Although recent studies have shown that adenosine-to-inosine (A-to-I) RNA editing occurs in microRNAs, its effects on tumor growth and metastasis are not well understood. We present evidence of CREB-mediated low expression of ADAR1 in metastatic melanoma cell lines and tumor specimens. Re-expression of ADAR1 resulted in the suppression of melanoma growth and metastasis in vivo. Consequently, we identified 3 miRs undergoing A-to-I editing in the low-metastatic melanoma but not in highly metastatic cell lines. One of these miRs, miR-455-5p has two A-to-I RNA editing sites. The biological function of edited miR-455-5p is different from the unedited form as it recognizes different set of genes. Indeed, w.t. miR-455-5p promotes melanoma metastasis via inhibition of the tumor suppressor gene CPEB1. Moreover, w.t. miR-455 enhances melanoma growth and metastasis in vivo while the edited form inhibits these features. These results demonstrate a previously unrecognized role of RNA editing in melanoma progression.

Summary Malignant melanoma is one of the most aggressive cancers and can disseminate from a relatively small primary tumor and metastasize to multiple sites, including the lung, liver, brain, bone, and lymph nodes. Elucidating the molecular and genetic changes that take place during the metastatic process has led to a better understanding of why melanoma is so metastatic. Herein, we describe the unique features that distinguish melanoma from other solid tumors and contribute to the malignant phenotype of melanoma cells. For example, although melanoma cells are highly antigenic, they are extremely efficient at evading host immune response. Melanoma cells share numerous cell surface molecules with vascular cells, are highly angiogenic, are mesenchymal in nature, and possess a higher degree of ‘stemness’ than do other solid tumors. Finally, analysis of melanoma mutations has revealed that the gene expression profile of malignant melanoma is different from that of other cancers. Elucidating these molecular and genetic processes in highly metastatic melanoma can lead to the development of improved treatment and individualized therapy options.

BackgroundThe pharmaceutical and biotechnology industries depend on findings from academic investigators prior to initiating programs to develop new diagnostic and therapeutic agents to benefit cancer patients. The success of these programs depends on the validity of published findings. This validity, represented by the reproducibility of published findings, has come into question recently as investigators from companies have raised the issue of poor reproducibility of published results from academic laboratories. Furthermore, retraction rates in high impact journals are climbing.Methods and FindingsTo examine a microcosm of the academic experience with data reproducibility, we surveyed the faculty and trainees at MD Anderson Cancer Center using an anonymous computerized questionnaire; we sought to ascertain the frequency and potential causes of non-reproducible data. We found that ∼50% of respondents had experienced at least one episode of the inability to reproduce published data; many who pursued this issue with the original authors were never able to identify the reason for the lack of reproducibility; some were even met with a less than “collegial” interaction.ConclusionsThese results suggest that the problem of data reproducibility is real. Biomedical science needs to establish processes to decrease the problem and adjudicate discrepancies in findings when they are discovered.

The molecular changes associated with the transition of melanoma cells from radial growth phase (RGP) to vertical growth phase (VGP) and the metastatic phenotype are not very well defined. However, some of the genes involved in this process and their transcriptional regulation are beginning to be elucidated. For example, the switch from RGP to VGP and the metastatic phenotype is associated with loss of the AP-2α transcription factor. AP-2α regulates the expression of c-KIT, MMP-2, VEGF, and the adhesion molecule MCAM/MUC18. Recently, we reported that AP-2α also regulates two G-protein coupled receptors (GPCR) PAR-1 and PAFR. In turn, the thrombin receptor, PAR-1, regulates the expression of the gap junction protein Connexin-43 and the tumor suppressor gene Maspin. Activation of PAR-1 also leads to overexpression and secretion of proangiogenic factors such as IL-8, uPA, VEGF, PDGF, as well certain integrins. PAR-1 also cooperates with PAFR to regulate the expression of the MCAM/MUC18 via phosphorylation of CREB. The ligands for these GPCRs, thrombin and PAF, are secreted by stromal cells, emphasizing the importance of the tumor microenvironment in melanoma metastasis. The metastatic phenotype of melanoma is also associated with overexpression and function of CREB/ATF-1. Loss of AP-2α and overexpression of CREB/ATF-1 results in the overexpression of MCAM/MUC18 which by itself contributes to melanoma metastasis by regulating the inhibitor of DNA binding-1 (Id-1). CREB/ATF-1 also regulates the angiogenic factor CYR-61. Our recent data indicate that CREB/ATF-1 regulates the expression of AP-2α, thus, supporting the notion that CREB is an important “master switch” in melanoma progression.

Melanoma is the deadliest form of skin cancer in which patients with metastatic disease have a five year survival-rate of less than 10%. Recently the over expression of a beta galactoside binding protein, galectin-3 (LGALS3), has been correlated with metastatic melanoma in patients. We have previously shown that silencing galectin-3 in metastatic melanoma cells reduces tumor growth and metastasis. Gene expression profiling identified the pro-tumorigenic gene autotaxin (ENPP2) to be down regulated after silencing galectin-3. Here we report that galectin-3 regulates autotaxin expression at the transcriptional level by modulating the expression of the transcription factor NFAT1 (NFATC2). Silencing galectin-3 reduced NFAT1 protein expression which resulted in decreased autotaxin expression and activity. Reexpression of autotaxin in galectin-3 silenced melanoma cells rescues angiogenesis, tumor growth and metastasis in vivo. Silencing NFAT1 expression in metastatic melanoma cells inhibited tumor growth and metastatic capabilities in vivo. Our data elucidate a previously unidentified mechanism by which galectin-3 regulates autotaxin, and assign a novel role for NFAT1 during melanoma progression.