mk-2206 and Cell-Transformation--Neoplastic

Gallbladder carcinoma (GBC) is the most common malignancy of the biliary tract with extremely poor prognosis. The malignant transformation of GBC is associated with cell proliferation, invasion, and epithelial-mesenchymal transition (EMT). However, the molecular mechanisms underlying GBC progression are poorly understood. We found that serine threonine tyrosine kinase 1 (STYK1) was elevated in GBC and was negatively correlated with clinical outcomes and prognosis. Overexpression of STYK1 in GBC cell lines gave rise to increased cell proliferation, colony formation, migration and invasion, thus committing cells to undergoing EMT. In contrast, silence of STYK1 led to opposite effects on cell transformation. Consistent with STYK1 gene knockdown, AKT specific inhibitor MK2206 abrogated tumor promoting action induced by STYK1, suggesting that PI3K/AKT pathway is essential for the oncogenic role of STYK1 in GBC. STYK1 shRNA in GBC cells inhibited development of xenografted tumors compared with control cells. Collectively, our findings suggest that STYK1 is a critical regulator of tumor growth and metastasis, and may serve as a potential target for GBC therapy.

Topics: Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Female; Gallbladder Neoplasms; Heterocyclic Compounds, 3-Ring; Humans; Male; Neoplasm Metastasis; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Receptor Protein-Tyrosine Kinases; Signal Transduction

Metabotropic glutamate receptor 1 (mGluR1/Grm1) is a member of the G-protein-coupled receptor superfamily, which was once thought to only participate in synaptic transmission and neuronal excitability, but has more recently been implicated in non-neuronal tissue functions. We previously described the oncogenic properties of Grm1 in cultured melanocytes in vitro and in spontaneous melanoma development with 100 % penetrance in vivo. Aberrant mGluR1 expression was detected in 60-80 % of human melanoma cell lines and biopsy samples. As most human cancers are of epithelial origin, we utilized immortalized mouse mammary epithelial cells (iMMECs) as a model system to study the transformative properties of Grm1. We introduced Grm1 into iMMECs and isolated several stable mGluR1-expressing clones. Phenotypic alterations in mammary acinar architecture were assessed using three-dimensional morphogenesis assays. We found that mGluR1-expressing iMMECs exhibited delayed lumen formation in association with decreased central acinar cell death, disrupted cell polarity, and a dramatic increase in the activation of the mitogen-activated protein kinase pathway. Orthotopic implantation of mGluR1-expressing iMMEC clones into mammary fat pads of immunodeficient nude mice resulted in mammary tumor formation in vivo. Persistent mGluR1 expression was required for the maintenance of the tumorigenic phenotypes in vitro and in vivo, as demonstrated by an inducible Grm1-silencing RNA system. Furthermore, mGluR1 was found be expressed in human breast cancer cell lines and breast tumor biopsies. Elevated levels of extracellular glutamate were observed in mGluR1-expressing breast cancer cell lines and concurrent treatment of MCF7 xenografts with glutamate release inhibitor, riluzole, and an AKT inhibitor led to suppression of tumor progression. Our results are likely relevant to human breast cancer, highlighting a putative role of mGluR1 in the pathophysiology of breast cancer and the potential of mGluR1 as a novel therapeutic target.

Topics: Animals; Apoptosis; Breast Neoplasms; Cell Proliferation; Cell Transformation, Neoplastic; Female; Gene Expression Regulation, Neoplastic; Heterocyclic Compounds, 3-Ring; Humans; Mammary Glands, Animal; MCF-7 Cells; Mice; Receptors, Metabotropic Glutamate; Riluzole; Xenograft Model Antitumor Assays

Clonal evolution and intratumoral heterogeneity drive cancer progression through unknown molecular mechanisms. To address this issue, functional differences between single T cell acute lymphoblastic leukemia (T-ALL) clones were assessed using a zebrafish transgenic model. Functional variation was observed within individual clones, with a minority of clones enhancing growth rate and leukemia-propagating potential with time. Akt pathway activation was acquired in a subset of these evolved clones, which increased the number of leukemia-propagating cells through activating mTORC1, elevated growth rate likely by stabilizing the Myc protein, and rendered cells resistant to dexamethasone, which was reversed by combined treatment with an Akt inhibitor. Thus, T-ALL clones spontaneously and continuously evolve to drive leukemia progression even in the absence of therapy-induced selection.

