Highly Expressed Granulocyte Colony-Stimulating Factor (G-CSF) and Granulocyte Colony-Stimulating Factor Receptor (G-CSFR) in Human Gastric Cancer Leads to Poor Survival

Abstract

BACKGROUND Chemotherapy for advanced gastric cancer (GC) patients has been the mainstay of therapy for many years. Although adding anti-angiogenic drugs to chemotherapy improves patient survival slightly, identifying anti-angiogenic therapy-sensitive patients remains challenging for oncologists. Granulocyte colony-stimulating factor (G-CSF) promotes tumor growth and angiogenesis, which can be minimized with the anti-G-CSF antibody. Thus, G-CSF might be a potential tumor marker. However, the effects of G-CSF and G-CSFR expression on GC patient survival remain unclear. MATERIAL AND METHODS Seventy GC tissue samples were collected for G-CSF and G-CSFR detection by immunohistochemistry. A total of 40 paired GC tissues and matched adjacent mucosa were used to measure the G-CSF and G-CSFR levels by ELISA. Correlations between G-CSF/G-CSFR and clinical characteristics, VEGF-A levels and overall survival were analyzed. Biological function and underlying mechanistic investigations were carried out using SGC7901 cell lines, and the effects of G-CSF on tumor proliferation, migration, and tube formation were examined. RESULTS The levels of G-CSFR were upregulated in GC tissues compared to normal mucosa tissues. Higher G-CSF expression was associated with later tumor stages and higher tumor VEGF-A and serum CA724 levels, whereas higher G-CSFR expression was associated with lymph node metastasis. Patients with higher G-CSF expression had shorter overall survival times. In vitro, G-CSF stimulated SGC7901 proliferation and migration through the JAK2/STAT3 pathway and accelerated HUVEC tube formation. CONCLUSIONS These data suggest that increased G-CSF and G-CSFR in tumors leads to unfavorable outcomes for GC patients by stimulating tumor proliferation, migration, and angiogenesis, indicating that these factors are potential tumor targets for cancer treatment.

Figure 1

G-CSF and G-CSFR expression in GC tissues. (A) The expression of G-CSF and G-CSFR was detected in cancer cells and tumor-infiltrating lymphocytes by immunohistochemistry. (B) G-CSF and G-CSFR protein concentrations were measured in 40 paired (cancer and normal tissue) gastric tissues by ELISA, and the G-CSFR levels in the cancer tissues were significantly higher than those in the normal tissues (P=0.045). (C) G-CSF and G-CSFR protein levels at different TNM stages by ELISA. * P<0.05.

Figure 2

G-CSF/G-CSFR correlation with GC patient survival. (A) The concentrations of G-CSF, G-CSFR and VEGF-A were measured in patients who had died and those who survived by ELISA. The G-CSF concentration was significantly higher in patients who had died than in those who had survived. (B) Kaplan-Meier postoperative survival analysis of OS for GC (n=70) using immunohistochemistry. High G-CSF expression was associated with poor OS. * P<0.05.

Figure 3

G-CSF promotes GC cell proliferation and migration via the JAK2/STAT3 pathway. (A) PCNA expression was detected in GC cells by Western blot. (B) GC cell proliferation with CCK8 assays. (C) G-CSF promotes GC cell migration in wound healing assays. (D) G-CSF promotes GC cell invasion in Transwell invasion assays. (E) The protein levels of STAT, phospho-STAT3 and JAK2 were examined by Western blotting with anti-STAT, anti-phospho-STAT3 and anti-JAK2 antibodies, respectively, in untreated (control) cells, in cells that were treated with G-CSF and in cells that were treated with anti-G-CSFR antibody before incubation with G-CSF. The expression levels of phospho-STAT3 and JAK2 were significantly increased in the G-CSF group compared to the control group and anti-G-CSFR group. * P<0.05.

Figure 4

G-CSFR expression evaluation in GC cells by qRT-PCR and Western blotting with anti-G-CSFR antibody. Cells treated with G-CSF (50 ng/mL) for 96 hours have significantly higher G-CSFR mRNA and protein levels than untreated (control) cells. * P<0.05 vs. control.

Figure 5

G-CSF promotes angiogenesis. (A) The VEGF-A protein concentrations for different TNM stages by ELISA. (B) The correlations of protein concentration between G-CSF/G-CSFR and VEGF-A. VEGF-A was significantly correlated with G-CSFR in GC tissues (P=0.001). (C) HUVEC network formation on matrix gel. G-CSF stimulated HUVEC network formation in a dose-dependent manner. (D) The total tube length was quantified. G-CSF (10, 50, and 100 ng/mL) significantly promoted tube formation compared to the control group. * P<0.05 vs. control.