RESUMO
Peritoneal metastases are the leading cause of morbidity and mortality in ovarian cancer. Cancer cells float in peritoneal fluid, named ascites, together with a definitely higher number of non neo-neoplastic cells, as single cells or multicellular aggregates. The aim of this work is to uncover the features that make these aggregates the metastasizing units. Immunofluorescence revealed that aggregates are made almost exclusively of ovarian cancer cells expressing the specific nuclear PAX8 protein. The same cells expressed epithelial and mesenchymal markers, such as EPCAM and αSMA, respectively. Expression of fibronectin further supported a hybrid epithelia-mesenchymal phenotype, that is maintained when aggregates are cultivated and proliferate. Hematopoietic cells as well as macrophages are negligible in the aggregates, while abundant in the ascitic fluid confirming their prominent role in establishing an eco-system necessary for the survival of ovarian cancer cells. Using ovarian cancer cell lines, we show that cells forming 3D structures neo-expressed thoroughly fibronectin and αSMA. Functional assays showed that αSMA and fibronectin are necessary for the compaction and survival of 3D structures. Altogether these data show that metastasizing units display a hybrid phenotype that allows maintenance of the 3D structures and the plasticity necessary for implant and seeding into peritoneal lining.
Assuntos
Ascite/patologia , Transição Epitelial-Mesenquimal , Neoplasias Ovarianas/patologia , Fenótipo , Biomarcadores Tumorais , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Transição Epitelial-Mesenquimal/genética , Feminino , Imunofluorescência , Humanos , Imuno-Histoquímica , Hibridização In Situ , Metástase Neoplásica , Neoplasias Ovarianas/metabolismo , Células Tumorais CultivadasRESUMO
The MET oncogene encodes a tyrosine kinase (TK) receptor. Its activation protects cells from death but also stimulates DNA damage response by triggering excess replicative stress. Transcriptomic classification of cancer cell lines based on MET expression showed that response to the PARP inhibitor (PARPi) olaparib is poorer in MET overexpressing cell lines. Accordingly, a high MET expressing lung carcinoma cell line was sensitized to PARPi by MET TK inhibition. This was not linked solely to MET overexpression: other MET overexpressing cell lines were biochemically but not functionally responsive to combined inhibition. Moreover, exogenously induced MET overexpression was unable to induce resistance to PARPi. The MET overexpressing cell line, responsive to the combined PARP and MET inhibition, carried a heterozygous mutation of the ATM gene and showed an attenuated response of ATM to PARPi. Among the downstream targets of ATM activation, NuMA was phosphorylated only in response to the combined PARP and MET inhibition. Given the role played by NuMA in mitosis, data show that the latter is affected by MET and PARP inhibition in cells with haploinsufficient ATM. This is important as ATM heterozygous mutation is frequently found in human cancer and in lung carcinomas in particular.
Assuntos
Antineoplásicos , Neoplasias Pulmonares , Antineoplásicos/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Haploinsuficiência , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Mutação , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologiaRESUMO
The tyrosine kinase encoded by the MET oncogene is activated by gene mutation or amplification in tumors, which in most instances maintain addiction, i.e., dependency, to MET activation. This makes MET an attractive candidate for targeted therapies. Here we show that, in 3/3 MET-addicted human gastric cancer cell lines, MET kinase inhibition resulted in a 3- to 4-fold increased expression of the antiapoptotic small heat-shock protein of 27 kDa (HSP27, HSPB1). HSP27 increase depended on the inhibition of the MEK/ERK pathway and on heat-shock factor 1 (HSF1) and hypoxia-inducible factor-1α (HIF-1α) regulation. Importantly, HSP27-silenced MET-addicted cells underwent 2- and 3-fold more apoptosis following MET inhibition in vitro and in vivo, respectively. Likewise, in human cancer cells susceptible to epidermal growth factor receptor (EGFR) inhibition, EGFR inhibitors induced HSP27 expression and were strengthened by HSP27 suppression. In control cell lines that were not affected by drugs targeting MET or EGFR, these drugs did not induce HSP27 increase. Therefore, in cancer therapies targeting the MET pathway, the induction of HSP27 might limit the efficacy of anti-MET agents. As HSP27 increase also impairs the effectiveness of EGFR inhibitors and is known to protect cells from chemotherapeutics, the induction of HSP27 by targeted agents might strongly affect the success of combination treatments.
