Such activation is frequently associated with down selleck bio regulation or loss of PTEN phosphatase that antagonises PI3K signalling. Ab sent or decreased PTEN expression occurs in up to 90% of primary melanomas with mutations of PTEN or loss of heterozygosity at the PTEN locus accounting for this deficiency. Transcriptional repression of the gene by promoter methylation also occurs in a high propor tion of metastatic melanomas. In addition to PTEN, other major elements of the PI3K/Akt pathway are found to be amplified or mutated in melanoma. Akt3 expression is increased as a result of elevated gene copy number in 50% and 70% of primary melanomas and metastatic melanomas, respectively. PI3K/Akt pathway activation in melanoma can also occur through mutational activation of PI3KCA along with the muta tional activation of upstream receptor tyrosine kinases such as c KIT and EGFR.

Given the previous links established between MIF and Akt signalling as de scribed in the Introduction, we focussed our efforts on in vestigating Akt as the likely major pathway downstream of MIF in melanoma. Akt activation can stimulate proliferation through mul tiple downstream targets that affect cell cycle regulation. For example, Akt can phosphorylate p27 cyclin dependent kinase inhibitor, preventing its localisa tion to the nucleus and attenuating its cell cycle inhibitory effects. In addition, Akt also serves to phosphoryl ate and inactivate GSK3. As GSK3 phosphorylates cyclin D and cyclin E and targets them for proteasomal degrad ation, inhibition of GSK3 by Akt thereby acts to stabilise key cyclins involved in cell cycle entry.

In the current study, MIF knockdown resulted in decreased Akt phosphorylation in all melanoma cell lines tested, albeit to varying degrees. This effect was accompanied by a reduc tion in expression of cyclin D1 and cyclin dependent kinase 4, and an increased expression of p27. Moreover there was a correlation between the effects of MIF knock down and the degree of Akt activation amongst individual cell lines. Collectively this suggests that activation of the Akt pathway is one of the major mechanisms whereby MIF contributes to cell cycle regulation in melanoma. Since the overall importance of the PI3K/Akt pathway to melanoma biology cannot be understated, these findings imply clinical significance of MIF signalling in this disease.

Supporting this notion, a previous study of melanocytic tumours showed MIF mRNA and protein levels were high in malignant Entinostat tumours while expression was significantly lower in benign naevi. Here we verified these data using independent expression microarray datasets where collectively these findings support the general concept that MIF is differentially expressed between non malignant and malignant lesions with increased expression during melanoma progression.