u-0126 and Mesothelioma

Mesothelioma is a notoriously chemotherapy-resistant neoplasm, as is evident in the dismal overall survival for patients with those of asbestos-associated disease. We previously demonstrated co-activation of multiple receptor tyrosine kinases (RTKs), including epidermal growth factor receptor (EGFR), MET, and AXL in mesothelioma cell lines, suggesting that these kinases could serve as novel therapeutic targets. Although clinical trials have not shown activity for EGFR inhibitors in mesothelioma, concurrent inhibition of various activated RTKs has pro-apoptotic and anti-proliferative effects in mesothelioma cell lines. Thus, we hypothesised that a coordinated network of multi-RTK activation contributes to mesothelioma tumorigenesis.. Activation of PI3K/AKT/mTOR, Raf/MAPK, and co-activation of RTKs were evaluated in mesotheliomas. Effects of RTK and downstream inhibitors/shRNAs were assessed by measuring mesothelioma cell viability/growth, apoptosis, activation of signalling intermediates, expression of cell-cycle checkpoints, and cell-cycle alterations.. We demonstrate activation of the PI3K/AKT/p70S6K and RAF/MEK/MAPK pathways in mesothelioma, but not in non-neoplastic mesothelial cells. The AKT activation, but not MAPK activation, was dependent on coordinated activation of RTKs EGFR, MET, and AXL. In addition, PI3K/AKT/mTOR pathway inhibition recapitulated the anti-proliferative effects of concurrent inhibition of EGFR, MET, and AXL. Dual targeting of PI3K/mTOR by BEZ235 or a combination of RAD001 and AKT knockdown had a greater effect on mesothelioma proliferation and viability than inhibition of individual activated RTKs or downstream signalling intermediates. Inhibition of PI3K/AKT was also associated with MDM2-p53 cell-cycle regulation.. These findings show that PI3K/AKT/mTOR is a crucial survival pathway downstream of multiple activated RTKs in mesothelioma, underscoring that PI3K/mTOR is a compelling target for therapeutic intervention.

Topics: Antineoplastic Agents; Butadienes; Cell Cycle; Cell Line, Tumor; Chromones; Drug Screening Assays, Antitumor; Enzyme Activation; Everolimus; Humans; Imidazoles; Indazoles; MAP Kinase Signaling System; Mesothelioma; Molecular Targeted Therapy; Morpholines; Neoplasm Proteins; Nitriles; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase Inhibitors; Quinolines; raf Kinases; Receptor Protein-Tyrosine Kinases; RNA Interference; RNA, Small Interfering; Signal Transduction; Sirolimus; Sulfonamides; TOR Serine-Threonine Kinases

Malignant pleural mesothelioma (MPM) is an aggressive malignancy for which there is no approved targeted therapy. We examined the therapeutic efficacy of the mitogen-activated protein kinase kinase (MEK) and phosphatidylinositol 3-kinase (PI3K) inhibitors against human MPM cell lines both in vitro and orthotopically inoculated into severe combined immunodeficient (SCID) mice. In addition, the molecular mechanisms of these agents were confirmed in vitro and in vivo. The MEK or the PI3K inhibitor suppressed MPM cell growth in vitro in a dose-dependent manner via induction of G1 cell cycle arrest and apoptosis. In addition, combined use of the MEK and PI3K inhibitors showed an additive or synergistic inhibitory effect on MPM cell growth compared to treatment with either individual drug. Treatment with MEK or PI3K inhibitor suppressed the production of thoracic tumors and pleural effusion and prolonged the survival time of EHMES-10 cell-bearing SCID mice. The combination therapy more effectively prolonged the survival time compared to treatment with either individual drug. Immunohistochemical and western blot analysis of thoracic tumors suggested that these agents induced cell cycle arrest, apoptosis and inhibition of tumor angiogenesis. Our results suggest that a combination of MEK and PI3K inhibitors is a promising therapeutic strategy for MPM.

