RESUMO
Engineered p53 mutant mice are valuable tools for delineating p53 functions in tumor suppression and cancer therapy. Here, we have introduced the R178E mutation into the Trp53 gene of mice to specifically ablate the cooperative nature of p53 DNA binding. Trp53R178E mice show no detectable target gene regulation and, at first sight, are largely indistinguishable from Trp53-/- mice. Surprisingly, stabilization of p53R178E in Mdm2-/- mice nevertheless triggers extensive apoptosis, indicative of residual wild-type activities. Although this apoptotic activity suffices to trigger lethality of Trp53R178E ;Mdm2-/- embryos, it proves insufficient for suppression of spontaneous and oncogene-driven tumorigenesis. Trp53R178E mice develop tumors indistinguishably from Trp53-/- mice and tumors retain and even stabilize the p53R178E protein, further attesting to the lack of significant tumor suppressor activity. However, Trp53R178E tumors exhibit remarkably better chemotherapy responses than Trp53-/- ones, resulting in enhanced eradication of p53-mutated tumor cells. Together, this provides genetic proof-of-principle evidence that a p53 mutant can be highly tumorigenic and yet retain apoptotic activity which provides a survival benefit in the context of cancer therapy.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Leucemia Mieloide Aguda/prevenção & controle , Linfoma/prevenção & controle , Mutação , Proteínas Proto-Oncogênicas c-mdm2/fisiologia , Proteína Supressora de Tumor p53/fisiologia , Animais , Carcinogênese/efeitos dos fármacos , Carcinogênese/metabolismo , Carcinogênese/patologia , Ciclo Celular , Modelos Animais de Doenças , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Linfoma/genética , Linfoma/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Tumorais CultivadasRESUMO
Cancer development is driven by activated oncogenes and loss of tumor suppressors. While oncogene inhibitors have entered routine clinical practice, tumor suppressor reactivation therapy remains to be established. For the most frequently inactivated tumor suppressor p53, genetic mouse models have demonstrated regression of p53-null tumors upon p53 reactivation. While this was shown in tumor models driven by p53 loss as the initiating lesion, many human tumors initially develop in the presence of wild-type p53, acquire aberrations in the p53 pathway to bypass p53-mediated tumor suppression, and inactivate p53 itself only at later stages during metastatic progression or therapy. To explore the efficacy of p53 reactivation in this scenario, we used a reversibly switchable p53 (p53ERTAM) mouse allele to generate Eµ-Myc-driven lymphomas in the presence of active p53 and, after full lymphoma establishment, switched off p53 to model late-stage p53 inactivation. Although these lymphomas had evolved in the presence of active p53, later loss and subsequent p53 reactivation surprisingly activated p53 target genes triggering massive apoptosis, tumor regression, and long-term cure of the majority of animals. Mechanistically, the reactivation response was dependent on Cdkn2a/p19Arf, which is commonly silenced in p53 wild-type lymphomas, but became reexpressed upon late-stage p53 inactivation. Likewise, human p53 wild-type tumor cells with CRISPR-engineered switchable p53ERTAM alleles responded to p53 reactivation when CDKN2A/p14ARF function was restored or mimicked with Mdm2 inhibitors. Together, these experiments provide genetic proof of concept that tumors can respond, in an ARF-dependent manner, to p53 reactivation even if p53 inactivation has occurred late during tumor evolution.
Assuntos
Carcinogênese/genética , Terapia Genética/métodos , Linfoma/genética , Proteína Supressora de Tumor p53/genética , Células 3T3 , Animais , Apoptose , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Células HCT116 , Humanos , Linfoma/terapia , Camundongos , Proteína Supressora de Tumor p53/metabolismoRESUMO
Mutations in the p53 tumor suppressor gene are the most frequent genetic alteration in cancer and are often associated with progression from benign to invasive stages with metastatic potential. Mutations inactivate tumor suppression by p53, and some endow the protein with novel gain of function (GOF) properties that actively promote tumor progression and metastasis. By comparative gene expression profiling of p53-mutated and p53-depleted cancer cells, we identified ectonucleoside triphosphate diphosphohydrolase 5 (ENTPD5) as a mutant p53 target gene, which functions as a uridine 5'-diphosphatase (UDPase) in the endoplasmic reticulum (ER) to promote the folding of N-glycosylated membrane proteins. A comprehensive pan-cancer analysis revealed a highly significant correlation between p53 GOF mutations and ENTPD5 expression. Mechanistically, mutp53 is recruited by Sp1 to the ENTPD5 core promoter to induce its expression. We show ENTPD5 to be a mediator of mutant p53 GOF activity in clonogenic growth, architectural tissue remodeling, migration, invasion, and lung colonization in an experimental metastasis mouse model. Our study reveals folding of N-glycosylated membrane proteins in the ER as a mechanism underlying the metastatic progression of tumors with mutp53 that could provide new possibilities for cancer treatment.
