Comparative analysis of the AIB1 interactome in breast cancer reveals MTA2 as a repressive partner which silences E-Cadherin to promote EMT and associates with a pro-metastatic phenotype.

Steroid regulated cancer cells use nuclear receptors and associated regulatory proteins to orchestrate transcriptional networks to drive disease progression. In primary breast cancer, the coactivator AIB1 promotes estrogen receptor (ER) transcriptional activity to enhance cell proliferation. The function of the coactivator in ER+ metastasis however is not established. Here we describe AIB1 as a survival factor, regulator of pro-metastatic transcriptional pathways and a promising actionable target. Genomic alterations and functional expression of AIB1 associated with reduced disease-free survival in patients and enhanced metastatic capacity in novel CDX and PDX ex-vivo models of ER+ metastatic disease. Comparative analysis of the AIB1 interactome with complementary RNAseq characterized AIB1 as a transcriptional repressor. Specifically, we report that AIB1 interacts with MTA2 to form a repressive complex, inhibiting CDH1 (encoding E-cadherin) to promote EMT and drive progression. We further report that pharmacological and genetic inhibition of AIB1 demonstrates significant anti-proliferative activity in patient-derived models establishing AIB1 as a viable strategy to target endocrine resistant metastasis. This work defines a novel role for AIB1 in the regulation of EMT through transcriptional repression in advanced cancer cells with a considerable implication for prognosis and therapeutic interventions.

A Functional Genomic Screen Identifies the Deubiquitinase USP11 as a Novel Transcriptional Regulator of ERα in Breast Cancer.

Approximately 70% of breast cancers express estrogen receptor α (ERα) and depend on this key transcriptional regulator for proliferation and differentiation. While patients with this disease can be treated with targeted antiendocrine agents, drug resistance remains a significant issue, with almost half of patients ultimately relapsing. Elucidating the mechanisms that control ERα function may further our understanding of breast carcinogenesis and reveal new therapeutic opportunities. Here, we investigated the role of deubiquitinases (DUB) in regulating ERα in breast cancer. An RNAi loss-of-function screen in breast cancer cells targeting all DUBs identified USP11 as a regulator of ERα transcriptional activity, which was further validated by assessment of direct transcriptional targets of ERα. USP11 expression was induced by estradiol, an effect that was blocked by tamoxifen and not observed in ERα-negative cells. Mass spectrometry revealed a significant change to the proteome and ubiquitinome in USP11-knockdown (KD) cells in the presence of estradiol. RNA sequencing in LCC1 USP11-KD cells revealed significant suppression of cell-cycle-associated and ERα target genes, phenotypes that were not observed in LCC9 USP11-KD, antiendocrine-resistant cells. In a breast cancer patient cohort coupled with in silico analysis of publicly available cohorts, high expression of USP11 was significantly associated with poor survival in ERα-positive (ERα+) patients. Overall, this study highlights a novel role for USP11 in the regulation of ERα activity, where USP11 may represent a prognostic marker in ERα+ breast cancer. SIGNIFICANCE: A newly identified role for USP11 in ERα transcriptional activity represents a novel mechanism of ERα regulation and a pathway to be exploited for the management of ER-positive breast cancer.

Transcriptome Characterization of Matched Primary Breast and Brain Metastatic Tumors to Detect Novel Actionable Targets.

BACKGROUND: Breast cancer brain metastases (BrMs) are defined by complex adaptations to both adjuvant treatment regimens and the brain microenvironment. Consequences of these alterations remain poorly understood, as does their potential for clinical targeting. We utilized genome-wide molecular profiling to identify therapeutic targets acquired in metastatic disease. METHODS: Gene expression profiling of 21 patient-matched primary breast tumors and their associated brain metastases was performed by TrueSeq RNA-sequencing to determine clinically actionable BrM target genes. Identified targets were functionally validated using small molecule inhibitors in a cohort of resected BrM ex vivo explants (n = 4) and in a patient-derived xenograft (PDX) model of BrM. All statistical tests were two-sided. RESULTS: Considerable shifts in breast cancer cell-specific gene expression profiles were observed (1314 genes upregulated in BrM; 1702 genes downregulated in BrM; DESeq; fold change > 1.5, Padj < .05). Subsequent bioinformatic analysis for readily druggable targets revealed recurrent gains in RET expression and human epidermal growth factor receptor 2 (HER2) signaling. Small molecule inhibition of RET and HER2 in ex vivo patient BrM models (n = 4) resulted in statistically significantly reduced proliferation (P < .001 in four of four models). Furthermore, RET and HER2 inhibition in a PDX model of BrM led to a statistically significant antitumor response vs control (n = 4, % tumor growth inhibition [mean difference; SD], anti-RET = 86.3% [1176; 258.3], P < .001; anti-HER2 = 91.2% [1114; 257.9], P < .01). CONCLUSIONS: RNA-seq profiling of longitudinally collected specimens uncovered recurrent gene expression acquisitions in metastatic tumors, distinct from matched primary tumors. Critically, we identify aberrations in key oncogenic pathways and provide functional evidence for their suitability as therapeutic targets. Altogether, this study establishes recurrent, acquired vulnerabilities in BrM that warrant immediate clinical investigation and suggests paired specimen expression profiling as a compelling and underutilized strategy to identify targetable dependencies in advanced cancers.

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.

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.