Figure 5.

APOL3 promotes the ubiquitylation-related degradation of LDHA and the antitumor ability of CD8+ T cells in CRC in vitro and in vivo. (A) Reciprocal IP-western analysis revealed that APOL3 directly interacted with LDHA; (B) we pretreated cells with CHX to determine the stability of LDHA and results showed that the half-life periods of LDHA were much longer in CRC cells with APOL3 knock-out compared to control cells; (C) Western blotting analysis found that LDHA was significantly increased in APOL3-knockout RKO cells, whereas suppressed LDHA levels were seen in cells with APOL3 overexpression, and this effect was diminished by treatment of MG132; (D) Co-IP using total K48-linked poly-ubiquitination antibody identified that overexpression of APOL3 promoted K48-linked ubiquitination of LDHA in HT29 and RKO cells; (E) IF analysis found APOL3 signal significantly overlapped with LDHA on cytoplasm in CRC cells; (F) In vivo analysis with subcutaneous xenograft models were constructed, APOL3-knockout of HCT116 cells promoted tumor growth when compared to their negative controls (left top); concentration of Fe²⁺ and MDA (%) in nude mice were not significantly altered (right top); with the treatment of ferroptosis inducer RSL3, APOL3 knockout significantly led to significant increase of tumor volume in vivo (left down); knock-out of APOL3 significantly inhibits the expression of Fe²⁺ and percent of MDA (right down); (G) In vitro, mouse-derived CT26 colon cells were indirectly co-cultured with CD8+ T cells with different treatment groups, overexpression of APOL3 markedly increased the efficacy of CD8+ T cell with higher lactate concentration and IFNG expression and can be diminished after co-overexpression of APOL3 and LDHA; (H) subcutaneous xenograft models of CT26 were created and knock-out of APOL3 promotes CT26 growth in both nude mice and BALB/c mice; the tumor volume difference was larger in BALB/c immune-competent mice; (I) APOL3-LDHA axis effect on BALB/c mice is shown, knockout of LDHA partly diminished tumor-promoting effect of APOL3-KO in CT26 cells with slight increase of IFNG mRNA expression and IFNγ+ CD8+ T cells; (J) IF analysis revealed similar results. Abbreviations: CRC, colorectal cancer; CHX, cycloheximide; IP, immunoprecipitation; IF, immunofluorescence; Co-IP, co-immunoprecipitation.