Assuntos
Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico HSP27/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Neoplasias Gástricas/tratamento farmacológico , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Western Blotting , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteínas de Choque Térmico HSP27/antagonistas & inibidores , Proteínas de Choque Térmico HSP27/genética , Proteínas de Choque Térmico , Humanos , Técnicas Imunoenzimáticas , Camundongos , Chaperonas Moleculares , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Gástricas/enzimologia , Neoplasias Gástricas/patologia , Células Tumorais Cultivadas , Regulação para Cima , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Titanium dioxide (TiO2) is widely used as ingredient in several products in the nanoform. TiO2-nanoparticles (NPs) are also currently studied for different medical applications. A large debate exists on possible adverse health effects related to their exposure. While there is some evidence of TiO2-NP central nervous system toxicity, their effects on peripheral neurons have been poorly explored. In this study we investigated the effects of TiO2-NPs on dorsal root ganglion (DRG) sensory neurons and satellite glial cells that may be reached by nanoparticles from the bloodstream. We found that TiO2-NPs are internalized in DRG cells and induce apoptosis in a dose dependent manner in both types of cells, ROS production and changes in expression of proinflammatory cytokine IL-1ß. Furthermore, we found that the axonal retrograde transport is altered in neurons upon exposure to TiO2-NPs. Overall, the results indicate a potential neurotoxic effect of TiO2-NPs on DRG cells. FROM THE CLINICAL EDITOR: Exposure to titanium dioxide nanoparticles is increasing in medical practice. Little is known about their potential toxic effects on the peripheral nervous system. The authors studied this aspect and showed that titanium nanoparticles might potentially cause toxicity over long term.
Assuntos
Apoptose/efeitos dos fármacos , Gânglios Espinais/metabolismo , Nanopartículas Metálicas , Titânio/metabolismo , Animais , Embrião de Galinha , Gânglios Espinais/citologia , Gânglios Espinais/efeitos dos fármacos , Interleucina-1beta/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Titânio/farmacologiaRESUMO
Platinum-based chemotherapy is widely used to treat various cancers, but many patients ultimately relapse due to drug resistance. We employed phosphoproteomic analysis and functional assays of the response of SK-OV-3 ovarian cancer cells to cisplatin as a strategy to identify kinases as candidate druggable targets to sensitize cells to platinum. A SILAC-based approach combined with TiO2-based phosphopeptide enrichment allowed the direct identification of ERK1/2, p90RSK, and ERBB2 as kinases whose phosphorylation is regulated by cisplatin. Bioinformatic analysis revealed enrichment in linear phosphorylation motifs predicted to be targets of p38MAPK, CDK2, and PIM2. All three PIM kinases were found expressed in a panel of 10 ovarian cancer cell lines, with the oncogenic PIM2 being the most commonly induced by cisplatin. Targeting PIM2 kinase by either biochemical inhibitors or RNA interference impaired cell growth, decreased cisplatin-triggered BAD phosphorylation, and sensitized ovarian cancer cells to drug-induced apoptosis. Overexpression of PIM2 triggered anchorage-independent growth and resulted in increased BAD phosphorylation and cell resistance to DNA damaging agents. The data show that the PIM2 kinase plays a role in the response of ovarian cancer cells to platinum drugs and suggest that PIM inhibitors may find clinical application as an adjunct to platinum-based therapies.
