Glycosyltransferase Gene Expression

Author Manuscript Published OnlineFirst on May 29, 2018; DOI: 10.1158/1078-0432.CCR-17-3533 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

1 Title: 2 Glycosyltransferase gene expression identifies a poor prognostic colorectal cancer subtype 3 associated with mismatch repair deficiency and incomplete glycan synthesis 4 5 Authors and affiliations: 6 Masaru Noda1,2, Hirokazu Okayama1, Kazunoshin Tachibana2, Wataru Sakamoto1, Katsuharu Saito1, 7 Aung Kyi Thar Min1, Mai Ashizawa1, Takahiro Nakajima1, Keita Aoto1, Tomoyuki Momma1, Kyoko 8 Katakura3, Shinji Ohki1, and Koji Kono1 9 10 1Department of Gastrointestinal Tract Surgery, 2Departmet of Breast Surgery, 3Department of 11 Gastroenterology, Fukushima Medical University School of Medicine, Japan 12 13 Running title: 14 Prognostic CRC subtypes based on glycosyltransferase profile 15 16 Keywords: 17 Colorectal cancer, glycosyltransferase, cancer-associated glycans, gene expression profiling, molecular 18 diagnosis and prognosis 19 20 Additional information: 21 This work was supported by JSPS KAKENHI Grant Numbers 15K10143 and 25870582. Hirokazu 22 Okayama and Masaru Noda were supported by Takeda Science Foundation. 23 24 Correspondence: Hirokazu Okayama, M. D., Ph. D. 25 Department of Gastrointestinal-tract Surgery, Fukushima Medical University School of Medicine 26 1 Hikarigaoka, Fukushima city, Fukushima, 960-1295, Japan. 27 TEL: +81-24-547-1259 28 FAX: +81-24-547-1980 29 E-mail: [email protected] 30 31 The authors declare no conflicts of interest associated with this manuscript.

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32 33 Translational Relevance 34 Here we report the identification and validation of a poor prognostic subgroup, displaying 35 mismatch repair deficiency (dMMR) and decreased GALNT6 levels, based upon glycosyltransferase 36 expression and methylation profiles in multiple cohorts containing a total of 4223 samples. We show that 37 downregulation of GALNT6 via epigenetic silencing occurs during transition from 38 precancerous/preinvasive neoplasia to invasive carcinoma in a certain subset of tumors that frequently 39 exhibit dMMR. Those transcriptional analyses were robustly recapitulated by immunohistochemistry on 40 403 specimens, where tumors lacking GALNT6 protein was associated with dMMR and poor patient 41 outcomes. Strikingly, loss of GALNT6 protein expression and decreased GALNT6 mRNA expression 42 each discriminated postoperative stage III patients with poor survival. Our study highlights the 43 possibility of GALNT6 as a novel prognostic biomarker for CRC and suggests its contribution to 44 colorectal carcinogenesis through incomplete glycan synthesis. 45

46 Abstract 47 Purpose: We aimed to discover glycosyltransferase gene (glycogene)-derived molecular subtypes of 48 colorectal cancer (CRC) associated with patient outcomes. 49 Experimental Design: Transcriptomic and epigenomic datasets of non-tumor, pre-cancerous, cancerous 50 tissues and cell lines with somatic mutations, mismatch repair status, clinicopathological and survival 51 information, were assembled (n=4223) and glycogene profiles were analyzed. Immunohistochemistry 52 for a glycogene, GALNT6, was conducted in adenoma and carcinoma specimens (n=403). The 53 functional role and cell surface glycan profiles were further investigated by in vitro loss-of-function 54 assays and lectin microarray analysis. 55 Results: We initially developed and validated a 15-glycogene signature that can identify a 56 poor-prognostic subtype, which closely related to deficient mismatch repair (dMMR) and GALNT6 57 downregulation. The association of decreased GALNT6 with dMMR was confirmed in multiple datasets 58 of tumors and cell lines, and was further recapitulated by immunohistochemistry, where approximately 59 15% tumors exhibited loss of GALNT6 protein. GALNT6 mRNA and protein was expressed in 60 premalignant/preinvasive lesions but was subsequently downregulated in a subset of carcinomas, 61 possibly through epigenetic silencing. Decreased GALNT6 was independently associated with poor 62 prognosis in the immunohistochemistry cohort and an additional microarray meta-cohort, by 63 multivariate analyses, and its discriminative power of survival was particularly remarkable in stage III 64 patients. GALNT6 silencing in SW480 cells promoted invasion, migration, chemoresistance and 65 increased cell surface expression of a cancer-associated truncated O-glycan, Tn-antigen. 66 Conclusions: The 15-glycogene signature and the expression levels of GALNT6 mRNA and protein 67 each serve as a novel prognostic biomarker, highlighting the role of dysregulated glycogenes in 68 cancer-associated glycan synthesis and poor prognosis. 69 70 71 Introduction 72 Despite major advances in diagnosis and treatment, colorectal cancer (CRC) remains one of 73 the leading causes of cancer-death worldwide (1, 2). CRC is commonly grouped into two categories: 74 tumors with microsatellite instability (MSI), caused by defective function of the DNA mismatch repair 75 (MMR) system, and tumors that are microsatellite stable but exhibiting chromosomal instability (CIN) 76 (3-5). The majority of CRC (~85%) follows the CIN pathway, often accompanied by KRAS mutations 77 and TP53 inactivation. Approximately 15% of CRCs that exhibit deficient MMR (dMMR) frequently 78 carry BRAF mutations (3, 5). Clinical trials implicated MMR status as a potential therapeutic classifier

79 for stage II patients in the adjuvant setting (6-8). In the metastatic setting, KRAS and BRAF mutations are 80 used for predicting unresponsiveness to EGFR-targeted therapies (4). Despite those increasing 81 knowledge, clinicopathological staging system remains the only prognostic classification currently used 82 in clinical practice. However, clinicopathologically similar tumors can strikingly differ in clinical 83 behaviors that likely reflect molecular heterogeneity. Although it is recommended that stage III patients 84 receive postoperative chemotherapy, approximately 30-40% of patients develop recurrence even after 85 standard treatment (9-12). 86 Glycosylation is a common post-translational modification that involves sequential addition of 87 single sugar residues to target structures, resulting in glycan elongation. Further chemical modifications 88 and branching can finally form a vast array of glycan structures (13). Those procedures are regulated by 89 the multienzymatic reaction of glycosyltransferases, whose encoding genes, namely “glycogenes”, are 90 equivalent to 1% of human genome. Cell surface glycans undergo changes during malignant 91 transformation and tumor progression accompanied by distinct biological functions and unique tumor 92 phenotypes, thereby making glycans as potential cancer biomarkers (13, 14). For instance, a 93 cancer-associated glycan epitope, CA19-9, called sialyl Lewis A (sLea), is routinely utilized as a serum 94 tumor marker (15). CA19-9 and several other cancer-associated glycans, including sialyl Lewis X (sLex), 95 sialyl Tn, Tn and T antigens, are associated with tumorigenesis and poor prognosis of CRC (13, 16). 96 Such glycans can be attributed to transcriptional dysregulation of glycosyltransferases that has been 97 postulated as two principal mechanisms, “incomplete synthesis” and “neo-synthesis” (13, 16-18). Some 98 glycogenes are repressed by epigenetic silencing during early stages of tumorigenesis, which lead to the 99 biosynthesis of truncated structures, such as Tn and STn expression, called incomplete synthesis. 100 Conversely, in the neo-synthesis process, transcriptionally induced glycogenes can result in the de novo 101 expression of cancer-antigens, such as sLea and sLex. 102 In the present study, with the aim to discover distinct classes of CRC on the basis of the 103 expression of glycosyltransferases, we compiled an extensive number of transcriptomic profiles obtained 104 from multiple cohorts by integrating other available data sources, including mutations, MMR status, 105 methylation, protein expression as well as non-tumor, pre-cancerous, preinvasive and cancerous samples. 106 We initially described a novel subtype based upon clustering analysis of genome-wide “glycogene” 107 expression patterns, and this led us to identify a glycogene, GALNT6 as a promising biomarker for 108 disease prognosis. Moreover, we found the functional characteristics of GALNT6 involved in tumor 109 progression and glycosylation, suggesting the contribution of epigenetic silencing of GALNT6 to 110 colorectal carcinogenesis, through the incomplete synthesis of cell surface glycans. 111

