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Proteasome inhibition represses ERalpha gene expression in ER+ cells: a new link between proteasome activity and estrogen signaling in breast cancer.

Powers GL, Ellison-Zelski SJ, Casa AJ, Lee AV, Alarid ET - Oncogene (2009)

Bottom Line: Unlike other laboratory proteasome inhibitors, bortezomib failed to stabilize ERalpha protein at a dose exceeding 90% inhibition of the chymotrypsin-like activity.This response can be explained by the fact that bortezomib induced a dramatic decrease in ERalpha mRNA because of direct transcriptional inhibition and loss of RNA polymerase II recruitment on the ERalpha gene promoter.Bortezomib treatment resulted in promoter-specific changes in estrogen-induced gene transcription that related with occupancy of ERalpha and RNA polymerase II (PolII) on endogenous promoters.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, University of Wisconsin-Madison, Madison, WI 53705, USA.

ABSTRACT
Estrogen receptor-alpha (ERalpha) is a major therapeutic target of hormonal therapies in breast cancer, and its expression in tumors is predictive of clinical response. Protein levels of ERalpha are tightly controlled by the 26S proteasome; yet, how the clinical proteasome inhibitor, bortezomib, affects ERalpha regulation has not been studied. Bortezomib selectively inhibits the chymotrypsin-like activity of the proteasome. Unlike other laboratory proteasome inhibitors, bortezomib failed to stabilize ERalpha protein at a dose exceeding 90% inhibition of the chymotrypsin-like activity. Unexpectedly, however, chronic bortezomib exposure caused a reduction of ERalpha levels in multiple ER+ breast cancer cell lines. This response can be explained by the fact that bortezomib induced a dramatic decrease in ERalpha mRNA because of direct transcriptional inhibition and loss of RNA polymerase II recruitment on the ERalpha gene promoter. Bortezomib treatment resulted in promoter-specific changes in estrogen-induced gene transcription that related with occupancy of ERalpha and RNA polymerase II (PolII) on endogenous promoters. In addition, bortezomib inhibited estrogen-dependent growth in soft agar. These results reveal a novel link between proteasome activity and expression of ERalpha in breast cancer and uncover distinct roles of the chymotrypsin-like activity of the proteasome in the regulation of the ERalpha pathway.

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Bortezomib results in a decrease in the nascent ERα mRNA transcript by decreasing RNA PolII on the ERα promoterMCF7 cells were treated for 30 minutes with 30 nM bortezomib and then for 24 hours with 10 nM E2. a) RNA isolation and qRT-PCR were performed as described in Figure 4 for the nascent ERα transcript. The data were analyzed relative to the average EtOH treatment and the mean of three independent experiments is shown. Statistics were performed to demonstrate significant differences (p< 0.05) between vehicle control and estrogen and are marked a and b, respectively. b) Chromatin immunoprecipitation (ChIP) was performed using antibodies for RNA PolII and IgG. Primers for non-specific (NS), the A promoter (A) and exon 1 (Ex1) regions of the ERα promoter were used with qRT-PCR to examine the occupancy at each site. Data are presented as the mean percent input of three independent experiments. Error bars represent the standard error of the mean.
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Figure 5: Bortezomib results in a decrease in the nascent ERα mRNA transcript by decreasing RNA PolII on the ERα promoterMCF7 cells were treated for 30 minutes with 30 nM bortezomib and then for 24 hours with 10 nM E2. a) RNA isolation and qRT-PCR were performed as described in Figure 4 for the nascent ERα transcript. The data were analyzed relative to the average EtOH treatment and the mean of three independent experiments is shown. Statistics were performed to demonstrate significant differences (p< 0.05) between vehicle control and estrogen and are marked a and b, respectively. b) Chromatin immunoprecipitation (ChIP) was performed using antibodies for RNA PolII and IgG. Primers for non-specific (NS), the A promoter (A) and exon 1 (Ex1) regions of the ERα promoter were used with qRT-PCR to examine the occupancy at each site. Data are presented as the mean percent input of three independent experiments. Error bars represent the standard error of the mean.

Mentions: To probe further into the mechanism of bortezomib-induced decrease in ERα, levels of nascent unspliced ERα mRNA were evaluated by qRT-PCR. Similar to total ERα mRNA, the nascent transcript was decreased approximately 90% ± 0.03 by bortezomib alone and was further reduced by an additional 7% ± 0.01 by E2. In total, ERα transcription was reduced to 3% of the levels in control cells by bortezomib in the presence of estrogen (Figure 5a). Chromatin immunoprecipitation (ChIP) was performed to examine RNA polymerase II (RNA PolII) occupancy as an independent measure of transcription. Previous studies in our laboratory and those of others demonstrated that the major promoter governing ERα gene expression in MCF7 breast cancer cells is the A promoter located near the transcription start site (Denger, et al., 2001; Ellison-Zelski, et al., 2009). Studies in Figure 5b show that RNA PolII is present on the A promoter and Exon 1, with negligible occupancy at a non-specific site at -940 basepairs upstream of the transcription start site. These results are consistent with the active transcription of the ERα gene in MCF7 cells. Paralleling the loss of nascent transcript, bortezomib and estrogen treatments both resulted in a decrease in RNA PolII occupancy at both the A promoter and Exon 1. These data indicate that bortezomib regulates ERα directly at the transcriptional level by reducing RNA PolII occupancy at the proximal promoter.


