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Project

[Molecular mechanism of bladder cancer invasion and metastasis focusing on invadopodia, exosome, and hyaluronidase]

Original in Japanese: Molecular mechanism of bladder cancer invasion and metastasis focusing on invadopodia, exosome, and hyaluronidase

Funder: Japan Society for the Promotion of Science

Funding period
JPY 4.2 M
USD 38 K
Funding amount
Abstract
[Recently, the role of exosomes in the invasion and metastasis of cancer cells has been drawing attention. The scaffold (invadopodia: Invadopodia) extended by bladder cancer cells is essential for invasion and metastasis, and the invasion process secretes exosomes. There are mRNAs, miRNAs, enzymes, etc. in exosomes, but there are still many unclear points. Exosomes may support the invasion and metastasis of cancer by preparing an environment (pre-metastatic niche) that allows easy survival. Since the exosome contains an enzyme that decomposes hyaluronan, which is a cell barrier, it is likely to be important for the transmission of exosome contents. However, since there are no studies investigating cancer invasion and metastasis in terms of exosomes and hyaluronidase, this study focuses on hyaluronidase in exosomes secreted by the infiltrates of bladder cancer cell lines, and determines the mechanism of bladder cancer invasion and metastasis. Elucidate. In this research, the following research was conducted in FY2018. (1) We established a method for preparing relatively large amounts of exosomes by ultracentrifugation from three types of urothelial cancer cell lines, KK47, RT-4, and YTS-1. The particle size and particle size of the prepared exosomes were measured using a nanoparticle multi-analyzer qNano. In addition, the expression of exosome markers CD9 and TSG10 was detected by Western blotting. From the above two data, it was confirmed that exosomes could be prepared. Furthermore, we established a method for fluorescently labeling exosomes with the fluorescent dye PKH67 and tracking the process of intracellular uptake when incubated with cells. (2) Using the urothelial carcinoma cell line YTS-1, we established the Cortactin KD YTS-1 strain in which the expression of cortactin, which is a protein essential for infiltrating process formation, was suppressed by shRNA. Of the four research projects, we completed the plan ① and established Cortactin KD YTS-1 cells. With the results so far, we are ready for an experiment to examine the role of exosomes listed in ② on the invasion and metastasis of cancer cells.]
Original in Japanese
近年、がん細胞の浸潤や転移におけるエクソソームの役割が注目されている。膀胱癌細胞が伸ばす足場(浸潤突起:Invadopodia)が浸潤・転移に必須であり、浸潤突起はエキソソームを分泌する。エキソソームの中にはmRNA、miRNA、酵素などが存在するが、未だ不明な点が多い。エキソソームはあらかじめ生存しやすい環境(前転移ニッチ)を整えて癌の浸潤・転移を補助している可能性がある。エキソソーム内には細胞バリアであるヒアルロン酸を分解する酵素がに含まれていることから、エキソソーム内容の伝達に重要である可能性が高い。しかしエキソソームとヒアルロニダーゼの点から癌の浸潤・転移を検討している研究はないため、本研究では膀胱癌細胞株の浸潤突起が分泌するエキソソーム内のヒアルロニダーゼに着目し膀胱癌浸潤・転移機序の解明を行う。本研究ではH30年度に以下の研究を行った。①3種の尿路上皮癌細胞株、KK47、RT-4、YTS-1から、超遠心により比較的大量にエキソソームを調製する方法を確立した。調製されたエキソソームの粒度・粒径を、ナノ粒子マルチアナライザーqNanoを使用して測定した。また、ウェスタン・ブロッティングによって、エキソソーム・マーカーであるCD9、TSG10の発現を検出した。上記2つのデータから確かにエキソソームの調製ができたことを確認した。さらに、蛍光色素PKH67を用いてエキソソームを蛍光標識し、細胞内とインキュベートした際に細胞内に取り込まれる過程を追跡する方法を確立した。②尿路上皮癌細胞株YTS-1を用いて、この浸潤突起形成に必須のタンパク質であるコータクチン(Cortactin)の発現を、shRNAにより抑制したCortactin KD YTS-1株を樹立した。本研究計画4つのうち、①の計画を完遂し、さらに、Cortactin KD YTS-1細胞を樹立した。ここまでの結果で、②で掲げたエキソソームが癌細胞の浸潤・転移に果たす役割を検討する実験の準備が整った。
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System

Categories
  • FOR (ANZSRC)

    0601 Biochemistry and Cell Biology

  • FOR (ANZSRC)

    1112 Oncology and Carcinogenesis

  • RCDC

    Cancer

  • RCDC

    Urologic Diseases

  • HRCS HC

    Cancer

  • HRCS RAC

    2.1 Biological and endogenous factors

  • Health Research Areas

    Biomedical

  • Broad Research Areas

    Basic Science