The Role And Mechanism Of POU2F1 In Promoting Colon Cancer Cell Proliferation And Oxaliplatin Resistance By Regulating Glucose Metabolic Reprogramming

Purposes:Metabolic reprogramming is an important feature of malignant tumors,and the occurrence and development of tumors are accompanied by abnormal expression and activity of various metabolism-related genes.Studies have shown that the glycolytic activity is abnormally enhanced in solid tumors such as colon cancer,lung cancer,breast cancer,esophageal cancer,gastric cancer,and renal cancer,which is related to tumor resistance to chemotherapy.Therefore,analyzing the functions of key genes that regulate tumor metabolic reprogramming can provide new strategies and targets for the diagnosis and treatment of tumor patients from the perspective of metabolism.POU domain class 2 transcription factor 1（POU2F1）can activate or inhibit the transcriptional expression of a variety of genes and is widely involved in the regulation of biological embryonic development,immune and inflammatory responses,energy metabolism and other processes.Recently,many studies have shown that POU2F1 plays a crucial role in the malignant transformation of cells,and is expected to become a new target for tumor-targeted therapy.However,the researches about the role and mechanism of POU2F1 in colon cancer progression is rare.We found that POU2F1 gene was highly expressed in colon cancer,which may be closely related to the malignant progression of colon cancer via bioinformatics analysis.Meanwhile,the gene enrichment analysis（GSEA）of GSE121842dataset showed that glycolysis and the pentose phosphate pathway（PPP）related genes were mainly enriched in the POU2F1 overexpression group,suggesting that POU2F1 may regulate the glycolysis and PPP to promote colon cancer cell proliferation.Therefore,the purpose of this study aimed to investigate the role and molecular mechanism of POU2F1 in glucose metabolic reprogramming and the proliferation of colon cancer cells.Method:Using GEO,TCGA database,and immunohistochemical staining analysis detected the expression and clinical significance of POU2F1 in colon cancer tissues.By constructing POU2F1 overexpressing/silencing colon cancer cell lines,and using experimental methods such as cell proliferation,clone formation,flow cytometry,glucose consumption,lactate production,tumor formation in nude mice,and ¹⁸F-FDG PET/CT,we analyzed the effect of alterd POU2F1 expression on the proliferation,glucose metabolism phenotype and oxaliplatin resistance of colon cancer cells in vitro and in vivo.q RT-PCR,Western Blot,immunofluorescence,Ch IP,dual-luciferase reporter gene and animal assays were used to explore the molecular mechanism of POU2F1 in regulating glucose metabolic reprogramming and promoting the proliferation and oxaliplatin resistance of colon cancer cells.Result:（1）Bioinformatics analysis of GEO and TCGA databases revealed that POU2F1 was significantly up-regulated in colon cancer tissues,and confirmed in colon cancer clinical samples and cell lines by immunohistochemistry,q RT-PCR and Western blot.Compared with the adjacent normal tissue and normal colon epithelial cells HCo Epi C,POU2F1 was significantly up-regulated in colon cancer tissue and colon cancer cell（P<0.05）.The high POU2F1 expression was significantly correlated with the TNM stage and metastasis（P<0.05）,regardless of gender,age and degree of differentiation.Kaplan-Meier analysis showed that the patients with high POU2F1 expression had significantly worse prognosis and shorter PFS and OS than patients with low POU2F1expression（P<0.05）.（2）We successfully constructed POU2F1 stable overexpressed/silenced colon cancer cell and corresponding blank control cell,respectively.CCK8 and clone formation assays showed that the proliferation ability of HCT116 cell was significantly enhanced after overexpressing POU2F1,and the proliferation ability of SW620 cell was significantly reduced after silencing POU2F1（P<0.05）.Nude mouse xenograft assays also showed that the proliferation ability of SW620 cell was significantly reduced after blocking POU2F1 expression in vivo.Overexpression of POU2F1 can significantly inhibit the apoptosis of colon cancer cells,down-regulate the expression of DNA damage markerγ-H₂AX,and up-regulate the expression of proliferation marker PCNA.Silencing POU2F1 can significantly promote colon cancer cell apoptosis,up-regulate the expression ofγ-H₂AX,down-regulate the expression of PCNA（P<0.05）.