ADAR1 regulates melanoma cell invasiveness by controlling beta3-integrin via microRNA-30 family members.

Melanoma cells utilize multiple mechanisms to exit the primary tumor mass, invade the surroundings and subsequently distant tissues. We have previously reported that the expression of the RNA editing enzyme ADAR1 (adenosine deaminase acting on RNA) is downregulated in metastatic melanoma, which facilitates proliferation and invasion. Here we show that ADAR1 controls melanoma invasiveness by regulating ITGB3 expression via miR-30a and miR-30d. ADAR1 overexpression or knockdown leads to an increase or decrease, respectively, in the expression of both microRNAs. The effect is independent of RNA-editing. Dual luciferase assays show that both microRNAs directly regulate the expression of the ITGB3 integrin. Overexpression of the miR-30a or miR-30d lead to a decrease in ITGB3 and a resultant decreased invasive and metastatic capacities. Neutralization of the endogenous miR-30a or miR-30d leads to the opposite effect. The microRNAs regulate ITGB3 levels probably through a post-transcriptional effect, as both mRNA and protein levels of ITGB3 are affected. These results further expand our knowledge on the ADAR1-ITGB3 network and its central role in acquisition of the invasive phenotype of metastatic melanoma.

ADAR1-mediated regulation of melanoma invasion.

Melanoma cells use different migratory strategies to exit the primary tumor mass and invade surrounding and subsequently distant tissues. We reported previously that ADAR1 expression is downregulated in metastatic melanoma, thereby facilitating proliferation. Here we show that ADAR1 silencing enhances melanoma cell invasiveness and ITGB3 expression. The enhanced invasion is reversed when ITGB3 is blocked with antibodies. Re-expression of wild-type or catalytically inactive ADAR1 establishes this mechanism as independent of RNA editing. We demonstrate that ADAR1 controls ITGB3 expression both at the post-transcriptional and transcriptional levels, via miR-22 and PAX6 transcription factor, respectively. These are proven here as direct regulators of ITGB3 expression. miR-22 expression is controlled by ADAR1 via FOXD1 transcription factor. Clinical relevance is demonstrated in patient-paired progression tissue microarray using immunohistochemistry. The novel ADAR1-dependent and RNA-editing-independent regulation of invasion, mediated by ITGB3, strongly points to a central involvement of ADAR1 in cancer progression and metastasis.

Regulation of CEACAM1 Protein Expression by the Transcription Factor ETS-1 in BRAF-Mutant Human Metastatic Melanoma Cells.

BRAF becomes constitutively activated in 50% to 70% of melanoma cases. CEACAM1 has a dual role in melanoma, including facilitation of cell proliferation and suppression of infiltrating lymphocytes, which are consistent with its value as a marker for poor prognosis in melanoma patients. Here we show that BRAFV600E melanoma cells treated with BRAF and MEK inhibitors (MAPKi) downregulate CEACAM1 mRNA and protein expression in a dose- and exposure time-dependent manners. Indeed, there is a significant correlation between the presence of BRAFV600E and CEACAM1 expression in melanoma specimens obtained from 45 patients. Vemurafenib-resistant cell systems reactivate the MAPK pathway and restore basal CEACAM1 mRNA and protein levels. These combined results suggest transcriptional regulation. Indeed, luciferase reporting assays show that CEACAM1 promoter (CEACAM1p) activity is significantly reduced by MAPKi. Importantly, we show that the MAPK-driven CEACAM1p activity is mediated by ETS1, a major transcription factor and downstream effector of the MAPK pathway. Phosphorylation mutant ETS1T38A shows a dominant negative effect over CEACAM1 expression. The data are consistent with independent RNAseq data from serial biopsies of melanoma patients treated with BRAF inhibitors, which demonstrate similar CEACAM1 downregulation. Finally, we show that CEACAM1 downregulation by MAPKi renders the cells more sensitive to T-cell activation. These results provide a new view on a potential immunological mechanism of action of MAPKi in melanoma, as well as on the aggressive phenotype observed in drug-resistant cells.

A novel immune resistance mechanism of melanoma cells controlled by the ADAR1 enzyme.

