Biofunctional study on chemoresistance in esophageal squamous carcinoma cells induced by missense mutation of NOTCH1 p.E450K

Abstract

Background: Neoadjuvant chemotherapy (nCT) combined with surgery is one of the main strategies for the treatment of resectable locally advanced esophageal squamous cell carcinoma (ESCC). However, nearly 40% of patients did not benefit from nCT, and the detection rate of NOTCH1 missense mutation was significantly increased in patients who did not respond to chemotherapy, suggesting that the missense mutation may be related to tumor chemoresistance. We aim to explore the effect of a NOTCH1 missense mutation on cell phenotype, to interpret the biofunctional changes in cell lines with a NOTCH1 missense mutation and to analyze the effect of a NOTCH1 missense mutation on drug resistance in ESCC cell lines.

Methods: Sanger sequencing was used to evaluate the exon mutations in the NOTCH1 ligand binding region of candidate ESCC cell lines. After screening, KYSE450 and KYSE140 cells were selected as the research objects, and point mutation cell lines [KYSE140-mutant-type (MT) and KYSE450-MT] were constructed by CRISPR/Cas9 technology. Then, functional experiments were performed with the four cell lines [KYSE450-MT/wild-type (WT) and KYSE140-MT/WT]. The drug resistance of ESCC cell lines was assessed with a drug sensitivity test, and the proliferation, invasion and migration of ESCC lines were evaluated by proliferation test, scratch test and Transwell test. The cell cycle status of ESCC cells was assessed using flow cytometry.

Results: Drug sensitivity tests showed that the NOTCH1 p.E450K point mutation caused chemotherapy resistance in KYSE140 and KYSE450 ESCC cell lines. Cell proliferation, Wound scratch and Transwell assays showed that the NOTCH1 p.E450K point mutation enhanced the proliferation, invasion and migration abilities of KYSE140 and KYSE450 cells. Flow cytometry analysis showed that the NOTCH1 p.E450K point mutation caused an increase in KYSE140 and KYSE450 cells in S phase.

Conclusions: The NOTCH1 p.E450K point mutation causes chemotherapy resistance in KYSE140 and KYSE450 ESCC cells. Cell functional experiments showed that the NOTCH1 p.E450K point mutation enhanced the proliferation, migration and invasion abilities of KYSE140 and KYSE450 cells and increased the number of cells in S phase.

Keywords: NOTCH1; chemotherapy resistance; esophageal squamous cell carcinoma (ESCC).