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. 2019 Feb 1;160(2):430-446.
doi: 10.1210/en.2018-00990.

Phosphorylated Progesterone Receptor Isoforms Mediate Opposing Stem Cell and Proliferative Breast Cancer Cell Fates

Thu H Truong  1 , Amy R Dwyer  1 , Caroline H Diep  1 , Hsiangyu Hu  1 , Kyla M Hagen  1 , Carol A Lange  1   2   3
Affiliations

Phosphorylated Progesterone Receptor Isoforms Mediate Opposing Stem Cell and Proliferative Breast Cancer Cell Fates

Thu H Truong et al. Endocrinology. .

Abstract

Progesterone receptors (PRs) are key modifiers of estrogen receptor (ER) target genes and drivers of luminal breast cancer progression. Total PR expression, rather than isoform-specific PR expression, is measured in breast tumors as an indicator of functional ER. We identified phenotypic differences between PR-A and PR-B in luminal breast cancer models with a focus on tumorsphere biology. Our findings indicated that PR-A is a dominant driver of cancer stem cell (CSC) expansion in T47D models, and PR-B is a potent driver of anchorage-independent proliferation. PR-A+ tumorspheres were enriched for aldehyde dehydrogenase (ALDH) activity, CD44+/CD24-, and CD49f+/CD24- cell populations relative to PR-B+ tumorspheres. Progestin promoted heightened expression of known CSC-associated target genes in PR-A+ but not PR-B+ cells cultured as tumorspheres. We report robust phosphorylation of PR-A relative to PR-B Ser294 and found that this residue is required for PR-A-induced expression of CSC-associated genes and CSC behavior. Cells expressing PR-A S294A exhibited impaired CSC phenotypes but heightened anchorage-independent cell proliferation. The PR target gene and coactivator, FOXO1, promoted PR phosphorylation and tumorsphere formation. The FOXO1 inhibitor (AS1842856) alone or combined with onapristone (PR antagonist), blunted phosphorylated PR, and tumorsphere formation in PR-A+ and PR-B+ T47D, MCF7, and BT474 models. Our data revealed unique isoform-specific functions of phosphorylated PRs as modulators of distinct and opposing pathways relevant to mechanisms of late recurrence. A clear understanding of PR isoforms, phosphorylation events, and the role of cofactors could lead to novel biomarkers of advanced tumor behavior and reveal new approaches to pharmacologically target CSCs in luminal breast cancer.

