SPOP suppresses prostate cancer through regulation of CYCLIN E1 stability

Abstract

SPOP is one of the important subunits for CUL3/SPOP/RBX1 complex tightly connected with tumorigenesis. However, its exact roles in different cancers remain debatable. Here, we identify CYCLIN E1, as a novel substrate for SPOP. SPOP directly interacts with CYCLIN E1 and specific regulates its stability in prostate cancer cell lines. SPOP/CUL3/RBX1 complex regulates CYCLIN E1 stability through poly-ubiquitination. CDK2 competes with SPOP for CYCLIN E1 interaction, suggesting that SPOP probably regulates the stability of CDK2-free CYCLIN E1. CYCLIN E1 expression rescued proliferation, migration, and tumor formation of prostate cancer cell suppressed by SPOP. Furthermore, we found SPOP selectively regulates the substrates' stability and signaling pathways in prostate cancer and CCRC cell lines, suggesting that complicated mechanisms exist for SPOP to regulate substrate specificity. Altogether, we have revealed a novel mechanism for SPOP in suppressing prostate cancer and provided evidence to show SPOP has dual functions in prostate cancer and CCRC.

Fig. 1

SPOP regulates the stability of exogenous expressed CYCLIN E1. a HEK293 cells were transfected with indicated plasmids for 24 h followed by treatment with 10 μM MG132 for 12 h, and co-IP was performed with anti-Flag or anti-HA antibodies. b His-SPOP and GST-CCNE1 were expressed in bacteria and affinity purified. GST-pulldown assay was performed to study the direct interactions between CYCLIN E1 and SPOP. c HEK293 cells were transfected with indicated plasmids. Twenty-four hours after transfection, cells were treated with 10 μM MG132 for 12 h and harvested for WB. d HEK293 cells were transfected with indicated plasmids. After 24 h, cells were treated with DMSO, MG132 (10 μM for 12 h), CQ (20 μM for 9 h), NH₄Cl (20 mM for 9 h), 3-MA (5 mM for 9 h), and harvested for WB. e DU145 cells were transfected with the indicated plasmids and treated with 100 μg/ml cycloheximide (CHX) 24 h later. Cells were then harvested at various time points for WB. CYCLIN E1 protein abundance was quantified by ImageJ and plotted as indicated. *means p-value < 0.05; **means p-value < 0.01

Fig. 2

SPOP regulates endogenous CYCLIN E1 specific in prostate and bladder cancer cell lines. a, b The indicated cell lines were transfected with control or SPOP-specific siRNAs. After 72 h, cells were harvested for WB. c After treatment with DMSO or MG132 (10 mM) for 12 h, DU145 lysates were prepared for co-IP with CYCLIN E1 antibody. d DU145 and PC3 cells were transfected with HA- or Myc-SPOP plasmid. After 24 h, cells were treated with DMSO or MG132 (10 mM) for 12 h and endogenous CYCLIN E1 level was assayed with WB. e Flag-SPOP (green) was expressed in PC3 cells and endogenous CYCLIN E1 (red) was assayed by immunofluorescent staining. f SPOP was knocked down in DU145 cells, and 48 h later cells were treated with 100 μg/ml cycloheximide (CHX), and harvested at various time points for WB. CYCLIN E1 protein abundance was quantified by ImageJ and plotted as indicated. *means p-value < 0.05

Fig. 3

SPOP/CUL3/RBX1 complex poly-ubiquitinates CYCLIN E1. a HEK293 cells were transfected with indicated plasmids for 24 h followed by MG132 (10 mM) treatment for 12 h. Immunoprecipitated Flag-CCNE1 were analyzed for ubiquitination with anti-HA WB. b SPOP/CUL3/RBX1 complex was expressed in insect cells and affinity purified. In vitro ubiquitination assay was performed together with bacteria-expressed E1, E2, and GST-CCNE1. c Flag-CCNE1 was expressed in HEK293 cells with SPOP wild-type or mutants. Twenty-four hours after transfection, cells were harvested for WB. d HEK293 cells were transfected with indicated plasmids. Co-immunoprecipitation was performed to study CYCLIN E1-SPOP interaction after treatment with 10 μM MG132 for 12 h. e HEK293 cells were transfected with indicated plasmids and treated with 10 μM MG132 for 12 h. Ubiquitination assay was performed to study the effect of SPOP mutants on CYCLIN E1 ubiquitination

