The Functional Role Of MiRNA-149~* In The Regulation Of Melanoma Under ER Stress

Melanoma is one of the most deadly cancers and has a number of distinctive biological properties, among which is the expression of the wild-type p53 that is frequently inactivated by mutations in many other cancer types. Another characteristic of melanoma is the increasing expression of the pro-survival Bcl-2 family protein Mcl-1 with progression of the disease. Mcl-1 is a major adaptive mechanism of melanoma cells to cellular stress conditions including endoplasmic reticulum (ER) stress, and plays an important role in resistance of melanoma to treatment.Since the expression of Mcl-1 is frequently regulated by post- transcriptional mechanisms, we sought to determine if microRNAs (miRNAs) are involved by use of expression profiling in the melanoma cell line Mel-RM under pharmacological ER stress that is known to cause up-regulation of Mcl-1. Amongst the changes in miRNAs triggered by the ER stress inducer tunicamycin (TM), an increase in miR-149* was the most pronounced and sustained.The effect of ER stress on miR-149* appeared specific for melanoma cells, as it was not observed in a number of other cell types including melanocytes, even though the ER stress response was triggered as evidenced by induction of Glucose-regulated protein 78 (GRP78). Analysis of 60 fresh metastatic melanoma isolates using qPCR showed that 59/60 samples expressed constitutively higher levels of miR-149* relative to melanocytes with an average～12 fold increase. Therefore, the expression of miR-149* is elevated in melanoma cells and can be further induced in response to ER stress.The tumour suppressor p53 is activated in response to cellular stress to prevent malignant transformation by activation of the DNA repair machinery to preserve the cell, or by induction of apoptosis to eliminate the cell should the damage prove irrevocable. The gene encoding p53 frequently undergoes inactivating mutations in many human cancers, but mutational inactivation of p53 is uncommon and wild-type p53 often expressed at high levels in melanoma, which, as judged from the malignant nature of the disease, fails to act as an effective tumour suppressor.Although some p53 downstream targets have been shown to be dysregulated, the mechanisms by which the tumour suppressing function of p53 is counteracted in melanoma remain to be elucidated.Here we show that p53 directly up-regulates microRNA-149* that in turn targets glycogen synthase kinase-3a??( GSK3a)? resulting in increased expression of Mcl-1 and resistance to apoptosis in melanoma cells. While deficiency in microRNA-149* undermines survival of melanoma cells and inhibits melanoma growth in a xenograft model, elevated expression of microRNA-149* is associated with decreased GSK3a? and increased Mcl-1 in fresh metastatic melanoma isolates. These results reveal a novel p53-dependent, microRNA-149*-mediated pathway that contributes to survival of melanoma cells and provide a rational explanation for the ineffectiveness of p53 to suppress melanoma. Moreover, they identify the expression of microRNA-149* as a mechanism involved in the increased expression of Mcl-1 in melanoma cells. We envisage that microRNA-149* may be a useful biomarker of melanoma progression and a therapeutic target for melanoma treatment.