Assuntos
Retículo Endoplasmático/metabolismo , Metástase Neoplásica , Proteínas Oncogênicas/metabolismo , Pirofosfatases/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/fisiologia , Animais , Apoptose , Calnexina/metabolismo , Calreticulina/metabolismo , Carcinogênese/metabolismo , Linhagem Celular Tumoral , Progressão da Doença , Feminino , Glicoproteínas/metabolismo , Glicosilação , Humanos , Masculino , Camundongos , Proteínas Mutantes/genética , Proteínas Mutantes/fisiologia , Mutação , Invasividade Neoplásica , Prognóstico , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Fator de Transcrição Sp1/metabolismoRESUMO
Inactivation of the p53 tumor suppressor by Mdm2 is one of the most frequent events in cancer, so compounds targeting the p53-Mdm2 interaction are promising for cancer therapy. Mechanisms conferring resistance to p53-reactivating compounds are largely unknown. Here we show using CRISPR-Cas9-based target validation in lung and colorectal cancer that the activity of nutlin, which blocks the p53-binding pocket of Mdm2, strictly depends on functional p53. In contrast, sensitivity to the drug RITA, which binds the Mdm2-interacting N terminus of p53, correlates with induction of DNA damage. Cells with primary or acquired RITA resistance display cross-resistance to DNA crosslinking compounds such as cisplatin and show increased DNA cross-link repair. Inhibition of FancD2 by RNA interference or pharmacological mTOR inhibitors restores RITA sensitivity. The therapeutic response to p53-reactivating compounds is therefore limited by compound-specific resistance mechanisms that can be resolved by CRISPR-Cas9-based target validation and should be considered when allocating patients to p53-reactivating treatments.
Assuntos
Sistemas CRISPR-Cas , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Furanos/farmacologia , Genes p53 , Terapia de Alvo Molecular/métodos , Cisplatino/farmacologia , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Regulação da Expressão Gênica , Genes p53/fisiologia , Células HCT116/efeitos dos fármacos , Humanos , Morfolinas/farmacologia , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Ubiquitina-Proteína LigasesRESUMO
Phosphorylation of the Wnt receptor low-density lipoprotein receptor-related protein 6 (LRP6) by glycogen synthase kinase 3ß (GSK3ß) and casein kinase 1γ (CK1γ) is a key step in Wnt/ß-catenin signalling, which requires Wnt-induced formation of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)). Here, we show that adenomatous polyposis coli membrane recruitment 1 (Amer1) (also called WTX), a membrane associated PtdIns(4,5)P(2)-binding protein, is essential for the activation of Wnt signalling at the LRP6 receptor level. Knockdown of Amer1 reduces Wnt-induced LRP6 phosphorylation, Axin translocation to the plasma membrane and formation of LRP6 signalosomes. Overexpression of Amer1 promotes LRP6 phosphorylation, which requires interaction of Amer1 with PtdIns(4,5)P(2). Amer1 translocates to the plasma membrane in a PtdIns(4,5)P(2)-dependent manner after Wnt treatment and is required for LRP6 phosphorylation stimulated by application of PtdIns(4,5)P(2). Amer1 binds CK1γ, recruits Axin and GSK3ß to the plasma membrane and promotes complex formation between Axin and LRP6. Fusion of Amer1 to the cytoplasmic domain of LRP6 induces LRP6 phosphorylation and stimulates robust Wnt/ß-catenin signalling. We propose a mechanism for Wnt receptor activation by which generation of PtdIns(4,5)P(2) leads to recruitment of Amer1 to the plasma membrane, which acts as a scaffold protein to stimulate phosphorylation of LRP6.
