Attenuation of γ‐secretase mediated activation of Notch signaling induces autophagic cell death in triple negative breast cancer cells

Abstract

Notch signaling is reported to be deregulated in several malignancies including breast. γ‐secretase, a multi‐subunit enzyme complex, catalyzes the cleavage of Notch receptor to release the Notch intracellular domain (NICD) in the cytosol, which then translocates into nucleus to activate the downstream signaling. Hence pharmacological inhibition of γ‐secretase might lead to the subsequent inhibition of Notch signaling in cancer cells.In search of novel γ‐secretase inhibitors (GSIs), we screened a series of triazole‐based compounds, for their potential to bind γ‐secretase and we observed that 3‐(3′4′,5′‐Trimethoxyphenyl)‐5‐(N‐methyl‐3′‐indolyl)‐1,2,4‐triazole compound (also known as NMK‐T‐057) can bind to γ‐secretase complex. Very interestingly, NMK‐T‐057 was found to inhibit proleiferation, colony forming ability, motility in various triple negative breast cancer cells such as MDA‐MB‐231, MDA‐MB‐468, 4T1 and also MCF‐7, with a negligible cytotoxicity against non‐cancerous MCF‐10A, PBMC and WI38. Moreover NMK‐T‐057 treatment resulted in the reprogramming of the EMT process in TNBCs, accompanied by the drastic reduction of CD44high/ CD24low population. NMK‐T‐057 also inhibited the spheroid forming ability of TNBCs and reduced the stem cell enriched side population. It was also observed that in NMK‐T‐057 treated cells, Notch‐1 and its downstream targets such as Hes‐1, pAkt, and Cyclin D1 were significantly down regulated. The in silico study revealing the affinity of NMK‐T‐057 towards γ‐secretase, was further validated by fluorescence based γ‐secretase activity assay, which confirmed the inhibition of γ‐secretase activity in NMK‐T‐057 treated TNBC cells.Autophagy plays a dual role in cancer depending on its role in the survival or death of cancer cells. The relationship between autophagy and Notch signaling is not clear and only a few reports have come across in highly reputed journals in last one year. We observed that NMK‐T‐057 induces significant autophagic responses in TNBCs and administration of the autophagy inhibitor 3‐MA, attenuates NMK‐T‐057 induced cell death. Similar results were observed on the treatment of TNBC cells with commercially available γ‐secretase inhibitor DAPT. Thus we might conclude that NMK‐T‐057 could be potential drug candidate against breast cancer, which can trigger autophagy mediated cell death in breast cancer cells by inhibiting the γ‐secretase – mediated activation of Notch‐signaling.Support or Funding InformationThis work is supported by Dr. D. S. Korhari Postdoctoral Fellowship Scheme, UGC, Govt. of India.