Wnt5a-mediated autophagy promotes radiation resistance of nasopharyngeal carcinoma

Abstract

Wnt signaling pathways and autophagy play an essential role in tumor progression. Canonical Wnt signaling pathways in radiation resistance have been studied in the past, but it remains unclear whether the noncanonical Wnt signaling pathways can affect tumor radiation resistance through protective autophagy. Nasopharyngeal carcinoma, a particular subtype of head and neck squamous cell carcinoma, relies on radiation therapy. In this study, we found that radioactive rays could significantly promote the expression of Wnt noncanonical signaling pathways ligands in nasopharyngeal carcinoma, among which Wnt5A was the most markedly altered. We have demonstrated that Wnt5a can reduce the radiation sensitivity of nasopharyngeal carcinoma in vitro and in vitro experiments. Meanwhile, we found much more greater autophagosomes in overexpressed-Wnt5A nasopharyngeal carcinoma cells by electron microscopy. Further mechanism exploration revealed that Beclin1 is the main target of Wnt5A, and knocking down Beclin1 can partially reduce Wnt5a-induced radiation resistance. By studying Wnt5A-mediated protective autophagy in promoting radiation resistance in nasopharyngeal carcinoma cells, we hope that the Wnt5A and Beclin1 can become effective targets for overcoming radiation resistance in the future.

Keywords: Head and Neck Squamous Cell Carcinoma; Wnt5a; autophagy; nasopharyngeal carcinoma; radiation resistance.

Figure 1

Wnt5a contributes to radiation resistance of nasopharyngeal carcinoma. (A) Western blotting assays were used to evaluate the expression of proteins in the Wnt5a in CNE-2 and 6-10B cells exposed to 4 Gy irradiation. (B-C) Nasopharyngeal carcinoma CNE2 and 6-10B cells were infected with lentivirus mediated Wnt5a or control cDNA and then subjected to puromycin screening for 2 weeks. Western blotting was used to examine the expression of Wnt5a protein in CNE2 and 6-10B cells. (D & F) Cells overexpressing Wnt5a or control cDNA were irradiated with the indicated doses of irradiation and clonogenic assays were obtained after 2 weeks. (E & G) Survival curves of cells in each group were determined by clonogenic assays. **p < 0.01; ***p < 0.001.

Figure 2

Exogenous rh-Wnt5a promotes radiation resistance to nasopharyngeal carcinoma. (A & C) Cells with medium added exogenous rh-Wnt5a and solvent were irradiated with the indicated doses of irradiation and clonogenic assays were obtained after 2 weeks. (B & D) Survival curves of cells in each group were determined by clonogenic assays. **p < 0.01; ***p < 0.001.

Figure 3

Wnt5a inhibits the levels of DNA damage. (A & C) Immunofluorescence staining was used to confirm the γ- H2AX foci in CNE2 and 6‐10B cells at 6 hours post‐irradiation with 4 Gy irradiation. (B & D) γH2AX foci were quantified by immunofluorescence staining. ***p < 0.001.

Figure 4

Wnt5a enhances radiation resistance in vivo. (A) Schematic flow of the experimental design. Nude mice were subcutaneously injected with 6‐10B cells overexpressing Wnt5a and then subjected to two consecutive 4 Gy irradiation treatments treatment when the tumor volume reached 100 mm³. Mice were finally killed at day 23 after tumor injection. (B) Tumor volumes were measured with callipers every 3‐4 days to monitor the growth pattern of mice. (C) The final gross tumors were captured. (D) Average tumor weight in each group was compared. *p < 0.05; **p < 0.01.

Figure 5

Wnt5a enhances autophagy. HNSCC CNE2 and 6‐10B cells were infected with lentivirus mediated Wnt5a or control cDNA and then subjected to puromycin screening for 2 weeks. (A) Representative images of autophagosomes (indicated by red arrows) observed by transmission electron microscopy in CNE2 and 6‐10B cells at 3 hours post 4 Gy irradiation exposure. (B) Western blotting was used to examine the expression of proteins associated with the Wnt5a and autophagic proteins (Beclin1 and LC3B). (C) The Pearson rank correlation coefficient validated the positive correlation between Wnt5a and Beclin1.

Figure 6

Wnt5a enhances radiation resistance to nasopharyngeal carcinoma through autophagy. HNSCC CNE2 and 6‐10B cells were infected witt lentivirus‐mediated Wnt5a or control cDNA and then infected with Beclin1 shRNA to inhibit its expression. (A & D) Western blotting assays were used to evaluate the transfection efficiency. (B & E) Cells in each group were irradiated with the indicated doses of irradiation and clonogenic assays were obtained after 2 weeks. (C & F) Survival curves of cells in each group were determined by clonogenic assays. *p < 0.05; **p < 0.01.