Glycosyltransferase Gene Expression

Published OnlineFirst May 29, 2018; DOI: 10.1158/1078-0432.CCR-17-3533

Personalized Medicine and Imaging Clinical Cancer Research Glycosyltransferase Gene Expression Identiﬁes a Poor Prognostic Colorectal Cancer Subtype Associated with Mismatch Repair Deﬁciency and Incomplete Glycan Synthesis Masaru Noda1,2, Hirokazu Okayama1, Kazunoshin Tachibana2, Wataru Sakamoto1, Katsuharu Saito1, Aung Kyi Thar Min1, Mai Ashizawa1, Takahiro Nakajima1, Keita Aoto1, Tomoyuki Momma1, Kyoko Katakura3, Shinji Ohki1, and Koji Kono1

Abstract

Purpose: We aimed to discover glycosyltransferase gene tumors and cell lines, and was further recapitulated by IHC, (glycogene)-derived molecular subtypes of colorectal cancer where approximately 15% tumors exhibited loss of GALNT6 associated with patient outcomes. protein. GALNT6 mRNA and protein was expressed in pre- Experimental Design: Transcriptomic and epigenomic malignant/preinvasive lesions but was subsequently down- datasets of nontumor, precancerous, cancerous tissues, and regulated in a subset of carcinomas, possibly through epige- cell lines with somatic mutations, mismatch repair status, netic silencing. Decreased GALNT6 was independently asso- clinicopathologic and survival information were assem- ciated with poor prognosis in the IHC cohort and an addi- bled (n ¼ 4,223) and glycogene profiles were analyzed. tional microarray meta-cohort, by multivariate analyses, and IHC for a glycogene, GALNT6, was conducted in adenoma its discriminative power of survival was particularly remark- and carcinoma specimens (n ¼ 403). The functional able in stage III patients. GALNT6 silencing in SW480 cells role and cell surface glycan profiles were further investi- promoted invasion, migration, chemoresistance, and gated by in vitro loss-of-function assays and lectin micro- increased cell surface expression of a cancer-associated trun- array analysis. cated O-glycan, Tn-antigen. Results: We initially developed and validated a 15-glyco- Conclusions: The 15-glycogene signature and the expres- gene signature that can identify a poor-prognostic subtype, sion levels of GALNT6 mRNA and protein each serve as a novel which closely related to deficient mismatch repair (dMMR) prognostic biomarker, highlighting the role of dysregulated and GALNT6 downregulation. The association of decreased glycogenes in cancer-associated glycan synthesis and poor GALNT6 with dMMR was confirmed in multiple datasets of prognosis. Clin Cancer Res; 1–14. 2018 AACR.

Introduction rectal cancer (85%) follows the CIN pathway, often accompanied by KRAS mutations and TP53 inactivation. Approximately Despite major advances in diagnosis and treatment, colorectal 15% of colorectal cancers that exhibit deficient MMR (dMMR) cancer remains one of the leading causes of cancer-related death frequently carry BRAF mutations (3, 5). Clinical trials implicated worldwide (1, 2). Colorectal cancer is commonly grouped into MMR status as a potential therapeutic classifier for stage II patients two categories: tumors with microsatellite instability (MSI), in the adjuvant setting (6–8). In the metastatic setting, KRAS and caused by defective function of the DNA mismatch repair (MMR) BRAF mutations are used for predicting unresponsiveness to system, and tumors that are microsatellite stable but exhibiting EGFR-targeted therapies (4). Despite increasing knowledge, clin- chromosomal instability (CIN; refs. 3–5). The majority of colo- icopathologic staging system remains the only prognostic classification currently used in clinical practice. However, clinicopath- ologically similar tumors can strikingly differ in clinical behaviors fl 1Department of Gastrointestinal Tract Surgery, Fukushima Medical University that likely re ect the molecular heterogeneity. Although it is School of Medicine, Fukushima City, Japan. 2Department of Breast Surgery, recommended that stage III patients receive postoperative che- Fukushima Medical University School of Medicine, Fukushima City, Japan. motherapy, approximately 30%–40% of patients develop recur- 3 Department of Gastroenterology, Fukushima Medical University School of rence even after standard treatment (9–12). Medicine, Fukushima City, Japan. Glycosylation is a common posttranslational modification that Note: Supplementary data for this article are available at Clinical Cancer involves sequential addition of single sugar residues to target Research Online (http://clincancerres.aacrjournals.org/). structures, resulting in glycan elongation. Further chemical mod- Corresponding Author: Hirokazu Okayama, Department of Gastrointestinal- ifications and branching can finally form a vast array of glycan tract Surgery, Fukushima Medical University School of Medicine, 1 Hikarigaoka, structures (13). Those procedures are regulated by the multi- Fukushima city, Fukushima 960-1295, Japan. Phone: 81-24-547-1259; Fax: 81-24- enzymatic reaction of glycosyltransferases, whose encoding genes, 547-1980; E-mail: [email protected] namely "glycogenes", are equivalent to 1% of human genome. doi: 10.1158/1078-0432.CCR-17-3533 Cell surface glycans undergo changes during malignant transfor- 2018 American Association for Cancer Research. mation and tumor progression accompanied by distinct

