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.
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Title:
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Glycosyltransferase gene expression identifies a poor prognostic colorectal cancer subtype
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associated with mismatch repair deficiency and incomplete glycan synthesis
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Authors and affiliations:
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Masaru Noda1,2, Hirokazu Okayama1, Kazunoshin Tachibana2, Wataru Sakamoto1, Katsuharu Saito1,
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Aung Kyi Thar Min1, Mai Ashizawa1, Takahiro Nakajima1, Keita Aoto1, Tomoyuki Momma1, Kyoko
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Katakura3, Shinji Ohki1, and Koji Kono1
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1
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Gastroenterology, Fukushima Medical University School of Medicine, Japan
Department of Gastrointestinal Tract Surgery,
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Departmet of Breast Surgery,
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Department of
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Running title:
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Prognostic CRC subtypes based on glycosyltransferase profile
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Keywords:
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Colorectal cancer, glycosyltransferase, cancer-associated glycans, gene expression profiling, molecular
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diagnosis and prognosis
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Additional information:
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This work was supported by JSPS KAKENHI Grant Numbers 15K10143 and 25870582. Hirokazu
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Okayama and Masaru Noda were supported by Takeda Science Foundation.
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Correspondence: Hirokazu Okayama, M. D., Ph. D.
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Department of Gastrointestinal-tract Surgery, Fukushima Medical University School of Medicine
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1 Hikarigaoka, Fukushima city, Fukushima, 960-1295, Japan.
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TEL: +81-24-547-1259
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FAX: +81-24-547-1980
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E-mail:
[email protected]
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The authors declare no conflicts of interest associated with this manuscript.
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Translational Relevance
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Here we report the identification and validation of a poor prognostic subgroup, displaying
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mismatch repair deficiency (dMMR) and decreased GALNT6 levels, based upon glycosyltransferase
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expression and methylation profiles in multiple cohorts containing a total of 4223 samples. We show that
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downregulation
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precancerous/preinvasive neoplasia to invasive carcinoma in a certain subset of tumors that frequently
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exhibit dMMR. Those transcriptional analyses were robustly recapitulated by immunohistochemistry on
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403 specimens, where tumors lacking GALNT6 protein was associated with dMMR and poor patient
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outcomes. Strikingly, loss of GALNT6 protein expression and decreased GALNT6 mRNA expression
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each discriminated postoperative stage III patients with poor survival. Our study highlights the
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possibility of GALNT6 as a novel prognostic biomarker for CRC and suggests its contribution to
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colorectal carcinogenesis through incomplete glycan synthesis.
of
GALNT6
via
epigenetic
silencing
occurs
during
transition
from
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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.
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Abstract
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Purpose: We aimed to discover glycosyltransferase gene (glycogene)-derived molecular subtypes of
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colorectal cancer (CRC) associated with patient outcomes.
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Experimental Design: Transcriptomic and epigenomic datasets of non-tumor, pre-cancerous, cancerous
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tissues and cell lines with somatic mutations, mismatch repair status, clinicopathological and survival
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information, were assembled (n=4223) and glycogene profiles were analyzed. Immunohistochemistry
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for a glycogene, GALNT6, was conducted in adenoma and carcinoma specimens (n=403). The
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functional role and cell surface glycan profiles were further investigated by in vitro loss-of-function
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assays and lectin microarray analysis.
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Results: We initially developed and validated a 15-glycogene signature that can identify a
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poor-prognostic subtype, which closely related to deficient mismatch repair (dMMR) and GALNT6
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downregulation. The association of decreased GALNT6 with dMMR was confirmed in multiple datasets
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of tumors and cell lines, and was further recapitulated by immunohistochemistry, where approximately
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15% tumors exhibited loss of GALNT6 protein. GALNT6 mRNA and protein was expressed in
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premalignant/preinvasive lesions but was subsequently downregulated in a subset of carcinomas,
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possibly through epigenetic silencing. Decreased GALNT6 was independently associated with poor
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prognosis in the immunohistochemistry cohort and an additional microarray meta-cohort, by
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multivariate analyses, and its discriminative power of survival was particularly remarkable in stage III
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patients. GALNT6 silencing in SW480 cells promoted invasion, migration, chemoresistance and
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increased cell surface expression of a cancer-associated truncated O-glycan, Tn-antigen.
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Conclusions: The 15-glycogene signature and the expression levels of GALNT6 mRNA and protein
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each serve as a novel prognostic biomarker, highlighting the role of dysregulated glycogenes in
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cancer-associated glycan synthesis and poor prognosis.
