NQO1 overexpression is associated with poor prognosis in squamous cell carcinoma of the uterine cervix

Abstract

Background: NQO1 (NAD(P)H: quinone oxidoreductase-1), located on chromosome 16q22, functions primarily to protect normal cells from oxidant stress and electrophilic attack. Recent studies have revealed that NQO1 is expressed at a high level in most human solid tumors including those of the colon, breast, pancreas, ovaries and thyroid, and it has also been detected following the induction of cell cycle progression and proliferation of melanoma cells. In this study, we aimed to investigate the clinicopathological significance of upregulated NQO1 protein expression in squamous cell carcinomas (SCCs) of the uterine cervix.

Methods: The localization of the NQO1 protein was determined in the SiHa cervical squamous cancer cell line using immunofluorescence (IF) staining, and immunohistochemical (IHC) staining performed on paraffin-embedded cervical SCC specimens from 177 patients. For comparison, 94 cervical intraepithelial neoplasia (CIN) and 25 normal cervical epithelia samples were also included. QRT-PCR was performed on RNA from fresh tissues to detect NQO1 mRNA expression levels, and HPV infection status was genotyped using oligonucleotide microarray. Disease-free survival (DFS) and 5-year overall survival (OS) rates for all cervical SCC patients were calculated using the Kaplan-Meier method, and univariate and multivariate analysis was performed using the Cox proportional hazards regression model.

Results: The NQO1 protein showed a mainly cytoplasmic staining pattern in cervical cancer cells, and only three cases of cervical SCC showed a nuclear staining pattern. The strongly positive rate of NQO1 protein expression was significantly higher in cervical SCCs and CINs than in normal cervical epithelia. High-level NQO1 expression was closely associated with poor differentiation, late-stage, lymph node metastasis and high-risk for HPV infection. Additionally, high-level NQO1 expression was associated with lower DFS and 5-year OS rates, particularly for patients with early-stage cervical SCCs. Furthermore, Cox analysis revealed that NQO1 expression emerged as a significant independent hazard factor for DFS rate in patients with cervical SCC.

Conclusions: NQO1 overexpression might be an independent biomarker for prognostic evaluation of cervical SCCs.

Figure 1

Immunofluorescence staining of the NQO1 protein in SiHa cervical SCC cells. The NQO1 protein was located in the cytoplasm of SiHa cervical SCC cells (red indicates NQO1 staining, blue indicates DAPI).

Figure 2

IHC staining of the NQO1 protein on tissue microarray of cervical lesions. (A) NQO1 protein was weakly positive in cervical SCC. (B) NQO1 protein showed diffuse and strong cytoplasmic-positive staining in cervical SCC. (C) NQO1 protein was negative in normal cervical epithelia. (D) NQO1 protein was positive in SCC.

Figure 3

NQO1 protein expression in cervical lesions using IHC. (A) NQO1 protein was negative in normal cervical epithelia. (B) NQO1 protein staining was positive in dysplastic cells of CIN-3, but negative in adjacent normal cervical glands (arrows). (C) NQO1 protein showed diffuse and strong cytoplasmic-positive staining in late-stage cervical SCC. (D) NQO1 was weakly positive in early-stage cervical SCC. (E) NQO1 protein was negative in cervical SCC without metastasis. (F) This case of SCC showed a rare nuclear staining pattern.

Figure 4

QRT-PCR analysis of NQO1 mRNA. Twelve cases of cervical SCC and eight cases of normal fresh cervical tissue were collected and subjected to qRT-PCR analysis of NQO1 mRNA levels. Data represent the mean of individual samples tested in triplicate relative to that of the normal control ± SD (P < 0.05).

Figure 5

Kaplan–Meier analysis of cervical SCC patient survival rates in relation to NQO1 protein expression. Disease-free survival (A) and overall survival rates (B) of patients with elevated (solid, n = 97) and low (dashed, n = 80) NQO1 expression.

Figure 6

Kaplan–Meier analysis of survival rates in patients with high- or low-level NQO1 expression and with early- or late-stage cervical SCC. Disease-free survival (A) and overall survival rates (B) were assessed in patients with early-stage cervical SCC concomitant with either high- (solid, n = 44) or low-level (dashed, n = 57) NQO1 expression. Disease-free survival (C) and overall (D) survival rates were also assessed in patients with late-stage cervical SCC concomitant with high- (solid, n = 53) or low-level (dashed, n = 23) NQO1 expression.