Valproic Acid Synergizes With Cisplatin and Cetuximab in vitro and in vivo in Head and Neck Cancer by Targeting the Mechanisms of Resistance.

Federica Iannelli,Tania Moccia,Elena Di Gennaro,Ernesta Cavalcanti,Francesco Caponigro,Rita Lombardi,Carlo Vitagliano,Maria Rita Milone,Maria Serena Roca,Alfredo Budillon,Francesca Bruzzese,Laura Grumetti,Alessandra Leone,Giuseppina Iachetta,Andrea Ilaria Zotti,Chiara Ciardiello,Salvatore Valiante,Franco Ionna,Rossella De Cecio

doi:10.3389/fcell.2020.00732

Abstract

Recurrent/metastatic head and neck squamous cell carcinoma (R/M HNSCC) is a devastating malignancy with a poor prognosis. The combination of cisplatin (CDDP) plus cetuximab (CX) is one of the standard first-line treatments in this disease. However, this therapeutic regimen is often associated with high toxicity and resistance, suggesting that new combinatorial strategies are needed to improve its therapeutic index. In our study, we evaluated the antitumor effects of valproic acid (VPA), a well-known antiepileptic agent with histone deacetylase inhibitory activity, in combination with CDDP/CX doublet in head and neck squamous cell carcinoma (HNSCC) models. We demonstrated, in HNSCC cell lines, but not in normal human fibroblasts, that simultaneous exposure to equitoxic doses of VPA plus CDDP/CX resulted in a clear synergistic antiproliferative and pro-apoptotic effects. The synergistic antitumor effect was confirmed in four different 3D-self-assembled spheroid models, suggesting the ability of the combined approach to affect also the cancer stem cells compartment. Mechanistically, VPA enhanced DNA damage in combination treatment by reducing the mRNA expression of ERCC Excision Repair 1, a critical player in DNA repair, and by increasing CDDP intracellular concentration via upregulation at transcriptional level of CDDP influx channel copper transporter 1 and downregulation of the ATPAse ATP7B involved in CDDP-export. Valproic acid also induced a dose-dependent downregulation of epidermal growth factor receptor (EGFR) expression and of MAPK and AKT downstream signaling pathways and prevent CDDP- and/or CX-induced EGFR nuclear translocation, a well-known mechanism of resistance to chemotherapy. Indeed, VPA impaired the transcription of genes induced by non-canonical activity of nuclear EGFR, such as cyclin D1 and thymidylate synthase. Finally, we confirmed the synergistic antitumor effect also in vivo in both heterotopic and orthotopic models, demonstrating that the combined treatment completely blocked HNSCC xenograft tumors growth in nude mice. Overall, the introduction of a safe and generic drug such as VPA into the conventional treatment for R/M HNSCC represents an innovative and feasible antitumor strategy that warrants further clinical evaluation. A phase II clinical trial exploring the combination of VPA and CDDP/CX in R/M HNSCC patients is currently ongoing in our institute.

Highlights

Head and neck squamous cell carcinoma (HNSCC) is the seventh most common cancer and a leading cause of cancerrelated mortality worldwide1
We used different models of spheroids in order to highlight different effects: (a) by evaluating treatments on 1st generation sphere formation, we investigated the capacity of treatment to prevent/reduce tumor formation; (b) by treating 2nd generation sphere formation, we evaluated the impact of treatment to prevent/reduce more aggressive tumors; (c) by treating formed-spheres, we evaluated the capacity of treatment to induce tumor regression
We have recently reported that the Histone deacetylase inhibitors (HDACi) vorinostat was able to inhibit EGFR phosphorylation and nuclear translocation induced by CDDP plus 5FU combination treatment in HNSCC cells, thereby preventing a mechanism of chemo-resistance and potentiating the antitumor effect (Piro et al, 2019)

Summary

Introduction

Head and neck squamous cell carcinoma (HNSCC) is the seventh most common cancer and a leading cause of cancerrelated mortality worldwide. Despite the development of multiple integrated approaches such as surgery, chemotherapy, radiotherapy, the antiepidermal growth factor receptor (EGFR) monoclonal antibody cetuximab (CX), and, recently, the use of immune checkpoint inhibitors (ICI), have improved outcome, the long-term survival rate is still poor, for recurrent or metastatic disease that develops in more than 65% of patients (Chow, 2020). The approval of CX introduced the first targeted therapy in HNSCC, thereby defining a new standard of care in first-line treatment of recurrent/metastatic head and neck squamous cell carcinoma (R/M HNSCC) to the EXTREME phase-III trial that demonstrated significantly improved survival and response rate by adding CX to platinum-based chemotherapy, compared to chemotherapy alone (Vermorken et al, 2008). No other EGFRblocking agent, including the anti-EGFR monoclonal antibody panitumumab, has matched the result of the EXTREME trial (Szturz and Vermorken, 2017)

Methods

Results

Conclusion