The Effect And Molecular Mechanisms Of TSSC3-induced Autophagy To Impede Tumorigenesis And Metastasis Of Osteosarcoma

Osteosarcoma is the most frequent type of primary malignancy of bone and is also the second most common cause of cancer-related death in young adolescents and children due to the highly malignant with highly aggressive behavior.Over the last two or three decades,the pace of development of treatments for osteosarcoma tends has been slow and the five-year or longer survival rate remains.Autophagy is an essential and highly conserved cellular process that targets selective proteins and abnormal organelles for lysosomal degradation.The role of autophagy in cancer is completed and controversial.To find out the effect of autophagy on different stage of osteosarcoma and the regulator of autophagy in osteosarcoma facilitating us to explore a new molecular target for osteosarcoma treatment.Previous study revealed that TSSC3 expression is downregulated during malignant transforming hFOB cell line and played as a tumor suppressor in osteosarcoma.However,the underlying mechanism by which TSSC3 suppresses the tumorigenesis and metastasis remains unclear.Recently,PHLDA1,the homologous gene of TSSC3 has been demonstrated to trigger autophagy,suggesting TSSC3 might have the ability to regulate autophagy.Moreover,the PI3K/Akt/mTOR signaling pathway,which is a classical pathway involved in the regulation of autophagy in several human tumors cells and can be activated by the Srcfamily kinases.Previous studies found that TSSC3 could inhibit thephosphorylation of Src in osteosarcoma cells;therefore,we speculated that autophagy might be involved in the anti-tumor effect of TSSC3 connected by Src.Thus,this study was aim to understand the mechanism by which TSSC3 suppresses the tumorigenesis and metastasis.These potential findings may be important for exploring the new molecular targets to treat osteosarcoma.As mentioned above,this study was divided into three parts.The first section was to investigate correlation of TSSC3 and autophgy in OS cell lines and human OS tissues and to find out the molecular mechanisms involved in the TSSC3 on inducing autophagy.The second section was to clarify the functional role of autophagy on tumorigenesis and metastasis of osteosarcoma in vitro and in vivo.Furthermore,we investigated the correlation between TSSC3,ATG5,and P62 expression and clinicopathological features of osteosarcoma and gave a checkout of their prognostic value.The main methods are as following: We investigated the dynamic expression patterns of TSSC3 and autophagy-related proteins(autophagy related 5(ATG5)and P62)in 33 human benign bone tumors and 58 osteosarcoma tissues using immunohistochemistry.We further investigated the correlations between TSSC3 and autophagy in osteosarcoma using western blotting and transmission electronic microscopy.CCK-8,Edu,and clone formation assays;wound healing and Transwell assays;PCR;immunohistochemistry;immunofluorescence;and western blotting were used to investigated the responses in TSSC3-overexpressing osteosarcoma cell lines,and in xenografts and metastasis in vivo models,with or without autophagy deficiency caused by chloroquine or ATG5 silencing.A list of the main results are as following:1.TSSC3 overexpression enhances autophagic flux inosteosarcoma cells1.1 First,we stably overexpressed TSSC3 using Lv-TSSC3 in MTF and SaOS2 cells and examined the formation of AVs by using TEM.The results demonstrated that there was a significant increased number of autophagosomes and autolysosomes in MTF or SaOS2 cells transfected with Lv-TSSC3(p<0.05).Consistent with this,qPCR,IF and WB analysis revealed an accumulation of ATG5,BECN1,and lipid-bound LC3-II in TSSC3-overexpressing cells1.2 The lysosomal autophagy inhibitor CQ was used,which resulted in increased LC3-II and P62 accumulation in TSSC3-overexpressing cells,supporting the notion that TSSC3 overexpression did not block autophagic flux but enhance autophagic flux in osteosarcoma cells in vitro.2.ATG5 expression correlates positively with TSSC3 expression in osteosarcoma tissues2.1 We observed higher rate of TSSC3 and ATG5 positivity in fibrous dysplasia(n=21)and osteoblastoma(n=12)compared with that in osteosarcoma(n=58)(P? <? 0.05),while the positive rate of P62 expression was lower but not statistically significant(P? >? 0.05)in fibrous dysplasia and osteoblastoma compared with that in osteosarcoma.2.2 Fifty eight human osteosarcoma tissues were used to investigate the relevance of TSSC3,ATG5,and P62 expression in vivo.We demonstrated that the TSSC3 expression is positively correlated with that of ATG5(P=0.003,R=0.395).However,we failed to detect a significant correlation between TSSC3 and P62 expression(P? >? 