RNA-seq Characterization of Melanoma Phenotype Switch in 3D Collagen after p38 MAPK Inhibitor Treatment.

Melanoma phenotype plasticity underlies tumour dissemination and resistance to therapy, yet its regulation is incompletely understood. In vivo switching between a more differentiated, proliferative phenotype and a dedifferentiated, invasive phenotype is directed by the tumour microenvironment. We found that treatment of partially dedifferentiated, invasive A375M2 cells with two structurally unrelated p38 MAPK inhibitors, SB2021920 and BIRB796, induces a phenotype switch in 3D collagen, as documented by increased expression of melanocyte differentiation markers and a loss of invasive phenotype markers. The phenotype is accompanied by morphological change corresponding to amoeboid-mesenchymal transition. We performed RNA sequencing with an Illumina HiSeq platform to fully characterise transcriptome changes underlying the switch. Gene expression results obtained with RNA-seq were validated by comparing them with RT-qPCR. Transcriptomic data generated in the study will extend the present understanding of phenotype plasticity in melanoma and its contribution to invasion and metastasis.

Targeting Mitochondrial Iron Metabolism Suppresses Tumor Growth and Metastasis by Inducing Mitochondrial Dysfunction and Mitophagy.

Deferoxamine (DFO) represents a widely used iron chelator for the treatment of iron overload. Here we describe the use of mitochondrially targeted deferoxamine (mitoDFO) as a novel approach to preferentially target cancer cells. The agent showed marked cytostatic, cytotoxic, and migrastatic properties in vitro, and it significantly suppressed tumor growth and metastasis in vivo. The underlying molecular mechanisms included (i) impairment of iron-sulfur [Fe-S] cluster/heme biogenesis, leading to destabilization and loss of activity of [Fe-S] cluster/heme containing enzymes, (ii) inhibition of mitochondrial respiration leading to mitochondrial reactive oxygen species production, resulting in dysfunctional mitochondria with markedly reduced supercomplexes, and (iii) fragmentation of the mitochondrial network and induction of mitophagy. Mitochondrial targeting of deferoxamine represents a way to deprive cancer cells of biologically active iron, which is incompatible with their proliferation and invasion, without disrupting systemic iron metabolism. Our findings highlight the importance of mitochondrial iron metabolism for cancer cells and demonstrate repurposing deferoxamine into an effective anticancer drug via mitochondrial targeting. SIGNIFICANCE: These findings show that targeting the iron chelator deferoxamine to mitochondria impairs mitochondrial respiration and biogenesis of [Fe-S] clusters/heme in cancer cells, which suppresses proliferation and migration and induces cell death. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/9/2289/F1.large.jpg.

Sustained Inflammatory Signalling through Stat1/Stat2/IRF9 Is Associated with Amoeboid Phenotype of Melanoma Cells.

The invasive behaviour of cancer cells underlies metastatic dissemination; however, due to the large plasticity of invasion modes, it is challenging to target. It is now widely accepted that various secreted cytokines modulate the tumour microenvironment and pro-inflammatory signalling can promote tumour progression. Here, we report that cells after mesenchymal-amoeboid transition show the increased expression of genes associated with the type I interferon response. Moreover, the sustained activation of type I interferon signalling in response to IFNß mediated by the Stat1/Stat2/IRF9 complex enhances the round amoeboid phenotype in melanoma cells, whereas its downregulation by various approaches promotes the mesenchymal invasive phenotype. Overall, we demonstrate that interferon signalling is associated with the amoeboid phenotype of cancer cells and suggest a novel role of IFNß in promoting cancer invasion plasticity, aside from its known role as a tumour suppressor.

High-throughput transcriptomic and proteomic profiling of mesenchymal-amoeboid transition in 3D collagen.

The plasticity of cancer cell invasion represents substantial hindrance for effective anti-metastatic therapy. To better understand the cancer cells' plasticity, we performed complex transcriptomic and proteomic profiling of HT1080 fibrosarcoma cells undergoing mesenchymal-amoeboid transition (MAT). As amoeboid migratory phenotype can fully manifest only in 3D conditions, all experiments were performed with 3D collagen-based cultures. Two previously described approaches to induce MAT were used: doxycycline-inducible constitutively active RhoA expression and dasatinib treatment. RNA sequencing was performed with ribo-depleted total RNA. Protein samples were analysed with tandem mass tag (TMT)-based mass spectrometry. The data provide unprecedented insight into transcriptome and proteome changes accompanying MAT in true 3D conditions.

