Search

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.

Table S1. Cell line authentication. Cell line DNA profiling was carried out using ATCC MCF7 DNA profile as reference. Table S2: List of ChIP and mRNA primers used for DNA and mRNA quantification on the Lightcycler (Roche). Table S3: Patient information for endocrine treated patients. Table S4. Over represented TF binding motifs in 666 ER ChIPseq peaks from LetR cells (using the Universe as background, q-value

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.

Table containing all brain metastatic cell lines in the BrMPanel

Figure S1. Characterization of MM134, SUM44, CAMA-1 and OCUB-M ILC cell lines.

Detailed description of the materials and methods

supplementary data figure legends updated to include wnt11 knockdown data in FigS3 (untracked)

Spread of cancer to the brain remains an unmet clinical need in spite of the increasing number of cases among patients with lung, breast cancer, and melanoma most notably. Although research on brain metastasis was considered a minor aspect in the past due to its untreatable nature and invariable lethality, nowadays, limited but encouraging examples have questioned this statement, making it more attractive for basic and clinical researchers. Evidences of its own biological identity (i.e., specific microenvironment) and particular therapeutic requirements (i.e., presence of blood–brain barrier, blood–tumor barrier, molecular differences with the primary tumor) are thought to be critical aspects that must be functionally exploited using preclinical models. We present the coordinated effort of 19 laboratories to compile comprehensive information related to brain metastasis experimental models. Each laboratory has provided details on the cancer cell lines they have generated or characterized as being capable of forming metastatic colonies in the brain, as well as principle methodologies of brain metastasis research. The Brain Metastasis Cell Lines Panel (BrMPanel) represents the first of its class and includes information about the cell line, how tropism to the brain was established, and the behavior of each model in vivo. These and other aspects described are intended to assist investigators in choosing the most suitable cell line for research on brain metastasis. The main goal of this effort is to facilitate research on this unmet clinical need, to improve models through a collaborative environment, and to promote the exchange of information on these valuable resources.

Figure S1. SRC-1 dependent transcriptome in endocrine resistant LY2 cells. Figure S2. Pathways enriched in SRC-1 repressed gene set. Figure S3. Repression gene set is directly regulated and suppressed by SRC-1. Figure S4. SRC-1 mediates endocrine treatment resistant phenotype Figure S5. Expression validation for functional experiments. Figure S6. Target gene set expression in untreated ER positive population Figure S7. RG-108 treatment can drive re-expression of suppressed target gene set. Figure S8: Nuclear receptor binding activity to the SRC-1 target genes.

Purpose: Acquired resistance to aromatase inhibitor (AI) therapy is a major clinical problem in the treatment of breast cancer. The detailed mechanisms of how tumor cells develop this resistance remain unclear. Here, the adapted function of estrogen receptor (ER) to an estrogen-depleted environment following AI treatment is reported.Experimental Design: Global ER chromatin immuno-precipitation (ChIP)-seq analysis of AI-resistant cells identified steroid-independent ER target genes. Matched patient tumor samples, collected before and after AI treatment, were used to assess ER activity.Results: Maintained ER activity was observed in patient tumors following neoadjuvant AI therapy. Genome-wide ER–DNA-binding analysis in AI-resistant cell lines identified a subset of classic ligand-dependent ER target genes that develop steroid independence. The Kaplan–Meier analysis revealed a significant association between tumors, which fail to decrease this steroid-independent ER target gene set in response to neoadjuvant AI therapy, and poor disease-free survival and overall survival (n = 72 matched patient tumor samples, P = 0.00339 and 0.00155, respectively). The adaptive ER response to AI treatment was highlighted by the ER/AIB1 target gene, early growth response 3 (EGR3). Elevated levels of EGR3 were detected in endocrine-resistant local disease recurrent patient tumors in comparison with matched primary tissue. However, evidence from distant metastatic tumors demonstrates that the ER signaling network may undergo further adaptations with disease progression as estrogen-independent ER target gene expression is routinely lost in established metastatic tumors.Conclusions: Overall, these data provide evidence of a dynamic ER response to endocrine treatment that may provide vital clues for overcoming the clinical issue of therapy resistance. Clin Cancer Res; 22(11); 2765–77. ©2016 AACR.

Supplementary Table S4: Patient information for patients A-L

Contains experimental therapies tested on brain metastatic cell lines and associated clinical trials

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.

Figure S1. Ligand independent ER drives growth of AI resistant cells. Figure S2. Confirmation of transient knockdown using siRNA. Figure S3: pSer118 ER expression significantly increases during neoadjuvant AI therapy. Figure S4: Androgen Receptor (AR) expression in patient tumours before and after endocrine therapy. Figure S5. Reduced ligand dependency is a feature of multiple endocrine resistant cells compared to endocrine sensitive MCF7 cells. Figure S6. FoxA1 expression and interactions with ER are comparable between MCF7 and LetR cells treated with either estrogen (E2) or androstenedione (A). Figure S7. Steroid-sensitive recruitment of ER to a selection of target genes in MCF7-Aro cells. q-PCR of ER ChIP in MCF7-Aro cells treated with vehicle (V) or steroid (S) for 45 minutes. IgG was used as an internal control. Bar charts show relative recruitment to each target gene with n=3. Heat map shows relative recruitment of ER to each target gene as a fold increase above IgG control. Figure S8. ER ChIPseq binding peak profiles in 7 genes of the ER target gene signature. ER binding activity is observed at the same location in both LY2 and LetR cells. Images were captured from UCSC Genome Browser. V, vehicle; E, estrogen; A, androstenedione. Figure S9. ER binding does not affect expression of PEBP4. Figure S10. A, EGR3 knockdown decreases cell growth in LetR cells. Figure S11: ER target gene signature is more accurate than ESR1 alone for predicting outcome on AI therapy.

Purpose: Despite the clinical utility of endocrine therapies for estrogen receptor–positive (ER) breast cancer, up to 40% of patients eventually develop resistance, leading to disease progression. The molecular determinants that drive this adaptation to treatment remain poorly understood. Methylome aberrations drive cancer growth yet the functional role and mechanism of these epimutations in drug resistance are poorly elucidated.Experimental Design: Genome-wide multi-omics sequencing approach identified a differentially methylated hub of prodifferentiation genes in endocrine resistant breast cancer patients and cell models. Clinical relevance of the functionally validated methyl-targets was assessed in a cohort of endocrine-treated human breast cancers and patient-derived ex vivo metastatic tumors.Results: Enhanced global hypermethylation was observed in endocrine treatment resistant cells and patient metastasis relative to sensitive parent cells and matched primary breast tumor, respectively. Using paired methylation and transcriptional profiles, we found that SRC-1–dependent alterations in endocrine resistance lead to aberrant hypermethylation that resulted in reduced expression of a set of differentiation genes. Analysis of ER-positive endocrine-treated human breast tumors (n = 669) demonstrated that low expression of this prodifferentiation gene set significantly associated with poor clinical outcome (P = 0.00009). We demonstrate that the reactivation of these genes in vitro and ex vivo reverses the aggressive phenotype.Conclusions: Our work demonstrates that SRC-1-dependent epigenetic remodeling is a ’high level’ regulator of the poorly differentiated state in ER-positive breast cancer. Collectively these data revealed an epigenetic reprograming pathway, whereby concerted differential DNA methylation is potentiated by SRC-1 in the endocrine resistant setting. Clin Cancer Res; 24(15); 3692–703. ©2018 AACR.

Table S3: Cox regression analysis on RATHER RNA-seq data

Table S1: 736 genes downregulated in LY2 shNT (Ensemble ID)

Supplementary Materials and Methods

Fixed-tissue ChIP-seq for H3K27 acetylation (H3K27ac) profiling (FiTAc-seq) is an epigenetic method for profiling active enhancers and promoters in formalin-fixed, paraffin-embedded (FFPE) tissues. We previously developed a modified ChIP-seq protocol (FiT-seq) for chromatin profiling in FFPE. FiT-seq produces high-quality chromatin profiles particularly for methylated histone marks but is not optimized for H3K27ac profiling. FiTAc-seq is a modified protocol that replaces the proteinase K digestion applied in FiT-seq with extended heating at 65 °C in a higher concentration of detergent and a minimized sonication step, to produce robust genome-wide H3K27ac maps from clinical samples. FiTAc-seq generates high-quality enhancer landscapes and super-enhancer (SE) annotation in numerous archived FFPE samples from distinct tumor types. This approach will be of great interest for both basic and clinical researchers. The entire protocol from FFPE blocks to sequence-ready library can be accomplished within 4 d. This protocol describes a ChIP-seq procedure optimized for profiling H3K27 acetylation in archived formalin-fixed, paraffin-embedded (FFPE) tissues sampled through whole or macrodissected sectioning or from punched cores.