0.05).3.TSSC3 overexpression induces autophagy in osteosarcoma cells via inactivating the Src-mediated PI3K/Akt/mTOR pathway3.1 The results showed that overexpression of TSSC3 did not affect the total Src,Akt and mTOR levels,but significantly decreased the levelsof their phosphorylated forms.3.2 We applied p-YEEI to activate Src signaling,Src or Akt/mTOR pathway was significantly activated,remarkably reversing the effect of TSSC3 overexpression on autophagy related proteins in MTF and SaOS2 cells.3.3 Following IGF-1 treatment,Akt/mTOR pathway was significantly activated,remarkably reversing the effect of TSSC3 overexpression on autophagy but no obvious increase of Src phosphorylation was found.3.4 We applied p-YEEI and BEZ235 together to activate Src signaling and inhibit PI3K/Akt/mTOR pathway.The result indicated that inhibition of Src phosphorylation without PI3K/Akt/mTOR inactivation could not decrease the ATG5 and LC3-II levels or increase the P62 levels in TSSC3-overexpressing MTF and SaOS2 cells.4.Crucial involvement of autophagy in TSSC3-mediated tumorigenesis in osteosarcoma cells4.1 We found that blocking autophagy with CQ attenuated the TSSC3-induced suppression of proliferation and colony-formation ability of MTF and SaOS2 cells,as assessed using CCK-8,EdU and colony formation assays(P? <? 0.05).In complementary experiments,We also found that blocking autophagy(ATG5 knockdown)reduced TSSC3-induced inhibition of cell proliferation and colony-formation ability in MTF and SaOS2 cells(P? <? 0.05).4.2 By using Hoechst and Annexin V-PE/7-AAD staining,we found inhibition of autophagy by CQ partly abrogated TSSC3-induced apoptosis in SaOS2 cells(P? <? 0.05),but not in MTF cells(P? >? 0.05).4.3 We found that TSSC3 overexpression significantly reduced tumorigenesis,as reflected by the tumor size,volume and tumor weight of the xenograft tumors.However,knockdown of ATG5 restoredtumorigenesis in TSSC3-overexpressing cells.The rate of positive Ki67 IHC staining was high in the control groups,decreased in the TSSC3-overexpressing group,and restored in the TSSC3-overexpressing and ATG5-knockdown groups(P? <? 0.05),suggesting that autophagy contributes to TSSC3-induced inhibition of malignant proliferation in vivo.5.TSSC3 inhibits osteosarcoma cells migration and invasion associated with autophagy5.1 By applying wound healing and transwell assays,the results showed that TSSC3 overexpression significantly decreased the motility and invasive ability of MTF and SaOS2 cells,the inhibitory effects of TSSC3 overexpression on cell motility and invasive ability were markedly attenuated by CQ treatment or knockdown of ATG5(P? <? 0.05).5.2 We observed that the epithelial cell marker E-cadherin was upregulated in TSSC3-overexpressing SaOS2 cells(but not in MTF cells),whereas the expression levels of mesenchymal markers N-cadherin,Vimentin,and MMP2 were inhibited.The N-cadherin,Vimentin,and MMP2 levels increased markedly when TSSC3-overexpressing MTF and SaOS2 cells were treated with CQ or knockdown of ATG5(P? <? 0.05).Consistent with these findings,the expression change of E-cadherin and Vimentin in subcutaneous xenograft tumors induced using transfected cells were measured using IHC staining,which indicated that TSSC3-induced autophagy blocked the EMT of osteosarcoma cells in vivo.5.3 We found that overexpression of TSSC3 notably reduced the ability of MTF cells to induce lung metastases,as reflected by the decreased incidence and number of metastatic nodules and the increased survival time of the mice.However,knockdown of ATG5 restored the ability of TSSC3-overexpressing cells to establish lung metastases.6.The correlations between TSSC3,ATG5,and P62 expressionand clinicopathological features of osteosarcoma and their prognostic value6.1 We found that expression of TSSC3 was significantly associated with a lower local recurrence rate(P? <? 0.05).Surprisingly,neither the expression of ATG5 nor P62 seemed to be associated with the clinicopathological features of osteosarcoma(P? >? 0.05).6.2 Kaplan-Meier curves showed positive expression of TSSC3 was significantly related to an improved overall survival(P? <? 0.05),while the differential expression of ATG5 and P62 had no notable impact on OS(P? >? 0.05).However,the patients with positive expression of both TSSC3 and ATG5 displayed a more favorable overall survival(P? <? 0.05).6.3 TSSC3 protein expression,tumor size,and metastasis were validated to be independent prognostic markers for overall survival of osteosarcoma by univariate and multivariate Cox regression(P? <? 0.05).In conclusion,we demonstrated,for the first time,that TSSC3 induces autophagy via inhibiting the Src-dependent PI3K/Akt/mTOR pathway in osteosarcoma.Furthermore,we found that TSSC3-induced autophagy contributes to suppressing tumorigenesis and metastasis in osteosarcoma,both in vitro and in vivo.In addition,ATG5 expression correlates positively with TSSC3 expression in osteosarcoma tissues.Moreover,TSSC3 is an independent prognostic marker in patients with osteosarcoma,and TSSC3-associated positive ATG5 expression might be a potential applicable predictor of favorable prognosis.