Phosphoglycerate Dehydrogenase (PHGDH) Inhibits Ferroptosis And Promot Malignant Progression By Upregulating SLC7A11 In Bladder Cancer

Background:Bladder urothelial carcinoma(bladder cancer)is one of the most common clinical malignant tumors.There are about 573,278 new cases of bladder cancer worldwide each year,and about 212,536 people die from bladder cancer.In china,bladder cancer is a common tumor of the urinary system,with an annual incidence rate of 5.80/100,000 and a mortality rate of 2.37/100,000.About 75% of patients with bladder cancer are non-muscle invasive bladder cancer(NMIBC),and muscle-invasive bladder cancer accounts for about 25%.The surgical method is radical cystectomy and urinary diversion.The 5-year survival rate after surgery is only 40-60%.About 50% of patients develop metastasis and eventually die.Therefore,it is urgent to elucidate the malignant progression mechanism of bladder cancer and find new therapeutic targets to curb the progression of bladder cancer and improve the prognosis of bladder cancer patients.Serine metabolism is an amino acid metabolic pathway that plays an important role in the synthesis of biological macromolecules.Serine metabolic reprogramming plays a key role in tumorigenesis and development,among which phosphoglycerate dehydrogenase(PHGDH)is a key enzyme affecting serine metabolism.PHGDH can catalyze 3-phosphoglycerate to 3-phosphodehydropyruvate,which is enzymatically converted to serine.In recent years,studies have found that PHGDH has received more and more attention in the occurrence and development of tumors.In this regard,we found that PHGDH,which is highly expressed in bladder cancer,plays a key role in regulating cell ferroptosis Ferroptosis is a new programmed cell death method discovered in recent years,which is cell death caused by iron-dependent excessive oxidation of polyunsaturated fatty acids.System Xc-is a cystine transporter on the cell membrane surface,consisting of a light chain subunit SLC7A11 and a heavy chain subunit SLC3A2.SLC7A11 is involved in the transport of cystine into cells for the synthesis of glutathione to resist cellular oxidative stress and is a key negative regulator of ferroptosis.Ferroptosis can inhibit tumor growth and play a key role in the occurrence and development of various tumors.However,the expression of PHGDH in bladder cancer and whether it regulates ferroptosis has not been reported.Methods:1.Firstly,the tissue samples of 90 bladder cancer patients in our center were sequenced to analyze the most significant differentially expressed pathway and the expression of PHGDH,and further analyze the correlation between PHGDH expression and prognosis in TCGA,GEO and other databases.Next,the expression of PHGDH in bladder cancer patients’ cancer tissues and normal tissues was analyzed by quantitative real time-PCR(q PCR),Western Blot(WB)and immunohistochemical(IHC)experiments.Likewise,we validated the expression of PHGDH in bladder cancer cell lines(RT4,T24,UMUC-3,J82 and 5637,etc.)and immortalized urothelial cells(SVHUC-1)by q PCR and WB.2.Through the above experiments,the cell line T24 with relatively high PHGDH expression was selected to construct a stable knockdown of PHGDH(sh-PHGDH)cell line,and the knockdown efficiency of PHGDH was detected by q PCR and WB.The RT4 cell line with relatively low PHGDH expression was selected to construct a stable cell line overexpressing PHGDH(oe-PHGDH),and the overexpression efficiency of PHGDH was verified by q PCR and WB.3.To further verify the biological function of PHGDH in bladder cancer.The CCK8 assay,plate cloning and EDU experiments were used to detect the proliferation ability of PHGDH;the effect of PHGDH on the migration and invasion was detected by transwell chamber,and flow cytometry was used to detect the effects of apoptosis.In vivo,bladder cancer cell lines with stable knockdown/overexpression of PHGDH and negative control were injected subcutaneously into nude mice to compare the in vivo proliferation ability of the two groups of cells.4.For RT4 cell line overexpressing PHGDH,it was found that SLC7A11,a key factor of ferroptosis,was significantly changed.The changes of related pathways after overexpression of PHGDH were further verified by GESA and enrichment analysis.The expression of SLC7A11 after knockdown or overexpression of PHGDH was detected by q PCR and WB,and the expression of SLC7A11 in subcutaneous tumors was verified by IHC.Other death mode inhibitors(such