MST1 Is a Multifunctional Caspase-Independent Inhibitor of Androgenic Signaling (original) (raw)

Tumor and Stem Cell Biology| June 14 2011

Authors' Affiliations: 1Departments of Medicine-Hematology/Oncology and Biomedical Sciences, Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center; 2Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, California; 3Urological Diseases Research Center, Children's Hospital Boston; Departments of Surgery and Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts; 4Bilkent University, Bilkent, Ankara, Turkey; 5University of Amsterdam, Amsterdam, The Netherlands; and 6Department of Microbiology and the Cancer Center, University of Virginia Health System, Charlottesville, Virginia

Corresponding Author: Bekir Cinar, Departments of Medicine-Hematology/Oncology and Biological Sciences, Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, University of California, 8750 Beverly Blvd., Atrium 103, Los Angeles, CA 90048. Phone: 310-423-8658; Fax: 310-423-8543; E-mail: bekir.cinar@cshs.org

Search for other works by this author on:

Filiz Kisaayak Collak;

Search for other works by this author on:

Delia Lopez;

Search for other works by this author on:

Seckin Akgul;

Search for other works by this author on:

Nishit K. Mukhopadhyay;

Search for other works by this author on:

Murat Kilicarslan;

Search for other works by this author on:

Daniel G. Gioeli;

Search for other works by this author on:

Michael R. Freeman

Search for other works by this author on:

Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Received: December 17 2010

Revision Received: March 08 2011

Accepted: April 01 2011

Online ISSN: 1538-7445

Print ISSN: 0008-5472

2011

Cancer Res (2011) 71 (12): 4303–4313.

Article history

Received:

December 17 2010

Revision Received:

March 08 2011

Split-Screen
Views Icon Views
Open the PDF for in another window
Tools Icon Tools
Search Site
Article Versions Icon Versions
- Version of Record June 14 2011
- Proof June 7 2011
- Accepted Manuscript April 21 2011

Abstract

The MST1 serine–threonine kinase, a component of the RASSF1-LATS tumor suppressor network, is involved in cell proliferation and apoptosis and has been implicated in cancer. However, the physiologic role of MST1 in prostate cancer (PCa) is not well understood. Here, we investigated the possibility of a biochemical and functional link between androgen receptor (AR) and MST1 signaling. We showed that MST1 forms a protein complex with AR and antagonizes AR transcriptional activity as shown by coimmunoprecipitation (co-IP), promoter reporter analysis, and molecular genetic methods. In vitro kinase and site-specific mutagenesis approaches indicate that MST1 is a potent AR kinase; however, the kinase activity of MST1 and its proapoptotic functions were shown not to be involved in inhibition of AR. MST1 was also found in AR–chromatin complexes, and enforced expression of MST1 reduced the binding of AR to a well-characterized, androgen-responsive region within the prostate-specific antigen promoter. MST1 suppressed PCa cell growth in vitro and tumor growth in mice. Because MST1 is also involved in regulating the AKT1 pathway, this kinase may be an important new link between androgenic and growth factor signaling and a novel therapeutic target in PCa. Cancer Res; 71(12); 4303–13. ©2011 AACR.

Introduction

The serine–threonine kinase MST1 or STK4 (mammalian sterile STE20-like kinase 1), a homolog of Hippo (Hpo/hpo) in Drosophila, was originally identified as a proapoptotic protein (1). MST1 is related to 3 paralogs (MST2, MST3, and MST4) with a conserved structure consisting of N-terminal catalytic (MST1-N) and C-terminal regulatory (MST1-C) domains and other functional sites, including caspase cleavage sites and nuclear export signals (2, 3). MST1 or MST2 can be activated by autophosphorylation of a unique threonine residue (Thr-183 in MST1 and Thr-180 in MST2) in the activation loop or by caspase-3 cleavage in response to a wide range of cell death stimuli (4).

In addition to their proapoptotic function, MST1 and its closest paralog MST2 have been shown to play an important role in mammalian development (5, 6), cell-cycle progression and tumorigenesis (7–10). For example, hpo deficiency in the developing Drosophila eye results in massive overgrowth due to an accelerated rate of proliferation and failure of developmental apoptosis (11–13). Likewise, MST1 or MST2 deficiency in mice is embryonically lethal (5). Loss or reduction of MST1 and MST2 expression has also been correlated with poor cancer prognosis (14). Recent genetic studies have indicated that liver-specific deletion of MST1 and MST2 in mice resulted in liver enlargement, cancer, and resistance to TNF-α induced apoptosis (7, 9, 10). Previous studies suggest that cross talk between androgen receptor (AR) and MST1 signaling may have important biological consequences in prostate cancer (PCa; refs. 15, 16). Therefore, we wanted to investigate whether MST1 functionally intersects with AR signaling and regulates the growth of PCa cells.

Here, we show that MST1 is a novel negative regulator of AR signaling. Our data suggest that MST1 attenuates AR activity via a mechanism that involves protein complex formation between AR and MST1 in a manner that is independent of its kinase activity. Furthermore, we provide evidence that enforced MST1 expression suppressed PCa cell growth, sensitized androgen-independent C4-2 cells to phosphatidylinositol 3-kinase (PI3K) inhibition, and attenuated tumor growth in vivo. These findings suggest that loss of MST1 signaling may promote hyperactivation of AR and may be associated with the emergence of the castration-resistant phenotype.

Materials and Methods

Plasmid constructions, antibodies, and reagents

The construction of HA- or Myc-tagged MST1-wt and Myc-MST1-N and Myc-MST1-C forms was described previously (15). For the construction of Doxycycline(Dox)-inducible HA-MST1 plasmid, PCR-amplified HA-tagged MST1-wt cDNA was inserted into BamH1 and MluI enzyme sites in the pRetro-X-Pur vector (Clontech Laboratories, Inc.), designated as pRXTP-HA-MST1. The construction of glutathione S-transferase (GST)-AR DBD/HR (AR DNA binding domain and hinge region) was described previously (17). MST1 and AR point mutations were generated by using the QuickChange site-directed mutagenesis kit (Stratagene). The orientation and fidelity of all constructs were confirmed by DNA sequencing. Note that names of antibodies and reagents and their sources are provided in the Supplementary Information section.