Invasive lobular carcinoma (ILC) is the second most common type of breast cancer after invasive ductal carcinoma (IDC), accounting for approximately 10-15% of all breast tumors. ILC is characterized by inactivation of E-Cadherin and neoplastic cells that invade the stroma in a "single-file" pattern. Women with ILC are usually older, have used hormone replacement therapy and are more likely to have hormone receptor-positive disease. ILCs have similar survival to IDCs at both five and 10 years, but despite this, the clinical course is distinct: ILCs are three times more likely to metastasize to the peritoneum, gastrointestinal tract, and ovaries and are more frequently bilateral. Therefore, tailored therapeutic options for this distinct, hard-to-treat subtype of breast cancer are required. As part of the RATHER FP7 HEALTH consortium (www.ratherproject.com), we carried out RNA-Seq analysis of 61 primary ILC samples and identified that high expression of the BET family protein Brd3 (uniquely among BRD family members) was associated with poor recurrence free survival (p=0.03, HR 8.63, CI 1.22-60.85). This observation was further validated in the independent METABRIC cohort (n=99), where again, high Brd3 expression (and not other BRD members) was associated with poor recurrence-free survival (p Citation Format: Darran P O'Connor, Louise Walsh, Kathryn Haley, Bruce Moran, Brian Mooney, Stephen Madden, Sudipto Das, Oscar Rueda, Catriona Dowling, Damir Vareslija, Suet-Feung Chin, Sabine Linn, Leonie Young, Karin Jirstrom, John P Crown, Rene Bernards, Carlos Caldas, William M Gallagher, Triona Ni Chonghaile. Combination of BET and FGFR inhibition as a rational therapeutic strategy for invasive lobular breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-10-06.

Purpose: Invasive lobular carcinoma (ILC) is a subtype of breast cancer accounting for 10% of breast tumors. The majority of patients are treated with endocrine therapy; however, endocrine resistance is common in estrogen receptor–positive breast cancer and new therapeutic strategies are needed. Bromodomain and extraterminal inhibitors (BETi) are effective in diverse types of breast cancer but they have not yet been assessed in ILC. Experimental Design: We assessed whether targeting the BET proteins with JQ1 could serve as an effective therapeutic strategy in ILC in both 2D and 3D models. We used dynamic BH3 profiling and RNA-sequencing (RNA-seq) to identify transcriptional reprograming enabling resistance to JQ1-induced apoptosis. As part of the RATHER study, we obtained copy-number alterations and RNA-seq on 61 ILC patient samples. Results: Certain ILC cell lines were sensitive to JQ1, while others were intrinsically resistant to JQ1-induced apoptosis. JQ1 treatment led to an enhanced dependence on antiapoptotic proteins and a transcriptional rewiring inducing fibroblast growth factor receptor 1 (FGFR1). This increase in FGFR1 was also evident in invasive ductal carcinoma (IDC) cell lines. The combination of JQ1 and FGFR1 inhibitors was highly effective at inhibiting growth in both 2D and 3D models of ILC and IDC. Interestingly, we found in the RATHER cohort of 61 ILC patients that 20% had FGFR1 amplification and we showed that high BRD3 mRNA expression was associated with poor survival specifically in ILC. Conclusions: We provide evidence that BETi either alone or in combination with FGFR1 inhibitors or BH3 mimetics may be a useful therapeutic strategy for recurrent ILC patients.

Introduction Brain metastasis (BrM) occurs in 10-30% of patients with advanced breast cancer (BC). BrM is increasing in incidence and confers a poor prognosis. We aimed to investigate the contribution of global epi-transcriptomic alterations in N6-methyladenosine (m6A) RNA-methylation as a therapeutic target in brain metastatic breast cancer. Method In preliminary studies we have demonstrated m6A demethylase – FTO as the main contributor to the progression of ER+ breast cancer. Furthermore an association between FTO and reduced disease-free-survival (n=870, p=0.018) was observed. Here we conducted an epigenetic inhibitor screen using two therapeutic agents, ethyl-ester-meclofenamic acid (MA2) and FB23-2 on matched 2D cell line, 3D organoid cultures and patient-derived xenografts (PDX) explant models of brain metastasis. Result Upon integration of mapped global RNA methylation landscape with matched proteomic analysis, we observed genome-wide RNA hypo-methylation of key pluripotency genes, including SOX2 and KLF4, as key players underlying tumour progression to the brain. Genetic and pharmacological inhibition of FTO in novel ex vivo models of BrM significantly reduced protein expression levels of KLF4 and SOX2. Moreover, pharmacological inhibition of FTO with MA2 and FB23-2, inhibited cell proliferation in endocrine-resistant BC and patient BrM cells. We translate our findings to the clinic by demonstrating the efficacy of anti-FTO therapies in several unique PDX and 3D organoid BrM models. Conclusion Our results reveal epi-transcriptional remodelling events as a key mechanism in BrM. This study establishes an early role for targeting RNA methylation in the management of disease progression and presents FTO as a potential therapeutic target in BrM. Take-home message This study establishes an early role for targeting RNA methylation in the management of disease progression and presents FTO as a potential therapeutic target in brain metastatic breast cancer.

