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AR ER protein densitometry and dose response to PI3K/mTORi

Gene changes in MCF7aro. The impact of cyp19 overexpression on SGK3 expression in an independent study. Impact of siRNA-AR/ER on SGK3 mRNA

Supplemental tables 1& 2

Impact of SiSGK3 in ZRaroLetR. MTS following siSGK3 under various hormonal stimuli.

Characterisation of ZR75aro-LetR. Validation of ER AR siRNA

Correlation between CYP19 amplification and SGK3 expression.

AR and ER binding events in the proximal promoters of SGK3, MYBL1, GREB1 and PKIB.

Supplemental methods

Divergent roles for androgen receptor (AR) in breast cancer have been reported. Following aromatase inhibitor (AI) treatment, the conversion of circulating androgens into estrogens can be diminished by >99%. We wished to establish whether the steroid environment can dictate the role of AR and the implications of this for subsequent therapy. This study utilizes models of AI resistance to explore responsiveness to PI3K/mTOR and anti-AR therapy when cells are exposed to unconverted weak androgens. Transcriptomic alterations driven by androstenedione (4AD) were assessed by RNA-sequencing. AR and estrogen receptor (ER) recruitment to target gene promoters was evaluated using ChIP, and relevance to patient profiles was performed using publicly available data sets. Although BEZ235 showed decreased viability across AI-sensitive and -resistant cell lines, anti-AR treatment elicited a decrease in cell viability only in the AI-resistant model. Serum and glucocorticoid-regulated kinase 3 (SGK3) and cAMP-dependent protein kinase inhibitor β (PKIB) were confirmed to be regulated by 4AD and shown to be mediated by AR; crucially, reexposure to estradiol suppressed expression of these genes. Meta-analysis of transcript levels showed high expression of SGK3 and PKIB to be associated with poor response to endocrine therapy (HR = 2.551, P = 0.003). Furthermore, this study found levels of SGK3 to be sustained in patients who do not respond to AI therapy. This study highlights the importance of the tumor steroid environment. SGK3 and PKIB are associated with poor response to endocrine therapy and could have utility in tailoring therapeutic approaches.

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.

Supplementary Figure S1 from Coassociation of Estrogen Receptor and p160 Proteins Predicts Resistance to Endocrine Treatment; SRC-1 is an Independent Predictor of Breast Cancer Recurrence

PDF file, 14KB, Supplementary Table 1: Demographics of patients according to AIB1 status.

PDF file, 320KB, Supplementary Figures 1-9 Supplementary Figure 1: Expression levels of aromatase in our cell lines and aromatase activity assay Supplementary Figure 2: Proliferation of Aro and LetR in response to treatment Supplementary Figure 3: Kaplan Meier estimates of disease free survival in the entire ER positive population Supplementary Figure 4:Interactions between ERalpha and AIB1 in letrozole sensitive Aro cells Supplementary Figure 5: Analysis of protein levels in Anastrozole resistant cell line (ANAR) Supplementary Figure 6: ChiP showing recruitment of ERalpha and AIB1 to distal region promoters Supplementary Figure 7: Basal protein levels of Myc and cyclinD1 protein and basal mRNA levels of pS2 Supplementary Figure 8:Effect of Lapatinib treatment on pS2 and Myc mRNA levels Supplementary Figure 9: Pathway analysis of ERalpha-AIB1 target genes.

