Abstract
TP53 gene mutations occur in 70% of oesophageal adenocarcinomas (OACs). Given the central role of p53 in controlling cellular response to therapy we investigated the role of mutant (mut-) p53 and SLC7A11 in a CRISPR-mediated JH-EsoAd1 TP53 knockout model. Response to 2 Gy irradiation, cisplatin, 5-FU, 4-hydroxytamoxifen, and endoxifen was assessed, followed by a TaqMan OpenArray qPCR screening for differences in miRNA expression. Knockout of mut-p53 resulted in increased chemo- and radioresistance (2 Gy survival fraction: 38% vs. 56%, p < 0.0001) and in altered miRNA expression levels. Target mRNA pathways analyses indicated several potential mechanisms of treatment resistance. SLC7A11 knockdown restored radiosensitivity (2 Gy SF: 46% vs. 73%; p = 0.0239), possibly via enhanced sensitivity to oxidative stress. Pathway analysis of the mRNA targets of differentially expressed miRNAs indicated potential involvement in several pathways associated with apoptosis, ribosomes, and p53 signaling pathways. The data suggest that mut-p53 in JH-EsoAd1, despite being classified as non-functional, has some function related to radio- and chemoresistance. The results also highlight the important role of SLC7A11 in cancer metabolism and redox balance and the influence of p53 on these processes. Inhibition of the SLC7A11-glutathione axis may represent a promising approach to overcome resistance associated with mut-p53.
Highlights
Oesophageal adenocarcinoma (OAC) carries a poor prognosis and is one of the deadliest cancers worldwide because of both its aggressive nature and the lack of early symptoms resulting in delayed diagnosis
We confirmed by Western blot that p53 was undetectable in p53-KO compared to parental cells (Figure 1A), and that there was no difference in the ability of mock-irradiated parentals and p53-KO cells to form colonies (p = 0.538). p53-KO resulted in significantly increased radioresistance: following 2 Gy irradiation, the survival fractions (SF) of the p53-KO and the parental cells were 56% (±SD 6.2%) and 38% (±SD 2.6%) respectively (p < 0.0001; Figure 1B)
Our data demonstrate that KO of p53 resulted in increased radio- and chemoresistance, suggesting that patients with a p53 missense mutation are potentially more likely to benefit from chemo- or radiotherapy than patients with a p53 null mutation
Summary
Oesophageal adenocarcinoma (OAC) carries a poor prognosis and is one of the deadliest cancers worldwide because of both its aggressive nature and the lack of early symptoms resulting in delayed diagnosis. The poor outcomes for OAC are primarily due to patients presenting with the disease at an advanced stage when curative treatments are less effective [2,3]. Patients with locally advanced disease are treated with neoadjuvant chemoradiotherapy (nCRT) before surgery [4]. Due to intrinsic or acquired therapy resistance, only around 30% of patients have a complete pathological response [5]. Non-responders are at risk of both the side effects from the neoadjuvant treatment, and the effects of delaying surgery [6,7]. The mechanisms by which OACs acquire therapy resistance are poorly understood, and there are to date no reliable biomarkers that can be used for routine clinical use to improve the personalization of treatment. Considerable attention has been paid to the tumour-suppressor gene TP53, which is the most frequently mutated gene across all cancers [8]
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