Retrospective studies have shown that patients with tobacco-related cancers who continue to smoke after their diagnoses have lower response rates and shorter median survival compared with patients who stop smoking. To provide insight into the biologic basis for these clinical observations, we tested whether two tobacco components, nicotine or the tobacco-specific carcinogen, 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK), could activate the Akt pathway and increase lung cancer cell proliferation and survival. Nicotine or NNK, rapidly and potently, activated Akt in non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC) cells. Nicotinic activation of Akt increased phosphorylation of multiple downstream substrates of Akt in a time-dependent manner, including GSK-3, FKHR, tuberin, mTOR and S6K1. Since nicotine or NNK bind to cell surface nicotinic acetylcholine receptors (nAchR), we used RT-PCR to assess expression of nine alpha and three beta nAchR subunits in five NSCLC cell lines and two types of primary lung epithelial cells. NSCLC cells express multiple nAchR subunits in a cell line-specific manner. Agonists of alpha3/alpha4 or alpha7 subunits activated Akt in a time-dependent manner, suggesting that tobacco components utilize these subunits to activate Akt. Cellular outcomes after nicotine or NNK administration were also assessed. Nicotine or NNK increased proliferation of NSCLC cells in an Akt-dependent manner that was closely linked with changes in cyclin D1 expression. Despite similar induction of proliferation, only nicotine decreased apoptosis caused by serum deprivation and/or chemotherapy. Protection conferred by nicotine was NFkappaB-dependent. Collectively, these results identify tobacco component-induced, Akt-dependent proliferation and NFkappaB-dependent survival as cellular processes that could underlie the detrimental effects of smoking in cancer patients.

The fact that small cell lung cancer (SCLC) is commonly incurable despite being initially responsive to chemotherapy, combined with disappointing results from a recent SCLC clinical trial with imatinib, has intensified efforts to identify mechanisms of SCLC resistance. Adhesion to extracellular matrix (ECM) is one mechanism that can increase therapeutic resistance in SCLC cells. To address whether adhesion to ECM increases resistance through modulation of signaling pathways, a series of SCLC cell lines were plated on various ECM components, and activation of two signaling pathways that promote cellular survival, the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway and the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK) pathway, was assessed. Although differential activation was observed, adhesion to laminin increased Akt activation, increased cellular survival after serum starvation, and caused the cells to assume a flattened, epithelial morphology. Inhibitors of the PI3K/ Akt/mTOR pathway (LY294002, rapamycin) but not the MEK/ERK pathway (U0126) abrogated laminin-mediated survival. SCLC cells plated on laminin were not only resistant to serum starvation-induced apoptosis but were also resistant to apoptosis caused by imatinib. Combining imatinib with LY294002 or rapamycin but not U0126 caused greater than additive increases in apoptosis compared with apoptosis caused by the inhibitor or imatinib alone. Similar results were observed when adenoviruses expressing mutant Akt were combined with imatinib, or when LY294002 was combined with cisplatin or etoposide. These studies identify laminin-mediated activation of the PI3K/Akt/mTOR pathway as a mechanism of cellular survival and therapeutic resistance in SCLC cells and suggest that inhibition of the PI3K/ Akt/mTOR pathway is one strategy to overcome SCLC resistance mediated by ECM. (Cancer Res 2005; 65(18): 8423-32)

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Janus kinase 2 (JAK2) plays a crucial role in the pathomechanism of myeloproliferative disorders and hematologic malignancies. A somatic mutation of JAK2 (Val617Phe) was previously shown to occur in 98% of patients with polycythemia vera and 50% of patients with essential thrombocythemia and primary myelofibrosis. Thus, effective JAK2 kinase inhibitors may be of significant therapeutic importance. Here, we applied a structure-based virtual screen to identify novel JAK2 inhibitors. One JAK2 inhibitor in particular, G6, demonstrated remarkable potency as well as specificity, which makes it as a potential lead candidate against diseases related to elevated JAK2 tyrosine kinase activity. KeywordsJanus kinase 2; Virtual screening; Inhibitor; Pharmacological evaluation Janus kinases (JAKs) are cytoplasmic protein kinases that play a crucial role in cytokinemediated signal transduction. In the absence of ligand, JAK kinases are constitutively bound to the cytoplasmic tails of cytokine receptors. The binding of ligand to the extracellular portion of the receptor results in a conformational change of the receptor itself and subsequent activation of the JAKs. Activated JAKs tyrosine autophosphorylate and phosphorylate specific tyrosine residues on the C-terminal end of the receptors. This results in the recruitment and activation of the Signal Transducers and Activators of Transcription (STAT) proteins. Activated STATs subsequently translocate into the nucleus where they alter specific gene transcription patterns. 1 JAKs contain seven conserved Janus homology (JH) 2 domains: a tyrosine kinase domain at the C-terminus (JH1), a pseudokinase domain (JH2) and five further * Corresponding author. Tel.: +36 1 4314605; fax: +36 1 4326002. gy.keseru@richter.hu (G.M. Keserű).