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Cytoplasmic PELP1 and ERRgamma protect human mammary epithelial cells from Tam-induced cell death.

Girard BJ, Regan Anderson TM, Welch SL, Nicely J, Seewaldt VL, Ostrander JH - PLoS ONE (2015)

Bottom Line: Expression of PELP1-cyto and ERRγ reduced Tam-induced LC3-II accumulation, and knockdown of ERRγ increased LC3-II levels in response to Tam.Our data indicate that cytoplasmic PELP1 induces signaling pathways that converge on ERRγ to promote cell survival in the presence of Tam.These data suggest that PELP1 localization and/or ERRγ activation could be developed as tissue biomarkers for Tam responsiveness.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota, 55455, United States of America.

ABSTRACT
Tamoxifen (Tam) is the only FDA-approved chemoprevention agent for pre-menopausal women at high risk for developing breast cancer. While Tam reduces a woman's risk of developing estrogen receptor positive (ER+) breast cancer, the molecular mechanisms associated with risk reduction are poorly understood. Prior studies have shown that cytoplasmic proline, glutamic acid and leucine rich protein 1 (PELP1) promotes Tam resistance in breast cancer cell lines. Herein, we tested for PELP1 localization in breast epithelial cells from women at high risk for developing breast cancer and found that PELP1 was localized to the cytoplasm in 36% of samples. In vitro, immortalized HMECs expressing a nuclear localization signal (NLS) mutant of PELP1 (PELP1-cyto) were resistant to Tam-induced death. Furthermore, PELP1-cyto signaling through estrogen-related receptor gamma (ERRγ) promoted cell survival in the presence of Tam. Overexpression of ERRγ in immortalized HMECs protected cells from Tam-induced death, while knockdown of ERRγ sensitized PELP1-cyto expressing HMECs to Tam. Moreover, Tam-induced HMEC cell death was independent of apoptosis and involved accumulation of the autophagy marker LC3-II. Expression of PELP1-cyto and ERRγ reduced Tam-induced LC3-II accumulation, and knockdown of ERRγ increased LC3-II levels in response to Tam. Additionally, PELP1-cyto expression led to the upregulation of MMP-3 and MAOB, known PELP1 and ERRγ target genes, respectively. Our data indicate that cytoplasmic PELP1 induces signaling pathways that converge on ERRγ to promote cell survival in the presence of Tam. These data suggest that PELP1 localization and/or ERRγ activation could be developed as tissue biomarkers for Tam responsiveness.

No MeSH data available.


Related in: MedlinePlus

Cytoplasmic PELP1 protects HMECs from Tam-induced cell death, independent of Akt and Erk1/2.A, Immunofluorescence of HMEC-hTERT cells stably expressing vector control (pLXSN), PELP1-wild type (wt), or PELP1-cyto. HMEC-hTERT (B) and 240Lp16sMY (D) cell lines stably expressing vector control (pLXSN), PELP1-wild-type (wt), or PELP1-cyto were examined by Western blotting of nuclear (NE) and cytoplasmic (CE) fractions with antibodies against PELP1, HDAC2, and p65., MTT assay of HMEC-hTERT (C) and 240Lp16sMY (E) cell lines expressing pLXSN, PELP1-wt, or PELP1-cyto were treated with 0.5 or 1.0 μM Tam for 3 days. One-way ANOVA was performed to test for statistical differences between cell lines treated with 0.5 μM Tam (HMEC-hTERT) and 0.5 μM and 1.0 μM Tam (240Lp16sMY). ** indicates p < 0.0001.
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pone.0121206.g002: Cytoplasmic PELP1 protects HMECs from Tam-induced cell death, independent of Akt and Erk1/2.A, Immunofluorescence of HMEC-hTERT cells stably expressing vector control (pLXSN), PELP1-wild type (wt), or PELP1-cyto. HMEC-hTERT (B) and 240Lp16sMY (D) cell lines stably expressing vector control (pLXSN), PELP1-wild-type (wt), or PELP1-cyto were examined by Western blotting of nuclear (NE) and cytoplasmic (CE) fractions with antibodies against PELP1, HDAC2, and p65., MTT assay of HMEC-hTERT (C) and 240Lp16sMY (E) cell lines expressing pLXSN, PELP1-wt, or PELP1-cyto were treated with 0.5 or 1.0 μM Tam for 3 days. One-way ANOVA was performed to test for statistical differences between cell lines treated with 0.5 μM Tam (HMEC-hTERT) and 0.5 μM and 1.0 μM Tam (240Lp16sMY). ** indicates p < 0.0001.

Mentions: To determine whether PELP1 localization has an effect on Tam-induced cell death in immortalized HMECs, we established stable cell lines that express vector control (pLXSN), PELP1-wild-type (wt), or PELP1-cyto (NLS mutant). Cells were selected for stable integration of PELP1 with G418. Clonal cell populations were screened for PELP1 localization by immunofluorescence (Fig. 2A) and Western blotting of cytoplasmic and nuclear fractions (Fig. 2B and 2D). Clonal cell lines expressing PELP1-cyto showed increased PELP1 in the cytoplasm compared to PELP1-wt and vector control cell lines. Western blotting for p65 and HDAC2 was performed as controls for cytoplasmic and nuclear fractionation, respectively (Fig. 2B and 2D). Vector control, PELP1-wt, and PELP1-cyto cells were tested for response to Tam by MTT assay (as described in Materials and Methods). HMEC-hTERT and 240Lp16sMY clonal cell lines expressing PELP1-cyto, but not PELP1-wt or vector control, were more resistant to Tam-induced cell death (Fig. 2C and 2E).


