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1.
Am J Cancer Res ; 11(6): 2911-2927, 2021.
Article in English | MEDLINE | ID: mdl-34249435

ABSTRACT

Acute myeloid leukemia (AML) is a highly heterogenous and aggressive disease with a poor prognosis, necessitating further improvements in treatment therapies. Recently, several targeted therapies have become available for specific AML populations. To identify potential new therapeutic targets for AML, we analyzed published genome wide CRISPR-based screens to generate a gene essentiality dataset across a panel of 14 human AML cell lines while eliminating common essential genes through integration analysis with core fitness genes among 324 human cancer cell lines and DepMap databases. The key glutathione metabolic enzyme, glutamate-cysteine ligase catalytic subunit (GCLC), met the selection threshold. Using CRISPR knockout, GCLC was confirmed to be essential for the cell growth, survival, clonogenicity, and leukemogenesis in AML cells but was comparatively dispensable for normal hematopoietic stem and progenitor cells (HSPCs), indicating that GCLC is a potential therapeutic target for AML. In addition, we evaluated the essentiality of GCLC in solid tumors and demonstrated that GCLC represents a synthetic lethal target for ARID1A-deficient ovarian and gastric cancers.

2.
Mol Cancer Ther ; 16(7): 1246-1256, 2017 07.
Article in English | MEDLINE | ID: mdl-28428442

ABSTRACT

Diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma are the most prevalent B-lymphocyte neoplasms in which abnormal activation of the Bruton tyrosine kinase (BTK)-mediated B-cell receptor signaling pathway contributes to pathogenesis. Ibrutinib is an oral covalent BTK inhibitor that has shown some efficacy in both indications. To improve ibrutinib efficacy through combination therapy, we first investigated differential gene expression in parental and ibrutinib-resistant cell lines to better understand the mechanisms of resistance. Ibrutinib-resistant TMD8 cells had higher BCL2 gene expression and increased sensitivity to ABT-199, a BCL-2 inhibitor. Consistently, clinical samples from ABC-DLBCL patients who experienced poorer response to ibrutinib had higher BCL2 gene expression. We further demonstrated synergistic growth suppression by ibrutinib and ABT-199 in multiple ABC-DLBCL, GCB-DLBCL, and follicular lymphoma cell lines. The combination of both drugs also reduced colony formation, increased apoptosis, and inhibited tumor growth in a TMD8 xenograft model. A synergistic combination effect was also found in ibrutinib-resistant cells generated by either genetic mutation or drug treatment. Together, these findings suggest a potential clinical benefit from ibrutinib and ABT-199 combination therapy. Mol Cancer Ther; 16(7); 1246-56. ©2017 AACR.


Subject(s)
Drug Resistance, Neoplasm/genetics , Lymphoma, Follicular/drug therapy , Lymphoma, Large B-Cell, Diffuse/drug therapy , Protein-Tyrosine Kinases/genetics , Proto-Oncogene Proteins c-bcl-2/genetics , Adenine/analogs & derivatives , Agammaglobulinaemia Tyrosine Kinase , Animals , Antineoplastic Combined Chemotherapy Protocols , Apoptosis/drug effects , Bridged Bicyclo Compounds, Heterocyclic/administration & dosage , Gene Expression Regulation, Neoplastic/drug effects , Humans , Lymphoma, Follicular/genetics , Lymphoma, Follicular/pathology , Lymphoma, Large B-Cell, Diffuse/genetics , Lymphoma, Large B-Cell, Diffuse/pathology , Mice , Piperidines , Protein Kinase Inhibitors/administration & dosage , Protein-Tyrosine Kinases/antagonists & inhibitors , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , Pyrazoles/administration & dosage , Pyrimidines/administration & dosage , Signal Transduction/drug effects , Sulfonamides/administration & dosage , Xenograft Model Antitumor Assays
3.
Am J Cancer Res ; 6(11): 2489-2501, 2016.
Article in English | MEDLINE | ID: mdl-27904766