112 Materials and Methods 113 Microarray data analysis, hierarchical clustering and assembly of the TCGA dataset 114 All microarray and methylation array data are publicly available in the Gene Expression 115 Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo) as shown in Supplementary Table S1. We 116 utilized the normalized expression values obtained from each dataset. If a gene is represented by 117 multiple probes, they were averaged. To generate a list of glycogenes, official gene symbols and Entrez 118 Gene IDs for 190 glycogenes were obtained from GGDB (GlycoGene DataBase; 119 http://acgg.asia/ggdb2/). Among 190 glycogenes, 185 unique genes were converted to 120 Affymetrix_3PRIME_IVT_ID using DAVID Bioinfomatics Resources6.7 121 (http://david.abcc.ncifcrf.gov/home.jsp) as shown in Supplementary Table S2. 122 Hierarchical clustering was initially performed using an Affymetrix dataset, GSE17536, 123 consisted of 177 CRC patients with survival information. Expression levels of 185 glycogenes were 124 median-centered, and then genes and samples were subjected to an unsupervised clustering by the 125 centroid linkage method using the Cluster3.0 and the Java Treeview program (19). Among 39 126 differentially expressed genes between two major clusters (Cluster A vs B, p<0.001 by t-test), 15 genes 127 exhibited significant differential expression between the subcluster (Cluster A1) and the remaining 128 subclusters (Clusters A2, B1 and B2) with stringent p-values at <0.0001 (Supplementary Table S3 and 129 S4). We then obtained two Affymetrix datasets, and 121 stage I-III patients in GSE41258 and 89 stage II 130 patients in GSE33113 with available survival information were used for hierarchical clustering. Based 131 on 3 independent clustering analyses, 3 glycogenes that were consistently upregulated or downregulated 132 between clusters with log2 fold-change>0.4 were identified. 133 Level 3 Illumina RNA-Seq data for colon and rectal adenocarcinoma (COADREAD) were 134 downloaded through cBioPortal (http://www.cbioportal.org/) (20). Clinicopathological and molecular 135 features were obtained from the TCGA data portal (http://tcga-data.nci.nih.gov/) in June 2015 (3). We 136 utilized two different versions of RNA-Seq data normalized either by RPKM or RSEM methods. These 137 two TCGA datasets, namely, RNA-Seq RPKM and RNA-Seq V2 RSEM, contained 193 and 361 CRC 138 samples, respectively, after removing 3 redundant samples from the latter dataset. Hierarchical clustering 139 based on the mRNA expression z-Scores for the 15 glycogenes was applied to each TCGA datasets as 140 described above. For the analysis of GALNT6, both mRNA expression z-Scores by RNA-Seq V2 RSEM 141 and DNA methylation β-values by Illumina Infinium HumanMethylation450 for 357 samples with 142 available MMR status were also downloaded from cBioPortal. 143 To analyze the relationship between glycogenes and molecular features, 9 additional datasets 144 were downloaded from GEO, including GSE39582, GSE39084, GSE42284, GSE75315, GSE26682,

145 GSE13294, GSE4554, GSE13067 and GSE18088 (Supplementary Table S1). They were discovered by 146 carefully searching the GEO database according to the availability of more than 10 dMMR samples in 147 each dataset. We also used an Illumina microarray dataset GSE59857, in which mutational and 148 transcriptional profiles of 151 CRC cell lines were available (Supplementary Table S1). 149 150 Precursor lesions 151 We obtained formalin-fixed paraffin-embedded (FFPE) specimens of endoscopically-resected 152 colorectal adenomas from 40 patients and surgically-resected colorectal adenomas from 20 patients 153 treated at Fukushima Medical University hospital. We also obtained 8 endoscopically-resected 154 specimens that were pathologically diagnosed as carcinoma in adenoma. In addition, transcriptomic and 155 epigenomic data from a total of 345 colon adenoma samples with 213 normal colon and 570 carcinoma 156 samples were analyzed. Briefly, we obtained datasets of colon biopsy specimens from normal colon, 157 adenoma and carcinoma (GSE4183, GSE77953, GSE37364, GSE20916, GSE41657 and GSE71187) and 158 four additional datasets (GSE45270, GSE79460, GSE4045 and GSE36758) of conventional tubular 159 adenomas/adenocarcinomas and serrated adenomas/adenocarcinomas (Supplementary Table S1). Also, 160 we utilized epigenome-wide data based on Illumina Infinium HumanMethylation450 BeadChip platform 161 for normal colon, adenoma and cancer tissues (GSE48684 and GSE77954), and 9 dMMR and 34 pMMR 162 CRC samples (GSE68060) (Supplementary Table S1). In those analyses, methylation levels were 163 reported as β-values or M-values, and we examined probe cg19265103 located in the GALNT6 164 promotor region, as it was utilized in the cBioPortal as described earlier. 165 166 CRC materials and survival analysis 167 We enrolled 368 consecutive patients with primary CRC, who underwent surgery between 168 1990 and 2010 in Fukushima Medical University hospital. Tumors were classified according to the TNM 169 classification of malignant tumors (21). After exclusion of patients who received preoperative 170 chemotherapy or radiotherapy, 335 stage 0 to IV patients with available FFPE tumor sections were used. 171 Adjacent normal mucosae from 304 sections were also available for evaluation. Clinical information was 172 retrospectively obtained by reviewing medical records, with the last follow-up in February 2016. For 173 survival analysis, 17 patients with stage 0 tumors (carcinoma in situ) were omitted, and 267 stage I to IV 174 patients who underwent curative resection (R0), with survival information, were utilized. We analyzed 175 disease-specific survival (DSS), disease-free survival (DFS) and overall survival (OS), which were 176 defined as time from the date of surgery to the date of disease recurrence, cancer death, and death from 177 any cause, respectively. The study was conducted in accordance with the Declaration of Helsinki and

