Characterization of tumor-associated MUC1 and its glycans expressed in mucoepidermoid carcinoma

Abstract

Mucoepidermoid carcinoma (MEC) is one of the most frequently misdiagnosed tumors. Glycans are modulated by malignant transformation. Mucin 1 (MUC1) is a mucin whose expression is upregulated in various tumors, including MEC, and it has previously been investigated as a diagnostic and prognostic tumor marker. The present study aimed to reveal the differences in the mucin glycans between MEC and normal salivary glands (NSGs) to discover novel diagnostic markers. Soluble fractions of salivary gland homogenate prepared from three MEC salivary glands and 7 NSGs were evaluated. Mucins in MEC and NSGs were separated using supported molecular matrix electrophoresis, and stained with Alcian blue and monoclonal antibodies. The glycans of the separated mucins were analyzed by mass spectrometry. MUC1 was found in MEC but not in NSGs, and almost all glycans of MUC1 in MEC were sialylated, whereas the glycans of mucins in NSGs were less sialylated. The core 2 type glycans, (Hex)₂(HexNAc)₂(NeuAc)₁ and (Hex)₂(HexNAc)₂(NeuAc)₂, were found to be significantly abundant glycans of MUC1 in MEC. MEC markedly produced MUC1 modified with sialylated core 2 glycans. These data were obtained from the soluble fractions of salivary gland homogenates. These findings provide a basis for the utilization of MUC1 as a serum diagnostic marker for the preoperative diagnosis of MEC.

Keywords: O-glycan; mucin; mucin 1; mucoepidermoid carcinoma; salivary gland.

Figure 1.

Mucins of mucoepidermoid carcinoma and normal salivary glands were separated by SMME. Arabic numerals indicate the sample numbers shown in Table I. PGM was used as a reference mixture of CS, HA, AM and NM. The migrating position of the neutral mucin of PGM was estimated from our previous study (18). HPAF-II lysate was used as a mixture containing MUC1. (A) Alcian blue staining of the SMME membranes. (B) Staining with anti-MUC1 antibody (MY.1E12). (C) Staining with hyaluronan binding protein (BC-40). AM, acidic mucin; CS, chondroitin sulfate; HA, hyaluronic acid; MUC1, mucin 1; NM, neutral mucin; PGM, porcine gastric mucin; SMME, supported molecular matrix electrophoresis.

Figure 2.

O-glycan profiles of mucins separated by SMME. (A) Position of the excised bands in the illustration derived from the SMME membranes stained with Alcian blue (Fig. 1A). Roman numerals indicate the excised band numbers. No bands appeared from sample 4. (B) Heatmap of O-glycan profiles of the excised bands. The color scale indicates the relative intensity (%) of each glycan to the sum of intensities of all 50 glycans (Table II). PGM, porcine gastric mucin; SMME, supported molecular matrix electrophoresis.

Figure 3.

Two-group comparisons of all glycan signals between MEC and NSGs. Bar height represents the mean of the relative intensity of the three bands (i, ii and iii) for MEC (white) and six bands (iv-ix) for NSGs (black). Error bars indicate the SEM. *P<0.001. (A) Sialoglycans and (B) neutral glycans. MEC, mucoepidermoid carcinoma; NSG, normal salivary gland.

Figure 4.

Proposed structures for the two significant glycans, glycan 8 (m/z, 1,344) and 16 (m/z, 1,793). (A) Possible structures for glycans 8 (m/z, 1,344) and 16 (m/z, 1,793) based on the biosynthetic pathway. (B) MS/MS spectrum of glycan 8 (m/z, 1,344). (C) MS/MS spectrum of glycan 16 (m/z, 1,793). The proposed structure and assignment of the fragment ions are indicated in the spectra. The structures in parentheses are considered to be minor components. MS/MS, tandem mass spectrometry.