TNFR1 signaling is positively regulated by Jak-2 and c-Src via tyrosine phosphorylation.

Background/aim: Tumor necrosis factor alpha (TNFα, a.k.a. TNF) is a pleiotropic cytokine that exerts most of its effects through type 1 TNF receptor (TNFR1). Following TNF binding, TNFR1 recruits TRADD (tumor necrosis factor receptor type 1-associated DEATH domain). This interaction triggers formation of signalosome complexes which have been claimed to induce apoptosis (via downstream caspase activations), inflammation (via NF-kappaB) and stress pathways (JNK & p38). However, the mechanism underlying TNF-induced ERK and AKT activation is not completely revealed. TNFR1 is known to constitutively bind c-Src and JAK2, and these enzymes were previously demonstrated to modulate TNF signaling. Therefore, we hypothesized that TNFR1 could be tyrosine phosphorylated by JAK2 and/or c-Src and TNF-induced ERK and Akt activation may be mediated by this phosphorylation. Materials and methods: Site-directed mutagenesis (SDM) was performed to substitute the two putative Tyrosine phosphorylation sites on TNFR1 (Y360 and Y401) with alanine (A) or with aspartic acid (D), to inhibit or mimic constitutive phosphorylation, respectively. In 293T cells transfected with mutated or wild type TNFR1, ERK and Akt activations were determined by western blot. TNFR1 interaction with c-Src, JAK2, p85 and Grb2 was examined by co-IP. NF-kB activation was measured by luciferase assay, while proliferation was measured by MTT and apoptosis was evaluated by colorimetric caspase 8/3 assays. For determination of necrosis rates, cellular DNA fragmentation ELISA was performed. Results: In this report, we show that TNFR1 is phosphorylated by JAK2 tyrosine kinase at Y401 and by c-Src at Y360 and Y401. Phosphorylation of Y360 and Y401 augments the interaction of Grb2 and PI3Kp85 with TNFR1. We also demonstrate that phosphomimetic mutations of Y360D and Y401D enhance ERK and Akt activation. Conclusion: TNFR1 is tyrosine phosphorylated by both c-Src and JAK2, triggering a "noncanonical" pathway, that activates ERK and Akt.

Investigating the physiological role of S199A and S199D mutants of PHF6 protein in T-cell acute lymphoblastic leukemia.

Background/aim: T-cell acute lymphoblastic leukemia (T-ALL) is a form of leukemia characterized by the proliferation of immature T lymphocytes. NOTCH1 is one of the most frequently mutated genes in T-ALL. NOTCH1 expression in T-cell development depends on plant homeodomain finger protein 6 (PHF6), which plays a tumor suppressor role in T-ALL. Several studies have shown that PHF6 expression is essential for NOTCH1 expression. Therefore, whether posttranslational modification of PHF6 plays a role in the regulation of NOTCH1 expression and T-ALL cell line proliferation was investigated herein. Materials and methods: The amino acid sequence of PHF6 was analyzed and it was found that a putative protein kinase A (PKA) phosphorylation motif RDRS199 was conserved in several vertebrate species and the S199 site was expected to be phosphorylated according to the PhosphoSite database. Therefore, an eukaryotic expression vector of human PHF6 was constructed, and the codon 199 was changed to the codon encoding the nonphosphorylatable alanine and the phosphorylation-mimicking aspartic acid via site-directed mutagenesis. After confirming the ectopic expressions of the PHF6 vectors by western blot analysis, the effects of these proteins were identified on the NOTCH1 expression using western blot analysis, leukemic cell proliferation using MTT assay, and expressions of the cell surface markers of T-cells using flow cytometry. Results: The ectopic expression of wild-type PHF6 stimulated the formation of CD4 + T-cells. While the expression of the wild-type PHF6 suppressed the growth of the leukemic cell line, this effect was diminished in both the alanine and aspartic acid mutants of PHF6. In addition, both mutants also seemed to negatively affect the NOTCH1 expression, although the effect of the alanine mutant was more severe. Conclusion: Taken together, the different biological activities exerted by the conserved S199 phosphorylation-site mutants shown in this study implicate that signaling pathway(s) leading to differential phosphorylation of this residue may have a substantial effect on the activity of PHF6, and thus may constitute a potential therapeutic target in T-ALL.

Positive regulation of TNFR1 signaling via SH3 recognition motif.

