Project description:Vanin1, a regulator of vitamin B5 metabolism, is expressed by sarcoma tumors. We evaluated its impact on sarcoma growth by using sarcoma cell lines derived from p16p19Vnn1-deficient mice and further transduced with an oncogenic RasV12 oncogene (R tumors) in the presence or not of a catalytically active (VR tumors) or mutated (VdR tumors) Vnn1 isoform. We used microarrays to detail the global programme of gene expression associated with the growth potential of various tumor cell lines grafted in Nude mice Overall design: Tumor cell lines were injected in Nude mice under normal diet (nd) and harvested between 2 and 3 weeks of growth. After RNA extraction, analysis of the transcriptome was perfomed using an Affymetrix platform at the Strasbourg
Project description:The role of Mithramycin as an anticancer drug has been well studied. Sarcoma is a type of cancer arising from cells of mesenchymal origin. Though incidence of sarcoma is not of significant percentage, it becomes vital to understand the role of Mithramycin in controlling tumor progression of sarcoma. In this article, we have analyzed the global gene expression profile changes induced by Mithramycin in two different sarcoma lines from whole genome gene expression profiling microarray data. We have found that the primary mode of action of Mithramycin is by global repression of key cellular processes and gene families like phosphoproteins, kinases, alternative splicing, regulation of transcription, DNA binding, regulation of histone acetylation, negative regulation of gene expression, chromosome organization or chromatin assembly and cytoskeleton.
Project description:To examine the addition of genetic or pharmacologic inhibition of hypoxia-inducible factor 1? (HIF-1?) to radiation therapy (RT) and vascular endothelial growth factor A (VEGF-A) inhibition (ie trimodality therapy) for soft-tissue sarcoma.Hypoxia-inducible factor 1? was inhibited using short hairpin RNA or low metronomic doses of doxorubicin, which blocks HIF-1? binding to DNA. Trimodality therapy was examined in a mouse xenograft model and a genetically engineered mouse model of sarcoma, as well as in vitro in tumor endothelial cells (ECs) and 4 sarcoma cell lines.In both mouse models, any monotherapy or bimodality therapy resulted in tumor growth beyond 250 mm(3) within the 12-day treatment period, but trimodality therapy with RT, VEGF-A inhibition, and HIF-1? inhibition kept tumors at <250 mm(3) for up to 30 days. Trimodality therapy on tumors reduced HIF-1? activity as measured by expression of nuclear HIF-1? by 87% to 95% compared with RT alone, and cytoplasmic carbonic anhydrase 9 by 79% to 82%. Trimodality therapy also increased EC-specific apoptosis 2- to 4-fold more than RT alone and reduced microvessel density by 75% to 82%. When tumor ECs were treated in vitro with trimodality therapy under hypoxia, there were significant decreases in proliferation and colony formation and increases in DNA damage (as measured by Comet assay and ?H2AX expression) and apoptosis (as measured by cleaved caspase 3 expression). Trimodality therapy had much less pronounced effects when 4 sarcoma cell lines were examined in these same assays.Inhibition of HIF-1? is highly effective when combined with RT and VEGF-A inhibition in blocking sarcoma growth by maximizing DNA damage and apoptosis in tumor ECs, leading to loss of tumor vasculature.
Project description:Alternative splicing of the Pkm gene product generates the PKM1 and PKM2 isoforms of the glycolytic enzyme pyruvate kinase. PKM2 expression is associated with embryogenesis, tissue regeneration, and cancer. PKM2 is also the pyruvate kinase isoform expressed in most wild-type adult tissues, with PKM1 restricted primarily to skeletal muscle, heart, and brain. To interrogate the functional requirement for PKM2 during tumor initiation in an autochthonous mouse model for soft tissue sarcoma (STS), we used a conditional Pkm2 allele (Pkm2fl ) to abolish PKM2 expression.PKM2 deletion slowed tumor onset but did not abrogate eventual tumor outgrowth. PKM2-null sarcoma cells expressed PKM1 with tumors containing a high number of infiltrating PKM2 expressing stromal cells. End-stage PKM2-null tumors showed increased proliferation compared to tumors with a wild-type Pkm2 allele, and tumor metabolite analysis revealed metabolic changes associated with PKM2 loss.While PKM2 is not required for soft tissue sarcoma growth, PKM2 expression may facilitate initiation of this tumor type. Because these data differ from what has been observed in other cancer models where PKM2 has been deleted, they argue that the consequences of PKM2 loss during tumor initiation are dependent on the tumor type.
Project description:MicroRNAs (miRNAs) can regulate tumor cell invasion and metastasis in a tumor-specific manner. We recently demonstrated that global downregulation of miRNAs after deleting dicer can promote development of distant metastases in a mouse model of primary soft tissue sarcoma (STS). In this study, we identified miRNAs that are differentially downregulated in metastatic STS in both human and mouse, and investigated the role of these miRNAs in metastasis. miRNA- TaqMan PCR arrays showed a global downregulation of miRNAs in metastatic human sarcomas. Similar analysis in mouse metastatic sarcomas revealed overlap for several downregulated miRNAs including miR-16, miR-103, miR-146a, miR-223, miR-342 and miR-511. Restoration of these downregulated miRNAs in mouse primary sarcoma cell lines showed that miR-16, but not other downregulated miRNAs, was able to significantly suppress both migration and invasion in vitro, without altering cell proliferation. In addition, orthotopic transplantation of a sarcoma cell line stably expressing miR-16 into the muscle of immunocompromised mice revealed that restoration of miR-16 can significantly decrease lung metastasis in vivo. However, no change in the rate of lung metastasis was observed when miR-16 was deleted in mouse primary sarcomas at sarcoma initiation. Taken together, these results indicate that miR-16 can have metastasis-suppressing properties both in vitro and in vivo. However, the loss-of-function experiments in autochthonous tumors indicate that loss of miR-16 is not sufficient to promote metastasis in vivo.
