Telephone: +44 (0)1613060813 AUTHOR FOOTNOTES *Co-corresponding authors RUNNING TITLE Dormant IL1R1-expressing ALDH+ breast cancer cells survive anti-estrogen therapies 3 SUMMARY Estrogen receptor-positive (ER+) breast tumours are often treated with antiestrogen (AE) therapies but frequently develop resistance. Cancer Stem Cells (CSCs) with high aldehyde dehydrogenase (ALDH) activity (ALDH+ cells) arereported to be enriched following AE treatment. Here we perform in vitro and in vivo functional CSC assays and gene expression analysis to characterise the ALDH+ population in AE resistant metastatic patient samples and an ER+ cell line. We show that the IL1 signalling pathway is activated in ALDH+ cells and data from single cells reveals that AE treatment selects for IL1R1-expressing ALDH+ cells. Importantly, we demonstrate that increased expression of IL1R1 is observed in the tumours of patients treated with AE therapy and predicts for treatment failure. Single-cell gene expression analysis revealed that at least 2 sub-populations exist within the ALDH+ population, one proliferative and one quiescent. Following AE therapy, the quiescent ALDH+IL1R1+ population is expanded, which suggests CSC dormancy as an adaptive strategy that facilitates treatment resistance. Supporting this, analysis of AE resistant dormant tumours reveals significantly increased expression of ALDH1A1, ALDH1A3 and IL1R1 genes. Thus, we propose that targeting of ALDH+IL1R1+ cells will reverse AE resistance, including in patients with minimal residual disease.
Background Interest in the ubiquitously expressed transmembrane receptor IL6ST (gp130) has developed as it has been identified as a predictive biomarker of endocrine treatment response in breast cancer patients and is included in the 'Endopredict' test. At least seven cytokines (IL-6, OSM, LIF, IL-11, CNTF, IL-27, CT-1) signal via IL6ST. Interleukin-6 (IL-6) can mediate effects via two signaling pathways; classic signaling (through the membrane-bound IL-6 receptor, IL-6R) and trans-signaling (via non-signaling membrane-bound soluble IL-6R, sIL-6R). Both pathways may occur in parallel and activate cells. Crosstalk between the ER signaling pathway and IL6ST through STAT3 has been identified and implicated in the conversion from ER responsiveness to non-responsiveness. Method A panel of 3 ERa+ luminal breast cancer cell lines (MCF-7, T47D, ZR-75-1) were chosen to examine IL6ST expression by western blot, gel electrophoresis and qRT-PCR. Proliferation assays (SRB) were carried out to investigate the effects of seven cytokines (IL-6, OSM, LIF, IL-11, CNTF, IL-27, CT-1) on cell growth. The action of both IL-6 and Oncostatin M (OSM) on cell migration and downstream signaling pathways (pSTAT3 and pERK1/2) was studied. The extent of trans-signaling occurrence and interaction between cytokines and estrogen was investigated using proliferation assays. Results Three cell lines (MCF-7, T47D, ZR-75-1 were shown to express varying levels of full-length IL6ST, with MCF-7 having the least expression. Gel electrophoresis and qRT-PCR confirmed the presence of all previously described IL6ST soluble forms in the three cell lines. Surprisingly, the growth response to the cytokines was variable across the cell lines. IL6 caused a modest increase in growth in MCF-7 but produced inhibition in ZR-75-1. OSM and LIF stimulated growth in MCF-7, whereas OSM inhibited ZR-75-1 and LIF had no effect. Interestingly, no significant effect on growth was seen in T47D with any of the cytokines except IL-11 which generated a significant growth effect on T47D cells. Both STAT3 and MAPK/ERK pathways were activated to different extents in the three cell lines as a result of OSM and IL-6 activation, whereas cells migration was only stimulated in ZR-75-1. The trans-signaling pathway significantly enhanced the growth inhibition in ZR-75-1. Estrogen eliminated the effect of both IL-6 and OSM on MCF-7 growth, while both cytokines decreased estrogen-induced cell proliferation in ZR-75-1. There was no effect in T47D. Conclusion These results indicate: · The presence of both classic and trans-signaling occurrence in breast cancer cell lines. · Differential patterns of pSTAT3 and pERK1/2 signaling which could help explain the variation in responses to the cytokines. · IL6ST full-length form is the most abundant form in all three cell lines under basal conditions. · The levels and roles of the different IL6ST soluble forms will be further studied after estrogen stimulation. · The effect of IL6ST silencing in the presence of estrogen and tamoxifen on cell growth and the gene expression currently being examined. Citation Format: Mosly DH, Turnbull AK, Langdon SP, Sims AH. A potential role for IL6ST mediating endocrine resistance in breast cancer via interaction with the ER signaling pathway [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-04-28.
