Fig 2: PKCα inhibition abolishes antitumor activity in immunocompetent mice. (A) Expression data for PRKCA were extracted from published gene expression data on breast cancer tissues available in the GEO database (GSE12790) (n = 5–17). Relative PRKCA transcript levels were measured using the 213093_at probe. (B) Expression data for PRKCA were extracted from published gene expression data on breast cancer cell lines available in the GEO database (GSE12790). Relative PRKCA transcript levels were measured using the 213093_at. (C–F) NCG mice (C, D) or BALB/c mice (E, F) were subcutaneously (s.c.) inoculated into mammary fat pads, and 4T1 cells were stably transfected with the indicated shRNA directed against Prkca or with a scrambled sequence as a control. The graphs depict the tumor volumes (C, E) or tumor weights (D, F) of mice given s.c. injections of 5 × 105 of the indicated cells (n = 6). (G–J) NCG mice (G, H) or BALB/c mice (I, J) were subcutaneously (s.c.) injected into mammary fat pads with 5 × 105 4T1 cells and then treated with the PKCα inhibitor Go6976 or vehicle. The graphs depict the tumor volumes (G, I) or tumor weights (H, J) (n = 6). (K) GSEA demonstrating the enrichment of gene sets related to hallmark genes in the ranked gene list of the PRKCA-High group (the top 10th percentile, n = 122) versus the PRKCA-Low group (the bottom 10th percentile, n = 122) of breast cancer patients expressing PRKCA from the TCGA database (n = 1218). The top ten pathways, together with their enrichment scores, are depicted. NES, normalized enrichment score. (L, M) GSEA demonstrating the enrichment of gene sets related to the TCR pathway (L) and interferon-gamma response (M) in the ranked gene list of the top 10th percentile (n = 122) versus the bottom 10th percentile (n = 122) of breast cancer patients expressing PRKCA from the TCGA database (n = 1218). NES, normalized enrichment score. FDR, false discovery rate. The results are presented as the mean ± SEM. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ns means not significant.

Fig 3: PKCα-mediated PD-L1 phosphorylation promotes PD-L1 degradation through β-TRCP. (A) Pearson's correlation analysis between PRKCA and CD274 mRNA expression across TCGA breast carcinoma (BRCA) datasets retrieved from the GEPIA website. (B) PD-L1 (CD274) and PKCα (PRKCA) mRNA expression in MDA-MB-231 cells transfected with Ctrl-shRNA or PKCα-shRNAs. (C) Effect of PKCα depletion on PD-L1 degradation in vitro. MDA-MB-231 cells transfected with Ctrl-shRNA or PKCα-shRNAs were incubated with 20 μmol/L cycloheximide (CHX) for the indicated times. (D) Co-IP of HEK293 cell lysates using NRS or an anti-HA Ab was performed for IB analyses probed with antibodies specific for phosphoserine, phosphothreonine, DDK, or HA. (E) Control or PKCα-DDK-expressing MDA-MB-468 cells were incubated with 200 nmol/L bafilomycin or 10 μmol/L MG132 for 8 h. The indicated proteins were analyzed using IB analysis. (F) Venn diagram indicating the overlap of PKCα-mediated PD-L1 phosphoprotein sites predicted by NetPhos 3.1 and GPS5.0. (G) Mapping the PD-L1 regions that bind to PKCα (upper). PKCα-DDK-expressing HEK293T cells were cotransfected with the indicated PD-L1-ECD-GFP or PD-L1-TM-ICD-GFP constructs. Cell extracts were immunoprecipitated with NRS or an anti-DDK Ab and then probed with an anti-GFP Ab or an anti-DDK Ab (bottom). (H) Lysates from HEK293T cells transfected with the indicated plasmids were immunoprecipitated with NRS or an anti-Myc Ab. IB shows the phosphorylation of PD-L1 and the PD-L1 S184A mutant. (I) Effect of β-TRCP and PKCα on PD-L1 ubiquitination. HEK293 cells transfected with the indicated plasmids were immunoprecipitated with NRS or an anti-HA Ab. The ubiquitination of PD-L1 was detected by IB. (J) IB showing the expression of PD-L1, PKCα, and β-TRCP in MDA-MB-468 cells transfected with Ctrl-shRNA or β-TRCP-shRNAs, as indicated at the top. The results are presented as the mean ± SEM. ∗∗∗P < 0.001.

