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T細胞リンパ腫におけるマイクロRNAによるがんとがん微小環境の制御

松山, 弘典 東京大学 DOI:10.15083/0002006124

2023.03.20

概要

【別紙2】

審査の結果の要旨
氏名



山 弘



本研究はがん遺伝子の一つで、チロシンキナーゼ活性を有するNPM-ALK陽性の未分化
大細胞リンパ腫(anaplastic large cell lymphoma、ALCL)において、NPM-ALKによるマイク
ロRNA(miRNA)の発現制御およびmiRNAのALK陽性ALCLの表現型への関与を解明する
ことを目的に研究を行い、下記の結果を得ている。
1.

既報においてALK陽性ALCLで発現が亢進しているとされたmiRNAの中で成熟型

miR-135bはヒト正常T細胞およびT細胞性白血病細胞株では発現が認められない一方で
ALK陽性ALCL細胞株では顕著に発現量が高いことが示された。miR-135b遺伝子はLEM
domain containing 1(LEMD1)遺伝子の第一イントロンに存在しており、LEMD1の発
現量もmiR-135bと同様にALK陽性ALCL細胞株において発現量が高いことが示された。
さらに、患者検体を用いた検討により、ALK陽性ALCLサンプルでは反応性リンパ節お
よびALK陰性ALCLサンプルと比較して、miR-135bの高発現が認められた。一方で、
ALKの異常を持つ肺がんおよび神経芽腫の細胞株ではその発現はALK陽性ALCL細胞株
と比較すると低いレベルであることが示された。
2. ALK阻害(低分子およびshRNAによる発現抑制)およびNPM-ALK野生型およびキナー
ゼ活性を失わせたNPM-ALK K210R変異体を用いた試験により、ALCL細胞でNPM-ALK
のキナーゼ活性を介してLEMD1とmiR-135bが誘導されていることが示唆された。次に、
NPM-ALKの下流で活性化されるシグナル経路であるSTAT3、Ras-Erk、およびPI3Kの
miR-135bの発現誘導に対する関与を検討したところ、STAT3が主にmiR-135bの発現に寄
与していることが示唆された。また、レポーター実験により、ALK陽性ALCL細胞株に
おいて内在性のmiR-135bが強い遺伝子制御活性を有していることを確認した。以上の結
果より、miR-135bはALK陽性ALCLにおいてNPM-ALK/STAT3シグナルによって誘導され
、高い活性を有していることが示された。
3.

ALK陽性ALCLにおけるmiR-135bの内在性かつ機能的な標的遺伝子の探索のため、in

silico標的遺伝子予測プログラムおよび3' UTRルシフェラーゼレポーターアッセイを組
み合わせた試験を実施し、LZTS1、LATS2、およびFOXO1が標的遺伝子候補であるこ
とが見出された。miRNAの機能を特異的に阻害するRNAデコイ(TuD RNA)システム
を用いた解析およびmiR-135bの導入実験により、miR-135bがFOXO1の発現を抑制し、
下流の細胞周期阻害因子p21とp27を調節することが明らかとなった。FOXOはDNAダ
メージを含む様々なストレスに対する増殖抑制反応における主要なメディエーターで
あることから、外因性miR-135b導入によるFOXO1のタンパク質発現量低下に伴う抗が
ん剤に対する反応性の変化を検討したところ、Cytosine -D-arabinofuranoside(Ara-C)
に対する耐性が上昇した。これらの結果より、ALK陽性ALCLにおいてmiR-135bは
FOXO1の発現制御を介して薬剤耐性に寄与している可能性が示唆された。
-1-

4.

さらなるmiR-135bの標的遺伝子を明らかとするために、shRNAを用いてNPM-ALK

の発現を抑制した場合としない場合の2通りのALCL細胞の遺伝子発現プロファイルの
比較データについて、miR-135bの予測標的遺伝子セットを用いたgene set enrichment
analysisとALCLを含む末梢T細胞リンパ腫-非特定型における遺伝子発現プロファイルの
再解析により、miR-135bの潜在的な標的にGATA3とSTAT6という2つのTh2マスター制
御因子が含まれていることを明らかとした。レポーターアッセイとTuD RNAシステム
を用いた検討により、STAT6およびGATA3がALK陽性ALCL細胞におけるmiR-135bの
内在性の標的であることを明らかとした。次に、TuD RNAシステムを用いてALK陽性
ALCL細胞株においてmiR-135bを抑制すると、IL-17AとIL-17Fの転写産物の発現レベル
が減弱し、IL-17Fはタンパク質産生の低下も認められた。さらに、Th17細胞への分化
の重要な制御因子として同定されたIB、炎症性サイトカインであるIL-6およびIL-8と
細胞傷害性分子であるgranzyme Bとperforin 1の発現低下も認められた。これらの結果か
ら、GATA3およびSTAT6を抑制することによってNPM-ALK/STAT3/miR-135b経路は
Th17細胞に似たIL-17産生性免疫学的表現型を誘導しALCLの免疫学的表現型に対して
幅広く影響を与えていることが示唆された。
5.

miR-135bによるIL-17産生性免疫学的表現型の形成がALCLにおけるがんの進展への

影響を検証するためにin vitroにおける非接触の共培養実験を実施し、ALCL細胞との共
培養によりヒト線維芽細胞の炎症促進性サイトカイン(IL-1、IL-6、およびIL-8)と
ケモカイン(CCL2、CCL7、CCL20、およびCXCL1/2)の発現が増強させること、かつ
その発現誘導がmiR-135bに依存していることが明らかとし、miR-135bがALCL細胞によ
る傍分泌型の炎症反応に関与していることが示唆された。さらに、in vivoでの検討にお
いて、miR-135bのアンチセンスオリゴヌクレオチドの腫瘍内への局所投与によりALCL
細胞株の皮下腫瘍の増殖が抑制された。この際、腫瘍内の血管新生の減少が認められ
た。これらの結果より、miR-135bによる傍分泌型炎症反応の調節が微小環境をがんに
とって有利な方向へと変化させていることが示唆された。

以上、本論文はALK陽性ALCLにおいて、NPM-ALK/STAT3経路がmiR-135bの発現を
誘導していること、高発現したmiR-135bがALK陽性ALCL細胞の免疫学的表現型の調節とそ
れに伴うがん微小環境の変化を誘導することおよびがん細胞の細胞周期関連因子を調節する
ことで、がんの増殖・進展に影響を及ぼしていると考えられる。
よって本論文は博士( 医学 )の学位請求論文として合格と認められる。

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