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1.
iScience ; 23(12): 101795, 2020 Dec 18.
Article in English | MEDLINE | ID: mdl-33305174

ABSTRACT

Aberrant activation of Wnt signaling triggered by mutations in either Adenomatous Polyposis Coli (APC) or CTNNB1 (ß-catenin) is a hallmark of colorectal cancers (CRC). As part of a program to develop epigenetic regulators for cancer therapy, we developed carboxamide-substituted benzhydryl amines (CBAs) bearing either aryl or heteroaryl groups that selectively targeted histone lysine demethylases (KDMs) and functioned as inhibitors of the Wnt pathway. A biotinylated variant of N-((5-chloro-8-hydroxyquinolin-7-yl) (4-(diethylamino)phenyl)-methyl)butyramide (CBA-1) identified KDM3A as a binding partner. KDM3A is a Jumonji (JmjC) domain-containing demethylase that is significantly upregulated in CRC. KDM3A regulates the demethylation of histone H3's lysine 9 (H3K9Me2), a repressive marker for transcription. Inhibiting KDM3 increased H3K9Me2 levels, repressed Wnt target genes, and curtailed in vitro CRC cell proliferation. CBA-1 also exhibited in vivo inhibition of Wnt signaling in a zebrafish model without displaying in vivo toxicity.

2.
BMC Bioinformatics ; 20(1): 620, 2019 Dec 02.
Article in English | MEDLINE | ID: mdl-31791231

ABSTRACT

BACKGROUND: Cancer arises through accumulation of somatically acquired genetic mutations. An important question is to delineate the temporal order of somatic mutations during carcinogenesis, which contributes to better understanding of cancer biology and facilitates identification of new therapeutic targets. Although a number of statistical and computational methods have been proposed to estimate the temporal order of mutations, they do not account for the differences in the functional impacts of mutations and thus are likely to be obscured by the presence of passenger mutations that do not contribute to cancer progression. In addition, many methods infer the order of mutations at the gene level, which have limited power due to the low mutation rate in most genes. RESULTS: In this paper, we develop a Probabilistic Approach for estimating the Temporal Order of Pathway mutations by leveraging functional Annotations of mutations (PATOPA). PATOPA infers the order of mutations at the pathway level, wherein it uses a probabilistic method to characterize the likelihood of mutational events from different pathways occurring in a certain order. The functional impact of each mutation is incorporated to weigh more on a mutation that is more integral to tumor development. A maximum likelihood method is used to estimate parameters and infer the probability of one pathway being mutated prior to another. Simulation studies and analysis of whole exome sequencing data from The Cancer Genome Atlas (TCGA) demonstrate that PATOPA is able to accurately estimate the temporal order of pathway mutations and provides new biological insights on carcinogenesis of colorectal and lung cancers. CONCLUSIONS: PATOPA provides a useful tool to estimate temporal order of mutations at the pathway level while leveraging functional annotations of mutations.


Subject(s)
Carcinogenesis/genetics , Molecular Sequence Annotation , Mutation/genetics , Probability , Signal Transduction/genetics , Computer Simulation , Databases, Genetic , Humans , Mutation Rate , Neoplasms/genetics , Reproducibility of Results , Time Factors
3.
J Med Chem ; 62(24): 11348-11358, 2019 12 26.
Article in English | MEDLINE | ID: mdl-31774672

ABSTRACT

The importance of upregulated Wnt signaling in colorectal cancers led to efforts to develop inhibitors that target ß-catenin in this pathway. We now report that several "Wnt inhibitors" that allegedly target ß-catenin actually function as mitochondrial proton uncouplers that independently activate AMPK and concomitantly inhibit Wnt signaling. As expected for a process in which mitochondrial uncoupling diminishes ATP production, a mitochondrial proton uncoupler, FCCP, and a glucose metabolic inhibitor, 2-DG, activated AMPK and inhibited Wnt signaling. Also consistent with these findings, a well-known "Wnt inhibitor", FH535, functioned as a proton uncoupler, and in support of this finding, the N-methylated analog, 2,5-dichloro-N-methyl-N-(2-methyl-4-nitrophenyl)benzenesulfonamide (FH535-M), was inactive as an uncoupler and Wnt inhibitor. Apart from suggesting an opportunity to develop dual Wnt inhibitors and AMPK activators, these findings provide a cautionary tale that claims for Wnt inhibition alone require scrutiny as possible mitochondrial proton uncouplers or inhibitors of the electron transport chain.