Cell transfections, reporter assays, and immunocytochemistry

LNCaP and C4-2 were cultured in RPMI 1640 medium and HEK 293T, and COS-7 cells were cultured in DMEM at 37°C in 5% CO2 incubator. Media were supplemented with 10% FBS and 1% penicillin/streptomycin. RNAi (siRNA) transfections with Dharma FECT 2 and plasmids transfections with Lipofectamine 2000 were carried out according to the manufacturer's instructions (Invitrogen). Luciferase reporter gene activities were measured by using the Luciferase Assay System from Promega and a BMG Labtech microplate reader. Immunocytochemistry (IHC) was done as described previously (15). Relative luciferase units were normalized to total protein and the result presented as luciferase (Luc) activity. Cells were imaged at 63× with a Plan-Apochromat oil immersion lens on an Axioplan 2 Apotome epifluorescence microscope (Zeiss). IHC was done by using reagents from DAKO and images were acquired at 20× with Nikon Imaging System.

Establishment of TetON-inducible cells

Retroviruses carrying Tet-repressor or HA-MST1 expression constructs were produced in HEK 293T cells expressing viral packaging proteins and then viral particles were concentrated by using PEG-it solution. LNCaP parental cells or its castration-resistant subline, C4-2, were first infected with retrovirus encoding pRetroX-TetON advanced plasmid, followed by selection with Geneticin (G418, 500 μg/mL) to generate the TetON cells. The LNCaP/ or C4-2/TetON cells were then infected with retrovirus encoding pRXTP-HA-MST1 vector, followed by Puromycin selection (3 μg/mL) to generate TetON inducible MST1 expressing cells. The inducible system allows fine control of MST1 expression. All protocols and procedures were done according to the manufacturer's instructions (Clontech Laboratories, Inc.).

Protein analyses

Cell lysis was conducted in buffer consisting of 20 mmol/L HEPES, pH 7.4, 150 mmol/L NaCl, 0.5% NP-40, 1 mmol/L EDTA, protease inhibitors, and phosphatase inhibitor. For immunoprecipitation, cleared lysates were incubated with antibody overnight at 4°C. Antibody–antigen complexes were collected on protein A- or G-sepharose and washed 3 times with cell lysis buffer. Immunoprecipitates were resolved by SDS-PAGE. PBST (0.1% Tween-20) containing 5% (w/v) skim milk powder or PBST containing 5% immunoglobulin G (IgG)-free bovine serum albumin (Sigma) was used in membrane blocking and antibody dilutions. Signals were visualized by chemiluminescence. GST-AR DBD/HR and its mutant forms were expressed in bacteria with IPTG (Isopropyl β-d-1-thiogalactopyranosid) induction and protein purification was done with GST-sepharose by using a standard protocol. Cytoplasmic and nuclear fractions were prepared as described (18).

Chromatin immunoprecipitation assays

Chromatin immunoprecipitation (ChIP) was done as described previously (19). Briefly, LNCaP or C4-2/HA-MST1 cells grown in serum-starved conditions were treated with R1881 (1 nmol/L) or EtOH (vehicle) overnight. Dox(−/+) used to induce MST1 expression in C4-2/HA-MST1 cells. DNA enriched with anti-MST1, anti-AR, or anti–Pol II antibody were quantified by semiquantitative PCR by using primer sets surrounding the AREIII region within the androgen-responsive element enhancer (ARE) core (AREc) or AREI of the prostate-specific antigen (PSA) promoter (18).

Kinase, cell death, and cell proliferation assays

For in vitro kinase assay, the recombinant, preactivated MST1 protein kinase was incubated with purified GST-AR DBD/HR fusion protein and 10 μCi 32P-γ-ATP or 100 μmol/l unlabeled-ATP. The reaction mixture was resolved on SDS-PAGE and autoradiographed. Cell Death ELISA and BrdU incorporation assays were conducted to assess cell death and cell proliferation, respectively, according to the manufacturer's instructions (Roche Molecular Diagnostics).

Animal studies

C4-2/Vector or C4-2/HA-MST1 cells mixed with Matrigel (1-to-1 ratio) were inoculated s.c. in athymic nude mice (CD-1 nu/nu; Charles River Laboratories). A total of 1 × 106 cells/100 μL were used per injection per site (right and left flanks). Mice were treated with Dox (0.5 mg/mL) in drinking water to induce MST1 expression for 12 weeks. Institutional Animal Care and Use Committee policies and guidelines were strictly applied. Tumor volumes were assessed by caliper according to procedures described previously (20). Mice were sacrificed and evaluated for tumor growth anatomically and tumor tissues extracted from mice were fixed in 10% formaldehyde for the construction of histologic sections or “snap” frozen at −80°C. The expression of MST1 was verified in histologic sections by IHC by using anti-HA antibody.

Statistics

Data are represented as mean ± SEM. Student t test (2-tailed) was used between the data pairs in which it is appropriate. A P value 0.05 or less was considered significant.

Results

MST1 binds and attenuates AR activity

To determine whether the MST1 kinase biochemically and functionally intersects with AR signaling, we employed cellular and biochemical approaches by using prostate and non-PCa cell lines that express either native, stable or transient AR. Coimmunoprecipitation (Co-IP) and Western blot experiments showed that MST1 interacts with endogenous AR in LNCaP cells (Fig. 1A, top panel) and ectopically expressed human AR in HEK 293 cells (Fig. 1A, bottom panel). Similarly, endogenous MST1 could also be found in the AR protein complex from PC3-hAR cells that were engineered to express near-physiologic levels of stable His-tagged human AR (18), as shown by Ni-NTA precipitation and Western blot experiments (Supplementary Fig. S1A and B).

Figure 1.

MST1 forms a protein complex with AR and antagonizes AR activity. A, co-IP of ectopically expressed human MST1 and endogenous AR in LNCaP (top) or exogenously expressed human AR in HEK 293 (bottom) cells. Co-IP and Western blots (WB) were done with corresponding antibodies. B, AR-responsive PSA promoter reporter (p61-Luc) activity in LNCaP under condition in which MST1 was knocked down (top) or enforced (bottom). An unpaired t test was conducted to analyze for differences between treatments. *, P ≤ 0.02. C, AR-responsive GRE4-Luc in LNCaP (top) and p61-Luc activity in COS-7 cells (bottom). Serum-starved cells were treated with (+) or without 1 nmol/L R1881, androgen analog, *, P ≤ 0.02. All assays were conducted at 36 hour. Relative luciferase units presented as luciferase (Luc) activity after normalization with total protein. Data are representative of multiple experiments.