Fig. 4

CDK2 inhibits CYCLIN E1 degradation promoted by SPOP. a PC3, DU145, SV-HUC-1 cells were transfected with SPOP/FBXW7/RhoBTB3 siRNAs. Seventy-two hours after transfection, cells were harvested for WB. b, c HEK293 cells were transfected with indicated plasmids. Twenty-four hours later, cells were harvested and Flag-CCNE1 protein level (b) and ubiquitination (c) were studied. d CCNE1 plasmids were constructed with mutation of CDK2 phosphorylation sites. Then CYCLIN E1 mutants were expressed in HEK293 with or without SPOP and western blotting was performed to study CYCLIN E1 protein level. e CYCLIN E1 mutants abolishing CDK2 interaction were generated and co-expressed with CDK2 wild-type or kinase dead mutant in HEK293. Western blotting was performed to study CYCLIN E1 protein stability. f Cells were transfected with indicated plasmids and the interaction between Flag-CCNE1 and HA-SPOP (MATH domain) was studied with co-IP. g GST-pulldown was performed with bacteria-expressed proteins. The addition of His-CDK2 inhibits the interaction between GST-CCNE1 and His-SPOP

Fig. 5

SPOP inhibits proliferation and migration of prostate cancer cell via CYCLIN E1. a SPOP stable knockdown cell line in DU145 was made with retroviral infection system. Cells were synchronized with double-thymidine block, and 8 h after release cells were pulse-labeled with 10 μM BrdU for 30 min and assayed with flow cytometry b Cell proliferation of SPOP knockdown cells in a, assayed with MTT. *means p-value < 0.05. c SPOP or CCNE1 was transiently knocked down with siRNAs in DU145 cells as indicated. BrdU-incorporation assay was carried out 4 h after release from synchronization. d, e SPOP and CYCLIN E1 stable expressed DU145 cells were generated with lentivirus expressing system. The real-time cell proliferation (d) and migration (e) were measured with RTCA assay according to the manufacturer’s protocol. f Plate colony formation assay of CYCLIN E1 and SPOP stable expressed DU145 cells. Colony numbers was counted and plotted as indicated. ***means p-value = 0.0005. g, h Xenograft experiments of SPOP and CYCLIN E1 stable expressed DU145 cells. In all, 5 × 10⁶ cells were injected subcutaneously into the nude mice and tumors were harvested 27 days later. Tumors were pictured (g) and tumor volumes were shown as mean ± SD, n = 9 (h). One tumor from each group was random picked and assayed with western blotting to confirm they are from original cell lines (g, right). * means p < 0.05

Fig. 6

SPOP plays opposite roles in PCa and CCRC tumorigenesis. a–c Xenograft experiments were performed in nude mice with DU145 or 769-P cells stable expressing SPOP. The resulted tumor tissues were pictured (a). Tumor volumes (b) and weights (c) of DU145 were shown as mean ± SEM, n = 9. ***means p < 0.005. d DU145, PC3, 769-P, and 786-O cells were transfected with control or two independent SPOP-specific siRNAs. Seventy-two hours after transfection, cells were harvested for WB. e DU145 or 769-P cells infected with lentiviral empty vector or SPOP was assayed with WB. f Venn-diagram shows the overlapped genes of DEGs between TCGA human cancer samples (PCa+CRCC) and DEGs of SPOP RNAi (DU145+769-P). g Heat map of the overlapped genes in f. Cancer-related genes were labeled with red color