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Phosphatidylserine (PS) receptors enhance infection of many enveloped viruses through virion-associated PS binding that is termed apoptotic mimicry. Here we show that this broadly shared uptake mechanism is utilized by SARS-CoV-2 in cells that express low surface levels of ACE2. Expression of members of the TIM (TIM-1 and TIM-4) and TAM (AXL) families of PS receptors enhance SARS-CoV-2 binding to cells, facilitate internalization of fluorescently-labeled virions and increase ACE2-dependent infection of SARS-CoV-2; however, PS receptors alone did not mediate infection. We were unable to detect direct interactions of the PS receptor AXL with purified SARS-CoV-2 spike, contrary to a previous report. Instead, our studies indicate that the PS receptors interact with PS on the surface of SARS-CoV-2 virions. In support of this, we demonstrate that: 1) significant quantities of PS are located on the outer leaflet of SARS-CoV-2 virions, 2) PS liposomes, but not phosphatidylcholine liposomes, reduced entry of VSV/Spike pseudovirions and 3) an established mutant of TIM-1 which does not bind to PS is unable to facilitate entry of SARS-CoV-2. As AXL is an abundant PS receptor on a number of airway lines, we evaluated small molecule inhibitors of AXL signaling such as bemcentinib for their ability to inhibit SARS-CoV-2 infection. Bemcentinib robustly inhibited virus infection of Vero E6 cells as well as multiple human lung cell lines that expressed AXL. This inhibition correlated well with inhibitors that block endosomal acidification and cathepsin activity, consistent with AXL-mediated uptake of SARS-CoV-2 into the endosomal compartment. We extended our observations to the related betacoronavirus mouse hepatitis virus (MHV), showing that inhibition or ablation of AXL reduces MHV infection of murine cells. In total, our findings provide evidence that PS receptors facilitate infection of the pandemic coronavirus SARS-CoV-2 and suggest that inhibition of the PS receptor AXL has therapeutic potential against SARS-CoV-2.
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COVID-19/etiologia , Receptores de Superfície Celular/fisiologia , SARS-CoV-2 , Enzima de Conversão de Angiotensina 2/fisiologia , Animais , Feminino , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas/fisiologia , Receptores Proteína Tirosina Quinases/fisiologia , Receptores de Superfície Celular/antagonistas & inibidores , Internalização do Vírus , Receptor Tirosina Quinase Axl , Tratamento Farmacológico da COVID-19RESUMO
BACKGROUND: The dose-response relationship is a fundamental pharmacological parameter necessary to determine therapeutic thresholds. Epi-allelic hypomorphic analysis using RNA interference (RNAi) can similarly correlate target gene dosage with cellular phenotypes. This however requires a set of RNAi triggers empirically determined to attenuate target gene expression to different levels. RESULTS: In order to improve our ability to incorporate epi-allelic analysis into target validation studies, we developed a novel flow cytometry-based functional screening approach (CellSelectRNAi) to achieve unbiased selection of shRNAs from high-coverage libraries that knockdown target gene expression to predetermined levels. Employing a Gaussian probability model we calculated that knockdown efficiency is inferred from shRNA sequence frequency profiles derived from sorted hypomorphic cell populations. We used this approach to generate a hypomorphic epi-allelic cell series of shRNAs to reveal a functional threshold for the tumor suppressor p53 in normal and transformed cells. CONCLUSION: The unbiased CellSelectRNAi flow cytometry-based functional screening approach readily provides an epi-allelic series of shRNAs for graded reduction of target gene expression and improved phenotypic validation.