Figure 1.

Close modal

To test whether the binding of MST1 has an impact on AR activity, we conducted AR-dependent promoter reporter assays (18, 21) by using knockdown and induction approaches. MST1 knockdown by a gene-specific siRNA (15) resulted in the upregulation of basal and androgen-stimulated AR-responsive PSA promoter reporter activation, by at least 2-fold in comparison with siRNA control (Fig. 1B, top panel). Enforced MST1 expression attenuated endogenous AR activity in LNCaP cells, as shown by the PSA promoter reporter (p61-Luc) assay (Fig. 1B, bottom panel). Enforced MST1 expression also attenuated AR-dependent GRE4-driven simple promoter reporter (pGRE4-TATA-Luc) activation in LNCaP (Fig. 1C, top panel) and p61-Luc promoter reporter activation in COS-7 (Fig. 1C, bottom panel) cells, in which AR expression was also enforced. Collectively, these observations indicate that MST1 is a binding partner and a physiologic negative regulator of AR signaling.

The full-length MST1 is a dominant AR suppressor

To map the AR binding domain on MST1, the full-length AR was coexpressed with vector, MST1-wt or MST1-N (residues 1–330) or MST1-C (residues 331–487) truncation mutants in COS-7 cells, followed by co-IP and Western blot analysis. The results showed that the full-length MST1 (MST1-wt) and MST1-N strongly interacted, whereas MST1-C displayed weak interaction with exogenous AR (Fig. 2A). The inhibition of AR activity by MST1-wt, MST1-N, or MST1-C coincided with the binding data, as revealed by the PSA promoter reporter assay (Fig. 2B).

Figure 2.

Caspase cleavage-deficient MST1 is a potent AR inhibitor. A, AR binding domains on MST1. AR with vector (V), MST1-wt (wild type) or MST1 domains (MST1-N or MST1-C) was transiently coexpressed in COS-7 cells. Co-IP and WB were done with antibodies to corresponding proteins. B, PSA promoter reporter activity in LNCaP cells with MST1-wt or MST1 truncation mutant in A, *, P ≤ 0.01; **, P ≤ 0.08. C, Western blots of MST1, AR or PSA protein in LNCaP cells transfected with Mock, MST1-wt, MST1-D326N (D/N), MST1-D349E (D/E), or MST1-D326N/D349E (DD/NE), followed by R1881 (+) or vehicle (−) treatment in serum-starved conditions. D, PSA promoter reporter activity with caspase-resistant MST1 constructs under the same conditions as described in C, *, P ≤ 0.01. Luciferase activity was determined at 36 hour. Data are representative of multiple experiments.

Figure 2.

Close modal

To determine which MST1 form (MST1-wt or the cleaved MST1-N) functions as a dominant AR inhibitor, we generated caspase-resistant single [D326N = D/N, Asp (D) →Asn (N) or D349E = D/E, Asp→Glu (E)] and double (D326N/D349E = DD/NE) MST1 mutants and assessed their effects on PSA protein levels or luciferase reporter activity mediated by AR. The results show (Fig. 2C and D) that the expression of each MST1 mutant is capable of inhibiting endogenous PSA protein levels and PSA promoter reporter activation induced by androgen, similar to the levels seen with MST1-wt. Neither the expression of MST1-wt nor these MST1 mutants affected AR protein levels, unless the cells were stimulated by androgen (Fig. 2C, AR blot). A similar level of AR-dependent PSA promoter reporter inactivation by MST1 was also obtained, even in the presence of increasing doses of a specific caspase inhibitor, Ac-DEVD-CHO (D-CHO; Supplementary Fig. S2A).

We then examined protein complexes between AR and the caspase-deficient MST1-DD/NE mutant. AR was coexpressed with vector, MST1-wt, or MST1-DD/NE mutant constructs in HEK 293 cells. As shown by co-IP/Western blot experiments, the MST1-DD/NE mutant maintained interaction with AR and the levels of interaction between AR and MST1-DD/NE were similar or even greater than that observed with MST1-wt (Supplementary Fig. S2B). These observations indicate that the caspase-deficient MST1 is a potent AR inhibitor.

MST1 attenuates AR activity in a Ser650 phosphorylation-independent manner

MST1 is a stress-induced kinase (2), and other stress-induced kinases such as c-Jun-N-Terminal Protein Kinase 1 (JNK1) or p38 mitogen-activated protein (MAP) kinase have been proposed to physically interact with and antagonize AR transcriptional activity by phosphorylating AR at Ser650 (17). To determine whether purified MST1 can also physically interact with and phosphorylate AR at this site, we conducted the GST-pulldown and an in vitro kinase assay by using recombinant, preactivated MST1, and purified GST-AR-DBD/HR as a substrate. The results of these experiments revealed that preactivated, recombinant MST1 physically interacted with (Fig. 3A) and specifically phosphorylated the purified GST-AR-DBD/HR fragment in vitro (Fig. 3B, left panel). Both binding and phosphorylation events occurred in a dose-dependent manner (Supplementary Fig. S3A and B, respectively). Using a site-specific phospho-AR antibody, we were able to identify the Ser650 residue as a target of the MST1 kinase activity, as revealed by nonradioactive in vitro kinase assay and Western blot experiments (Fig. 3B, right panel).

Figure 3.

Purified recombinant MST1 binds and phosphorylates GST-AR-D/HR fragment at Ser650. A and B, GST-pull down in A and in vitro kinase assay with bacterially expressed GST-AR-D/HR and recombinant, preactivated MST1 protein kinase in B, left. Right, WB probed with antibody to MST1 or to phospho-Ser650 AR in B. AR-D/HR: AR-DNA binding domain/hinge region. C, pGRE4-Luc promoter luciferase activity in COS-7 cells. Cells were cotransfected with pGRE4-Luc and corresponding constructs and treated with R1881 (+) or vehicle (−) in serum-starved conditions, *, **, P ≤ 0.01. Luciferase reporter assay was conducted at 36 hour following androgen treatment. D, immunofluorescence staining of AR protein (FITC, cytoplasm with EtOH vehicle and nuclear with R1881) with anti-AR antibody and DAPI (nucleus) in COS-7 cells transfected to express transient AR and stimulated with R1881 (+) or vehicle (−) for 6 hour in serum-starved conditions. The size bar represents 10 microns. Data are representative of multiple experiments.

