Expression of the uncharacterised isoform, BCL2β, in melanoma

There are two known isoforms of the anti-apoptotic protein BCL2. While BCL2α is well characterised and known predominantly for its role in apoptosis, BCL2β has not yet been described. We sought to confirm the presence of this isoform in melanoma and characterise its role in the apoptotic response. We were able to verify expression of the rare isoform at the protein level in cell lines using multiple reaction monitoring tandem mass spectrometry. We also examined the role of the isoforms in apoptosis and melanin synthesis by a) monitoring gene response to stress at the mRNA level and b) using siRNAs targetted to each individual transcript. We treated cells with a variety of stressors and then monitored their apoptotic response using flow cytometry. We also quantified melanin production in response to UVB. The BCL2α response to stress (i.e. downregulation prior to apoptosis) was matched across melanocyte and melanoma cell lines. However, BCL2β response was varied across the melanoma cell lines, but matched that of BCL2α in melanocytes. Knock-down of both isoforms (individually) resulted in increased apoptosis in melanoma cell lines. We quantified mRNA of both BCL2 isoforms in our cohort of 189 FFPE melanoma tumours using qPCR. We also quantified total BCL2 protein expression in this same cohort using immunohistochemistry. Interestingly, expression of the BCL2β isoform in tumours was significantly associated with increased overall survival (686.4 weeks, 95% CI 462.5-910.3). BCL2β expression was also elevated in metastatic tissue compared with primary. This pattern was actually seen in the reverse at the protein level; total BCL2 protein was elevated in primary compared with metastatic. Analysis of SNP rs3943258 revealed that possession of the mutant T allele in melanoma patients corresponded with increased BCL2β expression. This contrasts with previous observations from a different study on healthy controls. Our current understanding of the role of BCL2β is based on the concept that it lacks the C-terminal transmembrane domain, and is thus incapable of localising to target organelles. It is generally assumed that the isoform is of null function. However, these observations have been based based on studies of non-representative synthetic versions of BCL2β. This is the first time the naturally transcribed version of the rare isoform has been studied, and the first time its role has been investigated since the 1980s. We have demonstrated herein that BCL2β performs an anti-apoptotic role within the cell, and that its regulation may be disrupted in melanoma.