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Context Positron emission tomography imaging with 2-deoxy-2-[18F]-fluoro-D-glucose (FDG) is used clinically for initial staging, restaging, and assessing therapy response in breast cancer. Tumor FDG uptake in steroid hormone receptor–positive breast cancer and physiologic FDG uptake in normal breast tissue can be affected by hormonal factors such as menstrual cycle phase, menopausal status, and hormone replacement therapy. Objective The purpose of this study was to determine the role of the progesterone receptor (PR) in regulating glucose and FDG uptake in breast cancer cells. Methods and Results PR-positive T47D breast cancer cells treated with PR agonists had increased FDG uptake compared with ethanol control. There was no significant change in FDG uptake in response to PR agonists in PR-negative MDA-MB-231 cells, MDA-MB-468 cells, or T47D PR knockout cells. Treatment of T47D cells with PR antagonists inhibited the effect of R5020 on FDG uptake. Using T47D cell lines that only express either the PR-A or the PR-B isoform, PR agonists increased FDG uptake in both cell types. Experiments using actinomycin D and cycloheximide demonstrated the requirement for both transcription and translation in PR regulation of FDG uptake. GLUT1 and PFKFB3 mRNA expression and the enzymatic activity of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were increased after progestin treatment of T47D cells. Conclusion Thus, progesterone and progestins increase FDG uptake in T47D breast cancer cells through the classical action of PR as a ligand-activated transcription factor. Ligand-activated PR ultimately increases expression and activity of proteins involved in glucose uptake, glycolysis, and the pentose phosphate pathway.

Positron emission tomography imaging with 2-deoxy-2-[The purpose of this study was to determine the role of the progesterone receptor (PR) in regulating glucose and FDG uptake in breast cancer cells.PR-positive T47D breast cancer cells treated with PR agonists had increased FDG uptake compared with ethanol control. There was no significant change in FDG uptake in response to PR agonists in PR-negative MDA-MB-231 cells, MDA-MB-468 cells, or T47D PR knockout cells. Treatment of T47D cells with PR antagonists inhibited the effect of R5020 on FDG uptake. Using T47D cell lines that only express either the PR-A or the PR-B isoform, PR agonists increased FDG uptake in both cell types. Experiments using actinomycin D and cycloheximide demonstrated the requirement for both transcription and translation in PR regulation of FDG uptake.Thus, progesterone and progestins increase FDG uptake in T47D breast cancer cells through the classical action of PR as a ligand-activated transcription factor. Ligand-activated PR ultimately increases expression and activity of proteins involved in glucose uptake, glycolysis, and the pentose phosphate pathway.

Prolactin (PRL) and estrogen cooperate in lobuloalveolar development of the mammary gland and jointly regulate gene expression in breast cancer cells in vitro. Canonical PRL signaling activates STAT5A/B, homologous proteins that have different target genes and functions. Although STAT5A/B are important for physiological mammary function and tumor pathophysiology, little is known about regulation of their expression, particularly of STAT5B, and the consequences for hormone action. In this study, we examined the effect of two estrogenic ligands, 17β-estradiol (E2) and the clinical antiestrogen, ICI182,780 (ICI, fulvestrant) on expression of STAT5 isoforms and resulting crosstalk with PRL in normal and tumor murine mammary epithelial cell lines. In all cell lines, E2 and ICI significantly increased protein and corresponding nascent and mature transcripts for STAT5A and STAT5B, respectively. Transcriptional regulation of STAT5A and STAT5B by E2 and ICI, respectively, is associated with recruitment of estrogen receptor alpha and increased H3K27Ac at a common intronic enhancer 10 kb downstream of the Stat5a transcription start site. Further, E2 and ICI induced different transcripts associated with differentiation and tumor behavior. In tumor cells, E2 also significantly increased proliferation, invasion, and stem cell-like activity, whereas ICI had no effect. To evaluate the role of STAT5B in these responses, we reduced STAT5B expression using short hairpin (sh) RNA. shSTAT5B blocked ICI-induced transcripts associated with metastasis and the epithelial mesenchymal transition in both cell types. shSTAT5B also blocked E2-induced invasion of tumor epithelium without altering E2-induced transcripts. Together, these studies indicate that STAT5B mediates a subset of protumorigenic responses to both E2 and ICI, underscoring the need to understand regulation of its expression and suggesting exploration as a possible therapeutic target in breast cancer., STAT5B expression is regulated by ICI and mediates a subset of protumorigenic responses to ER ligands.

