We describe a strategy to comprehend signaling pathways active in lung cancer cells and targeted by dasatinib employing chemical proteomics to identify direct interacting proteins combined with immunoaffinity purification of tyrosine phosphorylated peptides corresponding to activated tyrosine kinases. We identified nearly 40 different kinase targets of dasatinib. These include SFK members (LYN, SRC, FYN, LCK, YES), non-receptor tyrosine kinases (FRK, BRK, ACK), and receptor tyrosine kinases (Ephrin receptors, DDR1, EGFR). Using quantitative phosphoproteomics we identified peptides corresponding to autophosphorylation sites of these tyrosine kinases that are inhibited in a concentration-dependent manner by dasatinib. Using drug resistant gatekeeper mutants, we show that SFK kinases, particularly SRC and FYN, as well as EGFR are relevant targets for dasatinib action. The combined mass spectrometry based approach described here provides a system-level view of dasatinib action in cancer cells and suggests both functional targets and rationale combinatorial therapeutic strategies.

Hyperleptinemia is a common feature of obese women who have a higher risk of endometrial cancer than women with normal weights, and epidemiologic studies have suggested a correlation between obesity and endometrial carcinoma. Therefore, understanding of the molecular mechanism involved in leptin signaling transduction is important in endometrial cancer prevention and treatment. In this study, both isoforms of the leptin receptor (Ob-R), the long form (Ob-Rb) and short form (Ob-Ra), were detected as being expressed in six endometrial cancer cell lines with various differentiation status by western blotting, and Ob-Ra was found to be more abundant than Ob-Rb in these cells. Moreover, the expressions of both isoforms were inversely correlated with histoprognostic grading. We also showed that leptin stimulated cell proliferation

Minibrain-related kinase (Mirk) is a member of the dual specificity tyrosine-phosphorylation-regulated kinase (Dyrk)/minibrain family of dual-specificity protein kinases and is identical to Dyrk1B. Mirk/Dyrk1B is a serine/threonine kinase that has been found to be upregulated in solid tumors and mediates cell survival in colon cancer, pancreatic ductal adenocarcinoma and rhabdomyosarcomas. There is little known about Mirk in lung cancer. In the present study, we showed that Mirk protein was widely overexpressed in 13 of 19 NSCLC cell lines. Mirk immunoprecipitation coupled with anti-phosphotyrosine western blotting confirmed tyrosine phosphorylation of Mirk in NSCLC cells. Mirk knockdown by small interfering RNA induced cell apoptosis concomitant with upregulation of Bak, a Bcl-2 family member, and downregulation of signal transducers and activators of transcription 3 (STAT3) tyrosine phosphorylation. Mirk knockdown led to decreased cell colony formation in vitro as well as delayed tumor growth in an orthotopic mouse model and sensitized cells to cisplatin-induced apoptosis. Using automated quantitative determination of the Mirk protein in tumor specimens of patients with early-stage lung cancer, overexpression of Mirk was found in nearly 90% of tumor specimens in both the cytoplasm and nucleus. These results suggest that Mirk is overexpressed in lung cancer, acts as a survival factor in lung cancer cells and may be a novel therapeutic target.

ndometrial cancer is one of the most common gynecologic cancers.

Serine/threonine kinase IKBKE is a newly identified oncogene; however, its regulation remains elusive. Here, we provide evidence that IKBKE is a downstream target of signal transducer and activator of transcription 3 (STAT3) and that tobacco components induce IKBKE expression through STAT3. Ectopic expression of constitutively active STAT3 increased IKBKE mRNA and protein levels, whereas inhibition of STAT3 reduced IKBKE expression. Furthermore, expression levels of IKBKE are significantly associated with STAT3 activation and tobacco use history in non-small cell lung cancer (NSCLC) patients examined. In addition, we show induction of IKBKE by two components of cigarette smoke, nicotine and nicotine-derived nitrosamine ketone (NNK). Upon exposure to nicotine or NNK, cells express high levels of IKBKE protein and mRNA, which are largely abrogated by inhibition of STAT3. Characterization of the IKBKE promoter revealed two STAT3-response elements. The IKBKE promoter directly bound to STAT3 and responded to nicotine and NNK stimulation. Notably, enforcing expression of IKBKE induces chemoresistance, whereas knockdown of IKBKE not only sensitizes NSCLC cells to chemotherapy but also abrogates STAT3- and nicotine-induced cell survival. These data indicate for the first time that IKBKE is a direct target of STAT3 and is induced by tobacco carcinogens through STAT3 pathway. In addition, our study also suggests that IKBKE is an important therapeutic target and could have a pivotal role in tobacco-associated lung carcinogenesis.