RESUMO
BACKGROUND: Vascular supply of tumors is one of the main targets for cancer therapy. Here, we investigated if plocabulin (PM060184), a novel marine-derived microtubule-binding agent, presents antiangiogenic and vascular-disrupting activities. METHODS: The effects of plocabulin on microtubule network and dynamics were studied on HUVEC endothelial cells. We have also studied its effects on capillary tube structures formation or destabilization in three-dimensional collagen matrices. In vivo experiments were performed on different tumor cell lines. RESULTS: In vitro studies show that, at picomolar concentrations, plocabulin inhibits microtubule dynamics in endothelial cells. This subsequently disturbs the microtubule network inducing changes in endothelial cell morphology and causing the collapse of angiogenic vessels, or the suppression of the angiogenic process by inhibiting the migration and invasion abilities of endothelial cells. This rapid collapse of the endothelial tubular network in vitro occurs in a concentration-dependent manner and is observed at concentrations lower than that affecting cell survival. The in vitro findings were confirmed in tumor xenografts where plocabulin treatment induced a large reduction in vascular volume and induction of extensive necrosis in tumors, consistent with antivascular effects. CONCLUSIONS: Altogether, these data suggest that an antivascular mechanism is contributing to the antitumor activities of plocabulin.
Assuntos
Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Microtúbulos/efeitos dos fármacos , Neovascularização Fisiológica/efeitos dos fármacos , Policetídeos/farmacologia , Pironas/farmacologia , Tubulina (Proteína)/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Feminino , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Camundongos Nus , Microtúbulos/metabolismo , Neoplasias/irrigação sanguínea , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Neovascularização Patológica/prevenção & controle , Policetídeos/metabolismo , Ligação Proteica , Pironas/metabolismo , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Loss of p53 function compromises genetic homeostasis in cells exhibiting deregulated DNA replication and/or DNA damage, and prevents normal cytotoxic responses to cancer therapies. Genetic and pharmacological approaches are being developed with the ultimate goal of restoring or controlling p53 functions in cancer patients. Progress has recently been made in the clinical use of replication-deficient virus carrying wt-TP53 (Ad5CMV-p53) and/or cancer-selective oncolytic adenoviruses (ONYX-015). These strategies demonstrated clinical activity as monotherapy and were synergistic with traditional chemotherapy agents in the treatment of some types of cancer. In addition, pharmacological methods are under development to either stimulate wild-type p53 protein function, or induce p53 mutant proteins to resume wild-type functions. These methods are based on small chemicals (CP-31388, PRIMA-1), peptides (CDB3) or single-chain Fv antibody fragments corresponding to defined p53 domains. Here, we discuss the mechanisms underlying these approaches and their perspectives for cancer therapy.
Assuntos
Adenoviridae , Antineoplásicos/uso terapêutico , Terapia Genética , Neoplasias/metabolismo , Neoplasias/terapia , Proteína Supressora de Tumor p53/metabolismo , Adenoviridae/genética , Adenoviridae/metabolismo , Proteínas E1B de Adenovirus , Animais , Antineoplásicos/química , Antineoplásicos/metabolismo , Dano ao DNA/genética , Replicação do DNA/genética , Desenho de Fármacos , Humanos , Neoplasias/genética , Ligação Proteica/genética , Estrutura Terciária de Proteína , Proteína Supressora de Tumor p53/genética , Vacinas ViraisRESUMO
The regulation of microtubule dynamics is critical to ensure essential cell functions. End binding protein 1 (EB1) is a master regulator of microtubule dynamics that autonomously binds an extended GTP/GDP-Pi structure at growing microtubule ends and recruits regulatory proteins at this location. However, negative regulation of EB1 association with growing microtubule ends remains poorly understood. We show here that microtubule-associated tumor suppressor ATIP3 interacts with EB1 through direct binding of a non-canonical proline-rich motif. Results indicate that ATIP3 does not localize at growing microtubule ends and that in situ ATIP3-EB1 molecular complexes are mostly detected in the cytosol. We present evidence that a minimal EB1-interacting sequence of ATIP3 is both necessary and sufficient to prevent EB1 accumulation at growing microtubule ends in living cells and that EB1-interaction is involved in reducing cell polarity. By fluorescence recovery of EB1-GFP after photobleaching, we show that ATIP3 silencing accelerates EB1 turnover at microtubule ends with no modification of EB1 diffusion in the cytosol. We propose a novel mechanism by which ATIP3-EB1 interaction indirectly reduces the kinetics of EB1 exchange on its recognition site, thereby accounting for negative regulation of microtubule dynamic instability. Our findings provide a unique example of decreased EB1 turnover at growing microtubule ends by cytosolic interaction with a tumor suppressor.