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S1. We utilized the normalized expression values obtained from Translational Relevance each dataset. If a gene is represented by multiple probes, they were Here we report the identification and validation of a poor averaged. To generate a list of glycogenes, official gene symbols prognostic subgroup, displaying mismatch repair deficiency and Entrez Gene IDs for 190 glycogenes were obtained from (dMMR) and decreased GALNT6 levels, based upon glycosyl- GGDB (GlycoGene DataBase; http://acgg.asia/ggdb2/). Among transferase expression and methylation profiles in multiple 190 glycogenes, 185 unique genes were converted to Affyme- cohorts containing a total of 4223 samples. We show that trix_3PRIME_IVT_ID using DAVID Bioinfomatics Resources6.7 downregulation of GALNT6 via epigenetic silencing occurs (http://david.abcc.ncifcrf.gov/home.jsp) as shown in Supple- during transition from precancerous/preinvasive neoplasia to mentary Table S2. invasive carcinoma in a certain subset of tumors that frequent- Hierarchical clustering was initially performed using an Affy- ly exhibit dMMR. Those transcriptional analyses were robustly metrix dataset, GSE17536, consisted of 177 colorectal cancer recapitulated by IHC on 403 specimens, where tumors lacking patients with survival information. Expression levels of 185 GALNT6 protein was associated with dMMR and poor patient glycogenes were median-centered, and then genes and samples outcomes. Strikingly, loss of GALNT6 protein expression and were subjected to an unsupervised clustering by the centroid decreased GALNT6 mRNA expression each discriminated post- linkage method using the Cluster3.0 and the Java Treeview operative stage III patients with poor survival. Our study program (19). Among 39 differentially expressed genes between highlights the possibility of GALNT6 as a novel prognostic two major clusters (Cluster A vs. B, P < 0.001 by t test), 15 genes biomarker for colorectal cancer and suggests its contribution exhibited significant differential expression between the subclus- to colorectal carcinogenesis through incomplete glycan ter (Cluster A1) and the remaining subclusters (Clusters A2, B1, synthesis. and B2) with stringent P values at <0.0001 (Supplementary Tables S3 and S4). We then obtained two Affymetrix datasets, and 121 stage I–III patients in GSE41258 and 89 stage II patients in GSE33113 with available survival information were used for biological functions and unique tumor phenotypes, thereby hierarchical clustering. On the basis of three independent clus- making glycans as potential cancer biomarkers (13, 14). For tering analyses, 3 glycogenes that were consistently upregulated or > instance, a cancer-associated glycan epitope, CA19-9, called sialyl downregulated between clusters with log2 fold-change 0.4 were fi Lewis A (sLea), is routinely utilized as a serum tumor marker (15). identi ed. CA19-9 and several other cancer-associated glycans, including Level 3 Illumina RNA-Seq data for colon and rectal adenocar- sialyl Lewis X (sLex), sialyl Tn, Tn, and T antigens, are associated cinoma (COADREAD) were downloaded through cBioPortal with tumorigenesis and poor prognosis of colorectal cancer (13, (http://www.cbioportal.org/; ref. 20). Clinicopathologic and 16). Such glycans can be attributed to transcriptional dysregula- molecular features were obtained from the TCGA data portal tion of glycosyltransferases that has been postulated as two (http://tcga-data.nci.nih.gov/) in June 2015 (3). We utilized two principal mechanisms, "incomplete synthesis" and "neosynth- different versions of RNA-Seq data normalized either by RPKM or esis" (13, 16–18). Some glycogenes are repressed by epigenetic RSEM methods. These two TCGA datasets, namely, RNA-Seq silencing during early stages of tumorigenesis, which lead to the RPKM and RNA-Seq V2 RSEM, contained 193 and 361 colorectal biosynthesis of truncated structures, such as Tn and STn expres- cancer samples, respectively, after removing three redundant sion, called incomplete synthesis. Conversely, in the neosynthesis samples from the latter dataset. Hierarchical clustering based on Z process, transcriptionally induced glycogenes can result in the de the mRNA expression -scores for the 15 glycogenes was applied novo expression of cancer antigens, such as sLea and sLex. to each TCGA datasets as described above. For the analysis of Z In this study, with the aim to discover distinct classes of GALNT6, both mRNA expression -scores by RNA-Seq V2 RSEM b fi colorectal cancer on the basis of the expression of glycosyltrans- and DNA methylation -values by Illumina In nium Human- ferases, we compiled an extensive number of transcriptomic Methylation450 for 357 samples with available MMR status were profiles obtained from multiple cohorts by integrating other also downloaded from cBioPortal. available data sources, including mutations, MMR status, meth- To analyze the relationship between glycogenes and molecular ylation, protein expression as well as nontumor, precancerous, features, 9 additional datasets were downloaded from GEO, preinvasive, and cancerous samples. We initially described a novel including GSE39582, GSE39084, GSE42284, GSE75315, subtype based upon clustering analysis of genome-wide "glyco- GSE26682, GSE13294, GSE4554, GSE13067, and GSE18088 gene" expression patterns, and this led us to identify a glycogene, (Supplementary Table S1). They were discovered by carefully GALNT6 as a promising biomarker for disease prognosis. More- searching the GEO database according to the availability of more over, we found the functional characteristics of GALNT6 involved than 10 dMMR samples in each dataset. We also used an Illumina in tumor progression and glycosylation, suggesting the contribu- microarray dataset GSE59857, in which mutational and transcrip- fi tion of epigenetic silencing of GALNT6 to colorectal carcinogen- tional pro les of 151 colorectal cancer cell lines were available esis, through the incomplete synthesis of cell surface glycans. (Supplementary Table S1).

Materials and Methods Precursor lesions We obtained formalin-ﬁxed parafﬁn-embedded (FFPE) speci- Microarray data analysis, hierarchical clustering, and assembly mens of endoscopically resected colorectal adenomas from of the TCGA dataset 40 patients and surgically resected colorectal adenomas from All microarray and methylation array data are publicly avail- 20 patients treated at Fukushima Medical University Hospital able in the Gene Expression Omnibus (GEO) database (http:// (Fukushima City, Japan). We also obtained 8 endoscopically www.ncbi.nlm.nih.gov/geo) as shown in Supplementary Table resected specimens that were pathologically diagnosed as

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carcinoma in adenoma. In addition, transcriptomic and epige- absence of nuclear staining of tumor cells in the presence of nomic data from a total of 345 colon adenoma samples with positive nuclear staining in normal colonic epithelium and lym- 213 normal colon and 570 carcinoma samples were analyzed. phocytes (6). Briefly, we obtained datasets of colon biopsy specimens from normal colon, adenoma, and carcinoma (GSE4183, GSE77953, Determination of MMR status GSE37364, GSE20916, GSE41657, and GSE71187) and four In the expression datasets, MSI testing data (MSI-H, MSI-L and additional datasets (GSE45270, GSE79460, GSE4045, and MSS) were obtained through the GEO or the TCGA data portal. GSE36758) of conventional tubular adenomas/adenocarcinomas Tumors demonstrating MSI-H or loss of at least one MMR protein and serrated adenomas/adenocarcinomas (Supplementary were collectively designated as dMMR, and tumors with MSS/MSI- Table S1). Also, we utilized epigenome-wide data based on L or intact MMR protein expression as proficient MMR (pMMR). Illumina Infinium HumanMethylation450 BeadChip platform for normal colon, adenoma and cancer tissues (GSE48684 and Prognostic validation of GALNT6 expression in an independent GSE77954), and 9 dMMR and 34 pMMR colorectal cancer sam- cohort ples (GSE68060; Supplementary Table S1). In those analyses, Stage II and III colorectal cancer samples from three indepen- methylation levels were reported as b-values or M-values, and we dent datasets were aggregated as an independent validation meta- examined probe cg19265103 located in the GALNT6 promotor cohort, herein termed microarray validation cohort (n ¼ 364). We region, as it was utilized in the cBioPortal as described earlier. utilized GSE37892, GSE24551 and GSE38832, because they were not used in the previous GALNT6 analyses of this study and had Colorectal cancer materials and survival analysis enough number of stage II and III colorectal cancer samples with We enrolled 368 consecutive patients with primary colorectal available DFS information. On the basis of the fact that 14.6% of cancer, who underwent surgery between 1990 and 2010 in colorectal cancer showed loss of GALNT6 protein by IHC, we Fukushima Medical University Hospital (Fukushima City, Japan). simply used the same percentile as cutoff, namely patients in the Tumors were classified according to the TNM classification of lowest 14.6th percentile of GALNT6 mRNA expression were malignant tumors (21). After exclusion of patients who received defined as GALNT6-low and the remaining patients were consid- preoperative chemotherapy or radiotherapy, 335 stage 0 to IV ered GALNT6-high within each dataset. patients with available FFPE tumor sections were used. Adjacent normal mucosae from 304 sections were also available for eval- Cell culture and reagents uation. Clinical information was retrospectively obtained by Short tandem repeat (STR)-authenticated colorectal cancer cell reviewing medical records, with the last follow-up in February lines, including SW480, SW620, and RKO, were purchased from 2016. For survival analysis, 17 patients with stage 0 tumors ATCC. SW837 and HCT116 were obtained from JCRB Cell Bank (carcinoma in situ) were omitted, and 267 stage I to IV patients and RIKEN Cell Bank, respectively. HCT15, SW48, LS180, and who underwent curative resection (R0), with survival informa- Colo205 were previously obtained and authenticated by STR tion, were utilized. We analyzed disease-specific survival (DSS), analysis (Promega). RKO and LS180 cells were maintained with disease-free survival (DFS), and overall survival (OS), which were DMEM; others with RPMI1640 containing 10% FBS and penicil- defined as time from the date of surgery to the date of disease lin/streptomycin (Thermo Fisher Scientific) at 37C in a humid- 0 recurrence, cancer-related death, and death from any cause, ified atmosphere of 5% CO2. A demethylation reagent, 5-aza-2 - respectively. The study was conducted in accordance with the deoxycytidine (5-aza-dC; Sigma-Aldrich) was dissolved in DMSO Declaration of Helsinki and was approved by the Institutional at 10 mmol/L and stored in aliquots at 80C until use. Review Board of Fukushima Medical University. Knockdown experiments were conducted using siRNA oligo- nucleotides of GALNT6 or scramble control with Lipofectamine IHC RNAiMAX Reagent, according to manufacturer's instructions IHC was performed as described previously (22), with primary (Ambion Silencer Select; s22154, s22155, and negative control rabbit polyclonal anti-GALNT6 antibody (HPA011762, Prestige #1, Thermo Fisher Scientific). Antibodies Powered by Atlas Antibodies, Sigma-Aldrich, Co. LLC), identified using the Human Protein Atlas database Quantitative real-time PCR (www.proteinatlas.org; ref. 23). Briefly, antigens were retrieved Total RNA was extracted using TRIzol Reagent, and 1 mg of total by autoclave, and anti-GALNT6 antibody was incubated in a RNA was reverse transcribed to cDNA using the SuperScript III 1:500 dilution at 4C overnight, and subsequently detected by First-Strand Synthesis System (Thermo Fisher Scientific) accord- a horseradish peroxidase (HRP)-coupled anti-rabbit polymer ing to the manufacturer's instructions. qRT-PCR was carried out followed by incubation with diaminobenzidine (EnVisionþ Sys- using TaqMan Gene Expression Master Mix on the 7500 Real- tem, Dako). IHC slides were evaluated by two independent Time PCR system in triplicate with TaqMan assays, including observers without knowledge of patients' clinical information. GALNT6 (Hs00926629_m1), MLH1 (Hs00179866_m1), and Several adenocarcinoma specimens from lung (24), pancreas ACTB (Hs99999903_m1; Thermo Fisher Scientific). Relative (25), breast (26), and stomach (27) were used as positive controls. expression levels were determined with SDS software by the DDC Each sections were considered positive for GALNT6 staining when 2 t method as described by the manufacturer. more than 10% of tumor cells were stained in the cytoplasm according to the procedure as described previously (24, 25). Western blotting IHC for MMR protein was performed as described elsewhere Total protein was extracted using RIPA lysis buffer supplemen- (28), with primary antibodies against MLH1 (ES05, 1:50, Dako), ted with Halt Protease Inhibitor Cocktail, and were boiled in Tris- MSH2 (FE11, 1:50, Dako), MSH6 (EP49, 1:200, Dako), and PMS2 Glycine SDS Sample Buffer (Thermo Fisher Scientific). Equal (EP51, 1:50, Dako). Loss of a MMR protein was defined as the amount of protein was loaded and separated by 10% SDS-PAGE

