3.1 Six transcripts match among the 100 most correlated with OS and DFS in GC

Using the GEPIA tool, we identified the top-100 transcripts most significantly associated with overall survival (OS) and disease-free survival (DFS) in the GC patients from the TCGA (n = 408) (Supplementary file 1). Among them, 6 were associated with both OS and DFS (Fig. 1A). These include the coding genes ANKRD6 (Ankyrin Repeat Domain 6), ITIH3 (inter-alpha-trypsin inhibitor heavy chain 3), SORCS3 (sortilin related VPS10 domain containing receptor 3), NPY1R (neuropeptide Y receptor Y1), CCDC178 (coiled-coil domain containing 178), and the long non-coding RNA AC002480.3. In particular, we focused on the 5 genes and we confirmed that their high individual expression was associated with reduced OS and DFS in the TCGA (Fig. 1B), results that were extended to the ACRG cohort (n = 299) (Fig. 1C). To further characterize their impact on survival, we performed a multivariate Cox proportional hazards regression model, adjusting for relevant factors including age and TNM tumor stage. This analysis revealed that the expression of the 5 genes represented an increased risk for OS in the TCGA patients (Fig. 1D), and these results were confirmed for ANKRD6, ITIH3, SORCS3 and NPY1R in the ACRG cohort (Fig. S1A). In relation to DFS, ANKRD6, ITIH3, NPY1R and CCDC178 represented also an increased risk for DFS in TCGA (Fig. S1B), and ANKRD6, ITIH3 and NPY1R in ACRG (Fig. S1C).

Fig. 1

Prognostic potential of ANKRD6, ITIH3, SORCS3, NPY1R and CCDC178 in GC. A ANKRD6, ITIH3, SORCS3, NPY1R, CCDC178 and the lncRNA AC002480 are the common transcripts among the top-100 transcripts most significantly associated with OS and DFS in the GC TCGA cohort (STAD dataset analyzed in GEPIA). Kaplan-Meier plots of OS and DFS according to the expression of the 5 cited coding genes in the TCGA (B) and ACRG (C) cohorts using the best cut-off method for expression level stratification. D Forest plots showing multivariate Cox regression analysis for ANKRD6, ITIH3, SORCS3, NPY1R and CCDC178 expression in association with OS in the TCGA cohort, adjusting for age and TNM stage. E Expression of the identified genes in normal gastric tissue versus the corresponding paired GC tissue in ACRG patients stratified into high and low expression subgroups according to the cut-offs used in the OS analysis from C (N = 100). Kaplan-Meier plots of OS (F) and DFS (G) according to the score of the 5-gene GSVA signature composed by ANKRD6, ITIH3, SORCS3, NPY1R and CCDC178 in the TCGA (left) and ACRG (right) cohorts using the best cut-off method for GSVA score stratification. H Forest plot showing multivariate Cox regression analysis for the 5-gene GSVA score in association with OS in the TCGA cohort, adjusting for age and TNM stage (I). 5-gene GSVA score in normal gastric tissue versus the corresponding paired GC tissue in ACRG patients stratified into high and low score subgroups defined in the OS analysis from F (right) (N = 100)

We next compared their expression in tumor and normal gastric samples. For this, we distributed the cases between patients with high or low tumor expression of the genes according to the cut-offs used for survival. For the 5 genes, the expression in normal tissue was similar between the groups of low or high survival, but the trend observed in the tumor samples was different between these groups. Noteworthy, overexpression of ANKRD6, ITIH3 and SORCS3 was detected in tumor compared with control tissue in the patients presenting poor survival (Fig. 1E). On the contrary, cases with extended survival displayed lower levels of the 5 genes in the tumor compared to healthy tissue (Fig. 1E).

