BeyoeyodteC C and the CENTRIC Trial – Integrin Targeting for … · 2015. 1. 29. · BeyoeyodteC...

OncoRay National Center forOncoRay – National Center forRadiation Research in Oncology, Dresden

Beyond the CENTRIC Trial –eyo d t e C C aIntegrin Targeting for Tumor Therapy

Sensitization

Nils Cordes

Cell adhesion and Survival

CCressPaula Cordes, Easter, 2008

Molecular and Cellular Radiobiology | Cordes | NCT Symposium 2013

Functions of the ECM

Fertilized egg

n

AdulthoodLife

span

Death

Rozario & DeSimone. Developmental Biology, 341, 1, 2010, 126–140 Fléchon et al. Reproductive Biology and Endocrinology 2003 1:124Nishida et al., Invest. Ophthalmol. Vis. Sci. 29:1887–1890, 1988


Integrin heterodimers

Hynes, Cell, 2000Legate et al., Genes Dev, 2009


Focal adhesion - SIGNALING

Extracellular space

ECMExtracellular space

Cell membrane

EGFR Integrin

PINCH1Nck2 ILKP i

Talin

Cell membrane

PP1MEK4/7 FAKGrb2Parvin Paxillin

C t lAkt p130CasJNK MEK1/2 Actin

Cytosol

Schaller and Parsons. Curr Opin Cell Biol. 1994Burridge and Chrzanowska-Wodnicka Annu Rev Cell

ERK1/2Wodnicka. Annu Rev Cell Dev Biol. 1996Legate et al., Nat Rev Mol Cell Biol. 2006Eke and Cordes, Radiother Oncol, 2011H hl t l BBA Survival, Proliferation, DifferentiationHehlgans et al., BBA reviews, 2007Vehlow and Cordes, BBA reviews, 2013


Overexpression of (various) Integrins

HNSCCLungLungPancreasProstateBreastBreastBrain….


Targeting beta1 integrin

Extracellular space

ECMExtracellular space

Cell membrane

EGFR beta1 Integrin

PINCH1Nck2 ILKP i

Talin

Cell membrane

XPP1MEK4/7 FAKGrb2

Parvin Paxillin

C t l

XAkt p130CasJNK MEK1/2 Actin

Cytosol XX

XXERK1/2

Radio- and Chemosensitization

XRadio and Chemosensitization

Anti-invasionMolecular and Cellular Radiobiology | Cordes | NCT Symposium 2013

beta1 integrin targeting - clonogenicity


Eke, Cordes et al., IJROBP, 2012

3D procedure

3D lrECM

Co = IgG1a AIIB2 = anti-1 integrin IgG mAb Eke, Cordes et al., JCI, 2012


AIIB2 cytotoxicity

Co = IgG1a AIIB2 = anti-1 integrin IgG mAb Eke, Cordes et al., JCI, 2012


1 integrin inhibition and radiosensitivity

* P < 0.05** P < 0.01

Co = IgG1a AIIB2 = anti-1 integrin IgG mAb

Eke, Cordes et al., JCI, 2012


Radiosensitization by 1 integrin inhibition

* P < 0.05** P < 0.01

Cal33

0 1

1

FaDu

0 1

1

HSC4

0 1

1

UTSCC5

0 1

1

* *

0 001

0.01

0.1

0 001

0.01

0.1

0 001

0.01

0.1

0 001

0.01

0.1

ctio

n

**

***

* ****

0 2 4 60.001

0 2 4 60.001

0 2 4 60.001

0 2 4 60.001

UTSCC14

1

UTSCC45

1

UTSCC8

1urvi

ving

frac

0.1

1

0 01

0.1

1

0 01

0.1

1Su

IgG#1 (1 µg/ml)AIIB2 (1 µg/ml)IgG#1 (10 µg/ml)AIIB2 (10 µg/ml)

***

***

**

0 2 4 60.001

0.01

0 2 4 60.001

0.01

0 2 4 60.001

0.01

Radiation dose [Gy]

( µg )**** *

Radiation dose [Gy]

Eke, Cordes et al., J Clin Invest, 2012


1 Integrin inhibition in vivo

UTSCC15 xenograft modelg(day 11; post 2.5 mg/kg mAb plus 20 Gy)

Time (d) to reach:

anti-1Co-IgG n = 129

( )

Co-Ab+ 20 Gy

anti-β1 Ab+ 20 Gy ER

26 [VB:19; 26] 56 [VB:28; 108] 5-fold starting volume

+ 20 Gy 20 Gy ER2.33

P < 0.025



DSB repair upon 1 integrin blocking

* P < 0,05** P < 0,01Dickreuter et al., in prep.