Assuntos
Proteínas Relacionadas a Receptor de LDL/metabolismo , Proteínas de Membrana/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Proteínas Wnt/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Western Blotting , Membrana Celular/metabolismo , Células Cultivadas , Imunofluorescência , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Humanos , Rim/citologia , Rim/metabolismo , Proteínas Relacionadas a Receptor de LDL/genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Proteínas de Membrana/genética , Fosforilação , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Proteínas Supressoras de Tumor , Proteínas Wnt/genética , beta Catenina/genética , beta Catenina/metabolismoRESUMO
The germline transmission of a mutation in the adenomatous polyposis coli (APC) gene leads to cancer of the gastro-intestinal tract upon somatic inactivation of the remaining allele in familial adenomatous polyposis (FAP) patients. APC mutations result in truncated products that have primarily lost the ability to properly regulate the level of the transcription factor ß-catenin. However, colorectal cancer cells from FAP patients always retain a truncated APC product and the reasons for this strong selective pressure are not understood. We describe here the surprising property for the transcriptional repressor C-terminal binding protein (CtBP) to promote the oligomerization of truncated APC through binding to the 15 amino acid repeats of truncated APC. CtBP can bind to either first, third or fourth 15 amino acid repeats, but not to the second. CtBP-mediated oligomerization requires both dimerization domains of truncated APC as well as CtBP dimerization. The analysis of the position of the mutations along the APC sequence in adenomas from FAP patients reveals that the presence of the first 15 amino acid repeat is almost always selected in the resulting truncated APC product. This suggests that the sensitivity of truncated APC to oligomerization by CtBP constitutes an essential facet of tumour development.
Assuntos
Proteína da Polipose Adenomatosa do Colo/química , Proteína da Polipose Adenomatosa do Colo/genética , Polipose Adenomatosa do Colo/metabolismo , Oxirredutases do Álcool/metabolismo , Neoplasias Colorretais/metabolismo , Proteínas de Ligação a DNA/metabolismo , Mutação , Proteínas do Tecido Nervoso/metabolismo , Polipose Adenomatosa do Colo/genética , Proteína da Polipose Adenomatosa do Colo/metabolismo , Oxirredutases do Álcool/química , Oxirredutases do Álcool/genética , Linhagem Celular , Linhagem Celular Tumoral , Proteínas Correpressoras , Neoplasias Colorretais/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Dimerização , Humanos , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Ligação Proteica , Multimerização Proteica , Estrutura Terciária de ProteínaRESUMO
Skin mast cells (MCs) are critical effector cells in acute allergic reactions, and they contribute to chronic dermatoses like urticaria and atopic and contact dermatitis. KIT represents the cells' crucial receptor tyrosine kinase, which orchestrates proliferation, survival, and functional programs throughout the lifespan. cAMP response element binding protein (CREB), an evolutionarily well-conserved transcription factor (TF), regulates multiple cellular programs, but its function in MCs is poorly understood. We recently reported that CREB is an effector of the SCF (Stem Cell Factor)/KIT axis. Here, we ask whether CREB may also act upstream of KIT to orchestrate its functioning. Primary human MCs were isolated from skin and cultured in SCF+IL-4 (Interleukin-4). Pharmacological inhibition (666-15) and RNA interference served to manipulate CREB function. We studied KIT expression using flow cytometry and RT-qPCR, KIT-mediated signaling using immunoblotting, and cell survival using scatterplot and caspase-3 activity. The proliferation and cycle phases were quantified following BrdU incorporation. Transient CREB perturbation resulted in reduced KIT expression. Conversely, microphthalmia transcription factor (MITF) was unnecessary for KIT maintenance. KIT attenuation secondary to CREB was associated with heavily impaired KIT functional outputs, like anti-apoptosis and cell cycle progression. Likewise, KIT-elicited phosphorylation of ERK1/2 (Extracellular Signal-Regulated Kinase 1/2), AKT, and STAT5 (Signal Transducer and Activator of Transcription) was substantially diminished upon CREB inhibition. Surprisingly, the longer-term interference of CREB led to complete cell elimination, in a way surpassing KIT inhibition. Collectively, we reveal CREB as non-redundant in MCs, with its absence being incompatible with skin MCs' existence. Since SCF/KIT regulates CREB activity and, vice versa, CREB is required for KIT function, a positive feedforward loop between these elements dictates skin MCs' fate.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico , Mastócitos , Humanos , Diferenciação Celular , Retroalimentação , PeleRESUMO
Biallelic mutation of the ADENOMATOUS POLYPOSIS COLI (APC) gene is a hallmark of sporadic colorectal cancer and colorectal, duodenal and desmoid tumours that develop in familial adenomatous polyposis (FAP) patients. The mutations affecting both APC alleles are interdependent, the position of the first APC mutation determining where the second hit will occur. This results in a complex pattern of mutation distribution in the APC sequence that translates into the stabilization of beta-catenin that in turn feeds the affected cells with a permanent mitogenic signal. We describe here a new APC domain, the beta-catenin inhibitory domain (CID) of APC located between the second and third 20 amino acid repeats and therefore present in many truncated APC products found in human tumours. In truncated APC, the CID is absolutely necessary to down-regulate the transcriptional activity and the level of beta-catenin, even when an axin/conductin binding site is present. The activity of the CID is dramatically reduced in several colon cancer cell lines and can be inhibited by shorter truncated APC lacking the CID. The CID is a direct target of the selective pressure acting on APC during tumourigenesis. It explains the interdependence of both APC mutations, not only in colorectal but also in duodenal and desmoid tumours.