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Citometria de Fluxo , Interferência de RNA , Alelos , Linhagem Celular Tumoral , Expressão Gênica/efeitos da radiação , Biblioteca Gênica , Células HL-60 , Células Endoteliais da Veia Umbilical Humana , Humanos , Distribuição Normal , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Radiação Ionizante , Análise de Sequência de DNA , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Soluble biomarkers are paramount to personalized medicine. However, the in vivo turnover and biodistribution of soluble proteins is seldom characterized. The cleaved extracellular domain of the AXL receptor (sAXL) is a prognostic biomarker in several diseases and a predictive marker of AXL targeting agents. Plasma sAXL reflects a balance between production in tissues with lymphatic transport into the circulation and removal from blood by degradation or excretion. It is unclear how this transport cycle affects plasma sAXL levels that are the metric for biomarker development. Radiolabeled mouse sAxl was monitored after intravenous injection to measure degradation and urinary excretion of sAxl, and after intradermal injection to mimic tissue or tumor production. sAxl was rapidly taken-up and degraded by the liver and kidney cortex. Surprisingly, intact sAxl was detectable in urine, indicating passage through the glomerular filter and a unique sampling opportunity. The structure of sAxl showed an elongated, flexible molecule with a length of 18 nm and a thickness of only 3 nm, allowing passage through the glomerulus and excretion into the urine. Intradermally injected sAxl passed through local and distant lymph nodes, followed by uptake in liver and kidney cortex. Low levels of sAxl were seen in the plasma, consistent with an extended transit time from local tissue to circulation. The rapid plasma clearance of sAxl suggests that steady-state levels in blood will sensitively and dynamically reflect the rate of production of sAxl in the tissues but will be influenced by perturbations of liver and kidney function.
Assuntos
Receptor Tirosina Quinase Axl , Biomarcadores , Proteínas Proto-Oncogênicas , Receptores Proteína Tirosina Quinases , Animais , Receptores Proteína Tirosina Quinases/metabolismo , Camundongos , Distribuição Tecidual , Biomarcadores/urina , Biomarcadores/sangue , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/urina , Proteínas Proto-Oncogênicas/sangue , Fígado/metabolismo , Humanos , FemininoRESUMO
Cellular behaviors are governed by combinations of systemic and microenvironmental factors; together, these regulate cell signaling responses to growth factors. This contextual microenvironmental influence also determines drug sensitivity. Hence using in vitro systems that model contextual cellular behavior is highly beneficial for effective therapeutic development. Angiogenesis (formation of blood vessels) is driven by a series of dynamic endothelial cell signaling responses to growth factors under the influence of the vascular extracellular matrix and adjacent pericytes. In vitro primary human vascular cell co-cultures self-assemble into capillary-like structures through reciprocal heterotypic interactions that mimic angiogenic context dynamics. By using temporal live-cell imaging-based analysis, unique angiogenic microenvironments can be delineated to quantify the contextual activity of compound inhibitors. We used this in vitro organotypic contextual screening approach to conduct structure-activity relationship analysis on a combretastatin A-4 analogue series to identify novel compounds with potent vascular disrupting activity in vivo.
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Inibidores da Angiogênese/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores da Angiogênese/química , Animais , Linhagem Celular , Técnicas de Cocultura/métodos , Células Endoteliais da Veia Umbilical Humana , Humanos , Artéria Pulmonar/citologia , Relação Estrutura-Atividade , Peixe-ZebraRESUMO
Metastasis underlies the majority of cancer-related deaths. Thus, furthering our understanding of the molecular mechanisms that enable tumor cell dissemination is a vital health issue. Epithelial-to-mesenchymal transitions (EMTs) endow carcinoma cells with enhanced migratory and survival attributes that facilitate malignant progression. Characterization of EMT effectors is likely to yield new insights into metastasis and novel avenues for treatment. We show that the presence of the receptor tyrosine kinase Axl in primary breast cancers independently predicts strongly reduced overall patient survival, and that matched patient metastatic lesions show enhanced Axl expression. We demonstrate that Axl is strongly induced by EMT in immortalized mammary epithelial cells that establishes an autocrine signaling loop with its ligand, Gas6. Epiallelic RNA interference analysis in metastatic breast cancer cells delineated a distinct threshold of Axl expression for mesenchymal-like in vitro cell invasiveness and formation of tumors in foreign and tissue-engineered microenvironments in vivo. Importantly, in two different optical imaging-based experimental breast cancer models, Axl knockdown completely prevented the spread of highly metastatic breast carcinoma cells from the mammary gland to lymph nodes and several major organs and increased overall survival. These findings suggest that Axl represents a downstream effector of the tumor cell EMT that is required for breast cancer metastasis. Thus, the detection and targeted treatment of Axl-expressing tumors represents an important new therapeutic strategy for breast cancer.
