Oncoprotein SND1 Promotes Tumour Immune Escape Through Inhibition Of MHC-i Antigen Presentation

Objectives:Tumour cells can escape immune response through loss of antigenicity and/or immunogenicity,or by coordinating a suppressive immune microenvironment.To explore the strategies for tumour immunotherapy highly dependent on the discovery of molecular mechanisms of tumour immune escape.Therefore,distinct therapeutic strategies may be required depending on the mechanisms.Tumour immunotherapy strategies mediated by T cells rely on the functional competence of multiple immunological elements.For example,defects in the surface expression of MHC class I molecules is one of the most important reasons of tumour immune escape due to decreased recognition by CD8⁺T cells,which has been found in approximately20-60%of common solid cancers including melanoma,lung,breast,renal,prostate,and bladder cancers.The molecular mechanisms underlying these changes vary according to the tumour type.These alterations can be genetic or regulatory,at the transcriptional or post transcriptional level.SND1(Staphylococcal Nuclease and Tudor Domain Containing 1)is a newly identified oncoprotein which is highly expressed in almost all the detected different tumour cells.It is a ubiquitously expressed and highly conserved protein in the mammals and plays important physiological roles in variety of cellular processes.Current studies have demonstrated that SND1 regulates the differentiation and migration of different tumour cells via variant signal pathways at the cellular level.However,the fundamental impact of SND1 on the tumourigenesis and immune microenvironment in vivo is largely unknown.Therefore,we aimed to investigate the correlation between SND1 and the tumour immune escape,and the molecular mechanism regulated by SND1 in MHC-I antigen presentation.Methods:This study was divided into three parts.Part 1:(1)Affinity purification and mass spectrometry were performed to identify SND1-associated proteins from cellular extracts of He La cells.(2)Co-immunoprecipitation(Co-IP)assay was performed to verify the association of SND1and HLA-A(heavy chain of human MHC-I)in He La cells,and the immunofluorescence assay and Duolink assay further confirmed the cellular co-localization of SND1 and HLA-A.(3)The bacterially produced GST-fusion protein containing different domains of SND1 or HLA-A were purified using Glutathione Agarose beads and employed to explore the interaction domain between SND1 and HLA-A by GST pull-down assay.The structural stability of the SND1-HLA-A complex was also analysed by I-TASSER and the Z-dock website,which could calculate the binding free energy and identify key residues to the association process.(4)To investigate whether the N-terminal signaling peptides of SND1 is a signaling peptide,an ER reporter assay by constructing the ER luminal reporter vector(GFG:GFP-FLAG-GFP)was developed.Then the co-localization of the X-S(N)P-GFG with ER marker protein was detected by immunofluorescence assay to verify the location of SND1.Part 2:(1)The flow cytometry and Western Blot(WB)were carried out to detect the protein level of MHC-I(HLA-A/B/C)on the surface of human cancer cells(He La and SKOV3 cell)with deletion of SND1 or ectopic overexpression of SND1.(2)The alteration of HLA-A at the m RNA level was ruled out by fluorescence quantitative polymerase chain reaction(q PCR).(3)To further investigate whether SND1 affected the degradation of HLA-A via ubiquitin mediated-proteasome pathway,the ubiquitilation of HLA-A was detected by immunoprecipitation after changing the expression of SND1.(4)Mass spectrometry,immunoprecipitation and Duolink assay were uesd to further explore the binding efficiency of HLA-A with its chaperones to clarify the molecular mechanism of degradation of HLA-A.Part 3:(1)The flow cytometry and Western Blot were used to detect the protein level of MHC-I(H2Kb)on the surface of mouse tumor cells(B16F10 and MC38 cell)with deletion of SND1.(2)At the animal level,mouse tumour cells were subcutaneously transplanted into the flank of C57BL/6 mice.The tumour growth was monitored and the infiltration of CD8⁺T cells in tumour tissues was detected by immunofluorescence and flow cytometry.(3)Cell proliferation,apoptosis or cell cycle in vitro were detected by cloning formation assay,CCK-8 assay and flow cytometry.(4)For xenograft experiments,RAG-1(recombination activating gene 1)knockout mice(Rag1^-/-mice)which lack mature T and B cells combined with C57BL/6 WT mice were used to investigate whether the absence of SND1 would affect tumour growth in vivo.(5)Transgenic OT-I mice were employed to further clarify the influence of SND1 on CD8⁺T cell mediated cellular immune responses in tumour.(6)The infiltration of CD8⁺T cells and the survival of patients were analysed with the TIMER database and Progno Scan database.Results:Part 1:(1)SND1 could physically interact with the immature form of HLA-A in partially folded/unfolded conformation.(2)Immunoprecipitation analysis in He La cells demonstrated that the SN3 region of SND1 was required for the interaction of HLA-A,and the electrostatic interaction between the basic and acidic amino acids on SN3 region and A3 domain might play an important role in the association process.(3)SND1 is an ER-associated protein anchored on ER membrane by binding SEC61A.Part 2:(1)The protein level of HLA-A was enhanced with deletion of SND1,while reduced with ectopic overexpression of SND1 in different cancer cells.(2)SND1leads to HLA-A degradating through the ubiquitin-proteasome pathway.(3)Immunoprecipitation assay and Duolink assay revealed that the binding efficiency of HLA-A to Calnexin,and HLA-A to?2m,was remarkably influenced by SND1.Moreover,glycosylation-site mutated HLA-A(N110Q)is not able to associate with both Calnexin and?2m,but sufficiently interacts with SND1.(4)The interaction of VCP,VIMP,and HRD1(E3 ligase)to HLA-A was remarkably increased in the presence of ectopically overexpressed SND1 but was largely decreased in the absence of SND1.Part 3:(1)Western blot and flow cytometry analysis demonstrated that the protein level of H2Kb(heavy chain of mouse MHC-I)was increased in B16F10 melanoma cells and MC38 colon adenocarcinoma cells with deletion of SND1.(2)Loss of SND1 in murine tumours limits tumour size in murine syngeneic tumour models.Comparing with the parental tumour tissue,there were more CD8⁺T cells infiltrated in SND1-KO tumour tissues.(3)SND1 deficiency in mice melanoma and colon carcinoma resulted in no significant changes in regulating cell proliferation,apoptosis or cell cycle in vitro and in vivo.(4)Moreover,the rescue of SN domain of SND1significantly increased the tumour growth through mobilizing less CD8⁺T cells infiltrating in tumours.(5)The results on transgenic OT-I mice further verified the impact of SND1 on MHC-I/antigenic peptides presenting in tumour cells and consequently affecting the infiltration of cytotoxic CD8⁺T cells in tumour tissue.(6)The TIMER database and the Progno Scan database showed that SND1 was negatively associated with the infiltration of CD8⁺T cells and the survival of patients in melanoma and colon adenocarcinoma.Conclusions:(1)We demonstrate that SND1 is an ER-associated protein anchored on ER membrane by binding SEC61A upon where catching the nascent HC of MHC-I.(2)SND1 sequestrates the nascent HC of MHC-I and reduces the binding efficiency of HC to Calnexin and?2m,which redirects it to the ERAD pathway for proteosomal degradation.(3)SND1 impaires tumour antigen presentation to cytotoxic CD8⁺T cells and sabotages the CD8⁺T mediated cellular immune response,which promotes the tumour growth in vivo.Moreover,SND1 is negatively associated with the infiltration of CD8⁺T cells in human tumour tissues and the survival of patients in melanoma and colon adenocarcinoma.