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Endometrial cancer-associated mutants of SPOP are defective in regulating estrogen receptor-α protein turnover.

Zhang P, Gao K, Jin X, Ma J, Peng J, Wumaier R, Tang Y, Zhang Y, An J, Yan Q, Dong Y, Huang H, Yu L, Wang C - Cell Death Dis (2015)

Bottom Line: Increasing amounts of evidence strongly suggests that dysregulation of ubiquitin-proteasome system is closely associated with cancer pathogenesis.Speckle-type POZ protein (SPOP) is an adapter protein of the CUL3-based E3 ubiquitin ligase complexes.It selectively recruits substrates for their ubiquitination and subsequent degradation.

View Article: PubMed Central - PubMed

Affiliation: 1] State Key Laboratory of Genetic Engineering, Collaborative Innovation Center for Genetics and Development, School of Life Sciences, Fudan University, 220 Handan Road, Shanghai 200433, China [2] Shanghai Cancer Center, Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, China.

ABSTRACT
Increasing amounts of evidence strongly suggests that dysregulation of ubiquitin-proteasome system is closely associated with cancer pathogenesis. Speckle-type POZ protein (SPOP) is an adapter protein of the CUL3-based E3 ubiquitin ligase complexes. It selectively recruits substrates for their ubiquitination and subsequent degradation. Recently, several exome-sequencing studies of endometrial cancer revealed high frequency somatic mutations in SPOP (5.7-10%). However, how SPOP mutations contribute to endometrial cancer remains unknown. Here, we identified estrogen receptor-α (ERα), a major endometrial cancer promoter, as a substrate for the SPOP-CUL3-RBX1 E3 ubiquitin ligase complex. SPOP specifically recognizes multiple Ser/Thr (S/T)-rich degrons located in the AF2 domain of ERα, and triggers ERα degradation via the ubiquitin-proteasome pathway. SPOP depletion by siRNAs promotes endometrial cells growth. Strikingly, endometrial cancer-associated mutants of SPOP are defective in regulating ERα degradation and ubiquitination. Furthermore, we found that SPOP participates in estrogen-induced ERα degradation and transactivation. Our study revealed novel molecular mechanisms underlying the regulation of ERα protein homeostasis in physiological and pathological conditions, and provided insights in understanding the relationship between SPOP mutations and the development of endometrial cancer.

