su-6656 and Cell-Transformation--Neoplastic

The Src kinase family (SKF) includes non‑receptor tyrosine kinases that interact with many cellular cytosolic, nuclear and membrane proteins, and is involved in the progression of cellular transformation and oncogenic activity. However, there is little to no evidence on the effect of SKF or its inhibitors on melanogenesis. Therefore, the present study investigated whether C‑terminal Src kinase inhibition can induce melanogenesis and examined the associated signaling pathways and mRNA expression of melanogenic proteins. First, whether stimulators of melanogenesis, such as ultraviolet B and α‑melanocyte‑stimulating hormone, can dephosphorylate Src protein was evaluated, and the results revealed that SU6656 and PP2 inhibited the phosphorylation of Src in G361 cells. Src inhibition by these chemical inhibitors induced melanogenesis in G361 cells and upregulated the mRNA expression levels of melanogenesis‑associated genes encoding microphthalmia‑associated transcription factor, tyrosinase‑related protein 1 (TRP1), TRP2, and tyrosinase. In addition, Src inhibition by small interfering RNA induced melanogenesis and upregulated the mRNA expression levels of melanogenesis‑associated genes. As the p38 mitogen‑activated protein kinase (MAPK) and cyclic adenosine monophosphate response element binding (CREB) pathways serve key roles in melanogenesis, the present study further examined whether Src mediates melanogenesis via these pathways. As expected, Src inhibition via SU6656 or PP2 administration induced the phosphorylation of p38 or CREB, as determined by western blotting analysis, and increased the levels of phosphorylated p38 or CREB, as determined by immunofluorescence staining. In addition, the induced pigmentation and melanin content of G361 cells by Src inhibitors was significantly inhibited by p38 or CREB inhibitors. Taken together, these data indicate that Src is associated with melanogenesis, and Src inhibition induces melanogenesis via the MAPK and CREB pathways in G361 cells.

Topics: alpha-MSH; Biomarkers; Cell Line, Tumor; Cell Transformation, Neoplastic; Cyclic AMP Response Element-Binding Protein; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Indoles; Melanins; Melanoma; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Pyrimidines; RNA, Small Interfering; src-Family Kinases; Sulfonamides; Ultraviolet Rays

Oncogenic mutants of c-Kit are often found in mastocytosis, gastrointestinal stromal tumors and acute myeloid leukemia. The activation mechanism of the most commonly occurring mutation, D816V in exon 17 of c-Kit, has been well-studied while other mutations remain fairly uncharacterized in this respect. In this study, we show that the constitutive activity of the exon 11 mutant V560D is weaker than the D816V mutant. Phosphorylation of downstream signaling proteins induced by the ligand for c-Kit, stem cell factor, was stronger in c-Kit/V560D expressing cells than in cells expressing c-kit/D816V. Although cells expressing c-Kit/V560D showed increased ligand-independent proliferation and survival compared to wild-type c-Kit-expressing cells, these biological effects were weaker than in c-Kit/D816V-expressing cells. In contrast to cells expressing wild-type c-Kit, cells expressing c-Kit/V560D were independent of Src family kinases for downstream signaling. However, the independence of Src family kinases was not due to a Src-like kinase activity that c-Kit/D816V displayed. Point mutations that selectively block the association of PI3 kinase with c-Kit/V560D inhibited ligand-independent activation of the receptor, while inhibition of the kinase activity of PI3 kinase with pharmacological inhibitors did not affect the kinase activity of the receptor. This suggests a lipid kinase-independent key role of PI3 kinase in c-Kit/V560D-mediated oncogenic signal transduction. Thus, PI3 kinase is an attractive therapeutic target in malignancies induced by c-Kit mutations independent of its lipid kinase activity.

Topics: Animals; Blotting, Western; Cell Line; Cell Proliferation; Cell Survival; Cell Transformation, Neoplastic; Chromones; HEK293 Cells; Humans; Indazoles; Indoles; Morpholines; Mutation, Missense; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Proto-Oncogene Proteins c-kit; Signal Transduction; src-Family Kinases; Stem Cell Factor; Sulfonamides

TrkC mediates many aspects of growth and development in the central nervous system. TrkC is expressed in a variety of non-neuronal tissues as well as human cancers. TrkC overexpression may drive tumorigenesis, invasion, and metastatic capability in cancer cells. However, relatively little is known about whether TrkC activity is also essential to maintain the malignant properties in human tumors. TrkC expression leads to the constitutive activation of two major effector pathways, namely the Ras-MAP kinase (MAPK) mitogenic pathway and the phosphatidylinositol 3-kinase (PI3K)-AKT pathway mediating cell survival. However, it remains unclear how TrkC activates Ras-Erk1/2 and/or PI3K-Akt cascades. Here we define some aspects of the molecular mechanisms regulating TrkC-dependent Ras-Erk1/2 and PI3K/Akt activation. We show that endogenous TrkC associated with c-Src in human and mouse cancer cells which express TrkC. TrkC-c-Src complexes were also detected in primary human breast cancer tissues. Suppression of c-Src by RNA interference in highly metastatic 4T1 mammary cancer cells, which express endogenous TrkC, resulted in markedly decreased expression of cyclin D1 and suppression of activation of Ras-Erk1/2 and PI3K-Akt. Moreover, inhibition of c-Src expression almost completely blocks colony formation of 4T1 cells in soft agar. Furthermore, in c-Src-deficient SYF cells, TrkC failed to activate the PI3K-Atk pathway, but not the Ras-Erk1/2 pathway. Therefore these data indicate that TrkC induces the PI3K-Akt cascade through the activation of c-Src.

