Receptor Subtypes Involved In LPA-induced Actions In Non-small Cell Lung Cancer And It’s Signal Mechanism

Lysophosphatidic acid(LPA),as a bioactive lipid,plays a variety of physiological and pathological roles via the activation of six types of G-protein-coupled LPA receptors(LPAR1–6).It has been reported that LPA accumulates in ascites of human ovarian cancer and pleural effusion of human lung cancer,and LPA is considered as a biomarker of ovarian cancer.Our finding showed that LPA promoted oncogenic effect of lung cancer cells.Therefore,this study aimed to determine the receptor subtypes involved in the targeting effects of LPA on non-small cell lung cancer and elucidate their signaling mechanism at the cellular,tissue and individual levels.As well as,to investigate whether LPA-mediated the receptor subtypes can be a potential small molecular target for lung cancer treatment based on our results in the study.In the study,lung cancer cell A549 was used as the cell model.CCK method,transwell chamber,and clone formation experiment were used to detect the effects of LPA on A549 cells.Pharmacological and molecular biology methods were used to measure LPA receptor subtypes in the process of LPA-induced cell proliferation and migration,respectively.Cloned human LPAR1 and LPAR3 genes,constructed LPAR1/3 gene overexpression vector,constructed LPAR1 gene interference vector,and LPAR1/3 overexpression cell line and LPAR1 knockdown cell line were constructed.The gene expression level was detected by q PCR,and the target protein expression was monitored by western blot.The concentration of intracellular c AMP was determined by ELISA.The lysophosphatidic acid receptors expression was measured in paracancer tissues and lung cancer tissues by immunohistochemistry analysis,and H-score was performed.A nude mouse xenograft tumor model of human lung cancer cell A549 was used to evaluate the tumorigenicity of lysophosphatidic acid receptors in vivo.We explored the signal transduction mechanism of LPA-induced effect on the A549 transgenic cell model.We found that LPA promoted proliferation,migration and clone formation of A549 cells.Lysophosphatidic acid receptor subtypes 1 and 3 were preferentially expressed in A549 cells.Both Ki16425(an antagonist of LPAR1/3)and Ono7300243(an antagonist of LPAR1)inhibited the proliferation and migration activity induced by LPA.LPA promoted proliferation and migration of LPAR1-overexpressed A549 cells,while overexpression of LPAR3 did not.LPAR1 knockdown reduced LPA-induced cell proliferation and migration.The expression of LPAR1 in lung cancer tissues was significantly higher than that in paracancer tissues.In the xenograft model of nude mice,the mass and volume of tumor blocks derived from LPAR1 stably over-expressed A549 cells were significantly higher than those of the control group.Meanwhile,the mass and volume of tumor blocks derived from nude mice with LPAR1 knockdown of A549 cells were significantly reduced.The tumor blocks of nude mice were measured by immunohistochemical analysis,which showed that LPAR1 and Ki-67 were highly expressed in LPAR1-overexpression tumor tissues and showed a positive correlation between them.In the study of signal pathways,we found that PTX as an inhibitor of Gi protein could significantly inhibit the proliferation and migration of A549 cells induced by LPA.STS,an inhibitor of PKC,could also inhibit LPA-induced migration of A549 cells,while H89(an inhibitor of PKA)has no obvious effect.LPA stimulated the phosphorylation of ERK1/2,JNK and p38 in LPAR1 overexpressing A549 lung cancer cells.PD98059(inhibitor of ERK1/2)significantly inhibited the proliferation of A549 cells in response to LPA,and SB203580(inhibitor of p38)significantly inhibited LPA-induced the migration of A549 cells.However,SP600125(inhibitor of JNK)was effective against LPA-induced A549 cells.The process of cell proliferation and migration has no effect.BAY11-7082,a NF-?B inhibitor,could simultaneously inhibited LPA/LPAR1-mediated A549 cell proliferation and migration.In conclusions,1.LPAR1 and LPAR3 were preferentially expressed in human lung cancer cell A549.LPAR1 promoted the proliferation,migration and clone formation of lung cancer cells induced by LPA,but LPAR3 did not involve in this process.LPAR1 was highly expressed in lung cancer tissues and can be considered as a potential molecular marker.2.LPAR1 was tumorigenic in vivo.3.The signal pathway involved in A549 lung cancer cells proliferation in response to LPA via LPAR1 was LPA/LPAR1/Gi/ ERK1/2/ NF-?B.And the pathway that LPA promoted migration was LPA/LPAR1/Gi/ PKC/JNK-p38/ NF-?B.Our research showed that LPAR1 might serve as a potential therapeutic target for lung cancer,and antagonists of LPAR1 were expected to be candidates for the treatment of lung cancer.