RESUMO
The epidermal growth factor receptor (EGFR) is involved in the regulation of various cellular processes and dysregulation of its signalling plays a critical role in the etiology of a variety of malignancies like breast cancer. At the same time, elevated levels of urokinase (uPA), its receptor uPAR, and other components of the plasminogen activation system are found to be correlated with a poor prognosis in breast cancer. Interestingly, EGFR appears to participate in transducing the signal generated upon binding of uPA to uPAR. However, whether uPA signalling would thereby interfere with ligand-driven EGFR signalling was not described before. Therefore, it was the aim of the present study to investigate the combined effects of uPA and EGF in the low invasive and high invasive breast adenocarcinoma cell lines MCF-7 and MDA-MB-231, respectively. Simultaneous exposure of cells to both signals negatively affected ERK1/2 and AKT activation whereas positive effects on p38 and Src kinase phosphorylation were noted in both cell lines. Furthermore, uPA attenuated the mitogenic effect of EGF on cellular proliferation, invasion and motility in both MCF-7 and MDA-MB-231 cells. Experiments with the uPA amino terminal fragment (ATF) revealed that the negative effects of uPA were independent from its protease activity. Together, these data suggest that enhanced levels of uPA in breast cancer modulate the mitogenic effects of EGF and thus, this knowledge may help to better understand breast cancer pathogenesis as well as to develop new therapeutic options.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Família de Proteínas EGF/farmacologia , Ativador de Plasminogênio Tipo Uroquinase/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Células MCF-7RESUMO
Triple-negative breast cancer (TNBC) is responsible for a disproportionate number of breast cancer patient deaths due to extensive molecular heterogeneity, high recurrence rates and lack of targeted therapies. Dysregulation of the phosphoinositide 3-kinase (PI3K)/AKT pathway occurs in approximately 50% of TNBC patients. Here, we performed a genome-wide CRISPR/Cas9 screen with PI3Kα and AKT inhibitors to find targetable synthetic lethalities in TNBC. Cholesterol homeostasis was identified as a collateral vulnerability with AKT inhibition. Disruption of cholesterol homeostasis with pitavastatin synergized with AKT inhibition to induce TNBC cytotoxicity in vitro, in mouse TNBC xenografts and in patient-derived, estrogen receptor (ER)-negative breast cancer organoids. Neither ER-positive breast cancer cell lines nor ER-positive organoids were sensitive to combined AKT inhibitor and pitavastatin. Mechanistically, TNBC cells showed impaired sterol regulatory element-binding protein 2 (SREBP-2) activation in response to single agent or combination treatment with AKT inhibitor and pitavastatin, which was rescued by inhibition of the cholesterol trafficking protein Niemann-Pick C1 (NPC1). NPC1 loss caused lysosomal cholesterol accumulation, decreased endoplasmic reticulum cholesterol levels, and promoted SREBP-2 activation. Taken together, these data identify a TNBC-specific vulnerability to the combination of AKT inhibitors and pitavastatin mediated by dysregulated cholesterol trafficking. These findings support combining AKT inhibitors with pitavastatin as a therapeutic modality in TNBC. .
RESUMO
COVID-19 remains a significant public health threat due to the ability of SARS-CoV-2 variants to evade the immune system and cause breakthrough infections. Although pathogenic coronaviruses such as SARS-CoV-2 and MERS-CoV lead to severe respiratory infections, how these viruses affect the chromatin proteomic composition upon infection remains largely uncharacterized. Here we used our recently developed integrative DNA And Protein Tagging (iDAPT) methodology to identify changes in host chromatin accessibility states and chromatin proteomic composition upon infection with pathogenic coronaviruses. SARS-CoV-2 infection induces TP53 stabilization on chromatin, which contributes to its host cytopathic effect. We mapped this TP53 stabilization to the SARS-CoV-2 spike and its propensity to form syncytia, a consequence of cell-cell fusion. Differences in SARS-CoV-2 spike variant-induced syncytia formation modify chromatin accessibility, cellular senescence, and inflammatory cytokine release via TP53. Our findings suggest that differences in syncytia formation alter senescence-associated inflammation, which varies among SARS-CoV-2 variants.