Increased Level of Long Non-Coding RNA MALAT1 is a Common Feature of Amoeboid Invasion.

The ability of cancer cells to adopt various migration modes (the plasticity of cancer cell invasiveness) is a substantive obstacle in the treatment of metastasis, yet still an incompletely understood process. We performed a comparison of publicly available transcriptomic datasets from various cell types undergoing a switch between the mesenchymal and amoeboid migration modes. Strikingly, lncRNA MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) was one of three genes that were found upregulated in all amoeboid cells analyzed. Accordingly, downregulation of MALAT1 in predominantly amoeboid cell lines A375m2 and A2058 resulted in decrease of active RhoA (Ras homolog family member A) and was accompanied by the amoeboid-mesenchymal transition in A375m2 cells. Moreover, MALAT1 downregulation in amoeboid cells led to increased cell proliferation. Our work is the first to address the role of MALAT1 in MAT/AMT (mesenchymal to amoeboid transition/amoeboid to mesenchymal transition) and suggests that increased MALAT1 expression is a common feature of amoeboid cells.

RNA-seq of macrophages of amoeboid or mesenchymal migratory phenotype due to specific structure of environment.

M2-polarized macrophages have been shown to adapt their 3D migration mode to physical properties of surrounding extracellular matrix. They migrate in the integrin-mediated adhesion and proteolytic activity-dependent "mesenchymal" mode in stiff matrices and in the integrin and protease-independent "amoeboid" mode in low density, porous environments. To find out what impact the switching between the migration modes has on expression of both protein-coding and non-coding genes we employed RNA sequencing of total RNA depleted of ribosomal RNA isolated from macrophages migrating in either mode in 3D collagens. Differentially expressed genes from both categories have been detected and the changes in expression of selected genes were further validated with RT-qPCR. The acquired data will facilitate better understanding of how mechanical properties of tissue microenvironment reflect in macrophage immune function and how the transitions between mesenchymal and amoeboid migratory modes are regulated at the gene expression level.

TRH-receptor mobility and function in intact and cholesterol-depleted plasma membrane of HEK293 cells stably expressing TRH-R-eGFP.

UNLABELLED: Here we investigated the effect of disruption of plasma membrane integrity by cholesterol depletion on thyrotropin-releasing hormone receptor (TRH-R) surface mobility in HEK293 cells stably expressing TRH-R-eGFP fusion protein (VTGP cells). Detailed analysis by fluorescence recovery after photobleaching (FRAP) in bleached spots of different sizes indicated that cholesterol depletion did not result in statistically significant alteration of mobile fraction of receptor molecules (Mf). The apparent diffusion coefficient (Dapp) was decreased, but this decrease was detectable only under the special conditions of screening and calculation of FRAP data. Analysis of mobility of receptor molecules by raster image correlation spectroscopy (RICS) did not indicate any significant difference between control and cholesterol-depleted cells. Results of our FRAP and RICS experiments may be collectively interpreted in terms of a "membrane fence" model which regards the plasma membrane of living cells as compartmentalized plane where lateral diffusion of membrane proteins is limited to restricted areas by cytoskeleton constraints. Hydrophobic interior of plasma membrane, studied by steady-state and time-resolved fluorescence anisotropy of hydrophobic membrane probe DPH, became substantially more "fluid" and chaotically organized in cholesterol-depleted cells. Decrease of cholesterol level impaired the functional coupling between the receptor and the cognate G proteins of Gq/G11 family. IN CONCLUSION: the presence of an unaltered level of cholesterol in the plasma membrane represents an obligatory condition for an optimum functioning of TRH-R signaling cascade. The decreased order and increased fluidity of hydrophobic membrane interior suggest an important role of this membrane area in TRH-R-Gq/G11α protein coupling.