Reduced mannosidase MAN1A1 expression leads to aberrant N-glycosylation and impaired survival in breast cancer

Abstract

Background: Alterations in protein glycosylation have been related to malignant transformation and tumour progression. We recently showed that low mRNA levels of Golgi alpha-mannosidase MAN1A1 correlate with poor prognosis in breast cancer patients.

Methods: We analysed the role of MAN1A1 on a protein level using western blot analysis (n=105) and studied the impact of MAN1A1-related glycosylation on the prognostic relevance of adhesion molecules involved in breast cancer using microarray data (n=194). Functional consequences of mannosidase inhibition using the inhibitor kifunensine or MAN1A1 silencing were investigated in breast cancer cells in vitro.

Results: Patients with low/moderate MAN1A1 expression in tumours showed significantly shorter disease-free intervals than those with high MAN1A1 levels (P=0.005). Moreover, low MAN1A1 expression correlated significantly with nodal status, grading and brain metastasis. At an mRNA level, membrane proteins ALCAM and CD24 were only significantly prognostic in tumours with high MAN1A1 expression. In vitro, reduced MAN1A1 expression or mannosidase inhibition led to a significantly increased adhesion of breast cancer cells to endothelial cells.

Conclusions: Our study demonstrates the prognostic role of MAN1A1 in breast cancer by affecting the adhesive properties of tumour cells and the strong influence of this glycosylation enzyme on the prognostic impact of some adhesion proteins.

Figure 1

Principle steps of N-glycosylation and functional role of α1,2-mannosidases. The transfer of an oligosaccharide precursor to a suitable asparagine residue within the nascent polypeptide is mediated by the oligosaccharyltransferase (OST) protein complex. Subsequent cleavage of three terminal glucose residues by DCS1 and GANAB and one mannose by the α-1,2-ER-mannosidase MAN1B1 leads to the formation of high-mannose glycan structures. Golgi α-1,2-mannosidases (MAN1A1, MAN1A2 and MAN1C1) cleave further α-1,2-bound mannose sugars from high-mannose glycans (Man8-9GlcNAc2), resulting in 5-mannose glycans (Man5GlcNAc2). This glycan structure is the substrate for various glycosyl transferases and mannosidases, which catalyse the formation of complex branched tri- or tetra-antennary N-glycans. ER=endoplasmatic reticulum; FuT=fucosyltransferases (i.e., FUT8); GalT=galactosyltransferases (i.e., B3GALT1); GnT=GlcNac transferases (i.e., MGAT family); ST=syalyltransferases (i.e., ST6GAL and ST3GAL).

Figure 2

MAN1A1 protein expression in clinical tumour tissue samples. (A) Representative western blot analysis showing MAN1A1 expression (ca. 70 and 60 kDa) in two breast cancer cell lines and 15 clinical tumour tissue samples. As a loading control, GAPDH expression is shown. (B, C) Kaplan–Meier analysis showing correlations of high MAN1A1 protein expression with longer disease-free survival (B) and overall survival (C). P-values after log-rank tests comparing four groups (quartiles Q1–Q4) or two groups (quartiles Q1–Q3 vs Q4) are shown. (D–I) Correlation of MAN1A1 protein expression with clinical and histological tumour parameters. P-values after χ²-tests are given. Significant associations of high MAN1A1 expression with negative nodal status (D) and high to moderate differentiation (G1/2; E), as well as a weak association with a luminal molecular subtype, are shown (F). In addition, low MAN1A1 levels correlated significantly with the appearance of brain metastases (G), whereas a trend was observed for bone and lung metastases (H, I).

Figure 3

Influence of MAN1A1 expression on the prognostic role of ALCAM and CD24. Expression of MAN1A1 and ALCAM (A, B) or MAN1A1 and CD24 (C, D) were analysed in clinical tumour tissue samples, based on cDNA microarray data. Regarding the ALCAM or CD24 expression data, the cases were divided into four quartiles for Kaplan–Meier analysis and log-rank tests, stratified for tumours with low (<median) or higher (>median) MAN1A1 mRNA expression. High CD24 and low ALCAM expression correlated significantly with shorter overall survival only in cases with a higher mannosidase MAN1A1 expression (B, D).

Figure 4

MAN1A1 protein expression in breast cancer cell lines. (A) Western blot analysis showing MAN1A1 expression in breast cancer cell lines: T47D and MCF7 (ER+ and PR+), SKBR3, AU565 and HCC1954 (HER2+) and MDA-MB-231, MDA-MB-231-BR and MDA-MB-231-SA (TNBC). As a loading control, GAPDH expression is shown. (B) MAN1A1 expression was quantified by densitometry, normalised to GAPDH expression and the relative expression of each sample was calculated in relation to MDA-MB231 cells, which was set as 1.

Figure 5

Influence of MAN1A1 expression on the adhesion of breast cancer cells to endothelial cells. (A–F) After treatment with different concentrations of the mannosidase inhibitor kifunensine, tumour cell adhesion to activated (+TNFα) and not activated (−TNFα) endothelial cells was analysed under static (MDA-MB-231: A, B; T47D; C, D) and dynamic conditions (MDA-MB-231: (E, F). (G) Western blot analysis showing a mass shift in ALCAM, ICAM-1 and BCAM after treatment of T47D and MDA-MB-231 cells with 10 μM kifunensine. (H) Western blot analysis (upper panel) and subsequent quantification (lower panel) of MAN1A1 knockdown in MDA-MB-231 cells, showing MAN1A1 expression in stably transfected MDA-MB-231 cells with scramble shRNA (nc) and two different MAN1A1 specific shRNA sequences (#2 and #3). (I–L) Tumour cell adhesion to activated (+TNFα) and control (−TNFα) endothelial cells in MAN1A1 knockdown MDA-MB-231 cells compared with the nc cells under static (I, J) and cell flow conditions (K, L). Values are means±s.d. (n=3). *P<0.05, **P<0.005.