SCP Phosphatases Suppress Renal Cell Carcinoma by ... · inducible factor (HIF) pathway. Recruitment of PML to KLHL20 requires a two-step posttranslational event, that is, phosphorylationfollowedbyPin1-dependent

Molecular and Cellular Pathobiology

SCP Phosphatases Suppress Renal Cell Carcinoma byStabilizing PML and Inhibiting mTOR/HIF Signaling

Yu-Ching Lin1,2, Li-Ting Lu1,2, Hsin-Yi Chen1,3, XueyanDuan4,5, Xia Lin4, Xin-Hua Feng4,5, Ming-Jer Tang6, andRuey-Hwa Chen1,2

AbstractThe tumor-suppressor protein promyelocytic leukemia (PML) is aberrantly degraded in multiple types of

human cancers through mechanisms that are incompletely understood. Here, we show that the phosphataseSCP1 and its isoforms SCP2/3 dephosphorylate PML at S518, thereby blocking PML ubiquitination anddegradation mediated by the prolyl isomerase Pin1 and the ubiquitin ligase KLHL20. Clinically, SCP1 and SCP3are downregulated in clear cell renal cell carcinoma (ccRCC) and these events correlated with PMLS518phosphorylation, PML turnover, and high-grade tumors. Restoring SCP1-mediated PML stabilization not onlyinhibited malignant features of ccRCC, including proliferation, migration, invasion, tumor growth, and tumorangiogenesis, but also suppressed themTOR–HIF pathway. Furthermore, blocking PML degradation in ccRCC bySCP1 overexpression or Pin1 inhibition enhanced the tumor-suppressive effects of the mTOR inhibitortemsirolimus. Taken together, our results define a novel pathway of PML degradation in ccRCC that involvesSCP downregulation, revealing contributions of this pathway to ccRCC progression and offering a mechanisticrationale for combination therapies that jointly target PML degradation and mTOR inhibition for ccRCCtreatment. Cancer Res; 74(23); 6935–46. �2014 AACR.

IntroductionThe promyelocytic leukemia (PML) was identified in the

patients with acute promyelocytic leukemia (1, 2) and elicitsmultiple tumor-suppressive functions, such as anti-prolifera-tion, apoptosis, senescence, anti-migration, anti-invasion, andanti-angiogenesis (3–6). These PML functions are attributed tothe regulation of a number of tumor promoting/suppressingpathways, including p53 (7, 8), PTEN/Akt (9, 10), andmTOR (3).PML knockout mice display an increased susceptibility totumor formation and/or progression (11–14). Clinically, PMLprotein, but not mRNA, is frequently downregulated in a broadspectrum of human cancers (15). Evidence has emerged that

the ubiquitin–proteasome pathway is a key mechanism forPML deregulation in tumors (16). For instance, a CK2/PIAS1-mediated PML ubiquitination pathway is hyperactivated insmall cell lung cancer (12, 17), and PML expression inverselycorrelates with its ubiquitin ligase E6AP in Burkitt lymphoma(18). Recently, we identified a PML destruction pathway medi-ated by a Cullin 3 (Cul3) ubiquitin ligase complex with KLHL20as the substrate-binding subunit (19). This pathway is hyper-activated in prostate cancers and contributes to tumor pro-gression partly through a feedback potentiation of the hypoxia-inducible factor (HIF) pathway. Recruitment of PML toKLHL20 requires a two-step posttranslational event, that is,phosphorylation followed by Pin1-dependent prolylisomeriza-tion on the S518-P519 residues. Given the dynamic regulationof protein phosphorylation state, the PMLS518-specific phos-phatase may govern PML stability in certain type of tumors.However, the identity of this phosphatase remains elusive.

Protein serine/threonine phosphatases can be divided intoPPM (PP2C-related), PPP (PP1/PP2A/PP2B-related), and FCP/SCP families. The FCP/SCP family consists of TFIIF-associatedC-terminal domain (CTD) phosphatase (FCP1) and three smallCTD phosphatases (SCP1, SCP2, and SCP3) and relies onaspartic acid of the sequence motif DxDxT/V for catalysis(20–23). SCPs negatively regulate RNA polymerase II by depho-sphorylating its CTD, which contributes to suppression ofneuronal genes in non-neuronal cells (24) and attenuation ofandrogen receptor transcriptional activity (25). In addition,SCPs dephosphorylate the linker region of Smad1/2/3, therebypotentiating TGFb and BMP pathways (26–28). Although SCPsare widely expressed in normal human tissues, deletion andmissense/nonsense mutations of SCP3 (also known as HYA22)

1Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan. 2Insti-tute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan.3Graduate Institute of Cancer Biology and Drug Discovery, Collegeof Medical Science and Technology, Taipei Medical University, Taipei,Taiwan. 4Michael E DeBakey Department of Surgery, Baylor College ofMedicine, Houston, Texas. 5Department of Molecular and Cellular Biology,Baylor College of Medicine, Houston, Texas. 6Department of Physiology,National Cheng Kung University Medical College, Tainan, Taiwan.

