SPOP targets oncogenic protein ZBTB3 for destruction to suppress endometrial cancer

Abstract

Dysregulation of the ubiquitin-proteasome pathway is closely associated with cancer initiation and progression. SPOP is an adapter protein of the CUL3-based E3 ubiquitin ligase complexes. Several whole genome/exome sequencing studies on endometrial cancers (ECs) revealed that the SPOP gene is frequently mutated. However, how SPOP mutations contribute to EC remains poorly understood. In this study, transcription factor ZBTB3 was identified as a proteolytic substrate for the SPOP-CUL3-RBX1 E3 ubiquitin ligase complex. SPOP specifically recognizes two Ser/Thr (S/T)-rich degrons located in ZBTB3 and triggers the degradation of ZBTB3 via the ubiquitin-proteasome pathway. By contrast, EC-associated SPOP mutants are defective in regulating ZBTB3 stability. SPOP inactivation promotes endometrial cell proliferation, migration, and invasion partly through ZBTB3 accumulation. Sonic hedgehog (SHH) was found to be a transcriptional target of ZBTB3. SPOP inactivation leads to ZBTB3-dependent SHH upregulation in EC cells. RUSKI-43, a small molecule inhibitor of SHH, suppresses cell proliferation, migration, and invasion in SPOP-depleted or EC-associated SPOP mutant-overexpressed EC cells. Our data indicate that pharmacological inhibition of SHH represents a possible treatment strategy for SPOP-mutated ECs.

Keywords: SPOP; ZBTB3; endometrial cancer; sonic hedgehog; ubiquitination.

Figure 1

Identification of ZBTB3 as a novel SPOP Interactor. Western blot of whole cell lysates (WCLs) and co-IP samples of anti-FLAG antibody obtained from 293 T cells transfected with indicated plasmids (A, B). (C) Western blot of WCLs and co-IP samples of anti-FLAG antibody obtained from ECC-1 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-ZBTB3 antibodies obtained from cell lysates of ECC-1 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 ZBTB3 is indicated with the symbol. (F) Western blot of WCLs and co-IP samples of anti-FLAG antibody obtained from 293 T cells transfected with indicated plasmids.

Figure 2

ZBTB3 is a bona fide substrate of the SPOP-CUL3-RBX1 E3 ubiquitin ligase complex. (A) Western blot of WCLs 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. (B) Western blot of WCLs of 293 T cells transfected with indicated plasmids. (C) Western blot of WCLs of ECC-1 cells infected with empty vector (EV) or lentivirus expressing wild-type or mutant SPOP. (D) Western blot of the WCLs of ECC-1 cells infected with control or lentivirus expressing SPOP-specific shRNAs (shSPOP#1,2). (E) Western blot of the WCLs of HEC-1-A cells infected with control or lentivirus expressing SPOP-specific shRNAs (shSPOP#1,2). (F) Quantitative RT-PCR measurement of SPOP and ZBTB3 mRNA levels in SPOP-depleted ECC-1 cells. GAPDH mRNA levels were used for normalization. Data are shown as means ± SD (n=3). *P < 0.01. (G) Western blot of WCLs of ECC-1 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. (H) At each time point, the intensity of each ZBTB3 protein was normalized to the intensity of actin and then to the value at 0 h. Western blot of the WCLs of ECC-1 cells transfected with control siRNAs or siRNAs towards Cul3 (I) or RBX1 (J). (K) 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.

Figure 3

Identification of degrons in ZBTB3 are required for SPOP-mediated ZBTB3 degradation. A. Diagram showing wild-type ZBTB3 proteins and SBC motif-deleted mutants. The SBC motif is depicted in red. B. Western blot of WCLs and co-IP samples of anti-FLAG antibody obtained from 293 T cells transfected with indicated plasmids. C. Western blot of WCLs of 293 T cells transfected with indicated plasmids. D. Western blots of WCLs 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. At each time point, the intensity of ZBTB3 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.

Figure 4

EC-associated mutants of SPOP are defective in promoting ZBTB3 degradation and ubiquitination. A. Distribution of the most common mutations in the SPOP gene found in endometrial cancer samples. B. Western blot of WCLs of 293 T cells transfected with indicated plasmids. C. Western blot of WCLs 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 the indicated proteins in ECC-1 cells infected with empty vector (EV) or lentivirus expressing wild-type or mutant SPOP. F. Western blot of WCLs and co-IP samples of anti-FLAG antibody obtained from 293 T cells transfected with indicated plasmids. G. Western blot of the in vivo ubiquitination assay in 293T cells transfected with the indicated plasmids and treated with 20 μM MG132 for 8 h.

