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High Expression of the Sd Synthase B4GALNT2 Associates with Good

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High Expression of the Sd Synthase B4GALNT2 Associates with Good cells Article High Expression of the Sda Synthase B4GALNT2 Associates with Good Prognosis and Attenuates Stemness in Colon Cancer Michela Pucci y, Inês Gomes Ferreira y, Martina Orlandani, Nadia Malagolini, Manuela Ferracin and Fabio Dall’Olio * Department of Experimental, Diagnostic and Specialty Medicine (DIMES), General Pathology Building, University of Bologna, Via San Giacomo 14, Via San Giacomo 14, 40126 Bologna, Italy; [email protected] (M.P.); [email protected] (I.G.F.); [email protected] (M.O.); [email protected] (N.M.); [email protected] (M.F.) * Correspondence: [email protected]; Tel.: +39-051-2094704 These authors have equal contribution. y Received: 25 February 2020; Accepted: 7 April 2020; Published: 11 April 2020 Abstract: Background: The carbohydrate antigen Sda and its biosynthetic enzyme B4GALNT2 are highly expressed in normal colonic mucosa but are down-regulated to a variable degree in colon cancer tissues. Here, we investigated the clinical and biological importance of B4GALNT2 in colon cancer. Methods: Correlations of B4GALNT2 mRNA with clinical data were obtained from The Cancer Genome Atlas (TCGA) database; the phenotypic and transcriptomic changes induced by B4GALNT2 were studied in LS174T cells transfected with B4GALNT2 cDNA. Results: TCGA data indicate that patients with high B4GALNT2 expression in cancer tissues display longer survival than non-expressers. In LS174T cells, expression of B4GALNT2 did not affect the ability to heal a scratch wound or to form colonies in standard growth conditions but markedly reduced the growth in soft agar, the tridimensional (3D) growth as spheroids, and the number of cancer stem cells, indicating a specific effect of B4GALNT2 on the growth in poor adherence and stemness. On the transcriptome, B4GALNT2 induced the down-regulation of the stemness-associated gene SOX2 and modulated gene expression towards an attenuation of the cancer phenotype. Conclusions: The level of B4GALNT2 can be proposed as a marker to identify higher- and lower-risk colorectal cancer patients. Keywords: glycosylation; sugar antigens; glycosyltransferases; gene expression; stemness; microarray analysis 1. Introduction Colorectal cancer (CRC) remains a worldwide leading cause of cancer deaths, notwithstanding the improved efficacy of the available therapies. Key factors for the success of the therapy include an early diagnosis and approaches tailored to the risk profile of each patient, sparing invasive and expensive therapies to lower-risk patients. Glycosylation is a very frequent post-translational modification of proteins, which undergoes profound changes during neoplastic transformation [1,2]. The Sda carbohydrate antigen is composed of an α2,3-sialylated galactose residue to which an N-acetylgalactosamine residue is β1,4 linked (Siaα2,3(GalNAcβ1,4) Gal-R), where R is the underlying carbohydrate structure (Figure1A). The last step of the Sd a antigen biosynthesis is mediated by the enzyme β1,4N-acetylgalactosaminyltransferase 2 (B4GALNT2) [3], the product of the B4GALNT2 gene [4–6]. Transcription of the B4GALNT2 gene generates at least two different transcripts that only differ in the first exon. These two transcripts contain a translational start site, from which two different transmembrane polypeptides originate: one, referred to as “long form”, contains an unusually Cells 2020, 9, 948; doi:10.3390/cells9040948 www.mdpi.com/journal/cells Cells 2020, 9, 948 2 of 22 only differ in the first exon. These two transcripts contain a translational start site, from which two different transmembrane polypeptides originate: one, referred to as “long form”, contains an unusually long cytoplasmic tail [5,6]; the second, known as “short form”, is provided with a cytoplasmic tail of conventional length [7]. Previous studies have shown a higher enzymatic activity Cellsof the2020 ,short9, 948 form compared with the long form [8]. The normal human colonic mucosa usually2 of 18 expresses very high levels of the B4GALNT2 mRNA and enzyme activity as well as high levels of the a a longSd antigen. cytoplasmic On the tail contrary, [5,6]; the in second, CRC tissues, known both as “short the B4GALNT2 form”, is provided [9,10] and with the aSd cytoplasmic antigen [8] tail are ofmarkedly conventional down length-regulated, [7]. Previousalthough studiesat a very have variable shown level a higher among enzymatic patients. activityIn fact, in of the the cancer short formtissues compared of the majority with the of long the formpatients, [8]. B4GALNT2 The normal is human virtually colonic undetectable, mucosa usually while in expresses a minority, very a highquite levels high ofactivity the