Damian McCartan

Interaction of Developmental Transcription Factor HOX11 with Steroid Receptor Coactivator SRC-1 Mediates Resistance to Endocrine Therapy in Breast Cancer

Mechanisms of acquired resistance to endocrine therapy in breast cancer, a major clinical challenge, are poorly understood. We have used a mass spectrometry-based screen to identify proteins that are associated with the endocrine-resistant phenotype. In this study, we report the identification of a novel pathway of resistance to endocrine therapy involving interactions of the developmental transcription HOXC11 with the steroid receptor coactivator protein SRC-1, which is a strong predictor of reduced disease-free survival in breast cancer patients. HOXC11 and SRC-1 cooperate to regulate expression of the calcium-binding protein S100beta in resistant breast cancer cells. Nuclear HOXC11 and S100beta were found to strongly predict poor disease-free survival in breast cancer patients (n = 560; hazard ratios: 5.79 and 5.82, respectively; P < 0.0001). Elevated serum levels of S100beta detected in patients also predicted reduced disease-free survival (n = 80; hazard ratio: 5.3; P = 0.004). Our findings define a biomolecular interaction network that drives an adaptive response to endocrine therapy with negative consequences for survival in breast cancer.

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.

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.

Prosaposin activates the androgen receptor and potentiates resistance to endocrine treatment in breast cancer.

IntroductionHOX genes play vital roles in growth and development, however, atypical redeployment of these genes is often associated with steroidal adaptability in endocrine cancers. We previously identified HOXC11 to be an indicator of poor response to hormonal therapy in breast cancer. In this study we aimed to elucidate genes regulated by HOXC11 in the endocrine resistant setting.MethodsRNA-sequencing paired with transcription factor motif-mapping was utilised to identify putative HOXC11 target genes in endocrine resistant breast cancer. Validation and functional evaluation of the target gene, prosaposin (PSAP), was performed in a panel of endocrine sensitive and resistant breast cancer cell lines. The clinical significance of this finding was explored in clinical cohorts at both mRNA and protein level.ResultsPSAP was shown to be regulated by HOXC11 in both tamoxifen and aromatase inhibitor (AI) resistant cell lines. Transcript levels of HOXC11 and PSAP correlated strongly in samples of primary breast tumours (r = 0.7692, n = 51). PSAP has previously been reported to activate androgen receptor (AR) in prostate cancer cells. In a panel of breast cancer cell lines it was shown that endocrine resistant cells exhibit innately elevated levels of AR compared to their endocrine sensitive counterparts. Here, we demonstrate that stimulation with PSAP can drive AR recruitment to a hormone response element (HRE) in AI resistant breast cancer cells. Functionally, PSAP promotes cell migration and invasion only in AI resistant cells and not in their endocrine sensitive counterparts. In a cohort of breast cancer patients (n = 34), elevated serum levels of PSAP were found to associate significantly with poor response to endocrine treatment (p = 0.04). Meta-analysis of combined PSAP and AR mRNA are indicative of poor disease-free survival in endocrine treated breast cancer patients (hazard ratio (HR): 2.2, P = 0.0003, n = 661).ConclusionThe HOXC11 target gene, PSAP, is an AR activator which facilitates adaptation to a more invasive phenotype in vitro. These findings have particular relevance to the development of resistance to AI therapy which is an emerging clinical issue. PSAP is a secreted biomarker which has potential in identifying patients failing to exhibit sustained response to hormonal treatment.

Developmental protein HOXC11 cooperates with SRC-1 in breast cancer: an adaptive response to endocrine therapy

The ability of a tumour to adapt and overcome targeted therapies has been recognised clinically for some time, but the molecular mechanisms driving this metamorphosis remain unclear. The steroid receptor coactivator protein, SRC-1, is a strong predictor of reduced disease-free survival in breast cancer patients. SRC-1 can also interact with nonsteroidal transcription factors, and defining these new transcriptional networks will uncover fresh strategies for managing endocrine resistance. Here we employed a mass spectrometry-based screen to identify proteins that are specific to the endocrine-resistant phenotype. The developmental protein, HOXC11, was identified and functionally validated as an interaction partner of SRC-1. We provide evidence that HOXC11 and SRC-1 cooperate to regulate expression of the calcium binding protein S100β in resistant breast cancer cells. Moreover, both nuclear HOXC11 and S100β were found to be strong predictors of poor disease-free survival in breast cancer patients (n = 560; hazard ratios = 5.79 and 5.82, respectively; P < 0.0001). Elevated serum levels of S100β detected in patients also predicted reduced disease-free survival (n = 80; hazard ratio = 5.3; P = 0.004). This translational study identifies a biomolecular interaction network central to the adaptive response to endocrine therapy with clear clinical applications.