（3）Combining GSEA analysis of GSE121842 datset and the results of transcriptional sequencing of SW620/sh-POU2F1 cells revealed that POU2F1 expression was closely related to the glycolysis and pentose phosphate pathways in colon cancer cells.Metabolic phenotype assaysshowed that POU2F1 overexpression promoted glucose consumption and lactate production and G6PD activity,but significantly down-regulated NADP⁺/NADPH ratio in HCT116 cells（P<0.05）,while silencing POU2F1 inhibited glucose consumption and lactate production and down-regulated G6PD activity,but significantly up-regulated NADP⁺/NADPH ratio in SW620 cells（P<0.05）.The results of“recovery experiments”further showed that restoring POU2F1 expression significantly weakened the inhibitory effects of silencing POU2F1 on proliferation,glucose consumption,lactate production,G6PD activity and intracellular NADPH levels in SW620 cell（P<0.05）.In addidtion,the ¹⁸F-FDG PET/CT of nude mouse xenograft exhibited that the maximum standard uptake value（SUV max）of the xenograft tumors of SW620/sh-POU2F1 groupwas significantly lower than the SW620/scramble group（P<0.001）.（4）Bioinformatics analysis by the GEPIA website（http://gepia.cancer-pku.cn/）showed that the expression of POU2F1 was positive associated withhexokinase 2（HK2）,fructose-6-phosphate-2-kinase/fructose-2,6-bisphosphatase 2（PFKFB2）,Adolase A（ALDOA）,Pyruvate kinase M1/2（PKM1/2）,Lactate dehydrogenase A（LDHA）,glucose-6-phosphate dehydrogenase（G6PD）and ribose 5-phosphate isomerase A（RPIA）expression.q RT-PCR and Western blot results showed that the expression levels of HK2,PFKFB2,ALDOA,PKM2,LDHA,G6PD and RPIA were significantly decreased in POU2F1-silenced SW620cell,especially ALDOA（P<0.05）.We also found that POU2F1 and ALDOA had the same expression pattern in colon cancer tissues,nude mouse xenografts and colon cancer cell lines,and the expression of the two is obviously positively correlated.Therefore,we focused on the regulating effect of POU2F1 on ALDOA expression in subsequent studies.（5）ALDOA was significantly up-regulated in colon cancer tissues,and significantly correlated with tumor TNM stage and metastasis（P<0.05）.High ALDOA expression had shorter lower PFS and OS（P<0.05）.Overexpression of ALDOA promoted proliferation,glucose consumption,lactate production,G6PD activity and intracellular NADPH level in HCT116 colon cancer cell（P<0.05）,while inhibition of ALDOA significantly inhibited proliferation,glucose consumption,lactate production,G6PD activity and intracellular NADPH level in SW620 cell（P<0.05）.（6）We found that POU2F1 could directly bind to the-514～-511 bp region of the ALDOA promoter and regulate the transcriptional expression of ALDOA through luciferase reporter gene assay and chromatin immunoprecipitation（Ch IP）assay.The results of cell proliferation and metabolic phenotype assays further showed that silencing ALDOA could significantly attenuate the promoting effects of POU2F1 overexpression on cell proliferation,glucose consumption,lactate production and G6PD activity,and reverse the effects of POU2F1 overexpression on HK2,PRKFB2,PKM2,LDHA,G6PD,RPIA,γ-H₂AX and PCNA expression.（7）Silencing POU2F1 promoted the drug sensitivity of colon cancer cells to oxaliplatin,but POU2F1 overexpression inhibited the drug sensitivity of cells to oxaliplatin.The expressions of POU2F1 and ALDOA in oxaliplatin-resistant cells HCT116/L were significantly higher than those in HCT116 cells（P<0.05）.We constructed oxaliplatin-resistant HCT116/L cell silencing POU2F1 expression with or without overexpressing ALDOA.We found that silencing POU2F1 inhibited the proliferation,glucose consumption,lactate production and G6PD activity while increased the NADP⁺/NADPH ratio and ROS level in oxaliplatin-resistant HCT116/L cell,but overexpression of ALDOA significantly reversed the effect of POU2F1 silencing on the above effects（P<0.05）.In addition,we observed that the expressions of HK2,PRKFB2,PKM2,LDHA,G6PD,and RPIA were significantly higher in HCT116/L cells than HCT116 cells,and these molecules expressions significantly decreased after POU2F1 inhibition but this effect could be reversed by overexpression of ALDOA.Conclusion1.POU2F1 is highly expressed in colon cancer tissue and is positively correlated with the poor prognosis of colon cancer patients.2.POU2F1 is involved in the regulation of glucose metabolic reprogramming,and promotes the proliferation and oxaliplatin resistance in colon cancer cells.3.POU2F1 can directly regulate the transcriptional expression of ALDOA,and the POU2F1-ALDOA axis promotes colon cancer cell proliferation and oxaliplatin resistance via regulating the reprogramming of glucose metabolism.