Ailis Fagan

Global characterization of the SRC-1 transcriptome identifies ADAM22 as an ER-independent mediator of endocrine resistant breast cancer

The development of breast cancer resistance to endocrine therapy results from an increase in cellular plasticity that permits the emergence of a hormone-independent tumor. The steroid coactivator protein SRC-1, through interactions with developmental proteins and other nonsteroidal transcription factors, drives this tumor adaptability. In this discovery study, we identified ADAM22, a non-protease member of the ADAM family of disintegrins, as a direct estrogen receptor (ER)-independent target of SRC-1. We confirmed SRC-1 as a regulator of ADAM22 by molecular, cellular, and in vivo studies. ADAM22 functioned in cellular migration and differentiation, and its levels were increased in endocrine resistant-tumors compared with endocrine-sensitive tumors in mouse xenograft models of human breast cancer. Clinically, ADAM22 was found to serve as an independent predictor of poor disease-free survival. Taken together, our findings suggest that SRC-1 switches steroid-responsive tumors to a steroid-resistant state in which the SRC-1 target gene ADAM22 has a critical role, suggesting this molecule as a prognostic and therapeutic drug target that could help improve the treatment of endocrine-resistant breast cancer.

Global Gene Repression by the Steroid Receptor Coactivator SRC-1 Promotes Oncogenesis

Transcriptional control is the major determinant of cell fate. The steroid receptor coactivator (SRC)-1 enhances the activity of the estrogen receptor in breast cancer cells, where it confers cell survival benefits. Here, we report that a global analysis of SRC-1 target genes suggested that SRC-1 also mediates transcriptional repression in breast cancer cells. Combined SRC-1 and HOXC11 ChIPseq analysis identified the differentiation marker, CD24, and the apoptotic protein, PAWR, as direct SRC-1/HOXC11 suppression targets. Reduced expression of both CD24 and PAWR was associated with disease progression in patients with breast cancer, and their expression was suppressed in metastatic tissues. Investigations in endocrine-resistant breast cancer cell lines and SRC-1(-/-)/PyMT mice confirmed a role for SRC-1 and HOXC11 in downregulation of CD24 and PAWR. Through bioinformatic analysis and liquid chromatography/mass spectrometry, we identified AP1 proteins and Jumonji domain containing 2C (JMD2C/KDM4C), respectively, as members of the SRC-1 interactome responsible for transcriptional repression. Our findings deepen the understanding of how SRC-1 controls transcription in breast cancers.

Adaptation to AI therapy in breast cancer can induce dynamic alterations in ER activity resulting in estrogen independent metastatic tumours

Purpose: Acquired resistance to aromatase inhibitor (AI) therapy is a major clinical problem in the treatment of breast cancer. The detailed mechanisms of how tumor cells develop this resistance remain unclear. Here, the adapted function of estrogen receptor (ER) to an estrogen-depleted environment following AI treatment is reported. Experimental Design: Global ER chromatin immuno-precipitation (ChIP)-seq analysis of AI-resistant cells identified steroid-independent ER target genes. Matched patient tumor samples, collected before and after AI treatment, were used to assess ER activity. Results: Maintained ER activity was observed in patient tumors following neoadjuvant AI therapy. Genome-wide ER–DNA-binding analysis in AI-resistant cell lines identified a subset of classic ligand-dependent ER target genes that develop steroid independence. The Kaplan–Meier analysis revealed a significant association between tumors, which fail to decrease this steroid-independent ER target gene set in response to neoadjuvant AI therapy, and poor disease-free survival and overall survival (n = 72 matched patient tumor samples, P = 0.00339 and 0.00155, respectively). The adaptive ER response to AI treatment was highlighted by the ER/AIB1 target gene, early growth response 3 (EGR3). Elevated levels of EGR3 were detected in endocrine-resistant local disease recurrent patient tumors in comparison with matched primary tissue. However, evidence from distant metastatic tumors demonstrates that the ER signaling network may undergo further adaptations with disease progression as estrogen-independent ER target gene expression is routinely lost in established metastatic tumors. Conclusions: Overall, these data provide evidence of a dynamic ER response to endocrine treatment that may provide vital clues for overcoming the clinical issue of therapy resistance. Clin Cancer Res; 22(11); 2765–77. ©2016 AACR.

