| 1. |
- Dolatabadi, Soheila, et al.
(författare)
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Cell Cycle and Cell Size Dependent Gene Expression Reveals Distinct Subpopulations at Single-Cell Level
- 2017
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Ingår i: Frontiers in Genetics. - : Frontiers Media SA. - 1664-8021. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- Cell proliferation includes a series of events that is tightly regulated by several checkpoints and layers of control mechanisms. Most studies have been performed on large cell populations, but detailed understanding of cell dynamics and heterogeneity requires single-cell analysis. Here, we used quantitative real-time PCR, profiling the expression of 93 genes in single-cells from three different cell lines. Individual unsynchronized cells from three different cell lines were collected in different cell cycle phases (GO/G1 - S - G2/M) with variable cell sizes. We found that the total transcript level per cell and the expression of most individual genes correlated with progression through the cell cycle, but not with cell size. By applying the random forests algorithm, a supervised machine learning approach, we show how a multi-gene signature that classifies individual cells into their correct cell cycle phase and cell size can be generated. To identify the most predictive genes we used a variable selection strategy. Detailed analysis of cell cycle predictive genes allowed us to define subpopulations with distinct gene expression profiles and to calculate a cell cycle index that illustrates the transition of cells between cell cycle phases. In conclusion, we provide useful experimental approaches and bioinformatics to identify informative and predictive genes at the single-cell level, which opens up new means to describe and understand cell proliferation and subpopulation dynamics.
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| 2. |
- Dolatabadi, Soheila, et al.
(författare)
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FUS-DDIT3 Fusion Oncoprotein Expression Affects JAK-STAT Signaling in Myxoid Liposarcoma
- 2022
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Ingår i: Frontiers in Oncology. - : Frontiers Media SA. - 2234-943X. ; 12
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Tidskriftsartikel (refereegranskat)abstract
- Myxoid liposarcoma is one of the most common sarcoma entities characterized by FET fusion oncogenes. Despite a generally favorable prognosis of myxoid liposarcoma, chemotherapy resistance remains a clinical problem. This cancer stem cell property is associated with JAK-STAT signaling, but the link to the myxoid-liposarcoma-specific FET fusion oncogene FUS-DDIT3 is not known. Here, we show that ectopic expression of FUS-DDIT3 resulted in elevated levels of STAT3 and phosphorylated STAT3. RNA sequencing identified 126 genes that were regulated by both FUS-DDIT3 expression and JAK1/2 inhibition using ruxolitinib. Sixty-six of these genes were connected in a protein interaction network. Fifty-three and 29 of these genes were confirmed as FUS-DDIT3 and STAT3 targets, respectively, using public chromatin immunoprecipitation sequencing data sets. Enriched gene sets among the 126 regulated genes included processes related to cytokine signaling, adipocytokine signaling, and chromatin remodeling. We validated CD44 as a target gene of JAK1/2 inhibition and as a potential cancer stem cell marker in myxoid liposarcoma. Finally, we showed that FUS-DDIT3 interacted with phosphorylated STAT3 in association with subunits of the SWI/SNF chromatin remodeling complex and PRC2 repressive complex. Our data show that the function of FUS-DDIT3 is closely connected to JAK-STAT signaling. Detailed deciphering of molecular mechanisms behind tumor progression opens up new avenues for targeted therapies in sarcomas and leukemia characterized by FET fusion oncogenes.
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| 3. |
- Dolatabadi, Soheila, et al.
