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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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Significant correlation between ERα and FOXM1 expression in human breast samplesExpression of ERα mRNA and FOXM1 mRNA in non-cancerous breast biopsies and malignant breast epithelial tissues. RNA was isolated from epithelial cells purified from normal breast tissues and primary tumours and subjected to qRT-PCR with FOXM1, ERα and L19 primers. A) Graph shows the FOXM1 and ERα mRNA levels of the tumour samples after normalisation with L19 RNA levels. No significant correlation is seen using Pearson correlation when considering all values (n=69; Spearman r=0.198; p=0.109). Significance is defined as p<0.05. Line is linear regression, shown for illustrative purposes. B) Box and whisker plot showing the distribution of values for FOXM1. Box edges represent 25th and 75th percentiles; middle line is the median, while plus shows the mean. The whiskers represent the 10th and 90th percentiles, while outliers are shown as dots. The 75th percentile for FOXM1 mRNA values is 0.7002. C) The right-hand graph shows the FOXM1 and ERα mRNA levels of the tumour samples with FOXM1/L19 mRNA levels below the upper quartile. Correlation analysis was performed between FOXM1 and ERα mRNA. A significant and positive association was found between FOXM1 and ERα mRNA levels in breast patient samples with FOXM1 mRNA level below the upper quartile (n=52; Spearman r=0.447; p=0.0001). P < 0.05 was considered statistically significant. D) The lower left-hand graph shows the FOXM1 and ERα mRNA levels of the tumour samples with FOXM1/L19 mRNA levels in the upper quartile (0.7) of high levels of FOXM mRNA expression. No significant correlation is found using Pearson correlation (n=17; Spearman r=0.186; p=0.474). Significance is defined as p≤0.05.
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Figure 4: Significant correlation between ERα and FOXM1 expression in human breast samplesExpression of ERα mRNA and FOXM1 mRNA in non-cancerous breast biopsies and malignant breast epithelial tissues. RNA was isolated from epithelial cells purified from normal breast tissues and primary tumours and subjected to qRT-PCR with FOXM1, ERα and L19 primers. A) Graph shows the FOXM1 and ERα mRNA levels of the tumour samples after normalisation with L19 RNA levels. No significant correlation is seen using Pearson correlation when considering all values (n=69; Spearman r=0.198; p=0.109). Significance is defined as p<0.05. Line is linear regression, shown for illustrative purposes. B) Box and whisker plot showing the distribution of values for FOXM1. Box edges represent 25th and 75th percentiles; middle line is the median, while plus shows the mean. The whiskers represent the 10th and 90th percentiles, while outliers are shown as dots. The 75th percentile for FOXM1 mRNA values is 0.7002. C) The right-hand graph shows the FOXM1 and ERα mRNA levels of the tumour samples with FOXM1/L19 mRNA levels below the upper quartile. Correlation analysis was performed between FOXM1 and ERα mRNA. A significant and positive association was found between FOXM1 and ERα mRNA levels in breast patient samples with FOXM1 mRNA level below the upper quartile (n=52; Spearman r=0.447; p=0.0001). P < 0.05 was considered statistically significant. D) The lower left-hand graph shows the FOXM1 and ERα mRNA levels of the tumour samples with FOXM1/L19 mRNA levels in the upper quartile (0.7) of high levels of FOXM mRNA expression. No significant correlation is found using Pearson correlation (n=17; Spearman r=0.186; p=0.474). Significance is defined as p≤0.05.

Mentions: To determine the clinical relevance of the relationship between ERα and FOXM1 expression, we analysed the FOXM1 and ERα mRNA levels in 69 frozen archival clinical samples, of which 21 were from benign disease patients, 39 from patients with breast tumours and 9 from healthy individuals (Fig. S3). Preliminary studies demonstrated that despite FOXM1 mRNA expression being associated with tumour status, grade, size, and menopausal status, the differences were not significant, possibly reflecting the relatively small sample size (Fig. S4). Analysis of unadjusted data revealed no significant correlation between the expression levels of FOXM1 and ERα mRNA (Fig. 4A). This is not entirely unexpected as high levels of FOXM1 mRNA expression (upper quartile) have previously been shown be associated with breast cancer progression, poor prognosis and endocrine resistance in a number of microarray studies (Martin et al., 2008). We next re-evaluated the data after excluding patient samples with high levels of FOXM1 expression. By leaving out patient samples with FOXM1 mRNA levels above the 75th percentile value (FOXM1/L19mRNA=>0.700) (Fig. 4B), we found a significant correlation between FOXM1 and ERα mRNA expression (Fig. 4C). In cases where there were high levels of FOXM1 mRNA expression, we also found that FOXM1 expression is independent of ERα mRNA levels, suggesting FOXM1 is no longer regulated by ERα (Fig. 4D). These results validate our tissue culture findings in breast patient samples, confirming the crucial relationship between ERα and FOXM1 expression. In breast cancer samples with high FOXM1 expression, the relationship between ERα and FOXM1 mRNA expression is uncoupled, suggesting ERα no longer controls FOXM1 expression. This also supports the conjecture that uncoupling of the regulation of FOXM1 by ERα may have a role in endocrine resistance.


