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Multi-modal molecular programs regulate melanoma cell state.

ABSTRACT: Melanoma cells display distinct intrinsic phenotypic states. Here, we seek to characterize the molecular regulation of these states using multi-omic analyses of whole exome, transcriptome, microRNA, long non-coding RNA and DNA methylation data together with reverse-phase protein array data on a panel of 68 highly annotated early passage melanoma cell lines. We demonstrate that clearly defined cancer cell intrinsic transcriptomic programs are maintained in melanoma cells ex vivo and remain highly conserved within melanoma tumors, are associated with distinct immune features within tumors, and differentially correlate with checkpoint inhibitor and adoptive T cell therapy efficacy. Through integrative analyses we demonstrate highly complex multi-omic regulation of melanoma cell intrinsic programs that provide key insights into the molecular maintenance of phenotypic states. These findings have implications for cancer biology and the identification of new therapeutic strategies. Further, these deeply characterized cell lines will serve as an invaluable resource for future research in the field.

SUBMITTER: Andrews MC

PROVIDER: S-EPMC9271073 | biostudies-literature | 2022 Jul

REPOSITORIES: biostudies-literature

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Multi-modal molecular programs regulate melanoma cell state.

Andrews Miles C MC Oba Junna J Wu Chang-Jiun CJ Zhu Haifeng H Karpinets Tatiana T Creasy Caitlin A CA Forget Marie-Andrée MA Yu Xiaoxing X Song Xingzhi X Mao Xizeng X Robertson A Gordon AG Romano Gabriele G Li Peng P Burton Elizabeth M EM Lu Yiling Y Sloane Robert Szczepaniak RS Wani Khalida M KM Rai Kunal K Lazar Alexander J AJ Haydu Lauren E LE Bustos Matias A MA Shen Jianjun J Chen Yueping Y Morgan Margaret B MB Wargo Jennifer A JA Kwong Lawrence N LN Haymaker Cara L CL Grimm Elizabeth A EA Hwu Patrick P Hoon Dave S B DSB Zhang Jianhua J Gershenwald Jeffrey E JE Davies Michael A MA Futreal P Andrew PA Bernatchez Chantale C Woodman Scott E SE

Nature communications 20220709 1

Melanoma cells display distinct intrinsic phenotypic states. Here, we seek to characterize the molecular regulation of these states using multi-omic analyses of whole exome, transcriptome, microRNA, long non-coding RNA and DNA methylation data together with reverse-phase protein array data on a panel of 68 highly annotated early passage melanoma cell lines. We demonstrate that clearly defined cancer cell intrinsic transcriptomic programs are maintained in melanoma cells ex vivo and remain highly ...[more]

PMID: 35810190

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Project description:The death and clearance of nurse cells is a consequential milestone in Drosophila melanogaster oogenesis. In preparation for oviposition, the germline-derived nurse cells bequeath to the developing oocyte all their cytoplasmic contents and undergo programmed cell death. The death of the nurse cells is controlled non-autonomously and is precipitated by epithelial follicle cells of somatic origin acquiring a squamous morphology and acidifying the nurse cells externally. Alternatively, stressors such as starvation can induce the death of nurse cells earlier in mid-oogenesis, manifesting apoptosis signatures, followed by their engulfment by epithelial follicle cells. To identify and contrast the molecular pathways underlying these morphologically and genetically distinct cell death paradigms, both mediated by follicle cells, we compared their genome-wide transcriptional, translational, and secretion profiles before and after differentiating to acquire a phagocytic capability, as well as during well-fed and nutrient-deprived conditions. By coupling the GAL4-UAS system to Translating Ribosome Affinity Purification (TRAP-seq) and proximity labeling (HRP-KDEL) followed by Liquid Chromatography tandem mass-spectrometry, we performed high-throughput screens to identify pathways selectively activated or repressed by follicle cells to employ nurse cell-clearance routines. We also integrated two publicly available single-cell RNAseq atlases of the Drosophila ovary to define the transcriptomic profiles of follicle cells. In this report, we describe the genes and major pathways identified in the screens and the striking consequences to Drosophila melanogaster oogenesis caused by RNAi perturbation of prioritized candidates. To our knowledge, our study is the first of its kind to comprehensively characterize two distinct apoptotic and non-apoptotic cell death paradigms in the same multi-cellular system. Beyond molecular differences in cell death, our investigation may also provide insights into how key systemic trade-offs are made between survival and reproduction when faced with physiological stress.

