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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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Related in: MedlinePlus

The SPOP-CUL3-RBX1 ubiquitin ligase complex targets ERα for ubiquitination and degradation. (a) SPOP regulates ERα protein levels through the proteasome pathway. The 293T cells were transfected with FH-ERα in combination with or without the Myc-SPOP constructs. After 24 h, cells were treated with MG132 (20 μM), Bortezomib (200 nM), chloroquine (100 mM), or DMSO for 4 h before cell lysates were prepared for WB analyzes. Actin, a loading control. (b) The BTB and MATH domains in SPOP are essential for SPOP-mediated degradation of ERα. FH-ERα and different amounts of Myc-SPOP-WT or deletion mutants (ΔMATH, ΔBTB) constructs were transfected into 293T cells. After 24 h, cell lysates were prepared for WB analyzes. (c) SPOP regulates endogenous ERα protein levels. Ishikawa cells were transfected with Myc-SPOP-WT, or deletion mutants (ΔMATH, ΔBTB) constructs. After 24 h, cell lysates were prepared for WB analyzes. (d) Knockdown of SPOP increases endogenous ERα protein levels. Ishikawa cells were transfected with control or two SPOP-specific siRNAs. After 48 h, cell lysates were prepared for WB analyzes. (e) Quantitative RT-PCR measurement of the mRNA levels of SPOP and ESR1 in SPOP-knockdown Ishikawa cells. The mRNA level of GAPDH was used for normalization. The mean values (S.D.) of three independent experiments are shown. (f,g) Knockdown of SPOP prolongs ERα protein half-life. Ishikawa cells were transfected with control or SPOP-specific siRNA. After 48 h, cells were chased with 30 μM cycloheximide (CHX). At the indicated time points, cell lysates were prepared for WB analyzes. (f) At each time point, the intensity of ERα was first normalized to the intensity of Actin (loading control) and then to the value of the 0-h time point (g). The mean values (S.D.) of three independent experiments are shown. (h) Knockdown of RBX1 or CUL3 increases endogenous ERα protein levels. Ishikawa cells were transfected with control siRNA or siRNAs for RBX1 or CUL3. After 48 h, cell lysates were prepared for WB analyzes. (i) SPOP promotes ERα polyubiquitination in vivo. FH-ERα, HA-Ub, and Myc-SPOP-WT or ΔBTB mutant constructs were co-transfected into 293T cells. After 24 h, cells were treated with 20 μM MG132 for 4 h. ERα proteins were immunoprecipitated with anti-FLAG antibody and resolved by SDS/PAGE. The ubiquitinated forms of ERα were analyzed by WB with anti-Ub antibody. (j) Knockdown of SPOP decreases ubiquitination of endogenous ERα. Ishiwaka cells were transfected control or SPOP-specific siRNA. After 48 h, cells were treated with 20 μM MG132 for 4 h and then the same procedure was performed as i. (k) Knockdown of SPOP promotes Ishikawa cells growth. Ishikawa cells were transfected with control or two SPOP-specific siRNAs. After 48 h, the cell growth was measured by CCK-8 assay at indicated days. The mean values (S.D.) of three independent experiments are shown
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fig2: The SPOP-CUL3-RBX1 ubiquitin ligase complex targets ERα for ubiquitination and degradation. (a) SPOP regulates ERα protein levels through the proteasome pathway. The 293T cells were transfected with FH-ERα in combination with or without the Myc-SPOP constructs. After 24 h, cells were treated with MG132 (20 μM), Bortezomib (200 nM), chloroquine (100 mM), or DMSO for 4 h before cell lysates were prepared for WB analyzes. Actin, a loading control. (b) The BTB and MATH domains in SPOP are essential for SPOP-mediated degradation of ERα. FH-ERα and different amounts of Myc-SPOP-WT or deletion mutants (ΔMATH, ΔBTB) constructs were transfected into 293T cells. After 24 h, cell lysates were prepared for WB analyzes. (c) SPOP regulates endogenous ERα protein levels. Ishikawa cells were transfected with Myc-SPOP-WT, or deletion mutants (ΔMATH, ΔBTB) constructs. After 24 h, cell lysates were prepared for WB analyzes. (d) Knockdown of SPOP increases endogenous ERα protein levels. Ishikawa cells were transfected with control or two SPOP-specific siRNAs. After 48 h, cell lysates were prepared for WB analyzes. (e) Quantitative RT-PCR measurement of the mRNA levels of SPOP and ESR1 in SPOP-knockdown Ishikawa cells. The mRNA level of GAPDH was used for normalization. The mean values (S.D.) of three independent experiments are shown. (f,g) Knockdown of SPOP prolongs ERα protein half-life. Ishikawa cells were transfected with control or SPOP-specific siRNA. After 48 h, cells were chased with 30 μM cycloheximide (CHX). At the indicated time points, cell lysates were prepared for WB analyzes. (f) At each time point, the intensity of ERα was first normalized to the intensity of Actin (loading control) and then to the value of the 0-h time point (g). The mean values (S.D.) of three independent experiments are shown. (h) Knockdown of RBX1 or CUL3 increases endogenous ERα protein levels. Ishikawa cells were transfected with control siRNA or siRNAs for RBX1 or CUL3. After 48 h, cell lysates were prepared for WB analyzes. (i) SPOP promotes ERα polyubiquitination in vivo. FH-ERα, HA-Ub, and Myc-SPOP-WT or ΔBTB mutant constructs were co-transfected into 293T cells. After 24 h, cells were treated with 20 μM MG132 for 4 h. ERα proteins were immunoprecipitated with anti-FLAG antibody and resolved by SDS/PAGE. The ubiquitinated forms of ERα were analyzed by WB with anti-Ub antibody. (j) Knockdown of SPOP decreases ubiquitination of endogenous ERα. Ishiwaka cells were transfected control or SPOP-specific siRNA. After 48 h, cells were treated with 20 μM MG132 for 4 h and then the same procedure was performed as i. (k) Knockdown of SPOP promotes Ishikawa cells growth. Ishikawa cells were transfected with control or two SPOP-specific siRNAs. After 48 h, the cell growth was measured by CCK-8 assay at indicated days. The mean values (S.D.) of three independent experiments are shown

Mentions: Then we explored whether the SPOP-CUL3-RBX1 E3 ubiquitin ligase complex can promote the ubiquitination and degradation of ERα. As shown in Figure 2a, expression of SPOP decreased the ectopically co-expressed ERα protein level in 293T cells in a dose-dependent manner. This effect was completely blocked when cells were treated with the proteasome inhibitors MG132 or Bortezomib (Figure 2a). In contrast, lysosome inhibitor chloroquine could not block SPOP-mediated ERα degradation. These results indicated that SPOP downregulates ERα protein via the proteasomal and not the lysosomal degradation pathway. Moreover, SPOP-WT, but not the SPOP-ΔBTB or SPOP-ΔMATH mutant, promoted ERα degradation (Figure 2b), indicating that the BTB and MATH domains are both required for SPOP-mediated ERα degradation. Next, we examined the effect of SPOP on the degradation of endogenous ERα. Similarly, as shown in Figure 2c, overexpression of SPOP-WT, but not the SPOP-ΔBTB or SPOP-ΔMATH mutant in Ishikawa cells resulted in a moderate decrease in the protein level of endogenous ERα. Moreover, knockdown of endogenous SPOP using two gene-specific siRNAs increased ERα protein levels in Ishikawa cells (Figure 2d). To exclude the possibility that ERα protein elevation resulted from transcriptional upregulation, we performed qRT-PCR to measure the mRNA levels of SPOP and ERα in SPOP-depleted Ishikawa cells. In contrast to the significant decrease of SPOP mRNA levels, the mRNA levels of ESR1 gene (encoding ERα) in SPOP-depleted Ishikawa cells stayed at a level similar to that of the control cells (Figure 2e), indicating that the effect of SPOP on ERα protein levels is not mediated through the upregulation of ERα mRNA levels. In addition, knockdown of SPOP remarkably prolonged the half-life of endogenous ERα protein in Ishikawa cells (Figures 2f and g), further suggesting that SPOP regulates ERα protein at the post-translational level.