178 was approved by the Institutional Review Board of Fukushima Medical University. 179 180 Immunohistochemistry (IHC) 181 IHC was performed as described previously (22), with primary rabbit polyclonal 182 anti-GALNT6 antibody (HPA011762, Prestige Antibodies Powered by Atlas Antibodies, Sigma-Aldrich, 183 Co. LLC. St. Louis, MO, USA), identified using the Human Protein Atlas database 184 (www.proteinatlas.org) (23). Briefly, antigens were retrieved by autoclave, and anti-GALNT6 antibody 185 was incubated in a 1:500 dilution at 4°C overnight, and subsequently detected by a horseradish 186 peroxidase (HRP)-coupled anti-rabbit polymer followed by incubation with diaminobenzidine 187 (EnVision+ System, Dako, Heverlee, Belgium). IHC slides were evaluated by two independent 188 observers without knowledge of patients’ clinical information. Several adenocarcinoma specimens from 189 lung (24), pancreas (25), breast (26) and stomach (27) were used as positive controls. Each sections were 190 considered positive for GALNT6 staining when more than 10% of tumor cells were stained in the 191 cytoplasm according to the procedure as previously described (24, 25). 192 IHC for MMR protein was performed as described elsewhere (28), with primary antibodies 193 against MLH1 (ES05, 1:50, Dako), MSH2 (FE11, 1:50, Dako), MSH6 (EP49, 1:200, Dako) and PMS2 194 (EP51, 1:50, Dako). Loss of a MMR protein was defined as the absence of nuclear staining of tumor 195 cells in the presence of positive nuclear staining in normal colonic epithelium and lymphocytes (6). 196 197 Determination of MMR status 198 In the expression datasets, MSI testing data (MSI-H, MSI-L and MSS) were obtained through 199 the GEO or the TCGA data portal. Tumors demonstrating MSI-H or loss of at least one MMR protein 200 were collectively designated as dMMR, and tumors with MSS/MSI-L or intact MMR protein expression 201 as proficient MMR (pMMR). 202 203 Prognostic validation of GALNT6 expression in an independent cohort 204 Stage II and III CRC samples from three independent datasets were aggregated as an 205 independent validation meta-cohort, herein termed microarray validation cohort (n=364). We utilized 206 GSE37892, GSE24551 and GSE38832, because they were not used in the previous GALNT6 analyses 207 of this study and had enough number of stage II and III CRC samples with available DFS information. 208 Based on the fact that 14.6% of CRC showed loss of GALNT6 protein by IHC, we simply used the same 209 percentile as cut-off, namely patients in the lowest 14.6th percentile of GALNT6 mRNA expression 210 were defined as GALNT6-low and the remaining patients were considered GALNT6-high within each

211 dataset. 212 213 Cell culture and reagents 214 Short tandem repeat (STR)-authenticated CRC cell lines, including SW480, SW620 and RKO, 215 were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). SW837 and 216 HCT116 were obtained from JCRB Cell Bank (Osaka, Japan) and RIKEN Cell bank (Ibaraki, Japan), 217 respectively. HCT15, SW48, LS180, and Colo205 were previously obtained and authenticated by STR 218 analysis (Promega, Madison, WI, USA). RKO and LS180 cells were maintained with DMEM; others 219 with RPMI-1640 containing 10% fetal bovine serum and penicillin/streptomycin (ThermoFisher

220 Scientific, , Waltham, MA, USA) at 37℃ in a humidified atmosphere of 5% CO2. A demethylation 221 reagent, 5-aza-2’-deoxycytidine (5-aza-dC) (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in 222 DMSO at 10 mM and stored in aliquots at −80°C until use. 223 Knockdown experiments were conducted using siRNA oligonucleotides of GALNT6 or 224 scramble control with Lipofectamine RNAiMAX Reagent, according to manufacturer’s instructions 225 (Ambion® Silencer Select; s22154, s22155 and negative control #1, ThermoFisher Scientific). 226 227 Quantitative real-time PCR 228 Total RNA was extracted using TRIzol Reagent, and one-μg of total RNA was reverse 229 transcribed to cDNA using the SuperScript III First-Strand Synthesis System (ThermoFisher Scientific) 230 according to the manufacturer’s instructions. qRT-PCR was carried out using TaqMan Gene Expression 231 Master Mix on the 7500 real time PCR system in triplicate with TaqMan assays, including GALNT6 232 (Hs00926629_m1), MLH1 (Hs00179866_m1), and ACTB (Hs99999903_m1) (ThermoFisher Scientific). 233 Relative expression levels were determined with SDS software by the 2-∆∆Ct method as described by 234 the manufacturer. 235 236 Western blotting 237 Total protein was extracted using RIPA lysis buffer supplemented with Halt Protease Inhibitor 238 Cocktail, and were boiled in Tris-Glycine SDS Sample Buffer (ThermoFisher Scientific). Equal amount 239 of protein was loaded and separated by 10% SDS-PAGE gel, and then transferred onto PVDF 240 membranes (ThermoFisher Scientific). The membrane was blocked with 5% non-fat dried skimmed milk 241 powder (Cell signaling Technology), and incubated with primary rabbit anti-GALNT6 (#HPA011762, 242 1:250, Atlas Antibodies) or mouse anti-β-actin (#SC-69879, 1:2000, Santa Cruz Biotechnology). The 243 membrane was incubated with goat anti-rabbit or anti-mouse HRP secondary antibody (Santa Cruz

244 Biotechnology), and developed with the SuperSignal West Pico chemiluminescent Substrate 245 (ThermoFisher Scientific) using LAS4000 imager (GE Healthcare). 246 247 Flow cytometry 248 Cell suspensions were incubated with mouse monoclonal anti-Tn antibody (MLS128, 1:100, 249 Wako, Osaka, Japan), followed by staining with goat anti-mouse IgG H&L (Alexa Fluor 488) (ab150113, 250 1:2000; Abcam, Cambridge, UK). The data were acquired on a FACSCanto II (Becton Dickinson, 251 Franklin Lakes, NJ, USA) and analyzed with FlowJo software (TOMY Digital Biology, Tokyo, Japan). 252 253 Cell proliferation assay, 5-FU cytotoxicity assay and detection of apoptosis 254 Cell proliferation was measured using the Cell Counting Kit-8 (CCK-8, DOJINDO, 255 Kumamoto, Japan) according to the manufacturer’s instructions. Cytotoxicity was assessed by CCK-8 256 assay using a series of 5-FU (Sigma-Aldrich) concentrations. We preliminarily applied a series of 5-FU 257 concentrations ranging from 0.1 to 1,000μg/ml or vehicle alone for generating dose-response curves. We 258 then used 1,5,10,50, and 100μg/ml of 5-FU for experiments. Apoptotic cells were detected using the 259 Annexin V-PE/7-AAD Apoptosis Detection Kit (BD Biosciences, Franklin Lakes, NJ, USA) according 260 to the manufacturer’s protocol. Annexin V-positive cells were regarded as apoptotic cells. 261 262 Wound-healing assay and transwell invasion assay 263 For wound-healing assay, cells were seeded on a 6-well plate and allowed to reach confluency. 264 After scratching the bottom of the well with a pipette tip, the monolayer of cells was washed, and the 265 wound closure photographs were captured at 0,6,12,18 and 24 hours using a phase contrast microscope. 266 The percent of wound closure was calculated as the cell migration distance to the initial wound distance. 267 Invasion assay was performed using Corning BioCoat 24-Multiwell Tumor Cell Invasion Systems 268 (Corning, NY, USA) according to the manufacturer’s protocol. Fluorescence of invaded cells labeled 269 with Calcein-AM (Corning) was measured using SkanIt RE for Varioskan Flash 2.4 (Thermo Fisher 270 Scientific). 271 272 Lectin Microarray 273 The lectin microarray was performed essentially as described elsewhere (29). Briefly, the 274 membrane fractions of cultured cells were obtained using the ProteoExtract Subcellular Proteome 275 Extraction kit (Merck Millipore, Darmstadt, Germany) and the total protein content was determined 276 using the Micro BCA Protein Assay kit (Thermo Fisher Scientific), and then Cy3-labeled proteins with