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gel, and then transferred onto polyvinylidene difluoride mem- Statistical analysis branes (Thermo Fisher Scientific). The membrane was blocked Fisher exact test, c2 test, unpaired t test, and Mann–Whitney with 5% nonfat dried skimmed milk powder (Cell Signaling U test were used to determine differences between two variables. Technology), and incubated with primary rabbit anti-GALNT6 Spearman correlation was used to evaluate the correlations (#HPA011762, 1:250, Atlas Antibodies) or mouse anti-b-actin between levels of expression and methylation. Cumulative sur- (#SC-69879, 1:2,000, Santa Cruz Biotechnology). The membrane vival was estimated by the Kaplan–Meier method, and differences was incubated with goat anti-rabbit or anti-mouse HRP secondary between the two groups were analyzed by log-rank test. Univariate antibody (Santa Cruz Biotechnology), and developed with the and multivariate models were computed using Cox proportional SuperSignal West Pico Chemiluminescent Substrate (Thermo hazards regression. All statistical analyses were two-sided and Fisher Scientific) using LAS4000 imager (GE Healthcare). were conducted using GraphPad Prism v6.0 (Graphpad Software Inc.) and SPSS Statistics version 24 (IBM Corporation). All Flow cytometry P values were two-sided, and P values less than 0.05 were con- Cell suspensions were incubated with mouse monoclonal anti- sidered statistically significant. Tn antibody (MLS128, 1:100, Wako), followed by staining with goat anti-mouse IgG H&L (Alexa Fluor 488; ab150113, 1:2,000; Results Abcam). The data were acquired on a FACSCanto II (Becton Transcriptional glycogene profiling demonstrated subgroups Dickinson) and analyzed with FlowJo software (TOMY Digital of colorectal cancer with distinct survival outcomes Biology). The overall study design is demonstrated in Supplementary Fig. S1. We initially conducted an unsupervised hierarchical Cell proliferation assay, 5-FU cytotoxicity assay, and detection clustering analysis in 177 patients from GSE17536 using the of apoptosis 185 glycogenes (Supplementary Table S2; Fig. 1A; Supplemen- Cell proliferation was measured using the Cell Counting Kit-8 tary Fig. S2A), resulting in two major clusters (Cluster A and B) (CCK-8, DOJINDO) according to the manufacturer's instructions. and four subclusters (Cluster A1, A2, B1, and B2; Supplemen- Cytotoxicity was assessed by CCK-8 assay using a series of 5-FU tary Table S5). Cluster A showed a clear tendency to be asso- (Sigma-Aldrich) concentrations. We preliminarily applied a series ciated with worse clinical outcomes (Supplementary Fig. S3A of 5-FU concentrations ranging from 0.1 to 1,000 mg/mL or and S3B). Moreover, patients segregating to Cluster A1 had vehicle alone for generating dose–response curves. We then significant poor DSS and DFS compared with the remaining used 1, 5, 10, 50, and 100 mg/mL of 5-FU for experiments. subclusters (Fig. 1B–E; Supplementary Fig. S3C and S3D). Apoptotic cells were detected using the Annexin V-PE/7-AAD Multivariate Cox analysis demonstrated that Cluster A1 was Apoptosis Detection Kit (BD Biosciences) according to the man- significantly associated with DSS [HR, 5.71; 95% confidence ufacturer's protocol. Annexin V–positive cells were regarded as interval (CI), 2.69–12.13; P ¼ 6.0E06], DFS (HR, 2.83; 95% apoptotic cells. CI, 1.38–5.80; P ¼ 0.005), and OS (HR, 3.71; 95% CI, 2.02– 6.82; P ¼ 2.5E05; Supplementary Table S6). Cluster A1 was Wound-healing assay and transwell invasion assay also associated with poorly differentiated histology (Supple- For wound-healing assay, cells were seeded on a 6-well plate mentary Table S5). and allowed to reach confluency. After scratching the bottom of As Cluster A and its subcluster Cluster A1 exhibited worse the well with a pipette tip, the monolayer of cells was washed, and survival outcome, we next sought to identify a minimum set of the wound closure photographs were captured at 0, 6, 12, 18, and genes whose expression was closely related to these poor 24 hours using a phase-contrast microscope. The percent of prognosis subgroups. Thirty-nine differentially expressed genes wound closure was calculated as the cell migration distance to between Cluster A and B were further narrowed down to 15 the initial wound distance. Invasion assay was performed using genes (GCNT3, FUT8, B3GAT2, GALNT6, POFUT1, GALNT1, Corning BioCoat 24-Multiwell Tumor Cell Invasion Systems B3GNT8, DPM1, HS3ST3B1, SLC35A1, MGAT2, GALNT5, (Corning) according to the manufacturer's protocol. Fluorescence GYLTL1B, MGAT5,andHS3ST1, designated the 15-glycogene of invaded cells labeled with Calcein-AM (Corning) was mea- signature) that were significantly altered between Cluster A1 sured using SkanIt RE for Varioskan Flash 2.4 (Thermo Fisher and the remaining subclusters (Supplementary Tables S3 Scientific). and S4). Lectin microarray The lectin microarray was performed essentially as described Prognostic validation of the 15-glycogene signature in two elsewhere (29). Briefly, the membrane fractions of cultured cells independent datasets were obtained using the ProteoExtract Subcellular Proteome To test the hypothesis that the 15-glycogene signature can Extraction kit (Merck Millipore) and the total protein content discriminate prognostic subgroups, independent datasets were was determined using the Micro BCA Protein Assay kit (Thermo utilized. Clustering analysis showed that 121 patients with stage I Fisher Scientific), and then Cy3-labeled proteins with Cy3 mono- to III diseases from GSE41258 were clearly separated into two reactive dye pack (GE Healthcare Life Science) were analyzed on a clusters, designated as 15-Glycogene Cluster A and 15-Glycogene lectin microarray glass slide (LecChip ver 1.0; GlycoTechnica). Cluster B, with significant DFS difference (Fig. 1F–H; Supplemen- Fluorescent images were acquired using an evanescent-field fluo- tary Fig. S2B). We next studied a homogeneous group of 89 stage II rescence scanner (GlycoStation Reader 1200; GlycoTechnica). The patients from GSE33113. This analysis verified the prognostic raw fluorescence intensity was first processed with the gain-merg- subgroups, demonstrating that 15-Glycogene Cluster A patients ing procedure, followed by average normalization (29). Data were had significant shorter DFS than that of 15-Glycogene Cluster B analyzed with GlycoStation Tools Pro Suite1.5 (GlycoTechnica). (Fig. 1I and J; Supplementary Fig. S2C, P ¼ 0.0080). Multivariate

