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.
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Introduction
Breast cancer is the most frequently diagnosed type of cancer in women worldwide [1]. Among all subtypes of breast cancer, the aggressive human epidermal growth factor 2-positive (HER2-positive) breast cancer, which is characterised by overexpressed or amplified HER2 receptor, accounts for approximately 20–30% of all annually diagnosed cases [2]. In the past century, patients diagnosed with HER2-amplified or -overexpressed (also referred as HER2-positive) breast cancer were frequently associated with the poorest prognosis and with the highest incidence of brain metastasis [3,4,5,6,7]. However, this is no longer the case following the relatively recent development of novel HER2-targeted therapies, including monoclonal antibody and small-molecule tyrosine kinase inhibitors (TKI), that more precisely target HER2-positive cells to inhibit the activation of downstream signalling while triggering cell death pathways [8].
Orally taken ATP-competitive TKIs, such as the pan-HER-TKI neratinib, have garnered considerable attention in recent years as an effective extended adjuvant HER2-targeted therapy for patients with HER2-positive breast cancers after receiving a combination of chemotherapy and monoclonal antibody trastuzumab [9]. Neratinib treatment significantly improves invasive disease-free survival rate while reducing long-term toxicity and complication rate. Mechanistically, neratinib forms a covalent bond with cysteine residues in the ATP-binding pocket of HER1 (also known as EGFR), HER2 and HER4 receptors to inhibit their downstream canonical Ras/ERK and Akt/mTOR pathways, suppressing cell proliferation, and triggering cell death [10, 11]. A previous study using in vitro HER2-positive breast cancer cell lines, which expressed wild-type oestrogen receptor, such as SKBR3, showed that neratinib treatment repressed the activity of nuclear factor-erythroid factor 2-related factor 2 (NRF2), a master regulator of antioxidant defence, to then promote oxidative-stress-dependent cell death [12]. Oxidative stress is a condition of sustained elevation of intracellular reactive oxygen species (ROS) that potentially leads to reversible and irreversible damage to biomolecules such as lipids, proteins, and nucleic acids [13]. ‘ROS’ is an umbrella term describing a collection of oxygen-derived radicals, such as singlet superoxide (O2• −) and hydroxyl radical (•OH), and species, such as hydrogen peroxide (H2O2) and lipid peroxide. Strikingly, a recent report from Nagpal and colleagues revealed that, rather than apoptosis as induced by other TKIs, such as lapatinib, neratinib promoted non-apoptotic cell death called ferroptosis in both human (SKBR3) and mouse (TBCP1) HER2-positive breast cancer cell lines [14]. Ferroptosis is a newly discovered type of cell death characterised by perturbed iron and redox homeostasis with excessive lipid peroxidation at phospholipid membrane [15]. Unless the rise of endogenous lipid peroxide is averted such as by the scavenging enzymatic activity of glutathione peroxidase 4 (GPX4), the accumulation of lethal membrane lipid peroxide results in the loss of membrane integrity and ultimately leads to cell death [16].