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

L.-T. Lu and H.-Y. Chen contributed equally to this article.

Current address for X. Duan: Department of Medicine, University of Texasat Houston, Houston, Texas.

Corresponding Author: Ruey-Hwa Chen, Institute of Biological Chemis-try, Academia Sinica, 128 Academia Road, Sec II, Nankang, Taipei, 115,Taiwan. Phone: 886-2-27855696, ext. 6020; Fax: 886-2-27889759; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-14-1330

�2014 American Association for Cancer Research.

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gene are observed in several epithelial cancers (29). Further-more, SCP3 overexpression in carcinoma cell lines suppressesproliferation and tumor formation.

Clear cell renal cell carcinoma (ccRCC) is the most frequentand malignant type of renal cancer (30, 31). ccRCC can occursporadically (>96%) or hereditarily (<4%). Almost all hereditaryccRCC and most sporadic ccRCC are characterized by loss/inactivation of the vonHippel-Lindau (VHL) tumor-suppressorgene (32). VHL is a substrate-binding subunit of Cullin 2ubiquitin ligase that targets HIF1/2a for degradation. VHLloss causes constitutive expression of HIF1/2, which, in turn,activates the expression of genes involving in metabolicchanges, angiogenesis, and other signaling events critical forthe growth of renal tumors (33). These important roles of theVHL–HIFpathway in ccRCCpathogenesis laid the groundworkfor the utilization of several antiangiogenic agents for ccRCCtreatment (34). However, primary and acquired resistance tothe antiangiogenic therapies occurs inevitably, indicating theneed for improved management of this disease.

In this study, we report that SCPs dephosphorylate PML toblock Pin1/KLHL20–mediated PML ubiquitination and deg-radation. The SCP–PMLaxis is downregulated in ccRCC, whichcontributes, in part, to HIF induction and ccRCC malignancy.We provide evidence indicating that the restoration of PMLstability may be combined with mTOR inhibitor for treatingccRCC.

Materials and MethodsCell culture and transfection

HeLa, 293T, and 293FT cells were cultured in Dulbecco'sModified Eagle Medium (DMEM) containing 10% fetal calfserum (FCS). HK-2 cells were cultured in DMEM-F12 mediumsupplemented with 10% FCS. A-498 cells were cultured inMinimal EssentialMedium containing 10%FCS and 1mmol/Lsodium pyruvate. 786-O cells were cultured in RPMI-1640medium supplemented with 10% FCS, 10 mmol/L HEPES,and 1 mmol/L sodium pyruvate. PC-3 cells were cultured inDMEM high-glucose medium supplemented with 10% FCS,nonessential amino acid, 1 mmol/L sodium pyruvate, and 20mmol/L L-glutamine. All cell lines were obtained from theAmerican Type Culture Collection or Biosource CollectionResearch Center, Taiwan. They were cultured for less than 6months of receipt or authenticated by short tandem repeatprofiling. Transfection was performed using Lipofectamine2000 reagent (Invitrogen) or calcium phosphate method. Forall transfection experiments, plasmid encoding GFP wasincluded to monitor transfection efficiency, which rangedfrom 80% to 90%. Stable cell lines were generated by lentivirustransduction. Hypoxia experiments were performed in aclosed chamber flushed with 1% O2/5% CO2/94% N2.

PlasmidsFlag-SCP1, Flag-SCP1 Dm (D96E, D98N), GST-SCP1, Flag-

SCP2, and Flag-SCP3were described previously (28). GST-SCP1Dm, Flag-SCP2 Dm (D107E, D109N), and Flag-SCP3 Dm(D101E, D103N) were generated by site-directed mutagenesis.HA-SCP1 and HA-SCP1 Dm were constructed by cloning SCP1WT and SCP1 Dm cDNAs into pcDNA3.0-HA vector, respec-

tively. Plasmids encoding HA-PML-I, Flag-PML-I, Flag-PML-I-S518A, myc-KLHL20, myc-ubiquitin, myc-Cul3, and myc-Roc1were described previously (19, 35). His-PML-I and His-PML-I-S518A were generated by replacing Flag tag of Flag-PML-I with6x His tag. For inducible gene expression, Flag-SCP1 and Flag-SCP1 Dm were subcloned to pAS4.1w.Ppuro-aOn obtainedfrom National RNAi Core Facility, Taiwan.