Supplementary Table S4: Patient information for patients A-L

Purpose: The use of aromatase inhibitors (AI) in the treatment of estrogen receptor (ER)-positive, postmenopausal breast cancer has proven efficacy. However, inappropriate activation of ER target genes has been implicated in the development of resistant tumors. The ER coactivator protein AIB1 has previously been associated with initiation of breast cancer and resistance to endocrine therapy.Experimental Design: Here, we investigated the role of AIB1 in the deregulation of ER target genes occurring as a consequence of AI resistance using tissue microarrays of patients with breast cancer and cell line models of resistance to the AI letrozole.Results: Expression of AIB1 associated with disease recurrence (P = 0.025) and reduced disease-free survival time (P = 0.0471) in patients treated with an AI as first-line therapy. In a cell line model of resistance to letrozole (LetR), we found ERα/AIB1 promoter recruitment and subsequent expression of the classic ER target genes pS2 and Myc to be constitutively upregulated in the presence of both androstenedione and letrozole. In contrast, the recruitment of the ERα/AIB1 transcriptional complex to the nonclassic ER target cyclin D1 and its subsequent expression remained sensitive to steroid treatment and could be inhibited by treatment with letrozole. Molecular studies revealed that this may be due in part to direct steroid regulation of c-jun-NH2-kinase (JNK), signaling to Jun and Fos at the cyclin D1 promoter.Conclusion: This study establishes a role for AIB1 in AI-resistant breast cancer and describes a new mechanism of ERα/AIB1 gene regulation which could contribute to the development of an aggressive tumor phenotype. Clin Cancer Res; 18(12); 3305–15. ©2012 AACR.

Supplementary Figures S1-7. Supplementary Figure S1: Gene expression profiles of classic biomarkers in matched tumour samples from 3 patients. Supplementary Figure S2: Network map showing the enriched KEGG pathways from upregulated differentially expressed genes (DEGs). Supplementary Figure S3: Network map showing the enriched KEGG pathways from downregulated differentially expressed genes (DEGs). Supplementary Figure S4: Protein expression in matched primary and metastatic tissue from endocrine treated breast cancer patients. Supplementary Figure S5: Phosphorylated mTOR signaling in endocrine resistant breast cancer tumours. Supplementary Figure S6: Interaction plot showing functional roles for 21 genes commonly upregulated in metastatic liver tumours of all three patients. Supplementary Figure S7: Genes upregulated in liver metastases are also strongly expressed in breast metastases to the brain.

Purpose: This study investigates the role of the p160 coactivators AIB1 and SRC-1 independently, and their interactions with the estrogen receptor, in the development of resistance to endocrine treatments.Experimental Design: The expression of the p160s and the estrogen receptor, and their interactions, was analyzed by immunohistochemistry and quantitative coassociation immunofluorescent microscopy, using cell lines, primary breast tumor cell cultures, and a tissue microarray with breast cancer samples from 560 patients.Results: Coassociation of the p160s and estrogen receptor α was increased in the LY2 endocrine-resistant cell line following treatment with tamoxifen in comparison with endocrine-sensitive MCF-7 cells. In primary cultures, there was an increase in association of the coactivators with estrogen receptor α following estrogen treatment but dissociation was evident with tamoxifen. Immunohistochemical staining of the tissue microarray revealed that SRC-1 was a strong predictor of reduced disease-free survival (DFS), both in patients receiving adjuvant tamoxifen treatment and untreated patients (P < 0.0001 and P = 0.0111, respectively). SRC-1 was assigned a hazard ratio of 2.12 using a Cox proportional hazards model. Endocrine-treated patients who coexpressed AIB1 with human epidermal growth factor receptor 2 had a significantly shorter DFS compared with all other patients (P = 0.03). Quantitative coassociation analysis in the patient tissue microarray revealed significantly stronger colocalization of AIB1 and SRC-1 with estrogen receptor α in patients who have relapsed in comparison with those patients who did not recur (P = 0.026 and P = 0.00001, respectively).Conclusions: SRC-1 is a strong independent predictor of reduced DFS, whereas the interactions of the p160 proteins with estrogen receptor α can predict the response of patients to endocrine treatment.

PDF file, 55KB, Supplementary table 2: Rate of incidence of disease recurrence according to AIB1, HER2, tumour grade, tumour size and nodal status in breast cancer patients treated with first-line AI.