277 Cy3 monoreactive dye pack (GE Healthcare Life Science, Pittsburgh, PA, USA) were analyzed on a 278 lectin microarray glass slide (LecChip ver 1.0; GlycoTechnica, Yokohama, Japan). Fluorescent images 279 were acquired using an evanescent-field fluorescence scanner (GlycoStation Reader 1200; 280 GlycoTechnica). The raw fluorescence intensity was first processed with the gain-merging procedure, 281 followed by average normalization (29). Data were analyzed with GlycoStation Tools Pro Suite1.5 282 (GlycoTechnica). 283 284 Statistical analysis 285 Fisher’s exact test, Chi-square test, unpaired t-test and Mann-Whitney U test were used to 286 determine differences between two variables. Spearman’s correlation was used to evaluate the 287 correlations between levels of expression and methylation. Cumulative survival was estimated by the 288 Kaplan-Meier method, and differences between the two groups were analyzed by log-rank test. 289 Univariate and multivariate models were computed using Cox proportional hazards regression. All 290 statistical analyses were two-sided and were conducted using Graphpad Prism v6.0 (Graphpad Software 291 Inc., La Jolla, CA, USA) and SPSS Statistics version 24 (IBM Corporation, NY, USA). All P-values 292 were two-sided, and P-values less than 0.05 were considered statistically significant. 293 294 295 Results 296 Transcriptional glycogene profiling demonstrated subgroups of CRC with distinct survival outcomes 297 The overall study design is demonstrated in Supplementary Figure S1. We initially conducted 298 an unsupervised hierarchical clustering analysis in 177 patients from GSE17536 using the 185 299 glycogenes (Supplementary Table S2, Figure 1A and Supplementary Figure S2A), resulting in two major 300 clusters (Cluster A and B) and four subclusters (Cluster A1, A2, B1 and B2) (Supplementary Table S5). 301 Cluster A showed a clear tendency to be associated with worse clinical outcomes (Supplementary Figure 302 S3A-B). Moreover, patients segregating to Cluster A1 had significant poor DSS and DFS compared to 303 the remaining subclusters (Figure 1B-E and Supplementary Figure S3C-D). Multivariate Cox analysis 304 demonstrated that Cluster A1 was significantly associated with DSS (HR 5.71; 95%CI 2.69-12.13; 305 P=6.0E-06), DFS (HR 2.83; 95%CI 1.38-5.80; P=0.005) and OS (HR 3.71; 95%CI 2.02-6.82; 306 P=2.5E-05) (Supplementary Table S6). Cluster A1 was also associated with poorly-differentiated 307 histology (Supplementary Table S5). 308 Since Cluster A and its subcluster Cluster A1 exhibited worse survival outcome, we next sought 309 to identify a minimum set of genes whose expression was closely related to these poor prognosis

310 subgroups. Thirty-nine differentially expressed genes between Cluster A and B were further narrowed 311 down to 15 genes (GCNT3, FUT8, B3GAT2, GALNT6, POFUT1, GALNT1, B3GNT8, DPM1, 312 HS3ST3B1, SLC35A1, MGAT2, GALNT5, GYLTL1B, MGAT5 and HS3ST1, designated the 15-glycogene 313 signature) that were significantly altered between Cluster A1 and the remaining subclusters 314 (Supplementary Table S3 and S4). 315 316 Prognostic validation of the 15-glycogene signature in two independent datasets 317 To test the hypothesis that the 15-glycogene signature can discriminate prognostic subgroups, 318 independent datasets were utilized. Clustering analysis showed that 121 patients with stage I to III 319 diseases from GSE41258 were clearly separated into two clusters, designated as 15-Glycogene Cluster A 320 and 15-Glycogene Cluster B, with significant DFS difference (Figure 1F-H and Supplementary Figure 321 S2B). We next studied a homogeneous group of 89 stage II patients from GSE33113. This analysis 322 verified the prognostic subgroups, demonstrating that 15-Glycogene Cluster A patients had significant 323 shorter DFS than that of 15-Glycogene Cluster B (Figure 1I-J and Supplementary Figure S2C, 324 P=0.0080). Multivariate analysis revealed that prognostic significance of 15-Glycogene Cluster A was 325 independent of clinical features in GSE41258 (HR 4.22; 95%CI 1.50-11.84; P=0.006) and in GSE33113 326 (HR 4.03; 95%CI 1.31-12.37; P=0.015) (Supplementary Table S7). In all 3 independent clustering 327 analyses, the expression of the 15 genes were each consistently altered between clusters (Supplementary 328 Figure 3E). Specifically, we identified upregulation of GCNT3 and FUT8, and downregulation of 329 GALNT6 as common features of Cluster A1 and 15-Glycogene Cluster A. Of note, the expression of 330 GCNT3 and FUT8, but not GALNT6, have been reported to be associated with prognosis in CRC (30, 331 31). 332 333 The 15-glycogene signature identified a subgroup exhibiting unique clinicopathological and genomic 334 profiles 335 We further analyzed the association between the 15-Glycogene Clusters and known molecular 336 markers, such as MMR, RAS, BRAF and TP53 status. In GSE41258, the 15-Glycogene Cluster A was 337 significantly associated with dMMR (P=0.003) and wild-type TP53 (P=0.050) (Figure 1K and 338 Supplementary Table S8). The same clustering procedure was applied to two independent RNA-Seq 339 datasets obtained from TCGA, consisting of RNA-Seq RPKM (n=193) and RNA-Seq V2 RSEM 340 (n=361). This validated the association of the 15-Glycogene Cluster A with dMMR and wild-type TP53 341 (Figure 1F, K-L, Supplementary Figure S2B,D-E and Supplementary Table S8). Intriguingly, we found 342 that proximal location, mucinous histology, mutant RAS and mutant BRAF were statistically significantly

343 enriched in the 15-Glycogene Cluster A. 344 345 Decreased expression of GALNT6 in dMMR tumors in 12 independent cohorts of CRC patients and a 346 dataset of CRC cell lines 347 We attempted to focus on single glycogenes, including GCNT3, FUT8 and GALNT6, altered 348 expression of which might be characteristics of tumors with dMMR or genetic alterations in BRAF, RAS 349 and TP53. Also, CpG island methylator phenotype (CIMP) was included in this analysis, since 350 CIMP-positive tumors are known to be closely related to dMMR and BRAF mutation (32). Nine 351 additional datasets were assembled and we were thus able to analyze 12 independent cohorts containing 352 a total of 2472 CRC patients (Figure 2). This revealed the association between GALNT6 expression and 353 MMR status with high reproducibility, where GALNT6 was statistically significantly downregulated in 354 dMMR tumors in all cohorts, comprised of 417 dMMR and 1800 pMMR tumors. It is worth noting that 355 the association of decreased GALNT6 with dMMR was clearly reproduced by the analysis of 151 CRC 356 cell lines (Figure 2 and Supplementary Figure S4A). This tight correlation between GALNT6 357 downregulation and dMMR prompted us to focus specifically on the significance of GALNT6, which 358 encodes one of the polypeptide GalNAc transferase (ppGalNAc-T) family enzymes involved in the 359 initiation of O-glycosylation. 360 361 GALNT6 was downregulated in a subset of carcinoma upon malignant transformation 362 Downregulation of glycogenes is an important step in CRC development and progression (14, 363 18). Thus, we hypothesized that downregulated GALNT6 is involved in carcinogenesis. To this end, we 364 analyzed multiple datasets containing normal colon (n=161, in total), colon adenoma (n=264, in total) 365 and carcinoma (n=387, in total) samples. In all 7 analyses, GALNT6 mRNA expression was significantly 366 higher in adenomas than that of normal colon, while it was significantly decreased in carcinomas, 367 compared to adenomas (Figure 3A-G). To further validate this finding, IHC for GALNT6 protein was 368 conducted using our large series of colorectal adenoma (n=60) and carcinoma specimens (n=335). IHC 369 demonstrated that GALNT6 protein expression was not detected in the vast majority of normal colon 370 mucosal cells (92.8% of 304 normal tissues were GALNT6-negative). Whereas, virtually all samples of 371 adenoma and carcinoma in situ (Tis) showed strong granular cytoplasmic staining of GALNT6 in tumor 372 cells essentially throughout the tumor area (98.3% of adenoma and 100.0% of Tis) (Figure 4A-C). 373 Likewise, intense GALNT6 staining was diffusely found in carcinoma cells (Figure 4D-E). However, 374 approximately 15% of carcinomas lacked GALNT6 protein expression (Figure 4F-G and Table 1). We 375 also examined the expression patterns of GALNT6 in adenoma-to-carcinoma transition within the same

