Src Inhibition with saracatinib reverses fulvestrant resistance in ER-positive ovarian cancer models in vitro and in vivo

Abstract

Purpose: More effective, less toxic treatments for recurrent ovarian cancer are needed. Although more than 60% of ovarian cancers express the estrogen receptor (ER), ER-targeted drugs have been disappointing due to drug resistance. In other estrogen-sensitive cancers, estrogen activates Src to phosphorylate p27 promoting its degradation and increasing cell-cycle progression. Because Src is activated in most ovarian cancers, we investigated whether combined Src and ER blockade by saracatinib and fulvestrant would circumvent antiestrogen resistance.

Experimental design: ER and Src were assayed in 338 primary ovarian cancers. Dual ER and Src blockade effects on cell cycle, ER target gene expression, and survival were assayed in ERα+ ovarian cancer lines, a primary human ovarian cancer culture in vitro, and on xenograft growth.

Results: Most primary ovarian cancers express ER. Src activity was greater in ovarian cancer lines than normal epithelial lines. Estrogen activated Src, ER-Src binding, and ER translocation from cytoplasm to nucleus. Estrogen-mediated mitogenesis was via ERα, not ERβ. While each alone had little effect, combined saracatinib and fulvestrant increased p27 and inhibited cyclin E-Cdk2 and cell-cycle progression. Saracatinib also impaired induction of ER-target genes c-Myc and FOSL1; this was greatest with dual therapy. Combined therapy induced autophagy and more effectively inhibited ovarian cancer xenograft growth than monotherapy.

Conclusions: Saracatinib augments effects of fulvestrant by opposing estrogen-mediated Src activation and target gene expression, increasing cell-cycle arrest, and impairing survival, all of which would oppose antiestrogen resistance in these ER+ ovarian cancer models. These data support further preclinical and clinical evaluation of combined fulvestrant and saracatinib in ovarian cancer.

Figure 1

Estrogen activates Src, ER/Src binding and ER translocation to the nucleus. A, Western shows ERα, total and activated Src, MEK and AKT in PEO1R, BG-1 and HOSE. (B-E) E2(10⁻⁸M) was added to E2 deprived PEO1R or BG-1 at t=0 and cells were recovered at indicated times. B, Westerns show phosphorylated or total Src (top). ERα immunoprecipitates were resolved and immunoblotted for pSrc or ERα (bottom). C, IF shows perinuclear ERα and pSrc co-localization 15 min after E2 addition. D, Sub-cellular fractions were blotted for ER and actin. E, ERα localization in BG-1at t=0 and after 4 hrs E2.

Figure 2

E2 stimulates proliferation via ERα and is required for PEO1R xenograft growth. A, PEO1R in 10% FBS were transferred to media with 10%cFBS, 0.1% cFBS alone or 0.1% cFBS together with E2 (10⁻⁹M), PPT (10⁻⁹M), THC (10⁻⁶M), DPN (10⁻⁹M), or [E2(10⁻⁹M)+ PHTPP(10⁻⁶M)] for 48hrs and cell cycle distribution analyzed. *p<.0001 for 10%FBS vs 0.1%cFBS, ‡ p <.0001 for 0.1%cFBS vs E2; ^ p=ns for E2, PPT and THC; + p=ns for DPN vs 0.1%cFBS. B, PEO1R were E2 deprived in 0.1%cFBS for 48hrs (T=0 hrs), then E2 (10⁻⁸M) was added and cells recovered for cell cycle analysis at indicated times (see also Supplemental Figure 1). C, BG-1 in 10%FBS were transferred to media with 10%cFBS +/− steroids as above for 48 hrs; * indicates p <.0001 for 10%FBS vs 10%cFBS, ‡ p <.0001 for 10%cFBS vs E2; ^ p=ns for E2, PPT and THC; +p=ns for DPN vs 10%cFBS. D, PEO1R^luc+ xenograft growth/time +/− E2 supplementation assayed by IVIS (normalized photon flux). (All graphs shown mean+/SEM).