ABSTRACT

Diffuse large B cell lymphoma (DLBCL) is a heterogeneous lymphoma and the most common subtype of non-Hodgkin lymphoma, accounting for roughly 30% of newly diagnosed cases in the United States. DLBCL can be separated into the activated B cell-like (ABC) and germinal center B cell-like (GCB) subtypes, with distinct gene expression profiles, oncogenic aberrations, and clinical outcomes. ABC-DLBCL is characterized by chronically active B-cell receptor (BCR) signaling that can be modulated by Bruton's tyrosine kinase (BTK) activity. Thus, BTK serves as an attractive therapeutic target in this type of B-cell malignancy. Ibrutinib, a first-in-class, orally available covalent BTK inhibitor, has demonstrated clinical activity in several B-cell leukemias and lymphomas. A phase 1/2 clinical trial of single-agent ibrutinib in relapsed and refractory DLBCL patients revealed an overall response rate of 37% in ABC-DLBCL patients. However, responses to kinase-directed therapies are often limited by emerging resistance mechanisms that bypass the therapeutic target. Here we report the discovery of point mutations within the kinase PIM1 that reduce sensitivity to ibrutinib in ABC-DLBCL. These mutations stabilize PIM1 and affect upstream regulators and downstream targets of NF-κB signaling. The introduction of mutant PIM1 into an ABC-DLBCL cell line, TMD8, increased colony formation and decreased sensitivity to ibrutinib. In addition, ibrutinib-resistant cell lines generated by prolonged ibrutinib exposure in vitro upregulated PIM1 expression, consistent with a role for PIM1 in antagonizing ibrutinib activity. The combination of a pan-PIM inhibitor with ibrutinib synergistically inhibited proliferation in vitro and tumor growth in vivo. Together, these data provide a rationale for combining BTK and PIM1 inhibition in the treatment of ABC-DLBCL.

4.
Cancer Cell ; 28(2): 198-209, 2015 Aug 10.
Article in English | MEDLINE | ID: mdl-26190263

ABSTRACT

The genetic programs that maintain leukemia stem cell (LSC) self-renewal and oncogenic potential have been well defined; however, the comprehensive epigenetic landscape that sustains LSC cellular identity and functionality is less well established. We report that LSCs in MLL-associated leukemia reside in an epigenetic state of relative genome-wide high-level H3K4me3 and low-level H3K79me2. LSC differentiation is associated with reversal of these broad epigenetic profiles, with concomitant downregulation of crucial MLL target genes and the LSC maintenance transcriptional program that is driven by the loss of H3K4me3, but not H3K79me2. The H3K4-specific demethylase KDM5B negatively regulates leukemogenesis in murine and human MLL-rearranged AML cells, demonstrating a crucial role for the H3K4 global methylome in determining LSC fate.


Subject(s)
Cell Transformation, Neoplastic/metabolism , Epigenesis, Genetic , Histones/metabolism , Leukemia/metabolism , Neoplastic Stem Cells/metabolism , Animals , Cell Line, Tumor , Cell Transformation, Neoplastic/genetics , Cells, Cultured , Gene Expression Profiling , Gene Expression Regulation, Leukemic , HEK293 Cells , Histone-Lysine N-Methyltransferase/genetics , Histone-Lysine N-Methyltransferase/metabolism , Humans , Interleukin Receptor Common gamma Subunit/deficiency , Interleukin Receptor Common gamma Subunit/genetics , Jumonji Domain-Containing Histone Demethylases/genetics , Jumonji Domain-Containing Histone Demethylases/metabolism , Leukemia/genetics , Leukemia/pathology , Lysine/metabolism , Methylation , Mice, Inbred C57BL , Mice, Inbred NOD , Mice, Knockout , Mice, SCID , Myeloid-Lymphoid Leukemia Protein/genetics , Myeloid-Lymphoid Leukemia Protein/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Oligonucleotide Array Sequence Analysis , RNA Interference , Repressor Proteins/genetics , Repressor Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Transplantation, Heterologous
5.
Cancer Cell ; 24(4): 423-37, 2013 Oct 14.
Article in English | MEDLINE | ID: mdl-24054986

ABSTRACT

MLL fusion proteins in leukemia induce aberrant transcriptional elongation and associated chromatin perturbations; however, the upstream signaling pathways and activators that recruit or retain MLL oncoproteins at initiated promoters are unknown. Through functional and comparative genomic studies, we identified an essential role for NF-κB signaling in MLL leukemia. Suppression of NF-κB led to robust antileukemia effects that phenocopied loss of functional MLL oncoprotein or associated epigenetic cofactors. The NF-κB subunit RELA occupies promoter regions of crucial MLL target genes and sustains the MLL-dependent leukemia stem cell program. IKK/NF-κB signaling is required for wild-type and fusion MLL protein retention and maintenance of associated histone modifications, providing a molecular rationale for enhanced efficacy in therapeutic targeting of this pathway in MLL leukemias.


