mk-2206 and Carcinoma--Non-Small-Cell-Lung

Non-small-cell lung cancer (NSCLC) with Kirsten RAt Sarcoma 2 viral oncogene homolog (KRAS) mutation has become a clinical challenge in cancer treatment as KRAS-mutant tumors are often resistant to conventional anti-tumor therapies. Activated CDC42-associated kinase 1 (ACK1), an activator of protein kinase B (AKT), is a promising target for KRAS-mutant tumor therapy, but the downstream ACK1 signaling remains poorly understood. The aim of this study was to evaluate the effectiveness of combined ACK1/AKT inhibition on the proliferation, migration, invasion, and apoptosis of KRAS-mutant NSCLC cell lines (NCI-H23, NCI-H358, and A549). The cells were treated with an inhibitor of either ACK1 (dasatinib or sunitinib) or AKT (MK-2206 or GDC-0068), and the optimal concentrations of the two yielding synergistic tumor-killing effects were determined by applying the Chou-Talalay equation for drug combinations. We showed that combined administration of ACK1 and AKT inhibitors at the optimal concentrations effectively suppressed NSCLC cell viability and promoted apoptosis while inducing cell cycle arrest at the G2 phase. Moreover, NSCLC cell migration and invasion were inhibited by combined ACK1/AKT inhibition. These phenomena were associated with the reduced phosphorylation levels of ACK1 and AKT (at Ser473 and Thr308), as well as alterations in caspase-dependent apoptotic signaling. Collectively, our results demonstrate the promising therapeutic potential of combined ACK1/AKT inhibition as a strategy against KRAS-mutant NSCLC. Our findings provide the basis for the clinical translation of biological targeted drugs (ACK1 and AKT inhibitors) and their rational combination in cancer treatment.

Topics: Carcinoma, Non-Small-Cell Lung; Cell Culture Techniques; Cell Line, Tumor; Cell Movement; Cell Proliferation; Dasatinib; Heterocyclic Compounds, 3-Ring; Humans; Lung Neoplasms; Piperazines; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins p21(ras); Pyrimidines; Sunitinib

Metformin is an antidiabetic drug that has been reported to have antitumor activity in many cancer types. This study investigated the molecular mechanisms underlying the antitumor effect of metformin.. We investigated the molecular mechanism of the antitumor effect of metformin alone and in combination with AKT serine/threonine kinase (AKT) inhibition via cell viability and western blot analyses.. Notably, metformin increased the phosphorylation of AKT at serine 473 using protein array screening. Metformin-induced AKT activation was markedly suppressed by siRNA targeting activating transcription factor 4 (ATF4) but not AMP-activated protein kinase α. These results indicate that AKT activation by metformin was induced in an ATF4-dependent and AMPKα-independent manner. Treatment using metformin combined with MK-2206, an AKT inhibitor, or a siRNA for AKT markedly reduced the viability of cells compared with those cells treated with these agents alone. In addition, MK-2206 increased cell sensitivity to the combination of metformin with ionizing radiation or cisplatin.. Inhibition of AKT can enhance the antitumor effect of metformin and would be a promising strategy to sensitize non-small-cell lung cancer to a combination of metformin with radiation or cisplatin.

Topics: AMP-Activated Protein Kinases; Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Cisplatin; Drug Resistance, Neoplasm; Heterocyclic Compounds, 3-Ring; Humans; Hypoglycemic Agents; Lung Neoplasms; Metformin; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt

AKT is a serine-threonine kinase implicated in tumorigenesis as a central regulator of cellular growth, proliferation, survival, and metabolism. Activated AKT is commonly overexpressed in non-small cell lung cancer (NSCLC) and accordingly AKT inhibitors are under clinical investigation for NSCLC treatment. Thus far, the AKT inhibitors being evaluated broadly target all three (1-3) AKT isoforms but recent evidence suggests opposing roles in lung tumorigenesis where loss of Akt1 inhibits while the loss of Akt2 enhances lung tumor development. Based on these findings, we hypothesized that selective inhibition of AKT-1 would be a more effective therapeutic strategy than pan-AKT inhibition for NSCLC treatment. Using six NSCLC cell lines, we found that the AKT-1 inhibitor, A-674563, was significantly more effective at reducing NSCLC cell survival relative to the pan-AKT inhibitor MK-2206. Comparison of the downstream effects of the inhibitors suggests that altered cell cycle progression and off-target CDK2 inhibition are likely vital to the improved efficacy of A-674563 over MK-2206.