Assuntos
Proteína da Polipose Adenomatosa do Colo/química , Proteína da Polipose Adenomatosa do Colo/genética , Polipose Adenomatosa do Colo/genética , Mutação , beta Catenina/metabolismo , Polipose Adenomatosa do Colo/metabolismo , Proteína da Polipose Adenomatosa do Colo/metabolismo , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Duodenais/genética , Neoplasias Duodenais/metabolismo , Fibromatose Agressiva/genética , Fibromatose Agressiva/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Modelos Genéticos , Ligação Proteica , Estrutura Terciária de Proteína , beta Catenina/genéticaRESUMO
The mutation cluster region (MCR) of adenomatous polyposis coli (APC) is located within the central part of the open reading frame, overlapping with the region encoding the 20 amino acid repeats (20R) that are beta-catenin-binding sites. Each mutation in the MCR leads to the synthesis of a truncated APC product expressed in a colorectal tumour. The MCR extends from the 3' border of the first 20R coding region to approximately the middle of the third 20R coding region, reflecting both positive and negative selections of the N- and C-terminal halves of the APC protein in colon cancer cells, respectively. In contrast, the second 20R escapes selection and can be either included or excluded from the truncated APC products found in colon cancer cells. To specify the functional outcome of the selection of the mutations, we investigated the beta-catenin binding capacity of the first three 20R in N-terminal APC fragments. We found in co-immunoprecipitation and intracellular co-localization experiments that the second 20R is lacking any beta-catenin binding activity. Similarly, we also show that the tumour-associated truncations abolish the interaction of beta-catenin with the third 20R. Thus, our data provide a functional definition of the MCR: the APC fragments typical of colon cancer are selected for the presence of a single functional 20R, the first one, and are therefore equivalent relative to beta-catenin binding.
Assuntos
Proteína da Polipose Adenomatosa do Colo/química , Proteína da Polipose Adenomatosa do Colo/metabolismo , Neoplasias do Colo/genética , Deleção de Sequência , beta Catenina/metabolismo , Proteína da Polipose Adenomatosa do Colo/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Linhagem Celular Tumoral , Neoplasias do Colo/metabolismo , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Família Multigênica , Ligação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Sequências Repetitivas de AminoácidosRESUMO
Posttranslational modifications are essential for regulating the transcription factor p53, which binds DNA in a highly cooperative manner to control expression of a plethora of tumor-suppressive programs. Here we show at the biochemical, cellular, and organismal level that the cooperative nature of DNA binding is reduced by phosphorylation of highly conserved serine residues (human S183/S185, mouse S180) in the DNA-binding domain. To explore the role of this inhibitory phosphorylation in vivo, new phosphorylation-deficient p53-S180A knock-in mice were generated. Chromatin immunoprecipitation sequencing and RNA sequencing studies of S180A knock-in cells demonstrated enhanced DNA binding and increased target gene expression. In vivo, this translated into a tissue-specific vulnerability of the bone marrow that caused depletion of hematopoietic stem cells and impaired proper regeneration of hematopoiesis after DNA damage. Median lifespan was significantly reduced by 20% from 709 days in wild type to only 568 days in S180A littermates. Importantly, lifespan was reduced by a loss of general fitness and increased susceptibility to age-related diseases, not by increased cancer incidence as often seen in other p53-mutant mouse models. For example, S180A knock-in mice showed markedly reduced spontaneous tumorigenesis and increased resistance to Myc-driven lymphoma and Eml4-Alk-driven lung cancer. Preventing phosphorylation of S183/S185 in human cells boosted p53 activity and allowed tumor cells to be killed more efficiently. Together, our data identify p53 DNA-binding domain phosphorylation as a druggable mechanism that balances tumorigenesis and aging. SIGNIFICANCE: These findings demonstrate that p53 tumor suppressor activity is reduced by DNA-binding domain phosphorylation to prevent aging and identify this phosphorylation as a potential target for cancer therapy.See related commentary by Horikawa, p. 5164.