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Figure 1. Identiﬁcation of glycosyltransferase gene (glycogene)-derived subtypes of colorectal cancer (CRC). A, Unsupervised clustering based on 185 glycogenes in 177 patients with stage I to IV colorectal cancer from GSE17536, demonstrating two major clusters, Cluster A and B, and four subclusters, Cluster A1, A2, B1, and B2. B–E, Cluster A1 was associated with poor prognosis compared with those in the remaining subclusters in DSS (B and C) and DFS (D and E) in GSE17536. F, Clustering analysis for prognostic validation using 15 glycogenes (designated as 15-glycogene signature) in 121 patients with stage I to III colorectal cancer from GSE41258. G and H, Patients segregating 15-Glycogene Cluster A were associated with poor DFS in GSE41258. I, Clustering analysis based on the 15-glycogene signature in 89 patients with stage II colorectal cancer from GSE33113. J, Patients segregating 15-Glycogene Cluster A had poor DFS in GSE33113. K and L, Clustering analysis using the 15- glycogene signature in two independent RNA-sequencing (RNA-Seq) datasets obtained from TCGA, consisting of RNA-Seq RPKM (n ¼ 193) and RNA-Seq V2 RSEM (n ¼ 361). Clinical and genetic features, including deﬁcient mismatch-repair (dMMR), mutations in RAS, BRAF and TP53, tumor location, disease recurrence, and histology are indicated. 185-gene (A) or 15-gene (F, I, K, L) columns are shown on the right-side of the heatmaps (red represents 15 glycogenes), with arrowheads indicating GALNT6 gene.

analysis revealed that prognostic significance of 15-Glycogene The 15-glycogene signature identified a subgroup exhibiting Cluster A was independent of clinical features in GSE41258 (HR, unique clinicopathologic and genomic profiles 4.22; 95% CI, 1.50–11.84; P ¼ 0.006) and in GSE33113 (HR, We further analyzed the association between the 15-Glycogene 4.03; 95% CI, 1.31–12.37; P ¼ 0.015; Supplementary Table S7). clusters and known molecular markers, such as MMR, RAS, BRAF, In all three independent clustering analyses, the expressions of and TP53 status. In GSE41258, the 15-Glycogene Cluster A was the 15 genes were each consistently altered between clusters significantly associated with dMMR (P ¼ 0.003) and wild-type (Supplementary Fig. S3E). Specifically, we identified upregulation TP53 (P ¼ 0.050; Fig. 1K; Supplementary Table S8). The same of GCNT3 and FUT8, and downregulation of GALNT6 as common clustering procedure was applied to two independent RNA-Seq features of Cluster A1 and 15-Glycogene Cluster A. Of note, the datasets obtained from TCGA, consisting of RNA-Seq RPKM (n ¼ expression of GCNT3 and FUT8, but not GALNT6, has been 193) and RNA-Seq V2 RSEM (n ¼ 361). This validated the reported to be associated with prognosis in colorectal cancer association of the 15-Glycogene Cluster A with dMMR and (30, 31). wild-type TP53 (Fig. 1F, K, and L; Supplementary Fig. S2B, SD

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and S2E; Supplementary Table S8). Intriguingly, we found that GALNT6 was downregulated in a subset of carcinoma upon proximal location, mucinous histology, mutant RAS, and mutant malignant transformation BRAF were statistically significantly enriched in the 15-Glycogene Downregulation of glycogenes is an important step in colo- Cluster A. rectal cancer development and progression (14, 18). Thus, we hypothesized that downregulated GALNT6 is involved in car- Decreased expression of GALNT6 in dMMR tumors in 12 cinogenesis. To this end, we analyzed multiple datasets con- independent cohorts of patients with colorectal cancer and a taining normal colon (n ¼ 161, in total), colon adenoma (n ¼ dataset of colorectal cancer cell lines 264, in total), and carcinoma (n ¼ 387, in total) samples. In all We attempted to focus on single glycogenes, including 7 analyses, GALNT6 mRNA expression was significantly higher GCNT3, FUT8,andGALNT6, altered expression of which might in adenomas than that of normal colon, while it was signifi- be characteristics of tumors with dMMR or genetic alterations cantly decreased in carcinomas, compared with adenomas in BRAF, RAS and TP53.Also,CpGislandmethylator pheno- (Fig. 3A–G). To further validate this finding, IHC for GALNT6 type (CIMP) was included in this analysis, as CIMP-positive protein was conducted using our large series of colorectal tumors are known to be closely related to dMMR and BRAF adenoma (n ¼ 60) and carcinoma specimens (n ¼ 335). IHC mutation (32). Nine additional datasets were assembled and demonstrated that GALNT6 protein expression was not we were thus able to analyze 12 independent cohorts contain- detected in the vast majority of normal colon mucosal cells ing a total of 2,472 patients with colorectal cancer (Fig. 2). This (92.8% of 304 normal tissues were GALNT6-negative). Where- revealed the association between GALNT6 expression and as, virtually all samples of adenoma and carcinoma in situ (Tis) MMR status with high reproducibility, where GALNT6 was showed strong granular cytoplasmic staining of GALNT6 in statistically significantly downregulated in dMMR tumors in tumor cells essentially throughout the tumor area (98.3% of all cohorts, comprised of 417 dMMR and 1,800 pMMR tumors. adenoma and 100.0% of Tis; Fig. 4A–C). Likewise, intense It is worth noting that the association of decreased GALNT6 GALNT6 staining was diffusely found in carcinoma cells with dMMR was clearly reproduced by the analysis of 151 (Fig. 4D and E). However, approximately 15% of carcinomas colorectal cancer cell lines (Fig. 2; Supplementary Fig. S4A). lacked GALNT6 protein expression (Fig. 4F and G; Table 1). We This tight correlation between GALNT6 downregulation and also examined the expression patterns of GALNT6 in adenoma- dMMR prompted us to focus specifically on the significance of to-carcinoma transition within the same lesion using 8 speci- GALNT6, which encodes one of the polypeptide GalNAc trans- mens of carcinoma-in-adenoma, showing that in one of 8 ferase (ppGalNAc-T) family enzymes involved in the initiation lesions (12.5%) GALNT6 staining was lacking in the carcinoma of O-glycosylation. component, but all the adenoma components exhibited