Topics: A549 Cells; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin-Dependent Kinase 2; Down-Regulation; Gene Expression Regulation, Neoplastic; Heterocyclic Compounds, 3-Ring; Humans; Indazoles; Lung Neoplasms; Pyridines

Accumulating evidence supports a role of the PI3K-AKT pathway in the regulation of cell motility, invasion and metastasis. AKT activation is known to promote metastasis, however under certain circumstances, it also shows an inhibitory activity on metastatic processes, and the cause of such conflicting results is largely unclear. Here we found that AKT1 is an important regulator of metastasis and down-regulation of its activity is associated with increased metastatic potential of A549 cells. Inhibition of AKT1 enhanced migration and invasion in KRAS- or EGFR-mutant non-small cell lung cancer (NSCLC) cells. The allosteric AKT inhibitor MK-2206 promoted metastasis of KRAS-mutated A549 cells in vivo. We next identified that the phosphorylation of Myristoylated alanine-rich C-kinase substrate (MARCKS) and LAMC2 protein level were increased with AKT1 inhibition, and MARCKS or LAMC2 knockdown abrogated migration and invasion induced by AKT1 inhibition. This study unravels an anti-metastatic role of AKT1 in the NSCLC cells with KRAS or EGFR mutations, and establishes an AKT1-MARCKS-LAMC2 feedback loop in this regulation.

Topics: A549 Cells; Carcinoma, Non-Small-Cell Lung; Cell Movement; Enzyme Inhibitors; ErbB Receptors; Heterocyclic Compounds, 3-Ring; Humans; Laminin; Models, Biological; Mutation; Myristoylated Alanine-Rich C Kinase Substrate; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins p21(ras); Signal Transduction

Lung cancer is one of the leading causes for cancer mortality. The poor therapeutic outcome of non-small cell lung carcinoma (NSCLC) is mainly due to late diagnosis and chemoresistance. In this study, we investigated the role of Musashi1 (MSI1) in NSCLC malignancy and chemoresistance.. Colony formation, MTT, glucose uptake and lactate production assays were employed to study lung cancer cell malignancy and chemoresistance. RT-PCR and Western blotting were performed to detect mRNA and protein expressions of genes. We used immunohistochemistry and Pearson correlation analysis to study the relationship of gene expression.. We demonstrated that MSI1 was able to promote the proliferation and glucose metabolism of NSCLC cells, and to mediate the sensitivity to chemotherapy drugs in NSCLC cells. Importantly, we found that MSI1 could regulate the activity of Akt signaling. The regulation of NSCLC proliferation, glucose metabolism and chemoresistance by MSI1 was dependent on the modulation of the activity of the Akt signaling pathway. We also found that MSI1 was a target of miR-181a-5p, a microRNA involved in the regulation of cancer development. The expression levels of MSI1 and miR-181a-5p were negatively correlated in NSCLC.. MSI1 promotes non-small cell lung carcinoma malignancy and chemoresistance via activating the Akt signaling pathway, which provides a new strategy for the therapy of NSCLC.

Topics: 3' Untranslated Regions; A549 Cells; Base Sequence; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Cisplatin; Drug Resistance, Neoplasm; Glucose; Heterocyclic Compounds, 3-Ring; Humans; Lung Neoplasms; MicroRNAs; Nerve Tissue Proteins; Oxygen Consumption; Proto-Oncogene Proteins c-akt; RNA Interference; RNA-Binding Proteins; RNA, Small Interfering; Sequence Alignment; Signal Transduction

Acquired resistance to erlotinib in patients with EGFR-mutant non-small cell lung cancer can result from aberrant activation of alternative receptor tyrosine kinases, such as the HGF-driven c-MET receptor. We sought to determine whether inhibition of AKT signaling could augment erlotinib activity and abrogate HGF-mediated resistance.. The effects of MK-2206, a selective AKT inhibitor, were evaluated in combination with erlotinib on a large panel of 13 lung cancer cell lines containing different EGFR or KRAS abnormalities. The activity of the combination was assessed using proliferation assays, flow cytometry and immunoblotting. The MEK inhibitor PD0325901 was used to determine the role of the MAP kinase pathway in erlotinib resistance.. The combination of MK-2206 and erlotinib resulted in synergistic growth inhibition independent of EGFR mutation status. In cell lines where HGF blocked the anti-proliferative and cytotoxic effects of erlotinib, MK-2206 could restore cell cycle arrest, but MEK inhibition was required for erlotinib-dependent apoptosis. Both AKT and MEK inhibition contributed to cell death independent of erlotinib in the T790M-containing H1975 and the EGFR-WT cell lines tested.. These findings illustrate the potential advantages and challenges of combined signal transduction inhibition as a generalized strategy to circumvent acquired erlotinib resistance.

Topics: Apoptosis; Benzamides; Carcinoma, Non-Small-Cell Lung; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cell Survival; Diphenylamine; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Drug Synergism; ErbB Receptors; Erlotinib Hydrochloride; Flow Cytometry; Hepatocyte Growth Factor; Heterocyclic Compounds, 3-Ring; Humans; Immunoblotting; Lung Neoplasms; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mutation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Quinazolines; ras Proteins

Preclinical modeling in non-small cell lung cancer (NSCLC) showed that stimulation with hepatocyte growth factor (HGF), the ligand for MET, could reverse the cytostatic and cytotoxic effects of the EGFR inhibitor erlotinib in erlotinib-sensitive cell lines. Inhibitors of AKT signaling mitigated this HGF-mediated resistance, partially restoring erlotinib activity. We conducted a phase II trial of erlotinib plus MK-2206, a highly selective inhibitor of AKT, in NSCLC patients.. Eligible patients must have progressed following prior benefit from erlotinib, defined as response or stable disease > 12 weeks. Treatment consisted of erlotinib 150 mg orally every day + MK-2206 45 mg orally every alternate day on a 28-day cycle. Primary endpoints were RECIST response rate > 30% (stratum 1: EGFR mutant) and disease control rate (DCR) > 20% at 12 weeks (stratum 2: EGFR wild-type).. Eighty patients were enrolled, 45 and 35 in stratum 1 and 2, respectively. Most common attributable adverse events (all grade 3) were rash, diarrhea, fatigue, and mucositis. Response and DCR were, respectively, 9% and 40% in stratum 1; 3% and 47% in stratum 2. Median progression-free survival was 4.4 months in stratum 1 and 4.6 months in stratum 2.. Combination MK-2206 and erlotinib met its primary endpoint in erlotinib-pretreated patients with EGFR wild-type NSCLC. Although activity was seen in EGFR-mutated NSCLC, this did not exceed a priori estimates. AKT pathway inhibition merits further clinical evaluation in EGFR wild-type NSCLC.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Non-Small-Cell Lung; ErbB Receptors; Erlotinib Hydrochloride; Female; Heterocyclic Compounds, 3-Ring; Humans; Kaplan-Meier Estimate; Lung Neoplasms; Male; Middle Aged; Neoplasm Staging; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Retreatment; Treatment Outcome

Vascular endothelial growth factor (VEGF) is a key stimulator of physiological and pathological angiogenesis. VEGF signals primarily through VEGF receptor 2 (VEGFR2), a receptor tyrosine kinase whose expression is found predominantly on endothelial cells. The purpose of this study was to determine the role of VEGFR2 expression in NSCLC cells. NSCLC cells and tissue sections were stained for VEGFR2 expression by immunohistochemistry (IHC). Immunoblotting and ELISA were used to determine the activation and inhibition of VEGFR2 and its downstream signalling pathways. Five-day proliferation assays were carried out in the presence or absence of VEGF. IHC analysis of NSCLC demonstrated tumour cell VEGFR2 expression in 20% of samples. Immunoblot analysis showed expression of VEGFR2 protein in 3/8 NSCLC cell lines that correlated with VEGFR2 mRNA expression levels. VEGF-dependent VEGFR2 activation was apparent in NSCLC cells, and was associated with increased tumor cell proliferation. Cediranib treatment or siRNA against VEGFR2 inhibited VEGF-dependent increases in cell proliferation. Inhibition of VEGFR2 tyrosine kinase activity using cediranib was more effective than inhibition of AKT (MK2206) or MEK (AZD6244) for overcoming VEGFR2-driven cell proliferation. VEGF treatment did not affect cell survival following treatment with radiation, cisplatin, docetaxel or gemcitabine. Our data suggest that a subset of NSCLC tumour cells express functional VEGFR2 which can act to promote VEGF-dependent tumour cell growth. In this tumour subset, therapies targeting VEGFR2 signalling, such as cediranib, have the potential to inhibit both tumour cell proliferation and angiogenesis.

