RESUMEN
Autophagy is a conserved cellular degradation process. While autophagy-related proteins were shown to influence the signaling and trafficking of some receptor tyrosine kinases, the relevance of this during cancer development is unclear. Here, we identify a role for autophagy in regulating platelet-derived growth factor receptor alpha (PDGFRA) signaling and levels. We find that PDGFRA can be targeted for autophagic degradation through the activity of the autophagy cargo receptor p62. As a result, short-term autophagy inhibition leads to elevated levels of PDGFRA but an unexpected defect in PDGFA-mediated signaling due to perturbed receptor trafficking. Defective PDGFRA signaling led to its reduced levels during prolonged autophagy inhibition, suggesting a mechanism of adaptation. Importantly, PDGFA-driven gliomagenesis in mice was disrupted when autophagy was inhibited in a manner dependent on Pten status, thus highlighting a genotype-specific role for autophagy during tumorigenesis. In summary, our data provide a mechanism by which cells require autophagy to drive tumor formation.
Asunto(s)
Neoplasias Encefálicas , Transducción de Señal , Ratones , Animales , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , AutofagiaRESUMEN
Glucocorticoids inhibit angiogenesis by activating the glucocorticoid receptor. Inhibition of the glucocorticoid-activating enzyme 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) reduces tissue-specific glucocorticoid action and promotes angiogenesis in murine models of myocardial infarction. Angiogenesis is important in the growth of some solid tumours. This study used murine models of squamous cell carcinoma (SCC) and pancreatic ductal adenocarcinoma (PDAC) to test the hypothesis that 11ß-HSD1 inhibition promotes angiogenesis and subsequent tumour growth. SCC or PDAC cells were injected into female FVB/N or C57BL6/J mice fed either standard diet, or diet containing the 11ß-HSD1 inhibitor UE2316. SCC tumours grew more rapidly in UE2316-treated mice, reaching a larger (P<0.01) final volume (0.158 ± 0.037 cm3) than in control mice (0.051 ± 0.007 cm3). However, PDAC tumour growth was unaffected. Immunofluorescent analysis of SCC tumours did not show differences in vessel density (CD31/alpha-smooth muscle actin) or cell proliferation (Ki67) after 11ß-HSD1 inhibition, and immunohistochemistry of SCC tumours did not show changes in inflammatory cell (CD3- or F4/80-positive) infiltration. In culture, the growth/viability (assessed by live cell imaging) of SCC cells was not affected by UE2316 or corticosterone. Second Harmonic Generation microscopy showed that UE2316 reduced Type I collagen (P<0.001), whilst RNA-sequencing revealed that multiple factors involved in the innate immune/inflammatory response were reduced in UE2316-treated SCC tumours. 11ß-HSD1 inhibition increases SCC tumour growth, likely via suppression of inflammatory/immune cell signalling and extracellular matrix deposition, but does not promote tumour angiogenesis or growth of all solid tumours.
Asunto(s)
Glucocorticoides , Neoplasias , Ratones , Femenino , Animales , Glucocorticoides/metabolismo , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/metabolismo , Inflamación , Neovascularización Patológica , FibrosisRESUMEN
The adhesion protein Kindlin-1 is over-expressed in breast cancer where it is associated with metastasis-free survival; however, the mechanisms involved are poorly understood. Here, we report that Kindlin-1 promotes anti-tumor immune evasion in mouse models of breast cancer. Deletion of Kindlin-1 in Met-1 mammary tumor cells led to tumor regression following injection into immunocompetent hosts. This was associated with a reduction in tumor infiltrating Tregs. Similar changes in T cell populations were seen following depletion of Kindlin-1 in the polyomavirus middle T antigen (PyV MT)-driven mouse model of spontaneous mammary tumorigenesis. There was a significant increase in IL-6 secretion from Met-1 cells when Kindlin-1 was depleted and conditioned media from Kindlin-1-depleted cells led to a decrease in the ability of Tregs to suppress the proliferation of CD8+ T cells, which was dependent on IL-6. In addition, deletion of tumor-derived IL-6 in the Kindlin-1-depleted tumors reversed the reduction of tumor-infiltrating Tregs. Overall, these data identify a novel function for Kindlin-1 in regulation of anti-tumor immunity, and that Kindlin-1 dependent cytokine secretion can impact the tumor immune environment.
