Project description:Recent evidence shows that partial mitochondrial permeabilization and non-lethal caspase activation occur under certain circumstances, though it remains unclear how failed apoptosis impacts established cancers. Using a cancer cell model to trigger non-lethal caspase activation based on either BH3-only protein expression or chemotherapy treatment, we found that melanoma cancer cells failing to undergo complete apoptosis have a particular transcriptomic signature associated with focal adhesions, transendothelial migration and modifications of actin cytoskeleton.
Project description:Purpose. Aggressiveness is a crucial issue related to cutaneous melanoma malignancy and its high metastatic potential. Aim of this work is to identify new pathways or molecules controlling melanoma cell aggressiveness. Proliferation, migration and invasion capability under serum stimulation were analyzed in 12 human metastatic melanoma cell lines to identify the most aggressive ones as a model. The most proliferating/invading (defines as the most aggressive) A375 cell line was compared to the less aggressive one, Sk mel 28 by means of different approaches: 1) transcriptomic analysis by ILLUMINA platform; 2) proteomic study through LC-MS/MS analysis; 3) multiplexed assay to measure secretion of cytokines in conditioned media bioinformatic analysis was then carried out. Two groups of cells significantly differing in aggressiveness were identified and 2 cell lines, namely A375 and SK-Mel-28 were selected as model of the most and the less aggressive phenotype, respectively. A multi-omic analysis of several experimental datasets derived from transcriptomic, proteomic (mass spectrometric) and cytokinomic data was then carried out via Ingenuity Pathway Analysis (IPA) software. Analysis of upstream regulators and network analysis, indicated that the expression of tumor necrosis factor (TNF) were significantly differently expressed and functioning. The involvement of these pathways was confirmed by functional validation studies as zymography and proliferation studies and the most significantly upregulated pathway (TNF-alfa) was tested. Five melanoma cell lines with different MAGS were treated with an anti-TNFα monoclonal antibody and the most aggressive ones were highly significantly affected.
Project description:Emerging evidences suggest that both function and position of organelles are pivotal for tumor cell dissemination. Among them, lysosomes stand out as they integrate metabolic sensing with gene regulation and secretion of proteases. Yet, how lysosomes function is linked to their position and thereby control metastatic progression remains elusive. Here, we analyzed lysosome subcellular distribution in micropatterned patient-derived melanoma cells and found that lysosome spreading scales with their aggressiveness. Peripheral lysosomes promote invadopodia-based matrix degradation and invasion of melanoma cells which is directly linked to their lysosomal and cell transcriptional programs. When controlling lysosomal positioning using chemo-genetical heterodimerization in melanoma cells, we demonstrated that perinuclear clustering impairs lysosomal secretion, matrix degradation and invasion. Impairing lysosomal spreading in a zebrafish metastasis model significantly reduces invasive outgrowth. Our study provides a mechanistic demonstration that lysosomal positioning controls cell invasion, illustrating the importance of organelle adaptation in carcinogenesis.
Project description:MicroRNA (miRNA) expression profiling identified miR-638 as one of the most significantly overexpressed miRNAs in metastatic lesions compared with primary melanomas. miR-638 enhanced the tumourigenic properties of melanoma cells in vitro and lung colonization in vivo. mRNA expression profiling of miR-638 and antagomir-transduced cells identified new candidate genes as miR-638 targets, the majority of which is involved in p53-mediated apoptosis regulation. miR-638 depletion stimulated expression of p53 and its downstream target genes and induced apoptosis and autophagy in melanoma cells. miR-638 promoter analysis revealed transcription factor associated protein 2-M-NM-1 (TFAP2A) as a direct negative regulator of miR-638. Further analyses provided strong evidence for a double negative regulatory feedback loop between miR-638 and TFAP2A. Taken together, miR-638 may support melanoma progression by suppressing p53-mediated apoptosis pathways and by targeting the transcriptional repressor TFAP2A. TaqManM-BM-. low-density arrays (TLDA; human microRNA Cards A v2.1 & B v2.0, Applied Biosystems, Darmstadt, Germany) were used for measuring the expression of 667 human miRNAs in primary melanomas (PM, n=8), lymph node metastases (LNM, n=9) or distant cutaneous metastases (DCM, n=10).
Project description:MicroRNA (miRNA) expression profiling identified miR-638 as one of the most significantly overexpressed miRNAs in metastatic lesions compared with primary melanomas. miR-638 enhanced the tumourigenic properties of melanoma cells in vitro and lung colonization in vivo. mRNA expression profiling of miR-638 and antagomir-transduced cells identified new candidate genes as miR-638 targets, the majority of which is involved in p53-mediated apoptosis regulation. miR-638 depletion stimulated expression of p53 and its downstream target genes and induced apoptosis and autophagy in melanoma cells. miR-638 promoter analysis revealed transcription factor associated protein 2-? (TFAP2A) as a direct negative regulator of miR-638. Further analyses provided strong evidence for a double negative regulatory feedback loop between miR-638 and TFAP2A. Taken together, miR-638 may support melanoma progression by suppressing p53-mediated apoptosis pathways and by targeting the transcriptional repressor TFAP2A. Whole genome cDNA microarray (Illumina Human HT-12 v4 Expression BeadChip Kit, San Diego, CA 92122 USA) analyses were performed in duplicates using RNA extracted from SK-Mel-147 cells transfected with a non-targeting control, miR-638 or antagomiR-638.
Project description:In the present work we propose a new therapy for NRAS mutant melanoma. Simultaneous inhibition of MEK and ROCK caused induction of BimEL , PARP, and Puma, and hence apoptosis. In vivo, MEK and ROCK inhibition suppressed growth of established tumors. Our findings warrant clinical investigation of the effectiveness of combinatorial targeting of MAPK/ERK and ROCK in NRAS mutant melanoma.
Project description:MicroRNA (miRNA) expression profiling identified miR-638 as one of the most significantly overexpressed miRNAs in metastatic lesions compared with primary melanomas. miR-638 enhanced the tumourigenic properties of melanoma cells in vitro and lung colonization in vivo. mRNA expression profiling of miR-638 and antagomir-transduced cells identified new candidate genes as miR-638 targets, the majority of which is involved in p53-mediated apoptosis regulation. miR-638 depletion stimulated expression of p53 and its downstream target genes and induced apoptosis and autophagy in melanoma cells. miR-638 promoter analysis revealed transcription factor associated protein 2-α (TFAP2A) as a direct negative regulator of miR-638. Further analyses provided strong evidence for a double negative regulatory feedback loop between miR-638 and TFAP2A. Taken together, miR-638 may support melanoma progression by suppressing p53-mediated apoptosis pathways and by targeting the transcriptional repressor TFAP2A.
Project description:MicroRNA (miRNA) expression profiling identified miR-638 as one of the most significantly overexpressed miRNAs in metastatic lesions compared with primary melanomas. miR-638 enhanced the tumourigenic properties of melanoma cells in vitro and lung colonization in vivo. mRNA expression profiling of miR-638 and antagomir-transduced cells identified new candidate genes as miR-638 targets, the majority of which is involved in p53-mediated apoptosis regulation. miR-638 depletion stimulated expression of p53 and its downstream target genes and induced apoptosis and autophagy in melanoma cells. miR-638 promoter analysis revealed transcription factor associated protein 2-α (TFAP2A) as a direct negative regulator of miR-638. Further analyses provided strong evidence for a double negative regulatory feedback loop between miR-638 and TFAP2A. Taken together, miR-638 may support melanoma progression by suppressing p53-mediated apoptosis pathways and by targeting the transcriptional repressor TFAP2A.