Topics: Benzimidazoles; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Movement; Cell Proliferation; Gene Expression Regulation, Neoplastic; Heterocyclic Compounds, 3-Ring; Humans; Lung Neoplasms; Protein Kinase Inhibitors; Quinazolines; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-2

Although non-small cell lung cancer (NSCLC) patients with EGFR mutation positive (EGFR M+) tumors initially respond well to EGFR tyrosine kinase inhibitor (TKI) monotherapy, the responses are usually incomplete. In this study we show that AKT inhibition, most importantly AKT2 inhibition, synergises with EGFR TKI inhibition to increase cell killing in EGFR M+ NSCLC cells. However, our data also suggest that the synergistic pro-apoptotic effects may be stunted due to a prosurvival autophagy response induced by AKT inhibition. Consequently, inhibiting autophagy with chloroquine significantly enhanced tumor cell death induced by gefitinib and AKT inhibitors in EGFR M+ cells in vitro, and produced greater tumor shrinkage in EGFR M+ xenografts in vivo. Together, our findings suggest that adding chloroquine to EGFR and AKT inhibition has the potential to improve tumor responses in EGFR M+ NSCLC, and that selective targeting of AKT2 may provide a new treatment option in NSCLC.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Autophagy; Blotting, Western; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Cell Survival; Chloroquine; Drug Synergism; ErbB Receptors; Female; Gefitinib; Heterocyclic Compounds, 3-Ring; Humans; Lung Neoplasms; Mice, Inbred BALB C; Mice, Nude; Mutation; Proto-Oncogene Proteins c-akt; Quinazolines; RNA Interference; Xenograft Model Antitumor Assays

The epidermal growth factor receptor (EGFR) is a central regulator of tumor progression in human cancers. Cetuximab is an anti-EGFR monoclonal antibody that has been approved for use in oncology. Despite clinical success the majority of patients do not respond to cetuximab and those who initially respond frequently acquire resistance. To understand how tumor cells acquire resistance to cetuximab we developed a model of resistance using the non-small cell lung cancer line NCI-H226. We found that cetuximab-resistant (Ctx (R) ) clones manifested strong activation of EGFR, PI3K/AKT and MAPK. To investigate the role of AKT signaling in cetuximab resistance we analyzed the activation of the AKT pathway effector molecules using a human AKT phospho-antibody array. Strong activation was observed in Ctx (R) clones for several key AKT substrates including c-jun, GSK3β, eIF4E, rpS6, IKKα, IRS-1 and Raf1. Inhibition of AKT signaling by siAKT1/2 or by the allosteric AKT inhibitor MK-2206 resulted in robust inhibition of cell proliferation in all Ctx (R) clones. Moreover, the combinational treatment of cetuximab and MK-2206 resulted in further decreases in proliferation than either drug alone. This combinatorial treatment resulted in decreased activity of both AKT and MAPK thus highlighting the importance of simultaneous pathway inhibition to maximally affect the growth of Ctx (R) cells. Collectively, our findings demonstrate that AKT activation is an important pathway in acquired resistance to cetuximab and suggests that combinatorial therapy directed at both the AKT and EGFR/MAPK pathways may be beneficial in this setting.