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Neoplasias da Mama/fisiopatologia , Células Epiteliais/citologia , Mesoderma/citologia , Metástase Neoplásica , Proteínas Oncogênicas/fisiologia , Receptores Proteína Tirosina Quinases/fisiologia , Animais , Feminino , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Invasividade Neoplásica , Prognóstico , Proteínas Proto-Oncogênicas , Interferência de RNA , Análise de Sobrevida , Engenharia Tecidual , Receptor Tirosina Quinase AxlRESUMO
OBJECTIVES: AXL, a transmembrane receptor tyrosine kinase, is highly expressed and associated with poor prognosis in non-small cell lung cancer (NSCLC). Bemcentinib (BGB324), a selective orally bioavailable small molecule AXL inhibitor, synergizes with docetaxel in preclinical models. We performed a phase I trial of bemcentinib plus docetaxel in previously treated advanced NSCLC. MATERIALS AND METHODS: Escalation of two dose levels of bemcentinib (200 mg load × 3 days then 100 mg daily, or 400 mg load × 3 days then 200 mg daily) in combination with docetaxel (60 or 75 mg/m2 every 3 weeks) followed a 3+3 study design. Due to hematologic toxicity, prophylactic G-CSF was added. Bemcentinib monotherapy was administered for one week prior to docetaxel initiation to assess pharmacodynamic and pharmacokinetic effects alone and in combination. Plasma protein biomarker levels were measured. RESULTS: 21 patients were enrolled (median age 62 years, 67% male). Median treatment duration was 2.8 months (range 0.7-10.9 months). The main treatment-related adverse events were neutropenia (86%, 76% ≥G3), diarrhea (57%, 0% ≥G3), fatigue (57%, 5% ≥G3), and nausea (52%, 0% ≥G3). Neutropenic fever occurred in 8 (38%) patients. The maximum tolerated dose was docetaxel 60 mg/m2 with prophylactic G-CSF support plus bemcentinib 400 mg load × 3 days followed by 200 mg daily thereafter. Bemcentinib and docetaxel pharmacokinetics resembled prior monotherapy data. Among 17 patients evaluable for radiographic response, 6 (35%) patients had partial response and 8 (47%) patients had stable disease as best response. Bemcentinib administration was associated with modulation of proteins involved in protein kinase B signaling, reactive oxygen species metabolism, and other processes. CONCLUSION: Bemcentinib plus docetaxel with G-CSF support demonstrates anti-tumor activity in previously treated, advanced NSCLC. The role of AXL inhibition in the treatment of NSCLC remains under investigation.
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Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Masculino , Pessoa de Meia-Idade , Feminino , Carcinoma Pulmonar de Células não Pequenas/patologia , Docetaxel/uso terapêutico , Neoplasias Pulmonares/patologia , Taxoides/uso terapêutico , Fator Estimulador de Colônias de Granulócitos , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Resultado do TratamentoRESUMO
Mutations in STK11/LKB1 in non-small cell lung cancer (NSCLC) are associated with poor patient responses to immune checkpoint blockade (ICB), and introduction of a Stk11/Lkb1 (L) mutation into murine lung adenocarcinomas driven by mutant Kras and Trp53 loss (KP) resulted in an ICB refractory syngeneic KPL tumor. Mechanistically this occurred because KPL mutant NSCLCs lacked TCF1-expressing CD8 T cells, a phenotype recapitulated in human STK11/LKB1 mutant NSCLCs. Systemic inhibition of Axl results in increased type I interferon secretion from dendritic cells that expanded tumor-associated TCF1+PD-1+CD8 T cells, restoring therapeutic response to PD-1 ICB in KPL tumors. This was observed in syngeneic immunocompetent mouse models and in humanized mice bearing STK11/LKB1 mutant NSCLC human tumor xenografts. NSCLC-affected individuals with identified STK11/LKB1 mutations receiving bemcentinib and pembrolizumab demonstrated objective clinical response to combination therapy. We conclude that AXL is a critical targetable driver of immune suppression in STK11/LKB1 mutant NSCLC.