PDF file, 213KB, Supplementary Table 3b ER/AIB1 overlap genelist - partial ERE.

Supplementary Materials and Methods

Supplementary Figures 1-3, Table 1 from Interaction of Developmental Transcription Factor HOXC11 with Steroid Receptor Coactivator SRC-1 Mediates Resistance to Endocrine Therapy in 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. Cancer Res; 72(1); 220–9. ©2011 AACR.

PDF file - 245K

Purpose: There is strong epidemiologic evidence indicating that estrogens may not be the sole steroid drivers of breast cancer. We hypothesize that abundant adrenal androgenic steroid precursors, acting via the androgen receptor (AR), promote an endocrine-resistant breast cancer phenotype. Experimental Design: AR was evaluated in a primary breast cancer tissue microarray (n = 844). Androstenedione (4AD) levels were evaluated in serum samples (n = 42) from hormone receptor–positive, postmenopausal breast cancer. Levels of androgens, progesterone, and estradiol were quantified using LC/MS-MS in serum from age- and grade-matched recurrent and nonrecurrent patients (n = 6) before and after aromatase inhibitor (AI) therapy (>12 months). AR and estrogen receptor (ER) signaling pathway activities were analyzed in two independent AI-treated cohorts. Results: AR protein expression was associated with favorable progression-free survival in the total population (Wilcoxon, P < 0.001). Pretherapy serum samples from breast cancer patients showed decreasing levels of 4AD with age only in the nonrecurrent group (P < 0.05). LC/MS-MS analysis of an AI-sensitive and AI-resistant cohort demonstrated the ability to detect altered levels of steroids in serum of patients before and after AI therapy. Transcriptional analysis showed an increased ratio of AR:ER signaling pathway activities in patients failing AI therapy (t test P < 0.05); furthermore, 4AD mediated gene changes associated with acquired AI resistance. Conclusions: This study highlights the importance of examining the therapeutic consequences of the steroid microenvironment and demonstrable receptor activation using indicative gene expression signatures.

In inclusive education, Disability Studies is a framework that has been useful for identifying gaps in theory, in practice and the spaces in between. Disability Studies in Education also provides n...

Narrative assessment is a strengths-based approach that stories children’s learning and makes visible teaching and relationships that are critical to quality education. This case study explores narrative assessment in the learning area of literacy in a New Zealand primary school. Listening to a child’s voice means, he can be an active participant in his learning and engage in the rich curricular opportunities alongside his peers. As his capabilities and communicative skills are made visible, meaningful relationships develop within the classroom community. In this case study, narrative assessment supports authentic belonging in a culture where care and respect are valued.

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.

This article describes the Ministry of Education’s Inclusive Education Capability Building Project (2013-2014). Project members were tasked with creating resources for professional development that would be used to grow inclusive practice in New Zealand schools. This article also shares the learning journey of some members of the project team as they engaged in the inquiry process around inclusive practice. A key understanding for this project was that all students can be successful learners and belong in The New Zealand Curriculum (Ministry of Education, 2007), also referred to in this article as NZC.

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.

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. Cancer Res; 74(9); 2533–44. ©2014 AACR.