Subject(s)
Epigenesis, Genetic , Gene Expression Regulation, Leukemic , Leukemia/metabolism , Myeloid-Lymphoid Leukemia Protein/physiology , NF-kappa B/metabolism , Animals , Cell Line, Tumor , Cell Survival , Chromatin/metabolism , Dose-Response Relationship, Drug , Genomics , Histone-Lysine N-Methyltransferase , Humans , I-kappa B Kinase/metabolism , Leukemia/genetics , Mice , Myeloid-Lymphoid Leukemia Protein/metabolism , Prognosis , Promoter Regions, Genetic , Signal Transduction , Time Factors , Transcription Factor RelA/metabolism , Transcription, Genetic
6.
Cancer Cell ; 23(6): 796-810, 2013 Jun 10.
Article in English | MEDLINE | ID: mdl-23764002

ABSTRACT

Epidermal growth factor receptor (EGFR) initiates a signaling cascade that leads to DNA synthesis and cell proliferation, but its role in regulating DNA replication licensing is unclear. Here, we show that activated EGFR phosphorylates the p56 isoform of Lyn, p56(Lyn), at Y32, which then phosphorylates MCM7, a licensing factor critical for DNA replication, at Y600 to increase its association with other minichromosome maintenance complex proteins, thereby promoting DNA synthesis complex assembly and cell proliferation. Both p56(Lyn) Y32 and MCM7 Y600 phosphorylation are enhanced in proliferating cells and correlated with poor survival of breast cancer patients. These results establish a signaling cascade in which EGFR enhances MCM7 phosphorylation and DNA replication through Lyn phosphorylation in human cancer cells.


Subject(s)
Breast Neoplasms/metabolism , Cell Cycle Proteins/physiology , DNA Replication/physiology , DNA-Binding Proteins/physiology , ErbB Receptors/physiology , Nuclear Proteins/physiology , src-Family Kinases/metabolism , Animals , Breast Neoplasms/pathology , Cell Cycle Proteins/metabolism , Cell Proliferation , DNA-Binding Proteins/metabolism , ErbB Receptors/genetics , ErbB Receptors/metabolism , Female , Humans , Mice , Minichromosome Maintenance Complex Component 7 , Nuclear Proteins/metabolism , Phosphorylation , Prognosis , Signal Transduction , Tyrosine/chemistry , Tyrosine/metabolism , src-Family Kinases/physiology
7.
Mol Cancer Ther ; 11(10): 2212-21, 2012 Oct.
Article in English | MEDLINE | ID: mdl-22826466

ABSTRACT

Clinical correlation studies have clearly shown that obesity is associated with breast cancer risk and patient survival. Although several potential mechanisms linking obesity and cancers have been proposed, the detailed molecular mechanism of obesity-mediated breast tumorigenesis has not yet been critically evaluated. In this study, we evaluated the effects of obesity on mammary tumor initiation and progression using mice with genetic and diet-induced obesity bearing mammary tumor xenografts and mouse mammary tumor virus-neu transgenic mice that were fed a high-fat diet. We show that obesity promoted mammary tumor growth and development in these animal models. Moreover, the expressions of TNFα, VEGF, IKKß, and mTOR are upregulated in mammary tumors of obese mice, suggesting that the IKKß/mTOR/VEGF signaling pathway is activated by TNFα in the tumors of obese mice. More importantly, inhibitors (rapamycin, bevacizumab, and aspirin) that target members of the pathway suppressed tumorigenesis and prolonged survival more effectively in obese mice than in nonobese mice. Here, we not only identified a specific signaling pathway that contributes to mammary tumorigenesis in obese mice but also a strategy for treating obesity-mediated breast cancer.


Subject(s)
Breast Neoplasms/etiology , I-kappa B Kinase/antagonists & inhibitors , Mammary Neoplasms, Animal/etiology , Molecular Targeted Therapy , Obesity/complications , TOR Serine-Threonine Kinases/antagonists & inhibitors , Vascular Endothelial Growth Factor A/antagonists & inhibitors , Animals , Breast Neoplasms/blood supply , Breast Neoplasms/pathology , Cell Proliferation , Cell Transformation, Neoplastic/pathology , Female , Humans , I-kappa B Kinase/metabolism , Mammary Neoplasms, Animal/blood supply , Mammary Neoplasms, Animal/pathology , Mammary Neoplasms, Animal/prevention & control , Mice , Mice, Inbred C57BL , Neovascularization, Pathologic , Obesity/pathology , Signal Transduction , Survival Analysis , TOR Serine-Threonine Kinases/metabolism , Tumor Necrosis Factor-alpha/metabolism , Vascular Endothelial Growth Factor A/metabolism , Xenograft Model Antitumor Assays
8.
Mol Cell ; 45(2): 171-84, 2012 Jan 27.
Article in English | MEDLINE | ID: mdl-22196886

ABSTRACT

Proinflammatory cytokine TNFα plays critical roles in promoting malignant cell proliferation, angiogenesis, and tumor metastasis in many cancers. However, the mechanism of TNFα-mediated tumor development remains unclear. Here, we show that IKKα, an important downstream kinase of TNFα, interacts with and phosphorylates FOXA2 at S107/S111, thereby suppressing FOXA2 transactivation activity and leading to decreased NUMB expression, and further activates the downstream NOTCH pathway and promotes cell proliferation and tumorigenesis. Moreover, we found that levels of IKKα, pFOXA2 (S107/111), and activated NOTCH1 were significantly higher in hepatocellular carcinoma tumors than in normal liver tissues and that pFOXA2 (S107/111) expression was positively correlated with IKKα and activated NOTCH1 expression in tumor tissues. Therefore, dysregulation of NUMB-mediated suppression of NOTCH1 by TNFα/IKKα-associated FOXA2 inhibition likely contributes to inflammation-mediated cancer pathogenesis. Here, we report a TNFα/IKKα/FOXA2/NUMB/NOTCH1 pathway that is critical for inflammation-mediated tumorigenesis and may provide a target for clinical intervention in human cancer.


