Molecular Response to Combined Molecular- and External Radiotherapy in Head and Neck Squamous Cell Carcinoma (HNSCC).

Treewut Rassamegevanon,Steffen Löck,Michael Baumann,Louis Feindt,Ariane Christel Kühn,Johannes Müller,Hans-Jürgen Pietzsch,Jörg Kotzerke,Cläre Von Neubeck,Robert Freudenberg,Wiebke Sihver,Antje Dietrich,Mechthild Krause,Lydia Koi,Enes Çevik,Annett Linge

doi:10.3390/cancers13225595

Abstract

Simple SummaryOur previous preclinical trial in a head and neck squamous cell carcinoma (HNSCC) xenograft model showed a high potential for the improvement of curative treatment outcome upon the combination treatment of a radiolabeled (Yttrium-90) anti-EGFR antibody (Cetuximab) and external radiotherapy. We aim to elucidate the molecular response of HNSCC tumors upon this combination. Here, we show that the combination treatment leads to an increasing number and complexity of DNA double strand breaks. The upregulation of p21cip1/waf1 expression and cleaved caspase-3 suggest a blockage of cell cycle transition and an induction of programmed cell death. Collectively, a complex interplay between molecular mechanisms involved in cell death induction, cell cycle arrest, and DNA double strand break repair accounts for the beneficial potential using Yttrium-90-Cetuximab in combination with external radiotherapy. Combination treatment of molecular targeted and external radiotherapy is a promising strategy and was shown to improve local tumor control in a HNSCC xenograft model. To enhance the therapeutic value of this approach, this study investigated the underlying molecular response. Subcutaneous HNSCC FaDuDD xenografts were treated with single or combination therapy (X-ray: 0, 2, 4 Gy; anti-EGFR antibody (Cetuximab) (un-)labeled with Yttrium-90 (90Y)). Tumors were excised 24 h post respective treatment. Residual DNA double strand breaks (DSB), mRNA expression of DNA damage response related genes, immunoblotting, tumor histology, and immunohistological staining were analyzed. An increase in number and complexity of residual DNA DSB was observed in FaDuDD tumors exposed to the combination treatment of external irradiation and 90Y-Cetuximab relative to controls. The increase was observed in a low oxygenated area, suggesting the expansion of DNA DSB damages. Upregulation of genes encoding p21cip1/waf1 (CDKN1A) and GADD45α (GADD45A) was determined in the combination treatment group, and immunoblotting as well as immunohistochemistry confirmed the upregulation of p21cip1/waf1. The increase in residual γH2AX foci leads to the blockage of cell cycle transition and subsequently to cell death, which could be observed in the upregulation of p21cip1/waf1 expression and an elevated number of cleaved caspase-3 positive cells. Overall, a complex interplay between DNA damage repair and programmed cell death accounts for the potential benefit of the combination therapy using 90Y-Cetuximab and external radiotherapy.

Highlights

HNSCC is the most common malignant disease arising in the oral cavity, oropharynx, hypopharynx, and larynx
To investigate the effect of 90 Y-Cetuximab inflicting DNA double strand breaks (DSB) damages, residual γH2AX foci were determined manually in nuclei located within three distance categories based on the distance from the nearest perfused vessel (100 μm, respectively)
The molecular response upon the combined molecular targeted and external beam radiotherapy was investigated via residual DNA DSB, mRNA expression of DDR genes and the corresponding protein expression of the two candidate genes that showed the highest upregulation upon the combination treatment, and the activation of cell death induction

Summary

Introduction

HNSCC is the most common malignant disease arising in the oral cavity, oropharynx, hypopharynx, and larynx. It is considered as the sixth most common cancer worldwide with approximately 900,000 newly diagnosed cases in 2020 [1]. Molecules coupled with tumor-associated antigenbinding components are systemically administered, allowing targeted internal irradiation to primary cancers as well as distant metastases [3]. By combining the precise and conformal dose delivery to the primary tumor via external beam radiotherapy with molecular targeted radiotherapy, an increased tumor dose and potential targeting of metastases can be achieved, improving treatment outcome in HNSCC [4]. A monoclonal antibody targeting EGFR conjugated with radionuclides showed a therapeutic efficacy in a broad set of tumor types as monotherapy or combination therapy with chemoor radiotherapy [4,7,8,9,10,11,12,13,14]

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