MOLECULAR CANCER THERAPEUTICS | CANCER BIOLOGYANDTRANSLATIONAL STUDIES

Serum- andGlucocorticoid-inducible Kinase 1 is Essentialfor Osteoclastogenesis and Promotes Breast CancerBone Metastasis A C

Zheng Zhang1, Qian Xu2, Chao Song1, Baoguo Mi3, Honghua Zhang1, Honglei Kang1, Huiyong Liu1,Yunlong Sun1, Jia Wang1, Zhuowei Lei1, Hanfeng Guan1, and Feng Li1

ABSTRACT◥

Bone metastasis is a severe complication associated withvarious carcinomas. It causes debilitating pain and pathologicfractures and dramatically impairs patients’ quality of life. Drugsaimed at osteoclast formation significantly reduce the incidenceof skeletal complications and are currently the standard treat-ment for patients with bone metastases. Here, we reported thatserum- and glucocorticoid-inducible kinase 1 (SGK1) plays apivotal role in the formation and function of osteoclasts byregulating the Ca2þ release-activated Ca2þ channel Orai1. Weshowed that SGK1 inhibition represses osteoclastogenesis in vitro

and prevents bone loss in vivo. Furthermore, we validated theeffect of SGK1 on bone metastasis by using an intracardiacinjection model in mice. Inhibition of SGK1 resulted in asignificant reduction in bone metastasis. Subsequently, theOncomine and the OncoLnc database were employed to verifythe differential expression and the association with clinicaloutcome of SGK1 gene in patients with breast cancer. Our datamechanistically demonstrated the regulation of the SGK1 in theprocess of osteoclastogenesis and revealed SGK1 as a valuabletarget for curing bone metastasis diseases.

IntroductionBone is a common target of metastatic spread in solid tumors. Bone

metastasis is generally incurable in patients because of limited ther-apeutic options (1, 2). Bonemetastasis can be regarded as a result of thecrosstalk between bone resorption and tumor growth (3). During theprocess of bone metastasis, cancer cells undergo epithelial–mesenchymal transition (EMT), get transported as circulating tumorcells (CTC), and home to target sites (4). Presently, bisphosphonatesand antibodies against receptor activator of NF-kB ligand (RANKL)are the standard of care in patients with bone metastases (5–7).

Once cancer cells infiltrate bones, themetastatic cells encounter nativebonemicroenvironmental cells.During the formationofbonemetastaticlesions, various osteolytic factors aiming at osteoclasts are secreted bytumor cells. Among these factors, macrophage CSF (M-CSF) andRANKL play a central role in this multistep process (8–10). M-CSFupregulates RANK expression on osteoclast precursors and promotes

osteoclast differentiation, whereas RANKL binds to RANK and causethe activation of nuclear transcriptional regulators (11). The recruitmentof variable adaptor molecules then activates downstream signalingpathways, includes the NF-kB and PI3K/AKT pathways, thereby ini-tiating osteoclast formation and activity (12–15).

In the terminal of osteoclasts differentiation, Ca2þ channels alsoinvolved. Hwang and colleagues (16) have shown that Orai1-mediatedcalcium entry is a major inducer and activator of nuclear factor ofactivated T cells c1 (NFATc1)-mediated RANKL signaling. RANKLevokes Ca2þ oscillations, leading to a selective and stable induction ofNFATc1, a Ca2þ-responsive transcription factor that drives osteoclastformation (16, 17).

Serum- and glucocorticoid-inducible kinase 1 (SGK1) belongs to asubfamily of serine/threonine kinases that is transcriptionally stimu-lated by serum and glucocorticoids (18). SGK1 expression is inducedby insulin and growth factors via PI3K, 3-phosphoinositide-dependentkinase 1 (PDK1), or mTOR complex-2 (19–21). In mammals, SGK1 isexpressed in all examined tissues and regulates awide variety of cellularmolecules, including signaling factors and ion channels (22–24). Pietroand colleagues (25) showed that SGK1 expressed in white adiposetissues promotes adipogenesis by regulating Foxo1 phosphorylation.In megakaryocytes, as a novel regulator of platelet function, SGK1upregulates Orai1 through the NF-kB pathway and increases store-operated Ca2þ entry (SOCE) in platelets (26). However, the role ofSGK1 in osteoclast formation remains unknown.

In this study, we demonstrated that SGK1 triggers osteoclastogen-esis through Orai1, and the inhibitor of SGK1 can reduce bonemetastasis in vitro. Thereby SGK1 is a promising therapeutic targetfor bone metastasis.

Materials and MethodsReagents

GSK650394 and BTP2 was purchased from Sigma-Aldrich. Solublerecombinant human M-CSF and mouse RANKL were obtained fromPeproTech.

