Tumor suppressor SPOP mediates the proteasomal degradation of progesterone receptors (PRs) in breast cancer cells

Abstract

Progesterone induces proliferation of breast cancer cells and contributes to the development of breast cancer. The effects of progesterone are mediated by progesterone receptors (PRs). However, it is still not fully understood how the proliferative effects of PR is regulated in vivo. Increasing amount of evidence strongly suggests that dysregulation of ubiquitin-proteasome system is closely associated with cancer pathogenesis. Speckle-type POZ protein (SPOP) is an adaptor protein of the CUL3-based E3 ubiquitin ligase complexes. SPOP represents one of the highest loci for loss of heterozygosity (LOH) in breast cancer. SPOP downregulation contributes to breast cancer cell growth and invasion. In this study, we revealed PR as a bona fide substrate for SPOP. SPOP interacts with PR in vivo and targets PR for ubiquitin-dependent proteasomal degradation. Moreover, SPOP suppresses progesteroneinduced PR transactivation, S phase entry, and Erk1/2 activation. Our study revealed novel molecular mechanisms underlying the regulation of PR protein homeostasis in breast cancer cells, and provided insights in understanding the relationship between SPOP inactivation and the development of breast cancer.

Keywords: Breast cancer; SPOP; degradation; progesterone receptor; ubiquitination.

Figure 1

SPOP interacts with PR in cells. A. 293T cells were co-transfected with Myc-SPOP and FH-PRA or PRB constructs. After 24 hr, cell lysates were prepared for co-IP with anti-Flag antibody and WB analyses; B. After treated with 20 µM MG132 for 4 hr, T47D cell lysates were prepared for co-IP with anti-PR antibody and WB analyses with indicated antibodies; C. Schematic representation of SPOP deletion mutants. Binding capacity of SPOP to PR is indicated with the symbol; D. 293T cells were co-transfected with FH-PRA and Myc-SPOP-WT or deletion mutants (ΔMATH, ΔBTB) constructs. After 24 hr, cell lysates were prepared for co-IP assay with anti-Myc antibody and WB analyses.

Figure 2

SPOP targets PR for ubiquitination and degradation. (A, B) 293T cells were transfected with FH-PRA (A) or PRB (B) in combination with or without Myc-SPOP constructs. After 24 hr, cells were treated with MG132 (20 µM), Bortezomib (200 nM), or DMSO for 4 hr before cell lysates were prepared for WB analyses; (C) FH-PRA/B and different amounts of Myc-SPOP-WT or deletion mutants (ΔMATH, ΔBTB) constructs were transfected into 293T cells. After 24 hr, cell lysates were prepared for WB analyses; (D) T47D cells were transfected with Myc-SPOP-WT, or deletion mutants (ΔMATH, ΔBTB) constructs. After 36 hr, cell lysates were prepared for WB analyses; (E) T47D cells were transfected with control siRNAs or two SPOP-specific siRNAs. After 48 hr, cell lysates were prepared for WB analyses; (F) Quantitative RT-PCR measurement of the mRNA levels of SPOP and PGR in SPOP-depleted T47D cells. The mRNA level of GAPDH was used for normalization. The mean values (S.D.) of three independent experiments are shown. (G, H) T47D cells were transfected with control or SPOP-specific siRNAs. After 48 hr, cells were chased with 30 μM cycloheximide (CHX). At the indicated time points, cell lysates were prepared for WB analyses; (G) At each time point, the intensity of PR was first normalized to the intensity of Actin (loading control) and then to the value of the 0-hr time point; (H) The mean values (S.D.) of three independent experiments are shown; (I) SPOP promotes PRA polyubiquitination in vivo. Flag-PRA, HA-Ub and Myc-SPOP-WT or ΔBTB mutant constructs were co-transfected into 293T cells. After 24 hr, cells were treated with 20 µM MG132 for 6 hr. PRA proteins were immunoprecipitated with anti-Flag antibody and resolved by SDS/PAGE. The ubiquitinated forms of PRA were analyzed by WB with anti-HA antibody.

Figure 3

SPOP inhibits progesterone-induced transcription. A. 293T Cells were transfected with MMTV luciferase reporter, and Myc-PRA, or Myc-SPOP constructs, alone or in combination as indicated. After 24 hr, cells were treated with vehicle ethanol (EtOH) or progesterone (10 nM) for 24 hr, and the luciferase activities were measured by luminometer; B. T47D cells were transfected with control siRNAs or SPOP siRNAs, After 24 hr, cells were co-transfected with pMMTV luciferase reporter for 24 hr, and then treated with vehicle ethanol (EtOH) or progesterone (10 nM) for 24 hr, and the luciferase activities were measured by luminometer; C. T47D cells were transfected with control siRNAs or SPOP-specific siRNA. After 48 hr, cells were then treated with the vehicle ethanol (EtOH) or progesterone (10 nM) for 18 hr. The mRNA level of PR target genes (KFBP5, Cyclin D1 and HSD2) was measured by qRT-PCR methods. 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); D. T47D cells were transfected with control or Myc-SPOP or SPOP-ΔBTB mutant constructs. After 24 hr, cells were treated with vehicle ethanol (EtOH) or progesterone (10 nM) for 24 hr. Cell lysates were prepared for WB analyses; E. T47D cells were transfected with control siRNAs or SPOP-specific siRNAs. After 48 hr, cells were then treated with the vehicle ethanol (EtOH) or progesterone (10 nM) for 24 hr. Cell lysates were prepared for WB analyses.

Figure 4

SPOP inhibits progesterone-induced S phase entry and Erk1/2 activation. (A) T47D cells were transfected with control or Myc-SPOP constructs. After 24 hr, cells were treated with the vehicle ethanol (EtOH) or progesterone (10 nM) for 24 hr. The cell cycle percentages was determined by PI staining and FACS; (B) T47D cells were transfected with control siRNAs or SPOP-specific siRNAs. After 48 hr, cells were treated and analyzed as in (A); (C) T47D cells were transfected with control or Myc-SPOP constructs. After 24 hr, cells were treated with the vehicle ethanol (EtOH) or progesterone (10 nM) for indicated times. Cell lysates were prepared for WB analyses; (D) T47D cells were transfected with control or SPOP-specific siRNA. After 48 hr, cells were treated and analyzed as in (C).