Sinead Cocchiglia

Ets-2 and p160 proteins collaborate to regulate c-Myc in endocrine resistant breast cancer

Associations between p160 coactivator proteins and endocrine resistance have been described. Though thought to primarily interact with steroid receptors, the p160 proteins can also interact with non-nuclear receptor transcription factors including the MAP kinase effector proteins Ets. Here, we observed that in breast cancer cells resistant and insensitive to endocrine treatment, the growth factor EGF induced Ets-2 but not Ets-1 transcriptional regulation of the oncogene myc. Ets-2 regulation of myc was found to be reliant on the p160 proteins SRC-1 and SRC-3. In support of these molecular observations, strong associations were observed between the transcription factor, Ets-2 and its coactivator SRC-1 (P<0.01) and the target gene myc (P<0.0001) in a cohort of breast cancer patients with locally advanced disease. Expression of Ets-2, SRC-1 and c-Myc individually all associated with reduced disease-free survival (P<0.001, P<0.001 and P=0.002 respectively). There was no association between SRC-3 and disease-free survival (P=0.707). SRC-1 can utilize MAP kinase effector transcription factor Ets-2 to regulate the production of the oncogene myc. These signalling mechanisms may be important in the development of steroid resistant/independent breast cancer.

Metastatic Progression with Resistance to Aromatase Inhibitors Is Driven by the Steroid Receptor Coactivator SRC-1

Aromatase inhibitors (AI) are a standard-of-care treatment for postmenopausal, estrogen receptor-positive breast cancers. Although tumor recurrence on AI therapy occurs, the mechanisms underlying acquired resistance to AIs remain unknown. In this study, we examined a cohort of endocrine-treated breast cancer patients and used a cell line model of resistance to the AI letrozole. In patients treated with a first-line AI, hormone receptor switching between primary and resistant tumors was a common feature of disease recurrence. Resistant cells exhibited a switch from steroid-responsive growth to growth factor-responsive and endocrine-independent growth, which was accompanied by the development of a more migratory and disorganized phenotype. Both the resistant cells and tumors from AI-resistant patients showed high expression of the steroid receptor coactivator SRC-1. Direct interactions between SRC-1 and the transcription factor Ets2 regulated Myc and MMP9. SRC-1 was required for the aggressive and motile phenotype of AI-resistant cells. Interestingly, SRC-1 expression in primary and/or recurrent tumors was associated with a reduction in disease-free survival in treated patients. Moreover, there was a significant association between SRC-1 and Ets2 in the recurrent tissue compared with the matched primary tumor. Together, our findings elucidate a mechanism of AI-specific metastatic progression in which interactions between SRC-1 and Ets2 promote dedifferentiation and migration in hormone-dependent breast cancer.

Growth factor receptor/steroid receptor cross talk in trastuzumab-treated breast cancer

Treatment with tyrosine kinase inhibitors (TKIs) including trastuzumab has revolutionized the management of HER2-positive breast cancer. Recent evaluation of clinical trial data suggests that a subset of HER2/ER double-positive cancers may not receive significant benefit from the TKI therapy. Here we investigate the cross talk between HER2 and ER in breast cancer and monitor the effect of trastuzumab on the tyrosine kinase effector transcription factor Myc. In HER2-positive breast cancer patients treated with neoadjuvant trastuzumab, steroid receptor-negative status (ER and PR negative) of pre-treatment biopsies predicted pathological complete response (pCR) (n=31 patients, P=0.0486), whereas elevated Myc protein inversely associated with pCR (P=0.0446). Liquid chromatography mass spectrometry identified the corepressor SMRT as a novel Myc-interacting protein. Trastuzumab treatment enhanced Myc–SMRT interactions in HER2-overexpressing breast cancer cells (LCC1) and inhibited expression of the Myc target gene survivin. In HER2-low, ER-positive steroid-dominant cells (MCF7), trastuzumab therapy repressed Myc–SMRT interactions and upregulated survivin expression. Trastuzumab treatment induced ER–CBP interactions, enhanced ER transcriptional activity and upregulated expression of the ER target gene pS2. The absence of pS2 expression in pre-treatment biopsies predicted pCR to neoadjuvant trastuzumab in breast cancer patients (n=25, P=0.0089) and pS2 expression associated with residual cancer burden (P=0.0196). Furthermore, metastatic tissues from patients who had failed trastuzumab therapy were pS2 positive. In HER2-overexpressing cells, trastuzumab treatment can repress Myc transcriptional activity and clinical response is favorable. However, with co-expression of the steroid pathway, this inhibition is lost and response to treatment is often poor.

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.

RuvBl2 cooperates with Ets2 to transcriptionally regulate hTERT in colon cancer

Human cancers utilise telomerase to maintain telomeres and prohibit cell senescence. Human telomerase reverse transcriptase (hTERT), an essential component of this complex, is regulated at the level of gene transcription. Using SILAC‐proteomic analysis and molecular studies, we identified the AAA+ ATPase, RuvBl2 as a transcriptional regulator of hTERT and established that this regulation is through cooperation with Ets‐2. In colon cancer patients, nuclear expression of RuvBl2 associated with nuclear expression of hTERT, pEts2 and advanced nodal disease (P < 0.01, P = 0.05 and P = 0.03 respectively, n = 170). These data firmly implicate RuvBl2 in Ets2 mediated regulation of hTERT in colon cancer which has functional and clinical consequences.

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.

Patient-Derived Xenografts of Breast Cancer

With the advancement of translational research, particularly in the field of cancer, it is now imperative to have models which more clearly reflect patient heterogeneity. Patient derived xenograft (PDX) models, which involve the orthotopic implantation of breast tumors into immune-compromised mice, recapitulate the native tumor biology. Despite the considerable challenges that establishing PDX models present, they are the ultimate model to study tumorigenesis of refractory disease and for assessing the efficacy of new pharmaceutical compounds.

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.

P5-12-01: Aromatase Inhibitor Specific Metastasis Is Driven by the Steroid Receptor Coactivator SRC-1.

Aromatase Inhibitors are currently one of the most promising therapies of estrogen-receptor positive breast cancer in postmenopausal women. Even though many women initially respond to the treatment, approximately 30–40% will acquire resistance and relapse within 5 years. The mechanisms involved in the development of resistance to AIs however are poorly understood as long term follow up is only now becoming available. To investigate the mechanisms involved in AI-specific tumour recurrence, we generated a cell line resistant to the AI Letrozole (LetR) and examined a cohort of endocrine treated breast cancer patients (n=150). In patients treated with a first-line AI (n=89), we found that hormone receptor switching between the primary tumour and the resistant metastasis was a common feature of disease recurrence. More so, this switch accompanied the development of a phenotype displaying an increase in migratory capacity and loss of organisation. Both the resistant cell model and AI resistant tumour samples expressed high levels of the steroid receptor coactivator SRC-1. We also found that the interaction between SRC-1 and the transcription factor Ets2 was involved in the regulation of Myc and MMP9 expression and that SRC-1 was required for the aggressive AI resistant phenotype. Expression of SRC-1 in the primary and/or recurrent tumour associated with poor disease free survival (p=0.01, n=89) in the AI treated population. A significant coassociation between SRC-1 and Ets2 in the nucleus of the recurrent tissue compared with the matched primary tumour was also observed (p=0.0004, n=3). These data describe a novel signaling mechanism of AI-specific metastatic progression where SRC-1 utilizes Ets2 to promote de-differentiation and migration to drive disease progression. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-12-01.