1Department of Orthopedics, Tongji Hospital of Tongji Medical College ofHuazhong University of Science and Technology,Wuhan, Hubei, China. 2Depart-ment of Hematology, Tongji Hospital of Tongji Medical College of HuazhongUniversity of Science and Technology, Wuhan, Hubei, China. 3Department ofSpine Surgery, Honghui Hospital, Xi'an Jiaotong University College of Medicine,Xi'an, Shaanxi, China.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

H. Guan and F. Li contributed equally to this article.

Corresponding Authors: Feng Li, Tongji Hospital of Tongji Medical College ofHuazhong University of Science and Technology, Wuhan, Hubei 430000, P.R.China. Phone: 158-0271-1777; E-mail: lifengmd@hust.edu.cn; and Hanfeng Guan,hguan@hust.edu.cn

Mol Cancer Ther 2020;19:650–60

doi: 10.1158/1535-7163.MCT-18-0783

�2019 American Association for Cancer Research.

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AnimalsC57BL/6 mice and BALB/c nu/nu mice were purchased from the

Experimental Animal Center of Tongji Medical College (Wuhan,China). Themice were housed in animal care facility of TongjiMedicalCollege at 25�C with 12-hour light/dark cycle and free access tostandard chow and water. All animal studies were approved by theInstitutional Animal Research Committee of Tongji Medical College.All animal experiments were carried out in accordance with theprotocols approved by the Institutional Animal Care and UseCommittee.

Intracardiac metastasis modelMDA-MB-231 cells were resuspended in sterile PBS at a con-

centration of 5 � 105 cells per 100 mL. We used a marked locationmidway between the sternal notch and the top of xiphoid process,and slightly left (anatomic) of the sternum. Four-week-old male(BALB/c nu/nu) mice were used for these experiments. A successfulintracardiac injection was determined on the basis of the systemicdistribution of bioluminescence throughout the animal at 12 hourspostinjection. Only mice demonstrating evidence of a successfulinjection were used for further analyses. Four weeks after inocu-lation, bone metastatic cells in hind limbs were isolated andcultured in DMEM supplemented with 10% FBS, penicillin (100U/mL), and streptomycin (100 mg/mL). Cultured bone metastaticcells were reinoculated into the left ventricle of the heart. Thisprocedure was repeated four times until no metastases weredetected in organs other than bones. These bone metastatic cellsin hind limbs, designated MBS4-TJGK cells, were isolated andcultured in DMEM.

Cell culture and transfectionMDA-MB-231 and RAW264.7 cell lines were purchased from the

Shanghai Institute of Life Sciences Cell Resource Center, Shanghai,China. Cell lines authentication were conducted by China Centerfor Type Culture Collection (CCTCC). STR data were comparedwith ATCC and DSMZ, all the locations were exactly matched.BMMs were obtained from femoral and tibial bone marrow of 6- to8-week-old C57BL/6 mice as described earlier (27). All cell cultureswere regularly checked for mycoplasma contamination. BMMsand RAW264.7 were cultured and induced to osteoclast asdescribed previously (15). Scrambled, SGK1-knockdown andOrai1-overexpression shRNA were obtained from ViGene Bios-ciences, Inc.

Western blot and immunofluorescence analysisAntibodies against the following targets were purchased from Cell

Signaling Technology: IkB kinase a (IKKa), p-IKKa, IkBa, p-IkBa,p65, p-p65, AKT, and phosphorylated AKT (p-AKT). Antibodiesagainst c-Fos, NFATc1, and SGK1was purchased fromAbcam. Rabbitantibodies against the PI3K catalytic subunit p110 and Orai1, theTRAP staining kit and all other reagents were purchased from Sigma-Aldrich. Mouse anti-b-actin and secondary antibodies were obtainedfrom Boster. Protein bands were visualized using a ChemiDoc XRSþSystem with Image Lab Software (Bio-Rad).

For IF analysis of cultured cells, BMMs were fixed with 4%paraformaldehyde for 15 minutes and then blocked with 5% bovineserum albumin and 0.1% Triton X-100 in PBS for 30 minutes atroom temperature. Immunostaining was performed using theappropriate primary and secondary antibodies. Nuclei were coun-terstained with DAPI. Images were captured using a fluorescencemicroscope.

