Progesterone and Estrogen Signaling Impact Each Other’s Transcriptional Activity in Endometrial Cancer

Endometrial cancer (EC) is the most common gynecologic cancer in the United States and is increasing in incidence. The most common form of EC is hormonally driven by excess estrogens unopposed by progesterone. Despite the use of progestins (synthetic progesterone) in early-stage fertility-sparing management of EC, the molecular action of progesterone receptor (PR) in suppressing EC remains poorly understood. To dissect the actions of PR and its relationship to estrogen signaling through estrogen receptor alpha (ER) in EC, we have engineered two EC models, Ishikawa and HCI-EC-23, to selectively express PR isoforms (PR-A or PR-B) or LacZ (control) in a doxycycline-inducible manner. Proliferation assays validated the utility of our models by demonstrating growth suppression by progesterone (P4) or progestins only when PR-A or PR-B is expressed. Importantly, PR-B expression had more growth suppression than PR-A in both models.

To identify the transcriptional targets of ER and PR, we performed RNA-seq in our models after treatment with E2, P4, combination E2 and P4, or vehicle. In Ishikawa cells, we identified hundreds of differentially expressed genes (DEGs) across all models and treatments. P4 induction led to hundreds of DEGs in PR-A or PR-B expressing cells, but not LacZ expressing cells, with the PR-B expressing cells having more DEGs. Nearly half of all P4 regulated genes were shared by both PR isoforms. Unexpectedly, the expression of unliganded PR (cells treated with E2 but not P4) expanded the number of E2-regulated genes. Expanded E2 DEGs common to both PR isoforms were enriched for terms relating to MYC targets. However, treatment with the combination of E2 and P4 significantly reduced the expression of E2 regulated genes, including MYC, which was more pronounced with PR-B expression. These results indicate that liganded PR opposes ER’s capacity to regulate transcription, which would explain progesterone’s ability to reduce EC growth. Preliminary ChIP-seq of ER and PR indicates that PR activation impacts ER genomic binding sites, suggesting that PR directly alters ER’s transcriptional targets.

To determine if similar gene expression changes are observed in patient samples, we performed RNA-seq on a cohort of 31 EC and endometrial hyperplasia patients who underwent progestin therapy. We found that genes upregulated in the patient cohort after successful progestin therapy significantly overlapped both PR-A and PR-B P4 DEGs, supporting the generalizability of the PR target genes in our EC models. Further, pre-treatment samples from responders exhibited a stronger estrogen driven gene expression signature than non-responders, which may prove to be a useful biomarker. By understanding how these two key steroid hormone receptors impact each other, we hope to improve the use of hormone therapy in EC patients.

Date of Presentation October 17, 2024