Assuntos
Cisplatino/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteômica/métodos , Proteínas Proto-Oncogênicas/metabolismo , Motivos de Aminoácidos , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Espectrometria de Massas em Tandem/métodos , Proteína de Morte Celular Associada a bcl/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The hepatocyte growth factor (HGF) also known as scatter factor activates cancer cell invasion and metastasis. We show that in ovarian cancer cells HGF induced the phosphorylation of the small heat shock protein of 27 kDa (HSP27) by activating the p38MAPK. HSP27 is increased in many cancers at advanced stage including ovarian cancer and associated with cancer resistance to therapy and poor patients' survival. The phosphorylation of HSP27 regulates both its chaperone activity and its control of cytoskeletal stability. We show that HSP27 was necessary for the remodeling of actin filaments induced by HGF and that motility in vitro depended on the p38MAPK-MK2 axis. In vivo, HSP27 silencing impaired the ability of the highly metastatic, HGF-secreting ovarian cancer cells to give rise to spontaneous metastases. This was due to defective motility across the vessel wall and reduced growth. Indeed, HSP27 silencing impaired the ability of circulating ovarian cancer cells to home to the lungs and to form experimental hematogenous metastases and the capability of cancer cells to grow as subcutaneous xenografts. Moreover, HSP27 suppression resulted in the sensitization of xenografts to low doses of the chemotherapeutic paclitaxel, likely because HSP27 protected microtubules from bundling caused by the drug. Altogether, these data show that the HSP27 is required for the proinvasive and prometastatic activity of HGF and suggest that HSP27 might be not only a marker of progression of ovarian cancer, but also a suitable target for therapy.
Assuntos
Movimento Celular , Resistencia a Medicamentos Antineoplásicos , Proteínas de Choque Térmico HSP27/metabolismo , Fator de Crescimento de Hepatócito/farmacologia , Neoplasias Pulmonares/secundário , Neoplasias Ovarianas/patologia , Animais , Antineoplásicos Fitogênicos/farmacologia , Apoptose , Western Blotting , Proliferação de Células , Feminino , Imunofluorescência , Proteínas de Choque Térmico HSP27/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Invasividade Neoplásica , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Paclitaxel/farmacologia , Fosforilação/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Glioblastoma (GBM) is known as an intractable, highly heterogeneous tumor encompassing multiple subclones, each supported by a distinct glioblastoma stem cell (GSC). The contribution of GSC genetic and transcriptional heterogeneity to tumor subclonal properties is debated. In this study, we describe the systematic derivation, propagation, and characterization of multiple distinct GSCs from single, treatment-naive GBMs (GSC families). The tumorigenic potential of each GSC better correlates with its transcriptional profile than its genetic make-up, with classical GSCs being inherently more aggressive and mesenchymal more dependent on exogenous growth factors across multiple GBMs. These GSCs can segregate and recapitulate different histopathological aspects of the same GBM, as shown in a paradigmatic tumor with two histopathologically distinct components, including a conventional GBM and a more aggressive primitive neuronal component. This study provides a resource for investigating how GSCs with distinct genetic and/or phenotypic features contribute to individual GBM heterogeneity and malignant escalation.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Humanos , Glioblastoma/patologia , Neoplasias Encefálicas/metabolismo , Amplificação de Genes , Células-Tronco Neoplásicas/metabolismo , Carcinogênese/patologia , Linhagem Celular TumoralRESUMO
Many nervous proteins are expressed in cancer cells. In this report, we asked whether the synaptic protein neuroligin 1 (NLGN1) was expressed by prostatic and pancreatic carcinomas; in addition, given the tendency of these tumors to interact with nerves, we asked whether NLGN1 played a role in this process. Through immunohistochemistry on human tissue microarrays, we showed that NLGN1 is expressed by prostatic and pancreatic cancer tissues in discrete stages and tumor districts. Next, we performed in vitro and in vivo assays, demonstrating that NLGN1 promotes cancer cell invasion and migration along nerves. Because of the established role of the neurotrophic factor glial cell line-derived neurotrophic factor (GDNF) in tumor-nerve interactions, we assessed a potential NLGN1-GDNF cooperation. We found that blocking GDNF activity with a specific antibody completely inhibited NLGN1-induced in vitro cancer cell invasion of nerves. Finally, we demonstrated that, in the presence of NLGN1, GDNF markedly activates cofilin, a cytoskeletal regulatory protein, altering filopodia dynamics. In conclusion, our data further prove the existence of a molecular and functional cross-talk between the nervous system and cancer cells. NLGN1 was shown here to function along one of the most represented neurotrophic factors in the nerve microenvironment, possibly opening new therapeutic avenues.
Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Neoplasias/metabolismo , Tecido Nervoso/metabolismo , Fatores de Despolimerização de Actina/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Camundongos Endogâmicos C57BL , Invasividade Neoplásica , Neoplasias/patologia , Tecido Nervoso/patologia , Ligação Proteica , Pseudópodes/metabolismoRESUMO
Compelling evidence shows that cancer persister cells represent a major limit to the long-term efficacy of targeted therapies. However, the phenotype and population dynamics of cancer persister cells remain unclear. We developed a quantitative framework to study persisters by combining experimental characterization and mathematical modeling. We found that, in colorectal cancer, a fraction of persisters slowly replicates. Clinically approved targeted therapies induce a switch to drug-tolerant persisters and a temporary 7- to 50-fold increase of their mutation rate, thus increasing the number of persister-derived resistant cells. These findings reveal that treatment may influence persistence and mutability in cancer cells and pinpoint inhibition of error-prone DNA polymerases as a strategy to restrict tumor recurrence.
Assuntos
Neoplasias Colorretais , Taxa de Mutação , Antibacterianos/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Humanos , Dinâmica PopulacionalRESUMO
The treatment of unresectable cholangiocarcinoma (CCA) is limited by the development of resistance to conventional first-line chemotherapy based on gemcitabine (GEM). In addition, a prior treatment with GEM frequently induces cross-resistance to other drugs employed in the second-line. Paclitaxel (PTX) is now emerging as an alternative option for the management of advanced/metastatic CCA. In the present work, we evaluate the antitumor activity of PTX in preclinical models of multidrug-resistant intrahepatic cholangiocarcinoma (iCCA). In vitro, PTX decreases tumor cell viability by affecting the cell cycle and inducing apoptosis and impairs the stem cell compartment. In vivo, a therapeutic regimen containing albumin-bound nanoparticle (Nab)-PTX overcomes drug resistance resulting in delayed tumor growth, impaired organization of the tumor vasculature, and reduced glucose uptake. Together, our results provide a rationale to consider PTX-based regimens in patients with iCCA who became refractory to conventional therapies.
RESUMO
In glioblastoma (GBM), the most frequent and lethal brain tumor, therapies suppressing recurrently altered signaling pathways failed to extend survival. However, in patient subsets, specific genetic lesions can confer sensitivity to targeted agents. By exploiting an integrated model based on patient-derived stem-like cells, faithfully recapitulating the original GBMs in vitro and in vivo, here, we identify a human GBM subset (â¼9% of all GBMs) characterized by ERBB3 overexpression and nuclear accumulation. ERBB3 overexpression is driven by inheritable promoter methylation or post-transcriptional silencing of the oncosuppressor miR-205 and sustains the malignant phenotype. Overexpressed ERBB3 behaves as a specific signaling platform for fibroblast growth factor receptor (FGFR), driving PI3K/AKT/mTOR pathway hyperactivation, and overall metabolic upregulation. As a result, ERBB3 inhibition by specific antibodies is lethal for GBM stem-like cells and xenotransplants. These findings highlight a subset of patients eligible for ERBB3-targeted therapy.
Assuntos
Glioblastoma/genética , MicroRNAs/metabolismo , Receptor ErbB-3/metabolismo , Anticorpos/metabolismo , Apoptose , Linhagem Celular Tumoral , Fator 2 de Crescimento de Fibroblastos , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Humanos , MicroRNAs/genética , Oligodendroglia/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Prognóstico , Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor ErbB-3/antagonistas & inibidores , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais , Esferoides Celulares/patologia , Serina-Treonina Quinases TOR/metabolismoRESUMO
Identifying cancer drivers and actionable mutations is critical for precision oncology. In epithelial ovarian cancer (EOC) the majority of mutations lack biological or clinical validation. We fully characterized 43 lines of Patient-Derived Xenografts (PDXs) and performed copy number analysis and whole exome sequencing of 12 lines derived from naïve, high grade EOCs. Pyrosequencing allowed quantifying mutations in the source tumours. Drug response was assayed on PDX Derived Tumour Cells (PDTCs) and in vivo on PDXs. We identified a PIK3R1W624R variant in PDXs from a high grade serous EOC. Allele frequencies of PIK3R1W624R in all the passaged PDXs and in samples of the source tumour suggested that it was truncal and thus possibly a driver mutation. After inconclusive results in silico analyses, PDTCs and PDXs allowed the showing actionability of PIK3R1W624R and addiction of PIK3R1W624R carrying cells to inhibitors of the PI3K/AKT/mTOR pathway. It is noteworthy that PIK3R1 encodes the p85α regulatory subunit of PI3K, that is very rarely mutated in EOC. The PIK3R1W624R mutation is located in the cSH2 domain of the p85α that has never been involved in oncogenesis. These data show that patient-derived models are irreplaceable in their role of unveiling unpredicted driver and actionable variants in advanced ovarian cancer.
