Direct Regulation of Estrogen Receptor Transcriptional Activity by NF1
Abstract
Background: The vast majority of breast cancers express the estrogen receptor-alpha (ER). Although great strides have been made in treating these ER+ tumors, relapse and death are common and are closely linked to resistance to ER-targeting endocrine therapies. To seek drivers for endocrine therapy resistance, we sequenced NF1 in >600 ER+ primary breast tumors from patients treated with 5 years of tamoxifen monotherapy (no chemo). Our preliminary results have revealed that truncating mutations in the NF1 (Neurofibromin 1) gene were strongly associated with poor outcome (Hazard Ratio approximately equal to 3). Our follow-up functional studies have demonstrated that NF1-deficiency can induce tamoxifen resistance and aggressive tumor behavior in vitro. In fact, tamoxifen acts as an agonist to stimulate the growth of NF1-silenced cells. Furthermore, these NF1-silenced cells also become hypersensitive to very low levels of estrogen, which do not support the growth of wild-type NF1 cells. Thus NF1-deficient tumors are likely to be aromatase inhibitor resistant as well. NF1 is already known to downregulate Ras activity, so that loss of this function leads to hyperactive Ras pathways. However, this large and highly conserved protein is likely to have other functions as well, since many cancer-borne NF1 mutants have an intact GAP domain. Following the lead that NF1-silenced cells are hypersensitive to estrogen, we searched mass spectrometry (MS) databases for ER and coregulator interactions with NF1. Thus far, this has led to the discovery that the ER co-activator SRC-1 binds NF1; in addition, NF1 silencing in ER+ breast cancer cells greatly enhances expression of ER-responsive genes, independent of Ras pathways. To uncover how NF1 regulates ER transcriptional activities, we have found evidence that NF1 can do so in two ways: (1) by controlling how much SRC-1 is in the nucleus and (2) by controlling how efficiently SRC-1 can be recruited to the ERE in a ligand-dependent manner. Indeed, NF1 appears to be recruited to the ER-ERE complex like a corepressor -- tamoxifen, as well as AZD9496, a new SERD (Selective ER Down-regulator), recruits more NF1 while reducing SRC-1 loading to the ER transcriptional complex. Objective and Hypothesis: Our overarching objectives are to define mechanisms of endocrine therapy resistance, and to design etiology-matched treatments to improve outcomes. This project will investigate the hypothesis that NF1 negatively regulates ER signaling via co-activator interactions, such that NF1 loss in tumors induces aggressive tumor behavior not only through activated Ras signaling but also by increased ER activity. Overarching Challenge: This project aims to identify and target a driver that induces aggressive tumor behavior, such as endocrine therapy resistance. As such, we will address one overarching challenge: "Identify what drives breast cancer growth; determine how to stop it." Aims and Study Design: In Aim 1, we will define how NF1 regulates expression of ER target genes by investigating a direct interaction between NF1 and canonical ER transcriptional co-regulators. We will first examine whether NF1 binds SRC-1 directly, and map the NF1 SRC-1 binding domain. We will measure whether NF1-silencing impacts SRC-1 half-life in the nucleus and in nuclear trafficking. Next, we will analyze NF1 control of co-regulator interactions with ER at the ERE in response to different ligands (E2, tamoxifen, AZD9496, and fulvestrant) as well as drugs targeting Ras and SRC-1, using an in vitro nuclear extract system. We will validate these interactions in ER+ breast cancer cells by analyzing proteins and DNA sequences associated with ER. In Aim 2, we will establish a strategy to effectively treat NF1-deficient ER+ breast cancers by rationally combining anti-Ras and anti-ER approaches. We will first determine what drug combination works best in vitro and will then further investigate this in viv
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Jan 31, 2017
- Source ID
- W81XWH1610539
Entities
People
- Matthew J Ellis
Organizations
- Baylor College of Medicine
- United States Army