Stress-induced nuclear speckle reorganization is linked to activation of immediate early gene splicing.

Current models posit that nuclear speckles (NSs) serve as reservoirs of splicing factors and facilitate posttranscriptional mRNA processing. Here, we discovered that ribotoxic stress induces a profound reorganization of NSs with enhanced recruitment of factors required for splice-site recognition, including the RNA-binding protein TIAR, U1 snRNP proteins and U2-associated factor 65, as well as serine 2 phosphorylated RNA polymerase II. NS reorganization relies on the stress-activated p38 mitogen-activated protein kinase (MAPK) pathway and coincides with splicing activation of both pre-existing and newly synthesized pre-mRNAs. In particular, ribotoxic stress causes targeted excision of retained introns from pre-mRNAs of immediate early genes (IEGs), whose transcription is induced during the stress response. Importantly, enhanced splicing of the IEGs ZFP36 and FOS is accompanied by relocalization of the corresponding nuclear mRNA foci to NSs. Our study reveals NSs as a dynamic compartment that is remodeled under stress conditions, whereby NSs appear to become sites of IEG transcription and efficient cotranscriptional splicing.

MCU controls melanoma progression through a redox-controlled phenotype switch.

Melanoma is the deadliest of skin cancers and has a high tendency to metastasize to distant organs. Calcium and metabolic signals contribute to melanoma invasiveness; however, the underlying molecular details are elusive. The MCU complex is a major route for calcium into the mitochondrial matrix but whether MCU affects melanoma pathobiology was not understood. Here, we show that MCUA expression correlates with melanoma patient survival and is decreased in BRAF kinase inhibitor-resistant melanomas. Knockdown (KD) of MCUA suppresses melanoma cell growth and stimulates migration and invasion. In melanoma xenografts, MCUA_KD reduces tumor volumes but promotes lung metastases. Proteomic analyses and protein microarrays identify pathways that link MCUA and melanoma cell phenotype and suggest a major role for redox regulation. Antioxidants enhance melanoma cell migration, while prooxidants diminish the MCUA_KD -induced invasive phenotype. Furthermore, MCUA_KD increases melanoma cell resistance to immunotherapies and ferroptosis. Collectively, we demonstrate that MCUA controls melanoma aggressive behavior and therapeutic sensitivity. Manipulations of mitochondrial calcium and redox homeostasis, in combination with current therapies, should be considered in treating advanced melanoma.

The Roles of Extracellular Vesicles in Malignant Melanoma.

Different types of cells, such as endothelial cells, tumor-associated fibroblasts, pericytes, and immune cells, release extracellular vesicles (EVs) in the tumor microenvironment. The components of EVs include proteins, DNA, RNA, and microRNA. One of the most important functions of EVs is the transfer of aforementioned bioactive molecules, which in cancer cells may affect tumor growth, progression, angiogenesis, and metastatic spread. Furthermore, EVs affect the presentation of antigens to immune cells via the transfer of nucleic acids, peptides, and proteins to recipient cells. Recent studies have also explored the potential application of EVs in cancer treatment. This review summarizes the mechanisms by which EVs regulate melanoma development, progression, and their potentials to be applied in therapy. We initially describe vesicle components; discuss their effects on proliferation, anti-melanoma immunity, and drug resistance; and finally focus on the effects of EV-derived microRNAs on melanoma pathobiology. This work aims to facilitate our understanding of the influence of EVs on melanoma biology and initiate ideas for the development of novel therapeutic strategies.

Protein Signatures of NK Cell-Mediated Melanoma Killing Predict Response to Immunotherapies.

Despite impressive advances in melanoma-directed immunotherapies, resistance is common and many patients still succumb to metastatic disease. In this context, harnessing natural killer (NK) cells, which have thus far been sidelined in the development of melanoma immunotherapy, could provide therapeutic benefits for cancer treatment. To identify molecular determinants of NK cell-mediated melanoma killing (NKmK), we quantified NK-cell cytotoxicity against a panel of genetically diverse melanoma cell lines and observed highly heterogeneous susceptibility. Melanoma protein microarrays revealed a correlation between NKmK and the abundance and activity of a subset of proteins, including several metabolic factors. Oxidative phoshorylation, measured by oxygen consumption rate, negatively correlated with melanoma cell sensitivity toward NKmK, and proteins involved in mitochondrial metabolism and epithelial-mesenchymal transition were confirmed to regulate NKmK. Two- and three-dimensional killing assays and melanoma xenografts established that the PI3K/AKT/mTOR signaling axis controls NKmK via regulation of NK cell-relevant surface proteins. A "protein-killing-signature" based on the protein analysis predicted NKmK of additional melanoma cell lines and the response of patients with melanoma to anti-PD-1 checkpoint therapy. Collectively, these findings identify novel NK cell-related prognostic biomarkers and may contribute to improved and personalized melanoma-directed immunotherapies. SIGNIFICANCE: NK-cell cytotoxicity assays and protein microarrays reveal novel biomarkers of NK cell-mediated melanoma killing and enable development of signatures to predict melanoma patient responsiveness to immunotherapies.

TIAR marks nuclear G2/M transition granules and restricts CDK1 activity under replication stress.

The G2/M checkpoint coordinates DNA replication with mitosis and thereby prevents chromosome segregation in the presence of unreplicated or damaged DNA Here, we show that the RNA-binding protein TIAR is essential for the G2/M checkpoint and that TIAR accumulates in nuclear foci in late G2 and prophase in cells suffering from replication stress. These foci, which we named G2/M transition granules (GMGs), occur at low levels in normally cycling cells and are strongly induced by replication stress. In addition to replication stress response proteins, GMGs contain factors involved in RNA metabolism as well as CDK1. Depletion of TIAR accelerates mitotic entry and leads to chromosomal instability in response to replication stress, in a manner that can be alleviated by the concomitant depletion of Cdc25B or inhibition of CDK1. Since TIAR retains CDK1 in GMGs and attenuates CDK1 activity, we propose that the assembly of GMGs may represent a so far unrecognized mechanism that contributes to the activation of the G2/M checkpoint in mammalian cells.