Inhibiting nicastrin (NCT) stabilizes expression of e-cadherin and has potential in inducing mesenchymal to epithelial transition in breast cancer (BC).

Abstract

Abstract Abstract #3066 Introduction: NCT is a member of the Gamma Secretase (GS) enzyme complex, responsible for activating multiple substrates (Notch proteins, Her4, E-cadherin , CD44 etc.), implicated in BC development and progression from primary tumour to metastasis. E-cadherin (E-cad) is a key protein regulating cell-to-cell adhesion. Loss of E-cad is a central feature of Epithelial-to-Mesenchymal (EMT) transition enabling cell migration and invasion, while restoration of E-cad expression indicates a less malignant cell phenotype.
 Materials and Methods: MCF-7 and MDA-MB-231 BC cell lines were used to investigate expression of NCT by Western Blot (WB) and RT-Q-PCR. Paraffin embedded BC tissue samples (n=126), 10 benign breast lesions (fibroadenoma) and 40 normal breast tissue specimens were analysed for NCT expression by Immunohistrochemistry (IHC). NCT was transiently silenced in vitro by siRNA and expression of E-cad, Beta-Catenin, Snail1 were analysed by WB and RT-Q-PCR. A commercially available kit was used to measure GS activity in vitro. Transmitted light microscopy was used to monitor cell motility using a model of a stable NCT knockout (KO) in mouse embryonic fibroblast (MEF) cells.
 Results: NCT was not expressed or was weakly expressed in normal breast tissue by IHC. Strikingly, NCT was highly expressed only in the myoepithelial layer of all 10 fibroadenomas analysed, while epithelial layer remained negative of weakly NCT positive (+). Breast carcinomas were graded as NCT 1+, 2+ or 3+. NCT siRNA (72 h) resulted in >90% reduction of both NCT mRNA and protein levels, as well as >50% reduction in GS activity in both MDA-MB-231 and MCF-7 cell lines. NCT siRNA did not affect E-cad, Beta-Catenin or Snail1 mRNA levels in these cell lines. However, NCT siRNA resulted in cellular redistribution of E-cad to the cell membrane, as visualized by immunofluorescence, and an increase in E-cad protein levels by WB in MCF-7 cells. The transient NCT silencing at 72 h did not result in change of morphology in MDA-MB-231 cells. Cell motility was, however, reduced by 20%: (0.01 µm/sec in parental MDA-MB-231 vs 0.008 µm/sec in MDA-MB-231 NCT siRNA cells). Stable NCT KO caused a notable change in cell phenotype, converting a spindle like wild type MEFs to rounded epitheloid-like morphology, coupled with a marked increase of E-cad localized to the membrane. Furthermore, cell motility was reduced by 3 fold in NCT KO MEFs (0.008 µm/sec in wild type MEFs vs 0.0025um/sec in the NCT KO MEFs).
 Conclusion: NCT is up-regulated in BC as compared to normal breast tissue. NCT is strongly expressed only in the myoepithelial cell compartment of breast fibroadenomas, suggesting possible involvement of NCT in EMT. Inhibiting NCT stabilizes E-cadherin protein levels in BC cells, at the post-translational level. Inhibiting NCT in BC may represent a potential strategy to reduce BC invasiveness and metastasis. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3066.