HDAC4 Mediated LHPP Deacetylation Enhances Its Destabilization And Promotes The Proliferation And Metastasis Of Nasopharyngeal Carcinoma

Background and objection: Nasopharyngeal Carcinoma(NPC)is the most common head and neck malignant tumor,and distant metastasis is the main cause of treatment failure and death in NPC patients.Research has shown that protein acetylation modification is comprehensively regulated by acetylases/deacetylases and plays an important role in tumor metastasis.Due to the fact that most tumor suppressor molecules are often deacetylated in malignant tumors,studying key molecules in NPC metastasis from the perspective of deacetylation modification is a meaningful topic.Phospholysine phosphohistidine inorganic pyrophosphate phosphatase(LHPP)is downregulated in some tumors and plays an anti-tumor role.However,the regulation of LHPP expression and its function in NPC remains unclear.Methods:(1)After treating NPC cells with a broad spectrum deacetylase inhibitor(HDACi)LBH589,the proteins with upregulated acetylation levels were analyzed by high-performance liquid chromatography tandem mass spectrometry;(2)After NPC cells HK1,5-8F were treated with HDACi LBH589 and PXD101,the direct acetylation level of LHPP was detected by immunoprecipitation and Western blot;(3)Western blot was used to detect the expression level of LHPP in NPC cells;(4)Immunohistochemical staining was used to detect the expression of LHPP in human NPC and adjacent tissues;(5)The effects of LHPP on the proliferation and invasion of NPC cells were detected using CCK8,cloning,scratch testing,and Transwell;(6)Deacetylase targeting LHPP was analyzed by immunoprecipitation,Western blot,and immunofluorescence;(7)Using half life and ubiquitination experiments,explore the molecular mechanism of histone deacetylase 4(HDAC4)regulating LHPP expression;(8)Using CCK8,cloning,scratch testing,Transwell,and repair testing,respectively,to analyze whether HDAC4 exerts biological functions through LHPP;(9)The downstream kinase regulated by LHPP in NPC was analyzed by high performance liquid chromatography tandem mass spectrometry,Western blot and immunofluorescence;(10)The expression and correlation of HDAC4,LHPP,P-TYK2,P-STAT1 in human NPC tissue were detected by immunohistochemical staining;(11)To further study the effect of HDAC4 on the proliferation and metastasis of NPC through LHPP in vivo,nude mouse models of transplanted tumor and metastatic tumor were constructed.Results:(1)LHPP was deacetylated in NPC;(2)LHPP is low expressed in NPC cells and human NPC tissues;(3)Overexpression of LHPP can inhibit the proliferation and invasion of NPC cells;(4)HDAC4is highly expressed in NPC,and IP experiments have verified that HDAC4 interacts directly with LHPP to promote the K6 site deacetylation of LHPP;(5)High performance liquid chromatography tandem mass spectrometry identified ubiquitin ligase Tripartite motif-containing 21(TRIM21)and kinase TYK2 as candidate interacting proteins for LHPP;(6)IP experiments have verified the direct interaction between LHPP and TRIM21,and the deacetylation of LHPP enhances its interaction with TRIM21;(7)Half life and ubiquitination experiments showed that knockdown of HDAC4 and knockdown of TRIM21 could increase the protein stability of LHPP;(8)IP experimental results showed that LHPP deacetylation promoted TRIM21 mediated ubiquitination of LHPP at position 48;(9)IP experiment verified that TYK2 is an interaction protein of LHPP;(10)LHPP inhibits the phosphorylation and activation of tyrosine kinase TYK2 in NPC cells;(11)In vivo level confirmed that HDAC4 further promotes the proliferation and metastasis of NPC by inhibiting the expression of LHPP and upregulating the phosphorylation and activation of TYK2.Conclusion: In NPC,high expressed HDAC4 deacetylated LHPP at K6,which promoted the degradation of LHPP by enhancing TRIM21 mediated K48-linked ubiquitination,thus losing the inhibition on TYK2 phosphorylation activation and upregulating STAT1 phosphorylation,further promoting the proliferation and metastasis of NPC.