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Estrogen potentiates SPOP-mediated degradation of ERα. (a) Estrogen enhances the SPOP-ERα interaction. FH-ERα and Myc-SPOP constructs were co-transfected into 293T cells. After 24 h, cells were treated with the vehicle ethanol (EtOH,−) or 10 nM 17β-estradiol (E2) for 4 h before cell lysates were prepared for co-IP and WB analyzes. (b) Estrogen enhances SPOP-mediated ERα degradation. The 293T cells were transfected with the indicated constructs. A small amount of Myc-SPOP constructs was used in transfection. After 24 h, cells were treated with the vehicle ethanol (EtOH) or 10 nM 17β-estradiol (E2) for 4 h before cells lysates were prepared for WB analyzes. The density of ERα was determined by normalizing to actin (loading control) first and then to the normalized value in mock-treated cells. (c) Knockdown of SPOP attenuates estrogen-induced degradation of ERα. Ishikawa cells were transfected with control or SPOP-specific siRNA. After 48 h, cells were then treated with the vehicle ethanol (EtOH,−) or 10 nM 17β-estradiol (E2) for 4 h before cell lysates were prepared for WB analyzes. (d) Estrogen potentiates SPOP-induced polyubiquitination of ERα. The 293T cells were transfected with the indicated constructs. After 24 h, cells were treated with the vehicle ethanol (EtOH,−) or 10 nM 17β-estradiol (E2). Cells were then treated with MG132 for 4 h before cell lysates were prepared for IP and WB analyzes. (e) Ishikawa cells lines that stably transfected with control, SPOP-WT or SPOP mutants constructs were treated with 10 nM 17β-estradiol (E2) for 24 h. The mRNA level of ERα target gene GREB1 was measured by qRT-PCR. The mRNA level of GAPDH was used for normalization. The mean values (S.D.) of three independent experiments are shown. **indicates statistical significance (**P<0.01). (f, g) Differential effects of estrogen on the protein level of ERα-WT and the SPOP degradation-resistant mutant (ERα-M4). The 293T cells were transfected with FH- ERα-WT or M4 mutant construct. After 24 h, cells were treated with vehicle ethanol (EtOH,−), 10 nM 17β-estradiol (E2), 10 nM Tamoxifen (Tam), and 10 nM Fulvestrant (Ful) for 4 h before cell lysates were prepared for WB analyzes
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fig5: Estrogen potentiates SPOP-mediated degradation of ERα. (a) Estrogen enhances the SPOP-ERα interaction. FH-ERα and Myc-SPOP constructs were co-transfected into 293T cells. After 24 h, cells were treated with the vehicle ethanol (EtOH,−) or 10 nM 17β-estradiol (E2) for 4 h before cell lysates were prepared for co-IP and WB analyzes. (b) Estrogen enhances SPOP-mediated ERα degradation. The 293T cells were transfected with the indicated constructs. A small amount of Myc-SPOP constructs was used in transfection. After 24 h, cells were treated with the vehicle ethanol (EtOH) or 10 nM 17β-estradiol (E2) for 4 h before cells lysates were prepared for WB analyzes. The density of ERα was determined by normalizing to actin (loading control) first and then to the normalized value in mock-treated cells. (c) Knockdown of SPOP attenuates estrogen-induced degradation of ERα. Ishikawa cells were transfected with control or SPOP-specific siRNA. After 48 h, cells were then treated with the vehicle ethanol (EtOH,−) or 10 nM 17β-estradiol (E2) for 4 h before cell lysates were prepared for WB analyzes. (d) Estrogen potentiates SPOP-induced polyubiquitination of ERα. The 293T cells were transfected with the indicated constructs. After 24 h, cells were treated with the vehicle ethanol (EtOH,−) or 10 nM 17β-estradiol (E2). Cells were then treated with MG132 for 4 h before cell lysates were prepared for IP and WB analyzes. (e) Ishikawa cells lines that stably transfected with control, SPOP-WT or SPOP mutants constructs were treated with 10 nM 17β-estradiol (E2) for 24 h. The mRNA level of ERα target gene GREB1 was measured by qRT-PCR. The mRNA level of GAPDH was used for normalization. The mean values (S.D.) of three independent experiments are shown. **indicates statistical significance (**P<0.01). (f, g) Differential effects of estrogen on the protein level of ERα-WT and the SPOP degradation-resistant mutant (ERα-M4). The 293T cells were transfected with FH- ERα-WT or M4 mutant construct. After 24 h, cells were treated with vehicle ethanol (EtOH,−), 10 nM 17β-estradiol (E2), 10 nM Tamoxifen (Tam), and 10 nM Fulvestrant (Ful) for 4 h before cell lysates were prepared for WB analyzes

Mentions: ERα is rapidly ubiquitinated and degraded through the ubiquitin-proteasome pathway after estrogen binding.21 However, the molecular mechanism by which estrogen downregulates ERα protein is not fully understood. To determine whether SPOP has a role in this process, we first examined whether estrogen treatment can affect SPOP-ERα interaction. FH-ERα and Myc-SPOP were overexpressed in 293T cells, and then the cells were treated with hormone 17β-estradiol (E2) or vehicle ethanol (EtOH) before harvesting. co-IP assay was performed to test the impact of estrogen treatment on SPOP-ERα interaction. As shown in Figure 5a, E2 treatment leads to a significant downregulation of overexpressed ERα. However, more SPOP was co-immunoprecipitated by ERα, suggesting that estrogen treatment enhances the interaction between ERα and SPOP. We further demonstrated that SPOP-induced downregulation of ERα protein was markedly enhanced by E2 treatment (Figure 5b). Furthermore, we demonstrated that knockdown of endogenous SPOP largely diminished E2-induced downregulation of endogenous ERα protein in Ishikawa cells (Figure 5c). Consistent with these findings, E2 treatment resulted an increase of SPOP-mediated ERα ubiquitination (Figure 5d). Finally, we demonstrated that stable overexpression of SPOP-WT but not the SPOP mutants diminished E2–dependent transactivation of ERα target gene GREB1 in Ishikawa cells (Figure 5e). Similar effects were observed in other two ERα target genes, Cyclin D1 and ABCA3 (Supplementary Figure S3A and 3B). We also demonstrated that SPOP-WT and mutants differentially regulated the protein levels of GREB1 and Cyclin D1 (Supplementary Figure S3C).