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Interleucina-6 , Neoplasias Mamarias Animales , Animales , Ratones , Proteínas Portadoras , Linfocitos T CD8-positivos/metabolismo , Línea Celular Tumoral , Interleucina-6/metabolismoRESUMEN
Targeting early-stage lung cancer is vital to improve survival. However, the mechanisms and components of the early tumor suppressor response in lung cancer are not well understood. In this report, we study the role of Toll-like receptor 2 (TLR2), a regulator of oncogene-induced senescence, which is a key tumor suppressor response in premalignancy. Using human lung cancer samples and genetically engineered mouse models, we show that TLR2 is active early in lung tumorigenesis, where it correlates with improved survival and clinical regression. Mechanistically, TLR2 impairs early lung cancer progression via activation of cell intrinsic cell cycle arrest pathways and the proinflammatory senescence-associated secretory phenotype (SASP). The SASP regulates non-cell autonomous anti-tumor responses, such as immune surveillance of premalignant cells, and we observe impaired myeloid cell recruitment to lung tumors after Tlr2 loss. Last, we show that administration of a TLR2 agonist reduces lung tumor growth, highlighting TLR2 as a possible therapeutic target.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Ratones , Animales , Humanos , Receptor Toll-Like 2/genética , Receptor Toll-Like 2/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/genética , Neoplasias Pulmonares/genética , Genes Supresores de Tumor , Pulmón/metabolismo , Senescencia Celular/genéticaRESUMEN
The MYC oncogene is a potent driver of growth and proliferation but also sensitises cells to apoptosis, which limits its oncogenic potential. MYC induces several biosynthetic programmes and primary cells overexpressing MYC are highly sensitive to glutamine withdrawal suggesting that MYC-induced sensitisation to apoptosis may be due to imbalance of metabolic/energetic supply and demand. Here we show that MYC elevates global transcription and translation, even in the absence of glutamine, revealing metabolic demand without corresponding supply. Glutamine withdrawal from MRC-5 fibroblasts depletes key tricarboxylic acid (TCA) cycle metabolites and, in combination with MYC activation, leads to AMP accumulation and nucleotide catabolism indicative of energetic stress. Further analyses reveal that glutamine supports viability through TCA cycle energetics rather than asparagine biosynthesis and that TCA cycle inhibition confers tumour suppression on MYC-driven lymphoma in vivo. In summary, glutamine supports the viability of MYC-overexpressing cells through an energetic rather than a biosynthetic mechanism.
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Apoptosis , Glutamina , Apoptosis/genética , Línea Celular Tumoral , Ciclo del Ácido Cítrico , Fibroblastos/metabolismo , Glutamina/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismoRESUMEN
Autophagy of the endoplasmic reticulum, or ER-phagy, maintains the homeostasis of the secretory pathway. This is particularly prominent in specialized secretory cells such as the acinar cells of the exocrine pancreas. The role for such a homeostatic pathway during ageing of mammals is modelled best by in vivo genetic or pharmacologic intervention in mice. This is due to the paucity of cellular models that can maintain acinar identity outside of an animal. Here we present methods for isolation of soluble and insoluble protein fractions of ER luminal proteins from the pancreas, alongside RNA. Analysis of these macromolecules allows inference of changes in ER luminal proteostasis upon autophagy-targeted interventions. These methods will likely be more widely applicable, beyond autophagy research.
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Células Acinares , Páncreas Exocrino , Animales , Autofagia , Retículo Endoplásmico/metabolismo , Estrés del Retículo Endoplásmico , Mamíferos , Ratones , SolubilidadRESUMEN
Cytoplasmic recognition of microbial lipopolysaccharides (LPS) in human cells is elicited by the caspase-4 and caspase-5 noncanonical inflammasomes, which induce a form of inflammatory cell death termed pyroptosis. Here we show that LPS-mediated activation of caspase-4 also induces a stress response promoting cellular senescence, which is dependent on the caspase-4 substrate gasdermin-D and the tumor suppressor p53. Furthermore, we found that the caspase-4 noncanonical inflammasome is induced and assembled in response to oncogenic RAS signaling during oncogene-induced senescence (OIS). Moreover, targeting caspase-4 expression in OIS showed its critical role in the senescence-associated secretory phenotype and the cell cycle arrest induced in cellular senescence. Finally, we observed that caspase-4 induction occurs in vivo in mouse models of tumor suppression and ageing. Altogether, we are showing that cellular senescence is induced by cytoplasmic LPS recognition by the noncanonical inflammasome and that this pathway is conserved in the cellular response to oncogenic stress.