Figure 3.

Close modal

To determine whether Ser650 phosphorylation has a role in the attenuation of AR activity by MST1, we generated phosphorylation-inactivating (Ser→Ala) or phosphomimetic (Ser→Glu) mutations and assessed their impact on MST1-mediated inhibition of AR activity. The data in Figure 3C show that enforced-MST1 expression is capable of inhibiting AR transcriptional activity regardless of the presence of phosphorylation-inactivating Ser650A or phosphomimetic Ser650D AR mutations. Similarly, other site-specific phosphorylation-inactivating mutations did not significantly affect AR inhibition by MST1 induction (Supplementary Fig. S3C). In addition, phosphorylation-inactivating or phosphomimetic mutations did not alter ligand-dependent nuclear localization of AR in COS-7 cells expressing exogenous AR (Fig. 3D). Furthermore, co-IP/Western blot analyses showed that neither type of mutation affected complex formation between the two proteins (Supplementary Fig. S3D). In addition, with the exception of the Ser308A mutation, these known phospho-site mutations do not significantly alter androgen-induced AR transcriptional activity in COS-7 cells (Supplementary Fig. S4A). These findings indicate that the phosphorylation of Ser650 by MST1 is not involved in the mediation of AR inhibition by MST1.

MST1 kinase activity is not required for the inhibition of AR activity

To determine whether MST1 kinase activity has an effect on the inhibition of AR transcriptional activity, we generated kinase-deficient MST1 mutants (MST1-K59R in the ATP binding pocket and MST1-T183A in the activation loop). Consistent with published data (3), neither of these MST1 mutants was able to induce apoptosis in COS-7 or in LNCaP cells, compared with MST1-wt (Fig. 4A). However, both mutants were capable of inhibiting AR-driven PSA promoter activation, similar to the levels observed with MST1-wt in LNCaP (Fig. 4B) and in its castration-resistant C4-2 subline (Supplementary Fig. S4B). We then examined the protein complexes between AR and these kinase-inactivated MST1 mutants. Mock, MST1-wt, MST1-K59R, or MST1-T183A constructs were coexpressed with AR in COS-7 cells and co-IP experiments were done by using total cell lysates. Western blot analysis with anti-AR antibody revealed that the kinase-inactivating mutation had no affect on the formation of the AR and MST1 complex (Fig. 4C). We also conducted the promoter reporter assay with MST2 and showed that transient expression of this close structural relative to MST1 also antagonized the AR transactivation function in LNCaP cells independently of its kinase activity (Fig. 4D). These results suggest that both kinases perform a similar inhibitory role with respect to the AR, and that neither the kinase activity nor the proapoptotic function of MST1 or MST2 is involved in the inhibition of AR activity.

Figure 4.

The kinase and apoptotic functions of MST1 are not involved in AR inhibition. A, apoptosis in LNCaP or in COS-7 cells transfected with MST1-wt or kinase deficient-MST1 mutant construct (MST1-K59R or MST1-T183A). Cell apoptosis was determined by using Cell Death ELISA. B, PSA promoter reporter activity in LNCaP cells with kinase intact or kinase-deficient MST1 as in A, followed by incubation with (+) or without (−) R1881. C, MST1 and AR protein complex in COS-7 cells expressing AR along with vector, MST-wt, or kinase-deficient MST1 mutant. Co-IP and Western blots were conducted in total cell lysates with antibodies to corresponding proteins. D, MST2-regulated PSA promoter reporter activity in LNCaP cells, *, P ≤ 0.01. p61-Luc (+) was cotransfected with MST2-wt or with kinase-deficient MST2 mutant (MST2-K56R), *, P ≤ 0.01. All assays were conducted at 36 hour. HC: IgG-heavy chain. Data are representative of multiple experiments.

Figure 4.

Close modal

MST1 suppresses AR activity by intersecting with AKT1 signaling and antagonizing formation of AR–chromatin complexes

An implication from the above findings is that additional mechanisms may be involved in MST1-mediated AR inhibition. MST1 was reported to inhibit AKT signaling (15), which is known to functionally intersect with (16, 22) and promote AR-driven PSA promoter activation (23). To test whether MST1 induction could suppress AR activation mediated by AKT1 signaling, we conducted promoter reporter assays and showed that enforced MST1 expression antagonized AKT1 mediated androgen-dependent and -independent AR activation (Fig. 5A, left panel). Co-IP experiments further revealed that MST1, AR, and AKT1 form a tri-partite complex in vivo (Fig. 5A, right panel), indicating that MST1 also intersects with AKT signaling to attenuate AR activity.

Figure 5.

MST1 antagonizes AKT-mediated AR activation and localizes to AR–chromatin complexes. A, graph: PSA promoter reporter activity modulated with MST1 and AKT signaling, *, **, P ≤ 0.01. LNCaP cells were transiently cotransfected with p61-Luc promoter reporter and AKT1 or MST1 alone or together with AKT1 and MST1, followed by androgen or vehicle treatment in serum-starved conditions. Blots: A tri-partite protein complex between AR, MST1, and AKT1 in COS-7 cells expressing transient MST1 along with AR or AR and AKT1. All assays were conducted at 36 hour. HC: IgG-heavy chain. B, co-IP and WB analysis of protein complexes between endogenous AR and MST1 in LNCaP cells grown in basal condition. C and D, ChIP assay in lysates of LNCaP cells. Protein-DNA complexes were precipitated with IgG (negative control), Pol II (positive control), AR, or MST1 antibody in total cell lysates. DNA interaction was assessed by semiquantitative PCR by using primers surrounding AREIII within the AREc and surrounding AREI and TATA region of the PSA promoter. Diagram showing AREc and proximal PSA promoter region where multiple AREs and TATA box, AREI, and AREII reside, respectively. Primer pair against PSA gene was used in input control. Data are representative of multiple experiments.

Figure 5.

