CD56 expression in breast cancer induces sensitivity to natural killer-mediated cytotoxicity by enhancing the formation of cytotoxic immunological synapse

Abstract

We examined the potential value of the natural killer (NK) cell line; NK-92, as immunotherapy tool for breast cancer (BC) treatment and searched for biomarker(s) of sensitivity to NK-92-mediated cytotoxicity. The cytotoxic activity of NK-92 cells towards one breast precancerous and nine BC cell lines was analyzed using calcein-AM and degranulation assays. The molecules associated with NK-92-responsiveness were determined by differential gene expression analysis using RNA-sequencing and validated by RT-PCR, immunostaining and flow cytometry. NK-target interactions and immunological synapse formation were assessed by fluorescence microscopy. Potential biomarker expression was determined by IHC in 99 patient-derived BC tissues and 10 normal mammary epithelial tissues. Most (8/9) BC cell lines were resistant while only one BC and the precancerous cell lines were effectively killed by NK-92 lymphocytes. NK-92-sensitive target cells specifically expressed CD56, which ectopic expression in CD56-negative BC cells induced their sensitivity to NK-92-mediated killing, suggesting that CD56 is not only a biomarker of responsiveness but actively regulates NK function. CD56 adhesion molecules which are also expressed on NK cells accumulate at the immunological synapse enhancing NK-target interactions, cytotoxic granzyme B transfer from NK-92 to CD56-expressing target cells and induction of caspase 3 activation in targets. Interestingly, CD56 expression was found to be reduced in breast tumor tissues (36%) with strong inter- and intratumoral heterogeneity in comparison to normal breast tissues (80%). CD56 is a potential predictive biomarker for BC responsiveness to NK-92-cell based immunotherapy and loss of CD56 expression might be a mechanism of escape from NK-immunity.

CD56 regulates human NK cell cytotoxicity through Pyk2

“…Previous reports on the role of CD56 in NK cell lytic function have focused on homotypic interactions between CD56 on NK cells and target cells and have led to conflicting conclusions about the role that CD56 plays in cytotoxicity (4,(30)(31)(32). To test the cytotoxic function of wild-type (WT) and CD56-knockout (KO) cell lines against CD56-negative target cells, we performed 51 Cr-release cytotoxicity assays.…”

“…Previous studies have demonstrated that CD56 can promote human NK cell cytotoxic function against some CD56 + target cells (29)(30)(31)(32), although in some cases lysis is not enhanced by target cell CD56 expression (4). As these studies focused on homotypic CD56-mediated interactions, the significance of CD56 binding to heterotypic ligands is unclear yet is likely relevant.…”

“…While CD56 has been implicated in NK cell development, migration and cytotoxicity (4,23,30,31,35), the signaling pathways that regulate its function in immune cells have not been described. Given signaling downstream of CD56 that is mediated by FAK in neuronal cells, one potential link between CD56 and IS formation is the closely related nonreceptor tyrosine kinase 2 (Pyk2), which is highly expressed in NK cells (36).…”

Systemic antitumor effect by regional hyperthermia combined with low-dose chemotherapy and immunologic correlates in an adolescent patient with rhabdomyosarcoma – a case report

“…aberrantly expressed CD56, also known as neural cell adhesion molecule-1 (NCAM-1). Aberrant CD56 expression is a common finding for rhabdomyosarcoma, including ARMS [26], and a recent study indicates an association between tumor-cell expression of CD56 and sensitivity to NK-cellmediated killing in breast cancer [27].…”

Bioinformatics Identified 17 Immune Genes as Prognostic Biomarkers for Breast Cancer: Application Study Based on Artificial Intelligence Algorithms

“…Toll-like receptor-activated plasma-like dendritic cells inhibited growth of BC cells (48). CD56 enhanced formation of cytotoxic immune synapses and strengthened sensitivity of cytotoxicity mediated by natural killer cells (49). CD4 and CD8 T cell tumor infiltration driven by HER2-dendritic cells improved survival of BC mice (50).…”