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Caspasas Iniciadoras , Inflamasomas , Animales , Caspasas Iniciadoras/inmunología , Senescencia Celular/inmunología , Citoplasma/inmunología , Humanos , Inmunidad Innata , Inflamasomas/inmunología , Lipopolisacáridos/farmacología , RatonesRESUMEN
SRC is a nonreceptor tyrosine kinase with key roles in breast cancer development and progression. Despite this, SRC tyrosine kinase inhibitors have so far failed to live up to their promise in clinical trials, with poor overall response rates. We aimed to identify possible synergistic gene-drug interactions to discover new rational combination therapies for SRC inhibitors. An unbiased genome-wide CRISPR-Cas9 knockout screen in a model of triple-negative breast cancer revealed that loss of integrin-linked kinase (ILK) and its binding partners α-Parvin and PINCH-1 sensitizes cells to bosutinib, a clinically approved SRC/ABL kinase inhibitor. Sensitivity to bosutinib did not correlate with ABL dependency; instead, bosutinib likely induces these effects by acting as a SRC tyrosine kinase inhibitor. Furthermore, in vitro and in vivo models showed that loss of ILK enhanced sensitivity to eCF506, a novel and highly selective inhibitor of SRC with a unique mode of action. Whole-genome RNA sequencing following bosutinib treatment in ILK knockout cells identified broad changes in the expression of genes regulating cell adhesion and cell-extracellular matrix. Increased sensitivity to SRC inhibition in ILK knockout cells was associated with defective adhesion, resulting in reduced cell number as well as increased G1 arrest and apoptosis. These findings support the potential of ILK loss as an exploitable therapeutic vulnerability in breast cancer, enhancing the effectiveness of clinical SRC inhibitors. SIGNIFICANCE: A CRISPR-Cas9 screen reveals that loss of integrin-linked kinase synergizes with SRC inhibition, providing a new opportunity for enhancing the clinical effectiveness of SRC inhibitors in breast cancer.
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Neoplasias de la Mama/genética , Proliferación Celular/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/genética , Familia-src Quinasas/antagonistas & inhibidores , Compuestos de Anilina/farmacología , Animales , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proliferación Celular/genética , Femenino , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica , Técnicas de Inactivación de Genes , Humanos , Estimación de Kaplan-Meier , Células MCF-7 , Ratones Noqueados , Nitrilos/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Quinolinas/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Ensayos Antitumor por Modelo de Xenoinjerto/métodos , Familia-src Quinasas/metabolismoRESUMEN
A more comprehensive understanding of how cells respond to drug intervention, the likely immediate signalling responses and how resistance may develop within different microenvironments will help inform treatment regimes. The nonreceptor tyrosine kinase SRC regulates many cellular signalling processes, and pharmacological inhibition has long been a target of cancer drug discovery projects. Here, we describe the in vitro and in vivo characterisation of the small-molecule SRC inhibitor AZD0424. We show that AZD0424 potently inhibits the phosphorylation of tyrosine-419 of SRC (IC50 ~ 100 nm) in many cancer cell lines; however, inhibition of cell viability, via a G1 cell cycle arrest, was observed only in a subset of cancer cell lines in the low (on target) micromolar range. We profiled the changes in intracellular pathway signalling in cancer cells following exposure to AZD0424 and other targeted therapies using reverse-phase protein array (RPPA) analysis. We demonstrate that SRC is activated in response to treatment of KRAS-mutant colorectal cell lines with MEK inhibitors (trametinib or AZD6244) and that AZD0424 abrogates this. Cell lines treated with trametinib or AZD6244 in combination with AZD0424 had reduced EGFR, FAK and SRC compensatory activation, and cell viability was synergistically inhibited. In vivo, trametinib treatment of mice-bearing HCT116 tumours increased phosphorylation of SRC on Tyr419, and, when combined with AZD0424, inhibition of tumour growth was greater than with trametinib alone. We also demonstrate that drug-induced resistance to trametinib is not re-sensitised by AZD0424 treatment in vitro, likely as a result of multiple compensatory signalling mechanisms; however, inhibition of SRC remains an effective way to block invasion of trametinib-resistant tumour cells. These data imply that SRC inhibition may offer a useful addition to MEK inhibitor combination strategies.
