Previously through a multi-center clinical proteomics study, we discovered that Connective Tissue Activating Peptide (CTAP-III) in serum was elevated among ovarian cancer patients in comparison to that in healthy women and patients with benign pelvic masses. A particular interesting observation was that among ovarian cancer patients who had a CA125 below 35U/ml, CTAP-III was significantly higher than that in the healthy controls (p<0.0005). Both CTAP-III and neutrophil activating peptide-2 (NAP-2) are processed forms of the CXC chemokine platelet basic protein (PBP). It is also known that NAP-2 shares the same chemokine receptor CXCR2 with another CXC chemokine GRO-alpha. As part of post-discovery research to understand the roles of discovered biomarkers in cancer biology, we observed that the inhibition of mitogen-activated protein kinase (MAPK) regulates the expression of both NAP-2 and GRO-alpha in the ovarian cancer cell line MPSC1 known to harbor a mutation in BRAF that activates MAPK. CI-1040 is a highly potent and selective inhibitor of MEK1/2 and has been used previously in experiments to prevent the activation (phosphorylation) of MAPK in MPSC1. In this study, NAP-2 and GRO-alpha expression in cell lysates prepared from MPSC1 cells treated with CI-1040 were compared to MPSC1 cells treated with DMSO as controls at multiple time points of 0, 24, 48 and 72 hours (later at more refined intervals of 0, 2, 4, 6 hours). Protein expression analysis were performed using immunoprecipitation/pull-down (by monoclonal Abs) followed by mass spectrometry. We observed that the expression level of both NAP-2 and GRO-alpha decreased significantly after only 2 hours, became barely noticeable after 24 hours, and were undetectable afterwards. Their expressions in the controls, however, were unchanged. Similar differences in protein expressions were observed when CI-1040 was replaced by two other drugs known to prevent the activation of MEK1/2. Our results were further corroborated at the gene expression level by quantitative real-time PCR. In conclusion, MAPK activation due to mutation of upstream regulators BRAF and KRAS has been implicated in many human cancers including ovarian cancer. Our results provided preliminary yet interesting leads to help us to understand the roles of CTAP-III/NAP-2, potential biomarkers for ovarian cancer that we had discovered through clinical proteomics studies, in cancer biology. These results including those of GRO-alpha further showed that discovery through large-scale molecular profiling of clinical samples should not necessarily be the end goal of research but rather the beginning of interesting hypothesis-driven research.
98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA