The Functional Role And Molecule Mechanisms Of Imprinted Gene TSSC3in Human Osteosarcoma

Osteosarcoma (OS) is the most common primary bone malignancy of children andyoung adolescents, characterized by their high metastasis, lung metastasis is the main cause ofOS-related death. Metastasis usually occurs in the early phase of osteosarcoma, and exists in85%~90%patients without overt meatstasis at diagnosis. Recently, the neoadjuvantchemotherapy has obviously decreased lung meatstasis, while5-year survival rates hasreached60%~80%. However, the long-term survival rate have since plateaued,chemoresistance is the main factor to influence chemotherapeutic effects. Therefore,metastasis and chemoresistance control have signified the improvement of survival rate inOS.Cancer metastasis is a mutistep biological process. Tumor cell acquired the malignantpotential to resist anoikis after detachment from their primary site becomes the foundationand the key step of successful metastasis. Cell cycle arrest and the disorder of apoptoticpathway are one of the main chemoresistance mechanisms. Therefore, the investigationunderlying mechanisms rendering tumor cells resistant to chemotherapy and anoikis willprovide meaningful theoretical and experimental evidence for OS meatstasis andchemoresistance.Our previous study found that the imprinted gene TSSC3was involved in malignanttransformation of human osteoblast hFOB1.19cells. TSSC3is the only apoptosis relatedimprinted gene. However, only a few studies have reported on the relationship betweenTSSC3and human cancers, including the loss of TSSC3expression in complete hydatidiformmoles, Wilms' tumors and brain cancer, and no reports on the TSSC3expression pattern, itsfunctional role and related mechanisms in pathogenesis of osteosarcoma have been published.Therefore, the article was divided into3parts. The first section was to examine theexpression pattern of TSSC3in OS cell lines and tissue, then to clarify its functional role in vitro and in vivo. The second section was to verify that the apoptotic signaling pathway ofTSSC3in osteosarcoma and further to investigate that the way and the mechanisms involvedin the influence of TSSC3on chemotherapeutic effects in osteosarcoma cells. Further more,the third section was to discuss the role and the mechanism of TSSC3underlying anoikisresisitance, migration, invasion and metastasis of OS.A list of experiment data and main conclusions are as following1. TSSC3is lowly expressed in human osteosarcoma cell lines and tissues,suggesting low expression of TSSC3was associated with OS transformation andprogression.(1) TSSC3is lowly expressed in human osteosarcoma cell lines:The results as follows was obtained by qRT-PCR, RT-PCR and immunofluorescence:1) TSSC3expression in MTF osteoblasts was significantly lower than that in hFOB1.19osteoblasts, and the difference was statistically significant.2) TSSC3expression in hFOB1.19osteoblasts was higher than that in all osteosarcomacell lines, and it appeared to be much higher in HOS or MG63cell lines (from low-gradeosteosarcoma) than in U2OS or SaOS2cell lines (from high-grade osteosarcoma). TSSC3was localized primarily to the cytoplasm at least in these cells.(2) TSSC3is lowly expressed in human osteosarcoma tissues:The results as follows was obtained by the RT-PCR and χ2test analysis:1) The expression of TSSC3in24OS cases (positive rate:45.8%, mean absorbanceintensity:0.18±0.03) was significantly lower than that of the matched non-tumor tissues(positive rate:79.1%, mean absorbance intensity:0.52±0.05), and the difference wasstatistically significant.2) The deletion rate of TSSC3in OS was54.2%, obviously higher than that of thematched non-tumor tissues (20.8%), and the difference was statistically significant. However,no notable associations between the loss of TSSC3expression and clinicopathologicalparameters, such as gender, age and histopathological classification were observed.2. Inhibition of cell proliferation in vitro and tumor growth in vivo by TSSC3(1) We used GeneSwitch Mifepristone-Regulated Expression System to generateTSSC3-overexpressing stable SaOS2cells (overTSSC3), as well as we used pSilencer5.1 Retro Retroviral Expression System to generate TSSC3-knockdown stable SaOS2cells(siTSSC3).