Resistance to Mitogen-Activated Protein Kinase Kinase (MEK) Inhibitors Correlates with Up-Regulation of the MEK/Extracellular Signal-Regulated Kinase Pathway in Hepatocellular Carcinoma Cells

Yip-Schneider, Michele; Klein, Patrick; Wentz, Sabrina; Zeni, Amer; Menze, Alex; Schmidt, C. Max

doi:10.1124/jpet.108.147306

Cited by 32 publications

(27 citation statements)

References 42 publications

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“…Similar to inhibitors of Akt kinase, inhibitors that occupy the nucleotide-binding pocket of other protein kinases also induce hyperphosphorylation of serine/ threonine residues in activation loops. For example, hyperphosphorylation of the Map2k MEK occurs upon exposure to many MEK inhibitors, including U0126, PD098059, and PD184161, Cl-1040, and AZD6244 (24)(25)(26). While these inhibitors are all allosteric and non-ATP-competitive, they all bind to a hydrophobic pocket adjacent to the ATP binding site (27).…”

Section: Discussionmentioning

confidence: 99%

Resistance of Akt kinases to dephosphorylation through ATP-dependent conformational plasticity

Chan

Zhang

Rodeck

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Phosphorylation of a threonine residue (T308 in Akt1) in the activation loop of Akt kinases is a prerequisite for deregulated Akt activity frequently observed in neoplasia. Akt phosphorylation in vivo is balanced by the opposite activities of kinases and phosphatases. Here we describe that targeting Akt kinase to the cell membrane markedly reduced sensitivity of phosphorylated Akt to dephosphorylation by protein phosphatase 2A. This effect was amplified by occupancy of the ATP binding pocket by either ATP or ATP-competitive inhibitors. Mutational analysis revealed that R273 in Akt1 and the corresponding R274 in Akt2 are essential for shielding T308 in the activation loop against dephosphorylation. Thus, occupancy of the nucleotide binding pocket of Akt kinases enables intramolecular interactions that restrict phosphatase access and sustain Akt phosphorylation. This mechanism provides an explanation for the "paradoxical" Akt hyperphosphorylation induced by ATP-competitive inhibitor, A-443654. The lack of phosphatase resistance further contributes insight into the mechanism by which the human Akt2 R274H missense mutation may cause autosomal-dominant diabetes mellitus. T he serine/threonine Akt protein kinases (Akt1, Akt2, and Akt3) affect multiple cellular functions related to cell growth and survival, differentiation, metabolism, and migration (reviewed in refs. 1, 2). Essential functions of Akt kinases in mammalian physiology are highlighted by retarded growth and increased neonatal mortality in Akt1 knockout mice, severe diabetes in Akt2 knockout mice, and reduced brain size in Akt3 knockout animals (reviewed in ref. 3). Furthermore, an inherited inactivating mutation in Akt2-R274 has been linked to autosomal-dominant severe insulin resistance and diabetes mellitus in humans (4).The activation state of Akt is regulated by controlling phosphorylation of two regulatory residues in its centrally located activation loop (T308 in Akt1) and in the carboxyl-terminal tail (S473 in Akt1) by two separate kinases (PDK1 and PDK2) (reviewed in refs. 1, 2). Phosphorylation of these regulatory sites is enabled by conformational changes induced by docking of the Akt pleckstrin homology (PH) domain to the membrane lipid products, PtdInsð3;4;5ÞP 3 or PtdInsð3;4ÞP 2 . PDK-dependent Akt1 phosphorylation is reversed by the abundantly expressed protein phosphatase 2A (PP2A) which dephosphorylates pT308 and, to a lesser extent, pS473 (5, 6). Specifically, Akt has been identified as the substrate of a complex containing the regulatory B55α or B56β subunits of PP2A in Caenorhabditis elegans, Drosophila melanogaster, and in mammalian cells (7-9). The functional relevance of B56β-containing PP2A to Akt regulation is further underscored by genetic studies in C. elegans demonstrating modulation of insulin/insulin-like growth factor effects on longevity, fat metabolism, and stress resistance by this phosphatase (9).Hyperactivation and phosphorylation of Akt kinases is commonly observed in diverse tumor types and has motivated the developmen...

