Tumor metastasis is a fundamental property of malignant cancer cells and the major cause of death in cancer patients. Recent studies indicate that tumor cell invasion and metastasis may be initiated by the formation of the actin-rich cell protrusions with ECM degradation activity, invadopodia. However, despite extensive research on the biology of invadopodia, very little is known about their specific inducers during tumor progression. Autotaxin (ATX) is a secreted lysophospholipase whose expression levels within tumors correlates strongly with their aggressiveness and invasiveness. ATX produces lyosophosphatidic acid (LPA), a phospholipid with known tumor promoting functions that acts through the G-protein coupled receptors, LPA[subscript 1-6] . Recently, overexpression of ATX and LPA receptors (LPA[subscript 1-3]) has been linked to increased tumor invasion and metastasis in vivo , however, the role of other LPA receptors (LPA[subscript 4-6]) as well as the exact mechanisms by which ATX induces tumor metastasis remain poorly characterized. In order to determine the involvement of ATX and LPA in invadopodia production, we used the fibrosarcoma HT-1080 cells stably transfected with ATX or shRNA targeting ATX in fluorescent matrix degradation assays. Our results demonstrate that ATX is implicated in the production of invadopodia resulting in an increase in both their formation and function. Using LPC or LPA, the substrate and product of ATX, we further show that invadopodia production is dependent on the production of LPA from LPC. Among the LPA receptors, LPA 4 has the highest expression in HT1080 cells. Using LPA[subscript 4] shRNA as well as agonists and inhibitors of the cAMP pathway, we provide evidence that LPA[subscript 4] signaling through the cAMP-EPAC-Rap1 axis, regulates invadopodia formation downstream of ATX. Furthermore, inhibition of Rac1, a known effector of Rap1 and invadopodia formation, abolished EPAC-induced invadopodia production, suggesting downstream participation of Rac1. Finally, results using LPA[subscript 4] shRNA support the requirement of this receptor for in vitro cell invasion and in vivo metastasis formation. Our results suggest that ATX through LPA[subscript 4] is a strong inducer of invadopodia formation that correlates with the ability of the cells to invade and metastasize. This study also revealed an unexpected signaling pathway for cell invasion involving LPA[subscript 4]-driven cAMP production and subsequent activation of the EPAC-Rap1-Rac1 axis.

京都大学 / 新制・課程博士 / 博士(薬学) / 甲第23847号 / 薬博第854号 / 新制||薬||242(附属図書館) / 京都大学大学院薬学研究科薬学専攻 / (主査)教授 金子 周司, 教授 土居 雅夫, 教授 竹島 浩 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM