O-glycans premature truncation affects cellular dynamics and alters gastric cancer cells morphology. Gastric cancer cell lines were genetically manipulated targeting COSMC gene. a) Mutations on COSMC gene lead to an interruption on the elongation of O-glycans and an increased expression of the precursor structure Tn and its sialylated glycoform, STn. b-e) Actin (red) and STn (green) (top) or actin (red) and tubulin (green) (bottom) immunolocalization were assessed in MKN45 and AGS SimpleCell lines (MKN45 SC and AGS SC) and wild-type controls (MKN45 WT and AGS WT). STn labeling was exclusively observed in MKN45 SC and AGS SC. STn induced expression by COSMC knock-out led to marked changes in the gastric cancer cell morphology, independently of the ECM component used to grow the cells, polymer surface (b), collagen IV (c), fibronectin (d) and poly-d-lysine (e). Both glycoengineered MKN45 SC and AGS SC exhibited a more elongated cell shape, displaying more cytoskeletal actin and tubulin projections, which also stained positive for STn antigen. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Premature truncation of O-glycans promotes an increased aggressiveness phenotypic behavior. a) Proliferation assay evaluated by BrdU labeling showed no differences in cell division rates among the glycoengineered cell lines and the wild-type controls. Results are shown as average ± SEM. Student t-test was used for statistical analysis. b) Adhesion assay revealed that MKN45 SC displayed significantly higher surface adhesion capacity than MKN45 WT, whereas AGS SC showed significant less surface adhesion when compared with the AGS WT. Results are shown as average ± SEM. Student t-test was used for statistical analysis. c) Wound-healing assay demonstrated that MKN45 SC presented significantly higher migration capacity than the MKN45 WT cell line, being able to close the wound in 58 h. d) MKN45 SC revealed an increased Fak activation when compared to MKN45 WT cells. Results are shown as average ± SEM. Student t-test was used for statistical analysis. e) Wound-healing assay showed that AGS SC and AGS WT cell lines did not reached a statistical significant difference in migration capacity, although a trend of AGS SC towards higher migration rates was observed leading to wound closer after 12 h. Results are shown as average ± SEM. Two-way ANOVA was used as statistical test. f) No differences in Fak activation were detected between AGS SC and AGS WT cells. Results are shown as average ± SEM. Student t-test was used for statistical analysis. g) Matrigel invasion assay revealed the enhanced capacity of the MKN45 SimpleCells to degrade and invade the ECM in comparison with the respective wild-type cell line. Results are shown as average ± SEM. Student t-test was used for statistical analysis. h) Matrigel invasion assay revealed that AGS SC have an enhanced capacity to degrade and invade the ECM than the respective wild-type cell line. Results are shown as average ± SEM. Student t-test was used for statistical analysis.

Transcriptomic expression profile of MKN45 SC and AGS SC discloses an enrichment in genes associated with development and motility. a) Venn diagram of upregulated (upper panel) and downregulated (lower panel) genes between MKN45 SC and MKN45 WT (in blue) and between AGS SC and AGS WT (in grey). The results are represented as gene count (and percentage) with the intersecting values indicating the common genes between MKN45 and AGS cell lines. Premature truncation of O-glycans lead to transcriptional changes in about 60% and 30% of both up- and down-regulated genes in MKN45 SC and in AGS SC model, respectively. Only about 3% of the genes were either commonly up- or down-regulated in both cell models. b) Functional enrichment analysis of GO Biological Processes for the set of differentially expressed genes between MKN45 SC and MKN45 WT (in blue) and between AGS SC and AGS WT (in grey). The results are represented as –log p-value (left axis) and gene frequency (right axis) per functional category with categories clustered based on term similarity. The genes differentially expressed were annotated to significantly enriched GO terms related to development, motility and organism processes. The statistical over-representation was calculated using a binomial test (release 2016.07.15) with Homo sapiens gene set as background reference and the results were considered significant at p < 0.05, after Bonferroni correction. c) Heatmap of the common differentially expressed genes between MKN45 and AGS cell lines. The results are represented as fold change ranked as percentile, indicated by the graded colour scale. SRPX2 and RUNX1 genes were the most upregulated genes in the two cell line models. Two biological replicates per cell line were used for transcriptomic analysis. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Glycoengineering of gastric cancer cells activates receptor tyrosine kinases that culminate in up-regulation of SRPX2 and RUNX1 genes. a) Induced O-glycosylation truncation on MKN45 cells led to both EGFR and ErbB2 RTKs phosphorylation. Results are shown as average ± SEM. Student t-test was used for the expression statistical analysis. b) Treating MKN45 WT cells with EGF ligand induced EGFR activation after 10 min. c) Activation of EGFR through EGF treatment for 4 h in MKN45 WT cells caused up-regulation of RUNX1 (not reaching statistical significance) and a significant up-regulation of SRPX2 gene. No effect was noted on the expression level of these genes when MKN45 SC were treated with EGF ligand in the same conditions as MKN45 WT cells. Results are shown as average ± SEM. Two-way ANOVA with Bonferroni correction was used for statistical analysis.

Premature truncation of O-glycans is associated in vivo with poor-survival. Two groups of mice were injected subcutaneously between the scapula with 1 × 10⁶ viable cells of MKN45 SC or MKN45 WT gastric cancer cell models. Five animals were injected with MKN45 WT and four animals were injected with MKN45 SC cells. a) During the experiment, MKN45 SC-injected mice lost a higher percentage of body weight than MKN45 WT-injected mice with a significant body weight loss 21 days after cells injection (red box). Mice body weight was recovered after tumor removal (red box) by surgery. Percentage of body weight loss was calculated taking into consideration the initial body weight of each mice. Results are shown as average ± SEM. Student t-test was used for body weight loss statistical analysis 21 days after cells injection. b) Analysis of tumorigeneses showed that the tumor formation between the two groups was not significantly different in size during the experience time course. Results are shown as average ± SEM. Two-way ANOVA was used as statistical test. c) Mice images were taken at day 21 (day of tumor removal by surgery). d) Mice tumor xenografts images after being removed by surgery, 21 days after cell injection. e) Measurement of tumor size after mice surgery confirmed no differences in tumor volume. Results are shown as average ± SEM. Student t-test was used for statistical analysis. f) Xenografts from MKN45 SC-injected mice clearly presented poorly cohesive cells (HE), showed overexpression of STn and a mislocalized E-cadherin when compared with xenografts from MKN45 WT-injected mice, together with higher expression of SRPX2 and RUNX1 proteins. Pictures were taken under 400× magnification. g) Tumor xenografts from mice injected with MKN45 SC cells showed significant less necrotic areas when compared with MKN45 WT tumors. Results are shown as average ± SEM. Student t-test was used for statistical analysis. h) Xenografts from MKN45 SC-injected tumors revealed fewer vessels, based on CD31 immunolabeling, when compared with MKN45 WT tumors. Results are shown as average ± SEM. Student t-test was used for statistical analysis. i) Survival curve analysis revealed that mice injected with MKN45 SC had a significant poor-survival rate when compared with mice injected with MKN45 WT. To be noticed that one of the MKN45 SC-injected mice died 18 days after cell injection. Kaplan-Meier method by means of Gehan-Breslow-Wilcoxon Test was used for survival curves statistical analysis. j) In vivo neoangiogenesis of MKN45 WT, MKN45 SC, AGS WT and AGS SC cells, was assessed by the CAM assay. Both MKN45 SC and AGS SC displayed significantly less vessel formation than MKN45 WT and AGS WT, respectively. Results are shown as average ± SEM. Student t-test was used for statistical analysis. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

O-glycans premature truncation revealed gastric cancer patients' survival biomarkers. a) Analysis of microarray expression data for SRPX2 and RUNX1 levels from the Oncomine database []. Expression values are represented as Log2 median-centered intensity. SRPX2 and RUNX1 expression levels were significantly higher in gastric cancer when compared with adjacent gastric mucosa. Student t-test was used for the expression statistical analysis. b) Microarray expression data for SRPX2 and RUNX1 levels from the Wang dataset [] extracted from the Oncomine database. Normalized expression values are shown for adjacent mucosa and tumor of the same patients. c) Correlation analysis based on Pearson statistical test demonstrated the highly significant correlation between SRPX2 and RUNX1 expression levels in gastric cancer. d) Correlation analysis based on Pearson statistical test demonstrated an inverse correlation between SRPX2 and C1GALT1C1 and between RUNX1 and C1GALT1C1 expression levels in gastric cancer. e) Immunohistochemistry of STn, SRPX2 and RUNX1 markers on adjacent mucosa, intestinal- and diffuse-type gastric carcinoma. Pictures were taken under 400× magnification. f) Overall survival analysis of gastric cancer patients based upon SRPX2 or RUNX1 expression levels. Analyses were performed on all cases of which gastric cancer patients' survival and gene expression data were available on Oncomine, namely 81 and 75 cases for SRPX2 and RUNX1, respectively []. Cohort was divided into high- (black line) and low- (green line) expressing subgroups. A log-rank test was used to evaluate differences between groups. Increased expression of both SRPX2 and RUNX1 genes significantly correlated with poor-prognosis in gastric cancer. Kaplan-Meier method was used for survival curves statistical analysis. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Truncation of O-glycans alters cancer cell biology promoting a poor-survival phenotype. During cancer progression micro-environmental changes, epigenetic and/or genetic mutations promote alterations in glycosyltransferases activity that culminate in several modifications in the cancer cell glycosylation profile. These glycophenotypic changes, namely premature truncation of O-glycans and concomitant STn overexpression occurring in gastric tumors, affected tumor cell morphology and ECM interactions leading to increased invasion. In our model, O-glycans truncation of gastric cancer cells promoted a switch in the cancer cell transcriptomic signature as well as activation of receptor tyrosine kinase (RTKs). We showed that EGFR and HER2 RTKs were activated after COSMC KO of gastric cancer cells, leading to up-regulation of SRPX2 and RUNX1 genes. These genes were further validated as highly expressed in gastric cancer patients, as co-expressed with STn and correlated with patients' poor-survival.