RESUMO
Coronavirus disease 2019 (COVID-19) remains a significant public health threat due to the ability of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants to evade the immune system and cause breakthrough infections. Although pathogenic coronaviruses such as SARS-CoV-2 and Middle East respiratory syndrome (MERS)-CoV lead to severe respiratory infections, how these viruses affect the chromatin proteomic composition upon infection remains largely uncharacterized. Here, we use our recently developed integrative DNA And Protein Tagging methodology to identify changes in host chromatin accessibility states and chromatin proteomic composition upon infection with pathogenic coronaviruses. SARS-CoV-2 infection induces TP53 stabilization on chromatin, which contributes to its host cytopathic effect. We mapped this TP53 stabilization to the SARS-CoV-2 spike and its propensity to form syncytia, a consequence of cell-cell fusion. Differences in SARS-CoV-2 spike variant-induced syncytia formation modify chromatin accessibility, cellular senescence, and inflammatory cytokine release via TP53. Our findings suggest that differences in syncytia formation alter senescence-associated inflammation, which varies among SARS-CoV-2 variants.
Assuntos
COVID-19 , Coronavírus da Síndrome Respiratória do Oriente Médio , Humanos , SARS-CoV-2 , Cromatina , Proteômica , Senescência Celular , Células Gigantes , Proteína Supressora de Tumor p53/genéticaRESUMO
The adaptor protein regulator for ubiquitous kinase/c-Cbl-interacting protein of 85kDa (Ruk/CIN85) was found to modulate HER1/EGFR signaling and processes like cell adhesion and apoptosis. Although these features imply a role in carcinogenesis, it is so far unknown how and by which molecular mechanisms Ruk/CIN85 could affect a certain tumor phenotype. By analyzing samples from breast cancer patients, we found high levels of Ruk(l)/CIN85 especially in lymph node metastases from patients with invasive breast adenocarcinomas, suggesting that Ruk(l)/CIN85 contributes to malignancy. Expression of Ruk(l)/CIN85 in weakly invasive breast adenocarcinoma cells deficient of Ruk(l)/CIN85 indeed converted them into more malignant cells. In particular, Ruk(l)/CIN85 reduced the growth rate, decreased cell adhesion, enhanced anchorage-independent growth, increased motility in both transwell migration and wound healing assays as well as affected the response to epidermal growth factor. Thereby, Ruk(l)/CIN85 led to a more rapid and prolonged epidermal growth factor-dependent activation of Src, Akt and ERK1/2 and treatment with the Src inhibitor PP2 and the PI3K inhibitor LY294002 abolished the Ruk(l)/CIN85-dependent changes in cell motility. Together, this study indicates that high levels of Ruk(l)/CIN85 contribute to the conversion of breast adenocarcinoma cells into a more malignant phenotype via modulation of the Src/Akt pathway.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Adesão Celular , Movimento Celular , Feminino , Humanos , Sistema de Sinalização das MAP Quinases , Proteína Oncogênica pp60(v-src)/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de SinaisRESUMO
We report a study of the attenuation spectra transformations for a series of Bismuth (Bi) doped silica fibers with various contents of emission-active Bi centers, which arise as the result of irradiation by a beam of high-energy electrons. The experimental data reveal a substantial decrease of concentration of the Bi centers, associated with the presence of Germanium in silica glass, at increasing the irradiation dose (the resonant-absorption bleaching effect in germano-silicate fiber). In contrast, the spectral changes that appear in Bi doped alumino-silicate fiber have through irradiation a completely different character, viz., weak growth of the resonant-absorption peaks ascribed to the Bi centers, associated with the presence of Aluminum in silica glass. These results demonstrating high susceptibility of Bi centers to electron irradiation while opposite routes of the irradiation-induced spectral changes in Bi doped germanate and aluminate fibers seem to be of worth notice for understanding the nature of these centers.
Assuntos
Bismuto/química , Bismuto/efeitos da radiação , Fibras Ópticas , Dióxido de Silício/química , Dióxido de Silício/efeitos da radiação , Elétrons , Desenho de Equipamento , Análise de Falha de Equipamento , Teste de Materiais , RefratometriaRESUMO
The tumor microenvironment (TME) is a complex neighborhood that consists of immune cells, fibroblasts, pericytes, adipocytes, endothelial and neuronal cells, and the extracellular matrix proteins. TME also consists of physical factors, such as oxygen availability, changing pH, interstitial fluid pressure, and tissue stiffness. As cancer progresses, the physical properties and the cells in the TME change significantly, impacting the efficacy of the therapies and modulating drug resistance. This has led to the development of several new treatments targeting the TME. This review focuses on recent advances on the role of TME in drug resistance, with a particular focus on the ongoing clinical trials aiming at disrupting the TME- and the extracellular matrix-mediated protection against therapies.