Subject(s)
Carcinoma, Hepatocellular/metabolism , Cell Transformation, Neoplastic/metabolism , Hepatocyte Nuclear Factor 3-beta/genetics , I-kappa B Kinase/metabolism , Liver Neoplasms/metabolism , Receptor, Notch1/metabolism , Animals , Carcinoma, Hepatocellular/pathology , Cell Proliferation , Gene Expression Regulation, Neoplastic , Hepatocyte Nuclear Factor 3-beta/metabolism , Humans , Liver Neoplasms, Experimental/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Models, Biological , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Phosphorylation , Receptor, Notch1/genetics , Signal Transduction , Tumor Necrosis Factor-alpha/metabolism
9.
J Clin Invest ; 121(11): 4526-36, 2011 Nov.
Article in English | MEDLINE | ID: mdl-21985787

ABSTRACT

Colorectal cancer is the second leading cause of death from cancer in the United States. Metastases in the liver, the most common metastatic site for colorectal cancer, are found in one-third of the patients who die of colorectal cancer. Currently, the genes and molecular mechanisms that are functionally critical in modulating colorectal cancer hepatic metastasis remain unclear. Here, we report our studies using functional selection in an orthotopic mouse model of colorectal cancer to identify a set of genes that play an important role in mediating colorectal cancer liver metastasis. These genes included APOBEC3G, CD133, LIPC, and S100P. Clinically, we found these genes to be highly expressed in a cohort of human hepatic metastasis and their primary colorectal tumors, suggesting that it might be possible to use these genes to predict the likelihood of hepatic metastasis. We have further revealed what we believe to be a novel mechanism in which APOBEC3G promotes colorectal cancer hepatic metastasis through inhibition of miR-29-mediated suppression of MMP2. Together, our data elucidate key factors and mechanisms involved in colorectal cancer liver metastasis, which could be potential targets for diagnosis and treatment.


Subject(s)
Colorectal Neoplasms/physiopathology , Cytidine Deaminase/physiology , Liver Neoplasms, Experimental/secondary , APOBEC-3G Deaminase , Animals , Base Sequence , Cell Line, Tumor , Colorectal Neoplasms/genetics , Cytidine Deaminase/genetics , Gene Expression Profiling , Humans , Liver Neoplasms, Experimental/genetics , Liver Neoplasms, Experimental/physiopathology , Matrix Metalloproteinase 2/genetics , Matrix Metalloproteinase 2/metabolism , Mice , Mice, Nude , MicroRNAs/genetics , MicroRNAs/metabolism , Neoplasm Transplantation , RNA, Messenger/genetics , RNA, Messenger/metabolism , Transplantation, Heterologous
10.
Nat Cell Biol ; 13(2): 174-81, 2011 Feb.
Article in English | MEDLINE | ID: mdl-21258366

ABSTRACT

Epidermal growth factor receptor (EGFR) can undergo post-translational modifications, including phosphorylation, glycosylation and ubiquitylation, leading to diverse physiological consequences and modulation of its biological activity. There is increasing evidence that methylation may parallel other post-translational modifications in the regulation of various biological processes. It is still not known, however, whether EGFR is regulated by this post-translational event. Here, we show that EGFR Arg 1175 is methylated by an arginine methyltransferase, PRMT5. Arg 1175 methylation positively modulates EGF-induced EGFR trans-autophosphorylation at Tyr 1173, which governs ERK activation. Abolishment of Arg 1175 methylation enhances EGF-stimulated ERK activation by reducing SHP1 recruitment to EGFR, resulting in augmented cell proliferation, migration and invasion of EGFR-expressing cells. Therefore, we propose a model in which the regulatory crosstalk between PRMT5-mediated Arg 1175 methylation and EGF-induced Tyr 1173 phosphorylation attenuates EGFR-mediated ERK activation.