Introduction AI’s are the front line treatment for endocrine postmenopausal breast cancer. However, response rates vary from 35%–70% and recurrence occurs in almost all advanced disease. In order to identify a target for overcoming AI treatment resistance, research has turned to the most widely expressed sex steroid receptor in breast cancer the AR. AI therapies, by their mechanism of action, create an unopposed androgenic steroid environment. 4AD is the major circulating steroid in postmenopausal women and AI therapy prevents it’s conversion to estrogens. We hypothesise that cancer cells adapt to utilise 4AD to promote resistance and survival in AI resistance. Material and methods Molecular changes in a breast cancer cell model which have transitioned from AI sensitive (MCF7 cells) to AI resistant (LetR cells) were assessed using RNA sequencing. Alterations in the steroid microenvironment occur as a result of AI therapy and responses to these steroids was confirmed using MTS, mammosphere and invasion assays. Mass spectrometry (LC–MS/MS) analysis identified androgen-mediated, AR protein interactions.The clinical relevance of AR in AI resistance was also evaluated in a cohort of primary breast cancer patients (n=375). Results and discussions LetR cell growth is AR dependent as the anti-AR drug Enzalutamide inhibits cell growth. Furthermore, Enzalutamide abrogates mammosphere formation and self-renewal of LetR cells. We have found LetR cells to be responsive to 4AD, and treatment with this steroid drives a more aggressive phenotype in vitro. RNA sequencing data highlighted a non-canonical cytoplasmic AR signalling mechanism. Our research suggests that 4AD initiates rapid second messenger signalling in the AI resistant setting. LC–MS/MS analysis identified androgen-mediated, AR protein interactors unique to our resistant model particularly associated with cell-cell adhesion and invasion. Evaluation of AR protein in a cohort of primary breast cancer patients found AR expression in 90% of patients, with 25% expressing high intensity AR staining. Analysis is ongoing into this subset and its association with clinical pathological parameters. Conclusion AR and its ligand 4AD may be a significant driving factor in AI resistance and may play a role in facilitating a more invasive phenotype. Further investigations into AR interactors identified will help elucidate mechanisms of resistance to AI therapy, and these novel AR protein partners may aid the identification of patients who would benefit from anti-AR therapy.

The heterogenous nature of breast cancer, exemplified by a wide range of subtype classifications, has been well documented. The identification of the novel molecular apocrine breast tumours (ER-, PR-ve, HER2-ve, AR+), with a gene expression profile reflective of ER+ luminal breast cancer, has led to the proposition of an alternative role for the androgen receptor as a pseudo ER. This proposition was borne out of global expression profiling and AR recruitment of molecular apocrine cells, demonstrating that more than 50% of AR binding to the genome mirrors the pattern of ER binding suggesting that in the absence of ER, AR can mimic ER in its DNA binding capabilities. Aromatase Inhibitor (AI) therapy is the gold standard first line therapy for postmenopausal breast cancer. AIs function by abrogating the activity of the enzyme Cyp 19 (aromatase), which is responsible for converting circulating androgens to estrogen. By preventing this conversion step, this eradicates the main steroid driving breast cancer growth and in turn results in a more androgenic environment. We hypothesise that AR may also act as a pseudo ER in our AI resistant (MCF7-letrozole resistant) cells. Preliminary data demonstrates that AR is recruited to some but not all classical ER target genes as well as some AR target genes in MCF7-LetR cells, therefore highlighting the potential of AR to act as an oncogene in this setting. Analysis of AR protein expression also demonstrates higher levels of AR in AI resistant cells versus sensitive cells. In the evaluation of the responsiveness of a range of breast cancer cell lines to the AR antagonist Bicalutamide, it was noted that our AI resistant cells demonstrated responsiveness to Bicalutamide treatment. RNA sequencing experiments following AR knockdown in AI resistant cells also provide crucial information as to the key genes differentially regulated by AR. Given that resistance to AI therapy is an emerging clinical issue, further elucidation of the potential mechanisms of AI resistance and potential AR targets is highly warranted. Citation Format: Leonie Young, Laura Creevey, Azlena Ali, Arnold DK Hill, Marie McIlroy. Investigation into the oncogenic potential of the androgen receptor in aromatase inhibitor resistant breast cancer [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 P3-05-21.

