The Role Of EFEMP2 In Human Osteosarcoma Invasion And Metastasis

Background:Osteosarcoma is a malignant tumor originating from mesenchymal tissues.It commonly arises in the distal femur and proximal tibia;it is highly malignant and has poor prognosis.Its clinical characteristics include early pulmonary metastasis,high disability rate,and high recurrence rate.Osteosarcoma treatment in 1970 was limited to amputation and radiotherapy,and most patients died of lung metastasis within two years with a 5-year survival rate of only 10%-20%.In the last 30 years,the wide application of combinatorial chemotherapy and active surgical resection has greatly improved the 5-year survival rate for osteosarcoma.However,though chemotherapy plays a pivotal role in osteosarcoma treatment,its overall efficacy is still around 60%.Thus;osteosarcoma therapy is now in a phase,where it is necessary to explore osteosarcoma markers to determine its biological characteristics and prognosis,and to determine novel clinical therapeutic targets for effective molecular therapy.EFEMP2(epidermalgrowth factor-containing fibulin-like extracellular matrix protein 2,also known as fibulin-4,MBP1,UPH1)is a member of the fibulin family of glycoproteins.Fibulin is a newly discovered ECM protein family,which currently includes seven members,namely,fbulin-1,-2,-3,-4,-5,-6,and-7.It is mainly involved inthe formation and stability of membranes,elastic fibers,and loose connective tissues.EFEMP2 is widely distributed in human tissues,and is closely associated with the basement membrane and the extracellular matrix of elastic fibers.EFEMP2 plays an important role in the stabilization of extracellular matrix structure.Fibulin family members are involved in the processes of cell morphologymaintenance,growth,adhesion,and movement,and are closely associated with the development of multiple tumors.Fibulin-1 expression is enhanced in ovarian cancer and breast cancer and promotes tumor development.However,in hepatocellular carcinoma,gastric cancer,and prostate cancer,fibulin-1 could suppress tumor growth,induce apoptosis,and inhibit tumor angiogenesis.Fibulin-2 as a tumor suppressor gene,could inhibit tumor cell growth and invasion in HCC and breast cancer,and could inhibit angiogenesis.Fibulin-3 is reported to be highly expressed in pancreatic cancer,cervical cancer,and glioma,and promotes tumor development.However,in nasopharyngeal carcinoma,breast cancer,and glioblastoma,fibulin-3 is downregulated,and can inhibit tumor cell proliferation,invasion,and metastasis.Fibulin-5 is widely considered a tumor suppressor gene that inhibits tumor growth,invasion,and angiogenesis in the development of most tumors.Fibulin-3,-4,-5 show high homology in structure and all of them have an epidermal growth factor(EGF like)region that binds to a calcium ion.At present,studies on the relationship between tumors and EFEMP2 are still in the initial stage,and few related studies are available.The EFEMP2 gene is located on chromosome 11q13,and the genes at end of this chromosome,including CCND1,FGF3,EMS1,FGF4,and FOSL1,often act as oncogenes in many cancers.Moreover,this region is also prone to chromosome translocation and rearrangement in tumor development.Therefore,EFEMP2 may play an important role in tumorigenesis.In this study,we investigated the function of EFEMP2 in human osteosarcoma invasion and metastasis.Objective1.To explore EFEMP2 in the tissue of osteosarcoma expression and its correlation with clinical significance.2.To Explore EFEMP2 impact on osteosarcoma invasion and migration ability.3.To Explore EFEMP2 impact on EMT in osteosarcoma cells.4.To Study the effects of EFEMP2 in osteosarcoma cell invasion and migration of related signaling pathway.Materialsand MethodsCell cultureOsteosarcoma cell lines(MG63 and U-20S)and the normal osteoblastic cell line,hFOB,were obtained from the Shanghai Institute for Biological Sciences,ChineseAcademy of Sciences.All cell lines were cultured in complete growth media containing DMEM/F12(Gibco BRL,Rockville,MD)supplemented with 10%FBS(Gibco BRL,Rockville,MD)and 1%antibiotics,and were maintained at 37℃in an incubator with 5%CO2.Isolation of MG63 cell subclonesMG63 cells at the logarithmic growth phase were collected and diluted to about 10 cells/ml,and were then seeded into a 96-well plate with 0.1 ml/well.Thus,as far as possible,there was only one cell in each