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Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Quinases Proteína-Quinases Ativadas por AMP , Animais , Linfócitos T CD8-Positivos/metabolismo , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Camundongos , Receptor de Morte Celular Programada 1/genética , Proteínas Serina-Treonina Quinases/genética , Receptor Tirosina Quinase AxlRESUMO
Carfilzomib is a proteasome inhibitor in clinical development that primarily targets the chymotrypsin-like (CT-L) subunits in both the constitutive proteasome (c20S) and the immunoproteasome (i20S). To investigate the impact of inhibiting the CT-L activity with carfilzomib, we set out to quantitate the levels of CT-L subunits beta5 from the c20S and LMP7 from the i20S in normal and malignant hematopoietic cells. We found that the i20S is a major form of the proteasome expressed in cells of hematopoietic origin, including multiple myeloma (MM) CD138+ tumor cells. Although specific inhibition of either LMP7 or beta5 alone was insufficient to produce an antitumor response, inhibition of all proteasome subunits was cytotoxic to both hematologic tumor cells and peripheral blood mononuclear cells. However, selective inhibition of both beta5 and LMP7 was sufficient to induce an antitumor effect in MM, non-Hodgkin lymphoma, and leukemia cells while minimizing the toxicity toward nontransformed cells. In MM tumor cells, CT-L inhibition alone was sufficient to induce proapoptotic sequelae, including proteasome substrate accumulation, Noxa and caspase 3/7 induction, and phospho-eIF2alpha suppression. These data support a hypothesis that hematologic tumor cells are uniquely sensitive to CT-L inhibition and provide a mechanistic understanding of the clinical safety profile and antitumor activity of proteasome inhibitors.
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Apoptose/efeitos dos fármacos , Neoplasias Hematológicas/tratamento farmacológico , Oligopeptídeos/farmacologia , Inibidores de Proteases/farmacologia , Inibidores de Proteassoma , Caspase 3/metabolismo , Caspase 7/metabolismo , Domínio Catalítico , Linhagem Celular Tumoral , Quimotripsina/antagonistas & inibidores , Quimotripsina/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Indução Enzimática/efeitos dos fármacos , Fator de Iniciação 2 em Eucariotos/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias Hematológicas/enzimologia , Humanos , Oligopeptídeos/uso terapêutico , Inibidores de Proteases/uso terapêutico , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismoRESUMO
Phosphatidylserine (PS) receptors are PS binding proteins that mediate uptake of apoptotic bodies. Many enveloped viruses utilize this PS/PS receptor mechanism to adhere to and internalize into the endosomal compartment of cells and this is termed apoptotic mimicry. For viruses that have a mechanism(s) of endosomal escape, apoptotic mimicry is a productive route of virus entry. We evaluated if PS receptors serve as cell surface receptors for SARS-CoV-2 and found that the PS receptors, AXL, TIM-1 and TIM-4, facilitated virus infection when low concentrations of the SARS-CoV-2 cognate receptor, ACE2, was present. Consistent with the established mechanism of PS receptor utilization by other viruses, PS liposomes competed with SARS-CoV-2 for binding and entry. We demonstrated that this PS receptor enhances SARS-CoV-2 binding to and infection of an array of human lung cell lines and is an under-appreciated but potentially important host factor facilitating SARS-CoV-2 entry.
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The successful progression to the clinic of angiogenesis inhibitors for cancer treatment has spurred interest in developing new classes of anti-angiogenic compounds. The resulting surge in available candidate therapeutics highlights the need for robust, high-throughput angiogenesis screening systems that adequately capture the complexity of new vessel formation while providing quantitative evaluation of the potency of these agents. Available in vitro angiogenesis assays are either cumbersome, impeding adaptation to high-throughput screening formats, or inadequately model the complex multistep process of new vessel formation. We therefore developed an organotypic endothelial-mural cell co-culture assay system that reflects several facets of angiogenesis while remaining compatible with high-throughput/high-content image screening. Co-culture of primary human endothelial cells (EC) and vascular smooth muscle cells (vSMC) results in assembly of a network of tubular endothelial structures enveloped with vascular basement membrane proteins, thus, comprising the three main components of blood vessels. Initially, EC are dependent on vSMC-derived VEGF and sensitive to clinical anti-angiogenic therapeutics. A subsequent phenotypic VEGF-switch renders EC networks resistant to anti-VEGF therapeutics, demarcating a mature vascular phenotype. Conversely, mature EC networks remain sensitive to vascular disrupting agents. Therefore, candidate anti-angiogenic compounds can be interrogated for their relative potency on immature and mature networks and classified as either vascular normalizing or vascular disrupting agents. Here, we demonstrate that the EC-vSMC co-culture assay represents a robust high-content imaging high-throughput screening system for identification of novel anti-angiogenic agents. A pilot high-throughput screening campaign was used to define informative imaging parameters and develop a follow-up dose-response scheme for hit characterization. High-throughput screening using the EC-vSMC co-culture assay establishes a new platform to screen for novel anti-angiogenic compounds for cancer therapy.
