Effect of ZCRB1 on the glycolysis via PKM2-HIF1α axis and promotion of immune tolerance in nasopharyngeal carcinoma.

130 Background: Nasopharyngeal carcinoma (NPC) is characterized by abundant lymphocyte infiltration. The failure of immune checkpoint inhibitors in NPC may be related to immune tolerance. Methods: Bioinformatics analysis was performed to identify the correlation between the ZCRB1 mRNA level and immune cell infiltration in NPC tissues. Quantitative real time PCR and immunoblotting were used to determine the mRNA and protein expression, respectively. Functional experiments (CCK8, colony formation assay, cell migration assay and tumor xenograft) were used to explore the effect of ZCRB1 on the malignant phenotype in NPC cells. Glucose and lactate measurement assays as well as Seahorse XF glycolysis stress assay, were conducted to investigate the effects of ZCRB1 on glycolysis in NPC cells. RNA sequencing, QPCR and minigene analysis were used to confirm the splicing switch between PKM1 and PKM2. Luciferase assay was performed to detect the transcription activity of HIF1-α. Finally, correlation analysis between the ZCRB1 expression and glycolysis level was performed to in NPC samples. Results: The expression of ZCRB1 was negatively correlated with checkpoint-related genes and immune regulation-related gene sets in head and neck cancer. Likewise, in NPC, the expression of ZCRB1 was negatively correlated with lymphocyte infiltration, tumor immune microenvironment score and tumor-infiltrating CD4+ T cells, CD8+ T cells, and dendritic cells. Low expression of ZCRB1 was associated with better prognosis. Functional assays demonstrated that ZCRB1 significantly promoted NPC cells growth, clonogenicity, migratory capacity and glycolysis. Mechanistically, ZCRB1 was mainly involved in the hypoxic signaling pathway. Cells with ZCRB1 downregulation downregulated glycolysis-related genes and significantly reduced the transcriptional activity of HIF-1α. Moreover, minigene assays showed that the intron 9 region spliced by PKM in cells with ZCRB1 downregulation. Finally, bioinformatics analysis showed that the mRNA expression of ZCRB1 was positively correlated with the immune infiltration score significantly during glycolysis. Conclusions: ZCRB1 promotes the growth and migration of nasopharyngeal carcinoma cells and is associated with immune tolerance. Further, ZCRB1 accelerates the glycolysis via enhancing the transcriptional activity of HIF-1α through increasing PKM2 isoform splicing, providing a scientific basis for tumor metabolic reprogramming to reshape the immune microenvironment.