Topics: Animals; Antineoplastic Agents; Apoptosis; Butadienes; Cell Proliferation; Chromones; Drug Synergism; G1 Phase Cell Cycle Checkpoints; Humans; Male; MAP Kinase Kinase Kinases; MAP Kinase Signaling System; Mesothelioma; Mice; Mice, SCID; Morpholines; Nitriles; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Pleural Neoplasms; Tumor Burden; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Members of the extracellular signal-regulated kinase (ERK) family may have distinct roles in the development of cell injury and repair, differentiation and carcinogenesis. Here, we show, using a synthetic small-molecule MEK1/2 inhibitor (U0126) and RNA silencing of ERK1 and 2, comparatively, that ERK2 is critical to transformation and homeostasis of human epithelioid malignant mesotheliomas (MMs), asbestos-induced tumors with a poor prognosis. Although MM cell (HMESO) lines stably transfected with shERK1 or shERK2 both exhibited significant decreases in cell proliferation in vitro, injection of shERK2 cells, and not shERK1 cells, into immunocompromised severe combined immunodeficiency (SCID) mice showed significant attenuated tumor growth in comparison to shControl (shCon) cells. Inhibition of migration, invasion and colony formation occurred in shERK2 MM cells in vitro, suggesting multiple roles of ERK2 in neoplasia. Microarray and quantitative real-time PCR analyses revealed gene expression that was significantly increased (CASP1, TRAF1 and FAS) or decreased (SEMA3E, RPS6KA2, EGF and BCL2L1) in shERK2-transfected MM cells in contrast to shCon-transfected MM cells. Most striking decreases were observed in mRNA levels of Semaphorin 3 (SEMA3E), a candidate tumor suppressor gene linked to inhibition of angiogenesis. These studies demonstrate a key role of ERK2 in novel gene expression critical to the development of epithelioid MMs. After injection of sarcomatoid human MM (PPMMill) cells into SCID mice, both shERK1 and shERK2 lines showed significant decreased tumor growth, suggesting heterogeneous effects of ERKs in individual MMs.

Topics: Animals; Apoptosis; Biomarkers, Tumor; Blotting, Western; Butadienes; Cell Adhesion; Cell Line, Tumor; Cell Movement; Cell Proliferation; Gene Expression Profiling; Humans; Immunoenzyme Techniques; Mesothelioma; Mice; Mice, SCID; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nitriles; Oligonucleotide Array Sequence Analysis; Pleural Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Xenograft Model Antitumor Assays

Malignant mesotheliomas (MM) have a poor prognosis, largely because of their chemoresistance to anti-cancer drugs such as doxorubicin (Dox). Here we show using human MM lines that Dox activates extracellular signal-regulated kinases (ERK1 and 2), causally linked to increased expression of ABC transporter genes, decreased accumulation of Dox, and enhanced MM growth. Using the MEK1/2 inhibitor, U0126 and stably transfected shERK1 and shERK2 MM cell lines, we show that inhibition of both ERK1 and 2 sensitizes MM cells to Dox.. U0126 significantly modulated endogenous expression of several important drug resistance (BCL2, ABCB1, ABCC3), prosurvival (BCL2), DNA repair (BRCA1, BRCA2), hormone receptor (AR, ESR2, PPARγ) and drug metabolism (CYP3A4) genes newly identified in MM cells. In comparison to shControl lines, MM cell lines stably transfected with shERK1 or shERK2 exhibited significant increases in intracellular accumulation of Dox and decreases in cell viability. Affymetrix microarray analysis on stable shERK1 and shERK2 MM lines showed more than 2-fold inhibition (p ≤ 0.05) of expression of ATP binding cassette genes (ABCG1, ABCA5, ABCA2, MDR/TAP, ABCA1, ABCA8, ABCC2) in comparison to shControl lines. Moreover, injection of human MM lines into SCID mice showed that stable shERK1 or shERK2 lines had significantly slower tumor growth rates in comparison to shControl lines after Dox treatment.. These studies suggest that blocking ERK1 and 2, which play critical roles in multi-drug resistance and survival, may be beneficial in combination with chemotherapeutic drugs in the treatment of MMs and other tumors.