376 lesion using 8 specimens of carcinoma-in-adenoma, showing that in one of 8 lesions (12.5%) GALNT6 377 staining was lacking in the carcinoma component, but all the adenoma component exhibited 378 positive-GALNT6 (Supplementary Figure S5A-H). GALNT6 staining was frequently lost in dMMR 379 tumors (52.0% were negative), although the majority of pMMR tumors showed positive-GALNT6 380 (11.6% were negative) (Figure 3H and Table 1). Collectively, in both mRNA and protein levels, 381 GALNT6 expression was the highest in precursor and preinvasive tumors, and was subsequently 382 downregulated or lost in a subset of carcinomas which was associated with dMMR tumors. 383 It has become apparent that more than 15% of CRC is known to originate from serrated 384 precursor lesions and is often characterized by activating BRAF mutations and CIMP that greatly 385 overlaps with dMMR tumors (4, 5, 33). Since decreased GALNT6 mRNA expression was associated not 386 only with MMR status but also with CIMP and BRAF mutations (Figure 2), it was speculated that 387 GALNT6 downregulation could be associated with the serrated neoplasia pathway. Indeed, dMMR, 388 CIMP and BRAF mutations were each highly enriched in tumors with decreased levels of GALNT6 389 expression in three cohorts (Supplementary Figure S6A-C). However, we observed no difference in 390 GALNT6 expression between serrated adenomas and conventional adenomas, or between serrated 391 adenocarcinomas and conventional adenocarcinomas in 4 datasets of histologically-confirmed adenoma 392 and adenocarcinoma samples (Supplementary Figure S7A-D). 393 394 Epigenetic silencing may contribute to GALNT6 downregulation 395 Since downregulation of some glycogenes results from epigenetic silencing mainly by DNA 396 hypermethylation upon malignant transformation (14), we addressed the possibility that DNA 397 methylation contributes to decreased GALNT6 expression. We observed significant inverse correlation 398 between mRNA expression and methylation of GALNT6 (Figure 3I, P<0.0001). Higher levels of 399 GALNT6 promotor methylation were observed in CRC tissues than adenomas in two additional cohorts 400 (Figure 3J-K), which was in clear contrast to the downregulated GALNT6 in CRC tissues compared to 401 adenomas (Figure 3A-F). Moreover, in CRC tissues, GALNT6 methylation levels were significantly 402 higher in dMMR tumors than that of pMMR (Figure 3L). To further confirm the methylation of GALNT6 403 in vitro, CRC cell lines, including HCT116, SW48 and RKO, which displayed relatively lower GALNT6 404 expression levels (Supplementary Figure S4B-D), were treated with a DNA methyltransferase inhibitor, 405 5-aza-dC. Demethylation treatment restored MLH1 expression in MLH1-methylated cell lines, including 406 RKO and SW48, while GALNT6 expression was induced only in SW48 cells (Figure 3M-N). 407 408 Lack of GALNT6 protein expression was associated with poor prognosis

409 We next examined the clinicopathological and prognostic significance of GALNT6 protein 410 expression in the FFPE cohort. Tumors lacking GALNT6 protein were associated with poorer 411 histological differentiation (P<0.0001), but exhibited no association with other clinical features (Table 1). 412 Intriguingly, negative-GALNT6 patients had significantly poorer DSS and OS, compared to those with 413 positive-GALNT6 (Supplementary Figure S8A-B, P=0.0038 and P=0.022, respectively). This remained 414 statistically significant when the analysis was conducted in 195 stage II and III patients (Figure 4H and 415 Supplementary Figure S8C, P=0.0008 and P=0.014, respectively). Multivariate Cox analysis 416 demonstrated that the lack of GALNT6 protein was significantly associated with poor DSS (HR 3.39; 417 95%CI 1.28-9.02; P=0.014) and OS (HR 2.34; 95%CI 1.08-5.05; P=0.031), independent of stage and 418 other conventional factors (Supplementary Table S9-10). Stratified analyses also showed that 419 negative-GALNT6 had significant prognostic impact on DSS and OS in stage III patients (P<0.0001 and 420 P=0.0016, respectively), but not evident in stage II patients (Figure 4I-J and Supplementary Figure 421 S8D-E). Concerning DFS, the prognostic values of GALNT6 expression showed only a trend, which did 422 not reach statistical significance (Supplementary Figure S8F-I). 423 Since GALNT6 was chosen from the 15 genes for detailed evaluation primarily because of its 424 tight relationship with MMR status, patients were further divided into four subgroups based on GALNT6 425 and MMR status to explore the clinicopathological and prognostic significance in those groups. 426 Interestingly, although dMMR tumors shared similar clinicopathological features irrespective of 427 GALNT6 expression (Supplementary Table S11), striking survival differences were found between 428 GALNT6-negative/dMMR and GALNT6-positive/dMMR subgroups. In stage II-III analysis, 429 GALNT6-negative/dMMR patients had significant poorer DSS, OS and DFS compared to those of 430 GALNT6-positive/dMMR (Supplementary Figure S9A-C). In addition, GALNT6-negative/pMMR 431 patients demonstrated significantly poorer prognosis than those of GALNT6-positive/pMMR, 432 particularly in stage III analyses (Supplementary Figure S9D-F). It appears that there was no 433 clinicopathological similarity between GALNT6-negative/dMMR and GALNT6-negative/pMMR, 434 although those two subgroups were sharing poor survival outcomes (Supplementary Table S11). 435 436 Decreased GALNT6 mRNA expression was associated with poor prognosis 437 Since loss of GALNT6 protein was associated with worse survival in stage II and III patients, 438 we hypothesized that decreased GALNT6 mRNA levels may also be prognostic. We assembled 3 439 additional datasets, combining them into a microarray validation meta-cohort containing 364 patients 440 with stage II and III CRC (Figure 4K and Supplementary Figure S10). Low GALNT6 mRNA was 441 significantly associated with worse DFS in patients with stage II and III CRC (Figure 4K, P=0.0241),

