Monitoring Autophagy Immunohistochemically and Ultrastructurally during Human Head and Neck Carcinogenesis. Relationship with the DNA Damage Response Pathway.

Sophia Havaki,Vassiliki Vlachou,Euthymios Kyrodimos,Christos Zampetidis,Dimitris Kletsas,Eleni Mavrogonatou,Vassilis Gorgoulis,Athanassios Kotsinas,Konstantinos Evangelou,Alexandra Giatromanolaki,Sophia Rizou,Platonas Selemenakis

doi:10.3390/ijms18091920

Abstract

Autophagy is a catabolic process that preserves cellular homeostasis. Its exact role during carcinogenesis is not completely defined. Specifically in head and neck cancer, such information from clinical settings that comprise the whole spectrum of human carcinogenesis is very limited. Towards this direction, we examined the in situ status of the autophagy-related factors, Beclin-1, microtubule-associated protein 1 light chain 3, member B (LC3B) and sequestosome 1/p62 (p62) in clinical material covering all histopathological stages of human head and neck carcinogenesis. This material is unique as each panel of lesions is derived from the same patient and moreover we have previously assessed it for the DNA damage response (DDR) activation status. Since Beclin-1, LC3B and p62 reflect the nucleation, elongation and degradation stages of autophagy, respectively, their combined immunohistochemical (IHC) expression profiles could grossly mirror the autophagic flux. This experimental approach was further corroborated by ultrastructural analysis, applying transmission electron microscopy (TEM). The observed Beclin-1/LC3B/p62 IHC patterns, obtained from serial sections analysis, along with TEM findings are suggestive of a declined authophagic activity in preneoplastic lesions that was restored in full blown cancers. Correlating these findings with DDR status in the same pathological stages are indicative of: (i) an antitumor function of autophagy in support to that of DDR, possibly through energy deprivation in preneoplastic stages, thus preventing incipient cancer cells from evolving; and (ii) a tumor-supporting role in the cancerous stage.

Highlights

Autophagy is a highly conserved catabolic process, which under physiological conditions contributes to the preservation and restoration of cellular homeostasis through the degradation of damaged intracellular proteins and organelles—the so-called autophagy cargo—in the lysosome [1]
A detailed view of the expression profile and in situ subcellular localization of Beclin-1, LC3B and p62 can be provided since all stages of carcinogenesis can be observed in the same section from the same patient
Given that Beclin-1, LC3B and p62 reflect in general the nucleation, elongation and degradation stages of autophagy respectively (Figure 1) [7,12], their immunohistochemical evaluation, corroborated by ultrastructural analysis, could mirror a gross estimation of the autophagic flux

Summary

Introduction

Autophagy is a highly conserved catabolic process, which under physiological conditions contributes to the preservation and restoration of cellular homeostasis through the degradation of damaged intracellular proteins and organelles—the so-called autophagy cargo—in the lysosome [1]. When the intracellular or the extracellular microenvironment is perturbed by chemical, physical or infectious factors that function as stressors, autophagy flux is activated to secure cellular fitness and to restore metabolism and homeostasis. In this respect, dysfunctional autophagic machinery has been associated with numerous diseases, such as neurodegeneration, cardiovascular disorders, infectious conditions, metabolic disorders and cancer [4,5]. Autophagy is classified into three types depending on the mechanism of cargo delivery to the lysosomes and the involved molecular components: microautophagy [6,7], chaperone-mediated autophagy [8,9] and macroautophagy [10,11]. ATGs are responsible for the biogenesis of double-membrane vesicles, called autophagosomes

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Results

Conclusion