Assuntos
Carcinoma Epitelial do Ovário/genética , Classe Ia de Fosfatidilinositol 3-Quinase/genética , Cistadenocarcinoma Seroso/genética , Neoplasias Ovarianas/genética , Animais , Carcinoma Epitelial do Ovário/tratamento farmacológico , Carcinoma Epitelial do Ovário/enzimologia , Linhagem Celular Tumoral , Classe Ia de Fosfatidilinositol 3-Quinase/metabolismo , Cistadenocarcinoma Seroso/tratamento farmacológico , Cistadenocarcinoma Seroso/enzimologia , Feminino , Xenoenxertos , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Terapia de Alvo Molecular , Mutação , Gradação de Tumores , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/enzimologia , Distribuição AleatóriaRESUMO
OBJECTIVE: Relapsed epithelial ovarian cancer (EOC) is frequently treated with pegylated liposomal doxorubicin (PLD). Unfortunately, most patients do not benefit from treatment. Prediction of response is crucial to optimize PLD use and avoid unnecessary toxicities. We aimed at assessing the value of topoisomerase II alpha (TOP2A) expression as predictive marker of response to PLD-based therapy in patients with relapsed EOCs. METHODS: We retrospectively analyzed Formalin Fixed Paraffin Embedded (FFPE) tissues from 101 patients with platinum resistant (PR) or partially platinum-sensitive (PPS) EOCs treated with PLD-based chemotherapy beyond second line in three referral cancer centers between January 2010 and June 2018. TOP2A expression was measured by immunohistochemistry (IHC): images of each sample were acquired by optical microscope and analyzed by using automatic counter software. Correlation between TOP2A expression and response to PLD was assessed. Since no cut-off for positivity has been validated yet, we dichotomized TOP2A expression based on a cut-off of 18% (mean value in this study). RESULTS: TOP2A expression beyond cut-off was not prognostic for primary platinum-free interval in our series (p = 0.77) neither for optimal cytoreduction (p = 0.9). TOP2A > 18% was associated with a longer time to progression (TTP) following PLD-treatment, although not statistically significant (p = 0.394). No difference was observed between PR and PPS patients' groups (p = 0.445 and p = 0.185, respectively). Not unexpectedly, patients with TOP2A expression > 18% treated with PLD monotherapy achieved a longer TTP compared with PLD-doublet therapy (p = 0.05). CONCLUSIONS: Our data suggest that TOP2A status might predict activity of PLD in patients with PR/PPS EOCs.
