Abstract P5-06-04: Nicastrin Reveals Gamma-Secretase Independent Function in Breast Cancer Cells and Can Be Targeted by a Blocking Monoclonal Antibody To Reduce Breast Cancer Cell Proliferation and Invasion
Authors: Filipovic A , Green A , Shao D , Yague E , Pannuti A , Yao K , Feng G , Lin S , Miele L , Coombes C. 
Published: April 2011

BACKGROUND: Nicastrin (NCT) is a structural and functional part of the gamma-secretase (GS) enzyme, implicated in tumorigenesis and resistance to treatment in multiple cancer types. However, its mechanism(s) of action and biological functions are poorly understood. We investigated functional and biological effects of inhibiting NCT in breast cancer (BC)cells by means of gene silencing and blocking antibodies, as well as the prognostic value of NCT expression in BC tissue. METHODS: NCT siRNA (Dahrmacon) was used at 40nM. A polyclonal antibody (pcAb) against NCT extracellular domain was developed at Biogenes (GE), and anti-NCT monoclonal antibodies (mAbs) at Genovac (GE). We tested the Abs for their ability to recognize endogenous NCT in BC cells by FACS and Western blot. Best Abs were tested in vitro, using a transwell cell invasion assay and proliferation assay. Anti-NCT Ab (Sigma, N1660) was used for immunohistochemistry (IHC) staining of BC tissues (n = 1050). Cases were scored as NCT negative (0), 1+, 2+ or 3+. Kaplan Meier analysis was used to investigate impact of NCT expression on survival. Illumina (Agilent) oligonucleotide arrays were used to investigate NCT siRNA effects on gene expression in BC cells. Both PcAb and mAbs recognized endogenous NCT in BC cells. In vitro, blocking NCT in MDA-MB231 BC cells with the PcAb, reduced cell proliferation in a dose dependent manner; by 50% and 70% at concentrations of 50μg/ml and 100μg/ml, respectively. mAbs reduced proliferation by 50% (clones 2H6 and 10C5) and 70% clone 10C11, (P&lt;0.01). In transwell cell invasion assay, anti-NCT pcAb induced a dose dependent reduction of invasion, by 35% at the 5μg/ml, peaking at &gt; 60% reduction at 50μg/ml (P&lt;0.001). mAbs reduced cell invasion by 40-50% (clone 10C5 and 10C11) and by &gt;65% (clone 2H6) (P&lt;001). IHC data showed that NCT was highly expressed (2+, 3+) in 47.5% of BC tissues. High NCT expression correlated with worse overall survival (OS) in ERnegative patients (p=0.05). To elucidate the effects of NCT loss, compared to those induced by GSIs or silencing Notch receptors, we examined cDNA from MDA231 treated independently with NCT, Notch1, Notch2, Notch4 siRNAs and GSI MK-003, on Illumina expression arrays. Thirteen of the 18 genes maximally affected by GSI were similarly affected by NCT siRNA. NCT knockdown also affected 1745 other genes that by large were not affected by GSI or Notch knockdowns. This suggests that NCT has GS-and Notch-independent cellular functions and may have different biological properties as a therapeutic target than GS. Among NCT siRNA affected genes, some are tumor suppressors: CST4 increased 8 fold; pro-invasive genes like GNG11 decreased 4 fold; cell adhesion genes: CTGF increased 4.5 fold etc.

CONCLUSIONS: NCT is overexpressed in 47.5% of BC cases where it infers adverse OS in the ER-vecohort of patients. Anti-NCT mAbs recognize endogenous NCT in BC cells and reduce BC cell proliferation and invasion. Silencing NCT in invasive BC cells induces extensive gene expression changes that only partially overlap with those caused by GSIs or Notch siRNAs. We conclude that NCT has GS-independent functions in BC cells and that an anti-NCT mAb may be a promising therapeutic approach for invasive BC.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P5-06-04.