as apoptosis inhibitors,autophagy inhibitors,etc.)add to PHGDH knockdown cell lines,and we verify and exclude whether only ferroptosis affects cell viability through CCK8 experiments.After using ferroptosis activator to overexpress PHGDH,the CCK8 assay was used to verify whether the cell viability could be restored.Cell ferroptosis was assessed by C11 probe detection of lipid ROS.Cell ferroptosis was also assessed by observing the effect of PHGDH on mitochondria by bioelectron microscopy.5.CO-IP and mass spectrometry were used to screen and analyze the proteins that PHGDH may bind,and find the differentially expressed protein PCBP2.Immunofluorescence co-localization experiments determined whether the localization of the two is the same.PCBP2 expression after knockdown of PHGDH were verified by q PCR and WB.The proteasome inhibitor MG132 was added to verify the expression of PCBP2 and protein ubiquitination after knockdown of PHGDH.The expression of PCBP2-specific ubiquitination levels were verified by CO-IP experiments by adding MG132.6.Using the antibody of PCBP2,the binding of PCBP2 to SLC7A11 was verified by RIP experiment.SLC7A11 m RNA stability after knockdown of PCBP2 were assessed by actinomycin experiments.Further through rescue experiments,PCBP2 was overexpressed in sh PHGDH,and the expression of SLC7A11 was verified by q PCR and WB.At the same time,the cell proliferation ability after the experiment were rescued by plate cloning,CCK8 and other experiments.Expression of SLC7A11 in tumor tissues of bladder cancer patients was verified by IHC.The role of PHGDH+SLC7A11 in evaluating the prognosis of patients was analyzed by the data of our center and TCGA data.7.To evaluate the effect of PHGDH specific inhibitor NCT-502 in bladder cancer cells.The IC50 of NCT-502 in bladder cancer cell lines was firstly determined by CCK8.A mouse subcutaneous tumor-bearing model was constructed,and NCT-502 was added to evaluate the effect of NCT-502 on bladder cancer in vivo.The expressions of PHGDH and SLC7A11 were detected by IHC experiments.The tissue organoids of bladder cancer patients were constructed and NCT-502 was added to evaluate the effect of NCT-502 on the proliferation of bladder cancer organoids.Plate cloning,bio-electron microscopy,etc.verified the effect of NCT-502 on ferroptosis of bladder cancer cells.Results:1.In the RNA sequencing analysis of 90 bladder cancer patients,gene set enrichment analysis showed that the top 10 biological pathways that were highly enriched in high-grade bladder cancer patients,serine metabolism was the only amino acid metabolism-related pathway,while,PHGDH was highly expressed in high-grade bladder cancer patients and muscle-invasive bladder cancer patients;further analysis of prognosis showed that the DFS time of bladder cancer patients with high PHGDH expression was significantly shorter than that of patients with low PHGDH expression.The TCGA database showed that the expression of PHGDH in bladder cancer tumor tissues was significantly higher than that in normal tissues;the OS time of bladder cancer patients with high PHGDH expression was significantly shorter than that of patients with low PHGDH expression.A multi-database prognostic meta-analysis of PHGDH showed that high expression of PHGDH gene was a risk factor for bladder cancer patients.Furthermore,in bladder cancer patient samples and cell lines,q PCR,WB and IHC confirmed that PHGDH was significantly high expressed2.Construction of bladder cancer cell lines with knockdown and overexpression of PHGDH.The efficiency of knockdown and overexpression of PHGDH was verified by q PCR and WB.Through CCK8 experiment,plate cloning and EDU experiments,it was found that the proliferation ability of sh-PHGDH was significantly decreased,whereas the proliferation ability of oe-PHGDH was significantly increased.Transwell results showed that after sh-PHGDH,the migration and invasion ability of cells decreased,whereas the migration and invasion ability of oe-PHGDH group increased significantly.The results of flow cytometry showed no significant changes in apoptosis whether PHGDH was knocked down or overexpressed.In vivo experiments showed that the proliferation of subcutaneous tumors in mice after knockdown of PHGDH slowed down,and the proliferation of subcutaneous tumors in mice was significantly increased after overexpression of PHGDH.3.RNA sequencing of RT4 cell lines overexpressing PHGDH showed that the expression of