442 and it was independent of clinical factors by multivariate analysis (Supplementary Table S12, HR 1.88; 443 95%CI 1.16-3.06; P=0.011). Consistent with IHC analysis, the prognostic value of GALNT6 mRNA 444 expression was clearly demonstrated in stage III patients (P=0.0036), but not in stage II (Figure 4L-M 445 and Supplementary Figure S10). 446 447 Lack of GALNT6 protein expression was associated with poor therapeutic response to 5-FU-based 448 adjuvant chemotherapy 449 It is well recognized that stage II and III patients with dMMR CRC may not benefit from 450 5-FU-based adjuvant chemotherapy (7, 34). We sought to determine if the expression of GALNT6 was 451 associated with response to adjuvant chemotherapy. Among 190 stage II and III patients in the IHC 452 cohort for which information on the administration of adjuvant chemotherapy was available, 114 patients 453 received intravenous or oral 5-FU-based adjuvant chemotherapy after surgery, while 76 patients were 454 treated by surgery alone. We conducted DFS analyses for GALNT6 expression by stratifying stage II 455 and III patients on the basis of adjuvant treatment history (Supplementary Figure S11A-F). Among 456 patients who received chemotherapy, negative-GALNT6 showed a nonsignificant trend towards worse 457 DFS (Supplementary Figure S11A). Notably, in stage III patients receiving adjuvant chemotherapy, 458 negative-GALNT6 was associated with poor therapeutic outcome (HR 5.56; 95%CI 1.57-19.69; 459 P=0.0079, Supplementary Figure S11C). This effect was not observed in stage III patients treated by 460 surgery alone (HR 0.31; 95%CI 0.04-2.68; P=0.290), although the number of patients in each group was 461 limited (Supplementary Figure S11D). There was no clear trend when stage II patients were analyzed 462 (Supplementary Figure S11E-F). 463 464 Depletion of GALNT6 enhanced invasion, migration and chemoresistance to 5-FU 465 To understand the biologic function of GALNT6, a pMMR cell line, SW480, with relatively 466 higher GALNT6 mRNA and protein expression were selected for further analyses (Supplementary 467 Figure S4B-D). We used two different siRNAs targeting GALNT6, demonstrating that GALNT6 was 468 effectively silenced, confirmed by qRT-PCR and western blotting (Figure 5A-B). Although silencing of 469 GALNT6 had no significant impact on cell proliferation (Figure 5C), it enhanced both cell migration and 470 invasion determined by wound healing assay and transwell invasion assay, respectively (Figure 5D-E 471 and Supplementary Figure S12). As we found the association between negative-GALNT6 and poor 472 response to 5-FU-based chemotherapy (Supplementary Figure S11), we tested the in vitro contribution of 473 GALNT6 expression to the sensitivity to 5-FU treatment. We found a moderate, but significant increase 474 of 5-FU resistance in GALNT6-knockdown cells as compared to cells treated with control siRNA (Figure

475 5F). Correspondingly, apoptosis was significantly suppressed in GALNT6-knockdown cells treated with 476 5-FU (Figure 5G). 477 478 Decreased GALNT6 resulted in the increase of cancer-associated truncated glycan, Tn antigen 479 Dysregulated glycogenes can result in alteration of cell surface glycosylation. Thus, we tested 480 to determine if the depletion of GALNT6 could affect the cell surface glycan profiles. Lectin microarray 481 analysis was conducted to examine the glycomic profiles of surface membranous fractions in SW480 482 cells. Compared to siRNA control, GALNT6-silenced cells demonstrated decreased lectin Jacalin and 483 ACA, each of which can bind to core 1 (Galβ1-3GalNAcα-Ser/Thr) and core 3 484 (GlcNAcβ1-3GalNAcα-Ser/Thr) extension of O-glycan, respectively (Supplementary Figure S13). 485 GALNT6 silencing also led to the increased signal intensity of lectin HPA that is highly specific to 486 GalNAcα-Ser/Thr, a truncated O-glycan structure, also known as Tn-antigen (Figure 5H and 487 Supplementary Figure S13) (35). We confirmed that GALNT6 knockdown increased the cell surface 488 expression of Tn-antigen by flow cytometry using a monoclonal antibody MLS128 (Figure 5I-J) (36). 489 Conversely, 5-aza-dc treatment in SW48 cells resulted in decreased expression of Tn antigen along with 490 the concomitant induction of GALNT6 (Figure 3N and Figure 5K-L). 491 492 Discussion 493 This study provides several lines of evidence that the expression of GALNT6 is a potential 494 biomarker for identifying a prognostic subgroup and is implicated in colorectal carcinogenesis. First, a 495 glycogene-derived transcriptional subtype, namely, the 15-Glycogene Cluster A, was identified and 496 validated using a total of 941 samples from multiple transcriptomic datasets. This novel subgroup, in 497 which GALNT6 was downregulated, was characterized by poor prognosis, poorly-differentiated 498 histology, proximal location, and dMMR. Moreover, strong association between decreased GALNT6 499 mRNA expression and dMMR was robustly confirmed in 12 patient cohorts and a dataset of cell lines, 500 followed by the analysis of a FFPE cohort at GALNT6 protein levels. Second, downregulation of 501 GALNT6 mRNA and protein seemed to occur during transition from adenoma to carcinoma, possibly 502 through epigenetic silencing, where GALNT6 was expressed in most of premalignant/preinvasive 503 lesions but was subsequently decreased in a subset of carcinomas. This suggests a crucial role of 504 GALNT6 in CRC, especially contributing to the mechanism referred to as incomplete synthesis of 505 glycans. Indeed, GALNT6 depletion not only increased the invasive and migratory potentials but also 506 upregulated the cancer-associated truncated glycan, Tn-antigen. By contrast, demethylation resulted in a 507 decrease of Tn-antigen along with GALNT6 reactivation. Third, decreased GALNT6 expression in both

508 mRNA and protein levels discriminated a poor prognostic subgroup that was largely consistent with that 509 of the 15-Glycogene Cluster A, reinforcing the notion that the glycogene-derived transcriptional subtype 510 is recapitulated by tumors lacking GALNT6 protein. 511 Our strategy integrated various gene expression platforms, including Affymetrix, Agilent and 512 Illumina microarrays and RNA-Seq, obtained from different laboratories, and even technologically 513 independent approach by IHC, consisting of a total of more than 4500 samples. Likewise, 514 downregulation and methylation of GALNT6 in carcinoma tissues, compared to adenoma tissues, was 515 clearly reproduced in multiple series of nonmalignant, premalignant and malignant lesions using 516 epigenomic and transcriptomic datasets and IHC analysis. This finally led us to identify a distinct 517 subgroup lacking GALNT6 protein expression in approximately 15% of CRC. Those integrated, 518 multistep analyses could minimize false-positive results. It is therefore unlikely that the presence of this 519 subgroup is related to false discoveries or batch effects from high-throughput data analyses. Notably, this 520 GALNT6-negative subgroup could be identified in both mRNA and protein levels, and its prognostic 521 values were statistically independent of clinical factors. Therefore, it is suggested that GALNT6 522 expression can be a robust prognostic biomarker for CRC. Since its prognostic performance was 523 particularly remarkable in stage III patients, GALNT6 expression may help guide clinical decisions, 524 including adjuvant chemotherapy and surveillance plans after curative surgery for patients with stage III 525 CRC. It is also important that IHC for GALNT6 protein is a practical assay that can be routinely 526 analyzed on readily-available FFPE specimens in clinical practice. 527 GALNT6 downregulation was originally identified to be tightly correlated with dMMR, and 528 finally we noticed that it had significant impact on prognosis. It is worth noting that GALNT6-negative 529 tumors shared poor survival outcomes even when the dMMR and pMMR tumors were analyzed 530 separately, but the prognostic impact of negative-GALNT6 seemed to be more remarkable in the 531 analysis of dMMR tumors. Therefore, we suggest that GALNT6 can be a promising prognostic 532 biomarker for both dMMR and pMMR tumors, and GALNT6 IHC combined with MMR may provide 533 more useful prognostic stratification that can discriminate an extremely poor prognostic subset 534 displaying negative-GALNT6/ dMMR, from GALNT6-positive/dMMR tumors with excellent prognosis. 535 These results warrant confirmation in large-scale prospective studies. 536 It is likely that GALNT6-negative patients receiving 5-FU-based adjuvant chemotherapy were 537 associated with poor therapeutic outcome. Despite the exploratory nature with small number of patients 538 and low number of events in each subgroup, the negative prognostic effect on DFS was evident in stage 539 III patients who received 5-FU-based adjuvant therapy, demonstrating a striking contrast to those who 540 treated by surgery alone. This was further supported by the finding that GALNT6 depleted CRC cells

