Prolonged cetuximab treatment promotes p27Kip1-mediated G1 arrest and autophagy in head and neck squamous cell carcinoma

Abstract

Cetuximab, an anti-epidermal growth factor receptor (EGFR) monoclonal antibody, is an efficient anti-tumor therapeutic agent that inhibits the activation of EGFR; however, data related to the cellular effects of prolonged cetuximab treatment are limited. In this study, the long-term cellular outcome of prolonged cetuximab treatment and the related molecular mechanism were explored in a head and neck squamous cell carcinoma cell line constitutively expressing a fluorescent ubiquitination-based cell cycle indicator. Fluorescent time-lapse imaging was used to assess clonal growth, cell motility, and cell-cycle progression. Western blot analysis was performed to measure the level of phosphorylation and protein-expression following cetuximab treatment. Over 5 days cetuximab treatment decreased cell motility and enhanced G1 phase cell arrest in the central region of the colonies. Significantly decreased phosphorylation of retinoblastoma, Skp2, and Akt-mTOR proteins, accumulation of p27^Kip1, and induction of type II LC3B were observed over 8 days cetuximab treatment. Results of the present study elucidate the cetuximab-dependent inhibition of cell migration, resulting in high cell density-related stress and persistent cell-cycle arrest at G1 phase culminating in autophagy. These findings provide novel molecular insights related to the anti-tumor effects of prolonged cetuximab treatment with the potential to improve future therapeutic strategy.

Figure 1

Delayed anti-tumor effect of cetuximab. (A) The anti-tumor effect of cetuximab (Cmab) was not apparent during the first few days of treatment, but dose-dependent growth inhibition was observed by the eighth day of treatment. *p < 0.001. (B) The serum concentration did not block the effect of the cetuximab antibody (10 µg/mL) on day 8. *p < 0.001. FBS, fetal bovine serum. (C) Total cells were counted, and the number of population doublings was calculated every 2 days. (D) The same numbers of cells were plated and cultured for 10 days. The control cells kept growing until they became confluent. In contrast, cells treated with cetuximab (10 µg/mL) showed delayed inhibition of cell growth, such that the arrested cells formed clonal colonies. Data are represented as means ± SD of three independent experiment. Scale bar, 500 µm.

Figure 2

Time-lapse and pedigree analysis of control- and cetuximab-treated SAS-Fucci cells. (A) Cetuximab was administered to SAS-Fucci cells 48 h after they were plated in cell culture dishes. Time-lapse analysis using the Fucci system revealed that cells in G1 phase (red) gradually accumulated especially in the central regions of colonies after 120 h of treatment. In contrast, the control cells showed stable growth. (B) Pedigree assays revealed that, after treatment with cetuximab (10 µg/mL), the cells in the central regions of colonies gradually became arrested in G1 phase, whereas the control cells continued to divide once per ~ 20 h. Red, yellow, green, and blue bars represented G1, S, G2, and M phase, respectively. Scale bar, 50 µm.

Figure 3

Movement velocities of control- and cetuximab-treated SAS-Fucci cells. (A) The movements of individual SAS-Fucci cells in small clusters were monitored for 10 h. (B) The velocity of cell movement, calculated using the TrackMate application, decreased significantly by the fifth day treatment with cetuximab (10 µg/mL). Data are represented as box-whisker plots showing outliers, distribution intervals, 25–75% interquartile range (box), and median. *p < 0.001. Scale bar, 50 µm.

Figure 4

The mKO2-Cdt1 expression levels in fifth-day control- and cetuximab-treated SAS-Fucci cells. The mKO2-Cdt1 expression levels in individual G1 phase-arrested cell were significantly higher after treatment with cetuximab (10 µg/mL) when compared to control cells. Data are represented as box-whisker plots showing outliers, distribution intervals, 25–75% interquartile range (box), and median. *p < 0.05.

Figure 5

Western blotting (WB) analysis of control- and cetuximab-treated SAS cells. WB revealed the phosphorylation levels of ERK1/2, which are regulated by the EGFR pathway, decreased slightly after 8 days of cetuximab treatment. There was also no evidence of p38 activation, based on the levels of phosphorylated p38. Those expression levels were evaluated with the number of pixels × density and analyzed with t test (control- versus cetuximab-treated group). The experiment was repeated 3 times. *p < 0.05, **p < 0.001.

Figure 6

RB and phosphorylated-RB protein levels decreased considerably after 8 or 10 days of cetuximab treatment. Thus, the expression levels of other CKIs were investigated. The p21 protein-expression level decreased as well. The p16 protein was not detected in control- or cetuximab-treated SAS-Fucci cells. Instead, strong p27^Kip1 expression was detected in SAS-Fucci cells treated with cetuximab (10 µg/mL) for 5 days. This examination revealed that prolonged cetuximab treatment inactivated the phosphorylation and function of Skp2. Those expression levels were evaluated with the number of pixels × density and analyzed with t test (control- versus cetuximab-treated group). The experiment was repeated 3 times. *p < 0.05, **p < 0.001.

Figure 7

Immunofluorescent staining also confirmed that p27^Kip1 was highly expressed in the central regions of colonies that formed after treatment with cetuximab (10 µg/mL) for 10 days, which was not observed in control cells following a 10-day treatment. This expression was not also detected in the same sized colonies which were established with 5-day incubation. Scale bar: 500 µm.

Figure 8

Examination of the Akt–mTOR pathway, which controls Skp2 activation. WB analysis revealed that cetuximab treatment (10 µg/mL) potently inhibited Akt phosphorylation at S473 and T308, which are related to Akt activation. In addition, cetuximab treatment suppressed mTOR phosphorylation at S2448 and S2481, which are related to mTOR activation. The expression levels of Atg12–Atg5 and LC3Bs, which are associated with autophagy, were also analyzed, which revealed markedly increased expression of the Atg12–Atg5 complex and type-II LC3B in cells treated with cetuximab for 8 or 10 days. Those expression levels were evaluated with the number of pixels × density and analyzed with t test (control- versus cetuximab-treated group). The experiment was repeated 3 times. *p < 0.05, **p < 0.001.

Figure 9

Mechanism of p27^Kip1 accumulation and autophagy induction following cetuximab treatment. Continuous inhibition of cell migration induced by prolonged cetuximab treatment resulted in decreased Akt and mTOR phosphorylation (red lines), which subsequently decreased Skp2 phosphorylation (blue line) and led to p27^Kip1 accumulation, due to the suppressed degradation of p27^Kip1. The down-regulation of mTOR phosphorylation was also associated with autophagy induction.