TNF is a pleiotropic cytokine and shows its biological function by binding to its receptors called TNFR1 and TNFR2. While TNFR1 induces apoptosis by activation of caspase-8 via the "death domain", it also activates IKKα/ß, MKK3/6, MKK4/7 by activation of TAK1. Although the TNFR1 signaling pathway is known by in large, it is not known how AKT and MAPKs p38, ERK1/2, and JNK1/2 are activated. The presence of a proline-rich PPAP region, (P448PAP451, a binding site for the SH3 domain-containing proteins) very close to the C-terminus promoted us to determine whether this region has any role in the TNFR1 signal transduction. To test this, the codons of P448 and P451 were changed to that of Alanin, GCG, via site-directed mutagenesis, and this plasmid was named as TNFR1-SH3-P/A. Subsequently, ectopically expressed the wild type TNFR1 and TNFR1-SH3-P/A in 293T cells and determined the levels of TNF-α-mediated phosphorylations of ERK, p38, JNK and AKT, NF-kB, and caspase-8 activation. While ectopic expression of our mutant diminished TNFα-mediated phosphorylations of p38, JNK, ERK and AKT, it increased NF-kB, and caspase-8 activations. In conclusion, TNFα-mediated ERK, AKT, JNK, p38 activations are affected by TNFR1 SH3 domain modifications.

Regulation of E2F1 activity via PKA-mediated phosphorylations.

E2F1 becomes activated during the G1 phase of the cell cycle, and posttranslational modifications modulate its activity. Activation of G-protein coupled receptors (GPCR) by many ligands induces the activation of adenylate cyclases and the production of cAMP, which activates the PKA enzyme. Activated PKA elicits its biological effect by phosphorylating the target proteins containing serine or threonine amino acids in the RxxS/T motif. Since PKA activation negatively regulates cell proliferation, we thought that activated PKA would negatively affect the activity of E2F1. In line with this, when we analyzed the amino acid sequence of E2F1, we found 3 hypothetical consensus PKA phosphorylation sites located at 127-130, 232-235, and 361-364 positions and RYET, RLLS, and RMGS sequences. After showing the binding and phosphorylation of E2F1 by PKA, we converted the codons of Threonine-130, Serine-235, and Serine-364 to Alanine and Glutamic acid codons on the eukaryotic E2F1 expression vector we had previously created. We confirmed the phosphorylation of T130, S235, and S364 by developing monoclonal antibodies against phospho-specific forms of these sites and showed that their phosphorylation is cell cycle-dependent. According to our results, PKA-mediated phosphorylation of E2F1 by PKA inhibits proliferation and glucose uptake and induces caspase-3 activation and senescence.

AKT-mediated phosphorylation of TWIST1 is essential for breast cancer cell metastasis.

Previously, it was shown that human TWIST1 (basic helix-loop-helix (b-HLH) is phosphorylated by Akt kinase at S42, T121, and S123. To show in vivo effect of these phosphorylations, we created mouse TWIST1 expression vector and converted the codons of S42, T125, and S127 to unphosphorylatable alanine and phosphorylation mimicking Glutamic acid. We hypothesized that alanine mutants would inhibit the metastatic ability of 4T1 cells while glutamic acid mutants would convert nonmetastatic 67NR cells into metastatic phenotype. To confirm this hypothesis, we created metastatic 4T1 and nonmetastatic 67NR cells expressing alanine mutants and glutamic acid mutants mouse TWIST1, respectively. Then, we injected 1 × 106 67NR and 1 × 105 4T1 cells overexpressing mutants of TWIST1 into the breast tissue of BALB/c mice. At the end of the 4th week, we sacrificed the animals, determined the numbers of tumors at lungs and liver. Although 67NR cells overexpressing wild-type TWIST1 did not show any metastasis, cells overexpressing S42E and T125E mutants showed 15-30 macroscopic metastasis to liver and lungs. Parallel to this, 4T1 cells expressing S42A and T125A mutants of TWIST1 showed no macroscopic metastasis. Our results indicate that phosphorylation of S42 and T125 by AKT is essential for TWIST1-mediated tumor growth and metastasis.

Negative Regulation of TNFR1 Signaling Via PKA-Mediated Phosphorylation of TNFR1.

