S100β as a serum marker in endocrine resistant breast cancer

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Trial registration: ClinicalTrials.gov, NCT01840293 ). Registered on 23 April 2013. Retrospectively registered.

Keywords: Biomarker; Breast cancer; Endocrine resistance; Estrogen receptor; S100β.

Figures

Fig. 1

Flow chart of patients with breast cancer recruited to the clinical studies. For prognostic tissue studies, FFPE breast tumor samples (n = 1079) were obtained from archival cases at St. Vincent’s University Hospital and Beaumont Hospital, Dublin, Ireland from 1987–1999 and from 2004–2008, respectively. For prognostic serum studies, blood samples were collected from breast cancer patients attending the National University Hospital Galway and Beaumont Hospital from 1993–2006 and 2008–2014, respectively, prior to initial surgery for the resection of primary breast tumor (n = 346). For monitoring serum studies, pre-operative and serial post-operative samples were collected from patients with breast cancer enrolled in a multicenter Cancer Trials Ireland-led trial (ICORG 09/07;NCT01840293/(ClinicalTrials.gov)) from 2008–2014 (n = 181)

Fig. 2

S100β associates with poor disease-free survival times in patients with endocrine-treated breast cancer. a S100β tissue expression strongly associated with poor disease-free survival in a TMA patient cohort of 509 ERα-positive, endocrine-treated patients as determined by Kaplan-Meier estimates of disease-free survival (HR 2.32, 95% CI is 1.58–3.40, p < 0.0001, Cox proportional hazard model). b 187 endocrine-treated ERα-positive patient serum samples were analyzed by ELISA for S100β using the established cut-off of 0.13 μg/L. S100β serum levels significantly associated with poor disease-free survival in the endocrine therapy-treated patient population (HR 3.09, 95% CI is 1.66–9.68, p = 0.002, Cox proportional hazard model). c Pre-operative elevated serum S100β returned to normal following surgical resection of the tumor in matched patient blood samples as measured by ELISA (55 individual patients, 6 pre-operative S100β ≥0.13 and 49 pre-operative S100β <0.13, paired t test error bars refer to 95% CI, p = 0.023)

Fig. 3

Metastasis forms in resistant breast cancer in vivo which is marked by S100β. a Endocrine sensitive MCF-7 and endocrine resistant LY2 cells were injected into the mammary fat pad of 6-week-old Balbc/SCID mice. Mice were treated with estrogen and with or without slow release tamoxifen pellets. Tumor volume was decreased significantly in the endocrine sensitive model with tamoxifen treatment (p = 0.0005, n = 3), and there was a small, but not significant, increase in tumor volume in the endocrine resistant model with tamoxifen treatment compared to the untreated endocrine resistant model (p = 0.068, n = 3). There was also a significant increase in tumor volume with tamoxifen treatment in the endocrine sensitive compared to endocrine resistant model (p = 0.0004, n = 3). b Metastasis was formed in the presence of tamoxifen treatment at week 2 which increased gradually until the experimental endpoint. Metastasis was visualized and quantified using in vivo and ex vivo IVIS imaging at the experimental endpoint (week 7). c Formation of metastasis in the organs was confirmed at experimental endpoint (week 7) by immunohistochemical (IHC) analysis. Representative images of primary tissue, lung, liver, and bone. H&E staining demonstrated the presence of tumor epithelial cells in the metastatic sites, and nuclear Ki67 indicated active proliferation. ERα expression was maintained throughout the experiment in the primary tumor and in the lungs, liver, and bone metastases and S100β expression in primary tissue, lung, liver, and bone. (Representative image n = 7.) d S100β levels in the serum of mice treated with tamoxifen increased concurrently with metastasis (non-parametric t test of blood between weeks 5 and 7, p = 0.0012). e In matched patients who recurred on endocrine therapy high expression of S100β in their primary tumor was maintained in metastatic tissue, while patients with low S100β in their primary tumor gained expression of S100β in their metastatic tissue at diagnosis as determined by IHC analysis (n = 4). f Post-operative monitoring levels of S100β was significantly higher in patients with metastatic disease (n = 96 Recurrence-Free and n = 22 Recurrence patients; error bars refer to 95% CI, p = 0.003)

Fig. 4

Endocrine treatment induces transcriptional regulation of S100β which is disrupted by tyrosine kinase inhibition. a Expression of ERα, PR, p-Src, c-Src, HOXC11, SRC-1, S100β, and β-actin in a panel of endocrine resistant (LY2, LetR) and sensitive (MCF-7, ARO) cell lines (representative blots from n = 3). b Chromatin immunoprecipitation of SRC-1 and HOXC11 of the S100β promoter region in LY2 cells treated with tamoxifen alone or in combination with dasatinib inhibits SRC-1 and HOXC11 recruitment to the S100β promoter (graphs are representative of the mean relative recruitment values relative to tamoxifen-treated controls, n = 3 ± SEM, p ≤ 0.05). c Inhibition of S100β protein expression with tyrosine kinase inhibitors PP2 or dasatinib in the endocrine resistant LY2 cells treated with tamoxifen (representative blots from n = 3). d Schematic representation of S100β pathway, showing long-term exposure to endocrine therapy induces expression of S100β in an Src-dependent manner

Fig. 5

Combined endocrine and Src-kinase inhibitor treatment decreases tumor burden, which is marked by reduction in S100β. a In vivo model of endocrine resistant xenograft treated with dasatinib (50 mg/kg/day) in combination with tamoxifen showed a significant inhibition of tumor growth (mean tumor volume from caliper measurement, n = 7, error bars refer to 95% CI, p < 0.05). Representative IVIS images of mice at week 1 and week 7. Representative IVIS ex vivo images of tumor metastasis to lung, liver, and bone. b IHC analysis of tamoxifen-treated primary tumor from the endocrine resistant xenograft showed ERα expression and inhibition of Ki67, p-Src, SRC-1, HOXC11, and S100β expression after treatment with dasatinib. c Serum levels of S100β reduced in the xenograft mice treated with dasatinib at week 7 (n = 5, p = 0.085 one-tailed t test). d IHC revealed that dasatinib decreased expression of S100β and the proliferative marker Ki67 while maintaining the expression of ERα in an ex vivo explant model of tumor tissue from endocrine resistant patients after treatment with letrozole in the presence or absence of dasatinib (n = 2)