Tumor cell-expressed IL-15Rα drives antagonistic effects on the progression and immune control of gastric cancer and is epigenetically regulated in EBV-positive gastric cancer

Purpose: Epstein-Barr virus associated gastric Cancer (EBVaGC) often exhibits a favorable prognosis that correlates with highly methylated viral and host genes and significant immune cell infiltration compared to EBV-negative gastric cancers (GCs). Previously, it has been reported that expression of the IL-15 Receptor α (IL-15Rα) is down-regulated in EBVaGC via promoter hypermethylation. In the present study, we offer a novel explanation for this puzzle by associating IL-15Rα expression with infiltration of lymphocytes in GC lesions.

Methods: We investigated the expression of IL-15Rα by RT-PCR, Western-blotting and immunohistochemistry in GC cell lines and primary tissues, respectively. IL-15Rα promoter methylation was analyzed using genomic methylation Sequencing. The growth behavior of GC cells was analyzed using MTT, flow cytometry, colony formation, transwell invasion and scratch wound healing assays. Demethylation of IL-15Rα was carried out using 5-Aza-CdR, and rIL-15 was added to evaluate growth promoting effects of the IL-15/IL-15Rα complex. Human peripheral blood mononuclear cells (PBMCs) were co-cultured with GC cells with/without the addition of rIL-15, after which the phosphorylation of STAT5 in PBMCs was evaluated using flow cytometry to estimate the activation of these immune cells through IL-15 binding to IL-2Rβ/γ receptors by in trans presentation.

Results: We found that EBV-positive GC cells (AE) expressed IL-15Rα at a significantly lower level than EBV-negative GC cells (AGS) due to promoter hypermethylation. In the absence of immune cells, IL-15Rα on the Cancer cell surface induced a malignant phenotype, including augmented cell growth, migration and invasion, and decreased Apoptosis. 5-Aza-CdR reverted AE cells to a more malignant phenotype similar to AGS cells, which may be attributed to activation of the STAT1, STAT3 and ERK1/2 pathways. However, when PBMCs were added to the GC cell cultures, these immune cells were activated as detected by increased pSTAT5 levels. Also, more GC cells underwent Apoptosis. These effects were enhanced by the addition of rIL-15 and, subsequently, confirmed in EBVaGC patient samples exhibiting increased expression of T cell surface markers and activation of immune co-stimulating pathways.

Conclusions: Our findings suggest a mechanistic explanation for the clinical association of EBVaGC with a lower IL-15Rα expression, a better prognosis and an increased lymphocyte infiltration. We propose that in highly infiltrated GCs the IL-15/IL-15Rα complex on the GC cell surface may present IL-15 in trans to IL-2Rβ/γ-expressing immune cells to activate these cells in the tumor microenvironment.