Quantitative real-time RT-PCRTotal RNA was extracted from cultured cells and cDNA was

synthesized with the Easy Script First-Strand cDNA Synthesis SuperMix Kit (TransGen Biotech). Real-time PCR was performed usingPower SYBRGreen PCRMasterMix (TransGen Biotech). The specificprimers used for PCR amplification were as follows (F, forward; R,reverse): SGK1, F 50-TGTGAAGTCCCTTCTGTGGA-30 and R 50-CCATCTTCGTACCCGTTTCT-30; TRAP, F 50-GATGCCAGCGA-CAAGAGGTT-30 and R 50-CATACCAGGGGATGTTGCGAA-30;NFATc1, F 50-CAACGCCCTGACCACCGATAG-30 and R 50-GGG-AAGTCAGAAGTGGGTGGA-30; CK, F 50-GAAGAAGACTCAC-CAGAAGCAG-30 and R 50-TCCAGGTTATGGGCAGAGATT-30;MMP9, F 50-CTGGACAGCCAGACACTAAAG-30 and R 50-CTCG-CGGCAAGTCTTCAGAG-30;GAPDH, F 50-CTCCCACTCTTCCA-CCTTCG-30 and R 50-TTGCTGTAGCCGTATTCATT-30.

Datasets of mRNA expression and clinical informationFollow-up data of 3,951 patients from Kaplan–Meier plotter

(kmplot.com/analysis/) and the GEO database were included in thisstudy, which was divided into two groups according to the expressionof SGK1 (28). Expression data of SGK1was drawn from theOncominedataset, which embodies large quantities of high-throughput data ofvaries disease.

Statistical analysisResults are presented as the means � SD. Every experiment had a

sample size greater than three and was independently performed threetimes, unless otherwise stated. Multigroup comparisons of the meanswere carried out by one-way ANOVA test with post hoc contrasts byDunnett multiple comparisons test. Cumulative survival time wascalculated by the Kaplan–Meier method and analyzed by the log-ranktest.

ResultsSuppression of SGK1 inhibits RANKL-inducedosteoclastogenesis and impairs osteoclast function in vitro

We evaluated the expression of SGK1 during RANKL-inducedosteoclastogenesis by quantitative real-time RT-PCR and immuno-blotting. Upon RANKL treatment, the mRNA and protein levels ofSGK1 were significantly increased (Fig. 1A and B; SupplementaryFig. S1A). We treated bone marrow mononuclear cells (BMM) once adaywith different concentrations ofGSK650394, an SGK1 inhibitor, inthe presence of RANKL (100 ng/mL) and M-CSF (30 ng/mL) for7 days (29). Osteoclast formation were inhibited in a dose-dependentmanner (Fig. 1C and D). Subsequently, the potential toxicity ofGSK650394 was evaluated by measuring the proliferation of BMMsvia the Cell Counting Kit-8 (CCK-8) after treating the cells withdifferent concentrations of GSK650394. However, the proliferationof BMMs was not markedly altered by GSK650394 at concentrationslower than 2 mmol/L (Fig. 1E).

To control the possible off-target effects of GSK650394, we knockeddown SGK1 in RAW264.7 cells using small interfering RNAs (siRNAs;Supplementary Fig. S1B). Moreover, three shRNA lentiviruses tested(sh1, sh2, sh3) were selected to create stable SGK1 knockdown in bothBMMs and RAW264.7 cells. The protein level of SGK1 was reduced asshown in Fig. 1F and Supplementary Fig. S1C. Upon RANKL treat-ment, osteoclast formation was significantly inhibited in cells witheither siRNA or shRNA-mediated knockdown of SGK1 (Fig. 1Gand H; Supplementary Fig. S1D–S1F). Therefore, SGK1 activity isupregulated during RANKL-induced osteoclastogenesis, and the

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Figure 1.

SGK1 is upregulated during osteoclast differentiation, and inhibition of SGK1 suppresses osteoclast differentiation and function. A and B, BMMs were cultured in thepresence ofM-CSF (30ng/mL) andRANKL (100ng/mL) andwere collected at the indicated timepoints to analyze SGK1 expressionvia quantitative real-timeRT-PCRand Western blotting. Immunoblots were quantified using ImageJ software. Experiments were performed in triplicate. � , P < 0.05; ��� , P < 0.001 (Dunnett t test)versus day0 group.C,BMMs (1.5� 104 cells/well) were incubated in completemedium containingM-CSF, RANKL, and varying concentrations of GSK650394 (0, 0.5,1, and 2mmol/L) for 7days. Then, the cellswerefixed and stained for TRAP.D,TRAP-positivemultinucleated (�3nuclei) osteoclastswere counted in eachwell of a 96-well plate, and the resultswere displayed in a histogram.Data are presented as themeans� SDs of three independent experiments. �� ,P<0.01 (Dunnett t test) versusthe vehicle-treated group.E,BMMs (4� 103 cells/well) were culturedwithM-CSF (30 ng/mL) and treatedwith varying concentrations of GSK650394 (0, 0.5, 1, 2, and5 mmol/L) for 5 days. Cell proliferation was assessed by the Cell Counting Kit-8. Data are presented as the means� SDs of three independent experiments. F, SGK1protein expression in BMMs was assessed by Western blotting. ACTB was used as a loading control. Immunoblots were quantified using ImageJ software.Experimentswere performed in triplicate.G, BMMs cellswere seeded in 96-well plates at a density of 1.0� 104 cells/well, transfected with SGK1-specific shRNAs andcultured in the presence of RANKL (100 ng/mL) for 5 days. TRAP stainingwas performed.H, TRAP-positive cells with�3 nuclei were counted. Data are presented asthemeans� SDs of three independent experiments. ��� , P < 0.001 (Dunnett t test) versus the vector group. ���� , P <0.0001 (Dunnett t test) versus the vector group.I, BMMs cultured in 96-well plates were treated with M-CSF (30 ng/mL) and RANKL (100 ng/mL) for 5 days and then seeded in Corning Osteo Assay Surface andincubated with different concentrations of GSK650394 for 72 hours. J, Bone resorption area was measured. Results shown are the percentage of eroded area of thetotal area. Data are presented as the means � SDs of three independent experiments. �� , P < 0.01 (Dunnett t test) versus the vehicle-treated group.