Figure 2. The association between the expression of GCNT3, FUT8,andGALNT6, and known molecular markers, including dMMR, mutations in BRAF, RAS, CIMP, and TP53. Twelve independent cohorts, comprised of 2,472 patients and a dataset of 151 cell lines were demonstrated. The red or blue colors in the heatmap represent glycogenes with statistically signiﬁcant upregulation or downregulation in tumors harboring dMMR, CIMP-positive, mutated BRAF, RAS,andTP53, respectively. The expression of GALNT6 was statistically signiﬁcantly downregulated in dMMR tumors in all 13 datasets.

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positive-GALNT6 (Supplementary Fig. S5A–S5H). GALNT6 sion was the highest in precursor and preinvasive tumors, and staining was frequently lost in dMMR tumors (52.0% were was subsequently downregulated or lost in a subset of carci- negative), although the majority of pMMR tumors showed nomas, which was associated with dMMR tumors. positive GALNT6 (11.6% were negative; Fig. 3H; Table 1). It has become apparent that more than 15% of colorectal cancer Collectively, in both mRNA and protein levels, GALNT6 expres- is known to originate from serrated precursor lesions and is often

Figure 3. Alteration of GALNT6 expression and methylation in colorectal carcinogenesis. A–E, GALNT6 mRNA levels in normal colon, colon adenoma, and carcinoma samples in GSE4183 (A), GSE77953 (B), GSE37364 (C), GSE20916 (D), GSE41657 (E), GSE71187 (F), and GSE41258 (G). Compared with adenomas, GALNT6 mRNA was downregulated in carcinomas, particularly in those with dMMR. H, GALNT6 protein expression by IHC in normal colon mucosa, colon adenoma, carcinoma in situ, and invasive carcinoma with MMR status. Loss of GALNT6 protein was found in carcinomas, particularly in those with dMMR. I, Inverse correlation between GALNT6 mRNA expression and GALNT6 methylation in 357 TCGA samples. J–L, GALNT6 promotor methylation levels in normal colon, colon adenoma, and carcinoma tissues in GSE48684 (J), GSE77954 (K), and GSE68060 (L) with MMR status. M and N, qRT-PCR analysis for MLH1 (M)orGALNT6 (N) expression in cell lines with and without MLH1 methylation, treated with a DNA demethylating agent, 5-aza-dc (, P < 0.05; , P < 0.01; , P < 0.001).

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Figure 4. Representative images of IHC for GALNT6 protein expression in colon adenoma and adjacent colon mucosa (A), nonneoplastic colon mucosa (B), colon adenoma (C), and colon carcinoma (D–G). Magniﬁcation: A, D, F 100; B, C, E, G 400. Intense granular staining of GALNT6 is found diffusely in the cytoplasm of adenoma and carcinoma cells (C, D, E), while a subset of colorectal cancer (CRC) lacks GALNT6 staining (F and G). Kaplan–Meier curves for DSS or DFS in stage II and III patients according to the expression of GALNT6 by IHC (H–J) or microarray (K–M). Analysis of stage II and III colorectal cancer (H and K), and stratiﬁed analysis of stage II (I and L), and stage III (J and M), respectively.

characterized by activating BRAF mutations and CIMP that greatly malignant transformation (14), we addressed the possibility overlaps with dMMR tumors (4, 5, 33). Because decreased that DNA methylation contributes to decreased GALNT6 GALNT6 mRNA expression was associated not only with MMR expression. We observed significant inverse correlation between status, but also with CIMP and BRAF mutations (Fig. 2), it was mRNA expression and methylation of GALNT6 (Fig. 3I; P < speculated that GALNT6 downregulation could be associated 0.0001). Higher levels of GALNT6 promotor methylation were with the serrated neoplasia pathway. Indeed, dMMR, CIMP, and observed in colorectal cancer tissues than adenomas in two BRAF mutations were each highly enriched in tumors with additional cohorts (Fig. 3J and K), which was in clear contrast decreased levels of GALNT6 expression in three cohorts (Sup- to the downregulated GALNT6 in colorectal cancer tissues plementary Fig. S6A–S6C).However,weobservednodifference compared with adenomas (Fig. 3A–F). Moreover, in colorectal in GALNT6 expression between serrated adenomas and con- cancer tissues, GALNT6 methylation levels were significantly ventional adenomas, or between serrated adenocarcinomas higher in dMMR tumors than thatofpMMR(Fig.3L).To and conventional adenocarcinomas in 4 datasets of histolog- further confirm the methylation of GALNT6 in vitro,colorectal ically confirmed adenoma and adenocarcinoma samples cancer cell lines, including HCT116, SW48, and RKO, which (Supplementary Fig. S7A–S7D). displayed relatively lower GALNT6 expression levels (Supple- mentary Fig. S4B–S4D), were treated with a DNA methyltrans- Epigenetic silencing may contribute to GALNT6 ferase inhibitor, 5-aza-dC. Demethylation treatment restored downregulation MLH1 expression in MLH1-methylated cell lines, including Because downregulation of some glycogenes results from RKO and SW48, while GALNT6 expression was induced only epigenetic silencing mainly by DNA hypermethylation upon in SW48 cells (Fig. 3M and N).

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Table 1. Clinicopathologic characteristics of patients with colorectal cancer according to GALNT6 expression by IHC GALNT6 Expression Negative Positive Total (n ¼ 335) n ¼ 49 (14.6%) n ¼ 286 (85.4%) P Age Mean SD 67.0 11.8 67.0 12.5 67.0 11.7 0.995 Gender 0.271 Male 198 25 (51.0) 173 (60.5) Female 137 24 (49.0) 113 (39.5) Location 0.105 Proximal colon 106 22 (44.9) 84 (29.4) Distal colon 103 11 (22.4) 92 (32.2) Rectum 126 16 (32.7) 110 (38.5) Differentiation <0.0001 Well 168 19 (38.8) 149 (52.1) Moderately 153 22 (44.9) 131 (45.8) Poorly 14 8 (16.3) 6 (2.1) Histology 0.189 Mucinous 20 5 (89.8) 15 (94.8) Non-mucinous 315 44 (10.2) 271 (5.2) Stage (UICC) 0.768 0 17 0 (0.0) 17 (5.9) I 62 10 (20.4) 52 (18.2) II 122 19 (38.8) 103 (36.0) III 89 15 (30.6) 74 (25.9) IV 45 5 (10.2) 40 (14.0) Tumor invasion 0.149 Tis (m) 17 0 (0.0) 17 (5.9) T1 (sm) 33 3 (6.1) 30 (10.5) T2 (mp) 49 12 (24.5) 37 (12.9) T3 (ss-a) 138 14 (28.6) 124 (43.4) T4 (se-si/ai) 98 20 (40.8) 78 (27.3) Lymph node metastasis 0.873 Absent 213 31 (63.3) 182 (63.6) Present 119 18 (36.7) 101 (35.3) Not available 3 0 (0.0) 3 (1.0) Distant metastasis 0.323 Absent 290 44 (89.8) 246 (86.0) Present 45 5 (10.2) 40 (14.0) MMR status <0.0001 pMMR 310 36 (73.5) 274 (95.8) dMMR 25 13 (26.5) 12 (4.2) , P < 0.05.