Cytoplasmic PELP1 and ERRgamma protect human mammary epithelial cells from Tam-induced cell death.

Girard BJ, Regan Anderson TM, Welch SL, Nicely J, Seewaldt VL, Ostrander JH - PLoS ONE (2015)

Cytoplasmic PELP1 protects HMECs from Tam-induced cell death, independent of Akt and Erk1/2.A, Immunofluorescence of HMEC-hTERT cells stably expressing vector control (pLXSN), PELP1-wild type (wt), or PELP1-cyto. HMEC-hTERT (B) and 240Lp16sMY (D) cell lines stably expressing vector control (pLXSN), PELP1-wild-type (wt), or PELP1-cyto were examined by Western blotting of nuclear (NE) and cytoplasmic (CE) fractions with antibodies against PELP1, HDAC2, and p65., MTT assay of HMEC-hTERT (C) and 240Lp16sMY (E) cell lines expressing pLXSN, PELP1-wt, or PELP1-cyto were treated with 0.5 or 1.0 μM Tam for 3 days. One-way ANOVA was performed to test for statistical differences between cell lines treated with 0.5 μM Tam (HMEC-hTERT) and 0.5 μM and 1.0 μM Tam (240Lp16sMY). ** indicates p < 0.0001.
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pone.0121206.g002: Cytoplasmic PELP1 protects HMECs from Tam-induced cell death, independent of Akt and Erk1/2.A, Immunofluorescence of HMEC-hTERT cells stably expressing vector control (pLXSN), PELP1-wild type (wt), or PELP1-cyto. HMEC-hTERT (B) and 240Lp16sMY (D) cell lines stably expressing vector control (pLXSN), PELP1-wild-type (wt), or PELP1-cyto were examined by Western blotting of nuclear (NE) and cytoplasmic (CE) fractions with antibodies against PELP1, HDAC2, and p65., MTT assay of HMEC-hTERT (C) and 240Lp16sMY (E) cell lines expressing pLXSN, PELP1-wt, or PELP1-cyto were treated with 0.5 or 1.0 μM Tam for 3 days. One-way ANOVA was performed to test for statistical differences between cell lines treated with 0.5 μM Tam (HMEC-hTERT) and 0.5 μM and 1.0 μM Tam (240Lp16sMY). ** indicates p < 0.0001.
Mentions: To determine whether PELP1 localization has an effect on Tam-induced cell death in immortalized HMECs, we established stable cell lines that express vector control (pLXSN), PELP1-wild-type (wt), or PELP1-cyto (NLS mutant). Cells were selected for stable integration of PELP1 with G418. Clonal cell populations were screened for PELP1 localization by immunofluorescence (Fig. 2A) and Western blotting of cytoplasmic and nuclear fractions (Fig. 2B and 2D). Clonal cell lines expressing PELP1-cyto showed increased PELP1 in the cytoplasm compared to PELP1-wt and vector control cell lines. Western blotting for p65 and HDAC2 was performed as controls for cytoplasmic and nuclear fractionation, respectively (Fig. 2B and 2D). Vector control, PELP1-wt, and PELP1-cyto cells were tested for response to Tam by MTT assay (as described in Materials and Methods). HMEC-hTERT and 240Lp16sMY clonal cell lines expressing PELP1-cyto, but not PELP1-wt or vector control, were more resistant to Tam-induced cell death (Fig. 2C and 2E).

Bottom Line: Expression of PELP1-cyto and ERRγ reduced Tam-induced LC3-II accumulation, and knockdown of ERRγ increased LC3-II levels in response to Tam.Our data indicate that cytoplasmic PELP1 induces signaling pathways that converge on ERRγ to promote cell survival in the presence of Tam.These data suggest that PELP1 localization and/or ERRγ activation could be developed as tissue biomarkers for Tam responsiveness.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota, 55455, United States of America.

ABSTRACT
Tamoxifen (Tam) is the only FDA-approved chemoprevention agent for pre-menopausal women at high risk for developing breast cancer. While Tam reduces a woman's risk of developing estrogen receptor positive (ER+) breast cancer, the molecular mechanisms associated with risk reduction are poorly understood. Prior studies have shown that cytoplasmic proline, glutamic acid and leucine rich protein 1 (PELP1) promotes Tam resistance in breast cancer cell lines. Herein, we tested for PELP1 localization in breast epithelial cells from women at high risk for developing breast cancer and found that PELP1 was localized to the cytoplasm in 36% of samples. In vitro, immortalized HMECs expressing a nuclear localization signal (NLS) mutant of PELP1 (PELP1-cyto) were resistant to Tam-induced death. Furthermore, PELP1-cyto signaling through estrogen-related receptor gamma (ERRγ) promoted cell survival in the presence of Tam. Overexpression of ERRγ in immortalized HMECs protected cells from Tam-induced death, while knockdown of ERRγ sensitized PELP1-cyto expressing HMECs to Tam. Moreover, Tam-induced HMEC cell death was independent of apoptosis and involved accumulation of the autophagy marker LC3-II. Expression of PELP1-cyto and ERRγ reduced Tam-induced LC3-II accumulation, and knockdown of ERRγ increased LC3-II levels in response to Tam. Additionally, PELP1-cyto expression led to the upregulation of MMP-3 and MAOB, known PELP1 and ERRγ target genes, respectively. Our data indicate that cytoplasmic PELP1 induces signaling pathways that converge on ERRγ to promote cell survival in the presence of Tam. These data suggest that PELP1 localization and/or ERRγ activation could be developed as tissue biomarkers for Tam responsiveness.

No MeSH data available.


Related in: MedlinePlus