Inhibition of Karyopherin beta 1 suppresses prostate cancer growth

Abstract

Prostate cancer (PCa) initiation and progression requires activation of numerous oncogenic signaling pathways. Nuclear-cytoplasmic transport of oncogenic factors is mediated by Karyopherin proteins during cell transformation. However, the role of nuclear transporter proteins in PCa progression has not been well defined. Here, we report that the KPNB1, a key member of Karyopherin beta subunits, is highly expressed in advanced prostate cancers. Further study showed that targeting KPNB1 suppressed the proliferation of prostate cancer cells. The knockdown of KPNB1 reduced nuclear translocation of c-Myc, the expression of downstream cell cycle modulators, and phosphorylation of regulator of chromatin condensation 1 (RCC1), a key protein for spindle assembly during mitosis. Meanwhile, CHIP assay demonstrated the binding of c-Myc to KPNB1 promoter region, which indicated a positive feedback regulation of KPNB1 expression mediated by the c-Myc. In addition, NF-κB subunit p50 translocation to nuclei was blocked by KPNB1 inhibition, which led to an increase in apoptosis and a decrease in tumor sphere formation of PCa cells. Furthermore, subcutaneous xenograft tumor models with a stable knockdown of KPNB1 in C42B PCa cells validated that the inhibition of KPNB1 could suppress the growth of prostate tumor in vivo. Moreover, the intravenously administration of importazole, a specific inhibitor for KPNB1, effectively reduced PCa tumor size and weight in mice inoculated with PC3 PCa cells. In summary, our data established the functional link between KPNB1 and PCa prone c-Myc, NF-kB, and cell cycle modulators. More importantly, inhibition of KPNB1 could be a new therapeutic target for PCa treatment.

Figure 1.. KPNB1 expression is associated with human prostate cancer progression.

A) The expression level of KPNB1 in human prostate cancer samples (N=498) and normal prostate tissues (N=52). Samples are from TCGA dataset. B) The expression level of KPNB1 human prostate cancer samples with different Gleason scores. Samples are from TCGA dataset. C) The expression level of KPNB1 human prostate cancer samples with different pathological stages. Samples are from TCGA dataset. D) The expression level of KPNB1 of localized and metastasis human prostate cancer samples. Samples are from GEO dataset. E) qPCR results showing relative expression level of KPNB1 RNA in indicated cell lines. FTH1 was used as internal control. F) Immunoblotting results showing protein level of KPNB1 in indicated cell lines. β-actin was used as internal control. G) Representative images of IHC staining of KPNB1 on human prostate cancer TMA slides. H) Statistical results of KPNB1 positive cells on the TMA slides. *p <0.05, **p<0.01, *** p<0.001, ****p<0.0001.

Figure 2.. KPNB1 knockdown inhibits PCa growth.

A and B) Crystal violet staining showing the growth of indicated cell lines with KPNB1 knockdown. Both cell lines were transfected with KPNB1 siRNA (50 nM) or control siRNA for 24 hours, 48 hours and 72 hours respectively. C) MTT assay showing the relative cell viabilities of indicated cell lines that were transfected with KPNB1 siRNA (50 nM) or control siRNA for 48 hours. D and E) Flow cytometry showing the cell cycle stage distribution of indicated cell lines that were transfected with KPNB1 siRNA (50 nM) or control siRNA for 48 hours. F) Immunoblotting results showing protein level of KPNB1, Cyclin D1, Cyclin B1, CDK1, RCC1 and pRCC1 of indicated cell lines that were transfected with KPNB1 siRNA (50 nM) or control siRNA for 48 hours. GAPDH was used as internal control. G) Immunoblotting results showing physical interaction between KPNB1 and c-Myc proteins by immunoprecipitation assay in both of C42B and PC3 cell lines. H) Immunoblotting results showing KPNB1 and c-Myc from nuclear extracts of C42B and PC3 that were transfected with KPNB1 siRNA (50 nM) or control siRNA for 48 hours. *p <0.05, **p<0.01, *** p<0.001, ****p<0.0001.

Figure 3.. c-Myc binds to the promoter regions of KPNB1.

A-D) qPCR showing the interaction of c-Myc with promoter regions of KPNB1. The indicated PCa cell lines were treated with importazole (20 μM) for 24 hours, DMSO was used as vehicle control. DNA of the treated cells were precipitated and purified by c-Myc antibody for CHIP assay. Polymerase II (POLR 2) antibody and control IgG antibody were used as positive and negative control, respectively. Relative amount of precipitated promoter DNA was analyzed using qPCR, and the PCR product was revealed by running an agarose gel. E-G) Analysis of the TCGA dataset to show the correlation of KPNB1 with c-Myc, Cyclin B1 and CDK1 in human prostate cancer samples. Correlations between indicated genes were analyzed by computing Pearson correlation coefficients. P value and R squared were revealed as well. H) Schematic model indicating the promoter region (c-Myc binding sites) of KPNB1. **p<0.01.

