Abstract 3311: Role of nicastrin in the maintenance of breast cancer stem cells and invasion

Abstract

Abstract Background: Cancer stem-cells may be involved in development and metastasis of tumours, including breast cancer. Initial identification of breast cancer stem cells (BCSCs) was based on a combination of CD44+, CD24- and Lin- markers. Later, several markers such as aldehyde dehydrogenase 1 (ALDH1) were defined. The notch signaling pathway has been implicated in BCSC survival. Gamma-secretase (GS) is an enzymatic multi-protein complex composed of presenilin 1 (PS1), nicastrin (NCT), anterior pharynx-defective phenotype 1 (APH-1), and the PS enhancer 2 (PEN-2) proteins, which activates Notch. We have previously shown that high levels of NCT observed in 47.5% of BC patients, confers worse overall survival in the ERα negative patients. Here, we have explored targeting NCT, the vital component of GS assembly and function, to determine its role in the maintenance of the BCSC pool. Methods: Stable NCT knock-down HCC1806 breast cancer cells (HCC1806 ShNCT) and control cells (HCC1806 ShLUC) were produced by lentiviral infections and puromicin selection. Stable NCT overexpressing MCF10A cells (MCF10ANCT) and control cells (MCF10ACTRL) were produced by retroviral infections and puromicin selection. The sulphorhodamine B and three-dimentional (3D) assay in Matrigel were used to measure the proliferation rate of these cells. Invasion assay was perfomed using Matrigel-coated Boyden chamber. Stem cell content was evaluated by the percentage of CD44+/CD24- and ALDEFLUORhigh cells and mammospheres formation assay. In vivo expreriments were performed by subcutaneous injection of HCC1806 ShLUC and ShNCT into nude mice. Results: Stable knockdown of NCT in HCC1806 breast cancer cells achieved the following effects: disruption of GS formation and GS/Notch activity, reduction of the pool of BCSC as defined by CD44+/CD24- and ALDH1+ phenotype and inhibition of BCSC cell invasion. Moreover, NCT silencing reduced breast cancer xenograft formation in vivo. Intriguingly, MCF10ANCT presented significant morphological alterations when cultured in a 3D context. Compared to control cells, the epithelial-mesenchymal (EMT) and invasion cell markers, vimentin, Twist and Snail are significantly up-regulated in MCF10ANCT cells. In addition, the over-expression of NCT promoted the generation of a BCSC phenotype characterized by the CD44+/CD24- phenotype and increased ALDH1 activity (up to 3 fold). Finally, using DAPT and Wortmannin as pharmacological inhibitors of Notch and AKT signalling pathways respectively, we found a reversion of the malignant phenotype of MCF10ANCT cells in terms of invasion and stem cell content. Conclusions: These results suggest that therapies directed against NCT may not only reduce tumor size but also the number and the invasive capacity of BCSC. Moreover, we have demonstrated that, even in normal breast epithelial cell line MCF10A, high expression of NCT can induce a Notch/pAKT-dependent EMT transition program. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3311. doi:1538-7445.AM2012-3311