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)

The SPOP-CUL3-RBX1 ubiquitin ligase complex targets ERα for ubiquitination and degradation. (a) SPOP regulates ERα protein levels through the proteasome pathway. The 293T cells were transfected with FH-ERα in combination with or without the Myc-SPOP constructs. After 24 h, cells were treated with MG132 (20 μM), Bortezomib (200 nM), chloroquine (100 mM), or DMSO for 4 h before cell lysates were prepared for WB analyzes. Actin, a loading control. (b) The BTB and MATH domains in SPOP are essential for SPOP-mediated degradation of ERα. FH-ERα and different amounts of Myc-SPOP-WT or deletion mutants (ΔMATH, ΔBTB) constructs were transfected into 293T cells. After 24 h, cell lysates were prepared for WB analyzes. (c) SPOP regulates endogenous ERα protein levels. Ishikawa cells were transfected with Myc-SPOP-WT, or deletion mutants (ΔMATH, ΔBTB) constructs. After 24 h, cell lysates were prepared for WB analyzes. (d) Knockdown of SPOP increases endogenous ERα protein levels. Ishikawa cells were transfected with control or two SPOP-specific siRNAs. After 48 h, cell lysates were prepared for WB analyzes. (e) Quantitative RT-PCR measurement of the mRNA levels of SPOP and ESR1 in SPOP-knockdown Ishikawa cells. The mRNA level of GAPDH was used for normalization. The mean values (S.D.) of three independent experiments are shown. (f,g) Knockdown of SPOP prolongs ERα protein half-life. Ishikawa cells were transfected with control or SPOP-specific siRNA. After 48 h, cells were chased with 30 μM cycloheximide (CHX). At the indicated time points, cell lysates were prepared for WB analyzes. (f) At each time point, the intensity of ERα was first normalized to the intensity of Actin (loading control) and then to the value of the 0-h time point (g). The mean values (S.D.) of three independent experiments are shown. (h) Knockdown of RBX1 or CUL3 increases endogenous ERα protein levels. Ishikawa cells were transfected with control siRNA or siRNAs for RBX1 or CUL3. After 48 h, cell lysates were prepared for WB analyzes. (i) SPOP promotes ERα polyubiquitination in vivo. FH-ERα, HA-Ub, and Myc-SPOP-WT or ΔBTB mutant constructs were co-transfected into 293T cells. After 24 h, cells were treated with 20 μM MG132 for 4 h. ERα proteins were immunoprecipitated with anti-FLAG antibody and resolved by SDS/PAGE. The ubiquitinated forms of ERα were analyzed by WB with anti-Ub antibody. (j) Knockdown of SPOP decreases ubiquitination of endogenous ERα. Ishiwaka cells were transfected control or SPOP-specific siRNA. After 48 h, cells were treated with 20 μM MG132 for 4 h and then the same procedure was performed as i. (k) Knockdown of SPOP promotes Ishikawa cells growth. Ishikawa cells were transfected with control or two SPOP-specific siRNAs. After 48 h, the cell growth was measured by CCK-8 assay at indicated days. The mean values (S.D.) of three independent experiments are shown
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fig2: The SPOP-CUL3-RBX1 ubiquitin ligase complex targets ERα for ubiquitination and degradation. (a) SPOP regulates ERα protein levels through the proteasome pathway. The 293T cells were transfected with FH-ERα in combination with or without the Myc-SPOP constructs. After 24 h, cells were treated with MG132 (20 μM), Bortezomib (200 nM), chloroquine (100 mM), or DMSO for 4 h before cell lysates were prepared for WB analyzes. Actin, a loading control. (b) The BTB and MATH domains in SPOP are essential for SPOP-mediated degradation of ERα. FH-ERα and different amounts of Myc-SPOP-WT or deletion mutants (ΔMATH, ΔBTB) constructs were transfected into 293T cells. After 24 h, cell lysates were prepared for WB analyzes. (c) SPOP regulates endogenous ERα protein levels. Ishikawa cells were transfected with Myc-SPOP-WT, or deletion mutants (ΔMATH, ΔBTB) constructs. After 24 h, cell lysates were prepared for WB analyzes. (d) Knockdown of SPOP