SLC7A11 was significantly increased(p<0.05,Fold Change>2).GESA and pathway enrichment analysis showed that after overexpressing PHGDH ferroptosis-related pathways were significantly changed.q PCR and WB showed that the level of SLC7A11 was significantly decreased in the sh-PHGDH group,and the expression of SLC7A11 was significantly increased in the oe-PHGDH group.The same conclusion was obtained by IHC verification of SLC7A11 expression in mouse subcutaneous tumors.After adding autophagy inhibitor CQ,necroptosis NEC-1 and ferroptosis inhibitor Fer-1 in sh-PHGDH group,it was found that only Fer-1 increased cell viability.Adding ferroptosis agonists(Erastin and RSL3)to the oe-PHGDH group,it was found that both could rescue the increased cell viability caused by overexpression.The C11 probe showed that ferroptosis was significantly increased in the sh-PHGDH group.Bio-electron microscopy showed that the mitochondria of cells in the shPHGDH group were smaller than normal mitochondria,the mitochondrial membrane density was increased,and the mitochondrial cristae were decreased.4.After the CO-IP experiment,silver staining found that there was a significantly different protein at 43 Kda.The mass spectrometry analysis found that the protein might be the RNA-binding protein PCBP2.Immunofluorescence co-localization showed the same localization of PHGDH and PCBP2.q PCR found that there was no significant change in PCBP2 in sh PHGDH,and Western Blot showed that PCBP2 was significantly decreased after knockdown of PHGDH.It is suggested that there may be posttranslational modification of PCBP2.Adding the proteasome inhibitor MG132 to the sh PHGDH group rescued the expression of PCBP2 in sh-PHGDH,and found that the level of total ubiquitinated protein was significantly increased in the shPHGDH+MG132 group.After the addition of MG132,the specific ubiquitination level of PCBP2 was also significantly increased by CO-IP experiments.5.Using the antibody of PCBP2,it was verified that PCBP2 can bind to SLC7A11 by RIP experiment.Actinomycin D experiment showed the increased the stability of SLC7A11 m RNA after knockdown of PCBP2.Rescue experiments showed that the expression of SLC7A11 was increased in the sh PHGDH+oe PCBP2 group.Plate cloning,CCK8 and other experiments confirmed that after the rescue experiment,the cell proliferation ability of the sh PHGDH+oe PCBP2 group was significantly increased.IHC demonstrated that the expression of SLC7A11 was significantly increased in tumor tissues of bladder cancer patients.Bioinformatic analysis of the data of our center and TCGA data showed that the effect of PHGDH+SLC7A11 in predicting the prognosis of bladder cancer patients was significantly stronger than that of TNM staging,and the construction of nomogra based on PHGDH+SLC7A11 could better predict and evaluate the prognosis of patients.6.IC50 test confirmed that the IC50 value of NCT-502 against bladder cancer cell line was 2.0μmol.In vivo experiments confirmed that NCT-502 could significantly slow down the growth of subcutaneous tumors in mice.IHC showed significantly reduced expression of PHGDH and SLC7A11 in mouse subcutaneous tumors treated with NCT-502.CCK8,plate clone showed that both NCT-502 and sh-PHGDH significantly reduced cell proliferation.Bio-electron microscopy showed that the mitochondria of the NCT-502 group were smaller than normal mitochondria,the mitochondrial membrane density was increased,and the mitochondrial cristae were decreased.Constructing bladder cancer patient organoids,adding NCT-502,and culturing for five days showed a slowdown in organoid growth.Conclusion:1.The RNA sequencing results of bladder cancer patients show that serine metabolism is abnormal in bladder cancer,and its key enzyme PHGDH is highly expressed,which is closely related to poor prognosis2.Knockdown of PHGDH can significantly affect the biological functions of bladder cancer,such as proliferation,but has no effect on apoptosis3.PHGDH sequencing showed that SLC7A11 changed significantly,and PHGDH could mediate ferroptosis by regulating the expression of SLC7A11.4.PHGDH binds to PCBP2 and affects its ubiquitination.As an RNA-binding protein,PCBP2 can regulate the stability of SLC7A11.5.PHGDH combined with SLC7A11 is better than TNM staging in predicting the prognosis of patients with bladder cancer6.PHGDH target inhibitor NCT-502 can affect the ferroptosis of bladder cancer cells and inhibit the malignant progression of bladder cancer.