p27 Is a Candidate Prognostic Biomarker andMetastatic Promoter in OsteosarcomaYiting Li1,2,3, Manjula Nakka1,3, Aaron J. Kelly1,4, Ching C. Lau1,2,3,4, Mark Krailo5,6,Donald A. Barkauskas5,6, John M. Hicks7, and Tsz-Kwong Man1,2,3,4
Abstract
Metastatic progression is the major cause of death in oste-osarcoma, the most common bone malignancy in children andyoung adults. However, prognostic biomarkers and efficacioustargeted treatments for metastatic disease remain lacking.Using an immunoproteomic approach, we discovered thatautoantibodies against the cell-cycle kinase inhibitor p27(KIP1, CDKN1B) were elevated in plasma of high-risk osteo-sarcoma patients. Using a large cohort of serum samples fromosteosarcoma patients (n ¼ 233), we validated that a higherlevel of the p27 autoantibody significantly correlated withpoor overall and event-free survival (P < 0.05). Immunohis-tochemical analysis also showed that p27 was mislocalized tothe cytoplasm in the majority of osteosarcoma cases and in
highly metastatic osteosarcoma cell lines. We demonstratedthat ectopic expression of cytoplasmic p27 promoted migra-tion and invasion of osteosarcoma cells, whereas shRNA-mediated gene silencing suppressed these effects. In addition,mutations at the p27 phosphorylation sites S10 or T198, butnot T157, abolished the migratory and invasive phenotypes.Furthermore, the development of pulmonary metastasesincreased in mice injected with cells expressing cytoplasmicp27 compared with an empty vector control. Collectively, ourfindings support further investigation of p27 as a potentialprognostic biomarker and therapeutic target in osteosarcomacases exhibiting aberrant p27 subcellular localization. CancerRes; 76(13); 4002–11. �2016 AACR.
IntroductionOsteosarcoma is the most common malignant bone tumor in
children. Approximately 400 to 900 new cases are diagnosed eachyear in the United States (1). The standard clinical treatmentconsists of a combination of surgery and chemotherapy.With thisregimen, the prognosis of localized osteosarcoma has improveddramatically when compared with surgery alone (2). The pressingclinical problem, however, is that the prognosis for metastatic,refractory, and recurrent osteosarcoma is still dismal (3). Approx-imately 20% of osteosarcoma patients have clinically detectablemetastasis at the time of diagnosis (2). Even with current treat-ment protocols, the progression-free survival rate for patientswithmetastatic osteosarcoma is still less than 20%, indicating that the
existing regimens are not effective for these patients (4, 5).Detection of patients with high metastatic potential or clinicalrisk earlymay help to prevent the use of ineffective therapy, whichallows extra time for the tumor to develop chemoresistance andmetastatic spread. In addition, identification and characterizationof key metastatic determinants may lead to the development ofnew targeting strategies and therapeutic options once thehigh-riskpatients are identified.
A vast amount of literature has indicated that cancer patientsoftenproduce autoantibodies to tumor-associated antigens (TAA)that are overexpressed, modified, or aberrantly cleaved or local-ized in tumor cells (6). Given that these autoantibodies areconsidered reporters of the immune response to an early anddeveloping tumor, they are excellent candidate biomarkers forearly cancer detection and may lead to identification of potentialtherapeutic targets. In fact, autoantibodies against TAAs have beenshown to be useful in both cancer diagnosis and prognosis (6).Besides being present in peripheral blood, the persistence andstability of autoantibodies in plasma is an added benefit overother potential markers, including TAAs themselves, which arereleased by tumor cells but are rapidly cleared after circulating inthe plasma for a limited amount of time (7). Emerging evidenceindicates that many TAAs detected by autoantibodies are impor-tant cellular proteins, such as ERBB2, MYC, and TP53, whoseaberrant regulation or alteration could be linked to malignancy(8). In this study, we adopted an immunoproteomic approach foridentifying important metastatic targets in osteosarcoma.
Although well-known tumor suppressor genes, such as TP53,RB1 and PTEN, have been implicated in osteosarcoma (9–11),little is known about p27. p27 or p27kip1 (encoded by CDKN1B)is a cyclin-dependent kinase (CDK) inhibitor that negativelyregulates cell proliferation through the inhibition of cell-cycleprogression (12). In a quiescent state, the p27 protein is expressed
1DepartmentofPediatrics, BaylorCollegeofMedicine, Houston,Texas.2Dan L. Duncan Cancer Center Baylor College of Medicine, Houston,Texas. 3Texas Children's Hematology and Oncology Centers, TexasChildren's Hospital, Houston, Texas. 4Program of Structural andComputational Biology and Molecular Biophysics, Baylor College ofMedicine, Houston, Texas. 5Children's Oncology Group, Monrovia,California. 6Department of Preventive Medicine, Keck School of Med-icine, University of Southern California, Los Angeles, California.7Department of Pathology, Baylor College of Medicine, Houston,Texas.
Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).
Y. Li and M. Nakka contributed equally to this article.
Corresponding Author: Tsz-Kwong Man, Section of Hematology andOncology,Department of Pediatrics, Baylor College of Medicine, 6621 Fannin St, MC3-3320, Houston, TX 77030-2399. Phone: 832-824-4682; Fax: 832-825-4038;E-mail: [email protected]
mainly in the nucleus and binds to the cyclin/CDK complex toinhibit cell-cycle progression. Degradation of the protein is reg-ulated predominantly through the ubiquitin pathway (13). Geneknockout studies have shown that mice lacking p27 developmultiorgan hyperplasia and tumorigenesis (14, 15). However,recent data indicate that p27 also has oncogenic properties, whichare distinct from the cell-cycle–regulatory functions (16). In thisstudy, we showed that p27 autoantibody is prognostically signif-icant and the mislocalization of the protein promotes tumor cellmigration and invasion as well as metastasis formation inosteosarcoma.
Materials and MethodsPatients and samples
To identify autoantibodies associated with high-risk in osteosar-coma, plasma samples frompatientswho developed localized (n¼26)ormetastatic disease (n¼ 13)withinone yearof follow-up, andanonymizedpatientswithnoncancerousdiseasesornodisease (n¼21), that is, child checkup, flu, constipation, gastroenteritis, orfebrile seizure, were analyzed by the ProtoArray and Luminexplatforms. These samples were collected at the time of diagnosisfrom Texas Children's Hospital (Houston, TX) and other collabo-rating institutions. Serum samples fromosteosarcoma patients andtheir survival information were provided by the Children's Oncol-ogy Group (COG). All patients gave consent to institutional reviewboard–approved protocols. The clinical information of patientsused in this study is summarized in Supplementary Table S1.
Autoantibody profiling analysisProtoArray Human Protein Array v4.0 (ThermoFisher Scientif-
ic) that contains 8,268 proteins was used to detect autoantibodiesin the plasma samples according to the manufacturer's instruc-tion. The detailed procedure is described in the SupplementaryMethods. The experiments were repeated thrice. Then, the back-ground subtracted intensities of the whole array were quantile-normalized and log-transformed. The threshold of the low inten-sity values was set at 5. The intensities of the duplicate spotsfor each protein were averaged and control spots were removed.P values were calculated by using unpaired, two-tailed t test. Theselection criteria for high-risk autoantibodies were as follows:(i) P < 0.001 in high-risk versus control and P < 0.05 in high-riskversus low-risk comparisons, and (ii) log fold change �1. TheProtoArray data (GSE78192) were deposited to the Gene Expres-sion Omnibus (GEO) repository.
ProtoPlex immune response assay to measure the p27autoantibody
The autoantibody validation assays were performed using theProtoPlex service from Thermo Fisher Scientific. The detailedprocedure is described in the Supplementary Methods. p27 auto-antibody fluorescence intensity values in the COG cohort werebackground corrected for BSA level, log-transformed, and binar-ized by median cutoff. Kaplan–Meier analysis was performed totest if a higher level of the p27 autoantibody correlated with pooroutcome and significance was evaluated using a one-tailed ver-sion of the log-rank statistic in overall and event-free survival. Inevent-free survival analysis, events indicated whether death orrelapse had occurred in each patient. To ensure p27 was anindependent prognostic factor, we confirmed its significance bycontrolling for the known prognostic factor, that is, metastasis at
diagnosis, using a multivariate Cox proportional hazard model.For all statistical tests, P < 0.05 is considered statisticallysignificant.
IHCOsteosarcoma cells were cultured on Lab-Tek chamber slides
(Nunc). After adhesion, cells were fixed with 2% formaldehyde(Sigma-Aldrich) andwashed three timeswith PBS (Thermo FisherScientific). For tissue microarrays, the arrays were incubated at60�C for one hour, deparaffinized with xylene, and rehydrated.Antigen retrievalwas performed at 95�C for 30minutes inAntigenUnmasking Solution (Vector Laboratories). Then, the slides wereincubated with 3% hydrogen peroxide and normal horse serum(Vector Laboratories) for 30 minutes. After that, slides wereincubated overnight with mouse anti-human p27 antibody (sc-56338, 1:200, Santa Cruz Biotechnology) at 4�C. A biotinylatedhorse anti-mouse IgG antibody (1:200) and the streptavidin–biotin–peroxidase reaction (Vector Laboratories) were addedsequentially. After color reaction with 3,3-diaminobenzidinesolution (Vector Laboratories), the slides were dehydrated andmounted. The staining results were examined and scored by ourpathologist (J.M. Hicks) to determine whether p27 was expressedin the tumor sections.
