Limits...
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.

Show MeSH

Related in: MedlinePlus

Proteasome inhibition results in gene specific effects on ERα target gene transcriptionMCF7 cells were treated for 30 minutes with or without 30 nM bortezomib and then treated for 24 hours with or without 10 nM E2. RNA isolation and qRT-PCR were performed as previously described using primers for a) PR or b) pS2. Data represent the means of a minimum of three independent experiments and error bars represent the standard error of the mean. Statistically significant differences (p< 0.05) relative to vehicle control (EtOH) or estrogen and are indicated with a or b, respectively. MCF7 cells were transfected with a 3× ERE-tk luciferase reporter plasmid and CMV-β-galactosidase (β-gal) plasmid. The next day the cells were treated for 24 hours with or without 30 nM bortezomib and 10 nM E2. c) Luciferase and β-galactosidase assays were performed on whole cell lysates. d) RNA isolation and qPCR was performed as previously described for luciferase mRNA. Data are presented relative to the EtOH control and error bars were calculated from the standard error of the mean of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2837136&req=5

Figure 7: Proteasome inhibition results in gene specific effects on ERα target gene transcriptionMCF7 cells were treated for 30 minutes with or without 30 nM bortezomib and then treated for 24 hours with or without 10 nM E2. RNA isolation and qRT-PCR were performed as previously described using primers for a) PR or b) pS2. Data represent the means of a minimum of three independent experiments and error bars represent the standard error of the mean. Statistically significant differences (p< 0.05) relative to vehicle control (EtOH) or estrogen and are indicated with a or b, respectively. MCF7 cells were transfected with a 3× ERE-tk luciferase reporter plasmid and CMV-β-galactosidase (β-gal) plasmid. The next day the cells were treated for 24 hours with or without 30 nM bortezomib and 10 nM E2. c) Luciferase and β-galactosidase assays were performed on whole cell lysates. d) RNA isolation and qPCR was performed as previously described for luciferase mRNA. Data are presented relative to the EtOH control and error bars were calculated from the standard error of the mean of three independent experiments.

Mentions: Next, the impact of bortezomib on ERα functional activity was assessed. The transcriptional activity of ERα was evaluated by analysis of endogenous progesterone receptor (PR) and pS2 gene expression. PR and pS2 are classical estrogen-responsive genes that are dependent on ERα for expression and are induced by estrogen (Feng, et al., 2007; Metivier, et al., 2003; Valley, et al., 2005). Chronic bortezomib treatment had opposite effects on these two estrogen receptor target genes. While bortezomib treatment resulted in a significant inhibition of estrogen-dependent activation of PR, it resulted in an enhancement of estrogen-dependent pS2 gene transcription (Figure 7a-b). In the absence of estrogen, bortezomib decreased pS2 and increased PR gene expression. An artificial reporter gene consisting of tandem estrogen response elements and a minimal thymidine kinase promoter was also analyzed to directly test the impact of bortezomib on ERα-dependent transcription. Luciferase protein and gene expression were significantly increased by estrogen treatment and this was unaffected by co-treatment with bortezomib (Figure 7c-d). Bortezomib also did not affect basal luciferase levels indicating that the thymidine kinase promoter was not sensitive to proteasome inhibition. These data suggest that bortezomib alters ERα-dependent gene transcription in a promoter-specific manner.


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)

Proteasome inhibition results in gene specific effects on ERα target gene transcriptionMCF7 cells were treated for 30 minutes with or without 30 nM bortezomib and then treated for 24 hours with or without 10 nM E2. RNA isolation and qRT-PCR were performed as previously described using primers for a) PR or b) pS2. Data represent the means of a minimum of three independent experiments and error bars represent the standard error of the mean. Statistically significant differences (p< 0.05) relative to vehicle control (EtOH) or estrogen and are indicated with a or b, respectively. MCF7 cells were transfected with a 3× ERE-tk luciferase reporter plasmid and CMV-β-galactosidase (β-gal) plasmid. The next day the cells were treated for 24 hours with or without 30 nM bortezomib and 10 nM E2. c) Luciferase and β-galactosidase assays were performed on whole cell lysates. d) RNA isolation and qPCR was performed as previously described for luciferase mRNA. Data are presented relative to the EtOH control and error bars were calculated from the standard error of the mean of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 7: Proteasome inhibition results in gene specific effects on ERα target gene transcriptionMCF7 cells were treated for 30 minutes with or without 30 nM bortezomib and then treated for 24 hours with or without 10 nM E2. RNA isolation and qRT-PCR were performed as previously described using primers for a) PR or b) pS2. Data represent the means of a minimum of three independent experiments and error bars represent the standard error of the mean. Statistically significant differences (p< 0.05) relative to vehicle control (EtOH) or estrogen and are indicated with a or b, respectively. MCF7 cells were transfected with a 3× ERE-tk luciferase reporter plasmid and CMV-β-galactosidase (β-gal) plasmid. The next day the cells were treated for 24 hours with or without 30 nM bortezomib and 10 nM E2. c) Luciferase and β-galactosidase assays were performed on whole cell lysates. d) RNA isolation and qPCR was performed as previously described for luciferase mRNA. Data are presented relative to the EtOH control and error bars were calculated from the standard error of the mean of three independent experiments.
Mentions: Next, the impact of bortezomib on ERα functional activity was assessed. The transcriptional activity of ERα was evaluated by analysis of endogenous progesterone receptor (PR) and pS2 gene expression. PR and pS2 are classical estrogen-responsive genes that are dependent on ERα for expression and are induced by estrogen (Feng, et al., 2007; Metivier, et al., 2003; Valley, et al., 2005). Chronic bortezomib treatment had opposite effects on these two estrogen receptor target genes. While bortezomib treatment resulted in a significant inhibition of estrogen-dependent activation of PR, it resulted in an enhancement of estrogen-dependent pS2 gene transcription (Figure 7a-b). In the absence of estrogen, bortezomib decreased pS2 and increased PR gene expression. An artificial reporter gene consisting of tandem estrogen response elements and a minimal thymidine kinase promoter was also analyzed to directly test the impact of bortezomib on ERα-dependent transcription. Luciferase protein and gene expression were significantly increased by estrogen treatment and this was unaffected by co-treatment with bortezomib (Figure 7c-d). Bortezomib also did not affect basal luciferase levels indicating that the thymidine kinase promoter was not sensitive to proteasome inhibition. These data suggest that bortezomib alters ERα-dependent gene transcription in a promoter-specific manner.

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