well.After 1 week at 37℃ with 5%CO2,a single clone from one well was selected and cultured as a subclone.Using acell electrophoretic instrument(DY-100,from College of Life Science,Shandong University,China),the electrophoretic migration rates of these subclones were measured individually.Moreover,the invasive and proliferative abilities of the highly invasive and low invasive subclones were analyzed by in vitro and in vivo functional assays.All data are expressed as mean ± standard error(SE).Osteosarcoma tissue samplesWith informed consent from patients,260 specimens were obtained from the Department of Pathology,Shandong Qilu Hospital and Shandong Province Hospital.None of these patients had undergone preoperative radiation or chemotherapy.All patients received regular follow-up.During the study period,contact with 15 patients was lost and 36 patients died.The follow-up period was from 2005 to 2014.This study was approved by the Institutional Medical Ethics Committee of Shandong University.Immunohistochemistry(IHC)and immunocytochemistry(ICC)For IHC,paraffin-embedded sections were dewaxed in xylene and rehydrated in ethanol.Heat-induced epitope retrieval was then performed using a pressure cooker in 0.01M citrate buffer at pH 6.0.The sections were incubated for 3 min after the cooker reached full pressure.For ICC,cells at 75-80%confluency were seeded into a cell culture dish containing coverslips.After 24 h,the coverslips were harvested,washed thrice with PBS,and fixed in 95%ethanol for 30 min.According to the procedure of the Streptavidin-Peroxidase Detection Kit(ZSGB-BIO),the following steps were common for both IHC and ICC.All sections and coverslips were treated with 3%hydrogen peroxide(H2O2)and goat serum for 30 min sequentially,to block endogenous peroxidase and the non-specific binding sites,and then incubated with rabbit anti-human EFEMP2 antibodies(ab125073,Abcam)at working dilutions of 1:200 overnight at 4℃.The sections and coverslips were then incubated with the anti-rabbit biotin-conjugated secondary antibody for 30 min at room temperature,stained for 1-5 min with the enzyme substrate 3 ’,3-diaminobenzidine tetrahydrochloride(DAB,Sigma-Aldrich,St.Louis,MO,USA),and counterstained for 5 min with hematoxylin.Paraffin-embedded sections of human ovarian cancer specimens(EFEMP2-positive)were used as positive controls[26],and the negative control was obtained by replacing the primary antibody with PBS.Brown granules in the cytoplasm or stroma were considered as positive EFEMP2 expression.Immunohistochemistry(IHC)and immunocytochemistry(ICC)analysisTo assess EFEMP2 expression in IHC and ICC experiments,the stained cell percentage and staining intensity were measured.The percentage of positively stained cells was scored from 0 to 4(score 0,0%cells stained;score 1,1-25%;score 2,26-50%;score 3,51-75%;or score 4,76-100%),whereas the staining intensity of EFEMP2 was scored as 0(negative),1(weak),2(moderate),or 3(strong).Taken together,the intensity and percentage scores made up the final staining score(0-7),and the scores of 0,1-3,4-5,and 6-7 were converted into sum indices-,+,++,and +++,respectively.For statistical analysis,low EFEMP2 expression was defined as-or +,whereas high EFEMP2 expression was indicated by ++ or +++.Each tissue section was independently analyzed by three pathologists.Lentivirus transfectionThe pLVX-EFEMP2 vector and EFEMP2 shRNA as well as a negative control were obtained from GeneChem Inc.(Shanghai,China).According to the manufacturer’ s instructions,prior to viral infection,target cells were plated at 0.5× 105 cells per well in a 24-well plate and incubated at 37℃ in a CO2 incubator for 24 h or 48 h until the cells were 60-80%confluent.The cells were then infected by adding the viral stock at a multiplicity of infection(MOI)of 100.In addition,a transduction well with negative control viral constructs was included.The cells were incubated overnight at 37℃ with 5%CO2;the transfection mixture was then replaced with normal complete growth medium to avoid cell toxicity.At the end of 48 h of incubation,the cells were assessed by fluorescence microscopy.The transfection efficiency was confirmed by western blotting,real-time quantitative RT-PCR,and immunocytochemistry(ICC).The siRNA sequence for EFEMP2 was:sense 5 ’-CAGAUCCGUGCUGGAAACUCG-3’ and antisense 3’-AGUUUCCAGCACGGAUCUGAA-5’.The negative control(scrambled