Close modal

Given that MST1 forms protein complexes with AR, we showed by using co-IP experiments that endogenous AR and MST1 form a complex preferentially in cell nuclei (Fig. 5B). These observations led us to investigate whether MST1 localizes within AR-transcriptional complexes. ChIP experiments showed that endogenous MST1 interacts with the PSA promoter region, which also binds AR in both serum- and androgen-free conditions and can be regulated by androgen in LNCaP cells (Fig. 5C). This segment of the PSA promoter, referred to as the ARE core, consists of multiple AREs and plays a prominent role in AR-driven PSA promoter regulation (18, 21). Additional data suggest that MST1 also binds ARE-I and the TATA region in the PSA promoter, which can also be regulated by androgen (Fig. 5D). Taken together, these findings suggest that MST1 reduces AR–chromatin complex formation to attenuate androgenic signals.

MST1 suppresses PCa cell growth in vitro and tumor growth in vivo

To address the impact of MST1 on cell growth, we established stable MST1 expressing LNCaP/HA-MST1 or C4-2/HA-MST1 cells by using a retroviral inducible system. LNCaP/HA-MST1 or C4-2/HA-MST1 cells were exposed to increasing doses of Dox. Dose-dependent induction of MST1 expression reduced the growth of LNCaP; however, castration-resistant LNCaP subline, C4-2, displayed resistance to the growth suppressive effects of enforced MST1 (Fig. 6A and B). The observed growth reduction originated from MST1 induction because the administration of the highest dose of Dox (4 μg/mL), which reduced the growth of LNCaP/HA-MST1 the most, had no effect on the growth of LNCaP/Vector cells (Supplementary Fig. S5A). In addition, enforced MST1 expression in C4-2/HA-MST1 cells dramatically reduced the number and size of C4-2 colonies grown in soft agar (Fig. 6C) and sensitized C4-2 cells to growth suppression induced by PI3K inhibitor LY294002 (Fig. 6D). C4-2 cells are relatively resistant to PI3K inhibitors (24) and less sensitive to the effects of MST1 compared with parental LNCaP cells (Fig. 6A). As expected, induction of MST1 prevented the growth of LNCaP cells regardless of the presence of PI3K inhibitor (Supplementary Fig. S5B).

Figure 6.

Enforced MST1 expression suppresses prostate tumor cell growth in monolayer culture and colony formation on soft agar. A, cell proliferation with BrdU incorporation assay, *, P ≤ 0.2; **, P ≤ 0.0009. B, stable MST1 expression by Western blot. LNCaP/HA-MST1 and C4-2/HA-MST1 cells treated with increasing doses (0, 0.5, 1, 2, and 4 μg/mL) of Dox, respectively, in A and B. Western blots probed with anti-HA antibody or anti–β- actin as a loading control. C, colony formation assay on soft agar. Equal numbers of C4-2/HA-MST1 cells were seeded on soft agar and grown for 14 days in the presence (0.5 μg/mL) and absence (−) of Dox in regular growth conditions with the media was replaced every 3 days; *, P ≤ 0.004. The graph represents the quantification of colonies and the micrograph represents colonies formed on soft agar. D, the growth of C4-2/HA-MST1 cells treated with LY294002 (20 μmol/L), a potent PI3K inhibitor or vehicle [dimethyl sulfoxide (DMSO)] in the presence (+) and absence (−) of Dox, *, P ≤ 0.08; **, P ≤ 0.007. All cell proliferation was determined at 24 hour. Data are representative of multiple experiments.

Figure 6.

Close modal

To assess the distribution of MST1, we analyzed levels of exogenous MST1 protein in cytoplasmic and nuclear fractions obtained from C4-2/HA-MST1 cells. The result revealed that although the majority of exogenous HA-MST1-wt localized in the cytoplasm, significant levels of exogenous HA-MST1-wt protein were also found in the nucleus (Fig. 7A, left panel). Immunofluorescence experiments confirmed these results (Supplementary Fig. S5C). Stable MST1 expression was capable of inhibiting AR-driven PSA promoter activation in C4-2/HA-MST1 cells (Fig. 7A, right panel). ChIP experiments by using lysates from C4-2/HA-MST1 cells further showed that loading of MST1 onto the ARE core region of the PSA promoter diminished the formation of AR–chromatin complexes (Fig. 7B).

Figure 7.

$Figure 7. Enforced MST1 expression suppresses PCa xenografts. A, left, WB analysis of ectopically expressed HA-MST1 in cytoplasmic and nuclear cell fractions obtained from C4-2/HA-MST1 cells treated with Dox (−) or Dox (+). HA-MST1 was probed with anti-HA antibody. Lamin A/C was used as a nuclear extraction control. Right, PSA promoter reporter (+) activity in C4-2/HA-MST1 cell treated with (+) or without (−) Dox and vehicle (EtOH) or androgen (R188) at 24 hour following treatment, *, P ≤ 0.01. B, ChIP assay in lysates from C4-2/HA-MST1 cells, respectively. Protein–DNA complexes were precipitated with HA-tag antibody. DNA interaction was assessed by semiquantitative PCR by using primers surrounding AREIII within the AREc region of the PSA promoter. Primer pair against PSA gene was used in input control. C, left, immunohistochemical staining of HA-MST1 with anti-HA antibody in histologic sections from tissue xenografts of C4-2/Vector or C4-2/HA-MST1 tumor. The size bar in C, left represents 100 microns. Right, the quantification of s.c. tumor growth in mice. Ten mice per group were used. Eight of 10 mice for C4-2/Vector or 2 of 10 mice for C4-2/HA-MST1 were developed tumors 12 weeks after s.c. inoculation of the cell. Data are representative of multiple experiments. D, diagram showing the points at which MST1 negatively regulates AR-mediated gene expression.$

Enforced MST1 expression suppresses PCa xenografts. A, left, WB analysis of ectopically expressed HA-MST1 in cytoplasmic and nuclear cell fractions obtained from C4-2/HA-MST1 cells treated with Dox (−) or Dox (+). HA-MST1 was probed with anti-HA antibody. Lamin A/C was used as a nuclear extraction control. Right, PSA promoter reporter (+) activity in C4-2/HA-MST1 cell treated with (+) or without (−) Dox and vehicle (EtOH) or androgen (R188) at 24 hour following treatment, *, P ≤ 0.01. B, ChIP assay in lysates from C4-2/HA-MST1 cells, respectively. Protein–DNA complexes were precipitated with HA-tag antibody. DNA interaction was assessed by semiquantitative PCR by using primers surrounding AREIII within the AREc region of the PSA promoter. Primer pair against PSA gene was used in input control. C, left, immunohistochemical staining of HA-MST1 with anti-HA antibody in histologic sections from tissue xenografts of C4-2/Vector or C4-2/HA-MST1 tumor. The size bar in C, left represents 100 microns. Right, the quantification of s.c. tumor growth in mice. Ten mice per group were used. Eight of 10 mice for C4-2/Vector or 2 of 10 mice for C4-2/HA-MST1 were developed tumors 12 weeks after s.c. inoculation of the cell. Data are representative of multiple experiments. D, diagram showing the points at which MST1 negatively regulates AR-mediated gene expression.

