Inhibition of IL-6-JAK/Stat3 signaling in castration-resistant prostate cancer cells enhances the NK cell-mediated cytotoxicity via alteration of PD-L1/NKG2D ligand levels

Abstract

To investigate whether IL-6 signaling affects the susceptibility of castration-resistant prostate cancer (CRPC) cells to cytotoxic action of natural killer (NK) cells, CRPC cell lines (having different IL-6 levels) were developed by lentiviral transduction. While observing no secreted IL-6 level in parental C4-2 and CWR22Rv1 cells, we found the IL-6 expression/secretion in these cells was induced after the transduction process and the IL-6 level difference in C4-2siIL-6/sc and CWR22siIL-6/sc cell CRPC cell sets could be detected. We then found that IL-6-knockdown cells were more susceptible to NK cell cytotoxicity than control cells due to lowered programmed death receptor ligand 1 (PD-L1) and increased NK group 2D (NKG2D) ligand levels. In animal studies, to concur with the in vitro results, we found that IL-6-expressing cell-derived tumors were more resistant to NK cell action than the tumors of IL-6-knockdown cells. Further, we discovered that JAK-Stat3 is the most critical IL-6 downstream signaling that modulates PD-L1/NKG2D ligand levels in CRPC cells. Furthermore, inhibition of the JAK or Stat3 signaling effectively increased the susceptibility of C4-2sc and CWRsc cells to NK cell cytotoxicity. We observed the most effective cytotoxicity when the PD-L1 Ab and JAK inhibitor (or Stat 3 inhibitor) were used together. These results suggest that the strategy of targeting IL-6 signaling (or its downstream signaling) may enhance the NK cell-mediated immune action to CRPC tumors, thus yielding clinical implications in developing future immunotherapeutics of exploiting this strategy to treat patients with CRPC.

Keywords: IL-6; JAK; NK cell cytotoxicity; NKG2D; Stat3; castration-resistant prostate cancer; programmed death receptor ligand 1.

Figure 1

IL‐6 signaling and CRPC cell resistance to NK cell cytotoxicity. (A) IL‐6 expressions after transduction of C4‐2 and CWR22Rv1 PCa cell lines with lentiviral system containing siIL‐6 or sc sequence. IL‐6 expressions (left panel, ELISA result; right panel, mRNA level) in C4‐2siIL‐6/sc and CWRsiIL‐6/sc cell lines were shown. (B) Results of LDH release‐based NK cell cytotoxicity tests to C4‐2siIL‐6/sc and CWRsiIL‐6si/sc cells (upper panel, data with primary NK cells; lower panel, data with NK92 cells). Left panels show data of C4‐2siIL‐6/sc cell set, and right panels show data of CWRsiIL‐6/sc cell set. Average value of triplicate in one experiment was obtained first and the final error bars were calculated from the average values obtained from three independent experiments. (C) NK cell cytotoxicity test by colony formation assay. The survival of C4‐2siIL‐6/sc and CWRsiIL‐6/sc cells after adding various ratios of NK cells was investigated. When colonies become visible, colonies were stained with crystal violet, and colony numbers were counted under the microscope. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2

In vivo mouse studies showing IL‐6‐mediated resistance of CRPC tumors to NK cell cytotoxic actions. (A) IL‐6 levels in luc‐C4‐2sc and luc‐C4‐2siIL‐6 cells injected into mice. (B) IVIS imaging of representative mice of each subgroup at indicated time points. Upper panel shows imaging of mice of non‐NK cell‐injected group, while lower panel shows imaging of NK cell‐injected mice (left panel, C4‐2sc xenografts; right panel, C4‐2siIL‐6 xenografts). (C) Tumors at sacrifice of mice of each group. Lower panel shows quantitation of the average weight of tumors obtained in mice of each group. (D) IL‐6 IHC staining of tumor tissues. Error bars and significance values were obtained by counting positively stained cells in one randomly chosen phase of slides of three different stains. Magnification, 20× (inlet, 100×). (E) Tumor growth analysis at each time point based on luminescence in IVIS. Luminescence (× 10⁷ radiance·s⁻¹·cm⁻²·sr⁻¹) was plotted as an indication of tumor growth. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3