We wondered whether the identified genes could represent a malignant signature associated to cancer and studied their prognostic potential in additional cancer types. For this, we took advantage of publicly available datasets of TCGA studies, and analyzed 32 cancer types. This analysis revealed interesting results, as some of the genes were associated with poor prognosis in several cancer types (Fig. S2). For instance, the expression of ANKRD6 and ITIH3 was associated with reduced OS in colon carcinoma (COAD) and kidney renal papillary cell carcinoma (KIRP), and the expression of CCDC178 in bladder urothelial carcinoma (BLCA) and thyroid carcinoma (THCA).

Next, we performed a Gene Set Variation Analysis (GSVA) to obtain a global score corresponding to the combined expression of the 5 genes in GC. We explored the potential of the score to predict prognosis in both TCGA and ACRG cohorts, and using Kaplan-Meier estimator, we identified that the high score of the signature was associated to reduced OS (Fig. 1F) and DFS (Fig. 1G). Besides, multivariate Cox regression analyses showed that the score of the signature constituted an independent prognostic factor for OS after adjusting for the risk factors age and tumor stage in both cohorts. Notably, the Hazard Ratios (HRs) of the signature were superior to those of the 5 genes individually (Figs. 1H and S1D). Similar results were obtained with DFS (Fig. S1E, F). Moreover, when we analyzed paired tumor versus gastric healthy tissue samples, the signature score was significantly higher in those subsets of patients presenting poor outcome (Fig. 1I). Concerning the other cancer types, there was not a general pattern linking the expression of the 5 genes to survival in additional cancer types (Fig. S2). These results reveal that the 5 identified genes individually or as a signature serve as age- and tumor stage- independent prognostic factors in GC.

3.2 High expression of the identified genes correlated with tumor recurrence and metastasis in GC

Next, we analyzed the expression of the identified genes in the different TNM stages of the disease. Overall, the expression of the 5 genes individually increased gradually according to the stage, being the differences in expression among stages significant for ANKRD6, ITIH3 and NPY1R (Figs. 2A and S3A). Similarly, the signature also displayed significant differences (Figs. 2B and S3B). We also studied the expression of the genes in relation to recurrence and found that the expression of all of them individually or as a signature was significantly higher in the biopsies of those patients that underwent recurrence (Fig. 2C, D). Then, we explored their expression in the different molecular subtypes of GC proposed by the TCGA and the ACRG. This study unveiled that for all the genes, the MSI subtype presented the lowest expression in both cohorts (Fig. 2E). Of note, this subtype displayed the best prognosis in the ACRG classification, further indicating the link of the 5 genes with GC malignancy. Consistent with this idea, the highest expression of the 5 genes in the ACRG was registered in the MSS/EMT subtype, that with the poorest outcome and more prone to recur [10]. In the TCGA, the highest expression was detected in the genomically stable (GS) subtype (Fig. 2E), the one which is classified principally as MSS/EMT when the ACRG criteria are applied to the TCGA patients [10]. These results were reproduced by the 5-gene signature (Fig. 2F) and suggest a link with the process of EMT.

Fig. 2

Association of ANKRD6, ITIH3, SORCS3, NPY1R and CCDC178 expression with clinic-pathological characteristics in TCGA and ACRG GC cohorts. Expression of the 5 genes individually (A) and as a GSVA signature (B) in the different tumor stages in the ACRG cohort (NI = 30, NII = 96, NIII = 95, NIV = 77). Expression of the 5 genes individually (C) and as a GSVA signature (D) in primary tumor tissue of patients who recurred (Yes) or not (No) after the primary therapy in the TCGA and ACRG cohorts (TCGA: NNo = 216, NYes = 58; ACRG: NNo = 157, NYes = 125). Expression of the 5 genes individually (E) and as a GSVA signature (F) in the different molecular subtypes of GC identified by the TCGA (top) and the ACRG (bottom). TCGA classification: MSI: GC with microsatellite instability (N = 55); EBV: Epstein-Barr virus-positive GC (N = 26) CIN: GC with chromosomal instability (N = 195), and GS: genomically stable GC (N = 42). ACRG classification: MSI: GC with microsatellite instability (N = 68); MSS/TP53+: microsatellite-stable with active TP53 (N = 79), MSS/TP53-: microsatellite-stable with inactive TP53 (N = 107); and EMT: microsatellite stable with epithelial-to-mesenchymal transition phenotype (N = 46). Expression of the 5 genes individually (G) and as a GSVA signature (H) in primary tumor tissue of patients presenting nodal dissemination (Node invasion) or not (N0) in ACRG. Node invasion group includes samples from N1 to N4 (NN0 = 38, NNode invasion = 262). I Spearman correlation between ANKRD6, ITIH3, SORCS3, NPY1R and CCDC178 in TCGA (left) and ACRG (right). Genes are grouped by hierarchical clustering. Red colour represents positive correlations (Colour figure online)

