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1.
Br J Cancer ; 130(1): 151-162, 2024 01.
Artigo em Inglês | MEDLINE | ID: mdl-37968472

RESUMO

BACKGROUND: Aberrant WNT/ß-catenin signaling drives carcinogenesis. Tankyrases poly(ADP-ribosyl)ate and destabilize AXINs, ß-catenin repressors. Tankyrase inhibitors block WNT/ß-catenin signaling and colorectal cancer (CRC) growth. We previously reported that 'short' APC mutations, lacking all seven ß-catenin-binding 20-amino acid repeats (20-AARs), are potential predictive biomarkers for CRC cell sensitivity to tankyrase inhibitors. Meanwhile, 'Long' APC mutations, which possess more than one 20-AAR, do not predict inhibitor-resistant cells. Thus, additional biomarkers are needed to precisely predict the inhibitor sensitivity. METHODS: Using 47 CRC patient-derived cells (PDCs), we examined correlations between the sensitivity to tankyrase inhibitors (G007-LK and RK-582), driver mutations, and the expressions of signaling factors. NOD.CB17-Prkdcscid/J and BALB/c-nu/nu xenograft mice were treated with RK-582. RESULTS: Short APC mutant CRC cells exhibited high/intermediate sensitivities to tankyrase inhibitors in vitro and in vivo. Active ß-catenin levels correlated with inhibitor sensitivity in both short and long APC mutant PDCs. PIK3CA mutations, but not KRAS/BRAF mutations, were more frequent in inhibitor-resistant PDCs. Some wild-type APC PDCs showed inhibitor sensitivity in a ß-catenin-independent manner. CONCLUSIONS: APC/PIK3CA mutations and ß-catenin predict the sensitivity of APC-mutated CRC PDCs to tankyrase inhibitors. These observations may help inform the strategy of patient selection in future clinical trials of tankyrase inhibitors.


Assuntos
Neoplasias Colorretais , Tanquirases , Animais , Camundongos , Humanos , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Tanquirases/genética , Tanquirases/metabolismo , Linhagem Celular Tumoral , beta Catenina/genética , beta Catenina/metabolismo , Camundongos Endogâmicos NOD , Via de Sinalização Wnt/genética , Biomarcadores , Classe I de Fosfatidilinositol 3-Quinases/genética , Classe I de Fosfatidilinositol 3-Quinases/metabolismo
2.
Biochem Biophys Res Commun ; 522(4): 945-951, 2020 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-31806370

RESUMO

Tankyrases (TNKS and TNKS2) are members of poly(ADP-ribose) polymerase (PARP) family proteins. Tankyrase has multiple ankyrin repeat cluster (ARC) domains, which recognize the tankyrase-binding motifs in proteins including the telomeric protein, TRF1 and Wnt signal regulators, AXINs. However, the functional significance of tankyrase interaction with many other putative binding proteins remains unknown. Here, we found that several proteins involved in microRNA (miRNA) processing have putative tankyrase-binding motifs and their functions are regulated by tankyrase. First, chemical inhibition of tankyrase PARP activity downregulated the expression levels of precursor miRNAs (pre-miRNAs) but not primary precursor miRNAs (pri-miRNAs). A subsequent reporter assay revealed that tankyrase inhibitors or PARP-dead mutant tankyrase overexpression repress pri-miRNA processing to pre-miRNA. Conversely, a PARP-1/2 inhibitor, olaparib, did not affect pri-miRNA processing. Tankyrase ARCs bound to DGCR8 and DROSHA, which are essential components for pri-miRNA processing and have putative tankyrase-binding motifs. These observations indicate that tankyrase binds to Microprocessor, DGCR8 and DROSHA complex and modulates pri-miRNA processing to pre-miRNA.


