Original ArticlesLPA-mediated migration of ovarian cancer cells involves translocalization of Gαi2 to invadopodia and association with Src and β-pix
Introduction
Lysophosphatidic acid (LPA) is a simple phospholipid that has been implicated in the progression of ovarian cancer and other cancers [1], [2], [3]. Importantly, the involvement of LPA in migration and invasion of ovarian and other types of cancer has been well documented by our lab and others [4], [5], [6], [7], [8], [9], [10], [11]. Therefore, understanding how LPA mediates ovarian cancer progression is of critical importance for elucidating the biology of ovarian cancer and the contributions of LPA and G proteins in ovarian and potentially other cancers. LPA binds to and activates at least six different G protein-coupled receptors (GPCRs). All of these LPA-specific GPCRs activate both the Gi and Gq family of proteins. Furthermore, all of the LPA receptors, except for LPA3, also activate the G12/13 family of proteins [12].
To date there have been very little studies that have looked at the role of G proteins in migration and even fewer studies have studied the spatiotemporal dynamics of G protein signaling. Therefore, in this report we sought to characterize the cellular localization and contribution that Gαi2, also known as the gip2 proto-oncogene, has on ovarian cancer cell migration. We chose to utilize ovarian cancer cells since these cells migrate robustly to LPA stimulation. Thus, by utilizing LPA to stimulate Gαi2-dependent invasive migration, we report here that Gαi2 directly associates with Src and β-pix in invadopodia. We demonstrate further that this interaction of Src and β-pix led to activation of Rac. In addition, our results indicate that Gαi2 could activate Rac1 through a pathway involving the scaffold protein p130Cas. Thus, our current study defines the spatiotemporal localization of Gαi2 in response to LPA and unravels the mechanism through which its downstream effectors could orchestrate invasive migration of cancer cells.
Section snippets
Reagents
The ovarian cancer cell lines HeyA8 were kindly provided by Dr. E. Premkumar Reddy (Mount Sinai School of Medicine, New York, NY) and SKOV3-ip cells were provided by Dr. Robert C. Bast (MD Anderson Cancer Center, Houston, TX). HeyA8 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) and SKOV3-ip cells were maintained in Roswell Park Memorial Institute (RPMI) 1640 media (Mediatech, Manassas, VA) containing 10% FBS (Gemini Bio-Products, West Sacramento, CA), 50 U/mL penicillin,
LPA stimulates the translocation of Gαi2 to the leading edge of ovarian cancer cells
To confirm our recent study [10] that indicated Gαi2 is necessary for LPA-meditated invasive migration we determined the effect of silencing Gαi2 on LPA-mediated invasive migration of SKOV3-ip cells. As shown in Fig. 1A, the ovarian cancer cells that had Gαi2 stably silenced had significantly diminished migration. Our recent study has established that the Gαi2-Src-p130Cas pathway is directly involved in inducing LPA-stimulated migration of ovarian cancer cells [10]. However, the spatiotemporal
Discussion
To date there has been little work done on how each specific G protein individually contributes to migration of cells. Importantly, our current study shows that Gαi2 is localized in invadopodia and that it directly interacts with Src and β-pix to activate Rac. Taken together with the previous findings that Src stimulates the phosphorylation of β-pix, which in turn activates Rac1 [31], it appears that Gαi2 plays an active role in directly initiating the invasive migration of ovarian cancer cells
Conflicts of interest
The authors have no conflicts of interests and declare no competing financial interests.
Acknowledgements
This work was supported by grants from the National Institutes of Health (CA116984, CA123233). OUHSC Flow Cytometry and Imaging Core is acknowledged for the use of confocal and epifluorescence microscopy. We thank Stephenson Cancer Center at the University of Oklahoma, Oklahoma City, OK and an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20 GM103639 for the immunostaining service.
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