Cell cultureSaOS-2, LM7, Dunn, K7, DLM8, and K7M3, were generously
provided by Dr. Eugenie S. Kleinerman from the M.D. AndersonCancerCenter at theUniversityof Texas (Houston, TX). SaOS-2 is ahuman osteosarcoma cell line with lowmetastatic potential (17).Themetastatic LM7 cell line was developed by repetitive cycling ofSaOS-2 cells through the lungs of nude mice seven times (18).DLM8 and K7M3 are metastatic murine osteosarcoma cell lines,whichwere derived fromeither repeated implantationor injectionof Dunn and K7, respectively (19, 20). Cells were cultured inDMEM supplemented with 10% FBS (Thermo Fisher Scientific) at37�C in a humidified 5% CO2 incubator. They were tested to bemycoplasma-free. Identities of the cell lines were validated by STRDNA fingerprinting using the AmpF_STR Identifiler kit accordingto the manufacturer's instruction (Thermo Fisher Scientific).
p27 constructs and transfectionp27 constructs harboring fusions with Nuclear Export
Sequence (NES) and enhanced yellow fluorescent protein (EYFP;Clontech) were stably expressed in lowly metastatic SaOS-2 cells.The p27 and control constructs (p27CK-, NES-p27CK- andpEYFP) were kindly provided by Dr. Catherine Denicourt withtheUniversity of Texas-Houston (Houston, TX). Specifically,NES-p27 was constructed by fusing two NES sequences of proteinkinase inhibitor (residues 35–49) in tandem to theNH2 terminus.The Cyclin–CDK complex binding domain of the p27 constructwas mutated (CK-) to control for the cell-cycle–inhibitory func-tion of p27 (15). The three p27 phosphomutants (NES-p27CK-S10A, -T157A, and -T198A) were generated by replacing serine 10(S10), threonine 157 (T157), and threonine 198 (T198) on thep27 cDNAwith alanine (A) using theQuikChange Lightning Site-Directed Mutagenesis Kit (Stratagene). They were subsequentlyconfirmed by Sanger Sequencing (Supplementary Fig. S1). Trans-fections were performed using FuGENE 6 Transfection Reagent(Roche Applied Sciences). Selection for stable transfectants wasstarted 48 hours after transfection using 1.25 mg/mL Geneticin(Thermo Fisher Scientific).
The Role of Mislocalized p27 in Osteosarcoma
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Knockdown of p27 expression in LM7Two specific MISSION TRC Lentivirus shRNAs for p27 and a
shRNA nontarget control were used in the silencing experiment(Sigma-Aldrich). Twenty-fivemicroliters of eachof thep27 shRNAviruses and 16 mg of hexadimethrine bromide (Santa Cruz Bio-technology) were added to each well containing 2mL of mediumand LM7 cells. Puromycin (1 mg/mL; Sigma-Aldrich) was used forselection at 48 hours after transduction. The sequences for the p27shRNAs are shown in the Supplementary Methods.
Migration and invasion assaysCytoSelect 24-well Migration and Invasion Assays (Cell Bio-
labs)were used tomeasure themigratory and invasive capabilitiesof the p27 mutant and control cells. A total of 3 � 105 cells inserum-depleted medium were added to the top chamber of atranswell. For the invasion assay, the top surface of a transwell wascoated with dried basement membrane matrix solution. Mediumcontaining 10% FBS was added to the bottom well. After 6–24hours of incubation, the migrated and invaded cells were stainedby the Cell Stain Solution, which contains crystal violet, andcounted in 3–4 fields under a microscope and used for statisticalanalysis.
Experimental metastasis assayTo measure the metastatic ability of cytoplasmic p27 in vivo,
we used a tail-vein injection mouse model. A total of 1.5 � 106
of SaOS-2 cells containing empty vector or NES-p27CK- as wellas metastatic LM7 cells were injected intravenously into thelateral tail vein of 4- to 6-week-old NOD/SCID mice of bothsexes. Five mice were injected in each group. The mice werebred and maintained in a specific pathogen-free animal facilityat the Texas Children's Hospital (Houston, Texas). Mice wereeuthanized either when they were moribund or 6 months afterthe injection of the tumor cells. The lungs of the mice wereharvested in 10% formalin, embedded in paraffin, and sec-tioned. The sections were then stained with hematoxylin andeosin, and examined for the occurrence of metastases under amicroscope by our pathologist (J.M. Hicks). All animal studieswere conducted according to Institutional Animal Care and UseCommittee protocols after approval was obtained from ourInstitutional Review Board.
Data analysis and other methodsUnless stated otherwise, all the statistical comparisons were
performed by Student unpaired t tests with P < 0.05 were con-
sidered significant. Additional details of the methods used aredescribed in the Supplementary Methods.
