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

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

Knockdown of ERRγ regulates PELP1-cyto genes.

<p>MAOB (<b>A</b>) and MMP-3(<b>B</b>) were measured by qRT-PCR in HMECs expressing pLXSN or PELP1-cyto and shControl or shERRγ. A student’s T-Test was performed to determine statistical significance for qRT-PCR experiments; p-values are shown for the indicated comparisons.</p

Cytoplasmic PELP1 protects HMECs from Tam-induced cell death, independent of Akt and Erk1/2.

<p><b>A</b>, Immunofluorescence of HMEC-hTERT cells stably expressing vector control (pLXSN), PELP1-wild type (wt), or PELP1-cyto. HMEC-hTERT (<b>B</b>) and 240Lp16sMY (<b>D</b>) 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 (<b>C</b>) and 240Lp16sMY (<b>E</b>) 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.</p

Human mammary epithelial cell lines are sensitive to Tam independent of ER expression.

<p><b>A</b>, MTT assay of mammary epithelial cell lines treated with increasing concentrations of Tam for 3 days. MTT assay of HMEC-hTERT (<b>B</b>) and MCF-7 (<b>C</b>) cell lines treated with increasing concentrations of either Tam or 4-OH-Tam for 3 days. <b>D</b>, qRT-PCR of ESR1 and TATA-binding protein 2 (TBP2) (internal control) mRNA levels in three mammary epithelial cell lines and MCF-7 cells. HMEC-hTERT and MCF-7 cells were treated with ethanol (control), 10 nM E2, 10 nM 4-OH-Tam, or 0.5 μM Tam for 24 hours. ER target genes pS2 (<b>E</b>) and Krt13 (<b>F</b>) were measured by qRT-PCR and averaged over values for glyceraldehyde-3-phosphate dehydrogenase (<i>GAPDH</i>) (internal control). A student’s T-Test was performed to determine statistical significance for qRT-PCR data. P-values are given for the indicated comparisons. A-D) Oneway ANOVA was performed to determine statistical significance of presented data. ** indicates p < 0.001.</p

PELP1-cyto does not activate Akt and Erk1/2 to promote cell survival of HMECs in the presence of Tam.

<p><b>A</b>, Western blotting for phospho-Akt and phospho-Erk1/2 in HMEC-hTERT and MCF-7 cells. Actin served as a loading control. <b>B</b> and <b>C</b>, MTT assay of HMECs expressing pLXSN or PELP1-cyto were treated with increasing concentrations of the Akt inhibitor LY294002 (LY) (<b>B</b>) or the MAPK inhibitor UO126 (UO)(<b>C</b>). The vehicle for LY and UO was DMSO.</p

Knockdown and overexpression of ERRγ alters response to Tam in HMECs.

<p><b>A</b>, qRT-PCR for ERRγ mRNA to verify knockdown in shControl and shERRγ stable cell lines. <b>B</b>, MTT assay of MCF-10A cell lines expressing either shRNA control (shControl) or shRNA against ERRγ (shERRγ). Cells were treated with 0.5 or 1.0 μM Tam for 3 days. <b>C</b>, qRT-PCR for ERRγ mRNA to verify knockdown in HMEC-LXSN and HMEC-Cyto cell lines stably expressing either shControl or shERRγ. <b>D</b>, MTT assay of HMEC-LXSN and HMEC-Cyto expressing either shControl or shERRγ. Cells were treated with increasing concentrations of Tam for 3 days. <b>E</b>, MTT assay of MCF-10A cell lines expressing vector control or ERRγ. Cells were treated with increasing concentrations of Tam. <b>F</b>, MTT assay of HMEC-hTERT cells stably expressing vector control or ERRγ. Cells were treated with increasing concentrations of Tam for 3 days. ERRγ overexpression in MCF-10A (<b>G</b>) and HMEC-hTERT (<b>H</b>) cell lines was verified by qRT-PCR of ERRγ. One-way ANOVA was performed to test for statistical differences between cell lines treated with Tam in MTT assays. Student’s T-test was performed to determine statistical significance for qRT-PCR experiments. P-values are shown for the indicated comparisons. ** indicates p<0.0001.</p

PELP1/SRC-3-dependent regulation of metabolic PFKFB kinases drives therapy resistant ER+ breast cancer.

Recurrence of metastatic breast cancer stemming from acquired endocrine and chemotherapy resistance remains a health burden for women with luminal (ER+) breast cancer. Disseminated ER+ tumor cells can remain viable but quiescent for years to decades. Contributing factors to metastatic spread include the maintenance and expansion of breast cancer stem cells (CSCs). Breast CSCs frequently exist as a minority population in therapy resistant tumors. In this study, we show that cytoplasmic complexes composed of steroid receptor (SR) co-activators, PELP1 and SRC-3, modulate breast CSC expansion through upregulation of the HIF-activated metabolic target genes PFKFB3 and PFKFB4. Seahorse metabolic assays demonstrated that cytoplasmic PELP1 influences cellular metabolism by increasing both glycolysis and mitochondrial respiration. PELP1 interacts with PFKFB3 and PFKFB4 proteins, and inhibition of PFKFB3 and PFKFB4 kinase activity blocks PELP1-induced tumorspheres and protein-protein interactions with SRC-3. PFKFB4 knockdown inhibited in vivo emergence of circulating tumor cell (CTC) populations in mammary intraductal (MIND) models. Application of PFKFB inhibitors in combination with ER targeted therapies blocked tumorsphere formation in multiple models of advanced breast cancer including tamoxifen (TamR) and paclitaxel (TaxR) resistant models, murine tumor cells, and ER+ patient-derived organoids (PDxO). Together, our data suggest that PELP1, SRC-3, and PFKFBs cooperate to drive ER+ tumor cell populations that include CSCs and CTCs. Identifying non-ER pharmacological targets offers a useful approach to blocking metastatic escape from standard of care ER/estrogen (E2)-targeted strategies to overcome endocrine and chemotherapy resistance