Lysophosphatidic Acid Receptors Determine Ovarian Cancer Aggressiveness
Lysophosphatidic Acid Receptors Determine Ovarian Cancer Aggressiveness
Background: Lysophosphatidic acid (LPA) acts through the cell surface G protein-coupled receptors, LPA1, LPA2, or LPA3, to elicit a wide range of cellular responses. It is present at high levels in intraperitoneal effusions of human ovarian cancer increasing cell survival, proliferation, and motility as well as stimulating production of neovascularizing factors. LPA2 and LPA3 and enzymes regulating the production and degradation of LPA are aberrantly expressed by ovarian cancer cells, but the consequences of these expression changes in ovarian cancer cells were unknown.
Methods: Expression of LPA1, LPA2, or LPA3 was inhibited or increased in ovarian cancer cells using small interfering RNAs (siRNAs) and lentivirus constructs, respectively. We measured the effects of changes in LPA receptor expression on cell proliferation (by crystal violet staining), cell motility and invasion (using Boyden chambers), and cytokines (interleukin 6 [IL-6], interleukin 8 [IL-8], and vascular endothelial growth factor [VEGF]) production by enzyme-linked immunosorbent assay. The role of LPA receptors in tumor growth, ascites formation, and cytokine production was assessed in a mouse xenograft model. All statistical tests were two-sided.
Results: SKOV-3 cells with increased expression of LPA receptors showed increased invasiveness, whereas siRNA knockdown inhibited both migration (P < .001, Student t test) and invasion. Knockdown of the LPA2 or LPA3 receptors inhibited the production of IL-6, IL-8, and VEGF in SKOV-3 and OVCAR-3 cells. SKOV-3 xenografts expressing LPA receptors formed primary tumors of increased size and increased ascites volume. Invasive tumors in the peritoneal cavity occurred in 75% (n = 4) of mice injected with LPA1 expressing SKOV-3 and 80% (n = 5) of mice injected with LPA2 or LPA3 expressing SKOV-3 cells. Metastatic tumors expressing LPA1, LPA2, and LPA3 were identified in the liver, kidney, and pancreas; tumors expressing LPA2 and LPA3 were detected in skeletal muscle; and tumors expressing LPA2 were also found in the cervical lymph node and heart. The percent survival of mice with tumors expressing LPA2 or LPA3 was reduced in comparison with animals with tumors expressing ß-galactosidase.
Conclusions: Expression of LPA2 or LPA3 during ovarian carcinogenesis contributes to ovarian cancer aggressiveness, suggesting that the targeting of LPA production and action may have potential for the treatment of ovarian cancer.
Lysophosphatidic acid (LPA, 1-acyl-2-lyso-SN-glycero-3-phosphate), the simplest glycerophospholipid, mediates a wide range of biologic actions, including stimulation of DNA synthesis, cell proliferation, cytoskeleton reorganization, cell survival, drug resistance, cell adhesion, migration, cytokine production, and ion transport. The biologic functions of extracellular LPA are mediated through specific G protein-coupled receptors (GPCRs), including Edg-2/LPA1, Edg-4/LPA2, and Edg-7/LPA3, that belong to the endothelial differentiation gene (Edg) family. Other members of the Edg family, Edg-1/S1P1, Edg-3/S1P3, Edg-5/S1P2, Edg-6/S1P4, and Edg-8/S1P5, are high-affinity receptors for the structurally related lysophospholipid sphingosine 1-phosphate (S1P). LPA receptors may heterodimerize, thereby increasing their selectivity and broadening their spectrum of activity. Recently, the GPCRs GPR23/p2y9 (LPA4), GPR92/93 (LPA5), GPR87/95 (LPA6), p2y5, and p2y10 have been reported to be novel, non-Edg family LPA receptors that have little sequence homology to LPA1-3. The physiological roles and the relevance of these new receptors to LPA function have not yet been determined. LPA can also bind to and activate the intracellular receptor PPARγ, leading to physiological and pathophysiological effects, in particular aberrations in thrombosis and atherogenesis.
LPA also increases the expression and production of neovascularizing factors such as interleukin 6 (IL-6), interleukin 8 (IL-8), vascular endothelial growth factor (VEGF), growth-regulated oncogene alpha (Gro-α), urokinase plasminogen activator (uPA), and other oncogenesis-related proteins by ovarian cancer cells. It has been extensively studied for its roles in signal transduction and physiological responses in the context of wound healing and multiple pathophysiological processes such as ischemia reperfusion injury, fibrosis, autoimmune disease, and cancer.
LPA was first implicated in human oncogenesis by our observation that LPA is present at high levels in the ascitic fluid of ovarian cancer patients. This could be due to the increased number of ovarian cancer cells present in the peritoneal cavity, as ovarian cancer cells can produce LPA. It could also be due to irritation of the peritoneal mesothelium (mesothelial cells produce LPA); aberrations in the production and action of LPA due to altered levels of autotaxin, the enzyme that produces LPA; or altered levels of lipid phosphate phosphohydrolases (LPPs), which metabolize LPA. Introduction of LPP3 into ovarian cancer cells decreases their growth both in vitro and in vivo.
Further evidence of a critical role for LPA in ovarian cancer was provided by the observation that LPA2 and LPA3 receptors are aberrantly overexpressed in the majority of ovarian cancer cells. We found that LPA1 mRNA levels are similar in normal and transformed ovarian epithelial cells; however, higher expression of LPA2 and LPA3 mRNA was detected in most ovarian cancer cell lines compared with normal ovarian epithelial cells. Using real-time reverse transcription-polymerase chain reaction (RT-PCR) we confirmed that LPA2 and LPA3 mRNAs are overexpressed in a substantial portion (15%-30% and 44%-49%, respectively) of ovarian cancers compared with tissues from normal ovaries, benign ovarian tumors, and other normal tissues of nonovarian origin. Aberrant expression of the LPA2 and/or LPA3 receptors, whether a cause or a consequence of cell transformation, has also been associated with other malignancies, including thyroid, colorectal and breast cancer. However, there is no evidence that the increased expression of LPA2 or LPA3 is causally linked to the malignant phenotype of cancer cells.
In this study, we investigated whether aberrant LPA receptor expression contributes to the malignant properties acquired by ovarian cancer cells, by exploring in vitro and in vivo systems of knockdown and overexpression of each Edg family LPA receptor in SKOV-3 cells, an ovarian cancer cell line exhibiting low to modest levels of endogenous LPA receptors.
Abstract and Introduction
Abstract
Background: Lysophosphatidic acid (LPA) acts through the cell surface G protein-coupled receptors, LPA1, LPA2, or LPA3, to elicit a wide range of cellular responses. It is present at high levels in intraperitoneal effusions of human ovarian cancer increasing cell survival, proliferation, and motility as well as stimulating production of neovascularizing factors. LPA2 and LPA3 and enzymes regulating the production and degradation of LPA are aberrantly expressed by ovarian cancer cells, but the consequences of these expression changes in ovarian cancer cells were unknown.
Methods: Expression of LPA1, LPA2, or LPA3 was inhibited or increased in ovarian cancer cells using small interfering RNAs (siRNAs) and lentivirus constructs, respectively. We measured the effects of changes in LPA receptor expression on cell proliferation (by crystal violet staining), cell motility and invasion (using Boyden chambers), and cytokines (interleukin 6 [IL-6], interleukin 8 [IL-8], and vascular endothelial growth factor [VEGF]) production by enzyme-linked immunosorbent assay. The role of LPA receptors in tumor growth, ascites formation, and cytokine production was assessed in a mouse xenograft model. All statistical tests were two-sided.
Results: SKOV-3 cells with increased expression of LPA receptors showed increased invasiveness, whereas siRNA knockdown inhibited both migration (P < .001, Student t test) and invasion. Knockdown of the LPA2 or LPA3 receptors inhibited the production of IL-6, IL-8, and VEGF in SKOV-3 and OVCAR-3 cells. SKOV-3 xenografts expressing LPA receptors formed primary tumors of increased size and increased ascites volume. Invasive tumors in the peritoneal cavity occurred in 75% (n = 4) of mice injected with LPA1 expressing SKOV-3 and 80% (n = 5) of mice injected with LPA2 or LPA3 expressing SKOV-3 cells. Metastatic tumors expressing LPA1, LPA2, and LPA3 were identified in the liver, kidney, and pancreas; tumors expressing LPA2 and LPA3 were detected in skeletal muscle; and tumors expressing LPA2 were also found in the cervical lymph node and heart. The percent survival of mice with tumors expressing LPA2 or LPA3 was reduced in comparison with animals with tumors expressing ß-galactosidase.
