MUC1 confers radioresistance in head and neck squamous cell carcinoma (HNSCC) cells

Abstract

Mucin 1 (MUC1), a transmembrane glycoprotein, has shown to be as the possible prognostic marker to predict the risk of aggressive head and neck squamous cell carcinoma (HNSCC). In the present study, we investigated the effect of MUC1 in HNSCC cells and the response to X-ray irradiation (IR). Here, we examined the impact of MUC1 overexpression or downexpression on clonogenic survival and apoptosis in response to X-ray irradiation (IR). Radioresistance and radiosensitivity were also observed in HNSCC cells that are MUC1 overexpression and MUC1 downexpression. This enhanced resistance to IR in MUC1-overexpressing cells is primarily due to increased the number of radiation-induced γH2AX/53BP1-positive foci and DNA double-strand break (DSB) repair kinetics. MUC1 overexpression repaired more than 90% of DSBs after 2 Gy radiation by 24 h compared to the empty vector overexpressing cells with less than 50% of DSB repair. However, MUC1 downexpression repaired less than 20% of DSBs compared to the empty vector-overexpresing cells. MUC1 overexpression inhibited proapoptotic protein expression, such as caspase-3, caspase-8, and caspase-9, and induced antiapoptotic protein Bcl-2, followed by resistance to IR-induced apoptosis. Our results showed that targeting MUC1 may be as a promising strategy to counteract radiation resistance of HNSCC cells.

Keywords: DNA double-strand break; Head and neck squamous cell carcinoma; MUC1; X-ray irradiation; radioresistance.

Figure 1.

Effect of shRNA or plasmid transfection on MUC1 expression in LSCC cells. (a) MUC1 protein expression was detected in Hep2, TU-212, TU686, TU177 and AMC-HN-8 cells by Western blot assay; (b) MUC1 mRNA was detected in Hep2, TU-212, TU686, TU177 and AMC-HN-8 cells by RT-PCR assay; (c) TU686 cells were transfected into MUC1 shRNA 1, 2, and 3 for 72 h, MUC1 protein expression was detected by Western blot assay; (d) MUC1 mRNA expression was detected by RT-PCR assay; E, Hep2 cells were transfected into pcDNA3.1 or pcDNA3.1 MUC1 for 72 h, MUC1 protein expression was detected by Western blot assay. vs untreated.**P < 0.01.

Figure 2.

Increased MUC1 expression increases radioresistance in Hep2 cells. (a) Clonogenic survival of Hep2/MUC1 and Hep2/NC. (b) Viability assay of Hep2/MUC1 and Hep2/NC cells in response to radiation; (c) Cell apoptosis was detected by flow cytometry assay. **P < 0.01.

Figure 3.

Decreased MUC1 expression increases radiosensitivity in TU686 cells. (a) Conogenic survival of MUC1 shRNA and NC shRNA transfected cells. (b) Viability assay of MUC1 shRNA and NC shRNA cells in response to radiation. (c) Cell apoptosis was detected by flow cytometry assay. *P < 0.05;**P < 0.01.

Figure 4.

Proapoptotic and antiapoptotic proteins expression in shRNA or plasmid-transfected LSCC cells. (a) Western blot analysis of proapoptotic and antiapoptotic proteins in MUC1 and NC cells in response to radiation. (b) Western blot analysis of proapoptotic and antiapoptotic proteins in MUC1 shRNA and NC shRNA cells in response to radiation. *P < 0.05;**P < 0.01.

Figure 5.

Accelerated DSB repair in MUC1-overexpressing cells after IR. (a) NC and MUC1 transfected cells were irradiated with 2 Gy and immunostained for 53BP1 (green) and phospho-γH2AX (red) foci at 24 h after radiation. (b) NC shRNA and MUC1 shRNA transfected cells were irradiated with 2 Gy and immunostained for 53BP1 (green) and phospho-γH2AX (red) foci at 24 h after radiation. Colocalized foci (yellow) were counted at 24 h (average, 50 nuclei). DNA repair kinetics between these two cells was obtained by plotting the percentage of remaining foci against time.