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Section: Discussionmentioning

confidence: 99%

Resistance of Akt kinases to dephosphorylation through ATP-dependent conformational plasticity

Chan

Zhang

Rodeck

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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“…Schmidt and coworkers showed that this upregulation of the MEK/ERK pathway was associated with drug resistance to MEK inhibitors in hepatocarcinoma cells. 45 This could suggest a negative feedback exerted by activated ERK1/2 on MEK1/2 phosphorylation, 46 that could be abolished by the MEK inhibitor or by ERK1 and ERK2 silencing. As observed by immunoblotting quantifications, ERK2 could play a major role in this process because of its increased expression in HuH7 cells.…”

Section: Cancer Cell Biologymentioning

confidence: 99%

RNAi‐mediated MEK1 knock‐down prevents ERK1/2 activation and abolishes human hepatocarcinoma growth in vitro and in vivo

Gailhouste

Ezan

Bessard

et al. 2009

Intl Journal of Cancer

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The mitogen-activated protein kinases MEK/ERK pathway regulates fundamental processes in malignant cells and represents an attractive target in the development of new cancer treatments especially for human hepatocarcinoma highly resistant to chemotherapy. Although gene extinction experiments have suggested distinct roles for these proteins, the MEK/ERK cascade remains widely considered as exhibiting an overlap of functions. To investigate the functionality of each kinase in tumorigenesis, we have generated stably knock-down clones for MEK1/2 and ERK1/2 isoforms in the human hepatocellular carcinoma line HuH7. Our results have shown that RNAi strategy allows a specific disruption of the targeted kinases and argued for the critical function of MEK1 in liver tumor growth. Transient and stable extinction experiments demonstrated that MEK1 isoform acts as a major element in the signal transduction by phosphorylating ERK1 and ERK2 after growth factors stimulation, whereas oncogenic level of ERK1/2 phosphorylation appears to be MEK1 and MEK2 dependent in basal condition. In addition, silencing of MEK1 or ERK2 abolished cell proliferation and DNA replication in vitro as well as tumor growth in vivo after injection in rodent. In contrast, targeting MEK2 or ERK1 had no effect on hepatocarcinoma progression. These results strongly corroborate the relevance of targeting the MEK cascade as attested by pharmacologic drugs and support the potential application of RNAi in future development of more effective cancer therapies. Our study emphasizes the importance of the MEK/ERK pathway in human hepatocarcinoma cell growth and argues for a crucial role of MEK1 and ERK2 in this regulation.The canonical signaling pathway known as Ras-dependent mitogen-activated protein kinase (MAPK) is involved in several biological processes such as proliferation, survival, motility and cellular differentiation.1,2 This cascade is under the control of numerous factors leading to the successive activation of Raf-family protein, MEK1/2 (mitogen-activated protein/ERK kinase) and ERK1/2 (extracellular signal-regulated kinase) ensuring signaling continuity by phosphorylation from the membrane receptors to downstream cytoplasmic and nuclear substrates. 3,4 The MEK/ERK pathway acts as an essential element in cellular responses induced by mitogen factors and contains several protooncogenes constitutively activated in more than 35% of a large variety of human cancers. This includes hepatocellular carcinoma (HCC), which is one of the most chemoresistant tumors.5-7

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“…For example, inhibitors of Map2k (MEK) have been found to induce activation loop hyperphosphorylation. [26][27][28] At first approximation, the effects of MEK inhibitors seem to be distinct from inhibitor-induced hyperphosphorylation in AGC kinases, because these inhibitors (U0126, PD098059, PD184161, Cl -1040 and AZD6244) are considered allosteric and non-ATP-competitive. [26][27][28] As in the case of Akt, drug-induced hyperphosphorylation of MEK kinases is thought to represent a homeostatic feedback mechanism to compensate for signal loss.…”

mentioning

confidence: 99%

“…[26][27][28] At first approximation, the effects of MEK inhibitors seem to be distinct from inhibitor-induced hyperphosphorylation in AGC kinases, because these inhibitors (U0126, PD098059, PD184161, Cl -1040 and AZD6244) are considered allosteric and non-ATP-competitive. [26][27][28] As in the case of Akt, drug-induced hyperphosphorylation of MEK kinases is thought to represent a homeostatic feedback mechanism to compensate for signal loss. 29 However, the MEK inhibitors listed, above all, bind to a hydrophobic pocket adjacent to the ATP binding site 30 that is similar to the space occupied by γ-phosphate/Mg 2+ in Akt and critical for ATP to support the phosphatase-resistant conformation.…”

mentioning

confidence: 99%

Autoregulation of kinase dephosphorylation by ATP binding in AGC protein kinases

et al. 2012

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Resistance to Mitogen-Activated Protein Kinase Kinase (MEK) Inhibitors Correlates with Up-Regulation of the MEK/Extracellular Signal-Regulated Kinase Pathway in Hepatocellular Carcinoma Cells

Cited by 32 publications

References 42 publications

Resistance of Akt kinases to dephosphorylation through ATP-dependent conformational plasticity

Resistance of Akt kinases to dephosphorylation through ATP-dependent conformational plasticity

RNAi‐mediated MEK1 knock‐down prevents ERK1/2 activation and abolishes human hepatocarcinoma growth in vitro and in vivo

Autoregulation of kinase dephosphorylation by ATP binding in AGC protein kinases

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