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Inibidores da Angiogênese/química , Descoberta de Drogas/métodos , Ensaios de Triagem em Larga Escala/métodos , Técnicas de Cocultura , Relação Dose-Resposta a Droga , Humanos , Concentração Inibidora 50RESUMO
INTRODUCTION: Acquired cancer therapy resistance evolves under selection pressure of immune surveillance and favors mechanisms that promote drug resistance through cell survival and immune evasion. AXL receptor tyrosine kinase is a mediator of cancer cell phenotypic plasticity and suppression of tumor immunity, and AXL expression is associated with drug resistance and diminished long-term survival in a wide range of malignancies, including NSCLC. METHODS: We aimed to investigate the mechanisms underlying AXL-mediated acquired resistance to first- and third-generation small molecule EGFR tyrosine kinase inhibitors (EGFRi) in NSCLC. RESULTS: We found that EGFRi resistance was mediated by up-regulation of AXL, and targeting AXL reduced reactivation of the MAPK pathway and blocked onset of acquired resistance to long-term EGFRi treatment in vivo. AXL-expressing EGFRi-resistant cells revealed phenotypic and cell signaling heterogeneity incompatible with a simple bypass signaling mechanism, and were characterized by an increased autophagic flux. AXL kinase inhibition by the small molecule inhibitor bemcentinib or siRNA mediated AXL gene silencing was reported to inhibit the autophagic flux in vitro, bemcentinib treatment blocked clonogenicity and induced immunogenic cell death in drug-resistant NSCLC in vitro, and abrogated the transcription of autophagy-associated genes in vivo. Furthermore, we found a positive correlation between AXL expression and autophagy-associated gene signatures in a large cohort of human NSCLC (n = 1018). CONCLUSION: Our results indicate that AXL signaling supports a drug-resistant persister cell phenotype through a novel autophagy-dependent mechanism and reveals a unique immunogenic effect of AXL inhibition on drug-resistant NSCLC cells.
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Neoplasias Pulmonares , Preparações Farmacêuticas , Autofagia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB , Humanos , Morte Celular Imunogênica , Neoplasias Pulmonares/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologiaRESUMO
BACKGROUND: Retroviruses are widely used to transfer genes to mammalian cells efficiently and stably. However, genetic elements required for high-level gene expression are incompatible with standard systems. The retroviral RNA genome is produced by cellular transcription and post-transcriptional processing within packaging cells: Introns present in the retroviral genomic transcript are removed by splicing, while polyadenylation signals lead to the production of ineffective truncated genomes. Furthermore strong enhancer/promoters within the retroviral payload lead to detrimental competition with the retroviral enhancer/promoter. RESULTS: By exploiting a new method of producing the retroviral genome in vitro it is possible to produce infectious retroviral particles carrying a high-level expression cassette that completely prohibits production of infectious retroviral particles by conventional methods. We produced an expression cassette comprising a strong enhancer/promoter, an optimised intron, the GFP open reading frame and a strong polyadenylation signal. This cassette was cloned into both a conventional MMLV retroviral vector and a vector designed to allow in vitro transcription of the retroviral genome by T7 RNA polymerase. When the conventional retroviral vector was transfected into packaging cells, the expression cassette drove strong GFP expression, but no infectious retrovirus was produced. Introduction of the in vitro produced uncapped retroviral genomic transcript into the packaging cells did not lead to any detectable GFP expression. However, infectious retrovirus was easily recovered, and when used to infect target primary human cells led to very high GFP expression - up to 3.5 times greater than conventional retroviral LTR-driven expression. CONCLUSION: Retroviral vectors carrying an optimized high-level expression cassette do not produce infectious virions when introduced into packaging cells by transfection of DNA. Infectious retrovirus carrying the same cassette is readily produced when packaging cells are transfected with in vitro transcribed retroviral genomic RNA. The applications of this technique are not limited to producing the higher levels of transgene expression demonstrated here. For example, novel reporters with alternatively spliced exon-intron configurations could readily be transduced into virtually any cell. Furthermore, because the in vitro transcripts are not translated within the packaging cells, retroviruses carrying genes lethal to the packaging cells can also be produced.