Transcriptomic Profiling of Sequential Tumors from Breast Cancer Patients Provides a Global View of Metastatic Expression Changes Following Endocrine Therapy

Purpose: Disease recurrence is a common problem in breast cancer and yet the mechanisms enabling tumor cells to evade therapy and colonize distant organs remain unclear. We sought to characterize global expression changes occurring with metastatic disease progression in the endocrine-resistant setting. Experimental Design: Here, for the first time, RNAsequencing has been performed on matched primary, nodal, and liver metastatic tumors from tamoxifen-treated patients following disease progression. Expression of genes commonly elevated in the metastases of sequenced patients was subsequently examined in an extended matched patient cohort with metastatic disease from multiple sites. The impact of tamoxifen treatment on endocrine-resistant tumors in vivo was investigated in a xenograft model. Results: The extent of patient heterogeneity at the gene level was striking. Less than 3% of the genes differentially expressed between sequential tumors were common to all patients. Larger divergence was observed between primary and liver tumors than between primary and nodal tumors, reflecting both the latency to disease progression and the genetic impact of intervening therapy. Furthermore, an endocrine-resistant in vivo mouse model demonstrated that tamoxifen treatment has the potential to drive disease progression and establish distant metastatic disease. Common functional pathways altered during metastatic, endocrine-resistant progression included extracellular matrix receptor interactions and focal adhesions. Conclusions: This novel global analysis highlights the influence of primary tumor biology in determining the transcriptomic profile of metastatic tumors, as well as the need for adaptations in cell–cell communications to facilitate successful tumor cell colonization of distant host organs. Clin Cancer Res; 21(23); 5371–9. ©2015 AACR.

Chemostat-based transcriptional analysis of growth rate change and BCL-2 over-expression in NS0 cells

We investigated the transcriptional response of NS0 cells undergoing controlled nutrient growth change in continuous chemostat culture using mouse microarrays. A 50% reduction in growth rate resulted in detectable alterations in the expression of 29 genes in NS0 cells. Notably, expression of genes in three major biological processes, namely transcriptional, translational, and protein processing functions, were modified. To further elucidate the advantage of the chemostat environment for establishment of “omic” data sets, an expression profile of the over‐expressed gene bcl‐2 in NS0 cells was probed. Functional analysis revealed that the underlying altered molecular mechanism was particularly associated with G1 cell cycle progression, protein synthesis, and apoptosis. Importantly, these findings agreed with the physical function of the cells. Despite an increase in survival rate, bcl‐2 over‐expression resulted in a decrease of specific productivity, glucose consumption, oxygen uptake rate and intracellular protein content, indicating a lower energy generating metabolism. Further, a prolongation of G1 cell cycle phase was evident on lowering the growth rate. Overall, the application of microarray analysis to chemostat‐grown cultures offers an excellent combination for the interpretation of transcriptomic profiles to elucidate the molecular mechanisms during nutrient growth change and bcl‐2 over‐expression. Biotechnol. Bioeng. 2011; 108:1603–1615.

Network analysis of SRC-1 reveals a novel transcription factor hub which regulates endocrine resistant breast cancer

Steroid receptor coactivator 1 (SRC-1) interacts with nuclear receptors and other transcription factors (TFs) to initiate transcriptional networks and regulate downstream genes which enable the cancer cell to evade therapy and metastasise. Here we took a top–down discovery approach to map out the SRC-1 transcriptional network in endocrine resistant breast cancer. First, rapid immunoprecipitation mass spectrometry of endogenous proteins (RIME) was employed to uncover new SRC-1 TF partners. Next, RNA sequencing (RNAseq) was undertaken to investigate SRC-1 TF target genes. Molecular and patient-derived xenograft studies confirmed STAT1 as a new SRC-1 TF partner, important in the regulation of a cadre of four SRC-1 transcription targets, NFIA, SMAD2, E2F7 and ASCL1. Extended network analysis identified a downstream 79 gene network, the clinical relevance of which was investigated in RNAseq studies from matched primary and local-recurrence tumours from endocrine resistant patients. We propose that SRC-1 can partner with STAT1 independently of the estrogen receptor to initiate a transcriptional cascade and control regulation of key endocrine resistant genes.