Assuntos
Neoplasias , Preparações Farmacêuticas , Matriz Extracelular , Humanos , Neoplasias/tratamento farmacológico , Células Estromais , Microambiente TumoralRESUMO
The EGFR adaptor protein, CIN85, has been shown to promote breast cancer malignancy and hypoxia-inducible factor (HIF) stability. However, the mechanisms underlying cancer promotion remain ill defined. Here we show that CIN85 is a novel binding partner of the main HIF-prolyl hydroxylase, PHD2, but not of PHD1 or PHD3. Mechanistically, the N-terminal SRC homology 3 domains of CIN85 interacted with the proline-arginine-rich region within the N-terminus of PHD2, thereby inhibiting PHD2 activity and HIF degradation. This activity is essential in vivo, as specific loss of the CIN85-PHD2 interaction in CRISPR/Cas9-edited cells affected growth and migration properties, as well as tumor growth in mice. Overall, we discovered a previously unrecognized tumor growth checkpoint that is regulated by CIN85-PHD2 and uncovered an essential survival function in tumor cells by linking growth factor adaptors with hypoxia signaling. SIGNIFICANCE: This study provides unprecedented evidence for an oxygen-independent mechanism of PHD2 regulation that has important implications in cancer cell survival. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/79/16/4042/F1.large.jpg.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Feminino , Células HEK293 , Humanos , Prolina Dioxigenases do Fator Induzível por Hipóxia/genética , Camundongos Nus , Domínios e Motivos de Interação entre Proteínas , Neoplasias de Mama Triplo Negativas/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Recent studies suggest that the ubiquitin-specific protease USP28 plays an important role in cellular repair and tissue remodeling, which implies that it has a direct role in carcinogenesis. The carcinogenic potential of USP28 was investigated in a comprehensive manner using patients, animal models, and cell culture. The findings demonstrate that overexpression of USP28 correlates with a better survival in patients with invasive ductal breast carcinoma. Mouse xenograft experiments with USP28-deficient breast cancer cells also support this view. Furthermore, lack of USP28 promotes a more malignant state of breast cancer cells, indicated by an epithelial-to-mesenchymal (EMT) transition, elevated proliferation, migration, and angiogenesis as well as a decreased adhesion. In addition to breast cancer, lack of USP28 in mice promoted an earlier onset and a more severe tumor formation in a chemical-induced liver cancer model. Mechanistically, the angio- and carcinogenic processes driven by the lack of USP28 appeared to be independent of HIF-1α, p53, and 53BP1.Implications: The findings of this study are not limited to one particular type of cancer but are rather applicable for carcinogenesis in a more general manner. The obtained data support the view that USP28 is involved in tumor suppression and has the potential to be a prognostic marker. Mol Cancer Res; 16(6); 1000-12. ©2018 AACR.
Assuntos
Neoplasias da Mama/irrigação sanguínea , Neoplasias da Mama/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias Hepáticas/irrigação sanguínea , Neoplasias Hepáticas/genética , Ubiquitina Tiolesterase/deficiência , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Ubiquitina Tiolesterase/metabolismoRESUMO
Clinical studies in breast cancer suggest important associations between intratumoral hypoxia, the upregulation of epidermal growth factor receptor (EGFR or HER1), hypoxia-inducible factor 1α (HIF-1α), and reduced patient survival. However, direct molecular links between EGFR and the hypoxia signaling system are not yet established. Since the oxygen sensor hypoxia-inducible factor prolyl hydroxylase 2 (PHD2) is considered to be the main HIF-1α regulator, we hypothesized that PHD2 and EGFR may be interconnected at the molecular level. By analyzing samples from 313 breast cancer patients, we found that EGFR is a first clinicopathological parameter positively correlating with PHD2. Mechanistically, we identified PHD2 as a direct binding partner of EGFR and show that PHD2 regulates EGFR stability as well as its subsequent signaling in breast carcinoma cells. Overall, we introduce for the first time the direct crosstalk between the oxygen sensor PHD2 and EGFR-mediated tumorigenesis in breast cancer.