Topics: Allosteric Regulation; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Cetuximab; Drug Evaluation, Preclinical; Drug Resistance, Neoplasm; Heterocyclic Compounds, 3-Ring; Humans; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Phosphorylation; Protein Processing, Post-Translational; Proto-Oncogene Proteins c-akt

Intrinsic resistance to agents targeting phosphoinositide 3-kinase (PI3K)/AKT pathway is one of the major challenges in cancer treatment with such agents. The objective of this study is to identify the genes or pathways that can be targeted to overcome the resistance of non-small cell lung carcinoma (NSCLC) to the AKT inhibitor MK2206, which is currently being evaluated in phase I and II clinical trials. Using a genome-wide siRNA library screening and biologic characterization, we identified that inhibition of thioredoxin reductase-1 (TXNRD1), one of the key antioxidant enzymes, with siRNAs or its inhibitor, auranofin, sensitized NSCLC cells to MK2206 treatment in vitro and in vivo. We found that simultaneous inhibition of TXNRD1 and AKT pathways induced robust reactive oxygen species production, which was involved in c-jun-NH2-kinase (JNK; MAPK8) activation and cell apoptosis. Furthermore, we found that the synthetic lethality interaction between the TXNRD1 and AKT pathways occurred through the KEAP1/NRF2 cellular antioxidant pathway. Finally, we found that synthetic lethality induced by TXNRD1 and AKT inhibitors relied on wild-type KEAP1 function. Our study indicates that targeting the interaction between AKT and TXNRD1 antioxidant pathways with MK2206 and auranofin, a U.S. Food and Drug Administration-approved drug, is a rational strategy to treat lung cancer and that KEAP1 mutation status may offer a predicative biomarker for such combination approaches.

Topics: Animals; Antirheumatic Agents; Apoptosis; Auranofin; Blotting, Western; Carcinoma, Non-Small-Cell Lung; Cell Proliferation; Fluorescent Antibody Technique; Genes, Lethal; Glutathione; Heterocyclic Compounds, 3-Ring; Humans; Immunoenzyme Techniques; Intracellular Signaling Peptides and Proteins; Kelch-Like ECH-Associated Protein 1; Luciferases; Lung Neoplasms; Mice; Microscopy, Fluorescence; Neoplasm Staging; NF-E2-Related Factor 2; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Thioredoxin Reductase 1; Tumor Cells, Cultured

TUSC2-defective gene expression is detected in the majority of lung cancers and is associated with worse overall survival. We analyzed the effects of TUSC2 re-expression on tumor cell sensitivity to the AKT inhibitor, MK2206, and explored their mutual signaling connections, in vitro and in vivo. TUSC2 transient expression in three LKB1-defective non-small cell lung cancer (NSCLC) cell lines combined with MK2206 treatment resulted in increased repression of cell viability and colony formation, and increased apoptotic activity. In contrast, TUSC2 did not affect the response to MK2206 treatment for two LKB1-wild type NSCLC cell lines. In vivo, TUSC2 systemic delivery, by nanoparticle gene transfer, combined with MK2206 treatment markedly inhibited growth of tumors in a human LKB1-defective H322 lung cancer xenograft mouse model. Biochemical analysis showed that TUSC2 transient expression in LKB1-defective NSCLC cells significantly stimulated AMP-activated protein kinase (AMPK) phosphorylation and enzymatic activity. More importantly, AMPK gene knockdown abrogated TUSC2-MK2206 cooperation, as evidenced by reduced sensitivity to the combined treatment. Together, TUSC2 re-expression and MK2206 treatment was more effective in inhibiting the phosphorylation and kinase activities of AKT and mTOR proteins than either single agent alone. In conclusion, these findings support the hypothesis that TUSC2 expression status is a biological variable that potentiates MK2206 sensitivity in LKB1-defective NSCLC cells, and identifies the AMPK/AKT/mTOR signaling axis as an important regulator of this activity.

Topics: AMP-Activated Protein Kinase Kinases; AMP-Activated Protein Kinases; Animals; Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Survival; Disease Models, Animal; Drug Resistance, Neoplasm; Gene Expression; Heterocyclic Compounds, 3-Ring; Humans; Lung Neoplasms; Mice; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; TOR Serine-Threonine Kinases; Tumor Burden; Tumor Stem Cell Assay; Tumor Suppressor Proteins; Xenograft Model Antitumor Assays

