Adk Hill

Coassociation of ERα and p160 proteins predicts resistance to endocrine treatment; SRC-1 is an independent predictor of breast cancer recurrence.

CTRC-AACR San Antonio Breast Cancer Symposium: 2008 Abstracts Abstract #3032 The p160 coactivators AIB1 and SRC-1 are known to play a critical role in modulating transcription in breast cancer cells in conjunction with ligand-bound estrogen receptor. The interactions of the p160 proteins with this nuclear receptor are also important in the development of resistance to endocrine treatments. Using quantitative coassociation immunofluorescent microscopy, the colocalisation of the p160s and ERα was increased in the LY2 endocrine resistant cell line following treatment with the anti-estrogen tamoxifen in comparison to the endocrine sensitive MCF-7 cell line. In cell cultures derived from patient tumours at the time of primary surgery prior to treatment, there was an increase in association of the coactivators with ERα following treatment with estrogen but dissociation was evident in the presence of tamoxifen. Immunohistochemical staining of a tissue microarray, constructed from 560 breast cancer patients, revealed that SRC-1 was a strong predictor of reduced disease-free survival, both in patients receiving adjuvant tamoxifen treatment and untreated patients (p<0.0001 and p=0.0111 respectively). AIB1 was not a significant independent predictor of disease recurrence. SRC-1 was assigned a hazard ratio of 2.12 when survival analysis using a Cox proportional hazards model was applied. Quantitative coassociation analysis of the p160 coactivators with ERα in the patient TMA revealed significantly stronger colocalisation of SRC-1 and ERα in patients who are known to have relapsed than those patients who did not recur (p=0.00001). This data suggests SRC-1 is pivotal in tumour aggressiveness and is a powerful predictor of progression of disease in breast cancer patients. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3032.

Abstract P2-05-03: Whole genome transcriptome analysis of sequential breast to brain metastasis uncovers new signalling pathways and druggable targets

The occurrence of brain metastasis (BM) in breast cancer (BC) is currently on the rise across all molecular subtypes with 10-30% reported incidence. The need to uncover the mechanisms underlying this clinically devastating complication is apparent, and in the current study we sought to identify BC cell mediators of BM. In our cohort of metastatic patients (n=196) we found that BM developed in 13% of the cases. Despite the previous reports of negative ER status being a risk factor for BM, the ER+ve patients accounted for 42% of all diagnosed BM. To elucidate the gene alterations required for successful colonisation of the brain we undertook RNA sequencing (RNA-seq) of sequential breast to brain metastasis of known receptor status (n=7).This study presents the first whole transcriptome next-generation RNA-seq analysis of resected BM and their matching primary breast tumours. We identified 500 differentially expressed genes (DEGs) ( ±1.5), accounting for those that were both upregulated and downregulated in BM compared to the primary. Analysis of protein-coding genes identified collective ER-specific metastatic pathways. Additionally, common functional pathways altered included ECM, cell adhesion and neuronal differentiation. Our analysis of the BM transcriptomic landscape and verification in cell line models that preferentially metastasise to the brain has unravelled a complex network of driver genes, cooperating with stromal derived factors, responsible for the organ-specific behaviour of the metastatic cells. Genes such as ANTRX1, THBS2, FAP, VCAN and TIMP2 were found to be part of the invasion and migration network that drives the extravasation of the BM cells. Furthermore, an EMT stemness signalling network driven by ANTRX1and WNT pathway driven RUNX was prominent in the cells acquiring the ability to migrate to the brain. Additional work is being carried out on uncovering the adaptations that re-activate the dormant brain metastatic cells and the contribution of the neuronal niche in the facilitating the colonisation by the MBC. This study highlights the requirement of unique gene sets for the invasion, migration and colonisation to the brain and that functional characterisation of the DEGs will enable the identification of novel molecular targets for prevention and treatment of breast cancer BM. Citation Format: Vareslija D, Fagan A, Buckley P, Farrell M, Hill A, Young L. Whole genome transcriptome analysis of sequential breast to brain metastasis uncovers new signalling pathways and druggable targets. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-05-03.

