ERα, HOXC11 and Breast Cancer: The Good, the Bad and the Ugly

Introduction: In Ireland breast cancer one of the most common causes of cancer related death for women. Upon diagnosis approximately two thirds of patients will present with a hormone receptor positive tumour (ER+). The ER pathway plays a pivotal role in breast cancer development and progression. Consequently endocrine therapies to block ER signalling are one of the most prevailing and effective treatment methods to date. However, a growing body of evidence suggests that within a heterogeneous breast tumour a sub-set of pluri-potent cells are capable of evading therapeutic treatment. Subsequently tumours metastasise and patients develop de novo or acquired resistance to endocrine therapies. Continuous activation of growth factor signalling pathways can provide tumours with altered proliferative and survival stimuli. Deregulated epigenetic machinery has also been implicated in the development of resistance. Translational studies from our group and others, has identified the steroid receptor co-activator SRC-1 to be master transcriptional regulator of breast cancer disease progression. Further studies identified the developmental protein and transcription factor HOXC11 as an interacting partner of SRC-1. Interactions between HOXC11 and SRC-1 in breast cancer cell lines and tissue have previously been reported. Their relationship has been shown to be important in tumour progression and metastasis in tamoxifen treated patients. Aberrant expression of HOXC11 has been associated with endocrine resistance. HOXC11 is known to drive the expression of the secreted protein S100P. Both HOXC11 and S100P have been described as tissue and serum biomarkers which can predict response to endocrine therapy. To date however, a functional role for HOXC11 in breast cancer has not been established. Hypothesis: The hypothesis of this thesis is to investigate a functional role for HOXC11 in an endocrine resistant breast cancer phenotype. It will investigate how this developmental transcription factor evades endocrine therapies in order to promote malignant transformation and it will explore the relationship between HOXC11 and ERa and the significance of this relationship at a clinical level. Results: Aberrant expression of HOXC11 we found confers a malignant phenotype similar to that of the known LY2 tamoxifen resistant cells. We found MCF7 endocrine sensitive breast cancer cells with overexpressed HOXC11 displayed a loss of differentiation and cellular polarity. HOXC11 alone induces proliferation, cell motility and anchorage independence. We observed a novel relationship between HOXC11 and the estrogen receptor alpha (ERa) and found that ERa could tame the aggressive malignant transformations induced by HOXC11. We observed that HOXC11 overexpression downregulates transcriptional regulation of ERa and propose a mechanism of epigenetic regulation in which HOXC11 may be silencing ERa activity. S100P a secreted protein and known HOXC11 target gene was also found to be a target of ERa and we observed HOXC11 and ERa compete for the regulation of S100P depending on the cellular microenvironment. Conclusion: HOXC11 can mediate malignant transformation and ERa in part can compete with HOXC11 to tame this aggressive phenotype. Tumour transformation is due in part to the altered therapeutic target. At a clinical level this is of significance as we have opened up new avenues in which resistant tumours could be targeted with alternative drug therapies

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

ADAM22/LGI1 complex as a new actionable target for breast cancer brain metastasis

Background: Metastatic breast cancer is a major cause of cancer-related deaths in woman. Brain metastasis is a common and devastating site of relapse for several breast cancer molecular subtypes, including oestrogen receptor-positive disease, with life expectancy of less than a year. While efforts have been devoted to developing therapeutics for extra-cranial metastasis, drug penetration of blood–brain barrier (BBB) remains a major clinical challenge. Defining molecular alterations in breast cancer brain metastasis enables the identification of novel actionable targets.Methods: Global transcriptomic analysis of matched primary and metastatic patient tumours (n = 35 patients, 70 tumour samples) identified a putative new actionable target for advanced breast cancer which was further validated in vivo and in breast cancer patient tumour tissue (n = 843 patients). A peptide mimetic of the target's natural ligand was designed in silico and its efficacy assessed in in vitro, ex vivo and in vivo models of breast cancer metastasis.Results: Bioinformatic analysis of over-represented pathways in metastatic breast cancer identified ADAM22 as a top ranked member of the ECM-related druggable genome specific to brain metastases. ADAM22 was validated as an actionable target in in vitro, ex vivo and in patient tumour tissue (n = 843 patients). A peptide mimetic of the ADAM22 ligand LGI1, LGI1MIM, was designed in silico. The efficacy of LGI1MIM and its ability to penetrate the BBB were assessed in vitro, ex vivo and in brain metastasis BBB 3D biometric biohybrid models, respectively. Treatment with LGI1MIM in vivo inhibited disease progression, in particular the development of brain metastasis.Conclusion: ADAM22 expression in advanced breast cancer supports development of breast cancer brain metastasis. Targeting ADAM22 with a peptide mimetic LGI1MIM represents a new therapeutic option to treat metastatic brain disease