Assuntos
Antibióticos Antineoplásicos/uso terapêutico , Carcinoma Epitelial do Ovário/tratamento farmacológico , DNA Topoisomerases Tipo II/metabolismo , Doxorrubicina/análogos & derivados , Recidiva Local de Neoplasia , Neoplasias Ovarianas/tratamento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica , Biomarcadores Tumorais/metabolismo , Carcinoma Epitelial do Ovário/metabolismo , Carcinoma Epitelial do Ovário/patologia , DNA Topoisomerases Tipo II/genética , Doxorrubicina/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Estimativa de Kaplan-Meier , Pessoa de Meia-Idade , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Platina/farmacologia , Polietilenoglicóis/uso terapêutico , Estudos RetrospectivosRESUMO
We have studied calcium signals and their role in the migration of neuronal and nonneuronal cells of embryonic chick ciliary ganglion (CG). In vitro, neurons migrate in association with nonneuronal cells to form cellular aggregates. Changes in the modulus of the velocity of the neuron-nonneuronal cell complex were observed in response to treatments that increased or decreased intracellular calcium concentration. In addition, both cell types generated spontaneous calcium activity that was abolished by removal of extracellular calcium. Calcium signals in neurons could be characterized as either spikes or waves. Neuronal spikes were found to be related to action potential generation whereas neuronal waves were due to voltage-independent calcium influx. Nonneuronal cells generated calcium oscillations that were dependent on calcium release from intracellular stores and on voltage-independent calcium influx. Application of thimerosal, a compound that stimulates calcium mobilization from internal stores, increased: (1) the amplitude of spontaneous nonneuronal oscillations; (2) the area of migrating nonneuronal cells; and (3) the velocity of the neuronal-nonneuronal cell complex. We conclude that CG cell migration is a calcium dependent process and that nonneuronal cell calcium oscillations play a key role in the modulation of velocity.
Assuntos
Sinalização do Cálcio/fisiologia , Movimento Celular/fisiologia , Gânglios Parassimpáticos/citologia , Gânglios Parassimpáticos/fisiologia , Animais , Embrião de Galinha , Neuroglia/fisiologia , Neurônios/fisiologiaRESUMO
The molecular mechanisms orchestrating peritoneal and hematogenous metastases of ovarian cancer cells are assumed to be distinct. We studied the p90RSK family of serine/threonine kinases that lie downstream the RAS-ERK/MAPK pathway and modulate a variety of cellular processes including cell proliferation, survival, motility and invasiveness. We found the RSK1 and RSK2 isoforms expressed in a number of human ovarian cancer cell lines, where they played redundant roles in sustaining in vitro motility and invasiveness. In vivo, silencing of both RSK1 and RSK2 almost abrogated short-term and long-term metastatic engraftment of ovarian cancer cells in the peritoneum. In addition, RSK1/RSK2 silenced cells failed to colonize the lungs after intravenous injection and to form hematogenous metastasis from subcutaneous xenografts. RSK1/RSK2 suppression resulted in lessened ovarian cancer cell spreading on endogenous fibronectin (FN). Mechanistically, RSK1/RSK2 knockdown diminished FN transcription, α5ß1 integrin activation and TGF-ß1 translation. Reduced endogenous FN deposition and TGF-ß1 secretion depended on the lack of activating phosphorylation of the transcription/translation factor YB-1 by p90RSK. Altogether data show how p90RSK activates a self-reinforcing cell autonomous pro-adhesive circuit necessary for metastatic seeding of ovarian cancer cells. Thus, p90RSK inhibitors might hinder both the hematogenous and the peritoneal metastatic spread of human ovarian cancer.
Assuntos
Neoplasias Pulmonares/genética , Neoplasias Ovarianas/genética , Neoplasias Peritoneais/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Animais , Western Blotting , Adesão Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Feminino , Fibronectinas/genética , Fibronectinas/metabolismo , Regulação Neoplásica da Expressão Gênica , Células HeLa , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Camundongos Endogâmicos NOD , Camundongos SCID , Microscopia Confocal , Invasividade Neoplásica , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Neoplasias Peritoneais/metabolismo , Neoplasias Peritoneais/secundário , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo , Transplante HeterólogoRESUMO
