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ERβ1: characterization, prognosis, and evaluation of treatment strategies in ERα-positive and -negative breast cancer.

Reese JM, Suman VJ, Subramaniam M, Wu X, Negron V, Gingery A, Pitel KS, Shah SS, Cunliffe HE, McCullough AE, Pockaj BA, Couch FJ, Olson JE, Reynolds C, Lingle WL, Spelsberg TC, Goetz MP, Ingle JN, Hawse JR - BMC Cancer (2014)

Bottom Line: In agreement with these observations, ERβ1 expression sensitized ERα-positive breast cancer cells to the anti-cancer effects of selective estrogen receptor modulators (SERMs).However, in the absence of ERα expression, ERβ-specific agonists potently inhibited cell proliferation rates while anti-estrogenic therapies were ineffective.However, the efficacy of SERMs and ERβ-specific agonists differ as a function of ERα expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Mayo Clinic, 16-01B Guggenheim Building, 200 First St, SW, Rochester, MN 55905, USA. hawse.john@mayo.edu.

ABSTRACT

Background: The role and clinical value of ERβ1 expression is controversial and recent data demonstrates that many ERβ antibodies are insensitive and/or non-specific. Therefore, we sought to comprehensively characterize ERβ1 expression across all sub-types of breast cancer using a validated antibody and determine the roles of this receptor in mediating response to multiple forms of endocrine therapy both in the presence and absence of ERα expression.

Methods: Nuclear and cytoplasmic expression patterns of ERβ1 were analyzed in three patient cohorts, including a retrospective analysis of a prospective adjuvant tamoxifen study and a triple negative breast cancer cohort. To investigate the utility of therapeutically targeting ERβ1, we generated multiple ERβ1 expressing cell model systems and determined their proliferative responses following anti-estrogenic or ERβ-specific agonist exposure.

Results: Nuclear ERβ1 was shown to be expressed across all major sub-types of breast cancer, including 25% of triple negative breast cancers and 33% of ER-positive tumors, and was associated with significantly improved outcomes in ERα-positive tamoxifen-treated patients. In agreement with these observations, ERβ1 expression sensitized ERα-positive breast cancer cells to the anti-cancer effects of selective estrogen receptor modulators (SERMs). However, in the absence of ERα expression, ERβ-specific agonists potently inhibited cell proliferation rates while anti-estrogenic therapies were ineffective.

Conclusions: Using a validated antibody, we have confirmed that nuclear ERβ1 expression is commonly present in breast cancer and is prognostic in tamoxifen-treated patients. Using multiple breast cancer cell lines, ERβ appears to be a novel therapeutic target. However, the efficacy of SERMs and ERβ-specific agonists differ as a function of ERα expression.

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Characterization of MDA-MB-231-ERβ1 expressing cells. A). Real-time PCR and Western blot analysis of MDA-MB-231-ERβ1 clonal cell lines # 4 and 12 indicating mRNA and protein expression levels of ERβ1 in the absence and presence of doxycycline (Dox). B). Real-time PCR analysis of the progesterone receptor (PR), trefoil factor 1 (PS2) and Kruppel like factor 10 (KLF10) following indicated treatments for 24 hours. P-values < 0.05 were considered to be statistically significant. *Denotes significant difference between indicated treatment and vehicle control treated cells.
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Fig4: Characterization of MDA-MB-231-ERβ1 expressing cells. A). Real-time PCR and Western blot analysis of MDA-MB-231-ERβ1 clonal cell lines # 4 and 12 indicating mRNA and protein expression levels of ERβ1 in the absence and presence of doxycycline (Dox). B). Real-time PCR analysis of the progesterone receptor (PR), trefoil factor 1 (PS2) and Kruppel like factor 10 (KLF10) following indicated treatments for 24 hours. P-values < 0.05 were considered to be statistically significant. *Denotes significant difference between indicated treatment and vehicle control treated cells.

Mentions: Since approximately 25% of TNBC were shown to express nuclear ERβ1 (Table 1; cohort 2), we next sought to determine whether expression of ERβ1 in MDA-MB-231 cells, a well-characterized model of TNBC, would alter the cell’s response to ERβ targeting treatments. Two clonal cell lines (#4 and #12) exhibiting robust doxycycline induced expression of ERβ1 mRNA and protein were chosen for further analysis (Figure 4A). To confirm that expression of ERβ1 was exclusively limited to the presence of doxycycline and that the expressed receptor was functional, cells were treated with vehicle, estrogen (1 nM) or estrogen plus ICI (100 nM) for 24 hours and the expression profiles of known ERβ1 target genes were examined. As shown in Figure 4B, these genes were significantly induced following estrogen treatment in the presence of doxycycline, an effect that was completely blocked by the addition of ICI. However, these genes were not induced by estrogen in the absence of doxycycline confirming that these cells do not endogenously express any form of the estrogen receptor (Figure 4B).Figure 4


ERβ1: characterization, prognosis, and evaluation of treatment strategies in ERα-positive and -negative breast cancer.