Figure 3

Effects of combined ER and Src inhibition on signaling and cell cycle regulators. (A-C). PEO1R, BG-1, and OC-E1P were treated with 10⁻⁶M Fulv (F), 10⁻⁶M saracatinib (SI) or both for 48 hrs, followed by cell cycle and protein analysis. A, PEO1R cell cycle distribution after 48 hrs +/−drug. * p<0.0001 for SI vs both, PEO1R showed synergism between the two drugs (p<.0001) (right panel mean %S phase). B, Western for ER, total and activated Src and p27 (left panel). Cyclin E was precipitated and associated proteins blotted (middle panel). Cyclin E precipitates were assayed for kinase activity and radioactivity in histone H1 substrate shown (right panel, top) and graphed as %max activity (right panel, bottom); * p<0.0003 SI vs both. C, BG-1 cell cycle distribution at 48 hrs +/−drug (left,*p<0.0001 Fulv vs both); Western in BG-1 (right). D, Western in asynchronous BG-1, PEO1R, and E1P (left). Cell cycle analysis of EIP +/− 48 hrs drug; * p=.005 SI vs both (right). E, PEO1R cells were stably transduced with shRNA-Src or shRNA-control and treated +/−Fulv (10⁻⁶M) 48 hrs. Western shows Src knockdown (left). Cell cycle distribution graphed, * indicates p=0.06 shSrc vs shSrc+Fulv (middle); QT-PCR for Src, Yes, Fyn and Lyn in PEO1R +/− Src knockdown. All data are graphed as mean +/−SEM (right).

Figure 4

Saracatinib and fulvestrant impair estrogen induced ERα target gene activation and cell cycle re-entry. (A-C) After 48hrs E2 deprivation in 0.1%cFBS for PEO1R and 10%cFBS for BG-1, cells were treated with E2 (10⁻⁸ M) +/−drug for 6hrs. c-MYC expression A, - in PEO1R, B, in BG-1 and C, FOSL in BG-1 were assayed by Q-PCR and graphed as mean fold-change vs control +/−SEM. For c-MYC in PEO1R, *p=.01 for E+SI vs both+E and ^p=0.02 for E+Fulv vs E+Both. For c-MYC in BG-1,*p<0.001 for E+SI vs E + Both and ^p=0.08 for E+Fulv vs E+Both. For FOSL in BG-1, *p=.03 for E+SI vs E+Both and ^p=0.001 for E+Fulv vs Both+E. D, Cell cycle distribution after 48 hrs in media with 0.1%cFBS, PEO1R followed by addition of E2(10⁻⁹M), or PPT (10⁻⁹M) alone or with indicated drugs for 20 hrs; *p<0.0001 for SI+PPT vs PPT+Both and ^p<0.0001 for Fulv+PPT vs PPT+Both.

Figure 5

Effects of ER and Src inhibition on apoptosis and autophagy. PEO1R and BG-1 were treated with 10⁻⁶M Fulv (F), 10⁻⁶M saracatinib (SI) or both drugs for 48 hrs. A, Western of LC3-II +/− drug. Lane 1 shows paclitaxel-treated 293T as positive control, lanes 2-5 PEO1R +/− indicated drugs. B, Profiling of Cyto-ID autophagy as histogram-overlays. C, Bar graph of % shift in Cyto-ID fluorescence (+/−SEM) from (B), indicates increase in autophagic vesicles *indicates p≤ 0.0007 (PEO1R) for SI and both vs control and ‡ p<0.006 (BG-1) for SI and both vs control. D, Western of PARP and cleaved PARP +/− drug; lane 1 shows 293T/taxol control, lanes 2-5 PEO1R +/− indicated drugs. E, AnnexinV labeling of PEO1R for early/late apoptosis by flow cytometry +/−drug (% AnnexinV+ cells shown). F. Percent cells with early/late AnnexinV staining +/−SEM from (E); *p=0.005 Ctrl vs SI; ^p=0.533 SI vs both for early apoptosis; late apoptosis all treatments p>0.05.

Figure 6

Effects of fulvestrant and saracatinib on OVCA xenografts. PEO1R^LUC+ was implanted under the renal capsule of estrogen supplemented nude mice. A, Drug initiated day 11 with Fulv (5mg/week), saracatinib (25mg/kg) or both. Tumor growth is graphed as mean normalized photon flux over time (+/−SEM). B, In a second in vivo experiment, treatments (Fulv 3.5 mg/week, saracatinib 25mg/kg; both) were initiated at week 3; tumors were harvested week 11. Mean normalized photon flux at week 11 is graphed +/− SEM for each; * indicates p=0.04 vs control: photographs show representative tumors.