Browsing by Subject "Myoblasts"
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Item EGFR and Akt Signaling in Rhabdomyosarcoma Pathogenesis(2018-07-25) Granados, Valerie Ann; Amatruda, James F.; Olson, Eric N.; Lum, Lawrence; Galindo, ReneRhabdomyosarcoma is an aggressive soft-tissue malignancy comprised microscopically of neoplastic skeletal muscle-lineage precursors that fail to exit the cell-cycle and fuse into syncytial muscle - the underlying pathogenetic mechanisms for which remain unclear. We previously identified that misregulated myoblast fusion signaling via the TANC1 adaptor molecule promotes neoplastic transformation in RMS cells. As TANC1 is not presently pharmacologically targetable, here we have turned to our Drosophila RMS-related model to identify myoblast fusion-related elements potentially targetable in RMS. Genetic modifier screening against the fly model revealed that decreased Epidermal Growth Factor Receptor (EGFR) activity, which regulates myoblast fusion programming in flies, suppresses PAX-FOXO1 (PF)-induced lethality. As EGFR is pharmacologically targetable, we demonstrate that EGFR inhibitors antagonize RMS in a ERMS-RD cell line, but that other RMS cell lines are resistant. Further interrogation finds that EGFR inhibitor-sensitive cells exhibit marked down-regulated activation of the Akt intracellular signaling transducer, but not MEK/MAPK or STAT3, suggesting that Akt promotes and/or sustains RMS. We then demonstrate that Akt pharmacologic inhibition antagonizes RMS in vitro and in vivo, including RMS cells resistant to EGFR inhibition. We additionally find that sustained Akt1 activity promotes RMS cell terminal differentiation-arrest. Together, these findings point towards Akt activity as a broad RMS underpinning and therapeutic vulnerability.Item Genetic and Molecular Dissection of Rhabdomyosarcoma Tumorigenesis(2011-11-15) Edelman, Lauren Alexis; Galindo, ReneRhabdomyosarcoma is a tumor of skeletal muscle-type histogenesis and the most common pediatric soft tissue cancer. Rhabdomyosarcoma is often caused by one of two chromosomal translocations, t(1;32)(q35;q14) or t(2;13)(p36;q14), that are rhabdomyosarcoma - specific and diagnostic, and both drive equivalent PAX-FKHR fusion oncogenic transcription factors. Despite aggressive multimodal therapy, the 5-year survival rate of patients with advance-staged rhabdomyosarcoma remains less than 30% and has not improved in three decades. We intend to genetically characterize the molecular underpinnings of rhabdomyosarcoma to find new potential drug targets for treatment. Since PAX biology is structurally and functionally conserved (as is syncytial muscle development and structure), we have generated a new transgenic PAX-FKHR Drosophila model, which we have used to conduct a forward unbiased genetic screen to identify dominant modifiers of PAX-FKHR pathogenesis when expressed in growing muscle tissue. We also performed microarray analysis of fly PAX-FKHR tissue versus control tissue. We are now actively profiling genetic loci of interest for phenotypes in mammalian murine C2C12 myoblasts. After testing a subset of candidate genes identified in the screen, we have found that the genes identified as genetic modifiers of PAX-FKHR pathogenicity in the fly screen are indeed active in mammalian myoblast biology and PAX-FKHR pathobiology. These genes include loci normally involved in myogenesis and genes not previously correlated with mammalian skeletal muscle development or PAX biology. Our results suggest that the PAX-FKHR Drosophila transgenic model and genetic screening are revealing previously unknown gene targets that will likely underlie rhabdomyosarcoma pathogenesis. The discovery of new genes seminal to rhabdomyosarcoma pathobiology will be a valuable tool in the conceptual design of new therapies to target rhabdomyosarcoma and thus improve treatment.Item Mitochondrial Fission with Function Impairment in Burn Serum Treated C2C12 Cells(2016-01-19) Sehat, Alvand; Song, Juquan; Kumar, Puneet; Cai, Anthony; Huebinger, Ryan M.; Carlson, Deborah, L.; Zang, Qun S.; Wolf, Steven E.BACKGROUND: Burn patients suffer muscle mass loss associated with a hypercatabolic status. Mitochondria dynamics cycle is affected by metabolic status, and mitochondrial fission mediated high glucose induced cell death. Mitochondria function impairment associated with muscle mass loss has been observed in severe burn patients. We hypothesize that severe burn impaired muscle mass loss is associated with increased mitochondria fission with function impairment. The study was to investigate mitochondrial dynamics in response to burn serum stimulation. METHODS: Murine myoblast C2C12 cells were treated with DMEM media containing 10% rat serum isolated either from 40% TBSA scald burn rats, or control rats. Mitochondria was labeled with 3nM of MitoTracker Green FM dye, and live cell images were taken sequentially under a Nikon Ti Eclipse Confocal microscope. Cell lysates were collected for molecular biological analysis. Mitochondrial function was evaluated with Enzo Mito-ID membrane potential cytotoxicity kit. Target protein signals from cell lysate were detected by SDS-PAGE and western blot analysis. RESULTS: Mitochondrial morphology maintained the elongated linear shape in C2C12 cells when treated with 10% control rat serum. In contrast, when cells were treated with 10% burn serum, mitochondria reduced the elongated linear shape at 24 to 48 hours, and the florescent dye diffused at 72 hours. The cell florescent images showed an increase in circularity and fragmentation of mitochondria in C2C12 cells with burn serum stimulation. Meanwhile mitochondrial membrane potential decreased with 6hr post-burn serum stimulation. Western blot data showed that mitofusion-1 (Mfn1) significantly decreased in C2C12 cells with burn serum stimulation, confirming the observation of mitochondrial fission in response