Targeting Src to treat antiestrogen resistant breast cancer breast - - PowerPoint PPT Presentation

Targeting Src to treat antiestrogen resistant breast cancer breast cancer

Joyce Slingerland

Braman Family Breast Cancer Institute Sylvester Comprehensive Cancer Center U of Miami Miller School of Medicine

p27- key mediator of G1 arrest

• binds and inhibits cyclin E-Cdk2
• levels fall as cells move through G1
• links extracellular growth regulators and

links extracellular growth regulators and the cell cycle

• reduced in up to 60% human cancers

Estrogens cause loss of p27 st oge s cause oss o p and G1 progression

22 16 12 9 6 3 Time (hrs) 27

44 44 12 68 26 6 78 16 6 92 3 5 93 2 5 92 2 6 92 3 5 G1 S G2/M

p27

p27 protein is reduced in 60% of Breast Ca

Nature Med 3: p227, 1997

Lymph node negative breast cancer p27 prognostic for poor DFS p27 prognostic for poor DFS

p27>25% p27<25%

n= 1015 P<0.0001

Disease Free Survival Node negative breast ca n=1015

Hazard Ratio (95% CI) p value

Node negative breast ca n=1015

Decreased p27 level

(<25% vs >25%) 1.53 (1.05, 2.23) 0.03

Tumor grade Tumor grade

( grade 2-3 vs 1) 3.37 (1.77, 6.39) 0.0002

Tumor size

5 2 3 01 (1 52 5 97) 0 002 >5 cm vs <2 cm 2-5 cm vs <2 cm 3.01 1.78 (1.52, 5.97) (1.2, 2.64) 0.002 0.004

Lymphatic invasion

1.76 (1.17, 2.65) 0.007

y p Estrogen receptor

(negative vs positive) 1.13 (0.67, 1.89) 0.36 NS

Progesterone receptor

(negative vs positive) 1.12 (0.71, 1.77) 0.63 NS

p27 degradation promotes S phase entry

Kinase X

g y

Cul 1 Skp1 Cul 1 Skp2 Cdc34 Cyclin E Cdk2

26S

P

p27

Cyclin E Cyclin E Cdk2 Cdk2

p27

P T187

G1 S

3 tyrosines in p27 interact with Cdk2 3 tyrosines in p27 interact with Cdk2 3 tyrosines in p27 interact with Cdk2 3 tyrosines in p27 interact with Cdk2

74 88 89

p27

74 88 89 KPSACRNLFGPVDHEELTRDLEKHCRDMEEASQRKWNFDFQNHKPLEGKYEWQEVEKGSLPEFYYRPPR

CDK2 BINDING CYCLIN A BINDING

25 93

CDK2 BINDING CYCLIN A BINDING

Russo, Nature, 1996

cSrc phosphorylates p27 in vitro cSrc phosphorylates p27 in vitro

Chu Cell 2007

S i d ti i ll l Y74 27 Src induction increases cellular pY74 p27

Chu Cell 2007

Effect of pY88 on binding of p27Kip1 to Cdk2

p27Kip1 p27Kip1 p27Kip1 p27Kip1

F80 F80 F82 F82

c-Src/Abl

Y88 pY88 L83 L83 V64 V64 L134 L134

Y88 interacts with hydrophobic aa V64, F80, F82, L83 and L134 pY88 ejected from hydrophobic pocket due to unfavorable thermodynamic interactions – destabilizes p27-Cdk2

pY pY-

• p27 has reduced inhibitory

p27 has reduced inhibitory activity toward cyclin E activity toward cyclin E Cdk2 Cdk2 activity toward cyclin E activity toward cyclin E-Cdk2 Cdk2

Effect of pY74 on binding of p27 to Cdk2

p27Kip1 p27Kip1 p27Kip1

Y74 pY74 V79 V79 V30 V30 L25 L25 L67 L67

c-Src/Abl

Y74 forms hydrophobic interactions with aa L25, V30, L67 and V79 In pY74 hydrophobic interactions lost: destabilizes Cdk2-p27

pY pY-

• p27 has reduced binding to

p27 has reduced binding to C li E C li E Cdk2 i it Cdk2 i it Cyclin E Cyclin E-Cdk2 in vitro Cdk2 in vitro

S l d 27 S l d 27 b d b d Src also reduces p27 Src also reduces p27-bound bound Cyclin E Cyclin E-

• Cdk2 in cells

Cdk2 in cells

Src siRNA or inhibitors increase p27 Src siRNA or inhibitors increase p27

Chu Cell 2007

Src inhibition increases p27 t1/2

Src induction decreases p27 t1/2

p27: inhibitor & substrate of Cyclin E-Cdk2

G1 S

pT187

p27

Cul 1 Skp2 cks Skp2 Cdc34

pY

26S

pY

Phosphorylation by Src promotes 27 d d ti p27 degradation

• Src phosphorylates p27
• Src phosphorylates p27
• Src-phosphorylated p27 is a poor Cdk2 inhibitor
• pYp27 has reduced binding to cyclin E-Cdk2
• Src induction increases p27pT187
• Src siRNA or inhibitors increase p27 levels
• Src siRNA or inhibitors increase p27 levels
• Src induction decreased p27 t1/2

Does Src promote p27 loss in cancers?

