AXL/PLCγ1/PKCα as a Central Axis for PD‐L1 Regulation in Triple‐Negative Breast Cancer

Abstract

Breast cancer is the most prevalent malignancy in women globally, accounting for 12% of all new annual cancer cases worldwide. Triple-negative breast cancer (TNBC), which is ER, PR and HER2 negative, represents 10-15% of breast cancers and displays an aggressive metastatic phenotype, with limited treatment options and poor patient prognosis. TNBC is associated with high expression levels of immune checkpoint programmed-death ligand 1 (PD-L1) compared with other breast cancer subtypes, and it is therefore amenable to immune checkpoint therapy. In this study, we analyzed the role of diacylglycerol (DAG)-regulated protein kinase C alpha (PKCα) in the control of PD-L1 expression in TNBC. Dataset analysis of 50 breast cancer cell lines revealed a prominent up-regulation of PKCα in TNBC, particularly in TNBC-B subtype, with a concomitant down-regulation of protein kinase C delta (PKCδ). This finding was authenticated in the TCGA-BRCA database. A significant correlation was found between PKCα expression and EMT markers, i.e., up-regulation of vimentin, Zeb1, TWIST1, SNAI2 and AXL, and E-cadherin down-regulation. High PKCα expression was also observed in TNBC patient-derived xenografts (PDXs). Immunofluorescence analysis revealed significant PKCα endogenous levels in the TNBC B cell line BT549, with strong "peripheral staining", a hallmark of its activated state. Conversely, a tenuous PKCα staining was detected in luminal breast cancer cells. Notably, TNBC-B cell lines express high levels of phosphorylated (active)-phospholipase C gamma 1 (PLCγ1), an enzyme responsible for DAG generation and PKCα activation. We also found a significant positive correlation between PKCα and PD-L1 expression in breast cancer cell lines. Notably, both RNAi silencing and pharmacological inhibition of PKCα or PLCγ1 reduced PD-L1 mRNA and protein levels in TNBC cells. We also show that AXL and PKCα mutually and positively control their expression in addition to up-regulating PD-L1, and they share a common gene expression signature that is highly enriched in cytokine, extracellular matrix, and motility pathways. Mechanistic analysis showed that inhibition of AXL, PLCγ1 or PKCα significantly reduced PD-L1 promoter activity in TNBC cells, suggesting a transcriptional control of the PDL1 (CD274) gene by this pathway. In summary, our results indicate that aberrant overactivation of AXL/PLCγ1/PKCα represents a cancer cell intrinsic mechanism for PD-L1 up-regulation in TNBC, suggesting a potential role for this axis in the control of tumor immunosurveillance. PKCα and AXL may be promising candidates for integrated targeted therapy in TNBC treatment.