Background
Uterine cervical cancer has been estimated to affect 500,000 women annually and cause 270,000 deaths worldwide [
1]. It is characterized by a range of minor to severe neoplastic changes in the epithelium, which typically advances locally and via the lymphatic route, sometimes recurrently [
2]. Advances in therapeutic methods and diagnostic tools have not improved the overall prognosis of patients with recurrent cervical cancer, and optimal treatment for recurrent disease is still open to debate [
3]. Therefore, understanding the molecular events and mechanisms underlying tumor initiation and progression, which could contribute to early detection, will be helpful in the prevention and treatment of cervical cancer.
The gene for NAD(P)H: quinone oxidoreductase-1 (NQO1), also known as DT-diaphorase, is located on chromosome 16q22 and consists of six exons and five introns [
4]. NQO1 is mainly a cytosolic enzyme that uses NADH or NADPH as substrates to catalyze the two-electron reduction of quinines to their hydroquinone forms [
5], thus bypassing toxic semiquinone intermediates, and these resultant hydroquinones are thus ready for further conjugation and excretion [
6]. However, conclusions on the biological functions of NQO1 in cancer have been contradictory. On the one hand, NQO1 is induced along with a battery of defensive genes that provide protection against different stresses to prevent organs from undergoing carcinogen-induced tumorigenesis. On the other hand, reductive activation of environmental carcinogens including dinitropyrenes and heterocyclic amines by NQO1 could contribute to carcinogenesis [
7]. Interestingly, NQO1 was found to be expressed at high levels in many solid tumors, including cholangiocarcinoma [
8], lung [
9] and pancreas [
10], and has also been detected following the induction of cell cycle progression and proliferation of melanoma cells [
11]. To date, the correlation between NQO1 expression and cervical cancer has not been adequately studied.
In this study, we aimed to analyze the expression status of NQO1 in squamous cell carcinomas (SCCs) of the uterine cervix, normal cervical epithelia and precancerous disease, investigate the relationship between it and clinicopathological parameters and discover its prognostic value in cervical SCC patients based on survival data.
Discussion
Professor Ernster first discovered NQO1 in 1958 [
17]. This ubiquitous flavoprotein has been found to be expressed in body tissues [
18], and has been localized primarily in the cytoplasm with lower levels being detected in the nucleus [
19]. Several functions of NQO1 have been proposed including xenobiotic detoxification, superoxide scavenging, maintenance of endogenous antioxidants, modulation of p53 and proteasomal degradation [
20]. It is conceivable that NQO1 is primarily involved in protecting normal cells from oxidant stress and electrophilic attack. These functions have also led to the suggestion that NQO1 plays an important role in cancer chemoprevention. Some studies have shown that the polymorphism in the NQO1 gene affects the translation of the NQO1 protein. The NQO1 C609T polymorphism has been reported to be associated with an increased risk of various cancers such as renal [
21], lung [
22], esophageal [
23], gastric [
24] and head and neck [
25]. Hu’ results indicated that functional polymorphisms in NQO1 SNP609 associate with the risk of cervical cancer especially in women infected with type 16- and/or type 18-related HPVs [
26].
It was also reported that the NQO1 protein and mRNA were abnormally elevated within many solid tumors. Awadallah
et al.[
10] and Lyn-Cook
et al.[
8] found that NQO1 was not only upregulated in pancreatic ductal adenocarcinoma (PDAC), but could also minimize the risk of false positive diagnosis by combining NQO1 expression with cellular morphology assessment. Mikami
et al.[
27] reported a close correlation between NQO1 enzyme activity and protein expression in both colon cancer cell lines and colorectal tumor samples. They also determined that tumors with nodal metastases showed significantly higher NQO1 protein levels than did tumors without metastasis, which suggested that NQO1 expression might be related to tumorigenesis and malignant progression of colorectal tumors.
Our previous study [
24] also showed that NQO1 was a significant prognostic or predictive marker in gastric cancer. In this study, we performed IHC staining and analysis of 177 cervical SCC samples, 94 precancerous disease samples and 25 normal epithelium tissues of the uterine cervix, and found that the strongly positive rate of NQO1 protein expression in both SCCs and CINs was significantly higher than in the normal cervix. Interestingly, the strongly positive rate of NQO1 protein was slightly higher in well-differentiated SCC (43.75%) than in CIN3 (40.74%) (P > 0.05), indicating that abnormal NQO1 expression might be an early event in the progression of cervical cancer. In addition, the qRT-PCR result also confirmed an increased level of NQO1 mRNA expression in SCCs compared with normal fresh cervical epithelium tissues.