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Figures

Figure 1.
Figure 1.
PR expression promotes tumorsphere formation in breast cancer cells. (A) Primary tumorsphere assays in MCF-7 cells. (B) Primary tumorsphere assays in BT-474 cells. Cells were treated with vehicle (veh; EtOH), R5020 (10 nM), E2 (1 nM), RU486 (100 nM), or onapristone (ona; 100 nM). (C) Tumorsphere assays in T47D-Y (PR-null) and T47D CO cells treated with vehicle (EtOH) or the indicated hormone treatment (R5020, 10 nM; P4, 10 nM; or E2, 1 nM). Representative images shown. (D) Primary and (E) secondary tumorspheres in T47D CO cells. (F) Primary and (G) secondary tumorsphere assays in T47D-Y cells. T47D-Y cells were pretreated with E2 (1 nM) for 48 hours, followed by tumorsphere assays with vehicle (EtOH) or the indicated hormone treatment. (F, Right Inset) mRNA levels of PR from primary tumorspheres in T47D-Y cells pretreated with E2. (H) Secondary tumorsphere assays in T47D-inducible EV control and PR-B cells. PR expression was induced within 24 hours after addition of AP21967 (1 nM); Right, Western blot. Graphed data represent the mean ± SD (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001.
Figure 2.
Figure 2.
PR isoforms have distinct phenotypes in breast cancer cells. (A) Primary and (B) secondary tumorsphere assays in T47D-YA and T47D-YB cells. Cells were treated with vehicle (veh; EtOH), R5020 (10 nM), P4 (10 nM), or E2 (1 nM). (C) Representative images of T47D (YA and YB) primary tumorspheres. (D) Average size of T47D (YA and YB) primary tumorspheres. (E) Primary tumorsphere assays in T47D-YA and T47D-YB short hairpin green fluorescent protein (shGFP) control or short hairpin PR (shPR) knockdown cells. (F) Primary and (G) secondary tumorsphere assays in T47D-YA and T47D-YB cells. Cells treated with vehicle (water) or EGF (20 ng/mL). (H) Soft agar colony formation and (I) average colony size in T47D-YA and T47D-YB cells were assessed in the presence of vehicle (EtOH) or the indicated hormone treatments (R5020, 10 nM; P4, 10 nM; or E2, 1 nM). Graphed data represent the mean ± SD (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001.
Figure 3.
Figure 3.
PR-A expression enhanced breast CSC populations and target gene expression. (A) ALDH+ activity in T47D (Y, YA, YB) tumorspheres was assessed using flow cytometry. (B) CD44+/CD24− populations from T47D (Y, YA, YB) tumorspheres. (C) CD49f+/CD24− populations from T47D-YA and T47D-YB tumorspheres. (D) mRNA levels of select genes (e.g., PTGES, p21, FOXO1, WNT4, KLF4, NOTCH2) in T47D-YA and T47D-YB cells. Cells were cultured in tumorsphere conditions and treated with vehicle (veh), R5020 (10 nM), or RU486 (100 nM). (E) ChIP assays showing PR recruitment to a PRE-containing region of the WNT4, KLF4, or NOTCH2 promoter. T47D-YA and T47D-YB cells were stimulated with vehicle (EtOH) or R5020 (10 nM) for 1 hour. Fixed lysates were subjected to ChIP assays using specific antibodies targeting PR (or IgG control). Graphed data represent the mean ± SD (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001.
Figure 4.
Figure 4.
PR isoforms are phosphorylated at S294. (A) Western blot of phosphorylated and total PR in T47D cells (CO, YA, YB). Cells were treated with vehicle (veh; EtOH) or R5020 (10 nM) for 60 minutes. Phosphorylated PR (pPR; S294) antibodies were used from the Lange laboratory [polyclonal antibody (pAb)] and the Edwards laboratory [monoclonal antibody (mAB)]. (B) Western blot of phosphorylated and total PR in a panel of luminal breast cancer cell lines. Cells were pretreated with E2 for 48 hours, followed by R5020 (10 nM) for 1 hour or EGF (30 ng/mL) for 15 minutes. (C) Western blot of phosphorylated and total PR and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; loading control) in T47D-YA and T47D-YB cells. The cells were treated with vehicle (veh; EtOH) or R5020 (10 nM) for the indicated times. (D) Western blot of phosphorylated and total PR and GAPDH (loading control) in T47D-YA and T47D-YB cells. The cells were cultured in tumorsphere conditions (i.e., suspension) and treated with vehicle (EtOH) or R5020 (10 nM). (E) ChIP assays showing phosphorylated PR (S294) recruitment to PRE-containing regions of the WNT4, KLF4, or NOTCH2 promoter. T47D-YA and T47D-YB cells were stimulated with vehicle (EtOH) or R5020 (10 nM) for 1 hour. Fixed lysates were subjected to ChIP assays using specific antibodies targeting phosphorylated PR (S294) or IgG control. Graphed data represent the mean ± SD (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001.
Figure 5.
Figure 5.
Ser294 contributes to PR-A–induced breast CSC phenotypes. (A) Primary tumorspheres from T47D (EV, WT PR-A, and S294A PR-A) cells. (B) ALDH activity in T47D (EV, WT PR-A, and S294A PR-A) primary tumorspheres. (C) Soft agar colony formation of T47D (EV, WT PR-A, and S294A PR-A) cells. (D) CD44+/CD24− populations in T47D (EV, WT PR-A, and S294A PR-A) primary tumorspheres. (E) mRNA levels of select genes (e.g.,PTGES, p21, FOXO1, WNT4, KLF4, NOTCH2) in T47D (EV, WT PR-A, and S294A PR-A) cells cultured in tumorsphere conditions. Cells were treated with vehicle (veh; EtOH) or R5020 (10 nM). (F) ChIP assays showing phosphorylated PR and (G) total PR recruitment to PRE-containing regions of the WNT4, KLF4, or NOTCH2 promoter. T47D (WT PR-A and S294 PR-A) cells were stimulated with vehicle (EtOH) or R5020 (10 nM) for 1 hour. Fixed lysates were subjected to ChIP assays using specific antibodies targeting phosphorylated PR (S294) or IgG control. Graphed data represent the mean ± SD (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001. Avg, average.
Figure 6.
Figure 6.
FOXO1 promoted breast CSC outgrowth in PR isoforms. (A) Western blot of FOXO1 (phosphorylated and total) and Akt (phosphorylated and total) in HeLa cells transfected with green fluorescent protein–tagged PR-A and with pcDNA FOXO1. Cells were treated with vehicle (veh; EtOH) or R5020 (10 nM) for the indicated times. shFOXO1 was included as a control. (B) Primary tumorsphere assays in T47D-YA and T47D-YB cells with FOXO1 knockdown. (C) Primary tumorsphere assays in T47D (YA and YB) cells stably expressing FOXO1. (D) ALDH activity in primary tumorspheres formed from T47D (YA and YB) FOXO1+ cells was assessed by flow cytometry. (E) Secondary tumorspheres in T47D (YA and YB) cells expressing WT FOXO1 or FOXO1-AAA. (F) Secondary tumorspheres in T47D (YA and YB) FOXO1+ cells treated with AS1842856 (100 nM). (Right) T47D (YA and YB) cells expressing FOXO1 were pretreated with AS1842856 (100 nM) for 60 minutes, followed by the indicated treatments with R5020 (10 nM). (G) Secondary tumorspheres in T47D (YA and YB) cells treated with AS1842856. (Right) T47D (YA and YB) cells were pretreated with AS1842856 (100 nM) for 60 minutes, followed by treatment with R5020 (10 nM). (H) MCF-7 and BT-474 primary tumorspheres treated with E2 (1 nM) or R5020 (10 nM), respectively, in the presence of onapristone (100 nM) or AS1842856 (100 nM), or both. Graphed data represent the mean ± SD (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001. Avg, average.
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