Platinum-based chemotherapy is the recommended first-line treatment for high-grade serous (HGS) epithelial ovarian cancer (EOC). However, most patients relapse because of platinum refractory/resistant disease. We aimed at assessing whether other drugs, commonly used to treat relapsed HGS-EOC and poorly active in this clinical setting, might be more effective against chemotherapy-naïve cancers. We collected couples of HGS-EOC samples from the same patients before and after neo-adjuvant platinum-based chemotherapy. Samples were propagated as Patient Derived Xenografts (PDXs) in immunocompromised mice ("xenopatients"). Xenopatients were treated in parallel with carboplatin, gemcitabine, pegylated liposomal doxorubicin (PLD) and trabectedin. PDXs derived from a naïve HSG-EOC showed responsiveness to carboplatin, trabectedin and gemcitabine. The PDXs propagated from a tumor mass of the same patient, grown after carboplatin therapy, did no longer respond to trabectedin and gemcitabine and showed heterogeneous response to carboplatin. In line, the patient experienced clinically platinum-sensitivity first and then discordant responses of different tumor sites to platinum re-challenge. Loss of PDX responsiveness to drugs was associated with 4-fold increase of NR2F2 gene expression. PDXs from another naïve tumor showed complete response to PLD, which was lost in the PDXs derived from a mass grown in the same patient after platinum-based chemotherapy. This patient showed platinum refractoriness and responded poorly to PLD as second-line treatment. PDX response to PLD was associated with high expression of TOP2A protein. PDXs demonstrated that chemotherapy-naïve HGS-EOC might display susceptibility to agents not used commonly as first line treatment. Data suggest the importance of personalizing also chemotherapy.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Ovarianas/tratamento farmacológico , Medicina de Precisão , Idoso , Animais , Apoptose , Carboplatina/administração & dosagem , Proliferação de Células , Desoxicitidina/administração & dosagem , Desoxicitidina/análogos & derivados , Dioxóis/administração & dosagem , Doxorrubicina/administração & dosagem , Doxorrubicina/análogos & derivados , Feminino , Humanos , Camundongos , Pessoa de Meia-Idade , Terapia Neoadjuvante , Neoplasias Ovarianas/patologia , Polietilenoglicóis/administração & dosagem , Tetra-Hidroisoquinolinas/administração & dosagem , Trabectedina , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto , GencitabinaRESUMO
Among patients with colorectal cancer who benefit from therapy targeted to the epidermal growth factor receptor (EGFR), stable disease (SD) occurs more frequently than massive regressions. Exploring the mechanisms of this incomplete sensitivity to devise more efficacious treatments will likely improve patients' outcomes. We tested therapies tailored around hypothesis-generating molecular features in patient-derived xenografts ("xenopatients"), which originated from 125 independent samples that did not harbor established resistance-conferring mutations. Samples from xenopatients that responded to cetuximab, an anti-EGFR agent, with disease stabilization displayed high levels of EGFR family ligands and receptors, indicating high EGFR pathway activity. Five of 21 SD models (23.8%) characterized by particularly high expression of EGFR and EGFR family members regressed after intensified EGFR blockade by cetuximab and a small-molecule inhibitor. In addition, a subset of cases in which enhanced EGFR inhibition was unproductive (6 of 16, 37.5%) exhibited marked overexpression of insulin-like growth factor 2 (IGF2). Enrichment of IGF2 overexpressors among cases with SD was demonstrated in the entire xenopatient collection and was confirmed in patients by mining clinical gene expression data sets. In functional studies, IGF2 overproduction attenuated the efficacy of cetuximab. Conversely, interception of IGF2-dependent signaling in IGF2-overexpressing xenopatients potentiated the effects of cetuximab. The clinical implementation of IGF inhibitors awaits reliable predictors of response, but the results of this study suggest rational combination therapies for colorectal cancer and provide evidence for IGF2 as a biomarker of reduced tumor sensitivity to anti-EGFR therapy and a determinant of response to combined IGF2/EGFR targeting.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Receptores ErbB/química , Fator de Crescimento Insulin-Like II/química , Animais , Anticorpos Monoclonais Humanizados/uso terapêutico , Biomarcadores Tumorais/metabolismo , Cetuximab , Ensaios Clínicos como Assunto , Resistencia a Medicamentos Antineoplásicos , Éxons , Humanos , Imuno-Histoquímica , Ligantes , Camundongos , Mutação , Transplante de NeoplasiasRESUMO
Embryonic chick ciliary ganglion (CG) neurons obtained from E7-E8 ganglia maintained in serum-free medium were stimulated with 50 microM muscarine. A fast hyperpolarization of the membrane potential was observed in 25% of the cells tested, that in some cases was associated with a slower depolarization. Accordingly, in voltage clamp experiments, either an outward current or a biphasic current response could be observed. Single-channel experiments provide evidence that these signals can be associated to the activation of a K(+) channel whose conductance is 20 pS.
