GPR155 Serves as a Predictive Biomarker for Hematogenous Metastasis in Patients with Gastric Cancer

Abstract

The prognosis of patients with gastric cancer (GC) with hematogenous metastasis is dismal. Identification of biomarkers specific for hematogenous metastasis is required to develop personalized treatments that improve patients' outcomes. Global expression profiling of GC tissues with synchronous hepatic metastasis without metastasis to the peritoneal cavity or distant lymph nodes was conducted using next-generation sequencing and identified the G protein-coupled receptor 155 (GPR155) as a candidate biomarker. GPR155 transcription was suppressed in GC cell lines compared with a nontumorigenic cell line. DNA methylation of the GPR155 promoter region was not detected, albeit 20% of GC cell lines harbored copy number loss at GPR155 locus. The expression levels of GPR155 mRNA correlated inversely with those of TWIST1 and WNT5B. Inhibition of GPR155 expression increased the levels of p-ERK1/2 and p-STAT1, significantly increased cell proliferation, and increased the invasiveness of a GC cell lines. GPR155 mRNA levels in GC clinical samples correlated with hematogenous metastasis and recurrence. Multivariate analysis revealed that reduced expression of GPR155 mRNA was an independent predictive marker of hematogenous metastasis. GPR155 may represent a biomarker for diagnosing and predicting hematogenous metastasis of GC.

Figure 1. Analysis of GPR155 expression and identification of candidate molecules that interact with GPR155: assays used indicate downstream effects.

(A) Reduced expression of GPR155 mRNA was detected in all GC cell lines compared with that of FHs74Int. Copy number loss was detected in AGS and SC-6-JCK cells. Methylation of the promoter region of GPR155 was not detected. (B) PCR array analysis showing that the expression level of GPR155 mRNA inversely correlated with those of TWIST1 and WNT5B mRNAs. (C) Cell signaling pathway analysis of AGS cells indicated that inhibition of GPR155 mRNA increased the levels of p-ERK1/2 and p-STAT1.

Figure 2. Phenotypes of AGS cells transfected with siGPR155.

(A,B) Knockdown of GPR155 expression increased cell proliferation and invasiveness. (C) There was no significant change in cell migration.

Figure 3. Expression of GPR155 in clinical samples, and the cumulative incidence of hematogenous recurrence in patients with Stage II/II GC.

(A) Significantly lower levels of GPR155 mRNA were detected in primary GC tissues compared with the corresponding noncancerous mucosal tissues. Patients with Stage IV GC with synchronous hematogenous metastasis had lower expression levels of GPR155 mRNA compared with those of patients without hematogenous metastasis. (B) The optimal cut-off value of GPR155 expression was determined at 0.0009. (C) The cumulative incidence of hematogenous recurrence was significantly higher in the low GPR155 group patients with Stage II/III GC. Abbreviations: Hem-rec; hematogenous recurrence, Hem; synchronous hematogenous metastasis.

Figure 4. IHC analysis of GPR155 expression in tissues of representative patients with GC and the survival curve of patients with Stage II/III GC.

(A) GPR155 staining intensity of GC tissues was generally low compared with the corresponding noncancerous mucosae. GPR155 staining intensity correlated significantly with synchronous and metachronous hematogenous metastasis. (B) There was not significant difference in overall survival between low GPR155 and high GPR155 groups with Stage II/III GC. (C) There was no significant difference in disease-free survival between the low GPR155 and high GPR155 groups with Stage II/III GC. Abbreviations: T; tumor tissue, N; non-cancerous component, GC; primary gastric cancer tissue, Hem/Hem-rec; hematogenous metastasis/recurrence.

Figure 5. Immunohistochemical staining for GPR155, TWIST and WNT5B proteins.

(A) Results in a representative case using tissues from primary GC and hepatic metastasis. (B) Correlations in staining patterns between GPR155, TWIST and WNT5B. Abbreviations: T; tumor tissue, N; non-cancerous component, GC; primary gastric cancer tissue.