Abstract P3-05-02: Global characterisation of the SRC-1 transcriptome and rational drug design results in the identification of a novel peptide targeting ADAM22 in endocrine resistance

In spite of therapeutic advances, up to 25% of luminal breast cancers will eventually develop resistance to endocrine therapy and develop metastatic disease. The underlying mechanism causing ER-positive, steroid responsive tumours to develop a resistant, metastatic phenotype remains unresolved. Previous work from our group and others has identified the P160 protein SRC-1 as a significant predictor of recurrence on endocrine therapy. The purpose of this study is to further examine downstream SRC-1 targets in the context of endocrine resistant breast cancer. We adopted a global approach to define the transcriptional targets of SRC-1. SRC-1 ChIP sequencing in endocrine resistant luminal B breast cancer cells was combined with SRC-1 gene expression array analysis. This identified a number of pathways significantly elevated following tamoxifen treatment, including a number involved in cellular adhesion. From these pathways, A Disintegrin And Metalloproteinase-22 (ADAM22) was selected for further study. Knockout studies confirmed ADAM22 as a tamoxifen dependent SRC-1 target gene. Functional assays including migration, three dimensional cell culture and adhesion independence growth assays confirmed a role for ADAM22 in promoting a migratory, aggressive phenotype. Samples from two separate TMAs comprising over 1,000 patients confirmed that ADAM22 is associated with poor disease free survival in breast cancer patients. LGI1 is a naturally occurring neuropeptide which acts on an inhibitory manner on ADAM22 in the central nervous system. Using molecular modelling, a novel peptide mimetic targeting the disintegrin binding domain of ADAM22 was designed. Treatment with this peptide mimetic restored endocrine resistant cells to a less aggressive, sensitive phenotype, similar to the effect seen with knockdown of ADAM22. Moreover in an endocrine resistant xenograft model, treatment with the LGI1 mimetic significantly reduced primary and metastatic tumour burden in tamoxifen treated animals. We have used next-generation sequencing techniques to identify a novel therapeutic target in endocrine resistant, metastatic breast cancer. Rational drug design has been used to manufacture a therapeutic peptide against ADAM22. A combination of in vitro, in vivo and patient studies has confirmed a role for ADAM22 in metastatic breast cancer. Our novel peptide mimetic may form a future basis for targeting ADAM22 in endocrine resistant disease. Citation Format: Jarlath C Bolger, Damian McCartan, Damir Vareslija, Ailis Fagan, Christopher Byrne, Marie McIlroy, Peadar O9Gaora, Arnold D Hill, Leonie S Young. Global characterisation of the SRC-1 transcriptome and rational drug design results in the identification of a novel peptide targeting ADAM22 in endocrine resistance [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-05-02.

Abstract P2-05-13: Transcriptomic profiling of patient sequential tumours provides cutting edge view of global metastatic expression changes following endocrine therapy

Disease recurrence is a common problem in breast cancer and yet the mechanisms enabling tumour cells to evade therapy and colonise distant organs remain unclear. Here, for the first time, RNAsequencing has been performed on matched primary, nodal and liver metastatic tumours from three tamoxifen-treated patients following metastatic disease progression. Despite all primary tumours being of a luminal subtype and all cancers metastasising to the liver, the extent of patient heterogeneity at the gene level was striking. Less than 3% of the genes differentially expressed between sequential tumours were common to all patients. Larger divergence was observed between primary and liver tumours than between primary and nodal tumours, reflecting both the latency time to disease progression and the genetic impact of endocrine therapy. Furthermore, a xenograft model demonstrated the ability of tamoxifen to drive disease progression and establish distant metastatic disease in the endocrine resistant setting. Common functional pathways altered during metastatic, endocrine-resistant progression included ECM receptor interactions and focal adhesions. This novel global analysis highlights the influence of primary tumour biology in determining the transcriptomic profile of metastatic tumours, as well as the need for adaptations in cell-cell communications in order for tumour cells to successfully colonise distant host organs. Citation Format: Jean McBryan, Ailis Fagan, Damian McCartan, Jarlath C Bolger, Fiona T Bane, Christopher Byrne, Marie McIlroy, Arnold D Hill, Leonie S Young. Transcriptomic profiling of patient sequential tumours provides cutting edge view of global metastatic expression changes following endocrine therapy [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-05-13.