ResultsThe p27 autoantibody is elevated in high-risk osteosarcomapatients
To identify dysregulated proteins in high-risk patients, we firstpooled the plasma samples from osteosarcoma patients, whodevelopedmetastasis and localized disease within the first year offollow-up into a high-risk group and a low-risk group, respec-tively. Then, the healthy or noncancerous pediatric patients werepooled into a control group. The pooled sample from each of thethree groups was hybridized with a high-density human proteinmicroarray (ProtoArray). The clinical characteristics of the cohortare listed in Supplementary Table S1. Our results showed that sixautoantibodies for TCP10, RPS6KB3,MAPK11, CDKN1B, TRPT1,and TFPT showed significantly higher intensities in the high-riskgroup than those in the low-risk and control groups (Supplemen-tary Table S2). p27 was selected for further investigation andvalidation, because it is a known tumor suppressor gene thatinhibits cell-cycle progression (Fig. 1A; ref. 12). Using an orthog-onal Luminex xMAP assay, we measured the p27 autoantibodylevel in individual, rather than pooled, plasma samples. Theresults show that the p27 autoantibody level was significantlyincreased in the high-risk groupwhen comparedwith the low-riskgroup (Fig. 1B, P¼ 0.0134), suggesting that it carries a prognosticvalue. Of note, a subset of patients had a much higher level of thep27 autoantibody within the high-risk group.
The p27 autoantibody predicts overall survivalAs the p27 autoantibody was elevated in the high-risk patients,
we further hypothesized that a higher level of the autoantibodypredicts a worse outcome in osteosarcoma patients. To test this,we used the Luminex xMAP assay to measure the amounts of thep27 autoantibody in a large osteosarcoma cohort (n ¼ 233)obtained from COG. Using the median abundance as a cutoff,a higher p27 autoantibody level was significantly associated withpoor overall (P ¼ 0.033, HR ¼ 1.46) and event-free (P ¼ 0.0079,HR ¼ 1.57) survival of osteosarcoma patients (Fig. 2). Patientswith a low level of the p27 autoantibody had a 5-year overallsurvival rate of 67% (95% CI, 59%–77%) and 5-year event-freesurvival of 57% (95% CI, 48%–67%), whereas patients with ahigh level of p27 autoantibody had a 5-year overall survival rate of52% (95% CI, 44%–62%) and 5-year event-free survival of 40%
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Figure 1.Identification of p27 as a tumor-associated antigen in high-riskosteosarcoma. A, the normalized logfluorescent intensity of the p27autoantibody in the pooled high-risksamples was significantly higher thanthose in the pooled samples from low-risk and noncancerous diseases (� , P <0.05) using ProtoArrays. B, theLuminex xMAP Assay validated thatthe p27 autoantibody levels in theindividual samples of the high-riskgroup were significantly higherthan those in the low-risk group (� , P <0.05). The lines in the plot denote theaverage intensity valuesof the groups.
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(95% CI, 31%–50%). After controlling for the known prognosticfactor at diagnosis, that is, initial metastasis or metastasis atdiagnosis, we found that the associations of the p27 autoantibodywith overall and event-free survival remained significant (P <0.05). When stratified by initial metastasis, p27 autoantibodylevels did not significantly correlate with overall or event-freesurvival (overall: P¼ 0.27, HR¼ 1.21; event-free: P¼ 0.18, HR¼1.29) in cases with initial metastasis, whereas in patients with nodetectable metastasis at diagnosis, a high p27 level significantlycorrelated with poor outcome in both overall and event-freesurvival (overall: P ¼ 0.04, HR ¼ 1.63; event-free: P ¼ 0.013, HR¼ 1.73; Fig. 2). These results indicate that the p27 autoantibodymay help to identify patients who currently would be classified as"low risk" because of their localized disease status, but actuallyhave significantly worse survival rates.
Cytoplasmic mislocalization of p27 in osteosarcoma tissuesand metastatic cell lines
To examine whether p27 is dysregulated in osteosarcoma, weperformed p27 IHC on two osteosarcoma tissue microarrays(TMA). One was from the COG and the other was from acommercial source (US BioMax). Our results showed that 82%of osteosarcoma cores showed either strong (30%) or weak(52%) cytoplasmic staining of p27 in the COG TMA. Similarly,80% of osteosarcoma cores had cytoplasmic staining of p27, inwhich 26 cases (52%) showed strong expressions and 14 cases(28%) showed weak expression in the US BioMax TMA (Fig. 3Aand B). No nuclear expression of p27 was observed in bothTMAs. These staining results suggest that p27 was expressed butmislocalized in the cytoplasm of the majority of osteosarcomacases. As cytoplasmic mislocalization of p27 has been impli-cated in metastasis of other solid tumors (16, 21), we per-formed p27 IHC on three pairs of isogenic osteosarcomametastatic cell lines. As shown in Fig. 3C, p27 expression was
either very low or restricted in nucleus in the lowly metastaticparental cell lines (SaOS-2, K7, Dunn). However, p27 wasexpressed relatively stronger in the cytoplasm of all threemetastatic sublines (LM7, K7M3, DLM8). These cell line resultssuggest that cytoplasmic expression of p27 may be linked tometastatic properties of osteosarcoma cells.