Assuntos
MicroRNAs/metabolismo , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA/genética , Tanquirases/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Humanos , Camundongos , MicroRNAs/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Ligação Proteica , Precursores de RNA/genética , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Ribonuclease III/química , Ribonuclease III/metabolismo
3.
Cancer Sci ; 109(12): 4003-4014, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30238564

RESUMO

Aberrant activation of Wnt/ß-catenin signaling causes tumorigenesis and promotes the proliferation of colorectal cancer cells. Porcupine inhibitors, which block secretion of Wnt ligands, may have only limited clinical impact for the treatment of colorectal cancer, because most colorectal cancer is caused by loss-of-function mutations of the tumor suppressor adenomatous polyposis coli (APC) downstream of Wnt ligands. Tankyrase poly(ADP-ribosyl)ates (PARylates) Axin, a negative regulator of ß-catenin. This post-translational modification causes ubiquitin-dependent degradation of Axin, resulting in ß-catenin accumulation. Tankyrase inhibitors downregulate ß-catenin and suppress the growth of APC-mutated colorectal cancer cells. Herein, we report a novel tankyrase-specific inhibitor RK-287107, which inhibits tankyrase-1 and -2 four- and eight-fold more potently, respectively, than G007-LK, a tankyrase inhibitor that has been previously reported as effective in mouse xenograft models. RK-287107 causes Axin2 accumulation and downregulates ß-catenin, T-cell factor/lymphoid enhancer factor reporter activity and the target gene expression in colorectal cancer cells harboring the shortly truncated APC mutations. Consistently, RK-287107 inhibits the growth of APC-mutated (ß-catenin-dependent) colorectal cancer COLO-320DM and SW403 cells but not the APC-wild (ß-catenin-independent) colorectal cancer RKO cells. Intraperitoneal or oral administration of RK-287107 suppresses COLO-320DM tumor growth in NOD-SCID mice. Rates of tumor growth inhibition showed good correlation with the behavior of pharmacodynamic biomarkers, such as Axin2 accumulation and MYC downregulation. These observations indicate that RK-287107 exerts a proof-of-concept antitumor effect, and thus may have potential for tankyrase-directed molecular cancer therapy.


Assuntos
Proteína da Polipose Adenomatosa do Colo/genética , Antineoplásicos/administração & dosagem , Neoplasias Colorretais/tratamento farmacológico , Inibidores Enzimáticos/administração & dosagem , Tanquirases/antagonistas & inibidores , Administração Oral , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/genética , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Células HCT116 , Humanos , Camundongos , Mutação , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Cancer Sci ; 104(3): 345-52, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23279575

RESUMO

Overexpression of the ErbB2/HER2 receptor tyrosine kinase contributes to tumorigenesis. However, mechanisms regulating ErbB2 protein levels remain largely unclear. Here, we identified novel mechanisms of ErbB2 downregulation. ErbB2 constitutively binds to an adaptor protein FRS2ß. We found that FRS2ß bound to CD2AP and CIN85, which induces endosomal trafficking that targets lysosomes. FRS2ß colocalized with CIN85 in the cytoplasm. Expression of wild type FRS2ß but not its CIN85 non-binding mutant, downregulated the ErbB2 protein and inhibited anchorage-independent cell growth. Moreover, the E3 ubiquitin-protein ligase Cbl was contained within a complex of FRS2ß and CIN85. Knockdown of both CIN85 and CD2AP or of Cbl, or treatment with lysosomal degradation inhibitors diminished FRS2ß downregulation of ErbB2. In addition, knockdown of endogenous FRS2ß caused upregulation of ErbB2 in primary neural cells. Finally, immunohistochemical analysis showed that human breast cancer tissues that overexpress ErbB2 expressed low levels of FRS2ß. Thus, an FRS2ß-CIN85/CD2AP-Cbl axis for downregulation of ErbB2 may regulate ErbB2 protein levels in physiological and pathological settings. Molecular targeting drugs that can increase or stabilize the ErbB2-FRS2ß-CIN85/CD2AP-Cbl axis may have promise for the control of ErbB2-overexpressing tumors.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fosfoproteínas/metabolismo , Receptor ErbB-2/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Motivos de Aminoácidos , Neoplasias da Mama/metabolismo , Células Cultivadas , Regulação para Baixo , Feminino , Técnicas de Silenciamento de Genes , Humanos , Neurônios/metabolismo , Fosfoproteínas/genética , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Transdução de Sinais
5.
J Biol Chem ; 286(34): 29848-60, 2011 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-21646355