Introduction The incidence of brain metastases is increasing despite longer survival rates for patients with advanced breast cancer. The identification of novel therapeutic targets for these patients is an urgent unmet clinical need. Sequencing of metastatic tumours have largely focused on mutations however gene fusions have an important, yet underappreciated role in tumorigenesis and disease progression. In this study, we investigate the role of gene fusions in brain metastatic disease and their impact on altered therapeutic responses. Method RNA sequencing was performed on the largest reported cohort of patient matched primary and resected brain metastatic tumours (45 patients n=90 samples). Expressed gene fusions were detected computationally using STAR-Fusion and Arriba. Result We identified differential gene fusion burden in brain metastatic tumours (medium of 58) vs. primary breast tumours (medium of 38) (p Conclusion These results highlight the significant role of gene fusions in breast cancer brain metastases. Abbreviations MAPK Mitogen Activated Protein Kinase, HER Human Epidermal Receptor, CDK12 Cyclin Dependent Kinase 12 Take-home message We highlight the significant role of gene fusions in breast cancer brain metastases and offer specific actionable genomic alterations to be exploited.

Introduction Metastatic breast cancer (MBC) is the main source of mortality in breast cancer patients largely due to lack of effective treatments. Our previous results suggest that tumour transcriptional heterogeneity drives therapy resistance and cancer relapse. While traditional in vitro human cancer cell line models have been widely used for disease modelling, they do not faithfully recapitulate the pathophysiology of MBC. Method In this study we developed patient-derived tumour organoid cultures from frozen patient-derived (PDX) models of MBC. Using those models we performed preclinical drug screening of investigational and FDA approved therapeutics previously uncovered by us as potentially clinically actionable in MBC. Result Our results reveal high heterogeneity in the responses to various targeted therapies among tested MBC organoids, which makes them a valuable tool for studying intra-tumor heterogeneity and drug response. Moreover, drug screening identified a divergent set of the breast to brain metastatic MBC organoids that showed high sensitivity to a new class of tyrosine kinase receptors, RET. Conclusion Taken together, our novel MBC models and methodology applied here provides an important modelling tool to assess the contribution of intra-tumour heterogeneity and microenvironment to drug response as they recapitulate the cellular, structural and biochemical complexity previously observed in our genomic characterisation of MBCs. Application of this type of translational research will enhance the development of new targeted precision medicine strategies and prelude stratification for clinical trials. Abbreviations MBC- Metastatic Breast Cancer; PDX- Patient-Derived Xenografts; FDA- Food and Drug Administration; RET- Receptor Tyrosine Kinase Take-home message Patient-derived tumour organoid cultures provide an important modelling tool to assess the contribution of intra-tumour heterogeneity and microenvironment to drug response as they recapitulate the cellular, structural and biochemical complexity previously observed in genomic characterisation of metastatic breast cancer. SURGICAL EDUCATION AND TRAINING

Funder: Breast Cancer Ireland GR 14-0883, 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.

Elevated levels of the anti-apoptotic BCL2 protein associate with favourable outcome in breast cancer. We investigated whether executioner caspase activation downstream of mitochondrial apoptosis was associated with, or independent, of BCL2's prognostic signature in breast cancer. Levels of pro- and anti-apoptotic BCL2 family proteins were quantified in triple negative breast cancer (TNBC) samples and utilised to calculate BCL2 profiles of 845 breast cancer patients. Biomarkers including single apoptosis proteins and network-enriched apoptosis system signatures were evaluated using uni- and multi-variate Cox-models. In both TNBC and non-TNBC breast cancer, the anti-apoptotic BCL2 protein was particularly abundant when compared to other solid tumours. High BCL2 protein levels were prognostic of favourable outcome across all breast cancers (HR 0.4, 95% CI 0.2-0.6, Wald p 0.0001). Although BCL2 and cleaved caspase-7 levels were negatively correlated, levels of cleaved caspase-7 were also associated with favourable outcome (HR 0.4, 95% CI 0.3-0.7, Wald p = 0.001). A combination of low BCL2 and low cleaved caspase-7 protein levels was highly prognostic of unfavourable outcome across all breast cancers (HR 11.29, 95% CI 2.20-58.23, Wald p = 0.01). A combination of BCL2 and cleaved caspase-7 levels is a promising prognostic biomarker in breast cancer patients.BCL2 levels are elevated in breast cancer where they are marker of good prognosis. BCL2 and active caspase levels correlate negatively; yet, active caspases indicate good outcome. Low BCL2 and low caspase-7 are highly prognostic of unfavourable outcome across all breast cancers. BCL2 levels indicate molecular subtype and tumour proliferation status in breast cancer.

