Human NK Cells Are Alerted to Induction of p53 in Cancer Cells by Upregulation of the NKG2D Ligands ULBP1 and ULBP2

Abstract

Natural killer (NK) cells are immune cells sensing and eliminating foreign, stressed, transformed, and senescent cells through specialized surface receptors, such as NKG2D, that interacts with several virus-or stressinducible ligands, including ULBP1 and -2, which are expressed on target cell surfaces. For example, induction of DNA damage or cellular senescence pathways in tumor cells led to upregulation of NKG2D ligands that activate NK cells. Although, both pathways activate p53, the relationship of p53 activation to upregulation of NKG2D ligands has not been addressed. In this study, we report that induction of wild-type p53, but not mutant p53, strongly upregulated mRNA and cell surface expression of ULBP1 and -2, whereas expression of other NK cell ligands was not affected. We defined intronic p53-responsive elements in these two novel p53 target genes. Coculture of wild-type p53-induced human tumor cells with primary human NK cells enhanced NKG2D-dependent degranulation and IFN-g production by NK cells. Accordingly, treatment of certain wild-type p53-expressing tumor cell lines with the p53-reactivating small molecular compound RITA resulted in upregulation of ULBP2 mRNA and cell surface protein expression. Taken together, our findings define the involvement of p53 in the regulation of specific NKG2D ligands that enhance NK cell-mediated target recognition. One implication of our work is that activating p53 after adoptive transfer of NK cells might constitute an effective combinatorial strategy of NK cell-based immunochemotherapy in cancers in which wild-type p53 function is preserved. Cancer Res; 71(18); 5998-6009. Ó2011 AACR.

The proto-oncogene Myc drives expression of the NK cell-activating NKp30 ligand B7-H6 in tumor cells

“…12 B7-H6 is expressed on the cell surface of many different tumor cell lines and on certain tumor cells in situ but not on hematopoietic cells from healthy individuals or primary keratinocytes. 12,15,25 So far, mechanisms that drive the expression of B7-H6 on tumors have not been elucidated. Several studies suggested that expression of activating NK cell ligands on tumors may be directly induced by oncogenes.…”

“…NK cell isolation from healthy donors was performed as previously described. 25 NK cells were cultured overnight in CellGro Stem Cell Growth Medium (CellGenix) with 10% human AB serum (PAA) and 200 U/ml recombinant human interleukin 2 (IL-2, NIH). All media were supplemented with 100 U/ml Penicillin and 100 mg/ml Streptomycin (Sigma-Aldrich) and cell lines were routinely tested for contamination by the Multiplex cell Contamination Testing service at the German Cancer Research Center.…”

“…Whole cell lysate preparation, SDS-PAGE and Western Blotting was performed as previously described 25 . Polyclonal rabbit antibodies specific for c-Myc or N-Myc (both Cell Signaling Technology, Danvers, MA, USA) were added at a 1:1000 final dilution in PBS/ 0.1% Tween 20/ 5% BSA for an overnight incubation.…”

“…Targets and effectors were incubated at a 1:1 ratio as previously described. 25 The effectors were analyzed for expression of CD107a by flow cytometry by gating on CD56C CD3-7-AAD-cells. The % specific degranulation was calculated by subtracting the % CD107aC NK cells in samples with NK cells by themselves from the % CD107aC NK cells cultured with targets.…”

Shaping of NK Cell Responses by the Tumor Microenvironment

“…Thus, up-regulation of activating ligands and/or down-regulation of inhibitory ligands on tumor cells would increase NK cell activation against tumors. For instance, we showed that activation of p53 with the small molecular compound, RITA, led to the upregulation of ULBP1 and ULBP2 in tumor cells resulting in enhanced NK cell-mediated target recognition [47]. Similarly, certain MIC and ULBP proteins can be upregulated by the oncogene Ras [48] or by application of histonedeacetylase inhibitors [49,50].…”