Fig 2: GPR50 Is Differentially Expressed in Various Cancer Types(A) Oncomine database Log2 median-centered expression intensities for GPR50 genes in various cancers, such as bladder (BLC; n = 9), brain and CNS cancer (BCC; n = 16), breast (BRC; n = 19), cervical (CEC; n = 7), colorectal (COC; n = 23), esophageal (ESC; n = 4), gastric (GAC; n = 5), head and neck (HNC; n = 6), kidney (KIC; n = 8), leukemia (LEU; n = 30), liver (HCC; n = 9), lung (LUC; n = 73), lymphoma (LYM; n = 38), melanoma (MEL; n = 12), myeloma (MYE; n = 5), ovarian (OVC; n = 5), pancreatic (PAC; n = 9), prostate (PRC; n = 3), and sarcoma (SAR; n = 17) cancers. (B) Analysis of GEO: GSE1477, GSE7803, GSE20347, GSE45436, and GSE2514 datasets for GPR50 mRNA expression in BRC (n = 28), CEC (n = 31), ESC (n = 34), HCC (n = 134), and LUC (n = 39) compared with normal breast, cervical, esophageal, liver, and lung tissue. Other GEO datasets for BRC, CEC, ESC, HCC, and LUC cancers were incorporated into Table S1. (C) Kaplan-Meier curves for clinical outcomes of patients with breast (n = 962), cervical (n = 191), esophageal (n = 184), liver (n = 361), and lung (n = 475) cancers, respectively, with high (red) and low (green) expression levels of GPR50. (D) GPR50 mRNA expression in HCC. Boxplot generated by the SurvExpress web shows GPR50 expression levels and the p value (t test of differences in TCGA RNA sequencing [RNA-seq] dataset). Low-risk (n = 191) and high-risk (n = 190) groups are shown in green and red, respectively. (E) In silico examination using cBioPortal reveals that 2.9% of samples had alterations in GPR50 expression in HCC TCGA PanCan data (n = 348). (F) GPR50 expression was analyzed by RT-PCR and western blotting in the indicated normal hepatic cell line and different HCC cell lines. GAPDH/actin were used as a loading control.

Fig 3: Overexpression of GPR50 Augments Notch Signaling via the Ligand-Independent ADAM17 Axis in HCC Cells(A) GPR50 overexpression was confirmed by RT-PCR and western blot analysis and normalized to that of GAPDH or actin. (B) Cell viability was analyzed over 3 days after overexpression of GPR50 using the EZ-cytox WST-1 assay. (C) Cell-sphere formation assay was performed using the noncoated culture dishes after GPR50 overexpression. Spheres were counted after 5 days of culture using crystal violet staining and presented as the percent (%) of colonies. Photos were acquired by inverted light microscopy. (D) Protein expression levels of ADAM17, NICD, and HES1 were analyzed by western blot analysis after GPR50 overexpression. (E and F) Notch signaling transcriptional activity was analyzed after GPR50 overexpression using the HES1 (E) and HES5 (F) luciferase assays. (G) Fold change in ADAM17 enzymatic activity was analyzed using the ADAM17 ELISA assay after GPR50 overexpression. (H) Protein expression levels of GPR50, ADAM17, NICD, and HES1 were analyzed by western blot analysis after GPR50 overexpression and treatment with/without DAPT (Notch signaling inhibitor) and/or marimastat (ADAM17 inhibitor). (I) Fold change in ADAM17 activity was analyzed using the ADAM17 ELISA assay after GPR50 overexpression and treatment with/without DAPT and/or marimastat. (J and K) Notch signaling transcriptional activity was analyzed after GPR50 overexpression and treatment with/without DAPT and/or marimastat using the HES1 (J) and HES5 (K) luciferase assay. ns, no significance; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Fig 4: Schematic Diagram Illustrating GPR50 Function in the Notch Signaling Pathway in HCCGPR50 regulates ADAM17 transcription via the AKT/SP1 axis and directly interacts with ADAM17 to induce ADAM17 enzymatic activity, which subsequently activates ligand-independent Notch signaling, mediating HCC progression.

Fig 5: GPR50 Directly Interacts with ADAM17 and Activates the Ligand-Independent Notch Signaling Pathway via ADAM17 in HCC(A) Protein expression levels of ADAM17 were analyzed using western blotting; actin was used as a loading control. (B) Fold change in ADAM17 activity was analyzed using the ADAM17 ELISA assay. (C) Lysates from the indicated cells were used for immunoprecipitation using Protein A/G Sepharose, as well as antibodies specific for GPR50, ADAM17, and normal IgG. The immunoprecipitates were analyzed by western blotting with the indicated antibodies. For inputs, lysates were analyzed by western blotting with the indicated antibodies. (D) Overexpression of FLAG-ADAM17 was confirmed by western blot analysis using FLAG antibody; actin was used as a loading control. (E and F) Fold change in Notch signaling transcriptional activity was analyzed using HES1 (E) and HES5 (F) luciferase assays after overexpression of ADAM17 and treatment with/without marimastat (4 μM). (G) Fold change in ADAM17 enzymatic activity was analyzed using the ADAM17 ELISA assay after overexpression of ADAM17 and treatment with/without marimastat (4 μM). (H) Cell viability was analyzed using the EZ-cytox 4-[3-(4iodophenyl)-2-(4- nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) assay after overexpression of ADAM17 and treatment with/without marimastat (4 μM) after 24 h. (I) Sphere formation assay was performed using the noncoated culture dishes after overexpression of ADAM17 and treatment with/without marimastat (4 μM). Spheres were counted after 5 days of culture using crystal violet staining and presented as fold change in colonies. ns, no significance; ∗∗p < 0.01, ∗∗∗∗p < 0.0001.