Topics: Animals; Animals, Genetically Modified; Antineoplastic Agents, Hormonal; Apoptosis; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Clonal Evolution; Dexamethasone; Disease Progression; Drug Resistance, Neoplasm; Enzyme Activation; Genetic Variation; Heterocyclic Compounds, 3-Ring; Humans; Mechanistic Target of Rapamycin Complex 1; Molecular Sequence Data; Multiprotein Complexes; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-myc; T-Lymphocytes; TOR Serine-Threonine Kinases; Zebrafish

In addition to overexpression and the occurrence of activating mutations, receptors can be aberrantly activated by impaired downregulation. In this study, we show that an oncogenic mutant of the ubiquitin ligase casitas B-lineage lymphoma (CBL; CBLΔexon8), which is found in acute myeloid leukemia patients, predominantly cooperates with receptor tyrosine kinase (RTK) class III receptors (PDGFRA, PDGFRB, KIT, and FLT3), but not with non-class III RTKs or cytokine receptors, to induce IL-3-independent growth of Ba/F3 cells. In cells coexpressing RTK class III/CBLΔexon8, receptor internalization was delayed, and cells were protected from apoptosis after cytokine withdrawal. Ligand-stimulated Ba/F3 cells and acute myeloid leukemia cell lines coexpressing the CBL deletion mutant and FLT3 showed enhanced AKT phosphorylation. Combined pharmacologic inhibition of the PI3K/AKT pathway and FLT3 had an additive effect on cell proliferation. The transforming potential of the CBL mutant was completely abolished by the mutation of the CBL PTB domain and was decreased by the mutation of tyrosines 589 and 591 in the juxtamembrane domain of FLT3. A constitutively active AKT1 mutant (E17K) recapitulated the phenotype induced by the CBL deletion mutant in Ba/F3 cells. This study reveals FLT3-CBL interaction sites and the AKT pathway as critical mediators of transformation by oncogenic CBL mutants.

Topics: Animals; Apoptosis; Benzothiazoles; Binding Sites; Blotting, Western; Cell Line; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Chromones; Flow Cytometry; fms-Like Tyrosine Kinase 3; Heterocyclic Compounds, 3-Ring; HL-60 Cells; Humans; Mice; Morpholines; Mutation; Phenylurea Compounds; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Protein Binding; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-cbl; Signal Transduction; Transfection; Tyrosine

The phosphoinositide 3-kinase (PI3K) pathway is targeted for frequent alteration in glioblastoma (GBM) and is one of the core GBM pathways defined by The Cancer Genome Atlas. Somatic mutations of PIK3R1 are observed in multiple tumor types, but the tumorigenic activity of these mutations has not been demonstrated in GBM. We show here that somatic mutations in the iSH2 domain of PIK3R1 act as oncogenic driver events. Specifically, introduction of a subset of the mutations identified in human GBM, in the nSH2 and iSH2 domains, increases signaling through the PI3K pathway and promotes tumorigenesis of primary normal human astrocytes in an orthotopic xenograft model. Furthermore, we show that cells that are dependent on mutant P85α-mediated PI3K signaling exhibit increased sensitivity to a small molecule inhibitor of AKT. Together, these results suggest that GBM patients whose tumors carry mutant PIK3R1 alleles may benefit from treatment with inhibitors of AKT.

Topics: Analysis of Variance; Astrocytes; Cell Survival; Cell Transformation, Neoplastic; Class Ia Phosphatidylinositol 3-Kinase; Dimethyl Sulfoxide; Dose-Response Relationship, Drug; Glioblastoma; Heterocyclic Compounds, 3-Ring; Humans; Immunoblotting; Mutagenesis; Mutation; Plasmids; Signal Transduction

