ABSTRACT
The cytokine interleukin-6 (IL6) and its downstream effector STAT3 constitute a key oncogenic pathway, which has been thought to be functionally connected to estrogen receptor α (ER) in breast cancer. We demonstrate that IL6/STAT3 signaling drives metastasis in ER+ breast cancer independent of ER. STAT3 hijacks a subset of ER enhancers to drive a distinct transcriptional program. Although these enhancers are shared by both STAT3 and ER, IL6/STAT3 activity is refractory to standard ER-targeted therapies. Instead, inhibition of STAT3 activity using the JAK inhibitor ruxolitinib decreases breast cancer invasion in vivo. Therefore, IL6/STAT3 and ER oncogenic pathways are functionally decoupled, highlighting the potential of IL6/STAT3-targeted therapies in ER+ breast cancer.
Subject(s)
Breast Neoplasms/genetics , Enhancer Elements, Genetic/genetics , Estrogen Receptor alpha/genetics , Interleukin-6/genetics , STAT3 Transcription Factor/genetics , Signal Transduction/genetics , Animals , Antineoplastic Agents, Hormonal/pharmacology , Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , Estrogen Receptor alpha/metabolism , Female , Fulvestrant/pharmacology , Gene Expression Profiling/methods , Gene Expression Regulation, Neoplastic , Humans , Interleukin-6/metabolism , Kaplan-Meier Estimate , MCF-7 Cells , Mice, Inbred NOD , Mice, Knockout , Mice, SCID , Neoplasm Metastasis , STAT3 Transcription Factor/metabolism , Xenograft Model Antitumor Assays/methodsABSTRACT
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
ABSTRACT
Using genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) screens to understand endocrine drug resistance, we discovered ARID1A and other SWI/SNF complex components as the factors most critically required for response to two classes of estrogen receptor-alpha (ER) antagonists. In this context, SWI/SNF-specific gene deletion resulted in drug resistance. Unexpectedly, ARID1A was also the top candidate in regard to response to the bromodomain and extraterminal domain inhibitor JQ1, but in the opposite direction, with loss of ARID1A sensitizing breast cancer cells to bromodomain and extraterminal domain inhibition. We show that ARID1A is a repressor that binds chromatin at ER cis-regulatory elements. However, ARID1A elicits repressive activity in an enhancer-specific, but forkhead box A1-dependent and active, ER-independent manner. Deletion of ARID1A resulted in loss of histone deacetylase 1 binding, increased histone 4 lysine acetylation and subsequent BRD4-driven transcription and growth. ARID1A mutations are more frequent in treatment-resistant disease, and our findings provide mechanistic insight into this process while revealing rational treatment strategies for these patients.
Subject(s)
Breast Neoplasms/drug therapy , Cell Cycle Proteins/metabolism , DNA-Binding Proteins/metabolism , Histone Deacetylase 1/metabolism , Transcription Factors/metabolism , Acetylation , Animals , Breast Neoplasms/mortality , Breast Neoplasms/pathology , Cell Cycle Proteins/genetics , Cell Proliferation , Clustered Regularly Interspaced Short Palindromic Repeats , DNA-Binding Proteins/genetics , Drug Resistance, Neoplasm/genetics , Estrogen Receptor alpha/genetics , Estrogen Receptor alpha/metabolism , Female , Gene Expression Regulation, Neoplastic , Hepatocyte Nuclear Factor 3-alpha/genetics , Hepatocyte Nuclear Factor 3-alpha/metabolism , Histone Deacetylase 1/genetics , Humans , MCF-7 Cells , Mice, Inbred NOD , Transcription Factors/genetics , Xenograft Model Antitumor AssaysABSTRACT
Estrogen Receptor alpha (ERα) plays a major role in most breast cancers, and it is the target of endocrine therapies used in the clinic as standard of care for women with breast cancer expressing this receptor. The two methods ChIP-seq (chromatin immunoprecipitation coupled with deep sequencing) and RIME (Rapid Immunoprecipitation of Endogenous Proteins) have greatly improved our understanding of ERα function during breast cancer progression and in response to anti-estrogens. A critical component of both ChIP-seq and RIME protocols is the antibody that is used against the bait protein. To date, most of the ChIP-seq and RIME experiments for the study of ERα have been performed using the sc-543 antibody from Santa Cruz Biotechnology. However, this antibody has been discontinued, thereby severely impacting the study of ERα in normal physiology as well as diseases such as breast cancer and ovarian cancer. Here, we compare the sc-543 antibody with other commercially available antibodies, and we show that 06-935 (EMD Millipore) and ab3575 (Abcam) antibodies can successfully replace the sc-543 antibody for ChIP-seq and RIME experiments.
Subject(s)
Antibodies , Chromatin Immunoprecipitation Sequencing/methods , Estrogen Receptor alpha/immunology , Immunoprecipitation/methods , Antibody Specificity , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Cell Line, Tumor , Estrogen Receptor alpha/genetics , Estrogen Receptor alpha/metabolism , Female , Humans , MCF-7 CellsABSTRACT
Considerable work has linked hormone receptors, such as estrogen receptor-alpha (ER), with the pioneer factor FOXA1. Altered FOXA1 levels contribute to endocrine-resistant breast cancer, where it maintains ER-chromatin interactions, even in contexts in which cells are refractory to ER-targeted drugs. A recent study controversially suggests that FOXA1 binding can be induced by hormonal pathways, including the estrogen-ER complex. We now show that the vast majority (>99%) of FOXA1 binding events are unaffected by steroid activation. A small number (<1%) of FOXA1 binding sites appear to be induced by estrogen, but these are created from chromatin interactions between ER binding sites and adjacent FOXA1 binding sites and do not represent genuine new FOXA1-pioneering elements. FOXA1 is therefore not regulated by estrogen and remains a bone fide pioneer factor that is entirely upstream of the ER complex.