(2) Inhibition of cell proliferation in vitro by TSSC3:1) The CCK8colorimetry showed that overTSSC3cells grew more slowly than thecontrols (SaOS2and overCON), and the difference was more pronounced after day2; Asexpected, TSSC3knockdown increased the SaOS2cell growth, and the difference betweensiTSSC3cells and the controls became significant on day5, and the difference wasstatistically significant.2) The flat colony forming experiments showed that cells overexpressing TSSC3hadremarkably reduced the efficiency in forming large colonies (＞100cells) from17.75±0.01%to0.67±0.01%, compared than the control; conversely, TSSC3knockdown significantlyincreased the efficiency of colonies, specially for the number in forming large colonies from7±2to107±21, and the difference was statistically significant.3) The immunofluorescence analysis of Ki67expression showed that TSSC3upregulation markedly reduced Ki67expression from both the intensity and positive rate;whereas TSSC3downregulation obviously enhanced it compared with the control, and thedifference was statistically significant.4) The cell cycle analysis showed that compared with the controls, accumulation ofoverTSSC3cells in the G0/G1phase increased, whereas TSSC3downregulation reduced thissubpopulation.(3) Inhibition of tumor growth in vivo by TSSC3:1) NOD/SCID mouse xenograft experiments showed that as for overTSSC3, theincidence was2/11, the onset was28days and the mean volume for7weeks was15.94±9.28mm3; while for the control (SaOS2and overCON), the incidence was6/7and4/6,respectively, the onset were14days and16days, respectively, and the mean volume for7weeks were90.17±15.42mm3and159.75±73.65mm3, and the difference was statisticallysignificant. Conversely, as for siTSSC3, the incidence was10/10, the onset was6days andthe mean volume for5weeks was153.45±42.38mm3; while for overCON, the incidence was4/6, the onset was6days and the mean volume for5weeks was66.10±24.64mm3, and thedifference was statistically significant. The results suggested TSSC3obviously reduced thetumorigenesis of OS. 2) HE staining of specimens obtained from subcutaneoustumors showed that thesiTSSC3cell-derived and untransfected SaOS2cell-derived tumors contained numeroushyperchromatic nuclei with karyokinesis, pleomorphism, necrosis and high density ofmicrovessels. In tumors formed by overTSSC3cells, no necrosis or karyokinesis wasobserved, and they had low microvessel densities. These results indicated that tumors derivedfrom siTSSC3cells displayed osteosarcoma features closer to that of clinical tumors.3) IHC staining for TSSC3and Ki67expression showed that tumors from theoverTSSC3groups most highly expressed TSSC3, which were weakly detected in tumorsfrom untransfected SaOS2cells and were not detected in tumors from siTSSC3cells.Similarly, the frequency and intensity of Ki67expression in tumor tissues derived fromsiTSSC3cells were significantly higher than that from overTSSC3and untransfected SaOS2cells, and the difference was statistically significant, suggesting that there was negativeexpression correlation between TSSC3and Ki67.3. Activation of mitochondrial apoptosis pathway by TSSC3in vitro and in vivo(1) Induction of apoptosis by TSSC3in vitro:1) Cells overexpressing TSSC3appeared rounded up and showed slower growth,decreased vitality and more detached apoptotic cells compared with controls by lightmicroscopy. The overTSSC3cells also showed apoptotic features by transmission electronmicroscopy. Nuclear membranes were irregular, and the crescent nuclei became condensedand fragmented, with vacuoles in the cytoplasm and apoptotic bodies.These morphologicalfeatures suggested that TSSC3may promote cell apoptosis.2) The annexin V/PI analysis showed that relative to the controls, early (lower right) andlate apoptotic (upper right) cells were both prominently increased from5.70and1.39%to14.70and9.97%, respectively, in cells overexpressing TSSC3. In contrast, TSSC3knockdown decreased the rate of early apoptosis by4.04-fold and late apoptosis by4.32-fold.Additionally, the percentage of cells in the sub-G/0phase representative of apoptotic cells,assayed by PI staining, was also reduced along with TSSC3downregulation. Results of theTUNEL assay also showed that the expression of FITC-positive cells in overTSSC3cellswere significantly higher than that in controls.(2) Induction of apoptosis by TSSC3in vivo: The immunofluorescence analysis andTUNEL assays were performed on tumors developed from overCON and overTSSC3cells. Compared with the effect of overCON cells, the overTSSC3-derived tumors had obviouslyincreased TSSC3and TUNEL staining while TSSC3expression and DNA fragmentationwere co-localized, indicating that TSSC3overexpression induced apoptosis in tumor tissues.