Lack of GALNT6 protein expression was associated with poor Because GALNT6 was chosen from the 15 genes for detailed prognosis evaluation primarily because of its tight relationship with We next examined the clinicopathologic and prognostic sig- MMR status, patients were further divided into four subgroups nificance of GALNT6 protein expression in the FFPE cohort. based on GALNT6 and MMR status to explore the clinicopatho- Tumors lacking GALNT6 protein were associated with poorer logic and prognostic significance in those groups. Interestingly, histologic differentiation (P < 0.0001), but exhibited no associ- although dMMR tumors shared similar clinicopathologic features ation with other clinical features (Table 1). Intriguingly, patients irrespective of GALNT6 expression (Supplementary Table S11), with negative GALNT6 had significantly poorer DSS and OS, striking survival differences were found between GALNT6-neg- compared with those with positive GALNT6 (Supplementary ative/dMMR and GALNT6-positive/dMMR subgroups. In Fig. S8A and S8B; P ¼ 0.0038 and P ¼ 0.022, respectively). This stage II–III analysis, GALNT6-negative/dMMR patients had remained statistically significant when the analysis was conducted significant poorer DSS, OS, and DFS compared with those in 195 stage II and III patients (Fig. 4H; Supplementary Fig. S8C; of GALNT6-positive/dMMR (Supplementary Fig. S9A–S9C). In P ¼ 0.0008 and P ¼ 0.014, respectively). Multivariate Cox analysis addition, GALNT6-negative/pMMR patients demonstrated sig- demonstrated that the lack of GALNT6 protein was significantly nificantly poorer prognosis than those of GALNT6-positive/ associated with poor DSS (HR, 3.39; 95% CI, 1.28–9.02; P ¼ pMMR, particularly in stage III analyses (Supplementary 0.014) and OS (HR, 2.34; 95% CI, 1.08–5.05; P ¼ 0.031), Fig. S9D–S9F). It appears that there was no clinicopathologic independent of stage and other conventional factors (Supple- similarity between GALNT6-negative/dMMR and GALNT6-neg- mentary Tables S9 and S10). Stratified analyses also showed that ative/pMMR, although those two subgroups were sharing poor negative GALNT6 had significant prognostic impact on DSS and survival outcomes (Supplementary Table S11). OS in stage III patients (P < 0.0001 and P ¼ 0.0016, respectively), but not evident in stage II patients (Fig. 4I and J; Supplementary Decreased GALNT6 mRNA expression was associated with poor Fig. S8D and S8E). Concerning DFS, the prognostic values of prognosis GALNT6 expression showed only a trend, which did not reach Because loss of GALNT6 protein was associated with worse statistical significance (Supplementary Fig. S8F–S8I). survival in stage II and III patients, we hypothesized that decreased

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GALNT6 mRNA levels may also be prognostic. We assembled Decreased GALNT6 resulted in the increase of cancer-associated three additional datasets, combining them into a microarray truncated glycan, Tn antigen validation meta-cohort containing 364 patients with stage II and Dysregulated glycogenes can result in alteration of cell surface III colorectal cancer (Fig. 4K; Supplementary Fig. S10). Low glycosylation. Thus, we tested to determine whether the depletion GALNT6 mRNA was significantly associated with worse DFS in of GALNT6 could affect the cell surface glycan profiles. Lectin patients with stage II and III colorectal cancer (Fig. 4K, P ¼ microarray analysis was conducted to examine the glycomic 0.0241), and it was independent of clinical factors by multivariate profiles of surface membranous fractions in SW480 cells. Com- analysis (Supplementary Table S12; HR, 1.88; 95% CI, 1.16–3.06; pared with siRNA control, GALNT6-silenced cells demonstrated P ¼ 0.011). Consistent with IHC analysis, the prognostic value of decreased lectin Jacalin and ACA, each of which can bind to core 1 GALNT6 mRNA expression was clearly demonstrated in stage III (Galb1-3GalNAca-Ser/Thr) and core 3 (GlcNAcb1-3GalNAca- patients (P ¼ 0.0036), but not in stage II (Fig. 4L and M; Ser/Thr) extension of O-glycan, respectively (Supplementary Fig. Supplementary Fig. S10). S13). GALNT6 silencing also led to the increased signal intensity of lectin HPA that is highly specific to GalNAca-Ser/Thr, a truncated O-glycan structure, also known as Tn-antigen (Fig. 5H; Lack of GALNT6 protein expression was associated with Supplementary Fig. S13; ref. 35). We confirmed that GALNT6 poor therapeutic response to 5-FU–based adjuvant knockdown increased the cell surface expression of Tn-antigen by chemotherapy flow cytometry using a mAb MLS128 (Fig. 5I and J; ref. 36). It is well recognized that stage II and III patients with dMMR Conversely, 5-aza-dc treatment in SW48 cells resulted in colorectal cancer may not benefit from 5-FU–based adjuvant decreased expression of Tn antigen along with the concomitant chemotherapy (7, 34). We sought to determine whether the induction of GALNT6 (Figs. 3N and 5K and L). expression of GALNT6 was associated with response to adjuvant chemotherapy. Among 190 stage II and III patients in the IHC cohort for which information on the administration of adjuvant Discussion chemotherapy was available, 114 patients received intravenous or This study provides several lines of evidence that the expression oral 5-FU–based adjuvant chemotherapy after surgery, while 76 of GALNT6 is a potential biomarker for identifying a prognostic patients were treated by surgery alone. We conducted DFS anal- subgroup and is implicated in colorectal carcinogenesis. First, yses for GALNT6 expression by stratifying stage II and III patients a glycogene-derived transcriptional subtype, namely, the 15- on the basis of adjuvant treatment history (Supplementary Fig. Glycogene Cluster A, was identified and validated using a total S11A–S11F). Among patients who received chemotherapy, neg- of 941 samples from multiple transcriptomic datasets. This novel fi ative-GALNT6 showed a nonsigni cant trend toward worse DFS subgroup, in which GALNT6 was downregulated, was character- (Supplementary Fig. S11A). Notably, in stage III patients receiving ized by poor prognosis, poorly differentiated histology, proximal adjuvant chemotherapy, negative-GALNT6 was associated with location, and dMMR. Moreover, strong association between poor therapeutic outcome (HR, 5.56; 95% CI, 1.57–19.69; P ¼ decreased GALNT6 mRNA expression and dMMR was robustly 0.0079, Supplementary Fig. S11C). This effect was not observed in confirmed in 12 patient cohorts and a dataset of cell lines, stage III patients treated by surgery alone (HR, 0.31; 95% CI, 0.04– followed by the analysis of a FFPE cohort at GALNT6 protein P ¼ 2.68; 0.290), although the number of patients in each group levels. Second, downregulation of GALNT6 mRNA and protein was limited (Supplementary Fig. S11D). There was no clear trend seemed to occur during transition from adenoma to carcinoma, when stage II patients were analyzed (Supplementary Fig. S11E possibly through epigenetic silencing, where GALNT6 was and S11F). expressed in most of premalignant/preinvasive lesions but was subsequently decreased in a subset of carcinomas. This suggests a Depletion of GALNT6 enhanced invasion, migration, and crucial role of GALNT6 in colorectal cancer, especially contribut- chemoresistance to 5-FU ing to the mechanism referred to as incomplete synthesis of To understand the biologic function of GALNT6, a pMMR glycans. Indeed, GALNT6 depletion not only increased the inva- cell line, SW480, with relatively higher GALNT6 mRNA and sive and migratory potentials but also upregulated the cancer- protein expression were selected for further analyses (Supple- associated truncated glycan, Tn-antigen. In contrast, demethyla- mentary Fig. S4B–S4D). We used two different siRNAs targeting tion resulted in a decrease of Tn-antigen along with GALNT6 GALNT6, demonstrating that GALNT6 was effectively silenced, reactivation. Third, decreased GALNT6 expression in both mRNA confirmed by qRT-PCR and Western blotting (Fig. 5A and B). and protein levels discriminated a poor prognostic subgroup that Although silencing of GALNT6 hadnosignificant impact on was largely consistent with that of the 15-Glycogene Cluster A, cell proliferation (Fig. 5C), it enhanced both cell migration and reinforcing the notion that the glycogene-derived transcriptional invasion determined by wound-healing assay and transwell subtype is recapitulated by tumors lacking GALNT6 protein. invasion assay, respectively (Fig. 5D and E; Supplementary Our strategy integrated various gene expression platforms, Fig. S12). As we found the association between negative including Affymetrix, Agilent, and Illumina microarrays and GALNT6 and poor response to 5-FU–based chemotherapy RNA-Seq, obtained from different laboratories, and even techno- (Supplementary Fig. S11), we tested the in vitro contribution logically independent approach by IHC, consisting of a total of of GALNT6 expression to the sensitivity to 5-FU treatment. We more than 4,500 samples. Likewise, downregulation and meth- found a moderate, but significant increase of 5-FU resistance in ylation of GALNT6 in carcinoma tissues, compared with adenoma GALNT6-knockdown cells as compared with cells treated with tissues, was clearly reproduced in multiple series of nonmalig- control siRNA (Fig. 5F). Correspondingly, apoptosis was sig- nant, premalignant, and malignant lesions using epigenomic and nificantly suppressed in GALNT6-knockdown cells treated with transcriptomic datasets and IHC analysis. This finally led us to 5-FU (Fig. 5G). identify a distinct subgroup lacking GALNT6 protein expression in