Subject(s)
AMP-Activated Protein Kinases/metabolism , Brain/drug effects , Colonic Neoplasms/drug therapy , Enzyme Activators/pharmacology , Hydrocarbons, Fluorinated/pharmacology , Mitochondria/drug effects , Urea/pharmacology , Wnt Proteins/antagonists & inhibitors , beta Catenin/antagonists & inhibitors , Animals , Brain/metabolism , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Energy Metabolism , Enzyme Activation , Enzyme Activators/chemistry , Gene Expression Regulation, Neoplastic , Humans , Hydrocarbons, Fluorinated/chemistry , Mitochondria/metabolism , Oxygen Consumption , Sulfonamides/chemistry , Sulfonamides/pharmacology , Tumor Cells, Cultured , Urea/analogs & derivatives , Urea/chemistry
4.
Sensors (Basel) ; 19(12)2019 Jun 14.
Article in English | MEDLINE | ID: mdl-31207972

ABSTRACT

Mobile crowdsourcing has been exploited to collect enough fingerprints for fingerprinting-based localization. Since the construction of a fingerprint database is time consuming, mobile users should be well motivated to participate in fingerprint collection task. To this end, a Walrasian equilibrium-based incentive mechanism is proposed in this paper to motivate mobile users. The proposed mechanism can eliminate the monopoly of the crowdsourcer, balance the supply and demand of fingerprint data, and maximize the benefit of all participators. In order to reach the Walrasian equilibrium, firstly, the social welfare maximization problem is constructed. To solve the original optimization problem, a dual decomposition method is employed. The maximization of social welfare is decomposed into the triple benefit optimization among the crowdsourcer, mobile users, and the whole system. Accordingly, a distributed iterative algorithm is designed. Through the simulation, the performance of the proposed incentive scheme is verified and analyzed. Simulation results demonstrated that the proposed iterative algorithm satisfies the convergence and optimality. Moreover, the self-reconstruction ability of the proposed incentive scheme was also verified, indicating that the system has strong robustness and scalability.

5.
Sci Rep ; 9(1): 6439, 2019 04 23.
Article in English | MEDLINE | ID: mdl-31015569

ABSTRACT

Structure-activity relationships (SAR) in the aurone pharmacophore identified heterocyclic variants of the (Z)-2-benzylidene-6-hydroxybenzofuran-3(2H)-one scaffold that possessed low nanomolar in vitro potency in cell proliferation assays using various cancer cell lines, in vivo potency in prostate cancer PC-3 xenograft and zebrafish models, selectivity for the colchicine-binding site on tubulin, and absence of appreciable toxicity. Among the leading, biologically active analogs were (Z)-2-((2-((1-ethyl-5-methoxy-1H-indol-3-yl)methylene)-3-oxo-2,3-dihydrobenzofuran-6-yl)oxy)acetonitrile (5a) and (Z)-6-((2,6-dichlorobenzyl)oxy)-2-(pyridin-4-ylmethylene)benzofuran-3(2H)-one (5b) that inhibited in vitro PC-3 prostate cancer cell proliferation with IC50 values below 100 nM. A xenograft study in nude mice using 10 mg/kg of 5a had no effect on mice weight, and aurone 5a did not inhibit, as desired, the human ether-à-go-go-related (hERG) potassium channel. Cell cycle arrest data, comparisons of the inhibition of cancer cell proliferation by aurones and known antineoplastic agents, and in vitro inhibition of tubulin polymerization indicated that aurone 5a disrupted tubulin dynamics. Based on molecular docking and confirmed by liquid chromatography-electrospray ionization-tandem mass spectrometry studies, aurone 5a targets the colchicine-binding site on tubulin. In addition to solid tumors, aurones 5a and 5b strongly inhibited in vitro a panel of human leukemia cancer cell lines and the in vivo myc-induced T cell acute lymphoblastic leukemia (T-ALL) in a zebrafish model.