RESUMO

Specific regulation of target genes by transforming growth factor-ß (TGF-ß) in a given cellular context is determined in part by transcription factors and cofactors that interact with the Smad complex. In this study, we determined Smad2 and Smad3 (Smad2/3) binding regions in the promoters of known genes in HepG2 hepatoblastoma cells, and we compared them with those in HaCaT epidermal keratinocytes to elucidate the mechanisms of cell type- and context-dependent regulation of transcription induced by TGF-ß. Our results show that 81% of the Smad2/3 binding regions in HepG2 cells were not shared with those found in HaCaT cells. Hepatocyte nuclear factor 4α (HNF4α) is expressed in HepG2 cells but not in HaCaT cells, and the HNF4α-binding motif was identified as an enriched motif in the HepG2-specific Smad2/3 binding regions. Chromatin immunoprecipitation sequencing analysis of HNF4α binding regions under TGF-ß stimulation revealed that 32.5% of the Smad2/3 binding regions overlapped HNF4α bindings. MIXL1 was identified as a new combinatorial target of HNF4α and Smad2/3, and both the HNF4α protein and its binding motif were required for the induction of MIXL1 by TGF-ß in HepG2 cells. These findings generalize the importance of binding of HNF4α on Smad2/3 binding genomic regions for HepG2-specific regulation of transcription by TGF-ß and suggest that certain transcription factors expressed in a cell type-specific manner play important roles in the transcription regulated by the TGF-ß-Smad signaling pathway.


Assuntos
Fator 4 Nuclear de Hepatócito/metabolismo , Proteínas de Homeodomínio/metabolismo , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Motivos de Aminoácidos , Células Hep G2 , Fator 4 Nuclear de Hepatócito/genética , Proteínas de Homeodomínio/genética , Humanos , Especificidade de Órgãos/fisiologia , Ligação Proteica , Proteína Smad2/genética , Proteína Smad3/genética , Fator de Crescimento Transformador beta/farmacologia
6.
J Med Chem ; 63(8): 4183-4204, 2020 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-32202790

RESUMO

Tankyrases (TNKS/TNKS2) belong to the poly(ADP-ribose) polymerase family. Inhibition of their enzymatic activities attenuates the Wnt/ß-catenin signaling, which plays an important role in cancer pathogenesis. We previously reported the discovery of RK-287107, a spiroindoline-based, highly selective, potent tankyrase inhibitor. Herein we describe the optimization process of RK-287107 leading to RK-582, which exhibits a markedly improved robust tumor growth inhibition in a COLO-320DM mouse xenograft model when orally administered. In addition to the dose-dependent elevation and attenuation of the levels of biomarkers AXIN2 and ß-catenin, respectively, results of the TCF reporter and cell proliferation studies on COLO-320DM are discussed.


Assuntos
Neoplasias do Colo/tratamento farmacológico , Desenho de Fármacos , Descoberta de Drogas/métodos , Inibidores Enzimáticos/administração & dosagem , Tanquirases/antagonistas & inibidores , Administração Oral , Animais , Linhagem Celular Tumoral , Neoplasias do Colo/enzimologia , Inibidores Enzimáticos/química , Feminino , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Estrutura Terciária de Proteína , Ratos , Tanquirases/química , Tanquirases/metabolismo , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
7.
Oncogene ; 38(14): 2580-2594, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30531840

