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. 2019 Mar 22;8(3):273.
doi: 10.3390/cells8030273.

N-Glycomic and Transcriptomic Changes Associated with CDX1 mRNA Expression in Colorectal Cancer Cell Lines

Stephanie Holst  1 , Jennifer L Wilding  2 , Kamila Koprowska  3 , Yoann Rombouts  4   5 , Manfred Wuhrer  6
Affiliations

N-Glycomic and Transcriptomic Changes Associated with CDX1 mRNA Expression in Colorectal Cancer Cell Lines

Stephanie Holst et al. Cells. .

Abstract

The caudal-related homeobox protein 1 (CDX1) is a transcription factor, which is important in the development, differentiation, and homeostasis of the gut. Although the involvement of CDX genes in the regulation of the expression levels of a few glycosyltransferases has been shown, associations between glycosylation phenotypes and CDX1 mRNA expression have hitherto not been well studied. Triggered by our previous study, we here characterized the N-glycomic phenotype of 16 colon cancer cell lines, selected for their differential CDX1 mRNA expression levels. We found that high CDX1 mRNA expression associated with a higher degree of multi-fucosylation on N-glycans, which is in line with our previous results and was supported by up-regulated gene expression of fucosyltransferases involved in antenna fucosylation. Interestingly, hepatocyte nuclear factors (HNF)4A and HNF1A were, among others, positively associated with high CDX1 mRNA expression and have been previously proven to regulate antenna fucosylation. Besides fucosylation, we found that high CDX1 mRNA expression in cancer cell lines also associated with low levels of sialylation and galactosylation and high levels of bisection on N-glycans. Altogether, our data highlight a possible role of CDX1 in altering the N-glycosylation of colorectal cancer cells, which is a hallmark of tumor development.

Keywords: HNF1A; N-glycosylation; caudal-related homeobox protein 1 (CDX1), differentiation; colorectal cancer cell lines; fucosylation; fucosyltransferase (FUT), hepatocyte nuclear factor (HNF)4A.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Figures