(författare)
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JAK–STAT signalling controls cancer stem cell properties including chemotherapy resistance in myxoid liposarcoma
- 2019
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Ingår i: International Journal of Cancer. - : Wiley. - 0020-7136 .- 1097-0215. ; 145:2, s. 435-449
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Tidskriftsartikel (refereegranskat)abstract
- Myxoid liposarcoma (MLS) shows extensive intratumoural heterogeneity with distinct subpopulations of tumour cells. Despite improved survival of MLS patients, existing therapies have shortcomings as they fail to target all tumour cells. The nature of chemotherapy-resistant cells in MLS remains unknown. Here, we show that MLS cell lines contained subpopulations of cells that can form spheres, efflux Hoechst dye and resist doxorubicin, all properties attributed to cancer stem cells (CSCs). By single-cell gene expression, western blot, phospho-kinase array, immunoprecipitation, immunohistochemistry, flow cytometry and microarray analysis we showed that a subset of MLS cells expressed JAK–STAT genes with active signalling. JAK1/2 inhibition via ruxolitinib decreased, while stimulation with LIF increased, phosphorylation of STAT3 and the number of cells with CSC properties indicating that JAK–STAT signalling controlled the number of cells with CSC features. We also show that phosphorylated STAT3 interacted with the SWI/SNF complex. We conclude that MLS contains JAK–STAT-regulated subpopulations of cells with CSC features. Combined doxorubicin and ruxolitinib treatment targeted both proliferating cells as well as cells with CSC features, providing new means to circumvent chemotherapy resistance in treatment of MLS patients. © 2019 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC
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| 4. |
- Dolatabadi, Soheila
(författare)
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The role of fusion oncogenes and cancer stem cells in myxoid liposarcoma
- 2017
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Myxoid liposarcoma (MLS) is characterised by the FUS-DDIT3, or the less common EWSR1-DDIT3 fusion oncogene and is the second most common type of liposarcoma. The fusion oncogenes encode chimeric transcription factors that are causal factors in tumourigenesis however, their functions are poorly known. Notwithstanding continuous progress in treating MLS patients, existing therapies suffer from a major flaw as they do not target the cancer stem cells (CSCs). Unique features of CSCs include self-renewal, tumour initiating capacity and increased resistance to radiotherapy- and chemotherapy-induced cell death. Thus, CSCs are crucial targets for successful therapy. The aims of this project were to define the role of fusion oncogenes in tumourigenesis and to define signalling pathways controlling CSC features in MLS. Here, we demonstrated that MLS has an intact TP53 system that may explain why this tumour entity is genetically stable. We investigated the regulatory mechanisms, expression levels and effects of FUS-DDIT3 in detail, and showed that FUS-DDIT3 was uniquely regulated at both transcriptional and post-translational level. We also screened 70 well-characterised kinase inhibitors and determined their effects on cell proliferation and FUS-DDIT3 expression at mRNA and protein levels. To facilitate these studies, we developed a novel direct lysis approach that enables us to quantify, cell proliferation, mRNA and protein expression in the same sample. This method allowed us to identify a number of previously unknown signalling pathways that regulated the expression of FUS-DDIT3. To study cell division and growth in detail, we applied single-cell analysis on unsynchronized cells at different cell cycle phases and cell sizes. We found that the total transcript level per cell and the expression of most individual genes correlated with progression of the cell cycle, but not with cell size. Detailed studies of cell cycle predictive genes revealed a previously unknown G1 subpopulation. Finally, we showed that MLS contains cells with CSC features and that JAK-STAT signalling controls their numbers. Leukaemia inhibitory factor stimuli increased the number of CSCs, while JAK inhibition depleted the CSC pool. Inhibition of JAK-STAT also showed synergistic effects when combined with chemotherapy in vitro. Our findings concerning FUS-DDIT3 function and CSCs have increased our molecular understanding of tumour development and therapy resistance in MLS that will facilitate development of specific treatment strategies.
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| 5. |
- Forootan, Amin, et al.
(författare)
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Identification of Distinct and Common Subpopulations of Myxoid Liposarcoma and Ewing Sarcoma Cells Using Self-Organizing Maps
- 2023
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Ingår i: Chemosensors. - : MDPI AG. - 2227-9040. ; 11:1
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Tidskriftsartikel (refereegranskat)abstract
- Myxoid liposarcoma and Ewing sarcoma are the two most common tumor types that are characterized by the FET (FUS, EWSR1 and TAF15) fusion oncogenes. These FET fusion oncogenes are considered to have the same pathological mechanism. However, the cellular similarities between cells from the different tumor entities remain unknown. Here, we profiled individual myxoid liposarcoma and Ewing sarcoma cells to determine common gene expression signatures. Five cell lines were analyzed, targeting 76 different genes. We employed unsupervised clustering, focusing on self-organizing maps, to identify biologically relevant subpopulations of tumor cells. In addition, we outlined the basic concepts of self-organizing maps. Principal component analysis and a t-distributed stochastic neighbor embedding plot showed gradual differences among all cells. However, we identified five distinct and robust subpopulations using self-organizing maps. Most cells were similar to other cells within the same tumor entity, but four out of five groups contained both myxoid liposarcoma and Ewing sarcoma cells. The major difference between the groups was the overall transcriptional activity, which could be linked to cell cycle regulation. We conclude that self-organizing maps are useful tools to define biologically relevant subpopulations and that myxoid liposarcoma and Ewing sarcoma exhibit cells with similar gene expression signatures.
