Limits...
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.

Show MeSH

Related in: MedlinePlus

Endometrial cancer-associated mutants of SPOP are defective in promoting ERα degradation and ubiquitination. (a) Distribution of the point mutations on the SPOP gene found in endometrial cancer samples. These mutations are exclusively located in the N-terminal MATH domain of SPOP. (b) Endometrial cancer-associated mutants of SPOP are defective in promoting ERα degradation. 293T cells were transfected with FH-ERα and wild-type or mutated SPOP constructs as indicated. After 24 h, cell lysates were prepared for WB analyzes. (c) Endometrial cancer-associated mutants of SPOP are defective in interacting with ERα. The 293T cells were transfected with the indicated constructs. After 24 h, cell lysates were prepared for co-IP assay with anti-FLAG antibody and WB analyzes. (d) Endometrial cancer-associated mutants of SPOP are defective in promoting ERα ubiquitination. The 293T cells were transfected with the indicated constructs. After 24 h, cells were treated with 20 μM MG132 for 4 h and cell lysates were prepared for immunoprecipitation and WB analyzes
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4385925&req=5

fig4: Endometrial cancer-associated mutants of SPOP are defective in promoting ERα degradation and ubiquitination. (a) Distribution of the point mutations on the SPOP gene found in endometrial cancer samples. These mutations are exclusively located in the N-terminal MATH domain of SPOP. (b) Endometrial cancer-associated mutants of SPOP are defective in promoting ERα degradation. 293T cells were transfected with FH-ERα and wild-type or mutated SPOP constructs as indicated. After 24 h, cell lysates were prepared for WB analyzes. (c) Endometrial cancer-associated mutants of SPOP are defective in interacting with ERα. The 293T cells were transfected with the indicated constructs. After 24 h, cell lysates were prepared for co-IP assay with anti-FLAG antibody and WB analyzes. (d) Endometrial cancer-associated mutants of SPOP are defective in promoting ERα ubiquitination. The 293T cells were transfected with the indicated constructs. After 24 h, cells were treated with 20 μM MG132 for 4 h and cell lysates were prepared for immunoprecipitation and WB analyzes

Mentions: SPOP was mutated in 5.7–10% of patients with endometrial cancer across multiple independent cohorts (Table 1). Moreover, SPOP mutations were detected in three major histological subtypes of endometrial cancer (endometrioid, clear cell and serous). Notably, all the SPOP mutations found in endometrial cancer far-exclusively occur in the MATH domain which is responsible for ERα binding (Figure 4a). Therefore, we propose that endometrial cancer-associated SPOP mutations may cause dysfunction in regulating ERα protein level. To test this, nine Myc-tagged endometrial cancer-associated mutants of SPOP were generated according to four large-scale exome-sequencing studies (Table 1), including E47K, E50K, G75R, S80R, P94A, M117V, M117I, R121Q, and D140G. We initially examined their interactions with ERα by co-IP assay. As shown in Figure 4b, mutations of the residues at MATH domain substantially decreased the capacity of SPOP to interact with ERα in vivo, suggesting the endometrial cancer-associated SPOP mutants are defective in binding to ERα. We then examined the capacities of endometrial cancer-associated SPOP mutants in promoting ERα degradation. As shown in Figure 4c, a group of SPOP mutants (SPOP-M117V, M117I, and R121Q) displayed reduced capacity to promote ERα degradation when compared with SPOP-WT, whereas other groups did not alter ERα protein level (SPOP-E47K, E50K, G75R, P94A, and D140G) or increased ERα protein level (SPOP-S80R), suggesting that endometrial cancer-associated SPOP mutants may differentially regulate ERα stability. Furthermore, in vivo ubiquitination assays indicated that some SPOP mutants (SPOP-E47K, E50K, G75R, S80R, and D140G) lost the capacity to promote ERα polyubiquitination, whereas SPOP-M117V, M117I, and R121Q mutants could generate partially polyubiquitinated form of ERα (Figure 4d). Taken together, our findings suggest that endometrial cancer-associated mutants of SPOP are defective in promoting ERα degradation and ubiquitination.


