Increased PD-L1 expression in radioresistant HNSCC cell lines after irradiation affects cell proliferation due to inactivation of GSK-3beta.

Daniela Schulz,Matthias Günther Hautmann,Irene Stancev,Torsten Erich Reichert,Gero Brockhoff,Philipp Beckhove,Richard Josef Bauer,Antonio Sorrentino,Ayse-Nur Menevse,Tobias Ettl

doi:10.18632/oncotarget.26542

Daniela Schulz, Matthias Günther Hautmann + Show 8 more

Open Access

https://doi.org/10.18632/oncotarget.26542

Copy DOI

Abstract

At present, targeting PD-1/PD-L1 axis for immune checkpoint inhibition has improved treatment of various tumor entities, including head and neck squamous cell carcinoma (HNSCC). However, one part of the patient cohort still shows little improvement or even hyperprogression. We established three radioresistant (RR) and three radiosensitive (RS) HNSCC cell lines. RR cells showed prolonged survival as well as delayed and diminished apoptosis after irradiation with vimentin expression but no E-cadherin expression, whereas RS cell lines died early and exhibited early apoptosis after irradiation and high vimentin expression. Here, we present results demonstrating differential basal PD-L1 gene and protein expression in RR and RS HNSCC cell lines. Moreover, we observed a radiation dose dependent increase of total PD-L1 protein expression in RR cell lines up to 96h after irradiation compared to non-irradiated (non-IRR) cells. We found a significant GSK-3beta phosphorylation, resulting in an inactivation, after irradiation of RR cell lines. Co-immunoprecipitation experiments revealed decreased interaction of GSK-3beta with PD-L1 in non-IRR compared to irradiated (IRR) RR cells leading to PD-L1 stabilization in RR cells. PD-L1 knockdown in RR cells showed a strong decrease in cell survival. In summary, our results suggest an irradiation dependent increase in basal PD-L1 expression in RR HNSCC cell lines via GSK-3beta inactivation.

Highlights

With more than 600.000 new diagnosis each year head and neck squamous cell carcinoma (HNSCC) is the 6th most common form of cancer worldwide with a strongly increasing incidence over the last 10 years [1]
To measure apoptosis in RS and RR cell lines, cells were incubated with the green fluorescent dye YOYO-1 which labels only cells with diminished membrane integrity
All RS cell lines revealed a strong increase in apoptosis with a minimum of 38h after irradiation and a median green object area of 6, 94x105 μm2/image (±1.69x105) 120h after irradiation (Figure 1H)

Summary

Introduction

With more than 600.000 new diagnosis each year head and neck squamous cell carcinoma (HNSCC) is the 6th most common form of cancer worldwide with a strongly increasing incidence over the last 10 years [1]. Patients suffering from localized HNSCC can be cured by radical surgical resection. HNSCC, a multidisciplinary approach, including surgical, chemotherapy and radiotherapy, is required. Despite improvement of these therapeutic interventions the survival rate has not increased remarkably over the last years [2]. During recent years immunotherapy by inhibition of checkpoint regulators has become an important part of successful treatment. The PD-1/ PD-L1 checkpoint plays a crucial role in the regulation of www.oncotarget.com

Methods

Results

Conclusion