Assuntos
Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Linhagem Celular , Polaridade Celular , Imunofluorescência , Humanos , Imunoprecipitação , TransfecçãoRESUMO
PM060184 belongs to a new family of tubulin-binding agents originally isolated from the marine sponge Lithoplocamia lithistoides. This compound is currently produced by total synthesis and is under evaluation in clinical studies in patients with advanced cancer diseases. It was recently published that PM060184 presents the highest known affinities among tubulin-binding agents, and that it targets tubulin dimers at a new binding site. Here, we show that PM060184 has a potent antitumor activity in a panel of different tumor xenograft models. Moreover, PM060184 is able to overcome P-gp mediated resistance in vivo, an effect that could be related to its high binding affinity for tubulin. To gain insight into the mechanism responsible of the observed antitumor activity, we have characterized its molecular and cellular effects. We have observed that PM060184 is an inhibitor of tubulin polymerization that reduces microtubule dynamicity in cells by 59%. Interestingly, PM060184 suppresses microtubule shortening and growing at a similar extent. This action affects cells in interphase and mitosis. In the first case, the compound induces a disorganization and fragmentation of the microtubule network and the inhibition of cell migration. In the second case, it induces the appearance of multipolar mitosis and lagging chromosomes at the metaphase plate. These effects correlate with prometaphase arrest and induction of caspase-dependent apoptosis or appearance of cells in a multinucleated interphase-like state unrelated to classical apoptosis pathways. Taken together, these results indicate that PM060184 represents a new tubulin binding agent with promising potential as an anticancer agent.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos/farmacologia , Policetídeos/farmacologia , Pironas/farmacologia , Moduladores de Tubulina/farmacologia , Animais , Apoptose , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Centrossomo/efeitos dos fármacos , Centrossomo/ultraestrutura , Feminino , Interfase , Camundongos Nus , Microtúbulos/efeitos dos fármacos , Microtúbulos/ultraestrutura , Prometáfase/efeitos dos fármacos , Fuso Acromático/efeitos dos fármacos , Fuso Acromático/ultraestrutura , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Metastasis, a fatal complication of breast cancer, does not fully benefit from available therapies. In this study, we investigated whether ATIP3, the major product of 8p22 MTUS1 gene, may be a novel biomarker and therapeutic target for metastatic breast tumors. We show that ATIP3 is a prognostic marker for overall survival among patients with breast cancer. Notably, among metastatic tumors, low ATIP3 levels associate with decreased survival of the patients. By using a well-defined experimental mouse model of cancer metastasis, we show that ATIP3 expression delays the time-course of metastatic progression and limits the number and size of metastases in vivo. In functional studies, ATIP3 silencing increases breast cancer cell migration, whereas ATIP3 expression significantly reduces cell motility and directionality. We report here that ATIP3 is a potent microtubule-stabilizing protein whose depletion increases microtubule dynamics. Our data support the notion that by decreasing microtubule dynamics, ATIP3 controls the ability of microtubule tips to reach the cell cortex during migration, a mechanism that may account for reduced cancer cell motility and metastasis. Of interest, we identify a functional ATIP3 domain that associates with microtubules and recapitulates the effects of ATIP3 on microtubule dynamics, cell proliferation, and migration. Our study is a major step toward the development of new personalized treatments against metastatic breast tumors that have lost ATIP3 expression.
Assuntos
Neoplasias da Mama/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Neoplasias da Mama/patologia , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular , Modelos Animais de Doenças , Progressão da Doença , Feminino , Humanos , Camundongos , Microscopia de Fluorescência/métodos , Microtúbulos/metabolismo , Metástase Neoplásica , Análise de Sequência com Séries de Oligonucleotídeos , Plasmídeos/metabolismo , Prognóstico , Estrutura Terciária de Proteína , Resultado do TratamentoRESUMO
Numerous works have questioned the pertinence of using ßII- and/or ßIII-tubulin expression as markers of prognosis and/or prediction of breast cancer response to chemotherapy containing microtubule-targeting agents. The rationale of such studies was essentially based on microtubule dynamics analysis using purified tubulin in vitro and cancer cell lines. Nonetheless, the significance of ßII- and ßIII-tubulin expression in the control of microtubule dynamics in normal mammary epithelium has never been addressed. Here we investigate the expression and the consequences of ßII- and/or ßIII-tubulin depletion in interphase microtubule dynamics in non-tumor human mammary epithelial cells. We find that both isoforms contribute to the tubulin isotype composition in primary and immortalized human mammary epithelial cells. Moreover, while ßII-tubulin depletion has limited effects on interphase microtubule behavior, ßIII-tubulin depletion causes a strong exclusion of microtubules from lamella and a severe suppression of dynamic instability. These results demonstrate that, while ßII-tubulin is dispensable, ßIII-tubulin is required for interphase microtubule dynamics in untransformed mammary epithelial cells. This strongly suggests that ßIII-tubulin is an essential regulator of interphase microtubule functions in normal breast epithelium cells.
