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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

Supplementary Table S4: Patient information for patients A-L

Supplementary Table S1: List of 31 genes which are upregulated in metastatic tumours relative to matched primary tumours for all three patients

Supplementary Table S2: List of 69 genes which are downregulated in metastatic liver tumours relative to matched primary tumours for all three patients

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.

Supplementary Table S3: List of 21 genes which are upregulated in the metastatic liver relative to both matched primary and matched node in all three patients (where available)

Supplementary Materials and Methods

PDF file - 93KB, PCR Primers and TaqMan qPCR Probes.

PDF file - 155KB

PDF file - 72KB, Positive controls for transfection assays.

PDF file - 198KB, Mammary tumor growth and metastasis in Tg(TVA)+RCAS-PyMT and Tg(NCOA1)x Tg(TVA)+RCAS-PyMT mice.

PDF file - 368KB, Mammary gland morphogenesis and epithelial proliferation of Tg(NCOA1) mice.