B3GNT3 Expression Is a Novel Marker Correlated with Pelvic Lymph Node Metastasis and Poor Clinical Outcome in Early-Stage Cervical Cancer

Abstract

Background: The β1,3-N-acetylglucosaminyltransferase-3 gene (B3GNT3) encodes a member of the B3GNT family that functions as the backbone structure of dimeric sialyl-Lewis A and is involved in L-selectin ligand biosynthesis, lymphocyte homing and lymphocyte trafficking. B3GNT3 has been implicated as an important element in the development of certain cancers. However, the characteristics of B3GNT3 in the development and progression of cancer remain largely unknown. Thus, our study aimed to investigate the expression pattern and the prognostic value of B3GNT3 in patients with early-stage cervical cancer.

Methods: The mRNA and protein levels of B3GNT3 expression were examined in eight cervical cancer cell lines and ten paired cervical cancer tumors, using real-time PCR and western blotting, respectively. Immunohistochemistry (IHC) was used to analyze B3GNT3 protein expression in paraffin-embedded tissues from 196 early-stage cervical cancer patients. Statistical analyses were applied to evaluate the association between B3GNT3 expression scores and clinical parameters, as well as patient survival.

Results: B3GNT3 expression was significantly upregulated in cervical cancer cell lines and lesions compared with normal cells and adjacent noncancerous cervical tissues. In the 196 cases of tested early-stage cervical cancer samples, the B3GNT3 protein level was positively correlated with high risk TYPES of human papillomavirus (HPV) infection (P = 0.026), FIGO stage (P < 0.001), tumor size (P = 0.025), tumor recurrence (P = 0.004), vital status (P < 0.001), concurrent chemotherapy and radiotherapy (P = 0.016), lymphovascular space involvement (P = 0.003) and most importantly, lymph node metastasis (P = 0.003). Patients with high B3GNT3 expression had a shorter overall survival (OS) and disease-free survival (DFS) compared with those with low expression of this protein. Multivariate analysis suggested that B3GNT3 expression is an independent prognostic indicator for cervical cancer patients.

Conclusions: Our study demonstrated that elevated B3GNT3 expression is associated with pelvic lymph node metastasis and poor outcome in early-stage cervical cancer patients. B3GNT3 may be a novel prognostic marker and therapeutic target for the treatment of cervical cancer.

Fig 1. Overexpression of B3GNT3 mRNA and protein in cervical cancer cell lines.

(a and b) Expression of B3GNT3 mRNA and protein in cervical cancer cell lines (C33a, Ca Ski, HeLa, HeLa 229, MS751, ME-180, SiHa and HCC 94) and normal cervical cell lines were examined by western blotting (a) and quantitative real-time PCR (qPCR) (b). The expression levels were normalized against α-tubulin and GAPDH, respectively. The error bars represent the standard deviation of the mean (SD) calculated from three parallel experiments. *P < 0.05.

Fig 2. Overexpression of B3GNT3 mRNA and protein in cervical cancer tissues.

(a) Representative images of western blotting analyses of the B3GNT3 protein expression in ten matched pairs of cervical cancer tissues (T) and adjacent noncancerous tissues (ANT). α-Tubulin was used as the loading control. (b) The average T/ANT ratios of B3GNT3 mRNA expression in the paired cervical cancer (T) and adjacent noncancerous tissues (ANT) were quantified by quantitative real-time PCR (qPCR) and normalized against GAPDH. The error bars represent the standard deviation of the mean (SD) calculated from three parallel experiments. (c) Immunohistochemical assay of B3GNT3 protein expression in ten pairs of matched cervical cancer tissues. *P < 0.05.

Fig 3. Immunohistochemical detection of B3GNT3 protein expression in paraffin-embedded tissues.

Positive B3GNT3 staining was observed mainly in the cytoplasm of cervical cancer cells. (A) a and b, B3GNT3 expression was not detected in normal cervical tissues; c and d, representative images of weak B3GNT3 staining in cervical cancer tissues; e and f, representative images of moderate B3GNT3 staining in cervical cancer tissues; g and h, representative images of strong B3GNT3 staining in cervical cancer tissues. (B) The statistical analyses of the average mean optical density (MOD) of B3GNT3 staining in the lymph node metastasis group and the lymph node metastasis-free group. *P < 0.05. (C) Kaplan-Meier curves of univariate analysis data (log-rank test).The overall survival (OS) and disease-free survival (DFS) for the patients with high versus low B3GNT3 expression.

Fig 4. Kaplan-Meier curves of univariate analysis data (log-rank test).

(a) The overall survival (OS) of the patients without lymph node metastasis with high versus low B3GNT3 expression. (b) The OS for the patients with squamous cell carcinoma antigen > 1.5ng/ml with high versus low B3GNT3 expression. (c) The OS for the patients with HPV infection with high versus low B3GNT3 expression. (d) The OS for the patients with deep stromal invasion with high versus low B3GNT3 expression. (e) The OS for the patients at stages Ib1-Ib2 with high versus low B3GNT3 expression. (f) The OS for the patients at stages IIa1-IIa2 with high versus low B3GNT3 expression. (g) The OS for the patients at differentiation 1–2 with high versus low B3GNT3 expression. (h) The OS for the patients at differentiation 2–3 with high versus low B3GNT3 expression. (i) The OS for the patients with high versus low B3GNT3 expression who received chemotherapy.