Tumor necrosis factor alpha (TNF-α) plays a paramount role in homeostasis by inducing tumor cytotoxicity and activating immune system. The signaling complexes formed by TNFR1 to activate JNK, p38, and nuclear factor-kappa B pathways and to subsequently induce apoptosis and necroptosis are well known. However, this "canonical TNF-α signaling" does not explain how ERK, AKT, and STAT3 can be activated by TNF-α. In addition, little to nothing is known about negative regulation of TNFR1 signaling. Because cyclic AMP-activated kinase (PKA) shows anti-TNF and anti-inflammatory activities, we postulated that PKA might affect TNF-α signaling by directly phosphorylating TNFR1. In line with this, we identified 2 putative PKA-phosphorylation motifs RRRT411 and REAT417 within the death domain of TNFR1, and investigated whether "canonical" and "noncanonical" TNFR1 signaling is regulated by modifications of T411 and T417. In this study, we demonstrate for the first time that PKA directly binds to and phosphorylates TNFR1 after TNF-α stimulation. To further support our hypothesis, we generated alanine and phosphomimetic (aspartic acid) mutants of TNFR1 at positions T411 and T417, ectopically expressed these mutants, and determined their influence on TNF-α-induced activations of ERKs, AKT, STAT3, p38α, and JNK1/2. Our results clearly showed that phosphomimetic mutants significantly suppressed and alanine mutants augmented TNF-α-induced phosphorylations of ERKs, AKT, Stat3, p38α, and JNKs. These findings strongly suggest that PKA-mediated phosphorylation of T411 and T417 of TNFR1 interferes with both "canonical" and "noncanonical" TNF-α signaling. [Figure: see text].

The regulation of circadian clock by tumor necrosis factor alpha.

All organisms display circadian rhythms which are under the control of the circadian clock located in the hypothalamus at the suprachiasmatic nucleus, (SCN). The circadian rhythms allow individuals to adjust their physiological activities and daily behavior for the diurnal changes in the living environment. To achieve these, all metabolic processes are aligned with the sleep/wake and fasting/feeding cycles. Subtle changes of daily behavior or food intake can result in misalignment of circadian rhythms. This can cause development of variety of metabolic diseases and even cancer. Although light plays a pivotal role for the activation of the master clock in SCN, the peripheral secondary clocks (or non-SCN), such as melatonin, growth hormone (GH), insulin, adiponectin and Ghrelin also are important in maintaining the circadian rhythms in the brain and peripheral organs. In recent years, growing body of evidence strongly suggest that CA2+ signaling, tumor necrosis factor alpha (TNFα) and transforming growth factor beta (TGFß) also play very important roles in the regulation of circadian rhythms by regulating the transcription of the clock genes.

Identification of novel mutations of Insulin Receptor Substrate 1 (IRS1) in tumor samples of non-small cell lung cancer (NSCLC): Implications for aberrant insulin signaling in development of cancer.

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.

TGF-ß-SMAD-miR-520e axis regulates NSCLC metastasis through a TGFBR2-mediated negative-feedback loop.

Transforming growth factor-ß (TGF-ß) pathway plays crucial roles during the carcinogenesis and metastasis. TGF-ß receptor 2 (TGFBR2) is a key molecule for the regulation of TGF-ß pathway and frequently downregulated or lost in several cancer types including non-small cell lung cancer (NSCLC), and TGF-ß pathway is often regulated by negative-feedback mechanisms, but little is known about the mechanism of TGFBR2 downregulation in NSCLC. Here, we found that the expression of miR-520e is upregulated in metastatic tumor tissues compared with non-metastatic ones, and its expression is inversely correlated with that of TGFBR2 in clinical samples. We also discovered that TGF-ß dramatically increased the expression of miR-520e, which targeted and downregulated TGFBR2, and the suppression of miR-520e significantly impaired TGF-ß-induced TGFBR2 downregulation. Chromatin immunoprecipitation-PCR experiments further showed that miR-520e is transcriptionally induced by SMAD2/3 in response to TGF-ß. Our findings reveal a novel negative-feedback mechanism in TGF-ß signaling and the expression level of miR-520e could be a predictive biomarker for NSCLC metastasis.

Downregulation of SATB2 is critical for induction of epithelial-to-mesenchymal transition and invasion of NSCLC cells.