Activated Src correlates with low p27 in breast cancers

n=482 p=0.02 Chu Cell 2007

39% of all cancers and 37% of ER+ cancers show Src activation

Antiestrogen Resistance in ER+ Breast Cancer

• Estrogens activate mammary cell proliferation
• 2/3 new breast cancers express ER protein
• >15 million women worldwide on tamoxifen or

aromatase inhibitors PROBLEM : RESISTANCE

p27 is required for p27 is required for antiestrogen mediated G1 arrest

• Estrogen stimulates cell cycle by

decreasing p27 g p

• Tam and fulvestrant cause G1 arrest

b i i 27 li E dk2 by increasing p27-cyclin E-cdk2

• Antisense p27 abrogates Tam arrest
• Antisense p27 abrogates Tam arrest

Cariou et al PNAS 2000 Cariou et al PNAS 2000

Since p27 is required for G1 arrest by tamoxifen arrest by tamoxifen and since Src activates p27 loss…… does Src mediate antiestrogen i t ? resistance?

Src inhibitor restores G1 arrest by Src inhibitor restores G1 arrest by Tamoxifen Tamoxifen Tamoxifen Tamoxifen

Chu Cell 2007

Anastrozole Anastrozole Anastrozole Anastrozole

• nonsteroidal aromatase inhibitor used for ER/PR +

breast cancer breast cancer

• blocks conversion of androstenedione to

estrogen estrogen

• reduces estrogen by >90% in postmenopausal

women

• e

aromatase

β-actin

AZD0530 and anastrozole cause G1 arrest

80 90 G0-G1% S% G2/M% 40 50 60 70 G2/M%

%S

10 20 30 40

phase no drug Src Inh AI Both A d t di

Asyn no E2

+ Androstendione

Drug effects on signaling kinases Drug effects on signaling kinases

p27 pSrc Src pMAPK MAPK pMAPK

AI & AZD0530 increase p27 AI & AZD0530 increase p27 binding to cyclin E-Cdk2

Cyclin E Cdk2 Cyclin E p27

Evidence for synergy between AZD0530 and anastrozole

20 no androstenedione no drug Ana SI + androstenedione 10 15

Relative T

SI Ana+SI 5 10

Tumor Volume

5 7 14

Week(s)

Molecular markers of tumor response

Drug effects on Src: g

• no inhibition in resistant tumors
• anastrozole alone activates Src
• combination inhibits Src

Src pY416

Proteomic data Resistance mechanism: mechanism: MEK pathway p y activation

Resistance mechanism: mechanism: PI3K pathway PI3K pathway activation

Growth Factors Estrogen RTK

P P

Shc

Ras

RTK

Raf P P

GDP

Ras

GTP

Sos

P

Shc

MEK

Grb2

ER Src

MAPK

AZD0530 AI/Tamoxifen p27 function AZD0530 AI/Tamoxifen p27 degradation

Rationale for AZD0530/anastrozole trials

• p27 mediates G1 arrest by AIs
• Src phosphorylates p27 to promote p27 loss
• AZD0530 cooperates with anastrozole in

p xenograft tumors

• p27 increase and Ki67 loss may predict response
• Anastrozole alone stimulates Src
• Rapid emergence of resistance to AZD0530 not
• Rapid emergence of resistance to AZD0530 not

seen with combination

• AZD0530 resistant tumors had MEK and PI3K

AZD0530 resistant tumors had MEK and PI3K activation

Src inhibitor plus AI clinical trial Src inhibitor plus AI clinical trial

Ph I t i l d bi ti i t t ti

• Phase I trial: drug combination in metastatic

disease

• Phase II trial: AZD0530/anastrozole for

postmenopausal LABC with pre-post biopsies:

– Tumor size and vascularity by MRI – IHC of p27 pSrc pMAPK pAkt Ki67 – RPPA Proteomic profiles – Src activation genomic profile – Mammosphere and TIC assays

Acknowledgements

Yi Chen

Slingerland Lab Collaborators

Mark Pegram (U Miami) Natalia Guggisberg Isabel Chu g Merce Jorda (U Miami) Ludger Hengst (Austria) B H

Jun Sun Mickey Tan Michelle Larrea

Bryan Hennessy Gordon Mills (MD Anderson)

AstraZeneca-drug only

Michelle Larrea Chendong Pan Feng Hong

Harriette Khan (U Toronto) Angel Arnaout (U Toronto) Steven Narod (U Toronto)

Thiago DaSilva

DOD Pre-doctoral award NCI R01 Avon/AACR Steven Narod (U Toronto) NCI R01 Avon/AACR BCRF Doris Duke Foundation

Evidence for synergy between Evidence for synergy between anastrozole and AZD0530

FTV FTV Combination

Anastrozole AZD0530

Expected FTVb Observed FTVc Combination Ratiod

0.66 1.07 0.71 0.45 1.57

a FTV, fractional tumor volume (mean final tumor volume

experimental)/(mean final tumor volume control). experimental)/(mean final tumor volume control).

b Expected FTV= (mean FTV of Anastrozole)× (mean FTV of AZD0530) c.Observed FTV= final tumor vol combined therapy/final tumor vol

androstendione alone

dCombination Ratio= Expected FTV/ Observed FTV. A ratio of > 1

indicates synergy

In vitro synergy between S I hibi d l Src Inhibitor and anastrozole

50 60

Ana Ana+SI

Anastrozole Anastrozole+AZD0530

30 40

S% S%

10 20 12.5 25 50 100 500 1000

Anastrozole (μM) Anastrozole (μM)

Crystal structure of cyclin A Crystal structure of cyclin A-

• Cdk2

Cdk2-

• p27

p27

Russo, Nature, 1996

Loss of p27 abrogates G1 arrest by Tamoxifen

3% S

3% S

22% S 2% S

Cariou et al PNAS 2000