order)was:sense 5 ’-AUCGACGAUCCCUAUUGGCGU-3 ’ and antisense 3 ’-GCC AAUAGGGAUCGUCGAUCU-5’.Quantitative real-time-polymerase chain reaction(qRT-PCR)TRIzol(?)Reagent(AmbionTM)was used to isolate total RNA from cell and tissue samples.Reverse Transcription was carried out with TaqMan(?)Reverse Transcription Reagents(Applied Biosystems Inc.;Thermo Fisher Scientific,Inc.).The procedure was based on the protocol provided by Invitrogen.The real-time PCR mixture volume was 25 μlincluding 12.5 μl SYBR Green Mix(Power SYBR(?)Green PCR Master Mix,Applied Biosystems Inc.),0.2 μl cDNA,1 μl primer pair mix(5 pmol/μl each primer),and 11.3 μl DNAse/RNAse-free H2O.The experiment was then set up with the following PCR program on ABI Prism SDS 7000(Applied Biosystems Inc.;Thermo Fisher Scientific,Inc.):50℃ for 2 min,1 cycle;95℃ for 10 min,1 cycle;40 cycles of 95 ℃ for 15 s->60 ℃ for 30 s->72℃ for 30 s;72℃ 10 min,1 cycle.Finally,the results were analyzed with SDS 7000 software.Specific primers were designed by LightCycler(?)Probe Design software(Roche Diagnostics,Basel,Switzerland)and were synthesized by Takara Biotechnology Co.,Ltd.Western blotCells were lysed on ice in RIPA(radioimmunoprecipitation assay)buffer with 1 mM PMSF(phenylmethylsulfonyl fluoride).From the cell lysate,40 μg of total protein was loaded into the wells of the SDS-PAGE(sodium dodecyl sulfate polyacrylamide gel electrophoresis)gel,along with a molecular weight marker.Electrophoresis was carried out for 1-2 h at 100 V,followed by transfer to PVDF(polyvinyl difluoride)membranes,which were then blocked with 5%BSA(bovine serum albumin).The membranes were then incubated overnight at 4℃ with primary antibodies(E-cadherin sc-8426,N-cadherin sc-7939,Vimentin sc-6260,Santa Cruz;EFEMP2 ab125073,Snail ab167609,Slug ab27568,Twist ab50887,PI3K ab 86714,p-PI3K ab 182651,AKT ab 8805,P-AKT 38449,mTOR ab 32028,p-mTOR ab 109268,Abcam)at working dilutions of 1:1000.After washing the membranes thrice with TBST for 5 min each,the membranes were incubated with conjugated secondary antibody diluted to 1:1000,at room temperature for 1 h.Blots were developed using the enhanced chemiluminescence method(PierceTM ECL Western Blotting Substrate;Thermo Fisher Scientific,Inc.).Growth curvesCells at the logarithmic phase were collected,seeded into the wells of a 24-well plate(1×104 cells/well)and cultured at 37℃ with 5%CO2.Three wells were harvested every day and the cells were counted and averaged.Growth curves were then plotted according to the average cell counts of 7 consecutive days.Soft agar colony formation assayDMEM with 20%FBS(1.5 ml)mixed with 1.5 ml of 1.2%agar was added to 3.5 cm dishes and solidified for the bottom layer.Next,1.5 ml of 0.7%agar was mixed with 1.5 ml DMEM(20%FBS)and 200 μl of cell suspension(containing 600 cells),and was immediately added to the above culture dishes.All the dishes were incubated for 2 weeks at 37℃ with 5%CO2.The assay was repeated in triplicate.Under an inverted microscope(Nikon Eclipse),the dish was divided into quadrants and in each quadrant,colonies with diameters of more than 2 mm were counted and the average was calculated.All the data are expressed as mean ± SE.Cell invasion assay and migration assayThe in vitro Matrigel invasion assay was performed as described previously[28].The polyvinylpyrrolidone-free polycarbonate(PVPF)membrane of Boyden chambers(BD Biosciences,Bedford,MA)were coated with 50 μl of Matrigel 1:3 diluted with serum-free media.Cell suspensions in volumes of 200 μl(2 ×105 cells)were seeded into the upper chambers,and 600 μl of serum-free culture supernatant of NIH3T3 cells was added to the lower chamber as a chemotactic faotor.The Boyden chambers were then incubated at 37℃ for 24 h.The non-invading cells on the upper surface of the membrane were removed,and the cells on the lower surface were fixed with 4%paraformaldehyde,stained with hematoxylin and eosin(H&E),and counted in five random high-power fields(HPF)under an inverted microscope.The cell migration assay was simultaneously performed with the above steps,without the Matrigel coating on the membrane and an incubation time of only 12h.The cell invasion and migration assays were both repeated in triplicate.All data are expressed as mean ± SE.Tumor xenografts in nude miceBALB/C-nu/nu nude mice were purchased from the NationalResourceCenter for Rodent Laboratory Animal of China.Each