RESUMO

Lung cancer is one of the major causes of cancer death and clarification of its molecular pathology is highly prioritized. The physiological importance of mRNA degradation through the CCR4-NOT deadenylase has recently been highlighted. For example, mutation in CNOT3, a gene coding for CNOT3 subunit of the CCR4-NOT complex, is found to be associated with T-cell acute lymphoblastic leukemia, T-ALL, though its contribution to other cancers has not been reported. Here, we provide evidence suggesting that CNOT3 is required for the growth of non-small cell lung cancer. Depletion of CNOT3 suppresses proliferation of A549 human non-small cell lung cancer cells with enhanced mRNA stability and subsequent elevated expression of p21. In addition, we identified the mRNA for Krüppel-like factor 2 transcription factor, an inducer of p21, as a novel mRNA degradation target of CNOT3 in non-small cell lung cancer cells. Aberrant up-regulation of Krüppel-like factor 2 by CNOT3 depletion leads to impairment in the proliferation of A549 cells. Consistent with these findings, elevated mRNA expression of CNOT3 in non-small cell lung cancer in comparison with the paired normal lung epithelium was confirmed through scrutinization of the RNA-sequencing datasets from The Cancer Genome Atlas. Moreover, we found an inverse correlation between CNOT3 and CDKN1A (encoding p21) mRNA expression using the combined datasets of normal lung epithelium and non-small cell lung cancer. Thus, we propose that the up-regulation of CNOT3 facilitates the development of non-small cell lung cancer through down-regulation of Krüppel-like factor 2 and p21, contrary to tumor suppressive functions of CNOT3 in T-ALL.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Ciclo Celular/genética , Neoplasias Pulmonares/genética , Fatores de Transcrição/genética , Células A549 , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Regulação para Baixo/genética , Regulação da Expressão Gênica/genética , Humanos , Fatores de Transcrição Kruppel-Like/genética , Neoplasias Pulmonares/patologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Estabilidade de RNA/genética , RNA Mensageiro/genética , Mucosa Respiratória/patologia , Regulação para Cima/genética
8.
J Med Chem ; 62(7): 3407-3427, 2019 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-30883102

RESUMO

The canonical WNT pathway plays an important role in cancer pathogenesis. Inhibition of poly(ADP-ribose) polymerase catalytic activity of the tankyrases (TNKS/TNKS2) has been reported to reduce the Wnt/ß-catenin signal by preventing poly ADP-ribosylation-dependent degradation of AXIN, a negative regulator of Wnt/ß-catenin signaling. With the goal of investigating the effects of tankyrase and Wnt pathway inhibition on tumor growth, we set out to find small-molecule inhibitors of TNKS/TNKS2 with suitable drug-like properties. Starting from 1a, a high-throughput screening hit, the spiroindoline derivative 40c (RK-287107) was discovered as a potent TNKS/TNKS2 inhibitor with >7000-fold selectivity against the PARP1 enzyme, which inhibits WNT-responsive TCF reporter activity and proliferation of human colorectal cancer cell line COLO-320DM. RK-287107 also demonstrated dose-dependent tumor growth inhibition in a mouse xenograft model. These observations suggest that RK-287107 is a promising lead compound for the development of novel tankyrase inhibitors as anticancer agents.


Assuntos
Inibidores Enzimáticos/farmacologia , Indóis/química , Indóis/farmacologia , Compostos de Espiro/farmacologia , Tanquirases/antagonistas & inibidores , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Ensaios de Triagem em Larga Escala , Humanos , Camundongos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Compostos de Espiro/química , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Sci Rep ; 7(1): 1166, 2017 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-28446749

RESUMO

Epithelial-mesenchymal transition (EMT) is induced by transforming growth factor (TGF)-ß and facilitates tumor progression. We here performed global mapping of accessible chromatin in the mouse mammary gland epithelial EpH4 cell line and its Ras-transformed derivative (EpRas) using formaldehyde-assisted isolation of regulatory element (FAIRE)-sequencing. TGF-ß and Ras altered chromatin accessibility either cooperatively or independently, and AP1, ETS, and RUNX binding motifs were enriched in the accessible chromatin regions of EpH4 and EpRas cells. Etv4, an ETS family oncogenic transcription factor, was strongly expressed and bound to more than one-third of the accessible chromatin regions in EpRas cells treated with TGF-ß. While knockdown of Etv4 and another ETS family member Etv5 showed limited effects on the decrease in the E-cadherin abundance and stress fiber formation by TGF-ß, gene ontology analysis showed that genes encoding extracellular proteins were most strongly down-regulated by Etv4 and Etv5 siRNAs. Accordingly, TGF-ß-induced expression of Mmp13 and cell invasiveness were suppressed by Etv4 and Etv5 siRNAs, which were accompanied by the reduced chromatin accessibility at an enhancer region of Mmp13 gene. These findings suggest a mechanism of transcriptional regulation during Ras- and TGF-ß-induced EMT that involves alterations of accessible chromatin, which are partly regulated by Etv4 and Etv5.