541 demonstrated an increase of chemoresistance to 5-FU treatment. This implicated that negative-GALNT6 542 may also have a predictive value for poor response to 5-FU-based adjuvant chemotherapy in stage III 543 CRC. Therefore, alternative therapeutic strategies, including combination regimens or targeted drugs, 544 may be more effective and appropriate for stage III patients with negative-GALNT6 tumor. Recent 545 clinical trials revealed that stage III dMMR patients may benefit from adjuvant 5-FU treatment 546 combined with oxaliplatin (37, 38). It would be interesting to address the effect of adding oxaliplatin 547 compared to the conventional 5-FU-based therapy alone, in relation to GALNT6 status, although no 548 patients in this study were treated with oxaliplatin in the adjuvant setting. Since dMMR CRC has 549 recently been reported to be effectively treated with anti-PD-1 immune checkpoint inhibitors (39, 40), 550 detailed analysis of down-regulated GALNT6 in relation to anti-tumor immunity may help to understand 551 the dMMR-CRC biology. 552 In normal tissues, GalNAc type O-glycans are modified by glycosyltransferases to generate 553 core structures, and core O-glycans are further extended and capped by the addition of sialylated and 554 fucosylated terminal structures (13, 41). The ppGalNAc-Ts, which catalyze the transfer of GalNAc to 555 Ser/Thr residues on substrate proteins, control the initiation step of GalNAc-type O-glycosylation. The 556 ppGalNAc-Ts form a family of 20 distinct isoenzymes expressed in a cell-type specific manner, with 557 different but overlapping substrate specificity, thus O-glycans are synthesized through concerted and 558 occasionally competitive action of ppGalNAc-Ts (41, 42). GALNT6 was reported to be expressed in 559 high percentages of adenocarcinoma cells from breast, lung, pancreas and renal cancer, whereas it was 560 undetectable or very weakly found in their normal counterpart (24-26, 43). We showed that GALNT6 561 staining was undetectable in normal colonic tissue, but was invariably overexpressed in virtually all 562 tumor cells of premalignant/preinvasive lesions. Transcriptomic and epigenomic data confirmed the 563 upregulation of GALNT6 mRNA along with demethylation of GALNT6 promotor in adenoma samples, 564 compared to normal colon. This suggests a role of GALNT6 in the early stage of tumorigenesis, where 565 GALNT6 may even support adenoma formation, irrespective of conventional or serrated carcinogenesis 566 pathways. GALNT6 expression in premalignant tumors seemed to be maintained in the majority of CRC 567 as well, with the exception of lower percentages (~15%) of CRC showing GALNT6 loss. Since 568 GALNT6 silencing could promote the capacity of invasion and migration in vitro, it is likely that in a 569 subset of CRC, decreased GALNT6 mRNA and loss of GALNT6 protein contribute to transition from 570 premalignant/preinvasive lesions to invasive carcinomas. 571 Altered expression of GALNT6 has been investigated in other tumor types, suggesting their 572 potential as cancer biomarkers (13). In pancreatic cancer, Li et al reported that loss of GALNT6 573 expression was associated with poor differentiation and poor OS (25). Conversely, the same group from

574 Li et al. has recently reported that GALNT6 expression predicted poor OS in lung adenocarcinoma (24). 575 In breast cancer, GALNT6 overexpression may contribute to mammary carcinogenesis through aberrant 576 glycosylation (26). Such conflict between different cancers has also been observed in several studies 577 investigating other ppGalNAc-Ts. For instance, GALNT3 expression correlated with poor survival in 578 ovarian cancer (44) and renal cell carcinoma (43), whereas it was associated with better survival in lung 579 adenocarcinoma (45), gastric cancer (46) and CRC (47). GALNT7 was shown to be targeted by 580 microRNA-214 in cervical (48) and esophageal cancer (49); its overexpression enhanced proliferation, 581 invasion and migration. By contrast, in melanoma cells, microRNA-30b/30d promoted invasion and 582 metastasis by direct suppression of GALNT7 (50). Although there is no direct explanation for the 583 contradictory influence of ppGalNAc-Ts in different cancers, these conflicting data among different 584 cancer types may indicate the complexity of O-glycosylation along with the diversity and distinct 585 substrate specificities of ppGalNAc-Ts that can confer specific roles in specific cellular contexts. Future 586 studies would be required to address this complexity of O-glycosylation associated with deregulated 587 ppGalNAc-Ts during tumorigenesis of various malignancies. 588 Upon malignant transformation, epigenetic alterations are recognized as key characteristics 589 that can cause dysregulation of glycogenes, resulting in aberrant expression of cell surface glycans (16). 590 We found an inverse correlation between the expression and methylation of GALNT6, and demethylation 591 treatment reactivated GALNT6 expression in CRC cell lines. Also, cell surface lectin microarray analysis 592 revealed that the levels of lectin HPA-recognized GalNAcα-Ser/Thr, known as cancer-associated Tn 593 antigen, were specifically increased in GALNT6-depleted cells, confirmed by using a monoclonal 594 antibody MLS128 (35, 36). This truncated O-glycan structure, Tn, is involved in tumor progression in 595 many types of cancer, including CRC (35, 51-53). Indeed, Tn antigen is known to be a marker of poorly 596 differentiated and mucinous adenocarcinoma, and poor patient prognosis in CRC (13, 42). GALNT6 597 knockdown promoted invasion and migration, which was in agreement with the tumor phenotype with 598 Tn antigen overexpression. Conversely, cell surface Tn antigen was diminished by DNA demethylating 599 agent along with GALNT6 induction. Those findings are highly consistent with the concept of 600 incomplete synthesis that glycan elongation in nonmalignant cells are impaired upon malignant 601 transformation by silencing of glycogenes, resulting in the expression of cancer-associated truncated 602 glycans (13, 16, 18). Taken together, our results suggest that GALNT6 expression is epigenetically 603 regulated during pre-invasive neoplasm-invasive carcinoma transition in a subgroup of colorectal tumors 604 that contribute to cancer progression possibly through the incomplete synthesis mechanism. 605 We found that not only dMMR but also CIMP and BRAF mutation were each enriched in 606 tumors showing decreased levels of GALNT6 expression, and thus they were overlapping considerably