Abstract P3-04-17: Global transcriptional repression by the coactivator SRC-1 mediates disease progression in treatment-resistant breast cancer

Despite the effectiveness of endocrine therapy in treating estrogen receptor (ER) positive breast cancer, nearly 40% of breast cancer patients may develop resistance which can lead to metastatic disease progression. Steroid receptor co-activator-1 (SRC-1), a key regulator of ER signalling, is overexpressed in 35% of breast cancer patients and is strongly associated with the development of endocrine resistant metastasis(1). Recent studies highlighted the ability of SRC-1 to also act as transcriptional repressor. SRC-1 can therefore bi-directionally regulate gene expression to promote the resistant phenotype (2). This study employed a genome-wide multi-omics sequencing approach to determine the SRC-1 repression signature in endocrine resistance and elucidate the mechanism by which SRC-1 mediates this repression. RNA-sequencing identified 736 genes significantly downregulated by SRC-1, with common functional pathways such as differentiation, cell morphogenesis and extracellular matrix enriched in the gene set. Parallel global methylation sequencing analysis revealed distinct differentially methylated regions specific to SRC-1 repressed target genes. Mechanistic studies in endocrine resistant cells revealed a role for methylation proteins, DNMTs and MBD, in SRC-1 directed repression. Through combined analysis of our global sequencing data we identified a network hub of five differentiation genes directly repressed by SRC-1. High expression of this signature predicts enhanced recurrence free survival in tamoxifen treated patients (n=335, p=0.032). A reduction in expression of these genes was shown to have functional output on proliferation, migration and mammosphere formation. Finally, we use tumour explant models to show that targeting DNA methylation can be used to reverse the SRC-1 driven suppression of this differentiation hub. Here we report a novel mechanism by which SRC-1 may be driving endocrine resistant tumorigenesis. Our genome-wide discovery approach revealed a global epigenetic re-programming pathway whereby concerted differential DNA methylation is potentiated by the activation of SRC-1 in the presence of tamoxifen in the endocrine resistant setting. This study suggests that therapeutic strategies of combined targeted epigenetic therapy with estrogen deprivation could be a successful strategy to prevent acquired resistance to endocrine therapy. Citation Format: Ward E, Vareslija D, Fagan A, Hill A, Young L. Global transcriptional repression by the coactivator SRC-1 mediates disease progression in treatment-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 P3-04-17.

Abstract P3-04-16: Steroid receptor co-activator 1 mediation of cancer cell reprogramming in endocrine resistant breast cancer

Cancer cells undergo dynamic and frequently reversible modifications and this cellular plasticity permits reprogramming in response to long term endocrine treatment. A well-established theory, the cancer stem cell theory; suggests that within an ER positive tumour cell population are a small number of stem cells which are capable of infinite self-renewal and are insensitive to treatment with standard endocrine regimes. However, emerging reports that pluripotent cells can be generated from adult somatic cells (Takahashi, 2007) alludes to an alternate mechanism for cancer cells to reprogramme and evade endocrine treatment. To investigate the role of tamoxifen in promoting cellular reprogramming and endocrine resistance in breast cancer, we isolated single luminal A breast cancer CD24+CD44- clones from endocrine sensitive MCF-7 cells. The clones were expanded in the presence and absence of tamoxifen. Extensive profiling of cells following long-term exposure to tamoxifen revealed a resistant phenotype similar to that reported in other established models. Steroid receptor co-activator 1 (SRC-1) is a master regulatory protein which has been shown to be central to the development of endocrine resistance. The mechanisms of which are still poorly understood (Walsh et al ., 2012). Utilising genome-wide transcriptomic sequencing this study investigated whether SRC-1 can regulate transcriptional networks which mediate reprogramming in individual cells to induce survival adaptability and drug resistance in breast cancer. RNA-sequencing was carried out in a model of endocrine resistance (shNon-Targeting versus shSRC-1). There were 1,731 genes up-regulated by SRC-1, of which 153 were identified as transcription factors (TFs)/chromatin remodellers. Combining the transcriptomic profiling with ChIP-sequencing we identified a transcriptional network pertinent to SRC-1. Molecular characterisation identified E2F7, NFIA, DEK, SMAD2, SMARCA1, ASCL1 and TRPS1 as key drivers of SRC-1 mediated endocrine resistance. Each TF was confirmed as a direct target of SRC-1, via promoter specific binding and were found to be drivers of endocrine resistant cell migration. Furthermore, all TFs were necessary in mammosphere formation and their promotion of cellular de-differentiation was observed by 3D acinar organisation and flow cytometry. To elucidate the core TFs required for cancer cell reprogramming, their effect on the pioneer reprogramming TFs (OCT4, SOX2, cMYC and KLF4) was investigated. SMAD2, SMARCA1, ASCL1 and TRPS1 have emerged as pivotal regulators of endocrine resistant cell reprogramming. This study has unravelled an SRC-1-mediated TF network responsible for promoting the cellular reprogramming of breast cancer cells. Concerted activity of this network is responsible for driving de-differentiation of cells and enhancing their stem-like, highly migratory and proliferative tumour initiation population. This study provides important information regarding the mechanism of cellular reprogramming in ER positive endocrine resistant cancer and may lead to potential novel therapeutic targets. Citation Format: Browne A, Fagan A, Vareslija D, Ward E, Hill A, Young L. Steroid receptor co-activator 1 mediation of cancer cell reprogramming 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 P3-04-16.