Project description:Project_description: We adapted proximity labeling to identify proteins trafficked through the endoplasmic reticulum in Drosophila follicle cells. To identify these proteins, we biochemically tagged and enriched proteins in the ER by expressing a genetically tagged fusion construct carrying horseradish peroxidase (HRP) fused to the ER-retention signal KDEL, along with secretion signal IgK leader sequence (ss). We expressed ss-HRP-KDEL-V5 in follicle cells, where it localized to the ER. We exposed dissected ovaries to biotin-phenol substrate and a brief pulse of H2O2, which catalyzed the biotinylation of the proteins in the vicinity of HRP. We identified 50 differentially abundant proteins in follicle cells in different states. Sample_processing_protocol: Freshly dissected ovaries were incubated in 300 uL of 500 uM biotin phenol for 30 minutes at room temperature rotating. Samples were then rinsed with 1X PBS twice and the biotinylation reaction was initiated by adding 1 mM H2O2 in PBS to the samples for 1 minute and rotating at room temperature. Ovaries were quickly washed with quencher solution (10 mM sodium ascorbate, 5 mM Trolox (Sigma-Aldrich), 10 mM sodium azide, then lysed in 100 uL RIPA buffer with quencher solution for 5 min on ice. RIPA buffer was composed of: 50 uL 1M Tris-HCl, 150 uL 5M NaCl, 50 uL of 10% SDS, 250 uL of 10% Sodium Deoxycholate, 500 uL of 10% TritonX-100, 50 uL of 100X Protease Inhibitor (Sigma-Aldrich - P8849), 50 uL of 100 mM PMSF, 3.550 mL of diH20. Tissue was homogenized by motorized pestle and centrifuged at 16.1g for 10 min at 4 deg. C. Clarified sample (clear middle layer) was transferred to a new tube and snap frozen in liquid nitrogen. Frozen protein samples were thawed on ice. Meanwhile, 50 uL streptavidin magnetic beads (Pierce 88817) were washed with 1 mL of RIPA lysis buffer twice. The beads were subsequently incubated with 90 uL of protein lysate (about 550 ug of protein) and an additional 500 uL of RIPA buffer was added to facilitate rotation for 1 hour at room temperature. Beads were pelleted on a magnetic rack and the supernatant (flow-through) was collected on ice. After each wash, the magnetic beads were transferred into new tubes and washed twice with 1 mL RIPA buffer, once with 1 mL KCl, once with 1 mL 0.1 M Na2CO3, once with 1 mL 2 M Urea in 10 mM Tris-HCl (pH 8), and twice with 1 mL RIPA buffer. RIPA buffer was removed, and the beads snap frozen in liquid nitrogen and stored at -80 deg. C. Beads from biotinylated protein pull-down were washed with 100 mM triethylammonium bicarbonate. Peptides were eluted from beads by on-bead trypsin digestion with 1ug Trypsin (Pierce) in 100 mM triethylammonium bicarbonate overnight rotating at 37 deg. C. Peptides were desalted using C18 ZipTip (Millipore) and subjected liquid chromatography coupled to tandem mass spectrometry on a Q Exactive HF-X (Thermo Fisher Scientific). Data-dependent fragmentation used collision-induced dissociation. Data_processing_protocol: RAW files were searched using MaxQuant under standard settings using the UniProt Drosophila melanogaster database, allowing for two missed trypsin cleavage sites, variable modifications for N-terminal acetylation, and methionine oxidation. Candidate peptides and protein identifications were filtered on the basis of a 1% false discovery rate. Raw intensities from the MaxQuant spectral database search engine were imported into R for downstream analysis. QC metrics such as number of unique peptides identified, percent of contaminants and reverse decoys detected, and total sum of intensities at the replicate level and condition level were computed and visualized using artMS v1.12.0. Spurious hits such as reverse decoys and potential contaminants were removed. R package DEP v1.16.0 was used to remove proteins not identified in both replicates simultaneously and variance stabilizing transformation was applied to normalize the intensities. Missing values were imputed using the k-nearest neighbors imputation method, followed by differential abundance estimation and multiple hypothesis testing correction in limma, all of which were implemented in DEP.

Dataset Information

Multi-modal molecular programs regulate melanoma cell state.

Publications

Multi-modal molecular programs regulate melanoma cell state.

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