CXCL14: A new molecular link between ROS and malignant behavior in breast cancer cells Free

LB-271

Breast cancer is a leading cause of cancer related death in women of the western world. Growing evidence suggests that cancer cells exhibit increased reactive oxygen species (ROS), due in part to oncogenic stimulation, increased metabolic activity, and mitochondrial malfunction. Elevated ROS can cause mutations and promote genetic instability and drug resistance. To evaluate the impact of ROS on malignant cancer cell behaviors, we have developed a unique experimental cell system from human breast cancer cells (MCF-7). Since electron leakage from the mitochondrial respiratory chain is a major source of ROS generation, we used rotenone, an agent known to disrupt the mitochondrial respiration by blocking complexe I and to increase ROS. MCF7 cells were treated with a sub-toxic concentration of rotenone in 3 repeat cycles, plated at low density to allow formation of colonies and several sub-clones with increased proliferation were isolated. Initial characterization showed that all clones had elevated ROS generation, and high cellular motility and invasive behaviors. By comparing gene expression profiles of these clones with their parental MCF7 cells using the microarray analysis, we discovered that the expression of CXCL14, a novel chemokine with yet undefined function, was up-regulated in all sub-clones. These findings were further confirmed by RT-PCR and western blot analyses. Conditioned medium from the CXCL14-overexpressing clones and recombinant CXCL14 protein were shown to stimulate migration and invasion of parental MCF7 cells. We further demonstrated that exogenous H₂O₂ could also promote CXCL14 expression, suggesting a possibility that the CXCL14 transcription may be controlled by a ROS-responsive element. Analysis of the CXCL14 promoter region revealed a binding site for AP-1. Using EMSA, we showed that all sub-clones exhibited constitutive increase in AP-1 DNA binding activity, and H₂O₂ also enhanced AP-1 activity in the parental MCF-7 cells. This DNA binding activity could be inhibited by thepharmacological JNK inhibitor, SP600125. Importantly, the elevated CXCL14 in the sub-clones or transfection of MCF7 cells with CXCL14 caused an increase in cytosolic Ca²⁺, which plays a critical role in cell motility and migration. Our data suggest that ROS stress in breast cancer promotes malignant cell behaviors through a novel pathway involving ROS→AP-1→CXCL14→Ca²⁺→motility. Interestingly, the malignant cells in long-term culture seem able to up-regulate their antioxidant capacity to offset the excessive ROS while still retain high CXCL14 expression, increased Ca²⁺, and elevated motility. These clones also exhibited significant alterations in energy metabolism, evident by lower mitochondrial oxygen consumption and higher glucose uptake.