RESUMO
The metastatic spread of cancer is achieved by the haematogenous dissemination of circulating tumour cells (CTCs). Generally, however, the temporal dynamics that dictate the generation of metastasis-competent CTCs are largely uncharacterized, and it is often assumed that CTCs are constantly shed from growing tumours or are shed as a consequence of mechanical insults1. Here we observe a striking and unexpected pattern of CTC generation dynamics in both patients with breast cancer and mouse models, highlighting that most spontaneous CTC intravasation events occur during sleep. Further, we demonstrate that rest-phase CTCs are highly prone to metastasize, whereas CTCs generated during the active phase are devoid of metastatic ability. Mechanistically, single-cell RNA sequencing analysis of CTCs reveals a marked upregulation of mitotic genes exclusively during the rest phase in both patients and mouse models, enabling metastasis proficiency. Systemically, we find that key circadian rhythm hormones such as melatonin, testosterone and glucocorticoids dictate CTC generation dynamics, and as a consequence, that insulin directly promotes tumour cell proliferation in vivo, yet in a time-dependent manner. Thus, the spontaneous generation of CTCs with a high proclivity to metastasize does not occur continuously, but it is concentrated within the rest phase of the affected individual, providing a new rationale for time-controlled interrogation and treatment of metastasis-prone cancers.
Assuntos
Neoplasias da Mama , Metástase Neoplásica , Sono , Animais , Neoplasias da Mama/patologia , Contagem de Células , Proliferação de Células , Modelos Animais de Doenças , Feminino , Glucocorticoides , Humanos , Insulina , Melatonina , Camundongos , Metástase Neoplásica/patologia , Células Neoplásicas Circulantes/patologia , RNA-Seq , Análise de Célula Única , Testosterona , Fatores de TempoRESUMO
Lung cancer is the leading cause of cancer deaths. Its high mortality is associated with high metastatic potential. Here, we show that the RAC1-selective guanine nucleotide exchange factor T cell invasion and metastasis-inducing protein 1 (TIAM1) promotes cell migration and invasion in the most common subtype of lung cancer, non-small-cell lung cancer (NSCLC), through an unexpected nuclear function. We show that TIAM1 interacts with TRIM28, a master regulator of gene expression, in the nucleus of NSCLC cells. We reveal that a TIAM1-TRIM28 complex promotes epithelial-to-mesenchymal transition, a phenotypic switch implicated in cell migration and invasion. This occurs through H3K9me3-induced silencing of protocadherins and by decreasing E-cadherin expression, thereby antagonizing cell-cell adhesion. Consistently, TIAM1 or TRIM28 depletion suppresses the migration of NSCLC cells, while migration is restored by the simultaneous depletion of protocadherins. Importantly, high nuclear TIAM1 in clinical specimens is associated with advanced-stage lung adenocarcinoma, decreased patient survival, and inversely correlates with E-cadherin expression.
Assuntos
Adenocarcinoma de Pulmão , Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Neoplasias Pulmonares/genética , Protocaderinas , Carcinoma Pulmonar de Células não Pequenas/genética , Caderinas/genética , Epigênese Genética , Proteína 28 com Motivo Tripartido , Proteína 1 Indutora de Invasão e Metástase de Linfoma de Células T/genéticaRESUMO
BACKGROUND: The role of pleiotrophin and its receptors RPTPß/ζ and Syndecan-3 during tumor metastasis remains unknown. RESULTS: RPTPß/ζ knockdown initiates EMT, promotes pleiotrophin-mediated migration and attachment through Syndecan-3 and induces in vivo metastasis. CONCLUSION: RPTPß/ζ plays a suppressor-like role in prostate cancer metastasis. SIGNIFICANCE: Boosting RPTPß/ζ or attenuating Syndecan-3 signaling pathways may lead to more effective therapeutic strategies in treating prostate cancer metastasis. Pleiotrophin is a growth factor that induces carcinogenesis. Despite the fact that many published reports focused on the role of pleiotrophin and its receptors, receptor protein tyrosine phosphatase (RPTPß/ζ), and syndecan-3 during tumor development, no information is available regarding their function in tumor metastasis. To investigate the mechanism through which pleiotrophin regulates tumor metastasis, we used two different prostate carcinoma cell lines, DU145 and PC3, in which the expression of RPTPß/ζ or syndecan-3 was down-regulated by the RNAi technology. The loss of RPTPß/ζ expression initiated epithelial-to-mesenchymal transition (EMT) and increased the ability of the cells to migrate and invade. Importantly, the loss of RPTPß/ζ expression increased metastasis in nude mice in an experimental metastasis assay. We also demonstrate that RPTPß/ζ counterbalanced the pleiotrophin-mediated syndecan-3 pathway. While the inhibition of syndecan-3 expression inhibited the pleiotrophin-mediated cell migration and attachment through the Src and Fak pathway, the inhibition of RPTPß/ζ expression increased pleiotrophin-mediated migration and attachment through an interaction with Src and the subsequent activation of a signal transduction pathway involving Fak, Pten, and Erk1/2. Taken together, these results suggest that the loss of RPTPß/ζ may contribute to the metastasis of prostate cancer cells by inducing EMT and promoting pleiotrophin activity through the syndecan-3 pathway.