Background: Apoptosis signalling is controlled by a complex interaction of pro- and anti-apoptotic BCL2 proteins and its dysregulation is believed to be a major contributor to therapy responses and resistance in cancer. We previously demonstrated that inhibition of cell death in cancer cell is associated to poor outcome in colorectal cancer (Lindner et al., Cancer Res, 2012) and to lower cell death in triple negative breast cancer (TNBC) cells in vitro (Lucantoni et al., in review). In ER+ breast cancer, the anti-apoptotic protein BCL2 is commonly overexpressed, but its expression is associated with improved clinical outcome. The aim of this study was to assess whether system modelling of BCL2 protein interactions stratifies low- and high-risk breast cancer patients, and to determine the contribution of apoptosis signalling in different molecular subtypes of breast cancer. Methods: Protein levels of BAK, BAX, BCL2 and BCL(X) were determined in fresh frozen, TNBC samples from the BREAST-PREDICT Irish Cancer Society Collaborative Research Centre cohort, using HeLa cells as standard in which absolute protein levels were previously determined. Clinical, protein level and gene expression datasets of 845 invasive breast carcinoma patients were accessed from The Cancer Genome Atlas project, and BCL2 protein profiles were calculated by linear regression based on the BREAST-PREDICT cohort. In both cohorts, profiles were used to calculate the stress dose required to induce mitochondrial apoptosis (η). Results: In contrast to experiments with TNBC cells in which a high η indicated lower rates of cell death in vitro (Lucantoni et al., in review), we found that in breast cancer patients, η ≤ 0 was associated with lower overall survival (OS) compare to η > 0 (HR 2.1, 95%CI 1.3-3.3, p < 0.01). η > 0 was associated with lower levels of cleaved caspase 7 compared to η ≤ 0 (ANOVA & Tukey post-hoc; p < 0.1). Cleaved caspase 7 levels > mean were associated with improved OS compared to levels ≤ mean (HR 0.4, 95%CI 0.3-0.7, p = 0.001). High values of η were significantly associated with lower proliferation in ER/PR+ cancer (ANOVA & Tukey post-hoc; p < 0.01), but not in HER2+ or TNBC. Next we performed hierarchical cluster analysis (ConsensusClusterPlus; Monti et al, Machine Learning, 2003) with 61 additional mRNAs and proteins which are not implemented in our systems modelling approach. We found a subgroup with high BAD, PUMA, BOK and TRADD mRNA expression levels in ER/PR+ breast cancer patients, independently of the value of η. ER/PR+, but not HER2+ or TNBC, patients with an averaged expression of these 4 mRNA > mean had significant higher OS compared with patients with an averaged expression ≤ mean (HR 0.4, 95%CI 0.2-0.9, p = 0.02). Conclusions: Impairment of apoptosis assessed solely on the levels of BAK, BAX, BCL2 and BCLX(L) proteins were not sufficient as prognostic marker in breast cancer patients. However, our analysis suggest that patients with ER/PR+ cancer cells potentially 'primed' towards apoptosis - via the expression of pro-apoptotic BAD, PUMA, BOK and TRADD - had a more favourable clinical outcome compared to patients with cancer cells lacking this priming. Citation Format: Lindner AU, Lucantoni F, Vareslija D, Resler A, Gallagher WM, Hill A, Young L, Prehn JHM. High expression of BAD, PUMA, BOK and TRADD mRNA is associated with higher overall survival in ER+ and PR+ breast cancer patients [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 P5-10-02.