group included 5 nude mice,each of which was inoculated subcutaneously with 5.0 × 106 cells.The mice were maintained in a sterile animal facility and monitored daily for tumor growth.Every week,the tumor volumes were measured using vernier calipers,and calculated according to the formula,V= length x width 2 x 0.25.After 2 months,the mice were sacrificed and the tumors were dissected and examined histologically.All data are expressed as mean ± SE.Statistical analysisIHC data were analyzed using a X2 test.A two-tailed t-test was used to compare the means between two sets,and a one-way analysis of variance was used to compare the means among three groups.By the Kaplan-Meier method and the log-rank test,survival curve analysis was performed to study the relationship between EFEMP2 and the prognosis of patients with osteosarcoma.The data were analyzed with SPSS software version 13.0(SPSS Inc.,Chicago,IL,USA).P<0.05(two-sided)was considered statistically significant.ResultsEFEMP2 expression in human osteosarcoma tissuesHigh EFEMP2 protein expression was detected in osteosarcoma tissues,mainly in the stroma and in the osteosarcoma cell cytoplasm.However,EFEMP2 immunoreactivity was very low in most normal tissues(Fig 1).Moreover,high EFEMP2 expression was positively associated with low differentiation and lymph node metastasis(Table 1).Similar results were also observed in the qRT-PCR experiment.High EFEMP2 mRNA expression was observed in osteosarcoma tissues and was correlated with low tumor differentiation and positive nodal metastasis(Table 2).Survival analysis was performed by Kaplan-Meier analysis.This result showed that patients with high EFEMP2 expression had poorer prognosis than those with low EFEMP2 expression(log rank,P<0.01;Fig 1E).Establishment of highly invasive and low invasive subclonesUsing the single cell cloning technique,31 subclones were obtained from MG63 cells.The subclone MG63-1,which had the highest migration rate(19.59 ±0.56 μm/s)showed higher proliferative and invasive abilities,compared to the subclone MG63-31,which showed the lowest migration rate(7.68 ± 0.13μm/s).In vivo,the subcutaneous tumor formation rate for the highly invasive subclone group was 100%,and was accompanied by rapid tumor growth.However,the tumor formation rate of the low invasive subclone group was only about 50%,with very slow tumor growth.The tumor volume for MG63-1 was 479.82 ± 34.31 mm3,much larger than that formed by MG63-31(35.91 ± 3.73 mm3,P<0.01).These results are shown in Fig 2.Different proliferation and invasion abilities of human osteosarcoma cell lines and the normalosteoblastic cell lineCompared to the normalosteoblastic cell line hFOB,the human osteosarcoma cell lines U-20S and MG63 showed stronger proliferative abilities(Fig 3A).In the soft agar colony formation assay,the number of colonies formed by MG63 and U-20S was also significantly greater than that formed by hFOB(Fig 3B,C).In the cell migration and Matrigel invasion assays,the average counts of migrating and invading MG63 and U-20S cells were both much higher than those of hFOB(Fig 3D,E,F).Upon comparing the two osteosarcoma cell lines,we found that MG63 had stronger proliferation and invasion abilities than those of U-20S.EFEMP2 expression in human osteosarcoma cell lines and in differently invasive subclonesAs shown in Fig 4,EFEMP2 was very weakly expressed in the normalosteoblastic cell line hFOB compared to in the human osteosarcoma cell lines MG63 and U-20S.The strongest EFEMP2 expression was detected in MG63,which showed the highest proliferation and invasion abilities.Similar results were also observed upon comparing subclones with differing invasive abilities.Compared with the low invasive subclone MG63-31,high EFEMP2 expression was detected in the highly invasive subclone MG63-1.These results indicate that high EFEMP2 expression might be positively associated with the proliferative and invasive abilities of osteosarcoma cells.Identification of downregulated and upregulated EFEMP2 expression in lentivirus transfection systemsTo further investigate the potential role of EFEMP2 inosteosarcoma cell proliferation and invasion,we decreased the expression of EFEMP2 in the highly invasive subclone,MG63-1,and increased EFEMP2 expression in the low invasive subclone MG63-31 by lentivirus transfection.After viral infection,real-time q-RT-PCR,western blotting,and ICC