Breast cancer has the highest incidence and second highest mortality rate among all cancers in women worldwide (1). The gold standard treatment for post-menopausal breast cancer is aromatase inhibitor (AI) therapy. Unfortunately, resistance is inevitable in about 30-60% of these patients (2). Research from our lab has previously identified a transcription factor, HOXC11, to be associated with breast cancer resistance and metastases (3). Prosaposin (PSAP) was then identified as a putative HOXC11 target gene in the AI resistant setting. PSAP is a secreted protein that also plays a role in metastatic prostate cancer in both a ligand dependent and independent fashion (4,5). We have previously shown PSAP to be an androgen responsive gene that can upregulate androgen receptor (AR) expression in a ligand dependent manner in AI resistance. An AR antagonist was effective at slowing down cell proliferation in AI resistant cells in vitro . However, since PSAP can also potentially act in a ligand independent fashion, this current study is focused on evaluating the role of PSAP in PI3K pathway activation in the development of AI resistance. Treatment of cells with recombinant PSAP protein (rhPSAP) resulted in increasing expression of p-AKT in a dose-dependent manner. To target this pathway of resistance, we used a pan-class PI3K/mTOR inhibitor BEZ235 on our AI resistant cells. Functional studies with BEZ235 treatment showed significant reduction in both cell motility as well as cell proliferation. Treatment of AI resistant cells with BEZ235 resulted in decreased expression of p-AKT, with little or no effect on AR expression. These findings suggest that to prevent resistance to AI therapy, combination treatment regimens including AR antagonists plus a PI3K inhibitor may ensure sustained response to therapy. References: 1. Servick K. Breast Cancer: a World of Differences. Science Magazine. Special Issue. March 2014 2. Kazi AA et al. Breast Nonhypoxic regulation and role of hypoxia-inducible factor 1 in aromatase inhibitor resistant breast cancer. Cancer Research. 2014; 16:R15 3. McIlroy M et al. Cancer Res.2010;70(4);1585-94 4. Koochekpour Set al. Prosaposin is an AR-target gene and its neurotrophic domain upregulates AR expression and activity in prostate stromal cells. J Cell Biochem. 2008; 104: 2272–2285. 5. Koochekpour S et al. Prosaposin is a novel androgen-regulated gene in prostate cancer cell line LNCaP. J Cell Biochem. 2007; 10: 631–641. Citation Format: Leonie S Young, Azlena Ali, Laura Creevey, Fiona Bane, Wan Afif Marzuki, Arnold DK Hill, Marie McIlroy. PI3K pathway as a novel target in androgen-driven aromatase inhibitor resistant breast cancer [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 P3-05-22.

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 O'Gaora, 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.

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. Cancer Res; 72(1); 220–9. ©2011 AACR.

In estrogen receptor positive breast cancer, endocrine therapy is the standard line of treatment and even though it results in reduced recurrence and mortality, a significant number of patients will eventually relapse. Early detection of metastatic disease would significantly enhance management of endocrine resistant breast cancer. Here we investigate the potential of the calcium-binding protein S100β as a predictive biomarker and monitoring tool in endocrine treated patients. Furthermore, the efficacy of S100β inhibition as therapy in patients that fail first line endocrine therapy was examined. Primary tumor tissue expression of S100β protein was assessed in a retrospective cohort of endocrine treated breast cancer patients. Expression of S100β indicated a significant reduction in time to disease recurrence (n=509, Wilcoxon p S100β protein is also detectable in serum of breast cancer patients and elevated levels of serum S100β prior to removal of primary tumor is associated with poor disease free survival in endocrine treated patients (n=190, Wilcoxon p=0.0367, hazard ratio 2.68, 95% C.I. is 1.12 to 6.41, p=0.026, Cox proportional hazard model). Serum levels of S100β are significantly reduced after primary tumor resection (n=19, p=0.0003). In serial samples taken during the treatment period, elevated levels of S100β significantly associated with disease progression and with the emergence of metastatic disease (p=0.0031). In an in-vivo model of endocrine resistant breast cancer, raised levels of S100β marked the emergence of disease progression. The oncogene steroid receptor co-activator 1 (SRC1) and its interaction with homeobox protein (HOXC11) regulates S100β production in a src-kinase dependent manner. Here, src-kinase inhibition reduced tumor burden with a concomitant reduction in serum S100β. We also observed a marked reduction in expression of proliferative marker Ki67 and S100β protein following the treatment of endocrine resistant patient tumor explants with src-kinase inhibitor. Associations between elevated levels of serum S100β and subsequent disease progression in endocrine treated patients, suggests S100β as a monitoring tool for early detection of disease progression. Additionally high level of S100β can be used as a potential companion diagnostic tool for stratifying patients on endocrine therapy suitable for treatment with small molecule src-kinase inhibitor. Citation Format: Charmsaz S, Hughes É, Byrne C, Bane F, Tibbitts P, McIlroy M, Hill AD, Young LS. S100β as a predictive biomarker and monitoring tool 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 P3-04-03.

