KPNA5 Suppresses Malignant Progression of Ovarian Cancer Through Importing the PTPN4 Into the Nucleus

Figures

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  FIGURE 1. Aberrantly repressed expression of KPNA5 and its positive association with prognostic outcomes in ovarian cancer. (A) Heatmap of significantly differential expressed karyopherin family member genes (KYPs) in ovarian tumor tissue samples versus normal ovary tissue samples from three microarray‐based GEO cohorts. (B, C) Replication of significant downregulation of KPNA5 in OC tumor samples from large RNA‐seq based OC cohorts: TCGA and GSE209964. (D, E) Negative association of KPNA5 expression and tumor stage in TCGA‐OV (D) and CPTAC2‐Ovary (E) cohort. (F, G) Higher expression of KPNA5 were associated with longer overall survival period in GSE26712 (F) and GSE63885 (G). (H) IHC staining analysis of KPNA5 protein expression levels in ovarian cancer tissues and normal ovarian tissues. (I) Correlation analysis the association between KPNA5 expression and patients' clinical stage and lymph node metastasis. (J) Lower expression level of KPNA5 is associated with shorter overall survival time of ovarian cancer patients in our cohort.

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  FIGURE 2. KPNA5 exerts tumor suppressive roles in ovarian cancer cells. (A) Western blot analysis was conducted to determine the overexpression efficiency of KPNA5 in SKOV3 and OVCAR3 cells. (B) CCK8 assay was conducted to determine the effect of KPNA5 overexpression on the cell proliferation of SKOV3 and OVCAR3 cells. (C) CCK8 assay was conducted to determine the effect of KPNA5 downregulation on the cell proliferation of SKOV3 and OVCAR3 cells. (D) colony formation assay was conducted to determine the effect of KPNA5 overexpression on the colony‐forming capacity of SKOV3 and OVCAR3 cells. (E) colony formation assay was conducted to determine the effect of KPNA5 downregulation on the colony‐forming capacity of SKOV3 and OVCAR3 cells. (F) Subcutaneous xenograft tumor model was generated to determine the effect of KPNA5 overexpression on SKOV3 cells derived tumor growth in vivo. (G) Weighing of the tumors from mice. (H) H&E and IHC staining analysis of the Ki67 and KPNA5 expression levels in the tumor tissues from the mice. Three individual experiments were performed, and error bars of the data indicate mean ± SEM, **p < 0.01; ***p < 0.001.

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  FIGURE 3. KPNA5 represses ovarian cancer cells migration and invasion in vitro. (A, B) Transwell assays were conducted to determine the effect of KPNA5 overexpression on SKOV3 and OVCAR3 cells migration. (C, D) Transwell assays were conducted to determine the effect of KPNA5 downregulation on SKOV3 and OVCAR3 cells migration. (E, F) Transwell assays were conducted to determine the effect of KPNA5 overexpression on SKOV3 and OVCAR3 cells invasion. (G, H) Transwell assays were conducted to determine the effect of KPNA5 downregulation on SKOV3 and OVCAR3 cells invasion. Three individual experiments were performed, and error bars of the data indicate mean ± SEM, **p < 0.01.

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  FIGURE 4. KPNA5 mediates the nuclear transporting of cargo protein PTPN4. (A) Silver‐stained image shows the protein bands interacting with KPNA5 in SKOV3 cells. (B, C) IP and Western blot analysis of the interaction between KPNA5 and PTPN4 in ovarian cancer cells. (D) Nucleus and cytoplasm fraction analysis of the effect of KPNA5 on PTPN4 nucleus translocation. (E) IF staining assays determine the effect of KPNA5 overexpression on PTPN4 protein distribution in SKOV3 and OVCAR3 cells. Three individual experiments were performed, and error bars of the data indicate mean ± SEM.

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  FIGURE 5. KPNA5 exerts tumor suppressive function through regulating PTPN4/STAT3 signaling pathway. (A) Western blot was conducted to determine the overexpression efficiency of wide‐type and mutant PTPN4 in ovarian cancer cells. (B) CCK8 assays were performed to evaluate the effect of PTPN4‐WT and PTPN4‐Mut expression on ovarian cancer cells proliferation. (C) Colony formation were performed to evaluate the effect of PTPN4‐WT and PTPN4‐Mut expression on ovarian cancer cells clone formation capacity. (D–G) Transwell assays were performed to evaluate the effect of PTPN4‐WT and PTPN4‐Mut expression on ovarian cancer cells migration and invasion. (H), Western blot was conducted to determine the overexpression efficiency of wide‐type and mutant PTPN4 on the STAT3 protein phosphorylation. Three individual experiments were performed, and error bars of the data indicate mean ± SEM, **p < 0.01; **p < 0.001; ns, no significant.