Subject(s)
Arginine/metabolism , ErbB Receptors/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , Tyrosine/metabolism , Enzyme Activation , ErbB Receptors/genetics , Extracellular Signal-Regulated MAP Kinases/genetics , HEK293 Cells , Humans , Methylation , Phosphorylation , Protein Methyltransferases/genetics , Protein Methyltransferases/metabolism , Protein-Arginine N-Methyltransferases
11.
Sci Signal ; 3(108): ra9, 2010 Feb 09.
Article in English | MEDLINE | ID: mdl-20145209

ABSTRACT

Mammalian target of rapamycin (mTOR) regulates various cellular functions, including tumorigenesis, and is inhibited by the tuberous sclerosis 1 (TSC1)-TSC2 complex. Here, we demonstrate that arrest-defective protein 1 (ARD1) physically interacts with, acetylates, and stabilizes TSC2, thereby repressing mTOR activity. The inhibition of mTOR by ARD1 inhibits cell proliferation and increases autophagy, thereby inhibiting tumorigenicity. Correlation between ARD1 and TSC2 abundance was apparent in multiple tumor types. Moreover, evaluation of loss of heterozygosity at Xq28 revealed allelic loss in 31% of tested breast cancer cell lines and tumor samples. Together, our findings suggest that ARD1 functions as an inhibitor of the mTOR pathway and that dysregulation of the ARD1-TSC2-mTOR axis may contribute to cancer development.


Subject(s)
Acetyltransferases/metabolism , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Signal Transduction , Tumor Suppressor Proteins/metabolism , Alleles , Animals , Autophagy , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Cell Line, Tumor , Cell Proliferation , Heterozygote , Humans , Mice , N-Terminal Acetyltransferase A , N-Terminal Acetyltransferase E , RNA, Small Interfering/metabolism , Tuberous Sclerosis Complex 2 Protein
12.
Am J Transl Res ; 2(1): 56-64, 2010 Jan 01.
Article in English | MEDLINE | ID: mdl-20182582

ABSTRACT

Arrest-defect-1 protein (ARD1), an acetyltransferase, catalyzes N-alpha-acetylation in yeast. In mammalian cells, both N-alpha-acetylation and epsilon-acetylation induced by ARD1 have been reported. Emerging evidence has revealed that ARD1 is involved in a variety of cellular functions, including proliferation, apoptosis, autophagy, and differentiation and that dysregulation of ARD1 is associated with tumorigenesis and neurodegenerative disorder. This review will discuss recent discoveries regarding variations among the different ARD1 isoforms, the associated biological functions of ARD1, and ARD1 localization in different cells. We will also discuss the potential upstream regulators and downstream targets of ARD1 to provide new avenues for resolving its controversial roles in cancer development.

13.
Mol Cell ; 36(1): 131-40, 2009 Oct 09.
Article in English | MEDLINE | ID: mdl-19818716

ABSTRACT

IkappaB kinase beta (IKKbeta) is involved in tumor development and progression through activation of the nuclear factor (NF)-kappaB pathway. However, the molecular mechanism that regulates IKKbeta degradation remains largely unknown. Here, we show that a Cullin 3 (CUL3)-based ubiquitin ligase, Kelch-like ECH-associated protein 1 (KEAP1), is responsible for IKKbeta ubiquitination. Depletion of KEAP1 led to the accumulation and stabilization of IKKbeta and to upregulation of NF-kappaB-derived tumor angiogenic factors. A systematic analysis of the CUL3, KEAP1, and RBX1 genomic loci revealed a high percentage of genome loss and missense mutations in human cancers that failed to facilitate IKKbeta degradation. Our results suggest that the dysregulation of KEAP1-mediated IKKbeta ubiquitination may contribute to tumorigenesis.


Subject(s)
I-kappa B Kinase/metabolism , Intracellular Signaling Peptides and Proteins/physiology , NF-kappa B/metabolism , Signal Transduction/physiology , Animals , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Line , Cell Line, Tumor , Cullin Proteins/genetics , Cullin Proteins/metabolism , DNA Copy Number Variations/genetics , Female , Gene Expression/drug effects , Gene Expression/genetics , Humans , I-kappa B Kinase/genetics , Interleukin-8/genetics , Kaplan-Meier Estimate , Kelch-Like ECH-Associated Protein 1 , Mice , Mutation/physiology , Neoplasms/genetics , Neoplasms/metabolism , Neovascularization, Physiologic/genetics , Protein Binding/physiology , Protein Interaction Domains and Motifs/physiology , RNA, Small Interfering/genetics , Signal Transduction/drug effects , Transcription Factor RelA/metabolism , Transfection , Tumor Necrosis Factor-alpha/pharmacology , Ubiquitination/physiology
14.
Biochem Biophys Res Commun ; 389(1): 156-61, 2009 Nov 06.
Article in English | MEDLINE | ID: mdl-19716809

ABSTRACT

IkappaB kinase beta (IKKbeta), a major kinase downstream of various proinflammatory signals, mediates multiple cellular functions through phosphorylation and regulation of its substrates. On the basis of protein sequence analysis, we identified arrest-defective protein 1 (ARD1), a protein involved in apoptosis and cell proliferation processes in many human cancer cells, as a new IKKbeta substrate. We provided evidence showing that ARD1 is indeed a bona fide substrate of IKKbeta. IKKbeta physically associated with ARD1 and phosphorylated it at Ser209. Phosphorylation by IKKbeta destabilized ARD1 and induced its proteasome-mediated degradation. Impaired growth suppression was observed in ARD1 phosphorylation-mimic mutant (S209E)-transfected cells as compared with ARD1 non-phosphorylatable mutant (S209A)-transfected cells. Our findings of molecular interactions between ARD1 and IKKbeta may enable further understanding of the upstream regulation mechanisms of ARD1 and of the diverse functions of IKKbeta.


Subject(s)
Acetyltransferases/metabolism , I-kappa B Kinase/metabolism , Acetyltransferases/genetics , Amino Acid Sequence , Animals , Cell Line, Tumor , Cell Proliferation , Conserved Sequence , Enzyme Stability , Humans , Mice , Molecular Sequence Data , N-Terminal Acetyltransferase A , N-Terminal Acetyltransferase E , Phosphorylation/genetics , Proteasome Endopeptidase Complex/metabolism
15.
Biochem Biophys Res Commun ; 389(4): 640-4, 2009 Nov 27.
Article in English | MEDLINE | ID: mdl-19766100

ABSTRACT

The transcription factor hypoxia-inducible factor 1alpha (HIF-1alpha) is regulated by oxygen availability as well as various inflammatory mediators, including tumor necrosis factor alpha (TNFalpha). Early work suggested that the phosphatidylinositol-3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling pathways are involved in TNFalpha-mediated HIF-1alpha accumulation and activation under normoxic conditions. Here, we provide evidence showing that IkappaB kinase beta (IKKbeta) is required for HIF-1alpha regulation by TNFalpha. We found that TNFalpha enhances HIF-1alpha protein expression in various breast cancer cell lines under either normoxic or hypoxia-mimicking conditions, but has little effect on the HIF-1alpha mRNA level. Increased HIF-1alpha expression was found in IKKbeta stable clones and transient transfectants, and depletion of IKKbeta consistently reduced the amount of HIF-1alpha protein. Treatment of cells with the IKKbeta inhibitor Bay 11-7082 reduced the TNFalpha-induced HIF-1alpha expression, suggesting that IKKbeta is required in this signaling pathway. Decreased expression of vascular endothelial growth factor (VEGF), a direct target of HIF-1alpha, was shown in IKKbeta-knockout mouse embryonic fibroblast cells. We further demonstrated a positive correlation between IKKbeta and VEGF expression in primary human breast cancer specimens. Our findings indicate that TNFalpha-induced HIF-1alpha accumulation is IKKbeta dependent, and may enable further understanding of the HIF-1alpha regulation by inflammatory signals.


Subject(s)
Breast Neoplasms/metabolism , Gene Expression Regulation , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , I-kappa B Kinase/metabolism , Tumor Necrosis Factor-alpha/metabolism , Animals , Breast Neoplasms/genetics , Cell Line, Tumor , Gene Knockout Techniques , Humans , I-kappa B Kinase/antagonists & inhibitors , I-kappa B Kinase/genetics , Mice , Transcription, Genetic , Tumor Necrosis Factor-alpha/pharmacology , Vascular Endothelial Growth Factor A/biosynthesis
16.
Mol Carcinog ; 48(11): 1048-58, 2009 Nov.
Article in English | MEDLINE | ID: mdl-19526459

ABSTRACT

MAX dimerization protein 1 (MAD1) is a transcription suppressor that antagonizes MYC-mediated transcription activation, and the inhibition mechanism occurs mainly through the competition of target genes' promoter MYC binding sites by MAD1. The promoter binding proteins switch between MYC and MAD1 affects cell proliferation and differentiation. However, little is known about MAD1's regulation process in cancer cells. Here, we present evidence that AKT inhibits MAD1-mediated transcription repression by physical interaction with and phosphorylation of MAD1. Phosphorylation reduces the binding affinity between MAD1 and its target genes' promoter and thereby abolishes its transcription suppression function. Mutation of the phosphorylation site from serine to alanine rescues the DNA-binding ability in the presence of activated AKT. In addition, AKT inhibits MAD1-mediated target genes (hTERT and ODC) transcription repression and promotes cell cycle and cell growth. However, mutated S145A MAD1 abrogates the inhibition by AKT. Thus, our results suggest that phosphorylation of MAD1 by AKT inhibits MAD1-mediated transcription suppression and subsequently activates the transcription of MAD1 target genes.