The blossom of immunotherapy in melanoma highlights the need to delineate mechanisms of immune resistance. Recently, we have demonstrated that the RNA editing protein, adenosine deaminase acting on RNA-1 (ADAR1) is down-regulated during metastatic transition of melanoma, which enhances melanoma cell proliferation and tumorigenicity. Here we investigate the role of ADAR1 in melanoma immune resistance.Importantly, knockdown of ADAR1 in human melanoma cells induces resistance to tumor infiltrating lymphocytes in a cell contact-dependent mechanism. We show that ADAR1, in an editing-independent manner, regulates the biogenesis of miR-222 at the transcription level and thereby Intercellular Adhesion Molecule 1 (ICAM1) expression, which consequently affects melanoma immune resistance. ADAR1 thus has a novel, pivotal, role in cancer immune resistance. Corroborating with these results, the expression of miR-222 in melanoma tissue specimens was significantly higher in patients who had no clinical benefit from treatment with ipilimumab as compared to patients that responded clinically, suggesting that miR-222 could function as a biomarker for the prediction of response to ipilimumab.These results provide not only novel insights on melanoma immune resistance, but also pave the way to the development of innovative personalized tools to enable optimal drug selection and treatment.

miR-17 regulates melanoma cell motility by inhibiting the translation of ETV1.

Melanoma is an aggressive malignancy with a high metastatic potential. microRNA-17 (miR-17) is a member of the oncogenic miR-17/92 cluster. Here we study the effect of miR-17 on melanoma cell motility. Over expression of the mature or pri-microRNA form of miR-17 in WM-266-4 and 624mel melanoma lines enhances cell motility, evident in both wound healing and transwell migration assays. TargetScan algorithm predicts the PEA3-subfamily member ETV1 as a direct target of miR-17. Indeed, a 3-4-fold decrease of ETV1 protein levels are observed following miR-17 transfection into the various melanoma lines, with no significant change in ETV1 mRNA expression. Dual luciferase experiments demonstrate direct binding of miR-17 to the 3'-untranslated region of ETV1, confirmed by abolishing point mutations in the putative binding site. These combined results suggest regulation of ETV1 by miR-17 by a direct translational repression. Further, in both melanoma cell lines ETV1 knockdown by selective siRNA successfully pheno-copies the facilitated cell migration, while overexpression of ETV1 inhibits cell motility and migration. Altered ETV1 expression does not affect melanoma net-proliferation. In conclusion, we show a new role for miR-17 in melanoma, facilitating cell motility, by targeting the translation of ETV1 protein, which may support the development of metastasis.

Differential regulation of aggressive features in melanoma cells by members of the miR-17-92 complex.

The various roles of microRNAs (miRNAs) in controlling the phenotype of cancer cells are the focus of contemporary research efforts. We have recently shown that miR-17 directly targets the ADAR1 gene and thereby enhances melanoma cell aggressiveness. miR-17 and miR-20a belong to the miR-17/92 complex, and their mature forms are identical except for two non-seed nucleotides. Nevertheless, here we show that these two miRNAs carry markedly different effects on melanoma cells. A strong positive correlation was observed between the expression of miR-17 and miR-20a among various melanoma cultures. Luciferase assays showed that miR-17 but not miR-20a directly targets the 3' untranslated region of the ADAR1 gene. Ectopic expression of these miRNAs in melanoma cells differentially alters the expression of five exemplar TargetScan-predicted target genes: ADAR1, ITGB8, TGFBR2, MMP2 and VEGF-A. Whole-genome expression microarrays confirm a markedly differential effect on the transcriptome. Functionally, over-expression of miR-20a but not of miR-17 in melanoma cells inhibits net proliferation in vitro. The differential functional effect was observed following ectopic expression of the mature miRNA or of the pre-miRNA sequences. This suggests that the two non-seed nucleotides dictate target sequence recognition and overall functional relevance. These miRNAs are clearly not redundant in melanoma cell biology.

MicroRNAs in cancer: lessons from melanoma.

Melanoma is a high-grade, poorly differentiated malignant tumor of pigment-producing cells (melanocytes), accounting for more than 70% of the skin cancer related deaths. Although new lines of targeted therapy and immunotherapy were introduced lately, durable responses are not common as it is hard to target the elusive metastatic phenotype. microRNAs (miRNAs) are short non-coding RNA molecules that function as specific epigenetic regulators of the transcriptome. miRNAs are involved in a broad spectrum of physiological and pathological processes, including cancer-related functions such as proliferation, cell cycle, migration, invasion, immune evasion and drug resistance. These functions are mostly regulated in melanoma through four molecular deregulated pathways, including the RAS/MAPK pathway, the MITF pathway, the p16INK4A-CDK4-RB pathway and the PI3K-AKT pathway. miRNAs provide a strong platform for delineation of cancer mechanisms. Here we review the diverse roles of miRNAs in melanoma cell biology. Studying miRNA-mediated regulation of aggressive and tumor related features is expected to provide novel mechanistic insights that may pave the way for new diagnostic, prognostic and predictive tools as well as new molecular targets for future therapy.

MicroRNA-mediated loss of ADAR1 in metastatic melanoma promotes tumor growth.