Assuntos
Gânglios Parassimpáticos/fisiologia , Neurônios/fisiologia , Canais de Potássio/fisiologia , Receptores Colinérgicos/fisiologia , Receptores Muscarínicos/fisiologia , Animais , Embrião de Galinha , Gânglios Parassimpáticos/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Muscarina/farmacologia , Agonistas Muscarínicos/farmacologia , Neurônios/efeitos dos fármacos , Técnicas de Patch-Clamp , Canais de Potássio/efeitos dos fármacos , Receptores Colinérgicos/efeitos dos fármacos , Receptores Muscarínicos/efeitos dos fármacosRESUMO
This study was aimed at characterizing the effect of purinergic transmission on migration of embryonic ciliary ganglion satellite glial cells. Application of adenosine significantly decreased the rate of migration of glial cells whereas no differences were observed in the presence of ATP. The A(2B) receptor antagonist reverted this action, but application of an A(2A) receptor antagonist or a cAMP-protein kinase inhibitor had no effect on the agonist's stimulation. Forskolin, which stimulates adenylate cyclase activity, and the cAMP analogue 8-CPT-2'-O-Me-cAMP, which selectively activates the guanine exchange factor Epac1, mimicked the effect of adenosine. In addition, intracellular calcium measurements studies revealed that application of either adenosine or ATP induced an increase in [Ca(2+)]i and that the adenosine-induced [Ca(2+)]i response was due to Ca(2+) entry and was blocked by an A(2A) receptor antagonist, SCH 58261, or by high Gd(3+) concentrations. Furthermore, forskolin, but not 8-CPT-2'-O-Me-cAMP, activated the Ca(2+) entry which was blocked by Gd(3+) and was independent of cAMP-protein kinase activity. These results demonstrate the involvement of purinergic P1 signalling in the regulation of cellular migration, and point to the importance of adenosine as a negative modulator of migration of peripheral developing glial cells and as an activator of Ca(2+) entry.
Assuntos
Adenosina/fisiologia , Cálcio/metabolismo , Movimento Celular/fisiologia , AMP Cíclico/fisiologia , Neuroglia/fisiologia , Transdução de Sinais/fisiologia , Adenosina/farmacologia , Trifosfato de Adenosina/farmacologia , Inibidores de Adenilil Ciclases , Animais , Movimento Celular/efeitos dos fármacos , Embrião de Galinha , Colforsina/farmacologia , AMP Cíclico/análogos & derivados , AMP Cíclico/metabolismo , AMP Cíclico/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Receptor A2B de Adenosina/fisiologiaRESUMO
Ghrelin is a hormone regulating energy homeostasis via interaction with its receptor, GHSR-1a. Ghrelin activities in dorsal root ganglia (DRG) cells are unknown. Herein we show that ghrelin induces a change of cytosolic calcium concentration in both glia and neurons of embryonic chick DRG. Both RT-PCR and binding studies performed with fluorescent ghrelin in the presence of either unlabeled ghrelin or GHSR-1a antagonist D-Lys(3)-GHRP-6, indicate that DRG cells express GHSR-1a. In glial cells the response is characterized by a rapid transient rise in [Ca(2+)](i) followed by a long lasting rise. The calcium elevation is dependent on calcium release from thapsigargin-sensitive intracellular stores and on activation of two distinct Ca(2+) entry pathways, a receptor activated calcium entry and a store operated calcium entry. Surprisingly, D-Lys(3)-GHRP-6 exerts several activities in the absence of exogenous ghrelin: (i) it activates calcium release from thapsigargin-sensitive intracellular stores and calcium entry via voltage-operated channels in non-neuronal cells; (ii) it inhibits calcium oscillations in non-neuronal cells exhibiting spontaneous Ca(2+) activity and iii) it promotes apoptosis of DRG cells, both neurons and glia. In summary, we provide the first evidence for ghrelin activity in DRG, and we also demonstrate that the widely used D-Lys(3)-GHRP-6 ghrelin antagonist features ghrelin independent activities.