0076 Evaluation of simulation for peripheral arterial examination teaching to medical students

Background Physical examination is a critical component of medical practice yet skills are falling into disuse resulting in unfamiliarity with signs and an increased reliance on laboratory and imaging tests.1,2 The focus on safety and quality of care and efficient patient turnover has significantly impacted on the availability of patients with clinical findings willing to be examined by students. It is our experience and reported by others that skills’ teaching is not consistent across clinical rotations.2 The need for standardisation and a shortage of training capacity have contributed to the increasing use simulation techniques. Methodology 2nd year students were divided into two groups, one taught peripheral arterial examination on a SimMan 3G and the second group on simulated patients (SPs) and assessed by OSCE with patients presenting with peripheral arterial disease. Feedback was obtained from students in focus group discussions. Results (Tables with results to be presented in the poster) There was no difference in the pass rate at OSCE between the two groups. SimMan better facilitated repeated practice, group learning, peer teaching and discussion which were highly valued by the students. Students felt that their spatial cognition, pattern recognition and development of mental spatial signs were not best facilitated during the SimMan tutorial. They also felt unprepared to deal with real patients in terms of having practiced appropriate language to issue commands, to put patients at ease or to reassure patients and felt embarrassed when faced with an intimate examination of a real patient for the first time. Conclusion Both methods of simulation teaching have distinct merits and the ideal approach maybe to use the SimMan in combination with SP tutorials. References Schwind CJ, Boehler ML, Folse R, Dunnington G, Markwell SJ. Development of physical examination skills in a third-year surgical clerkship. Am J Surg. 2001;181:338–40 Mangione S, Burdick WP, Peitzman SJ. Physical diagnosis skills of physicians in training: a focused assessment. Acad Emerg Med.1995;2:622–9 Ramani S. Twelve tips for excellent physical examination teaching. Med Teach. 2008;30:851–856

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.

Abstract P6-04-21: AIB1 expression specifically predicts breast cancer patient response to aromatase inhibitor therapy

