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

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.

<p>Autophagy-Related Beclin 1 and Head and Neck Cancers</p>

“…98 In addition, nuclear Beclin 1 had nonautophagic functions in DNA repair according to the results of Havaki et al, who reported that Beclin 1 expression in the nucleus in successive stages of laryngeal carcinogenesis was gradually decreased, which might indicate an impaired DNA repair process that increases genomic instability. 65 In 43 ESCCs, the expression levels of Beclin-1, LC3B, and CASP-3 proteins as detected by IHC were not significantly correlated with clinicopathological factors. Beclin 1 expression was also not associated with the prognoses of ESCCs; however, high LC3B expression and low CASP-3 expression were significantly associated with a poor prognosis for ESCC.…”

“…The authors reviewed past reports and suggested that nucleocytoplasmic translocation of Beclin 1 was associated with its autophagic function and tumor suppressive roles. 65…”

Integrating the DNA damage and protein stress responses during cancer development and treatment

“…Genome damage results in the activation of proteostatic modules, and crosstalk between the DDR/R machinery and autophagy has been established (supplementary material, Figure S6A) . Prominent connections include those between ATM and PARP1 with AMPK*, one of the central metabolic regulators in eukaryotes.…”

“…However, the link between DDR/R and the PN or the PDR warrants further investigation, especially during carcinogenesis (supplementary material, Figure S6C), as this interrelationship is not straightforward. Specifically, recent in vivo data indicate an inverse relationship in precancerous lesions, whereas, at the cancerous stage, PN modules and the DDR operate in parallel .…”

“…Particular attention should be given to the p16 INK4A –Rb–E2F pathway, which is a major ‘molecular crossroad’ that most mitogenic/oncogenic signals converge to. Its phosphorylation status determines whether the cell will bypass the ‘restriction point’ of G 1 phase, committing the cell to progress to S phase without the requirement for extracellular stimulants …”