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

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

Semaphorin-3A is expressed by tumor cells and alters T-cell signal transduction and function

An important aspect of tumor progression is the ability of cancer cells to escape detection and clearance by the immune system. Recent studies suggest that several tumors express soluble factors interfering with the immune response. Here, we show that semaphorin-3A (Sema-3A), a secreted member of the semaphorin family involved in axonal guidance, organogenesis, and angiogenesis, is highly expressed in several tumor cells. Conditioned media of Sema-3A-transfected COS-7 cells or human recombinant Sema-3A inhibited primary human T-cell proliferation and cytokines production under anti-CD3 plus anti-CD28 stimulating conditions. Sema-3A also inhibited the activation of nonspecific cytotoxic activity in mixed lymphocyte culture (MLC), as measured against K-562 cells. In contrast, suppression of Sema-3A in tumor cells with a small interfering RNA (siRNA) augmented T-cell activation. The inhibitory effect of Sema-3A in T cells is mediated by blockade of Ras/mitogen-activated protein kinase (MAPK) signaling pathway. The presence of Sema-3A increased the activation of the Ras family small GTPase Rap1 and introduction of the dominant-negative mutant of Rap1 (Rap1N17) blunted the immunoinhibitory effects of Sema-3A. These results suggest that Sema-3A inhibits primary T-cell activation and imply that it can contribute to the T-cell dysfunction in the tumor microenvironment. \ua9 2006 by The American Society of Hematology

Deubiquitinase Activities Required for Hepatocyte Growth Factor-Induced Scattering of Epithelial Cells

The scattering response of epithelial cells to activation of the Met receptor tyrosine kinase represents one facet of an “invasive growth” program [1, 2]. It is a complex event that incorporates loss of cell-cell adhesion, morphological changes, and cell motility. Ubiquitination is a reversible posttranslational modification that may target proteins for degradation or coordinate signal transduction pathways [3, 4]. There are ∼79 active deubiquitinating enzymes (DUBs) predicted in the human genome [5, 6]. Here, via a small interfering RNA (siRNA) library approach, we have identified 12 DUBs that are necessary for aspects of the hepatocyte growth factor (HGF)-dependent scattering response of A549 cells. Different phenotypes are evident that range from full loss of scattering, similar to receptor knockdown (e.g., USP30, USP33, USP47), to loss of cell-cell contacts even in the absence of HGF but defective motility (e.g., USP3, ATXN3L). The knockdowns do not incur defective receptor, phosphatidylinositol 3-kinase, or MAP kinase activation. Our data suggest widespread involvement of the ubiquitin system at multiple stages of the Met activation response, implying significant crosstalk with phosphorylation-based transduction pathways. Development of small-molecule inhibitors of particular DUBs may offer a therapeutic approach to contain metastasis