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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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SPOP interacts with ERα in cells. (a,b) Ectopically expressed SPOP and ERα interact with each other. The 293T cells were co-transfected with FLAG-HA (FH)-SPOP and Myc-ERα constructs. After 24 h, cell lysates were prepared for co-IP with anti-FLAG antibody and WB analyzes. (b) co-IP assay was performed between ectopically expressed FH-ERα and Myc-SPOP. (c) Endogenous SPOP and ERα proteins interact with each other in Ishikawa cells. After being treated with 20 μM MG132 for 4 h, cell lysates were prepared for co-IP with anti-ERα antibody and WB analyzes with indicated antibodies. (d) Schematic representation of SPOP deletion mutants. Binding capacity of SPOP to ERα is indicated with the symbol. (e) ERα binds to the MATH domain of SPOP. The 293T cells were co-transfected with FH-ERα and Myc-SPOP-WT or deletion mutants (ΔMATH, ΔBTB) constructs. After 24 h, cell lysates were prepared for co-IP assay with anti-FLAG antibody and WB analyzes. (f) Schematic representation of MATH domain deletion mutants of SPOP. Binding capacity of SPOP to ERα is indicated with the symbol. (g) The integrity MATH domain of SPOP is crtical for ERα binding. The 293T cells were co-transfected with FH-ERα and Myc-SPOP-WT or deletion mutants (D1–D8) constructs. After 24 h, cell lysates were prepared for co-IP assay with anti-Myc antibody and WB analyzes
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fig1: SPOP interacts with ERα in cells. (a,b) Ectopically expressed SPOP and ERα interact with each other. The 293T cells were co-transfected with FLAG-HA (FH)-SPOP and Myc-ERα constructs. After 24 h, cell lysates were prepared for co-IP with anti-FLAG antibody and WB analyzes. (b) co-IP assay was performed between ectopically expressed FH-ERα and Myc-SPOP. (c) Endogenous SPOP and ERα proteins interact with each other in Ishikawa cells. After being treated with 20 μM MG132 for 4 h, cell lysates were prepared for co-IP with anti-ERα antibody and WB analyzes with indicated antibodies. (d) Schematic representation of SPOP deletion mutants. Binding capacity of SPOP to ERα is indicated with the symbol. (e) ERα binds to the MATH domain of SPOP. The 293T cells were co-transfected with FH-ERα and Myc-SPOP-WT or deletion mutants (ΔMATH, ΔBTB) constructs. After 24 h, cell lysates were prepared for co-IP assay with anti-FLAG antibody and WB analyzes. (f) Schematic representation of MATH domain deletion mutants of SPOP. Binding capacity of SPOP to ERα is indicated with the symbol. (g) The integrity MATH domain of SPOP is crtical for ERα binding. The 293T cells were co-transfected with FH-ERα and Myc-SPOP-WT or deletion mutants (D1–D8) constructs. After 24 h, cell lysates were prepared for co-IP assay with anti-Myc antibody and WB analyzes

Mentions: It was previously reported that SPOP regulates AR stability.19 Because ERα is the most extensively studied biomarker and the best predictor for response to endocrine therapy in patients with endometrial cancer, we explored the possibility that SPOP regulates ERα protein stability via a similar mechanism as that of AR stability. Accordingly, we first examined whether SPOP interacts with ERα in cells. To do this, FLAG-HA (FH)-SPOP and Myc-ERα constructs were co-expressed in 293T cells. Cell lysates were subsequently prepared for co-immunoprecipitation (co-IP) with anti-FLAG antibody. As shown in Figure 1a, Myc-ERα was immunoprecipitated by FH-SPOP, suggesting an interaction between these two proteins. Similar results were also obtained in the reciprocal co-IP experiment in which FH-ERα was able to immunoprecipiate Myc-SPOP (Figure 1b). Next, we decided to extend our analysis by investigating whether endogenous SPOP and ERα can interact with each other. In this case, we chose Ishikawa cells, an ERα-positive human endometrial adenocarcinoma cell line, for subsequent study. Sanger sequencing of SPOP exon6/7 of Ishikawa cells revealed no mutation in MATH domain (data not shown). Immunoprecipitation using anti-ERα antibody was performed using cell lysates prepared from Ishikawa cells. As shown in Figure 1c, endogenous SPOP was efficiently immunoprecipitated by ERα, suggesting these two proteins can also interact with each other at endogenous levels.


