Novel selective estrogen mimics for the treatment of tamoxifen-resistant breast cancer

Abstract

Endocrine-resistant breast cancer is a major clinical obstacle. The use of 17β-estradiol (E2) has reemerged as a potential treatment option following exhaustive use of tamoxifen or aromatase inhibitors, although side effects have hindered its clinical usage. Protein kinase C alpha (PKCα) expression was shown to be a predictor of disease outcome for patients receiving endocrine therapy and may predict a positive response to an estrogenic treatment. Here, we have investigated the use of novel benzothiophene selective estrogen mimics (SEM) as an alternative to E2 for the treatment of tamoxifen-resistant breast cancer. Following in vitro characterization of SEMs, a panel of clinically relevant PKCα-expressing, tamoxifen-resistant models were used to investigate the antitumor effects of these compounds. SEM treatment resulted in growth inhibition and apoptosis of tamoxifen-resistant cell lines in vitro. In vivo SEM treatment induced tumor regression of tamoxifen-resistant T47D:A18/PKCα and T47D:A18-TAM1 tumor models. T47D:A18/PKCα tumor regression was accompanied by translocation of estrogen receptor (ER) α to extranuclear sites, possibly defining a mechanism through which these SEMs initiate tumor regression. SEM treatment did not stimulate growth of E2-dependent T47D:A18/neo tumors. In addition, unlike E2 or tamoxifen, treatment with SEMs did not stimulate uterine weight gain. These findings suggest the further development of SEMs as a feasible therapeutic strategy for the treatment of endocrine-resistant breast cancer without the side effects associated with E2.

Figure 1. PKCα-overexpression in breast cancer cells correlates with sensitivity to E2-induced growth inhibition

A) Effect of E2 and 4-OHT on T47D:A18-TAM1 proliferation in vitro. DNA assays were performed as described in Materials and Methods. Graph shows mean ± SEM and is representative of three independent experiments. B) Effect of TAM and E2 on T47D:A18-TAM1 xenograft growth. 2 mice/group were bilaterally injected with T47D:A18-TAM1 cells and either left untreated or were given oral TAM (1.5 mg/day). Dashed line represents the start of E2 treatment. C) Western blot analysis of PKCα expression in E2-stimulated (T47D:A18/neo, T47D:A18, MCF-7:WS8) and E2-inhibited (T47D:A18/PKCα, T47D:A18-TAM1, and MCF-7:5C) cell lines. D) Densitometric quantification of three western blots from three independent cell lysates. Student’s t-test was used to compare each E2-inhibited cell line to the E2-stimulated counterpart. *, P ≤ 0.05.

Figure 2. Effect of BTC and TTC-352 on proliferation of T47D:A18/neo and T47D:A18/PKCα cells in vitro

Structures of BTC (A) and TTC-352 (B). Effect of BTC and TTC-352 treatment on the growth T47D:A18/neo cells (C and D respectively), T47D:A18/PKCα cells (E and F respectively) and T47D:A18-TAM1 cells (G and H respectively). DNA assays were performed as described in Materials and Methods. Graphs show mean ± SEM and are representative of three independent experiments.

Figure 3. BTC and TTC-352 inhibit T47D:A18/PKCα and T47D:A18-TAM1 colony formation in 3D Matrigel^™ and induce apoptosis in MCF-7:5C cells in vitro

Colonies were established as described in Materials and Methods and treated for 10 days (Control [0.1% DMSO], E2 1 nM, BTC 100 nM, TTC-352 100 nM). A) T47D:A18/neo. B) T47D:A18/PKCα. C) T47D:A18-TAM1. Proliferation assay was performed following 9 days of treatment. D) MCF-7:WS8. E) MCF-7:5C. F) Apoptosis was assessed in MCF-7:5C cells following 6 days of treatment as described in Materials and Methods. *, P ≤ 0.05 versus DMSO. **, P ≤ 0.01 versus DMSO. ***, P ≤ 0.001 versus DMSO. Graph shows mean ± SEM of 3 independent experiments.

Figure 4. BTC and TTC-352 induce ERα transcriptional activity in T47D:A18/neo, T47D:A18/PKCα and T47D:A18-TAM1 cell lines

A) T47D:A18/neo, B) T47D:A18/PKCα and C) T47D:A18-TAM1 cell lines. Data is expressed normalized to E2 (100%).*, P ≤ 0.05 versus DMSO. **, P ≤ 0.01 versus DMSO. ***, P ≤ 0.001 versus DMSO. Graph shows mean ± SEM of 3 independent experiments.

Figure 5. BTC and TTC-352 inhibit T47D:A18/PKCα and T47D:A18-TAM1 xenograft tumors

A) BTC and TTC-352 treatment result in regression of T47D:A18/PKCα tumors. Graph shows percentage of tumor regression (100% ~0.5 cm²). Dotted line indicates when treatment was ended. Arrow designates where tumors from (B) were obtained. *, P ≤ 0.001 versus control. Graph shows mean ± SEM. B) ERα localization in T47D:A18/PKCα tumors by immunofluorescence staining. Total magnification: 630X.C) T47D:A18-TAM1 tumors regress when treated with BTC and TTC-352. Dotted lines represents start of treatment.

Figure 6. BTC and TTC-352 have no effect on T47D:A18/neo tumor growth, body weight or uterine weights of ovariectomized mice

A) BTC and TTC-352 do not result in growth of T47D:A18/neo tumors. B) Body weights of treated mice from (A). C) Uterine weights from mice in (A). Weights are reported as uterine weight (mg)/body weight (g).***, P ≤ 0.001 versus control. Graphs show mean ± SEM.