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Introduction
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Despite major advances in diagnosis and treatment, colorectal cancer (CRC) remains one of
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the leading causes of cancer-death worldwide (1, 2). CRC is commonly grouped into two categories:
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tumors with microsatellite instability (MSI), caused by defective function of the DNA mismatch repair
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(MMR) system, and tumors that are microsatellite stable but exhibiting chromosomal instability (CIN)
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(3-5). The majority of CRC (~85%) follows the CIN pathway, often accompanied by KRAS mutations
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and TP53 inactivation. Approximately 15% of CRCs that exhibit deficient MMR (dMMR) frequently
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carry BRAF mutations (3, 5). Clinical trials implicated MMR status as a potential therapeutic classifier
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for stage II patients in the adjuvant setting (6-8). In the metastatic setting, KRAS and BRAF mutations are
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used for predicting unresponsiveness to EGFR-targeted therapies (4). Despite those increasing
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knowledge, clinicopathological staging system remains the only prognostic classification currently used
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in clinical practice. However, clinicopathologically similar tumors can strikingly differ in clinical
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behaviors that likely reflect molecular heterogeneity. Although it is recommended that stage III patients
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receive postoperative chemotherapy, approximately 30-40% of patients develop recurrence even after
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standard treatment (9-12).
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Glycosylation is a common post-translational modification that involves sequential addition of
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single sugar residues to target structures, resulting in glycan elongation. Further chemical modifications
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and branching can finally form a vast array of glycan structures (13). Those procedures are regulated by
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the multienzymatic reaction of glycosyltransferases, whose encoding genes, namely “glycogenes”, are
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equivalent to 1% of human genome. Cell surface glycans undergo changes during malignant
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transformation and tumor progression accompanied by distinct biological functions and unique tumor
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phenotypes, thereby making glycans as potential cancer biomarkers (13, 14). For instance, a
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cancer-associated glycan epitope, CA19-9, called sialyl Lewis A (sLea), is routinely utilized as a serum
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tumor marker (15). CA19-9 and several other cancer-associated glycans, including sialyl Lewis X (sLex),
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sialyl Tn, Tn and T antigens, are associated with tumorigenesis and poor prognosis of CRC (13, 16).
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Such glycans can be attributed to transcriptional dysregulation of glycosyltransferases that has been
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postulated as two principal mechanisms, “incomplete synthesis” and “neo-synthesis” (13, 16-18). Some
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glycogenes are repressed by epigenetic silencing during early stages of tumorigenesis, which lead to the
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biosynthesis of truncated structures, such as Tn and STn expression, called incomplete synthesis.
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Conversely, in the neo-synthesis process, transcriptionally induced glycogenes can result in the de novo
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expression of cancer-antigens, such as sLea and sLex.
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In the present study, with the aim to discover distinct classes of CRC on the basis of the
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expression of glycosyltransferases, we compiled an extensive number of transcriptomic profiles obtained
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from multiple cohorts by integrating other available data sources, including mutations, MMR status,
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methylation, protein expression as well as non-tumor, pre-cancerous, preinvasive and cancerous samples.
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We initially described a novel subtype based upon clustering analysis of genome-wide “glycogene”
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expression patterns, and this led us to identify a glycogene, GALNT6 as a promising biomarker for
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disease prognosis. Moreover, we found the functional characteristics of GALNT6 involved in tumor
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progression and glycosylation, suggesting the contribution of epigenetic silencing of GALNT6 to
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colorectal carcinogenesis, through the incomplete synthesis of cell surface glycans.
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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.
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Materials and Methods
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Microarray data analysis, hierarchical clustering and assembly of the TCGA dataset
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All microarray and methylation array data are publicly available in the Gene Expression
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Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo) as shown in Supplementary Table S1. We
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utilized the normalized expression values obtained from each dataset. If a gene is represented by
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multiple probes, they were averaged. To generate a list of glycogenes, official gene symbols and Entrez
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Gene
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http://acgg.asia/ggdb2/).
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Affymetrix_3PRIME_IVT_ID
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(http://david.abcc.ncifcrf.gov/home.jsp) as shown in Supplementary Table S2.
IDs
for
190
glycogenes Among
were
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obtained
glycogenes,
using
from
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DAVID
GGDB
unique
(GlycoGene
genes
were
Bioinfomatics
DataBase;
converted
to
Resources6.7
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Hierarchical clustering was initially performed using an Affymetrix dataset, GSE17536,
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consisted of 177 CRC patients with survival information. Expression levels of 185 glycogenes were
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median-centered, and then genes and samples were subjected to an unsupervised clustering by the
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centroid linkage method using the Cluster3.0 and the Java Treeview program (19). Among 39
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differentially expressed genes between two major clusters (Cluster A vs B, p
