GPR137 inactivates Hippo signaling to promote gastric cancer cell malignancy

Abstract

As the fifth most common cancer in the world, gastric cancer (GC) ranks as the third major cause of cancer-related death globally. Although surgical resection and chemotherapy still remains the mainstay of potentially curative treatment for GC, chemotherapy resistance and adverse side effects limit their clinical applications. Thus, further investigation of the mechanisms of carcinogenesis in GC and discovery of novel biomarkers is of great concern. We herein report that the elevated expression of GPR137 is correlated with GC. Overexpression of GPR137 potentiates human gastric cancer AGS cell malignancy, including proliferation, migration, invasion, colony formation and xenograft growth in nude mice in vivo, whereas knockout of GPR137 by CRISPR/Cas9 gene editing exerts the opposite effects. Mechanistically, GPR137 could bind to MST, the upstream kinases in Hippo pathway, which disrupts the association of MST with LATS, subsequently activating the transcriptional co-activators, YAP and TAZ, and thereby triggering the target transcription and the alterations in GC cell biological actions consequently. Therefore, our findings may provide with the evidence of developing a potentially novel treatment method with specific target for GC.

Keywords: GPR137; Gastric cancer; Hippo; MST.

Fig. 1

Up-regulated GPR137 expression is associated with GC. A Expression of GPR137 in STAD tissues and control groups was revealed by GEPIA database. *p < 0.05; error bar, SD. B Immunohistochemistry staining for GPR137 by using paraffin-embedded sections of cancer tissues and adjacent normal gastric epithelial tissues from patients with GC. N = 10, bar = 40 μm. C IHC score for GPR137. N = 10. D The protein expression levels of GPR137 in human normal gastric epithelial GES-1 cells, human gastric cancer AGS cells and SGC-7901 cells. One representative of 3 independent blots is shown

Fig. 2

GPR137 promotes AGS cell malignancy. A CCK-8 assays of AGS cells transfected with GPR137 or an empty vector (control) and cultured for different periods. **p < 0.01; error bar, SD. N = 3. B Wound healing assays of AGS cells transfected with GPR137 or an empty vector (control) at 24 h. Bar = 100 μm. N = 3. C Statistical analysis of unoccupied area in (B). **p < 0.01; error bar, SD. D Matrigel invasion assays of AGS cells transfected with GPR137 or an empty vector (control) for 24 h. Bar = 100 μm. N = 3. E Quantitative analysis of (D). **p < 0.01; error bar, SD. F Colony formation assays of AGS cells transfected with GPR137 or an empty vector (control). N = 3. G Quantitative analysis of (F). **p < 0.01; error bar, SD. H Nude mice with xenografts derived from AGS cells infected with GPR137-expressing lentiviruses (GPR137) or control viruses (Control). I, J Tumor weight (I) and tumor volume (J) in (H). N = 6; **p < 0.01; error bar, SD

Fig. 3

Transcriptome analysis of GPR137-regulated genes. A Protein levels of GPR137 in AGS cells transfected with GPR137 (GPR137, +) or an empty vector (GPR137, -) for 24 h were detected by western blot. The number of genes whose expression was altered by GPR137 is summarized in the table, and GO analysis was performed. One representative of 3 independent blots is shown. B KEGG was conducted to compare the gene expression from GPR137 group and the control. C Pathway enrichment analysis of (B). D Top cancer-related pathways were shown. E Immunohistochemistry staining for TAZ by using paraffin-embedded sections of cancer tissues and adjacent normal gastric epithelial tissues from patients with GC. N = 10, bar = 40 μm

Fig. 4

GPR137 inhibits Hippo signaling activity. A TEAD-luciferase assays in AGS cells transfected with GPR137 or an empty vector (control) for 24 h. **p < 0.01; error bar, SD. N = 3. B Protein levels of GPR137 in AGS cells transfected with GPR137 shRNA (shGPR137, +) or control scrambled shRNA (shGPR137, -) for 72 h. One representative of 3 independent blots is shown. C TEAD-luciferase assays in AGS cells transfected with GPR137 shRNA or control scrambled shRNA and culture for 72 h. **p < 0.01; error bar, SD. N = 3. D, E qRT-PCR assays for mRNA levels of CTGF (D) and CYR61 (E) in AGS cells transfected with GPR137 or an empty vector (control) for 24 h. **p < 0.01; error bar, SD. N = 3. F Protein levels of GPR137 in SGC-7901 cells transfected with GPR137 (GPR137, +) or an empty vector (GPR137, -) for 24 h. One representative of 3 independent blots is shown. G, H) qRT-PCR assays for mRNA levels of CTGF G and CYR61 (H) in SGC-7901 cells transfected with GPR137 or an empty vector (control) for 24 h. **p < 0.01; error bar, SD. N = 3. I, J qRT-PCR assays for mRNA levels of YAP (I) and TAZ (J) in AGS cells transfected with GPR137 or an empty vector (control) for 24 h. Error bar, SD. N = 3. K, L GEPIA database displayed the correlation between GPR137 and CTGF (K) as well as GPR137 and CYR61 (L) in STAD