Assuntos
Metástase Neoplásica , Neoplasias da Próstata/enzimologia , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores/deficiência , Animais , Linhagem Celular Tumoral , Movimento Celular , Humanos , Masculino , Camundongos , Camundongos Knockout , Camundongos Nus , Fosforilação , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores/genética , Transdução de Sinais , Sindecana-3/genética , Sindecana-3/metabolismo , Regulação para CimaRESUMO
Despite advances in uncovering vulnerabilities, identifying biomarkers, and developing more efficient treatments, cancer remains a threat because of its ability to progress while acquiring resistance to therapy. The circadian rhythm governs most of the cellular functions implicated in cancer progression, and its exploitation therefore opens new promising directions in the fight against metastasis. In this review we summarize the role of the circadian rhythm in tumor development and progression, with emphasis on the circadian rhythm-regulated elements that control the generation of circulating tumor cells (CTCs) and metastasis. We then present data on chronotherapy and discuss how circadian rhythm investigations may open new paths to more effective anticancer treatments.
Assuntos
Ritmo Circadiano , Células Neoplásicas Circulantes , Humanos , Cronoterapia/métodos , BiomarcadoresRESUMO
Blood-borne metastasis of breast cancer involves a series of tightly regulated sequential steps, including the growth of a primary tumor lesion, intravasation of circulating tumor cells (CTC), and adaptation in various distant metastatic sites. The genes orchestrating each of these steps are poorly understood in physiologically relevant contexts, owing to the rarity of experimental models that faithfully recapitulate the biology, growth kinetics, and tropism of human breast cancer. Here, we conducted an in vivo loss-of-function CRISPR screen in newly derived CTC xenografts, unique in their ability to spontaneously mirror the human disease, and identified specific genetic dependencies for each step of the metastatic process. Validation experiments revealed sensitivities to inhibitors that are already available, such as PLK1 inhibitors, to prevent CTC intravasation. Together, these findings present a new tool to reclassify driver genes involved in the spread of human cancer, providing insights into the biology of metastasis and paving the way to test targeted treatment approaches. SIGNIFICANCE: A loss-of-function CRISPR screen in human CTC-derived xenografts identifies genes critical for individual steps of the metastatic cascade, suggesting novel drivers and treatment opportunities for metastatic breast cancers.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Células Neoplásicas Circulantes/metabolismo , Animais , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/sangue , Neoplasias da Mama/patologia , Sistemas CRISPR-Cas , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Metástase Neoplásica , Células Neoplásicas Circulantes/patologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo , RNA-Seq/métodos , Análise de Sobrevida , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Quinase 1 Polo-LikeRESUMO
BACKGROUND: Pleiotrophin, also known as HARP (Heparin Affin Regulatory Peptide) is a growth factor expressed in various tissues and cell lines. Pleiotrophin participates in multiple biological actions including the induction of cellular proliferation, migration and angiogenesis, and is involved in carcinogenesis. Recently, we identified and characterized several pleiotrophin proteolytic fragments with biological activities similar or opposite to that of pleiotrophin. Here, we investigated the biological actions of P(122-131), a synthetic peptide corresponding to the carboxy terminal region of this growth factor. RESULTS: Our results show that P(122-131) inhibits in vitro adhesion, anchorage-independent proliferation, and migration of DU145 and LNCaP cells, which express pleiotrophin and its receptor RPTPß/ζ. In addition, P(122-131) inhibits angiogenesis in vivo, as determined by the chicken embryo CAM assay. Investigation of the transduction mechanisms revealed that P(122-131) reduces the phosphorylation levels of Src, Pten, Fak, and Erk1/2. Finally, P(122-131) not only interacts with RPTPß/ζ, but also interferes with other pleiotrophin receptors, as demonstrated by selective knockdown of pleiotrophin or RPTPß/ζ expression with the RNAi technology. CONCLUSIONS: In conclusion, our results demonstrate that P(122-131) inhibits biological activities that are related to the induction of a transformed phenotype in PCa cells, by interacing with RPTPß/ζ and interfering with other pleiotrophin receptors. Cumulatively, these results indicate that P(122-131) may be a potential anticancer agent, and they warrant further study of this peptide.