The role of IL‐6 signaling in the upregulation of PD‐L1 and downregulation of NKG2D ligands in CRPC cells. (A) PD‐L1 level in C4‐2siIL‐6/sc and CWRsiIL‐6/sc cell lines (left panel, mRNA level; right panel, protein level). (B) PD‐L1 IHC staining of tumor tissues. Error bars and significance values were obtained by counting positively stained cells in one randomly chosen phase of slides of three different stains. Magnification, 20× (inlet, 100×). (C) Blocking of IL‐6 Ab by neutralizing Ab of IL‐6 and the effect on PD‐L1 level in C4‐2sc and CWRsc cells. Cells were treated with either IL‐6 Ab or control IgG, total RNA extracted, cDNA converted, and the expression of PD‐L1 was compared in qPCR analyses. (D) PD‐L1 level in parental C4‐2 and CWR22Rv1 cells upon the addition of rhIL‐6. Parental cells (C4‐2P and CWR22Rv1P) were treated with rhIL‐6 (20 ng·mL⁻¹) and PD‐L1 mRNA level was analyzed. (E) IHC staining of CRPC patient tumor samples. Two sets of adjacent tumor tissues (both samples, CRPC stage, Gleason score 8, patient age 70, Ningbo hospital in China) were stained with IL‐6 and PD‐L1. Arrows indicate the area showing positive staining of two molecules. (F) NKG2D ligand levels in IL‐6‐expressing cells and in IL‐6‐knockdown cells. Levels of five NKG2D ligands in C4‐2siIL‐6/sc and CWRsiIL‐6/sc cells were analyzed in qPCR analyses. (G) NKG2D ligand levels in parental C4‐2 and CWR22Rv1 cells upon the addition of rhIL‐6. Parental cells (C4‐2P and CWR22Rv1P) were treated with rhIL‐6 (20 ng·mL⁻¹) and the NKG2D ligand levels (mRNA) were analyzed. (H) Flow cytometric analyses of NKG2D and PD‐1 on NK cells. Left two panels, primary NK cells were stained with PE‐NKG2D or APC‐PD‐1 and positive staining was analyzed. Right two panels, flow cytometric analyses of PD‐1 on NK cells, after coculture with tumor cells (6 h of incubation). Primary NK cells were added into tumor cells (1 : 1 ratio, tumor cells/NK cells) and collected after 6 h of incubation. PD‐1 levels in the collected NK cells were analyzed in flow cytometric analysis (using APC‐PD‐1 Ab). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4

IL‐6 downstream signaling JAK/Stat mediates PD‐L1/NKG2D ligand expressions in CRPC cells. (A) Western blot analyses showing the expression/activation of several IL‐6 downstream signaling molecules in C4‐2siIL‐6/sc and CWRsiIL‐6/sc cells. (B) qPCR analyses in C4‐2siIL‐6/sc and CWRsiL‐6/sc cells with inhibitors of individual candidate signaling pathway. Inhibitors of JAK/Stat (AG490), PI3K/Akt (LY294002), JAK (JAK inhibitor 1), MAPK (SB203580), MEK/Ark (U0126), and Stat3 (Stattic) were used. (C) qPCR analysis of NKG2D ligands in C4‐2siIL‐6/sc and CWRsiIL‐6/sc cells upon treatment with JAK (JAK inhibitor 1) or Stat3 inhibitor (Stattic). Upper panel shows data with C4‐2siIL‐6/sc cell set, and lower panel shows data with CWRsiIL‐6/sc cell set. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5

NK cell cytotoxicity to C4‐2sc and CWRsc cells with (A) PD‐L1 Ab, (B) JAK inhibitor, or (C) Stat 3 inhibitor added to cocultures of tumor cells/NK cells. In performing experiments in (A), both primary NK cells (upper panel) and NK92 cells (lower panel) were used. In (B) and (C), primary NK cells were used. Red lines show data with C4‐2sc and CWRsc cells, while black lines show data with C4‐2siIL‐6 and CWRsiIL‐6 cells. Solid lines show data with control IgG or vehicle, while dotted lines show data with PD‐L1 Ab or inhibitors. In all data presentation, C4‐2 cell data are shown in left panels and CWR22Rv1 cell data are shown on right. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6

NK cell cytotoxicity to C4‐2sc and CWRsc cells with (A) PD‐L1 Ab and JAK inhibitor or (B) PD‐L1 Ab and Stat 3 inhibitor added together to cocultures of tumor cells/NK cells vs. with PD‐L1 Ab or inhibitor alone. In all experiments, primary NK cells were used as a NK cell source. Upper panels show data with C4‐2sc and CWRsc cells, and lower panel shows data with C4‐2siIL‐6 and CWRsiIL‐6 cells. Red lines show data with combined use of (A) PD‐L1Ab plus JAK inhibitor (JAK inhibitor 1) and (B) PD‐L1 Ab plus Stat 3 inhibitor (Stattic). In all data presentation, C4‐2 cell data are shown in left panels and CWR22Rv1 cell data are shown on right. (C) Summary diagram of the results. *P < 0.05, **P < 0.01, ***P < 0.001.