Figure 7.

Close modal

To determine the physiologic relevance of our in vitro findings, we carried out xenograft experiments. C4-2/HA-MST1 or C4-2/Vector cells were inoculated s.c. into immunodeficient male mice and the animals were then treated with Dox in the drinking water. Immunohistochemical analyses of the resultant tumors verified the expression of HA-MST1 in histologic sections from C4-2/HA-MST1 or C4-2/Vector tumor xenografts (Fig. 7C, micrograph). Consistent with observations in vitro, enforced MST1 expression suppressed the growth by 35-fold and the tumorigenic rates by 4-fold of C4-2/HA-MST1 cells compared with C4-2/Vector counterparts (Fig. 7C, graph and tumor mass image).

Discussion

In this study, we show that the serine–threonine kinase MST1 is a physiologic negative regulator of AR signaling. We provide evidence that MST1 forms protein complexes in vitro and in vivo with AR and antagonizes AR activity in multiple cell backgrounds. Although both the full-length MST1 and the cleaved MST1-N forms bound and inhibited AR activity, caspase-resistant MST1 was the most potent AR inhibitor. We found that the kinase activity of MST1 was not required for the attenuation of AR activity. Similarly, the proapoptotic function of MST1 is not involved in AR inhibition because kinase deficient MST1, which failed to induce cell death, was capable of interacting with AR and inhibiting AR transcriptional activity. Furthermore, promoter reporter and ChIP experiments revealed that enforced MST1 antagonized AKT-mediated AR activation and reduced binding of AR to its cognate DNA binding site. On the basis of these observations, we propose that MST1 antagonizes AR-dependent gene expression by forming inhibitory protein and/or transcriptional complexes with AR, thereby suppressing prostate tumor growth.

Posttranslational modifications, such as phosphorylation (17), palmitoylation (25), ubiquitination (26), acetylation (27), or SUMOylation (28) play important roles in the regulation of AR activity. Several phosphorylation sites at serine residues and a tyrosine residue have been identified in AR (29, 30). These modifications negatively or positively regulate AR activity in a context-dependent manner (24), and their functional significance in PCa is beginning to emerge (31). For example, the phosphorylation of AR at Tyr-534 by c-Src was shown to enhance AR activation and AR-dependent gene expression, which was shown to be correlated with hormone-refractory PCa (30). On the other hand, phosphorylation of AR at Ser650 by JNK1 or p38 MAP kinase was shown to inhibit AR activity (17). Here, we showed that MST1 is an AR kinase and phosphorylates AR at Ser650 (Fig. 3C). However, phosphorylation of Ser650 by MST1 had no effect on the observed inhibition of AR-transcriptional activity. Nevertheless, the role of phospho-Ser650 on mechanisms of AR action deserves further investigation, given that phosphorylation-inactivating Ser650A or phosphomimetic Ser650D AR mutations are able to alter the cellular distribution of AR in comparison to that observed with AR-wt under androgen-free conditions (Fig. 3D). Our data do not rule out the possibility that the existence of other potential MST1 phosphorylation sites might play a role in the inhibition of AR activity by MST1. In addition, the induction of MST1 was shown to activate JNK1 (32), which is known to inhibit AR (17). We did not observe JNK1 activation in response to MST1 induction in LNCaP, unless cells were treated with phorbol ester (Supplementary Fig. S2C). This indicates that MST1 attenuates AR signaling by a distinct mechanism apart from JNK or p38 MAP kinase activation.

In addition to posttranslational modifications, transcriptional coregulators (i.e., corepressors or coactivators) play an essential role in the modulation of AR activity, and their altered expression has been shown in prostate tumor progression (33). For example, the recruitment of nuclear corepressor (N-CoR) (34) or silencing mediator for thyroid and retinoid receptors (35) into the AR transcriptional complex was shown to antagonize AR activity by a mechanism involving protein–protein interaction. Similarly, displacement of coactivators such as p300 from the holo-AR transcriptional complex, or recruitment to the complex of histone deacetylase, which modifies chromatin structure to a transcriptionally inactive form (36), have also been shown to attenuate AR activity (35). Given that MST1 attenuates AR activity by forming protein complexes, the localization of MST1 into the holo-AR transcriptional complex could also attenuate the AR. Our data in Figures 5C, D and 7B support this conclusion; however, comprehensive studies are needed to elucidate precisely how MST1 alters AR–chromatin complexes.

MST1 and its downstream effectors, such as WW45 or LATS1/2, have been implicated in cancer, including PCa (37). For example, the liver specific knockout of MST1/2 expression in mice has been associated with hepatocellular carcinoma, which has been linked to the activation of YAP (7, 9, 10), and YAP is normally attenuated by the MST–LATS signaling network (10). The loss or reduced expression of LATS2 was reported in PCa and this was shown to be associated with hyperactivation of AR and upregulation of AR-dependent gene expression (38), with protein products known to promote PCa cell survival and inhibit apoptosis. In addition, mice lacking WW45 expression displayed hyperplasia in several organ sites (39). Here, we found that the induction of MST1 expression is sufficient to antagonize AR-driven gene expression and suppress PCa cell growth. Moreover, we found that the growth suppressive effects of MST1 significantly declined in castration-resistant C4-2 cells in comparison with the effects of MST1 in castration-sensitive LNCaP parental cells, though both cell models expressed similar levels of MST1 protein. MST1 was identified as a negative regulatory component of PI3K-AKT signaling, and reduced MST1 expression was shown to correlate with PCa progression to the hormone-refractory metastatic state, which coincides with AKT activation (15). Our data are consistent with this observation and indicate that the induction of MST1 expression sensitized C4-2 cells to growth suppression induced by PI3K inhibition. Our findings raise the possibility that deregulated-MST function may be associated with the emergence of the castration-resistant disease phenotype. The use of MST1/2 knockout mouse models will address the hypothesis of whether one or both of these kinases have a direct role in prostate carcinogenesis and emergence of castration-resistance.

