Ferroptosis - a potential feature underlying neratinib-induced colonic epithelial injury

Abstract

Purpose: Neratinib, a small-molecule tyrosine kinase inhibitor (TKI) that irreversibly binds to human epidermal growth factor receptors 1, 2 and 4 (HER1/2/4), is an approved extended adjuvant therapy for patients with HER2-amplified or -overexpressed (HER2-positive) breast cancers. Patients receiving neratinib may experience mild-to-severe symptoms of gut toxicity including abdominal pain and diarrhoea. Despite being a highly prevalent complication in gut health, the biological processes underlying neratinib-induced gut injury, especially in the colon, remains unclear.

Methods: Real-time quantitative polymerase chain reaction (RT-qPCR) and histology were integrated to study the effect of, and type of cell death induced by neratinib on colonic tissues collected from female Albino Wistar rats dosed with neratinib (50 mg/kg) daily for 28 days. Additionally, previously published bulk RNA-sequencing and CRISPR-screening datasets on human glioblastoma SF268 cell line and glioblastoma T895 xenograft, and mouse TBCP1 breast cancer cell line were leveraged to elucidate potential mechanisms of neratinib-induced cell death.

Results: The severity of colonic epithelial injury, especially degeneration of surface lining colonocytes and infiltration of immune cells, was more pronounced in the distal colon than the proximal colon. Sequencing showed that apoptotic gene signature was enriched in neratinib-treated SF268 cells while ferroptotic gene signature was enriched in neratinib-treated TBCP1 cells and T895 xenograft. However, we found that ferroptosis, but less likely apoptosis, was a potential histopathological feature underlying colonic injury in rats treated with neratinib.

Conclusion: Ferroptosis is a potential feature of neratinib-induced colonic injury and that targeting molecular machinery governing neratinib-induced ferroptosis may represent an attractive therapeutic approach to ameliorate symptoms of gut toxicity.

Keywords: Colonic injury; Ferroptosis; Gut toxicity; Neratinib.

Fig. 1

Histopathological features of neratinib-induced injury in proximal and distal colon. a, Representative images of H&E panels of proximal and distal colon proximal and distal colon treated with either vehicle control or neratinib at 20X and 40X magnification. In the distal colon of neratinib-treated rat, (*) indicates denuded mucosal lining is covered by attenuated, surviving enterocytes in an attempt to repair the injured surface lining colonocytes and (Δ) indicates a marked lymphocytic infiltrate in the lamina propria. Scale bar, 100 mm. Representative images of IHC (CA1 and Ki67) panels of proximal and distal colon of rats treated with either vehicle control or neratinib. Scale bar, 50 mm (proximal colon) and 100 mm (distal colon) b, Quantification of histopathological scoring. c, Quantification of crypt length. d, Quantification of the percentage of Ki67-postive cells per crypt. For b, c, and d, n = 6 rats in each treatment group. Unpaired Student’s t-test was used for statistical analysis, where P values below 0.05 were deemed statistical significance. The centre line represents the mean, and the error bar represents s.e.m

Fig. 2

Whether apoptosis or ferroptosis is induced following neratinib treatment may be cell-type specific. a, Volcano plot for upregulated key markers of ferroptosis analysed from published bulk RNA-sequencing data of TBCP-1 cell line and TS895 xenograft following 24 h and 3 h of neratinib treatment, respectively. b, A table of enriched gene sets in autophagy and positive regulation of autophagy following neratinib treatment in TBCP-1 cell line (24 h) and TS895 xenograft (3 h). c, Differential gene expression of selected markers for iron transport and ferritinophagy in ferroptotic pathway from KEGG pathway analysis in TS895 xenograft. Unpaired Student’s t-test was used for statistical analysis, where P values below 0.05 were deemed statistical significance. The centre line represents the mean, and the error bar represents s.e.m. d, A table of enriched gene sets in apoptotic process following neratinib treatment in SF268 cell line (72 h). e, Gene candidates corresponding to neratinib resistant phenotype identified from SF268 CRISPR screen experiment

Fig. 3

Ferroptosis is a potential underlying histopathological feature of neratinib-induced injury in the distal colon. a, Representative images of IHC-stained (Caspase-3 and FTH1) distal colon treated with either vehicle HPMC or neratinib. Arrowhead indicates Caspase-3-positive cell. Scale bar, 100 mm. b, The quantification of positive Caspase-3 cells per crypt. c, The gene expression levels of markers for iron metabolism (Tfrc and Fth1). d, The gene expression levels of markers for lipid peroxidation, namely Gpx4, Alox15, and Acsl4. For b, c, and d, n = 4 rats in vehicle-treated group, and n = 5 in neratinib-treated group. The gene expression levels were determined by RT-qPCR and were shown relative to Ubc housekeeping gene. Except non-parametric Mann-Whitney test was used for Tfrc, Gpx4, and Acsl4, unpaired Student’s t-test was used for statistical analysis, where P values below 0.05 were considered significant. The centre line represents the mean, and the error bar represents s.e.m