Despite therapeutic effectiveness against HER2-positive breast cancers, patients receiving neratinib can experience mild to severe symptoms of gut toxicity, such as abdominal pain and diarrhoea, which can lead to early dose reduction or treatment discontinuation [17, 18]. Interestingly, a recent randomised controlled trial conducted by Sonnichsen and colleagues reported that though serum levels of neratinib were higher in patients receiving 240 mg of neratinib once daily (QD) than those receiving 120 mg of neratinib twice daily (BID), patients in the QD group experienced less severe diarrhoea [18]. This suggested that diarrhoea is potentially a consequence of local exposure to neratinib rather than systemic exposure. In support, starting patients on a reduced dose of neratinib for the first two weeks is also associated with greatly reduced levels of diarrhoea and treatment discontinuations [19]. Furthermore, an escalating dose of neratinib resulted in more severe symptoms of gut toxicity observed in our preclinical rat models and other clinical trials [20, 21]. We therefore postulate that inhibition of locally expressed HER receptors in the intestinal epithelial cells which subsequently triggers epithelial cell death is likely a cause of gut toxicity. This is plausible due to the gut epithelium naturally expressing HER receptors and being strongly dependent on HER downstream signalling pathways for cell survival, differentiation, and proliferation [22,23,
RNA-sequencing analysis
RNA-sequencing analysis on previously published datasets was carried out to examine which biological pathways were significantly enriched following neratinib treatment. Processed count tables for protein encoded genes from neratinib-treated mouse TBCP-1 breast cancer and human glioblastoma SF268 and TS895 cell lines were kindly provided by Dr Normand Pouliot (Olivia Newton-John Cancer Research Institute) and Dr Colin Tang (Weill Cornell Medicine) [14, 31]. Differential gene expression between neratinib and vehicle control groups was performed using DESeq2 (v1.38.3) statistical package in R [35]. Unless otherwise stated, a master list of differentially expressed (DE) genes was generated when the P-adjusted value less than 0.05. Subsequently, a list of significantly upregulated genes with log2FoldChange values above 0.1 was generated from the master DE gene list with P-adjusted values or false discovery rate (FDR) below 0.05. Subsequently, this gene list was inserted into the online database for annotation, visualisation, and integrated discovery (DAVID) bioinformatic resource website for gene ontology (GO) terms and KEGG pathway analyses as previously described [36,37,38]. The computational algorithm used to determine statistical significance of individual pathways was detailed in the original papers [36, 39]. Here, GO terms and KEGG pathways were deemed as significantly enriched with P values below 0.05.
CRISPR-screening analysis
As previously described in Tang et al. [31], a pool CRISPR screen was performed on neratinib-treated SF268 human glioblastoma cell line to identify genes contributing to neratinib sensitivity. A count table of differentially expressed single-guided RNA and an output file of DrugZ were kindly provided by Dr Colin Tang from Weill Cornell Medicine. Using R script publicly available generated by Dr Colin Tang, drugZ rank plot was generated for normalised Z score against sgRNA rank 95. The positive or negative normalised Z score of a gene with FDR below 0.05 confers with either neratinib sensitivity or resistance, respectively.
Data analysis and statistics
Descriptions for sample size and statistical tests are detailed in figure legends. Unless otherwise specified, all statistical analyses were carried out using unpaired Student’s t-test in GraphPad Prism 9 (version 9.2.0; USA). Statistical values were reported in mean ± standard error of the mean (mean ± SEM). Statistical significance, which was reported as exact P values, was considered as followed – for all histological assessment, RT-qPCR, gene ontology and KEGG pathway analyses, P values below 0.05 were deemed significant while for differential gene expression and Drug-Z analyses, P-adjusted values, or false discovery rate (FDR) below 0.05 were deemed significant.
Data availability
The data generated in this study are available within the article and its supplementary data files, with further raw data available upon request. Further information and requests for resources should be directed to and will be fulfilled by the corresponding authors, Joanne M. Bowen (joanne.bowen@adelaide.edu.au) and Triet PM. Nguyen(phuminhtrietthomas.nguyen@unimelb.edu.au).
Results
Neratinib-induced injury is spatially located in the rat colon
As previously reported from our group, all rats dosed with neratinib experienced common signs of gut toxicity including weight loss and moderate diarrhoea [32]. To determine the spatial effect of neratinib on the rat colon, we performed histopathological assessment using haematoxylin and eosin (H&E) and immunohistochemical (IHC) staining on proximal and distal colon. Overall, we observed that following neratinib treatment, the most severe morphological damage was found in the distal colon as reflected by a significantly higher histopathological score and increased crypt length (Fig. 1a, b, and c). In sharp contrast, despite a significant increase in proliferative Ki67-positive cells, the proximal colon of neratinib-treated rats only displayed a few, small, focal areas of mild mucosal damage without apparent sign of injury in the surface lining colonocytes expressing carbonic anhydrase 1 (CA1) (Fig. 1a-d).
