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FOXM1 is a transcriptional target of ERalpha and has a critical role in breast cancer endocrine sensitivity and resistance.

Millour J, Constantinidou D, Stavropoulou AV, Wilson MS, Myatt SS, Kwok JM, Sivanandan K, Coombes RC, Medema RH, Hartman J, Lykkesfeldt AE, Lam EW - Oncogene (2010)

Bottom Line: Our data also revealed that upon OHT treatment ERalpha recruits histone deacetylases to the ERE site of the FOXM1 promoter, which is associated with a decrease in histone acetylation and transcription activity.Conversely, ectopic expression of FOXM1 abrogated the cell cycle arrest mediated by the anti-estrogen OHT.OHT repressed FOXM1 expression in endocrine sensitive but not resistant breast carcinoma cell lines.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery and Cancer, Imperial College London, London W12 0NN, UK.

ABSTRACT
In this study, we investigated the regulation of FOXM1 expression by estrogen receptor alpha (ERalpha) and its role in hormonal therapy and endocrine resistance. FOXM1 protein and mRNA expression was regulated by ER-ligands, including estrogen, tamoxifen (OHT) and fulvestrant (ICI182780; ICI) in breast carcinoma cell lines. Depletion of ERalpha by RNA interference (RNAi) in MCF-7 cells downregulated FOXM1 expression. Reporter gene assays showed that ERalpha activates FOXM1 transcription through an estrogen-response element (ERE) located within the proximal promoter region. The direct binding of ERalpha to the FOXM1 promoter was confirmed in vitro by mobility shift and DNA pull-down assays and in vivo by chromatin immunoprecipitation (ChIP) analysis. Our data also revealed that upon OHT treatment ERalpha recruits histone deacetylases to the ERE site of the FOXM1 promoter, which is associated with a decrease in histone acetylation and transcription activity. Importantly, silencing of FOXM1 by RNAi abolished estrogen-induced MCF-7 cell proliferation and overcame acquired tamoxifen resistance. Conversely, ectopic expression of FOXM1 abrogated the cell cycle arrest mediated by the anti-estrogen OHT. OHT repressed FOXM1 expression in endocrine sensitive but not resistant breast carcinoma cell lines. Furthermore, qRT-PCR analysis of breast cancer patient samples revealed that there was a strong and significant positive correlation between ERalpha and FOXM1 mRNA expression. Collectively, these results show FOXM1 to be a key mediator of the mitogenic functions of ERalpha and estrogen in breast cancer cells, and also suggest that the deregulation of FOXM1 may contribute to anti-estrogen insensitivity.

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Effects of ERα and FOXM1 silencing on the expression of FOXM1 and response to E2 induction in MCF-7 cellsA) MCF-7 cells were transiently transfected with ERα, FOXM1 or control smart pool siRNA, and 72 h after transfection cells were analysed by western blot using specific antibodies as indicated and by qRT-PCR. B) MCF-7 cells were transiently transfected with smart pool siRNA against FOXM1, incubated with E2 and analysed by western blotting. SRB assays were also performed on these cells, indicating that the knockdown of FOXM1 decreases the cell proliferation rate and renders MCF-7 cells unresponsive to E2 stimulation. C) OHT-resistant TAMR4 MCF-7 cells were transiently transfected with smart pool siRNA against FOXM1 or control siRNA pool (non-specific/n.s. siRNA) and analysed by western blotting (upper panel). These transfected cells were incubated with or without E2 (middle panel), and with or without OHT in the presence of E2 (lower panel). SRB assays were performed on these cells, indicating that the knockdown of FOXM1 sensitizes the resistant TAMR4 MCF-7 cells to OHT and diminishes their responsiveness to E2 stimulation. Statistical analysis was performed on the proliferation results at 72 h. ** denotes very significant, P<0.01 and * significant, P<0.05. The results show mean+SEM of triplicate measurements.
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Figure 5: Effects of ERα and FOXM1 silencing on the expression of FOXM1 and response to E2 induction in MCF-7 cellsA) MCF-7 cells were transiently transfected with ERα, FOXM1 or control smart pool siRNA, and 72 h after transfection cells were analysed by western blot using specific antibodies as indicated and by qRT-PCR. B) MCF-7 cells were transiently transfected with smart pool siRNA against FOXM1, incubated with E2 and analysed by western blotting. SRB assays were also performed on these cells, indicating that the knockdown of FOXM1 decreases the cell proliferation rate and renders MCF-7 cells unresponsive to E2 stimulation. C) OHT-resistant TAMR4 MCF-7 cells were transiently transfected with smart pool siRNA against FOXM1 or control siRNA pool (non-specific/n.s. siRNA) and analysed by western blotting (upper panel). These transfected cells were incubated with or without E2 (middle panel), and with or without OHT in the presence of E2 (lower panel). SRB assays were performed on these cells, indicating that the knockdown of FOXM1 sensitizes the resistant TAMR4 MCF-7 cells to OHT and diminishes their responsiveness to E2 stimulation. Statistical analysis was performed on the proliferation results at 72 h. ** denotes very significant, P<0.01 and * significant, P<0.05. The results show mean+SEM of triplicate measurements.

