogenesis

Glycolysis

TCA cycle


Collagen binding

Focal adhesión

Senescence (SASP)

Hypoxia


Focal adhesion

Pyrimidine biosynthesis

Gluconeogenesis

TCA cycle

Pentose Phosphate Pathway

Cellular respiration

Senescence (SASP)

Hypoxia

Promoter enrichment:

MAZ/MYC/MAX

Promoter enrichment:

ERRα

PGC1α

positive

La

be

l F

ree

LC

-MS

/MS

Pro

teo

mic

s A

na

lysis


Urine

IL from prostate

tumors

SN of cells

Metabolic regulation of the metastatic niche in PCa

OXPHOS-PGC1α high


Functional and Molecular

Characterization

EVs/SF EVs/SF


Cell autonomous

function


Has PGC1α a non-cell autonomous function?

Is the TS of PGC1α “transferable”?

EVs/SF

PGC1α lost induces stromal reaction at early stages of PCa

Focal

invasion Metastasis

Pten/Pgc1a lox/lox ; Pb-Cre

?

Pten lox/lox Pten lox/lox Pgc1a lox/lox


Focal

invasion Metastasis


Mireia Castillo, PhD. F. Champalimaud, Lisbon

Pten lox/lox Pten lox/lox Pgc1a lox/lox


Focal

invasion Metastasis


Mireia Castillo, PhD. F. Champalimaud, Lisbon

Stroma

Epithelia

TIL

TAKE HOME MESSAGE

In the light of our data we propose that the anti-proliferative activity of PGC1α goes

beyond the cell-autonomous activation of catabolic processes presenting the

transcriptional co-regulator as a key metabolic player that modulates secretome

composition and its impact on prostate cancer agressiveness.

Thank you! Gracie! Milesker! Gràcies! Gracias!

Bilbao, Mikel Azkalgorta, Féliz Elortza- Proteomics Platform CICbioGUNE

Santander, Fernando Calvo- IBBITEC

Madrid, María Mazariegos, Alberto Hernández, Héctor Peinado-CNIO

Barcelona, Roger Gomis- IRB

London, UK, Eva Crosas, Irene Hernández, Victoria Sanz Moreno-Barts Cancer Institute

Cambridge, UK, Christian Frezza

SECRETOME

=

soluble factors (proteins, lipids, metabolites…) + Extracellular vesicles

668 proteins/genes

Cortazar et al. 2017

12 candidates

Biogenesis (20) and secretion (10)

2 candidates

Criterial: N-PT-Mets & DFS

PCa cell lines PCa cell line

TRIPZ-DPT

No dox

Dox

Basal levels LV-TRIPZ-DPT

PCa cell line

pLKO or

CRISPR/Cas9

CTHRC1

No dox

Dox

LV-pLKO tet on

LV-CRISPR/Cas9

PCa cell lines • Basal levels, pLKO

• Complete deletion,

CRISPR/Cas9

Ove

r-e

xp

res

sio

n

Sil

en

cin

g/d

ele

tio

n

Functional and mechanistical analysis

Cell autonomous

and

non-cell autonomous

effects

deciphering the secret(ome)s of cell communication along ...€¦ · madrid . hanahan &...

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