Central nervous system (CNS), notably brain, metastases are most prevalent in lung cancer (20–56% of patients), breast cancer (5–20%) and melanoma (7–16%). Lesions occur in both the brain parenchyma and the meninges. To mechanistically understand CNS metastasis formation and develop preventive and therapeutic strategies, it is essential to use model systems that, as much as possible, faithfully recapitulate the clinical disease process. Furthermore, the complexities of brain metastases dictate that studies should utilize multiple model systems in various stages of brain metastases progression. To facilitate brain metastasis research, 19 laboratories around the world have compiled comprehensive information on their brain metastasis mouse models. Each lab has provided details on the cell lines that they have generated or characterized as being capable of forming metastatic colonies in the brain, as well as principle methodologies of brain metastasis research. This Brain Metastasis Cell Lines Panel (BrMPanel, https://apps.cnio.es/app/BrainMetastasis/CellLines) represents the first of its class and includes information about each cell line, how tropism to the brain was established, and the behavior of each model in vivo. The BrMPanel is composed of 60 cell lines, derived from patients (32 cell lines, 53%), mouse (27, 45%) or rat (1, 2%), and represent the three main cancer types that result in brain metastasis: breast cancer (38 cell lines, 63%), lung cancer (8, 13%) and melanoma (14, 23%). This resource is intended to assist investigators in choosing the most suitable model for research on brain metastasis, and is available to the entire scientific community. The ultimate goal of this effort is to facilitate research on this unmet clinical need, to improve models through a collaborative environment, and to promote the exchange of information on these valuable resources. We invite other collaborators to contribute their models to the BrMPanel to grow this resource.

Spread of cancer to the brain remains an unmet clinical need in spite of the increasing number of cases among patients with lung, breast cancer, and melanoma most notably. Although research on brain metastasis was considered a minor aspect in the past due to its untreatable nature and invariable lethality, nowadays, limited but encouraging examples have questioned this statement, making it more attractive for basic and clinical researchers. Evidences of its own biological identity (i.e., specific microenvironment) and particular therapeutic requirements (i.e., presence of blood–brain barrier, blood–tumor barrier, molecular differences with the primary tumor) are thought to be critical aspects that must be functionally exploited using preclinical models. We present the coordinated effort of 19 laboratories to compile comprehensive information related to brain metastasis experimental models. Each laboratory has provided details on the cancer cell lines they have generated or characterized as being capable of forming metastatic colonies in the brain, as well as principle methodologies of brain metastasis research. The Brain Metastasis Cell Lines Panel (BrMPanel) represents the first of its class and includes information about the cell line, how tropism to the brain was established, and the behavior of each model in vivo. These and other aspects described are intended to assist investigators in choosing the most suitable cell line for research on brain metastasis. The main goal of this effort is to facilitate research on this unmet clinical need, to improve models through a collaborative environment, and to promote the exchange of information on these valuable resources.

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.

Breast cancer brain metastasis (BCBM) is an area of unmet clinical need. MicroRNAs (miRNAs) have been linked to the metastatic process in breast cancer (BC). In this study, we aim to determine differentially-expressed miRNAs utilising primary BCs that did not relapse (BCNR, n = 12), primaries that relapsed (BCR) and their paired (n = 40 pairs) brain metastases (BM) using the NanoString™ nCounter™ miRNA Expression Assays. Significance analysis of microarrays identified 58 and 11 differentially-expressed miRNAs between BCNR vs BCR and BCR vs BM respectively and pathway analysis revealed enrichment for genes involved in invasion and metastasis. Four miRNAs, miR-132-3p, miR-199a-5p, miR-150-5p and miR-155-5p, were differentially-expressed within both cohorts (BCNR-BCR, BCR-BM) and receiver-operating characteristic curve analysis (p = 0.00137) and Kaplan-Meier survival method (p = 0.0029, brain metastasis-free survival; p = 0.0007, overall survival) demonstrated their potential use as prognostic markers. Ingenuity pathway enrichment linked them to the MET oncogene, and the cMET protein was overexpressed in the BCR (p

6-Hydroxydopamine (6-OHDA), which is a neurotoxin that selectively destroys catecholaminergic nerves in sympathetically innervated tissues, has been used to provide a model of Parkinson's disease in experimental animals. It is rapidly autoxidised to yield potentially toxic products and reactive oxygen species. Its ability to release Fe(II) from protein storage sites also results in the formation of hROS. This account will consider how this family of toxic products may contribute to the observed effects of 6-OHDA.

