Abstract 829: Role of nicastrin in the mediating chemoresistance of breast cancer cells.

Abstract

Abstract Background: Nicastrin (NCT) is the gate keeper of the gamma secretase (GS) enzyme complex. We have previously shown that high levels of NCT are observed in breast cancer patients. Moreover, we recently highlighted the importance of NCT expression for the expansion of breast cancer stem cells and their invasive features. The involvement of NCT in development of chemoresistance has been suggested by the evidence that 1) NCT is increased in response to oxaliplatin, irinotecan and 5-FU in colon cancer and 2) NCT regulates cell death within the p53 and AKT signalling pathway in human embryonic kidney (HEK293) cells. Here, we have explored the potential role of NCT in the chemoresistance of breast cancer cells. Methods: Stable NCT overexpressing MCF10A cells and control cells were produced by retroviral infections. siRNA strategy was used to silence NCT expression in a panel of p53wt and p53mut breast cancer cell lines. The sulphorhodamine B and Annexin V/PI staining were used to measure proliferation and apoptosis rate in breast cancer cells unstressed or treated with chemotherapeutic agents alone or in combination with DAPT, Wortmannin or Nutlin. p53 levels and activity were assessed by Western blotting, RT-qPCR and gene reporter assays. Results: Stable overexpression of NCT in MCF10A cells induced resistance to doxorubicin and cisplatin treatments as demonstrated by a decreased early and late apoptotis. Moreover, NCT lowers p53 expression, its phosphorylation at the phosphoSer15 site and its transcriptional activity in both unstressed cells and doxorubicin-treated cells. Using DAPT and Wortmannin as pharmacological inhibitors of Notch and AKT signalling pathways and Nutlin to inhibit Mdm2-mediated p53 degradation, we confirmed that NCT mediated chemoresistance in a p53 dependent manner partially through the PI3K/AKT pathways, while it is role is completely independent from GS activity. NCT knockdown was able to sensitize several breast cancer cell lines (MCF7, T47D, ZR75) with a wild type p53 status to doxorubicin. Moreover, NCT overexpression reduced the sensitivity to cisplatin in p53wt HTC116 colon cancer cells while it had no effect on p53mut cells. In order to translate these results to clinical settings, we have developed anti-NCT monoclonal antibodies which have been showed to inhibit proliferation and invasion of breast cancer cells. These antibodies used in combination with chemotherapy mirrored the positive effect of NCT siRNA in terms of response to doxorubicin in p53wt breast cancer cell lines. Conclusions: Our study indicates that in breast cancer cells NCT controls chemotherapy-induced cell death in a p53-dependent manner through the regulation of AKT pathway. This function is independent from the GS. We aim to investigate the association of NCT expression and p53 mutation status with clinical outcome in breast cancers in order to support potential combination of anti-NCT monoclonal antibodies with chemotherapy. Citation Format: Ylenia Lombardo, Aleksandra Filipovic, Monica Faronato, Raoul Charles Coombes. Role of nicastrin in the mediating chemoresistance of breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 829. doi:10.1158/1538-7445.AM2013-829