The Functional Roles And Molecular Mechanisms Of MEF2D In Immune Escape Of Hepatocellular Carcinoma

Targeted therapies against hepatocellular carcinoma?HCC?,one of the most common and lethal cancers worldwide,provide only modest benefit.The myocyte enhancer factor 2?MEF2?family of transcription factors comprises key regulators of muscle and neuronal development.MEF2D contains an N-terminal minichromosome maintenance 1 homolog,agamous,deficiens,and serum response factor?MADS?/myocyte enhancer factor 2?MEF2?domain that mediates dimerization,DNA binding,and cofactor interactions,and C-terminal transcriptional activation domains that differ from those of other family members.We and others have found that MEF2D is overexpressed in many solid tumors,especially HCC,and predicts malignant progression and poor patient prognosis.MEF2D can promote tumor growth,metastasis and angiogenesis,affecting tumor cells and even the tumor microenvironment.Notably,HCC is an immunogenic liver lesion indicated by the presence of tumor-infiltrating lymphocytes.Some of the lymphocytes-mediated anti-tumor immunity retards HCC progression.Although recent studies have focused on the interaction between MEF2D and the tumor microenvironment,the methods used in these studies could not take the potential effect of immune cells into consideration.In addition,previous study has demonstrated that MEF2 acetylation enhances its DNA binding activity and promotes the transactivation of target genes.Several acetyltransferases and deacetylases,including p300and histone deacetylase 3,regulate MEF2 protein acetylation.The role of MEF2D or MEF2D acetylation in regulation of anti-tumor immunity and its regulatory mechanisms in HCC remain largely unknown.Immunotherapies targeting programmed cell death 1?PD-1?and its ligand programmed cell death 1 ligand 1?PD-L1?have been approved for several cancers,with durable clinical benefit.However,the majority of patients with HCC are unresponsive.The response to PD-L1/PD-1 blockade might be correlated with tumor PD-L1 expression levels.Recent studies revealed that silenced tumoral PD-L1 expression in many HCCs could be significantly induced under interferon gamma?IFNG?stimulation,targeted therapies?e.g.,cell cycle-related kinase inhibition?,hepatitis B virus infection,and MYC activation,which attenuated anti-tumor immunity and promoted HCC progression.Hence,understanding the pathways controlling inducible HCC PD-L1 expression may provide a molecular basis to improve the efficacy of PD-L1/PD-1 blockade.PD-L1 expression regulation in tumors is complex,with protein levels being affected by the trafficking mode and post-translational modifications,along with transcriptional and RNA stability regulation.Adaptive PD-L1expression mediated in part by IFN-regulatory factors?IRFs?upon exposure to cytokines-especially IFNG-secreted by metastasis-infiltrating lymphocytes,is critical for tumor immune evasion.However,whether other regulator?s?affect constitutive and induced PD-L1 expression in HCC remains unknown.We wonder whether and how MEF2D regulates immune escape of hepatocellular carcinoma and whether PD-L1 plays a role in this process.We also wish to provide some novel HCC treatment strategies based on the results of above questions.Therefore,we start this study.The main experiment designs and results of this study are shown as follows:Experiment designs:We analyzed immune cells from 20 fresh HCC tissues by flow cytometry.We analyzed 225 fixed HCC tissues?from 2 cohorts?from patients in China by immunohistochemistry and obtained survival data.We created mice with liver-specific knockout of MEF2D(MEF2D^LPC-KO)mice.We knocked out or knocked down MEF2D,E1A binding protein p300?p300?,or sirtuin 7?SIRT7?in SMMC-7721,Huh7,H22,and Hepa1-6 HCC cell lines,some incubated with IFNG.We analyzed liver tissues form mice and cell lines by RNA sequence,immunoblot,dual luciferase reporter,and chromatin precipitation assays.MEF2D protein acetylation and proteins that interact with MEF2D were identified by co-immunoprecipitation and pull-down assays.H22 cells,with MEF2D knockout or without?controls?were transplanted into BALB/c mice,some mice were given antibodies to deplete T cells.Mice bearing orthotopic tumors grown from HCC cells with or without knockout of SIRT7,were given injections of an antibody against PD-1.Growth of tumors was measured and tumors were analyzed by immunohistochemistry and flow cytometry.Results:1.In human HCC specimens,we found an inverse correlation between level of MEF2D and numbers of CD4⁺and CD8⁺T cells;level of MEF2D correlated with percentages of PD-1-positive or TIM3-positive CD8⁺T cells.Knockout of MEF2D from H22 cells reduced their growth as allograft tumors in immune-competent mice,but not in immune-deficient mice or mice with depletion of CD8⁺T cells.When MEF2D-knockout cells were injected into immune-competent mice,they formed smaller tumors that had increased infiltration and activation of T cells,compared with control HCC cells.2.In human and mouse HCC cells,MEF2D knockdown or knockout reduced expression of PD-L1.MEF2D bound the promoter region of the CD274 gene?encodes PD-L1?and activated its transcription.Overexpression of p300 in HCC cells,or knockout of SIRT7,promoted acetylation of MEF2D and increased its binding,along with acetylated histones,to the promoter region of the CD274.Exposure of HCC cells to IFNG induced expression of p300 and its binding MEF2D,which reduced the interaction between MEF2D and SIRT7.MEF2D-induced expression of PD-L1 upon IFNG exposure was independent of interferon-regulatory factor 1?IRF1?or IRF9.In HCC cells not exposed to IFNG,SIRT7formed a complex with MEF2D that attenuated expression of PD-L1.3.Knockout of SIRT7 reduced proliferation of HCC cells and growth of tumors in immune-deficient mice.Compared with allograft tumors grown from control HCC cells,in immune-competent mice,tumors grown from SIRT7-knockout HCC cells expressed higher level of PD-L1 and had reduced infiltration and activation of T cells.In immune-competent mice given antibodies to PD-1,allograft tumors grew more slowly from SIRT7-knockout HCC cells than from control HCC cells.The main conclusions of this study are:1.MEF2D disruption promotes CD8⁺T cell-mediated anti-tumor immunity that suppresses HCC growth.2.MEF2D attenuates anti-tumor immunity by transactivating PD-L1 under IFNG stimulation in HCC.3.p300-induced MEF2D acetylation is a key mediator of IFNG-induced PD-L1expression in HCC cells.4.Deacetylase SIRT7 interacts with and deacetylates MEF2D to suppress PD-L1expression in the absence of IFNG.5.MEF2D regulation of PD-L1 mediates enhanced HCC immune escape caused by SIRT7 disruption.6.SIRT7 inhibition combined with PD-1 blockade therapy enhances HCC therapeutic efficacy.Collectively,expressing MEF2D by HCC cells increases their expression of PD-L1,which prevents CD8⁺T cell-mediated anti-tumor immunity.When HCC cells are exposed to IFNG,p300 acetylates MEF2D,causing it to bind the CD274 gene promoter and upregulate PD-L1 expression.In addition to promoting HCC cell proliferation,SIRT7reduced acetylation of MEF2D and expression of PD-L1 in HCC cells not exposed to IFNG.Strategies to manipulate this pathway might increase the efficacy of immune therapies for HCC.