Some solid tumors have reduced posttranscriptional RNA editing by adenosine deaminase acting on RNA (ADAR) enzymes, but the functional significance of this alteration has been unclear. Here, we found the primary RNA-editing enzyme ADAR1 is frequently reduced in metastatic melanomas. In situ analysis of melanoma samples using progression tissue microarrays indicated a substantial downregulation of ADAR1 during the metastatic transition. Further, ADAR1 knockdown altered cell morphology, promoted in vitro proliferation, and markedly enhanced the tumorigenicity in vivo. A comparative whole genome expression microarray analysis revealed that ADAR1 controls the expression of more than 100 microRNAs (miRNAs) that regulate many genes associated with the observed phenotypes. Importantly, we discovered that ADAR1 fundamentally regulates miRNA processing in an RNA binding­dependent, yet RNA editing­independent manner by regulating Dicer expression at the translational level via let-7. In addition, ADAR1 formed a complex with DGCR8 that was mutually exclusive with the DGCR8-Drosha complex that processes pri-miRNAs in the nucleus. We found that cancer cells silence ADAR1 by overexpressing miR-17 and miR-432, which both directly target the ADAR1 transcript. We further demonstrated that the genes encoding miR-17 and miR-432 are frequently amplified in melanoma and that aberrant hypomethylation of the imprinted DLK1-DIO3 region in chromosome 14 can also drive miR-432 overexpression.

Insulin-like growth factor-binding protein-7 (IGFBP7) transcript: A-to-I editing events in normal and cancerous human keratinocytes.

Non-melanoma skin cancers (NMSC) are the most common malignancies in caucasians worldwide. Insulin-like growth factor-binding protein-7 (IGFBP7) was suggested to function as a tumor suppressor gene in several cancers, and to play a role in the proliferation of keratinocytes. A-to-I RNA editing is a post-transcriptional mechanism frequently used to expand and diversify transcriptome and proteome repertoire in eukaryotic cells. A-to-I RNA editing can alter codons, substitute amino acids and affect protein sequence, structure, and function. Two editing sites were identified within the IGFBP7 transcript. To evaluate the expression and editing of IGFBP7 mRNA in NMSC compared to normal epidermis. We examined the expression and mRNA editing level of IGFBP7 in 22 basal cell carcinoma (BCC), 15 squamous cell carcinoma (SCC), and 18 normal epidermis samples that were surgically removed from patients by the Mohs Micrographic Surgery procedure. We studied the effect of IGFBP7 editing on an immortalized HaCaT keratinocyte cell model. IGFBP7 mRNA is over expressed in BCC and SCC compared to normal epidermis. Moreover, the IGFBP7 transcript is highly edited in normal epidermis, but its editing is significantly reduced in BCC and SCC. The edited form of IGFBP7 can inhibit proliferation and induce senescence in cultured keratinocytes. This study describes for the first time A-to-I editing in the coding sequence of a tumor suppressor gene in humans, and suggests that IGFBP7 editing serves as a fine-tuning mechanism to maintain the equilibrium between proliferation and senescence in normal skin.

Regulation of cancer aggressive features in melanoma cells by microRNAs.

MicroRNAs (miRNAs) are small non-coding RNAs with regulatory roles, which are involved in a broad spectrum of physiological and pathological processes, including cancer. A common strategy for identification of miRNAs involved in cell transformation is to compare malignant cells to normal cells. Here we focus on identification of miRNAs that regulate the aggressive phenotype of melanoma cells. To avoid differences due to genetic background, a comparative high-throughput miRNA profiling was performed on two isogenic human melanoma cell lines that display major differences in their net proliferation, invasion and tube formation activities. This screening revealed two major cohorts of differentially expressed miRNAs. We speculated that miRNAs up-regulated in the more-aggressive cell line contribute oncogenic features, while the down-regulated miRNAs are tumor suppressive. This assumption was further tested experimentally on five candidate tumor suppressive miRNAs (miR-31, -34a, -184, -185 and -204) and on one candidate oncogenic miRNA (miR-17-5p), all of which have never been reported before in cutaneous melanoma. Remarkably, all candidate Suppressive-miRNAs inhibited net proliferation, invasion or tube formation, while miR-17-5p enhanced cell proliferation. miR-34a and miR-185 were further shown to inhibit the growth of melanoma xenografts when implanted in SCID-NOD mice. Finally, all six candidate miRNAs were detected in 15 different metastatic melanoma specimens, attesting for the physiological relevance of our findings. Collectively, these findings may prove instrumental for understanding mechanisms of disease and for development of novel therapeutic and staging technologies for melanoma.