OBJECTIVES: The epithelial-to-mesenchymal transition (EMT) is considered as a key step in invasion of cancer cells. There are several regulator proteins responsible for induction of EMT, but underlying mechanisms are still unclear. SATB2 is an epigenetic regulator involved in osteoblastic differentiation. The role of SATB2 in EMT and invasion of NSCLC cells is unknown. Therefore, we aimed to explain roles of SATB2 with underlying molecular mechanisms in EMT and invasion of NSCLC cells. MATERIALS AND METHODS: We used A549 and NCI-H1650 cells as a model to evaluate the effects of SATB2 in EMT and invasion of NSCLC cells. Cell culture, western blot analysis, siRNA-mediated gene knockdown, and invasion assay were performed in this study. RESULTS AND CONCLUSION: In this study, we investigated the regulatory role of SATB2 expression in TGF-ß-induced EMT and invasion of NSCLC cells, and found that SATB2 is downregulated in A549 cells and TGF-ß can induce EMT in these cells, however, TGF-ß can not induce EMT in SATB2 expressing cells such as H1650, PC3, II-18, Hcc78 and Hcc193. Our results demonstrated that SATB2 knockdown is sufficient to induce generation of fibroblast-like morphology, EMT and invasion of NSCLC cells by upregulating the expressions of Slug, Twist and Zeb1. Moreover, SATB2 knockdown promotes TGF-ß-induced EMT and invasion in NSCLC cells. These results strongly suggest that SATB2 prevents induction of EMT by suppressing expression of EMT-inducing transcription factors in NSCLC cells. Furthermore, SATB2 could inhibit tumour initiation by suppressing stemness marker genes such as CD44, Nanog, Oct-4A and Sox-2. Consequently, our results clearly indicate that SATB2 plays pivotal role in EMT, invasion and stemness of NSCLC cells.

Differential expression and activation of epidermal growth factor receptor 1 (EGFR1), ERK, AKT, STAT3, and TWIST1 in nonsmall cell lung cancer (NSCLC).

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.

Type 1 TNF receptor forms a complex with and uses Jak2 and c-Src to selectively engage signaling pathways that regulate transcription factor activity.

The type 1 TNFR (TNFR1) contains a death domain through which it interacts with other death-domain proteins to promote cellular responses. However, signaling through death-domain proteins does not explain how TNFR1 induces the tyrosine phosphorylation of intracellular proteins, which are important to cellular responses induced by TNFR1. In this study, we show that TNFR1 associates with Jak2, c-Src, and PI3K in various cell types. Jak2 and c-Src constitutively associate with and are constitutively active in the TNFR1 complex. Stimulation with TNF induces a time-dependent change in the level of Jak2, c-Src, and PI3K associated with TNFR1. The tyrosine kinase activity of the complex varies with the level of tyrosine kinase associated with TNFR1. TNFR1/c-Src plays a role in activating Akt, but not JNK or p38 MAPK, whereas TNFR1/Jak2 plays a role in activating p38 MAPK, JNK, and Akt. TNFR1/c-Src, but not TNFR1/Jak2, plays an obligate role in the activation of NF-kappaB by TNF, whereas TNFR1/Jak2, but not TNFR1/c-Src, plays an obligate role in the activation of STAT3. Activation of TNFR1 increased the expression of vascular endothelial growth factor, p21(WAF1/CIP1), and manganese superoxide dismutase in MCF7 breast cancer cells, and increased the expression of CCl2/MCP-1 and IL-1beta in THP-1 macrophages. Inhibitors of Jak2 and c-Src impaired the induction of each of these target proteins. These observations show that TNFR1 associates with and uses nonreceptor tyrosine kinases to engage signaling pathways, activate transcription factors, and modulate gene expression in cells.

Lack of BCL-2 confers interferon-alpha sensitivity to B-cell lymphomas.

Hairy cell leukemia (HCL) is a chronic B-cell lymphoproliferative disorder with pathological manifestations usually including splenomegaly and pancytopenia. Interferons (IFNs), specifically of the alpha subtypes, have shown a significant anti-tumor effect in HCL patients, with improvement of hematological parameters within the first few months of treatment. However, the therapeutic effect of IFN-alpha is still rather limited. The mechanisms responsible for the beneficial action of IFN-alpha in HCL patients are unclear. A continuous line of cells (Eskol) from a patient diagnosed with HCL was established and shown to have several properties of HCL. Even though, Eskol cells are very resistant to anti-proliferative activity of IFN-alpha, Daudi cells, another human B-cell-derived cell line, are very sensitive to anti-proliferative activity of IFN-alpha and are commonly used as a model cell to test anti-proliferative effect of IFN-alpha. To understand the molecular reason(s) behind the observed obvious differences to IFN sensitivity of above cells, we have analyzed the expression levels of BCL2, caspase-1, Laminin and PARP in these cells. We found that Daudi cells do not express BCL2 at all, and probably because of that, these cells have constantly cleaved, and probably activated form of caspase-1. However, when we over-expressed BCL2 in these cells, they lost processed form of caspase-1 and became resistant to anti-proliferative activity of IFN-alpha. These results let us to suggest that IFN-alpha sensitivity of B-cell lymphomas, once again, depends on the presence or absence of BCL2.

Compound heterozygosity for two beta chain variants: the mildly unstable Hb Tyne (codon 5 Pro→Ser) and HbS (codon 6 Glu→Val).