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suppression of SGK1 inhibits RANKL-induced osteoclastogenesisin vitro.

Given that osteoclast formation was impaired in SGK1-knockdownosteoclast precursors, we next investigated the effect of GSK650394 onbone resorption. Mature osteoclasts were seeded in calcium phos-phate-coated plates in the presence of RANKL andM-CSF and treatedwith different concentrations of GSK650394 for 3 days. Comparedwith that in control cells, the formation of resorption pits in vitro wassignificantly reduced in GSK650394-treated osteoclasts (Fig. 1I and J).The luciferase reporter gene assays also suggested that GSK650394treatment inhibited NF-kB luciferase reporter in BMMs (Supplemen-tary Fig. S2A). Thus, impaired SGK1 activity inhibits the resorptionfunction of mature osteoclasts.

SGK1 modulates the NF-kB and PI3K/AKT pathways inosteoclastogenesis

The NF-kB and PI3K/AKT transcription factor families are bothdownstream of TRAF6, which play pivotal roles in osteoclastogenesis.SGK1 is known to modulate transcription by upregulating NF-kBactivity through the phosphorylation and activation of IKKa, whereasin the PI3K/AKTpathway, SGK1 is highly homologous toAKTand is acritical downstream mediator of PDK1 (24). Specific inhibitors of thePI3K/AKTpathway result in the reciprocal activation of SGK1 (20). Toexplore the potential mechanisms by which SGK1 modulates osteo-clastogenesis, we analyzed the expression of proteins associated withosteoclast formation in BMMs. Western blot analysis showed that theinhibition of SGK1 via GSK650394 dramatically suppressed theRANKL-induced phosphorylation and degradation of IKKa, IkBa,and p65 approximately 60 minutes after stimulation with RANKL(Fig. 2A; Supplementary Fig. S2A). Moreover, regarding to the AKT/PI3K pathway, GSK650394 treatment decreased the levels of p-AKTand the PI3K catalytic subunit p110 (Fig. 2B; Supplementary Fig. S2B).Therefore, the suppression of SGK1 inhibits RANKL-induced NF-kBand PI3K/AKT activation.

Suppression of SGK1 inhibits RANKL-induced c-Fos and NFATc1expression and downregulates the expression of osteoclastmaker genes

To determine whether the suppression of SGK1 inhibits the expres-sion of c-Fos and NFATc1, which are essential transcription factors inosteoclast formation, we analyzed the expression of these two factorsby Western blot analysis. As shown in Fig. 2C and SupplementaryFig. S2B, the protein levels of c-Fos and NFATc1 were upregulatedduring RANKL-induced osteoclast differentiation, which can be pre-vented efficiently by GSK650394 treatment.

We then tested whether GSK650394 suppresses the expression ofosteoclast marker genes such as tartrate-resistant acid phosphatase(TRAP), MMP9, NFATc1, and cathepsin K (CK); notably, TRAP,MMP9, and CK are target genes of NFATc1 (30). As shown in Fig. 2Dand E, GSK650394 inhibited the mRNA expression of MMP9 andCK during late stages of osteoclastogenesis and inhibited TRAPand NFATc1 at both early and late stages of osteoclastogenesis,respectively.

