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Proteomic profiling of HT1080 fibrosarcoma cells of mesenchymal or amoeboid migratory phenotype

ABSTRACT: Amoeboid and mesenchymal migration of cancer cells both contribute to metastatic spreading of tumors. To characterize proteome changes underlying the different migratory modes, we performed mass spectrometry profiling of HT1080 fibrosarcoma cells undergoing mesenchymal-amoeboid transition induced by either doxycycline-inducible constitutively active RhoA or dasatinib treatment. Cells were kept in three-dimensional collagen gels with or without induction for 48 hours, lysed and processed for mass spectrometry analysis. In case of the inducible RhoA system, we also performed parallel two-dimensional experiments with cells on top of a dense layer of collagen (it was not feasible with dasatinib treatment).

OTHER RELATED OMICS DATASETS IN: E-MTAB-6823

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

SUBMITTER: Karel Harant

LAB HEAD: Jan Brábek

PROVIDER: PXD010425 | Pride | 2020-04-23

REPOSITORIES: Pride

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	D1_170107152223.raw	Raw
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	D4_170107104914.raw	Raw
	D5_170108050200.raw	Raw

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Publications

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

Čermák Vladimír V Gandalovičová Aneta A Merta Ladislav L Harant Karel K Rösel Daniel D Brábek Jan J

Scientific data 20200527 1

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: ...[more]

PMID: 32461585

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Transcriptomic profiling of HT1080 fibrosarcoma cells of mesenchymal or amoeboid migratory phenotype

Project description:Amoeboid and mesenchymal migration of cancer cells both contribute to metastatic spreading of tumors. To characterize gene expression profiles underlying the different migratory modes, we performed RNA sequencing of HT1080 fibrosarcoma cells undergoing mesenchymal-amoeboid transition induced by either doxycycline-inducible constitutively active RhoA or dasatinib treatment. Cells were kept in three-dimensional collagen gels with or without induction for 48 hours. RNA was isolated with a modified Chomczynski protocol. RNA-seq libraries were constructed from DNase I treated, rRNA depleted total RNA and sequenced with Illumina 2000/2500 sequencers.

2019-07-29 | E-MTAB-6823 | biostudies-arrayexpress

Dynamic T. vaginalis secretome and secretion of β-amalyses

Project description:During infection of the urogenital tract, the parasite Trichomonas vaginalis is challenged by factors such as nutrient limitation, the host immune response and coexistence with other members of the microbial fauna. Thus establishment of trichomonad infection depends on multifactorial host-parasite interactions, that involve both contact-dependent mechanisms, such as adherence to vaginal epithelial cells, contact-dependent extracellular killing of host cells, and active phagocytosis of host cells and bacteria, as well as contact independent mechanisms that include secretion of soluble biologically active molecules. To get more insight into the secreted proteome of T. vaginalis we detected and quantified released proteins at consecutive time points. Out of 2072 detected proteins, 89 were estimated to significantly increase in time. These include leishmaniolysin-like metallopeptidases, cathepsin-like cystein peptidases and a number of proteins involved in sugar metabolism, most notably beta-amylases, which may be significant in utilization of host-cell derived glycogen. We further discovered a large group of secreted hypothetical proteins with predicted triple tyrosine domain that may facilitate interaction with the extracellular matrix of the host epithelium.

2017-12-19 | PXD007034 | Pride

Evolution Anaerobic peroxisome in Mastigamoeba balamuthi

Project description:Adaptation of eukaryotic cells to anaerobic condition is reflected by deep changes in mitochondrial metabolism and their functional reduction. The most modified types of mitochondria are hydrogenosomes that generate molecular hydrogen with concomitant ATP synthesis and mitosome that completely lost energy metabolism. The reduction of mitochondria is associated with loss of peroxisomes that evolved from ER to compartmentalize pathways generating reactive oxygen species (ROS) and thus prevent cellular oxidative damage. Biogenesis and function of peroxisomes is tightly coupled with mitochondria. They share the fission machinery, pathways of oxidative metabolism, ROS scavenging, and metabolic products. The loss of peroxisomes in anaerobic eukaryotes with reduced mitochondria is thus not unexpected. Surprisingly, we identified peroxisomes in anaerobic, hydrogenosome bearing protist Mastigamoeba balamuthi. Initially, we identified conserved set of peroxisomal proteins peroxins that are required for protein import, peroxisomal growth and division. Key membrane associated peroxins (MbPex3, MbPex11, and MbPex14) were visualized in numerous vesicles that were distinct from hydrogenosomes, ER and Golgi body. Proteomic analysis of cellular fractions and prediction of peroxisomal targeting signals (PTS1/PTS2) allows identification of 51 putative peroxisomal matrix proteins. Expression of selected proteins in Saccharomyces cerevisiae revealed that they are specifically targeted to yeast peroxisomes. Matrix protein includes components of acyl CoA and carbohydrate metabolism, pyrimidine and CoA biosynthesis, whereas neither components of β-oxidation nor catalase were present. In conclusion, we identified new subclass of peroxisomes named “anaerobic” peroxisome that shifts the current paradigm and rises attention to reductive evolution of peroxisomes in anaerobic organisms.