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Neoplasias , Quinazolinas , Animales , Línea Celular Tumoral , Proliferación Celular , Humanos , Ratones , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Neoplasias/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Quinazolinas/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
ISG15 is an ubiquitin-like modifier that is associated with reduced survival rates in breast cancer patients. The mechanism by which ISG15 achieves this however remains elusive. We demonstrate that modification of Rab GDP-Dissociation Inhibitor Beta (GDI2) by ISG15 (ISGylation) alters endocytic recycling of the EGF receptor (EGFR) in non-interferon stimulated cells using CRISPR-knock out models for ISGylation. By regulating EGFR trafficking, ISGylation enhances EGFR recycling and sustains Akt-signalling. We further show that Akt signalling positively correlates with levels of ISG15 and its E2-ligase in basal breast cancer cohorts, confirming the link between ISGylation and Akt signalling in human tumours. Persistent and enhanced Akt activation explains the more aggressive tumour behaviour observed in human breast cancers. We show that ISGylation can act as a driver of tumour progression rather than merely being a bystander.
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Neoplasias de la Mama/metabolismo , Citocinas/genética , Citocinas/metabolismo , Inhibidores de Disociación de Guanina Nucleótido/metabolismo , Ubiquitinas/genética , Ubiquitinas/metabolismo , Neoplasias de la Mama/genética , Sistemas CRISPR-Cas , Línea Celular Tumoral , Endocitosis , Receptores ErbB/metabolismo , Femenino , Técnicas de Inactivación de Genes , Humanos , Fosforilación , Pronóstico , Proteómica , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Análisis de SupervivenciaRESUMEN
Despite the approval of several multikinase inhibitors that target SRC and the overwhelming evidence of the role of SRC in the progression and resistance mechanisms of many solid malignancies, inhibition of its kinase activity has thus far failed to improve patient outcomes. Here we report the small molecule eCF506 locks SRC in its native inactive conformation, thereby inhibiting both enzymatic and scaffolding functions that prevent phosphorylation and complex formation with its partner FAK. This mechanism of action resulted in highly potent and selective pathway inhibition in culture and in vivo. Treatment with eCF506 resulted in increased antitumor efficacy and tolerability in syngeneic murine cancer models, demonstrating significant therapeutic advantages over existing SRC/ABL inhibitors. Therefore, this mode of inhibiting SRC could lead to improved treatment of SRC-associated disorders. SIGNIFICANCE: Small molecule-mediated inhibition of SRC impairing both catalytic and scaffolding functions confers increased anticancer properties and tolerability compared with other SRC/ABL inhibitors.
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Neoplasias Óseas/tratamiento farmacológico , Neoplasias de la Mama/tratamiento farmacológico , Quinasa 1 de Adhesión Focal/antagonistas & inhibidores , Piperidinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-abl/antagonistas & inhibidores , Pirazoles/farmacología , Pirimidinas/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Familia-src Quinasas/antagonistas & inhibidores , Animales , Apoptosis , Neoplasias Óseas/metabolismo , Neoplasias Óseas/secundario , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proliferación Celular , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Conformación Proteica , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto , Familia-src Quinasas/química , Familia-src Quinasas/metabolismoRESUMEN
We mutated the focal adhesion kinase (FAK) catalytic domain to inhibit binding of the chaperone Cdc37 and ATP, mimicking the actions of a FAK kinase inhibitor. We reexpressed mutant and wild-type FAK in squamous cell carcinoma (SCC) cells from which endogenous FAK had been deleted, genetically fixing one axis of a FAK inhibitor combination high-content phenotypic screen to discover drugs that may synergize with FAK inhibitors. Histone deacetylase (HDAC) inhibitors represented the major class of compounds that potently induced multiparametric phenotypic changes when FAK was rendered kinase-defective or inhibited pharmacologically in SCC cells. Combined FAK and HDAC inhibitors arrest proliferation and induce apoptosis in a subset of cancer cell lines in vitro and efficiently inhibit their growth as tumors in vivo Mechanistically, HDAC inhibitors potentiate inhibitor-induced FAK inactivation and impair FAK-associated nuclear YAP in sensitive cancer cell lines. Here, we report the discovery of a new, clinically actionable, synergistic combination between FAK and HDAC inhibitors.
