Dual targeting of estrogen receptor α and estrogen-related receptor α: a novel endocrine therapy for endometrial cancer

Abstract

Background: Endometrial cancer (EC) is a hormone dependent carcinoma that may involve complex molecular mechanisms. Endocrine therapy by blocking the estrogen and estrogen receptor α (ERα) has been effective in breast cancer, while it is still controversial in EC. Recently, estrogen-related receptor α (ERRα) was proven to be another endocrine therapy target.

Methods: The anti-tumor effect of selective estrogen receptor modulators (SERMs) and XCT790 (XCT) used alone or in combination were evaluated in both of ERα-positive (ERα+) and ERα-negative (ERα-) EC cells. ERα and ERRα mRNA were tested by qPCR, while the protein was detected by Western blot. The proliferation was tested by MTS and cell cycle, apoptosis rate were analyzed by flow cytometry.

Results: A relatively high dose (10 μM) of tamoxifen (TAM) suppressed the expression of ERα and ERRα in two types of EC cells. However, 10 μM raloxifene (RAL) exhibited no effect on ERα and ERRα, while 10 μM XCT down regulated ERRα specifically, but not ERα in all EC cells. When dual targeting on ERα and ERRα by combining TAM with XCT, the proliferation inhibitory effect and apoptosis reached the strongest in all EC cells (P<0.05). Moreover, the inhibitory effect of proliferation was attributed significantly to the G0/G1 arrest (P<0.05). Interestingly, the apoptosis induced by combining TAM with XCT were obviously higher in ERα+ EC cells than ERα- EC cells (P<0.05).

Conclusion: Taken together, the results indicate that dual targeting on ERα and ERRα represents a better anti-tumor effect, which provides a novel endocrine based therapy strategy for EC.

Keywords: ERRα; ERα; SERM; XCT790; anti-tumor effect.

Figure 1

Expression of ERα and ERRα following treatment of EC cells with SERMs and/or XCT790. The mRNA levels of ERα and ERRα after treatment of RL952 (A), ECC-1 (B), HEC-1A (C), and HEC-1B (D) cells with 10 μM tamoxifen (TAM), tamoxifen combined with XCT790 (T+X), raloxifene (RAL), raloxifene combined with XCT790 (R+X), or XCT790 (XCT) for 24 h, as determined by real-time PCR. Levels in all five treatment groups were compared with those in cells treated with 0.1% DMSO as a control (CON). Data represent means ± SEM. All experiments were repeated in triplicate. *P<0.05.

Figure 2

Protein expression of ERα and ERRα following treatment of EC cells with SERMs and/or XCT790. The protein levels of ERα and ERRα after treatment of RL952 (A), ECC-1 (B), HEC-1A (C), and HEC-1B (D) cells with 10 μM TAM, T+X, RAL, R+X, or XCT for 24 h as determined by Western blotting. Data represent means ± SEM. All experiments were repeated in triplicate. *P<0.05.

Figure 3

Effect of SERMS and/or XCT790 treatment on EC cell proliferation. The proliferative capacities of RL952 (A), ECC-1 (B), HEC-1A (C), and HEC-1B (D) cells were evaluated by MTS assay following TAM, T+X, RAL, R+X, or XCT treatment for 0, 24, 48, 72, or 96 h. Data represent means ± SEM. All experiments were repeated in triplicate.

Figure 4

Effect of SERMS and/or XCT790 treatment on cell cycle progression of EC cells. Cell cycle changes were assessed by flow cytometry (FCM) in RL952 (A), ECC-1 (B), HEC-1A (C), and HEC-1B (D) cells treated with TAM, T+X, RAL, R+X, or XCT for 24 h. Cell distributions in each phase of the cell cycle were determined in RL952 (E), ECC-1 (F), HEC-1A (G), and HEC-1B (H) cells. All experiments were repeated in triplicate. *P<0.05.

Figure 5

Effect of SERMS and/or XCT790 treatment on EC cell apoptosis. Cell apoptosis was assessed by flow cytometry (FCM) in RL952 (A), ECC-1 (B), HEC-1A (C), and HEC-1B (D) cells treated with TAM, T+X, RAL, R+X, or XCT for 24 h. Rates of cell apoptosis were determined in RL952 (E), ECC-1 (F), HEC-1A (G), and HEC-1B (H) cells. Data represent means ± SEM. All experiments were repeated in triplicate. *P<0.05.

Figure 6

Effects of different doses of TAM on ERα and ERRα. High dose TAM inhibited ERα expression, which mediated the down regulation of ERRα, while low dose TAM exerted the opposite effect on ERα and ERRα in ERα+ EC cells. High dose TAM inhibited ERRα though these signaling pathways crosstalked with estrogen-ER signaling, while low dose TAM exerted the opposite effect on ERRα in ER- EC cell.