2019 Fiscal Year Annual Research Report
エネルギー・栄養感知と細胞分化・増殖シグナルハブとしてのライソゾーム新機能の解明
| Project/Area Number |
19J15484
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| Research Institution | Kumamoto University |
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Principal Investigator |
馬 文娟 熊本大学, 医学教育部, 特別研究員(DC2)
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| Project Period (FY) |
2019-04-25 – 2021-03-31
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| Keywords | FLCN / TFE3 / nutrient / hypoxia / HIF / lysosome |
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| Outline of Annual Research Achievements |
① Regulation of hypoxia response pathway by aberrantly activated TFE3 transcription factor.
We have reported that FLCN-TFE3 axis regulates cell proliferation and differentiation through the metabolic reprogramming. However, precise downstream of FLCN-TFE3 has not been clarified thoroughly. To elucidate the critical downstream of activated TFE3, I performed RNA sequencing on chimeric TFE3 inducible cell lines and found that hypoxia signature genes were significantly enriched in chimeric TFE3 induced cells. The HIF1a and HIF2a mRNA level was elevated in chimeric TFE3 expressing cells. Next, ChIP-qPCR demonstrated that chimeric TFE3 bound to the M-BOX in HIF1a and HIF2a regulatory sequences. I also confirmed that chimeric TFE3 directly transcribe HIF1a and HIF2a by performing Luciferase reporter assay.
② Involvement of TFE3 in mTORC1 activation on lysosome.
I have analyzed the relationship between TFE3 and mTORC1 activity and found that mTORC1 is activated in chimeric TFE3 induced cells. To elucidate the mTORC1 activation mechanism, I have looked at the mTORC1 regulatory proteins on lysosome and found some of them, which constitute nutrient sensing machinery, expressed abundantly in chimeric TFE3 expression dependent manner.
③ A feedback loop among TFE3-mTORC1-HIF1a pathway under hypoxia and nutrient starvation
I treated chimeric TFE3 induced cell with variable starvation conditions and found that amino acid starvation and hypoxia stress can activate chimeric TFE3 and HIFa transcription activity.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
I have found that FLCN complex localizing on the lysosome regulates TFE3 transcriptional activity, which in turn upregulate nutrient sensing machinery and activate mTORC1. In addition, I found that HIF1a and HIF2a were direct transcriptional targets of activated TFE3. Furthermore, I have obtained a clue to support a new concept that FLCN-TFE3 axis coordinate nutrient and hypoxia signaling.
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| Strategy for Future Research Activity |
(1) Clarify essential target genes involved in FLCN-TFE3 axis
I will clarify the essential genes involved in FLCN-TFE3 axis which sense the amino acid signal and coordinate with the hypoxia and low nutrient stress signals. We developed Xp11.2 tRCC mouse model, expressing chimeric TFE3 specifically in kidney epithelial cells. We conditionally knocked out HIF1α and/or HIF2α on chimeric TFE3 KI mice. The histology and RNA-Seq of mouse kidneys indicated HIF1α knockout and HIF1/2α knockout rescued malignant phenotype of PRCC-TFE3 KI, KSP-Cre mice. On the other hand, HIF2α knockout did not rescued it. Thus, HIF1α may have essential roles in Xp11.2 tRCC development. We’re trying to find out essential genes and pathways which are specifically targeted by HIF1α and chimeric TFE3 by analyzing RNA-seq on mouse kidneys.
(2) Elucidate important inhibitors to perform experiments in vivo and vitro
Once we get crucial genes and pathways from Plan (1), we will perform cell proliferation assay on cancer cell line to check the inhibition effects of inhibitors under hypoxia and low nutrient conditions. We will also use the inhibitors to treat tRCC xenograft mice to confirm inhibition effects.
(3) Write the manuscript for publication
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Research Products
(4 results)
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[Journal Article] TFE3 Xp11.2 translocation renal cell carcinoma mouse model reveals novel therapeutic targets and identifies GPNMB as a diagnostic marker for human disease.2019
Author(s)
Baba M, Furuya M, Motoshima T, Lang M, Funasaki S, Ma W, Sun H, Hasumi H, Huang Y, Kato I, Kadomatsu T, Satou Y, Morris N, Karim BO, Ileva L, Kalen JD, Wilan Krisna LA, Hasumi Y, Sugiyama A, Kurahashi R, Nishimoto K, Oyama M, Nagashima Y, Kuroda N, Araki K, Eto M, Yao M, Kamba T, Suda T, Oike Y, Schmidt LS, Linehan WM.
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Journal Title
Molecular Cancer Research
Volume: 17
Pages: 1613～1626
DOI
Peer Reviewed / Open Access / Int'l Joint Research
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[Presentation] Regulation of hypoxia response pathway by chimeric TFE3 transcription factors found in Xp11.2 translocation renal cell carcinoma2019
Author(s)
Wenjuan Ma, Takanobu Motoshima, Yorifumi Satou, Paola Miyazato, Hisashi Hasumi, Tsuyoshi Kadomatsu, Mitsuko Furuya, Yoshiaki Kubota, Masahiro Yao, W. Marston Linehan, Yuichi Oike, Tomomi Kamba, Masaya Baba
Organizer
先端モデル動物プラットフォーム 2019年度若手支援技術講習会