Activation of Orai1 abolishes the inhibition ofosteoclastogenesis by GSK650394

Orai1 is a SOCE channel on membrane, which regulates Ca2þ

oscillations and NFATc1 transactivation during osteoclastogen-esis (31). Immunofluorescence (IF) andWestern blot analysis showedthat the inhibition of SGK1 decreased Orai1 protein expression duringosteoclastogenesis (Fig. 3A, C and D). Ca2þ imaging analysis con-

firmed essentially complete inhibition of SOCE in BMMs treated withGSK650394 compared with control (Fig. 3E).We therefore speculatedthat Orai1mightmediate the effect of SGK1 on osteoclast. To elucidatethe role of Orai1 in the SGK1-mediated inhibition of osteoclastformation, we overexpressed Orai1 in BMMs (Fig. 3B). Scrambledor Orai1 overexpressed BMMs were treated with or withoutGSK650394 in the presence of RANKL (75 ng/mL) for 5 days,respectively. As shown in Fig. 3F and G, the inhibitory effects ofGSK650394 on osteoclast differentiation were markedly alleviated inOrai1 overexpression group, indicating thatOrai1 is a key downstreammediator of the terminal differentiation of osteoclasts by SGK1(Supplementary Fig. S2C).

GSK650394 prevents ovariectomy-induced bone lossTo investigate the effects of SGK1 on bone loss, we analyzed the

bone mass of femurs in ovariectomized (OVX) mouse model. Theresult revealed that the reduction of intrabecular bone volume/tissuevolume (BV/TV) and trabecular number (Tb.N) in the OVXmice wasdramatically relieved by GSK650394 treatment (50 mg/kg; Fig. 4Aand B). Furthermore, we performed TRAP staining in femoral sec-tions. The number and size of osteoclasts in OVX mice were signif-icantly higher than those in sham-operated mice. In response toGSK650394 treatment, the OVX mice demonstrated dramaticallyreduced ovariectomy-induced osteoclast activity (Fig. 4C). Histomor-phometric analyses confirmed that the osteoclast surface/bone surface(Oc.S/BS) ratio was strikingly higher in OVX mice than in sham-operated mice (Fig. 4D). Furthermore, compared with those ofuntreated OVX mice, these osteoclastic parameters were significantlydecreased in GSK650394-treated OVX mice (Fig. 4B).

In addition, compared with OVX group GSK650394 treatmentsignificantly suppress the ovariectomy-induced elevation in type Icollagen cross-linked C-terminal telopeptide (CTX-I) and RANKLlevels in serum (Fig. 4E). Moreover, we tested the levels of OPG byELISA. The results are similar (Fig. 4E). Therefore, comparedwith thatin the untreated OVX mice, the RANKL/OPG ratio was markedlysuppressed in the GSK650394-treated OVX mice (Fig. 4E).

SGK1 serves as a promising therapeutic target for bonemetastasis

To further verify the effect of GSK650394 on the formation of bonemetastases, we used a model based on the intracardiac inoculation oftumor cells (32). A bone-seeking clone (MBS4-TJGK) of the humanbreast cancer cell line MDA-MB-231 was established by repeatedsequential intracardiac inoculation of the cells into nude mice andfurther culture of metastatic cells obtained from bone metastasesin vitro. After four cycles, MBS4-TJGK metastasize exclusively tobones. Then, MBS4-TJGK cells were inoculated intracardially. Nudemice were intraperitoneally injected with GSK650394 (50 mg/kg, fivetimes per week) for 4 weeks. Bioluminescence imaging showed thatcompared with the DMSO-treated group, hind limb and spine tumorburden in mice was significantly decreased in the GSK650394-treatedgroup (Fig. 5A and B). Micro-CT imaging confirmed that the micetreated with GSK650394 exhibited a reduction in bone lesions(Fig. 5C).

Because SGK1 is also highly expressed in heart and gastrointestinaltract, we then evaluate the toxicity of GSK650394. Mice were eutha-nized 3 weeks after injection and plasma biochemistry analyses wereperformed. The indicators for heart and liver injuries, as well as kidneyfunctions did not change significantly at the concentration of 50mg/kg(Fig. 5D and E). However, when lifted dose to 75 mg/kg, lacticdehydrogenase (LDH-L) dropped in treated group mice (Fig. 5D).

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These results suggest that intraperitoneal injection of GSK650394 inour model did not lead to reductions in hepatic and renal function butmay have off-target influence on myocardial cells damaging with highdose.

Then, direct effect of GSK650394 treatment on MDA-MB-231 celllines was tested by flow cytometry and CCK-8 (Fig. 5F and G).Apoptotic and necrosis cells were detected using FITC-AnnexinV/propidium iodide (PI). Cells incubated with GSK650394 at theconcentration of 5 mmol/L showed higher apoptotic rate among othergroups. The proliferation of MDA-MB-231 cells were altered byGSK650394 in a dose-dependent manner.