Aromatase inhibitors (AI) have evolved over the last decade into an effective therapeutic regime for postmenopausal women with primary or advanced breast cancer. Despite their remarkable success in the clinic, intrinsic resistance to therapy occurs in a proportion of patients, while other patients who respond initially to treatment will relapse with recurrent disease. Previous studies suggest that this may be due, at least in part to estrogen receptor (ER) hypersensitivity. We undertook ER ChIPseq analysis on AI resistant cell model and data for this suggests that ER transcriptional regulation alone may not be responsible for the development of the resistant phenotype. We examined the role of the established ER coactivator protein AIB1. AIB1 has previously been associated with initiation of breast cancer and resistance to endocrine therapy. In tamoxifen treated patients, expression of AIB1 in conjunction with an activated HER2 cascade has been associated with treatment resistance and early disease recurrence. By contrast, we have observed that AIB1 alone can predict response to AIs. In our TMA the expression of AIB1 associated with disease recurrence (p = 0.025) and reduced disease free survival time (p = 0.0471) in patients treated with AIs as first-line therapy. Reflecting increased growth factor activity reported in AI resistance, AIB1 expression associated with the growth factor second messenger signaling proteins, p-Src and pERK1/2, but not the receptor HER2. These results suggest that AIB1 may utilize additional transcription factors other than ER to drive endocrine resistance. Additionally, we show that AIB1 is highly expressed in AI resistant metastases; therefore, monitoring AIB1 expression may be useful to screen for disease progression and detect disease advancement before metastases appear. Our studies of cell line models of AI resistance suggest that AIB1 may play a functional role in aggressive, migratory, phenotype of AI resistance. We have generated cell line models of resistance to letrozole (LetR) and anastrozole (AnaR). Our resistance models have higher levels of AIB1 and have increased migratory capacity. Interestingly, knockdown of AIB1 reduces the migratory capacity of the resistant cells. Furthermore, we have observed that AIB1 regulation of ER target genes is selectively enhanced in AI resistant cells in a promoter specific context. AIB1 recruitment to ER target genes such as pS2 and Myc becomes insensitive to letrozole. By contrast, AIB1 recruitment to cyclinD1 retained letrozole sensitivity. Our evidence suggests that steroidal regulation of transcription factors such as Jun and Fos may contribute to this promoter-specific regulation of ER target genes. We establish a role for AIB1 in AI-resistant breast cancer and describe a new mechanism of ERalpha/AIB1 gene regulation which could contribute to the development of an aggressive tumour phenotype. We provide evidence of a central role for AIB1 in regulating selective ER transcriptional activity and driving tumour recurrence in AI treated patients. Tackling the emerging problem of AI resistance in a timely fashion will enable us to tailor existing therapies and improve outcome in specific patient groups before disease recurrence becomes a clinical issue. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-04-21.

Characterising an aromatase inhibitor resistant breast cancer cell line

Results Let-R cells have higher levels of ERa compared to Aros. CyclinD1 basal levels are elevated in Aro cells compared to Let-Rs. Levels of JNK and c-jun are higher in Let-R cells compared to Aros. A highly effective ERa knockdown in Let-R cells was optimized. Conclusions The significant ERa knockdown achieved in the Let-R cells will enable further examination of knockdown effects on protein levels and mRNA expression. Elevated expression of ERa in the Let-R cells observed is consistent with the association between AI resistance and ER hypersensitivity. In resistant cells, the decrease in basal levels of CyclinD1 may be partly responsible for the lack of regulation of classical genes like pS2, previously detected by the lab. The elevated levels of JNK and c-jun observed in Let-R cells supports the theory of crosstalk between the ER and growth factor pathways in resistance. Furthermore, it would be of interest to accompany AI treatments with growth factor inhibitors to further our understanding of endocrine resistance.

The role of cardiac glycosides in influencing breast cancer cell proliferation

Cardiac Glycosides (CGs) are commonly used to treat congestive heart failure. CGs inhibit the Sodium Potassium ATPase (Na+/K+ ATPase) pump. Interestingly, CGs have been suggested to inhibit proliferation and migration of breast cancer cells. A pool of non-pumping Na+/K+ ATPase reportedly localizes in specific membrane organelles, caveolae, by interacting with the structural protein caveolin-1. It has been postulated that Na+/K+ ATPase forms a complex with caveolin-1 and the tyrosine kinase Src, and that binding of CGs to Na+/K+ ATPase activates Src-dependent signalling cascades. In this project we explored whether CGs reduce proliferation in breast cancer cells and whether these effects might involve Src and ERK (Extracellular-signal-Regulated Kinases).

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.