ONECUT2 mRNA expression level was upregulated in GC tissues
To identify the underlying role of ONECUT2 in GC, the expression status of ONECUT2 was analyzed firstly. Although previous study has reported that higher ONECUT2 mRNA level was concluded in tumor tissues than normal tissues from their local GC samples[14], a largely comprehensive statistic still needed to be performed to validate that. Herein, the data comprising 174 normal stomach samples in The Genotype-Tissue Expression (GTEx) and 36 normal stomach samples plus 414 primary Stomach Adenocarcinoma (STAD) samples in The Cancer Genome Atlas (TCGA) were downloaded from UCSC Xena website, which was a visualized online exploration tool[15]. After the integration and analysis of these samples, the results showed that the expression of ONECUT2 was significantly higher in the tumor group (Figure 1A). Consistently, higher expression level of ONECUT2 in tumor tissues was further concluded by analyzing 19 pairs of GC samples (Figure 1B). These findings demonstrated that ONECUT2 was upregulated in GC tumor tissues.
ONECUT2 expression predicted poor DFI and new tumor events in GC
Previous study has reported that their GC patients with high ONECUT2 expression level had poor overall survival (OS) outcomes[14]. However, OS situation of GC patients in TCGA cohort had no significantly correlation with ONECUT2 level (Figure 1C). When categorized by Disease-Free Interval (DFI), higher ONECUT2 expression predominantly contributed to worse DFI (Figure 1D). Moreover, GC patients with high ONECUT2 expression level were inclined to getting new tumor events (Figure 1E), indicating that ONECUT2 somewhat affected the efficacy of therapy for GC patients.
ONECUT2 was a risk factor for tumor recurrence in chemotherapeutic GC patients
To unveil the unknown role of ONECUT2 in GC chemotherapy, we sorted out 102 patients once enduring with pharmaceutical therapy. The correlation between several clinical characteristics and ONECUT2 expression level of tumor tissues were investigated by χ2 test. The results showed that only new neoplasm event had statistically significant positive with high ONECUT2 level (Table 1). Meanwhile, as also categorized by median ONECUT2 expression value, multivariable Cox regression analyses showed that ONECUT2 expression was an independent predictor of chemotherapeutically treated patients with a significant HR for worse DFI (Figure 1F). Taken together, these results implied that ONECUT2 was an indicator of chemotherapeutic insensitivity in GC and making patients more susceptible to cancer relapse.
High ONECUT2 expression level was positively correlated with drug metabolism
To explore the mechanism of drug resistance induced by ONECUT2, we performed GSEA using gene expression RNA-sequencing data of 100 STAD tumor samples acquired from UCSC website. In Hallmarks database, high ONECUT2 group containing 50 samples is positively correlated with 8 gene sets listed as Normalized Enrichment Score (NES) ranking, all of which have statistical significance (normalized P value, P<0.05; and false discovery rate, FDR<0.25) (Figure 2A), including striking alteration in xenobiotic metabolism (Figure 2B). Consistently, KEGG database analysis in the same grouping presents similarly supporting results that two xenobiotic metabolism pathways are enriched (Figure 2C-E). These GSEA processing results show that the ability of drug catabolism is widely regulated by ONECUT2.
Targeting ONECUT2 increases drug sensitivity of 5-FU and cisplatin in GC cells
To further validate that ONECUT2 was a key regulator for drug resistance, we measured half maximal inhibitory concentration (IC50) of different drugs treated GC cells in vitro. Above all, one normal human gastric epithelial cell line (GSE-1) and 7 human GC cell lines were used to detect ONECUT2 mRNA expression level. Real-time qualitative PCR confirmed that ONECUT2 was upregulated in tumor cell lines (Figure 3A). Then, HGC27 cells and MGC803 cells were infected with short hairpin (sh)-ONECUT2 lentivirus and overexpressed (oe)-ONECUT2 lentivirus to establish stable ONECUT2 knockdown and overexpression cell lines respectively. And the efficiency of changed expression of ONECUT2 was estimated by real-time qualitative PCR (Figure 3B and C). Next, we performed CCK8 assays by treating established GC cells with gradient concentrations of 5-FU or cisplatin to measure IC50 influenced by differentially expressed ONECUT2, and illustrated IC50 fitting curves based on relative CCK8 values. These graphs showed that knocking down ONECUT2 decreased IC50 value of 5-FU and cisplatin of HGC27 cells (Figure 3D and E), but ONECUT2 overexpression prompted the survivability of MGC803 cells (Figure 3F and G). Additionally, the specific inhibitor for ONECUT2 (CSRM617) reversed the tolerance to 5-FU or cisplatin caused by ONECUT2 overexpression in MGC803 cells (Figure 3H and I). To sum up, ONECUT2 was a functional factor promoting GC cells growth under pharmaceutical drugs stress in vitro.
ONECUT2 level negatively correlated with therapeutic efficacy of 5-FU in relapsed GC patients
To further validate the role of ONECUT2 in chemotherapy, 30 surgically resected biopsies from GC patients were collected, who were only treated with 5-FU (400mg/m2) for 8 weeks on account of tumor reoccurrence. Real-time qualitative PCR was performed to measure ONECUT2 mRNA level and 30 patients were split into low or high group (Figure 4A). Then the correlation between ONECUT2 level and reduced tumor volume caused by 5-FU was assessed using linear regression analysis. Supportively, as the mRNA level of ONECUT2 increased, the efficacy of 5-FU treatment to GC patients tended to be obviously worse in low (Figure 4B) or total ONECUT2 group (Figure 4D). However, as for high ONECUT2 group, the tumor volume was contrarily increased in some patients (Figure 4C). These results demonstrated that higher ONECUT2 expression impeded the curative effect of 5-FU in GC patients with relapsed tumors.