Interactions of tumor necrosis factor receptor-associated factor 4 and pyruvate kinase muscle isoform 2 promote malignant behavior and aerobic glycolysis in colorectal cancer cells.

OBJECTIVE

Colorectal cancer (CRC) is a malignant tumor of the digestive system, and the main causes of death are metastasis and recurrence. Tumor necrosis factor receptor-associated factor 4 (TRAF4) is associated with the development of various tumors, but its role in CRC development is limited, especially glycolysis. Therefore, TRAF4's role in the regulation of cell malignant behavior and glycolysis and its specific mechanism were explored in CRC.

MATERIAL AND METHODS

The TRAF4 or pyruvate kinase muscle isoform 2 (PKM2) gene expression was inhibited or promoted by short hairpin ribonucleic acid (sh- RNA) or overexpression (oe) plasmids in Lovo cells. Transfection efficiency was detected by Western blot and real-time quantitative polymerase chain reaction. Cell growth and colony formation were assessed using 5-ethynyl-2'-deoxyuridine and clone formation assays, respectively, and cell migration and invasion ability were observed by scratch healing and Transwell assay. Glucose uptake and lactate production were measured with a kit and used in evaluating the glycolysis capacities of the cells. The levels of TRAF4, PKM2, and glycolytic-related and wingless-type (Wnt)/beta (β)-catenin pathway-related proteins were detected by Western blot, and co-immunoprecipitation (Co-IP) verified TRAF4 and PKM2 interaction in CRC cells.

RESULTS

TRAF4 expression increased in CRC cell lines ( 0.05, 0.001, 0.0001). After sh-TRAF4, oeTRAF4, or oe-PKM2 transfection, TRAF4 or PKM2 expression levels in the Lovo cells decreased or increased ( 0.05, 0.01, 0.001, and 0.0001). TRAF4 knockdown inhibited cell malignant behavior, glucose uptake, lactate production, and glucose transporter type 1 (GLUT1), hexokinase 2 (HK2), PKM2, and lactate dehydrogenase A (LDHA) protein expression levels in CRC cells ( 0.01, 0.001, 0.0001). Co-IP experiment showed that TRAF4 was bound to PKM2. PKM2 protein level decreased after TRAF4 knockdown ( 0.0001), and PKM2 protein expression increased when TRAF4 was overexpressed ( 0.001). PKM2 overexpression offset the effect of TRAF4 knockdown on cell malignant behavior and aerobic glycolysis ( 0.05, 0.01, 0.001, and 0.0001). Moreover, Wnt/β-catenin pathway proteins were inhibited after TRAF4 knockdown and were restored by PKM2 overexpression ( 0.01 and 0.0001). Notably, the effects of TRAF4 or PKM2 overexpression on cell malignant behavior, glucose uptake, lactate production, and GLUT1, PKM2, HK2, and LDHA protein expression levels were partially offset by the Wnt/β-catenin signaling suppressor XAV939 ( 0.05, 0.01, 0.001, and 0.0001).

CONCLUSION

TRAF4 and PKM2 are associated with CRC development. TRAF4 binds to PKM2 and promotes CRC malignant behavior and glycolysis through the Wnt/β-catenin signaling pathway.