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

Funder: Breast Cancer Ireland GR 14-0883Abstract: 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

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

Funder: Breast Cancer Ireland GR 14-0883Abstract: 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

S100β as a serum marker in endocrine resistant breast cancer

Abstract Background Endocrine therapy is standard treatment for estrogen receptor (ER)-positive breast cancer. However, its efficacy is limited by intrinsic and acquired resistance. Here the potential of S100β as a biomarker and inhibition of its signaling network as a therapeutic strategy in endocrine treated patients was investigated. Methods The expression of S100β in tissue and serum was assessed by immunohistochemistry and an enzyme-linked immunosorbent assay, respectively. The S100β signaling network was investigated in cell line models of endocrine resistance by western blot, PCR, immunoprecipitation, and chromatin-immunoprecipitation. Endocrine resistant xenografts and tumor explants from patients with resistant tumors were treated with endocrine therapy in the presence and absence of the p-Src kinase inhibitor, dasatinib. Results Tissue and serum levels of S100β were found to predict poor disease-free survival in endocrine-treated patients (n = 509, HR 2.32, 95% CI is 1.58–3.40, p < 0.0001 and n = 187, HR 4.009, 95% CI is 1.66–9.68, p = 0.002, respectively). Moreover, elevated levels of serum S100β detected during routine surveillance over the patient treatment period significantly associated with subsequent clinically confirmed disease recurrence (p = 0.019). In vivo studies demonstrated that endocrine treatment induced transcriptional regulation of S100β which was successfully disrupted with tyrosine kinase inhibition. In endocrine resistant xenografts and tumor explants from patients with endocrine resistant breast cancer, combined endocrine and dasatinib treatment reduced tumor proliferation and down-regulated S100β protein expression in comparison to endocrine treatment alone. Conclusions S100β has potential as a new surveillance tool for patients with ER-positive breast cancer to monitor ongoing response to endocrine therapy. Moreover, endocrine resistant breast cancer patients with elevated S100β may benefit from combined endocrine and tyrosine-kinase inhibitor treatment. Trial registration ClinicalTrials.gov,  NCT01840293 ). Registered on 23 April 2013. Retrospectively registered

S100β as a serum marker in endocrine resistant breast cancer

Background: Endocrine therapy is standard treatment for estrogen receptor (ER)-positive breast cancer. However, its efficacy is limited by intrinsic and acquired resistance. Here the potential of S100 beta as a biomarker and inhibition of its signaling network as a therapeutic strategy in endocrine treated patients was investigated. Methods: The expression of S100 beta in tissue and serum was assessed by immunohistochemistry and an enzyme-linked immunosorbent assay, respectively. The S100 beta signaling network was investigated in cell line models of endocrine resistance by western blot, PCR, immunoprecipitation, and chromatin-immunoprecipitation. Endocrine resistant xenografts and tumor explants from patients with resistant tumors were treated with endocrine therapy in the presence and absence of the p-Src kinase inhibitor, dasatinib. Results: Tissue and serum levels of S100 beta were found to predict poor disease-free survival in endocrine-treated patients (n = 509, HR 2.32, 95% CI is 1.58-3.40, p &amp;lt; 0.0001 and n = 187, HR 4.009, 95% CI is 1.66-9.68, p = 0.002, respectively). Moreover, elevated levels of serum S100 beta detected during routine surveillance over the patient treatment period significantly associated with subsequent clinically confirmed disease recurrence (p = 0.019). In vivo studies demonstrated that endocrine treatment induced transcriptional regulation of S100 beta which was successfully disrupted with tyrosine kinase inhibition. In endocrine resistant xenografts and tumor explants from patients with endocrine resistant breast cancer, combined endocrine and dasatinib treatment reduced tumor proliferation and down-regulated S100 beta protein expression in comparison to endocrine treatment alone. Conclusions: S100 beta has potential as a new surveillance tool for patients with ER-positive breast cancer to monitor ongoing response to endocrine therapy. Moreover, endocrine resistant breast cancer patients with elevated S100 beta may benefit from combined endocrine and tyrosine-kinase inhibitor treatment