Surface PCNA enables pancreatic and colon cancer stem cells to inhibit NK cell effector function

Purpose: Cancer stem cells (CSC) are a subset of tumor cells that have a stem-cell-like phenotype and are thought to facilitate metastasis by evading peripheral NK cell effector function. NK cell function is regulated by the balance of activating and inhibitory receptors binding to ligands on the surface of target cells. Cancer cells may escape NK cell killing by expressing or secreting ligands for NK cell inhibitory receptors. NKp44, a member of the natural cytotoxicity receptor family, can function as either an activating or an inhibitory receptor depending on ligand interaction. Proliferating cell nuclear antigen (PCNA) associates with MHC class I and forms an inhibitory ligand for NKp44, resulting in the inhibition of NK cytotoxicity. We hypothesize cell surface PCNA can be used as a marker for CSC and as a potential immunotherapeutic target for pancreatic and colon cancer. Methods: Pancreatic (Panc-1) and colon (HCT 116) cancer cell lines were labeled with antibodies against PCNA, CD44, and CD133 and flow cytometry was performed to determine surface expression. CSC were identified as being CD44+CD133+. Cells were labeled and sorted via FACS; CSC transcription factors NANOG, SOX2, and Oct-4 were analyzed by qRT-PCR from sorted populations. NK receptor-ligand interactions were blocked by incubating cells with anti-PCNA, anti-NKp44, or control antibodies; interferon gamma and chromium release assays were performed. Results: In both Panc-1 and HCT 116 cells, a PCNA+CD44+CD133+ population was detected and enriched in naturally detached cells. Furthermore, cell sorting and qRT-PCR determined cells with cell surface PCNA have increased expression of CSC transcription factors compared to cells lacking surface PCNA. Blocking the PCNA-NKp44 interaction enhanced the specific lysis of target cells by NK cells and altered the release of interferon gamma. Conclusions: Cell surface PCNA is associated with co-expression of CD44 and CD133 as well as increased CSC transcription factor expression. Additionally, cell surface PCNA alters interferon gamma secretion and facilitates escape from NK cell killing. Our data suggest that blocking NKp44-PCNA interactions may provide a novel immunotherapeutic target for pancreatic and colon cancer stem cells and prevent metastasis.