EZH2 Regulates Tumor Growth In HNSCC In Vitro And In Vivo

Head and neck squamous cell carcinoma(HNSCC) is the sixth most common cancer worldwide, and known for its rapid clinical progression and poor prognosis. In recent years, although research on the head and neck squamous cell carcinoma has made some progress, but the treatment effect is still unsatisfactory. Thus, it’s urgent to develop novel diagnostic and therapeutic targets for HNSCC.The enhancer of Zeste Homologue 2(EZH2),a key catalytic subnit of polycomb repressive complex 2(PRC2), as a histone methyltransferases Lysine 27 of Histone H3. The mechanism of EZH2 regulated tumor cell apoptosis is unclear, and there’s no reports about EZH2 affected cell apoptosis and proliferation in HNSCC. MICU1(mitochondrial calcium uptake1) is a negative regulator of mitochondria dependent cell-death pathway. Thus, we employed two human HNSCC cell lines, Cal27 and SCC25; used EZH2 inhibitor DZNEP inhibited EZH2 function., and MICU1 sh RNA knockdown the expression of MICU1. Research is divided into three parts:Part I: EZH2 expression in a Chinese HNSCC cohort of 97 cases by using IHC assay. Human HNSCC displayed positive expression of EZH2 in tumor cell nuclei.Kaplan-Meier survival analysis showed that the patients with upper quantile EZH2 expression showed shorter survival comparing with the rest patients. EZH2 expression of different tumor grades displayed obvious difference. Western blot assayed the expression of EZH2 and MICU1 in 5 HNSCC cell lines.Part II: IC50 value of DZNEP in the two cell lines was determined by MTT assay. Flow cytometry analysis measured cell apoptosis and cell cycle. SA-β-gal activity staining assay detected cell senescence. JC-1 probe was used to measure mitochondrial membrane potentials. Fluo 3-AM used to determine the free Ca2+ level was induced by EZH2 inhibition. Western blot detected apoptosis related proteins and cell cycle related proteins. The above experimental reseach DZNEP inhibitied the growth of HNSCC cell lines in vitro. Next, we employed a MICU1 sh RNA(sh MICU1) vector to further validate the role of targeting EZH2/MICU1 in regulating cell apoptosis in HNSCC cells. Moreover, we introduced MICU1 expression vector(GV230-MICU1) into Hep-2 cell that had a low endogenous MICU1 expression level,detected the change of apoptosis.Part III: Tumor-bearing nude mouse models were established by subcutaneous implantation of Cal27 cells into 16 mice. Observed tumor growth closely, measured tumor valume and weight. Immunohistochemistry(IHC) detected expressions of MICU1, MCU and apoptosis related proteins. TUNEL assay was used to investigate active cell death(apoptosis).Results:Cal27 and SCC25 with both higher HOTAIR and MICU1 expressed than the other HNSCC cell lines. DZNEP blocked the level of EZH2 expression significantly in Cal27 and SCC25 cell lines. IC50 of Cal27 and SCC25 cell lines were 6 and 3μmol/L. Compared with control cells, DZNEP inhibited cell proliferation and induced apoptosis in both cell lines; mitochondrial transemembrane potential changed significantly; free Ca2+ level was induced by EZH2 inhibition; ΔΨm was drastically reduced respectively in both cell lines and Ca2+ indicator suggested free Ca2+ level induced by MICU1 targeting;Western blot analysis showed that Bcl-2 was decreased and BAX or Cleaved caspase-3 was increased by sh MICU1 treatment in both cell lines. Compared with the DMSO group, the tumor size and weight of DZNEP group were significantly reduced. TUNEL assay data showed that more apoptotic nuclei were found in EZH2 inhibition groups.Conclusions:1. HOTAIR si RNA decreased MICU1 expression, induced HNSCC mitochondrial related apoptosis, and inhibited cell proliferation.2. DZNEP inhibited the fuction of EZH2, decreased the expression of MICU1,induced HNSCC mitochondrial related apoptosis, and inhibited cell proliferation.3. DZNEP inhibit Cal27-xenograft tumor growth. EZH2 inhibition may be served as a new target with therapeutic potential for HNSCC.