The novel Akt inhibitor, API-1, induces apoptosis through undefined mechanisms. The current study focuses on revealing the mechanisms by which API-1 induces apoptosis.. API-1 rapidly and potently reduced the levels of Mcl-1 primarily in API-1-senstive lung cancer cell lines. Ectopic expression of Mcl-1 protected cells from induction of apoptosis by API-1. API-1 treatment decreased the half-life of Mcl-1, whereas inhibition of the proteasome with MG132 rescued Mcl-1 reduction induced by API-1. API-1 decreased Mcl-1 levels accompanied with a rapid increase in Mcl-1 phosphorylation (S159/T163). Moreover, inhibition of GSK3 inhibited Mcl-1 phosphorylation and reduction induced by API-1 and antagonized the effect of API-1 on induction of apoptosis. Knockdown of either FBXW7 or β-TrCP alone, both of which are E3 ubiquitin ligases involved in Mcl-1 degradation, only partially rescued Mcl-1 reduction induced by API-1. However, double knockdown of both E3 ubiquitin ligases enhanced the rescue of API-1-induced Mcl-1 reduction.. API-1 induces GSK3-dependent, β-TrCP- and FBXW7-mediated Mcl-1 degradation, resulting in induction of apoptosis.

Topics: Apoptosis; beta-Transducin Repeat-Containing Proteins; Carcinoma, Non-Small-Cell Lung; Cell Cycle Proteins; Cell Line, Tumor; F-Box Proteins; F-Box-WD Repeat-Containing Protein 7; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Glycogen Synthase Kinase 3; Heterocyclic Compounds, 3-Ring; Humans; Leupeptins; Myeloid Cell Leukemia Sequence 1 Protein; Nucleosides; Phosphorylation; Proteasome Endopeptidase Complex; Proto-Oncogene Proteins c-akt; Ubiquitin-Protein Ligases

Increasing evidence has suggested that high expression level of cyclooxygenase-2 (Cox-2) is associated with the malignancies of non-small cell lung cancer (NSCLC), leading to a rationale of applying Cox-2 inhibitors as adjuvant therapy in the treatment of NSCLC. However, the addition of celecoxib, a selective Cox-2 inhibitor, to chemotherapy in clinical trials failed to benefit the survival of NSCLC patients, which urges the investigation to re-evaluate this strategy for NSCLC treatment. In this study, we observed that celecoxib treatment at clinically relevant concentrations induced epithelial-mesenchymal transition (EMT) in NSCLC cells regardless of Cox-2 status, which, however, was not recapitulated using another Cox-2 inhibitor, etodolac. Celecoxib-stimulated EMT in turn promoted cell invasion and rendered cells resistant to chemotherapy. Further mechanistic investigation by disrupting the integrity of signaling pathways using specific inhibitors or RNA interference revealed that celecoxib-induced EMT in NSCLC cells is indispensable of transforming growth factor-β1/Smad signaling. Instead, the activated MEK/ERK/SNAIL1 signaling largely accounted for celecoxib-induced EMT. Taken together, our study reveals the diverse impacts of Cox-2 inhibitors on EMT in NSCLC cells independent of Cox-2 inhibition, where celecoxib treatment leads to metastasis and chemoresistance via EMT induction. These findings reveal the increased risks of cancer metastasis and chemoresistance by applying Cox-2 inhibitors, celecoxib in particular, in clinical trials of NSCLC treatment and urge intensive preclinical assessment before proceeding to clinical application.

Topics: Animals; Antineoplastic Agents; Benzimidazoles; Carcinoma, Non-Small-Cell Lung; Celecoxib; Cell Line, Tumor; Cell Movement; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Dogs; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; Gene Knockdown Techniques; Heterocyclic Compounds, 3-Ring; Humans; Lung Neoplasms; MAP Kinase Kinase 1; MAP Kinase Kinase 2; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Proto-Oncogene Proteins c-akt; Pyrazoles; RNA, Small Interfering; Smad Proteins; Sulfonamides; Transforming Growth Factor beta1