607 each other. Since CIMP-positive tumors are known to have poor prognosis and are frequently 608 accompanied by BRAF mutation, our study might raise the possibility that the prognostic impact of 609 GALNT6 can be at least in part attributed to CIMP phenotype. Although the underlying driver biology 610 of GALNT6 loss remains inconclusive, it appears that CIMP might be one of the putative mechanisms 611 that can explain the epigenetic silencing of GALNT6. Further investigation would be required for 612 understanding the pathogenesis of GALNT6-negative CRC associated with promotor methylation of 613 GALNT6 and methylator phenotype. 614 This study had several limitations, including its retrospective nature and lack of IHC validation 615 in independent cohorts. In addition, CIMP status and mutational profiles, such as RAS and BRAF, were 616 unavailable in the FFPE cohort. Therefore, those results presented here would need to be validated in the 617 future investigations, by combining GALNT6 IHC with other genetic and epigenetic biomarkers, for 618 instance, BRAF, RAS, CIMP, and PIK3CA (4, 5). Concerning the in vitro loss-of-function experiments, 619 the present study might not provide conclusive evidence that epigenetically silenced GALNT6 could 620 directly contribute to tumor progression. Thus, we suggest that functional assays using panels of cell 621 lines harboring several genetic profiles and in vivo tumorigenicity assays would be interesting future 622 directions. 623 In conclusion, we developed and validated the 15-glycogene signature that can identify a 624 genomically distinct subgroup exhibiting dMMR, decreased GALNT6 expression and poor outcomes. 625 Also, GALNT6 expression can be a novel prognostic biomarker that can be applied to FFPE specimens. 626 GALNT6 downregulation, in part due to epigenetic silencing, may contribute to the incomplete 627 O-glycan synthesis and increased expression of the cancer-associated Tn antigen, highlighting the 628 possible role of GALNT6 in colorectal carcinogenesis and poor prognosis. 629 630 Acknowledgements 631 This work was supported by JSPS KAKENHI Grant Numbers 15K10143 and 25870582. 632 Hirokazu Okayama and Masaru Noda were supported by Takeda Science Foundation. The authors thank 633 Dr. Yuuichirou Kiko for providing pathological advice. 634 635 636 Figure Legends 637 Figure 1: Identification of glycosyltransferase gene (glycogene)-derived subtypes of colorectal cancer 638 (CRC). (A) Unsupervised clustering based on 185 glycogenes in 177 patients with stage I to IV CRC 639 from GSE17536, demonstrating two major clusters, Cluster A and B, and four subclusters, Cluster A1,

640 A2, B1 and B2. (B,C,D,E) Cluster A1 was associated with poor prognosis compared to those in the 641 remaining subclusters in disease-specific survival (DSS) (B,C) and disease-free survival (DFS) (D,E) in 642 GSE17536. (F) Clustering analysis for prognostic validation using 15 glycogenes (designated as 643 15-glycogene signature) in 121 patients with stage I to III CRC from GSE41258. (G,H) Patients 644 segregating 15-Glycogene Cluster A were associated with poor DFS in GSE41258. (I) Clustering 645 analysis based on the 15-glycogene signature in 89 patients with stage II CRC from GSE33113. (J) 646 Patients segregating 15-Glycogene Cluster A had poor DFS in GSE33113. (K,L) Clustering analysis 647 using the 15-glycogene signature in two independent RNA sequence (RNA-Seq) datasets obtained from 648 TCGA, consisting of RNA-Seq RPKM (n=193) and RNA-Seq V2 RSEM (n=361). Clinical and genetic 649 features, including deficient mismatch-repair (dMMR), mutations in RAS, BRAF and TP53, tumor 650 location, disease recurrence and histology, are indicated. 185-gene (A) or 15-gene (F,I,K,L) columns are 651 shown on the right-side of the heatmaps (red represents 15 glycogenes), with arrowheads indicating 652 GALNT6 gene. 653 654 Figure 2: The association between the expression of GCNT3, FUT8 and GALNT6, and known molecular 655 markers, including deficient mismatch-repair (dMMR), mutations in BRAF, RAS, CIMP and TP53. 656 Twelve independent cohorts, comprised of 2472 patients and a dataset of 151 cell lines were 657 demonstrated. The red or blue colors in the heatmap represent glycogenes with statistically significant 658 upregulation or downregulation in tumors harboring dMMR, CIMP-positive, mutated BRAF, RAS and 659 TP53, respectively. The expression of GALNT6 was statistically significantly downregulated in dMMR 660 tumors in all 13 datasets. 661 662 Figure 3: Alteration of GALNT6 expression and methylation in colorectal carcinogenesis. (A,B,C,D,E) 663 GALNT6 mRNA levels in normal colon, colon adenoma and carcinoma samples in GSE4183 (A), 664 GSE77953 (B), GSE37364 (C), GSE20916 (D), GSE41657 (E), GSE71187 (F), and GSE41258 (G). 665 Compared to adenomas, GALNT6 mRNA was downregulated in carcinomas, particularly in those with 666 deficient mismatch-repair (dMMR). (H) GALNT6 protein expression by immunohistochemistry in 667 normal colon mucosa, colon adenoma, carcinoma in situ, and invasive carcinoma with MMR status. 668 Loss of GALNT6 protein was found in carcinomas, particularly in those with dMMR. (I) Inverse 669 correlation between GALNT6 mRNA expression and GALNT6 methylation in 357 TCGA samples. 670 (J,K,L) GALNT6 promotor methylation levels in normal colon, colon adenoma and carcinoma tissues in 671 GSE48684 (J), GSE77954 (K) and GSE68060 (L) with MMR status. (M,N) qRT-PCR analysis for 672 MLH1 (M) or GALNT6 (N) expression in cell lines with and without MLH1 methylation, treated with a

673 DNA demethylating agent, 5-aza-dc. *P<0.05, **P<0.01, ***P<0.001. 674 675 Figure 4: Representative images of immunohistochemistry for GALNT6 protein expression in colon 676 adenoma and adjacent colon mucosa (A), non-neoplastic colon mucosa (B), colon adenoma (C), and 677 colon carcinoma (D-G). Magnification: A,D,F x100; B,C,E,G x400. Intense granular staining of 678 GALNT6 is found diffusely in the cytoplasm of adenoma and carcinoma cells (C,D,E), while a subset of 679 CRC lacks GALNT6 staining (F,G). Kaplan-Meier curves for disease-specific survival or disease-free 680 survival in stage II and III patients according to the expression of GALNT6 by immunohistochemistry 681 (H-J) or microarray (K-M). Analysis of stage II and III CRC (H, K), and stratified analysis of stage II (I, 682 L), and stage III (J, M), respectively. 683 684 Figure 5: Biological characteristics of GALNT6 silencing. (A,B) Transfection of siRNAs targeting 685 GALNT6 or scramble control in SW480 cells was confirmed by qRT-PCR (A) and western blotting (B). 686 (C) Cell proliferation assay at different time points. (D) Cell invasion determined by transwell invasion 687 assay. (E) Cell migration determined by wound-healing assay. (F) Dose-dependent effect of 5-FU 688 treatment on cell viability. (G) In 10µM 5-FU treated cells, apoptosis was analyzed by flow cytometry 689 labeled with Annexin V-PE and 7-AAD. (H,I,J) Identification and confirmation of increased cell surface 690 Tn-antigen expression in GALNT6-depleted SW480 cells recognized by lectin HPA using lectin 691 microarray (H) and flow cytometry with monoclonal antibody MLS128 (I,J). (K,L) Treatment with 692 5-aza-dc reduced cell surface Tn-antigen expression in SW48 cells. Data are expressed as mean±S.D. of 693 three independent experiments (C,D,E,F,G), normalized signal intensity of triplicate measurements in 694 lectin microarray (H), or mean fluorescence intensity (MFI) from three independent flow cytometric 695 analyses (J,L) ; *P<0.05, **P<0.01. 696 697 698 References 699 1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2016. CA Cancer J Clin. 700 2016;66:7-30. 701 2. Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, et al. Cancer 702 incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. 703 Int J Cancer. 2015;136:E359-86. 704 3. Network TCGA. Comprehensive molecular characterization of human colon and rectal 705 cancer. Nature. 2012;487:330-7.

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Table 1. Clinicopathological characteristics of colorectal cancer patients according to GALNT6 expression by immunohistochemistry GALNT6 expression Total (n=335) Negative Positive P n=49 (14.6%) n=286 (85.4%) Age Mean±SD 67.0±11.867.0±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)

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.

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