Introduction: IL6ST is regarded as a putative ER target gene. Recently it has been recognised as a key biomarker for prediction of response to endocrine therapy (ET), having been included as the primary biomarker in our EA2Clin test and as an ER-signalling gene in the EndoPredict test. In both tests higher IL6ST expression is associated with a better response to ET and better prognosis. Despite its importance as a biomarker, little is known about its functional role in breast cancer (BC). Methods: Pre- and on-treatment (at 14-days and at surgery) samples were collected from 102 post-menopausal women with ER+ BC, treated with 3-6 months of neoadjuvant ET. RNA was extracted for whole-genome expression analysis. From a subset with available fresh frozen tissue (28 patients, 83 samples) protein was extracted and proteome analysis using mass spectrometry is currently underway – results available for SABCS 2017. Immunohistochemistry was performed on FFPE tissue microarrays (TMAs) comprising pre-treatment samples from 102 patients. Cytoplasmic/membrane staining was scored using a graduated scale (0-3+) and nuclear staining was graded using an Immunoscore. Results: IL6ST exists in membrane-bound and soluble forms of varying size. The full-length membrane bound molecule comprises 8 domains: 6 extracellular, 1 transmembrane and 1 cytoplasmic. In the EA2Clin test, pre-treatment BC tissues are stained for IL6ST with an antibody specific for a region spanning the transmembrane and cytoplasmic domains. TMAs were stained for IL6ST with both this and a second antibody binding the extracellular part, detecting both full-length and most soluble isoforms. Levels of both were correlated (R=0.82, P<0.0001). IL6ST is known to mediate the action of cytokines including IL6, OSM and LIF via downstream regulation of pathways such as JAK/STAT. TMAs were stained for antibodies against IL6ST, OSM, IL6, total STAT3, pSTAT3 (Tyr705) and pSTAT3 (Ser727). IL6ST was scored as low (0/1+) or high (2+/3+). There was a positive association between levels of IL6ST and IL6 (P=0.02) and total STAT3 (P=0.003). There was no association between IL6ST and OSM or either pSTAT3. Supervised gene expression analysis comparing pre-treatment samples with high and low IL6ST levels revealed increased levels of STAT3-regulated genes: cell cycle (CEBPD, CDKN1B), apoptosis (NFIL3, ATF3, BCL2), extracellular matrix remodelling (ADM, SEPRINE1-3) and interferon signalling (IFIT1, IFI44, IFI27). Unsupervised gene enrichment analysis revealed increased expression of genes involved with JAK/STAT, PI3K, mTOR and ERBB1 signalling in tumours expressing higher IL6ST levels. Lower levels were associated with increased energy generation, cellular metabolism and epithelial-mesenchymal transition. Conclusions: • This is the first matched whole-genome and mass spectrometry proteome analysis of sequential ET-treated BC patients • IL6ST predicts response to ET – it is used in2 independent assays • Levels of full-length IL6ST appear to be the most important for ET response prediction • IL6ST may have an active role in BC cells, mediating signalling of cytokines such as IL6 through the JAK/STAT pathway and subsequent downstream transcriptional regulation. Citation Format: Turnbull AK, Fernando A, Martinez-Perez C, Finch AJ, von Kriegsheim A, Wills J, Quinn N, Selli C, Mosley D, Langdon SP, Sims AH, Dixon JM. Understanding the mechanisms of action underlying the role of IL6ST, a key biomarker for prediction of response to endocrine therapy [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 P4-08-02.
Purpose: Neo-Adjuvant chemotherapy treatment is increasingly being used in breast cancer to preoperatively shrink tumour volumes and facilitate surgical plans. These datasets however, are still scarce, making it difficult to assess the relative value of multiple time point biopsies compared to diagnostic only sampling. This study aims to identify sequential samplings intrinsic value. Method: A total of 97 samples from a cohort of 50 neoadjuvant chemotherapy treated primary breast cancer patients (aged 29-76 at diagnosis, Allred status 47:53% +/-, Her2 status 80:20% +/-, mixed grade and menopausal status) taken pre- treatment, at 2 weeks on-treatment, mid chemotherapy and at resection were sequenced with Ion Ampliseq transcriptome yielding expression values for 12,635 genes. Differential expression analysis was performed across response groups (16 Responders, 34 Non-Responders) as defined by Pathological Complete Response and over treatment time to identify significantly differentially expressed genes, pathways and markers indicative of response status. Results: An on-treatment marker for response was identified (AAGAB), which resulted in a testing accuracy of 100% and a validation accuracy of 78% in the I-SPY 1 Trial. AAGAB was predictive of long term survival (p = 0.048 testing, p = 0.031 validation) in both chemotherapy cohorts at the same expression level as defined for treatment response. The single gene on-treatment biomarker, AAGAB proves more performant than established prognostic tests, Mammaprint (Edinburgh NEO trial, pre-treatment 61%, on-treatment 63%. I-SPY 1 trial, pre-treatment 60%, on-treatment 66%) and Pam50 RORS (neo trial pre-treatment 50%, on treatment 58%, Magbanua trial pre-treatment 56%, on-treatment 64%) Conclusion: Changes in gene expression of on-treatment chemotherapy breast cancer resulted in the identification of a novel gene marker that was as effective in predicting prognostic status as established prognostic tests. These results support the use of on-treatment testing in breast cancer to improve the accuracy of tumour response prediction. Citation Format: Bownes RJ, Turnbull AK, Cameron D, Sims AH, Oikonomidou O. On-treatment biomarkers can improve prediction of response to neoadjuvant chemotherapy in breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-11-13.
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