Figure 4.. Inhibition of KPNB1 using importazole suppresses PCa growth.

A and B) Crystal violet staining showing the growth of indicated cell lines that were treated with 10 μM or 20 μM of importazole for 24 hours, 48 hours or 72 hours. DMSO was used as vehicle control. C) MTT assay showing relative cell viabilities of PC3 or C42B that was treated with importazole of indicated concentrations for 48 hours. DMSO was used as vehicle control. D and E) Flow cytometry showing the cell cycle stage distribution of indicated cell lines that were treated with 10 μM or 20 μM of importazole for 48 hours. DMSO was used as vehicle control. F) Immunoblotting results showing protein level of Cyclin B1, CDK1, pRCC1 and Cyclin D1 of indicated cell lines that were treated with DMSO, 10 μM or 20 μM of importazole for 48 hours. β-actin was used as internal control. G) Immunoblotting results showing nuclear distribution of c-Myc of indicated cell lines that were treated with 10 μM or 20 μM of importazole for 48 hours. DMSO was used as vehicle control. Lamin A/C was used as loading control of nuclear protein. *p <0.05, **p<0.01, *** p<0.001, ****p<0.0001.

Figure 5.. Inhibition of KPNB1 using importazole induces apoptosis and suppresses sphere formation of PCa through NF-κB signaling.

A) Immunoblotting results showing nuclear NF-κB p50 of PC3 or C42B that was transfected with KPNB1 siRNA (50 nM) or control siRNA for 48 hours. Lamin A/C was used as internal control. B and C) Immunoblotting results showing nuclear NF-κB p50 of PC3 or C42B that was treated with indicated concentrations of importazole or DMSO. Lamin A/C was used as internal control. D and E) Flow cytometry showing the apoptosis of PC3 or C42B that was transfected KPNB1 siRNA (50 nM) or control siRNA for 48 hours. Annexin V positive cells were counted as apoptotic population. F and G) Flow cytometry showing the apoptosis of PC3 or C42B that was treated with indicated concentrations of importazole or DMSO. Annexin V positive cells were counted as apoptotic population. Sphere formation assay of PC3 or C42B that was H) treated with indicated concentrations of importazole or DMSO, or I) transfected with KPNB1 siRNA (50 nM) or control siRNA for 1 week. *p <0.05, **p<0.01, ****p<0.0001.

Figure 6.. KPNB1 knockdown attenuates PCa tumor growth in vivo.

A) Crystal violet staining showing the growth of C42B-scramble or C42B-shKPNB1 cell line. B) Immunoblotting results showing protein level of KPNB1, Cyclin B1, CDK1, pRCC1 of C42B-scramble and C42B-shKPNB1 cell lines. α-Tubulin was used as internal control. C). Immunoblotting results showing NF-κB p50 within nuclear of C42B-scramble and C42B-shKPNB1. Lamin A/C was used as internal control. D) IHC staining of KPNB1, Cyclin D1, Cyclin B1, CDK1, pRCC1 of tumor sections. E) Statistical results of positive stained cells of tumor sections. F and G) Tumor burdens of C42B-scramble and C42B-shKPNB1 through subcutaneous injection were dissected and weighted. *p <0.05, **p<0.01, *** p<0.001.

Figure 7.. Importazole suppresses PCa tumor growth in vivo.

A) Schematic model showing the procedure of importazole treatment in vivo. One million PC3 cells were inoculated to nude mice through subcutaneous injection. After one week, importazole was encapsulated into PEG-PLGA nanoparticle and injected intravenously twice a week for 3 weeks. B) Tumor growth curve of indicated groups. Tumor sizes of both groups were measured weekly. Tumor burdens of indicated groups were C) dissected and D) weighted. E) IHC staining of Cyclin D1, Cyclin B1, CDK1, pRCC1 of tumor sections. F) Statistical results of positive stained cells of tumor sections. IHC staining of G-H) c-Myc and I-J) p50 using tumor tissue form mice treated with NP-IPZ or NP vehicle. Positive cells were quantified using Image J software. *p <0.05, **p<0.01.

Figure 8.. Schematic model of the role of KPNB1 in PCa progression.

Dysregulation of KPNB1 in PCa facilitates the nuclear translocation of NF-κB p50, by which it can promote tumorigenesis and cell survival PCa cells. In addition, KPNB1-c-Myc positive feedback loop increases the expression level of Cyclin D1, Cyclin B1 and CDK1. The complex of Cyclin B1 and CDK1 can further enhances phosphorylation level of RCC1, by which it promotes PCa cell proliferation through accelerating cell cycle and mitosis.