increases endogenous ERα protein levels. Ishikawa cells were transfected with control or two SPOP-specific siRNAs. After 48 h, cell lysates were prepared for WB analyzes. (e) Quantitative RT-PCR measurement of the mRNA levels of SPOP and ESR1 in SPOP-knockdown Ishikawa cells. The mRNA level of GAPDH was used for normalization. The mean values (S.D.) of three independent experiments are shown. (f,g) Knockdown of SPOP prolongs ERα protein half-life. Ishikawa cells were transfected with control or SPOP-specific siRNA. After 48 h, cells were chased with 30 μM cycloheximide (CHX). At the indicated time points, cell lysates were prepared for WB analyzes. (f) At each time point, the intensity of ERα was first normalized to the intensity of Actin (loading control) and then to the value of the 0-h time point (g). The mean values (S.D.) of three independent experiments are shown. (h) Knockdown of RBX1 or CUL3 increases endogenous ERα protein levels. Ishikawa cells were transfected with control siRNA or siRNAs for RBX1 or CUL3. After 48 h, cell lysates were prepared for WB analyzes. (i) SPOP promotes ERα polyubiquitination in vivo. FH-ERα, HA-Ub, and Myc-SPOP-WT or ΔBTB mutant constructs were co-transfected into 293T cells. After 24 h, cells were treated with 20 μM MG132 for 4 h. ERα proteins were immunoprecipitated with anti-FLAG antibody and resolved by SDS/PAGE. The ubiquitinated forms of ERα were analyzed by WB with anti-Ub antibody. (j) Knockdown of SPOP decreases ubiquitination of endogenous ERα. Ishiwaka cells were transfected control or SPOP-specific siRNA. After 48 h, cells were treated with 20 μM MG132 for 4 h and then the same procedure was performed as i. (k) Knockdown of SPOP promotes Ishikawa cells growth. Ishikawa cells were transfected with control or two SPOP-specific siRNAs. After 48 h, the cell growth was measured by CCK-8 assay at indicated days. The mean values (S.D.) of three independent experiments are shown
Mentions: Then we explored whether the SPOP-CUL3-RBX1 E3 ubiquitin ligase complex can promote the ubiquitination and degradation of ERα. As shown in Figure 2a, expression of SPOP decreased the ectopically co-expressed ERα protein level in 293T cells in a dose-dependent manner. This effect was completely blocked when cells were treated with the proteasome inhibitors MG132 or Bortezomib (Figure 2a). In contrast, lysosome inhibitor chloroquine could not block SPOP-mediated ERα degradation. These results indicated that SPOP downregulates ERα protein via the proteasomal and not the lysosomal degradation pathway. Moreover, SPOP-WT, but not the SPOP-ΔBTB or SPOP-ΔMATH mutant, promoted ERα degradation (Figure 2b), indicating that the BTB and MATH domains are both required for SPOP-mediated ERα degradation. Next, we examined the effect of SPOP on the degradation of endogenous ERα. Similarly, as shown in Figure 2c, overexpression of SPOP-WT, but not the SPOP-ΔBTB or SPOP-ΔMATH mutant in Ishikawa cells resulted in a moderate decrease in the protein level of endogenous ERα. Moreover, knockdown of endogenous SPOP using two gene-specific siRNAs increased ERα protein levels in Ishikawa cells (Figure 2d). To exclude the possibility that ERα protein elevation resulted from transcriptional upregulation, we performed qRT-PCR to measure the mRNA levels of SPOP and ERα in SPOP-depleted Ishikawa cells. In contrast to the significant decrease of SPOP mRNA levels, the mRNA levels of ESR1 gene (encoding ERα) in SPOP-depleted Ishikawa cells stayed at a level similar to that of the control cells (Figure 2e), indicating that the effect of SPOP on ERα protein levels is not mediated through the upregulation of ERα mRNA levels. In addition, knockdown of SPOP remarkably prolonged the half-life of endogenous ERα protein in Ishikawa cells (Figures 2f and g), further suggesting that SPOP regulates ERα protein at the post-translational level.

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