Compound heterozygosity for Hb Tyne and HbS, that is very rare, was identified by direct DNA sequencing of the beta-globin gene in a Turkish patient. Hematological investigation of a girl at the age of 9 due to the presence of HbS (40.7%) led to the identification of a compound heterozygosity at codons 5-6. This was found to be the result of substitution of cytosine (C) for thymidine (T) at the fifth position and a substitution of adenine (A) for thymidine (T) at the sixth position of the beta globin gene. As a result of these mutations, the order of amino acids at codons 5-6 was changed from Pro-Glu to Ser-Val, respectively. Since the co-inheritance of Hb Tyne and HbS had not been reported in literature before, our case set an example for identification of coinheritance of Hb Tyne and HbS for the first time. Therefore, such cases may be considered as an important example for understanding the structural variants of hemoglobin and may provide important clues for critical amino acids responsible for stabilization of hemoglobin tetrameric structure and genetic counseling.

Tumor necrosis factor activates CRE-binding protein through a p38 MAPK/MSK1 signaling pathway in endothelial cells.

Tumor necrosis factor (TNF) promotes immunity and modulates cell viability, in part, by promoting alterations of cellular gene expression. The mechanisms through which TNF communicates with the nucleus and alters gene expression are incompletely understood. Incubation of human umbilical vein endothelial cells (HUVEC) with TNF induces phosphorylation of the CRE-binding protein (CREB) transcription factor on serine 133 and increases CREB DNA binding and transactivation. Dominant negative CREB, an antagonist antibody directed against the type 1 TNF receptor, or pharmacological inhibition of p38 MAPK signaling blocked TNF-induced CREB activation as determined by phosphorylation and gene reporter assays. From among the kinases that can activate CREB, we found that downstream of p38 MAPK, MSK1 is activated by TNF to promote CREB activation. These observations show that CREB is activated by TNF/TNFR1 signaling through a p38MAPK/MSK1 signaling pathway.

Suppression of TNF-alpha mediated apoptosis by EGF in TNF-alpha sensitive human cervical carcinoma cell line.

The tumor suppressor protein p53 is the most frequently mutated gene in human cancer. The function of p53 is not restricted to "guarding" against oncogenic stress, but also p53 can guard against the presence of DNA damage. One of the principal mechanisms by which cells achieve this is by regulating the p53 protein level although its phosphorylation and cellular localization also contribute to the regulation of its function. Since many tumors secrete growth factor(s) that inhibit apoptosis and support the growth of cancer cells, we investigated the effects of human epidermal growth factor (EGF) on human TNF-alpha-mediated induction of p53 and its transcriptional target, p21 in TNF-alpha sensitive human cervical carcinoma cell line, ME180S. We found that TNF-alpha can increase the cellular levels of p53, p21 and induce apoptosis in ME180S cells. However, pretreatment of cells with EGF can suppress all these effects of TNF-alpha. To determine which kinase(s) pathway was utilized by EGF to show these suppressive effects, cells were pretreated with inhibitors of MAPK, PI3K and PKC pathways. Among these only PKC inhibitor reversed all the suppressive effects of EGF. We also found that ME180S cells express only zeta, lambda, epsilon, iota, delta, theta, beta PKC subtypes and among these EGF treatment activate only PKC-delta redistribution to the membrane from the cytosol. An inhibitor of PKC, GF 109203X inhibited EGF-mediated suppression of TNF-alpha-induced accumulation of p53, p21 and induction of apoptosis. In summary, we concluded that EGF can protect ME180S cells from TNF-alpha-induced apoptosis through activation of PKC-delta.

Tumor necrosis factor-alpha-induced accumulation of tumor suppressor protein p53 and cyclin-dependent protein kinase inhibitory protein p21 is inhibited by insulin in ME-180S cells.

The tumor suppressor protein p53 plays an important role in the protection against the development of cancer and is inactivated in many human malignancies. Since p53 is an important inhibitor of cell growth, keeping p53 function under control is critical for survival of cell. One of the principal mechanisms by which cells achieve this is by regulating the p53 protein level, although its phosphorylation and cellular localization also contribute to the regulation of its function. Since many tumors secrete growth factor(s) that inhibit apoptosis and support the growth of cancer cells, we wanted to know whether insulin would have an effect on antitumor and p53-inducing activities of human tumor necrosis factor-alpha (TNF-alpha). Here we show that treatment of human cervical carcinoma cell line, ME-180S, with TNF-alpha results in time-dependent accumulation of p53 and its transcriptional target, p21. However, pretreatment of these cells with insulin inhibits TNF-alpha-dependent cell killing, induction of p53, p21 and apoptosis.