2020-03-04 | PXD014205 | Pride

scRNA-seq and differential expression analyses of metastatic melanoma cells before, during and after tumour escape

Project description:Highly invasive integrin and protease-independent amoeboid migration is often employed by cancer cells at the invasive front, though little is known about the transcriptomic changes underlying the switch to an amoeboid migratory mode. Using a metastatic melanoma cell line expressing photoconvertible Dendra2 protein (WM983c-D2), tumour spheroids were cultured in 3D collagen matrices and imaged over time. Spheroids grown from WM983c-D2 cells generate cells of three distinct phenotypes: 1) compact, non-invading cells organised at the spheroid surface with no visual cell protrusions, hereafter termed ‘epithelial’; 2) cells still attached to the spheroid periphery that have commenced tumour escape, exhibiting cellular protrusions and an elongated phenotype, hereafter termed ‘escaping’; 3) singly migrating cells exhibiting a rounded phenotype and no lamellipodia consistent with amoeboid cell migration, hereafter termed 'amoeboid'. Individual amoeboid and escaping cells, as well as groups of epithelial cells at the spheroid edge were photoconverted and single cell sorted via FACS following enzymatic digestion of the collagen matrix. 10 Epithelial, 12 escaping and 13 amoeboid cells were subjected to scRNA-seq and differential expression analyses in order to identify changes in gene expression as melanoma cells convert from a non-invasive epithelial state to invasive amoeboid cells.

2022-05-21 | E-MTAB-11722 | biostudies-arrayexpress

RhoA-ROCK competes with YAP to regulate amoeboid breast cancer cell migration in response to lymphatic-like flow

Project description:Lymphatic drainage generates force that induces prostate cancer cell motility via activation of Yes-associated protein (YAP), but whether this response to fluid force is conserved across cancer types is unclear. Here, we show that shear stress corresponding to fluid flow in the initial lymphatics modifies taxis in breast cancer. Whereas some cell lines employ rapid amoeboid migration behavior in response to fluid flow, a separate subset decrease movement. Positive responders displayed transcriptional profiles typical of an amoeboid cell state. Regulation of the HIPPO tumor suppressor pathway and YAP activity also differed between breast subsets and prostate cancer. Although subcellular localization of YAP to the nucleus positively correlated with overall velocity of locomotion, YAP gain- and loss-of-function demonstrates that YAP inhibits breast cancer motility but is outcompeted by other pro-taxis mediators in the context of flow. Specifically, we show that RhoA dictates response to flow. GTPase activity of RhoA, but not Rac1 or Cdc42 Rho family GTPases, is elevated in cells that positively respond to flow and is unchanged in cells that decelerate under flow. Disruption of RhoA or the RhoA effector, Rho-associated kinase (ROCK), blocked shear stress-induced motility. Collectively, these findings demonstrate stratification of breast cancer subsets by flow-sensing mechanotransduction pathways and point to a role for biophysical force in regulation of an amoeboid cell state.

2022-03-27 | GSE191142 | GEO

Three types of Leishmania mexicana amastigotes: proteome comparison by quantitative proteomic analysis

Project description:Leishmania is the unicellular parasite transmitted by phlebotomine sand fly bite. It exists in two different forms; extracellular promastigotes, occurring in the gut of sand flies, and intracellular, round-shaped amastigotes residing mainly in vertebrate macrophages. As amastigotes originating from infected animals are often present in insufficient quality and quantity, two alternative types of amastigotes were introduced for laboratory experiments: axenic amastigotes and amastigotes from macrophages infected in vitro. Nevertheless, there is very little information about the degree of similarity/difference among these three types of amastigotes on proteomic level, whose comparison is crucial for assessing the suitability of using alternative types of amastigotes in experiments. In this study, L. mexicana amastigotes obtained from lesion of infected BALB/c mice were proteomically compared with alternatively cultivated amastigotes (axenic and macrophage-derived ones). Amastigotes of all three types were isolated, individually treated and analysed by LC-MS/MS proteomic analysis with quantification using TMT10-plex isobaric labeling. Significant differences were observed in the abundance of metabolic enzymes, virulence factors and proteins involved in translation and condensation of DNA. The most pronounced differences were observed between axenic amastigotes and lesion-derived amastigotes, macrophage-derived amastigotes were mostly intermediate between axenic and lesion-derived ones.