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Expressão Gênica/fisiologia , Melhoramento Genético/métodos , Vetores Genéticos/genética , Engenharia de Proteínas/métodos , Proteínas Recombinantes/metabolismo , Retroviridae/genética , Transfecção/métodosRESUMO
Retroviral vectors are powerful tools to study gene function. However, conventional methods require a cellular transcription step to generate the genomic RNA for viral production. This limits the scope of genetic elements that may be transferred by these vectors, excluding many key gene regulatory signals, including RNA editing motifs, alternative splicing, and various promoter/enhancer constellations, as well as cytotoxic genes. To address this problem, we devised a simple approach where in vitro-synthesized vector genomic RNA is transfected into the cytoplasm of a packaging cell, allowing immediate viral particle assembly. We demonstrate that high-titer retroviruses that efficiently transduce mammalian cell lines and primary cells are readily generated. Importantly, we show that an intron-containing expression cassette can be transferred by this method, leading to increased expression levels in the target cell. Further, we demonstrate that the cap structure is not required for retroviral packaging, thus avoiding translation of vector-encoded genes in the packaging cell. This allows the retroviral transfer of cytotoxic genes or proteins that otherwise inhibit viral production.
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Técnicas de Transferência de Genes , Vetores Genéticos , Retroviridae/genética , Sequência de Bases , Expressão Gênica , Técnicas In Vitro , Íntrons , Dados de Sequência Molecular , Capuzes de RNA , RNA Viral/biossíntese , Fatores de Tempo , Transdução Genética , Transfecção , Montagem de VírusRESUMO
Axl, a receptor tyrosine kinase belonging to the Tyro/Axl/Mer (TAM) family, has been shown to be overexpressed in breast cancer with poor outcome. Moreover, Axl was associated with a basal-like phenotype (BLP) in these tumors. Our aim was to investigate Axl expression in breast cancers from an African population since these tumors are known to be aggressive and have a high frequency of the basal-like phenotype. We studied 170 paraffin-embedded breast carcinoma cases by tissue microarrays and immunohistochemical methods. In total, 128 tumor cases (75%) had strong Axl expression and 42 cases (25%) had weak or negative staining. Strong expression of Axl was associated with high tumor grade (p < 0.0005), estrogen receptor (ER) negativity (p = 0.024), p53 expression (p = 0.004), P-cadherin positivity (p = 0.017), and basal-like phenotypic profiles BLP2 (p = 0.033) and BLP3 (p = 0.022). In addition, Axl overexpression also showed an association with markers of tumor cell proliferation and tumor angiogenesis. In conclusion, our findings indicate strong expression of Axl in a high proportion of breast cancer cases among African women and associations with markers of aggressive features, indicating poor prognosis. These findings suggest Axl as a potential therapeutic target in this population.
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Neoplasias da Mama/diagnóstico , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , População Negra/genética , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Humanos , Pessoa de Meia-Idade , Neovascularização Patológica , Prognóstico , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Receptor Tirosina Quinase AxlRESUMO
Changes in mitochondrial amount and shape are intimately linked to maintenance of cell homeostasis via adaptation of vital functions. Here, we developed a new live-cell reporter strategy to simultaneously monitor mitochondrial biogenesis and morphology. This was achieved by making a genetic reporter construct where a master regulator of mitochondrial biogenesis, nuclear respiratory factor 1 (NRF-1), controls expression of mitochondria targeted green fluorescent protein (mitoGFP). HeLa cells with the reporter construct demonstrated inducible expression of mitoGFP upon activation of AMP-dependent protein kinase (AMPK) with AICAR. We established stable reporter cells where the mitoGFP reporter activity corresponded with mitochondrial biogenesis both in magnitude and kinetics, as confirmed by biochemical markers and confocal microscopy. Quantitative 3D image analysis confirmed accordant increase in mitochondrial biomass, in addition to filament/network promoting and protecting effects on mitochondrial morphology, after treatment with AICAR. The level of mitoGFP reversed upon removal of AICAR, in parallel with decrease in mtDNA. In summary, we here present a new GFP-based genetic reporter strategy to study mitochondrial regulation and dynamics in living cells. This combinatorial reporter concept can readily be transferred to other cell models and contexts to address specific physiological mechanisms.