Subject(s)
Neoplasm Proteins/metabolism , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma , Prostatic Neoplasms , Protein Multimerization/drug effects , Tubulin/metabolism , Zebrafish/metabolism , Animals , Benzofurans/chemical synthesis , Benzofurans/chemistry , Benzofurans/pharmacology , Binding Sites , Colchicine , Humans , Male , Mice , Mice, Nude , PC-3 Cells , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/pathology , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Xenograft Model Antitumor Assays
6.
Cancer Epidemiol Biomarkers Prev ; 28(2): 348-356, 2019 02.
Article in English | MEDLINE | ID: mdl-30377206

ABSTRACT

BACKGROUND: Lung cancer is the leading cause of cancer mortality in the United States (U.S.). Squamous cell carcinoma (SQCC) represents 22.6% of all lung cancers nationally, and 26.4% in Appalachian Kentucky (AppKY), where death from lung cancer is exceptionally high. The Cancer Genome Atlas (TCGA) characterized genetic alterations in lung SQCC, but this cohort did not focus on AppKY residents. METHODS: Whole-exome sequencing was performed on tumor and normal DNA samples from 51 lung SQCC subjects from AppKY. Somatic genomic alterations were compared between the AppKY and TCGA SQCC cohorts. RESULTS: From this AppKY cohort, we identified an average of 237 nonsilent mutations per patient and, in comparison with TCGA, we found that PCMTD1 (18%) and IDH1 (12%) were more commonly altered in AppKY versus TCGA. Using IDH1 as a starting point, we identified a mutually exclusive mutational pattern (IDH1, KDM6A, KDM4E, JMJD1C) involving functionally related genes. We also found actionable mutations (10%) and/or intermediate or high-tumor mutation burden (65%), indicating potential therapeutic targets in 65% of subjects. CONCLUSIONS: This study has identified an increased percentage of IDH1 and PCMTD1 mutations in SQCC arising in the AppKY residents versus TCGA, with population-specific implications for the personalized treatment of this disease. IMPACT: Our study is the first report to characterize genomic alterations in lung SQCC from AppKY. These findings suggest population differences in the genetics of lung SQCC between AppKY and U.S. populations, highlighting the importance of the relevant population when developing personalized treatment approaches for this disease.


Subject(s)
Carcinoma, Squamous Cell/genetics , Isocitrate Dehydrogenase/genetics , Lung Neoplasms/genetics , Mutation , Protein D-Aspartate-L-Isoaspartate Methyltransferase/genetics , Adult , Aged , Aged, 80 and over , Appalachian Region , Carcinoma, Squamous Cell/metabolism , Female , Genomics , Humans , Kentucky , Lung Neoplasms/metabolism , Male , Middle Aged , White People/genetics , Exome Sequencing
7.
Org Biomol Chem ; 15(36): 7623-7629, 2017 Sep 20.
Article in English | MEDLINE | ID: mdl-28868548

ABSTRACT

Cytisine-linked isoflavonoids (CLIFs) inhibited PC-3 prostate and LS174T colon cancer cell proliferation by inhibiting a peroxisomal bifunctional enzyme. A pull-down assay using a biologically active, biotin-modified CLIF identified the target of these agents as the bifunctional peroxisomal enzyme, hydroxysteroid 17ß-dehydrogenase-4 (HSD17B4). Additional studies with truncated versions of HSD17B4 established that CLIFs specifically bind the C-terminus of HSD17B4 and selectively inhibited the enoyl CoA hydratase but not the d-3-hydroxyacyl CoA dehydrogenase activity. HSD17B4 was overexpressed in prostate and colon cancer tissues, knocking down HSD17B4 inhibited cancer cell proliferation, suggesting that HSD17B4 is a potential biomarker and drug target and that CLIFs are potential probes or therapeutic agents for these cancers.