2023-03-11 | PXD036664 | Pride

pH stress in Desulfovibrio vulgaris Hildenborough

Project description:Using data from microarray experiments, we investigated the effects of excess NaCl on D. vulgaris. Keywords: stress response, time course Comparison of cells treated with pH10 pH to untreated cells at times of 0,120,240,30,60 min.

2007-08-31 | E-GEOD-8197 | biostudies-arrayexpress

Proteome responses of Grapevine to Downy mildew

Project description:A comparative proteomic analysis of grapevine leaves from the resistant genotype V. davidii ‘LiuBa-8’ (LB) and susceptible genotype V. vinifera ‘Pinot Noir’ (PN) at 12 hpi was conducted to understand the complex relationship between grapevine and P. viticola and the molecular mechanisms between difference resistant vitis genotypes at the early stage of infection. A total of 444 and 349 differentially expressed proteins (DEPs) were identified in LB and PN respectively at 12 hpi by iTRAQ. MapMan analysis showed that the majority of these DEPs were related to photosynthesis, metabolism, stress and redox. More up-expressed DEPs involved in photosynthesis and less down-expressed DEPs involved in metabolism contribute to the resistance in LB. PR10.2, PR10.3, HSF70.2 and HSP90.6 are proposed to be key proteins in both compatible and incompatible interactions. Accumulation H2O2 established the ROS signaling in incompatible interaction and APXs, GSTs maybe associated with the resistance of grapevine against downy mildew. Moreover, we verified four proteins to ensure the accuracy of proteome data using PRM. Overall, these data provide new insights into molecular events and provide valuable candidate proteins that could be used to illuminate molecular mechanisms underlying the incompatible and compatible interaction in early stage and eventually to be exploited to develop new protection strategy against downy mildew in grapevine.

2021-09-09 | PXD018845 | Pride

Transcriptomic profiles of CD4+ T lymphocytes from RhoA wild type (WT) and RhoA G17V mice were generated by deep sequencing in duplicate, using Illumina poly-A RNA seq.

Project description:The goal of this study is to identify transcriptomic effects of RhoA G17V expression in mouse CD4+ T lymphocytes. To this end, mice conditionally expressing RhoA G17V were generated by replacing exon 3 of RhoA with a minigene containing RhoA exons 3 to 6 flanked by loxP sites followed by a neomycin selection cassette and a mutant Rhoa exon 3 encoding the G17V substitution. These mice were then crossed with the CD4CreERT2 line (Tg(Cd4-cre/ERT2)11Gnri, Jackson Laboratories), which expresses a 4-hydroxi-tamoxifen-inducible form of the Cre recombinase under the control of the mouse Cd4 promoter to generate conditional inducible CD4 specific RhoA G17V mice (Rhoaco-G17V/+ CD4CreERT2). Mice were treated with vehicle only or Tamoxifen to induce expression of the RhoA G17V in CD4+ T cells. CD4CreERT2 mice were included as a negative control. CD4+ T cells were isolated from single cell suspensions of spleen by negative selection using the mouse naive CD4+ T Cell Isolation Kit (Milteny#130-104-453) according to the manufacturer’s protocol. Total RNA was extracted and standard Illumina poly-A RNA seq was performed. Naive mouse CD4+ T cells expressing RhoA G17V displayed an enrichment in a Tfh gene expression program.

2017-09-01 | GSE101340 | GEO

RNA-seq of human melanoma cell line A375m2 treated with SB202190 OR BIRB796 against DMSO-treated controls

Project description:Melanoma cells can switch between a more differentiated, proliferative phenotype and a dedifferentiated, invasive phenotype in response to signals from the environment. We found that p38 MAPK inhibitors SB202190 and BIRB796 induce phenotype switching from the dedifferentiated, invasive phenotype to the more differentiated phenotype in A375m2 melanoma cells cultured in three-dimensional collagen. The phenotype switch is accompanied by a morphological transition from rounded, blebby, amoeboid shape to elongated, spindle-like morphology. We performed transcriptome profiling with RNA-seq to uncover the underlying gene expression changes. Cells kept in 3D collagen were treated either with 10 µM inhibitor or with 0.1% DMSO (the vehicle) only for 48 hours and total RNA was isolated using a TRIzol-like procedure. Sequencing libraries were prepared from poly(A) RNA and sequenced with Illumina HiSeq 2500 instrument.

2020-11-28 | E-MTAB-9273 | biostudies-arrayexpress

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