Proliferation, migration, and invasion assaysProliferation was assayed using Cell Proliferation ELISA

BrdU kit (Roche). Transwell migration assay was performedas described previously (36). To distinguish migration effectfrom proliferation effect, the same number of cells was seededon a regular culture plate. At the end of incubation, cells thathad migrated onto the lower membrane surface of the Trans-well plate were fixed by 4% formaldehyde, stained with DAPI,counted, and normalized with the number of cells appearing inthe regular plate. For invasion assays, the Transwellmembranewas coated with Matrigel.

Tissue specimens and IHC analysisTissue microarrays were obtained from Biomax Inc. and

Pantomics Inc. Studies involving these tissues were approvedby Institutional Review Boards at Academia Sinica (Taipei,Taiwan). For immunohistochemical (IHC) staining, endoge-nous peroxidase activity was blocked by incubation in 3%H2O2

at room temperature for 20 minutes. Antigen retrieval wasperformed by heat denaturation of paraffin sections with10 mmol/L sodium citrate buffer (pH 6.0) for 30 minutes andnonspecific binding was blocked by PBS containing 10% goatserum or Avidin/Biotin Blocking kit (Invitrogen). The slideswere incubated with antibody to SCP1 (1:150), SCP3 (1:100),PML (1:60), pS518PML (1:300), or KLHL20 (1:400) at 4�C forovernight. The bound antibody was detected by the Super-Picture Polymer Detection Kit (Invitrogen). The couterstainingwas performed with hematoxylin. Protein expression wasscored semiquantitatively as high or low expression based onstaining intensity and percentage of cells staining positive.

For IHC analysis on xenograft tumors, paraffin-embeddedtumors were cut to 5-mm sections, deparaffinized, hydrated,and rinsed with PBS. IHC staining was performed as describedabove with antibody to CD31 (1:100) or Ki67 (1:100). Thepositive signal was quantified by the ImageJ software.

Cell viability (MTS) assayCellswere seeded at a density of 1� 103 cells in 96-well plates

and treated with various inhibitors for 48 hours. CellTiter 96AQueous One Solution Reagent (Promega) was added andincubated for 50 minutes at 37�C, followed by absorbancemeasurement at 490 nm.

Xenograft modelSix- to eight-week-old female BALB/c nude mice were

purchased from National Laboratory Animal Center (Taipei,Taiwan). For subcutaneous xenografts,micewere transplantedwith 5.5 � 106 786-O cell derivatives mixed with PBS andMatrigel. For conditional SCP1-WT or SCP1-Dm expression,mice at 2 weeks after transplantation were randomly grouped

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Cancer Res; 74(23) December 1, 2014 Cancer Research6936




and given drinking water (containing 50 g sucrose/L) with orwithout 2 mg/mL doxycycline. Drinking water was exchangedevery 3 days. For inhibitor treatment, once the transplantedtumors reached approximately 50 mm3, mice were selectedand randomly divided into four groups for intraperitonealinjection daily with DMSO, temsirolimus (0.1 mg/kg), Juglone(1 mg/kg), or a combination of temsirolimus and Juglone inapproximately 200 mL PBS with 0.5% DMSO. Body weight andtumor size of the drug-injected mice were measured every 2 or3 days. Allmice experiments were conductedwith the approvalfrom the Experimental Animal Committee, Academia Sinica.For additional details, refer to Supplementary Materials and

Methods.

ResultsSCP1/2/3 dephosphorylate PML at S518Consistent with a crucial role of S518 phosphorylation in

PML degradation (19), PMLS518A mutant was more resistantto proteasome inhibitor than wild-type PML (SupplementaryFig. S1A). To identify phosphatases targeting this residue, weundertook an expression screening strategy by transfectingcells with a panel of Ser/Thr phosphatases together withPML-I. Immunoblot with a pS518-specific antibody (19)revealed that SCP1, SCP2, and SCP3 significantly decreasedPML-I S518 phosphorylation (Supplementary Fig. S1B and Fig.1A). However, the catalytically inactive mutants (Dmmutants)of SCP1, SCP2, and SCP3 did not alter PML S518 phosphory-lation (Fig. 1A). Because SCP1 is the prototype of this family ofphosphatases, we mainly focused on SCP1 in the followingstudies. SCP1 also decreased S518 phosphorylation on endog-enous PML (Fig. 1B). Furthermore, in vitro analysis revealedthat recombinant GST-SCP1 readily dephosphorylated puri-fied PML at S518, whereas SCP1 Dm did not elicit this effect(Fig. 1C). This result demonstrated a direct action of SCP1 onPML. Importantly, simultaneous depletion of SCP1, SCP2, andSCP3 elevated S518 phosphorylation on endogenous PML (Fig.1D, top), even though the total level of PMLwas decreased (dueto PML destabilization, see below; Fig. 1D, middle). Our datasupport a physiologic role of these SCPs in dephosphorylatingPML at S518.