Fig 4: PKCα interacts with PD-L1 and suppresses PD-L1 expression. (A) Immunoblotting (IB) and quantitative analyses of the expression of immune checkpoints in MDA-MB-231 cells transfected with Ctrl-shRNA or PKCα-shRNAs, as indicated at the top. (B) Coimmunoprecipitation (co-IP) of MDA-MB-231 cell lysates using normal rabbit serum (NRS) or an anti-PD-L1 antibody (Ab) was performed for immunoblotting analyses probed with an antibody specific for PKCα or PD-L1. (C) The colocalization of PKCα with PD-L1 was detected by immunofluorescence staining of MDA-MB-231 cells (n = 6) and primary breast tumor tissues (n = 20). The quantification of PKCα/PD-L1 colocalization is shown as the Pearson's coefficient. (D) Quantitative real-time PCR analysis of PRKCA in MDA-MB-231, BT549, and MDA-MB-468 cells. (E) IB and quantitative analyses of protein lysates from MDA-MB-468 or BT549 cells (as indicated at the bottom) transfected with the indicated overexpression plasmids or shRNAs (as indicated at the top). (F) The expression of PD-L1 in allograft tumors from mice implanted with 4T1 cells transfected with either Ctrl-shRNA or Pkcα-shRNAs was detected by immunofluorescence staining. (G) IB and quantitative analyses of the expression of PD-L1 and PKCα in MDA-MB-231 cells that received three different PKCα inhibitor treatments, as indicated at the top. (H) PD-1 binding assay in MDA-MB-231 cells transfected with Ctrl-shRNA or PKCα-shRNAs, as indicated at the top. (I, J) Representative plots (I) and statistical analyses (J) of the results of the T-cell-mediated tumor cell killing assay in 4T1 OVA cells transfected with the indicated plasmids. The results are shown as the percentages of 7-AAD+ tumor cells. The results are presented as the mean ± SEM. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ns means not significant.

Fig 5: PKCα inhibition synergistically enhances anti-PD-L1 mAb activity in breast cancer cells. (A–C) BALB/c mice implanted with 4T1 cells transfected with either Ctrl-shRNA or Pkca-shRNA1 were treated with an anti-PD-L1 mAb or an IgG2b isotype control (n = 6). (A) A schematic view of the treatment plan. (B) Tumor volumes over time (days). (C) Weights of the tumors excised from each group. (D–F) Growth and weight of 4T1 tumors in female BALB/c mice after treatment with a single agent or in combination (n = 6). (D) A schematic view of the treatment plan. (E) Tumor volumes over time (days). (F) Weights of the tumors excised from each group. (G, H) The expression of PD-L1 and CD8α in allograft tumors from mice implanted with 4T1 cells transfected with either Ctrl-shRNA or Pkcα-shRNA1 was detected by immunofluorescence staining. Representative images with 50 μm scale bars (G) and statistical quantification (H) are shown. (I) Flow cytometric quantification of the percentages of IFNγ+ CD8+ cells (left) and granzyme B+ CD8+ cells (right) among CD8+ T cells from allograft tumors derived from 4T1 tumor-bearing mice treated with a single agent or combination therapy. (J) IB (top) and quantitative analyses (bottom) of ERK and AKT signaling, PD-L1, and PKCα from 4T1 tumor-bearing mice treated with a single agent or combination therapy. The results are presented as the mean ± SEM. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ns means not significant.