Correlation between KAT6A and PD-L1 expression and role of KAT6A in colorectal cancer

Figure 1 KAT6A enhances the proliferation, invasion, and migration of colorectal cancer cells. SW480 and SW620 cells were transfected with KAT6A siRNAs. A: Protein level of KAT6A was measured by Western blotting assay; B: Cell viability of SW480 and SW620 cells was measured by cell counting kit-8 assay; C: Cell migration and invasion were detected by using Transwell experiment; D: Protein levels of E-cadherin and Vimentin were detected by Western blotting assay. ^bP < 0.01.

Figure 2 KAT6A induces immune evasion in a colorectal cancer cell/T cell co-culture system. Colorectal cancer (CRC) cells treated with KAT6A siRNAs were co-cultured with unstimulated or anti-CD3/CD28-activated T cells (5 μg/mL each antibody, 72 hours of stimulation). A: T cell-mediated apoptosis of CRC cells was measured by flow cytometry; B: The expression of GZMB and perforin in T cells was analyzed by Western blot; C: The levels of PD-L1 in CRC cells were analyzed by Western blotting analysis.

Figure 3 KAT6A-mediated H3K23pr activates PD-L1 expression in colorectal cancer cells. SW480 and SW620 cells were transfected with KAT6A siRNAs. A: The levels of H3K23pr were detected by Western blotting assay; B: The enrichment of H3K23pr on PD-L1 promoter was analyzed by ChIP-PCR; C: The mRNA expression of PD-L1 was measured by quantitative PCR. ^bP < 0.01.

Figure 4 KAT6A contributes to progression and immune evasion of colorectal cancer cells by PD-L1. SW480 and SW620 cells were transfected with KAT6A siRNAs, or co-treated with PD-L1 overexpression plasmids. A: Cell migration and invasion were detected by using Transwell experiment; B: The expression of GZMB and perforin in T cells was detected Western blotting assay; C: The levels of PD-L1 in colorectal cancer (CRC) cells were analyzed by Western blotting assay; D: CRC cells were co-cultured with unstimulated or activated T cells. The T cell-mediated apoptosis on CRC cells was measured by flow cytometry.

Figure 5 Pharmacological inhibition of KAT6A recapitulates genetic knockdown phenotypes. A: Dose-dependent suppression of H3K23pr and PD-L1 by WM-3835. Western blot (left) and densitometric quantification (right) showed H3K23pr and PD-L1 protein levels in SW480/SW620 cells treated with WM-3835 (0, 5, or 10 μM) for 48 hours; B: PD-L1 transcriptional downregulation as revealed by quantitative PCR analysis of PD-L1 mRNA levels; C: Enhanced T cell-mediated apoptosis as shown by flow cytometry quantification of apoptotic colorectal cancer cells co-cultured with activated T cells; D: Activation of T cell cytotoxic markers demonstrated by Western blot analysis of GZMB and perforin in T cells. Data represent the mean ± SEM (n = 3). ^aP < 0.05, ^bP < 0.01, ^cP < 0.001 vs DMSO (one-way ANOVA with Dunnett’s test). siKAT6A data from Figures 1 and 2 are shown as positive controls.

Figure 6 Epigenetic editing of H3K23pr at PD-L1 promoter directly modulates immune evasion. A: CRISPR/dCas9-mediated H3K23pr editing efficiency evaluated by ChIP-qPCR analysis of H3K23pr enrichment at PD-L1 promoter in SW480 cells transfected with dCas9-p300 or dCas9-LSD1 and PD-L1-targeting sgRNA (5’-GCGCTCCAGCTCCGACCTGA-3’). Non-targeting sgRNA and empty vector served as controls; B: PD-L1 transcriptional regulation shown by quantitative PCR analysis of PD-L1 mRNA levels; C: Functional validation of immune evasion. Transwell invasion (left) and T cell-mediated apoptosis (right) assays; D: Rescue experiment in KAT6A-depleted cells. PD-L1 mRNA (left) and apoptosis (right) in siKAT6A cells co-transfected with dCas9-p300. Data represent the mean ± SEM (n = 3). ^cP < 0.001 vs non-targeting sgRNA (one-way ANOVA with Dunnett’s test). ^dP < 0.01.