Fixed-tissue ChIP-seq for H3K27 acetylation (H3K27ac) profiling (FiTAc-seq) is an epigenetic method for profiling active enhancers and promoters in formalin-fixed, paraffin-embedded (FFPE) tissues. We previously developed a modified ChIP-seq protocol (FiT-seq) for chromatin profiling in FFPE. FiT-seq produces high-quality chromatin profiles particularly for methylated histone marks but is not optimized for H3K27ac profiling. FiTAc-seq is a modified protocol that replaces the proteinase K digestion applied in FiT-seq with extended heating at 65 °C in a higher concentration of detergent and a minimized sonication step, to produce robust genome-wide H3K27ac maps from clinical samples. FiTAc-seq generates high-quality enhancer landscapes and super-enhancer (SE) annotation in numerous archived FFPE samples from distinct tumor types. This approach will be of great interest for both basic and clinical researchers. The entire protocol from FFPE blocks to sequence-ready library can be accomplished within 4 d.

BACKGROUND: Metastasis is the major cause of mortality in breast cancer (BrCa) patients. Our understanding of brain metastasis (BrM) is limited, reflected by a lack of effective treatments. We aimed to (1) determine BrCa gene signature differences between primary tumors and matched BrM and (2) uncover BrM-specific alterations that may be clinically actionable. MATERIALS and METHODS: NanoString expression profiling of 127 genes from 5 major prognostic tests (MammaPrint, EndoPredict, PAM50, OncotypeDX, MGI) was performed on 20 patient-matched primary (10 ER-neg, 10 ER-pos) and metastatic brain tumors. Subtype classification was performed using genefu. Protein changes in ER and HER2 (ERBB2) were confirmed by IHC. BrM-specific ERBB2 gains were corroborated in a publicly available dataset of 18 additional patient-matched cases (dbGAP phs000730.v1.p1). To test whether ERBB2 amplification and base pair mutation is metastasis-site specific, we further analyzed an expanded cohort of 7,884 breast tumors enriched for metastatic samples (52%) including liver (16.7%), lung (4.3%), bone (3.6%), and brain (2.0%) using comprehensive hybrid-capture sequencing of ERBB2. RESULTS: 17/20 BrM retained the PAM50 subtype of the primary BrCa. Despite this concordance, 17/20 BrM harbored expression changes (< or > 2-fold) in clinically actionable genes including gains of FGFR4 (30%), FLT1 (20%), AURKA (10%) and loss of ESR1 expression (45%). The most recurrently upregulated gene was ERBB2, showing a >2-fold expression increase in 35% of BrM. 3 of 13 (23.3%) cases originally HER2-negative, and thus HER2-therapy naive, in the primary BrCa were IHC-positive (3+) in the paired BrM with an observed metastasis-specific amplification of the ERBB2 locus. In an independent dataset, 2 of 9 (22.2%) HER2-negative BrCa switched to HER2-positive with one BrM acquiring ERBB2 amplification and the other showing metastastic enrichment of the activating V777L ERBB2 mutation. Analysis of a large cohort of breast tumors (n=7,884) showed that across all organs ERBB2 amplification and/or base pair mutation was similar (p=0.18) between primary (13%) and metastatic disease (12%), however, a strong and significant enrichment was seen for BrM (primary 13% vs BrM 24%, p CONCLUSIONS: Taken together, these results demonstrate that the majority (85%) of patient-matched BrM retain the intrinsic subtype of the primary cancer. However, despite this transcriptional similarity, alterations in clinically actionable genes are common, with BrM acquiring ERBB2 amplifications and/or base pair mutations at a frequency of ∼20%, even in HER2-therapy naive tumors. In a large cohort of primary and metastatic breast cancers, there is also a unique enrichment for ERBB2 alterations in BrM. This study provides a strong rationale to molecularly profile metastatic lesions to both better understand biological mechanisms of metastases and to perhaps refine therapeutic decision-making in advanced cancers. Citation Format: Priedigkeit N, Hartmaier RJ, Chen Y, Vareslija D, Basudan A, Thomas R, Leone JP, Lucas PC, Bhargava R, Hamilton RL, Chmielecki J, Davidson NE, Oesterreich S, Brufsky AM, Young L, Lee AV. Breast cancer brain metastases show limited intrinsic subtype switching, yet exhibit acquired ERBB2 amplifications and activating mutations [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 PD1-05.

