Abstract 4762: Anti-Nicastrin antibodies for the treatment of endocrine resistant breast cancer .
Authors: Faronato Monica , Lombardo Ylenia , Filipovic Aleksandra , Ali Simak , Coombes R.Charles 
Published: August 2014

Background: Resistance to endocrine therapy occurs in at least 30% of patients with breast cancer (BC) and remains a major clinical problem. Among the mechanisms underpinning endocrine resistance is the enrichment of epithelial-to-mesenchymal transition (EMT) features and signalling pathways such as EGFR and Notch. Notch receptors are activated by the gamma-secretase (GS) enzyme composed of nicastrin, presenilin, PEN2 and Aph-1. Gamma secretase inhibitors are being tested as novel therapeutics for cancer treatment, predominantly in combination with anti-hormonal agents as well as chemotherapy, however treatment related toxicities are apparent. Nicastrin is upregulated in BC and we have developed anti-nicastrin monoclonal antibodies (MAbs) to target its functions and block GS. Here, we show that nicastrin may be a relevant therapeutic target in endocrine resistance and demonstrate the effects of anti-nicastrin MAbs.

Materials and Methods: We used two cell line models of MCF7 cells which developed endocrine resistance: TamR cells (Hiscox et al) and MLET2/MLET5 developed in our laboratory. We raised anti-nicastrin MAbs (MAb1 and MAb2) against nicastrin extracellular domain. Western blotting, RT-qPCR, transwell invasion and 3D matrigel growth assays were used to demonstrate their molecular and functional effects. Gamma secreatse inhibitor RO4929097 was used for comparison.

Results: We demonstrate that nicastrin is overexpressed at both mRNA and protein level in endocrine resistant cell lines (&gt; 4-fold). These cells concomitantly exhibit elevated molecular markers of EMT: Vimentin (3-fold), CD44 (&gt; 2-fold), EGFR (&gt; 4-fold), RhoGTPase Cdc42 target genes IQGAP1 (2-fold) and IQGAP2 (&gt;100-fold); as well as Notch4 (&gt;2-fold), Notch-targets Hey1 and Hes1 (4-fold), while Notch1 is suppressed (&gt; 2-fold). Anti-nicastrin MAb1, MAb2 and RO4929097 were equally potent in inhibiting Notch target genes Hey1 and Hes1, as well as growth of TAMR and MLET acini in 3D Matrigel. Interestingly, effective inhibition of vimentin, CD44 and IQGAPs was observed only upon treatment with anti-nicastrin MAbs, while RO4929097 had no effect. Interestinly, a clear distinction was noted between anti-nicastrin MAbs and RO4929097 in terms of their effect on Notch4. MAb1 and MAb2 attenuated Notch4 transcription and reduced activated Notch4 and nicastrin levels, while RO4929097 achieved an opposite effect. Furthermore, only anti-nicastrin MAbs were also potent inhibitors of TAMR cell invasion (by &gt;50%, p&lt;0.01).

Conclusion: Nicastrin is overexpressed in endocrine resistant breast cancer cells. Anti-nicastrin monoclonal antibodies inhibit Notch4 signalling, attenuate expression of pro-invasive molecules such as vimentin, IQGAPs and CD44 and reduce cellular invasiveness and growth in 3D Matrigel. Therefore, targeting nicastrin may be a valid therapeutic strategy in breast cancer patients that relapse of endocrine therapy.

Citation Format: Monica Faronato, Ylenia Lombardo, Aleksandra Filipovic, Simak Ali, R.Charles Coombes. Anti-Nicastrin antibodies for the treatment of endocrine resistant breast cancer . [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 4762. doi:10.1158/1538-7445.AM2013-4762