In the distal colon, degeneration of surface lining CA1+ colonocytes with occasional exfoliation of epithelial cells into the lumen leaving a residual denuded surface was observed (Fig. 1a). Migration of flattened, attenuated enterocytes across the denuded mucosal surface was also found which suggested that surviving epithelial cells attempted to cover the surface defects to maintain the integrity of the epithelial lining (Fig. 1a). A significant increase in the proliferative crypt compartment containing Ki67-positive cells further suggested that a reparative mechanism was in place to regenerate the injured surface epithelium (Fig. 1d). A markedly increased inflammatory infiltrate, which primarily comprised of lymphocytes, but also a few granular leukocytes, mostly eosinophils, were observed in the lamina propria layer of the distal colon following the exposure to neratinib compared to vehicle control (Fig. 1a). Together, these histopathological findings appeared to consistent with features of microscopic colitis, specifically lymphocytic colitis [40]. Our current histopathological data collectively substantiated our observations that the injury in the distal colon and its surface lining colonocytes appeared to be more severe than in the proximal colon on day 28 of neratinib treatment.
Enriched gene signatures for apoptosis or ferroptosis may be cell-type specific following neratinib treatment
To gain insight into the biological processes underlying neratinib-induced cell death, we performed comprehensive analyses on differential gene expression, KEGG pathway and GO on previously published RNA-sequencing datasets [14, 31]. These datasets included TBCP-1 cells, a mouse HER2-overexpressing breast cancer cell line capable of metastasising to the brain, treated with neratinib for 24 h, and intracranial human EGFR/HER1-mutant TS895 glioblastoma xenograft treated with neratinib for 3 h [14, 31]. We decided to utilise these datasets for this analysis because of the absence of -omics studies in neratinib-treated healthy colonocytes or at least in colorectal cancer cell lines in vitro. Our KEGG pathway analysis revealed that ferroptotic cell death, which is characterised by perturbed iron homeostasis and lipid peroxidation [41], was significantly enriched in both model systems (Fig. 2a). As a previous study by Nagpal and colleagues also confirmed that ferroptosis was induced by neratinib in HER2-postive human (SKBR3) and mouse (TBCP1) breast cancer cell lines [14], we hypothesised that neratinib-induced ferroptosis might extend beyond the context of breast cancer.
Gene Ontology (GO) analyses for enriched biological processes further uncovered gene signatures involved in autophagy and positive regulation of autophagy among all biological processes in the list of significantly upregulated genes in both neratinib-treated TBCP1 and TS895 cells (Fig. 2b and Suppl. Table 1). Autophagy is a process mediated by the lysosomal pathway [42] and was previously related to the induction of ferroptosis, especially in the form of ferritinophagy [Full size image
We next aimed to determine the presence of ferroptosis by measuring key markers of iron homeostasis and lipid peroxidation. Through IHC staining, we observed a significant reduction of endogenous ferritin heavy chain 1 (FTH1) in the distal colon of rats treated with neratinib (Fig. 3a and c). A significant upregulation of expression of iron absorption gene, Tfrc, and downregulation of iron storage gene, Fth1, measured by RT-qPCR further suggested that neratinib perturbed the pool of intracellular iron storage leading to an increase in cytosolic reactive ferrous (Fe2+) ion in the rat colon (Fig. 3d). Although neratinib did not alter gene expression of Gpx4, which encodes a lipid peroxide scavenging enzyme contributing to ferroptotic resistance [16, 46], we found a significant upregulation of gene expression of Acsl4, which esterifies polyunsaturated fatty acids (PUFA) making them competent for ferroptosis, and arachidonate 15-lipoxygenase (Alox15), which directly causes lipid peroxidation [47, 48] (Fig. 3e). Collectively, the current findings support the notion that ferritinophagy-mediated ferroptosis, but not apoptosis, is the potential histopathological feature underlying neratinib-induced colonic epithelial injury.