Figure 1
Figure 1
N-glycans and Lewis antigens. (A) N-glycans are attached to an asparagine (N) in the consensus sequence N-X-S/T with X any amino acid except proline, followed by serine (S) or threonine (T). Main monosaccharides involved in N-glycosylation are mannoses (Man), galactoses (Gal), N-acetylglucosamine (GlcNAc), fucoses (Fuc), and N-aceylneuraminic acid (NeuAc). N-glycans share a common core-structure, consisting of two GlcNAc and three Man. Depending on the elongation, three N-glycan types are differentiated, as follows: i) High-mannose type N-glycans, ii) complex-type N-glycans, and iii) hybrid-type N-glycans. The illustrated glycans represent examples. The number of monosaccharides added can vary and complex type glycans can exhibit more than two antennae. A detailed description of N-glycosylation is given by Stanley [4]. (B) Depiction of different Lewis-antigens and involved glycosyltransferase genes. Fucosyltransferase genes FUT3,4,5,6,7,9 are involved in fucosylation α1,3- and α1,4-fucosylation of antennae GlcNAc, while FUT1,2 attach α1,2-fucosylation to Gal-residues. Activity of several α2,3-sialyltransferase (ST3GAL genes) attach NeuAc residues to galactoses to form sialyl Lewis antigens.
Figure 2
Figure 2
Exemplary MALDI-TOF-MS spectra. MALDI-TOF-MS spectrum of CDX1-low expressing cell line COLO678 (upper spectrum), and of CDX1-high expressing cell line HCA46 (lower spectrum). Spectra were recorded in positive ion reflectron mode on a Bruker UltrafleXtreme mass spectrometer. On the y-axis, the relative intensity is given with 100% corresponding to the highest peak in each spectrum. The spectrum range of m/z 2350 to m/z 4600 is enlarged in the inset. Main peaks are annotated with glycan cartoons, representing compositions, and the presence of additional structural isomers cannot be excluded. To simplify the cartoons, repeating epitopes are indicated as “nx”. Green circle = mannose, Man; yellow circle = galactose, Gal; blue square = N-acetylglucosamine, GlcNAc; white square = N-acetylhexosamine, HexNAc; red triangle = fucose, Fuc; purple diamond = sialic acid, N-acetylneuraminic acid, NeuAc. Differences in N-acetylneuraminic acid linkages are indicated using different angles.
Figure 3
Figure 3
Principle component analysis of N-glycomic profiles. Principle component analysis (PCA) was performed to explore differences in N-glycosylation between CDX1-high and CDX1-low expressing cells. (A) Score plot of principal components (PC) 1 vs. 2 explaining 25.8% and 19.4% of the data, respectively. (B) Corresponding loading plot with color indications for multi- (green) and mono-fucosylated (purple) individual N-glycans, and (C) the same loading plot with color indications for α2,3-sialylated (rose) and α2,6-sialylated (blue) N-glycans. Red triangle = fucose, Fuc; purple diamond = sialic acid, N-acetylneuraminic acid, NeuAc. Differences in N-acetylneuraminic acid linkages are indicated using different angles.
Figure 4
Figure 4
Differentially expressed N-glycan traits. Derived N-glycan traits were calculated to evaluate glycosylation characteristics associated with CDX1 mRNA expression. Differential expressions were observed for (A) multi-fucosylation (more than one fucose) indicative for antenna fucosylation, (B) overall sialylation (C), α2,3-sialylation, (D) N-glycans with more or equal N-acetylhexosamines than hexoses (HexNAc ≥ Hex), (E) galactosylation per antenna, and (F) N-glycans with seven or more HexNAcs. Differences were evaluated by Mann–Whitney test for derived traits and significances (p-value < 0.05) after multiple testing corrections are indicated (*), ns = non-significant. Boxplots consistently indicate the median and interquartile range.
Figure 5
Figure 5
Differentially expressed genes related to glycosylation. N-glycomic phenotypes of the eight CDX1-high vs. eight CDX1-low cell lines were further compared to transcriptomic data of the same cell lines obtained from a gene expression microarray analysis using the Human genome U133+2 Affimetrix chips. The Y axis shows the linear normalized fluorescent intensities of the respective probesets. Differential expressions were observed for fucosyltransferase genes (A) FUT3, (B) FUT6, and (C) GMDS, for (D) MGAT3 (bisecting GlcNAc) and (E) B4GALNT3 (LacdiNAc), as well as (F) B3GNT3 (poly-LacNAc), (G) B3GNT8 (poly-LacNAc), (H) MGAT4A (α1-3-branching), and (I) α2,3-sialyltransferase gene ST3GAL6. Furthermore, transcription factors (J) hepatocyte nuclear factor (HNF)1A, and (K) HNF4A, as well as (L) soluble galectin 4 (LGALS4) showed increased expression with high CDX1 expression in the set of 16 colorectal cancer cell lines. Differences were evaluated by a t-test. Boxplots indicate the median and interquartile range. Significances after multiple testing correction are indicated and correspond to * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001; ns non-significant.
Figure 6
Figure 6
Model for regulation of fucosylation. Based on our findings as well as reports from literature (Lauc, G. et al. (2010) PLoS Genet 6; Guo, R. J., Suh, E. R. & Lynch, J. P. (2004) Cancer Biol Ther 3, 593-601) we hypothesize that antenna-fucosylation is regulated by an interplay of several transcription factors, especially CDX1, CDX2, HNF1A, and HNF4A, which can be affected by several signaling pathways, promotor methylation, as well as treatment with epidermal growth factor (EGF)/bFGF (Sakuma, K., Aoki, M. & Kannagi, R. (2012) Proceedings of the National Academy of Sciences of the United States of America 109, 7776-7781) or under hypoxia (Belo, A. I. et al. (2015) FEBS Lett 589, 2359-2366).
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