RÉSUMÉ
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.
Sujet(s)
Tumeurs colorectales , Tankyrases , Animaux , Souris , Humains , Tumeurs colorectales/traitement médicamenteux , Tumeurs colorectales/génétique , Tumeurs colorectales/métabolisme , Tankyrases/génétique , Tankyrases/métabolisme , Lignée cellulaire tumorale , bêta-Caténine/génétique , bêta-Caténine/métabolisme , Souris de lignée NOD , Voie de signalisation Wnt/génétique , Marqueurs biologiques , Phosphatidylinositol 3-kinases de classe I/génétique , Phosphatidylinositol 3-kinases de classe I/métabolismeRÉSUMÉ
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.
Sujet(s)
Tumeurs du côlon/traitement médicamenteux , Conception de médicament , Découverte de médicament/méthodes , Antienzymes/administration et posologie , Tankyrases/antagonistes et inhibiteurs , Administration par voie orale , Animaux , Lignée cellulaire tumorale , Tumeurs du côlon/enzymologie , Antienzymes/composition chimique , Femelle , Humains , Souris , Souris de lignée NOD , Souris SCID , Structure tertiaire des protéines , Rats , Tankyrases/composition chimique , Tankyrases/métabolisme , Résultat thérapeutique , Tests d'activité antitumorale sur modèle de xénogreffe/méthodesRÉSUMÉ
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.
Sujet(s)
microARN/métabolisme , Précurseurs des ARN/métabolisme , Maturation post-transcriptionnelle des ARN/génétique , Tankyrases/métabolisme , Séquence d'acides aminés , Animaux , Lignée cellulaire , Humains , Souris , microARN/génétique , Poly(ADP-ribose) polymerases/métabolisme , Liaison aux protéines , Précurseurs des ARN/génétique , Protéines de liaison à l'ARN/composition chimique , Protéines de liaison à l'ARN/métabolisme , Ribonuclease III/composition chimique , Ribonuclease III/métabolismeRÉSUMÉ
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.
Sujet(s)
Antienzymes/pharmacologie , Indoles/composition chimique , Indoles/pharmacologie , Spiranes/pharmacologie , Tankyrases/antagonistes et inhibiteurs , Animaux , Antinéoplasiques/pharmacologie , Lignée cellulaire tumorale , Tests de criblage à haut débit , Humains , Souris , Bibliothèques de petites molécules/composition chimique , Bibliothèques de petites molécules/pharmacologie , Spiranes/composition chimique , Tests d'activité antitumorale sur modèle de xénogreffeRÉSUMÉ
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.
Sujet(s)
Carcinome pulmonaire non à petites cellules/génétique , Cycle cellulaire/génétique , Tumeurs du poumon/génétique , Facteurs de transcription/génétique , Cellules A549 , Carcinome pulmonaire non à petites cellules/anatomopathologie , Lignée cellulaire tumorale , Prolifération cellulaire/génétique , Inhibiteur p21 de kinase cycline-dépendante/génétique , Régulation négative/génétique , Régulation de l'expression des gènes/génétique , Humains , Facteurs de transcription Krüppel-like/génétique , Tumeurs du poumon/anatomopathologie , Leucémie-lymphome lymphoblastique à précurseurs T/génétique , Leucémie-lymphome lymphoblastique à précurseurs T/anatomopathologie , Stabilité de l'ARN/génétique , ARN messager/génétique , Muqueuse respiratoire/anatomopathologie , Régulation positive/génétiqueRÉSUMÉ
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.
Sujet(s)
Protéine de la polypose adénomateuse colique/génétique , Antinéoplasiques/administration et posologie , Tumeurs colorectales/traitement médicamenteux , Antienzymes/administration et posologie , Tankyrases/antagonistes et inhibiteurs , Administration par voie orale , Animaux , Antinéoplasiques/composition chimique , Antinéoplasiques/pharmacologie , Lignée cellulaire tumorale , Prolifération cellulaire/effets des médicaments et des substances chimiques , Survie cellulaire/effets des médicaments et des substances chimiques , Tumeurs colorectales/enzymologie , Tumeurs colorectales/génétique , Antienzymes/composition chimique , Antienzymes/pharmacologie , Cellules HCT116 , Humains , Souris , Mutation , Résultat thérapeutique , Tests d'activité antitumorale sur modèle de xénogreffeRÉSUMÉ
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.
Sujet(s)
Facteur de transcription Zeb1/métabolisme , Animaux , Séquence nucléotidique , Marqueurs biologiques tumoraux/métabolisme , Lignée cellulaire tumorale , Prolifération cellulaire/effets des médicaments et des substances chimiques , Chimiokines/métabolisme , Milieux de culture conditionnés/pharmacologie , ADN tumoral/métabolisme , Femelle , Fibroblastes/effets des médicaments et des substances chimiques , Fibroblastes/métabolisme , Fibroblastes/anatomopathologie , Régulation de l'expression des gènes tumoraux , Humains , Cancers du sein inflammatoires/génétique , Cancers du sein inflammatoires/métabolisme , Cancers du sein inflammatoires/anatomopathologie , Souris de lignée BALB C , Souris nude , Cellules myéloïdes/effets des médicaments et des substances chimiques , Cellules myéloïdes/métabolisme , Communication paracrine/effets des médicaments et des substances chimiques , Phénotype , Transduction du signal/effets des médicaments et des substances chimiques , Facteur de croissance transformant bêta/pharmacologie , Facteur de transcription Zeb2/métabolisme , Facteur de transcription Zeb1/génétiqueRÉSUMÉ
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.