Validation of the role of SGK1 from databaseComparing the mRNA expression level of SGK1 in normal breast

tissues and breast cancer tissues (Fig. 6A), we found SGK1 is signif-icantly increased in breast cancer (33, 34, Perou and colleagues). It

validated our results of RT-PCR. To further determine the clinicalsignificance of SGK1, we examined whether SGK1 expression wasassociatedwith the clinical outcome of breast cancer patients (Fig. 6B).The survival analysis based on large population included in the KMplotter showed no significant prognostic performance of SGK1 onbreast cancer patients (HR¼ 0.92; 95% CI, 0.82–1.02; P¼ 0.120). Butin other datasets GSE2603 (HR¼ 2.24; 95% CI, 1.05–4.79; P¼ 0.033)and GSE12276 (HR ¼ 1.74; 95% CI, 1.30–2.34; P < 0.001) we foundhigh expression of SGK1 could predict poor prognosis, indicating thepotential oncogenic effect of SGK1 (35, 36).

DiscussionCa2þ signaling is essential for diverse biological processes and is

known to be involved in osteoclast formation and function (37–43).Ca2þ release from intracellular stores and Ca2þ entry across the cell

Figure 2.

GSK650394 inhibits RANKL-induced activation of the NF-kB, PI3K pathways, and suppresses the expression of c-Fos/NFATc1 and osteoclast marker genes.A,GSK650394 inhibits RANKL-induced phosphorylation of NF-kB. BMMswere starved in 0.5% FBS ina-MEM for 12 hours before treatment and then pretreatedwithor without GSK650394 (2 mmol/L) for 24 hours followed by RANKL (100 ng/mL) stimulation for the indicated times. Cell lysates were extracted for immunoblottingwith the indicated antibodies. Immunoblots were quantified using ImageJ software. Experiments were performed in triplicate. B, Effect on PI3K activation. BMMswere treated as described above, and total cell protein was fractionated for immunoblotting with the indicated antibodies. Immunoblots were quantified usingImageJ software. Experiments were performed in triplicate. C, GSK650394 inhibits RANKL-induced protein expression of c-Fos and NFATc1. BMMs treated with orwithout GSK650394 (2 mmol/L) were cultured in the presence of M-CSF (30 ng/mL) and RANKL (100 ng/mL) and collected at the indicated time points for theanalysis of c-fos and NFATc1 protein expression. Immunoblots were quantified using ImageJ software. Experiments were performed in triplicate. D and E,GSK650394 suppresses RANKL-induced mRNA expression of TRAP, NFATc1, CK, and MMP9. BMMs were treated with or without GSK650394 (2 mmol/L) for 2 or5 days. Total RNA was isolated and analyzed by quantitative real-time RT-PCR. Data represent the means � SDs. � , P < 0.05; �� , P < 0.01 (Tukey test).

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membrane both accomplish Ca2þ in cytoplasm. SOCE, a majorCa2þ influx pathway in most nonexcitable cell types, is mediated bythe Ca2þ release-activated Ca2þ channel, which consist of Orai1, 2,or 3 binding to their regulators STIM 1 or 2 (31). Previous work hasidentified SGK1 as a powerful stimulator of several ion channels

including Orai1. Some clinical studies have been undertaken tocorroborate the relevance of SGK1 in diseases such as hypertensionand cancer. The mechanism can be related to coordinate withdifferent signaling pathways include FoxO1 and NF-kB and leadto target organ inflammation (44). However, it was unknown

Figure 3.

Orai1 is upregulated during RANKL-induced osteoclastogenesis and can be suppressed by GSK650394. A, BMMs treated with or without GSK650394 (2 mmol/L)were cultured in the presence of M-CSF (30 ng/mL) and RANKL (100 ng/mL) and collected at the indicated time points for the analysis of Orai1 protein expression.Immunoblots were quantified using ImageJ software. Experiments were performed in triplicate. B, SGK1 protein expression in BMMs was assessed by Westernblotting. ACTB was used as a loading control. Immunoblots were quantified using ImageJ software. Experiments were performed in triplicate. C, IF of Orai1 proteinlevels in BMMs from the control, RANKL-treated andRANKL/GSK650394 cotreated groups. Imageswere captured using afluorescencemicroscope. Red, SGK1; blue,nuclei. D,Orai1 protein on themembrane was analyzed flow cytometry. GSK650394 at the concentrations of 2 mmol/L was used. E, Ca2þ imaging of RANKL inducedBMMs treated with or without GSK650394. Cells were loaded with 5 mmol/L Fluo-3/AM, and SOCE was revealed by readdition of 2 mmol/L Ca2þ after TG-inducedCa2þ release fromERandmeasuredbyMulti-ModeMicroplate Reader of intracellular Ca2þ concentration.F,Control orOrai1-overexpressedBMMs inducedbyRANKL(50 ng/mL) treated with or without GSK650394 (2 mmol/L) for 5 days. Then, cells were fixed for TRAP staining. G, TRAP-positive cells with�3 nuclei were counted.Data are presented as the means � SDs of three independent experiments. ���� , P < 0.0001 (Tukey test).