Aromatase inhibitors (AI) are the recommended first line therapy used to treat postmenopausal breast cancer. These compounds work by inhibiting the aromatase enzyme thus preventing the conversion of circulating androgens to estrogen; as a consequence they alter the tumour intracrinology and create an unopposed highly androgenic steroid environment. We have previously reported androgen receptor (AR) protein levels to be up-regulated in an AI resistant cell line (LetR cells), and subsequently identified a mechanism by which AR drives a more aggressive phenotype in AI resistant breast cancer [1]. In this current study, LetR cells are shown to be responsive to the weak androgen androstenedione (4AD), and treatment with this steroid drives an invasive phenotype in vitro. In support of this data, clinical studies have also reported increases in the serum levels of 4AD in patients that recur on AI therapy [2]. In the canonical pathway, androgens bind to the AR which results in a conformational change to an active state. Non-canonical AR activation occurs when ligand-transformed AR interacts with molecular partners within the cytosol to induce rapid intracellular signalling cascades. These events do not depend upon AR mediated gene transcription and occur extremely quickly within a manner of minutes [3]. In vitro studies using western blot analysis and co-localisation experiments have indicated 4AD treatment potentiates a resistant phenotype through non-genomic AR actions initiated by rapid second messenger signalling within the cytoplasm. Mass spectrometry (LC–MS/MS) analysis has identified androgen-mediated, rapid cytoplasmic AR protein interactions, resulting in the identification of AR partners unique to our resistant model. Of note, evaluation of AR protein and p-ERK1/2 in a cohort of primary breast cancer patients (n=363) demonstrated that high levels of cytoplasmic AR significantly diminished survival in ER+ PR- patients (p=0.023, Fisher's exact). Elevated pERK1/2 when concomitant with increased levels of cytoplasmic AR result in a significant decrease in the period of disease free survival (p=0.018). Further investigations into these AR interactors will help elucidate mechanisms of resistance to AI therapy, and in turn these novel AR protein partners will aid the identification of patients who would benefit from anti-AR therapy. References 1. Ali, A., et al., Prosaposin activates the androgen receptor and potentiates resistance to endocrine treatment in breast cancer. Breast Cancer Research : BCR, 2015. 17(1): p. 123. 2. Elliott, K.M., et al., Effects of aromatase inhibitors and body mass index on steroid hormone levels in women with early and advanced breast cancer. British Journal of Surgery, 2014. 101(8): p. 939-948. 3. Foradori, C.D., M.J. Weiser, and R.J. Handa, Non-genomic actions of androgens. Front Neuroendocrinol, 2008. 29(2): p. 169-81. Citation Format: Bleach RM, Creevey L, Hill ADK, Madden S, Young LS, Pennington SR, McIlroy M. Androstenedione initiates rapid non-genomic signalling mediated by cytoplasmic androgen receptor in aromatase inhibitor resistant breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P4-04-13.