SCP1 interacts with PMLA bona fide phosphatase should interact with its substrate.

Accordingly, immunoprecipitation analysis demonstrated theinteraction of Flag-SCP1with both PML-I andPML-IV (Fig. 1E).With a SCP1-specific antibody (Supplementary Fig. S2), wedetected the interaction between endogenous PML and endog-enous SCP1 by reciprocal immunoprecipitation (Fig. 1F). Fur-thermore, in vitropull-down analysis showed that recombinantGST-SCP1 and GST-SCP1 Dmwere able to bind purified PML-I(Fig. 1G). These findings indicate a direct and physical asso-ciation of PML with SCP1, further substantiating SCP1 as thePMLS518 phosphatase.

SCP1 inhibits Pin1/KLHL20–mediated PMLubiquitination and degradation through PML S518Our previous study indicates that PMLS518 phosphorylation

is required for interaction with Pin1, which catalyzes PML

prolylisomerization to facilitate PML recruitment to theKLHL20-containing ubiquitin ligase (19). Consistent with therole of SCP1 in PMLS518 dephosphorylation, overexpression ofSCP1, but not SCP1 Dm, compromised PML interaction withPin1 and KLHL20 (Fig. 2A and B) and suppressed PML-Iubiquitination mediated by KLHL20-based ubiquitin ligase(Fig. 2C). In addition, preincubation of purified PML-I withGST-SCP1 for dephosphorylation suppressed its ubiquitina-tion in vitro by KLHL20-based E3 ligase (Fig. 2D). These datacollectively indicate that SCP1-mediated PML dephosphory-lation disrupts PML interaction with Pin1 and KLHL20, there-by attenuating KLHL20-mediated PML polyubiquitination.Wenext evaluated the role of SCP1 in PML stability. The turnoverof PMLwas decreased by overexpression of SCP1, but not SCP1Dm (Fig. 2E). Furthermore, the steady-state level of endoge-nous PMLwas elevated by SCP1 overexpression (Fig. 2B, lysate)and was decreased by SCP1/2/3 knockdown (Fig. 1D, middle).Consistent with our previous finding that KLHL20-dependentPML ubiquitination is responsible for hypoxia-induced PMLdestruction (19), overexpression of SCP1, but not SCP1 Dm,completely reversed hypoxia-induced PML destruction (Fig.2F). Thus, SCP-mediated PML dephosphorylation stabilizesPML in both normoxia and hypoxia conditions.

Previous study revealed that Pin1 interacts with PMLthroughmultiple pS/T-Pmotifs (37), we thus evaluated wheth-er the effect of SCP1 on PML is mediated specifically by S518dephosphorylation. Importantly, the ubiquitination and abun-dance of PMLS518Amutant were not affected by SCP1 (Fig. 2Gand H). Accordingly, SCP1 failed to regulate the turnover ofPMLS518A (Fig. 2E), which had an elevated stability due toresistant to Pin1/KLHL20-mediated degradation (19). Our datasupport a pivotal and specific role of S518 dephosphorylationin SCP1-mediated PML stabilization.

Deregulation of SCP–PML axis in ccRCC correlates withdisease progression

Next, we investigated the relevance of SCP-dependent PMLregulation to human cancers. IHC analysis on human tissuemicroarray derived frommultiple types of patients with cancerrevealed a possible downregulation of SCP1 in ccRCC (Sup-plementary Table S1). Furthermore, SCP3 downregulation wasreported in certain RCC cell lines (29). We therefore accessedthe expression of SCP1, SCP3, PML, and pS518PML in 36 ccRCCtissues and their matched adjacent normal tissues. AlthoughSCP1, SCP3, and PML displayed high expression in normaltissues, they were all downregulated in ccRCCs (Fig. 3A and Band Supplementary Fig. S3). Conversely, PMLS518 phosphor-ylation was increased in ccRCCs. Furthermore, SCP1 and SCP3expressions correlated positively with PML expression andnegatively with PMLS518 phosphorylation in this cohort ofnormal and tumor tissues (Fig. 3C and D), supporting theexistence of SCP–PML axis. Consistent with the activation ofHIF1 in ccRCC, high expression of KLHL20, aHIF1 effector (19),was observed in the majority (88.9%) of ccRCC tissues (Fig. 3Aand B). However, the expression of KLHL20 did not show aninverse correlation with that of PML and pS518PML (Fig. 3Cand D). These findings indicate that SCP1 and SCP3 play moreimportant roles than KLHL20 in determining the expression of

SCP–PML Axis Suppresses ccRCC and mTOR/HIF Signaling

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PML in ccRCC and therefore underscore the clinical relevanceof SCP–PML axis to the pathogenesis of ccRCC. To determinethe correlation of SCP1, SCP3, PML, and pS518PML expressionwith tumor stages, we performed IHC analysis on anothercohort of ccRCC patients, which contains certain patients inadvanced disease stages. By combining data fromboth cohorts,we found that donwregulations of SCP1, SCP3, and PML andupregulation of pS518PML were more frequently observed instage III/V patients than stage I/II patients (Fig. 3E). Our dataindicate that downregulations of SCP1 and SCP3 are associatedwith PMLS518 phosphorylation, PML downregulation, andhigh-grade ccRCCs.