Subject(s)
Alkaloids/pharmacology , Antineoplastic Agents/pharmacology , Enzyme Inhibitors/pharmacology , Isoflavones/pharmacology , Peroxisomal Multifunctional Protein-2/antagonists & inhibitors , Alkaloids/chemistry , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Azocines/chemistry , Azocines/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Humans , Isoflavones/chemical synthesis , Isoflavones/chemistry , Molecular Structure , Peroxisomal Multifunctional Protein-2/metabolism , Quinolizines/chemistry , Quinolizines/pharmacology , Structure-Activity Relationship
9.
Mol Cell Biol ; 35(19): 3301-11, 2015 Oct.
Article in English | MEDLINE | ID: mdl-26169834

ABSTRACT

Wnt signaling plays important roles in development and tumorigenesis. A central question about the Wnt pathway is the regulation of ß-catenin. Phosphorylation of ß-catenin by CK1α and GSK3 promotes ß-catenin binding to ß-TrCP, leading to ß-catenin degradation through the proteasome. The phosphorylation and ubiquitination of ß-catenin have been well characterized; however, it is unknown whether and how a deubiquitinase is involved. In this study, by screening RNA interference (RNAi) libraries, we identified USP47 as a deubiquitinase that prevents ß-catenin ubiquitination. Inactivation of USP47 by RNAi increased ß-catenin ubiquitination, attenuated Wnt signaling, and repressed cancer cell growth. Furthermore, USP47 deubiquitinates itself, whereas ß-TrCP promotes USP47 ubiquitination through interaction with an atypical motif in USP47. Finally, in vivo studies in the Drosophila wing suggest that UBP64E, the USP47 counterpart in Drosophila, is required for Armadillo stabilization and plays a positive role in regulating Wnt target gene expression.


Subject(s)
Drosophila Proteins/physiology , Ubiquitin Thiolesterase/physiology , Ubiquitin-Specific Proteases/physiology , Ubiquitination , Wnt Signaling Pathway , beta Catenin/metabolism , Amino Acid Sequence , Animals , Binding Sites , Drosophila melanogaster , HEK293 Cells , Humans , Molecular Sequence Data , Proteolysis , Wings, Animal/enzymology , beta-Transducin Repeat-Containing Proteins/metabolism
10.
J Med Chem ; 57(14): 6083-91, 2014 Jul 24.
Article in English | MEDLINE | ID: mdl-24950374

ABSTRACT

Inhibition of the catalytic subunit of the heterodimeric methionine S-adenosyl transferase-2 (MAT2A) with fluorinated N,N-dialkylaminostilbenes (FIDAS agents) offers a potential avenue for the treatment of liver and colorectal cancers where upregulation of this enzyme occurs. A study of structure-activity relationships led to the identification of the most active compounds as those with (1) either a 2,6-difluorostyryl or 2-chloro-6-fluorostyryl subunit, (2) either an N-methylamino or N,N-dimethylamino group attached in a para orientation relative to the 2,6-dihalostyryl subunit, and (3) either an N-methylaniline or a 2-(N,N-dimethylamino)pyridine ring. These modifications led to FIDAS agents that were active in the low nanomolar range, that formed water-soluble hydrochloride salts, and that possessed the desired property of not inhibiting the human hERG potassium ion channel at concentrations at which the FIDAS agents inhibit MAT2A. The active FIDAS agents may inhibit cancer cells through alterations of methylation reactions essential for cancer cell survival and growth.


Subject(s)
Aniline Compounds/pharmacology , Antineoplastic Agents/pharmacology , Catalytic Domain/drug effects , Enzyme Inhibitors/pharmacology , Methionine Adenosyltransferase/antagonists & inhibitors , Pyridines/pharmacology , Pyrimidines/pharmacology , Stilbenes/pharmacology , Aniline Compounds/chemical synthesis , Aniline Compounds/chemistry , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Biocatalysis/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , HEK293 Cells , Humans , Methionine Adenosyltransferase/metabolism , Molecular Structure , Protein Subunits/drug effects , Pyridines/chemical synthesis , Pyridines/chemistry , Pyrimidines/chemical synthesis , Pyrimidines/chemistry , Stilbenes/chemical synthesis , Stilbenes/chemistry , Structure-Activity Relationship , Tumor Cells, Cultured
11.
J Biol Chem ; 289(13): 8881-90, 2014 Mar 28.
Article in English | MEDLINE | ID: mdl-24532795

ABSTRACT

Wnt signaling plays an important role in colorectal cancer (CRC). Although the mechanisms of ß-catenin degradation have been well studied, the mechanism by which ß-catenin activates transcription is still not fully understood. While screening a panel of DNA demethylases, we found that thymine DNA glycosylase (TDG) up-regulated Wnt signaling. TDG interacts with the transcription factor TCF4 and coactivator CREB-binding protein/p300 in the Wnt pathway. Knocking down TDG by shRNAs inhibited the proliferation of CRC cells in vitro and in vivo. In CRC patients, TDG levels were significantly higher in tumor tissues than in the adjacent normal tissues. These results suggest that TDG warrants consideration as a potential biomarker for CRC and as a target for CRC treatment.