The serine/threonine kinase Akt lies at a critical signaling node downstream of phosphatidylinositol-3-kinase and is important in promoting cell survival and inhibiting apoptosis. An Akt inhibitor may be particularly useful for cancers in which increased Akt signaling is associated with reduced sensitivity to cytotoxic agents or receptor tyrosine kinase inhibitors. We evaluated the effect of a novel allosteric Akt inhibitor, MK-2206, in combination with several anticancer agents. In vitro, MK-2206 synergistically inhibited cell proliferation of human cancer cell lines in combination with molecular targeted agents such as erlotinib (an epidermal growth factor receptor inhibitor) or lapatinib (a dual epidermal growth factor receptor/human epidermal growth factor receptor 2 inhibitor). Complementary inhibition of erlotinib-insensitive Akt phosphorylation by MK-2206 was one mechanism of synergism, and a synergistic effect was found even in erlotinib-insensitive cell lines. MK-2206 also showed synergistic responses in combination with cytotoxic agents such as topoisomerase inhibitors (doxorubicin, camptothecin), antimetabolites (gemcitabine, 5-fluorouracil), anti-microtubule agents (docetaxel), and DNA cross-linkers (carboplatin) in lung NCI-H460 or ovarian A2780 tumor cells. The synergy with docetaxel depended on the treatment sequence; a schedule of MK-2206 dosed before docetaxel was not effective. MK-2206 suppressed the Akt phosphorylation that is induced by carboplatin and gemcitabine. In vivo, MK-2206 in combination with these agents exerted significantly more potent tumor inhibitory activities than each agent in the monotherapy setting. These findings suggest that Akt inhibition may augment the efficacy of existing cancer therapeutics; thus, MK-2206 is a promising agent to treat cancer patients who receive these cytotoxic and/or molecular targeted agents.

Topics: Allosteric Regulation; Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Blotting, Western; Camptothecin; Carboplatin; Carcinoma, Non-Small-Cell Lung; Caspases; Cell Line, Tumor; Cell Proliferation; Drug Synergism; Enzyme Activation; ErbB Receptors; Erlotinib Hydrochloride; Heterocyclic Compounds, 3-Ring; Humans; Lapatinib; Lung Neoplasms; Mice; Molecular Structure; Neoplasms; Proto-Oncogene Proteins c-akt; Quinazolines; Xenograft Model Antitumor Assays

AZD6244 and MK2206 are targeted small-molecule drugs that inhibit MEK and AKT respectively. The efficacy of this combination in lung cancer is unknown. Our previous work showed the importance of activated AKT in mediating resistance of non-small cell lung cancer (NSCLC) to AZD6244. Thus we hypothesized that dual inhibition of both downstream MEK and AKT pathways would induce synergistic antitumor activity. In this study, we evaluated the efficacy of AZD6244 and MK2206 individually on a large panel of lung cancer cell lines. Then, we treated 28 human lung cancer cell lines with a combination of AZD6244 and MK2206 at clinically applicable drug molar ratios. The AZD6244-MK2206 combination therapy resulted in a synergistic effect on inhibition of lung cancer cell growth compared to the results of single drug treatment alone. MK2206 enhanced AZD6244-induced Bim overexpression and apoptosis in A549 and H157 cells. When we tested the combination of AZD6244 and MK2206 at ratios of 8∶1, 4∶1, 2∶1, and 1∶8, we found that the synergistic effect of the combination therapy was ratio-dependent. At ratios of 8∶1, 4∶1, and 2∶1, the drug combination consistently demonstrated synergy, whereas decreasing the ratio to 1∶8 resulted in a loss of synergy and produced an additive or antagonistic effect in most cell lines. Furthermore, the AZD6244-MK2206 combination therapy showed synergy in the suppression of A549 and H157 xenograft tumor growth and increased mean animal survival time. The AZD6244-MK2206 combination therapy resulted in effective inhibition of both p-ERK and p-AKT expression in tumor tissue. In addition, a significant increase of apoptosis was detected in tumor tissue from mice treated with AZD6244-MK2206 compared with that from the single agent treated mice. Our study suggests that the combination of AZD6244 and MK2206 has a significant synergistic effect on tumor growth in vitro and in vivo and leads to increased survival rates in mice bearing highly aggressive human lung tumors.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Benzimidazoles; Carcinoma, Non-Small-Cell Lung; Cell Cycle; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Drug Synergism; Female; Heterocyclic Compounds, 3-Ring; Humans; Lung Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Mitogen-Activated Protein Kinase Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction; Survival Analysis; Tumor Burden; Xenograft Model Antitumor Assays