Purpose: Acquired resistance to aromatase inhibitor (AI) therapy is a major clinical problem in the treatment of breast cancer. The detailed mechanisms of how tumor cells develop this resistance remain unclear. Here, the adapted function of estrogen receptor (ER) to an estrogen-depleted environment following AI treatment is reported. Experimental Design: Global ER chromatin immuno-precipitation (ChIP)-seq analysis of AI-resistant cells identified steroid-independent ER target genes. Matched patient tumor samples, collected before and after AI treatment, were used to assess ER activity. Results: Maintained ER activity was observed in patient tumors following neoadjuvant AI therapy. Genome-wide ER–DNA-binding analysis in AI-resistant cell lines identified a subset of classic ligand-dependent ER target genes that develop steroid independence. The Kaplan–Meier analysis revealed a significant association between tumors, which fail to decrease this steroid-independent ER target gene set in response to neoadjuvant AI therapy, and poor disease-free survival and overall survival (n = 72 matched patient tumor samples, P = 0.00339 and 0.00155, respectively). The adaptive ER response to AI treatment was highlighted by the ER/AIB1 target gene, early growth response 3 (EGR3). Elevated levels of EGR3 were detected in endocrine-resistant local disease recurrent patient tumors in comparison with matched primary tissue. However, evidence from distant metastatic tumors demonstrates that the ER signaling network may undergo further adaptations with disease progression as estrogen-independent ER target gene expression is routinely lost in established metastatic tumors. Conclusions: Overall, these data provide evidence of a dynamic ER response to endocrine treatment that may provide vital clues for overcoming the clinical issue of therapy resistance. Clin Cancer Res; 22(11); 2765–77. ©2016 AACR.

BACKGROUND: Brain metastatic disease occurs in 10-30% of metastatic breast cancer cases. The incidence of brain metastases is increasing, yet overall survival remains < 2 years. Treatment of brain metastases is limited with current clinical practice centered on radiation, chemotherapy and surgery. Although these treatments may prolong survival in the short term, targeting oncogenic alterations in brain metastases may deliver a more sustained clinical benefit. In this multicenter study, we comprehensively characterized DNA and RNA alterations that aberrantly drive specific oncogenic pathway activity pertinent to breast cancer brain metastases (BCBM). METHODS: RNA sequencing was performed on a cohort of patient-matched primary and resected brain metastatic tumours (45 patients; N=90 samples). Whole exome DNA sequencing (WXS) was performed for 18/45 patients (54 trios consisting of primary tumor, brain metastasis and matched normal tissue). An independent brain metastatic WXS cohort (N=21 patients) (PMID: 26410082) was also analysed resulting in a total of 39 patient samples. Recurrent somatic copy number alterations (SCNA), somatic single nucleotide variants (SNVs) and mutational signatures were identified from WXS data. Expressed gene fusions were detected computationally from RNA-Seq (N=45 cases) RESULTS: Of the 45 BCBM patients, median age at diagnosis was 51 years [25, 67], median overall survival 57 months (range 18-255) with median brain metastases free survival 34 months (range 5-216).Clinical molecular subtype of the primary tumour included 13 ER+/HER2- (29%), 16 HER2+ (35.5%) and 16 TNBC (35.5%). Regions of significant recurrent amplifications and deletions in BCBM (N=39 patients) were identified in 4q12, 10q11.21, 8p11.23, 8q23.3 and 17q12 (FDR < 0.10). Recurrent expressed gene fusions identified in known cancer driver genes were associated with chromatin modification, MAPK and HER signaling pathways. Mutational signature analysis of SNVs identified signatures associated with ageing, mismatch repair and homologous recombination deficiency (HRD) mutational processes. The relative contribution of the HRD signature was significantly increased in brain metastases compared to matched primary tumour (p < 0.05). Concordantly increased HRD in the brain metastatic transcriptome was confirmed in these patients by gene set variation analysis (GSVA) of homologous recombination pathway genes from KEGG database in RNA-Seq data. Moreover, in the extended BCBM RNA-Seq (N=45 patients) GSVA pathway scores were elevated in brain metastases relative to primary tumours, validating the functional significance of altered DNA repair defects in brain metastases CONCLUSIONS: Here, we report recurrent genetic drivers, supported by altered functional transcriptome, unique to brain metastasis that may have clinical implications for prognosis and treatment choice. Specifically, targeting defects in the homologous recombination repair mechanism may represent new therapeutic strategies and management opportunities for breast cancer brain metastases patients. Citation Format: Nicola S Cosgrove, Damir Varešlija, Stephen Keelan, Simon Furney, Jodi M Carter, Steven N Hart, Siddhartha Yadav, Arnold DK Hill, Steffi Oesterreich, Adrian V Lee, Fergus J Couch, Leonie Young. Homologous recombination deficiency represents a new therapeutic strategy for breast cancer brain metastases [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD13-01.