The online version of this article (doi:10.1186/s12916-017-0836-2) contains supplementary material, which is available to authorized users.
Endocrine therapy is standard treatment for estrogen receptor (ER)-positive breast cancer. However, its efficacy is limited by intrinsic and acquired resistance. Here the potential of S100β as a biomarker and inhibition of its signaling network as a therapeutic strategy in endocrine treated patients was investigated.
The expression of S100β in tissue and serum was assessed by immunohistochemistry and an enzyme-linked immunosorbent assay, respectively. The S100β signaling network was investigated in cell line models of endocrine resistance by western blot, PCR, immunoprecipitation, and chromatin-immunoprecipitation. Endocrine resistant xenografts and tumor explants from patients with resistant tumors were treated with endocrine therapy in the presence and absence of the p-Src kinase inhibitor, dasatinib.
Tissue and serum levels of S100β were found to predict poor disease-free survival in endocrine-treated patients (n = 509, HR 2.32, 95% CI is 1.58–3.40, p < 0.0001 and n = 187, HR 4.009, 95% CI is 1.66–9.68, p = 0.002, respectively). Moreover, elevated levels of serum S100β detected during routine surveillance over the patient treatment period significantly associated with subsequent clinically confirmed disease recurrence (p = 0.019). In vivo studies demonstrated that endocrine treatment induced transcriptional regulation of S100β which was successfully disrupted with tyrosine kinase inhibition. In endocrine resistant xenografts and tumor explants from patients with endocrine resistant breast cancer, combined endocrine and dasatinib treatment reduced tumor proliferation and down-regulated S100β protein expression in comparison to endocrine treatment alone.
S100β has potential as a new surveillance tool for patients with ER-positive breast cancer to monitor ongoing response to endocrine therapy. Moreover, endocrine resistant breast cancer patients with elevated S100β may benefit from combined endocrine and tyrosine-kinase inhibitor treatment.
ClinicalTrials.gov, NCT01840293). Registered on 23 April 2013. Retrospectively registered.
Additional file 1: Figure S1. Training set to establish S100β elevated cut-off. Serum S100β levels were determined using a commercial ELISA kit (Diasorin) according to the manufacturer’s instructions. To determine an appropriate S100β cut-off level, a training set was constructed comprising ten breast cancer patients with no relapse (No Recurrence), ten patients who went on to have confirmed disease recurrence (Recurrence), and ten aged matched controls (Normal). The upper limit of normal was calculated (mean + (t0.975,n-1 x √(n+1/n) x SD)), 0.13 μg/L was considered the upper end of normal and was taken as the cut-off. Patient clinicopathological parameters and treatment details are provided for No Recurrence and Recurrence patients. Median age of control patients is 62.8 years. Figure S2. Site of recurrence in patients with elevated S100β. Site of recurrence in patients with elevated pre-operative or monitoring serum S100β levels (n = 13). Figure S3. Dasatinib successfully inhibits p-Src expression in endocrine resistant breast cancer. (A) Expression of p-AKT, AKT, P-ERK, and ERK in panel of endocrine resistant (LY2, LetR) and sensitive (MCF-7, ARO) cell lines. (B) Tamoxifen resistant cells LY2 expressed increased levels of p-Src when treated with tamoxifen which was inhibited with PP2 (10 μM) and dasatinib (0.1 μM). Letrozole resistant cells LetR expressed increased levels of p-Src when treated with EGF which was inhibited with PP2 (10 μM) and dasatinib (0.1 μM). (C) Interactions between SRC-1 and the transcription factor HOXC11 were increased by 4-OHT (1 × 10–7 M) in tamoxifen resistant LY2 cells and by EGF (10 ng/ml) in letrozole resistant LetR cells. These interactions were inhibited in both cell lines by pre-treatment with the p-SRC inhibitors PP2 (10 μM) and dasatinib (0.1 μM). (D) mRNA expression of the HOXC11/SRC-1 complex’s downstream target gene S100β increased with tamoxifen (T) treatment compared with vehicle (V). This tamoxifen-induced expression was successfully inhibited when treated with dasatinib (D). Figure S4. Proliferation in resistant breast cancer cell lines following dasatinib treatment. (A) Dasatinib treatment (1 μM) for 72 h inhibits both estrogen- and tamoxifen-driven proliferation of the tamoxifen resistant cell line LY2 and AI resistant LetR measured by MTS assay (n = 3). (B) Expression of nuclear Ki67 significantly decreased in the dasatinib-treated xenograft primary tumors (p = 0.01). Studying the architecture of the epithelial cells in the primary tumors by H&E staining revealed that dasatinib-treated tumors had a more organized cell population than that of the controls. Figure S5. SRC-1 and HOXC11 scoring of primary tumors from endocrine resistant xenograft model. Primary tumors from vehicle- and dasatinib-treated mice were stained for SRC-1 and HOXC11 and analyzed for total positivity using Aperio Imagescope software. Results are expressed as mean protein positivity ± SEM, p ≤ 0.05. The expression of SRC-1 and HOXC11 was decreased in the dasatinib-treated group compared to that of the vehicle-treated group. Figure S6. Migratory potential which can be inhibited by dasatinib. (A) The migratory potential of endocrine resistant cell lines (LY2 and LetR) is elevated in comparison to that of their parental, the non-migratory cell line MCF-7. This migration was comparable to that of the highly metastatic triple negative breast cancer cell line MDA-MB-231. Treatment with dasatinib significantly inhibited this migration (p < 0.05). (B) Endocrine sensitive MCF-7 cells were stably transfected with SRC-1 and HOXC11 vectors to assay the effects of these proteins on migratory potential. The successful overexpression was confirmed by western blot (n = 3). (C) Overexpression of the transcription factor HOXC11 and SRC-1 significantly increased the migration of MCF-7 compared to the empty vector transfected cells (p < 0.05). Dasatinib treatment (1 μM) significantly inhibited both HOXC11- and SRC-1-driven cellular migration (p < 0.001). (PPTX 670 kb)
Additional file 2: Table S1. (A) S100β validation set (n = 76 ER-positive and ER-negative patients). (B) S100β validation set (n = 59 ER-positive patients). Association of S100β status with clinicopathological variables and disease recurrence using Fisher’s exact test. Table S2. Pre-operative and post-operative S100β serum levels in 55 ER-positive patients. Association of S100β status with clinicopathological variables using Fisher’s exact test. Table S3. S100β tissue expression in matched primary and metastatic tissue from ER-positive patients. Table S4. Patient details from explant study. Explant endocrine resistant tumor tissue (n = 2) was treated with AI therapy (letrozole) in the presence and absence of dasatinib. (PPTX 93 kb)
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- S100β as a serum marker in endocrine resistant breast cancer
Fiona T. Bane
Bryan T. Hennessy
Róisín M. Dwyer
Michael J. Kerin
Arnold D. Hill
Leonie S. Young
- BioMed Central
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