Subject(s)
Colorectal Neoplasms/pathology , Thymine DNA Glycosylase/metabolism , Wnt Proteins/metabolism , Wnt Signaling Pathway , Amino Acid Sequence , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/metabolism , Cell Line, Tumor , Cell Proliferation , Gene Expression Regulation, Neoplastic , Humans , Peptide Fragments/metabolism , Protein Transport , Sialoglycoproteins/metabolism , Sumoylation , Thymine DNA Glycosylase/chemistry , Thymine DNA Glycosylase/genetics , Transcription Factor 4 , Transcription Factors/metabolism , Up-Regulation
12.
ACS Chem Biol ; 8(4): 796-803, 2013 Apr 19.
Article in English | MEDLINE | ID: mdl-23363077

ABSTRACT

Methionine S-adenosyltransferase 2A (MAT2A) is the catalytic subunit for synthesis of S-adenosylmethionine (SAM), the principal methyl donor in many biological processes. MAT2A is up-regulated in many cancers, including liver cancer and colorectal cancer (CRC) and is a potentially important drug target. We developed a family of fluorinated N,N-dialkylaminostilbene agents, called FIDAS agents, that inhibit the proliferation of CRC cells in vitro and in vivo. Using a biotinylated FIDAS analogue, we identified the catalytic subunit of MAT2A as the direct and exclusive binding target of these FIDAS agents. MAT2B, an associated regulatory subunit of MAT2A, binds indirectly to FIDAS agents through its association with MAT2A. FIDAS agents inhibited MAT2A activity in SAM synthesis, and depletion of MAT2A by shRNAs inhibited CRC cell growth. A novel FIDAS agent delivered orally repressed CRC xenografts in athymic nude mice. These findings suggest that FIDAS analogues targeting MAT2A represent a family of novel and potentially useful agents for cancer treatment.


Subject(s)
Colonic Neoplasms/prevention & control , Methionine Adenosyltransferase/drug effects , Stilbenes/pharmacology , Animals , Biocatalysis , Colonic Neoplasms/enzymology , Fluorine/chemistry , Humans , Methionine Adenosyltransferase/metabolism , Mice , Models, Molecular , Stilbenes/chemistry , Transplantation, Heterologous
13.
Carcinogenesis ; 33(6): 1239-46, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22491752

ABSTRACT

Kruppel-like factor 4 (KLF4) is a transcription factor that is highly expressed in differentiated epithelial cells including that of the skin. It is critical for specification or function of differentiated epithelial cells. Moreover, KLF4 functions either as a tumor suppressor or an oncogene depending on different cellular contexts. However, the role of KLF4 in skin tumorigenesis remains controversial. To address this issue, we first examined KLF4 expression using a cohort of samples from patients with skin squamous cell carcinoma and basal cell carcinoma and found that in 21 of 24 tumor tissues (87.5%), KLF4 expression as assayed by immunohistochemistry was absent when compared with that in normal tissues. In addition, knockdown of KLF4 in human epidermal squamous cell carcinoma SCC13 cells was accompanied by increased cell growth. Further analysis revealed that KLF4 deficiency promoted cell migration and adhesion, which are the important properties of tumor cells. These observations were supported by the effect upon overexpression of KLF4 in SCC13 cells. Furthermore, we generated a novel tamoxifen-inducible KLF4/CreER and KLF4(flox) double transgenic mouse model to examine the role of KLF4 in skin cancer development. Consistent with in vitro studies, KLF4 deficiency increased the ability of migration and adhesion of mouse primary skin keratinocytes. Moreover, KLF4 knockout led to increased cell proliferation and skin carcinogenesis in a classical DMBA/TPA mouse skin cancer model. Taken together, our data suggest that KLF4 inhibits cell proliferation, migration and adhesion and that loss of KLF4 promotes skin tumorigenesis.


Subject(s)
Carcinoma, Basal Cell/metabolism , Carcinoma, Squamous Cell/metabolism , Kruppel-Like Transcription Factors/deficiency , Kruppel-Like Transcription Factors/metabolism , Skin Neoplasms/metabolism , Skin Neoplasms/pathology , 9,10-Dimethyl-1,2-benzanthracene , Animals , Carcinoma, Basal Cell/genetics , Carcinoma, Basal Cell/pathology , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/pathology , Cell Adhesion/genetics , Cell Differentiation , Cell Movement/genetics , Cell Proliferation , Cell Transformation, Neoplastic , Cells, Cultured , Female , Gene Expression Regulation, Neoplastic , Humans , Keratinocytes/metabolism , Kruppel-Like Factor 4 , Kruppel-Like Transcription Factors/genetics , Mice , Mice, Transgenic , RNA Interference , RNA, Small Interfering , Skin Neoplasms/chemically induced , Tamoxifen/pharmacology , Tetradecanoylphorbol Acetate
14.
PLoS One ; 7(2): e32492, 2012.
Article in English | MEDLINE | ID: mdl-22384261

ABSTRACT

Krüppel-like factor 4 (KLF4) is a zinc finger transcription factor that plays a vital role in regulating cell lineage differentiation during development and maintaining epithelial homeostasis in the intestine. In normal intestine, KLF4 is predominantly expressed in the differentiated epithelial cells. It has been identified as a tumor suppressor in colorectal cancer. KLF4 knockout mice demonstrated a decrease in number of goblet cells in the colon, and conditional ablation of KLF4 from the intestinal epithelium led to altered epithelial homeostasis. However, the role of KLF4 in differentiated intestinal cells and colon cancer cells, as well as the mechanism by which it regulates homeostasis and represses tumorigenesis in the intestine is not well understood. In our study, KLF4 was partially depleted in the differentiated intestinal epithelial cells by a tamoxifen-inducible Cre recombinase. We found a significant increase in the number of goblet cells in the KLF4-deleted small intestine, suggesting that KLF4 is not only required for goblet cell differentiation, but also required for maintaining goblet cell numbers through its function in inhibiting cell proliferation. The number and position of Paneth cells also changed. This is consistent with the KLF4 knockout study using villin-Cre [1]. Through immunohistochemistry (IHC) staining and statistical analysis, we found that a stem cell and/or tuft cell marker, DCAMKL1, and a proliferation marker, Ki67, are affected by KLF4 depletion, while an enteroendocrine cell marker, neurotensin (NT), was not affected. In addition, we found KLF4 depletion altered the morphology and polarity of the intestinal epithelial cells. Using a three-dimensional (3D) intestinal epithelial cyst formation assay, we found that KLF4 is essential for cell polarity and crypt-cyst formation in human colon cancer cells. These findings suggest that, as a tumor suppressor in colorectal cancer, KLF4 affects intestinal epithelial cell morphology by regulating proliferation, differentiation and polarity of the cells.


Subject(s)
Epithelial Cells/cytology , Gene Expression Regulation, Neoplastic , Intestines/cytology , Kruppel-Like Transcription Factors/physiology , Animals , Caco-2 Cells , Chromosomes, Artificial, Bacterial , Colonic Neoplasms/metabolism , DNA, Complementary/metabolism , Homeostasis , Humans , Immunohistochemistry/methods , Intestinal Neoplasms/metabolism , Kruppel-Like Factor 4 , Kruppel-Like Transcription Factors/metabolism , Mice , Mice, Knockout , Mice, Transgenic
15.
J Biol Chem ; 287(6): 3760-8, 2012 Feb 03.
Article in English | MEDLINE | ID: mdl-22170051

ABSTRACT

B lymphoma Mo-MLV insertion region 1 (Bmi1) is a Polycomb Group (PcG) protein important in gene silencing. It is a component of Polycomb Repressive Complex 1 (PRC1), which is required to maintain the transcriptionally repressive state of many genes. Bmi1 was initially identified as an oncogene that regulates cell proliferation and transformation, and is important in hematopoiesis and the development of nervous systems. Recently, it was reported that Bmi1 is a potential marker for intestinal stem cells. Because Wnt signaling plays a key role in intestinal stem cells, we analyzed the effects of Wnt signaling on Bmi1 expression. We found that Wnt signaling indeed regulates the expression of Bmi1 in colon cancer cells. In addition, the expression of Bmi1 in human colon cancers is significantly associated with nuclear ß-catenin, a hallmark for the activated Wnt signaling. Krüppel-like factor 4 (KLF4) is a zinc finger protein highly expressed in the gut and skin. We recently found that KLF4 cross-talks with Wnt/ß-catenin in regulating intestinal homeostasis. We demonstrated that KLF4 directly inhibits the expression of Bmi1 in colon cancer cells. We also found that Bmi1 regulates histone ubiquitination and is required for colon cancer proliferation in vitro and in vivo. Our findings further suggest that Bmi1 is an attractive target for cancer therapeutics.


