Prostate cancer-associated mutations in speckle-type POZ protein (SPOP) regulate steroid receptor coactivator 3 protein turnover - PubMed (original) (raw)

. 2013 Apr 23;110(17):6997-7002.

doi: 10.1073/pnas.1304502110. Epub 2013 Apr 4.

Bin He, Limei Xu, Christopher E Barbieri, Vijay Kumar Eedunuri, Sue Anne Chew, Martin Zimmermann, Richard Bond, John Shou, Chao Li, Mirjam Blattner, David M Lonard, Francesca Demichelis, Cristian Coarfa, Mark A Rubin, Pengbo Zhou, Bert W O'Malley, Nicholas Mitsiades

Affiliations

Prostate cancer-associated mutations in speckle-type POZ protein (SPOP) regulate steroid receptor coactivator 3 protein turnover

Chuandong Geng et al. Proc Natl Acad Sci U S A. 2013.

Erratum in

Abstract

The p160 steroid receptor coactivators (SRCs) SRC-1, SRC-2 [nuclear receptor coactivator (NCOA)2], and SRC-3 [amplified in breast cancer 1 (AIB1)/NCOA3] are key pleiotropic "master regulators" of transcription factor activity necessary for cancer cell proliferation, survival, metabolism, and metastasis. SRC overexpression and overactivation occur in numerous human cancers and are associated with poor clinical outcomes and resistance to therapy. In prostate cancer (PC), the p160 SRCs play critical roles in androgen receptor transcriptional activity, cell proliferation, and resistance to androgen deprivation therapy. We recently demonstrated that the E3 ubiquitin ligase adaptor speckle-type poxvirus and zinc finger (POZ) domain protein (SPOP) interacts directly with SRC-3 and promotes its cullin 3-dependent ubiquitination and proteolysis in breast cancer, thus functioning as a potential tumor suppressor. Interestingly, somatic heterozygous missense mutations in the SPOP substrate-binding cleft recently were identified in up to 15% of human PCs (making SPOP the gene most commonly affected by nonsynonymous point mutations in PC), but their contribution to PC pathophysiology remains unknown. We now report that PC-associated SPOP mutants cannot interact with SRC-3 protein or promote its ubiquitination and degradation. Our data suggest that wild-type SPOP plays a critical tumor suppressor role in PC cells, promoting the turnover of SRC-3 protein and suppressing androgen receptor transcriptional activity. This tumor suppressor effect is abrogated by the PC-associated SPOP mutations. These studies provide a possible explanation for the role of SPOP mutations in PC, and highlight the potential of SRC-3 as a therapeutic target in PC.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

PC-associated SPOP mutants lack the capacity of SPOPWT to interact with SRC-3. (A) Distribution on the SPOP gene of the most common mutations found in PC. These recurrent mutations are clustered to the N-terminal MATH domain, which is necessary for SPOP binding to substrates (including SRC-3). (B) HEK293T cells were transfected with pcDNA3.1-HA-SPOP (WT or each individual mutant; described in Materials and Methods) or FLAG-tagged SRC-3 expression vectors for 2 d. Cell lysates containing approximately equal amounts of each expressed HA-SPOP (WT or mutant) and SRC-3 protein were mixed overnight at 4 °C and immunoprecipitated with anti-FLAG M2 antibody. SDS/PAGE and immunoblotting were used to detect HA-SPOPs and FLAG-SRC-3. The input was loaded at 1/10 of the total lysate amount subjected to each immunoprecipitation experiment. SPOPWT was immunoprecipitated together with FLAG-tagged SRC-3. This interaction between SPOP and SRC-3 was attenuated in the case of the PC-associated SPOP mutants.

Fig. 2.

Fig. 2.

SPOPWT, but not PC-associated SPOP mutants, promotes degradation of SRC-3 protein through a mechanism that involves ubiquitination and requires the SPOP BTB domain. (A) HEK293T cells were cotransfected with 0.6 μg pCMV-FLAG 2B-SRC-3 and different amounts (0, 0.2, 0.4, 0.8, 1.6, and 2.0 μg) of pcDNA3.1-Hygro-HA-SPOP or each of the pcDNA3.1-Hygro-HA-SPOP mutants. After 72 h, the FLAG-SRC-3 and HA-SPOPs expressed in these cells were analyzed by immunoblotting. Actin was used as a loading control. SPOP_WT_ efficiently promoted degradation of SRC-3 in a dose-dependent manner. None of the PC-associated SPOP mutants could promote degradation of SRC-3 protein. (B) SRC-3 interacts with SPOP or SPOP(ΔBTB), but not PC-associated F133V mutant. 293T cells were transiently transfected with FLAG-tagged SRC-3, together with FLAG-HA–tagged SPOP, SPOP(F133V), or SPOP(ΔBTB). SPOP proteins were immunoprecipitated with anti-HA antibody and immunoblotted with anti-FLAG antibody. WB, Western blot. (C) SRC-3 degradation in PC cells is promoted by SPOP but not SPOP(ΔBTB). PC-3 cells were transiently transfected with SRC-3 and different doses of FLAG-HA-tagged (FH)-SPOP or FH-SPOP(ΔBTB), and subjected to immunoblotting with antibodies against SRC-3 or FLAG. Vsp34 served as loading control. o, nonspecific band. (D) PC-associated SPOP mutants are defective at promoting SRC-3 ubiquitination. 293T cells were transiently transfected with 3 µg His-Ub and 5 µg FLAG-SRC-3, together with 10 µg FLAG-HA–tagged wild-type SPOP or HA-tagged SPOP mutants [PC-associated mutants or SPOP(ΔBTB)] for 48 h, and then treated with proteasome inhibitor (20 µM MG132 for 2 h). Cells were harvested under denaturing conditions. His-Ub–modified cellular proteins were purified by Ni-NTA agarose resin and subjected to SDS/PAGE. Ubiquitinated SRC-3 was detected by immunoblotting with anti–SRC-3 antibody (topmost panel). Cell extracts were also probed with antibodies against FLAG for F-SRC-3, HA for wild-type or SPOP mutants, or tubulin (loading control).