Abstract PD3-5: Survival benefit conferred by the androgen receptor is lost in aromatase inhibitor treated breast cancer

Aromatase Inhibitors have proven to be most effective in the treatment of post-menopausal breast cancer. Their mode of action is to inhibit the synthesis of estrogen (estrone) by the aromatase enzyme Cyp 19 thereby blocking ligand-dependent activation of the estrogen receptor. What has not been addressed to date is how cells that are deprived of estrogen, may potentially, adapt to the more androgenic environment resulting from long-term treatment with AI therapy. Research from our lab has identified the homeobox protein, HOXC11, to be an indicator of poor response to endocrine therapy and development of metastasis. To further our understanding of HOXC11 and its role in the development of endocrine-resistance and metastatic spread we undertook an RNA-seq experiment to identify its target genes in resistant breast cancer. This analysis identified PSAP, IFIT1 and HSP90AA1. Both PSAP (an androgen agonist) and HSP90AA1 (AR chaperone) are closely associated with AR which led to further investigation into the role of HOXC11 in the development of steroidal adaptability in Letrozole-resistant breast cancer. We hypothesize that HOXC11 regulated expression of PSAP results in oncogenic activation of AR in an AI resistant setting. Our findings have shown that AI-resistant cell lines in vitro have significantly elevated levels of AR and that loss of HOXC11 results in concommitant decrease in AR mRNA. In AI resistance expression of HOXC11 results in upregulation/stabilization of AR by PSAP thus enabling the tumour to adapt to use androgenic steroids for cell proliferation. The anti-androgen, Bicalutamide, reduces cell proliferation and cell motility in AI resistant cell lines. Survival analysis of AR in a TMA (n = 488) indicates that AR confers a survival benefit in the tamoxifen treated population. This protective effect is diminished in patients receiving AI therapy and is reflected in the altered Hazard Ratio of AR from the total population (HR: 0.485) to the AI treated cohort (HR: 1.197). Secreted PSAP was readily detectable in breast cancer patient serum and associates significantly with expression of HOXC11 in matched patient tissue (∼20). PSAP is associated with poor response to endocrine therapy and metastatic spread of prostate cancer and as it is secreted it could potentially be used to monitor patients on AI who might benefit from dual targeted therapy treatment. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr PD3-5.

P4-01-10: The Role of the Steroid Receptor Coactivator SRC1 and Its Functional Partner HOXC11 in the Development of Endocrine Resistant Breast Cancer.

Background: The steroid receptor coactivator; SRC1, has been well described in the development of endocrine resistant breast cancer. SRC1 associates with clinically aggressive tumours and promotion of distant metastasis. It directly interacts with the developmental transcription factor, HOXC11 and together they are found to strongly predict poor disease-free survival in breast cancer patients (hazard ratios: 5.79; P Materials and Methods: Cells which are resistant to tamoxifen (LY2s) have enhanced SRC1 and HOXC11 mRNA and protein expression in comparison to their endocrine sensitive parent cells (MCF-7s). ChIP-sequencing data for SRC1- and HOXC11- DNA interactions in conjunction with DNA microarray and RNA-sequencing data identified potential signalling targets at play in the LY2 model of endocrine resistance. Real-time analysis and flow cytometry confirmed these interactions at a transcriptional and protein level. These observations were further confirmed in primary breast cancer cultures using flow cytometry. Results: SRC1 and HOXC11 interactions are driven in tamoxifen treated LY2 resistant cells. Combined SRC1 ChIP-sequencing and expression array data analysed in conjunction with HOXC11 ChIP-sequencing and RNA-sequencing data reveal that the SRC1/HOXC11 transcriptional process can orchestrate the loss of luminal cell markers such as ERα, CD24 and PTCH1 whilst concomitantly upregulating mediators of tumourigenicity such as CD44 and MSI2. Primary breast cancer cultures confirm the loss of CD24 in tamoxifen resistant patients. In these patients, loss of CD24 was accompanied by loss of steroid receptor expression (ERα and PR) and by a gain of the basal marker CD44. Discussion: Here, we describe a new signalling pathway where SRC1 and HOXC11 regulate two distinct but complementary mechanisms to drive tumour adaptability. Silencing of luminal cell markers and a concomitant increase in a basal cell phenotype has the potential to alter the survival mechanism of breast cancer cells to evade targeted therapy. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-01-10.