Mentions: To provide further evidence for the regulation of FOXM1 by ERα, we investigated the effect of ERα silencing on FOXM1 expression in proliferating MCF-7 cells. As shown in Figure 5A, the ERα siRNA effectively knocked down the expression of ERα, which in turn also resulted in a down-regulation of FOXM1 at the protein level. Conversely, FOXM1 knockdown also resulted in a decrease in ERα expression, consistent with our previous finding (Madureira et al., 2006). qRT-PCR analysis of the knocked-down cells also revealed a substantial decrease in FOXM1 mRNA level (Fig. 5A). FOXM1 mRNA expression was not completely abolished by ERα silencing; this is likely to be due to the fact that FOXM1 transcription is regulated by other proliferative signals as well (McGovern et al., 2009). The complete down-regulation of FOXM1 at the protein level suggests the possibility of alternative mechanisms of regulation of FOXM1 expression by ERα (Luscher-Firzlaff et al., 2006; Ma et al., 2005; Major et al., 2004; Wierstra and Alves, 2006b). We next investigated the role of FOXM1 in mediating the proliferative function of ERα by studying the effects of FOXM1 knockdown on estrogen-dependent growth in MCF-7 cells. MCF-7 cells were estrogen-starved for 48 h then induced to proliferate with E2 in the presence or absence of FOXM1 siRNA. Western blot analysis showed that FOXM1 was successfully silenced by the siRNA, and that in the FOXM1 knocked-down cells there was also a significant decrease in ERα expression (Fig. 5B). This is in agreement with our previous finding that FOXM1 regulates ERα expression (Madureira et al., 2006). The results of the cell proliferation assay showed that FOXM1 silencing completely abolished the mitogenic effects of E2 in the ERα positive MCF-7 cells, indicating that FOXM1 is essential for the estrogen-dependent growth of MCF-7 cells, and mediates the proliferative function of ERα (Fig. 5B). To investigate the potential role of FOXM1 in hormonal resistance, we silenced FOXM1 expression using siRNA in the MCF-7 TAMR4 cells, an OHT-resistant MCF-7 clone (Lykkesfeldt et al., 1986; Lykkesfeldt et al., 1994), and studied its effects on cell proliferation (Fig. 5C). Interestingly, the proliferation assays showed that the MCF-7 TAMR4 was responsive to E2 re-stimulation, and knockdown of FOXM1 decreased the ability of E2 to induce the proliferation of the MCF-7 TAMR4 cells. Notably, these cells with FOXM1 knockdown cultured in the absence or presence of E2 had the same proliferation rate as steroid-depleted cells, suggesting E2 targets FOXM1 to induce proliferation in this system. In addition, our data also demonstrated that OHT could antagonise the proliferative function of E2, and that FOXM1 knockdown could be combined with OHT to cause further decreases in the rate of cell proliferation. Together these results clearly show that the knockdown of FOXM1 sensitizes the resistant cells to OHT and diminishes the responsiveness to E2.