Epigenome-wide SRC-1–Mediated Gene Silencing Represses Cellular Differentiation in Advanced Breast Cancer

Purpose: Despite the clinical utility of endocrine therapies for estrogen receptor–positive (ER) breast cancer, up to 40% of patients eventually develop resistance, leading to disease progression. The molecular determinants that drive this adaptation to treatment remain poorly understood. Methylome aberrations drive cancer growth yet the functional role and mechanism of these epimutations in drug resistance are poorly elucidated. Experimental Design: Genome-wide multi-omics sequencing approach identified a differentially methylated hub of prodifferentiation genes in endocrine resistant breast cancer patients and cell models. Clinical relevance of the functionally validated methyl-targets was assessed in a cohort of endocrine-treated human breast cancers and patient-derived ex vivo metastatic tumors. Results: Enhanced global hypermethylation was observed in endocrine treatment resistant cells and patient metastasis relative to sensitive parent cells and matched primary breast tumor, respectively. Using paired methylation and transcriptional profiles, we found that SRC-1–dependent alterations in endocrine resistance lead to aberrant hypermethylation that resulted in reduced expression of a set of differentiation genes. Analysis of ER-positive endocrine-treated human breast tumors (n = 669) demonstrated that low expression of this prodifferentiation gene set significantly associated with poor clinical outcome (P = 0.00009). We demonstrate that the reactivation of these genes in vitro and ex vivo reverses the aggressive phenotype. Conclusions: Our work demonstrates that SRC-1-dependent epigenetic remodeling is a ’high level’ regulator of the poorly differentiated state in ER-positive breast cancer. Collectively these data revealed an epigenetic reprograming pathway, whereby concerted differential DNA methylation is potentiated by SRC-1 in the endocrine resistant setting. Clin Cancer Res; 24(15); 3692–703. ©2018 AACR.

Abstract P2-05-03: Whole genome transcriptome analysis of sequential breast to brain metastasis uncovers new signalling pathways and druggable targets

The occurrence of brain metastasis (BM) in breast cancer (BC) is currently on the rise across all molecular subtypes with 10-30% reported incidence. The need to uncover the mechanisms underlying this clinically devastating complication is apparent, and in the current study we sought to identify BC cell mediators of BM. In our cohort of metastatic patients (n=196) we found that BM developed in 13% of the cases. Despite the previous reports of negative ER status being a risk factor for BM, the ER+ve patients accounted for 42% of all diagnosed BM. To elucidate the gene alterations required for successful colonisation of the brain we undertook RNA sequencing (RNA-seq) of sequential breast to brain metastasis of known receptor status (n=7).This study presents the first whole transcriptome next-generation RNA-seq analysis of resected BM and their matching primary breast tumours. We identified 500 differentially expressed genes (DEGs) ( ±1.5), accounting for those that were both upregulated and downregulated in BM compared to the primary. Analysis of protein-coding genes identified collective ER-specific metastatic pathways. Additionally, common functional pathways altered included ECM, cell adhesion and neuronal differentiation. Our analysis of the BM transcriptomic landscape and verification in cell line models that preferentially metastasise to the brain has unravelled a complex network of driver genes, cooperating with stromal derived factors, responsible for the organ-specific behaviour of the metastatic cells. Genes such as ANTRX1, THBS2, FAP, VCAN and TIMP2 were found to be part of the invasion and migration network that drives the extravasation of the BM cells. Furthermore, an EMT stemness signalling network driven by ANTRX1and WNT pathway driven RUNX was prominent in the cells acquiring the ability to migrate to the brain. Additional work is being carried out on uncovering the adaptations that re-activate the dormant brain metastatic cells and the contribution of the neuronal niche in the facilitating the colonisation by the MBC. This study highlights the requirement of unique gene sets for the invasion, migration and colonisation to the brain and that functional characterisation of the DEGs will enable the identification of novel molecular targets for prevention and treatment of breast cancer BM. Citation Format: Vareslija D, Fagan A, Buckley P, Farrell M, Hill A, Young L. Whole genome transcriptome analysis of sequential breast to brain metastasis uncovers new signalling pathways and druggable targets. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-05-03.

Abstract P3-04-17: Global transcriptional repression by the coactivator SRC-1 mediates disease progression in treatment-resistant breast cancer