To characterize a potential interaction with metastasis, we investigated their link with lymph node dissemination and ascertained that the expression of the identified genes was higher in the patients of the ACRG exhibiting lymph node affectation, being the difference significant for ANKRD6 and ITIH3 (Fig. 2G). In the TCGA cohort, the differences, although less remarkable, confirmed the higher expression of ANKRD6 when the nodes are invaded (Fig. S3C). For the 5-gene signature we found a significantly higher expression in samples from patients exhibiting positive nodes in the ACRG cohort (Figs. 2H and S3D).

To further explore the association between the identified genes, we analyzed the Spearman correlation between the 5 genes in the TCGA and ACRG cohorts, finding a positive correlation for all the pairs of genes in both of them (Fig. 2I). Moreover, the highest correlations were observed between ANKRD6 with ITIH3 and NPY1R (Fig. 2I). Overall, these results reveal an association of the identified genes with metastasis, and postulate ANKRD6 and ITIH3 as the strongest genes within the signature.

3.3 Integrated analysis with data from 2000 GC patients across 9 independent cohorts confirms association with metastasis and recurrence

Next, with the aim of extending the study of the 5 genes in GC metastasis, we developed an analysis pipeline and we jointly analyzed available datasets corresponding to a total of 1908 GC patients from 9 independent cohorts (Table 1). We assessed the expression of the 5 genes in relation to metastasis, detecting significantly higher expression of ANKRD6 and ITIH3 in the samples belonging to patients suffering from metastatic disease (Fig. 3A). Furthermore, higher expression of ANKRD6, ITIH3 and NPY1R was also detected in the samples of the patients exhibiting recurrent disease (Fig. 3B). Since recurrence and therapy resistance are related processes, we explored the association of the genes with the survival specifically in treated patients, revealing reduced OS in treated patients with high ANKRD6, ITIH3 and NPY1R expression (Fig. 3C), and being the expression of ANKRD6 and NPY1R independent prognostic factors in treated patients (Fig. S4). We also studied in this large set of patients the association with survival and tumor stage to confirm the data obtained in the TCGA and ACRG cohorts. Notably, high levels of ANKRD6, ITIH3 and NPY1R significantly correlated with reduced OS, whilst high expression of ANKRD6, ITIH3, SORCS3 and NPY1R was associated with lower DFS (Fig. 3D). Moreover, the multivariate Cox regression analysis showed that ANKRD6, ITIH3 and NPY1R constituted independent prognostic factors for OS (Fig. S5) and DFS (Fig. S6). In relation to expression, we observed a progressive and significant increase of ANKRD6 and ITIH3 across the tumor stages (Fig. 3E). These results highlight the usefulness of the pipeline, validate the results obtained in TCGA and ACRG, and denote the clinical impact of the identified genes, with special emphasis for ANKRD6 and ITIH3 in GC malignancy, recurrence and metastasis.