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)

SPOP interacts with ERα in cells. (a,b) Ectopically expressed SPOP and ERα interact with each other. The 293T cells were co-transfected with FLAG-HA (FH)-SPOP and Myc-ERα constructs. After 24 h, cell lysates were prepared for co-IP with anti-FLAG antibody and WB analyzes. (b) co-IP assay was performed between ectopically expressed FH-ERα and Myc-SPOP. (c) Endogenous SPOP and ERα proteins interact with each other in Ishikawa cells. After being treated with 20 μM MG132 for 4 h, cell lysates were prepared for co-IP with anti-ERα antibody and WB analyzes with indicated antibodies. (d) Schematic representation of SPOP deletion mutants. Binding capacity of SPOP to ERα is indicated with the symbol. (e) ERα binds to the MATH domain of SPOP. The 293T cells were co-transfected with FH-ERα and Myc-SPOP-WT or deletion mutants (ΔMATH, ΔBTB) constructs. After 24 h, cell lysates were prepared for co-IP assay with anti-FLAG antibody and WB analyzes. (f) Schematic representation of MATH domain deletion mutants of SPOP. Binding capacity of SPOP to ERα is indicated with the symbol. (g) The integrity MATH domain of SPOP is crtical for ERα binding. The 293T cells were co-transfected with FH-ERα and Myc-SPOP-WT or deletion mutants (D1–D8) constructs. After 24 h, cell lysates were prepared for co-IP assay with anti-Myc antibody and WB analyzes
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4385925&req=5

fig1: SPOP interacts with ERα in cells. (a,b) Ectopically expressed SPOP and ERα interact with each other. The 293T cells were co-transfected with FLAG-HA (FH)-SPOP and Myc-ERα constructs. After 24 h, cell lysates were prepared for co-IP with anti-FLAG antibody and WB analyzes. (b) co-IP assay was performed between ectopically expressed FH-ERα and Myc-SPOP. (c) Endogenous SPOP and ERα proteins interact with each other in Ishikawa cells. After being treated with 20 μM MG132 for 4 h, cell lysates were prepared for co-IP with anti-ERα antibody and WB analyzes with indicated antibodies. (d) Schematic representation of SPOP deletion mutants. Binding capacity of SPOP to ERα is indicated with the symbol. (e) ERα binds to the MATH domain of SPOP. The 293T cells were co-transfected with FH-ERα and Myc-SPOP-WT or deletion mutants (ΔMATH, ΔBTB) constructs. After 24 h, cell lysates were prepared for co-IP assay with anti-FLAG antibody and WB analyzes. (f) Schematic representation of MATH domain deletion mutants of SPOP. Binding capacity of SPOP to ERα is indicated with the symbol. (g) The integrity MATH domain of SPOP is crtical for ERα binding. The 293T cells were co-transfected with FH-ERα and Myc-SPOP-WT or deletion mutants (D1–D8) constructs. After 24 h, cell lysates were prepared for co-IP assay with anti-Myc antibody and WB analyzes
Mentions: It was previously reported that SPOP regulates AR stability.19 Because ERα is the most extensively studied biomarker and the best predictor for response to endocrine therapy in patients with endometrial cancer, we explored the possibility that SPOP regulates ERα protein stability via a similar mechanism as that of AR stability. Accordingly, we first examined whether SPOP interacts with ERα in cells. To do this, FLAG-HA (FH)-SPOP and Myc-ERα constructs were co-expressed in 293T cells. Cell lysates were subsequently prepared for co-immunoprecipitation (co-IP) with anti-FLAG antibody. As shown in Figure 1a, Myc-ERα was immunoprecipitated by FH-SPOP, suggesting an interaction between these two proteins. Similar results were also obtained in the reciprocal co-IP experiment in which FH-ERα was able to immunoprecipiate Myc-SPOP (Figure 1b). Next, we decided to extend our analysis by investigating whether endogenous SPOP and ERα can interact with each other. In this case, we chose Ishikawa cells, an ERα-positive human endometrial adenocarcinoma cell line, for subsequent study. Sanger sequencing of SPOP exon6/7 of Ishikawa cells revealed no mutation in MATH domain (data not shown). Immunoprecipitation using anti-ERα antibody was performed using cell lysates prepared from Ishikawa cells. As shown in Figure 1c, endogenous SPOP was efficiently immunoprecipitated by ERα, suggesting these two proteins can also interact with each other at endogenous levels.

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