The immediate molecular mechanisms behind invasive melanoma are poorly understood.,Recent studies implicate microRNAs (miRNAs) as important agents in melanoma and other cancers.,To investigate the role of miRNAs in melanoma, we subjected human melanoma cell lines to miRNA expression profiling, and report a range of variations in several miRNAs.,Specifically, compared with expression levels in melanocytes, levels of miR-211 were consistently reduced in all eight non-pigmented melanoma cell lines we examined; they were also reduced in 21 out of 30 distinct melanoma samples from patients, classified as primary in situ, regional metastatic, distant metastatic, and nodal metastatic.,The levels of several predicted target mRNAs of miR-211 were reduced in melanoma cell lines that ectopically expressed miR-211.,In vivo target cleavage assays confirmed one such target mRNA encoded by KCNMA1.,Mutating the miR-211 binding site seed sequences at the KCNMA1 3′-UTR abolished target cleavage.,KCNMA1 mRNA and protein expression levels varied inversely with miR-211 levels.,Two different melanoma cell lines ectopically expressing miR-211 exhibited significant growth inhibition and reduced invasiveness compared with the respective parental melanoma cell lines.,An shRNA against KCNMA1 mRNA also demonstrated similar effects on melanoma cells. miR-211 is encoded within the sixth intron of TRPM1, a candidate suppressor of melanoma metastasis.,The transcription factor MITF, important for melanocyte development and function, is needed for high TRPM1 expression.,MITF is also needed for miR-211 expression, suggesting that the tumor-suppressor activities of MITF and/or TRPM1 may at least partially be due to miR-211's negative post transcriptional effects on the KCNMA1 transcript.,Given previous reports of high KCNMA1 levels in metastasizing melanoma, prostate cancer and glioma, our findings that miR-211 is a direct posttranscriptional regulator of KCNMA1 expression as well as the dependence of this miRNA's expression on MITF activity, establishes miR-211 as an important regulatory agent in human melanoma.
The question whether tumorigenic cancer stem cells exist in human melanomas has arisen recently1.,Here we show that in melanomas, tumor stem cells (MTSC) can be isolated prospectively as a highly enriched CD271+ MTSC population using a process that maximizes viable cell transplantation1,6.,In this study the tumors sampled were taken from a broad spectrum of sites and stages.,High viability FACS isolated cells resuspended in a matrigel vehicle were implanted into T, B, and NK deficient Rag2−/− γc−/− mice (RG) mice.,The CD271+ subset of cells was the tumor initiating population in 9/10 melanomas tested.,Transplantation of isolated melanoma cells into engrafted human skin or bone in RG mice resulted in melanoma from CD271+ but not CD271− cells.,We also showed that tumors transplanted by CD271+ patient cells were capable of metastasis in-vivo.,Importantly, CD271+ melanoma cells lacked expression of TYR, MART and MAGE in 86%, 69% and 68% of melanoma patients respectively suggesting why T cell therapies directed at these antigens usually result in only temporary tumor shrinkage.