Introduction: mTOR and its downstream effectors the 4E-binding protein 1 (4EBP1) and the p70 ribosomal S6 kinases (S6K1 and S6K2) are frequently upregulated in breast cancer, and assumed to be driving forces in tumourigenesis, in close connection with oestrogen receptor (ER) networks. Here, we investigated these factors as clinical markers in five different cohorts of breast cancer patients. Methods: The prognostic significance of 4EBP1, S6K1 and S6K2 mRNA expression was assessed with real-time PCR in 93 tumours from the treatment randomised Stockholm trials, encompassing postmenopausal patients enrolled between 1976 and 1990. Three publicly available breast cancer cohorts were used to confirm the results. Furthermore, the predictive values of 4EBP1 and p4EBP1_S65 protein expression for both prognosis and endocrine treatment benefit were assessed by immunohistochemical analysis of 912 node-negative breast cancers from the Stockholm trials. Results: S6K2 and 4EBP1 mRNA expression levels showed significant correlation and were associated with a poor outcome in all cohorts investigated. 4EBP1 protein was confirmed as an independent prognostic factor, especially in progesterone receptor (PgR)-expressing cancers. 4EBP1 protein expression was also associated with a poor response to endocrine treatment in the ER/PgR positive group. Cross-talk to genomic as well as non-genomic ER/PgR signalling may be involved and the results further support a combination of ER and mTOR signalling targeted therapies. Conclusion: This study suggests S6K2 and 4EBP1 as important factors for breast tumourigenesis, interplaying with hormone receptor signalling. We propose S6K2 and 4EBP1 as new potential clinical markers for prognosis and endocrine therapy response in breast cancer.

Introduction: Endocrine therapies targeting cell proliferation and survival mediated by estrogen receptor α (ERα) are among the most effective systemic treatments for ERα-positive breast cancer. However, most tumors initially responsive to these therapies acquire resistance through mechanisms that involve ERα transcriptional regulatory plasticity. Herein we identify VAV3 as a critical component in this process. Methods: A cell-based chemical compound screen was carried out to identify therapeutic strategies against resistance to endocrine therapy. Binding to ERα was evaluated by molecular docking analyses, an agonist fluoligand assay and short hairpin (sh)RNA–mediated protein depletion. Microarray analyses were performed to identify altered gene expression. Western blot analysis of signaling and proliferation markers, and shRNA-mediated protein depletion in viability and clonogenic assays, were performed to delineate the role of VAV3. Genetic variation in VAV3 was assessed for association with the response to tamoxifen. Immunohistochemical analyses of VAV3 were carried out to determine its association with therapeutic response and different tumor markers. An analysis of gene expression association with drug sensitivity was carried out to identify a potential therapeutic approach based on differential VAV3 expression. Results: The compound YC-1 was found to comparatively reduce the viability of cell models of acquired resistance. This effect was probably not due to activation of its canonical target (soluble guanylyl cyclase), but instead was likely a result of binding to ERα. VAV3 was selectively reduced upon exposure to YC-1 or ERα depletion, and, accordingly, VAV3 depletion comparatively reduced the viability of cell models of acquired resistance. In the clinical scenario, germline variation in VAV3 was associated with the response to tamoxifen in Japanese breast cancer patients (rs10494071 combined P value = 8.4 × 10⁻⁴). The allele association combined with gene expression analyses indicated that low VAV3 expression predicts better clinical outcome. Conversely, high nuclear VAV3 expression in tumor cells was associated with poorer endocrine therapy response. Based on VAV3 expression levels and the response to erlotinib in cancer cell lines, targeting EGFR signaling may be a promising therapeutic strategy. Conclusions: This study proposes VAV3 as a biomarker and a rationale for its use as a signaling target to prevent and/or overcome resistance to endocrine therapy in breast cancer.

EPH/ephrin profile and EPHB2 expression predicts patient survival in breast cancer

Purpose The protein tyrosine phosphatase PTPN2 dephosphorylates several tyrosine kinases in cancer-related signalling pathways and is thought to be a tumour suppressor. As PTPN2 is not frequently studied in breast cancer, we aimed to explore the role of PTPN2 and the effects of its loss in breast cancer. Methods Protein expression and gene copy number of PTPN2 were analysed in a cohort of pre-menopausal breast cancer patients with immunohistochemistry and droplet digital PCR, respectively. PTPN2 was knocked down in three cell lines, representing different breast cancer subtypes, with siRNA transfection. Several proteins related to PTPN2 were analysed with Western blot. Results Low PTPN2 protein expression was found in 50.2% of the tumours (110/219), gene copy loss in 15.4% (33/214). Low protein expression was associated with a higher relapse rate in patients with Luminal A and HER2-positive tumours, but not triple-negative tumours. In vitro studies further suggested a subtype-specific role of PTPN2. Knockdown of PTPN2 had no effect on the triple-negative cell line, whilst knockdown in MCF7 inhibited phosphorylation of Met and promoted that of Akt. Knockdown in SKBR3 led to increased Met phosphorylation and decreased Erk phosphorylation as well as EGF-mediated STAT3 activation. Conclusion We confirm previous studies showing that the PTPN2 protein is lost in half of the breast cancer cases and gene deletion occurs in 15–18% of the cases. Furthermore, the results suggest that the role of PTPN2 is subtype-related and should be further investigated to assess how this could affect breast cancer prognosis and treatment response.

The tyrosine kinase receptor EphB4 and its ligand ephrin-B2 interact through cell-to-cell contacts. Upon interaction, EphB4 transmits bidirectional signals. A forward signal inside EphB4-expressing cells is believed to suppress tumor growth, while inside the ephrin-expressing cells, an oncogenic reverse signal arises. In breast cancer cells with a high EphB4 receptor expression the forward signal is low, in part due to the low expression of the ligand ephrin-B2. Therefore, we hypothesized that by re-introducing the ligand in EphB4-positive cells, tumor suppression could be induced by the stimulation of the forward signal. This question was addressed in vitro by the stable lentiviral infection of breast cancer cells with either wild-type EFNB2 or with a mutant EFNB2-5F, unable to transmit reverse signaling. Furthermore, we investigated ephrin-B and EphB4 protein expression in 216 paraffin-embedded tumors using immunohistochemistry. The in vitro results indicated that ephrin-B2 expression was associated with a lower cell proliferation, migration and motility compared with the control cells. These effects were more pronounced when the cells lacked the ability to transmit the reverse signal (B2-5F). In clinical material, ephrin-B protein expression was associated with a positive estrogen receptor (ER) status, a low HER-2 expression and was negatively associated with Nottingham histologic grade (NHG) III. Ephrin-B expression indicated a good prognosis, whereas EphB4 expression was associated with a shorter metastasis-free survival in univariate and multivariate analysis. Furthermore, the prognostic value of EFNB2 and EPHB4 was confirmed at the gene expression level in public datasets. Thus, on the whole, the findings of this study suggest that ephrin-B2 expression is associated with less proliferation and lower motility of breast cancer cells and with a longer patient survival in breast cancer.