Amano Yosuke, Ishikawa Rie, Sakatani Toshio, Ichinose Junji, Sunohara Mitsuhiro, Watanabe Kousuke, Kage Hidenori, Nakajima Jun, Nagase Takahide, Ohishi Nobuya, Takai Daiya
Department of Respiratory Medicine, The University of Tokyo Hospital, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan.
Department of Cardiothoracic Surgery, The University of Tokyo Hospital, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan.
Biochem Biophys Res Commun. 2015 Feb 13;457(3):457-60. doi: 10.1016/j.bbrc.2015.01.014. Epub 2015 Jan 14.
Inflammatory myofibroblastic tumor (IMT) is a mesenchymal tumor that can arise from anywhere in the body. Anaplastic lymphoma kinase (ALK) gene rearrangements, most often resulting in the tropomyosin 3 (TPM3)-ALK fusion gene, are the main causes of IMT. However, the mechanism of malignant transformation in IMT has yet to be elucidated. The purpose of this study was to clarify the role of the TPM3 region in the transformation of IMT via TPM3-ALK. Lentivirus vectors containing a TPM3-ALK fusion gene lacking various lengths of TPM3 were constructed and expressed in HEK293T and NIH3T3 cell lines. Focus formation assay revealed loss of contact inhibition in NIH3T3 cells transfected with full-length TPM3-ALK, but not with ALK alone. Blue-native polyacrylamide gel electrophoresis (BN-PAGE) revealed that TPM3-ALK dimerization increased in proportion to the length of TPM3. Western blot showed phosphorylation of ALK, ERK1/2, and STAT3 in HEK293T cells transfected with TPM3-ALK. Thus, the coiled-coil structure of TPM3 contributes to the transforming ability of the TPM3-ALK fusion protein, and longer TPM3 region leads to higher dimer formation.
炎性肌纤维母细胞瘤（IMT）是一种间叶性肿瘤，可发生于身体的任何部位。间变性淋巴瘤激酶（ALK）基因重排，最常见的是导致原肌球蛋白3（TPM3）-ALK融合基因，是IMT的主要病因。然而，IMT恶性转化的机制尚未阐明。本研究的目的是阐明TPM3区域在IMT通过TPM3-ALK转化中的作用。构建了含有缺失不同长度TPM3的TPM3-ALK融合基因的慢病毒载体，并在HEK293T和NIH3T3细胞系中表达。集落形成试验显示，用全长TPM3-ALK转染的NIH3T3细胞失去接触抑制，而单独用ALK转染的细胞则没有。蓝色天然聚丙烯酰胺凝胶电泳（BN-PAGE）显示，TPM3-ALK二聚化与TPM3的长度成正比增加。蛋白质免疫印迹法显示，用TPM3-ALK转染的HEK293T细胞中ALK、ERK1/2和STAT3发生磷酸化。因此，TPM3的卷曲螺旋结构有助于TPM3-ALK融合蛋白的转化能力，且TPM3区域越长，二聚体形成越高。