Subject(s)
Biomarkers, Tumor/metabolism , Colonic Neoplasms/metabolism , Gene Expression Regulation, Neoplastic , Intestinal Mucosa/metabolism , Kruppel-Like Transcription Factors/metabolism , Neoplasm Proteins/metabolism , Nuclear Proteins/biosynthesis , Proto-Oncogene Proteins/biosynthesis , Repressor Proteins/biosynthesis , Wnt Signaling Pathway , beta Catenin/metabolism , Animals , Biomarkers, Tumor/genetics , Colonic Neoplasms/genetics , Colonic Neoplasms/pathology , Histones , Humans , Intestines/pathology , Kruppel-Like Factor 4 , Kruppel-Like Transcription Factors/genetics , Mice , Mice, Nude , Neoplasm Proteins/genetics , Neoplasm Transplantation , Nuclear Proteins/genetics , Polycomb Repressive Complex 1 , Proto-Oncogene Proteins/genetics , Repressor Proteins/genetics , Stem Cells/metabolism , Stem Cells/pathology , Transplantation, Heterologous , Ubiquitination/genetics , beta Catenin/genetics
16.
J Med Chem ; 54(5): 1288-97, 2011 Mar 10.
Article in English | MEDLINE | ID: mdl-21291235

ABSTRACT

Colorectal cancer (CRC) is the second leading cause of cancer-related mortality in the United States. CRC is initiated by mutations of the tumor suppressor gene, adenomatous polyposis coli (APC), or ß-catenin gene. These mutations stabilize ß-catenin and constitutively activate Wnt/ß-catenin target genes, such as c-Myc and cyclin D1, ultimately leading to cancer. Naturally occurring stilbene derivatives, resveratrol and pterostilbene, inhibit Wnt signaling and repress CRC cell proliferation but are ineffective at concentrations less than 10 µM. To understand the structure--activity relationship within these stilbene derivatives and to develop more efficacious Wnt inhibitors than these natural products, we synthesized and evaluated a panel of fluorinated N,N-dialkylaminostilbenes. Among this panel, (E)-4-(2,6-difluorostyryl)-N,N-dimethylaniline (4r) inhibits Wnt signaling at nanomolar levels and inhibits the growth of human CRC cell xenografts in athymic nude mice at a dosage of 20 mg/kg. These fluorinated N,N-dialkylaminostilbenes appear to inhibit Wnt signaling downstream of ß-catenin, probably at the transcriptional level.


Subject(s)
Colonic Neoplasms/drug therapy , Stilbenes/chemical synthesis , Wnt Proteins/physiology , Animals , Cell Line, Tumor , Cell Proliferation/drug effects , Colonic Neoplasms/pathology , Drug Screening Assays, Antitumor , Humans , Mice , Mice, Nude , Neoplasm Transplantation , Resveratrol , Signal Transduction/drug effects , Stereoisomerism , Stilbenes/chemistry , Stilbenes/pharmacology , Structure-Activity Relationship , Transplantation, Heterologous
17.
J Biol Chem ; 284(34): 22649-56, 2009 Aug 21.
Article in English | MEDLINE | ID: mdl-19556239

