Abstract 3775: Lysophosphatidic acid receptor-3 pathways are involved in up-regulation of cell migration and invasion activity of human sarcoma cells.

Abstract

Abstract Lysophosphatidic acid (LPA) acts through G-protein coupled transmembrane receptors, and at least 6 types of LPA receptors (LPARs) have been identified. They regulate many different cellular responses, such as proliferation, survival, differentiation, migration, cytoskeletal changes and calcium influx through these pathways, and may act as a positive or negative regulator of cancer cell behaviors in a receptor specific manner which depends on cell types. Gene expression profiles analyzed in rat sarcoma model showed highly metastatic osteosarcoma cells expressing abundant LPA receptor-3 (LPAR3) compared to mesenchymal stem cells and non-metastatic MFH cells. In the current study, we generated LPAR3-knockdown cells from human fibrosarcoma HT1080 and osteosarcoma HOS cells by transfection with shRNA plasmids and investigated the involvement of LPA3 pathway for cell migration and invasion abilities. Both sarcoma cells expressed through LPAR1 to 5 except for LPAR1 in osteosarcoma HOS. Firstly, we tested the effects of chemical inhibitors of downstream effector of G-proteins, and results suggested that downstream of Gi and Gq protein inhibition suppressed cell motility in both sarcoma cells, while downstream of G12/13 inhibition did not. Then, we generated LPAR3 knockdown cells by transfection of shRNA for LPAR3, which resulted in effective knockdown of the LPAR3 expression in those cells. Expression of autotaxin, a mediator for LPA production, was suppressed in HT1080 but not in HOS by shRNA, indicating the difference of LPA production loop between those cells. LPAR3 knockdown did not affect cellular growth rates in both sarcoma cells. Moreover, LPA treatments stimulated cell proliferation in a dose dependent manner even in LPAR-3 knockdown cells, suggesting that LPA signaling on cell proliferation could be regulated by other LPAR pathways rather than LPAR3 in these cells. On the contrary, LPAR3-knockdown cells showed significantly lower cell motility than control cells both in cell motility assay and in scrape assay. Invasion assay with the Matrigel-coated filter also showed significantly lower invasion activities in LPAR3-knockdown HT1080 cells, which originally showed strong invasive capacity. While, there were no differences observed in matrix metalloproteinase (MMP) -2 and 9 activities on gelatin zymography, suggesting that the control of invasiveness through LPAR3 signaling pathways was independently regulated from MMPs. These results suggest that a part of the abilities for cell migration and invasion in human sarcoma cells were possibly regulated through the downstream of LPAR3, Gi and Gq protein signaling pathways, and those pathways could be the candidates for molecular targeted therapy against sarcomas. Further elucidation of detailed mechanisms underlying in these complex pathways will be required for future therapeutic approach. Citation Format: Kanya Honoki, Hiromasa Tsujiuchi, Akira Kido, Shinji Tsukamoto, Yasuhito Tanaka, Toshifumi Tsujiuchi. Lysophosphatidic acid receptor-3 pathways are involved in up-regulation of cell migration and invasion activity of human sarcoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3775. doi:10.1158/1538-7445.AM2013-3775