Table 1 Cohorts characteristicsFig. 3

Association of ANKRD6, ITIH3, SORCS3, NPY1R and CCDC178 expression with metastasis, recurrence and survival on a total of 1908 GC patients from 9 publicly available GC data sets. A Expression of the 5 genes in primary tumor tissue of GC patients presenting distant metastasis (M) or not (M0) (NM0 = 513, NM1 = 45). B Expression of the 5 genes individually in primary tumor tissue of patients who recurred (Yes) or not (No) after the primary therapy (NNo = 573, NYes = 423). C Kaplan-Meier plots of OS according to the expression of the 5 genes in patients treated with adjuvant chemotherapy (N = 1036). The best cut-off method was used for expression level stratification. D Kaplan-Meier plots of OS (top) and DFS (bottom) according to the expression of the 5 genes. The best cut-off method was used for expression level stratification. E Expression of the 5 genes in the different tumor stages (NI = 217, NII = 495, NIII = 879, NIV = 316)

3.4 ANKRD6 and ITIH3 in GC progression, metastasis and EMT

Given the higher clinical impact of ANKRD6 and ITIH3, we explored the score of their combined expression and compared its potential with respect to the 5 genes individually and the 5-gene signature. First, we detected that a high score of this 2-gene signature in ACRG and TCGA cohorts predicted worse prognosis (Figs. 4A and S7A), and was an age- and stage- independent prognostic factor (Figs. 4B and S7B), in the former cohort with HRs higher than those obtained with the genes individually. The 2-gene score was significantly higher in tumor versus paired normal gastric tissue in patients presenting poor outcome (Fig. 4C), and was also higher in advanced disease stages (Figs. 4D and S7C) and in patients with recurrence (Figs. 4E and S7D). Moreover, the score was higher in the MSS/EMT and GS molecular subtypes (Figs. 4F and S7E), in samples from patients presenting node invasion (Figs. 4G and S7F), and in metastatic cases in TCGA and ACRG (Figs. 4H and S7G). In the metastatic patients, the score of the 5-gene signature was not significantly higher neither in the TCGA cohort nor in the ACRG cohort (Fig. S8A), reinforcing the relevance of ANKRD6 and ITIH3 in metastasis. Moreover, using the Cancer Cell Line Encyclopedia (CCLE), we determined that among our identified genes, in GC cells, ANKRD6 showed the highest levels on average, with the metastatic cell lines displaying higher expression than those cells derived from primary cases (Fig. S8B, S8C).

Fig. 4

Association of the GSVA score of ANKRD6 and ITIH3 with prognosis and clinic-pathological characteristics, and correlation of the 5 genes with hallmark gene sets in the ACRG cohort. A Kaplan-Meier plots of OS and DFS according to the ANKRD6 and ITIH3 GSVA score in the ACRG cohort using the best cut-off method for GSVA score stratification. B Forest plots showing multivariate Cox regression analysis for the ANKRD6 and ITIH3 GSVA score in association with OS (left) and DFS (right), adjusting for age and TNM stage, in the and ACRG cohort. C ANKRD6 and ITIH3 GSVA score in normal gastric tissue versus the corresponding paired GC tissue in ACRG patients stratified into high and low score subgroups according to the cut-off defined in (A). ANKRD6 and ITIH3 GSVA score according to tumor stage (D), recurrence (E), molecular subtype (F), N stage (G), and M stage (H). I Correlation of ANKRD6, ITIH3, SORCS3, NPY1R and CCDC178 with the GSVA scores of the 50 hallmark gene sets defining biological states or processes from The Molecular Signatures Database (MSigDB) in the ACRG cohort. Genes and hallmarks are grouped by hierarchical clustering. Blue colour represents negative correlations and red colour positive correlations. Spearman’s rho correlation coefficient is indicated in significant correlations. Correlation of ANKRD6, ITIH3, SORCS3, NPY1R and CCDC178 with epithelial and mesenchymal markers, and EMT inductors (J), and with CSC markers (K) in the ACRG cohort. In (J), markers are clustered within each marker category, and in (K), genes in columns and rows are grouped by hierarchical clustering. Blue colour represents negative correlations and red colour positive correlations. Spearman’s rho correlation coefficient is indicated in significant correlations (Colour figure online)

Then, with the aim of exploring the biological significance of the identified genes in GC, we studied their relationship with described pathways. For this, we took advantage of the GSVA signature score of the hallmark gene sets included in MsigDb (https://www.gsea-msigdb.org/gsea/msigdb/). The Spearman correlation analysis showed that the 5 genes presented positive correlation with the hallmarks of Epithelial mesenchymal transition (EMT), KRAS, Hedgehog signaling, or myogenesis in both TCGA and ACRG cohorts (Figs. 4I and S7H). In contrast, there was negative correlation with G2M checkpoint or DNA repair hallmarks (Figs. 4I and S7H). These analyses also showed differences in the pattern of correlations of the genes, differentiating ANKRD6 and ITIH3 in one side, from SORCS3, NPY1R and CCDC178. Thus, TGF beta, STAT3 and TNFA signaling and apoptosis were pathways specifically correlated with ANKRD6 and ITIH3 (Figs. 4I and S7H). Moreover, ANKRD6 and ITIH3 were the genes most positively correlated with the hallmark of EMT in both cohorts, with Spearman coefficients of 0.46 and 0.35 for ANKRD6 in ACRG and TCGA; and coefficients of 0.44 and 0.49 for ITIH3 in the corresponding cohorts (p-value < 0.001 in all cases) (Figs. 4I and S7H).

To further characterize the connection of these genes with the process of EMT, we explored the correlations established between them and canonical epithelial and mesenchymal markers, whose expression decrease and increase in cells undergoing EMT respectively, as well as with EMT inducers. This study revealed negative correlations with epithelial genes, such as the epithelial cell adhesion protein E-Cadherin (CDH1), the cytoskeletal genes coding the keratins 8, 18 and 19 (KRT8, KRT18 and KRT19) and desmoglein 3 (DSG3) in both ACRG and TCGA cohorts (Figs. 4J and S7I). On the contrary, positive correlations with mesenchymal markers such as N-Cadherin (CDH2), the cytoskeletal protein Vimentin (VIM), the matrix metallopeptidase 2 (MMP2), proteins belonging to the extracellular matrix (ECM) such as Fibronectin 1 (FN1) and Matrilin-3 (MATN3), or related to its composition such as the serpin family H member 1 (SERPINH1), were detected (Figs. 4J and S7I). Finally, positive correlations were found between the 5 genes with the expression of different EMT inducers including ZEB1/2, SNAI1/2, TWIST1/2, PRRX1 and STAT3 (Figs. 4J and S7I). Notably, the strongest correlations and with the highest number of markers of each group were detected for ANKRD6 and ITIH3.

Since metastasis and EMT, as well as recurrence and therapy resistance, are attributed to cancer stem cells (CSCs) [31, 32], we studied the correlation of the 5 genes with regulators of CSCs. This analysis revealed that several stem cell markers were positively and significantly correlated with the 5 genes in both cohorts (Figs. 4K and S7J). As in the case of biological pathways and EMT markers/inducers, ANKRD6 and ITIH3 were the genes, specially the first one, presenting the strongest patterns of correlation and with a higher number of stem markers, exhibiting ANKRD6 significant correlations with SOX2, BMI1, KLF8, LINGO2, CXCR4, CD90 and YAP1 (Figs. 4K and S7J). These results associate high levels of ANKRD6 and ITIH3 with the EMT process.

