Cellular and soluble immune checkpoint signaling forms PD-L1 and PD-1 in renal tumor tissue and in blood.

Immune checkpoint blockade therapy is a treatment option of various metastatic cancer diseases including renal cell carcinoma (RCC). Approved antibody drugs target the co-inhibitory signaling of Programmed Cell Death Ligand-1 (PD-L1) and its receptor Programmed Cell Death-1 (PD-1). The combined evaluation of PD-L1 and PD-1 at the mRNA and protein levels in tumor tissue with differentiation of tumor and immune cells as well as of soluble forms (sPD-L1) and (sPD-1) in blood is of basic interest in assessing biomarker surrogates. Here, we demonstrate that PD-L1 determined as fraction of stained tumor cells (TPS-score) correlates with PD-L1-mRNA in tumor tissue, reflecting the predominant expression of PD-L1 in tumor cells. Conversely, PD-1 in immune cells of tumor tissue (IC-score) correlated with PD-1-mRNA tissue levels reflecting the typical PD-1 expression in immune cells. Of note, sPD-L1 in blood did not correlate with either the TPS-score of PD-L1 or with PD-L1-mRNA in tumor tissue. sPD-L1 released into the supernatant of cultured RCC cells closely followed the cellular PD-L1 expression as tested by interferon γ (IFNG) induction and siRNA knockdown of PD-L1. Further analysis in patients revealed that sPD-L1 significantly increased in blood following renal tumor resection. In addition, sPD-L1 correlated significantly with inflammation marker C-reactive protein (CRP) and with PD-L1 mRNA level in whole blood. These results indicate that the major source of sPD-L1 in blood may be peripheral blood cells and not primarily tumor tissue PD-L1.

c-Src mediated tyrosine phosphorylation of plakophilin 3 as a new mechanism to control desmosome composition in cells exposed to oxidative stress.

Plakophilins (PKP1 to PKP3) are essential for the structure and function of desmosomal junctions as demonstrated by the severe skin defects observed as a result of loss-of-function mutations in mice and men. PKPs play additional roles in cell signaling processes, such as those controlling the cellular stress response and cell proliferation. A key post-translational process controlling PKP function is phosphorylation. We have discovered that reactive oxygen species (ROS) trigger the c-Src kinase-mediated tyrosine (Tyr)-195 phosphorylation of PKP3. This modification is associated with a change in the subcellular distribution of the protein. Specifically, PKP3 bearing phospho-Tyr-195 is released from the desmosomes, suggesting that phospho-Tyr-195 is relevant for the control of desmosome disassembly and function, at least in cells exposed to ROS. Tyr-195 phosphorylation is transient under normal physiological conditions and seems to be strictly regulated, as the activation of particular growth factor receptors results in a modification at this site only when tyrosine phosphatases are inactivated by pervanadate. We have identified Tyr-195 of PKP3 as a phosphorylation target of epidermal growth factor receptor signaling. Interestingly, this PKP3 phosphorylation also occurs in certain poorly differentiated adenocarcinomas of the prostate, suggesting a possible role in tumor progression. Our study thus identifies a new mechanism controlling PKP3 and hence desmosome function in epithelial cells.

An alternative promoter of the human plakophilin-3 gene controls the expression of the new isoform PKP3b.

The plakophilin family (PKP1 to PKP3) is an essential component of the desmosomal adhesion complex with differentiation-dependent and partially overlapping expression and possible participation of the corresponding genes in malignant transformation. Here, we describe a new protein variant of the human PKP3 gene, namely PKP3b, which differs from the published PKP3a only at the amino-terminus by the splicing in of the newly identified exon 1b. Specific antibodies have demonstrated differential expression patterns of the two variants. Whereas PKP3a is broadly expressed among epithelial cells, PKP3b is abundant in the desmosomes of stratified epithelial cells, such as HaCaT but absent or heterogeneous in simple epithelial cells such as CaCo2 or MCF7. The differential expression of the PKP3 variants has been observed in a similar manner in selected normal human tissues and carcinomas derived thereof. Both variants are localized to the desmosomes of all cells of stratified tissues, whereas the new PKP3b is heterogeneously expressed in the colon and its tumors. Therefore, we assume that both variants are controlled by alternative promoters. Reporter gene assays have confirmed that a fragment upstream of exon 1b exhibits transcriptional activity only in HaCaT cells but not in CaCo2 cells and thus has been identified as an alternative promoter driving the expression of PKP3b. Finally, by using electromobility shift assays, we found a potential binding site in the PKP3b promoter for transcription factor C/EBP regulating keratinocyte differentiation and probably also PKP3b expression. We discuss the properties of the new variant PKP3b as a possible marker protein for the analyses of differentiation and malignant transformation.