Assuntos
Interfase , Glândulas Mamárias Humanas/citologia , Microtúbulos/metabolismo , Tubulina (Proteína)/metabolismo , Células Cultivadas , Feminino , Técnicas de Silenciamento de Genes , Humanos , Cultura Primária de Células , Interferência de RNA , Análise de Célula Única , Tubulina (Proteína)/genéticaRESUMO
Despite its frequent inactivation in human breast cancers, the role of p21(Cip1) (p21) in morphological plasticity of normal mammary epithelial cells is still poorly understood. To address this question, we have investigated the consequences of p21 silencing in two-dimensional (2D) morphogenesis of untransformed human mammary epithelial cells. Here we show that p21 inactivation causes a reduction of 2D cell spreading and suppresses focal adhesion. In order to investigate the cytoskeletal modifications associated with this altered morphology, we have analyzed the microtubule dynamics in interphase p21-depleted cells. Our results demonstrate that interphase microtubule dynamic instability is strongly increased by p21 silencing. This alteration correlates with severe microtubule hypoacetylation. Next, we show that these microtubule defects in p21-depleted cells can be reversed by the use of the small molecule tubacin, a specific inhibitor of the α-tubulin deacetylase HDAC6. Tubacin-induced microtubule dynamics decrease also correlates with a partial recovery of cell spreading and focal adhesion in those cells. Collectively, these data indicate that p21 regulates the morphological plasticity of normal mammary epithelial cells by modulating dynamics of key cytoskeletal components.
Assuntos
Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Células Epiteliais/metabolismo , Adesões Focais/metabolismo , Interfase , Glândulas Mamárias Humanas/metabolismo , Microtúbulos/metabolismo , Anilidas/farmacologia , Adesão Celular , Proliferação de Células , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21/genética , Citoesqueleto/metabolismo , DNA/biossíntese , Células Epiteliais/citologia , Técnicas de Silenciamento de Genes , Inativação Gênica , Desacetilase 6 de Histona , Histona Desacetilases/metabolismo , Humanos , Ácidos Hidroxâmicos/farmacologia , Glândulas Mamárias Humanas/citologia , Proteínas rho de Ligação ao GTP/antagonistas & inibidoresRESUMO
The SNAIL and SLUG transcription factors play important roles in embryogenesis owing to their anti-apoptotic properties and their ability to promote morphogenetic changes by inducing epithelial-mesenchymal transitions (EMT). These characteristics provide many of the proteins in these families with oncogenic and pro-metastatic capabilities when reactivated in cancers. The SCRATCH subgroup of the SNAIL superfamily, including SCRATCH1 and SCRATCH2, display distinct embryonic functions and diverge early in evolution. Despite the described overexpression of SCRT1 (encoding for SCRATCH1) in a small subset of human lung cancers, there is little data supporting a role of SCRATCH proteins in tumorigenesis. To further explore this possibility, we assessed SNAI1 (SNAIL), SNAI2 (SLUG) and SCRT1 (SCRATCH1) expression in a wide panel of human and murine tumors encompassing 151 primary tumors and 6 different cancer types, including melanomas and multiple different carcinomas. Whereas SNAI1 and SNAI2 are widely expressed in human and murine tumors, our results reveal that SCRT1 transcripts are undetectable in nearly all of the examined tumors suggesting that SCRATCH1 plays a minor role, if any, in tumorigenesis. Our data therefore suggest that oncogenic properties are not shared by all SNAIL superfamily members but instead are specifically allotted to the SNAIL subgroup supporting the conclusions that SNAIL and SCRATCH subgroups are functionally divergent and strengthening the hypothesis that the oncogenic potential of SNAIL and SLUG proteins relies on the hijacking of their embryonic functions.
Assuntos
Neoplasias/genética , Fatores de Transcrição/genética , Animais , Carcinoma/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Mamárias Animais/genética , Melanoma/genética , Camundongos , Fatores de Transcrição da Família Snail , Fatores de Transcrição/fisiologiaRESUMO
We investigated the mechanisms responsible for paclitaxel resistance in HME-1 cells (human mammary epithelial cells immortalized with hTERT). These cells were exposed to paclitaxel (10 pM for 7 days) and 20 cellular surviving populations (PSP) were obtained. PSP demonstrated high levels of resistance to paclitaxel cytotoxicity as compared with HME-1 cells. Activation of mdr-1 gene expression was observed in 2 PSP. Protein expression analysis using a C-terminal targeted antibody showed that 13 PSP were negative for p21/WAF1 expression after ionizing radiation (6 Gy) or doxorubicin (100 nM) treatment. Sequencing of the 3 exons of the CDKN1A gene revealed that 13 PSP contained a point mutation in exon 2. This mutation consisted in a T insertion at codon 104 leading to a premature STOP codon appearance. Mismatch amplification mutation assay and RFLP-PCR confirmed the presence of the mutation in 16 PSP. Western blot using an N-terminal targeted antibody demonstrated that the C-terminal-truncated p21/WAF1 protein (14 kDa) was indeed expressed in the 13 PSP. Our data suggest that p21/WAF1 inactivation may confer a strong resistance to paclitaxel in noncancerous breast epithelial cells harboring a p21/WAF1 mutant.