Osman Nidai Özeş

Tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) by the insulin receptor permits this docking protein to interact with signaling proteins that promote insulin action. Serine phosphorylation uncouples IRS-1 from the insulin receptor, thereby inhibiting its tyrosine phosphorylation and insulin signaling. For this reason, there is great interest in identifying serine/threonine kinases for which IRS-1 is a substrate. Tumor necrosis factor (TNF) inhibited insulin-promoted tyrosine phosphorylation of IRS-1 and activated the Akt/protein kinase B serine-threonine kinase, a downstream target for phosphatidylinositol 3-kinase (PI 3-kinase). The effect of TNF on insulin-promoted tyrosine phosphorylation of IRS-1 was blocked by inhibition of PI 3-kinase and the PTEN tumor suppressor, which dephosphorylates the lipids that mediate PI 3-kinase functions, whereas constitutively active Akt impaired insulin-promoted IRS-1 tyrosine phosphorylation. Conversely, TNF inhibition of IRS-1 tyrosine phosphorylation was blocked by kinase dead Akt. Inhibition of IRS-1 tyrosine phosphorylation by TNF was blocked by rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), a downstream target of Akt. mTOR induced the serine phosphorylation of IRS-1 (Ser-636/639), and such phosphorylation was inhibited by rapamycin. These results suggest that TNF impairs insulin signaling through IRS-1 by activation of a PI 3-kinase/Akt/mTOR pathway, which is antagonized by PTEN.

Lung cancer is the leading cause of cancer-related death, and NSCLC constitutes nearly 85%-90% of all cases. The IRS proteins function as adaptors and transmit signals from multiple receptors. Upon binding of insulin to the insulin receptor (IR), IRS1 is phosphorylated at several YXXM motifs creating docking sites for the binding of PI3Kp85, which activates AKT kinase. Therefore, we thought that gain of function mutantions of IRS1 could be related to development of lung cancer. In line with this, we wanted determine whether the IRS1 gene was mutated in the coding regions surrounding YXXM motifs. We sequenced the coding regions surrounding YXXM motifs of IRS1 using tumor samples of 42 NSCLC patients and 40 matching controls and found heterozygote p.S668T mutation in nine of 42 samples and four of nine also had the p.D674H mutation. We generated IRS1 expression vectors harboring p.S668T, p.D674H and double mutants. Expression of the mutants differentially affected insulin-induced phosphorylation of IRS1, AKT, ERK, and STAT3. Also, our mutants induced proliferation, glucose uptake, inhibited the migration of 293T cells and affected the responsiveness of the cells to cisplatin and radiation. Our results suggest that these novel mutations play a role in the phenotype of lung cancer.

Lung cancer is the leading cause of death of both men and women across the world. Overexpression and activating mutations of the epidermal growth factor receptor-1 (EGFR1) are frequently observed and associated with poor prognosis. To inhibit the function of EGFR1, multiple antibodies and small-molecule tyrosine kinase inhibitors (TKI) that target EGFR1 have been developed. Even though some patients respond to these TKI, subsequent studies reveal that this is not the case for all nonsmall cell lung cancer (NSCLC) patients. In this study, we determine whether activation and expression levels of EGFR1, ERK, AKT, STAT3, and TWIST1 are dependent on the activating mutations of EGFR1. Protein lysates and DNA have been isolated from tumor and corresponding normal tissues of 16 NSCLC patients. Genomic-DNA is used to sequence the exons 18, 19, and 21 of EGFR1, and exon 2 of k-RAS. Protein lysates were used to determine the expression or phosphorylation levels of EGFR, STAT3, ERK, AKT, and TWIST1. Our results revealed that 16 tumor samples of NSCLC patients showed no mutation in any of the indicated exons of EGFR1 and k-RAS albeit significant levels of activation or expression of the above-mentined oncogenes. In NSCLC patients, the tumor micro-environment can be as important as the activating mutations of EGFR1. TK therapy may also be considered for patients who show high levels of activation of EGFR1 even in the absence of activating mutations.

diseases in the world, is an autosomal recessive disease generally caused by point mutations in the β-globin gene that is located as a cluster on the short arm of chromosome 11 (1-3). Modern molecular techniques have provided a wealth of information about the nature of mutations and their distributions within the world population. More than 200 different mutations affecting diverse levels of β-globin gene expression have so far been identified (2,4,5). These mutations such as frameshifts of the insertion/deletion type and/or nucleotide substitutions have been reported to interfere with the transcription of the β-globin gene, splicing procedures and translation of β-globin mRNA (1-5). All these mutations result in either an absence or reduction of synthesis of β-globin chains (3,6). Insertion or deletion of one or more nucleotides in the coding region of the βglobin gene disrupts the normal reading frame and causes the frameshift. As a result of frameshift the β-globin chain is synthesized as either elongated or truncated (7, 8).