AMBRA1 and Loricrin as prognostic biomarkers to identify high-risk melanoma patient subgroups and as companion biomarkers for novel stratified treatment to prevent TGF-[Beta]2 mediated tumour metastasis

Abstract

Cutaneous melanoma remains an increasing world health problem, emphasised by the lack of credible biomarkers able to identify the risk of disease progression of seemingly low risk tumours, or stratify such patients for early novel adjuvant precision-based therapies to prevent metastasis. Data leading to the present study identified the loss of epidermal differentiation proteins, AMBRA1 and Loricrin as putative prognostic biomarkers for AJCC stage I melanomas and their associated loss of expression with increased tumoural secretion of isoform specific TGF-β2. The aim of the present study was to validate the combined loss of AMBRA1 and Loricrin (AMLo) overlying AJCC stage I melanomas as a prognostic and companion biomarker marker to stratify high risk tumour subsets for novel adjuvant treatment strategies targeting TGF-β2 signalling. Semi-quantitative immunohistochemical analysis of AMLo expression in 236 AJCC stage I melanomas identified a high risk subset (defined as tumours with complete loss of AMBRA1 and any break in epidermal Loricrin expression), for which univariate analysis demonstrated a significant decrease in disease free survival in tumours stratified as high risk. Addtionally, semi-quantitative immunohistochemical analysis of TGF-β2 in high risk AJCC stage I melanomas revealed the significant correlation between increased tumoural TGF-β2 expression and loss of epidermal AMLo. Furthermore, high risk AJCC stage I melanomas were associated with a concurrent loss of peri-tumoural endothelial AMBRA1 where treatment of endothelial cells with exogenous TGF-β2 induced the significant downregulation of AMBRA1 and junctional proteins VE-Cadherin or Claudin-5, the effect of which was partially rescued by the stable knockdown of TGF-β2 in melanoma cells. Combined bioinformatics of the AMBRA1 promoter, the analysis of TGF-β2 receptor expression and Smad signalling in endothelial cells revealed the contribution of non-canonical TGF-β2 signalling to enhanced proliferation and loss of endothelial integrity. Finally, combined studies of ALK1 and ALK5 inhibition suggest, targeting ALK1 in high risk AJCC stage I melanomas may provide a novel adjuvant therapeutic strategy to prevent metastasis, for which the loss of AMLo is a companion biomarker.