Abnormal Expression of c-Myc Oncogene in NK Cells in Patients with Cancer

Abstract

Natural killer (NK) cells have received a lot of attention in recent years for the roles they play in immunity and particularly in antitumor immune responses. Although defects in NK cell functions are recognized as important mechanisms for immune evasion of malignant cells, molecular pathways regulating NK cell dysfunction and exhaustion in cancer are largely unknown. Here we tested whether the c-myc proto-oncogene, known to promote cell proliferation, growth, differentiation, and apoptosis by regulating the expression of numerous target genes, may be involved in the mechanism of NK cell abnormalities in patients with lung and gastric cancer. Analysis of c-myc mRNA and protein expression in peripheral blood NK cells, mitogen-activated protein kinase (MAPK) activity, cell cycle, and cell longevity revealed a significantly decreased expression of c-myc mRNA and protein and mitotic arrest of NK cells in different phases of cell cycle. In addition, a significant decrease of NK cell death was also detected. These data allow the suggestion that defects of NK cell-mediated tumor surveillance may be associated with disturbed c-myc expression in NK cells in cancer patients. A better understanding of the mechanisms of NK cell dysfunction in cancer will help in the NK cell-mediated therapeutic eradication of primary and metastatic cancer cells and prolong patient survival.

Keywords: MAPK; NK cells; c-myc; gastric cancer; lung cancer.

Figure 1

Differences in c-myc mRNA expression in NK cells harvested from healthy donors and cancer patients. NK cells were isolated from the peripheral blood samples by negative selection using Dynabeads, incubated in complete medium for 20 h and c-myc expression was determined by Smart Flare method as described in M&M. (A) Data of mean fluorescent intensity (MFI) are shown as the mean ± SEM (ANOVA). (B) C-myc-mRNA expression in peripheral NK cells from one of 10 representative healthy donors. (C) C-myc-mRNA expression in peripheral NK cells from one of 7 representative patients with lung cancer. (D) C-myc-mRNA expression in peripheral NK cells from one of 12 representative patients with gastric cancer. (B–D) The relative expression was determined by flow cytometry on stained NK cells.

Figure 2

Differences in c-Myc protein expression in NK cells harvested from healthy donors and cancer patients. NK cells were isolated from the peripheral blood samples by negative selection using Dynabeads. C-myc-protein expression was determined as described in M&M. (A) Data of mean fluorescent intensity (MFI) are shown as the mean ± SEM (ANOVA). (B) C-myc-protein expression in peripheral NK cells from one of 10 representative healthy donors. (C) C-myc-protein expression in peripheral NK cells from one of 7 representative patients with lung cancer. (D) C-myc-protein expression in peripheral NK cells from one of 12 representative patients with gastric cancer.

Figure 3

Differences in the total and phosphorylated level of ERK in NK cells harvested from healthy donors and cancer patients. NK cells were isolated from the peripheral blood samples by negative selection using Dynabeads. The total and phosphorylated levels of ERK were determined as described in M&M. (A) Data of MFI of total levels of ERK are shown as the mean ± SEM. (B) Data of MFI of phosphorylated levels of ERK are shown as the mean ± SEM. (** p < 0.01).

Figure 4

Differences in % NK cells in cell cycle phases harvested from healthy donors and cancer patients. NK cells were isolated from the peripheral blood samples by negative selection using Dynabeads. Cell cycle was determined as described in M&M. (A) Data of % NK cells in G0/G1 stage are shown as the mean ± SEM (ANOVA). (B) Data of % NK cells in S and G2/M stage are shown as the mean ± SEM (ANOVA). (C) Cell cycle of NK cells from one of 10 representative healthy donors. (D) Cell cycle of NK cells from one of 7 representative patients with lung cancer. (E) Cell cycle of NK cells from one of 12 representative patients with gastric cancer. (* p < 0.05 and *** p < 0.001).