Assuntos
Proteínas de Transporte/química , Citocinas/química , Fragmentos de Peptídeos/farmacologia , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores/metabolismo , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , HumanosRESUMO
Circulating tumor cells are tumor-derived pioneers responsible for the metastatic spread of cancer. Here, we outline recent discoveries, challenges, and future trends for circulating tumor cell investigations, arguing that the time is coming for translation of this work into clinical practice.
Assuntos
Biomarcadores Tumorais , Células Neoplásicas Circulantes , Pesquisa Translacional Biomédica , Biomarcadores Tumorais/imunologia , Biomarcadores Tumorais/metabolismo , Humanos , Células Neoplásicas Circulantes/imunologia , Células Neoplásicas Circulantes/metabolismo , Células Neoplásicas Circulantes/patologiaRESUMO
The perinuclear actin cap is an important cytoskeletal structure that regulates nuclear morphology and re-orientation during front-rear polarisation. The mechanisms regulating the actin cap are currently poorly understood. Here, we demonstrate that STEF/TIAM2, a Rac1 selective guanine nucleotide exchange factor, localises at the nuclear envelope, co-localising with the key perinuclear proteins Nesprin-2G and Non-muscle myosin IIB (NMMIIB), where it regulates perinuclear Rac1 activity. We show that STEF depletion reduces apical perinuclear actin cables (a phenotype rescued by targeting active Rac1 to the nuclear envelope), increases nuclear height and impairs nuclear re-orientation. STEF down-regulation also reduces perinuclear pMLC and decreases myosin-generated tension at the nuclear envelope, suggesting that STEF-mediated Rac1 activity regulates NMMIIB activity to promote stabilisation of the perinuclear actin cap. Finally, STEF depletion decreases nuclear stiffness and reduces expression of TAZ-regulated genes, indicating an alteration in mechanosensing pathways as a consequence of disruption of the actin cap.
Assuntos
Proteínas de Capeamento de Actina/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Membrana Nuclear/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Células A549 , Aciltransferases , Animais , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Humanos , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Miosina não Muscular Tipo IIB/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismoRESUMO
A member of the ribonucleaseâ A superfamily, human angiogenin (hAng) is a potent angiogenic factor. Heteronuclear NMR spectroscopy combined with induced-fit docking revealed a dual binding mode for the most antiangiogenic compound of a series of ribofuranosyl pyrimidine nucleosides that strongly inhibit hAng's angiogenic activity inâ vivo. While modeling suggests the potential for simultaneous binding of the inhibitors at the active and cell-binding sites, NMR studies indicate greater affinity for the cell-binding site than for the active site. Additionally, molecular dynamics simulations at 100â ns confirmed the stability of binding at the cell-binding site with the predicted protein-ligand interactions, in excellent agreement with the NMR data. This is the first time that a nucleoside inhibitor is reported to completely inhibit the angiogenic activity of hAng inâ vivo by exerting dual inhibitory activity on hAng, blocking both the entrance of hAng into the cell and its ribonucleolytic activity.
Assuntos
Nucleosídeos de Pirimidina/química , Ribonuclease Pancreático/antagonistas & inibidores , Animais , Sítios de Ligação , Linhagem Celular , Embrião de Galinha , Membrana Corioalantoide/irrigação sanguínea , Membrana Corioalantoide/efeitos dos fármacos , Simulação por Computador , Humanos , Simulação de Dinâmica Molecular , Neovascularização Fisiológica/efeitos dos fármacos , Ressonância Magnética Nuclear Biomolecular , Relação Estrutura-AtividadeRESUMO
In this study, a novel anticancer reagent based on polyplexes nanoparticles was developed. These nanoparticles are obtained by mixing negatively charged polyelectrolytes with the antitumour cationically-charged pseudopeptide N6L. Using two in vivo experimental tumor pancreatic models based upon PANC-1 and mPDAC cells, we found that the antitumour activity of N6L is significantly raised via its incorporation in polyplexed nanoparticles. Study of the mechanism of action using affinity isolation and si-RNA experiments indicated that N6L-polyplexes are internalized through their interaction with nucleolin. In addition, using a very aggressive model of pancreatic cancer in which gemcitabine, a standard of care for this type of cancer, has a weak effect on tumour growth, we observed that N6L-polyplexes administration has a stronger efficacy than gemcitabine. Biodistribution studies carried out in tumour-bearing mice indicated that N6L-polyplexes localises in tumour tissue, in agreement with its antitumour effect. These results support the idea that N6L nanoparticles could develop into a promising strategy for the treatment of cancer, especially hard-to-treat pancreatic cancers.