Fig. 3.

Fig. 3.

PC-associated SPOP mutants lack the capacity of SPOPWT to attenuate the coactivator function of SRC-3 on AR transcriptional activity. HepG2 cells were transfected with expression vectors for AR, SRC-3, and SPOP (WT or mutant) or the corresponding empty vectors, as well as PSA61-Luc reporter vector (that carries the promoter and upstream enhancer region of the PSA gene). The cells were treated 24 h later with 10 nM R1881 or vehicle (EtOH). Luciferase activity was determined 24 h later using the Promega Luciferase Assay Kit according to the manufacturer’s protocols. Error bars represent SD.

Fig. 4.

Fig. 4.

SPOPWT, but not its PC-associated mutants, binds SRC-3 and suppresses endogenous SRC-3 protein levels in PC cells. (A) LNCaP-Abl PC cells stably transfected with tetracycline-inducible constructs encoding for SPOPWT or PC-associated SPOP mutants were treated with 200 ng/mL doxycycline for a total of 48 h. During the last 24 h of the incubation, the cells were also treated with a proteasome inhibitor (PS-341, 250 nM) or vehicle (DMSO). The cells were then lysed in lysis buffer, as in Fig. 1_B_. Immunoprecipitation was performed using antibodies against endogenous SRC-3 or HA or control IgG. In cells treated with the proteasome inhibitor, SRC-3 could coprecipitate SPOPWT, whereas its capacity to bind the PC-associated SPOP mutants was found to be impaired. (B) LNCaP-Abl (Abl) PC cells stably transfected with tetracycline-inducible constructs encoding for SPOPWT or each of the PC-associated SPOP mutants were treated with 0, 50, or 500 ng/mL doxycycline (Dox) for 72 h, lysed, and immunoblotted using antibodies against the HA tag (recognizing only the transfected WT or mutant SPOP), SPOP itself (recognizing both endogenous and transfected SPOPs), SRC-3, and actin.

Fig. 5.

Fig. 5.

SPOPWT, but not its PC-associated mutants, suppresses endogenous AR transcriptional activity and is a potent tumor suppressor in PC cells. (A) Abl cells stably transfected with tetracycline-inducible constructs encoding for SPOPWT or each of the PC-associated SPOP mutants were treated with 0, 100, or 200 ng/mL doxycycline for 48 h. Expression of the AR-dependent gene KLK3 (PSA) was quantified by RT-qPCR. **P < 0.01 for doxycycline-treated versus vehicle-treated cells. (B) MTT assay was used to determine the proliferation of LNCaP-Abl cells when SPOPWT or SPOP mutants were expressed in these cells. Cells were treated with 0, 200, or 300 ng/mL doxycycline for 6 d. Experiments were repeated at least three times, with each experimental condition repeated at least in quadruplicate per experiment. **P < 0.01 for doxycycline-treated versus vehicle-treated cells.

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References

    1. de Bono JS, et al. COU-AA-301 Investigators Abiraterone and increased survival in metastatic prostate cancer. N Engl J Med. 2011;364(21):1995–2005. - PMC - PubMed
    1. Scher HI, et al. AFFIRM Investigators Increased survival with enzalutamide in prostate cancer after chemotherapy. N Engl J Med. 2012;367(13):1187–1197. - PubMed
    1. Mitsiades N, et al. Distinct patterns of dysregulated expression of enzymes involved in androgen synthesis and metabolism in metastatic prostate cancer tumors. Cancer Res. 2012;72(23):6142–6152. - PMC - PubMed
    1. Agoulnik IU, et al. Androgens modulate expression of transcription intermediary factor 2, an androgen receptor coactivator whose expression level correlates with early biochemical recurrence in prostate cancer. Cancer Res. 2006;66(21):10594–10602. - PubMed
    1. Taylor BS, et al. Integrative genomic profiling of human prostate cancer. Cancer Cell. 2010;18(1):11–22. - PMC - PubMed

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