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Figure 5. Biological characteristics of GALNT6 silencing. A and B, Transfection of siRNAs targeting GALNT6 or scramble control in SW480 cells was confirmed by qRT-PCR (A) and Western blotting (B). C, Cell proliferation assay at different time points. D, Cell invasion determined by transwell invasion assay. E, Cell migration determined by wound-healing assay. F, Dose-dependent effect of 5-FU treatment on cell viability. G, In 10 mmol/L 5-FU–treated cells, apoptosis was analyzed by flow cytometry labeled with Annexin V-PE and 7-AAD. H–J, Identification and confirmation of increased cell surface Tn-antigen expression in GALNT6-depleted SW480 cells recognized by lectin HPA using lectin microarray (H)andflow cytometry with mAb MLS128 (I and J). K and L, Treatment with 5-aza-dc reduced cell surface Tn- antigen expression in SW48 cells. Data are expressed as mean SD of three independent experiments (C–G), normalized signal intensity of triplicate measurements in lectin microarray (H), or MFI from three independent flow cytometric analyses (J and L; , P < 0.05; , P < 0.01). approximately 15% of colorectal cancer. Those integrated, mul- patients with stage III colorectal cancer. It is also important that tistep analyses could minimize false-positive results. It is therefore IHC for GALNT6 protein is a practical assay that can be routinely unlikely that the presence of this subgroup is related to false analyzed on readily available FFPE specimens in clinical practice. discoveries or batch effects from high-throughput data analyses. GALNT6 downregulation was originally identified to be tightly Notably, this GALNT6-negative subgroup could be identified in correlated with dMMR, and finally we noticed that it had signif- both mRNA and protein levels, and its prognostic values were icant impact on prognosis. It is worth noting that GALNT6- statistically independent of clinical factors. Therefore, it is sug- negative tumors shared poor survival outcomes even when the gested that GALNT6 expression can be a robust prognostic bio- dMMR and pMMR tumors were analyzed separately, but the marker for colorectal cancer. Because its prognostic performance prognostic impact of negative GALNT6 seemed to be more was particularly remarkable in stage III patients, GALNT6 expres- remarkable in the analysis of dMMR tumors. Therefore, we suggest sion may help guide clinical decisions, including adjuvant che- that GALNT6 can be a promising prognostic biomarker for both motherapy and surveillance plans after curative surgery for dMMR and pMMR tumors, and GALNT6 IHC combined with

