Enhancement of Neuroblastoma NK-Cell-Mediated Lysis through NF-kB p65 Subunit-Induced Expression of FAS and PVR, the Loss of Which Is Associated with Poor Patient Outcome.

Elisa Brandetti,Valentina Folgiero,Doriana Fruci,Nicola Cotugno,Loredana Cifaldi,Chiara Focaccetti,Marzia Ognibene,Monica Benvenuto,Roberto Bei,Vittorio Manzari,Paolo Rossi,Paolo Palma,Annalisa Pezzolo

doi:10.3390/cancers13174368

Abstract

Simple SummaryNeuroblastoma (NB) cells adopt several molecular strategies to evade the Natural Killer (NK)-mediated response. Herein, we found that the overexpression of the NF-kB p65 subunit in NB cell lines upregulates the expression of both the death receptor FAS and the activating ligand PVR, thus rendering NB cells more susceptible to NK-cell-mediated apoptosis, recognition, and killing. These data could provide a clue for a novel NK-cell-based immunotherapy of NB. In addition, array CGH analysis performed in our cohort of NB patients showed that loss of both the FAS and PVR genes correlated with low survival, thus revealing a novel biomarker predicting the outcome of NB patients.High-risk neuroblastoma (NB) is a rare childhood cancer whose aggressiveness is due to a variety of chromosomal genetic aberrations, including those conferring immune evasion. Indeed, NB cells adopt several molecular strategies to evade recognition by the immune system, including the downregulation of ligands for NK-cell-activating receptors. To date, while molecular strategies aimed at enhancing the expression of ligands for NKG2D- and DNAM-1-activating receptors have been explored, no evidence has been reported on the immunomodulatory mechanisms acting on the expression of death receptors such as Fas in NB cells. Here, we demonstrated that transient overexpression of the NF-kB p65 subunit upregulates the surface expression of Fas and PVR, the ligand of DNAM-1, thus making NB cell lines significantly more susceptible to NK-cell-mediated apoptosis, recognition, and killing. In contrast, IFNγ and TNFα treatment, although it induced the upregulation of FAS in NB cells and consequently enhanced NK-cell-mediated apoptosis, triggered immune evasion processes, including the strong upregulation of MHC class I and IDO1, both of which are involved in mechanisms leading to the impairment of a proper NK-cell-mediated killing of NB. In addition, high-resolution array CGH analysis performed in our cohort of NB patients revealed that the loss of FAS and/or PVR genes correlated with low survival independently of the disease stage. Our data identify the status of the FAS and PVR genes as prognostic biomarkers of NB that may predict the efficacy of NK-cell-based immunotherapy of NB. Overall, restoration of surface expression of Fas and PVR, through transient upregulation of NF-kB, may be a clue to a novel NK-cell-based immunotherapy of NB.

Highlights

To efficiently eradicate neuroblastoma (NB), the most common extracranial solid tumor occurring in childhood [1], many efforts are still required
In the search for molecular strategies leading to increased expression of ligands for Natural Killer (NK)-cellactivating receptors in NB cells, the restoration of p53 function led to the upregulation of the ligands ULBP1 and ULBP2 for NKG2D [9] and, as we previously reported, of the ligand PVR (CD155) for DNAM-1 [10]
High-resolution array CGH (a-CGH) analysis revealed that the FAS gene was lost in SK-N-AS, SK-N-BE(2)c, and LA-N-1, it was gained in SH-EP and IMR-32, and it was present in a single copy in the remaining NB cell lines (Table 1)

Summary

Introduction

To efficiently eradicate neuroblastoma (NB), the most common extracranial solid tumor occurring in childhood [1], many efforts are still required. NK cells mediate the anti-tumor cytotoxicity through two main mechanisms: (i) on the one hand, the recognition of ligands expressed on cancer cells by NK-cell-activating receptors, such as NKG2D and DNAM-1, which triggers lytic granule production; and (ii) on the other hand, the recognition of death receptors expressed on cancer cells by NK cells’ apoptotic ligands, which triggers the apoptotic signaling [5]. PVR is a type I transmembrane glycoprotein belonging to the nectin and nectin-like family of immunoglobulin-like molecules that is expressed in several solid tumors [11] whose recognition by DNAM-1 [12] contributes to the triggering of the NK-cell-mediated release of lytic granules [13]. In order to strongly enhance the susceptibility of NB to NK cells, other immunomodulatory mechanisms should be explored

Methods

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Discussion

Conclusion