Cytoplasmic p27 promotes the metastatic potential ofosteosarcoma cells in vitro
To test whether cytoplasmic mislocalization of p27 canincrease the metastatic potential of osteosarcoma cells, a p27construct harboring a fusion with a NES and EYFP was stablyexpressed in the lowly metastatic SaOS-2 cell line with lowcytoplasmic p27 expression. The Cyclin–CDK complex bindingdomain of the p27 construct was mutated (CK-) to control forthe cell-cycle–inhibitory function of p27 (Fig. 4A). In the stablecell line containing the empty vector control (pEYFP), thefluorescent protein was expressed in both nucleus and cyto-plasm of the tumor cell (Fig. 4B). In contrast, p27 was expressedin the cytoplasm of the cells harboring NES-p27CK- (Fig. 4B).To examine the effect of cytoplasmic p27, in vitromigration andinvasion assays were performed on the NES-p27CK-mutant andcontrols. As shown in Fig. 4C, ectopic expression of cytoplasmicp27 significantly increased the numbers of migrated and invad-ed cells relative to the empty vector control (P ¼ 2 � 10�7 formigration and P ¼ 2 � 10�4 for invasion). Similar results wereobserved in the repeated experiment (Supplementary Fig. S2).The results suggest that cytoplasmic mislocalization of p27 wassufficient to promote the motility and invasiveness of osteo-sarcoma cells. The migratory and invasive abilities of NES-p27cells without the CK mutation behaved similarly to the CK-mutant, suggesting that the prometastatic effect of cytoplasmicp27 is not associated with its Cyclin–CDK inhibitory function(Supplementary Fig. S3).
All patients Met-dx No Met-dx
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Figure 2.The Kaplan–Meier curves of theLuminex xMAP Assay of the p27autoantibody in 233 osteosarcomaserum samples. Using a medianintensity cutoff, a higher level of thep27 autoantibody significantlycorrelated with the poor overallsurvival (top) and event-free survival(bottom) of the osteosarcomapatients (left). The middle and theright panels show the Kaplan–Meiercurves when stratified by the initialmetastasis status. Met-dx, metastasisat diagnosis.
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The effect of phosphorylation on the migration/invasionfunction of cytoplasmic p27
Phosphorylation plays an important role in regulating theprotein activity and subcellular location of p27 (22–24). To testwhether p27 phosphorylation has any effects on the migration/invasion function of p27 when it is localized in the cytoplasm ofosteosarcoma, we created three stably transfected mutants har-boring an alanine mutation in each of the three major phosphor-ylation sites, namely S10, T157, and T198, on the NES-p27CK-construct (Fig. 4A). Evidence that thesemutated phosphoproteinswere cytoplasmically localized are shown in Fig. 4B. The transwellassay showed that both S10A and T198Amutations abolished thepromoting effect of NES-p27CK- on cell migration andinvasion, while the migratory or invasive abilities of the T157Amutant were similar to the controls (Fig. 4C). The growth rates ofNES-p27CK- and all phosphomutants were similar to the
control cells, suggesting the changes of metastatic potential inthe NES-p27CK- cells and the phosphomutants were not due todifferences in growth rates (Supplementary Fig. S4). These resultsindicate that the S10 and T198 phosphorylations are not justimportant for subcellular localization of p27, but are also criticalfor the prometastatic function of cytoplasmic p27 in osteosarco-ma cells.
To investigate the prometastatic mechanism of cytoplasmicp27, we examined RHOA-GTPase activity, which has beenreported to be a downstream effector protein of cytoplasmicp27 for cell motility in other tumors (23, 25). As shown in Fig.4D, the RHOA activity, as measured by the level of GTP-boundRHOA, decreased significantly in NES-p27CK- cells comparedwith that in the empty vector control. Among the three phospho-mutants, we found that the S10A mutation restored the RHOAactivity, which could explain the observation that the cell motility
Figure 3.The p27 protein exhibits cytoplasmiclocalization in osteosarcoma cases andmetastatic cell lines. A,�10 IHC imagesof p27 staining in two osteosarcomatumor tissue microarrays (COG,Children's Oncology Group and USBioMax). B, a table to show the numberand percentage of nuclear andcytoplasmic staining results in thetissue microarrays. C, �40immunocytochemistry images showthat p27 was expressed higher in thecytoplasm of the three osteosarcomametastatic cell lines (LM7, K7M3, andDLM8) when compared with theparental cells (SaOS-2, K7, and Dunn).
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was lower in this mutant. As expected from the transwell assays,the RHOA activity of the T157 mutant was similar to that in theNES-p27CK- cells. Interestingly, despite the decrease of cellmigra-tion and invasion in the T198Amutant, it showed little inhibitoryeffect on the RHOA activity (Fig. 4D).