PCa is the most commonly diagnosed cancer among men and the second leading cause of cancer death in Western countires (40, 41). Evidence indicates that cooperative AR and PI3K/AKT-mTOR pathway signaling is critical to human prostate tumor development and progression to the metastatic phenotype (42–44). On the basis of published studies (15, 16) and our present findings, we propose a model (Fig. 7D) in which MST1/2 attenuates AR-dependent gene expression by interacting with the AR, which may lead to the alterations of the AR protein and/or transcriptional complexes, as well as by targeting upstream of the AR signal. An important implication from these observations is that deregulation of MST1 may account for the upregulation of AR and AKT signaling regulating cell survival. Therefore, disruption of the AR-AKT oncogenic network by MST1/2 alone and/or in combination with chemotherapeutic agents that target AR and PI3K/AKT-mTOR may have important therapeutic implications.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Leland Chung, Rosalyn Adam, and Haiyen Zhau for their critical review of the data during the course of the study. We also thank Paul Guthrie, Mustafa Kupeli, Xiaojian Yang, and Yueming Wang for their technical assistance and Gary Mawyer for editorial suggestions.

Grant Support

This study was supported by the Edwin Beer Fellowship grant (R24D00) from the New York Academy of Medicine and the Garber Foundation Award from Cedars-Sinai Medical Center (B. Cinar), NIH grants R01 CA124706 (D. Gioeli), R01 CA143777 and R01 CA112303 (M.R. Freeman), and U.S. Department of Defense grant W81XWH-08–1-0150 (M.R. Freeman).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

References

Creasy

Chernoff

Cloning and characterization of a member of the MST subfamily of Ste20-like kinases

Gene

1995

;

167

303

–

Ling

Yuan

Lai

Biosignaling of mammalian Ste20-related kinases

Cell Signal

2008

;

1237

–

Ura

Masuyama

Graves

Gotoh

Caspase cleavage of MST1 promotes nuclear translocation and chromatin condensation

Proc Natl Acad Sci U S A

2001

;

10148

–

Teraishi

Guo

Zhang

Dong

Davis

Sasazuki

, et al

Activation of sterile20-like kinase 1 in proteasome inhibitor bortezomib-induced apoptosis in oncogenic K-ras-transformed cells

Cancer Res

2006

;

6072

–

Lee

Kim

Hwang

, et al

Crucial role for Mst1 and Mst2 kinases in early embryonic development of the mouse

Mol Cell Biol

2009

;

6309

–

Huang

Dong

Pan

Hippo encodes a Ste-20 family protein kinase that restricts cell proliferation and promotes apoptosis in conjunction with salvador and warts

Cell

2003

;

114

445

–

Kim

Bossuyt

Liu

Qiu

, et al

Hippo signaling is a potent in vivo growth and tumor suppressor pathway in the mammalian liver

Proc Natl Acad Sci U S A

2010

;

107

1437

–

Praskova

Xia

Avruch

MOBKL1A/MOBKL1B phosphorylation by MST1 and MST2 inhibits cell proliferation

Curr Biol

2008

;

311

–

Song

Mak

Topol

Yun

Garrett

, et al

Mammalian Mst1 and Mst2 kinases play essential roles in organ size control and tumor suppression

Proc Natl Acad Sci U S A

2010

;

107

1431

–

Zhou

Conrad

Xia

Park

Payer

Yin

, et al

Mst1 and Mst2 maintain hepatocyte quiescence and suppress hepatocellular carcinoma development through inactivation of the Yap1 oncogene

Cancer Cell

2009

;

425

–

Harvey

Pfleger

Hariharan

The Drosophila Mst ortholog, hippo, restricts growth and cell proliferation and promotes apoptosis

Cell

2003

;

114

457

–

Jia

Zhang

Wang

Trinko

Jiang

The Drosophila Ste20 family kinase dMST functions as a tumor suppressor by restricting cell proliferation and promoting apoptosis

Genes Dev

2003

;

2514

–

Udan

Kango-Singh

Nolo

Tao

Halder

Hippo promotes proliferation arrest and apoptosis in the Salvador/Warts pathway

Nat Cell Biol

2003

;

914

–

Seidel

Schagdarsurengin

Blumke

Wurl

Pfeifer

Hauptmann

, et al

Frequent hypermethylation of MST1 and MST2 in soft tissue sarcoma

Mol Carcinog

2007

;

865

–

Cinar

Fang

Lutchman

Di Vizio

Adam

Pavlova

, et al

The pro-apoptotic kinase Mst1 and its caspase cleavage products are direct inhibitors of Akt1

EMBO J

2007

;

4523

–

Cinar

Mukhopadhyay

Meng

Freeman

Phosphoinositide 3-kinase-independent non-genomic signals transit from the androgen receptor to Akt1 in membrane raft microdomains

J Biol Chem

2007

;

282

29584

–

Gioeli

Black

Gordon

Spencer

Kesler

Eblen

, et al

Stress kinase signaling regulates androgen receptor phosphorylation, transcription, and localization

Mol Endocrinol

2006

;

503

–

Cinar

Yeung

Konaka

Mayo

Freeman

Zhau

, et al

Identification of a negative regulatory cis-element in the enhancer core region of the prostate-specific antigen promoter: implications for intersection of androgen receptor and nuclear factor-kappaB signalling in prostate cancer cells

Biochem J

2004

;

379

421

–

Mukhopadhyay

Cinar

Mukhopadhyay

Lutchman

Ferdinand

Kim

, et al

The zinc finger protein ras-responsive element binding protein-1 is a coregulator of the androgen receptor: implications for the role of the Ras pathway in enhancing androgenic signaling in prostate cancer

Mol Endocrinol

2007

;

