Oxytocin Receptor Regulates the Hippo/YAP Axis to Drive Hepatocarcinogenesis

Abstract

Dysregulation of Hippo signaling, especially the downstream effector YAP, is a critical driver of hepatocellular carcinoma (HCC). Therefore, identifying therapeutic targets to block Hippo signaling could help improve survival outcomes for patients with HCC. In this study, we conducted an unbiased siRNA screen on G protein-coupled receptors targeted by drugs approved in the United States and strongly associated with the Hippo/YAP pathway and identified the oxytocin receptor (OXTR) as an important activator of the Hippo/YAP axis in HCC. The OXTR was correlated with the Hippo gene signature and poor survival outcomes in HCC, and OXTR activation promoted HCC progression through the Hippo/YAP axis. The OXTR antagonist atosiban blocked the growth of HCC in xenograft, patient-derived explant, organoid, and MST1/2 double-knockout mouse models. Molecular studies revealed that activation of the OXTR facilitated YAP dephosphorylation, nuclear accumulation, and transcriptional activation in HCC. OXTR interacted with Gαq/11 at several important sites (R137, I141, and I227) and induced YAP activation through the Gαq/11/Rho-associated protein kinase/LATS axis. Chromatin immunoprecipitation assays showed that YAP bound to the enhancer region of the OXTR and facilitated its transcription, creating a positive feedback loop. Together, this study uncovered the interplay between Hippo signaling and the OXTR pathway in hepatocarcinogenesis and established OXTR inhibition with atosiban as a promising strategy for treating HCC.

Significance: The FDA-approved oxytocin receptor antagonist atosiban can ameliorate Hippo signaling dysfunction in liver cancer to suppress tumor growth, providing an effective and rapidly translatable therapy for hepatocellular carcinoma.

Graphical abstract

Figure 1.

Identification of the OXTR as a novel mediator of the Hippo/YAP pathway in HCC. A, Comparison of 75 GPCRs strongly associated with the Hippo/YAP pathway and 134 GPCRs targeted by drugs approved in the United States according to GSEA (NES >1.5; P < 0.05). B, The flowchart shows the siRNA screening procedure involved in modulating Hippo/YAP signaling for the 28 GPCRs shown in A. C, The relative expression level of CTGF in cells that were transfected with siGPCRs in the screening library. The qRT-PCR results of the siControl group were normalized to one. ns, nonsignificant. D, Relative RNA levels of OXTRs in HCC tumor samples (n = 371) vs. normal samples (n = 50) from the TCGA database (https://www.genome.gov/). FC = 2.16. E, IHC was used to detect OXTR expression in 90 liver cancer tissues and 60 normal liver tissues, and statistical analysis of OXTR expression was performed. Scale bar, 100 μm. F, Kaplan–Meier analysis showing overall survival depending on OXTR expression levels in HCC tumor samples from the TCGA database. G, GSEA revealed a significant positive correlation between the OXTR and YAP target gene signature in HCC samples from the TCGA database, with a threshold criterion of P < 0.05. H, Heatmap of the relationships between the OXTR and differentially expressed Hippo/YAP pathway–related genes in the TCGA database with threshold criteria of P < 0.05 and a FC >1.5. I, Volcano map of RNA-seq data from SNU449 cell lines treated with siControl or siOXTR. |log₂ FC| > 1 and P < 0.05 were set as screening criteria. NoDiff, no difference; Sig_Down, significant downregulation; Sig_Up, significant upregulation. J, Kyoto Encyclopedia of Genes and Genomes analysis of downregulated genes in RNA-seq data from SNU449 cell lines treated with siControl or siOXTR with threshold criteria of P < 0.001 and a FC > 1.5. K, GSEA showing enrichment of the CORDENONSI_YAP_CONSERVED_SIGNATURE in RNA-seq data from SNU449 cell lines treated with siControl or siOXTR. L and M, IHC was used to detect OXTR and YAP expression in 90 liver cancer tissues (L). Statistical analysis of OXTR and YAP expression in L (M). Scale bar, 100 μm. N, Correlations between the OXTR expression level in HCC tumor samples and the clinicopathologic characteristics of the corresponding patients. Data analysis revealed that OXTR expression was correlated with sex (P = 0.005), tumor invasion (P = 0.031), and lymph node metastasis (P = 0.027). All data are presented as the means ± SDs. ns, nonsignificant; *, P < 0.05; **, P < 0.01; ***, P < 0.001 for comparisons. B, Created in BioRender. Yang, H. (2025) https://BioRender.com/exsenbv.

