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 Time (hrs) 0 3 6 9 12 16 22 G1 92 92 93 92 78 68 44 S 3 2 2 3 16 26 44 G2/M 5 6 5 5 6 6 12 p27 27
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 Node negative breast ca n=1015 Hazard Ratio (95% CI) p value 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 cm vs <2 cm 5 2 3 01 3.01 (1 52 5 97) (1.52, 5.97) 0 002 0.002 2-5 cm vs <2 cm 1.78 (1.2, 2.64) 0.004 Lymphatic invasion y p 1.76 (1.17, 2.65) 0.007 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 g y Kinase X Skp1 Cul 1 Cul 1 Skp2 Cyclin E 26S Cdc34 p27 Cdk2 P Cyclin E Cyclin E p27 Cdk2 Cdk2 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 p27 74 74 88 89 88 89 KPSACRNLFGPVDHEELTRDLEKHCRDMEEASQRKWNFDFQNHKPLEGK Y EWQEVEKGSLPEF YY RPPR 25 93 CDK2 BINDING CDK2 BINDING CYCLIN A BINDING CYCLIN A BINDING Russo, Nature, 1996
Chu Cell 2007 cSrc phosphorylates p27 in vitro cSrc phosphorylates p27 in vitro
27 Src induction increases cellular pY74 p27 Chu Cell 2007 Y74 ll l i ti i d S
Effect of pY88 on binding of p27Kip1 to Cdk2 p27Kip1 p27Kip1 p27Kip1 p27Kip1 F80 F80 F82 F82 Y88 pY88 L83 c-Src/Abl V64 L134 L83 V64 L134 pY88 ejected from hydrophobic pocket due to Y88 interacts with hydrophobic aa V64, unfavorable thermodynamic interactions – destabilizes p27-Cdk2 F80, F82, L83 and L134
pY pY- -p27 has reduced inhibitory p27 has reduced inhibitory activity toward cyclin E activity toward cyclin E Cdk2 activity toward cyclin E activity toward cyclin E-Cdk2 Cdk2 Cdk2
Effect of pY74 on binding of p27 to Cdk2 p27Kip1 p27Kip1 p27Kip1 L25 L25 Y74 pY74 V79 V79 V30 V30 L67 L67 c-Src/Abl Y74 forms hydrophobic interactions In pY74 hydrophobic interactions lost: with aa L25, V30, L67 and V79 destabilizes Cdk2-p27
pY- pY -p27 has reduced binding to p27 has reduced binding to Cyclin E-Cdk2 in vitro C C Cyclin E li li E E Cdk2 i Cdk2 i Cdk2 in vitro it it S S Src also reduces p27-bound Src also reduces p27 l l d d 27 b 27 b bound d d Cyclin E Cyclin E- -Cdk2 in cells Cdk2 in cells
Chu Cell 2007 Src siRNA or inhibitors increase p27 Src siRNA or inhibitors increase p27
Src inhibition increases p27 t1/2 Src induction decreases p27 t1/2
26S p27: inhibitor & substrate of Cyclin E-Cdk2 S Cul 1 Cdc34 Skp2 Skp2 cks pT187 pY pY p27 G1
Phosphorylation by Src promotes p27 degradation 27 d d ti • 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 by increasing p27-cyclin E-cdk2 b i i 27 li E dk2 • 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 resistance? i t ?
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 o e aromatase β -actin
AZD0530 and anastrozole cause G1 arrest G0-G1% 90 S% 80 G2/M% G2/M% 70 60 50 %S 40 40 phase 30 20 10 0 no drug Src Inh AI Both Asyn no E 2 + Androstendione A d t di
Drug effects on signaling kinases Drug effects on signaling kinases pSrc MAPK pMAPK pMAPK p27 Src
AI & AZD0530 increase p27 AI & AZD0530 increase p27 binding to cyclin E-Cdk2 Cyclin E Cyclin E Cdk2 p27
Evidence for synergy between AZD0530 and anastrozole 20 no androstenedione no drug + androstenedione Ana SI SI 15 Ana+SI Relative Tumor T 10 10 Volume 5 5 0 0 0 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 RTK P P Ras Shc Shc GDP Ras P P P GTP Sos Raf Grb2 ER MEK Src MAPK AZD0530 AZD0530 AI/Tamoxifen AI/Tamoxifen p27 function 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 • Phase I trial: drug combination in metastatic Ph I t i l d bi ti i t t ti 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 Slingerland Lab Collaborators Yi Chen Mark Pegram (U Miami) g Merce Jorda (U Miami) Natalia Guggisberg Ludger Hengst (Austria) Isabel Chu Bryan Hennessy B H Jun Sun Gordon Mills (MD Anderson) Mickey Tan AstraZeneca-drug only Michelle Larrea Michelle Larrea Harriette Khan (U Toronto) Chendong Pan Angel Arnaout (U Toronto) Feng Hong Steven Narod (U Toronto) Steven Narod (U Toronto) Thiago DaSilva DOD Pre-doctoral award NCI R01 NCI R01 Avon/AACR Avon/AACR BCRF Doris Duke Foundation
Evidence for synergy between Evidence for synergy between anastrozole and AZD0530 Combination FTV FTV Expected Observed Combination Anastrozole AZD0530 FTV b FTV c Ratio d 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 d Combination Ratio= Expected FTV/ Observed FTV. A ratio of > 1 indicates synergy
In vitro synergy between S Src Inhibitor and anastrozole I hibi d l 60 Anastrozole Ana Anastrozole+AZD0530 50 Ana+SI 40 S% S% 30 20 10 0 0 12.5 25 50 100 500 1000 Anastrozole ( μ M) Anastrozole ( μ M)
Crystal structure of cyclin A Crystal structure of cyclin A- -Cdk2 Cdk2- -p27 p27 Russo, Nature, 1996
Cariou et al PNAS 2000 Loss of p27 abrogates G1 arrest by Tamoxifen 2% S 22% S 3% S 3% S
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