NGFR Increases the Chemosensitivity of Colorectal Cancer Cells by Enhancing the Apoptotic and Autophagic Effects of 5-fluorouracil via the Activation of S100A9

Abstract

Colorectal cancer (CRC) is currently the third leading cause of cancer-related deaths worldwide, and 5-fluorouracil (5-FU)-based chemotherapies serve as important adjuvant therapies before and after surgery for CRC. However, the efficacy of CRC chemotherapy is limited by chemoresistance, and therefore the discovery of novel markers to indicate chemosensitivity is essential. Nerve growth factor receptor (NGFR), a cell surface receptor, is involved in cell death and survival. Our previous study indicated that NGFR acts as a tumor suppressor, and high expression is associated with better outcomes in patients receiving 5-FU-based adjuvant chemotherapy after surgery. The aim of this study was to investigate the effect of NGFR on the chemotherapeutic response in CRC. Chemosensitivity was investigated using DLD1 and HCT8 cells after NGFR transfection. Apoptosis was investigated by flow cytometry. Autophagy was assessed using GFP-LC3B transient transfection. Gene expression was measured using an mRNA microarray. Beclin-1 and Bcl-2 protein expressions were assessed by western blot. NGFR and S100 calcium-binding protein A9 (S100A9) expressions in CRC patients were investigated by immunohistochemistry. The results showed that the half maximal inhibitory concentration of NGFR-transfected cells was lower than that of controls in DLD1 and HCT8 cells after 5-FU treatment, and cell viability was lower than in empty-vector cells. Tumor sizes were also smaller than in empty-vector cells in vivo. The percentages of apoptotic and autophagic cells were higher in NGFR-transfected cells. NGFR elevated the expression of S100A9 after 5-FU treatment. The combination of Bcl-2 and Beclin-1 was significantly suppressed by overexpressed NGFR. Five-year overall and disease-free survival in NGFR+/S100A9+ patients was better than in NGFR-/S100A9- patients. This study's findings suggest that NGFR may serve as a marker predicting CRC patients' chemosensitivity.

Keywords: NGFR; S100A9; apoptosis; autophagy; chemosensitivity; colorectal cancer.

Figure 1

NGFR contributed to colorectal cancer cell suppression after 5-FU treatment in vitro. (A) An MTS assay was performed to investigate the viability of overexpressed NGFR–transfected DLD1 and HCT8 cells at different 5-FU concentrations (0–256 µmol/L). (B) A colony formation assay was performed using overexpressed NGFR–transfected DLD1 and HCT8 cells. (C) The IC50 values were determined as indicated on the plot (t-test; P < 0.001 for DLD1 cells, P < 0.01 for HCT8 cells). (D) Colony formation assays indicated that NGFR inhibited overexpressed NGFR–transfected DLD1 and HCT8 cell colony formation (cell viability) with increasing 5-FU concentrations (0–200 µM for DLD1 cells, 0–100 µM for HCT8 cells; t-test; P < 0.01 for DLD1 cells, P < 0.001 for HCT8 cells). *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 2

NGFR increases the chemosensitivity of CRC cells to 5-FU by elevating 5-FU-induced apoptosis. (A, B) Real-time cellular analysis confirmed the results of the colony formation assays regarding the inhibition of overexpressed NGFR–transfected DLD1 and HCT8 cell growth with 5-FU treatment (t-test; P < 0.01 for DLD1 cells). Overexpressed NGFR–transfected (C) DLD1 and (D) HCT8 cells treated with 5-FU for 48 h. Cell apoptosis was quantitated on the graph using an Annexin V:PE apoptosis detection kit and a flow cytometer. The apoptosis rate was significantly increased in overexpressed NGFR–transfected (E) DLD1 and (F) HCT8 cells after 5-FU treatment (Q4, early apoptosis; Q2 + Q4, total apoptosis; t-test; P < 0.01 for DLD1 cells, P < 0.01 for HCT8 total apoptosis cells, P < 0.05 for HCT8 early apoptosis cells). *P < 0.05; **P < 0.01.

Figure 3

NGFR promoted 5-FU-induced autophagy of CRC cells. Overexpressed NGFR–transfected DLD1 and HCT8 cells were treated with 5-FU for 48 h. The cellular autophagy induced by (A) 200 µM of 5-FU for DLD1 and (B) 100 µM for HCT8 cells was examined by confocal microscopy with transfection with a GFP-LC3B vector. (C) Autophagosome-expressing LC3B (green dot) was quantitated in overexpressed NGFR–transfected compared to empty vector–transfected DLD1 and HCT8 cells treated with 5-FU (t-test; ***P < 0.001).

Figure 4

NGFR overexpression enhanced 5-FU-induced apoptosis and autophagy of CRC cells. Apoptosis markers (Bcl-2, caspase-3, and cleaved caspase-3) were detected in (A) DLD1 and (B) HCT8 cells treated with 5-FU (0, 100, and 200 µM for DLD1 cells; 0, 50, and 100 µM for HCT8 cells) for 48 h by western blot. Autophagy markers (Beclin-1, LC3A [upper band], and LC3B [lower band]) were detected in (C) DLD1 and (D) HCT8 cells treated with 5-FU (0, 100, and 200 µM for DLD1 cells; 0, 50, and 100 µM for HCT8 cells) for 48 h by western blot. The role of NGFR in tumor suppression of CRC with 5-FU-based therapy was investigated in vivo. The tumor (E) volume and (F) size of a xenograft mouse model in overexpressed NGFR–transfected HCT8 cells were smaller than those with empty vector–transfected HCT8 cells after 5-FU treatment (t-test; ***P < 0.001).

Figure 5

RNA sequencing revealed potential downstream NGFR signaling. (A) The top 10 upregulated genes (red) and top 10 downregulated genes (green). S100A8 and S100A9 are marked with red frame. (B, C) Gene Ontology analysis of altered genes involved in biological process functions. (D) Gene Set Enrichment Analysis revealed enrichment of NGFR-associated genes. S100A9 upregulation shown in graphs of overexpressed NGFR–transfected (E) DLD1 and (F) HCT8 cells after 5-FU treatment. t-test; **P < 0.01; ***P < 0.001.

Figure 6

Low expressions of NGFR and S100A9 were associated with poor prognosis in patients with CRC after 5-FU-based chemotherapy. (A) Samples of CRC patients stained by immunohistochemistry. The expressions of NGFR and S100A9 are shown in the cytoplasm (brown stain) of CRC cells. A Kaplan-Meier plot showed that NGFR and S100A9 low co-expression was associated with poor (B) overall survival and (C) disease-free survival in patients with CRC after 5-FU-based chemotherapy. (D) DLD1 and SW480 cells were transfected with an NGFR overexpression plasmid and an shRNA plasmid following a 48-h 5-FU treatment. Cell lysates were then extracted for immunoprecipitation with anti-Beclin-1 or anti-Bcl-2 antibodies, and precipitates were immunoblotted with anti-Bcl-2 or anti-Beclin-1 antibodies. The upper band shows that the combination of Bcl-2 and Beclin-1 was considerably suppressed by overexpressed NGFR. The lower band shows that when NGFR was downregulated, the combination of Bcl-2 and Beclin-1 was markedly elevated.