| Project/Area Number |
22K06224
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Multi-year Fund |
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| Section | 一般 |
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| Review Section |
Basic Section 44010:Cell biology-related
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| Research Institution | Juntendo University |
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Principal Investigator |
ニバ タベ・エマ・エコ 順天堂大学, 大学院医学研究科, 准教授 (00727810)
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| Co-Investigator(Kenkyū-buntansha) |
篠原 正和 神戸大学, 医学研究科, 教授 (80437483)
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| Project Period (FY) |
2022-04-01 – 2025-03-31
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| Project Status |
Granted (Fiscal Year 2023)
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| Budget Amount *help |
¥4,290,000 (Direct Cost: ¥3,300,000、Indirect Cost: ¥990,000)
Fiscal Year 2024: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000)
Fiscal Year 2023: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
Fiscal Year 2022: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000)
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| Keywords | dystrophin / tumor suppressor / mdx mouse / rhabdomyosarcoma / metabolomics / alanine / spindle cell sarcoma / slc1a5 / serine / taurine / DMD / tumor / Epithelial/mesenchymal |
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| Outline of Research at the Start |
Dystrophin gene cause Duchenne muscular dystrophy (DMD), a skeletal muscle wasting disease. Dystrophin model mouse (mdx) and some DMD patients spontaneously develop tumors but the mechanism is not clear. Here, we want to identify some metabolites that might contribute to tumor growth in mdx mouse.
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| Outline of Annual Research Achievements |
In FY2023, the applicants identified many pathways that were enriched in cultured cells derived from spindle cell sarcoma (SCS) as well as rhabdomyosarcoma (RMS) through metabolomic analysis. One of them, the alanine, aspartate and glutamate biosynthetic pathway was the most impacted pathway with four components (alanine, L-aspartic acid, pyruvic acid and L-glutamic acid). Alanine showed significant 50% reduction in SCS when compared to RMS. Comparison of the tumor types for the other three components of this pathway showed statistically significant fold changes in L-aspartic acid and pyruvic acid, but not in L-glutamic acid. Next, the applicants selected alanine as the key metabolite, and performed functional assays to understand its significance in SCS tumor progression. Through amino acid supplementation, growth and invasion assays, transcription, and translation of EMT markers, and alanine transporter-knockdown experiments, the applicants observed that alanine was significant for growth, invasion, and increased expression of mesenchymal markers. They also demonstrated that the effect of alanine on growth was through the ERK/AMPK pathway.
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| Current Status of Research Progress |
Current Status of Research Progress
3: Progress in research has been slightly delayed.
Reason
The research was slightly delayed. There were some obstacles as explained below.
Design of shRNA for the alanine serine cysteine transporter (ASCT2/slc1a5) took some time. In addition, establishing a working concentration of metabolites and inhibitors for metabolite supplementation and inhibition assays was also time consuming. Manuscript submission and revision consumed a lot of time so some experiments were put on hold.
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| Strategy for Future Research Activity |
In FY2024, the applicants plan to extend their study to investigate the significance of increased intracellular alanine concentration in mdx mouse-derived RMS tumor cultured cells. They will study the effects of knockdown of alanine intracellular transporter, slc1a5 in growth, invasion and clonogenicity.
With the cultured cells established from three mdx mouse derived RMS, the applicants are now preparing stock of cells for various analyses. They are also preparing amino acid and inhibitor stock solutions for supplementation assay and silencing assays, respectively. In addition, to analyze metabolite uptake after silencing of amino acid transporter, they are preparing radiolabeled amino acid molecules. Finally, the applicants are also preparing manuscript for submission as a research article to American Journal of Cell Physiology- Cell physiology.
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