SCP1 stabilizes PML in ccRCC to suppress ccRCCmalignant characters

Because the clinical data support a role of SCP–PML axis inccRCC, we evaluated its impact on tumor-related features by

using ccRCC cell lines. Overexpression of SCP1, but not SCP1Dm, in ccRCC cell lines 786-O and A-498 reduced PMLS518phosphorylation and increased endogenous PML expression(Fig. 4A). Similar results were obtained with SCP2 and SCP3(Fig. 4B). Furthermore, proteasome inhibitor induced agreater PML upregulation in control or SCP1 Dm-expressingcells than in SCP1-expressing cells, supporting a role of SCP1in blocking PML proteasomal degradation (Fig. 4C). Con-versely, knockdown of SCP1/2/3 in HK-2 normal renalepithelial cells increased PMLS518 phosphorylation andreduced PML expression (Fig. 4D). Thus, the SCP–PML axisis manifested in ccRCC. To investigate the effect of SCP–PML axis on ccRCC malignancy, we established 786-O andA-498 stable lines carrying doxycycline-inducible SCP1 orSCP1 Dm. Then, PML shRNA or control shRNA was intro-duced to SCP1-inducible cells (Fig. 4E). These cells were usedfor assaying several malignant traits of ccRCC. Although

Figure 1. SCPs bind PML and dephosphorylate PML at S518. A and B, immunoblot analysis of S518 phosphorylation of PML-I (A) or endogenous PML (B) in293T cells transfected with indicated constructs. C, in vitro dephosphorylation of baculovirally purified PML-I by GST-SCP1 or its mutant purified fromEscherichia coli. The reaction products were analyzed by immunoblot. D, immunoblot analysis of PMLS518 phosphorylation and PML level in 293T cellstransfected with control siRNA or cotransfected with SCP1, SCP2, and SCP3 siRNAs (at a ratio of 1:1:1). E, immunoprecipitation analysis of the interactionbetween SCP1 and PML-I/PML-IV in 293T cells transfected with indicated constructs. F, immunoprecipitation analysis of the interaction betweenendogenous SCP1 and endogenous PML in 293T cells. G, in vitro pull down of baculovirally purified PML-I by GST-SCP1 or its mutant.

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Figure2. SCP1 inhibitsKLHL20- andPin1-mediatedPMLubiquitination anddegradation throughPMLS518. A, immunoprecipitation analysis of the interactionbetween PML-I and Pin1 in 293T cells transfected with indicated constructs. B, immunoprecipitation analysis of the interaction between KLHL20 andendogenous PML inHeLa cells transfectedwith indicated constructs. C andG,His-PML-I (C) or His-PML-I-S518A (G) purified under denaturing conditions byNi-NTA agarose from 293T cells transfected with indicated constructs was analyzed by immunoblot for its ubiquitination. D, baculovirally purified PML-I waspreincubated with GST or GST-SCP1 protein in the dephosphorylation reaction followed by in vitro ubiquitination assay and immunoblot analysis. E,immunoblot analysis of PML-I or PML-I-S518A stability in HeLa cells transfected with indicated constructs and treated with cycloheximide for indicated timepoints. The expression of SCP1 or its mutant was shown on the upper right panel. F, immunoblot analysis of PML expression in HeLa cells transfected withindicated constructs and cultured in normoxia or hypoxia. H, immunoblot analysis of PML-I or PML-I-S518A expression in 293T cells transfected withindicated constructs.






expression of SCP1 Dm in 786-O and A-498 cells did not elicitany significant effect, SCP1 expression suppressed prolifer-ation, migration, and invasion (Fig. 4F and G). Importantly,these tumor-promoting functions were all abrogated by PMLknockdown. To test the effects of SCP–PML axis on tumorgrowth in vivo and tumor angiogenesis, we subcutaneouslyinjected 786-O derivatives into nude mice. Induction of SCP1expression by doxycycline led to a significant suppression of

tumor growth in vivo and this effect was blocked by PMLknockdown (Fig. 5A). Induction of the expression of SCP1Dm, however, did not affect tumor growth (Fig. 5B). IHCstaining on tumor tissues revealed that SCP1 expressionstrongly suppressed tumor angiogenesis, which was againreversed by PML knockdown (Fig. 5C). These findings indi-cate a significant role of SCP1-induced PML stabilization insuppressing ccRCC malignant characters.

