Les tumeurs ont la peau dure : régulation de leurs ...pataclope83.com/wp-content/blogs.dir/3/files/2013/... · 15e Colloque de la Recherche de la Ligue contre le cancer, Nice, 24-25

15e Colloque de la Recherche de la Ligue contre le cancer, Nice, 24-25 janvier 2013

Les tumeurs ont la peau dure : régulation de leurs propriétés mécaniques par CD98hc

Etienne BoulterIRCAN - INSERM U1081, CNRS UMR 7284 – Equipe AVENIR Chloé Féral - Nice


Sommaire de la présentation

• Propriétés mécaniques des tissus et mécanotransduction• La peau comme modèle d’étude• Rôle de CD98hc dans la régulation des propriétés

mécaniques


Des concepts émergents : propriétés mécaniques des tissus et mécanotransduction

La mécanotransduction est le processus de transformation d’un stimulus mécanique en signal chimique

Cellules ciliées de la cochlée ou du saccule dans l’oreille interneCanaux ioniques activés sous l’effet du Stress

Cellules endothéliales qui ressentent la pression du flux sanguinPECAM1, VE-Cadherine

Nature Reviews | Molecular Cell Biology

p

Endothelial cellVascular smoothmuscle cellBlood flow

HyperlipidaemiaThe state of blood carryin ghigh levels of lipoproteins tha tcontain cholesterol an dtriglycerides.

’Laplace s lawThis law states that tension i nthe vessel wall equals th edifference in pressure acros sthe vessel times the radius o fthe vessel, divided by th ethickness of the wall. Thus, higher blood pressure o rvessels of larger radius requir ethicker walls to b e

mechanically stable.

Mechanotransduction in vessel physiologyPeriodic contractions of the heart cause large, pulsatile changes in blood pressure on the arterial side of the circu-lation. Large arteries respond to blood pressure passively owing to their intrinsic elasticity. These arteries, especially the aorta, expand at each peak of pressure from the heart (systole) and then gradually deflate as pressure from the heart drops (diastole), which releases blood downstream. The elasticity of large vessels therefore dampens the peri-odic variations in pressure, which evens out the blood flow in smaller, less elastic vessels during the cardiac cycle. The resultant cyclic stretching of artery walls promotes a quiescent, contractile state in which VSMCs express a full range of differentiation markers5.

Blood pressure is determined mainly by the diameter of smaller resistance arteries that lead into capillary beds. VSMCs in these vessels actively respond to acute changes in blood pressure through a mechanism called the myo-genic effect6. Elevated pressure triggers VSMC contraction, which narrows small resistance arteries to keep blood flow constant in downstream capillaries. If pressure remains elevated over longer periods, VSMCs remodel, which causes the vascular wall to thicken to resist these forces. Indeed, arterial wall thicknesses seem to be calibrated to resist wall tension according to a simple physical law,

’Laplace s law, which governs wall tension as a function of vessel radius and pressure7. However, under pathological conditions in which pressures remain high, this remod-elling can eventually compromise vessel elasticity, which decreases their ability to accommodate sudden changes in pressure8.

ECs also respond to stretch, but fluid shear stress seems to be the main determinant of EC function. ECs in arteries respond to increased blood flow by causing the relaxation of the surrounding smooth muscle. They do so by producing substances, such as nitric oxide (NO),

prostacyclin and a poorly characterized arachidonic acid metabolite that induces smooth muscle hyper polarization, and by releasing K+ through membrane channels9–11. Hyperpolarization is associated with relaxation — it makes the VSMCs less likely to activate the voltage- dependent calcium channels that open when the cell depolarizes. These channels admit calcium ions to trigger the activation of myosin and thus cell contraction. VSMC relaxation in response to blood flow occurs over seconds to minutes, widening arterial diameters to restore wall shear stresses to initial levels. If high flow persists, flow-dependent signals from the ECs lead to remodelling of the artery wall over weeks to months to enlarge the artery lumens12.

