CRISP3-PSP94 complex regulates P2RX7 mediated signalling in prostate cancer cells and macrophages via CITED2

CRISP3 (Cysteine rich secretory protein 3) and PSP94 (Prostate secretory protein of 94 amino acids) are evolutionarily conserved proteins that form high affinity complexes. They show an inverse expression pattern during prostate tumorigenesis and are linked to poor prognosis. Apart from prostate, they are present in various body fluids and are speculated to contribute to innate immunity, however their role in tumor immune microenvironment remains uninvestigated. In our earlier study, P2RX7, an ATP-gated ion channel and an important player in the inflammasome pathway, was amongst the genes upregulated upon CRISP3 knockdown. We investigated it further and demonstrated that exogenously added rhCRISP3 downregulated P2RX7 levels in PC3 cells and THP1 macrophages. Interestingly, this effect was abrogated when it was complexed with PSP94. CRISP3 mediated P2RX7 downregulation also reduced ATP-induced cytotoxicity and IL-1β secretion, which was reversed in the presence of PSP94. PSP94 also affected endocytosis of CRISP3 and its interaction with flotillin-2. Using an antibody array, CITED2, a transcriptional coregulator was identified as a downstream mediator of CRISP3. Overexpression of CITED2 also downregulated P2RX7. Since CITED2 modulates transcription through p300 availability, presence of p300 at P2RX7 promoter in PC3 cells and THP1 macrophages was confirmed by CUT&RUN assay. CITED2 overexpression led to reduced p300 levels which could be the reason for downregulation of P2RX7. Our findings unravel a novel mechanism linking CRISP3-PSP94-P2RX7 with CITED2. CRISP3 overexpression and PSP94 downregulation in prostate tumors may influence the tumor immune microenvironment by regulating P2RX7 and CITED2 levels in tumor cells and tumor-associated macrophages.