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. 2022 Nov 17:9:1040552.
doi: 10.3389/fvets.2022.1040552. eCollection 2022.

Anti-EGFR monoclonal antibody Cetuximab displays potential anti-cancer activities in feline oral squamous cell carcinoma cell lines

Gennaro Altamura  1 , Giuseppe Borzacchiello  1
Affiliations

Anti-EGFR monoclonal antibody Cetuximab displays potential anti-cancer activities in feline oral squamous cell carcinoma cell lines

Gennaro Altamura et al. Front Vet Sci. .

Abstract

Feline oral squamous cell carcinoma (FOSCC) is a malignant tumor characterized by an aggressive behavior and poor prognosis, for which no fully effective therapies are available. Studies of comparative oncology suggest that epidermal growth factor receptor (EGFR) may be a therapeutic target in FOSCC, similarly to human head and neck SCC (HNSCC), where the use of anti-EGFR monoclonal antibody Cetuximab has entered the clinical practice. The aim of this study was to assess the efficacy of Cetuximab in three validated preclinical models of FOSCC (SCCF1, SCCF2, SCCF3). Sequencing of tyrosine kinase domain of EGFR in the cell lines revealed a wild-type genotype, excluding the presence of activating mutations. Western blotting experiments demonstrated that Cetuximab inhibited activation of EGFR and its downstream kinase Akt in SCCF1, SCCF2 and SCCF3 along with HNSCC cell line CAL 27 included as control. Importantly, CCK-8 and trypan blue exclusion assays revealed that treatment with Cetuximab caused a decrease in cell proliferation and cell viability in all cell lines, with a general dose- and time-dependent trend. Cell death induced by Cetuximab was associated with cleavage of PARP, indicating occurrence of apoptosis. Taken together, our data suggest that Cetuximab exerts potential anti-cancer activities in FOSCC, paving the way for future translational studies aimed at assessing its employment in the therapy of this lethal cancer of cats.

Keywords: Akt; Cetuximab; EGFR; cat; molecular targeted therapy; oral squamous cell carcinoma.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Inhibition of EGFR pathway by Cetuximab in feline oral squamous cell carcinoma cell lines (SCCF1, SCCF2, SCCF3). (A) Cells were incubated with Cetuximab (CTX) at 100 μg/mL for 12 h and analyzed by western blotting (WB) for EGFR, phospho-EGFR (pEGFR), Akt and phospho-Akt (pAkt). Cetuximab-sensitive CAL 27 cells were included as control. The treatment (+) induced reduction of pEGFR levels in all cell lines with respect to untreated control (–), along with decreased EGFR in SCCF2 and its accumulation in SCCF3 and SCCF1. Impairment of EGFR by Cetuximab was accompanied by a decrease of pAkt levels. WB for β-actin antibody ensured comparable protein loading and allowed normalization. (B) Densitometric analysis of EGFR expressed as densitometric ratio with β-actin. (C,D) Densitometric analysis of pEGFR and pAkt expressed as densitometric ratio pEGFR/EGFR and pAkt/Akt, respectively. Standard deviations are from three repeated, independent experiments (*: statistically significant by t-test, P ≤ 0.05; **: statistically significant by t-test, P ≤ 0.01).
Figure 2
Figure 2
Impact of Cetuximab on cell proliferation in feline oral squamous cell carcinoma cell lines. Cells treated with Cetuximab at 50 and 100 μg/mL were analyzed by CCK-8 assay after 24, 48, and 72 h. The treatment induced a dose- and time-dependent impairment of cell proliferation in SCCF1 (A), SCCF2 (B) and SCCF3 (C). Data are presented as percentage (%) compared with the respective control (CTRL) and represent mean ± SD from three replicates. Paired Student's t-tests were used to compare each treatment to control (*: statistically significant by t-test, P ≤ 0.05; **: statistically significant by t-test, P ≤ 0.01). ANOVA analysis showed also statistically significant difference between all group means at 72 h in SCCF2 (F = 34.7, P < 0.01), 48 (F = 8.644, P < 0.05) and 72 h (F = 9.376, P < 0.01) in SCCF3.
Figure 3
Figure 3
Impact of Cetuximab on cell viability in feline oral squamous cell carcinoma cell lines. Cells treated with Cetuximab at 50 and 100 μg/mL were analyzed by trypan blue exlclusion assay after 48 and 72 h. The treatment induced decrease in cell viability in SCCF1 (A), SCCF2 (B) and SCCF3 (C). Data are presented as percentage (%) compared with the respective control (CTRL) and represent mean ± SD from three independent experiments. Paired Student's t-tests were used to compare treatments to control; differences were not statistically significant, except were indicated (*P ≤ 0.05).
Figure 4
Figure 4
Cleavage of the proapoptotic marker PARP in feline oral squamous cell carcinoma cell lines (SCCF1, SCCF2, SCCF3) treated with Cetuximab. (A) Cells treated with Cetuximab (CTX) at 100 μg/mL were analyzed by western blotting (WB) for PARP after 48/72 h. Cetuximab-sensitive CAL 27 cells were included as control. The treatment (+) induced an increase of cleaved PARP in all cell lines compared to untreated control (–). WB for β-actin antibody ensured comparable protein loading and allowed normalization. Paired boxes for each cell line are cut from the same membrane at the same exposure time and properly aligned according to the molecular marker loaded onto the gel. Full scans from original gels are shown in Supplementary Figure 8. (B) Densitometric analysis of cleaved PARP expressed as densitometric ratio with β-actin. Standard deviations are from two repeated, independent experiments yielding comparable results.
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