In this study, we sought to assess whether radiation exposure would activate CBR1 and if CBR1 inhibition could enhance radiation sensitivity. The findings of this study are as follows: [
1] IR increased the activity of CBR1; [
2] CBR1 inhibition increased the radiation sensitivity measured by clonogenic assay in vitro; [
3] overexpression of CBR1 protected the HNSCC cells against IR; [
4] CBR1 inhibition resulted in accumulated intracellular ROS levels leading to an increase in mitotic catastrophe and mitotic arrest; and [
5] of the patients treated with radiation, patients with low CBR1 expression showed better prognosis. These results suggest that CBR1 is essential for the survival of cancer cells after IR and can be a good target in developing radiosensitisers.
HNSCC is challenging to treat effectively while maintaining the function of vital healthy structures. Radical surgical resection of the primary tumour and regional cervical lymph nodes used to be the standard of treatment. Concurrent chemoradiotherapy (CCRT) and radiotherapy after radical surgery improves the survival of locally advanced head and cancer patients [
17,
18]. More recently, organ-preserving strategies using either radiation alone or CCRT have become a treatment option for HNSCC patients and have been the focus of much investigation. The systematic clinical investigation of organ-preserving radiotherapy and CCRT regimens suggested that these regimens could produce overall survival results as good as surgical resection for patients with advanced HNSCC; thus, radiation has become a cornerstone of treatment for patients with advanced HNSCC [
19‐
22]. Despite advances in radiotherapy techniques, such as intensity-modulated radiation therapy, treatment outcomes have not improved.
One of the biggest challenges in radiation therapy is that IR affects both normal tissue and solid tumours. Thus, effective radiation therapy is a way to maximise cancer cell-killing ability within acceptable ranges where adjacent healthy normal tissue can withstand radiation damage leading to improving the survival rate of HNSCC patients. Recent studies have focused on upregulation of ROS generation to induce cancer cell death or cell growth inhibition as a therapeutic strategy for increasing radiation sensitivity [
23‐
26]. In this aspect, Nrf2, a well-studied antioxidant protein in various tumours [
25,
27], is a very useful candidate to increase the ROS level. Downstream genes of Nrf2, such as CBR1, can be effective targets to regulate intracellular ROS levels in diverse tumours including HNSCC. Human CBR1 is expressed in a large variety of tissues, with high levels found in the liver, placenta, and CNS [
28], consistent with a possible protective role against toxic carbonyls. CBR1 reduces highly reactive lipid aldehydes formed through oxidative stress, such as ONE, HNE, and acrolein, and catalyses a variety of endogenous and xenobiotic carbonyl compounds [
7]. CBR1 affects the resistance to arsenic trioxide in leukaemia and CBR1 overexpression was sufficient to protect cells against arsenic trioxide through modulation of the generation of ROS [
9]. CBR1 overexpression enhanced cell survival by decreasing oxidative stress under hypoxia, cisplatin, and doxorubicin treatment in hepatocellular carcinoma [
11]. (−)-epigallocatechin gallate, a promising inhibitor of CBR1, enhanced the antitumor activity of daunorubicinol against hepatocellular carcinoma cells expressing high levels of CBR1 and corresponding xenografts [
29]. There is increasing evidence that cancer cells produce higher basal levels of ROS than normal cells [
30]. When endogenous oxidative stress persists, cancer cells become resistant to exogenous oxidants by enhancing endogenous antioxidant capacity [
31,
32]. It is well-established that cell-killing after exposure to IR and a subset of cytotoxic chemotherapeutics is partially mediated by free radicals [
33]. Therefore, we hypothesised that CBR1 could also regulate the ROS produced by IR and the inhibition of CBR1 could increase the radiation sensitivity via accumulation of ROS. First, we examined the relationship between the expression of CBR1 and IR in HNSCC cells. The mRNA and protein expression of CBR1 was increased dose-dependently by IR, suggesting that they are transcriptionally regulated by IR. The Nrf2-antioxidant DNA response element was important to maintain resistance to IR [
34]. Nrf2 is known to be a transcriptional regulator of CBR1 genes under oxidative stress [
10]. Consistently, our results showed that the inhibition of Nrf2 decreased the expression of CBR1 after IR. This result demonstrates that CBR1 modulates the sensitivity of radiation therapy, as a downstream molecule of Nrf2. Second, we examined whether CBR1 promotes the effect of radiation therapy for HNSCC. Our results showed that the combination of CBR1 inhibition and IR reduced colony formation in vitro and suppressed tumour growth in vivo in xenograft models. In our patient cohort, HNSCC patients with low CBR1 expression showed better survival than patients with high CBR1 expression after radiation therapy. In 174 HNSCC patients from a publicly available open database, low-expression groups of CBR1 had a better disease-free survival. These results concretely suggest that CBR1 expression can be a predictor for radiation therapy efficacy in HNSCC patients. Next, we sought to determine the mechanism by which CBR1 promotes the efficacy of radiation therapy. The suppression of CBR1 with IR resulted in IR-induced DSBs, increased ROS generation, G2-M arrest, mitotic catastrophe, and eventually a reduction in colony formation. There is a limitation in this study. HNSCC patients enrolled in this study and from publicly available database were treated with various treatment modalities rather than only radiotherapy. There are 2 purpose in the treatment for HNSCC: cure of cancer and preservation of function (breathing, speech and swallowing). Therefore, there are various treatment methods for the treatment of head and neck cancer such as concurrent chemoradiation, surgery followed by radiation, surgery followed by chemoradiation. Patients receiving only radiation are very limited such as T1, T2 larynx cancer or T1, T2 oropharynx cancer. Inevitably, it is very difficult to recruit patients with the same treatment in head and neck cancer research.