Acy1 Coenzyme A:cholesterol Acyltransferase 1 Inhibits The Proliferation And Metastasis Of Nasophayrngeal Carcinoma

BackgroundNasopharygeal carcinoma(NPC)is a malignant tumor deriving from nasopharynx epithelium,with distinct geographical,ethnicity and ethnic distribution.NPC is prevalent in southern China and Southeast Asia,with an incidence of 30 per 100000 in males and 10 per 100000 in females.It has been shown that genetic susceptibility,Epstein-Barr virus(EBV)infection,environmental factors and dietary carcinogens are the etiology of NPC,the pathogenesis is a complicated process with multi-steps and multi-factors.Because of no significant symptoms at early stage,most of the firstly diagnosed NPC patients are already at stage II-IVb.The characteristic of NPC patients is a mass in the neck,suggesting an existence of lymph node metastasis.Currently,the treatment options for NPC are limited.The conventional treatment of NPC patients is Cisplatin-based chemotherapy combined with intensity-modulated radiotherapy(IMRT).However,around 20% patients failed due to distant metastasis and local recurrence mainly.Therefore,it is necessary to investigate the possible mechanisms for NPC metastasis and improve the therapeutic approach targeting metastasis.Abnormal energy metabolism is one of the characteristics of tumor,which provides survival advantages for tumor cells.Abnormal energy metabolism is closely related to the occurrence and progression of tumors.Normal neurons and glial cells can effectively metabolic ketone body and through the ketone body energy metabolism,and a variety of tumor cells,including glioma,cannot use of ketone body as an energy substrate,but as the precursor of lipid synthesis.In contrast,in breast and prostate cancer,the cancer cells use ketone body as an energy source,the expression of ketone body metabolism related enzymes is thought to be associated with invasive phenotype prognostic markers.In addition,the effect of ketogenic diet on tumor is also controversial.In colorectal and pancreatic cancers,the ketogenic diet suppresses tumor size and the progression of systemic inflammation in tumor-burdened mice.In gliomas and cervical cancers,the ketogenic diet is ineffective and may even promote tumor growth,resulting in reduced survival of tumor-burdened mice.Our previous study found that HMGCL,a key gene involved in ketogenic pathway,was down-regulated in NPC.The overexpression of HMGCL increased the content of betahydroxybutyrate in NPC cells and was positively correlated with the reversal of EMT.Endogenous and exogenous beta-hydroxybutyrate inhibits proliferation and metastasis of NPC by activating ROS.Acy1 Coenzyme A:Cholesterol Acyltransferases1(ACAT1),an upstream catalytic enzyme of HMGCL,is also a key gene in the ketone body metabolism pathway and has not been reported in NPC.Objective:To investigate the abnormality of transcription and expression of ACAT1 gene in NPC.To reveal the effect of ACAT1 expression on ketone body metabolism,malignant biological behavior of NPC cells,and the underlying molecular mechanisms.Methods: 1.Quantitative real-time PCR(qPCR)and immunohistochemical(IHC)staining were used to verify the transcription and expression of ACAT1 in NPC tissues and normal nasopharyngeal epitheliums,NPC cells and normal nasopharyngeal epithelial cells.2.Establishing ACAT1 stably overexpressed NPC cells,and evaluating the biological function of ACTA1 gene in NPC cells by performing proliferation assay,colony formation assay,tumor formation in nude mice,2D cell migration assay,and 3D cell invasion assay.3.Western blot was used to detect the expression of E-cadherin,Vimentin and ?-catenin,and to determine whether ACAT1 affects the metastasis of cancer cells through epithelial-mesenchymal transition(EMT)signaling pathway.4.MTT assay was used to verify the effect of exogenous ketone bodies on proliferation of NPC cells by using exogenous ketone bodies to treat NPC cell lines.Results: 1.ACAT1 gene is down-regulated in NPC.We verified the transcription level of ACAT1 gene in NPC cell lines(HK1?HONE1?CNE1?5-8F?6-10B?TW03?C666-1)and immortalized normal nasopharyngeal epithelial cell line(NP69)by qPCR and found that ACAT1 gene was downregulated in NPC cell lines compared to normal nasopharyngeal epithelial cell lines.Further,we also verified the transcription level of ACAT1 gene in NPC tissues and normal nasopharyngeal tissues.The results suggested that ACAT1 gene was down-regulated in NPC.Immunohistochemical staining to compare 19 cases of NPC and 15 cases of normal nasopharyngeal tissue found that in the cancer tissue ACAT1 protein is mainly distributed in the cytoplasm of tumor cells;in normal nasopharyngeal epithelial tissue,ACAT1 protein is mainly distributed in submucosal glandular epithelial cells in the cytoplasm.One case of nasopharyngeal carcinoma was negative(0 point),5 cases were positive(1 and 2 points),and 9 cases were positive(3 points or more).In addition,1 case of normal nasopharyngeal tissue was suspected to be positive(2 points).18 cases were all positive(more than 3 points),NPC score was 3.387±0.6766,and normal nasopharyngeal tissue score was 7.611±0.4077.There was a statistically significant difference between the two groups.The results suggest that ACAT1 protein is mainly expressed in normal nasopharyngeal epithelial cells,and it shows low or no expression in NPC tissues.2.Exogenous overexpression of ACAT1 inhibits proliferation,migration and metastasis of NPC cells.Through CCK8 cell proliferation assay,plate cloning experiment,wound healing assay and Transwell invasion assay,it was found that exogenous overexpression of ACAT1 gene can inhibit the proliferation,colony formation,cell migration and invasion ability of NPC cells.It suggests that ACAT1 gene inhibits the malignant biological behavior of NPC cells.3.Exogenous overexpression of ACAT1 inhibits epithelial-mesenchymal transition in NPC cells.The expression of Vimentin was down-regulated in NPC cells transfected with ACAT1,while the expression of E-cadherin was up-regulated,suggesting that overexpression of ACAT1 inhibits epithelial-mesenchymal transition of NPC cells.4.Exogenous overexpression of ACAT1 inhibits proliferation of NPC cells by ?-hydroxybutyrate.The increase of beta-hydroxybutyrate content in NPC cells transfected with ACAT1 gene suggests that ACAT1 is involved in the production of betahydroxybutyrate(?-HB).Exogenous ?-HB treatment on NPC cells inhibits proliferation of NPC cells in a concentration-dependent manner,suggesting that ACAT1 affects the proliferation of NPC by regulating ?-HB production.Conclusion: 1.The transcription and expression of ACAT1 is downregulated in NPC.2.Overexpression of ACAT1 can inhibit the malignant biological behavior of NPC cells.3.ACAT1 inhibits the metastasis of NPC by inhibiting the epithelial-mesenchymal transition pathway of NPC cells,and inhibits the proliferation of NPC cells by ?-HB.In summary,ACAT1 gene might be a candidate tumor suppressor gene in NPC.Taken together with our previous results,ACAT1 inhibit the proliferation and metastasis of NPC cells by promoting the production of ?-HB.