Subject(s)
Cell Cycle Proteins/antagonists & inhibitors , Nuclear Proteins/antagonists & inhibitors , Proto-Oncogene Proteins c-akt/metabolism , Transcription, Genetic , Cell Cycle Proteins/genetics , Cell Line, Tumor , Chromatin Immunoprecipitation , Gene Expression Regulation , Genes, myc , Humans , Mass Spectrometry , Nuclear Proteins/genetics , Phosphorylation , Reverse Transcriptase Polymerase Chain Reaction
17.
Int J Mol Med ; 22(5): 633-8, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18949383

ABSTRACT

The proinflammatory cytokine TNFalpha is one of the factors that links obesity-derived chronic inflammation with insulin resistance. Activation of mTOR signaling pathway has been found to suppress insulin sensitivity through serine phosphorylation and the inhibition of IRS1 by mTOR and its downstream effector, S6K1. It remains elusive that whether the mTOR pathway has a role in TNFalpha-mediated insulin resistance. In the present study, we demonstrated that TNFalpha-IKKbeta-mediated inactivation of TSC1 resulted in increasing phosphorylation of IRS1 serine 307 and serine 636/639, impaired insulin-induced glucose uptake, tyrosine phosphorylation of IRS1, and the association between IRS1 and PI3K p85. Furthermore, a higher expression of pIKKbeta (S181), pTSC1(S511), and pS6(S240/244) was found in livers obtained from both C57BL/6J mice on a high-fat diet and B6.V-Lepob/J mice. Collectively, dysregulation of the TSC1/ TSC2/mTOR signaling pathway by IKKbeta is a common molecular switch for both cancer pathogenesis and diet- and obesity-induced insulin resistance.


Subject(s)
Carrier Proteins/metabolism , I-kappa B Kinase/metabolism , Insulin Resistance , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Signal Transduction , Tumor Suppressor Proteins/metabolism , Animals , Cell Line , Chronic Disease , Inflammation , Insulin Receptor Substrate Proteins/metabolism , Male , Mice , Mice, Obese , Phosphatidylinositol 3-Kinases/metabolism , Ribosomal Protein S6 Kinases, 90-kDa/metabolism , TOR Serine-Threonine Kinases , Tuberous Sclerosis Complex 1 Protein , Tuberous Sclerosis Complex 2 Protein , Tumor Necrosis Factor-alpha/metabolism
18.
Cancer Res ; 68(15): 6109-17, 2008 Aug 01.
Article in English | MEDLINE | ID: mdl-18676833

ABSTRACT

Myeloid cell leukemia-1 (Mcl-1), a Bcl-2-like antiapoptotic protein, plays a role in cell immortalization and chemoresistance in a number of human malignancies. A peptidyl-prolyl cis/trans isomerase, Pin1 is involved in many cellular events, such as cell cycle progression, cell proliferation, and differentiation through isomerizing prophosphorylated substrates. It has been reported that down-regulation of Pin1 induces apoptosis, and that Erk phosphorylates and up-regulates Mcl-1; however, the underlying mechanisms for the two phenomena are not clear yet. Here, we showed that Pin 1 stabilizes Mcl-1, which is required for Mcl-1 posphorylation by Erk. First, we found expression of Mcl-1 and Pin1 were positively correlated and associated with poor survival in human breast cancer. We then showed that Erk could phosphorylate Mcl-1 at two consensus residues, Thr 92 and 163, which is required for the association of Mcl-1 and Pin1, resulting in stabilization of Mcl-1. Moreover, Pin1 is also required for the up-regulation of Mcl-1 by Erk activation. Based on this newly identified mechanism of Mcl-1 stabilization, two strategies were used to overcome Mcl-1-mediated chemoresistance: inhibiting Erk by Sorafenib, an approved clinical anticancer drug, or knocking down Pin1 by using a SiRNA technique. In conclusion, the current report not only unravels a novel mechanism to link Erk/Pin1 pathway and Mcl-1-mediated chemoresistance but also provides a plausible combination therapy, Taxol (Paclitaxel) plus Sorafenib, which was shown to be effective in killing breast cancer cells.


