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

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.

Prognostic values of the mRNA expression of natural killer receptor ligands and their association with clinicopathological features in breast cancer patients.

BACKGROUND: Natural killer (NK) cells are lymphocytes of the innate immune system that have potent cytotoxic activity against tumor cells. NK cell recognition and activity towards cancer cells are regulated by an integrated interplay between numerous inhibitory and activating receptors acting in concert to eliminate tumor cells expressing cognate ligands. Despite strong evidence supporting the role of NK cells in breast cancer (BC) control, BC still develops and progresses to form large tumors and metastases. A major mechanism of BC escape from NK immunity is the alteration of the expression of NK receptor ligands. The aim of this study was to determine whether NK receptor ligands' mRNA expression might influence prognosis in BC patients and whether these effects differ by molecular subtypes and clinicopathological features. METHODS: We used the KM plotter platform to analyze the correlation between mRNA expression of 32 NK receptor ligands and relapse-free survival (RFS) and overall survival (OS) in 3951 and 1402 BC patients, respectively. The association with tumor subtypes and clinicopathological features was determined. BC samples were split into high and low expression groups according to the best cutoff value and the two patient cohorts were compared by Kaplan-Meier survival plots. The hazard ratios with 95% confidence intervals and log rank P values were calculated and FDR-adjusted for multiple testing correction. The data was considered to be statistically significant when FDR-adjusted P value < 0.05. RESULTS: High mRNA expression of around 80% of ligands for NK activating and inhibitory receptors associated with better RFS, which correlated with longer OS for only about half of the NK-activating ligands but for most NK-inhibitory ligands. Also, five NK-activating ligands correlated with worse prognosis. These prognostic values were differentially associated with the BC clinical criteria. In addition, the favorable prognostic influence of NK-activating ligands' upregulation, as a whole, was mainly significantly associated with HER2-positive and basal-like subtypes, lymph node positive phenotype, and high-grade tumors. CONCLUSIONS: NK receptor ligands appear to play an important role in defining BC patient prognosis. Identification of a group of patients with worse prognosis expressing high levels of NK-activating ligands and low levels of NK-inhibitory ligands makes them ideal potential candidates for NK-based immunotherapy to eliminate residual tumor cells, prevent relapse and improve patient survival.