Conversely, decreased flow induces vessel narrowing that is also mediated by signals from the endothelium13. In severe cases, low flow leads to complete vessel regression, which involves apoptosis of the ECs14,15. Interestingly, flow is a potent survival signal for ECs because it has effects on multiple signalling pathways, which include the phospho-inositide 3-kinase (PI3K), extracellular signal-regulated kinase 5 (ERK5; also known as mitogen-activated pro-tein kinase 7 (MAPK7)) and NO pathways16. In summary, both ECs and VSMCs respond to mechanical forces to modulate artery diameters so that the blood flow meets the demands of the tissues.

Potential mechanotransducersMost of the responses to flow mentioned above are restricted to ECs, which indicates that ECs must express specific mechanotransducers that convert physical stresses into biochemical signals. Many putative mechanotrans-ducers have been proposed to function in sensing flow, including ion channels, integrins, receptor Tyr kinases, the apical glycocalyx, primary cilia, heterotrimeric G proteins, platelet/endothelial cell-adhesion molecule 1 (PECAM1) and vascular endothelial (VE)-cadherin16,17 ( .FIG )3 . However, we do not understand in detail how these various components orchestrate responses to shear stress.

C y t o s k e l e t o n . The cytoskeleton has an important role in EC responses to shear. Microtubules, actin and interme-diate filaments physically connect different regions of the EC to transmit forces from the apical domain, where shear is applied, to the basal or lateral domains, where mechano-transduction events have been observed18. Imaging of a green fluorescent protein fusion of the intermediate filament protein vimentin showed that flow induces non-homogeneous displacements of these filaments in the cells; regions of high strain were usually observed at lateral and basal structures, which suggested the transmission of forces to these sites19. These results therefore support models in which cell–matrix or cell–cell adhesions medi-ate mechanotransduction (see the Review by Geiger, Spatz and Bershadsky107 in this issue). Consistent with the idea that force from flow is transmitted through the cytoskele-ton, inhibition of actin, microtubules or intermediate filaments by drugs or genetic methods blocks many EC responses to flow20–22. However, there is little evidence that the cytoskeleton is a direct mechanotransducer of shear stress p e r s e .

Figure 1 | Mechanical forces on the vessel wall. A section of an artery wall shows the endothelial cells that form the inner lining and align longitudinally, and vascular smooth muscle cells that form the outer layers and align circumferentially. Pressure (p ) is normal to the vessel wall, which results in circumferential stretching of the vessel wall. Shear stress ( ) is parallel to the vessel wall and is exerted longitudinally in the direction of blood flow.

R E V I E W S

54 | JAN UARY 2009 | VOLUME 10 www.nature.com/reviews/molcellbio

R E V I E W S

Toute cellule adhérente qui évalue la rigidité du tissu Intégrines, RhoA, actine

Matrice extracellulaire

Microtubules


De nombreuses maladies ont pour origine la dérégulation des propriétés mécaniques

• La surdité• L’ostéoporose• Le glaucome et la myopie axiale• L’asthme• Des défauts de la fonction rénale• …• Le Cancer


Nature Reviews | Molecular Cell Biology

AdipocyteNeuron

MSC

OsteoblastSkeletal muscle cell

Endothelial cells Apoptosis ProliferationMesenchymal stem cells Adipogenesis OsteogenesisKeratinocytes Self-renewal

Small adhesive areaa

b

c

Large adhesive area

50 200 400 800 1,200 2,000 3,000 5,000 12,000 20,000 2–4 × 109

Elastic modulus (Pa)

170 Pa >1,200 Pa

Stromal cell Collagen I

Acini

YAP and TAZ transduce mechanical cuesGiven that the actomyosin cytoskeleton is required for mechanotransduction, how are mechanical signals transduced into biological outcomes and how do these signals ultimately affect gene expression? The transcriptional co-activators YAP and