Serine/threonine kinase Akt regulates key cellular processes such as cell growth, proliferation, and survival. Activation of Akt by mitogenic factor depends on phosphatidylinositol 3-kinase (PI3K). Here, we report that IKBKE (also known as IKKε and IKKi) activates Akt through a PI3K-independent pathway. IKBKE directly phosphorylates Akt-Thr308 and Ser473 independent of the pleckstrin homology (PH) domain. IKBKE activation of Akt was not affected by inhibition of PI3K, knockdown of PDK1 or mTORC2 complex. Further, this activation could be inhibited by Akt inhibitors MK-2206 and GSK690693 but not the compounds (perifosine and triciribine) targeting the PH domain of Akt. Expression of IKBKE largely correlates with activation of Akt in breast cancer. Moreover, inhibition of Akt suppresses IKBKE oncogenic transformation. These findings indicate that IKBKE is an Akt-Thr308 and -Ser473 kinase and directly activates Akt independent of PI3K, PDK1, and mTORC2 as well as PH domain. Our data also suggest that Akt inhibitors targeting the PH domain have no effect on the tumors in which hyperactive Akt resulted from elevated IKBKE.

Topics: 3-Phosphoinositide-Dependent Protein Kinases; Animals; Breast Neoplasms; Cell Line, Tumor; Cell Transformation, Neoplastic; Enzyme Activation; Female; Gene Deletion; HEK293 Cells; Heterocyclic Compounds, 3-Ring; Humans; I-kappa B Kinase; Mammary Neoplasms, Animal; Mice; Mice, Knockout; NIH 3T3 Cells; Oxadiazoles; Phosphatidylinositol 3-Kinases; Phosphorylation; Phosphorylcholine; Protein Serine-Threonine Kinases; Protein Structure, Tertiary; Proto-Oncogene Proteins c-akt; Ribonucleosides; Trans-Activators; Transcription Factors

MK-2206 is an orally active, allosteric inhibitor of AKT, a component of the phosphatidylinositol-3 kinase (PI3K) pathway. The PI3K-AKT pathway is a downstream signaling pathway that has recently been found to play an important role in head and neck squamous cell carcinoma (HNSCC). The objective of this study is to examine the role AKT inhibition may play in treatment of HNSCC.. In vivo and in vitro study.. Cell migration after 24-hour treatment with subtherapeutic doses of MK-2206 was assessed using an enzyme-linked immunosorbent assay in four HNSCC cell lines: CAL27, FaDu, SCC-1, and SCC-5. In vitro effect of MK-2206 on cell migration was assessed by making linear scratches in culture plates after cell lines were grown to confluency. Images were taken at 8, 16, and 24 hours. In vivo analysis was performed on nude mice with human SCC1-orthotopic tongue tumors. After tumors were allowed to grow for 7 days, mice were treated with oral dosing of 120 mg/kg of MK-2206 every other day for 2 weeks. Tumor size was assessed after each treatment using a pair of digital calipers. At the end of the treatment period, mice were sacrificed and cervical lymph nodes were assessed for metastasis using fluorescent imaging of tumor cell markers.. Subtherapeutic doses of MK-2206 were sufficient to significantly reduce cell migration in FaDu, SCC-1, and SCC-5 cell lines (P < .001) but not in Cal27 (P = .09). In vitro scratch test results in SCC-1 cells yielded significant reduction in cell movement at 8, 16, and 14 hours (P < .001). In vivo orthotopic model yielded significant reduction in primary tumor size (P = .04) and reduction in positive cervical lymph nodes (P = .01) between treatment and control mice. In addition we found 100% survival of MK-2206 treated mice after 2 weeks of treatment compared with 70% survival in our control group (P = .03).. Treatment with MK-2206 is sufficient to inhibit HNSCC chemotaxis and migration in vitro. In an orthotopic model, treatment with MK-2206 reduces primary tumor size and cervical metastasis while improving survival. MK-2206 currently is being used in phase II clinical trials for combination treatment of metastatic solid tumors and may be useful for treating HNSCC as well.

Topics: Administration, Oral; Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Apoptosis; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; Cell Transformation, Neoplastic; Cetuximab; Female; Heterocyclic Compounds, 3-Ring; Humans; In Vitro Techniques; Lymphatic Metastasis; Mice; Mice, Nude; Neoplasm Transplantation; Otorhinolaryngologic Neoplasms; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Tongue Neoplasms; Tumor Burden