Topics: Animals; Antibiotics, Antineoplastic; Butadienes; Cell Line, Tumor; Doxorubicin; Flow Cytometry; Humans; Mesothelioma; Mice; Mice, SCID; Microscopy, Fluorescence; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Multidrug Resistance-Associated Protein 2; Nitriles; Reverse Transcriptase Polymerase Chain Reaction; Xenograft Model Antitumor Assays

Mutations in Ras family genes are rare in malignant mesothelioma. The role of activation of the Ras signaling pathway in the pathogenesis of mesothelioma is not clear.. We studied the activation status of the Ras pathway and the status of other Ras-associated kinases in a panel of human mesothelioma cell lines. In addition, we tested the effect of inhibition of several kinase pathways on mesothelioma cell proliferation. The potential role of kinase signaling on the regulation of cap-dependent translation was also studied.. In general, Ras-guanosine triphosphate (GTP) was higher in mesothelioma cell lines when compared with a nontransformed mesothelial cell line (LP9). Furthermore, known Ras effectors such as extracellular-regulated kinase 1/2, p38 mitogen-activated protein kinase, and c-Jun N-terminal kinase were found to be active in most of the mesothelioma cell lines tested. Exposure to specific inhibitors of extracellular-regulated kinase 1/2 (U0126) and c-Jun N-terminal kinase (SP600125) significantly decreased the proliferation of H2596 and H2373 cells compared with mock-treated cells. SP600125-mediated c-Jun N-terminal kinase inhibition, but not extracellular-regulated kinase 1/2 inhibition, resulted in a decrease in phosphorylation of 4E-BP1, consequently decreasing cap-dependent activation.. These experiments provide a rationale for targeting Ras and associated signaling pathways in mesothelioma and also suggest cap-dependent translation as one mechanism by which Ras induces proliferation in this disease.

Topics: Adaptor Proteins, Signal Transducing; Anthracenes; Apoptosis; Blotting, Western; Butadienes; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; DNA, Neoplasm; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Genes, ras; Humans; Immunoprecipitation; JNK Mitogen-Activated Protein Kinases; Mesothelioma; Mitogen-Activated Protein Kinase 3; Mutation; Nitriles; p38 Mitogen-Activated Protein Kinases; Phosphoproteins; Phosphorylation; Polymerase Chain Reaction; Repressor Proteins; Signal Transduction

The majority of malignant pleural mesotheliomas (MPMs) aberrantly express the epidermal growth factor receptor (ErbB1). We examined the efficacy of GW572016 (lapatinib), a dual inhibitor of ErbB1/ErbB2 with a panel of 10 MPM cell lines. Two of the 10 MPM cell lines, H2373 and H2452, underwent G1/S cell cycle arrest and growth inhibition with an IC(50) of 1 muM and 0.8 muM, respectively. There was no relationship between the presence or the amount of ErbB1, phospho-ErbB1, phospho-ErbB2, ErbB3, ErbB4, phospho-Akt, and Akt or the ability of lapatinib to inhibit phospho-ErbB1 in these cell lines compared to those that did not respond to lapatinib. The sensitive cell lines had a time-dependent decrease in phospho-Akt and/or ERK1/2, and an increase in p27 and when treated with lapatinib. The combination of lapatinib with U0126, LY294002 or rapamycin caused greater growth inhibition than either drug alone in the sensitive cell lines while this did not occur in the resistant cell lines. Our findings suggest that ErbB1 alone is a therapeutic target for the minority of mesotheliomas and that combining ErbB1 inhibitors with signal transduction inhibitors in mesothelioma will enhance their effectiveness. Furthermore, combinations of growth factor and signal transduction inhibitors may be needed to inhibit the growth of the majority of MPM cell lines, and therefore patients with MPM.

Topics: Antibiotics, Antineoplastic; Butadienes; Cell Cycle; Chromones; Enzyme Inhibitors; ErbB Receptors; Gene Expression Regulation, Neoplastic; Humans; Lapatinib; Mesothelioma; Mitogen-Activated Protein Kinase 3; Morpholines; Nitriles; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Pleural Neoplasms; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Quinazolines; Receptor, ErbB-2; Receptor, ErbB-3; Receptor, ErbB-4; Signal Transduction; Sirolimus; Transforming Growth Factor alpha; Tumor Cells, Cultured