Proteasome inhibition represses ERalpha gene expression in ER+ cells: a new link between proteasome activity and estrogen signaling in breast cancer.

Powers GL, Ellison-Zelski SJ, Casa AJ, Lee AV, Alarid ET - Oncogene (2009)

Bortezomib results in a decrease in the nascent ERα mRNA transcript by decreasing RNA PolII on the ERα promoterMCF7 cells were treated for 30 minutes with 30 nM bortezomib and then for 24 hours with 10 nM E2. a) RNA isolation and qRT-PCR were performed as described in Figure 4 for the nascent ERα transcript. The data were analyzed relative to the average EtOH treatment and the mean of three independent experiments is shown. Statistics were performed to demonstrate significant differences (p< 0.05) between vehicle control and estrogen and are marked a and b, respectively. b) Chromatin immunoprecipitation (ChIP) was performed using antibodies for RNA PolII and IgG. Primers for non-specific (NS), the A promoter (A) and exon 1 (Ex1) regions of the ERα promoter were used with qRT-PCR to examine the occupancy at each site. Data are presented as the mean percent input of three independent experiments. Error bars represent the standard error of the mean.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2837136&req=5

Figure 5: Bortezomib results in a decrease in the nascent ERα mRNA transcript by decreasing RNA PolII on the ERα promoterMCF7 cells were treated for 30 minutes with 30 nM bortezomib and then for 24 hours with 10 nM E2. a) RNA isolation and qRT-PCR were performed as described in Figure 4 for the nascent ERα transcript. The data were analyzed relative to the average EtOH treatment and the mean of three independent experiments is shown. Statistics were performed to demonstrate significant differences (p< 0.05) between vehicle control and estrogen and are marked a and b, respectively. b) Chromatin immunoprecipitation (ChIP) was performed using antibodies for RNA PolII and IgG. Primers for non-specific (NS), the A promoter (A) and exon 1 (Ex1) regions of the ERα promoter were used with qRT-PCR to examine the occupancy at each site. Data are presented as the mean percent input of three independent experiments. Error bars represent the standard error of the mean.
Mentions: To probe further into the mechanism of bortezomib-induced decrease in ERα, levels of nascent unspliced ERα mRNA were evaluated by qRT-PCR. Similar to total ERα mRNA, the nascent transcript was decreased approximately 90% ± 0.03 by bortezomib alone and was further reduced by an additional 7% ± 0.01 by E2. In total, ERα transcription was reduced to 3% of the levels in control cells by bortezomib in the presence of estrogen (Figure 5a). Chromatin immunoprecipitation (ChIP) was performed to examine RNA polymerase II (RNA PolII) occupancy as an independent measure of transcription. Previous studies in our laboratory and those of others demonstrated that the major promoter governing ERα gene expression in MCF7 breast cancer cells is the A promoter located near the transcription start site (Denger, et al., 2001; Ellison-Zelski, et al., 2009). Studies in Figure 5b show that RNA PolII is present on the A promoter and Exon 1, with negligible occupancy at a non-specific site at -940 basepairs upstream of the transcription start site. These results are consistent with the active transcription of the ERα gene in MCF7 cells. Paralleling the loss of nascent transcript, bortezomib and estrogen treatments both resulted in a decrease in RNA PolII occupancy at both the A promoter and Exon 1. These data indicate that bortezomib regulates ERα directly at the transcriptional level by reducing RNA PolII occupancy at the proximal promoter.

Bottom Line: Unlike other laboratory proteasome inhibitors, bortezomib failed to stabilize ERalpha protein at a dose exceeding 90% inhibition of the chymotrypsin-like activity.This response can be explained by the fact that bortezomib induced a dramatic decrease in ERalpha mRNA because of direct transcriptional inhibition and loss of RNA polymerase II recruitment on the ERalpha gene promoter.Bortezomib treatment resulted in promoter-specific changes in estrogen-induced gene transcription that related with occupancy of ERalpha and RNA polymerase II (PolII) on endogenous promoters.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, University of Wisconsin-Madison, Madison, WI 53705, USA.

ABSTRACT
Estrogen receptor-alpha (ERalpha) is a major therapeutic target of hormonal therapies in breast cancer, and its expression in tumors is predictive of clinical response. Protein levels of ERalpha are tightly controlled by the 26S proteasome; yet, how the clinical proteasome inhibitor, bortezomib, affects ERalpha regulation has not been studied. Bortezomib selectively inhibits the chymotrypsin-like activity of the proteasome. Unlike other laboratory proteasome inhibitors, bortezomib failed to stabilize ERalpha protein at a dose exceeding 90% inhibition of the chymotrypsin-like activity. Unexpectedly, however, chronic bortezomib exposure caused a reduction of ERalpha levels in multiple ER+ breast cancer cell lines. This response can be explained by the fact that bortezomib induced a dramatic decrease in ERalpha mRNA because of direct transcriptional inhibition and loss of RNA polymerase II recruitment on the ERalpha gene promoter. Bortezomib treatment resulted in promoter-specific changes in estrogen-induced gene transcription that related with occupancy of ERalpha and RNA polymerase II (PolII) on endogenous promoters. In addition, bortezomib inhibited estrogen-dependent growth in soft agar. These results reveal a novel link between proteasome activity and expression of ERalpha in breast cancer and uncover distinct roles of the chymotrypsin-like activity of the proteasome in the regulation of the ERalpha pathway.

Show MeSH
Related in: MedlinePlus