Knockdown of p27 decreases cell migration and invasion andrestores RHOA activity
Next, we tested whether cytoplasmic p27 was necessary for theprometastatic effect. Two shRNAs targeting p27 were stably trans-duced to highlymetastatic LM7 cells that express cytoplasmic p27(Fig. 3C). The p27 mRNA levels were significantly reduced in theshRNA-1 and shRNA-2 cells comparedwith the parental LM7 cells
and LM7 cells with control shRNAs (P < 0.05, Fig. 5A). The growthrates of the two p27-silenced mutants were significantly higherthan the scramble and parental controls (P < 0.05, Fig. 5B).Transwellmigration and invasion assays showed that cellmotilityand invasiveness of the p27-shRNA mutants were significantlylower than those in the scramble control (P < 0.05, Fig. 5C).Similar results were observed in a repeated experiment andusing atwo-dimensional migration assay (Supplementary Figs. S5 andS6). Furthermore, the RHOA activities of the p27-silencedmutants were higher than those in the controls (Fig. 5D). Takentogether, the results of the NES and shRNA mutants showed thatcytoplasmic p27 was both sufficient and necessary for promotingcell motility and invasiveness of osteosarcoma cells in vitro.
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Figure 4.Functional analysis of cytoplasmic expression of p27 in osteosarcoma. A, the NES constructs used in this study. All the NES-p27 constructs contain mutations in CDKbinding domain (CK-) as indicated by the asterisks. NES and EYFP denote Nuclear Export Sequence and enhanced yellow fluorescent protein, respectively.The mutations in S10A, T157A, and T198A are highlighted in green. B, the fluorescent images (�40) indicate the subcellular localization of the empty vector(EYFP), NES-p27CK-, and phosphomutant proteins in the stable clones. C, the results of the transwell cell migration and invasion assays indicate that the number ofmigrated and invaded cells in the NES-p27CK- and T157A cells were significantly higher than those in the empty vector control (� , P < 0.05), as well as in the S10Aand T198A mutants. Migrated and invaded cells were quantified by counting stained cells in three independent microscopic fields at 6 and 24 hours, respectively. Thestaining was done with Cell Staining Solution included in the Cell Migration and Invasion Kit. Error bars and asterisks represent SDs and statistical significance(� , P < 0.05), respectively. D, the RHOA-GTPase activity assay indicates the effects of cytoplasmic p27 and phosphomutations on the inhibition of the RHOA activity.
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However, the exact mechanism of how various p27 phosphoryla-tions affect this phenotype still needs further investigation.
p27mislocalization increases the formation of lungmetastasesin an animal model
To confirm cytoplasmic p27 can promote the development ofmetastasis, we injected SaOS-2 cells containing NES-p27CK- intoNOD/SCID–deficient mice via lateral tail vein. SaOS-2 cells withempty vector and LM7 cells were used as negative and positivecontrols, respectively. The result showed that only 1 of 5 mice
injected with cells containing the empty vector developed lungmetastasis, while 3 of 5 mice and 4 of 5 mice developed lungmetastases after injection with cells containing NES-p27CK- andhighly metastatic LM7 cells, respectively (Fig. 6A). In addition,the total number of lungmetastases inmice injectedwith theNES-p27CK- cells was significantly higher than the controls (P <0.05, Fig. 6A). The metastatic lung nodules in the mice withNES-p27CK- and LM7 cells were visible macroscopically (Fig.6B) and were confirmed under themicroscope by our pathologist(Fig. 6C). Additional images of other lung metastases are shown
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Figure 5.Functional analysis of shRNA-mediated gene silencing of p27 in osteosarcoma. A, p27 mRNA (top) and protein (bottom) expressions were measured inLM7 cells alone and with stably transfected Ctrl-shRNA (scramble sequence), p27 shRNA-1, and p27 shRNA-2. The p27 mRNA expression was normalized to 18Sribosomal RNA. B, the growth curves show that p27-shRNA silenced mutants grew faster than the parental and control cells. The plot is in a semi-log scale,where x-axis represents days of incubation. Asterisks denote statistical significancewhen comparing the number of cells in shRNAmutantswith LM7 and control cellsat each time point (�, P < 0.05). C, transwell migration and invasion assays show that the numbers of migrated and invaded cells in the p27-silcencedcells were significantly lower than the control cells. Error bars and asterisks represent SDs and statistical significance (� , P <0.05), respectively. D, the RHOA-GTPaseactivity assay shows that RHOA activation in the p27-silcenced cells was higher than those in the parental or control cells.
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in Supplementary Fig. S7. These results indicate that cytoplasmicp27 is sufficient to generate a higher number of lungmetastasis invivo, likely by increasing the motility and invasiveness of thetumor cells.