Subject(s)
Antineoplastic Agents/pharmacology , Benzenesulfonates/pharmacology , Breast Neoplasms/metabolism , Down-Regulation , MAP Kinase Signaling System , Peptidylprolyl Isomerase/metabolism , Proto-Oncogene Proteins c-bcl-2/physiology , Pyridines/pharmacology , Breast Neoplasms/pathology , Cell Line, Tumor , Humans , Mutagenesis, Site-Directed , Myeloid Cell Leukemia Sequence 1 Protein , NIMA-Interacting Peptidylprolyl Isomerase , Niacinamide/analogs & derivatives , Phenylurea Compounds , Phosphorylation , Sorafenib
19.
Cancer Res ; 68(8): 2632-40, 2008 Apr 15.
Article in English | MEDLINE | ID: mdl-18413730

ABSTRACT

Barrett's esophagus, a columnar metaplasia of the lower esophagus epithelium related to gastroesophageal reflux disease, is the strongest known risk factor for the development of esophageal adenocarcinoma (EAC). Understanding the signal transduction events involved in esophageal epithelium carcinogenesis may provide insights into the origins of EAC and may suggest new therapies. To elucidate the molecular pathways of bile acid-induced tumorigenesis, the newly identified inflammation-associated signaling pathway involving I kappaB kinases beta (IKK beta), tuberous sclerosis complex 1 (TSC1), and mammalian target of rapamycin (mTOR) downstream effector S6 kinase (S6K1) was confirmed to be activated in immortalized Barrett's CPC-A and CPC-C cells and esophageal cancer SEG-1 and BE3 cells. Phosphorylation of TSC1 and S6K1 was induced in response to bile acid stimulation. Treatment of these cells with the mTOR inhibitor rapamycin or the IKK beta inhibitor Bay 11-7082 suppressed bile acid-induced cell proliferation and anchorage-independent growth. We next used an orthotopic rat model to evaluate the role of bile acid in the progression of Barrett's esophagus to EAC. Of interest, we found high expression of phosphorylated IKK beta (pIKK beta) and phosphorylated S6K1 (pS6K1) in tumor tissues and the Barrett's epithelium compared with normal epithelium. Furthermore, immunostaining of clinical EAC tissue specimens revealed that pIKK beta expression was strongly correlated with pS6K1 level. Together, these results show that bile acid can deregulate TSC1/mTOR through IKK beta signaling, which may play a critical role in EAC progression. In addition, Bay 11-7082 and rapamycin may potentially be chemopreventive drugs against Barrett's esophagus-associated EAC.


Subject(s)
Adenocarcinoma/genetics , Barrett Esophagus/complications , Bile Acids and Salts/pharmacology , Esophageal Neoplasms/etiology , Esophageal Neoplasms/genetics , Gene Expression Regulation, Neoplastic , Protein Kinases/genetics , Tumor Suppressor Proteins/genetics , Adenocarcinoma/pathology , Barrett Esophagus/physiopathology , Cell Division , Chenodeoxycholic Acid/pharmacology , Esophageal Neoplasms/pathology , Gastroesophageal Reflux/complications , Gastroesophageal Reflux/genetics , Gastroesophageal Reflux/physiopathology , Humans , Inflammation/complications , Inflammation/physiopathology , NF-kappa B/antagonists & inhibitors , Nitriles/pharmacology , RNA, Small Interfering/genetics , Sirolimus/pharmacology , Sulfones/pharmacology , TOR Serine-Threonine Kinases , Tuberous Sclerosis Complex 1 Protein , Ursodeoxycholic Acid/pharmacology
20.
Nat Cell Biol ; 10(2): 138-48, 2008 Feb.
Article in English | MEDLINE | ID: mdl-18204439

ABSTRACT

The RAS-ERK pathway is known to play a pivotal role in differentiation, proliferation and tumour progression. Here, we show that Erk downregulates Forkhead box O 3a (FOXO3a) by directly interacting with and phosphorylating FOXO3a at Ser 294, Ser 344 and Ser 425, which consequently promotes cell proliferation and tumorigenesis. The ERK-phosphorylated FOXO3a degrades via an MDM2-mediated ubiquitin-proteasome pathway. However, the non-phosphorylated FOXO3a mutant is resistant to the interaction and degradation by murine double minute 2 (MDM2), thereby resulting in a strong inhibition of cell proliferation and tumorigenicity. Taken together, our study elucidates a novel pathway in cell growth and tumorigenesis through negative regulation of FOXO3a by RAS-ERK and MDM2.


Subject(s)
Cell Transformation, Neoplastic/metabolism , Extracellular Signal-Regulated MAP Kinases/physiology , Forkhead Transcription Factors/antagonists & inhibitors , Proto-Oncogene Proteins c-mdm2/physiology , Amino Acid Sequence , Animals , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Line , Cell Line, Tumor , Cell Proliferation , Down-Regulation , Extracellular Signal-Regulated MAP Kinases/antagonists & inhibitors , Female , Forkhead Box Protein O3 , Forkhead Transcription Factors/biosynthesis , Humans , Mass Spectrometry , Mice , Mice, Nude , Molecular Sequence Data , Neoplasm Transplantation , Phosphorylation , Protein Binding , Proto-Oncogene Proteins c-mdm2/biosynthesis , Serine/metabolism , Signal Transduction , Transplantation, Heterologous
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