TAZ recently emerged as key mediators of the biological effects that are observed in response to ECM elasticity and cell shape8,9. YAP and TAZ localize in the nucleus and are transcriptionally active in cells cultured on a stiff ECM, whereas YAP and TAZ are excluded from the nucleus and functionally

inhibited in cells cultured on a soft ECM8

( .FIG 2). A similar regulation of the activity and nuclear–cytoplasmic shuttling of YAP and TAZ occurs in cells that are grown on micro patterned ECMs with the same stiffness but that show different degrees of cell spreading: cells that are seeded on large fibronectin islands, which enable cell spreading, have active YAP and TAZ, whereas cells that are confined to small adhesive islands have the inactive forms of YAP and TAZ8,9 ( .FIG 2). Importantly, the activity of YAP and TAZ ultimately deter-mines the biological response to mechanical cues. Knockdown of YAP and TAZ in cells grown on large adhesive areas or on a stiff ECM produced a phenotype that is typical for cells grown on small adhesive areas or on soft ECM; vice versa, overexpression of YAP and TAZ was sufficient to ‘trick’ cells into behaving as they would on a stiff matrix even in the presence of a soft matrix8.

In agreement with the idea that mechano transduction is tightly linked to the integrity of the actomyosin cytoskeleton, YAP and TAZ are inactivated when F-actin is disrupted or when RHO is inhibited8–10,12. On the other hand, F-actin polymerization resulting from overexpression of the RHO-regulated F-actin nucleator diaphanous correlates with increased activity of YAP and TAZ8,10. Finally, blunting of endogenous tensile forces through the inhibition of the myosi n regulators ROCK and MLCK or myosin itself results in YAP and TAZ inacti-vation8,9. This leads to cell behaviour similar to that observed in the presence of a soft ECM or following cell size restriction24,28. Thus, YAP and TAZ not only respond to mechanical cues, but they are also mediators of mechanical signals.

In line with these data, genetic experi-ments in fly embryos showed that the expression of actin polymerization antago-nists is required to restrain the activity of Yorkie in developing tissues10,11. Severe overgrowth of imaginal discs, which are the larval single-cell layer epithelial struc-tures that give rise to wings, legs and other appendages, was shown to be induced by loss of function of capping proteins (leading to excessive growth of F-actin at barbed ends), deletion of adenylyl cyclase-associated proteins (CAPs; leading to increased association of actin monomers into new actin filaments), or by overexpres-sion of an activated version of Diaphanous. These overgrown imaginal discs showed increased levels of Yorkie activity, and their phenotype was strikingly similar to that caused by loss of Hippo signalling

Figure 1 | Influence of mechanical and physical properties of the EC M on cell behaviour. a | Microprinting techniques enable the design of extracellular matrix (ECM) areas of defined shape and dimensions, on which cells adhere by conforming to the substrate geometry. In endothelial cells, cell geometry is sufficient to change the response to growth factors from apoptosis to proliferation24,26,49.

commitment from adipocytes to osteoblasts27,28. In keratinocytes, restriction of cell shape induces ter-minal differentiation26,49. b | Cells within tissues experience very different degrees of ECM elasticity, ranging from very soft surroundings (such as those found in the brain and adipose tissue) to very stiff and rigid environments (such as those found within bones or at the bone surface). By recapitulating these different ECM elasticities , it was found that MSCs differentiate optimally into neurons, adipocytes, skeletal muscle cells or osteoblasts at elasticities that match the physiological ECM stiffness of their corresponding natural niche (shown as coloured lines, with peaks indicating maximal differen-tiation)28. Similarly, muscle stem cells maintain their self-renewal and regenerative capacities only when expanded on substrates mimicking the elasticity of skeletal muscle 30 (not shown). c | Mammary epithelial cells (shown in blue) grown embedded in a soft basement membrane form growth-arrested and well polarized acinar structures36 (left). Increasing type I collagen concentration and crosslinking, and thus ECM stiffness, compromises tissue organization, inhibits apoptosis and lumen formation and destabilizes adherens junctions (centre), favouring the acquisition of migratory and invasive behaviour s37,38 (right). Pa, Pascal.