Assuntos
Transformação Celular Neoplásica , Cromatina/metabolismo , Células Epiteliais/fisiologia , Transição Epitelial-Mesenquimal , Glândulas Mamárias Animais/citologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , DNA/metabolismo , Regulação da Expressão Gênica , Camundongos , Ligação Proteica
10.
Oncotarget ; 8(29): 47902-47915, 2017 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-28615517

RESUMO

Activation of Wnt/ß-catenin signaling is essential for colorectal carcinogenesis. Tankyrase, a member of the poly(ADP-ribose) polymerase (PARP) family, is a positive regulator of the Wnt/ß-catenin signaling. Accordingly, tankyrase inhibitors are under preclinical development for colorectal cancer (CRC) therapy. However, Wnt-driven colorectal cancer cells are not equally sensitive to tankyrase inhibitors, and cellular factors that affect tankyrase inhibitor sensitivity remain elusive. Here, we established a tankyrase inhibitor-resistant cell line, 320-IWR, from Wnt/ß-catenin-dependent CRC COLO-320DM cells. 320-IWR cells exhibited resistance to tankyrase inhibitors, IWR-1 and G007-LK, but remained sensitive to a PARP-1/2 inhibitor, olaparib, and several anti-CRC agents. In 320-IWR cells, nuclear localization of active ß-catenin was decreased and expression of ß-catenin target genes was constitutively repressed, suggesting that these cells repressed the Wnt/ß-catenin signaling and were dependent on alternative proliferation pathways. 320-IWR cells exhibited upregulated mTOR signaling and were more sensitive to mTOR inhibition than the parental cells. Importantly, mTOR inhibition reversed resistance to tankyrase inhibitors and potentiated their anti-proliferative effects in 320-IWR cells as well as in CRC cell lines in which the mTOR pathway was intrinsically activated. These results indicate that mTOR signaling confers resistance to tankyrase inhibitors in CRC cells and suggest that the combination of tankyrase and mTOR inhibitors would be a useful therapeutic approach for a subset of CRCs.


Assuntos
Neoplasias Colorretais/metabolismo , Resistencia a Medicamentos Antineoplásicos , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Tanquirases/antagonistas & inibidores , Proteínas Wnt/metabolismo , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Biologia Computacional/métodos , Relação Dose-Resposta a Droga , Humanos , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/metabolismo
11.
Mol Oncol ; 11(9): 1241-1262, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28618162

RESUMO

Zinc finger E-box binding protein 1 (ZEB1) and ZEB2 induce epithelial-mesenchymal transition (EMT) and enhance cancer progression. However, the global view of transcriptional regulation by ZEB1 and ZEB2 is yet to be elucidated. Here, we identified a ZEB1-regulated inflammatory phenotype in breast cancer cells using chromatin immunoprecipitation sequencing and RNA sequencing, followed by gene set enrichment analysis (GSEA) of ZEB1-bound genes. Knockdown of ZEB1 and/or ZEB2 resulted in the downregulation of genes encoding inflammatory cytokines related to poor prognosis in patients with cancer, including IL6 and IL8, therefore suggesting that ZEB1 and ZEB2 have similar functions in terms of the regulation of production of inflammatory cytokines. Antibody array and ELISA experiments confirmed that ZEB1 controlled the production of the IL-6 and IL-8 proteins. The secretory proteins regulated by ZEB1 enhanced breast cancer cell proliferation and tumor growth. ZEB1 expression in breast cancer cells also affected the growth of fibroblasts in cell culture, and the accumulation of myeloid-derived suppressor cells in tumors in vivo. These findings provide insight into the role of ZEB1 in the progression of cancer, mediated by inflammatory cytokines, along with the initiation of EMT.