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)

Endometrial cancer-associated mutants of SPOP are defective in promoting ERα degradation and ubiquitination. (a) Distribution of the point mutations on the SPOP gene found in endometrial cancer samples. These mutations are exclusively located in the N-terminal MATH domain of SPOP. (b) Endometrial cancer-associated mutants of SPOP are defective in promoting ERα degradation. 293T cells were transfected with FH-ERα and wild-type or mutated SPOP constructs as indicated. After 24 h, cell lysates were prepared for WB analyzes. (c) Endometrial cancer-associated mutants of SPOP are defective in interacting with ERα. The 293T cells were transfected with the indicated constructs. After 24 h, cell lysates were prepared for co-IP assay with anti-FLAG antibody and WB analyzes. (d) Endometrial cancer-associated mutants of SPOP are defective in promoting ERα ubiquitination. The 293T cells were transfected with the indicated constructs. After 24 h, cells were treated with 20 μM MG132 for 4 h and cell lysates were prepared for immunoprecipitation and WB analyzes
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig4: Endometrial cancer-associated mutants of SPOP are defective in promoting ERα degradation and ubiquitination. (a) Distribution of the point mutations on the SPOP gene found in endometrial cancer samples. These mutations are exclusively located in the N-terminal MATH domain of SPOP. (b) Endometrial cancer-associated mutants of SPOP are defective in promoting ERα degradation. 293T cells were transfected with FH-ERα and wild-type or mutated SPOP constructs as indicated. After 24 h, cell lysates were prepared for WB analyzes. (c) Endometrial cancer-associated mutants of SPOP are defective in interacting with ERα. The 293T cells were transfected with the indicated constructs. After 24 h, cell lysates were prepared for co-IP assay with anti-FLAG antibody and WB analyzes. (d) Endometrial cancer-associated mutants of SPOP are defective in promoting ERα ubiquitination. The 293T cells were transfected with the indicated constructs. After 24 h, cells were treated with 20 μM MG132 for 4 h and cell lysates were prepared for immunoprecipitation and WB analyzes
Mentions: SPOP was mutated in 5.7–10% of patients with endometrial cancer across multiple independent cohorts (Table 1). Moreover, SPOP mutations were detected in three major histological subtypes of endometrial cancer (endometrioid, clear cell and serous). Notably, all the SPOP mutations found in endometrial cancer far-exclusively occur in the MATH domain which is responsible for ERα binding (Figure 4a). Therefore, we propose that endometrial cancer-associated SPOP mutations may cause dysfunction in regulating ERα protein level. To test this, nine Myc-tagged endometrial cancer-associated mutants of SPOP were generated according to four large-scale exome-sequencing studies (Table 1), including E47K, E50K, G75R, S80R, P94A, M117V, M117I, R121Q, and D140G. We initially examined their interactions with ERα by co-IP assay. As shown in Figure 4b, mutations of the residues at MATH domain substantially decreased the capacity of SPOP to interact with ERα in vivo, suggesting the endometrial cancer-associated SPOP mutants are defective in binding to ERα. We then examined the capacities of endometrial cancer-associated SPOP mutants in promoting ERα degradation. As shown in Figure 4c, a group of SPOP mutants (SPOP-M117V, M117I, and R121Q) displayed reduced capacity to promote ERα degradation when compared with SPOP-WT, whereas other groups did not alter ERα protein level (SPOP-E47K, E50K, G75R, P94A, and D140G) or increased ERα protein level (SPOP-S80R), suggesting that endometrial cancer-associated SPOP mutants may differentially regulate ERα stability. Furthermore, in vivo ubiquitination assays indicated that some SPOP mutants (SPOP-E47K, E50K, G75R, S80R, and D140G) lost the capacity to promote ERα polyubiquitination, whereas SPOP-M117V, M117I, and R121Q mutants could generate partially polyubiquitinated form of ERα (Figure 4d). Taken together, our findings suggest that endometrial cancer-associated mutants of SPOP are defective in promoting ERα degradation and ubiquitination.

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