Anti-nicastrin monoclonal antibodies elicit pleiotropic anti-tumour pharmacological effects in invasive breast cancer cells

Abstract

The goal of targeted cancer therapies is to specifically block oncogenic signalling, thus maximising efficacy, while reducing side-effects to patients. The gamma-secretase (GS) complex is an attractive therapeutic target in haematological malignancies and solid tumours with major pharmaceutical activity to identify optimal inhibitors. Within GS, nicastrin (NCSTN) offers an opportunity for therapeutic intervention using blocking monoclonal antibodies (mAbs). Here we explore the role of anti-nicastrin monoclonal antibodies, which we have developed as specific, multi-faceted inhibitors of proliferation and invasive traits of triple-negative breast cancer cells. We use 3D in vitro proliferation and invasion assays as well as an orthotopic and tail vail injection triple-negative breast cancer in vivo xenograft model systems. RNAScope assessed nicastrin in patient samples. Anti-NCSTN mAb clone-2H6 demonstrated a superior anti-tumour efficacy than clone-10C11 and the RO4929097 small molecule GS inhibitor, acting by inhibiting GS enzymatic activity and Notch signalling in vitro and in vivo. Confirming clinical relevance of nicastrin as a target, we report evidence of increased NCSTN mRNA levels by RNA in situ hybridization (RNAScope) in a large cohort of oestrogen receptor negative breast cancers, conferring independent prognostic significance for disease-free survival, in multivariate analysis. We demonstrate here that targeting NCSTN using specific mAbs may represent a novel mode of treatment for invasive triple-negative breast cancer, for which there are few targeted therapeutic options. Furthermore, we propose that measuring NCSTN in patient samples using RNAScope technology may serve as companion diagnostic for anti-NCSTN therapy in the clinic.Electronic supplementary materialThe online version of this article (doi:10.1007/s10549-014-3119-z) contains supplementary material, which is available to authorized users.

Effect of nicastrin on hepatocellular carcinoma proliferation and apoptosis through PI3K/AKT signalling pathway modulation

“…Furthermore, Lombardo et al [6] reported that NCSTN regulates Akt activation in breast cancer cells and that NCSTN inhibition in malignant breast cells can inhibit breast tumour formation in vivo. Studies have also demonstrated that anti-NCSTN monoclonal antibodies exert antitumour effects in invasive breast cancer cells [8]. Notably, these results are consistent with not only the KEGG pathway results for GSEA based on TCGA data but also our observation that NCSTN depletion could induce apoptosis and reduce proliferation in HCC cell lines, partly through the PI3K/Akt pathway.…”

“…NCSTN, the largest constituent of the γ-secretase complex [24], has a single transmembrane domain and a large extracellular domain with functional regions [8]. Intriguingly, it has been reported that NCSTN could exert its function alone rather than participate in the γ-secretase complex in mouse muscle [25], which indicates that NCSTN is likely to exert some functions independently.…”

“…NCSTN, one of the main constituents and the gatekeeper of the γ-secretase complex, modulates the composition of this complex, maintains enzyme stability and executes GS substrate recognition [7]. Moreover, NCSTN has a GS-independent function in the process of controlling cell death through the Akt pathway [8]. Consistent with the biological correlation of NCSTN in malignant diseases, studies have shown that NCSTN is upregulated in patients with colon cancer who underwent chemotherapy [9] and that stable knockdown of NCSTN enhances the antitumour effect of EGFR inhibitors by blocking the Notch and Akt signalling pathways [10].…”

Notch Signaling in Development, Tissue Homeostasis, and Disease

“…Blocking of Jagged1 prevented and reversed lung goblet cell metaplasia in mouse models (and these effects were enhanced when Jagged2 was simultaneously inhibited), raising the provocative possibility that such treatments might be clinically deployed for respiratory disease characterized by excess mucus secretions (384). In addition to antibodies to receptors and ligands, antibodies to Nicastrin have been explored preclinically (190,263), although they, like GSIs, are likely to affect all ␥-secretase-cleaved proteins (244). Antibodies can also be used for GVHD, with few reported side effects (723; for review, see Ref.…”

Endocrine therapy resistance and epithelial to mesenchymal transition are driven by Nicastrin and Notch4 cooperation in MCF7 breast cancer cells

“…Haywashi and colleagues synthesized a monoclonal antibody against the extracellular domain of Nicastrin reporting the neutralisation of GS activity. We fully characterized two mAbs showing tumor reduction in vivo of triple negative breast cancer cell lines ( [16] and [17] ). In this paper, we highlight our recent study [18] on the efficacy of GSI PF03084014 and anti-Nicastrin mAbs to partially revert the EMT process and re-sensitize ETR breast cancer cells.…”