Overexpression Of HepaCAM Inhibits Reprogramming Of Glutamine Metabolism And Proliferation Of Prostate Cancer By Suppressing PIK3CA

Objective: To predict the expression of hepatocyte adhesion molecule(HepaCAM),and phosphatidylinositol-4,5-diphosphate3-kinase catalytic subunit α(PIK3CA)in tumor and tissue,and their correlation with clinicopathological parameters.To explore the concentration of common amino acids in blood,and to study the specific mechanism of HepaCAM on glutamine(Gln)metabolic reprogramming and cell proliferation in prostate cancer(PCa)cells.Methods: Bioinformatics database was used to analyze the m RNA expression of HepaCAM and PIK3CA in each dataset,the correlation between m RNA expression and the difference of expression between normal samples and PCa samples,and the levels of amino acids in blood samples were detected by liquid chromatography-tandem mass spectrometry and gas chromatography-mass spectrometry.The expression levels of HepaCAM and PIK3CA in Benign prostatic hyperplasia(BPH)and PCa tissues were detected by immunohistochemical staining and H&E technique,and the m RNA and protein expressions of HepaCAM,PIK3CA,glutamine metabolism-related genes and proliferation related genes in PCa cells were detected by RT-q PCR and Western blot after the treatment of adenovirus transfection,deprivation of glutamine and PIK3CA inhibitor respectively.Clone formation assay was used to detect the clone ability of PCa cells,and cell counting kit(CCK)-8 assay was used to detect the proliferative activity of cells.The distribution of cell cycle was analyzed by Flow cytometry.Glutamine metabolism-related genes include glutaminase(GLS),solute carrier family 1 member 5(SLC1A5),and proliferation-related genes include cyclin D1 and proliferating cell nuclear antigen(PCNA).Results:(1)Bioinformatics analysis emerged a significant difference to expression of HepaCAM and PIK3CA between the normal and PCa patients,which was verified at the clinical tissue,and correlated with the Gleason score of clinical related parameters.(2)The levels of glutamine in blood of PCa patients were significantly lower than the control group and converted to glutamate(Glu).(3)Transfection of adenovirus of HepaCAM(Ad-HepaCAM)could overexpress HepaCAM and down-regulate the expression of PIK3CA,glutamine metabolism-related genes and proliferation-related genes in PCa cells,which could also inhibit the clone formation ability and proliferation activity of PCa cells.(4)Deprivation of glutamine in cell culture conditions can cause a certain degree of stress resistance in cell metabolism,which can be eliminated by overexpression of HepaCAM,and the combined treatment of both can further inhibit the ability of cell proliferation.(5)The metabolic stress resistance induced by the deprivation of glutamine was significantly counteracted by the addition of PIK3CA inhibitor alpelisib,and the down-regulation of PIK3CA,GLS,SLC1A5 and proliferation-related genes induced by adenovirus overexpression of HepaCAM was enhanced,and the inhibitory effect was the most obvious after the combination between them.Additionally,Deprivation of glutamine and alpelisib had no significant effect on HepaCAM.(6)The combination of alpelisib and overexpressed HepaCAM could significantly inhibit the proliferation and metabolism of PCa cells and block the cell cycle.Conclusion: There is a significant difference in the expression of HepaCAM and PIK3CA between the normal and PCa patients.There is a significant correlation between HepaCAM and PIK3CA in PCa tissues,which is closely related to Gleason score.The concentration of glutamine in blood of PCa patients is abnormally decreased.Overexpression of HepaCAM can block glutamine metabolic reprogramming and cell proliferation,which is achieved by regulating the expression of PIK3CA.In addition,deprivation of glutamine could induce metabolic stress resistance in PCa cells,and HepaCAM could eliminate this phenomenon by inhibiting the expression of PIK3CA.