Cytochrome P450 1B1 (CYP1B1) mRNA is constitutively expressed in most normal extra-hepatic tissues; however the protein is not detectable in these tissues but is expressed in a wide variety of tumors. CYP1B1 is responsible for the activation of a number of carcinogens present in tobacco smoke and food. A surgical model of rat esophageal tumorigenesis, promoted by gastric or duodenal reflux was used to determine CYP1B1 expression in premalignant esophageal tissue. Immunohistochemistry was performed using a modified amplified fluorescein tyramide protocol. CYP1B1 was not observed in normal esophageal mucosa, submucosa, or muscularis mucosa. Animals exposed to gastric reflux developed mild hyperplasia. Varying degrees of hyperplasia were observed in the duodenal reflux group. All regions of hyperplasia showed moderate or strong CYP181 immunoreactivity. Duodenal reflux induced a small number of premalignant changes: immunoreactivity was absent from the epithelium of squamous dysplasia (0/10), Barrett's esophagus (0/7), and majority of dysplastic Barrett's esophagus (1/4). Moderate or strong immunoreactivity was observed in the majority (7/8) of squamous cell carcinomas (SCCs) in situ. Immunoreactivity was also observed in the lamina propria and submucosa in association with inflammation, regardless of the severity of inflammation. The expression of CYP1B1 in hyperplasia, SCCs in situ, or in association with inflammation may increase the production of carcinogenic metabolites, which may promote esophageal tumorigenesis. (C) 2008 Wiley-Liss, Inc.

Cyclooxygenase-2 (COX-2) is associated with breast tumour progression. Clinical and molecular studies implicate human epidermal growth factor receptor 2 (HER2) in the regulation of COX-2 expression. Recent reports raise the possibility that HER2 could mediate these effects through direct transcriptional mechanisms. The relationship between HER2 and COX-2 was investigated in a cohort of breast cancer patients with or without endocrine treatment. A tissue microarray comprising tumours from 560 patients with 10-year follow-up was analysed for HER2, ERK1/2, polyoma enhancer activator 3 (PEA3) and COX-2 expression. Subcellular localisation of HER2 was assessed by immunofluorescence and confocal microscopy. Expression of markers examined was analysed in relation to classic clinicopathological parameters and disease-free survival in the presence and absence of tamoxifen. COX-2 expression associated with both membranous and nuclear expression of HER2 (P=0.0033 and P

Introduction HOX 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. Methods RNA-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. Results PSAP 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). Conclusion The 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. Electronic supplementary material The online version of this article (doi:10.1186/s13058-015-0636-6) contains supplementary material, which is available to authorized users.

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.

Resistance to aromatase inhibitors (AIs) is a major clinical problem in the treatment of estrogen receptor (ER)-positive breast cancer. In two breast cancer cell line models of AI resistance, we identified widespread DNA hyper- and hypomethylation, with enrichment for promoter hypermethylation of developmental genes. For the homeobox gene HOXC10, methylation occurred in a CpG shore, which overlapped with a functional ER binding site, causing repression of HOXC10 expression. Although short-term blockade of ER signaling caused relief of HOXC10 repression in both cell lines and breast tumors, it also resulted in concurrent recruitment of EZH2 and increased H3K27me3, ultimately transitioning to increased DNA methylation and silencing of HOXC10. Reduced HOXC10 in vitro and in xenografts resulted in decreased apoptosis and caused antiestrogen resistance. Supporting this, we used paired primary and metastatic breast cancer specimens to show that HOXC10 was reduced in tumors that recurred during AI treatment. We propose a model in which estrogen represses apoptotic and growth-inhibitory genes such as HOXC10, contributing to tumor survival, whereas AIs induce these genes to cause apoptosis and therapeutic benefit, but long-term AI treatment results in permanent repression of these genes via methylation and confers resistance. Therapies aimed at inhibiting AI-induced histone and DNA methylation may be beneficial in blocking or delaying AI resistance.

As New Zealand teachers in the twenty first century we work within complex and contradictory environments. Political actions continue to focus on neoliberal approaches to education. The introduction of national standards dominates the primary school classroom.