TSSC3/RanBP9 Regulates Anoikis And EZH2 Promote Tumor Growth By Repressing The Expression Of TSSC3 In Human Osteosarcoma

Osteosarcoma is the most common primary malignant bone tumor, and is the second highest cause of cancer-related death in children and young adolescents. In recent years, the emergence of neoadjuvant chemotherapy dramatically increased the five-year survival rate. However, osteosarcoma remains a malignancy with a very high case fatality rate and disability rate. The progress of osteosarcoma treatment has been painfully slow for the past 20 years. Therefore, developing more targeted treatment is imperative.Cancer metastasis is a complicated mutistep biological process. Suppression of anoikis is a prerequisite for tumor cell metastasis and correlates with chemoresistance and poor prognosis. Thus, to investigate the potential mechanisms rendering tumor cells anoikis resistance, chemoresistance and lung metastasis will provide further meaningful theoretical and biological experiment evidence for osteosarcoma meatstasis and chemoresistance. Our previous study showed tumor-suppressing STF cDNA 3(TSSC3) acts as a tumor suppressor in osteosarcoma. And TSSC3 could inhibit cell proliferation in vitro and in vivo in our previous study, however the related mechanism was not fully elucidated. TSSC3 is an imprinting gene, the expression of which is mainly regulated by epigenetic mechanisms, which aroused our attention. The enhancer of zeste homolog 2(EZH2) methyltransferase is the catalytic subunit of polycomb repressive complex 2(PRC2) and an important epi genetic regulation factor. The association between EZH2 and TSSC3 expression has not been investigated in osteosarcoma.The main results are listed as two parts:1. Yeast two-hybrid screening of a human fetal brain library using full-length TSSC3 c DNA as bait identified RanBP9 as a novel putative binding partner for TSSC3; co-immunoprecipitation of Ran BP9 and TSSC3 protein; immunoprecipitation assays using mutant Ran BP9 and TSSC3 constructs of TSSC3 demonstrated that SPRY domain of Ran BP9 and PH domain of TSSC3 mediated the interaction of these two; identification of the role of TSSC3/Ran BP9 in osteosarcoma anoikis and distant metastasis and related mechanisms.2. Immunohistochemistry(IHC),immunofluorescence and q RT-PCR were applied to determine the expression of EZH2 and TSSC3 in human osteosarcoma; CCK-8, cell cycle analysis, soft agar assay, cell migration and invasion assays and chromatin Immunoprecipitation(Ch IP) were used to detect the biological effect of EZH2 in osteosarcoma and its epigenetic regulation of imprinting gene TSSC3.The main conclusions are listed as follows:The novel RanBP9-TSSC3 cooperates to suppress anoikis resistance and metastasis via inhibiting Src-mediated Akt signaling in osteosarcoma1. We characterized a novel interaction between TSSC3/RanBP9 in osteosarcoma that requires the Ran BP9 SPRY domain and TSSC3 PH domain and exerts transcriptional and post-translation regulation.2. TSSC3/RanBP9 complex negatively correlated with a highly migratory, invasive and anoikis-resistant phenotype in osteosarcoma cells.3. TSSC3/RanBP9 complex also negatively correlated with metastasis in human osteosarcoma.4. RanBP9 cooperated with TSSC3 to inhibit anchorage-independent growth, migration and invasion and promote anoikis in vitro and suppress lung metastasis in vivo.5. The Ran BP9 SPRY domain was required for TSSC3/RanBP9 complex- mediated anoikis resistance.6. Mechanistic studies demonstrated Ran BP9 forms a ternary complex with TSSC3 and Src and scaffolds this interaction, which suppresses Src and Src-dependent Akt pathway activation and facilitates mitochondrial- associated anoikis.In summary, we identified a novel protein Ran BP9, which could interact with TSSC3. And they cooperate to repress the anoikis resistance and metastasis in human osteosarcoma via inhibiting Src-dependent PI3K/Akt signaling. It suggests TSSC3 and Ran BP9 both act as tumor-suppressor in osteosarcoma. Results of this part may offer preliminary theoretical basis for the therapy of osteosarcoma based on TSSC3 and Ran BP9.EZH2 silencing inhibits tumor growth and lung metastasis of human osteosarcoma by facilitating re-expression of TSSC31. EZH2 is overexpressed in human osteosarcoma tissues and cell lines.2.Association between EZH2 expression and the clinicopathologic features of osteosarcoma patients: EZH2 expression is significantly associated with the Enneking stage of osteosarcoma(P < 0.01), grade and lung metastasis.3. EZH2 knockdown inhibits cell growth in vitro and in vivo.4.EZH2 knockdown induces apoptosis, inhibits migration and invasion in osteosarcoma.5. Inhibition of in vivo lung metastasis by siEZH2.6. Enhanced sensitivity of osteosarcoma cells to chemotherapeutic agents by EZH2 silencing through growth inhibition and promotion of apoptosis.7. EZH2 silencing enhances expression of TSSC3, and TSSC3 is a direct target of H3K27me3.8. Repression of TSSC3 is potentially involved in the oncogenic function of EZH2.In summary, we found EZH2 was abnormally elevated in human osteosarcoma. And EZH2 knockdown could notably inhibit the tumor growth and lung metastasis in osteosarcoma. Therefore EZH2 has the potential of being oncogene for osteosarcoma. Its epigenetic repression of TSSC3 is involved in the biological effect of its silence. Results of this part may offer preliminary theoretical basis for the epigenetic therapy of osteosarcoma by targeting EZH2.