Figure 5

SPOP suppresses cell proliferation, migration and invasion partially dependent on ZBTB3. (A) Western blot (left panel) and cell proliferation assay (right panel) of ECC-1 cells infected with lentivirus expressing the indicated shRNAs. Standard deviation (S.D.) of at least three independent experiments is shown to indicate statistical significance. *P < 0.05. (B) Western blot (left panel) and cell proliferation assay (right panel) of ECC-1 cells infected with empty vector or lentivirus expressing HA-SPOP-G75R in combination with control shRNA or ZBTB3-spefic shRNAs. Data are shown as means ± SD (n=3). *P < 0.05. (C) Cell colony formation assay of ECC-1 cells infected with lentivirus expressing the indicated shRNAs. All data shown are mean values ± SD from three replicates. *P < 0.05. (D) Cell colony formation assay of ECC-1 cells infected with empty vector or lentivirus expressing SPOP-G75R in combination with control shRNA or ZBTB3-spefic shRNAs. Cell migration (E) and invasion (F) assay of ECC-1 cells infected with lentivirus expressing the indicated shRNAs. Data are shown as means ± SD (n=3). *P < 0.05. (G, H) Cell migration (G) and invasion (H) assay of ECC-1 cells with lentivirus expressing FLAG-SPOP-G75R in combination with control shRNA or ZBTB3-spefic shRNAs. Data are shown as means ± SD (n=3). *P < 0.05.

Figure 6

ZBTB3 regulates the mRNA expression of a subset genes. (A) Hierarchical clustering of the differentially expressed genes in sh-control and ZBTB3-depleted ECC-1 cells. (B) Quantitative data of up- and down-regulated genes shown in (A). (C) RT-qPCR measurement of the mRNA expression of differentially expressed genes in sh-control and ZBTB3-depleted ECC-1 or HEC-1-A cells. Data are shown as means ± SD (n=3). *P < 0.05; **P < 0.01. (D) RT-qPCR measurement of the mRNA expression of differentially expressed genes in EV, SPOP-WT or SPOP-M3 mutant overexpressed ECC-1 or HEC-1-A cells. Data are shown as means ± SD (n=3). *P < 0.05; **P < 0.01. (E) RT-qPCR measurement of the mRNA expression of differentially expressed genes in sh-control, SPOP/ZBTB3-depleted ECC-1 or HEC-1-A cells. Data are shown as means ± SD (n=3). *P < 0.05.

Figure 7

SPOP suppresses ZBTB3-SHH signaling. (A) Western blot of the indicated proteins in WCLs from ECC-1 or HEC-1-A cells infected with indicated shRNAs. (B) Western blot of the indicated proteins in WCLs from ECC-1 infected with the indicated shRNAs. (C) Western Blot of the indicated proteins in WCLs from of EV and FLAG-SPOP-G75R-overexpressing ECC-1 cells. (D) Quantitative RT-qPCR measurement of mRNA expression of SHH from of EV and FLAG-SPOP-G75R-overexpressing ECC-1 cells. Data are shown as means ± SD (n=3). *P < 0.05. (E) Schematic representation of ZBTB3 deletion mutants. (F) Western blot of the indicated proteins in WCLs obtained from ECC-1 cells transfected with indicated plasmids. (G) Quantitative RT-qPCR measurement of mRNA expression of SHH in ECC-1 cells infected with the indicated plasmids. Data are shown as means ± SD (n=3). *P < 0.05. (H) 293T cells were transfected with the SHH-luc reporter, pTKgalactosidase (internal control), and indicated plasmids. After 24 hr, the luciferase activities were measured by luminometer. Data are shown as means ± SD (n=3). *P < 0.05; **P < 0.01. (I) Western blot and cell proliferation assay of ECC-1 cells infected with lentivirus expressing EV or FLAG-SPOP-G75R, and treated with DMSO or RUSKI-43 (10 μM). Data are shown as means ± SD (n=3). *P < 0.05; **P < 0.01. (J) Cell colony formation assay of ECC-1 cells infected with lentivirus expressing EV or FLAG-SPOP-G75R, and treated with DMSO or RUSKI-43 (10 μM). Data are shown as means ± SD (n=3). *P < 0.05. Cell migration (K) and invasion (L) assay of ECC-1 cells infected with lentivirus expressing EV or FLAG-SPOP-G75R, and treated with DMSO or RUSKI-43 (10 μM). Data are shown as means ± SD (n=3). *P < 0.05.

Figure 8

Schematic of the proposed mechanism through which SPOP mutants enhance ZBTB3-SHH axis-induced malignant transformation in SPOP-mutated endometrial cancer.