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| 6. |
- Jonasson, Emma, 1987, et al.
(författare)
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Total mRNA Quantification in Single Cells: Sarcoma Cell Heterogeneity.
- 2020
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Ingår i: Cells. - : MDPI AG. - 2073-4409. ; 9:3
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Tidskriftsartikel (refereegranskat)abstract
- Single-cell analysis enables detailed molecular characterization of cells in relation to cell type, genotype, cell state, temporal variations, and microenvironment. These studies often include the analysis of individual genes and networks of genes. The total amount of RNA also varies between cells due to important factors, such as cell type, cell size, and cell cycle state. However, there is a lack of simple and sensitive methods to quantify the total amount of RNA, especially mRNA. Here, we developed a method to quantify total mRNA levels in single cells based on global reverse transcription followed by quantitative PCR. Standard curve analyses of diluted RNA and sorted cells showed a wide dynamic range, high reproducibility, and excellent sensitivity. Single-cell analysis of three sarcoma cell lines and human fibroblasts revealed cell type variations, a lognormal distribution of total mRNA levels, and up to an eight-fold difference in total mRNA levels among the cells. The approach can easily be combined with targeted or global gene expression profiling, providing new means to study cell heterogeneity at an individual gene level and at a global level. This method can be used to investigate the biological importance of variations in the total amount of mRNA in healthy as well as pathological conditions.
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| 7. |
- Kroneis, Thomas, et al.
(författare)
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Global preamplification simplifies targeted mRNA quantification
- 2017
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- The need to perform gene expression profiling using next generation sequencing and quantitative real-time PCR (qPCR) on small sample sizes and single cells is rapidly expanding. However, to analyse few molecules, preamplification is required. Here, we studied global and target-specific preamplification using 96 optimised qPCR assays. To evaluate the preamplification strategies, we monitored the reactions in real-time using SYBR Green I detection chemistry followed by melting curve analysis. Next, we compared yield and reproducibility of global preamplification to that of target-specific preamplification by qPCR using the same amount of total RNA. Global preamplification generated 9.3-fold lower yield and 1.6-fold lower reproducibility than target-specific preamplification. However, the performance of global preamplification is sufficient for most downstream applications and offers several advantages over target-specific preamplification. To demonstrate the potential of global preamplification we analysed the expression of 15 genes in 60 single cells. In conclusion, we show that global preamplification simplifies targeted gene expression profiling of small sample sizes by a flexible workflow. We outline the pros and cons for global preamplification compared to target-specific preamplification.
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| 8. |
- Lindén, Malin, et al.
(författare)
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FET family fusion oncoproteins target the SWI/SNF chromatin remodeling complex
- 2019
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Ingår i: EMBO Reports. - : EMBO. - 1469-221X .- 1469-3178. ; 20
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Tidskriftsartikel (refereegranskat)abstract
- Members of the human FET family of RNA-binding proteins, comprising FUS, EWSR1, and TAF15, are ubiquitously expressed and engage at several levels of gene regulation. Many sarcomas and leukemias are characterized by the expression of fusion oncogenes with FET genes as 5′ partners and alternative transcription factor-coding genes as 3′ partners. Here, we report that the N terminus of normal FET proteins and their oncogenic fusion counterparts interact with the SWI/SNF chromatin remodeling complex. In contrast to normal FET proteins, increased fractions of FET oncoproteins bind SWI/SNF, indicating a deregulated and enhanced interaction in cancer. Forced expression of FET oncogenes caused changes of global H3K27 trimethylation levels, accompanied by altered gene expression patterns suggesting a shift in the antagonistic balance between SWI/SNF and repressive polycomb group complexes. Thus, deregulation of SWI/SNF activity could provide a unifying pathogenic mechanism for the large group of tumors caused by FET fusion oncoproteins. These results may help to develop common strategies for therapy. © 2019 The Authors. Published under the terms of the CC BY 4.0 license
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| 9. |
- Ranji, Parmida, et al.
