AMBRA1 as a Biomarker and its Functional Crosstalk with Autophagy and Epidermal Differentiation in Cutaneous Squamous Cell Carcinoma Tumourigenesis

Abstract

Cutaneous Squamous Cell Carcinoma (cSCC) is a skin cancer with an increasing worldwide incidence. While most patients have an excellent prognosis, a subset of patients develop disease recurrence/metastasis, emphasising the need for novel reliable prognostic biomarkers, as well as an improved understanding of the cellular signalling mechanisms underlying cSCC tumourigenesis and progression. Autophagy is essential for cellular homeostasis and keratinocyte differentiation, with the deregulation of both processes being associated with cSCC tumourigenesis. As a key protein to both autophagy and keratinocyte differentiation, the aim of the current study was to define crosstalk between AMBRA1 and the deregulation of these processes in cSCC development and progression and its potential, together with the associated autophagy cargo protein SQSTM1 (p62), as prognostic biomarkers. Biomarker assay development and analysis in a cohort of primary cSCC tumours revealed that loss of cytoplasmic AMBRA1 expression in the tumour growth front, in combination with loss of cytoplasmic p62 expression in the peritumoural epidermis, as putative prognostic biomarkers for cSCC reoccurrence and metastasis, independent of tumour differentiation status. Importantly, the combined loss of these proteins also identified moderately/poorly differentiated primary cSCC tumours at high risk of metastasis. Studies of the potential contribution of cullin E3 ligase-mediated degradation or TGF-β2-mediated downregulation of AMBRA1 in cSCC cell lines revealed only increased levels of TGF-β2 secretion correlated with loss of AMBRA1 expression. Furthermore, although chemical inhibition of TGF-β signalling inhibited cSCC cell proliferation in vitro, no effect on AMBRA1 expression levels was observed, suggesting an undefined TGF-β2 independent-mediated mechanism of AMBRA1 loss in cSCC. Studies investigating AMBRA1 involvement in keratinocyte differentiation and autophagy further demonstrated AMBRA1 expression in keratinocytes initially relies on autophagy activation but is later maintained by epidermal differentiation-related calcium signalling. Additional studies also revealed that this calcium-signalling mediated regulation of AMBRA1 expression is lost during cSCC tumourigenesis, likely resulting in the maintenance of a dedifferentiated cell phenotype, facilitating sustained tumour cell proliferation. This further highlights that loss of AMBRA1 expression as a key event in the uncoupling of autophagy and keratinocyte differentiation in cSCC development. Collectively these data highlight the tumour suppressive role of AMBRA1 in cSCC and its loss of expression in the tumour growth front, in combination with the loss of peritumoural epidermal p62 expression, as a novel prognostic biomarker for cSCC reoccurrence and metastasis.