Figure 3. SCP1 and SCP3 downregulations in ccRCC correlate with PML phosphorylation, PML downregulation, and disease progression. A, IHC analysis ofindicated protein expression in a representing ccRCC specimen and its adjacent normal specimen. Bar, 50 mm. B, summary of the SCP1, SCP3, PML,pS518PML, and KLHL20 expression profiles in 36 ccRCC specimens and their adjacent normal specimens. C and D, correlations of PML expression (C) andpS518PML expression (D) with SCP1 and SCP3 expression, but not with KLHL20 expression in all specimens. E, correlation of PML, pS518PML, SCP1, andSCP3 expression with tumor stage.

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SCP–PML axis downregulates HIF signaling in ccRCCThe pathology of ccRCC is tightly associated with aberrant

elevation of HIF signaling resulted from VHL deficiency (32).PML, however, negatively regulates HIF expression throughmTOR repression (3, 19). Thus, inhibition of the SCP–PMLaxis could serve as an alternative route to enhance HIFexpression and signaling in ccRCC. In line with this notion,overexpression of SCP1, but not SCP1 Dm, in 786-O andA-498 cells attenuated mTOR activity (measured by phos-

phorylation of S6 kinase) and HIF2a expression (786-O andA-498 cells do not express HIF1a). Importantly, these effectswere reversed by PML knockdown (Fig. 6A and B). Conse-quently, the levels of HIF targets VEGF and GLUT1 weredecreased by overexpression of SCP1 but not SCP1 Dm (Fig.6C). Conversely, depletion of SCP1/2/3 in HK-2 increasedmTOR activity and HIF1a/HIF2a expression (Fig. 6D). Thesedata support a cross-talk of SCP–PML axis with mTOR/HIFpathway in ccRCC.

Figure 4. SCP–PML axis ismanifested in ccRCC and inhibitsmultiple malignant phenotypes ofccRCC. A and B, immunoblotanalysis of PMLS518phosphorylation and PMLabundance in ccRCC cellstransfected with indicatedconstructs. C, immunoblotanalysis of 786-O cells expressingdoxycycline-inducible SCP1 orSCP1 Dm treated with doxycyclineand/or MG132. The induction ofSCP1bydoxycyclinewas shown inE. D, immunoblot analysis of HK-2cells transfected with indicatedsiRNAs. E, immunoblot analysis ofPML expression in indicated 786-Oor A-498 stable cells. F and G,SCP–PML axis suppressesmalignant phenotypes of ccRCC.786-O (F) and A-498 (G) cells stablyexpressing inducible SCP1 or itsmutant and constitutive PMLshRNAwere treatedwith or withoutdoxycycline and analyzed for cellproliferation, migration, andinvasion. Data are mean � SD(�, P < 0.05; ��, P < 0.005;��, P < 0.0005; n ¼ 3).






Combined targeting of mTOR pathway and PMLdegradation pathway provides better tumor-suppressiveeffects

The uncovering of a cross-talk between SCP–PML axis andHIF signaling, togetherwith the ability of PML to regulate othertumor-promoting or -suppressing factors, suggest a beneficialeffect for targeting PML degradation pathway on ccRCC ther-apy. In linewith this notion, SCP1 overexpression increased thesensitivity of 786-O cells to mTOR inhibitor temsirolimus,which correlated with enhanced effects on PML stabilization,

mTOR inhibition, and VEGF downregulation (SupplementaryFig. S4A and S4B). To select a druggable target in the SCP–PMLpathway, we chose to block the activity of Pin1, which isrequired for targeting S518 phosphorylated PML for degrada-tion (19). We found that treatment of 786-O cells with Pin1inhibitor PiB or Juglone at 0.1 or 1 mmol/L, respectively, did notsignificantly inhibit cell viability but enhanced the killing effectof temsirolimus (Fig. 7A, left). A similar effect was observedwith Pin1 knockdown (Supplementary Fig. S4C). Importantly,the enhanced killing effect by combined treatment was

Figure 5. SCP–PML axis inhibits ccRCC tumor growth and angiogenesis. A and B, tumor growth rates of 786-O derivatives in nudemice. Data aremean�SD;�, P < 0.05; ��, P < 0.005; n ¼ 5 for A and n ¼ 6 for B. Mice and tumors at 83 days after injection are shown on the right. C, similar-sized tumors derivedfrom indicated cell lines were analyzed for IHC staining with anti-CD31 antibody. Representative images are shown on the left and quantitation datafor CD31-positive areas are shown on the right. Bar, 50 mm. Data are mean � SD (��, P < 0.005; ��, P < 0.0005; n ¼ 3).