Topics: Adaptor Proteins, Signal Transducing; Animals; Cell Line; Cell Line, Tumor; Cell Transformation, Neoplastic; Cyclin D1; Enzyme Activation; Fibroblasts; Humans; Indoles; Insulin Receptor Substrate Proteins; MAP Kinase Kinase 1; Mice; Phosphatidylinositol 3-Kinases; Phosphorylation; Protein Binding; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins pp60(c-src); Receptor, trkC; RNA, Small Interfering; Sulfonamides; Tumor Stem Cell Assay

The ETV6-NTRK3 (EN) chimeric tyrosine kinase, a potent oncoprotein expressed in tumors derived from multiple cell lineages, functions as a constitutively active protein-tyrosine kinase. ETV6-NTRK expression leads to the constitutive activation of two major effector pathways of wild-type NTRK3, namely, the Ras-mitogen-activated protein kinase (MAPK) mitogenic pathway and the phosphoinositide-3-kinase (PI3K)-Akt pathway mediating cell survival, and both are required for EN transformation. However, it remains unclear how ETV6-NTRK3 activates Ras-Erk1/2 and/or PI3K-Akt cascades. Here, we define some aspects of the molecular mechanisms regulating ETV6-NTRK-dependent Ras-Erk1/2 and PI3K-Akt activation. We show that ETV6-NTRK3 associates with c-Src, and that treatment with SU6656, a c-Src inhibitor, completely blocks ETV6-NTRK-transforming activity. Treatment of NIH3T3 cells expressing ETV6-NTRK3 with SU6656 attenuated the activation of Ras-Erk1/2 and PI3K-Akt. Suppression of c-Src by RNA interference in NIH3T3-ETV6-NTRK3 cells resulted in markedly decreased expression of cyclin D1 and suppression of activation of Ras-Erk1/2 and PI3K-Akt. However, in Src-deficient cells, the ETV6-NTRK3 failed to activate the PI3K-Atk pathway, but not the Ras-Erk1/2 pathway. Therefore, these data indicate that ETV6-NTRK3 induces the PI3K-Akt cascade through the activation of c-Src.

Topics: Animals; Cell Line; Cell Transformation, Neoplastic; CSK Tyrosine-Protein Kinase; Enzyme Activation; Humans; Indoles; Mice; NIH 3T3 Cells; Oncogene Proteins, Fusion; Phosphatidylinositol 3-Kinases; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-akt; RNA, Small Interfering; src-Family Kinases; Sulfonamides

The proto-oncogene c-Src has been implicated in the development and progression of a number of human cancers including those of colon and breast. Accumulating evidence indicates that activated alleles of Src may induce cell transformation through Ras-ERK-dependent and -independent pathways. Here we show that Rac1 activity is strongly elevated in Src-transformed cells and that this small G protein is a critical component of the pathway connecting oncogenic Src with cell transformation. We further show that Vav2 and the ubiquitously expressed Rac1 guanine nucleotide exchange factor Tiam1 are phosphorylated in tyrosine residues in cells transfected with active and oncogenic Src. Moreover, phosphorylation of Tiam1 in cells treated with pervanadate, a potent inhibitor of tyrosine phosphatases, was partially inhibited by the Src inhibitor SU6656. Using truncated mutants of Tiam1, we demonstrate that multiple sites can be tyrosine-phosphorylated by Src. Furthermore, Tiam1 cooperated with Src to induce activation of Rac1 in vivo and the formation of membrane ruffles. Similarly, activation of JNK and the c-jun promoter by Src were also potently increased by Tiam1. Together, these results suggest that Vav2 and Tiam1 may act as downstream effectors of Src, thereby regulating Rac1-dependent pathways that participate in Src-induced cell transformation.

Topics: 3T3 Cells; Alleles; Animals; Binding Sites; Catalysis; Cell Line; Cell Transformation, Neoplastic; DNA; Enzyme Activation; Enzyme Inhibitors; Genes, Reporter; GTP Phosphohydrolases; Guanine Nucleotide Exchange Factors; Humans; Indoles; JNK Mitogen-Activated Protein Kinases; MAP Kinase Kinase 4; Mice; Microscopy, Fluorescence; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Mutation; Oncogene Proteins; Phosphorylation; Plasmids; Promoter Regions, Genetic; Proteins; Proto-Oncogene Mas; Proto-Oncogene Proteins c-jun; Proto-Oncogene Proteins c-vav; Proto-Oncogene Proteins pp60(c-src); rac1 GTP-Binding Protein; Sulfonamides; T-Lymphoma Invasion and Metastasis-inducing Protein 1; Time Factors; Transfection; Tyrosine; Vanadates