FOXM1 is a transcriptional target of ERalpha and has a critical role in breast cancer endocrine sensitivity and resistance.

Millour J, Constantinidou D, Stavropoulou AV, Wilson MS, Myatt SS, Kwok JM, Sivanandan K, Coombes RC, Medema RH, Hartman J, Lykkesfeldt AE, Lam EW - Oncogene (2010)

Effects of ERα and FOXM1 silencing on the expression of FOXM1 and response to E2 induction in MCF-7 cellsA) MCF-7 cells were transiently transfected with ERα, FOXM1 or control smart pool siRNA, and 72 h after transfection cells were analysed by western blot using specific antibodies as indicated and by qRT-PCR. B) MCF-7 cells were transiently transfected with smart pool siRNA against FOXM1, incubated with E2 and analysed by western blotting. SRB assays were also performed on these cells, indicating that the knockdown of FOXM1 decreases the cell proliferation rate and renders MCF-7 cells unresponsive to E2 stimulation. C) OHT-resistant TAMR4 MCF-7 cells were transiently transfected with smart pool siRNA against FOXM1 or control siRNA pool (non-specific/n.s. siRNA) and analysed by western blotting (upper panel). These transfected cells were incubated with or without E2 (middle panel), and with or without OHT in the presence of E2 (lower panel). SRB assays were performed on these cells, indicating that the knockdown of FOXM1 sensitizes the resistant TAMR4 MCF-7 cells to OHT and diminishes their responsiveness to E2 stimulation. Statistical analysis was performed on the proliferation results at 72 h. ** denotes very significant, P<0.01 and * significant, P<0.05. The results show mean+SEM of triplicate measurements.
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Figure 5: Effects of ERα and FOXM1 silencing on the expression of FOXM1 and response to E2 induction in MCF-7 cellsA) MCF-7 cells were transiently transfected with ERα, FOXM1 or control smart pool siRNA, and 72 h after transfection cells were analysed by western blot using specific antibodies as indicated and by qRT-PCR. B) MCF-7 cells were transiently transfected with smart pool siRNA against FOXM1, incubated with E2 and analysed by western blotting. SRB assays were also performed on these cells, indicating that the knockdown of FOXM1 decreases the cell proliferation rate and renders MCF-7 cells unresponsive to E2 stimulation. C) OHT-resistant TAMR4 MCF-7 cells were transiently transfected with smart pool siRNA against FOXM1 or control siRNA pool (non-specific/n.s. siRNA) and analysed by western blotting (upper panel). These transfected cells were incubated with or without E2 (middle panel), and with or without OHT in the presence of E2 (lower panel). SRB assays were performed on these cells, indicating that the knockdown of FOXM1 sensitizes the resistant TAMR4 MCF-7 cells to OHT and diminishes their responsiveness to E2 stimulation. Statistical analysis was performed on the proliferation results at 72 h. ** denotes very significant, P<0.01 and * significant, P<0.05. The results show mean+SEM of triplicate measurements.
Mentions: To provide further evidence for the regulation of FOXM1 by ERα, we investigated the effect of ERα silencing on FOXM1 expression in proliferating MCF-7 cells. As shown in Figure 5A, the ERα siRNA effectively knocked down the expression of ERα, which in turn also resulted in a down-regulation of FOXM1 at the protein level. Conversely, FOXM1 knockdown also resulted in a decrease in ERα expression, consistent with our previous finding (Madureira et al., 2006). qRT-PCR analysis of the knocked-down cells also revealed a substantial decrease in FOXM1 mRNA level (Fig. 5A). FOXM1 mRNA expression was not completely abolished by ERα silencing; this is likely to be due to the fact that FOXM1 transcription is regulated by other proliferative signals as well (McGovern et al., 2009). The complete down-regulation of FOXM1 at the protein level suggests the possibility of alternative mechanisms of regulation of FOXM1 expression by ERα (Luscher-Firzlaff et al., 2006; Ma et al., 2005; Major et al., 2004; Wierstra and Alves, 2006b). We next investigated the role of FOXM1 in mediating the proliferative function of ERα by studying the effects of FOXM1 knockdown on estrogen-dependent growth in MCF-7 cells. MCF-7 cells were estrogen-starved for 48 h then induced to proliferate with E2 in the presence or absence of FOXM1 siRNA. Western blot analysis showed that FOXM1 was successfully silenced by the siRNA, and that in the FOXM1 knocked-down cells there was also a significant decrease in ERα expression (Fig. 5B). This is in agreement with our previous finding that FOXM1 regulates ERα expression (Madureira et al., 2006). The results of the cell proliferation assay showed that FOXM1 silencing completely abolished the mitogenic effects of E2 in the ERα positive MCF-7 cells, indicating that FOXM1 is essential for the estrogen-dependent growth of MCF-7 cells, and mediates the proliferative function of ERα (Fig. 5B). To investigate the potential role of FOXM1 in hormonal resistance, we silenced FOXM1 expression using siRNA in the MCF-7 TAMR4 cells, an OHT-resistant MCF-7 clone (Lykkesfeldt et al., 1986; Lykkesfeldt et al., 1994), and studied its effects on cell proliferation (Fig. 5C). Interestingly, the proliferation assays showed that the MCF-7 TAMR4 was responsive to E2 re-stimulation, and knockdown of FOXM1 decreased the ability of E2 to induce the proliferation of the MCF-7 TAMR4 cells. Notably, these cells with FOXM1 knockdown cultured in the absence or presence of E2 had the same proliferation rate as steroid-depleted cells, suggesting E2 targets FOXM1 to induce proliferation in this system. In addition, our data also demonstrated that OHT could antagonise the proliferative function of E2, and that FOXM1 knockdown could be combined with OHT to cause further decreases in the rate of cell proliferation. Together these results clearly show that the knockdown of FOXM1 sensitizes the resistant cells to OHT and diminishes the responsiveness to E2.

Bottom Line: Our data also revealed that upon OHT treatment ERalpha recruits histone deacetylases to the ERE site of the FOXM1 promoter, which is associated with a decrease in histone acetylation and transcription activity.Conversely, ectopic expression of FOXM1 abrogated the cell cycle arrest mediated by the anti-estrogen OHT.OHT repressed FOXM1 expression in endocrine sensitive but not resistant breast carcinoma cell lines.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery and Cancer, Imperial College London, London W12 0NN, UK.

ABSTRACT
In this study, we investigated the regulation of FOXM1 expression by estrogen receptor alpha (ERalpha) and its role in hormonal therapy and endocrine resistance. FOXM1 protein and mRNA expression was regulated by ER-ligands, including estrogen, tamoxifen (OHT) and fulvestrant (ICI182780; ICI) in breast carcinoma cell lines. Depletion of ERalpha by RNA interference (RNAi) in MCF-7 cells downregulated FOXM1 expression. Reporter gene assays showed that ERalpha activates FOXM1 transcription through an estrogen-response element (ERE) located within the proximal promoter region. The direct binding of ERalpha to the FOXM1 promoter was confirmed in vitro by mobility shift and DNA pull-down assays and in vivo by chromatin immunoprecipitation (ChIP) analysis. Our data also revealed that upon OHT treatment ERalpha recruits histone deacetylases to the ERE site of the FOXM1 promoter, which is associated with a decrease in histone acetylation and transcription activity. Importantly, silencing of FOXM1 by RNAi abolished estrogen-induced MCF-7 cell proliferation and overcame acquired tamoxifen resistance. Conversely, ectopic expression of FOXM1 abrogated the cell cycle arrest mediated by the anti-estrogen OHT. OHT repressed FOXM1 expression in endocrine sensitive but not resistant breast carcinoma cell lines. Furthermore, qRT-PCR analysis of breast cancer patient samples revealed that there was a strong and significant positive correlation between ERalpha and FOXM1 mRNA expression. Collectively, these results show FOXM1 to be a key mediator of the mitogenic functions of ERalpha and estrogen in breast cancer cells, and also suggest that the deregulation of FOXM1 may contribute to anti-estrogen insensitivity.

Show MeSH
Related in: MedlinePlus