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MMR may provide more useful prognostic stratification that can colorectal cancer, decreased GALNT6 mRNA and loss of discriminate an extremely poor prognostic subset displaying GALNT6 protein contribute to transition from premalignant/ negative GALNT6/dMMR, from GALNT6-positive/dMMR tumors preinvasive lesions to invasive carcinomas. with excellent prognosis. These results warrant confirmation in Altered expression of GALNT6 has been investigated in other large-scale prospective studies. tumor types, suggesting their potential as cancer biomarkers (13). It is likely that GALNT6-negative patients receiving 5-FU– In pancreatic cancer, Li and colleagues reported that loss of based adjuvant chemotherapy were associated with poor ther- GALNT6 expression was associated with poor differentiation and apeutic outcome. Despite the exploratory nature with small poor OS (25). Conversely, the same group from Li and colleagues number of patients and low number of events in each sub- has recently reported that GALNT6 expression predicted poor OS group, the negative prognostic effect on DFS was evident in in lung adenocarcinoma (24). In breast cancer, GALNT6 over- stage III patients who received 5-FU–based adjuvant therapy, expression may contribute to mammary carcinogenesis through demonstrating a striking contrast to those who were treated by aberrant glycosylation (26). Such conflict between different can- surgery alone. This was further supported by the finding that cers has also been observed in several studies investigating other GALNT6-depleted colorectal cancer cells demonstrated an ppGalNAc-Ts. For instance, GALNT3 expression correlated with increase of chemoresistance to 5-FU treatment. This implicated poor survival in ovarian cancer (44) and renal cell carcinoma (43), that negative GALNT6 may also have a predictive value for poor whereas it was associated with better survival in lung adenocar- response to 5-FU–based adjuvant chemotherapy in stage III cinoma (45), gastric cancer (46), and colorectal cancer (47). colorectal cancer. Therefore, alternative therapeutic strategies, GALNT7 was shown to be targeted by miRNA-214 in cervical including combination regimens or targeted drugs, may be (48) and esophageal cancer (49); its overexpression enhanced more effective and appropriate for stage III patients with proliferation, invasion, and migration. In contrast, in melanoma negative-GALNT6 tumor. Recent clinical trials revealed that cells, microRNA-30b/30d promoted invasion and metastasis by stage III dMMR patients may benefitfromadjuvant5-FU direct suppression of GALNT7 (50). Although there is no direct treatment combined with oxaliplatin (37, 38). It would be explanation for the contradictory influence of ppGalNAc-Ts in interesting to address the effect of adding oxaliplatin compared different cancers, these conflicting data among different cancer with the conventional 5-FU–based therapy alone, in relation to types may indicate the complexity of O-glycosylation along with GALNT6 status, although no patients in this study were treated the diversity and distinct substrate specificities of ppGalNAc-Ts with oxaliplatin in the adjuvant setting. Because dMMR colo- that can confer specific roles in specific cellular contexts. Future rectal cancer has recently been reported to be effectively treated studies would be required to address this complexity of O-gly- with anti-PD-1 immune checkpoint inhibitors (39, 40), cosylation associated with deregulated ppGalNAc-Ts during detailed analysis of downregulated GALNT6 in relation to tumorigenesis of various malignancies. ant-tumor immunity may help to understand the dMMR– Upon malignant transformation, epigenetic alterations are colorectal cancer biology. recognized as key characteristics that can cause dysregulation of In normal tissues, GalNAc type O-glycans are modified by glycogenes, resulting in aberrant expression of cell surface glycans glycosyltransferases to generate core structures, and core O- (16). We found an inverse correlation between the expression and glycans are further extended and capped by the addition of methylation of GALNT6, and demethylation treatment reacti- sialylated and fucosylated terminal structures (13, 41). The vated GALNT6 expression in colorectal cancer cell lines. Also, cell ppGalNAc-Ts, which catalyze the transfer of GalNAc to Ser/Thr surface lectin microarray analysis revealed that the levels of lectin residues on substrate proteins, control the initiation step of HPA-recognized GalNAca-Ser/Thr, known as cancer-associated GalNAc-type O-glycosylation. The ppGalNAc-Ts form a family Tn antigen, were specifically increased in GALNT6-depleted cells, of 20 distinct isoenzymes expressed in a cell-type–specific confirmed by using a mAb MLS128 (35, 36). This truncated O- manner, with different but overlapping substrate specificity, glycan structure, Tn, is involved in tumor progression in many thus O-glycans are synthesized through concerted and occa- types of cancer, including colorectal cancer (35, 51–53). Indeed, sionally competitive action of ppGalNAc-Ts (41, 42). GALNT6 Tn antigen is known to be a marker of poorly differentiated and was reported to be expressed in high percentages of adenocar- mucinous adenocarcinoma, and poor patient prognosis in colo- cinoma cells from breast, lung, pancreas, and renal cancer, rectal cancer (13, 42). GALNT6 knockdown promoted invasion whereas it was undetectable or very weakly found in their and migration, which was in agreement with the tumor pheno- normal counterpart (24–26, 43). We showed that GALNT6 type with Tn antigen overexpression. Conversely, cell surface Tn staining was undetectable in normal colonic tissue, but was antigen was diminished by DNA demethylating agent along with invariably overexpressed in virtually all tumor cells of prema- GALNT6 induction. Those findings are highly consistent with the lignant/preinvasive lesions. Transcriptomic and epigenomic concept of incomplete synthesis that glycan elongation in non- data confirmed the upregulation of GALNT6 mRNA along with malignant cells are impaired upon malignant transformation by demethylation of GALNT6 promotor in adenoma samples, silencing of glycogenes, resulting in the expression of cancer- compared with normal colon. This suggests a role of GALNT6 associated truncated glycans (13, 16, 18). Taken together, our in the early stage of tumorigenesis, where GALNT6 may even results suggest that GALNT6 expression is epigenetically regulated support adenoma formation, irrespective of conventional or during preinvasive neoplasm-invasive carcinoma transition in a serrated carcinogenesis pathways. GALNT6 expression in pre- subgroup of colorectal tumors that contribute to cancer progres- malignant tumors seemed to be maintained in the majority of sion possibly through the incomplete synthesis mechanism. colorectal cancer as well, with the exception of lower percen- We found that not only dMMR but also CIMP and BRAF tages (15%) of colorectal cancer showing GALNT6 loss. mutation were each enriched in tumors showing decreased levels Because GALNT6 silencing could promote the capacity of of GALNT6 expression, and thus they were overlapping consid- invasion and migration in vitro, it is likely that in a subset of erably each other. Because CIMP-positive tumors are known to

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have poor prognosis and are frequently accompanied by BRAF associated Tn antigen, highlighting the possible role of GALNT6 mutation, our study might raise the possibility that the prognostic in colorectal carcinogenesis and poor prognosis. impact of GALNT6 can be at least in part attributed to CIMP phenotype. Although the underlying driver biology of GALNT6 Disclosure of Potential Conflicts of Interest loss remains inconclusive, it appears that CIMP might be one of No potential conflicts of interest were disclosed. the putative mechanisms that can explain the epigenetic silencing of GALNT6. Further investigation would be required for under- Authors' Contributions standing the pathogenesis of GALNT6-negative colorectal cancer Conception and design: M. Noda, H. Okayama, K. Kono associated with promotor methylation of GALNT6 and methyl- Development of methodology: M. Noda, H. Okayama, K. Tachibana, K. Kono ator phenotype. Acquisition of data (provided animals, acquired and managed patients, This study had several limitations, including its retrospective provided facilities, etc.): M. Noda, H. Okayama, K. Saito, T. Nakajima, T. Momma, K. Katakura, S. Ohki, K. Kono nature and lack of IHC validation in independent cohorts. In Analysis and interpretation of data (e.g., statistical analysis, biostatistics, addition, CIMP status and mutational profiles, such as RAS computational analysis): M. Noda, H. Okayama, K. Saito, T. Nakajima, K. Aoto, and BRAF, were unavailable in the FFPE cohort. Therefore, K. Kono thoseresultspresentedherewouldneedtobevalidatedinthe Writing, review, and/or revision of the manuscript: M. Noda, H. Okayama, future investigations, by combining GALNT6 IHC with other K. Kono genetic and epigenetic biomarkers, for instance, BRAF, RAS, Administrative, technical, or material support (i.e., reporting or organizing PIK3CA in vitro data, constructing databases): H. Okayama, K. Tachibana, W. Sakamoto, CIMP, and (4, 5). Concerning the loss-of- A.K. Thar Min, M. Ashizawa, T. Momma function experiments, this study might not provide conclusive Study supervision: H. Okayama, K. Saito, K. Kono evidence that epigenetically silenced GALNT6 could directly contribute to tumor progression. Thus, we suggest that func- Acknowledgments tional assays using panels of cell lines harboring several genetic This work was supported by JSPS KAKENHI grant numbers 15K10143 and profiles and in vivo tumorigenicity assays would be interesting 25870582. H. Okayama and M. Noda were supported by Takeda Science future directions. Foundation. The authors thank Dr. Yuuichirou Kiko for providing pathologic In conclusion, we developed and validated the 15-glycogene advice. signature that can identify a genomically distinct subgroup exhi- The costs of publication of this article were defrayed in part by the payment of biting dMMR, decreased GALNT6 expression and poor outcomes. page charges. This article must therefore be hereby marked advertisement in Also, GALNT6 expression can be a novel prognostic biomarker accordance with 18 U.S.C. Section 1734 solely to indicate this fact. that can be applied to FFPE specimens. GALNT6 downregulation, in part, due to epigenetic silencing, may contribute to the incom- Received December 2, 2017; revised March 18, 2018; accepted May 22, 2018; plete O-glycan synthesis and increased expression of the cancer- published first May 29, 2018.

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OF14 Clin Cancer Res; 2018 Clinical Cancer Research

Glycosyltransferase Gene Expression Identifies a Poor Prognostic Colorectal Cancer Subtype Associated with Mismatch Repair Deficiency and Incomplete Glycan Synthesis

Masaru Noda, Hirokazu Okayama, Kazunoshin Tachibana, et al.

Clin Cancer Res Published OnlineFirst May 29, 2018.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-17-3533

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