AKT can modulate the in vitro response of HNSCC cells to irreversible EGFR inhibitors

Abstract

Epidermal growth factor receptor (EGFR) is overexpressed in up to 90% of head and neck squamous cell carcinoma (HNSCC) tumors. Cetuximab is the first targeted (anti-EGFR) therapy approved for the treatment of HNSCC patients. However, its efficacy is limited due to primary and secondary resistance, and there is no predict biomarkers of response. New generation of EGFR inhibitors with pan HER targeting and irreversible action, such as afatinib and allitinib, represents a significant therapeutic promise. In this study, we intend to compare the potential cytotoxicity of two anti-EGFR inhibitors (afatinib and allitinib) with cetuximab and to identify potential predictive biomarkers of response in a panel of HNSCC cell lines. The mutational analysis in the eight HNSCC cell lines revealed an EGFR mutation (p.H773Y) and gene amplification in the HN13 cells. According to the growth inhibition score (GI), allitinib was the most cytotoxic drug, followed by afatinib and finally cetuximab. The higher AKT phosphorylation level was associated with resistance to anti-EGFR agents. Therefore, we further performed drug combinations with anti-AKT agent (MK2206) and AKT1 gene editing, which demonstrated afatinib and allitinib sensitivity restored. Additionally, in silico analysis of TCGA database showed that AKT1 overexpression was present in 14.7% (41/279) of HNSCC cases, and was associated with perineural invasion in advanced stage. In conclusion, allitinib presented a greater cytotoxic profile when compared to afatinib and cetuximab. AKT pathway constitutes a predictive marker of allitinib response and combination with AKT inhibitors could restore response and increase treatment success.

Rationale for Using Irreversible Epidermal Growth Factor Receptor Inhibitors in Combination with Phosphatidylinositol 3-Kinase Inhibitors for Advanced Head and Neck Squamous Cell Carcinoma

“…Our data are consistent with previous studies showing the benefit of PI3K-and EGFR-inhibitor combination therapies (Rebucci et al, 2011;Young et al, 2013;D'Amato et al, 2014;Lattanzio et al, 2015;Michmerhuizen et al, 2016;Anisuzzaman et al, 2017;Silva-Oliveira et al, 2017) and also extend that work by discovering that PI3K inhibitors are much more effective in combination with irreversible than reversible EGFR inhibitors in HNSCC. In prior work comparing the classes of EGFR-targeting monotherapies in this cancer type, preclinical data demonstrated that irreversible EGFR inhibitors are superior to other EGFR-targeting agents, including cetuximab (Ather et al, 2013;Silva-Oliveira et al, 2017) and reversible inhibitor gefitinib (Young et al, 2015). Similarly, previous work has shown that the addition of ERBB2-targeting antibodies pertuzumab (Erjala et al, 2006) or trastuzumab (Kondo et al, 2008) to gefitinib enhances its efficacy in HNSCC cell lines; however, our findings demonstrated that the broader specificity of irreversible inhibitors alone cannot explain these differences in sensitivity, as administering ERBB2-inhibitor CP-724714 with gefitinib and HS-173 did not enhance drug effects (Fig.…”

“…In this study, pharmacologic inhibition or siRNA knockdown of AKT resulted in improved sensitivity to afatinib and allitinib (a second irreversible EGFR inhibitor) in HN13 cells (Silva-Oliveira et al, 2017). The need to suppress AKT phosphorylation in responses to PI3K 1 EGFR drug combinations is supported by studies of both EGFR-targeting antibodies (Benavente et al, 2009;Rebucci et al, 2011) and reversible inhibitors (Benavente et al, 2009;Rebucci et al, 2011;Young et al, 2013;Silva-Oliveira et al, 2017). In lungcancer models, irreversible EGFR inhibitors have sustained reductions in EGFR phosphorylation and an improved ability to decrease effector AKT phosphorylation compared with reversible inhibitors (Kwak et al, 2005).…”

“…Preclinical data indicate that dual therapies directed against both PI3K and EGFR pathways might improve responses in HNSCC (Rebucci et al, 2011;Young et al, 2013;D'Amato et al, 2014;Lattanzio et al, 2015;Michmerhuizen et al, 2016;Anisuzzaman et al, 2017;Silva-Oliveira et al, 2017). Given these promising data, several clinical trials assessing the combination in HNSCC have been opened, most of which use the EGFR-targeting antibody cetuximab in combination with various inhibitors of PI3K (e.g., NCT01816984, NCT2282371, NCT02822482).…”

Coronarin D Induces Apoptotic Cell Death and Cell Cycle Arrest in Human Glioblastoma Cell Line

“…After washing with PBS (1 mL/well, twice), cells were lysed with lysis buffer (70 µL/well); Tris 50 mM pH 7.6-8, NaCl 150 mM, EDTA 5 mM, Na 3 VO 4 1 mM, NaF 10 mM, Na pyrophosphate 10 mM, and 1% NP-40 and supplemented with a cocktail of inhibitors (DTT-Dithiothreitol, leupeptin hemisulfate, aprotinin, PSMF-Phenylmethylsulfonyl fluoride, and EDTA) for 1 h, followed by centrifugation (4 • C, 15 min, 13,000 rpm). The protein concentration was determined by Bradford reagent [50]. All samples (untreated and Coronarin d-treated cells) were total cellular protein (20 µg protein/sample) and separated by electrophoresis on 10% gradient gels in SDS-PAGE and blotted onto nitrocellulose membranes by electroblotting in transfer buffer (Trizma base, glycine, distilled water, and methanol).…”

Metabolic Imaging Using Hyperpolarized Pyruvate–Lactate Exchange Assesses Response or Resistance to the EGFR Inhibitor Cetuximab in Patient-Derived HNSCC Xenografts

“…However, only a minority of patients derive long-term benefit from anti-EGFR mAbs (5,6). Resistance mechanisms to EGFR inhibition for HNSCC remain unclear (7), although multiple mechanisms of acquired and de novo resistance have been suggested (8,9), paving the way for trials of combination strategies (7,10,11). Optimal HNSCC patient selection for anti-EGFR-based therapy remains a challenge (7).…”