Overexpressed Raf-1 and phosphorylated cyclic adenosine 3'-5'-monophosphatate response element-binding protein are early markers for lung adenocarcinoma

BACKGROUND: Pulmonary adenocarcinoma (PAC) is the leading type of lung cancer and has a high mortality. The tobacco carcinogen nicotine-derived nitrosamine 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) stimulates the proliferation of human PAC cells and small airway epithelial cells through beta-1 adrenorecptor-mediated transactivation of the epidermal growth factor receptor (EGFR). METHODS: Using the NNK hamster PAC model and human PAC tissue arrays with matched and unmatched normal lung tissues, the authors tested the hypothesis that Raf-1, an effector of the EGFR, and P-CREB, an effector of the beta-adrenoreceptor, are overexpressed in a significant subset of human PACs and are early markers of PAC development. Western blots from respiratory epithelial cells and microadenomas harvested by laser-capture microdissection from hamster lungs accompanied by immunostains were used to monitor the expression levels of Raf-1 and P-CREB after 5 weeks, 10 weeks, and 20 weeks of NNK treatment. Expression levels of these markers in human PAC tissue arrays were assessed by immunostains. Reverse-phase proteomics, Western blot analysis, and immunoprecipitation in immortalized human small-airway epithelial cells and in a human PAC cell line in the presence and absence of dominant-negative Raf were used to determine Raf dependence of extracellular signal-regulated kinase 1 and 2 (ERK1/2) activation in response to NNK or isoproterenol. RESULTS: The data showed a time-dependent increase in the expression of Raf-1 and P-CREB after NNK treatment in small-airway epithelial cells and microadenomas of hamsters. The majority of human lung adenocarcinomas simultaneously overexpressed Raf-1 and P-CREB. Dominant-negative Raf completely abrogated ERK1/2 activation by NNK and isoproterenol. CONCLUSIONS: The current results indicated that RAF-1 and P-CREB may contribute to the development of a significant subset of human lung adenocarcinomas and may offer promising targets for early detection and treatment.