Endometrial cancer-associated mutants of SPOP are defective in regulating estrogen receptor-α protein turnover.

Zhang P, Gao K, Jin X, Ma J, Peng J, Wumaier R, Tang Y, Zhang Y, An J, Yan Q, Dong Y, Huang H, Yu L, Wang C - Cell Death Dis (2015)

Estrogen potentiates SPOP-mediated degradation of ERα. (a) Estrogen enhances the SPOP-ERα interaction. FH-ERα and Myc-SPOP constructs were co-transfected into 293T cells. After 24 h, cells were treated with the vehicle ethanol (EtOH,−) or 10 nM 17β-estradiol (E2) for 4 h before cell lysates were prepared for co-IP and WB analyzes. (b) Estrogen enhances SPOP-mediated ERα degradation. The 293T cells were transfected with the indicated constructs. A small amount of Myc-SPOP constructs was used in transfection. After 24 h, cells were treated with the vehicle ethanol (EtOH) or 10 nM 17β-estradiol (E2) for 4 h before cells lysates were prepared for WB analyzes. The density of ERα was determined by normalizing to actin (loading control) first and then to the normalized value in mock-treated cells. (c) Knockdown of SPOP attenuates estrogen-induced degradation of ERα. Ishikawa cells were transfected with control or SPOP-specific siRNA. After 48 h, cells were then treated with the vehicle ethanol (EtOH,−) or 10 nM 17β-estradiol (E2) for 4 h before cell lysates were prepared for WB analyzes. (d) Estrogen potentiates SPOP-induced polyubiquitination of ERα. The 293T cells were transfected with the indicated constructs. After 24 h, cells were treated with the vehicle ethanol (EtOH,−) or 10 nM 17β-estradiol (E2). Cells were then treated with MG132 for 4 h before cell lysates were prepared for IP and WB analyzes. (e) Ishikawa cells lines that stably transfected with control, SPOP-WT or SPOP mutants constructs were treated with 10 nM 17β-estradiol (E2) for 24 h. The mRNA level of ERα target gene GREB1 was measured by qRT-PCR. The mRNA level of GAPDH was used for normalization. The mean values (S.D.) of three independent experiments are shown. **indicates statistical significance (**P<0.01). (f, g) Differential effects of estrogen on the protein level of ERα-WT and the SPOP degradation-resistant mutant (ERα-M4). The 293T cells were transfected with FH- ERα-WT or M4 mutant construct. After 24 h, cells were treated with vehicle ethanol (EtOH,−), 10 nM 17β-estradiol (E2), 10 nM Tamoxifen (Tam), and 10 nM Fulvestrant (Ful) for 4 h before cell lysates were prepared for WB analyzes
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Related In: Results  -  Collection

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fig5: Estrogen potentiates SPOP-mediated degradation of ERα. (a) Estrogen enhances the SPOP-ERα interaction. FH-ERα and Myc-SPOP constructs were co-transfected into 293T cells. After 24 h, cells were treated with the vehicle ethanol (EtOH,−) or 10 nM 17β-estradiol (E2) for 4 h before cell lysates were prepared for co-IP and WB analyzes. (b) Estrogen enhances SPOP-mediated ERα degradation. The 293T cells were transfected with the indicated constructs. A small amount of Myc-SPOP constructs was used in transfection. After 24 h, cells were treated with the vehicle ethanol (EtOH) or 10 nM 17β-estradiol (E2) for 4 h before cells lysates were prepared for WB analyzes. The density of ERα was determined by normalizing to actin (loading control) first and then to the normalized value in mock-treated cells. (c) Knockdown of SPOP attenuates estrogen-induced degradation of ERα. Ishikawa cells were transfected with control or SPOP-specific siRNA. After 48 h, cells were then treated with the vehicle ethanol (EtOH,−) or 10 nM 17β-estradiol (E2) for 4 h before cell lysates were prepared for WB analyzes. (d) Estrogen potentiates SPOP-induced polyubiquitination of ERα. The 293T cells were transfected with the indicated constructs. After 24 h, cells were treated with the vehicle ethanol (EtOH,−) or 10 nM 17β-estradiol (E2). Cells were then treated with MG132 for 4 h before cell lysates were prepared for IP and WB analyzes. (e) Ishikawa cells lines that stably transfected with control, SPOP-WT or SPOP mutants constructs were treated with 10 nM 17β-estradiol (E2) for 24 h. The mRNA level of ERα target gene GREB1 was measured by qRT-PCR. The mRNA level of GAPDH was used for normalization. The mean values (S.D.) of three independent experiments are shown. **indicates statistical significance (**P<0.01). (f, g) Differential effects of estrogen on the protein level of ERα-WT and the SPOP degradation-resistant mutant (ERα-M4). The 293T cells were transfected with FH- ERα-WT or M4 mutant construct. After 24 h, cells were treated with vehicle ethanol (EtOH,−), 10 nM 17β-estradiol (E2), 10 nM Tamoxifen (Tam), and 10 nM Fulvestrant (Ful) for 4 h before cell lysates were prepared for WB analyzes
Mentions: ERα is rapidly ubiquitinated and degraded through the ubiquitin-proteasome pathway after estrogen binding.21 However, the molecular mechanism by which estrogen downregulates ERα protein is not fully understood. To determine whether SPOP has a role in this process, we first examined whether estrogen treatment can affect SPOP-ERα interaction. FH-ERα and Myc-SPOP were overexpressed in 293T cells, and then the cells were treated with hormone 17β-estradiol (E2) or vehicle ethanol (EtOH) before harvesting. co-IP assay was performed to test the impact of estrogen treatment on SPOP-ERα interaction. As shown in Figure 5a, E2 treatment leads to a significant downregulation of overexpressed ERα. However, more SPOP was co-immunoprecipitated by ERα, suggesting that estrogen treatment enhances the interaction between ERα and SPOP. We further demonstrated that SPOP-induced downregulation of ERα protein was markedly enhanced by E2 treatment (Figure 5b). Furthermore, we demonstrated that knockdown of endogenous SPOP largely diminished E2-induced downregulation of endogenous ERα protein in Ishikawa cells (Figure 5c). Consistent with these findings, E2 treatment resulted an increase of SPOP-mediated ERα ubiquitination (Figure 5d). Finally, we demonstrated that stable overexpression of SPOP-WT but not the SPOP mutants diminished E2–dependent transactivation of ERα target gene GREB1 in Ishikawa cells (Figure 5e). Similar effects were observed in other two ERα target genes, Cyclin D1 and ABCA3 (Supplementary Figure S3A and 3B). We also demonstrated that SPOP-WT and mutants differentially regulated the protein levels of GREB1 and Cyclin D1 (Supplementary Figure S3C).

Bottom Line: Increasing amounts of evidence strongly suggests that dysregulation of ubiquitin-proteasome system is closely associated with cancer pathogenesis.Speckle-type POZ protein (SPOP) is an adapter protein of the CUL3-based E3 ubiquitin ligase complexes.It selectively recruits substrates for their ubiquitination and subsequent degradation.

View Article: PubMed Central - PubMed

Affiliation: 1] State Key Laboratory of Genetic Engineering, Collaborative Innovation Center for Genetics and Development, School of Life Sciences, Fudan University, 220 Handan Road, Shanghai 200433, China [2] Shanghai Cancer Center, Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, China.

ABSTRACT
Increasing amounts of evidence strongly suggests that dysregulation of ubiquitin-proteasome system is closely associated with cancer pathogenesis. Speckle-type POZ protein (SPOP) is an adapter protein of the CUL3-based E3 ubiquitin ligase complexes. It selectively recruits substrates for their ubiquitination and subsequent degradation. Recently, several exome-sequencing studies of endometrial cancer revealed high frequency somatic mutations in SPOP (5.7-10%). However, how SPOP mutations contribute to endometrial cancer remains unknown. Here, we identified estrogen receptor-α (ERα), a major endometrial cancer promoter, as a substrate for the SPOP-CUL3-RBX1 E3 ubiquitin ligase complex. SPOP specifically recognizes multiple Ser/Thr (S/T)-rich degrons located in the AF2 domain of ERα, and triggers ERα degradation via the ubiquitin-proteasome pathway. SPOP depletion by siRNAs promotes endometrial cells growth. Strikingly, endometrial cancer-associated mutants of SPOP are defective in regulating ERα degradation and ubiquitination. Furthermore, we found that SPOP participates in estrogen-induced ERα degradation and transactivation. Our study revealed novel molecular mechanisms underlying the regulation of ERα protein homeostasis in physiological and pathological conditions, and provided insights in understanding the relationship between SPOP mutations and the development of endometrial cancer.

Show MeSH
Related in: MedlinePlus