Assuntos
Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo , Animais , Sequência de Bases , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Quimiocinas/metabolismo , Meios de Cultivo Condicionados/farmacologia , DNA de Neoplasias/metabolismo , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Inflamatórias Mamárias/genética , Neoplasias Inflamatórias Mamárias/metabolismo , Neoplasias Inflamatórias Mamárias/patologia , Camundongos Endogâmicos BALB C , Camundongos Nus , Células Mieloides/efeitos dos fármacos , Células Mieloides/metabolismo , Comunicação Parácrina/efeitos dos fármacos , Fenótipo , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta/farmacologia , Homeobox 2 de Ligação a E-box com Dedos de Zinco/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genética
12.
Mol Cancer Ther ; 16(4): 752-762, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28179481

RESUMO

In most colorectal cancers, Wnt/ß-catenin signaling is activated by loss-of-function mutations in the adenomatous polyposis coli (APC) gene and plays a critical role in tumorigenesis. Tankyrases poly(ADP-ribosyl)ate and destabilize Axins, a negative regulator of ß-catenin, and upregulate ß-catenin signaling. Tankyrase inhibitors downregulate ß-catenin and are expected to be promising therapeutics for colorectal cancer. However, colorectal cancer cells are not always sensitive to tankyrase inhibitors, and predictive biomarkers for the drug sensitivity remain elusive. Here we demonstrate that the short-form APC mutations predict the sensitivity of colorectal cancer cells to tankyrase inhibitors. By using well-established colorectal cancer cell lines, we found that tankyrase inhibitors downregulated ß-catenin in the drug-sensitive, but not resistant, colorectal cancer cells. The drug-sensitive cells showed higher Tcf/LEF transcriptional activity than the resistant cells and possessed "short" truncated APCs lacking all seven ß-catenin-binding 20-amino acid repeats (20-AARs). In contrast, the drug-resistant cells possessed "long" APC retaining two or more 20-AARs. Knockdown of the long APCs with two 20-AARs increased ß-catenin, Tcf/LEF transcriptional activity and its target gene AXIN2 expression. Under these conditions, tankyrase inhibitors were able to downregulate ß-catenin in the resistant cells. These results indicate that the long APCs are hypomorphic mutants, whereas they exert a dominant-negative effect on Axin-dependent ß-catenin degradation caused by tankyrase inhibitors. Finally, we established 16 patient-derived colorectal cancer cells and confirmed that the tankyrase inhibitor-responsive cells harbor the short-form APC mutations. These observations exemplify the predictive importance of APC mutations, the most common genetic alteration in colorectal cancers, for molecular targeted therapeutics. Mol Cancer Ther; 16(4); 752-62. ©2017 AACR.


Assuntos
Proteína da Polipose Adenomatosa do Colo/genética , Biomarcadores Tumorais/genética , Neoplasias Colorretais/genética , Inibidores Enzimáticos/farmacologia , Tanquirases/antagonistas & inibidores , Via de Sinalização Wnt/efeitos dos fármacos , Proteína da Polipose Adenomatosa do Colo/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/enzimologia , Células HCT116 , Células HT29 , Compostos Heterocíclicos com 3 Anéis/farmacologia , Humanos , Imidas/farmacologia , Ligação Proteica , Quinolinas/farmacologia , Sulfonas/farmacologia , Triazóis/farmacologia
13.
Stem Cell Reports ; 6(1): 64-73, 2016 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-26771354

RESUMO

Bone morphogenetic protein (BMP) signaling exerts paradoxical roles in pluripotent stem cells (PSCs); it sustains self-renewal of mouse embryonic stem cells (ESCs), while it induces differentiation in other PSCs, including human ESCs. Here, we revisit the roles of BMP-4 using mouse ESCs (mESCs) in naive and primed states. SMAD1 and SMAD5, which transduce BMP signals, recognize enhancer regions together with KLF4 and KLF5 in naive mESCs. KLF4 physically interacts with SMAD1 and suppresses its activity. Consistently, a subpopulation of cells with active BMP-SMAD can be ablated without disturbing the naive state of the culture. Moreover, Smad1/5 double-knockout mESCs stay in the naive state, indicating that the BMP-SMAD pathway is dispensable for it. In contrast, the MEK5-ERK5 pathway mediates BMP-4-induced self-renewal of mESCs by inducing Klf2, a critical factor for the ground state pluripotency. Our study illustrates that BMP exerts its self-renewing effect through distinct functions of different Krüppel-like factors.