In estrogen receptor (ER) positive breast cancers, there is a continual balance between growth and differentiation. Upon activation with estrogen, ER is phosphorylated at serine 118 (pS118-ER). High pS118-ER expression has been observed in low grade and well differentiated tumors. However, this phospho-variant presents a paradox as high expression has also been detected in therapy resistant metastatic tumors. Recently our lab conducted a comprehensive genome wide analysis of ER and pS118-ER through a ChIP-seq study. DNA motif analysis revealed enrichment of the GRHL2 motif near pS118-ER occupancy sites over ER occupancy sites. GRHL2 is a transcription factor that has also been implicated in epithelial differentiation and has been shown to have roles as both a tumor suppressor and oncogene in breast cancer. However, its role in facilitating pS118-ER transcriptional activity is unclear. In these studies, we examine the role of GRHL2 in pS118-ER occupancy through conventional ChIP at GRHL2/pS118-ER co-occupancy sites. GRHL2 occupancy can increase upon estrogen treatment at sites where pS118-ER is also present, but that increase has not been seen at sites lacking pS118-ER occupancy. Using a CRISPR knockout of GRHL2, the role of GRHL2 in pS118-ER DNA binding is being investigated further. In addition, we are using luminal cultures to model breast ducts and investigate the role of GRHL2 in breast cancer epithelial differentiation. These lumens maintain their estrogen responsiveness and GRHL2 expression, which will allow for the study of GRHL2 in a biologically relevant structure. These studies provide the foundation for understanding GRHL2 function in breast cancer.

Post-translational modifications are key regulators of protein function, providing signals that alter protein interactions and activity. Phosphorylation of estrogen receptor-α (ER) at serine 118 (pS118-ER) occurs in response to multiple stimuli and is involved in modulating ER-dependent gene transcription. While the effects of pS118 on ER-DNA interactions have been investigated at specific genomic sites, the genome-wide binding profile (cistrome) of pS118-ER remains to be studied. To determine and characterize the pS118-ER cistrome, chromatin immunoprecipitation sequencing (ChIP-seq) was performed on both pS118-ER and ER and their binding profiles were compared. A subset of ER sites was found to be occupied by pS118-ER and although these sites were not found to be enriched in promoter regions relative to all ER sites, the pS118-ER sites were associated with the active enhancer mark H3K27ac as well as genes upregulated, but not downregulated, by estrogen. Additionally, pS118-ER sites were found to be enriched in the DNA binding motif for GRHL2 as well as the estrogen response element (ERE) relative to all ER sites. Utilizing a DNA binding microarray, pS118-ER was found to be more commonly associated with direct DNA binding events compared to indirect binding events. These results suggest a role for pS118-ER at active enhancers and as a regulator of direct ER-DNA interactions. Prevention of this phosphorylation event could serve as a potential treatment for ER-positive breast cancers by reducing the transcriptional activity of ER and decreasing its occupancy on DNA.

The Grainyhead-like protein family, composed of GRHL1, GRHL2, and GRHL3, are nuclear transcription factors that regulate epithelial differentiation. GRHL2 has been associated with several nuclear hormone receptors, including progesterone receptor (PR), androgen receptor (AR), and more recently, estrogen receptor (ER). In breast cancer, GRHL2 has been shown to both activate ER-dependent enhancers through FOXA1 and MLL3-mediated deposition of the activating histone mark H3K4me1 and repress enhancers via inhibition of the histone acetyltransferase p300. Cistromic analysis by our group of ER phosphorylated at serine 118 (pS118-ER), a form of transcriptionally active ER, found an enrichment of the GRHL motif near pS118-ER binding sites. Despite these findings, the direct relationship between GRHL2 and ER transcriptional function and how that relationship influences ER-positive breast cancer growth and differentiation is not well-defined. To explore the relationship between GRHL2 and pS118-ER further, we used transcriptomic and cistromic analysis of ER-positive cells lacking GRHL2 to determine the impact of the loss of GRHL2 on cellular and transcriptional responses to estrogen. This analysis identified a subset of genes that are controlled by both GRHL2 and estrogen. In addition, CRISPR engineered T47D cells lacking a portion of the GRHL2 transactivation domain (ΔTAD) demonstrate reduced nuclear ER levels and reduced ER chromatin occupancy. Gene expression analysis of ΔTAD-GRHL2 cells showed increased GRHL3 expression, and ChIP analysis revealed increased Pol II occupancy at the GRHL3 promoter, suggesting that there may be a compensatory mechanism within the GRHL family to regulate the transcriptome. Finally, ΔTAD-GRHL2 mutants reduced growth and colony formation relative to wild-type controls. Together, this work will provide an understanding of how transcriptionally active ER and GRHL2 selectively cooperate to regulate transcription, growth, and differentiation in ER-positive breast cancer.