We are pursuing the hypothesis that prostate tumors from current, past, and never smokers exhibit differences in their gene expression profiles that are consistent with distinct oncogenic molecular alterations in tumors of current smokers. We are also exploring the effects of nicotine in human prostate cancer cells and TRAMP mice, and are evaluating whether those resemble smoking-associated alterations in prostate tumors. This research is aimed at identifying the mechanisms by which cigarette smoking induces prostate cancer progression, and to define the specific role of nicotine in this process. This proposal combines novelty with a high-impact concept. If we find that nicotine induces disease metastasis, the results could have significant public health implications. Tobacco smoke contains numerous chemicals, including many that are DNA-damaging and carcinogenic. Nitrosamines that are produced from the alkaloid nicotine during post-harvest processing and the burning process of cigarettes are an important group of carcinogens in tobacco smoke. Recently, nicotine and nitrosamines were found to activate signaling pathways in non-neuronal mammalian cells by receptor-mediator mechanisms. Several of these pathways are cancer-related and promote cell survival, angiogenesis, and metastasis. For example, nicotine activates the Akt pathway, which is a key pathway in the development and progression of many cancers, including prostate cancer. In addition, nicotine can reach high nanomolar steady-state concentrations in the blood of current smokers that may activate a signaling pathway in organs other than lung if the appropriate receptors are expressed by the target cells. We collected 67 prostate tumors from 16 current, 28 past, and 23 never smokers for the study, which we obtained from the NCI CPCTR, our resource contract, and Johns Hopkins Medical Institutions. The clinical characteristics of these tumors are similar among current, past, and never smokers. In a pilot, we analyzed the gene expression profiles of tumors from 9 current, 21 past, and 17 never smokers. This analysis revealed a very distinct signature that differentiated tumors from current smokers from those of never and past smokers. Because the first dataset contained tumors from only 9 current smokers, additional prostate tumors were collected at the Department of Urology, Johns Hopkins Medical Institutions (in collaboration with Jun Luo and William Isaacs) and combined with the existing samples to increase the statistical power of our study to identify additional genes that are differentially expressed between current and never/past smokers. The comparison of tumors from current and never smokers yielded 98 transcripts encoding 73 differentially expressed genes. A second comparison, current versus past/never smokers, resulted in a shorter list of only 70 transcripts encoding 40 differentially expressed genes. Likely a residual effect of smoking on the tumor gene signature in past smokers, this study yielded fewer genes when the current and combined past/never smokers were compared. Many of the differentially expressed genes have known immune-regulatory functions. Several others of the differentially genes were found to have an association with hepatocyte growth factor (HGF). The latter is intriguing because both nicotine and HGF activate common pathways, e.g., the PI3 kinase-Akt axis. Some of our observations are preliminary and will need further validation. Nevertheless, the data show that a current smoking status generates a gene signature in prostate tumors that could reveal the mechanism by which smoking causes the metastatic spread of prostate cancer. These observations are being studied. Specifically, we are investigating whether the smoking signature in prostate tumors is partially a nicotine signature. Nicotine activates the Akt pathway and enhances NFkappaB signaling, which are features that it shares with HGF. Nicotine exerts these effects by binding to nicotinic acetylcholine receptors. We examined whether human prostate tissue and prostate cancer cell lines express non-neuronal nicotinic acetylcholine receptors and found that normal and cancerous prostate and human prostate cancer cell lines express several functionally significant nAChR subtypes. We have begun treating human prostate cancer cells (e.g., 22Rv1, DU145, LNCaP, PC3) with physiologically relevant concentrations of nicotine, HGF, or both, in the presence and absence of nAChR antagonists. These experiments will examine whether nicotine alone or in combination with HGF activates oncogenic pathways and induces an invasive phenotype. Preliminary results with nicotine and HGF have revealed a synergistic effect of the two in phenotypic assays that are consistent with the promotion of metastasis. These data support our hypothesis that nicotine has oncogenic properties in prostate cancer. To further advance our observations from tumors and human cancer cells, we will study the effect of nicotine on prostate cancer progression in TRAMP mice. These mice develop localized prostate cancer. Here, we will evaluate immunologic, angiogenic, and hepatocyte growth factor-related tumor markers and phenotypic markers of extraprostatic extension and disease metastasis in nicotine-treated animals. Ultimately, we want to test whether nicotine induces disease metastasis and leads to alterations of tumor markers that are consistent with those in human tumors of current smokers and nicotine-treated human cancer cells. The proposed animal study will be conducted in collaboration with Arthur Hurwitz, M.D. (Laboratory of Molecular Immunoregulation, CCR). The study will be performed under a modification to Dr. Hurwitz's protocol, 06-060.