P E R S P E C T I V E S

594 | SEPTEMBER 2012 | VOLUME 13 www.nature.com/reviews/molcellbio

© 2012 Macmillan Publishers Limited. All rights reserved

Nature Reviews | Cancer

Plastic/glass

Neuron

2–4 GPa50 200 400 800 1,200 2,000 3,000 5,000 20,000

Increasing stiffness

Elastic modulus (Pa)

Breast

Lung

Skeletal muscle

Smoothmuscle OsteoblastFibroblast

Endothelialcell

12,000

Increasing stiffness in breast tumours

Fluid: blood or mucus

BoneChondrocyte

within a compliant reconstituted basement membrane (rBM) or in response to mechanical loading is associ-ated with the repression and induction of hundreds of genes, and we determined that human mammary epithelial cells (HMEC) respond to matrix stiffness by altering the expression of at least 1,500 genes that span multiple functional categories86 (K. C. Tsai e t a l . , unpublished data). Likewise, although we showed that breast tumour progression in the HMT-3522 human breast cancer model is associated with specific genomic alterations, the accompanying gene expression profile differs markedly between those cells grown on either a rigid tissue culture plastic or stiff rBM-conjugated polyacrylamide gels and those within compliant rBM or on soft rBM-conjugated polyacrylamide gels87. This suggests that additional gene regulatory mechanisms, possibly linked to chromatin remodelling, must also be regulated by force.

A direct mechanical link from the ECM to nuclear chromatin could dynamically alter gene expression in response to exogenous force1 through a solid-state signalling mechanism that is governed by the princi-ples of ‘tensegrity’ (tensional integrity). The tensegrity model implies that integrins are linked to the nucleus through the cytoskeleton, that an applied force is trans-mitted to the DNA through the cytoskeleton by nuclear lamins and nuclear envelope receptor complexes, and that this then modulates gene expression by inducing

conformational changes in chromatin either by altering the nature of the protein complexes at the telomeres of chromosomes or by changing the activity of DNA remodelling enzymes88–92. Support for this paradigm has come from studies demonstrating how application of force on the integrin–ECM interface can induce nuclear and chromatin distortion93, that tension can alter DNA wrapping94, and that speared chromatin can be excised from the nucleus as a continuum that remains physi-cally linked to the cytoskeleton and adhesion interface95. Alternately, epigenetic changes regulate gene expression during embryogenesis and tissue-specific develop-ment. Given that force also modulates these processes, it follows that mechanotransduction might influence chromatin remodelling to regulate histone acetyla-tion and methylation. For example, HMEC morpho-genesis and differentiation in a compliant rBM but not on a stiff two-dimensional substrate is associated with pronounced chromatin remodelling, changes in his-tone deacetylase (HDAC) expression and activity, and increased expression of the methyl-CpG-binding pro-tein MECP2 (REFS , )96 97 (Tsai e t a l . , unpublished data). In addition, we and others have found that rBM compli-ance dictates the response of differentiated HMEC acini to the methylation inhibitor 5-azacytidine or the HDAC inhibitor trichostatin A. Only on compliant matrices do these inhibitors induce gene expression to sensitize a mammary epithelium to exogenous growth and death

Figure 1 | Cells are tuned to the materials properties of their matrix. All cells, including those in traditionally mechanically static tissues, such as the breast or the brain, are exposed to isometric force or tension that is generated locally at the nanoscale level by cell–cell or cell–extracellular matrix interactions and that influences cell function through actomyosin contractility and actin dynamics. Moreover, each cell type is specifically tuned to the specific tissue in which it resides. The brain, for instance, is infinitely softer than bone tissue. Consequently, neural cell growth, survival and differentiation are favoured by a highly compliant matrix. By contrast, osteoblast differentiation and survival occurs optimally on stiffer extracellular matrices with material properties more similar to newly formed bone. Normal mammary epithelial cell growth, survival, differentiation and morphogenesis are optimally supported by interaction with a soft matrix. Following transformation, however, breast tissue becomes progressively stiffer and tumour cells become significantly more contractile and hyper-responsive to matrix compliance cues. Normalizing the tensional homeostasis of tumour cells, however, can revert them towards a non-malignant phenotype6, thereby illustrating the functional link between matrix materials properties, cellular tension and normal tissue behaviour. Importantly, however, although breast tumours are much stiffer than the normal breast, the materials properties of a breast tumour remain significantly softer than those of muscle or bone, emphasizing the critical association between tissue phenotype and matrix rigidity.