Figure 2.

OXTR depletion slows HCC progression in vivo and in vitro. A, Immunoblot analysis showing the expression level of the OXTR in SNU449 cell lines transfected with siControl or two independent OXTR siRNAs for 48 hours. β-Actin was used as the internal control. B, A CCK-8 assay was used to detect the viability of SNU449 cells transfected with siControl or siOXTR for 24 hours, and the results were tested at the indicated time points. C and D, Colony formation of SNU449 cell lines transfected with siControl or two independent OXTR siRNAs for 24 hours. D, Quantitative analysis of the colony formation assay results. E‒H, Transwell and wound-healing assays were used to detect the migration ability of SNU449 cells treated as indicated. F and H, The results of the quantitative analysis. I and J, Imaging (I) and number (J) of orthotopic xenograft HCC tumors harvested from four mice per group. K and L, IHC was used to detect Ki67 expression in orthotopic xenograft HCC tumors (I), and a statistical analysis of Ki67 expression (L) was performed. Scale bar, 40 μm. M‒O, Imaging (M), tumor weights (N), and tumor volumes (O) in BALB/c nude mice subcutaneously inoculated with stably transfected shControl or shOXTR SNU449 cells. P, Lysates of tumor tissues from M were subjected to immunoblotting analyses with the indicated antibodies. Q–S, IHC was used to detect OXTR, YAP, p-YAP, and Ki67 expression in M (Q), and statistical analysis of YAP (R) and Ki67 expression (S) was performed. Scale bar, 150 μm. Three independent experiments were conducted to obtain the results shown in A‒H. All data are presented as the means ± SDs. *, P < 0.05; **, P < 0.01; ***, P < 0.001 for comparisons (Student t test).

Figure 3.

The OXTR antagonist atosiban inhibits HCC progression in vivo and in vitro. A, Immunoblot analysis showing the expression level of the OXTR in SNU449 cell lines treated with DMSO or atosiban for 24 hours. β-Actin was used as the internal control. B, A CCK-8 assay was used to detect the viability of SNU449 cells treated with DMSO or atosiban for 24 hours at the indicated time points. C and D, Colony formation of SNU449 cell lines treated with DMSO or atosiban for 24 hours. D, Quantitative analysis of the colony formation assay results. E‒H, Transwell and wound-healing assays were used to detect the migration ability of SNU449 cells treated with DMSO or atosiban for 24 hours. F and H The results of the quantitative analysis. I‒L, Flowchart (I), imaging (J), tumor weights (K), and tumor volume (L) in BALB/c nude mice subcutaneously inoculated with stably transfected shControl or shOXTR SNU449 cells. Error bars, means ± SDs. M‒O, IHC was used to detect hematoxylin and eosin (H&E), YAP, p-YAP, and Ki67 expression in J (M), and statistical analysis of YAP (N) and Ki67 expression (O) was performed. Error bars, means ± SDs. Scale bar, 150 μm. Three independent experiments were conducted to obtain the results shown in A‒H. All data are presented as the means ± SDs. **, P < 0.01; ***, P < 0.001 for comparisons (Student t test). I, Image of mice created in BioRender. Yang, H. (2025) https://BioRender.com/twioz0d.

Figure 4.

