Expression of Cancer Stem Cell (CSC) Specific Transcription Factors and Cell Surface PCNA and Their Role in CSC Escape From NK Cell Effector Function

Purpose: Natural Killer (NK) cells participate in the innate immune response against cancer and infection without prior sensitization. NK cell function depends on a balance of signals transmitted from activating and inhibitory receptors interacting with ligands on the surface of target cells. Cancer cells may evade NK-mediated killing by expressing or secreting ligands for NK cell inhibitory receptors. The Natural Cytotoxicity Receptor (NCR) family, comprised of NKp30, NKp44, and NKp46, is classically described as a group of activating receptors that induce NK cell activation and cytotoxicity. Notably, NKp44 functions as an activating or inhibitory receptor depending on ligand interaction. Proliferating cell nuclear antigen (PCNA) associates with Human Leukocyte Antigen I (HLA I) and forms the inhibitory ligand for NKp44, resulting in the inhibition of NK function. Cancer stem cells (CSC), a unique subset of tumor cells, possess a stem-cell-like phenotype and are thought to facilitate metastasis by escaping NK cell effector function. Methods: Diffuse B cell lymphoma (DB) cells were labeled and sorted for cell surface PCNA expression via fluorescence activated cell sorting (FACS). Total RNA was isolated, converted to cDNA, and the transcription factors NANOG, SOX2, and Oct-4, which are associated with CSC phenotype, were analyzed by qRT-PCR from sorted PCNA+ and PCNA- cells. NK receptor-ligand interactions were blocked by incubating DB cells with anti-PCNA, anti-NKp44, or control antibodies and a chromium release killing assay was performed. Results: Cell sorting and qRT-PCR confirmed DB cells with cell surface PCNA have increased expression of transcription factors compared to PCNA- cells. Blocking the interaction of NKp44 and PCNA enhanced the killing of DB by NK cells. Conclusions: Cell surface PCNA is associated with increased CSC transcription factor expression. Additionally, cell surface PCNA on CSC may facilitate escape from NK cell killing by interacting with NKp44 and transmission of an inhibitory signal. Characterization of stem cell transcription factors and cell surface PCNA may provide novel immunotherapeutic targets to destroy CSC and thus prevent cancer metastasis.