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Mitocôndrias/fisiologia , Dinâmica Mitocondrial , Adenilato Quinase/metabolismo , Biomarcadores/metabolismo , Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Células HeLa , Humanos , Mitocôndrias/ultraestrutura , Fator 1 Nuclear Respiratório/metabolismo , Biogênese de Organelas , Análise de Célula ÚnicaRESUMO
Repurposing "old" drugs can facilitate rapid clinical translation but necessitates novel mechanistic insight. Warfarin, a vitamin K "antagonist" used clinically for the prevention of thrombosis for more than 50 years, has been shown to have anticancer effects. We hypothesized that the molecular mechanism underlying its antitumor activity is unrelated to its effect on coagulation, but is due to inhibition of the Axl receptor tyrosine kinase on tumor cells. Activation of Axl by its ligand Gas6, a vitamin K-dependent protein, is inhibited at doses of warfarin that do not affect coagulation. Here, we show that inhibiting Gas6-dependent Axl activation with low-dose warfarin, or with other tumor-specific Axl-targeting agents, blocks the progression and spread of pancreatic cancer. Warfarin also inhibited Axl-dependent tumor cell migration, invasiveness, and proliferation while increasing apoptosis and sensitivity to chemotherapy. We conclude that Gas6-induced Axl signaling is a critical driver of pancreatic cancer progression and its inhibition with low-dose warfarin or other Axl-targeting agents may improve outcome in patients with Axl-expressing tumors.
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Carcinoma Ductal Pancreático/tratamento farmacológico , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Proteínas de Neoplasias/fisiologia , Neoplasias Pancreáticas/tratamento farmacológico , Proteínas Proto-Oncogênicas/fisiologia , Receptores Proteína Tirosina Quinases/fisiologia , Varfarina/farmacologia , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Apoptose/efeitos dos fármacos , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Desoxicitidina/administração & dosagem , Desoxicitidina/análogos & derivados , Desoxicitidina/uso terapêutico , Progressão da Doença , Sinergismo Farmacológico , Feminino , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Invasividade Neoplásica , Metástase Neoplásica , Proteínas de Neoplasias/antagonistas & inibidores , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , RNA Interferente Pequeno/farmacologia , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/genética , Transdução de Sinais/efeitos dos fármacos , Organismos Livres de Patógenos Específicos , Varfarina/administração & dosagem , Varfarina/uso terapêutico , Ensaios Antitumorais Modelo de Xenoenxerto , Gencitabina , Receptor Tirosina Quinase AxlRESUMO
The carboxy-terminal truncated p53 alternative spliced isoforms, p53ß and p53γ, are expressed at disparate levels in cancer and are suggested to influence treatment response and therapy outcome. However, their functional role in cancer remains to be elucidated. We investigated their individual functionality in the p53(null) background of cell lines H1299 and SAOS-2 by stable retroviral transduction or transient transfection. Expression status of p53ß and p53γ protein was found to correlate with increased response to camptothecin and doxorubicin chemotherapy. Decreased DNA synthesis and clonogenicity in p53ß and p53γ congenic H1299 was accompanied by increased p21((CIP1/WAF1)), Bax and Mdm2 proteins. Chemotherapy induced p53 isoform degradation, most prominent for p53γ. The proteasome inhibitor bortezomib substantially increased basal p53γ protein level, while the level of p53ß protein was unaffected. Treatment with dicoumarol, a putative blocker of the proteasome-related NAD(P)H quinone oxidoreductase NQO1, effectively attenuated basal p53γ protein level in spite of bortezomib treatment. Although in vitro proliferation and clonogenicity assays indicated a weak suppressive effect by p53ß and p53γ expression, studies of in vivo subcutaneous H1299 tumor growth demonstrated a significantly increased growth by expression of either p53 isoforms. This study suggests that p53ß and p53γ share functionality in chemosensitizing and tumor growth enhancement but comprise distinct regulation at the protein level.
Assuntos
Antineoplásicos/farmacologia , Deleção de Genes , Neoplasias Pulmonares/patologia , Osteossarcoma/patologia , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genética , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica , Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Camundongos , Osteossarcoma/genética , Inibidores de Proteases/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Isoformas de Proteínas/deficiência , Isoformas de Proteínas/genéticaRESUMO
The ability to visualize reporter gene expression in vivo has revolutionized all facets of biologic investigation and none more so than imaging applications in oncology. Near-infrared reporter gene imaging may facilitate more accurate evaluation of chemotherapeutic response in preclinical models of orthotopic and metastatic cancers. We report the development of a cell permeable, quenched squarine probe (CytoCy5S), which is reduced by Escherichia coli nitroreductase (NTR), resulting in a near-infrared fluorescent product. Time-domain molecular imaging of NTR/CytoCy5S reporter platform permitted noninvasive monitoring of disease progression in orthotopic xenografts of disseminated leukemia, lung, and metastatic breast cancer. This methodology facilitated therapeutic evaluation of NTR gene-directed enzymatic prodrug therapy with conventional metronidazole antibiotics. These studies show NTR/CytoCy5S as a near-infrared gene reporter system with broad preclinical and prospective clinical applications within imaging, and gene therapy, of cancer.