RESUMO
Aberrant WNT signaling drives colorectal cancer (CRC). Here, we identify TIAM1 as a critical antagonist of CRC progression through inhibiting TAZ and YAP, effectors of WNT signaling. We demonstrate that TIAM1 shuttles between the cytoplasm and nucleus antagonizing TAZ/YAP by distinct mechanisms in the two compartments. In the cytoplasm, TIAM1 localizes to the destruction complex and promotes TAZ degradation by enhancing its interaction with ßTrCP. Nuclear TIAM1 suppresses TAZ/YAP interaction with TEADs, inhibiting expression of TAZ/YAP target genes implicated in epithelial-mesenchymal transition, cell migration, and invasion, and consequently suppresses CRC cell migration and invasion. Importantly, high nuclear TIAM1 in clinical specimens associates with increased CRC patient survival. Together, our findings suggest that in CRC TIAM1 suppresses tumor progression by regulating YAP/TAZ activity.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Movimento Celular , Neoplasias Colorretais/metabolismo , Células Epiteliais/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Mucosa Intestinal/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fosfoproteínas/metabolismo , Transporte Ativo do Núcleo Celular , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Células CACO-2 , Proteínas de Ciclo Celular , Núcleo Celular/metabolismo , Proliferação de Células , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Citoplasma/metabolismo , Células Epiteliais/patologia , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Predisposição Genética para Doença , Fatores de Troca do Nucleotídeo Guanina/deficiência , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Mucosa Intestinal/patologia , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Invasividade Neoplásica , Fenótipo , Fosfoproteínas/genética , Proteólise , Interferência de RNA , Proteína 1 Indutora de Invasão e Metástase de Linfoma de Células T , Transativadores , Fatores de Transcrição , Transcrição Gênica , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional , Transfecção , Via de Sinalização Wnt , Proteínas de Sinalização YAP , Peixe-Zebra/embriologia , Proteínas Contendo Repetições de beta-Transducina/genética , Proteínas Contendo Repetições de beta-Transducina/metabolismoRESUMO
Centrosome separation is critical for bipolar spindle formation and the accurate segregation of chromosomes during mammalian cell mitosis. Kinesin-5 (Eg5) is a microtubule motor essential for centrosome separation, and Tiam1 and its substrate Rac antagonize Eg5-dependent centrosome separation in early mitosis promoting efficient chromosome congression. Here we identify S1466 of Tiam1 as a novel Cdk1 site whose phosphorylation is required for the mitotic function of Tiam1. We find that this phosphorylation of Tiam1 is required for the activation of group I p21-activated kinases (Paks) on centrosomes in prophase. Further, we show that both Pak1 and Pak2 counteract centrosome separation in a kinase-dependent manner and demonstrate that they act downstream of Tiam1. We also show that depletion of Pak1/2 allows cells to escape monopolar arrest by Eg5 inhibition, highlighting the potential importance of this signalling pathway for the development of Eg5 inhibitors as cancer therapeutics.
Assuntos
Centrossomo/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fuso Acromático/metabolismo , Animais , Proteína Quinase CDC2/metabolismo , Cães , Células HEK293 , Humanos , Cinesinas/metabolismo , Células Madin Darby de Rim Canino , Camundongos , Fosforilação , Proteína 1 Indutora de Invasão e Metástase de Linfoma de Células T , Proteínas rac de Ligação ao GTPRESUMO
Lamprey gonadotropin-releasing hormone type III (lGnRH-III) is an isoform of GnRH isolated from the sea lamprey (Petromyzon marinus) with negligible endocrine activity in mammalian systems. Data concerning the superior direct anticancer activity of lGnRH-III have been published, raising questions on the structure-activity relationship. We synthesized 21 lGnRH-III analogs with rational amino acid substitutions and studied their effect on PC3 and LNCaP prostate cancer cell proliferation. Our results question the importance of the acidic charge of Asp6 for the antiproliferative activity and indicate the significance of the stereochemistry of Trp in positions 3 and 7. Furthermore, conjugation of an acetyl-group to the side chain of Lys8 or side chain cyclization of amino acids 1-8 increased the antiproliferative activity of lGnRH-III demonstrating that the proposed salt bridge between Asp6 and Lys8 is not crucial. Conformational studies of lGnRH-III were performed through NMR spectroscopy, and the solution structure of GnRH-I was solved. In solution, lGnRH-III adopts an extended backbone conformation in contrast to the well-defined ß-turn conformation of GnRH-I.