Conclusions: Expression of LPA2 or LPA3 during ovarian carcinogenesis contributes to ovarian cancer aggressiveness, suggesting that the targeting of LPA production and action may have potential for the treatment of ovarian cancer.
Introduction
Lysophosphatidic acid (LPA, 1-acyl-2-lyso-SN-glycero-3-phosphate), the simplest glycerophospholipid, mediates a wide range of biologic actions, including stimulation of DNA synthesis, cell proliferation, cytoskeleton reorganization, cell survival, drug resistance, cell adhesion, migration, cytokine production, and ion transport. The biologic functions of extracellular LPA are mediated through specific G protein-coupled receptors (GPCRs), including Edg-2/LPA1, Edg-4/LPA2, and Edg-7/LPA3, that belong to the endothelial differentiation gene (Edg) family. Other members of the Edg family, Edg-1/S1P1, Edg-3/S1P3, Edg-5/S1P2, Edg-6/S1P4, and Edg-8/S1P5, are high-affinity receptors for the structurally related lysophospholipid sphingosine 1-phosphate (S1P). LPA receptors may heterodimerize, thereby increasing their selectivity and broadening their spectrum of activity. Recently, the GPCRs GPR23/p2y9 (LPA4), GPR92/93 (LPA5), GPR87/95 (LPA6), p2y5, and p2y10 have been reported to be novel, non-Edg family LPA receptors that have little sequence homology to LPA1-3. The physiological roles and the relevance of these new receptors to LPA function have not yet been determined. LPA can also bind to and activate the intracellular receptor PPARγ, leading to physiological and pathophysiological effects, in particular aberrations in thrombosis and atherogenesis.
LPA also increases the expression and production of neovascularizing factors such as interleukin 6 (IL-6), interleukin 8 (IL-8), vascular endothelial growth factor (VEGF), growth-regulated oncogene alpha (Gro-α), urokinase plasminogen activator (uPA), and other oncogenesis-related proteins by ovarian cancer cells. It has been extensively studied for its roles in signal transduction and physiological responses in the context of wound healing and multiple pathophysiological processes such as ischemia reperfusion injury, fibrosis, autoimmune disease, and cancer.
LPA was first implicated in human oncogenesis by our observation that LPA is present at high levels in the ascitic fluid of ovarian cancer patients. This could be due to the increased number of ovarian cancer cells present in the peritoneal cavity, as ovarian cancer cells can produce LPA. It could also be due to irritation of the peritoneal mesothelium (mesothelial cells produce LPA); aberrations in the production and action of LPA due to altered levels of autotaxin, the enzyme that produces LPA; or altered levels of lipid phosphate phosphohydrolases (LPPs), which metabolize LPA. Introduction of LPP3 into ovarian cancer cells decreases their growth both in vitro and in vivo.
Further evidence of a critical role for LPA in ovarian cancer was provided by the observation that LPA2 and LPA3 receptors are aberrantly overexpressed in the majority of ovarian cancer cells. We found that LPA1 mRNA levels are similar in normal and transformed ovarian epithelial cells; however, higher expression of LPA2 and LPA3 mRNA was detected in most ovarian cancer cell lines compared with normal ovarian epithelial cells. Using real-time reverse transcription-polymerase chain reaction (RT-PCR) we confirmed that LPA2 and LPA3 mRNAs are overexpressed in a substantial portion (15%-30% and 44%-49%, respectively) of ovarian cancers compared with tissues from normal ovaries, benign ovarian tumors, and other normal tissues of nonovarian origin. Aberrant expression of the LPA2 and/or LPA3 receptors, whether a cause or a consequence of cell transformation, has also been associated with other malignancies, including thyroid, colorectal and breast cancer. However, there is no evidence that the increased expression of LPA2 or LPA3 is causally linked to the malignant phenotype of cancer cells.
In this study, we investigated whether aberrant LPA receptor expression contributes to the malignant properties acquired by ovarian cancer cells, by exploring in vitro and in vivo systems of knockdown and overexpression of each Edg family LPA receptor in SKOV-3 cells, an ovarian cancer cell line exhibiting low to modest levels of endogenous LPA receptors.
