Oncogenic Signaling Pathways Activated by Lysophosphatidic Acid (LPA) in Ovarian Carcinoma

Abstract

Ovarian cancer is currently the most fatal gynecologic cancer and the fifth leading cause of fatal cancer in women overall. As compared to the better-characterized malignancies, such as such as prostate, breast and colorectal cancers, there have been no major changes in methods of detection or treatment of ovarian cancers since the 1970's. As a result, the incidence and age-adjusted death rates for this disease have improved only marginally since that time. The molecular changes required for ovarian cancer pathogenesis remain poorly defined. Lysophosphatidic acid (LPA) has emerged as a biomarker present in the ascitic fluid and serum of ovarian cancer patients. Subsequent studies have identified LPA as an agonist for G protein coupled receptors (GPCRs). LPA has been well characterized as a pro-migratory factor in ovarian cancer and other cell systems. However, the role of LPA in mediating a proliferative response in ovarian cancer cells has yet to be fully characterized. In addition, the identity of the G protein pathways involved in this proliferative response remains a major unresolved question in the field. To investigate the mitogenic role of LPA in ovarian cancers, a panel of representative human ovarian cancer cells was assembled. A series of immunoblot and RT-PCR analyses was used to profile the LPA receptors and Gα-subunits expressed in these cells. In addition to verifying the migratory effect of LPA in these cells, a series of proliferation assays were used to investigate the potential role for LPA as a mitogen. The results indicate that stimulation with LPA results in a robust and statistically significant proliferative response. This response was quantified using multiple approaches. In addition, the proliferative response was observed in three independent ovarian cancer cell lines using concentrations of LPA within the range found in vivo in the ascitic fluid of ovarian cancer patients. Taken together, these data for the first time validate the role of LPA as a mitogen in ovarian cancer cells. To gain further insight into the oncogenic signaling response stimulated by LPA, activation of the mitogen activated protein kinase (MAPK) modules was determined. Using a series of immunoblot analyses and kinase assays, LPA was found to stimulate ERK as well as JNK modules. To investigate the functional roles of these pathways, a series of proliferation assays were carried out using inhibitors of ERK and JNK signaling. Consistent with the role of ERK as a crucial regulator of growth-factor induced proliferation in other cell systems, the results demonstrated a significantly attenuated growth response to LPA with ERK inhibition. Moreover, additional studies demonstrated for the first time that inhibition of JNK signaling significantly attenuates the proliferative response to LPA. In order to investigate the potential role of Gα12 in mediating the oncogenic response to LPA, the activation status of Gα12 was monitored in ovarian cancer cells stimulated with LPA. These studies demonstrate rapid activation of Gα12 with LPA stimulation. Finally to investigate the functional role of LPA-Gα12 signaling, a series of cell lines was established which express a dominant negative form of Gα12. Expression of this construct induced complete inhibition of Gα12 activation by LPA. These cells were then used to determine the effects of Gα12 inhibition on the oncogenic response to LPA. Consistent with the role of G12 family members in mediating cell migration, these cells demonstrated an attenuated migratory response to LPA. In addition, inhibition of Gα12 resulted in an attenuated proliferative response to serum. Finally, to investigate the role of Gα12 in mediating the proliferative response to LPA, a series of proliferation assays was carried out. The results indicated a significant > 50% inhibition in multiple ovarian cancer cell lines. Taken together, these results, presented here for the first time, establish that LPA is a potent mitogen that induces a proliferative response in human ovarian carcinoma cells. Although LPA had previously been shown to induce a proliferative response in multiple other cell types, it had not been known if LPA activates specific oncogenic pathways. This thesis tested the hypothesis that LPA, which is crucially involved in the pathophysiology of ovarian carcinoma, induces the activation of Gα12. In this context, the data presented here demonstrating a novel role for Gα12- which has been defined as the gep oncogene - in mediating this proliferative response in ovarian carcinoma, represents a major finding in the field.