The E3 ubiquitin ligase NEDD4 regulates chemoresistance to 5-fluorouracil in colorectal cancer cells by altering JNK signalling

Abstract

Colorectal cancer (CRC) is the second leading cause of cancer deaths. Though chemotherapy is the main treatment option for advanced CRC, patients invariably acquire resistance to chemotherapeutic drugs and fail to respond to the therapy. Although understanding the mechanisms regulating chemoresistance has been a focus of intense research to manage this challenge, the pathways governing resistance to drugs are poorly understood. In this study, we provide evidence for the role of ubiquitin ligase NEDD4 in resistance developed against the most commonly used CRC chemotherapeutic drug 5-fluorouracil (5-FU). A marked reduction in NEDD4 protein abundance was observed in a panel of CRC cell lines and patient-derived xenograft samples that were resistant to 5-FU. Knockout of NEDD4 in CRC cells protected them from 5-FU-mediated apoptosis but not oxaliplatin or irinotecan. Furthermore, NEDD4 depletion in CRC cells reduced proliferation, colony-forming abilities and tumour growth in mice. Follow-up biochemical analysis highlighted the inhibition of the JNK signalling pathway in NEDD4-deficient cells. Treatment with the JNK activator hesperidin in NEDD4 knockout cells sensitised the CRC cells against 5-FU. Overall, we show that NEDD4 regulates cell proliferation, colony formation, tumour growth and 5-FU chemoresistance in CRC cells.

Fig. 1. Abundance of NEDD4 is low in CRC cells resistant to 5-FU.

A Apoptosis of LIM1215 wild-type (WT) and resistant (Res) cells following 5-FU treatment was analysed using flow cytometry (n = 3). B and C Western blot analysis of WT and Res LIM1215 cell lysates for NEDD4 and β-actin. Quantification of NEDD4 intensity normalised to β-actin (n = 3). D and E Western blot analysis of WT and Res CRC cell lysates for NEDD4 and β-actin. Quantification of NEDD4 intensity normalised to β-actin (n = 3). F and G Western blot analysis of WT and Res PDX lysates for NEDD4 and β-actin. Quantification of NEDD4 intensity normalised to β-actin (n = 3). H Relative mRNA expression of NEDD4 normalised to GAPDH in WT and Res CRC cells is depicted (n = 3). All data is represented as mean ± SEM. Significance is determined by two-tailed t-test.

Fig. 2. Knockout of NEDD4 protects CRC cells from 5-FU-induced apoptosis.

A Western blot analysis for the confirmation of NEDD4 KO in LIM1215 cell line. B FACS-based apoptosis was performed on LIM1215 WT and NEDD4 KO treated with 5-FU (5, 25 and 50 µM) for 72 h. Percentage of cell death is represented as percent sub-G1 (n = 4). C 5-FU (5, 25 and 50 µM) treated WT and NEDD4 KO LIM1215 cells were analysed for cell cycle profile. All data are represented as mean ± SEM. Significance is determined by two-tailed t-test.

Fig. 3. Knockout of NEDD4 does not show cross-resistance to oxaliplatin and irinotecan-induced apoptosis.

FACS-based apoptosis was performed on LIM1215 WT and NEDD4 KO cells treated with or without chemotherapeutic drug A oxaliplatin (n = 4) (0.5, 1 and 2 µM) and B irinotecan (n = 3) (25 and 50 µM) for 72 h. Percentage of cell death is represented as percent sub-G1. C, D Oxaliplatin and irinotecan-treated WT and NEDD4 KO LIM1215 cells, respectively, were analysed for cell cycle profile. All data are represented as mean ± SEM.

Fig. 4. Loss of NEDD4 altered cell morphology and reduced cell proliferation.

A NEDD4 knockout in CRC cells leads to altered morphology. Representative phase contrast micrographs of LIM1215 WT and NEDD4 KO cells are shown. B Cell proliferation was measured by MTS assay at 24, 48 and 72 h (n = 4). C Reduced cell growth in NEDD4 KO clones was analysed using trypan blue assay which was performed at 24 and 48 h (n = 3). D Defects in two NEDD4 KO clones were analysed using colony formation assay. E Graphical representation of number of colonies counted (n = 3). F Representative image of wound healing assay LIM1215 WT and NEDD4 KO cells. The wound was created after the cells reached 100% confluency, migration was assessed at T0 and 16 h post-wounding. Images were taken under the ×4 objective of the light microscope. G Quantification of wound closure is showed (n = 3). All data are represented as mean ± SEM.

Fig. 5. Loss of NEDD4 alters JNK signalling pathway.

A Western blot analysis of epithelial marker E-cadherin and mesenchymal marker YBX1. B Western blot analysis of autophagy markers p62 and Atg5. β-actin was used for a loading control. C Western blot analysis of autophagy marker LC3. D Western blot analysis of pro-apoptotic markers p53, caspase 3, caspase 8, DNA damage repair marker gamma-H2AX and anti-apoptotic marker BCL2 and enzyme thymidylate synthase (TS) was performed following 5-FU treatment. E Western blot analysis of β-catenin and Wnt target genes Axin 2 and Cyclin D1 following 5-FU treatment. F Western blot analysis of cellular signalling pathways pMAPK, PI3K/AKT, pSTAT3 and pJNK following 5-FU treatment. G Quantification of pJNK intensity normalised to total JNK (n = 3). All data is represented as mean ± SEM. Significance is determined by two-tailed t-test.

Fig. 6. Loss of NEDD4 reduces primary tumour burden.

A Diagrammatic depiction of the method used for mouse xenograft establishment. Athymic nude mice, at 6–8 weeks of age, received subcutaneous transplantation of either WT or NEDD4 KO LIM1215 cells. Following tumour growth mice were treated with 5-FU. It was hypothesised that the NEDD4 KO LIM1215 tumour would be smaller and resistant to 5-FU based on the in vitro data. B Tumour volume was measured using a digital calliper post-5-FU treatment (n = 8). C Tumour volume with or without 5-FU treatment over the course of the experiment (n = 8). All data are represented as mean ± SEM.

Fig. 7. JNK signalling activation sensitises CRC cells to 5-FU.

A Western blot analysis of pJNK and total JNK following treatment with JNK activator hesperidin of two independent NEDD4 KO clones in LIM1215 cells. B Quantitative representation of pJNK in Western blots (n = 3). C FACS-based apoptosis was performed on LIM1215 WT and NEDD4 KO treated with 5-FU (5, 25 and 50 µM) and/or hesperidin (125 µM) for 72 h. Percentage of cell death is represented as percent sub-G1 (n = 3). All data are represented as mean ±SEM.