Reactive oxygen species (ROS) are implicated in the signaling pathways that control the growth of ovarian cancer cells and their resistance to chemotherapy. However, the molecular mechanisms by which ROS control cell function remain elusive. We have shown that lysophosphatidic acid (LPA) stimulates ROS production in ovarian cancer cells and that disrupting this signaling results in growth inhibition and apoptosis. The Raf-MEK-ERK pathway and NF-B appear to be two major targets of regulation by ROS. NF-B dependent signaling is major determinant of resistance to chemotherapy. Thus, higher levels of ROS, which are a characteristic of cancer cells, may stimulate NF-B dependent synthesis of anti-apoptotic proteins. The two main chemotherapeutic drugs for the treatment of ovarian cancer are cisplatin and taxanes. Both of these drugs stimulate ROS production and their anti-neoplastic effects are altered by antioxidants. Thus, ROS signaling is a critical determinant of cellular control of growth and apoptosis. Our goal is to identify the targets of ROS that control these fates. We have developed novel methods for analyzing the cysteine sulfenic acid modification that can be used to identify proteins modified in response to ROS. We will use this approach to determine the ROS-responsive signaling pathways in ovarian cancer cells. We will analyze the response to growth and apoptotic signals, which we have determined involve ROS signaling. Lysophosphatidic acid (LPA) is a lipid mediator of proliferative signaling and cisplatin and taxanes are chemotherapeutic agents which stimulate apoptosis by a ROS-dependent mechanism. The goal of this project is to determine how ROS- dependent signaling in response to LPA, cisplatin and taxanes can lead to either proliferation or apoptosis in ovarian cancer cells. The overall strategy is to use treatment conditions which lead to a growth response or apoptosis and determine what proteins are oxidized in a functionally critical way. How do these oxidations lead to changes in NF-kB activity and how does NF-kB-dependent transcription affect the cells fate? The specific aims are: 1. To determine the role of redox state in the control growth and apoptosis. 2. To determine the role of ROS in the control of NF-kB dependent transcriptional regulation and resistance to chemotherapy. 3. To determine the downstream targets of ROS-signaling in resistance to apoptosis. We will use our newly developed reagents to identify and characterize proteins in the pathways that lead to apoptosis or resistance to apoptosis. This approach involves labeling modified proteins with biotin derivatives of dimedone, followed by avidin pull-down and analysis by western blotting and mass spectrometry. We will use this approach to determine the ROS-responsive signaling pathways in ovarian cancer cells. In summary, the experiments outlined here will contribute to our understanding of how apoptosis and cell growth are controlled by ROS in ovarian cancer cells. This will allow for a better understanding of how ROS play a role in the normal growth control and in resistance to chemotherapy.

R01CA142838

2009-12-11

2014-11-30