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)

Significant correlation between ERα and FOXM1 expression in human breast samplesExpression of ERα mRNA and FOXM1 mRNA in non-cancerous breast biopsies and malignant breast epithelial tissues. RNA was isolated from epithelial cells purified from normal breast tissues and primary tumours and subjected to qRT-PCR with FOXM1, ERα and L19 primers. A) Graph shows the FOXM1 and ERα mRNA levels of the tumour samples after normalisation with L19 RNA levels. No significant correlation is seen using Pearson correlation when considering all values (n=69; Spearman r=0.198; p=0.109). Significance is defined as p<0.05. Line is linear regression, shown for illustrative purposes. B) Box and whisker plot showing the distribution of values for FOXM1. Box edges represent 25th and 75th percentiles; middle line is the median, while plus shows the mean. The whiskers represent the 10th and 90th percentiles, while outliers are shown as dots. The 75th percentile for FOXM1 mRNA values is 0.7002. C) The right-hand graph shows the FOXM1 and ERα mRNA levels of the tumour samples with FOXM1/L19 mRNA levels below the upper quartile. Correlation analysis was performed between FOXM1 and ERα mRNA. A significant and positive association was found between FOXM1 and ERα mRNA levels in breast patient samples with FOXM1 mRNA level below the upper quartile (n=52; Spearman r=0.447; p=0.0001). P < 0.05 was considered statistically significant. D) The lower left-hand graph shows the FOXM1 and ERα mRNA levels of the tumour samples with FOXM1/L19 mRNA levels in the upper quartile (0.7) of high levels of FOXM mRNA expression. No significant correlation is found using Pearson correlation (n=17; Spearman r=0.186; p=0.474). Significance is defined as p≤0.05.
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Figure 4: Significant correlation between ERα and FOXM1 expression in human breast samplesExpression of ERα mRNA and FOXM1 mRNA in non-cancerous breast biopsies and malignant breast epithelial tissues. RNA was isolated from epithelial cells purified from normal breast tissues and primary tumours and subjected to qRT-PCR with FOXM1, ERα and L19 primers. A) Graph shows the FOXM1 and ERα mRNA levels of the tumour samples after normalisation with L19 RNA levels. No significant correlation is seen using Pearson correlation when considering all values (n=69; Spearman r=0.198; p=0.109). Significance is defined as p<0.05. Line is linear regression, shown for illustrative purposes. B) Box and whisker plot showing the distribution of values for FOXM1. Box edges represent 25th and 75th percentiles; middle line is the median, while plus shows the mean. The whiskers represent the 10th and 90th percentiles, while outliers are shown as dots. The 75th percentile for FOXM1 mRNA values is 0.7002. C) The right-hand graph shows the FOXM1 and ERα mRNA levels of the tumour samples with FOXM1/L19 mRNA levels below the upper quartile. Correlation analysis was performed between FOXM1 and ERα mRNA. A significant and positive association was found between FOXM1 and ERα mRNA levels in breast patient samples with FOXM1 mRNA level below the upper quartile (n=52; Spearman r=0.447; p=0.0001). P < 0.05 was considered statistically significant. D) The lower left-hand graph shows the FOXM1 and ERα mRNA levels of the tumour samples with FOXM1/L19 mRNA levels in the upper quartile (0.7) of high levels of FOXM mRNA expression. No significant correlation is found using Pearson correlation (n=17; Spearman r=0.186; p=0.474). Significance is defined as p≤0.05.
Mentions: To determine the clinical relevance of the relationship between ERα and FOXM1 expression, we analysed the FOXM1 and ERα mRNA levels in 69 frozen archival clinical samples, of which 21 were from benign disease patients, 39 from patients with breast tumours and 9 from healthy individuals (Fig. S3). Preliminary studies demonstrated that despite FOXM1 mRNA expression being associated with tumour status, grade, size, and menopausal status, the differences were not significant, possibly reflecting the relatively small sample size (Fig. S4). Analysis of unadjusted data revealed no significant correlation between the expression levels of FOXM1 and ERα mRNA (Fig. 4A). This is not entirely unexpected as high levels of FOXM1 mRNA expression (upper quartile) have previously been shown be associated with breast cancer progression, poor prognosis and endocrine resistance in a number of microarray studies (Martin et al., 2008). We next re-evaluated the data after excluding patient samples with high levels of FOXM1 expression. By leaving out patient samples with FOXM1 mRNA levels above the 75th percentile value (FOXM1/L19mRNA=>0.700) (Fig. 4B), we found a significant correlation between FOXM1 and ERα mRNA expression (Fig. 4C). In cases where there were high levels of FOXM1 mRNA expression, we also found that FOXM1 expression is independent of ERα mRNA levels, suggesting FOXM1 is no longer regulated by ERα (Fig. 4D). These results validate our tissue culture findings in breast patient samples, confirming the crucial relationship between ERα and FOXM1 expression. In breast cancer samples with high FOXM1 expression, the relationship between ERα and FOXM1 mRNA expression is uncoupled, suggesting ERα no longer controls FOXM1 expression. This also supports the conjecture that uncoupling of the regulation of FOXM1 by ERα may have a role in endocrine resistance.

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