Assuntos
Neoplasias , Proteína Supressora de Tumor p53 , Envelhecimento/genética , Animais , Carcinogênese/genética , DNA , Dano ao DNA , Longevidade/genética , Camundongos , Neoplasias/genética , Fosforilação , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Cancer cells have a characteristic metabolism, mostly caused by alterations in signal transduction networks rather than mutations in metabolic enzymes. For metabolic drugs to be cancer-selective, signaling alterations need to be identified that confer a druggable vulnerability. Here, we demonstrate that many tumor cells with an acquired cancer drug resistance exhibit increased sensitivity to mechanistically distinct inhibitors of cancer metabolism. We demonstrate that this metabolic vulnerability is driven by mTORC1, which promotes resistance to chemotherapy and targeted cancer drugs, but simultaneously suppresses autophagy. We show that autophagy is essential for tumor cells to cope with therapeutic perturbation of metabolism and that mTORC1-mediated suppression of autophagy is required and sufficient for generating a metabolic vulnerability leading to energy crisis and apoptosis. Our study links mTOR-induced cancer drug resistance to autophagy defects as a cause of a metabolic liability and opens a therapeutic window for the treatment of otherwise therapy-refractory tumor patients.
Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Animais , Apoptose/efeitos dos fármacos , Autofagia/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Desoxiglucose , Tratamento Farmacológico , Feminino , Humanos , Neoplasias Pulmonares , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Wnt signalling is prevented by the proteosomal degradation of ß-catenin, which occurs in a destruction complex containing adenomatous polyposis coli (APC), APC-like (APCL), Axin and Axin2. Truncating mutations of the APC gene result in the constitutive stabilisation of ß-catenin and the initiation of colon cancer, although tumour cells tolerate the expression of wild-type APCL. Using the colocalisation of overexpressed Axin, APC and APCL constructs as a readout of interaction, we found that Axin interacted with the second twenty amino acid repeat (20R2) of APC and APCL. This interaction involved a domain adjacent to the C-terminal DIX domain of Axin. We identified serine residues within the 20R2 of APCL that were involved in Axin colocalisation, the phosphorylation of truncated APCL and the down-regulation of ß-catenin. Our results indicated that Axin, but not Axin2, displaced APC, but not APCL, from the cytoskeleton and stimulated its incorporation into bright cytoplasmic dots that others have recognised as ß-catenin destruction complexes. The SAMP repeats in APC interact with the N-terminal RGS domain of Axin. Our data showed that a short domain containing the first SAMP repeat in truncated APC was required to stimulate Axin oligomerisation. This was independent of Axin colocalisation with 20R2. Our data also suggested that the RGS domain exerted an internal inhibitory constraint on Axin oligomerisation. Considering our data and those from others, we discuss a working model whereby ß-catenin phosphorylation involves Axin and the 20R2 of APC or APCL and further processing of phospho-ß-catenin occurs upon the oligomerisation of Axin that is induced by binding the SAMP repeats in APC.