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abrogated by PML knockdown (Fig. 7A, right), indicating a keyrole of PML in determining the effect of such treatment.Consistent with the cell viability data, we found that the twoPin1 inhibitors or Pin1 shRNAs not only stabilized PML, butalso enhanced the effect of temsirolimus on suppressingmTORactivity and VEGF expression (Fig. 7B and Supplementary Fig.S4D). To examine the effect of combined treatment in vivo, wesubcutaneously inoculated nude mice with 786-O cells andadministrated mice with temsirolimus and/or Juglone aftertumor formation. Although treatment of temsirolimus orJuglone modestly reduced tumor growth, combined treatmentcompletely suppressed tumor growth (Fig. 7C). IHC analysis ontumor tissues revealed that Juglone potentiated the antipro-liferation and antiangiogenesis effects of temsirolimus (Fig. 7Dand E and Supplementary Fig. S5A). Notably, neither did singlenor combined treatment affect mice body weight (Supplemen-tary Fig. S5B), arguing against the induction of toxicity effect bythese treatments. Our data suggest a beneficial effect bycombined targeting of mTOR pathway and PML degradationpathway for ccRCC therapy.

DiscussionPhosphorylation at the S518 residue is crucial for targeting

PML to a destruction pathway mediated by Pin1 followed by

KLHL20-containing ubiquitin ligase (19). In this study, weidentify SCP1, SCP2, and SCP3 as PMLS518 phosphatases.Using SCP1 as amodel, we show that SCP1-dependent dephos-phorylation stabilizes PML by blocking its interaction withPin1 and KLHL20-based ubiquitin ligase. Furthermore, deple-tion of SCP1/2/3 increases PMLS518 phosphorylation to down-regulate PML. These findings indicate a physiologic role ofSCPs in PML stabilization and suggest a tumor-suppressivefunction of these phosphatases. Indeed,we show that SCP1 andSCP3 are downregulated in ccRCC and their downregulationscorrelate with PMLS518 phosphorylation, PML downregula-tion, and high-grade tumor. Interestingly, although KLHL20 isalso highly expressed in ccRCC, an inverse correlation betweenthe expression of KLHL20 and PML is not observed. Thesefindings underscore a more important role of SCP1 and SCP3than KLHL20 in determining the expression level of PML inrenal tissues. These clinical observations are further strength-ened by preclinical data demonstrating that the SCP–PML axissuppresses many features of aggressive ccRCC, such as pro-liferation,migration, invasion, tumor growth in vivo, and tumorangiogenesis. Our study thus uncovers a SCP–PML pathway inccRCC and indicates a crucial role of this pathway in suppres-sing ccRCC progression. Notably, IHC analysis revealed apossible SCP1 downregulation in prostate cancer (Supplemen-tary Table S1) and SCP1 induced PMLS518 dephosphorylation

Figure 6. SCP–PML axis inhibitsmTOR/HIF signaling. A,immunoblot analysis of 786-O andA-498 cells transfected with SCP1or SCP1 Dm. B, immunoblotanalysis of 786-O cells stablyexpressing inducible SCP1 andconstitutive PML shRNA andtreated with or without doxycyclinefor 20 hours. C, qPCR analysis of786-O or A-498 cells carryinginducible SCP1 or its mutanttreated with or withoutdoxycycline. D, immunoblotanalysis of HK-2 cells transfectedwith indicated siRNAs. Data aremean � SD (�, P < 0.05;��, P < 0.005; ��, P < 0.0005;n ¼ 3).






and PML upregulation in prostate cancer cell line PC-3 (Sup-plementary Fig. S6), suggesting the significance of SCP–PMLaxis in this cancer type.

In addition to demonstrating the tumor-suppressive effectsof SCP–PML axis on ccRCC, our study reveals a cross-talk ofthis pathwaywithHIF pathway. Previous studies indicated thatPML blocks mTOR activation (3), thereby suppressing theexpression of HIF1a and HIF2a (3, 19). Consistent with thesefindings, we show that the SCP–PML axis suppresses mTORactivity and HIF expression, leading to the downregulation ofHIF effectors, such as GLUT1 and VEGF. These observationsnot only point out additional impacts of this PML degradationpathway on ccRCC, such as altering metabolism and promot-ing angiogenesis, but also highlight a coordinated action of SCPdownregulation and VHL deficiency in potentiating HIF sig-

naling in ccRCC. Given the importance of HIF signaling inccRCC pathology and the pleiotropic functions of PML intumor suppression, we reason that the SCP downregulation-induced PML degradation may be considered as a therapeutictarget for combining with a current FDA-approved regimen forccRCC therapy. In support of this notion, we show thattargeting of this PML degradation pathway by SCP1 over-expression or Pin1 inhibition enhances the tumor-suppressiveeffects of temsirolimus. Interestingly, although Pin1 has beenreported to elicit tumor-promoting effects through multipleeffectors, PML likely plays a major role in ccRCC, as PMLdepletion abolishes the beneficial effect of Pin1 inhibitors.Thus, our study suggests a new option for ccRCC targetedtherapy and further highlights a key role of PML in suppressingccRCC.