To further illustrate that NQO1 was a potential effective predictor of poor prognosis, we analyzed the correlation between NQO1 expression and clinicopathological features of cervical SCCs, and found that high-level expression of the NQO1 protein was significantly correlated with poor differentiation, late clinical stage, and the presence of lymph node metastasis (P < 0.05). These results indicated that NQO1 played a potentially predictive role in tumor progression of cervical SCCs.
The presence of high-risk HPV infection has been found to be the main cause of cervical cancer [
28]. So far, more than 200 HPV types have been reported and many HPVs have been identified in healthy individuals who have no clinical symptoms, while the path from initial infection to severe epithelial lesion still remains unknown [
29]. Recent studies suggested that microRNAs have important effects in the manifestation of HPV infections in target epithelial cells [
30]. Geiger
et al.[
31] found that during the very early stages of transformation in HPV16-transformed keratinocytes, many epithelial features were gradually eliminated and some mesenchymal traits emerged. In the present study, the positive rate of high-risk HPV infection was 65.52% in CIN-1, 76.32% in CIN-2, 74.07% in CIN-3 and 86.44% in SCC of the cervix, according to ther HPV-DNA chip results. The strongly positive rate of NQO1 protein expression was significantly higher in HPV-positive cervical SCCs (58.17%) than in HPV-negative cases (33.33%). Therefore, it is possible that abnormal expression of NQO1 may make the cervix more prone to HPV infection, and subsequently, HPV infection could accelerate NQO1 overexpression and lead to invasion and metastasis of cervical SCCs [
26].
Regarding survival, we found that cervical SCC patients with high-level NQO1 expression had lower DFS (P < 0.01) and 5-year OS rates (P < 0.01) than patients with low-level NQO1 expression. In early-stage cervical SCC, patients with high-level NQO1 expression had lower DFS and 5-year OS rates compared with those with low-level NQO1 expression (P < 0.05, respectively). Moreover, along with clinical stage and lymph node metastasis, multivariate survival analysis demonstrated that NQO1 expression emerged as a significant independent hazard factor for DFS but not for 5-year OS in patients with cervical SCC. These results indicated that NQO1 was a potential predictor of poor prognosis, especially in patients with early-stage cervical SCC.
Overall, our present work implies that NQO1 might be a new biomarker for early diagnosis and prognostic evaluation as well as a potential molecular target in patients with cervical SCC. However, NQO1 was upregulated as a part of the oxidative stress response and inexplicably overexpressed in particular types of tumors, whose function has not yet been elucidated [
32]. Marco
et al.[
11] demonstrated that the expression of NQO1 significantly induced cell cycle progression via the upregulation of cyclin A2, B1 and D1 that led to the proliferation of melanoma cells, which may account for the overexpression of NQO1 in primary melanoma. Lau
et al.[
33] postulated that NQO1 overexpression was accompanied by an increase in other antioxidant enzymes, such as HMOX-1 and GST, providing tumors with increased protection against cytotoxic agents allowing for rapid cancer progression.
Recent studies on the regulation of NQO1 gene expression have shown that a complex molecular pathway was involved. In addition, studies of Yao
et al. showed that induction of the NQO1 gene by hypoxia and mitomycin C in human colon adenocarcinoma HT29 and human hepatoma HepG2 cells was mediated through a mechanism involving the NF-κB signaling pathway [
34,
35], which has been shown to play an important role in proliferation, resistance to apoptosis, invasion, and metastasis of HeLa cells [
36,
37]. Even in light of this recent information, the molecular mechanism of NQO1 responsible for cervical tumor progression remains to be elucidated, and additional studies are warranted to further our understanding of the role that NQO1 plays in cervical tumorigenesis.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
YM, JK, TJ and DJ participated in the study conception, design, case selection and experiments. GY, XR and YM carried out data collection. TJ, LL and ZL performed the data analysis and wrote the manuscript. All authors read and approved the final manuscript.