to burn serum. Cell death marker caspase 3 increased its expression in C2C12 cells with burn serum stimulation, suggesting a superfluous cell death in skeletal muscle after burn. CONCLUSION: Our results show an increase in the mitochondria fission/fusion ratio in C2C12 cells stimulated with burn serum isolated 6 hours after burn. The mechanism of mitochondrial fission with function impairment leading to muscle death is under investigation.Item A Role for POU3F3 in Myocyte Differentiation: Exploring New Frontier in Alveolar Rhabdomyosarcoma Development(2015-01-26) Denegre, Amelia; Granados, Valerie; Avirneni, Usha; Galindo, ReneSUMMARY: Children who are diagnosed with pediatric rhabdomyosarcoma (RMS), a mesenchymal-derived soft tissue cancer that comprises 3.5% of childhood cancers, are often delivered a bleak prognosis with little hope of a future. Despite significant advances illuminating transcription factor signaling in RMS onset and progression, research is still needed to precisely understand RMS pathogenesis on a molecular level in order to develop targeted treatment options. OBJECTIVE: The goal of this project is to explore the role of POU3F3 in myogenesis, particularly in relation to cell fusion and myocyte differentiation. METHODS: Immunofluorescence: POU3F3 knockdown cells were differentiated in 2% horse serum. On day 2 of differentiation, cells were probed with POU3F3 primary and red immunofluorescent secondary antibody, allowing for imaging of POU3F3 localization during myoblast differentiation. Western Blot: Three knock-down shPOU3F3 C2C12 cell-line constructs were tested. Western blots were performed that compared C2C12 control, POU3F3 overexpression, and POU3F3 knockdown cells. Crystal Violet: After differentiation, POU3F3 knock-down and control cell lines were stained with crystal violet stain to visualize the effect of POU3F3 knockdown on differentiation. RESULTS: Immunofluorescence: We confirmed that in knockdown C2C12 cells, POU3F3 localizes like control C2C12, in the periphery. Previous research has shown that in overexpressed POU3F3 C2C12 cell lines, POU3F3 localizes to the nucleus. The implication of the disconnect between POU3F3 location in controls and knock-downs versus overexpressed cell lines is an area that is an opportunity for further research. Western Blot: Western Blot analysis confirmed that POU3F3 knockdown was successful, and provides a platform for further POU3F3 interrogation. Crystal Violet: Crystal violet staining suggests that POU3F3 participates in a myoblast differentiation, as the control cells fuse into myotubes, while POU3F3-silenced cells do not. CONCLUSION: These initial results suggest that POU3F3 participates in muscle differentiation. Next, the Galindo lab will be probing POU3F3 function in myogenesis in greater depth, insights they will next apply to RMS.Item Role of NPHS Gene in Mammalian Myogenesis and Rhabdomyosarcoma(2019-03-27) Lawson, Kelly; Galindo, Rene; Castrillon, Diego H.; Le, Lu Q.BACKGROUND: Rhabdomyosarcoma (RMS) is an aggressive soft tissue sarcoma comprised histologically of neoplastic skeletal muscle-lineage precursors that fail to terminally differentiate or fuse into syncytial muscle. The underlying RMS pathogenetic mechanisms that impede differentiation and promote neoplastic transformation remain largely unclear. The lab has shown misregulated myoblast fusion activity as a molecular underpinning of RMS pathogenesis. Data point to Immunoglobulin Superfamily transmembrane Receptors family members KIRREL and NPHS as fusion receptors. OBJECTIVE: We hypothesize that KIRREL and NPHS are essential for muscle development, with alterations underlying RMS pathogenesis. METHODS: Here, we look at pattern of expression of NPHS throughout myogenesis and its effects on differentiation and fusion. We use a C2C12 murine myoblast cell line to evaluate the role of NPHS in muscle development. Pattern of expression is evaluated through a qPCR analysis to quantify levels of NPHS RNA throughout the six-day mammalian myogenesis process. Loss-of- function studies are achieved by transfecting C2C12 cells with vectors containing sequences necessary to express shRNA directed against NPHS RNA, effectively knocking down the NPHS gene product in these cells. Gain-of-function studies are achieved by transfecting C2C12 cells with vectors containing the NPHS gene. The myoblasts with NPHS knockdown and overexpression are observed throughout the six-day myogenesis process to assess for alteration in differentiation, fusion, or both differentiation and fusion. To better visualize the results, crystal violet stains and immunofluorescent stains are used. RESULTS: Quantification of NPHS RNA illustrates the precisely regulated expression of NPHS during myogenesis. Specifically, upregulation of the gene is seen during key points in differentiation and fusion, with a dramatic increase in NPHS expression on days three and six of the six-day myogenesis process. NPHS loss-of-function studies show a decrease in myoblast differentiation and fusion. When viewed on day four, myoblasts with reduced expression of NPHS showed a significant decrease (p < 0.01) in differentiation and fusion when compared to controls. NPHS gain-of-function studies show enhanced differentiation and fusion. Day four and day six myoblasts with increased expression of NPHS had significantly increased levels of differentiation and fusion when compared to the control myoblasts (p < 0.01). CONCLUSION: These results lead us to conclude that NPHS is critical for proper muscle development, and potentiates RMS, making it a prospective treatment target. With this knowledge as a foundation, the pathways of myogenesis and the RMS pathogenesis will continue to be elucidated. This information will allow for novel targeted treatments of RMS that will not carry the morbidity and mortality of traditional treatments such as surgery, radiation, and chemotherapy.