R E V I E W S

112 | FEBRUARY 2009 | VOLUME 9 www.nature.com/reviews/cancer

Les tumeurs plus agressives sont plus rigides

Rigidité croissante

D’après Halder et al., 2012, Nat Rev Mol Cell Biol

Rigidité croissante

Rigidité croissante dans les tumeurs du sein

Cellule stromale Fibres de collagène

NeuronePoumon

Sein

Cellule endothéliale

Fibroblaste

Cellule musculaire

lisse

Cellule musculaire striée

Chondrocyte OstéoblasteOs

Verre/plastiqueSang ou

mucus

Pa : Pascal (unité de mesure de la pression)


La peau : un modèle d’étude du cancer aux propriétés mécaniques essentielles

D’après www.aroma-zone.com

Copyright © 2012, A.D.A.M., Inc.

Carcinome spinocellulaire

Carcinome basocellulaire

• Une fonction primordiale de la peau est de constituer une barrière résistante et étanche à l’environnement.

Sa résistance mécanique est donc essentielle.

• Deux principaux types de tumeurs d’origine épithéliale peuvent se développer

http://www.ncbi.nlm.nih.gov/pubmedhealth/copyright/


CD98hc, une protéine au carrefour de plusieurs fonctions

• Sucres• Lipides• Acides aminés

• Adhérence à la matrice• Migration cellulaire• Invasion

Intégrines

Transporteurs d’acides aminés

CD98hc


Rôle de CD98hc dans la tumorigenèse

Souris modèle : Inhibition de CD98hc de façon inductible au niveau de l’épiderme.

L’expression de CD98hc est abolie dans l’épiderme par un traitement local de la peau (traitement « 4OHT »).

Génération de tumeurs cutanées par traitement chimique local répétéPapillomes carcinome spinocellulaire

(grain de beauté)

4OHT(6 appl. total)

Carcinogenèse chimique induite par le DMBA et le TPA (semaines)-4 0

DMBATPA

(2 applications par semaine)

29-2 1


Rôle de CD98hc dans la tumorigénèse

Weeks after DMBA treatment

8 10 12 14 16 18 20 22 24

Aver

age

ppm

/mou

se

0

5

10

15

20

25

30

ACETONE4OHTCtrl4OHT

4OHT ou Ctrl(6 appl. total)

0

TPA(2 applications per week)

292

DMBA

12N

ombr

e de

lési

ons

par a

nim

al

Durant la formation de tumeurs


10 15 20 25 30

Aver

age

ppm

/mou

se

0

2

4

6

8

10

12

14

16 ACETONE4OHTCtrl4OHT

Nombre de semaines après traitement au DMBA

Nom

bre

de lé

sion

s pa

r ani

mal

Avec CD98hc

Sans CD98hc


Carcinogenèse chimique induite par le DMBA et le TPA

(semaines)

0


292

DMBA

12

4OHTou Ctrl

(6 appl. total)

-4 -2Carcinogenèse chimique

induite par le DMBA et le TPA (semaines)

Avant l’apparition de tumeurs




10 15 20 25 30 35

Aver

age

tum

ors/

mou

se

0

2

4

6

8

10

12

14 Ace4OHTCtrl4OHT

Ctrl

4OHT

Fibres de collagèneAprès l’apparition de tumeurs

Nom

bre

de tu

meu

rs m

oyen

ne p

ar s

ouris


4OHT ou Ctrl(6 appl. total)

0


292

DMBA

12


Réponse cellulaire aux variations de rigidité

Rigidité support

Rigidité cellule/contraction

Milieu liquide Plastique

Prolifération Dissémination

Cellule normale

Cellule transformée


CD98hc régule la mécanotransduction

CD98hc

ContractionModification du tissu

Membrane

Augmentation de la rigidité



Mesure de la rigidité

Contractilité Rigidification du tissu

Prolifération

Perte de CD98hcPrésence de CD98hc

Pas de mesure de la rigidité Contractilité Rigidification

du tissu

Prolifération


Documents

Les tumeurs ont la peau dure : régulation de leurs ...pataclope83.com/wp-content/blogs.dir/3/files/2013/... · 15e Colloque de la Recherche de la Ligue contre le cancer, Nice, 24-25