1 Integrin in radioresistance: The mechanisms

PKA Calpha/beta T197

JNK T183/Y185

Phosphoproteome array analysis

XF354fl2 SAS UTSCC15

in vitro

AIIB2IgG#2UTSCC15

in vivo

FAK S843

FAK S722

FAK S910

FAK Y397

PKA Cbeta S338

PKCbeta1/2 T500

p

FAK

pFAK Y397

AIIB2IgG#2

GSK3beta S216

GSK3alpha Y279

GSK3alpha S21

Src Y529

PAK1/2/3 S144/S141/S154

PKCzeta/lambda T410/T403

FAK S843

pJNK2 T183/Y185

JNK2

pJNK1 T183/Y185

JNK1

Raf1 S259

ERK2 T185/Y187

GRK2 S670

GSK3beta S9

GSK3beta S216

0 0.5 1 1.5 2 2.5 3 3.5

-Actin

JNK2

Fold change upon 1 integrin inhibition(normalized to total protein expression)



1 integrin-FAK interaction

In situ Proximity ligation assay



1 Integrin inhibition affects FAK

Gabarra-Niecko et al., Biochem J. 2002Eke, Cordes et al., JCI, 2012


1 Integrin inhibition affects FAK

* P < 0.05** P < 0.01

*

Increase of radioresistanceReduction of AIIB2 susceptibility

Co = IgG1a AIIB2 = anti-1 integrin IgG mAb

Gabarra-Niecko et al., Biochem J. 2002Eke, Cordes et al., JCI, 2012


Cell rounding upon 1 integrin blocking



FAK interacts with Cortactin

UTSCC15IP: FAKIP: IgG

UTSCC15

FAK

CTTN-Actin

CTTN

pCTTN Y421

CTTN

Eke et al., J Clin Invest. 2012 Apr 2;122(4):1529-40


Modified Cortactin by 1 integrin inhibition

In situ Proximity ligation assay

IP M S hIP – Mass Spec approach

Cortactin : Cortical actin binding protein:

- cytoplasmic expression

- polymerization and rearrangement ofpolymerization and rearrangement ofactin cytoskeleton



Proper FAK-Cortactin interaction critical forradiation cell survival

Kinase domain

P873A/P876A/P879A

FERM FAT GFPKinase domain

P371A/P374A

FERM FAT GFP FAK-Cort 3FAK-Cort 1

Kinase domain

P973A/P976A


P642A/P645A/P648A


UTSCC15 UTSCC15

1

FAK

CTTN

GFPwtFAK-Cort 1FAK-Cort 2

* *

g fr

actio

n

0.60.81.01.2

el. C

TTN

pr

ecip

itatio

n

** ** **

n.s.FAK-Cort 2FAK-Cort 3FAK-Cort 4

***

Surv

ivin

g0.00.20.4re

Co-

p

** **0 2 4 6

0.1 **

Radiation dose [Gy]S

Eke et al., J Clin Invest. 2012 Apr 2;122(4):1529-40


Proper FAK-Cortactin interaction critical forradiation cell survival

Kinase domain

P873A/P876A/P879A


P371A/P374A


Kinase domain

P973A/P976A


P642A/P645A/P648A


UTSCC15 UTSCC15

1

FAK

CTTN

GFPwtFAK-Cort 1FAK-Cort 2

* *

g fr

actio

n

0.60.81.01.2

el. C

TTN

pr

ecip

itatio

n

** ** **

n.s.FAK-Cort 2FAK-Cort 3FAK-Cort 4

***

Surv

ivin

g0.00.20.4re

Co-

p

** **0 2 4 6

0.1 **

Radiation dose [Gy]S

Eke et al., J Clin Invest. 2012 Apr 2;122(4):1529-40*p < 0.05; **p < 0.01


Our model


Additional Data

No effects of beta1 integrin inhibition in normal No effects of beta1 integrin inhibition in normal cells Eke, Cordes et al., JCI, 2012

No irritation of mice parameters such as weight, behavior Eke, Cordes et al., JCI, 2012

No signs of hypoxia, modified perfusion, vascular d it d t t i i idensity and metastasis in vivo

Modulation of radiation induced oral mucositis


Modulation of radiation-induced oral mucositis (mouse) by selective inhibition of beta1 integrin

Albert, Cordes, Dörr, et al.,Radiother Oncol 2012Radiother Oncol, 2012


Head & neck cancer,Lung cancerLung cancer,

Pancreatic cancer,,Breast cancer,

L k iLeukemia,Glioblastoma multiformeGlioblastoma multiforme …


Take home messages

Cell-ECM interactions essentially impact on tumor cell survival mechanisms and therapy resistance

beta1 integrin mediated radioresistance is conferred beta1 integrin-mediated radioresistance is conferred by FAK-Cortactin-JNK1 signaling in HNSCC

beta1 integrins seem to be potential targets in various tumor entities – adjuvant targeting to radiochemotherapy might be beneficial


AcknowledgementsMolecular and Cellular RadiobiologyEllen DickreuterIris EkeClaudia Förster

MPI Dresden (GER)Anna Shevchenko

Institute of Pathology, UKD (GER)Julia HennigInga LangeMandy PoschauAndre SagererChristina Schorradt

gy ( )Gustavo Baretton

MPI of Biochemistry (GER)Reinhard FässlerFabio Stanchi

Ahmed SoffarAnne SteglichKatja StorchAnne VehlowNicole Wachtel

UCSF (USA)David Schlaepfer

West Virginia University (USA)Nicole WachtelSuse WiedemannElisa ZienertKatja ZscheppangNils Cordes

West Virginia University (USA)Michael D. Schaller

NIH-NIDCR (USA)Kenneth M. Yamada

OncoRay Research GroupsMichael Baumann, Mechthild Krause

Turku University Central Hospital(Finland)R. Grenman


BeyoeyodteC C and the CENTRIC Trial – Integrin Targeting for … · 2015. 1. 29. · BeyoeyodteC...

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