Assuntos
Proteína Morfogenética Óssea 4/metabolismo , Autorrenovação Celular/fisiologia , Fatores de Transcrição Kruppel-Like/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Animais , Western Blotting , Proteína Morfogenética Óssea 4/genética , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Autorrenovação Celular/genética , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Células Hep G2 , Humanos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Sistema de Sinalização das MAP Quinases/genética , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Camundongos Knockout , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Smad1/genética , Proteína Smad1/metabolismo , Proteína Smad5/genética , Proteína Smad5/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo
14.
Sci Signal ; 9(442): ra84, 2016 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-27555661

RESUMO

The p53 family of transcription factors includes p63, which is a master regulator of gene expression in epithelial cells. Determining whether p63 is tumor-suppressive or tumorigenic is complicated by isoform-specific and cellular context-dependent protein associations, as well as antagonism from mutant p53. ΔNp63 is an amino-terminal-truncated isoform, that is, the predominant isoform expressed in cancer cells of epithelial origin. In HaCaT keratinocytes, which have mutant p53 and ΔNp63, we found that mutant p53 antagonized ΔNp63 transcriptional activity but that activation of Ras or transforming growth factor-ß (TGF-ß) signaling pathways reduced the abundance of mutant p53 and strengthened target gene binding and activity of ΔNp63. Among the products of ΔNp63-induced genes was dual-specificity phosphatase 6 (DUSP6), which promoted the degradation of mutant p53, likely by dephosphorylating p53. Knocking down all forms of p63 or DUSP6 and DUSP7 (DUSP6/7) inhibited the basal or TGF-ß-induced or epidermal growth factor (which activates Ras)-induced migration and invasion in cultures of p53-mutant breast cancer and squamous skin cancer cells. Alternatively, overexpressing ΔNp63 in the breast cancer cells increased their capacity to colonize various tissues upon intracardiac injection in mice, and this was inhibited by knocking down DUSP6/7 in these ΔNp63-overexpressing cells. High abundance of ΔNp63 in various tumors correlated with poor prognosis in patients, and this correlation was stronger in patients whose tumors also had a mutation in the gene encoding p53. Thus, oncogenic Ras and TGF-ß signaling stimulate cancer progression through activation of the ΔNp63 transcriptional program.


Assuntos
Neoplasias da Mama/metabolismo , Movimento Celular , Proteína Oncogênica p21(ras)/metabolismo , Transdução de Sinais , Transcrição Gênica , Fator de Crescimento Transformador beta/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Transformada , Fosfatase 6 de Especificidade Dupla/genética , Fosfatase 6 de Especificidade Dupla/metabolismo , Feminino , Células HEK293 , Humanos , Proteína Oncogênica p21(ras)/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
15.
J Biochem ; 148(6): 733-41, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20965945

RESUMO

Arkadia is a positive regulator of transforming growth factor (TGF)-ß signalling that induces ubiquitin-dependent degradation of several inhibitory proteins of TGF-ß signalling through its C-terminal RING domain. We report here that, through yeast-two-hybrid screening for Arkadia-binding proteins, the µ2 subunit of clathrin-adaptor 2 (AP2) complex was identified as an interacting partner of Arkadia. Arkadia was located in both the nucleus and the cytosol in mammalian cells. The C-terminal YXXΦ-binding domain of the µ2 subunit associated with the N-terminal YALL motif of Arkadia. Arkadia ubiquitylated the µ2 subunit at Lys130. In addition, Arkadia interacted with the AP2 complex, and modified endocytosis of epidermal growth factor receptor (EGFR) induced by EGF. Arkadia thus appears to regulate EGF signalling by modulating endocytosis of EGFR through interaction with AP2 complex.


Assuntos
Complexo 2 de Proteínas Adaptadoras , Endocitose/fisiologia , Receptores ErbB , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Nucleares , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína/fisiologia , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta/metabolismo , Ubiquitina-Proteína Ligases , Ubiquitinação/fisiologia , Complexo 2 de Proteínas Adaptadoras/metabolismo , Animais , Linhagem Celular , Receptores ErbB/metabolismo , Feminino , Haplorrinos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Transfecção , Técnicas do Sistema de Duplo-Híbrido , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
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