Despite the effectiveness of endocrine therapy in treating estrogen receptor (ER) positive breast cancer, nearly 40% of breast cancer patients may develop resistance which can lead to metastatic disease progression. Steroid receptor co-activator-1 (SRC-1), a key regulator of ER signalling, is overexpressed in 35% of breast cancer patients and is strongly associated with the development of endocrine resistant metastasis(1). Recent studies highlighted the ability of SRC-1 to also act as transcriptional repressor. SRC-1 can therefore bi-directionally regulate gene expression to promote the resistant phenotype (2). This study employed a genome-wide multi-omics sequencing approach to determine the SRC-1 repression signature in endocrine resistance and elucidate the mechanism by which SRC-1 mediates this repression. RNA-sequencing identified 736 genes significantly downregulated by SRC-1, with common functional pathways such as differentiation, cell morphogenesis and extracellular matrix enriched in the gene set. Parallel global methylation sequencing analysis revealed distinct differentially methylated regions specific to SRC-1 repressed target genes. Mechanistic studies in endocrine resistant cells revealed a role for methylation proteins, DNMTs and MBD, in SRC-1 directed repression. Through combined analysis of our global sequencing data we identified a network hub of five differentiation genes directly repressed by SRC-1. High expression of this signature predicts enhanced recurrence free survival in tamoxifen treated patients (n=335, p=0.032). A reduction in expression of these genes was shown to have functional output on proliferation, migration and mammosphere formation. Finally, we use tumour explant models to show that targeting DNA methylation can be used to reverse the SRC-1 driven suppression of this differentiation hub. Here we report a novel mechanism by which SRC-1 may be driving endocrine resistant tumorigenesis. Our genome-wide discovery approach revealed a global epigenetic re-programming pathway whereby concerted differential DNA methylation is potentiated by the activation of SRC-1 in the presence of tamoxifen in the endocrine resistant setting. This study suggests that therapeutic strategies of combined targeted epigenetic therapy with estrogen deprivation could be a successful strategy to prevent acquired resistance to endocrine therapy. Citation Format: Ward E, Vareslija D, Fagan A, Hill A, Young L. Global transcriptional repression by the coactivator SRC-1 mediates disease progression in treatment-resistant breast cancer [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 P3-04-17.

Abstract P3-04-16: Steroid receptor co-activator 1 mediation of cancer cell reprogramming in endocrine resistant breast cancer

Cancer cells undergo dynamic and frequently reversible modifications and this cellular plasticity permits reprogramming in response to long term endocrine treatment. A well-established theory, the cancer stem cell theory; suggests that within an ER positive tumour cell population are a small number of stem cells which are capable of infinite self-renewal and are insensitive to treatment with standard endocrine regimes. However, emerging reports that pluripotent cells can be generated from adult somatic cells (Takahashi, 2007) alludes to an alternate mechanism for cancer cells to reprogramme and evade endocrine treatment. To investigate the role of tamoxifen in promoting cellular reprogramming and endocrine resistance in breast cancer, we isolated single luminal A breast cancer CD24+CD44- clones from endocrine sensitive MCF-7 cells. The clones were expanded in the presence and absence of tamoxifen. Extensive profiling of cells following long-term exposure to tamoxifen revealed a resistant phenotype similar to that reported in other established models. Steroid receptor co-activator 1 (SRC-1) is a master regulatory protein which has been shown to be central to the development of endocrine resistance. The mechanisms of which are still poorly understood (Walsh et al ., 2012). Utilising genome-wide transcriptomic sequencing this study investigated whether SRC-1 can regulate transcriptional networks which mediate reprogramming in individual cells to induce survival adaptability and drug resistance in breast cancer. RNA-sequencing was carried out in a model of endocrine resistance (shNon-Targeting versus shSRC-1). There were 1,731 genes up-regulated by SRC-1, of which 153 were identified as transcription factors (TFs)/chromatin remodellers. Combining the transcriptomic profiling with ChIP-sequencing we identified a transcriptional network pertinent to SRC-1. Molecular characterisation identified E2F7, NFIA, DEK, SMAD2, SMARCA1, ASCL1 and TRPS1 as key drivers of SRC-1 mediated endocrine resistance. Each TF was confirmed as a direct target of SRC-1, via promoter specific binding and were found to be drivers of endocrine resistant cell migration. Furthermore, all TFs were necessary in mammosphere formation and their promotion of cellular de-differentiation was observed by 3D acinar organisation and flow cytometry. To elucidate the core TFs required for cancer cell reprogramming, their effect on the pioneer reprogramming TFs (OCT4, SOX2, cMYC and KLF4) was investigated. SMAD2, SMARCA1, ASCL1 and TRPS1 have emerged as pivotal regulators of endocrine resistant cell reprogramming. This study has unravelled an SRC-1-mediated TF network responsible for promoting the cellular reprogramming of breast cancer cells. Concerted activity of this network is responsible for driving de-differentiation of cells and enhancing their stem-like, highly migratory and proliferative tumour initiation population. This study provides important information regarding the mechanism of cellular reprogramming in ER positive endocrine resistant cancer and may lead to potential novel therapeutic targets. Citation Format: Browne A, Fagan A, Vareslija D, Ward E, Hill A, Young L. Steroid receptor co-activator 1 mediation of cancer cell reprogramming in endocrine resistant breast cancer [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 P3-04-16.