Fig 2: Expression and pathological significance of RSPO2 in ovarian cancer(A) Relative mRNA expression levels of RSPO2 in ovarian cancer cell lines. RSPO2 mRNA levels were determined using RT-qPCR. mRNA expression levels were normalized to those in normal ovarian epithelial cells (IOSE80).(B) Representative images of immunohistochemical staining for RSPO2 in a tissue microarray containing thirty ovarian tumors (T) and paired normal ovarian (N) tissues. Scale bar, 50 µm.(C) Immunohistochemical staining score for RSPO2 in the tissue microarray (n = 30). **p < 0.01, calculated by two-tailed unpaired t-test.(D) Kaplan-Meier survival analysis of ovarian cancer patients with RSPO2-high vs. RSPO2-low tumors from the TCGA cohort.(E) Kaplan-Meier survival analysis of ovarian cancer patients with RSPO2-high vs. RSPO2-low tumors from the GSE26193 cohort.

Fig 3: Schematic diagram illustrating the dual mechanism of RSPO2-mediated FAK/Src signaling activationIn the presence of RSPO2, endocytic degradation of LGR4 is attenuated, which increases the accumulation of LGR4 on the plasma membrane, leading to plasma membrane translocation and autophosphorylation of Src. RSPO2 can also increase the stability of integrins by directly binding to integrin ß3, leading to enhanced FAK phosphorylation. Constitutive activation of FAK/Src signaling promotes the growth and metastasis of ovarian cancer.

Fig 4: RSPO2 promotes ovarian cancer growth and metastasis(A) Representative images (upper panel) and quantification (lower panel) of the colony formation results of A2780 and OVCAR3 cells after RSPO2 overexpression. Cells stably overexpressing RSPO2 (RS2, pooled) or empty vector (Vec) were used. ***p < 0.001 vs. Vec; two-tailed Student’s t-test. Error bars indicate mean ± SD.(B) Colony formation assay in A2780 and OVCAR3 cells after shRNA-mediated RSPO2 knockdown. The lentiviral vector containing shRNA against RSPO2 (shRS2) was designed using the most effective interference sequence, siRS2-2#. **p < 0.01 and ***p < 0.001 vs. control (shNC); two-tailed Student’s t-test. Error bars indicate mean ± SD.(C and D) Representative images (upper panel) and quantification (lower panel) of the Transwell assay results show that the overexpression of RSPO2 enhanced the migration (C) and invasion (D) of ovarian cancer cells.(E) Wound healing assay showing increased wound closure in the RSPO2-overexpressing cell group.(F) Representative images of tumor nodules in the peritoneum (upper panel) and ovary (lower panel) in the orthotopic mouse model. A2780 and OVCAR3 cells stably overexpressing RSPO2 (RS2, pooled) or empty vector (Vec) were inoculated into the ovary (n = 5 per group). The white dashed circles indicate the metastatic nodules and the black arrow shows the tumor formed in ovarian in situ.(G–I) Disseminated tumor nodules and ascites fluid in the peritoneal cavity were harvested and measured after mice were sacrificed. The total number of nodules (G), aggregate tumor weight (H), and ascites weight (I) were calculated for each mouse.**p < 0.01 and ***p < 0.001 vs. Vec; two-tailed Student’s t-test. Error bars indicate mean ± SD.

Fig 5: RSPO2 promotes ovarian cancer growth and metastasis through FAK/Src signaling cascades(A) KEGG enrichment analysis of the pathways affected by overexpression of RSPO2 in A2780 cells.(B) Effects of RSPO2 overexpression on the phosphorylation of FAK (Tyr397), Src (Tyr416, Y416), Src (Tyr527, Y527), EGFR (Tyr1068), and Akt (Ser473). Proteins were extracted from A2780 and OVCAR3 cells stably overexpressing RSPO2 (RS2, pooled) or empty vector (Vec) and were analyzed by Western blotting.(C) The effects of RSPO2 knockdown on the phosphorylation of FAK, Src, and Akt were analyzed by Western blotting. Cells were infected with lentivirus containing shRS2 or nontargeting shNC for 48 h.(D) Representative images (left panel) and quantification (right panel) of Transwell assay results showing that the FAK inhibitor defactinib (Def) abolished the promotive effect of RSPO2 overexpression on cell migration. Cells were treated with DMSO or 1.0 µM defactinib for 3 h.(E) Representative images (left panel) and quantification (right panel) of colony formation assay results showing that the Src inhibitor saracatinib (Sar) abolished the promotive effect of RSPO2 overexpression on cell proliferation. Cells were treated with DMSO or saracatinib (Sar, 10 µM) for 1 h.(F) Defactinib abolished the effect of RSPO2 on the expression of EMT markers and MMP7. A2780 and OVCAR3 cells stably overexpressing RSPO2 (RS2, pooled) or empty vector (Vec) were treated with DMSO or 1.0 µM defactinib for 3 h.(G) Saracatinib blocked RSPO2-induced EGFR/Akt phosphorylation.Cells were treated with DMSO or saracatinib (Sar, 10 µM) for 24 h n.s., not significant; p*** <0.001; two-tailed Student’s t test. Error bars indicate mean ± SD.