Assuntos
Proteína da Polipose Adenomatosa do Colo/genética , Polipose Adenomatosa do Colo/genética , Proteína Axina/genética , Proteínas do Citoesqueleto/genética , Regulação Neoplásica da Expressão Gênica , beta Catenina/genética , Polipose Adenomatosa do Colo/metabolismo , Polipose Adenomatosa do Colo/patologia , Proteína da Polipose Adenomatosa do Colo/química , Proteína da Polipose Adenomatosa do Colo/metabolismo , Proteína Axina/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Células HEK293 , Humanos , Dados de Sequência Molecular , Fosforilação , Ligação Proteica , Multimerização Proteica , Estrutura Terciária de Proteína , Sequências Repetitivas de Aminoácidos , Alinhamento de Sequência , Transdução de Sinais , beta Catenina/metabolismoRESUMO
Truncating mutations affect the adenomatous polyposis coli (APC) gene in most cases of colon cancer, resulting in the stabilization of ß-catenin and uncontrolled cell proliferation. We show here that colon cancer cell lines express also the paralog APC-like (APCL or APC2). RNA interference revealed that it controls the level and/or the activity of ß-catenin, but it is less efficient and binds less well to ß-catenin than APC, thereby providing one explanation as to why the gene is not mutated in colon cancer. A further comparison indicates that APCL down-regulates the ß-catenin level despite the lack of the 15R region known to be important in APC. To understand this discrepancy, we performed immunoprecipitation experiments that revealed that phosphorylated ß-catenin displays a preference for binding to the 15 amino acid repeats (15R) rather than the first 20 amino acid repeat of APC. This suggests that the 15R region constitutes a gate connecting the steps of ß-catenin phosphorylation and subsequent ubiquitination/degradation. Using RNA interference and domain swapping experiments, we show that APCL benefits from the 15R of truncated APC to target ß-catenin for degradation, in a process likely involving heterodimerization of the two partners. Our data suggest that the functional complementation of APCL by APC constitutes a substantial facet of tumour development, because the truncating mutations of APC in colorectal tumours from familial adenomatous polyposis (FAP) patients are almost always selected for the retention of at least one 15R.
Assuntos
Proteína da Polipose Adenomatosa do Colo/metabolismo , Polipose Adenomatosa do Colo/metabolismo , Proteínas do Citoesqueleto/metabolismo , beta Catenina/metabolismo , Polipose Adenomatosa do Colo/química , Polipose Adenomatosa do Colo/genética , Proteína da Polipose Adenomatosa do Colo/genética , Linhagem Celular Tumoral , Colo/metabolismo , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Regulação Neoplásica da Expressão Gênica , Genes APC , Humanos , Fosforilação , Estrutura Terciária de Proteína , Proteólise , Reto/metabolismo , Sequências Repetitivas de Aminoácidos , Ativação Transcricional , beta Catenina/genéticaRESUMO
The tumour suppressor gene adenomatous polyposis coli (APC) is mutated in most colorectal cancer cases, leading to the synthesis of truncated APC products and the stabilization of ß-catenin. Truncated APC is almost always retained in tumour cells, suggesting that it serves an essential function. Here, RNA interference has been used to down-regulate truncated APC in several colorectal cancer cell lines expressing truncated APCs of different lengths, thereby performing an analysis covering most of the mutation cluster region (MCR). The consequences on proliferation in vitro, tumour formation in vivo and the level and transcriptional activity of ß-catenin have been investigated. Down-regulation of truncated APC results in an inhibition of tumour cell population expansion in vitro in 6 cell lines out of 6 and inhibition of tumour outgrowth in vivo as analysed in one of these cell lines, HT29. This provides a general rule explaining the retention of truncated APC in colorectal tumours and defines it as a suitable target for therapeutic intervention. Actually, we also show that it is possible to design a shRNA that targets a specific truncated isoform of APC without altering the expression of wild-type APC. Down-regulation of truncated APC is accompanied by an up-regulation of the transcriptional activity of ß-catenin in 5 out of 6 cell lines. Surprisingly, the increased signalling is associated in most cases (4 out of 5) with an up-regulation of ß-catenin levels, indicating that truncated APC can still modulate wnt signalling through controlling the level of ß-catenin. This control can happen even when truncated APC lacks the ß-catenin inhibiting domain (CiD) involved in targeting ß-catenin for proteasomal degradation. Thus, truncated APC is an essential component of colorectal cancer cells, required for cell proliferation, possibly by adjusting ß-catenin signalling to the "just right" level.