Assuntos
Carbocianinas/metabolismo , Neoplasias/metabolismo , Nitrorredutases/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Anti-Infecciosos/farmacologia , Carbocianinas/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Luciferases/genética , Luciferases/metabolismo , Imageamento por Ressonância Magnética/métodos , Metronidazol/metabolismo , Metronidazol/farmacologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Microscopia de Fluorescência/métodos , Metástase Neoplásica , Neoplasias/tratamento farmacológico , Neoplasias/genética , Nitroimidazóis/metabolismo , Nitroimidazóis/farmacologia , Nitrorredutases/química , Nitrorredutases/genética , Reprodutibilidade dos Testes , TransfecçãoRESUMO
The transcription factor p63 is central for epithelial homeostasis and development. In our model of epithelial to mesenchymal transition (EMT) in human prostate cells, p63 was one of the most down-regulated transcription factors during EMT. We therefore investigated the role of p63 in EMT. Over-expression of the predominant epithelial isoform ΔNp63α in mesenchymal type cells of the model led to gain of several epithelial characteristics without resulting in a complete mesenchymal to epithelial transition (MET). This was corroborated by a reciprocal effect when p63 was knocked down in epithelial EP156T cells. Global gene expression analyses showed that ΔNp63α induced gene modules involved in both cell-to-cell and cell-to-extracellular-matrix junctions in mesenchymal type cells. Genome-wide analysis of p63 binding sites using ChIP-seq analyses confirmed binding of p63 to regulatory areas of genes associated with cell adhesion in prostate epithelial cells. DH1 and ZEB1 are two elemental factors in the control of EMT. Over-expression and knock-down of these factors, respectively, were not sufficient alone or in combination with ΔNp63α to reverse completely the mesenchymal phenotype. The partial reversion of epithelial to mesenchymal transition might reflect the ability of ΔNp63α, as a key co-ordinator of several epithelial gene expression modules, to reduce epithelial to mesenchymal plasticity (EMP). The utility of ΔNp63α expression and the potential of reduced EMP in order to counteract metastasis warrant further investigation.
Assuntos
Próstata/citologia , Fatores de Transcrição/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Antígenos CD , Sequência de Bases , Caderinas/metabolismo , Linhagem Celular , Forma Celular , Sequência Consenso , Epigênese Genética , Transição Epitelial-Mesenquimal , Matriz Extracelular/metabolismo , Expressão Gênica , Ontologia Genética , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Junções Intercelulares/metabolismo , Laminina/genética , Masculino , Fenótipo , Regiões Promotoras Genéticas , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de ZincoRESUMO
The success of tissue engineering depends on the rapid and efficient formation of a functional blood vasculature. Adult blood vessels comprise endothelial cells and perivascular mural cells that assemble into patent tubules ensheathed by a basement membrane during angiogenesis. Using individual vessel components, we characterized intra-scaffold microvessel self-assembly efficiency in a physiological in vivo tissue engineering implant context. Primary human microvascular endothelial and vascular smooth muscle cells were seeded at different ratios in poly-L-lactic acid (PLLA) scaffolds enriched with basement membrane proteins (Matrigel) and implanted subcutaneously into immunocompromised mice. Temporal intra-scaffold microvessel formation, anastomosis and perfusion were monitored by immunohistochemical, flow cytometric and in vivo multiphoton fluorescence microscopy analysis. Vascularization in the tissue-engineering context was strongly enhanced in implants seeded with a complete complement of blood vessel components: human microvascular endothelial and vascular smooth muscle cells in vivo assembled a patent microvasculature within Matrigel-enriched PLLA scaffolds that anastomosed with the host circulation during the first week of implantation. Multiphoton fluorescence angiographic analysis of the intra-scaffold microcirculation showed a uniform, branched microvascular network. 3D image reconstruction analysis of human pulmonary artery smooth muscle cell (hPASMC) distribution within vascularized implants was non-random and displayed a preferential perivascular localization. Hence, efficient microvessel self-assembly, anastomosis and establishment of a functional microvasculture in the native hypoxic in vivo tissue engineering context is promoted by providing a complete set of vascular components.