# NEDD4L affects KLF5 stability through ubiquitination to control ferroptosis and radiotherapy resistance in oesophageal squamous cell carcinoma

## TL;DR

This study shows how NEDD4L controls KLF5 levels through ubiquitination, affecting ferroptosis and making oesophageal cancer cells more sensitive to radiotherapy.

## Contribution

The novel finding is that NEDD4L regulates KLF5 stability via ubiquitination, influencing ferroptosis and radiotherapy resistance in ESCC.

## Key findings

- Low KLF5 expression increases lipid peroxidation and ferroptosis markers in ESCC cells.
- NEDD4L ubiquitinates and degrades KLF5, reducing ferroptosis and DNA damage while enhancing radiosensitivity.
- NEDD4L/KLF5 inhibition promotes cancer cell survival and resistance to radiation in vitro and in vivo.

## Abstract

Oesophageal squamous cell carcinoma (ESCC) contributes to high mortality. Modulating ferroptosis may reverse resistance to radiotherapy. This article was to explore the ubiquitination modification of KLF5 and its effect on ferroptosis in ESCC. KLF5 was under‐expressed by shRNA plasmids in the cells and ROS levels were analysed by flow cytometry, ferroptotic gene expression was detected by qRT‐PCR, MDA and GSH levels were determined by ELISA, cell morphology was observed by transmission electron microscopy, and Fe ion levels were analysed by immunofluorescence. Cells were treated with Ferrostatin‐1 and NAC and analysed for cell proliferation by colony formation assay, cell migration and invasion by Transwell assays, and apoptosis by flow cytometry. DNA damage in cells was also analysed using comet assay, EdU doping assay, γH2AX fluorescence, DNA‐PKcs and PCR. NEDD4L and KLF5 binding was analysed by immunoprecipitation. Changes in ferroptosis, DNA damage and resistance were analysed in cells with both silencing NEDD4L and KLF5. Changes in tumour resistance to radiation were analysed in mice underexpressing NEDD4L and KLF5. Low expression of KLF5 significantly promotes cellular lipid peroxidation levels, with decreased expression of SOD and GPX4, and increased expression of ACSL4. Concurrently, MDA levels deplete GSH, and cells exhibit typical ferroptotic morphology with increased Fe2+ content. KLF5 inhibition results in enhanced cellular clonogenicity, migration and invasion activities, reduced apoptosis, increased tail DNA, nuclear EdU incorporation, nuclear γH2AX foci and elevated expression of DNA‐PKcs, LIG4, RAD9B and BMI1. Ferrostatin‐1 and NAC reverse these effects. NEDD4L ubiquitination modifies and degrades KLF5, with NEDD4L/KLF5 inhibition mitigating cellular ferroptosis and DNA damage, thereby promoting radiosensitivity both in vitro and in vivo. NEDD4L increases radiosensitivity by accelerating cellular ferroptosis via ubiquitination modification of KLF5.