B4GALNT2 is detectable. mRNA Both and normal enzyme and activitycancerous as wellcolonic ashigh tissues levels express of the mainly, Sda antigen. if not Onexclusively, the contrary, the inshort CRC form tissues, of B4GALNT2. both the B4GALNT2 The α2,3 [ 9,sialylated10] and the type Sd a 2antigen sugar [chains8] are markedlyon which a down-regulated,B4GALNT2 elaborates although the at aSd very antigen variable can level also among be utilized patients. by In fact,fucosyltransferases in the cancer tissues (mainly of x thefucosyltransferase majority of the 6 patients, (FUT6)) B4GALNT2for the biosynthesis is virtually of the undetectable, cancer-associated while antigen in a minority, sialyl Lewis a quite X (sLe high) activity[11–13]. is Our detectable. group [8] Both and normalothers [14] and showed cancerous that colonic the forced tissues expression express of mainly, B4GALNT2 if not exclusively,in CRC cell x a thelines short partially form ofreplaces B4GALNT2. the sLe The withα2,3 the sialylated Sd antigen. type 2In sugar gastrointestinal chains on which cell lines, B4GALNT2 this modification elaborates thehas Sd beena antigen shown can to also reduce be utilized the metastatic by fucosyltransferases potential [14,15 (mainly]. However, fucosyltransferase the clinical implications 6 (FUT6)) for of theB4GALNT2 biosynthesis expression of the cancer-associated in CRC have antigennever been sialyl investigated. Lewis X (sLe xTo)[ 11obtain–13]. Oursignificant group [8clinical] and otherscorrelations [14] showed between that gene the expression forced expression and clinical of B4GALNT2 parameters, in it CRC is necessary cell lines to partially access large replaces cohorts the sLeof xfullywith char theacterized Sda antigen. patients, In gastrointestinal such as The cellCancer lines, Genome this modification Atlas (TCGA), has been which shown contains to reduce gene theexpression metastatic and potential clinical [data14,15 from]. However, hundreds the of clinical patients. implications Owing to the of B4GALNT2 well-recognized expression importance in CRC of haveglycosylation never been in cancer, investigated. we used To TCGA obtain data significant to compare clinical the prognostic correlations predicti betweenve potential gene expression of several andglycosyltransferases clinical parameters, involved it is necessary in the tobiosynthesis access large of cohorts cancer of-associated fully characterized glycans. In patients, a preliminary such as Thesurvey Cancer of TCGA, Genome we Atlas noticed (TCGA), that whichamong contains various gene glycosyltransferases expression and clinical involved data in from colon hundreds cancer, ofB4GALNT2 patients. Owingdisplayed to thea very well-recognized good predictive importance potential of in glycosylation that patients in retaining cancer, wehigher used levels TCGA of dataB4GALNT2 to compare mRNA the prognosticdisplayed a predictive much longer potential overall of survival. several glycosyltransferasesIn particular, all long involved-time survivals in the biosynthesisdisplayed high of cancer-associated levels of B4GALNT2 glycans. mRNA. In a preliminary To obtain survey insight of TCGA,into the we mechanisms noticed that amonglinking a variousB4GALNT2/Sd glycosyltransferases expression to involved the CRC in phenotype, colon cancer, we B4GALNT2analyzed the displayed phenotype a and very the good transcriptome predictive potentialof LS174T in cells that patientstransfected retaining with the higher short levels form of of B4GALNT2 B4GALNT2. mRNA We found displayed that B4GALNT2 a much longer expression overall survival.reduces the In particular,clonogenic all ability long-time in non survivals-adherent displayed conditions high and levels the ofstemness B4GALNT2 of the mRNA. cells through To obtain the insightmodulation into the of the mechanisms gene expression. linking B4GALNT2/Sda expression to the CRC phenotype, we analyzed the phenotype and the transcriptome of LS174T cells transfected with the short form of B4GALNT2. We found that B4GALNT2 expression reduces the clonogenic ability in non-adherent conditions and the stemness of the cells through the modulation of the gene expression. B4GALNT2 β1,4 α2,3 A R Sda N-acetylglucosamine (GlcNAc) β1,4 Galactose (Gal) β1,4 α2,3 Sialic acid (Sia) R N-acetylgalactosamine (GalNAc) β1,4 α2,3 Fucose (Fuc) R sLex FUT6 α1,3 B C Anti-Sda Anti-sLex activity MW 12 mRNA 50 prot activity 9 mRNA 25 250 /h mg 6 130 3 pmol ( B4GALNT2 B4GALNT2 B4GALNT2 B4GALNT2 Neo S2 S11 Anti β-actin Figure 1. Biochemical characterization of B4GALNT2-transfected cell lines. (A) the Sda and the sLex antigens derive from alternative and mutually exclusive terminations of a common α2,3-sialylated type 2 structure. (B) both the enzymatic activity (dark gray) and the mRNA (light gray) of B4GALNT2 were negligible in Neo cells, but strongly
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