Dataset Information

Translational control mechanisms in cutaneous malignant melanoma: the role of eIF2?.

ABSTRACT: BACKGROUND:Melanoma cells develop adaptive responses in order to cope with particular conditions of tumor microenvironment, characterized by stress conditions and deregulated proliferation. Recently, the interplay between the stress response and the gene expression programs leading to metastatic spread has been reported. METHODS:We evaluated levels and localization of eIF2?/peIF2? in V600BRAF and wtBRAF metastatic melanoma cell lines by means of western blot and confocal microscopy analyses. Furthermore, we performed a sequence analyses and structure and dynamics studies of eIF2? protein to reveal the role of eIF2? and its correlations in different pathways involved in the invasive phase of melanoma. RESULTS:We found peIF2? both in cytoplasm and nucleus. Nuclear localization was more represented in V600BRAF melanoma cell lines. Our studies on eIF2? protein sequence indicated the presence of a predicted bipartite NLS as well as a nuclear export signal NES and an S1 domain, typical of RNA interacting proteins. Furthermore, we found high levels of transcription factor EB (TFEB), a component of the MiT/TFE family, and low ?-catenin levels in V600BRAF cells. CONCLUSIONS:Based on our results, we suggest that peIF2? nuclear localization can be crucial in ER stress response and in driving the metastatic spread of melanoma, through lysosomal signaling and Wnt/?-catenin pathway. In conclusion, this is the first evidence of nuclear localization of peIF2?, representing a possible target for future therapeutic approaches for metastatic melanoma.

SUBMITTER: Maida I

PROVIDER: S-EPMC6329103 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Publications

Translational control mechanisms in cutaneous malignant melanoma: the role of eIF2α.

Maida Immacolata I Zanna Paola P Guida Stefania S Ferretta Anna A Cocco Tiziana T Palese Luigi Leonardo LL Londei Paola P Benelli Dario D Azzariti Amalia A Tommasi Stefania S Guida Michele M Pellacani Giovanni G Guida Gabriella G

Journal of translational medicine 20190111 1

<h4>Background</h4>Melanoma cells develop adaptive responses in order to cope with particular conditions of tumor microenvironment, characterized by stress conditions and deregulated proliferation. Recently, the interplay between the stress response and the gene expression programs leading to metastatic spread has been reported.<h4>Methods</h4>We evaluated levels and localization of eIF2α/peIF2α in V600BRAF and wtBRAF metastatic melanoma cell lines by means of western blot and confocal microscop ...[more]

PMID: 30634982

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Project description:Perturbations in microRNA (miRNA) expression profiles has been reported for cutaneous malignant melanoma (CMM). With regards to a rapidly growing number of newly discovered miRNA sequences, the availability of up-to-date miRNA expression profiles for primary cutaneous malignant melanoma (PCMM), cutaneous malignant melanoma metastases (CMMM) and benign melanocytic naevi (BMN) is limited. Patients with PCMM (n=9), CMMM (n=4) and BMN (n=8) were included in the study. Specimens were obtained during surgery from the center of the tumors (lesional). An exploratory microarray analysis was performed by miRNA expression profiling based on miRBase V.16. Additionally, the expression levels of selected miRNA candidates were confirmed by TaqMan real-time quantitative polymerase chain reaction (RT-PCR). New miRNA candidates previously not described to be associated with CMM were found to be potentially dysregulated in CMM. Nine patients with PCMM (PCMM1-PCMM9), four patients with CMMM (CMMM1-CMMM4) and eight patients with BMN (BMN1-BMN8) were enrolled in the present study. All cutaneous specimens were harvested in the operating room, immediately stored in RNAlater (Quiagen, Hilden, Germany) and kept at - 80 °C until RNA isolation. Total RNA including miRNAs was isolated using the miRNeasy Mini Kit (Quiagen, Hilden, Germany) according to the manufacturer’s protocol. RNA concentration and purity was determined by NanoDrop ND-1000 spectral photometer (Peqlab, Erlangen, Germany). RNA integrity control (RIN) was determined by means of capillary electrophoresis with the 2100 Bioanalyzer and the RNA 6000 NanoLabCHip Kits (both Agilent Technologies, Santa Clara, USA). To be further considered for Microarray analysis a RIN ≥ 7.0 indicating medium to good RNA quality was defined as inclusion criteria. For assessment of labeling and hybridization efficiencies total RNA samples were spiked with in-vitro synthesized oligonucleotides by using the MicroRNA Spike-In Kit (Agilent Technologies, Santa Clara, USA). A total of 100 ng total RNA per sample were introduced into a labeling reaction. The spiked total RNA was treated with alkaline calf intestine phosphatase (CIP). The dephosphorylated RNA was labeled with the miRNA Complete Labeling and Hyb Kit (Agilent Technologies, Santa Clara, USA) according to the manufacturer´s instructions using T4 RNA ligase, incorporating Cyanine 3-Cytidine biphosphate (pCp). The Cyanine-3-labeled miRNA samples were prepared for One-Color based hybridization with Complete miRNA Labeling and Hyb Kit (Agilent Technologies, Santa Clara, USA) according to the manufacturer´s instructions. Labeled miRNA samples were hybridized at 55°C for 20 hrs on separate Human miRNA Microarrays Release 16.0 (8x60K format, (Agilent Technologies, Santa Clara, USA). Afterwards, microarrays were washed with increasing stringency using Gene Expression Wash Buffers (Agilent Technologies, Santa Clara, USA) followed by drying with acetonitrile (Sigma-Aldich, St.Louis, USA). Fluorescent signal intensities were detected with Scan Control A.8.4.1 Software (Agilent Technologies, Santa Clara, USA) on the Agilent DNA Microarray Scanner and extracted from the images using Feature Extraction 10.7.3.1 Software (Agilent Technologies, Santa Clara, USA). All the steps described were carried out according to the manufacturer´s instructions.

Dataset Information

Translational control mechanisms in cutaneous malignant melanoma: the role of eIF2?.

Publications

Translational control mechanisms in cutaneous malignant melanoma: the role of eIF2α.

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