(3) Activation of mitochondrial apoptosis pathway by TSSC3in SaOS2cells:1) qRT-PCR analysis indicated that upregulation of TSSC3obviously increased theexpression of caspase-3and Cytc, as well as the Bax:Bcl-2ratio; as for death receptorsignaling molecules, Fas was reduced, whereas Flip was increased in overTSSC3cells,compared with that in overCON cells and the difference was statistically significant.Conversely, downregulation of TSSC3decreased caspase-3, Cytc, the Bax:Bcl-2ratio andFlip as well as increased Fas, and the difference was statistically significant. The resultssuggested activation of mitochondrial apoptosis pathway at the transcriptional level byTSSC3.2) The immunofluorescence assay showed that overTSSC3cells expressed increasedlevels of cleaved caspase-3and decreased procaspase-9or Bcl-2relative to the control. Incontrast, procaspase-9and Bcl-2were upregulated in siTSSC3cells. However, procaspase-8expression was not obviously different between overTSSC3and overCON cells. Western blotresults showed that upregulation of TSSC3obviously increased the expression of cleavedcaspase-3and Cytc, as well as decreased Fas; in contrast, siTSSC3cells expressed decreasedlevels of Cytc relative to the control, and the difference was statistically significant. Theresults suggested activation of mitochondrial apoptosis pathway at the protein level byTSSC3.3) JC-1staining results showed that overCON cells showed orange–red spots, indicatinghealthy mitochondria with an aggregated phenotype, whereas overTSSC3cells showeddiffuse green fluorescence approximate to the positive control because of decrease in ΔΨ,refiecting the transformation of JC-1aggregates into monomers when mitochondrialmembrane becomes depolarized. The overTSSC3cells had a significantly increased ratio ofgreen/red fluorescence intensities of JC-1from0.90±0.04to4.31±0.87and difference wasstatistically significant, indicating that TSSC3triggers a disruption of ΔΨ resulting inmembrane permeabilization and activation of mitochondrial apoptosis pathway.4. Enhanced sensitivity of SaOS2cells to chemotherapeutic agents by TSSC3through activation of mitochondrial apoptosis pathway. (1) Constitutive TSSC3expression clearly enhanced the sensitivity of osteosarcomacells to chemotherapeutic agents by increasing apoptosis:1) Annexin V/PI analysis showed that the intensity of annexin V staining was stronglyincreased in overTSSC3cells relative to the control, especially for late apoptotic cells. Incontrast, siTSSC3cells became more resistant to chemotherapeutic agents (cisplatin andepirubicin) as shown by annexin V/PI staining.2) The TUNEL assay also showed that the intensity of FITC staining of overTSSC3cells was significantly higher than that of overCON cells after chemotherapeutic agentsexposure.(2) Enhanced sensitivity of SaOS2cells to chemotherapeutic agents by TSSC3through activation of mitochondrial apoptosis pathway:1) The immunofluorescence assay showed that the overTSSC3cells displayed a higherfluorescence intensity of cleaved caspase-3than the controls after chemotherapeutic agentsexposure. Western blot analysis also showed an obvious increase in expression of Cytc,cleaved caspase-3and cleaved PARP in overTSSC3cells relative to those in overCON cells;in contrast, siTSSC3cells expressed decreased levels of Cytc relative to the control and thedifference was statistically significant.2) JC-1staining showed that overTSSC3cells after chemotherapeutic agents exposuresignificantly increased JC-1monomeric forms, which apparently improved the ratio ofgreen/red fluorescence intensities of JC-1compared with the control (from1.33±0.67to6.73±1.65), and the difference was statistically significant. Enhanced disruption of themitochondrial membrane potential combined with the increased expression of Cytc inoverTSSC3cells after chemotherapeutic agents exposure confirmed that TSSC3increasedcell apoptosis after chemotherapeutic agents exposure through activation of mitochondrialapoptosis pathway.5. TSSC3gradual downregulation was accompanied with gradual enhancedanoikis resistance.(1) Anoikis resistance model was succeeded to establish in OS:1) The anoikis resistant OS cells model establishment by sequential cycles of culturingunder adherent condition and under suspended condition: SaOS2cells were maintained undersuspended condition for3days, then cultured under adherent condition to form monolayers, then were suspended again for14days, finally cultured again under adherent condition toform monolayers, which means anoikis resistant SaOS2cells and designated as SaOSar.When suspended, individual cells rounded up, then formed small and loose aggregatesinitially, along with the time, formed large and compact aggregates.2) The anoikis resistant OS cells model identification: Annexin V/PI analysis showedthat the apoptotic rate (only for early apoptotic rate) of adherent SaOS2cell was4.34%, andreached to20.36%most rapidly when cell suspened for24h, then reached to33.76%and49.36%, respectively when cell suspened for3day and7day, while the apoptotic rateincreased slowly after cell suspened for7day, reached to55.28%when cell suspened for14day, thereafter no notable changes for apoptotic rate after cell suspened over14day wereobserved. Early and late apoptotic rate in SaOS2cells suspened for24h were14.39%and21.79%respectively, while in SaOSar cells suspened for24h were5.35%和10.07%respectively.