Atosiban inhibits HCC progression in Mst1^flox/flox; Mst2^flox/flox mice and patient-derived explant and organoid models. A, PCR analysis of Cre, Mst1, and Mst2 from the indicated genotypes. B, Gross images of livers from Mst1^wt/wt; Mst2^wt/wt; Alb-Cre and Mst1^flox/flox; Mst2 ^flox/flox; Alb-Cre mice at 2 months of age. C, Liver weight‒to‒body weight ratios in Mst1^wt/wt; Mst2^wt/wt; Alb-Cre and Mst1^flox/flox; Mst2 ^flox/flox; Alb-Cre mice at 2 months of age. Statistical significance (unpaired t test) is indicated. D, Flowchart of atosiban or saline solution treatment experiments in Mst1^flox/flox; Mst2 ^flox/flox; Alb-Cre mice. E and F, Hepatoma formation following the intraperitoneal administration of normal saline or 1 mg/kg atosiban (E). F, The relative tumor number is indicated. G, Immunoblot analysis showing the expression levels of Mst1, Mst2, p-YAP, YAP, p-LATS1, and LATS1 in the indicated mice subjected to the indicated treatments. β-Actin was used as the internal control. H, CTGF and CYR61 mRNA levels were determined via qRT-PCR in the indicated mice subjected to the indicated treatments. I, Nucleoplasm separation by immunoblotting was used to detect YAP in the indicated mice subjected to the indicated treatments. J and K, IHC was used to detect Ki67 expression, as shown in E (J), and to perform a statistical analysis of Ki67 expression (K). Error bars, means ± SDs. Scale bar, 40 μm. L‒N, In the patient-derived explant assay, atosiban treatment inhibited the proliferation potential of HCC tumors. HCC tumor xenografts were treated with vehicle or 10 μmol/L atosiban. The samples were fixed and stained for OXTR, YAP, p-YAP, and Ki67. The YAP- and Ki67-positive cells were counted for analysis (M and N). Scale bar, 150 μm. O and P, Single organoid expanding on days 3, 6, 9, 12, and 15 after treatment with vehicle or 10 μmol/L atosiban. Scale bar, 40 μm (O). Statistical analysis of organoid viability (P). ns, nonsignificant. Q, Heatmap of the relationship of relative mRNA expression (log₂-FC) of 45 Hippo/YAP target genes in RNA-seq data (atosiban vs. vehicle, GSEA266229) in three different HCC organoids treated with vehicle or 10 μmol/L atosiban. The upregulation and downregulation of mRNA levels are indicated by red and green, respectively, with threshold criteria of P < 0.05 and a FC > 1.5. R‒T, GSEA showing enrichment of the YAP target gene signature in RNA-seq data from three different HCC organoids treated with vehicle or 10 μmol/L atosiban (GSEA266229). All data are presented as the means ± SDs. *, P < 0.05; **, P < 0.01; ***, P < 0.001 for comparisons (Student t test). D, Image of mice created in BioRender. Yang, H. (2025) https://BioRender.com/twioz0d.

Figure 5.

The OXTR activates the Hippo/YAP axis by decreasing YAP phosphorylation. A‒D, Immunoblot analysis showing the expression levels of the OXTR, YAP phospho-tag, and total YAP in SNU449 and SNU761 cells treated with the indicated methods for 48 hours. β-Actin was used as the internal control. E‒H, CTGF and CYR61 mRNA levels were determined by qRT-PCR in SNU449 and SNU761 cells treated with the indicated method for 48 hours. I‒L, TEAD response element transcriptional activity was detected in SNU449 and SNU761 cells treated with the indicated method for 48 hours. M‒P, Nucleoplasm separation by immunoblotting confirmed that YAP was located in the cytoplasm and nucleus after 48 hours of incubation with the indicated methods. Q‒T, Immunofluorescence staining assay showing the localization patterns of YAP. OXTR overexpression and carbetocin promote the translocation of YAP from the cytoplasm to the nucleus. Endogenous YAP (green) and nuclei (blue) were stained with specific antibodies and DAPI, respectively. Scale bar, 10 μm. Quantification of the subcellular localization of YAP in at least 100 randomly selected cells. Three independent experiments were conducted to obtain the results. All data are presented as the means ± SDs. **, P < 0.01; ***, P < 0.001 for comparisons (Student t test).

Figure 6.

The OXTR activates YAP through the Gα_q/11–ROCK–LATS axis in HCC. A and B, OXTR activation decreases YAP phosphorylation through Gα_q/11. SNU761 cells were transiently transfected with siControl, siGα_q/11, or siGα_i. After 24 hours, Flag-OXTR or carbetocin was added. The mixture was incubated for another 24 hours. The levels of YAP, phosphorylated YAP, Gα_q/11, Gα_i, and Flag were determined by immunoblotting. C‒F, OXTR activation induces YAP nuclear localization through Gα_q/11. Endogenous YAP (green) and nuclei (blue) were stained with specific antibodies and DAPI, respectively. Scale bar, 10 μm. Quantification of the subcellular localization of YAP in at least 100 randomly selected cells. D and F are presented as the means ± SDs. ***, P < 0.001 for comparisons (Student t test). ns, nonsignificant. G and H, OXTR activation inhibits LATS1 activity. SNU761 cells were treated via the indicated method. LATS1 was immunoprecipitated. Phosphorylation of YAP by LATS1 was determined with a phospho-YAP antibody. I and J, Ectopic expression of LATS1 blocks YAP dephosphorylation induced by the OXTR. SNU761 cells were transiently transfected with control, LATS1 wild-type (WT), or kinase-dead mutant (K/R) strains. Twenty-four hours later, the cells were treated with the OXTR plasmid or carbetocin for another 24 hours. The phosphorylation and protein levels of YAP were determined by immunoblotting. K and L, Rho GTPase is involved in the dephosphorylation of YAP induced by the OXTR. SNU761 cells were transiently transfected with control, Myc-Rho-L63, or C3. Twenty-four hours later, the cells were treated with the OXTR plasmid or carbetocin for another 24 hours. Total and phosphorylated YAP protein levels were determined by immunoblotting. M and N, ROCK is required for OXTR-induced YAP activation. SNU761 cells were pretreated with the OXTR plasmid or carbetocin for another 24 hours, followed by treatment with GSK429286 (1 mmol/L) or Y27632 (1 mmol/L) for 4 hours. The total and phosphorylated YAP protein levels were determined by immunoblotting. Three independent experiments were conducted to obtain the results.