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whether SGK1 affects osteoclast differentiation and bone resorptionvia Ca2þ channel through Orai1 until now.

In our study, we observed SGK1 expression was upregulated uponthe initiation of osteoclastogenesis. Both pharmacologic inhibition andknockdown of SGK1 by shRNA suppressed osteoclast differentiationand function. In addition, Orai1 protein abundance appeared aswell asthe significant decrease of Ca2þ influx after GSK650394 treatment(Fig. 3A, C–E). Moreover, overexpressing Orai1 in BMMs counter-acted the suppressive effect of GSK650394 on osteoclast formation. Onthe basis of these data, we concluded that SGK1 expression is closelyrelated to osteoclastogenesis and has a strong effect on Orai1. Tofurther confirm SGK1 inhibition on ovariectomy-induced bone lossin vivo, we used GSK650394 to prevent bone loss in OVX mouse

model. We observed attenuated ovariectomy-induced bone loss inGSK650394 treated group, which was consistent with results in vitro.In the previous work, SGK1 was showed to be evolved in nucleartranslocation and activation of NF-kB (24, 45, 46). SGK1 couldphosphorylate IKKa, which lead to IKKb activation. The IKK complexand the NF-kB pathway would be activated similarly, which wereconfirmed in our study. In addition, we demonstrated that Orai1es-tablished an previously unknown link between SGK1 and osteoclastgenesis.

SGK1 expressed heavily in several tumor cells, including coloncancer, myeloma, medulloblastoma, prostate cancer, and ovariantumors (47, 48). Some research has shown that SGK1 contributes tothe glucocorticoid-induced resistance of breast cancer cells to

Figure 4.

GSK650394 inhibits ovariectomy-induced bone loss. A, Representative micro-CT images near the distal femoral of mice from the sham-operated (SHAM),ovariectomized (OVX), and OVX þ GSK650394-treated groups. Mice were sacrificed after 8 weeks of GSK650394 treatment. B, Histograms represent theparameters of the distal femur: Trabecular bone volume/tissue volume (BV/TV), trabecular separation (Tb.Sp), trabecular number (Tb.N), and trabecular thickness(Tb.Th). Data represent themeans� SDs, n¼ 6. � , P < 0.05; �� , P < 0.01 (Tukey test). C, TRAP staining was performed in sections of themetaphyseal regions of distalfemurs from SHAM, OVX, and OVXþGSK650394 groups. D, Osteoclast surface/bone surface (Oc.S/BS, %) of TRAP-stained sections was measured. Data arepresented as themeans� SDs, n¼ 6. �� ,P <0.01 (Tukey test).E,Micewere sacrificed after 8weeks of GSK650394 treatment. Serum levels of CTX-I, RANKL, andOPGweremeasured by ELISA. Data are presented as themeans� SDs, n¼ 6. Statistically significant differences between the treatment and control groups are indicatedas �P < 0.05 and ��P < 0.01 (Tukey test).

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Figure 5.

GSK650394 prevents bone metastasis in an intracardiac inoculation model. A, Representative images of whole-body in vivo bioluminescence imaging (BLI) ofmice at day 30 after the intracardiac injection of MBS-TJGK4 cells. B, Quantitation of bioluminescence images at day 30 (n ¼ 6). Statistically significantdifferences between the treatment and control groups are indicated as ���P < 0.0003 (Student t test). C, Representative micro-CT images of tibia from theDMSO- and GSK650394-treated groups. D, The myocardial cells injury after intraperitoneal injection of DMSO, GSK650394 (50 mg/kg), or GSK650394(75 mg/kg) were determined by blood chemistry tests. LDH1, lactic dehydrogenase isoenzyme 1; LDH-L, lactic dehydrogenase; CK-MB, creatine kinase-MB.� , P < 0.05 (Dunnett t test) versus the vehicle-treated group. E, The hepatic and renal function of mice after intraperitoneal injection of DMSO, GSK650394(50 mg/kg), or GSK650394 (75 mg/kg) were determined by blood chemistry tests. ALT, alanine aminotransferase; AST, aspartate aminotransferase; CR,creatinine. F, Apoptosis was analyzed using an Annexin V/PI Staining Kit. GSK650394 concentrations of 1, 2, and 5 mmol/L were used. G, MDA-MB-231 cellswere treated with varying concentrations of GSK650394 (0, 1, 2, and 5 mmol/L) for 5 days. Cell proliferation was assessed by the Cell Counting Kit-8. Data arepresented as the means � SDs of three independent experiments.

