Mechanism Of B4GALNT4 Promoting The Progression Of Prostate Cancer By Mediating PDK1 Glycosylation To Activate PI3K-Akt Signaling Pathway

Background:Prostate cancer(PCa)is a malignant tumor of the acinar epithelium of the prostate,which is very common tumor in men.In recent years,with the aging of the Chinese population,the incidence and mortality of prostate cancer have been rising quickly.Prostate cancer threatens male life and health seriously.Currently,the treatment difficulty of prostate cancer in our country mainly lies in:advanced prostate cancer is prone to progression into castration-resistant prostate cancer(CRPC),and the current drug therapies are not effective for CRPC,so it is urgent to develop new targeted therapeutic drugs.Currently,the clinical targeted therapeutic drugs for prostate cancer are all developed based on the genotypes of European and American populations.However,the developed targeted drugs may not be applicable to the Chinese population.The genotypes of prostate cancer in Chinese population are urgently needed for the development of new targeted therapeutic drugs.In recent years,glycogenes have become a hot topic in the research of tumor development.Glycogenes include glycosylation-related genes,such as glycosyltransferase,ribonucleotide synthesis and transporter genes etc.Numerous studies have found that glycogenes are involved in the initiation,progression and metastasis of pancreatic,ovarian,colorectal,bladder and other tumor cells and drug resistance mechanisms.However,the relationship between glycogenes and prostate cancer has not been fully studied.In order to break the dilemma of prostate cancer treatment,we explored the possible mechanisms involving glycogenes in the occurrence and development of prostate cancer utilizing the Chinese prostate cancer genome map previously studied by our team.The potential therapeutic targets of prostate cancer were analyzed to provide new ideas for the development of targeted therapeutic drugs for prostate cancer.Purpose:Our research team analyzed the correlation between the identified glycogenes and prostate cancer from the Chinese prostate cancer genome as the research starting point.Through rigorous bioinformatics methods in the database of TCGA and CPGEA(previous research results of our group),glycogene B4GALNT4 was finally confirmed as a gene closely related to the malignancy degree and poor prognosis of prostate cancer.The effects of B4GALNT4 were investigated on cell phenotype and PI3K-Akt signaling pathway in prostate cancer cells.To further explore the mechanism of how glycogene B4GALNT4 mediates glycosylation of PDK1 protein to regulate PI3K-Akt signaling pathway and thus affect the progression of prostate cancer cells.Methods:In this study,bioinformatics methods were used to screen glycogenes closely related to the malignancy degree of prostate cancer.Immunohistochemical methods were used to verify the correlation between the expression of glycogene B4GALNT4 and the malignancy degree and clinical prognosis of prostate cancer.Constructed stably transfected prostate cancer cells of B4GALNT4 knockdown were used to analyze the effects of the changes of B4GALNT4 on the phenotype of prostate cancer cells.The downstream signaling pathways that may be affected by B4GALNT4 were analyzed based on RNA-seq technology and protein spectrometry.The association between B4GALNT4 and targeted PDK1 protein was verified by immunohistochemistry and Co-IP experiments.The function recovery experiment of exogenous overexpression of PDK1 protein confirmed that B4GALNT4 may affect the proliferation,apoptosis and invasion of prostate cancer cells by regulating PI3K-Akt pathway through the action of PDK1 protein.The effect of B4GALNT4 on protein glycosylation in prostate cancer cells was further verified by Western blot and immunofluorescence.The functional effects of glycosyltransferase B4GALNT4 on the glycosylation modification of PDK1 protein were verified by CHX protein tracking assay and protein degradation pathway experiment.Finally,the analysis of online glycosylation site database and liquid chromatography-mass spectrometry were used to investigate the glycosylation modification sites of PDK1 protein.Results:Bioinformatics analysis confirmed that glycogene B4GALNT4 is closely related to malignant degree and clinical prognosis of prostate cancer.Immunohistochemical analysis of 67 patients with prostate cancer in Changhai Hospital showed that the expression level of B4GALNT4 protein was closely correlated with the tumor malignant index of PSA(P=0.029),Gleason score(P < 0.001),prostatic extravasal invasion(P=0.04),seminal vesicle invasion(P=0.02)and pathological T stage.The level of B4GALNT4 protein can be used as an effective indicator for preliminary screening and prognosis of patients with prostate cancer.We constructed stably transfected cell lines of B4GALNT4 knockdown from prostate cancer C4-2 and LNCa P cells with high expression of B4GALNT4.It was found that high expression of glycogene B4GALNT4 can promote the proliferation and invasion of prostate cancer cells and inhibit apoptosis.Further mechanism studies revealed that B4GALNT4 in prostate cancer cells mainly affects the PI3K-Akt signaling pathway.PDK1 protein,as the core factor of the PI3K-Akt signaling pathway,may be the target protein of glycosylation modification by B4GALNT4.The interaction between B4GALNT4 protein and PDK1 protein was also confirmed by immunohistochemistry of prostate cancer tissues and Co-IP experiments of prostate cancer cells.Through exogenous overexpression of PDK1 protein,the negative effect of B4GALNT4 knockdown on the function of prostate cancer cells was reversed.It was found that B4GALNT4 could improve the expression of PDK1 protein,then affect the PI3K-Akt signaling pathway and promote the malignant development of prostate cancer cells.B4GALNT4 as glycosyltransferase can effectively improve the glycosylation level of protein in prostate cancer,that was confirmed through various methods with prostate cancer tissues and cells.It was further confirmed that B4GALNT4 protein increased the expression of PDK1 protein through post-translational modification rather than regulating the expression of PDK1 m RNA in prostate cancer cells.We also found that the glycosyltransferase B4GALNT4 enhanced the stability of PDK1 protein through glycosylation modification to avoid its degradation by proteasome and lysosome.Finally,we found that the amino acid site of PDK1 protein(N531)was modified by the glycosyltransferase B4GALNT4 by using online prediction database of glycosylation site and liquid chromatoc-mass spectrometry.The N531 point mutation will lead to the degradation of PDK1 protein due to its inability to be glycosylated.It further indicated that B4GALNT4 can glycosylate the amino acid site of PDK1 protein N531 to maintain the protein stability and stimulate the downstream pathway to promote the malignant development of prostate cancer cells.Conclusion:We have discovered that glycogene B4GALNT4 can serve as a clinical predictor for prostate cancer screening and prognosis.Furthermore,we have confirmed that glycosyltransferase B4GALNT4 can enhance the stability of PDK1 through glycosylation modification of the PDK1 protein.This,in turn,activates the PI3K-Akt signaling pathway to facilitate the proliferation and invasion of prostate cancer cells.Finally,we have identified a glycoylated amino acid site,N531,in the PDK1 protein that may offer a new approach to developing targeted drugs for prostate cancer patients with high expression of B4GALNT4.In summary,our multi-level studies have established the role and mechanism of glycogene B4GALNT4 in the development and progression of prostate cancer.These findings provide a new theoretical foundation for the diagnosis and treatment of prostate cancer patients,and may offer fresh ideas on how to develop targeted drugs for prostate caner.