Sujet(s)
Tumeurs colorectales/métabolisme , Résistance aux médicaments antinéoplasiques , Inhibiteurs de poly(ADP-ribose) polymérases/pharmacologie , Transduction du signal , Sérine-thréonine kinases TOR/métabolisme , Tankyrases/antagonistes et inhibiteurs , Protéines de type Wingless/métabolisme , Antinéoplasiques/pharmacologie , Lignée cellulaire tumorale , Prolifération cellulaire/effets des médicaments et des substances chimiques , Biologie informatique/méthodes , Relation dose-effet des médicaments , Humains , Voie de signalisation Wnt/effets des médicaments et des substances chimiques , bêta-Caténine/métabolismeRÉSUMÉ
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.
Sujet(s)
Transformation cellulaire néoplasique , Chromatine/métabolisme , Cellules épithéliales/physiologie , Transition épithélio-mésenchymateuse , Glandes mammaires animales/cytologie , Protéines proto-oncogènes p21(ras)/métabolisme , Facteur de croissance transformant bêta/métabolisme , Animaux , Sites de fixation , Lignée cellulaire , ADN/métabolisme , Régulation de l'expression des gènes , Souris , Liaison aux protéinesRÉSUMÉ
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.
Sujet(s)
Protéine de la polypose adénomateuse colique/génétique , Marqueurs biologiques tumoraux/génétique , Tumeurs colorectales/génétique , Antienzymes/pharmacologie , Tankyrases/antagonistes et inhibiteurs , Voie de signalisation Wnt/effets des médicaments et des substances chimiques , Protéine de la polypose adénomateuse colique/métabolisme , Sites de fixation , Lignée cellulaire tumorale , Prolifération cellulaire/effets des médicaments et des substances chimiques , Tumeurs colorectales/traitement médicamenteux , Tumeurs colorectales/enzymologie , Cellules HCT116 , Cellules HT29 , Composés hétérocycliques 3 noyaux/pharmacologie , Humains , Imides/pharmacologie , Liaison aux protéines , Quinoléines/pharmacologie , Sulfones/pharmacologie , Triazoles/pharmacologieRÉSUMÉ
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.
Sujet(s)
Tumeurs du sein/métabolisme , Mouvement cellulaire , Protéine oncogène p21(ras)/métabolisme , Transduction du signal , Transcription génétique , Facteur de croissance transformant bêta/métabolisme , Tumeurs du sein/génétique , Tumeurs du sein/anatomopathologie , Lignée de cellules transformées , Dual Specificity Phosphatase 6/génétique , Dual Specificity Phosphatase 6/métabolisme , Femelle , Cellules HEK293 , Humains , Protéine oncogène p21(ras)/génétique , Facteurs de transcription/génétique , Facteurs de transcription/métabolisme , Facteur de croissance transformant bêta/génétique , Protéine p53 suppresseur de tumeur/génétique , Protéine p53 suppresseur de tumeur/métabolisme , Protéines suppresseurs de tumeurs/génétique , Protéines suppresseurs de tumeurs/métabolismeRÉSUMÉ
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.
Sujet(s)
Protéine morphogénétique osseuse de type 4/métabolisme , Auto-renouvellement cellulaire/physiologie , Facteurs de transcription Krüppel-like/métabolisme , Cellules souches embryonnaires de souris/métabolisme , Animaux , Technique de Western , Protéine morphogénétique osseuse de type 4/génétique , Différenciation cellulaire/génétique , Différenciation cellulaire/physiologie , Auto-renouvellement cellulaire/génétique , Régulation de l'expression des gènes au cours du développement , Cellules HEK293 , Cellules HepG2 , Humains , Facteur-4 de type Kruppel , Facteurs de transcription Krüppel-like/génétique , Système de signalisation des MAP kinases/génétique , Système de signalisation des MAP kinases/physiologie , Souris , Souris knockout , Cellules souches embryonnaires de souris/cytologie , Cellules souches pluripotentes/cytologie , Cellules souches pluripotentes/métabolisme , Liaison aux protéines , RT-PCR , Protéine Smad-1/génétique , Protéine Smad-1/métabolisme , Protéine Smad-5/génétique , Protéine Smad-5/métabolisme , Danio zébré/embryologie , Danio zébré/génétique , Danio zébré/métabolismeRÉSUMÉ
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.
Sujet(s)
Protéines adaptatrices de la transduction du signal/métabolisme , Phosphoprotéines/métabolisme , Récepteur ErbB-2/métabolisme , Protéines adaptatrices de la transduction du signal/génétique , Motifs d'acides aminés , Tumeurs du sein/métabolisme , Cellules cultivées , Régulation négative , Femelle , Techniques de knock-down de gènes , Humains , Neurones/métabolisme , Phosphoprotéines/génétique , Protéines proto-oncogènes c-cbl/métabolisme , Transduction du signalRÉSUMÉ
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.
Sujet(s)
Facteur nucléaire hépatocytaire HNF-4/métabolisme , Protéines à homéodomaine/métabolisme , Protéine Smad2/métabolisme , Protéine Smad-3/métabolisme , Motifs d'acides aminés , Cellules HepG2 , Facteur nucléaire hépatocytaire HNF-4/génétique , Protéines à homéodomaine/génétique , Humains , Spécificité d'organe/physiologie , Liaison aux protéines , Protéine Smad2/génétique , Protéine Smad-3/génétique , Facteur de croissance transformant bêta/pharmacologieRÉSUMÉ
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.