2056

–

Cinar

Koeneman

Edlund

Prins

Zhau

Chung

Androgen receptor mediates the reduced tumor growth, enhanced androgen responsiveness, and selected target gene transactivation in a human prostate cancer cell line

Cancer Res

2001

;

7310

–

Cleutjens

Van Der Korput

van Eekelen

van Rooij

Faber

Trapman

An androgen response element in a far upstream enhancer region is essential for high, androgen-regulated activity of the prostate-specific antigen promoter

Mol Endocrinol

1997

;

148

–

Sun

Yang

Feldman

Sun

Bhalla

Jove

, et al

Activation of phosphatidylinositol 3-kinase/Akt pathway by androgen through interaction of p85alpha, androgen receptor, and Src

J Biol Chem

2003

;

278

42992

–

3000

Wang

Kreisberg

Ghosh

Cross-talk between the androgen receptor and the phosphatidylinositol 3-kinase/Akt pathway in prostate cancer

Curr Cancer Drug Targets

2007

;

591

–

604

Lin

Adam

Santiestevan

Freeman

The phosphatidylinositol 3′-kinase pathway is a dominant growth factor-activated cell survival pathway in LNCaP human prostate carcinoma cells

Cancer Res

1999

;

2891

–

Pedram

Razandi

Sainson

Kim

Hughes

Levin

A conserved mechanism for steroid receptor translocation to the plasma membrane

J Biol Chem

2007

;

282

22278

–

Shimelis

Linn

Jiang

Yang

Sun

, et al

Regulation of androgen receptor transcriptional activity and specificity by RNF6-induced ubiquitination

Cancer Cell

2009

;

270

–

Rao

Wang

Zhang

Hessien

, et al

The androgen receptor acetylation site regulates cAMP and AKT but not ERK-induced activity

J Biol Chem

2004

;

279

29436

–

Nishida

Yasuda

PIAS1 and PIASxalpha function as SUMO-E3 ligases toward androgen receptor and repress androgen receptor-dependent transcription

J Biol Chem

2002

;

277

41311

–

Gioeli

Ficarro

Kwiek

Aaronson

Hancock

Catling

, et al

Androgen receptor phosphorylation. Regulation and identification of the phosphorylation sites

J Biol Chem

2002

;

277

29304

–

Guo

Dai

Jiang

Xie

Kim

, et al

Regulation of androgen receptor activity by tyrosine phosphorylation

Cancer Cell

2006

;

309

–

McCall

Gemmell

Mukherjee

Bartlett

Edwards

Phosphorylation of the androgen receptor is associated with reduced survival in hormone-refractory prostate cancer patients

Br J Cancer

2008

;

1094

–

101

Ura

Masuyama

Graves

Gotoh

MST1-JNK promotes apoptosis via caspase-dependent and independent pathways

Genes Cells

2001

;

519

–

Heemers

Tindall

Androgen receptor (AR) coregulators: a diversity of functions converging on and regulating the AR transcriptional complex

Endocr Rev

2007

;

778

–

808

Hodgson

Astapova

Cheng

Lee

Verhoeven

Choi

, et al

The androgen receptor recruits nuclear receptor CoRepressor (N-CoR) in the presence of mifepristone via its N and C termini revealing a novel molecular mechanism for androgen receptor antagonists

J Biol Chem

2005

;

280

6511

–

Liao

Chen

Zhang

Godavarthy

Xia

Ghosh

, et al

Regulation of androgen receptor activity by the nuclear receptor corepressor SMRT

J Biol Chem

2003

;

278

5052

–

Guenther

Barak

Lazar

The SMRT and N-CoR corepressors are activating cofactors for histone deacetylase 3

Mol Cell Biol

2001

;

6091

–

101

Zeng

Hong

The emerging role of the hippo pathway in cell contact inhibition, organ size control, and cancer development in mammals

Cancer Cell

2008

;

188

–

Powzaniuk

McElwee-Witmer

Vogel

Hayami

Rutledge

Chen

, et al

The LATS2/KPM tumor suppressor is a negative regulator of the androgen receptor

Mol Endocrinol

2004

;

2011

–

Lee

Kim

Yang

Koo

Lee

, et al

A crucial role of WW45 in developing epithelial tissues in the mouse

EMBO J

2008

;

1231

–

Scher

Sawyers

Biology of progressive, castration-resistant prostate cancer: directed therapies targeting the androgen-receptor signaling axis

J Clin Oncol

2005

;

8253

–

Mohler

Castration-recurrent prostate cancer is not androgen-independent

Adv Exp Med Biol

2008

;

617

223

–

Edwards

Krishna

Witton

Bartlett

Gene amplifications associated with the development of hormone-resistant prostate cancer

Clin Cancer Res

2003

;

5271

–

Ghosh

Malik

Bedolla

Wang

Mikhailova

Prihoda

, et al

Signal transduction pathways in androgen-dependent and -independent prostate cancer cell proliferation

Endocr Relat Cancer

2005

;

119

–

King

Wongvipat

Hieronymus

Carver

Leung

, et al

Cooperativity of TMPRSS2-ERG with PI3-kinase pathway activation in prostate oncogenesis

Nat Genet

2009

;

524

–

2011

Supplementary data

701 Views

31 Web of Science

30 Crossref

MST1 Is a Multifunctional Caspase-Independent Inhibitor of Androgenic Signaling (original) (raw)

Abstract

Introduction

Materials and Methods

Plasmid constructions, antibodies, and reagents

Cell transfections, reporter assays, and immunocytochemistry

Establishment of TetON-inducible cells

Protein analyses

Chromatin immunoprecipitation assays

Kinase, cell death, and cell proliferation assays

Animal studies

Statistics

Results

MST1 binds and attenuates AR activity

The full-length MST1 is a dominant AR suppressor

MST1 attenuates AR activity in a Ser650 phosphorylation-independent manner

MST1 kinase activity is not required for the inhibition of AR activity

MST1 suppresses AR activity by intersecting with AKT1 signaling and antagonizing formation of AR–chromatin complexes

MST1 suppresses PCa cell growth in vitro and tumor growth in vivo

Discussion

Disclosure of Potential Conflicts of Interest

Acknowledgments

Grant Support

References

Supplementary data

Citing articles via

Email alerts