Assuntos
Proteína da Polipose Adenomatosa do Colo/genética , Proteína da Polipose Adenomatosa do Colo/metabolismo , Deleção de Sequência , beta Catenina/metabolismo , Proteína da Polipose Adenomatosa do Colo/deficiência , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias Colorretais/patologia , Regulação para Baixo/genética , Humanos , Isoformas de Proteínas/deficiência , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Interferência de RNA , Transcrição Gênica/genética , beta Catenina/genéticaRESUMO
Most colon cancer cells express truncated versions of the tumour suppressor Adenomatous Polyposis Coli (APC). These molecules are selected during tumourigenesis for impaired beta-catenin degrading activity. In this study, we describe that truncated APC can still control the activity of beta-catenin in colon cancer cell lines via its first 20 amino acid repeat. First, we show that both endogenous and ectopically expressed truncated APC molecules can bind to beta-catenin. Second, reduction of the levels of truncated APC by RNA interference increases the activity of a beta-catenin-dependent reporter gene and stimulates the expression of the beta-catenin target gene AXIN2/conductin. This occurs without alterations of the amounts of cytosolic beta-catenin. Conversely, ectopic expression of truncated APC decreases beta-catenin-dependent transcription without affecting the intensity of immunofluorescence staining of beta-catenin in transfected cells. Third, we reveal that the APC level increases when cells reach the G1-S boundary during cell cycle progression. Simultaneously, the amount of beta-catenin bound to APC increases and the transcriptional activity of beta-catenin drops in an APC-dependent manner. Again, this occurs independently of the amounts of either total or phosphorylated cytosolic beta-catenin. Together, these results indicate that truncated APC controls the ability of beta-catenin to activate transcription. As we also show that the inhibition involves the first 20 amino acid repeat of APC, our data suggest that colon cancer cells retain a truncated APC molecule containing at least the first 20 amino acid repeat to modulate the transcriptional activity of beta-catenin in a cell cycle-dependent manner.
Assuntos
Proteína da Polipose Adenomatosa do Colo/genética , Ciclo Celular/genética , Neoplasias do Colo/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , beta Catenina/metabolismo , Proteína da Polipose Adenomatosa do Colo/metabolismo , Proteína Axina , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Proteínas do Citoesqueleto/metabolismo , Imunofluorescência , Humanos , Mutação/genética , Oligonucleotídeos , Interferência de RNA , Sequências Repetitivas de Aminoácidos/genética , beta Catenina/genéticaRESUMO
APC is a multifunctional tumor suppressor protein that negatively controls Wnt signaling, but also regulates cell adhesion and migration by interacting with the plasma membrane and the microtubule cytoskeleton. Although the molecular basis for the microtubule association of APC is well understood, molecular mechanisms that underlie its plasma membrane localization have remained elusive. We show here that APC is recruited to the plasma membrane by binding to APC membrane recruitment 1 (AMER1), a novel membrane-associated protein that interacts with the ARM repeat domain of APC. The N-terminus of AMER1 contains two distinct phosphatidylinositol(4,5)-bisphosphate [PtdIns(4,5)P(2)]-binding domains, which mediate its localization to the plasma membrane. Overexpression of AMER1 increases APC levels and redirects APC from microtubule ends to the plasma membrane of epithelial cells. Conversely, siRNA-mediated knockdown of AMER1 reduces the overall levels of APC, promotes its association with microtubule ends in cellular protrusions and disturbs intercellular junctions. These data indicate that AMER1 controls the subcellular distribution of APC between membrane- and microtubule-associated pools, and might thereby regulate APC-dependent cellular morphogenesis, cell migration and cell-cell adhesion.
Assuntos
Proteína da Polipose Adenomatosa do Colo/metabolismo , Membrana Celular/metabolismo , Proteínas de Membrana/metabolismo , Microtúbulos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Proteína da Polipose Adenomatosa do Colo/genética , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Humanos , Proteínas de Membrana/genética , Dados de Sequência Molecular , Alinhamento de Sequência , Proteínas Supressoras de Tumor , Técnicas do Sistema de Duplo-HíbridoRESUMO
The tumour suppressor APC is truncated in most colon cancers, which leads to the stabilization of beta-catenin and to the constitutive activation of Wnt signalling. However, it is not clear why colon cancer cells retain the truncated APC fragment. Here, we show that a decrease of APC levels achieved by RNA interference impairs cell proliferation and DNA replication, not only in 293 cells that express a wild-type protein, but also in SW480 colon cancer cells that express exclusively a truncated APC fragment. This correlates with a reduction of the levels of cyclin A, cyclin A-dependent kinase activity, p27(kip1) and the catalytic subunit of DNA polymerase delta. Thus, our data suggest that colon cancer cells retain a truncated APC fragment because it is essential for cell proliferation.