Reese JM, Suman VJ, Subramaniam M, Wu X, Negron V, Gingery A, Pitel KS, Shah SS, Cunliffe HE, McCullough AE, Pockaj BA, Couch FJ, Olson JE, Reynolds C, Lingle WL, Spelsberg TC, Goetz MP, Ingle JN, Hawse JR - BMC Cancer (2014)

Characterization of MDA-MB-231-ERβ1 expressing cells. A). Real-time PCR and Western blot analysis of MDA-MB-231-ERβ1 clonal cell lines # 4 and 12 indicating mRNA and protein expression levels of ERβ1 in the absence and presence of doxycycline (Dox). B). Real-time PCR analysis of the progesterone receptor (PR), trefoil factor 1 (PS2) and Kruppel like factor 10 (KLF10) following indicated treatments for 24 hours. P-values < 0.05 were considered to be statistically significant. *Denotes significant difference between indicated treatment and vehicle control treated cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4196114&req=5

Fig4: Characterization of MDA-MB-231-ERβ1 expressing cells. A). Real-time PCR and Western blot analysis of MDA-MB-231-ERβ1 clonal cell lines # 4 and 12 indicating mRNA and protein expression levels of ERβ1 in the absence and presence of doxycycline (Dox). B). Real-time PCR analysis of the progesterone receptor (PR), trefoil factor 1 (PS2) and Kruppel like factor 10 (KLF10) following indicated treatments for 24 hours. P-values < 0.05 were considered to be statistically significant. *Denotes significant difference between indicated treatment and vehicle control treated cells.
Mentions: Since approximately 25% of TNBC were shown to express nuclear ERβ1 (Table 1; cohort 2), we next sought to determine whether expression of ERβ1 in MDA-MB-231 cells, a well-characterized model of TNBC, would alter the cell’s response to ERβ targeting treatments. Two clonal cell lines (#4 and #12) exhibiting robust doxycycline induced expression of ERβ1 mRNA and protein were chosen for further analysis (Figure 4A). To confirm that expression of ERβ1 was exclusively limited to the presence of doxycycline and that the expressed receptor was functional, cells were treated with vehicle, estrogen (1 nM) or estrogen plus ICI (100 nM) for 24 hours and the expression profiles of known ERβ1 target genes were examined. As shown in Figure 4B, these genes were significantly induced following estrogen treatment in the presence of doxycycline, an effect that was completely blocked by the addition of ICI. However, these genes were not induced by estrogen in the absence of doxycycline confirming that these cells do not endogenously express any form of the estrogen receptor (Figure 4B).Figure 4

Bottom Line: In agreement with these observations, ERβ1 expression sensitized ERα-positive breast cancer cells to the anti-cancer effects of selective estrogen receptor modulators (SERMs).However, in the absence of ERα expression, ERβ-specific agonists potently inhibited cell proliferation rates while anti-estrogenic therapies were ineffective.However, the efficacy of SERMs and ERβ-specific agonists differ as a function of ERα expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Mayo Clinic, 16-01B Guggenheim Building, 200 First St, SW, Rochester, MN 55905, USA. hawse.john@mayo.edu.

ABSTRACT

Background: The role and clinical value of ERβ1 expression is controversial and recent data demonstrates that many ERβ antibodies are insensitive and/or non-specific. Therefore, we sought to comprehensively characterize ERβ1 expression across all sub-types of breast cancer using a validated antibody and determine the roles of this receptor in mediating response to multiple forms of endocrine therapy both in the presence and absence of ERα expression.

Methods: Nuclear and cytoplasmic expression patterns of ERβ1 were analyzed in three patient cohorts, including a retrospective analysis of a prospective adjuvant tamoxifen study and a triple negative breast cancer cohort. To investigate the utility of therapeutically targeting ERβ1, we generated multiple ERβ1 expressing cell model systems and determined their proliferative responses following anti-estrogenic or ERβ-specific agonist exposure.

Results: Nuclear ERβ1 was shown to be expressed across all major sub-types of breast cancer, including 25% of triple negative breast cancers and 33% of ER-positive tumors, and was associated with significantly improved outcomes in ERα-positive tamoxifen-treated patients. In agreement with these observations, ERβ1 expression sensitized ERα-positive breast cancer cells to the anti-cancer effects of selective estrogen receptor modulators (SERMs). However, in the absence of ERα expression, ERβ-specific agonists potently inhibited cell proliferation rates while anti-estrogenic therapies were ineffective.

Conclusions: Using a validated antibody, we have confirmed that nuclear ERβ1 expression is commonly present in breast cancer and is prognostic in tamoxifen-treated patients. Using multiple breast cancer cell lines, ERβ appears to be a novel therapeutic target. However, the efficacy of SERMs and ERβ-specific agonists differ as a function of ERα expression.

Show MeSH
Related in: MedlinePlus