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| Status |
Public on Apr 10, 2017 |
| Title |
Posttranslationally modified progesterone receptors direct ligand-specific expression of breast cancer stem cell-associated gene programs |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by array
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| Summary |
Background
Estrogen and progesterone are potent breast mitogens. In addition to steroid hormones, multiple signaling pathways input to estrogen receptor (ER) and progesterone receptor (PR) actions via posttranslational events. Protein kinases commonly activated in breast cancers phosphorylate steroid hormone receptors (SRs) and profoundly impact their activities.
Methods
To better understand the role of modified PRs in breast cancer, we measured total and phospho-Ser294 PRs in 209 human breast tumors represented on 2754 individual tissue spots within a tissue microarray and assayed the regulation of this site in human tumor explants cultured ex vivo. To complement this analysis, we assayed PR target gene regulation in T47D luminal breast cancer models following treatment with progestin (promegestone; R5020) and antiprogestins (mifepristone, onapristone, or aglepristone) in conditions under which the receptor is regulated by Lys388 SUMOylation (K388 intact) or is SUMO-deficient (via K388R mutation to mimic persistent Ser294 phosphorylation). Selected phospho-PR-driven target genes were validated by qRT-PCR and following RUNX2 shRNA knockdown in breast cancer cell lines. Primary and secondary mammosphere assays were performed to implicate phospho-Ser294 PRs, epidermal growth factor signaling, and RUNX2 in breast cancer stem cell biology.
Results
Phospho-Ser294 PR species were abundant in a majority (54%) of luminal breast tumors, and PR promoter selectivity was exquisitely sensitive to posttranslational modifications. Phospho-PR expression and target gene programs were significantly associated with invasive lobular carcinoma (ILC). Consistent with our finding that activated phospho-PRs undergo rapid ligand-dependent turnover, unique phospho-PR gene signatures were most prevalent in breast tumors clinically designated as PR-low to PR-null (luminal B) and included gene sets associated with cancer stem cell biology (HER2, PAX2, AHR, AR, RUNX). Validation studies demonstrated a requirement for RUNX2 in the regulation of selected phospho-PR target genes (SLC37A2). In vitro mammosphere formation assays support a role for phospho-Ser294-PRs via growth factor (EGF) signaling as well as RUNX2 as potent drivers of breast cancer stem cell fate.
Conclusions
We conclude that PR Ser294 phosphorylation is a common event in breast cancer progression that is required to maintain breast cancer stem cell fate, in part via cooperation with growth factor-initiated signaling pathways and key phospho-PR target genes including SLC37A2 and RUNX2. Clinical measurement of phosphorylated PRs should be considered a useful marker of breast tumor stem cell potential. Alternatively, unique phospho-PR target gene sets may provide useful tools with which to identify patients likely to respond to selective PR modulators that block PR Ser294 phosphorylation as part of rational combination (i.e., with antiestrogens) endocrine therapies designed to durably block breast cancer recurrence.
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| Overall design |
The study contains 3 different cell lines (PR-null, PRB-wildtype, PRB-K388R) tested with 8 different treatments in triplicate (8 x 3 x 3 = 72 individual samples). We also included 12 previously published samples (GSE34148) with the same treatments, which increased our total number of samples to 84. From parental T47D-Y (PR-null) human breast cancer cell lines, we created stable clones expressing either (1) an empty vector (pIRESneo3) or (2) the wild type progesterone receptor isoform B (pIRESneo3-PR-B), or (3) pIRESneo3-PR-B-K388R. These three cell lines were co-treated with either (1) vehicle control (ethanol) or (2) progesterone/R5020 10e-8 M, (3) RU486 10e-7 M, (4) onapristone 10e-7 M, (5) aglepristone 10e-7 M, (6) progesterone plus RU486, (7) progesterone plus onapristone, or (8) progesterone plus aglepristone. Treatments were for 6 hours before total RNA harvest. Standard Illumina HT-12v4 chip was used for gene expression analysis.
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| Web link |
https://jhoonline.biomedcentral.com/articles/10.1186/s13045-017-0462-7
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| Contributor(s) |
Knutson TP, Lange CA |
| Citation(s) |
28412963 |
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| Submission date |
Feb 01, 2017 |
| Last update date |
Aug 13, 2018 |
| Contact name |
Todd P Knutson |
| E-mail(s) |
knut0297@umn.edu
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| Phone |
612-626-8911
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| Organization name |
University of Minnesota
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| Department |
Minnesota Supercomputing Institute
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| Street address |
117 Pleasant St SE
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| City |
Minneapolis |
| State/province |
MN |
| ZIP/Postal code |
55455 |
| Country |
USA |
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| Platforms (1) |
| GPL10558 |
Illumina HumanHT-12 V4.0 expression beadchip |
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| Samples (84)
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| Relations |
| BioProject |
PRJNA369476 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE94363_RAW.tar |
26.2 Mb |
(http)(custom) |
TAR |
| GSE94363_non-normalized_matrix.txt.gz |
23.1 Mb |
(ftp)(http) |
TXT |
| GSE94363_normalized_gene-level_matrix.txt.gz |
11.1 Mb |
(ftp)(http) |
TXT |
| GSE94363_normalized_probe-level-matrix.txt.gz |
16.1 Mb |
(ftp)(http) |
TXT |
| Processed data included within Sample table |
| Processed data are available on Series record |
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