br Conflicts of interest br Acknowledgements br
Conflicts of interest
Lung cancer continues to be the leading cause of cancer deaths in the US and worldwide., Non–small-cell lung cancer (NSCLC) remains the predominant form of lung cancer (approximately 85%)., With some improvements in surgical techniques and combined therapies over the past several decades, the relative survival rate for lung cancer has increased slightly. However, lung cancer remains extremely lethal, with a 5-year survival rate of only about 15% in the US. Unclear molecular mechanisms, lack of early diagnostic biomarkers, and deficiency of targeted therapy in lung cancer are some of the major reasons that its incidence, diagnosis, and prognosis remain relatively unchanged. Evidence shows that 80–90% of lung cancers are directly or indirectly traceable to tobacco use., , More than 60 known carcinogens have been identified in cigarette smoke,, among which N-nitrosamines play major roles in carcinogenesis. NNK [4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone] is an important nitrosamine with highly carcinogenic activities and a consistent presence in relatively considerable amounts in cigarette smoke. NNK has been shown to have lung-selective toxicity and induce primarily lung adenocarcinoma in a variety of laboratory animals., The development of lung cancer has been extensively investigated in the past 40 years. Some of these studies, including our previous studies,, , have revealed a frequent occurrence of mutations in several proto-oncogenes and tumor suppressor genes, including p53 gene, and such alterations have been associated with the initiation and progression of lung cancer. In addition to mutations in oncogenes and tumor suppressor genes, accumulated evidence has also shown that stage-specific genes turn on or off during the process of cancer development. For instance, in eukaryotic cells nuclear-cytoplasmic transport is critical for normal biological functions, such as transcription and hpdp regulation., Chromosome region maintenance 1 (CRM1), the best characterized nuclear export receptor, was first identified in the yeast and has been found as a conserved gene in eukaryotes. CRM1 protein, facilitated by Ran, plays an essential role in nuclear export signal (NES)-dependent nuclear export of various cancer-associated “cargo” proteins,, , , including both tumor suppressors and pro-oncogenes, which control genomic stability, cell cycle arrest, and apoptosis, such as p53, epidermal growth factor receptor (EGFR), protein kinase 1 (Akt1), survivin, and so on. The structure of CRM1 protein contains a highly conserved central region involved in RanGTP-dependent NES recognition and cysteine residue covalently modified by leptomycin B (LMB). LMB, an antifungal agent, is a highly specific and potent inhibitor of CRM1 function by irreversibly reacting with a Cys residue (Cys529) near or within the cargo binding domain of CRM1. Elevated CRM1 protein expression has been identified in various human tumors,, , , , but no study has been conducted on lung cancer. Data from our previous studies have suggested that decreased CRM1 plays an important role in the initial response of lung epithelial cell to tobacco carcinogen and the tumor formation of a bi-transgenic lung tumor model., To get further insight into the mechanisms by which CRM1 is involved in late phase of lung cancer development, in this study we have analyzed CRM1 expression in lung tumor tissues from lung cancer patients, lung cancer cells, and NNK-treated mice and human lung epithelial cells. In addition, the therapeutic potential of targeting CRM1 in lung cancer was also investigated. MATERIALS AND METHODS
DISCUSSION Increased CRM1 expression has been reported previously in other tumor types, including cervical, ovarian, and pancreatic cancers, glioma, and osteosarcoma and was associated with a negative prognosis. To the best of our knowledge, this is the first study showing CRM1 overexpression in lung cancer. It is now well known that CRM1 is involved in nuclear-cytoplasmic transport of various cancer-associated “cargo” proteins, such as p53, and other proteins, including p21, p27, EGFR, Akt1, and survivin.17, 18, 19, 20 The observed CRM1 overexpression could in turn lead to dysfunction/inactivation of tumor suppressor proteins or activation of pro-oncogenes by shuttling them out of nucleus to cytoplasm. For instance, besides p53 mutation, another important pathway to p53 regulation/dysregulation is through posttranslational modifications, including phosphorylation of wild-type p53 and a subsequent alteration in its subcellular localization and function., Phosphorylation at some of such sites is critical for shuttling p53 from the nucleus to cytoplasm to assume its diverse functions. For instance, the phosphorylation at Thr55 is required for MDM2 to promote the CRM1 and p53 interaction and the export of p53 to the cytoplasm, leading to p53 degradation and a decrease in G1 arrest of the cell cycle, whereas inhibition of Thr55 phosphorylation restored the p53 nuclear localization. We showed that Thr55 of p53 protein was phosphorylated not only in lung tumors from NNK-treated mice but also in BEAS-2B cells that were transformed following NNK-exposure. Furthermore, these tumors and transformed cells showed CRM1 overexpression, suggesting a mechanism of lung carcinogenesis involving CRM1 overexpression and inactivation of p53 by posttranslational phosphorylation of Thr55 in lung carcinogenesis.