Abstract 3684: Basal Notch1 and Notch4 activation as potential markers of aggressiveness and sensitivity to Notch inhibition in human cancer cell lines
Authors: Astorgues-Xerri Lucile , Martinet Matthieu , Raymond Eric , Tijeras-Raballand Annemilaï 
Published: September 2020

Background. Notch pathway is involved in tumor biology including cell proliferation, migration, drug resistance, and epithelial-to-mesenchymal transition (EMT). Notch activation leads to a proteolytic cleavage, followed by the release of the Notch intracellular domain (NICD), which translocates into the nucleus and activates target genes. Notch1 and Notch4 expressions were described as mutually exclusive in some tumors, with an increased aggressiveness linked to low Notch1/high Notch4 expression phenotype. What about the role of their basal activation in cancers? The aim of this work is to characterize the role of basal Notch1 and Notch4 pathway on cell proliferation, motility and sensitivity to Notch inhibition in human pancreatic (PDAC), head&neck (H&N), colorectal (CRC), cholangio (CK), and hepato (HCC) carcinoma cell lines.

Materials and Methods. Cell lines were selected regarding their basal expression of NICD4 using western blot (one low/one high Notch4 basal activation) for each tumor type. Chosen cell lines were then characterized, by western blot and/or RT-qPCR, for Notch signaling (NICD1, HES1, NUMB), EMT status (vimentin, E-cadherin) and survival pathways (pAKT, pERK). Basal cell proliferation and motility were studied using MTT and wound-healing assay. The effects of a Notch inhibitor, PF-03084014, were assessed on cell proliferation and Notch signaling pathway by MTT and western blot.

Results. In PDAC, H&N, and CRC cell lines, high NICD4 expression was associated with low NICD1, HES1, and NUMB expressions, whereas the opposite was observed in low NICD4 expressing cells. Interestingly, all high NICD4/low NICD1 cells displayed a mesenchymal phenotype with high vimentin and low E-cadherin expressions. This phenotype was also associated with an increased proliferation rate and basal cell motility in all tumor types, except for HCC. In addition, basal ERK phosphorylation was increased in the aggressive cells (high NICD4/low NICD1). In PDAC, H&N, and CRC cell lines, PF-03084014 showed higher antiproliferative effects in high NICD4/low NICD1 cells compared to low NICD4/high NICD1 cells. In all cell lines, PF-03084014 displayed no effect on Notch4 activation, but abrogated Notch1 activation in all low NICD4/high NICD1 cells. Interestingly, PF-03084014 decreased HES1 expression in all cells, with increased effects in the most sensitive cells (high NICD4/low NICD1). Further analysis of PF-04084014 effects on cell signaling pathways will be displayed at the conference.

Conclusions. In this study, high Notch4 activation is shown to be correlated with low NICD1, HES1 and NUMB expressions. This phenotype is associated with an increased aggressiveness of human cancer cell lines, and an increased sensitivity to Notch inhibition. As expected, the strongest HES1 inhibition was observed in cell lines with an increased sensitivity to Notch inhibition. Since the inhibition of Notch pathway is an interesting topic for anticancer therapy, this study could help to select tumor types that may be good candidate for Notch inhibitors in the clinics.

Citation Format: Lucile Astorgues-Xerri, Matthieu Martinet, Eric Raymond, Annemilaï Tijeras-Raballand. Basal Notch1 and Notch4 activation as potential markers of aggressiveness and sensitivity to Notch inhibition in human cancer cell lines [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3684.