SPOP promotes ATF2 ubiquitination and degradation to suppress prostate cancer progression

Abstract

Background: Next-generation sequencing of the exome and genome of prostate cancers has identified numerous genetic alterations. SPOP (Speckle-type POZ Protein) is one of the most frequently mutated genes in primary prostate cancer, suggesting that SPOP may be a potential driver of prostate cancer. The aim of this work was to investigate how SPOP mutations contribute to prostate cancer development and progression.

Methods: To identify molecular mediators of the tumor suppressive function of SPOP, we performed a yeast two-hybrid screen in a HeLa cDNA library using the full-length SPOP as bait. Immunoprecipitation and Western Blotting were used to analyze the interaction between SPOP and ATF2. Cell migration and invasion were determined by Transwell assays. Immunohistochemistry were used to analyze protein levels in patients' tumor samples.

Results: Here we identified ATF2 as a bona fide substrate of the SPOP-CUL3-RBX1 E3 ubiquitin ligase complex. SPOP recognizes multiple Ser/Thr (S/T)-rich degrons in ATF2 and triggers ATF2 degradation via the ubiquitin-proteasome pathway. Strikingly, prostate cancer-associated mutants of SPOP are defective in promoting ATF2 degradation in prostate cancer cells and contribute to facilitating prostate cancer cell proliferation, migration and invasion.

Conclusion: SPOP promotes ATF2 ubiquitination and degradation, and ATF2 is an important mediator of SPOP inactivation-induced cell proliferation, migration and invasion.

Keywords: ATF2; Prostate cancer; Proteasomal degradation; SPOP; Ubiquitination.

Fig. 1

Identification of ATF2 as a novel SPOP Interactor. a and b Western blot of whole cell lysates(WCL) and co-IP samples of anti-FLAG antibody obtained from 293 T cells transfected with indicated plasmids. c Western blot of WCL and co-IP samples of anti-FLAG antibody obtained from C4–2 cells infected with lentivirus expressing FLAG-SPOP or control. The cells were treated with 20 μM MG132 for 8 h before harvesting. d Western blot of co-IP samples of IgG or anti-ATF2 antibodies obtained from cell lysates of C4–2 cells. The cells were treated with 20 μM MG132 for 8 h before harvesting. e Schematic representation of SPOP deletion mutants. Binding capacity of SPOP to ATF2 is indicated with the symbol. f Western blot of WCL and co-IP samples of anti-FLAG antibody obtained from 293 T cells transfected with indicated plasmids

Fig. 2

ATF2 is a bona fide substrate of the SPOP-CUL3-RBX1 E3 ubiquitin ligase complex. a Western blot of WCL from 293 T cells transfected with the indicated plasmids. and treated with MG132 (20 μM), Bortezomib (200 nM), Chloroquine (100 mM) or DMSO for 8 h. Actin was used as a loading control. b Western blot of WCL of 293 T cells transfected with indicated plasmids. c Western blot of WCL of C4–2 cells transfected with indicated plasmids. d Western blot of WCL of 293 T cells transfected with indicated plasmids. e Western blot of the WCL of C4–2 infected with control or lentivirus expressing SPOP-specific shRNAs (shSPOP#1,2). f Quantitative RT-PCR measurement of SPOP and ATF2 mRNA levels in SPOP-depleted C4–2 cells. GAPDH mRNA levels were used for normalization. Standard deviation (S.D.) of at least three independent experiments is shown to indicate statistical significance. **p<0.01. g Western blot of WCL of C4–2 cells infected with control or lentivirus expressing SPOP-specific shRNAs and then treated with 50 μg/ml cycloheximide (CHX) and harvested at different time points (upper panel). At each time point, the intensity of each BET protein was normalized to the intensity of actin and then to the value at 0 h (lower panel). h Western blot of the WCL of C4–2 cells infected with control or lentivirus expressing CUL3 or RBX1-specific shRNAs (sh-CUL3#1,2; sh-RBX1#1,2). i Western blots of the products of in vivo ubiquitination assays performed using cell lysate from 293 T cells transfected with the indicated plasmids and treated with 20 μM MG132 for 8 h

Fig. 3

Multiple Ser/Thr (S/T)-rich motifs in ATF2 are required for SPOP-mediated ATF2 degradation. a Diagram showing wild-type ATF2 proteins and S/T rich motif-deleted mutants. The S/T rich motif is depicted in red. b Western blot of WCL of 293 T cells transfected with indicated plasmids. c Western blot of WCL and co-IP samples of anti-FLAG antibody obtained from 293 T cells transfected with indicated plasmids. d and e Western blots of WCL from 293 T cells transfected with the indicated constructs, treated with 50 μg/ml cycloheximide (CHX) and harvested at different time points. e Quantification of the western blots carried out in d. At each time point, the intensity of ATF2 protein was normalized to the intensity of actin and then to the value at 0 h. f Western blots of the products of in vivo ubiquitination assays performed using cell lysate from 293 T cells transfected with the indicated plasmids and treated with 20 μM MG132 for 8 h

Fig. 4

Prostate cancer-associated mutants of SPOP are defective in promoting ATF2 degradation and ubiquitination. a Distribution of the most common mutations in the SPOP gene found in prostate cancer. b Western blot of WCL of 293 T cells transfected with indicated plasmids. c Western blot of WCL and co-IP samples of anti-FLAG antibody obtained from 293 T cells transfected with indicated plasmids. d Western blots of the products of in vivo ubiquitination assays performed using cell lysate from 293 T cells transfected with the indicated plasmids and treated with 20 μM MG132 for 8 h. e Western blot of WCL and co-immunoprecipitation of anti-FLAG antibody in lysates from 293 T cells transfected with the indicated plasmids. f Western blot of the in vivo ubiquitination assay in 293 T cells transfected with the indicated plasmids and treated with 20 μM MG132 for 8 h

Fig. 5

SPOP suppresses cell proliferation, invasion and migration partially dependent on ATF2. a Cell proliferation assay (left panel) and western blot (right panel) of C4–2 cells infected with lentivirus expressing the indicated shRNAs. All data shown are mean values ± SD (error bar) from three independent experiments. *p < 0.05 in C4–2 cells at day 7. b Assays shown in (A) were performed in C4–2 cells infected with empty vector or lentivirus expressing HA-SPOP-F133 V in combination with control shRNA or ATF2-spefic shRNAs. c and d Cell migration (C) and invasion (D) assay of C4–2 cells infected with lentivirus expressing the indicated shRNAs. All data shown are mean values ± SD from three replicates. *p < 0.05. e Cell migration and invasion f assay of C4–2 cells infected with lentivirus expressing HA-SPOP-F133 V in combination with control shRNA or ATF2-spefic shRNAs. All data shown are mean values ± SD from three replicates. *p < 0.05. g Quantitative RT-qPCR measurement of mRNA expression of three ATF2 transcriptional targets SOX9, MMP9, and TGFB2 in C4–2 cells. GAPDH mRNA levels were used for normalization. Standard deviation (S.D.) of at least three independent experiments is shown to indicate statistical significance. *p<0.05,**p<0.01, **p<0.001

Fig. 6

ATF2 protein expression is elevated in SPOP mutant-expressing prostate cancer specimens. a Representative images of ATF2 IHC from 90 cases of prostate cancer expressing wild-type SPOP or mutant SPOP (MUT). b Quantitative data of ATF2 protein staining in a. Statistical significance was determined by Wilcoxon rank-sum test