Fig. 5

GPR137 suppresses Hippo signaling by MST. A Protein levels of pMST1, MST1, pLATS1, LATS1, pLATS2 and LATS2 in AGS cells transfected with GPR137 (GPR137, +) or an empty vector (GPR137, -) for 24 h. One representative of 3 independent blots is shown. B Protein levels of pTAZ, TAZ, pYAP, YAP, CTGF and CYR61 in AGS cells transfected with GPR137 (GPR137, +) or an empty vector (GPR137, -) for 24 h. One representative of 3 independent blots is shown. C Co-immunoprecipitation of HA-tagged GPR137 and Flag-tagged MST1 in HEK293T cells. IP: Flag; WB: HA. N = 3. D Co-immunoprecipitation of endogenous GPR137 and MST1 in AGS cells. IP: MST1; WB: GPR137. IgG was used as a negative control. One representative of 3 independent blots is shown. E Co-immunoprecipitation of endogenous MST1 and LATS1 in the presence of GPR137 in AGS cells. IP: MST1; WB: LATS1. IgG was used as a negative control. One representative of 3 independent blots is shown. F Co-immunoprecipitation of endogenous MST1 and LATS1 in the presence of XMU-MP-1 in AGS cells. IP: MST1; WB: LATS1. IgG was used as a negative control. One representative of 3 independent blots is shown. G Protein expression examination of MST1 and MST2 in MST1/2-knockout (MST1/2-KO) AGS cells or control AGS cells. One representative of 3 independent blots is shown. H Nucleo-cytoplasmic separation assays in MST1/2-knockout AGS (MST1/2-KO) cells or control cells, and protein levels of YAP and TAZ were detected. Proteins in nucleus (left) and in cytoplasm (right). One representative of 3 independent blots is shown. I Nucleo-cytoplasmic separation assays in GPR137-transfected AGS (GPR137, +) cells or control cells (GPR137, -), and protein levels of YAP and TAZ were detected. Proteins in nucleus (upper) and in cytoplasm (lower). One representative of 3 independent blots is shown. J TEAD-luciferase assays in MST1/2-knockout (MST1/2-KO) AGS cells or control AGS cells transfected with GPR137 or an empty vector (control). **p < 0.01; error bar, SD. N = 3. K TEAD-luciferase assays in MST1/2-knockout (MST1/2-KO) AGS cells or control AGS cells transfected with GPR137 shRNA or control scrambled shRNA. **p < 0.01; error bar, SD. N = 3

Fig. 6

GPR137 loss declines AGS cell malignancy but potentiates Hippo activity. A Protein expression examination of GPR137 in GPR137-knockout (GPR137-KO) AGS cells or control AGS cells. One representative of 3 independent blots is shown. B CCK-8 assays of GPR137-knockout AGS cells or control cells at different periods. **p < 0.01; error bar, SD. N = 3. C Wound healing assays of GPR137-knockout (GPR137-KO) AGS cells or control cells at 24 h. Bar = 100 μm. N = 3. D Statistical analysis of unoccupied area in (C). **p < 0.01; error bar, SD. E Matrigel invasion assays of GPR137-knockout (GPR137-KO) AGS cells or control cells at 24 h. Bar = 100 μm. N = 3. F Quantitative analysis of (E). **p < 0.01; error bar, SD. G Colony formation assays of GPR137-knockout (GPR137-KO) AGS cells or control cells. N = 3. H Quantitative analysis of (G). **p < 0.01; error bar, SD. I Protein levels of pMST1 and MST1 in GPR137-knockout (GPR137-KO) AGS cells or control cells. One representative of 3 independent blots is shown. J Protein levels of pTAZ, TAZ, pYAP and YAP in GPR137-knockout (GPR137-KO) AGS cells or control cells. One representative of 3 independent blots is shown. K Nucleo-cytoplasmic separation assays in GPR137-knockout AGS (GPR137-KO) cells or control cells, and protein levels of YAP and TAZ were detected. Proteins in nucleus (upper) and in cytoplasm (lower). One representative of 3 independent blots is shown. L, M qRT-PCR assays for mRNA levels of CTGF (L) and CYR61 (M) in GPR137-knockout (GPR137-KO) AGS cells or control cells. **p < 0.01; error bar, SD. N = 3. N Immunofluorescent staining for HA-derived signal in GPR137-knockout (GPR137-KO) AGS cells or control cells transfected with HA-TAZ-S89A (upper) or HA-YAP-S127A (lower). Nuclei were stained with DAPI. Bar = 5 μm. N = 3

Fig. 7

GPR137 elevates AGS cell malignancy through Hippo signaling. A Protein expression examination of YAP (upper) and TAZ (lower) in YAP-shRNA or TAZ-shRNA transfected AGS cells. One representative of 3 independent blots is shown. B TEAD-luciferase assays in AGS cells transfected with GPR137 or an empty vector (control) in combination with YAP shRNA, TAZ shRNA, or YAP shRNA and TAZ shRNA for 72 h. **, ^##, p < 0.01; error bar, SD. N = 3. C CCK-8 assays in AGS cells transfected with GPR137 or an empty vector (control) in combination with YAP shRNA and TAZ shRNA at different periods. **p < 0.01; ^#p < 0.05; error bar, SD. N = 3. D Wound healing assays in AGS cells transfected with GPR137 or an empty vector (control) in combination with YAP shRNA and TAZ shRNA (GPR137 + shY/T). N = 3. E Quantitative analysis of (D). **, ^##, p < 0.01; error bar, SD. F Matrigel invasion assays in AGS cells transfected with GPR137 or an empty vector (control) in combination with YAP shRNA and TAZ shRNA (GPR137 + shY/T). N = 3. G Quantitative analysis of (F). **p < 0.01; ^#p < 0.05; error bar, SD. (H) Quantitative analysis for colony formation assays in AGS cells transfected with GPR137 or an empty vector (control) in combination with YAP shRNA and TAZ shRNA (GPR137 + shY/T). **p < 0.01; ^#p < 0.05; error bar, SD. N = 3