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chemotherapy, and SGK1 silencing could increase the toxicity ofchemotherapeutic drugs. On the basis of the Oncomine database, wefound the expression of SGK1 was significantly upregulated in differ-ent types of breast cancer compared with normal breast tissue.However, the result of further survival analysis in breast cancer variesfrom different database. To investigate the exact role of SGK1 inprognosis of breast cancer, further research should be conducted inspecific population cohort with confounding factors adjusted.

SGK1-dependent regulation of Orai1 has been shown to play acritical role in regulation of cell proliferation (17). In this study, weinvestigated the effect of SGK1 on bone metastasis in breast cancerusing an intracardiac injection-induced model of bone metastasis inmice. Our results demonstrated the effects of GSK650394 in theinhibition of breast cancer bone metastasis. Besides the effects on thecell proliferation, more application of SGK1 inhibition has beenexplored.

On the basis of this study, SGK1 is a powerful regulator inosteoclastogenesis and bone metastasis. SGK1 inhibitors might be apromising therapeutic approach target for various pathologic condi-tions including bone metastasis. Because we are just exploring theeffect of SGK1 on osteoclastogenesis, the possible osteoblastic effectalso needs to be considered. Previous study has confirmed that SGK1

couldmediate osteoinductive effects of phosphate in the vascular tissueand trigger vascular calcification. SGK1 leads to IkBaphosphorylationand NF-kB activation, which results in osteo-/chondrogenic transdif-ferentiation of VSMCs. Further study needs to be done in thefuture (49–51).

There are several limitations to our study. Because SOCE is not theonly calcium entry pathway, the calcium influx could be through t-typecalcium channel, TRP channels, and so on. We used a selective SOCE-specific inhibitor BTP2 and monitored calcium influx after intracel-lular store depletion (52). The drug abolished the Ca2þ transientamplitude but cannot be fully rescued by Orai1 overexpression(Supplementary Fig. S1B). Also, upregulation of Orai1 is not the onlyway SGK1 modify Ca2þ activity. SGK1 can also have effects onactivation of Kþ channel or Ca2þ-permeable TRP channels whichcould hyperpolarize the cell membrane and influence the Ca2þ chan-nels. Our results only focused on Orai1, not on the whole family ofOrai. It is also possible that different levels of other isoforms, Orai2 andOrai3, are present in the cells in both groups (53, 54). It highlights theneed for future deeper studies to analyze the differences and connec-tions among the three. Another limitation is that the effects of theinhibitor is not specific for bonemetastasis, but also impair the survivalof tumor cells. Although in our experiment, GSK650394 did not show a

Figure 6.

Validation of SGK1 from database. A, SGK1 mRNA expression was significantly higher in breast malignancy tissues (IDBC, LBC) compared with normal breast tissuesbased on theOncomine database. � , P <0.05; �� ,P <0.01 (Dunnett t test) versus normal group.B,Overall survival curves of SGK1 according to its expression based onKaplan–Meier plotter and datasets GSE2603 and GSE12276 from the GEO database.

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direct effect on breast cancer apoptosis (Fig 5F), high levels of SGK1have been found as compensation upon PI3K or AKT pathway andcontribute to the resistance to AKT inhibitors in breast cancer cells,whereas loss of SGK1 profoundly impacts thyroid cancer cell prolif-eration and survival despite intact PI3K and AKT activity (50, 55, 56).Therefore, the effect of GSK650394 to bone metastasis may becomposed of complex factors.

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

Authors’ ContributionsConception and design: Z. Zhang, C. Song, H. Guan, F. LiDevelopment of methodology: Z. Zhang, Q. Xu, B. Mi, H. Zhang, H. Liu, Y. Sun,H. Guan, F. LiAcquisition of data (provided animals, acquired and managed patients, providedfacilities, etc.): Z. Zhang, Q. Xu, B. Mi, H. Zhang, H. Liu, Y. Sun, F. Li

Analysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): Z. Zhang, Q. Xu, C. Song, J. Wang, Z. Lei, H. Guan, F. LiWriting, review, and/or revision of the manuscript: Z. Zhang, Q. Xu, H. Zhang,H. Guan, F. LiAdministrative, technical, or material support (i.e., reporting or organizing data,constructing databases): Z. Zhang, Q. Xu, B. Mi, H. Kang, J. Wang, Z. Lei, F. LiStudy supervision: H. Guan, F. Li

AcknowledgmentsThis work was supported by National Natural Science Foundation of China

(no. 81572857, to H. Guan; no. 81401762, to J. Wang; and 81472133 to F. Li)

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

Received July 16, 2018; revised December 30, 2018; accepted October 31, 2019;published first November 6, 2019.

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