ABSTRACT

A central question in Wnt signaling is the regulation of beta-catenin phosphorylation and degradation. Multiple kinases, including CKI alpha and GSK3, are involved in beta-catenin phosphorylation. Protein phosphatases such as PP2A and PP1 have been implicated in the regulation of beta-catenin. However, which phosphatase dephosphorylates beta-catenin in vivo and how the specificity of beta-catenin dephosphorylation is regulated are not clear. In this study, we show that PP2A regulates beta-catenin phosphorylation and degradation in vivo. We demonstrate that PP2A is required for Wnt/beta-catenin signaling in Drosophila. Moreover, we have identified PR55 alpha as the regulatory subunit of PP2A that controls beta-catenin phosphorylation and degradation. PR55 alpha, but not the catalytic subunit, PP2Ac, directly interacts with beta-catenin. RNA interference knockdown of PR55 alpha elevates beta-catenin phosphorylation and decreases Wnt signaling, whereas overexpressing PR55 alpha enhances Wnt signaling. Taken together, our results suggest that PR55 alpha specifically regulates PP2A-mediated beta-catenin dephosphorylation and plays an essential role in Wnt signaling.


Subject(s)
Protein Phosphatase 2/metabolism , beta Catenin/metabolism , Animals , Axin Protein , Blotting, Western , Cell Line , Cell Line, Tumor , Drosophila melanogaster , Enzyme Inhibitors/pharmacology , Glycogen Synthase Kinase 3/antagonists & inhibitors , Glycogen Synthase Kinase 3/metabolism , Humans , Immunoprecipitation , Lithium Chloride/pharmacology , Okadaic Acid/pharmacology , Phosphorylation/drug effects , Protein Binding , Protein Phosphatase 2/antagonists & inhibitors , Protein Phosphatase 2/genetics , Protein Subunits , Pyrans/pharmacology , RNA, Small Interfering , Repressor Proteins/genetics , Repressor Proteins/metabolism , Spiro Compounds/pharmacology
18.
J Biol Chem ; 282(8): 5842-52, 2007 Feb 23.
Article in English | MEDLINE | ID: mdl-17179143

ABSTRACT

The essentially infinite expansion potential and pluripotency of human embryonic stem cells (hESCs) makes them attractive for cell-based therapeutics. In contrast to mouse embryonic stem cells (mESCs), hESCs normally undergo high rates of spontaneous apoptosis and differentiation, making them difficult to maintain in culture. Here we demonstrate that p53 protein accumulates in apoptotic hESCs induced by agents that damage DNA. However, despite the accumulation of p53, it nevertheless fails to activate the transcription of its target genes. This inability of p53 to activate its target genes has not been observed in other cell types, including mESCs. We further demonstrate that p53 induces apoptosis of hESCs through a mitochondrial pathway. Reducing p53 expression in hESCs in turn reduces both DNA damage-induced apoptosis as well as spontaneous apoptosis. Reducing p53 expression also reduces spontaneous differentiation and slows the differentiation rate of hESCs. Our studies reveal the important roles of p53 as a critical mediator of human embryonic stem cells survival and differentiation.


Subject(s)
Apoptosis/physiology , Cell Differentiation/physiology , DNA Damage , Embryonic Stem Cells/metabolism , Gene Expression Regulation/physiology , Tumor Suppressor Protein p53/biosynthesis , Animals , Cells, Cultured , Embryonic Stem Cells/cytology , Humans , Mice , Mitochondria/metabolism
19.
Biochem Biophys Res Commun ; 346(1): 131-9, 2006 Jul 21.
Article in English | MEDLINE | ID: mdl-16753134

ABSTRACT

Traditionally, undifferentiated human embryonic stem cells (hESCs) are maintained on mouse embryonic fibroblast (MEF) cells or on matrigel with an MEF-conditioned medium (CM), which hampers the clinical applications of hESCs due to the contamination by animal pathogens. Here we report a novel chemical-defined medium using DMEM/F12 supplemented with N2, B27, and basic fibroblast growth factor (bFGF) [termed NBF]. This medium can support prolonged self-renewal of hESCs. hESCs cultured in NBF maintain an undifferentiated state and normal karyotype, are able to form embryoid bodies in vitro, and differentiate into three germ layers and extraembryonic cells. Furthermore, we find that hESCs cultured in NBF possess a low apoptosis rate and a high proliferation rate compared with those cultured in MEF-CM. Our findings provide a novel, simplified chemical-defined culture medium suitable for further therapeutic applications and developmental studies of hESCs.


Subject(s)
Cell Culture Techniques/methods , Culture Media/chemistry , Fibroblast Growth Factor 2 , HLA-B27 Antigen , Pluripotent Stem Cells/cytology , Apoptosis , Cell Differentiation , Humans
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