Assuntos
Neoplasias da Mama/enzimologia , Receptores ErbB/metabolismo , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Transdução de Sinais , Hipóxia Tumoral , Microambiente Tumoral , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/agonistas , Receptores ErbB/genética , Feminino , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Prolina Dioxigenases do Fator Induzível por Hipóxia/genética , Fosforilação , Ligação Proteica , Estabilidade Proteica , Proteólise , Interferência de RNA , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , TransfecçãoRESUMO
Plasminogen activator inhibitor-1 (PAI-1) is the major and most specific acting urokinase (uPA) and tissue plasminogen activator (tPA) inhibitor. Apart from its function in the fibrinolytic system, PAI-1 was also found to contribute to processes like tissue remodelling, angiogenesis, and tumour progression. However, the role of PAI-1 in those processes remains largely controversial with respect to the influence of PAI-1 on cell signalling pathways. Although PAI-1 does not possess its own cellular receptor, it can be bound to low-density lipoprotein receptor-related protein 1 (LRP1) which was proposed to modulate the ß-catenin pathway. Therefore, we used wild-type mouse embryonic fibroblasts (MEFs), and MEFs deficient of LRP1 to study PAI-1 as modulator of the ß-catenin pathway. We found that PAI-1 influences MEF proliferation and motility in a LRP1-dependent manner and that ß-catenin is important for that response. In addition, expression of ß-catenin and ß-catenin-dependent transcriptional activity were induced by PAI-1 in wild type MEFs, but not in LRP1-deficient cells. Moreover, PAI-1-induced ERK1/2 activation was more prominent in the LRP1-deficient cells and interestingly knockdown of ß-catenin abolished this effect. Together, the data of the current study show that PAI-1 can promote cell migration via LRP1-dependent activation of the ß-catenin and ERK1/2 MAPK pathway which may be important in stage-specific treatment of human diseases associated with high PAI-1 levels.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Receptores de LDL/metabolismo , Serpina E2/metabolismo , Ativador de Plasminogênio Tecidual/metabolismo , Proteínas Supressoras de Tumor/metabolismo , beta Catenina/metabolismo , Animais , Movimento Celular , Proliferação de Células , Fibroblastos/metabolismo , Células HEK293 , Humanos , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Camundongos , RNA Interferente Pequeno/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais , Transcrição Gênica , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
Increased levels of plasminogen activator inhibitor-1 (PAI-1) indicate an enhanced risk of ischaemic/hypoxic cardiovascular events and a poor prognosis. The expression of PAI-1 can be induced by various stimuli including hypoxia, insulin and insulin-like growth factor 1 (IGF-1). The hypoxia-inducible factor-1 (HIF-1) is critical for hypoxia or insulin/IGF-1 mediated PAI-1 induction, but the components involved in merging the signals are not known so far. The adaptor/scaffold protein Ruk/CIN85 may be a candidate since it plays important roles in the regulation of processes associated with cardiovascular and oncological diseases such as downregulation of receptor tyrosine kinases, apoptosis, adhesion and invasion. Therefore, it was the aim of this study to investigate the involvement of Ruk/CIN85 in the regulation of PAI-1 expression. It was found that Ruk/CIN85 induced PAI-1 mRNA and protein expression both under normoxia and hypoxia. The induction of PAI-1 expression by Ruk/CIN85 occurred at the transcriptional level since the half-life of PAI-1 mRNA was not affected in cells overexpressing Ruk/CIN85 and reporter gene assays using wild-type and mutant human PAI-1 promoter luciferase constructs showed that the hypoxia responsive element was responsible for Ruk/CIN85 effects. Further, knocking down HIF-1alpha abolished not only the hypoxia-dependent but also the Ruk/CIN85-dependent PAI-1 induction. In addition, transient or stable overexpression of Ruk/CIN85 also induced HIF-1alpha protein levels and HIF-1 activity and knocking down Ruk/CIN85 reversed these effects. Thereby, Ruk/CIN85 interfered with the proline hydroxylation-dependent HIF-1alpha protein destabilisation. Together, these results provide the first evidence that Ruk/CIN85 induces PAI-1 expression via modulation of HIF-1alpha stability.