Regulation of Super Enhancers by Phosphorylated Progesterone Receptors Drives Breast Cancer Stem Cell Expansion

Current treatments for estrogen receptor (ER) positive breast cancer largely target rapidly dividing tumor cells. However, aggressive breast cancers often contain long-lived and weakly proliferative cancer stem cells (CSCs) that readily escape treatments aimed at cycling cells. Furthermore, up to 40% of endocrine responsive ER+ patients experience latent recurrence, often after 5 years to decades after completion of their ER-targeted therapies. We previously reported that progesterone receptors (PR) and their target genes aid the growth of CSCs and help them evade anti-estrogen therapy. As proliferation and stemness are directed by separate signaling pathways that lead to the expression of distinct gene programs, it's crucial to understand how PR facilitates this cellular transformation at the genomic level. Establishing a gene expression program that may limit proliferation but encourages stemness requires coordination between transcription factors and their coregulators to alter the nuclear microenvironment and chromatin landscape.

To reveal these mechanisms, we performed integrated analysis of RNA-seq data and CUT&RUN genomic binding data for PR, ER, CTCF and H3K27ac inT47D luminal breast cancer cells grown in ultra-low attachment (3D) mammosphere conditions. We used machine learning to carry out a logistic regression analysis of our RNA-seq data with backward elimination to generate a high-confidence PR-induced gene signature that is predictive of poor long-term survival (<10 years) for patients in the METABRIC cohort, suggesting that tracking PR activity is highly relevant to outcomes for patients with late recurrence. Our analysis of genomic binding profiles displayed a pattern of PR/ER co-occupancy at distant sites more than 10 kilobases away from the nearest transcriptional start site. We used Rank Ordering of Super Enhancer (ROSE) analysis on these PR/ER binding sites to predict which are likely super enhancer elements capable of coordinating the regulation of multi-gene subsets. We tested the function of novel enhancer sites linked to PR-target genes by disrupting the PR/ER binding with a dCas9-KRAB construct directed to the predicted enhancer elements. We propose that abnormal growth factor signaling in breast cancer cells permits PR/ER complex formation which promotes expression of genes that induce endocrine resistance and CSC expansion through regulation of super enhancer elements. Understanding the intricacies of PR/ER crosstalk is the crucial next step in developing new therapies that target PR-driven processes in ER+ breast cancers.

Date of Presentation October 17, 2024