3.5 ANKRD6 is required for gastric cancer cell activity

ANKRD6 was the most robustly associated with poor outcome, recurrence and metastasis among the 5 genes. Moreover, its impact on GC cell activity remained unexplored. Therefore, we selected this gene in order to address its functional role in GC cells. For this, we silenced the expression of ANKRD6 using 2 independent short hairpin RNAs in the metastatic GC cell line MKN45, which expresses high levels of ANKRD6, as observed in CCLE (Fig. S8B). Once we validated the significant inhibition of ANKRD6 using both short hairpins (Fig. 5A), we analyzed the phenotype of the ANKRD6-silenced cells, detecting multiple cells de-attached in sh1 and sh2 conditions, indicative that they could undergo apoptosis. To confirm this idea, immunofluorescence of the apoptosis markers active Caspase-3 and proteolyzed PARP-1 were performed. Noteworthy, we detected an increase of around 5–10 fold in ANKRD6-silenced cells compared to control cells. In particular, the proportion of cells positive for active Caspase-3 was 3.37% ± 0.31 and 4.61% ± 0.54 in sh1 and sh2 cells compared to 0.56% ± 0.14 in controls (Fig. 5B). Similarly, proteolyzed PARP-1-positive cells were 2.64% ± 0.55 and 4.00% ± 0.32 in ANKRD6-silenced compared to 1.03% ± 0.27 in controls (Fig. 5C). Additionally, flow cytometry analysis unveiled a significant increase in the proportion of cells in the subG1 phase in ANKRD6-silenced cells (sh1; 11.87% ± 0.25; sh2: 13.39% ± 0.99) respect to control cells (0.77% ± 0.45) (Fig. 5D). Moreover, we observed an increase in the percentage of cells in the G2M phase, whilst the proportion of cells decreased in G1 and S phases (Fig. 5D), suggesting cell cycle arrest. To test the effect on cell proliferation, we performed cell count experiments, which revealed a significant and progressive reduction in the number of cells over time in both sh conditions compared to control cells (Fig. 5E). Concretely, at day 5, the number of sh1 and sh2 cells was reduced by ~60% (Fig. 5E).

Fig. 5

Role of ANKRD6 in gastric cancer cell activity. A ANKRD6 mRNA expression in MKN45 GC cell line lentivirally transduced with pLKO (control) or ANKRD6 silencing (sh1 and sh2) plasmids (n ≥ 3). Quantification and representative images of active Caspase-3-positive cells (B) and proteolyzed PARP-1-positive cells (C) in pLKO and shANKRD6 MKN45 cells analyzed by immunofluorescence (n = 3). D Cell cycle distribution in pLKO and shANKRD6 MKN45 cells assessed through the study of cell DNA content by flow cytometry (n = 3). E Growth curves representing the number of pLKO and shANKRD6 MKN45 cells counted at days 1, 3 and 5 after seeding (n ≥ 3). F Volume at the indicated time points and image of subcutaneous tumors generated by pLKO (control) and shANKRD6 MKN45 cells in immunodeficient FOXn1nu (nu/nu) mice. G Mass of subcutaneous tumors represented in (F). Representative images and quantification of cells presenting active Caspase-3 (H) and Ki67 positive staining (I) assessed by IHC in subcutaneous tumors from (F). Scale bar: 100 µm

Then, we moved to the in vivo setting and established subcutaneous xenografts of control and ANKRD6-silenced cells in immunodeficient mice. ANKRD6-silenced cells displayed a significant reduction in tumor growth with respect to control cells (Fig. 5F). The decrease in tumor volume was 67.2% in sh1 tumors and 66.8% in sh2 at day 18. Consistent with this, the tumor weight was significantly lower in those derived from ANKRD6-silenced cells (Fig. 5G). Furthermore, we explored Caspase-3 and the proliferation marker Ki67 in these tumors by IHC, finding a 2–3 fold increase in the number of active Caspase-3 positive cells (Fig. 5H), and a decrease in the proportion of Ki67-positive cells (Fig. 5I) in sh conditions, which confirmed in vivo the requirement of ANKRD6 for tumor cell survival and proliferation. Overall, these results demonstrate that ANKRD6 is required for GC metastatic cell activity and malignancy.