were used to confirm the altered expression of EFEMP2 at both mRNA and protein levels,indicating the high efficiency of the lentivirus transfections(Fig 5).Effect of EFEMP2 knockdown and overexpression on osteosarcoma cell proliferationDownregulated EFEMP2 markedly inhibited cell proliferation of the highly invasive subclone MG63-1,whereas upregulated EFEMP2 significantly promoted cell proliferation of the low invasive subclone MG63-31(Fig 6A).In the soft agar colony formation assay,the colony forming efficiencies of EFEMP2-silenced cells were decreased,and conversely,upregulation of EFEMP2 could increase the colony forming efficiency of the low invasive subclone(Fig 6B,C).No significant differences were observed in the uninfected and negative control groups.Effect of EFEMP2 knockdown and overexpression onosteosarcomacell migration and invasionAs shown in Fig 7,EFEMP2 knockdown inhibited osteosarcoma cell invasion and migration.The average counts of migrating and invading EFEMP2 shRNA infected cells were much lower than those of the negative controls and uninfected groups(P<0.05).Meanwhile,EFEMP2 overexpression promoted the invasion and migration of osteosarcoma cells.The average counts of migrating and invading pLVX-EFEMP2 infected cells were much higher than those of the negative controls and the uninfected groups(P<0.05).There were no significant differences between the negative controls and uninfected groups.Effects of EFEMP2 knockdown and overexpression on tumor growth in a xenograft modelThe EFEMP2 shRNA infected cells,pLVX-EFEMP2 infected cells,negative control MG63-1,and negative control MG63-31 were each inoculated subcutaneously in 5 nude mice respectively.The tumor formation rate of the negative control MG63-1 was 100%,whereas the tumor formation rate in the EFEMP2 shRNA infected groupwas only 60%.Moreover,the average volumes of the tumors formed in the EFEMP2 shRNA infected group were much lower than those formed by the negative control MG63-1.EFEMP2 knockdown inhibited tumor formation in nude mice.Simultaneously,EFEMP2 overexpression promoted tumor growth in nude mice.The tumor formation rate of pLVX-EFEMP2 infected cells was 100%,whereas the tumor formation rate of the negative control MG63-31 was only 40%.Moreover,the average volumes of the tumors formed by pLVX-EFEMP2 infected cells were much higher than those formed by the negative control MG63-31(Fig 8).Effects of EFEMP2 on key EMT genesEMT is significantly associated with the invasion and metastasis of various cancers.Therefore,we wondered if EFEMP2 knockdown and upregulation affected several key EMT genes including E-cadherin,N-cadherin,vimentin,Snail,Slug,and Twist,using real-time q-RT-PCR and western blot.The results showed that EFEMP2 knockdown significantly inhibited the process of EMT,accompanied with increased E-cadherin expression and decreased expression of N-cadherin,vimentin,Snail,Slug,and Twist;in contrast,EFEMP2 upregulation could induce EMT,with decreased E-cadherin expression,and increased expression of N-cadherin,vimentin,Snail,Slug,and Twist(Fig 9).In conclusion,EFEMP2 could promote the invasion and metastasis of osteosarcoma cells by inducing the process of EMT.Effects of EFEMP2 on the PI3K/AKT signaling pathwayPI3K-AKT-mTOR is one of the major signaling pathways identified as important in cancer.We determined the expression of the main signaling molecules PI3K,AKT,and mTOR,and the changes in their phosphorylation levels.The results revealed that EFEMP2 knockdown could reduce the PI3K,AKT,and mTOR phosphorylation levels;in contrast,EFEMP2 upregulation could increase their phosphorylation levels.Usingthe PI3K/AKTsignaling pathway inhibitor LY294002(5,10,and 20 μmol/L),we treated the pLVX-EFEMP2-infected cells for 48 h.We found that the inhibitor could significantly inhibit the PI3K/AKT pathway,and EMT,both of which were activated by EFEMP2 upregulation.Concentration-dependent increases were observed with increasing doses(Fig 10).In summary,EFEMP2 could induce EMT to promote osteosarcoma invasion and metastasis by the PI3K/AKTsignaling pathway.Conclusion:1.High EFEMP2 expression was associated with poor prognosis in human osteosarcoma and the malignant phenotype of osteosarcoma cells.2.EFEMP2 has the ability to promote proliferation,invasion,and metastasis of osteosarcoma cells.3.EFEMP2 could induce EMT via the PI3K/AKT/mTOR signaling pathway.