Abstract P3-05-03: A novel progesterone receptor (PR)-RNA polymerase III association represses estrogen-dependent growth in breast tumor patient-derived xenografts
Authors: Finlay-Schultz J , Gillen AE , Brechbuhl HM , Ivie J , Bentley DL , Kabos P , Sartorius CA 
Published: March 2017

Background: Progesterone (P) is an important hormone for development and normal function of the breast; however, its role in established breast cancers is less clear. P has been implicated in regulating tumor cell growth, signaling, differentiation state, and stem/progenitor properties in breast cancer cells. Progesterone receptors (PRs) are considered positive prognostic indicators yet potential targets for treatment, although the dilemma of positive or negative targeting persists. P can either inhibit or stimulate breast cancer cell growth in the absence of estrogens (E), and usually blocks E mediated growth. Recent studies using breast cancer cell lines have deciphered a mechanism by which P suppresses E dependent growth through modulation of the estrogen receptor (ER) cistrome through a physical ER/PR association. However, the ER/PR association is weak to undetectable in our ER+PR+ breast cancer patient-derived xenografts (PDX). We therefore hypothesize that PR can regulate E-dependent breast cancer growth independent of direct interference of ER transcription. The purpose of this study was to use unbiased genomics and proteomics of breast cancer PDX to uncover additional mechanisms of P repression of breast cancer growth.

Methods: These studies used two luminal ER+PR+ PDX (UCD4 and UCD65) that contain high levels of ER and PR (&gt;90%), and where P inhibits E-dependent growth. Tumors were grown in vivo in female NSG mice under continuous placebo, E (17b-estradiol), E plus P, or E plus the synthetic progestin medroxyprogesterone acetate (MPA) for 8-10 weeks. RNAseq, chromatin immunoprecipitation sequencing (ChIPseq), and rapid immunoprecipitation followed by mass spectrometry of endogenous proteins (RIME) were performed to analyze differential transcriptional, cistromic, and protein-protein interactions in E compared to E plus P or MPA treated tumors. Co-immunoprecipitation (co-IP) and ChIP were used to verify results.

Results: Both P and MPA potently inhibited E-dependent growth of both tumor lines. Gene expression studies found that both hormones reversed transcription of over one third of the estrogen/ER transcriptome in both tumors. RIME for PR uncovered significant interactions between PR and multiple RNA polymerase III subunits (POLR3). Co-IP using POLR3A and POLR3B confirmed PR associates with the POLR3 holoenzyme. Furthermore, ChIPseq revealed that PR binds to one third of POLR3 regulated tRNA genes. PR also associated with Maf1 in one tumor, a known POLR3 suppressor.

Conclusions: Here we describe a novel association of PR with POLR3 in two luminal breast cancer PDX, an interaction not described in breast cancer cell lines. Our data suggest this is a negative regulatory interaction that may occur through recruiting a POLR3 repressor. These data implicate multifaceted P control of E dependent tumor growth; in addition to antagonizing E regulated genes at the transcription level, P can regulate translation though depletion of amino acid carrying tRNAs, thus slowing protein synthesis. Identifying which tumors utilize this growth-suppressive mechanism may pinpoint appropriate candidates for progestin therapy and/or provide a prognostic tool for predicting tumor progression.

Citation Format: Finlay-Schultz J, Gillen AE, Brechbuhl HM, Ivie J, Bentley DL, Kabos P, Sartorius CA. A novel progesterone receptor (PR)-RNA polymerase III association represses estrogen-dependent growth in breast tumor patient-derived xenografts [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-05-03.