493 Melanoma secretion of TGFβ2 mediates epidermal AMBRA1 and Claudin1 downregulation, loss of epidermal integrity and tumour ulceration

Ulceration is an adverse prognostic factor in melanoma for which the underlying cause remains undefined. Recently, the combined loss of epidermal AMBRA1 and Loricrin (AMLo) as a prognostic biomarker for AJCC Stage I melanoma has been reported to be mediated by a melanoma secretory mechanism (Ellis et al. 2019). Coupled with bioinformatic data revealing TGFβ responsive elements in the AMBRA1 promotor and the known association of increased isoform specific TGFβ2 with metastatic melanomas, these data led to the current hypothesis that melanoma TGFβ2 secretion results in loss of AMBRA1, epidermal integrity and tumour ulceration. Semi-quantitative automated immunohistochemistry in a cohort of 108 all AJCC primary melanomas was used to correlate tumoural TGFβ2 with overlying epidermal AMBRA expression, as well as AMLo status and metastasis in a sub-cohort of 35 AJCC stage I melanomas. qPCR was used to determine mRNA expression of TGFβ receptors, ALK1 and ALK5 in the keratinocyte cell line, CCD1106. Calcium-induced differentiation of CCD1106 cells, prior to treatment for 72 hr with recombinant TGF-β2 and Western blotting for AMBRA1, Claudin1, total and psmad 2/3 and 1/5/9 was used to determine the impact of TGFβ2 on epidermal integrity. Results demonstrated a significant correlation between increased melanoma TGFβ2 secretion and loss of epidermal AMBRA1, with increased TGFβ2 secretion by AJCC stage I melanomas also significantly associated with a high-risk AMLo status and tumour metastasis. qPCR analysis of ALK1/5 mRNA revealed only ALK5 expression by CCD1106 cells while treatment of differentiated CCD1106 cells with exogenous TGFβ2 resulted in the downregulation of AMBRA1 and Claudin1 and increased expression of psmad 2/3. Collectively these data suggest secretion of TGFβ2 by high-risk melanomas results in canonical mediated-down regulation of AMBRA1 and Claudin1, the loss of epidermal integrity and tumour ulceration.