Effect of colony‐stimulating factor‐1 receptor overexpression on the growth of nasopharyngeal carcinoma xenografts in nude mice and its mechanism of action

Although nasopharyngeal carcinoma (NPC) is sensitive to radiotherapy, local recurrence and distant metastasis still occur in a proportion of patients due to radioresistance. Our previous research confirmed that colony‐stimulating factor‐1 receptor (CSF‐1R) promotes the proliferation, migration, and invasion of 6‐10B cells through the phosphoinositide 3‐kinase (PI3K) pathway in vitro. The objective of the present study was to investigate the effects of colony‐stimulating factor‐1 receptor (CSF‐1R) on proliferation, apoptosis, and autophagy in the human nasopharyngeal carcinoma 6‐10B cell line in vivo, and the possible underlying mechanisms.


INTRODUCTION
Nasopharyngeal carcinoma (NPC) is a highly prevalent malignancy with a significant geographic incidence, and approximately 80% of NPCs occur in southern China and southeastern Asia. 1 is usually sensitive to radiotherapy, local recurrence and distant metastasis still occur in a proportion of patients due to radioresistance. 3 Previously, we have compared gene expression profiles between 12 radioresistant NPC patients and eight radiosensitive patients using DNA chip technology, and observed that the colony-stimulating factor-1 receptor (CSF-1R) showed a distinct difference between the two groups. Its expression in tissues of radiation-resistant patients was higher than that in radiation-sensitive patients. 4 Therefore, we hypothesize that CSF-1R might induce radiotherapy resistance in NPC patients, and hence there is an urgent need to decipher the molecular mechanism of CSF-1R in NPC to formulate better treatment plans.
CSF-1R is a 972 amino acid long single-chain transmembrane glycoprotein belonging to the tyrosine kinase receptor family. CSF-1R is produced in the osteoblasts, bone marrow stromal cells, endothelial cells, fibroblasts, and epithelial cells. It is also widely expressed during tumor progression, thus playing a key role in the process. 5 In vivo, CSF-1R, and CSF-1 are phosphorylated upon binding to activate their phosphokinase domain, which then activates the mononuclear macrophage system to promote tumor growth, neovascularization, and extracellular matrix decomposition. CSF-1R thus enables tumor cells to drive tumor development. Studies have also shown that CSF-1R directly affects tumor cells, 6 and this could be associated with CSF-1R being overexpressed in individual tumors. [7][8][9] Hence, CSF-1R detected in circulating blood is identified as a tumor marker in certain malignancies. However, there is little evidence on the effect of CSF-1R on NPC.
Our previous in vitro studies confirmed the role of CSF-1R in promoting the proliferation, migration, and invasion of 6-10B cells through the phosphoinositide 3-kinase (PI3K) pathway. 10 Furthermore, in this study, we developed a nude mouse model of NPC, and sequentially evaluated the expression of CSF-1R and other proliferation-, invasion-, apoptosis-, autophagy-, and PI3K/protein kinase B (Akt) pathwayrelated factors in the xenograft model. We sought to determine the molecular mechanism of CSF-1R in the progression of NPC through in vivo experiments to unravel the possible causes of radiotherapy resistance seen in NPC cases. CSF-1R might serve as a potential candidate for NPC gene therapy, and we hope to provide experimental evidence to identify new molecular targets for NPC treatment in the future.

Cell culture and animals
The NPC cell line 6-10B was obtained from Sun Yat-Sen Univer-  Thereafter, reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blot analysis were used to verify the mRNA and protein expression in the control vector (NC group)treated, CSF-1R overexpression vector (transfection group)-treated, and untreated 6-10B cells.

Reverse transcription-quantitative polymerase chain reaction
Total RNA was extracted from frozen tissues or cells using TRIzol reagent (Ambion; Thermo Fisher Scientific) following the manufacturer's protocol. Single-stranded cDNA was generated using a

Western blot analysis
Protein samples were prepared either from the cancerous tissues of nude mice or the cells. Tissues or cells were harvested and lysed in lysis buffer (Beyotime Institute of Biotechnology, Haimen, China), containing a protease inhibitor cocktail (100:1) and then kept on ice TA B L E 1 Quantitative polymerase chain reaction primers

In vivo tumor growth assay
Three groups of 4-week-old nude mice were used. 6

Statistical analysis
The data obtained were from at least three independent experiments, and all statistical analyses were carried out using GraphPad version

Effect of CSF-1R overexpression in 6-10B cells on tumor growth in vivo
To test whether CSF-1R played an important role in vivo, we used a xenograft mouse model. The tumorigenicity of 6-10B cells after transfection was compared with the mock group and the NC group in the nude mice model. Each mouse was inoculated with 2 × 10 6 cells. Five days post-inoculation, tumors were noted in the three groups of inoculated mice. Initially, we only observed a small white pit of the inoculation site, while the tumor gradually turned into a solid soft nodule, and the formation rate increased to 100% (Figure 2a). Subsequently, the length and width of the tumors were measured every 3 days using microcalipers (Table 2). Finally, the mice in each group were killed

mRNA expression level of CSF-1R
The RT-PCR method showed that the relative expression of CSF-1R mRNA in the experimental group was 6.161 ± 0.319, which was significantly higher than that of the NC and blank control groups at 0.119 ± 0.100 and 0.076 ± 0.044, respectively. The values and differences were statistically significant (F = 970.046, P < 0.001).

CSF-1R protein expression
As shown in Figure 4, the relative expression of CSF-1R in the blank control group was 0.023 ± 0.009, whereas that in the NC group and experimental transfection group was 0.025 ± 0.009 and 1.711 ± 0.201, respectively. Thus, the experimental protein expression was significantly higher as compared with that in the other groups (F = 210.049, P = 0.013).

Expression of related proteins in nude mice xenografts
The relative expression levels of cyclin D1 in the blank control, NC, and experimental groups were 0.748 ± 0.163, 0.778 ± 0.150, and Data presented as the mean ± SD in mm 3 (n = 5). * P < 0.05 compared with the mock group. # P < 0.05 compared with the negative control (NC) group. BC, blank control; CSF-1R, colony-stimulating factor-1 receptor.   18 Song et al. observed that autophagy is responsible for paclitaxel resistance seen in nasopharyngeal carcinoma cells. 19 Makowska et al. 20 found that the autophagy levels of NPC cell CNE-2R are negatively correlated with apoptosis and radiation resistance. 21 Pang et al. believe that emodin methyl ether can induce the ROS/miR-27a/ZBTB10 signaling axis by targeting Sp, and induce apoptosis and autophagy in nasopharyngeal carcinoma cells. 22 Gao et al. confirmed that overexpression of miR-138-5p enhances the sensitivity of nasopharyngeal carcinoma cells to radiation by targeting EIF4-EBP1. 23 Although the aforementioned studies have explored the relationship between autophagy and apoptosis in nasopharyngeal carcinoma on cancer cell proliferation, tumor growth, and radiotherapy reactivity, the conclusions are still not uniform.

F I G U R E 3
However inhibitors might be an effective treatment for NPC.