(2) TSSC3gradual downregulation was accompanied with gradual enhancedanoikis resistance:The results as follows was obtained by qRT-PCR and immunofluorescence:1) TSSC3gradual downregulation was accompanied with gradual enhanced anoikisresistance: The expression of TSSC3in cells suspened for1day and7day decreased mostrapidly, both decreased65%compared with that of cells suspened for their prior time point,respectively. The expression of TSSC3in cells suspened for14day decreased95%relative tothat of adherent SaOS2cells and the difference was statistically significant.2) TSSC3mRNA and protein expression in SaOSar cells were downregulated56%and55%respectively, compared with that of SaOS2cells. The difference was statisticallysignificant.3) The expression of TSSC3in MTF was downregulated67%compared with that ofhFOB1.19osteoblast, while the expression of TSSC3in MTFar was downregulated15%compared with that of MTF. The difference was statistically significant.Taken together, The results above suggested that inhibiton of TSSC3was involved inanoikis resistance, therefore promoted OS progression.6. Induction of MET and inhibiton of migration, invasion, metastasis potential byTSSC3regulating the expression of EMT and metastasis related molecules (1) Inhibiton of migration by TSSC3: the scratch assay showed that the distance ofoverTSSC3and overTSSC3ar cells migrated into the scratch was significantly shorter thantheir control cells, respectively, while siTSSC3cell got the reverse results relative to thecontrol.(2) Inhibiton of invasion by TSSC3: the transwell invasion assay showed that the cellnumber in lower chamber of overTSSC3and overTSSC3ar was8±2and40±6, respectively,significantly lower than their control (overCON:105±16and overCONar:179±26), and thedifference was statistically significant. While the cell number in lower chamber of siTSSC3was158±11much more than that of siCON (30±5), and the difference was statisticallysignificant.(3) The influence of TSSC3on EMT and metastasis related molecules in OS:The results as follows was obtained by qRT-PCR:1)TSSC3overexpression upregulated E-cadherin, a epithelial marker anddownregulated N-cadherin, Vimentin and Fibronectin, the mesenchymal markers, alsodownregulated Twist, Snail, Slug, Smad1, Smad4, ZEB1and ZEB2, the EMT relatedtranscription factors. The difference was statistically significant, suggesting that TSSC3overexpression made OS cells MET.2) TSSC3overexpression decreased CD44s, CXCR4, the metastasis related genes andMMP2, MMP7, MMP9, MMP14, the metalloproteases family. We also got the same resultsin overTSSC3ar cells. The difference was statistically significant. Conversely, TSSC3downregulation increased the expression of CD44s, CXCR4, MMP7, and MMP14. Thedifference was statistically significant, suggesting that TSSC3inhibited the invasion andmetastasis potential at the transcriptional level in OS.7. Enhanced anoikis and sensitivity of anoikis cells to chemotherapeutic agents byTSSC3through inhibition of integrin mediated PI3K/Akt anoikis resistance signalingpathways in OS(1) TSSC3increased anoikis by mitochondrial pathway through inhibition ofintegrin mediated PI3K/Akt anoikis resistance signaling pathways in OS:1) Compared with the control, overTSSC3cells suspended for3day formed small andloose aggregates, also displayed more detached apoptotic cells by light microscopy. AnnexinV/PI analysis for transfectants suspended for3day showed that the apoptotic rate (early) of SaOS2, overCON and overTSSC3was31.20%,31.00%and47.90%, respectively. Asexpected, siTSSC3cells suspended for1day formed large and compact aggregates, lessscattered apoptotic cells relative to the control by light microscopy. Annexin V/PI analysis fortransfectants suspended for1day showed that the apoptotic rate (early) of siCON andsiTSSC3was22.70%and3.52%, respectively, furthermore the anoikis rate of siTSSC3wascloser to that of SaOSar(8.55%, indicating that TSSC3increased OS anoikis.2) JC-1flow cytometric detection showed that after cells suspended for3day, the JC-1monomeric ratio of overTSSC3was36.5%, obviously higher than that of overCON (14.4%)and SaOS2(13.9%). Western blot analysis showed that the Cytc expression in the cytosolicfraction of overTSSC3was much higher than that of overCON and the difference wasstatistically significant, suggesting that a disruption of ΔΨ and