3.6 ANKRD6 is required for GC metastatic traits

Then, to unravel the molecular mechanisms associated to the activity of ANKRD6, we performed RNA sequencing in control and ANKRD6-silenced cells. 450 genes were differentially expressed with respect to control cells, with a fold-change cut-off of >1.5 and p-adjusted values <0.05. Among them, 198 genes were up-regulated and 252 down-regulated. The gene set enrichment analysis (GSEA) identified statistically significant downregulation of 11 pathways in ANKRD6-silenced cells (Fig. 6A). Among them, the hallmark of EMT was identified, as well as other hallmarks such as G2M checkpoint or KRAS signaling up, which is in agreement with the results obtained above. Consequently, we studied functional and molecular events linked to EMT and metastasis in ANKRD6-silenced cells. First, we detected an increased expression of the epithelial markers E-Cadherin and Keratin-18 (KRT18) in ANKRD6-silenced cells using IHC, western blot and immunofluorescence techniques in vivo and in vitro (Fig. 6B–D). In line with these findings at cellular level, the expression of ANKRD6 inversely correlated with both epithelial markers in the GC samples from the ACRG and TCGA patients (Figs. 4J and S7I). Moreover, the capacity of GC cells for migration and invasion was significantly impaired by ANKRD6 silencing (Fig. 6E, F). Thus, the migration capacity of ANKRD6-silenced cells compared to controls represented the 60.8 ± 11.2% and the 43.7 ± 4.3% for sh1 and sh2 cells, respectively (Fig. 6E). Similarly, the invasive potential of sh cells was reduced in around 30% (Fig. 6F).

Fig. 6

Pro-metastatic role of ANKRD6 in gastric cancer. A GSEA ridge plots depicting the enrichment of signal pathways altered in shANKRD6 cells respect to pLKO cells. B Representative image and quantification of E-Cadherin determined by IHC in subcutaneous tumors derived from pLKO and shANKRD6 MKN45 cells. Scale bar: 100 µm. Representative western blot (C) and immunofluorescence (D) of E-Cadherin and KRT18 in pLKO and sh2 ANKRD6 MKN45 cells. Scale bar: 100 µm. Representative images and relative migration (E) and invasion (F) of ANKRD6-silenced cells respect to control cells determined by transwell assays (n ≥ 3). G Genes belonging to the Epithelial mesenchymal transition (EMT) hallmark gene set that are differentially expressed in shANKRD6 cells respect to pLKO cells. Genes down-regulated are represented in purple and up-regulated in green. H Correlation in TCGA and ACRG cohorts between the expression of ANKRD6 and the DEGs from the EMT hallmark. I Expression of MATN3, GPC1, TIMP1 and DKK1 in normal gastric tissue (grey) versus the corresponding paired GC tissue (red) in TCGA (Ntumor = 32, Nnormal = 375) and ACRG (Ntumor = 98, Nnormal = 98). J Kaplan-Meier plots of OS (left) and DFS (right) according to the expression of MATN3, TIMP1 and DKK1 in TCGA, ACRG and the integrated cohort. K Expression of MATN3, TIMP1 and DKK1 according to M stage in the integrated cohort (NM0 = 513, NM1 = 45). L mRNA expression of MATN3, GPC1, TIMP1 and DKK1 determined by qPCR in shANKRD6 respect to pLKO cells (n ≥ 3) (Colour figure online)

We observed that within the hallmark of EMT, 8 genes were significantly down-regulated and 4 were up-regulated in ANKRD6-silenced cells (Fig. 6G). The down-regulated genes were PMEPA1, a native regulator of TGF-beta; DKK1, an antagonist of the canonical Wnt signaling pathway; CAPG, which is involved in actin dynamics; and genes coding components of the ECM (MATN3 and LAMC2) or ECM-interacting proteins (ITGA2, GPC1 and TIMP1) (Fig. 6G). We moved back to the patients and found that MATN3, GPC1, TIMP1 and DKK1 were positively and significantly correlated with the expression of ANKRD6 in the GC patients from both the TCGA and the ACRG cohorts (Fig. 6H). Additionally, their expression was higher in GC tissue respect to normal gastric tissue in both cohorts (Fig. 6I), whereas high expression of MATN3, TIMP1 and DKK1 was significantly associated with reduced OS and DFS in GC patients from the TCGA, ACRG and the additional cohorts used in this study (Fig. 6J), their expression being also significantly elevated in the samples of those patients presenting metastasis (Fig. 6K). Finally, we detected that the expression of the four genes was significantly downregulated in shANKRD6 cells validating the in silico results (Fig. 6L). These findings, overall, reveal a role of ANKRD6 in GC progression, metastasis and EMT.