Novel insights into the molecular mechanisms of endocrine resistance in ERα positive breast cancer

ERα transcriptional activity drives tumour development and metastasis in more than 70% of breast cancer cases. Tamoxifen is the most widely and successfully used endocrine treatment for pre-menopausal women with ERα positive breast cancer. However, subgroups of patients are resistant to this drug. Investigation into the mechanisms of the endocrine refractory phenotype would therefore open possibilities for novel targeted therapies. One key aspect of ERα gene regulation is its accessibility to compacted chromatin. FOXA1 is a pioneer transcription factor that has the ability to bind to ‘closed’ chromatin and open it up for ERα subsequent binding, thereby creating the regulatory elements that are used by ERα. In this thesis, the dependence of the ERα hormone receptor on FOXA1 is reinforced and the latter is confirmed as a bone fide pioneer transcription factor and a promising drug target in hormone-dependent cancers. Moreover, novel molecular mechanisms of Tamoxifen resistance were investigated using quantitative multiplexed rapid immunoprecipitation mass spectrometry of endogenous proteins (qPLEX-RIME) in multiple in vitro and in vivo breast cancer models. The results showed that the two key proteins ERα and FOXA1 are enriched in the resistant phenotype, together with their newly-identified interactor ETV6. The role of ETV6 in endocrine resistance was confirmed using an independent siRNA screen. In addition, the direct contribution of ETV6 to breast cancer progression was proved by the promoting effects of ETV6 overexpression on colony formation ability of endocrine sensitive cell lines. Furthermore, chromatin immunoprecipitation followed by sequencing (ChIP-seq) analysis revealed that Tamoxifen resistance is associated with a global redistribution of FOXA1, ERα, ETV6- DNA interactions and altered genomic landscape. This differential binding of the three transcription factors also results in compromised transcriptional programmes in endocrine resistance, as assessed by RNA-seq. In addition, inhibition of MAPK pathway reduced breast cancer progression and modulated ETV6-chromatin interactions. Importantly, the clinical significance of ETV6 copy number amplifications was assessed in the METABRIC cohort (Curtis et al., 2012). They correlate with significantly reduced disease-free survival in Luminal B breast cancer subtype, which is the more aggressive ERα positive subtype and is more likely to metastasise. Moreover, by conducting a screen of 1000 FDA-approved drugs, potential candidates for the treatment of hormone-refractory breast cancer were identified. Further in vitro and in vivo validation would consolidate these findings. Taken together, the data presented in this dissertation reinforces FOXA1 independence of hormonal signalling, it identifies ETV6 as a novel ERα and FOXA1 co-factor that drives a more proliferative phenotype and proposes alternative therapies for the endocrine refractory phenotype.Cancer Research U

Comprehensive Genomic Analysis Reveals that the Pioneering Function of FOXA1 Is Independent of Hormonal Signaling.

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.ERC Consolidator award (Project number 646876), CRUK funding and a Komen Scholarship

Identification of ChIP-seq and RIME grade antibodies for Estrogen Receptor alpha

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

Comprehensive Genomic Analysis Reveals that the Pioneering Function of FOXA1 Is Independent of Hormonal Signaling

Summary: 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. : Glont et al. show that FOXA1 binding sites are not regulated by hormones. A small number (<1%) of FOXA1 binding events appear to be estrogen regulated, but these are shadow peaks that are created via pre-existing binding sites that form chromatin loops

Identification of ChIP-seq and RIME grade antibodies for Estrogen Receptor alpha.

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

IL6/STAT3 Signaling Hijacks Estrogen Receptor α Enhancers to Drive Breast Cancer Metastasis.

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

IL6/STAT3 Signaling Hijacks Estrogen Receptor α Enhancers to Drive Breast Cancer Metastasis.

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

ARID1A influences HDAC1/BRD4 activity, intrinsic proliferative capacity and breast cancer treatment response.

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.ERC Consolidator award (646876), Susan G Komen leadership grant and CRUK Core Fundin