enhanced release of Cytc byTSSC3.3) qPCR analysis showed that TSSC3overexpression negatively regulated the deathreceptor signaling by reducing Fas and inducing Flip. TSSC3upregulation increased theexpression of Puma, Noxa, Bim, Bax, Bak, Cytc, Apaf-1and caspase-3, simultaneouslydecreased the expression of Bcl-2and Bcl-xL, suggesting promoting anoikis at every step ofthe mitochondrial signaling pathway. The difference was statistically significant. In contrast,TSSC3downregulation decreased the expression of Puma, Noxa, Bax, Cytc and caspase-3,simultaneously increased the expression of Bcl-2. The difference was statistically significant.In addition, the ratio of Bax/Bcl-2in overTSSC33day cells was5.97, while that of overCON3day was0.40, indicating that TSSC3enhanced the sensitivity of OS cells to anoikis by themitochondrial signaling pathway.4) qPCR analysis showed that TSSC3upregulation decreased integrinβ4, Ezrin andincreased integrinα2, integrinβ1, furthermore TSSC3positively related to Caveolin-1. Thedifference was statistically significant. The results demonstrated that TSSC3regulated severalmolecules of the OS anoikis resistance signaling pathways.5) Western blot analysis showed that TSSC3overexpression sequently resulted in cellssuspended a decrease of Src phosphorylation, inhibition of PI3K/Akt anoikis resistancesignaling pathways by a decrease of Akt phosphorylation and increase of Akt, activation ofproapoptotic Bad by dephosphorylation, then enhanced the release of Cytc to the cytosol,finally activated caspase-3by cleaving. The results demonstrated that TSSC3increased anoikis by mitochondrial pathway through inhibition of integrin mediated PI3K/Akt anoikisresistance signaling pathways in OS.(2) TSSC3enhanced the sensitivity of anoikis cells to chemotherapeutic agentsthrough inhibition of integrin mediated PI3K/Akt anoikis resistance signaling pathwaysin OS:1) Annexin V/PI analysis for transfectants exposure to3μg/ml cisplatin suspended for3day showed that the apoptotic rate (early) of SaOS2, overCON and overTSSC3was59.90%,56.90%and78.60%, respectively, indicating that TSSC3upregulation notably enhanced thesensitivity of anoikis cells to chemotherapeutic agents.2)JC-1flow cytometric detection for transfectants exposure to3μg/ml cisplatinsuspended for3day showed that the ratio of JC-1monomers in SaOS2, overCON andoverTSSC3was42.8%,35.6%and63.4%, respectively, indicating that TSSC3enhanced thesensitivity of anoikis cells to chemotherapeutic agents by mitochondrial signaling pathway.3)Western blot analysis for transfectants exposure to3μg/ml cisplatin suspended for3day showed that TSSC3overexpression sequently resulted in Src and Akt dephosphorylation,then increased the expression of cleaved caspase-3, demonstrating that TSSC3enhanced thesensitivity of anoikis cells to chemotherapeutic agents through inhibition of integrin mediatedPI3K/Akt anoikis resistance signaling pathways in OS.In conclusion, the above data suggested that:1. TSSC3is lowly expressed in human osteosarcoma. It plays a tumor suppressor rolethrough growth inhibition, apoptosis induction, and the underlying mechanisms may enhancethe basic apoptosis and chemotherapeutic effects by induction of mitochondrial apoptosispathway in human osteosarcoma.2. Induction of MET and inhibiton of migration, invasion, metastasis potential byTSSC3regulating the expression of EMT and metastasis related molecules.3. TSSC3gradual downregulation was accompanied with gradual enhanced anoikisresistance. The underlying mechanisms may enhances anoikis and sensitivity of anoikis cellsto chemotherapeutic agents by through inhibition of integrin protein transduction kinase Srcmediated PI3K/Akt anoikis resistance signaling pathways in human OS.In summary, Our study reported for the first time that the expression pattern, thefounctional role and the underlying mechanisms of the imprinted gene TSSC3, moreover to discuss the mechanisms of anoikis resistance and chemoresistance. We suggested that TSSC3played a tumor suppressor role through growth inhibition, apoptosis induction (includinganoikis), and induction of MET and inhibiton of migration, invasion, metastasis potential byTSSC3regulating the expression of EMT and metastasis related molecules. The underlyingmechanisms may promote apoptosis and anoikis through induction of mitochondrial apoptosispathway and inhibition of integrin protein transduction kinase Src mediated PI3K/Akt anoikisresistance signaling pathways, respectively. Taken together, TSSC3played an important roleduring OS development and progression, and our study provide the experimental andtheoretical foundation for TSSC3targeted therapy and epigenetic regulation in OS.