Figure 7.

The OXTR interacts with Gα_q/11 via several important sites (R137, I141, and I227) and promotes YAP activity via the Gα_q/11–ROCK–LATS axis. A and B, Overall structure of the OXTR bound to oxytocin (PDB code: 7RYC). Oxytocin, the OXTR, Gα, Gβ, and Gγ are yellow, brown, cyan, green, and purple, respectively. The critical interactions between the OXTR and Gα are magnified (right), and the residues referred to in the text are labeled. Software: All the structure figures were analyzed and rendered via PyMOL (https://pymol.org/2/). Four mutant OXTR plasmids were constructed according to the potential binding sites. C, Immunoprecipitation assay showing the interaction between mutant OXTRs and Gα_q/11. D, Immunoblot analysis showing the expression levels of total YAP and p-YAP¹²⁷ in SNU761 cells treated with the indicated plasmids for 48 hours. β-Actin was used as the internal control. E and F, Immunofluorescence staining assay showing the localization patterns of YAP. Endogenous YAP (green) and nuclei (blue) were stained with specific antibodies and DAPI, respectively; scale bar, 10 μm. Quantification of the subcellular localization of YAP in at least 100 randomly selected cells. G, Nucleoplasm separation experiments by immunoblotting confirmed that I227, I141, and R137 of the OXTR were important for promoting YAP nuclear accumulation in MGC SNU761 cells. H, CTGF and CYR61 mRNA levels were determined by qRT-qPCR in SNU761 cells treated with the indicated method for 48 hours. I, TEAD response element transcriptional activity was detected in SNU761 cells treated with the indicated method for 48 hours. J and K, Colony formation (left) of SNU761 cells treated with the indicated method for 24 hours. L‒O, Transwell and wound-healing assays were used to detect the migration ability of SNU761 cells treated with the indicated method for 24 hours. All data are presented as the means ± SDs. ns, nonsignificant; *, P < 0.05; **, P < 0.01; ***, P < 0.001 for comparisons (Student t test).

Figure 8.

YAP transcriptionally regulates OXTR expression, which forms a positive feedback loop between Hippo/YAP and the OXTR. A, OXTR expression was significantly correlated with that of CCN1 (CYR61) and CCN2 (CTGF) in HCC samples from the TCGA database. B, OXTR genome schematic and database analysis of the binding region of YAP or TEAD to the OXTR. Sites 1‒4 CRISPRi sgRNAs were designed to target the four OXTR sites. C and D, YAP ChIP‒qPCR and qRT-qPCR with or without CRISPRi in YAP-overexpressing SNU449 cells. E‒G, qRT-qPCR indicated that siRNA, VP, or VT104 treatment via the indicated method in SNU449 cells decreased the CTGF and CYR61 mRNA levels. H‒J, Immunoblot analysis showing the expression level of the OXTR in SNU449 cells treated with siYAP, VP, or VT104 via the indicated methods. Treatment inhibited OXTR protein expression and mRNA expression. K‒M, RT-qPCR showing the mRNA expression levels of the OXTR with siYAP, VP, or VT104 treatment via the indicated method in SNU449 cells. N and O, Immunofluorescence imaging of the OXTR (red) and DAPI (blue) in SNU449 cells subjected to the indicated treatments. Scale bar, 10 μm. All data are presented as the means ± SDs. ns, nonsignificant; **, P < 0.01; ***, P < 0.001 for comparisons (Student t test).