Study On The Function And Molecular Mechanism Of STUB1 Regulating PSPH Protein Homeostasis In Non-small Cell Lung Cancer

Background and purpose:Lung cancer is the most common tumor and an important cause of tumor-related death worldwide,of which non-small cell lung cancer is the main type of lung cancer,accounting for about 85%.According to the Global Cancer Statistics 2020,lung cancer remains the leading cause of death as high as 18%.Although the clinical treatment of patients with lung cancer has been greatly improved,the 5-year survival rate of patients with non-small cancer worldwide is only about 5%.Therefore,it is of great clinical significance to explore the potential therapeutic target in non-small cell lung cancer.Serine is one of the most utilized amino acids by cancer cells,second only to glutamine.As an important source of intracellular carbon units,serine plays an important role in the synthesis of nucleotides,ATP,SAM and NAPDH.The synthesis of serine requires the participation of three enzymes,namely phosphoglycerate dehydrogenase(PHGDH),phosphoserine aminotransferase 1(PSAT1)and phosphoserine phosphatase(PSPH).PSPH,as the key enzyme in the last step of serine synthesis,catalyzes the transformation of 3-phosphoserine into serine.At present,little is known about the function of PSPH in tumor,and its molecular mechanism of maintaining protein homeostasis in tumor has not been reported.This study aims to explore the molecular mechanism of PSPH maintaining protein homeostasis in non-small cell lung cancer and clarify the role of PSPH in non-small cell lung cancer.The study is divided into three parts.The first part is to clarify the role of PSPH in proliferation and migration in non-small cell lung cancer;the second part is to find the molecular mechanism of regulating PSPH homeostasis;the third part is to find the key site that regulates PSPH protein homeostasis and clarify the role of this site on cell proliferation and migration.These findings lay a solid theoretical foundation for the development of novel anticancer drug targeting PSPH.PartⅠ PSPH promotes the proliferation and migration of NSCLC cells Methods:1.The expression of PSPH in LUAD and LUSC and its correlation with survival rate were analyzed by TCGA database.2.The tumor tissues and adjacent normal tissues of NSCLC patients were collected,and the expression of PSPH at the protein level was detected by Western blot;normal bronchial epithelial cell(BEAS-2B)and non-small cell lung cancer cells(A549,H1299,PC9,H359,HCC827,H292)were cultured,the total protein and RNA were extracted to detect the expression of PSPH at protein level and m RNA level by Western blot and q-PCR respectively.3.Specific si RNA was used to knock down the expression of PSPH in non-small cell lung cancer cells.The effects of PSPH on the proliferation and migration of non-small cell lung cancer cells were detected by proliferation assay,wound healing assay and transwell assay.Results:1.In TCGA database,the expression of PSPH in non-small cell lung cancer tissues was higher than that in adjacent normal tissues.In LUAD,high PSPH expression significantly reduced patient survival probability,while in LUSC,high PSPH expression had no significant effect on patient survival probability.2.At the tissue level,the expression of PSPH in tumor tissues was significantly higher than that in adjacent normal tissues;at the cellular level,the protein and m RNA expression of PSPH in 6 non-small cell lung cancer cells were significantly higher than that in bronchial epithelial cells.3.Knockdown of PSPH inhibited the proliferation and migration of non-small cell lung cancer cells.PartⅡ STUB1 promotes ubiquitination and degradation of PSPH protein Methods:1.BEAS-2B cells and A549 cells were treated with cycloheximide CHX,and the samples were collected at different time points.The protein degradation rate of PSPH was detected by Western blot;at the same time,CHX combined with proteasome degradation pathway inhibitor MG132 or combined with lysosomal degradation pathway inhibitor CQ were used to clarify the degradation pathway of PSPH through the recovery of protein expression.2.Immunocoprecipitation was used to detect the binding between PSPH and STUB1;the co-localization of PSPH and STUB1 in cells was detected by immunofluorescence assay.3.STUB1 was overexpressed or knocked down in A549 cells.The m RNA and protein expression level,protein degradation rate and ubiquitination modification level of PSPH were detected by q-PCR and Western blot.Results:1.The degradation rate of PSPH in A549 cells was significantly slower than BEAS-2B,and the expression of PSPH protein in A549 cells recovered after MG132 and CQ treatment,while the recovery of PSPH in MG132 treatment group was more significant.Meanwhile,MG132 treatment significantly increased the ubiquitination level of PSPH,but there was no significant change in the ubiquitination level after CQ treatment.2.Immunocoprecipitation showed that STUB1 combined with PSPH;immunofluorescence experiment showed that STUB1 co-localized with PSPH.3.Overexpression of STUB1 in A549 cells significantly down regulated the expression of PSPH protein and accelerated the degradation rate of PSPH,but had no significant effect on the m RNA level of PSPH.4.STUB1 promoted the K48-linked ubiquitination of PSPH,but did not affect its k63-linked ubiquitination.Part Ⅲ Lysine 158 of PSPH is the key ubiquitination site to regulate its degradation and inhibiting ubiquitination at this site promotes tumor proliferation and migration Methods:1.A549 was transfected with wild-type PSPH and mutant PSPH-K158 R respectively,and its protein ubiquitination level and protein degradation rate were detected.2.The stable cell lines overexpressing wild-type PSPH and mutant PSPH-K158 R were constructed.The cell proliferation and migration were detected by cell clonal colony formation assay and transwell assay.Results:1.Compared with wild-type PSPH,the ubiquitination level of mutant PSPH158 R decreased significantly and the protein degradation rate slowed down significantly.2.The proliferation and migration of mutant PSPH-K158 R cells were significantly enhanced.Conclusion:1.PSPH promotes the proliferation and migration of non-small cell lung cancer.2.STUB1 ubiquitinates PSPH and promotes its protein degradation.3.Inhibition of PSPH-K158 R ubiquitination promotes the proliferation and migration of non-small cell lung cancer cells.