Assuntos
Antineoplásicos/síntese química , Hormônio Liberador de Gonadotropina/síntese química , Ácido Pirrolidonocarboxílico/análogos & derivados , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células , Hormônio Liberador de Gonadotropina/química , Hormônio Liberador de Gonadotropina/farmacologia , Humanos , Espectroscopia de Ressonância Magnética , Masculino , Neoplasias da Próstata/tratamento farmacológico , Conformação Proteica , Ácido Pirrolidonocarboxílico/síntese química , Ácido Pirrolidonocarboxílico/química , Ácido Pirrolidonocarboxílico/farmacologia , Relação Estrutura-AtividadeRESUMO
Analogs of GnRH, including [DLeu6, desGly1o]-GnRH-NHEt (leuprolide, commercial product), have been widely used in oncology to induce reversible chemical castration. Several studies have provided evidence that, besides their pituitary effects, GnRH analogs may exert direct antiproliferative effects on tumor cells. To study the effect of modifications in positions 4 and 6 of leuprolide on prostate cancer cell proliferation, we synthesized 12 new leuprolide analogs. All GnRH analogs lacked the carboxy-terminal Gly10-amide of GnRH, and an ethylamide residue was added to Pro9. Gly6 was substituted by DLys, Nepsilon-modified DLys, Glu, and DGlu. To improve the enzymatic stability, NMeSer was incorporated in position 4, and the rate of hydrolysis by alpha-chymotrypsin and subtilisin was investigated. Our results demonstrate that this incorporation increases enzymatic stability in all analogs of GnRH, whereas the antiproliferative effect on PC3 and LNCaP prostate cancer cells is similar to that of leuprolide. Conformational studies were performed to elucidate structural changes occurring on substitution of native residues and to study structure-activity relationship for these analogs. The solution models of [DLeu6, desGly10]-GnRH-NHEt (leuprolide), [NMeSer4, DGlu6, desGly10]-GnRH-NHEt, [Glu6, desGly10]-GnRH-NHEt, and [DGIu6, desGly10]-GnRH-NHEt peptides were determined through two-dimensional nuclear magnetic resonance spectroscopy in dimethylsulfoxide. Nuclear magnetic resonance data provide experimental evidence for the U-turn-like structure appeared in all four analogs, which could be characterized as beta-hairpin conformation. The most stable analog [NMeSer4, DGlu6, desGly10]-GnRH-NHEt against proteolytic cleavage forms a second extra backbone turn observed for residues 1-4.
Assuntos
Antineoplásicos Hormonais/farmacologia , Proliferação de Células/efeitos dos fármacos , Leuprolida/análogos & derivados , Leuprolida/farmacologia , Antineoplásicos Hormonais/química , Linhagem Celular Tumoral , Humanos , Leuprolida/química , Masculino , Ressonância Magnética Nuclear Biomolecular , Estrutura Secundária de Proteína , Relação Estrutura-AtividadeRESUMO
Heparin affin regulatory peptide (HARP) is an 18 kDa heparin-binding protein that plays a key role in tumor growth. We showed previously that the synthetic peptide P(111-136) composed of the last 26 HARP amino acids inhibited HARP-induced mitogenesis. Here, to identify the exact molecular domain involved in HARP inhibition, we investigated the effect of the shorter basic peptide P(122-131) on DU145 cells, which express HARP and its receptor protein tyrosine phosphatase beta/zeta (RPTPbeta/zeta). P(122-131) was not cytotoxic; it dose-dependently inhibited anchorage-independent growth of DU145 cells. Binding studies using biotinylated P(122-131) indicated that this peptide interfered with HARP binding to DU145 cells. Investigation of the mechanisms involved suggested interference, under anchorage-independent conditions, of P(122-131) with a HARP autocrine loop in an RPTPbeta/zeta-dependent fashion. Thus, P(122-131) may hold potential for the treatment of disorders involving RPTPbeta/zeta.