(författare)
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Deciphering the role of FUS::DDIT3 expression and tumor microenvironment in myxoid liposarcoma development
- 2024
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Ingår i: Journal of Translational Medicine. - : BioMed Central Ltd. - 1479-5876. ; 22
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Tidskriftsartikel (refereegranskat)abstract
- Background: Myxoid liposarcoma (MLS) displays a distinctive tumor microenvironment and is characterized by the FUS::DDIT3 fusion oncogene, however, the precise functional contributions of these two elements remain enigmatic in tumor development. Methods: To study the cell-free microenvironment in MLS, we developed an experimental model system based on decellularized patient-derived xenograft tumors. We characterized the cell-free scaffold using mass spectrometry. Subsequently, scaffolds were repopulated using sarcoma cells with or without FUS::DDIT3 expression that were analyzed with histology and RNA sequencing. Results: Characterization of cell-free MLS scaffolds revealed intact structure and a large variation of protein types remaining after decellularization. We demonstrated an optimal culture time of 3 weeks and showed that FUS::DDIT3 expression decreased cell proliferation and scaffold invasiveness. The cell-free MLS microenvironment and FUS::DDIT3 expression both induced biological processes related to cell-to-cell and cell-to-extracellular matrix interactions, as well as chromatin remodeling, immune response, and metabolism. Data indicated that FUS::DDIT3 expression more than the microenvironment determined the pre-adipocytic phenotype that is typical for MLS. Conclusions: Our experimental approach opens new means to study the tumor microenvironment in detail and our findings suggest that FUS::DDIT3-expressing tumor cells can create their own extracellular niche.
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| 10. |
- Safavi, Setareh, et al.
(författare)
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HSP90 inhibition blocks ERBB3 and RET phosphorylation in myxoid/round cell liposarcoma and causes massive cell death in vitro and in vivo
- 2016
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Ingår i: OncoTarget. - : Impact Journals, LLC. - 1949-2553. ; 7:1, s. 433-445
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Tidskriftsartikel (refereegranskat)abstract
- Myxoid sarcoma (MLS) is one of the most common types of malignant soft tissue tumors. MLS is characterized by the FUS-DDIT3 or EWSR1-DDIT3 fusion oncogenes that encode abnormal transcription factors. The receptor tyrosine kinase (RTK) encoding RET was previously identified as a putative downstream target gene to FUS-DDIT3 and here we show that cultured MLS cells expressed phosphorylated RET together with its ligand Persephin. Treatment with RET specific kinase inhibitor Vandetanib failed to reduce RET phosphorylation and inhibit cell growth, suggesting that other RTKs may phosphorylate RET. A screening pointed out EGFR and ERBB3 as the strongest expressed phosphorylated RTKs in MLS cells. We show that ERBB3 formed nuclear and cytoplasmic complexes with RET and both RTKs were previously reported to form complexes with EGFR. The formation of RTK hetero complexes could explain the observed Vandetanib resistence in MLS. EGFR and ERBB3 are clients of HSP90 that help complex formation and RTK activation. Treatment of cultured MLS cells with HSP90 inhibitor 17-DMAG, caused loss of RET and ERBB3 phosphorylation and lead to rapid cell death. Treatment of MLS xenograft carrying Nude mice resulted in massive necrosis, rupture of capillaries and hemorrhages in tumor tissues. We conclude that complex formation between RET and other RTKs may cause RTK inhibitor resistance. HSP90 inhibitors can overcome this resistance and are thus promising drugs for treatment of MLS/RCLS.
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