DiscussionIdentifying prognostic biomarkers and therapeutic targets for
osteosarcoma has been a difficult task and amajor obstacle in thefield. The reason, in part, is due to the highly rearranged genomesthat occur in osteosarcoma.Despite intensive studies fromvariousgroups, identifying potential biomarkers and targets from thegenomic space has not significantly improved the clinical out-comes of osteosarcoma patients (26–29). In this study, weshowed that higher levels of p27 autoantibody significantlycorrelated with both poor overall and event-free survival. Most
interestingly, patients with localized disease at diagnosis but highp27 autoantibody levels had poor overall and event-free survival.This suggests that thep27autoantibodymaybeuseful in detectinga subset of either localized patients with a higher clinical risk orpatients with mcirometastases too small to be detected by con-ventional imaging techniques at diagnosis. These patients, togeth-er with metastatic patients, could be treated more aggressively orwith an alternative approach to improve their outcomes.
Previous studies have shown that many different human can-cers exhibit reduced levels of p27 proteins (13, 30–35). Further-more, cytoplasmic mislocalization of p27 has been shown in anumber of human tumors, which include esophageal carcinomas(36), colorectal carcinoma (37), invasive and metastatic mela-nomas (16), andbreast carcinomas (38, 39). AKT1 activationwithp27 mislocalization was observed in approximately 40% ofprimary breast cancers (22, 38, 40). Prognostically, cytoplasmicp27 was associated with poor survival and predicts poor prog-nosis in breast carcinomas (22), acute myelogenous leukemia(41), pancreatic cancer (42), and ovarian carcinomas (43). OurTMA results show that a major proportion of osteosarcomapatients had cytoplasmic mislocalization of p27, indicating thatthey may have a higher metastatic potential at diagnosis. Thevariation in the percentage of cases with high and low staining ofcytoplasmic p27 between the two TMAsmay be due to differencesin fixation and decalcification procedures used in preparing theTMAs. Nonetheless, the large proportion of cases with p27 mis-localization (both low and high staining) fits very well withhistoric data that a large proportion of osteosarcoma patientsdevelop metastatic disease when only surgery is used (2). One ofour future goals is to examine whether p27 degradation ormislocalization in osteosarcoma confers a poorer outcome inosteosarcoma using clinically annotated cases.
Although p27 dysregulation has been described in osteoblastcells and osteosarcoma, little is known about its role inmetastasis(44, 45). The discovery of p27 as a TAA in high-risk osteosarcomais a novel finding. Our result shows for the first time thatcytoplasmic mislocalization of p27 is sufficient and necessary forpromoting metastatic potential in osteosarcoma. In addition, thep27 shRNAs reduced both nuclear and cytoplasmic expressions ofp27 in LM7. We believe that the increase of cell growth in theshRNA mutants was due to decreased nuclear expression of p27,while the decrease of cell motility and invasiveness was due to thedecreased cytoplasmic expression of p27. We further show thatosteosarcoma cells harboring cytoplasmic p27 increase the occur-rence of pulmonarymetastases in amousemodel, suggesting thatthe increase of migration and invasion in vitro can lead to thedevelopment of metastasis in vivo. Mechanistically, we demon-strate that the increase in cell motility and invasiveness is relatedto the decrease of RHOA-GTPase activity. These results are con-sistent with previous findings that p27 mislocalization can pro-mote cell motility in different cell types and metastasis in mel-anoma (16, 25, 46, 47). Thus, cytoplasmic p27 acquires onco-genic functions that are independent of cell-cycle regulation,potentially through effects on RHOA, in osteosarcoma and othercancers.
Three phosphosites were selected for mutation analysis tounderstand their contributions to the prometastatic phenotypeofmislocalized p27. The S10 residue, which is phosphorylated byhuman kinase-interacting stathmin (hKIS) and other kinases, isknown to control p27 nuclear export, XPO1 binding and proteindegradation (24). The T157 residue, which is phosphorylated by
A
B
C
LM7NES-p27CK-
Empty NES-p27CK- LM70
5
10
15 *N
umbe
r of m
etas
tase
s
NES-p27CK- LM7
Figure 6.Cytoplasmic p27 promotes the development ofmetastasis in an experimentalmouse model. A, the number of pulmonary metastases in mice injected withSaOS-2 containing NES-p27CK- was significantly increased when comparedwith the empty vector control (� , P < 0.05, one-tailed t test). The lines in theplot denote the average intensity values of the groups. B, macroscopicexamination of lungs derived from the mice injected with SaOS-2 cells withNES-p27CK- and LM7 cells. C, histologic examination of metastasesdeveloped in the lung sections dissected from SaOS-2 cells with NES-p27CK-(left) and LM7 cells (right) and after hematoxylin and eosin staining. Arrows,the metastatic nodules.