Figure 7. Targeting of PML degradation pathway enhances the tumor suppressive effects of mTOR inhibitor. A, viability of 786-O cells expressing indicatedshRNAs treatedwith temsirolimus in the presence or absence of PiB or Juglone. B, immunoblot and RT/qPCR analyses of 786-O cells treatedwith 0.1 nmol/Ltemsirolimus, 0.1 mmol/L PiB, and/or 1 mmol/L Juglone for 4 hours (for immunoblot) or 6 hours (for RT/qPCR). C, mice bearing 786-O cell–derived tumor weretreatedwith indicated inhibitors and assayed for tumor growth. Data aremean�SD (�,P < 0.05; ��,P < 0.005; n¼ 5 for DMSO; n¼ 6 for single treatment; n¼ 7for combined treatment). Tumors at 14 days after treatment are shown on the bottom. D and E, IHC staining of tumors derived frommice treatedwith indicatedagents. Representative images are shown inSupplementary Fig. S5A. F, schematic presentation of the regulation SCP–PMLaxis in normal kidney and ccRCCand its cross-talk with the mTOR–HIF pathway. Data in B–E are mean � SD (�, P < 0.05; ��, P < 0.005; ��, P < 0.0005; n ¼ 3).


Lin et al.




The tumor-suppressive function of PML in kidney wasobserved by studies with mice models. In compoundPml�/�Tsc2þ/� mice, PML loss in the Tsc2þ/� backgroundfurther increases mTOR activity and accelerates kidney tumorprogression without affecting tumor initiation (11). Theseobservations are consistent with our finding that aberrantdownregulation of SCP–PMLaxis is associatedwith high-gradeccRCC. Thus, even though the tumor-suppressive functionsof PML could be context-dependent (38), data derived frommice models, human ccRCC specimens, and ccRCC cell linesprovide definitive evidence for a suppressive role of PML inthis cancer type.SCP1 has been reported to dephosphorylate other substrates

such as RNA polymerase II, Smad1/2/3, and Snail (23, 26–28, 39). Similar to PML, the residues targeted by SCP1 in thesesubstrates are all within the pS/T-P motif. This implies thatSCPs are specific to pS/T-Pmotif, even though identification ofmore substrates is needed to confirm this notion. The pS/T-Pmotif is also recognized by Pin1 for prolylisomerization. Inter-estingly, the first identified SCP substrate, RNA polymerase II,is also a substrate of Pin1 and Pin1-dependent modificationregulates the binding of phosphorylated RNA polymerase II tocertain nuclear factors (40). Thus, SCP-mediated dephosphor-ylation may antagonize certain biologic functions elicited byPin1 and proline-directed serine/threonine kinases.In summary, our study identifies a PML degradation path-

way mediated by downregulation of SCPs and demonstratesa role of this pathway in promoting mTOR/HIF signaling

(Fig. 7F). This pathway contributes to multiple aggressivecharacters of ccRCC and is associated with advanced ccRCC.We propose that targeting of this pathway in combination ofmTOR inhibitor is an option for ccRCC treatment.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: Y.-C. Lin, X.-H. Feng, R.-H. ChenDevelopment of methodology: Y.-C. Lin, H.-Y. Chen, X. Duan, X. LinAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Y.-C. Lin, L.-T. Lu, H.-Y. Chen, X. Duan, X.-H. FengAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): Y.-C. Lin, L.-T. Lu, X. Duan, R.-H. ChenWriting, review, and/or revision of the manuscript: Y.-C. Lin, X.-H. Feng,M.-J. Tang, R.-H. ChenAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): Y.-C. Lin, L.-T. Lu, M.-J. TangStudy supervision: Y.-C. Lin, X.-H. Feng, R.-H. Chen

AcknowledgmentsThe authors thank National RNAi Core Facility for RNAi, Histopathology Core

Facility for tissue processing, and Taiwan Mouse Clinics for slide scanning.

Grant SupportThis work was supported by NHRI grant NHRI-Ex102-10205NI.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 inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received May 1, 2014; revised August 7, 2014; accepted September 8, 2014;published OnlineFirst October 7, 2014.

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Lin et al.




2014;74:6935-6946. Published OnlineFirst October 7, 2014.Cancer Res Yu-Ching Lin, Li-Ting Lu, Hsin-Yi Chen, et al. PML and Inhibiting mTOR/HIF SignalingSCP Phosphatases Suppress Renal Cell Carcinoma by Stabilizing

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