Project description:Sarcomas are rare mesenchymal malignant tumors with unique biological and clinical features. Given their diversity, heterogeneity, complexity, and rarity, the clinical management of sarcomas is quite challenging. Cell lines have been used as indispensable tools for both basic research and pre-clinical studies. However, empirically, sarcoma cell lines are not readily available. To understand the present status of sarcoma cell lines and identify their current challenges, we systematically reviewed reports on sarcoma cell lines. We searched the cell line database, Cellosaurus, and categorized the sarcoma cell lines according to the WHO classification. We identified the number and availability of sarcoma cell lines with a specific histology. We found 844 sarcoma cell lines in the Cellosaurus database, and 819 of them were named according to the WHO classification. Among the 819 cell lines, 36 multiple and nine single cell lines are available for histology. No cell lines were reported for 133 of the histological subtypes. Among the 844 cell lines, 148 are currently available in public cell banks, with 692 already published. We conclude that there needs to be a larger number of cell lines, with various histological subtypes, to better benefit sarcoma research.
Project description:Driver tyrosine kinase mutations are rare in sarcomas, and patterns of tyrosine phosphorylation are poorly understood. To better understand the signaling pathways active in sarcoma, we examined global tyrosine phosphorylation in sarcoma cell lines and human tumor samples. Anti-phosphotyrosine antibodies were used to purify tyrosine phosphorylated peptides, which were then identified by liquid chromatography and tandem mass spectrometry. The findings were validated with RNA interference, rescue, and small-molecule tyrosine kinase inhibitors. We identified 1,936 unique tyrosine phosphorylated peptides, corresponding to 844 unique phosphotyrosine proteins. In sarcoma cells alone, peptides corresponding to 39 tyrosine kinases were found. Four of 10 cell lines showed dependence on tyrosine kinases for growth and/or survival, including platelet-derived growth factor receptor (PDGFR)?, MET, insulin receptor/insulin-like growth factor receptor signaling, and SRC family kinase signaling. Rhabdomyosarcoma samples showed overexpression of PDGFR? in 13% of examined cases, and sarcomas showed abundant tyrosine phosphorylation and expression of a number of tyrosine phosphorylated tyrosine kinases, including DDR2, EphB4, TYR2, AXL, SRC, LYN, and FAK. Together, our findings suggest that integrating global phosphoproteomics with functional analyses with kinase inhibitors can identify drivers of sarcoma growth and survival.
Project description:To expand the available tools for investigating human sarcomas, we characterized the primary properties of 22 common, uncommon, and newly characterized sarcoma cell lines representing eight different histological subtypes. Throughout the characterization process we noticed that in vitro markers and assays are poor indicators of tumorigenicity and that generated xenografts often bear little resemblance to the original histopathology. In vitro properties examined included morphology, proliferation rate, cell cycle characteristics, invasiveness, and immunohistochemical expression of p53 and phospho-AKT. In vivo properties examined included days to tumor formation in NOD/SCID mice, xenograft morphology in several locations and immunohistochemical expression of Ki67, p53 and phospho-AKT. We believe that such an in depth comparison of a large cohort of sarcoma cell lines will be useful in both designing and interpreting experiments aimed at elucidating both the molecular biology and efficacy of therapeutic agents in sarcomas. However, that data generated also suggests a small set of sarcoma cell lines may be inappropriate for generalizations regarding biological behavior of specific sarcoma subtypes. Integration of functional genomics or other more sophisticated assays of cell lines may help bridge the differences in vitro and in vivo.
Project description:Current therapies for sarcomas are often inadequate. This study sought to identify actionable gene targets by selective targeting of the molecular networks that support sarcoma cell proliferation. Silencing of asparagine synthetase (ASNS), an amidotransferase that converts aspartate into asparagine, produced the strongest inhibitory effect on sarcoma growth in a functional genomic screen of mouse sarcomas generated by oncogenic Kras and disruption of Cdkn2a. ASNS silencing in mouse and human sarcoma cell lines reduced the percentage of S phase cells and impeded new polypeptide synthesis. These effects of ASNS silencing were reversed by exogenous supplementation with asparagine. Also, asparagine depletion via the ASNS inhibitor amino sulfoximine 5 (AS5) or asparaginase inhibited mouse and human sarcoma growth in vitro, and genetic silencing of ASNS in mouse sarcoma cells combined with depletion of plasma asparagine inhibited tumor growth in vivo. Asparagine reliance of sarcoma cells may represent a metabolic vulnerability with potential anti-sarcoma therapeutic value.