The Role of Mislocalized p27 in Osteosarcoma
www.aacrjournals.org Cancer Res; 76(13) July 1, 2016 4009
SGK1 and AKT1, is known to impair nuclear import, affectcytoplasmic localization, and increase cyclin D1–CDK4–p27complex formation (48). On the other hand, the T198 residuecan be phosphorylated by RPS6KA1 and AKT1. This residue canaffect cytoplasmic localization of the protein and protein stability(23). However, the functions of these phosphosites in relation tothe cytoplasmic metastatic function of p27 are largely unknown.Our results indicate that the S10Aor T198Amutation candecreasethe cell motility and invasiveness of the tumor cells harboringcytoplasmic p27. These results suggest that targeting S10 or T198phosphorylation may provide a new therapeutic approach formetastatic osteosarcoma cells harboring cytoplasmic p27. Ourresults also suggest that the effects of S10A and T198A mutationson RHOA are not the same. The S10 mutation increased RHOA-GTPase activity, while the T198A mutation had no effect onRHOA. The mutation of T198A may affect a downstream playerof the RHOA pathway or a RHOA-independent metastatic path-way, such as Rac (49). However, the T198 mutation has beenshown to affect RHOA binding and activity in breast cancer cells(23), suggesting that these phosphorylation sites onmislocalizedp27 may have cell-type–specific effects. This notion is supportedby previous studies of fibrosarcoma and trophoblast cells, wherecytoplasmic p27 inhibits migration by binding to stathmin(50, 51). Further investigations of the function of mislocalizedp27 in different tumor types may shed light on this issue. Inaddition, as human kinase interacting stathmin (hKIS) is knownto phosphorylate the S10 residue of p27, identifying a smallmolecule to inhibit hKISmay provide a new therapeutic targetingstrategy for metastatic osteosarcoma. Development of such atargeting strategy for cytoplasmic p27 may have a widespreadapplication in cancer research.
Although the relationship between p27 autoantibody levelsand p27 mislocalization in tumor cells is still not known, ourstudy has provided a solid foundation for future associationstudies between the p27 autoantibody and the subcellularlocalization of the protein in osteosarcoma or other cancers.It is possible that p27 might be expressed on the cancer cellsurface to elicit an antibody response. Alternatively, the immu-noreactivity of p27 may be due to release of the protein into thebloodstream following cancer cell death. Further characteriza-tion of the p27 mislocalization process will help us to under-stand how the body's immune system reacts to the protein,which may provide a new angle for immunotherapy in oste-osarcoma. In summary, our results suggest that further studiesof the role of p27 in osteosarcoma may lead to a betterprognostication method at diagnosis, so that high-risk osteo-sarcoma patients can be treated more appropriately to improve
their chance of survival. Our study also suggests that develop-ment of a novel targeted therapy approach, such as inhibitionof phosphorylation of the S10 residue, may provide an effectiveand rational treatment for patients who harbor the p27 mis-localization in the tumor.
Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.
Authors' ContributionsConception and design: C.C. Lau, J.M. Hicks, T.-K. ManDevelopment of methodology: Y. Li, M. Nakka, J.M. Hicks, T.-K. ManAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Y. Li, M. Nakka, C.C. Lau, D.A. Barkauskas, J.M. Hicks,T.-K. ManAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): Y. Li, M. Nakka, A.J. Kelly, M. Krailo, J.M. Hicks,T.-K. ManWriting, review, and/or revision of the manuscript: Y. Li, M. Nakka, C.C. Lau,D.A. Barkauskas, J.M. Hicks, T.-K. ManAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): C.C. Lau, M. KrailoStudy supervision: T.-K. Man
AcknowledgmentsThe authors thank the Bone SarcomaCommittee with COG for their support
of this study, Dr. Shixia Huang and Myra Costello with the Proteomics SharedResource at Baylor College ofMedicine for scanning the ProtoArrays,Drs. NancyWeigel and Neha Parikh for their technical assistance, and Dr. Xiao-Nan Li forhelp in the animal study. The authors also thank Drs. Lisa Wang, Lazlo Perlaky,Jeffery Murray, and William Myers for their assistance in collecting the plasmasamples as well as Carolyn Pena for proofreading the manuscript.
Grant SupportThis work was partly supported by the Multi-Investigator Research Award
(RP101335-P02/RP140022-P2) and Baylor College of Medicine Comprehen-sive Cancer Training Program (RP140102) by Cancer Prevention and ResearchInstitute of Texas, Bear Necessities Pediatric Cancer Foundation, and NationalInstitute of Children's Health and Development (R01 HD074553). This workwas also supported by the Chair's Grant U10 CA98543 and Human SpecimenBanking Grant U24 CA114766 of the Children's Oncology Group from theNational Cancer Institute, NIH (Bethesda, MD), and the WWWW (QuadW)Foundation, Inc. (www.QuadW.org). Additional support was provided by theNational Cancer Institute Cancer Center Support Grant (P30CA125123) of theProteomics Shared Resource at Baylor College of Medicine.
The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.
Received November 18, 2015; revised March 3, 2016; accepted March 31,2016; published OnlineFirst April 19, 2016.
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2016;76:4002-4011. Published OnlineFirst April 19, 2016.Cancer Res Yiting Li, Manjula Nakka, Aaron J. Kelly, et al. in Osteosarcomap27 Is a Candidate Prognostic Biomarker and Metastatic Promoter
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