Asunto(s)
Proteína-Tirosina Quinasas de Adhesión Focal/uso terapéutico , Inhibidores de Histona Desacetilasas/uso terapéutico , Neoplasias/tratamiento farmacológico , Animales , Proliferación Celular , Sinergismo Farmacológico , Humanos , Ratones , Transducción de SeñalRESUMEN
PURPOSE: Targeted therapies have resulted in major advances in the treatment of HER2-positive breast cancers. Despite this, up to 70% of patients will develop resistance to treatment within 2 years and new strategies for targeting resistant disease are needed. METHODS: To identify potential resistance mechanisms, we used the mouse MMTV-NIC-PTEN+/- spontaneous model of HER2-positive breast cancer and the pan-HER family kinase inhibitor sapatinib. Vehicle and sapatinib-treated tumors were evaluated by immunohistochemistry and proteomic analysis. In vitro studies were carried out to define the role of heme oxygenase 1 (HO-1) and autophagy in resistance to sapatinib and lapatinib, another pan-HER family kinase inhibitor. RESULTS: Treatment of tumor-bearing MMTV-NIC-PTEN+/- mice with sapatinib resulted in delayed tumor progression and increased survival. However, tumors eventually progressed on treatment. Proteomic analysis identified proteins associated with cellular iron homeostasis as being upregulated in the sapatinib-treated tumors. This included HO-1 whose overexpression was confirmed by immunohistochemistry. Overexpression of HO-1 in HER2-expressing SKBR3 breast cancer cells resulted in reduced sensitivity to both pan-HER family kinase inhibitors sapatinib and lapatinib. This was associated with increased autophagy in the HO-1 over-expressing cells. Furthermore, increased autophagy was also seen in the sapatinib-treated tumors. Treatment with autophagy inhibitors was able to increase the sensitivity of the HO-1 over-expressing cells to both lapatinib and sapatinib. CONCLUSION: Together these data indicate a role for HO-1-induced autophagy in resistance to pan-HER family kinase inhibitors.
Asunto(s)
Autofagia/efectos de los fármacos , Neoplasias de la Mama/tratamiento farmacológico , Hemo-Oxigenasa 1/metabolismo , Lapatinib/farmacología , Proteínas de la Membrana/metabolismo , Quinazolinas/farmacología , Receptor ErbB-2/antagonistas & inhibidores , Animales , Antineoplásicos/farmacología , Neoplasias de la Mama/patología , Línea Celular Tumoral , Evaluación Preclínica de Medicamentos , Resistencia a Antineoplásicos , Femenino , Humanos , Ratones , Ratones Transgénicos , Terapia Molecular Dirigida , Inhibidores de Proteínas Quinasas/farmacología , Receptor ErbB-2/metabolismoRESUMEN
This study investigated the antitumour effects of two dual mTOR/PI3K inhibitors, gedatolisib (WYE-129587/PKI-587/PF-05212384) and PF-04691502 against a panel of six human patient derived ovarian cancer xenograft models. Both dual mTOR/PI3K inhibitors demonstrated antitumour activity against all xenografts tested. The compounds produced tumour stasis during the treatment period and upon cessation of treatment, tumours re-grew. In several models, there was an initial rapid reduction of tumour volume over the first week of treatment before tumour stasis. No toxicity was observed during treatment. Biomarker studies were conducted in two xenograft models; phospho-S6 (Ser235/236) expression (as a readout of mTOR activity) was reduced over the treatment period in the responding xenograft but expression increased to control (no treatment) levels on cessation of treatment. Phospho-AKT (Ser473) expression (as a readout of PI3K) was inhibited by both drugs but less markedly so than phospho-S6 expression. Initial tumour volume reduction on treatment and regrowth rate after treatment cessation was associated with phospho-S6/total S6 expression ratio. Both drugs produced apoptosis but minimally influenced markers of proliferation (Ki67, phospho-histone H3). These results indicate that mTOR/PI3K inhibition can produce broad spectrum tumour growth stasis in ovarian cancer xenograft models during continuous chronic treatment and this is associated with apoptosis.
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Morfolinas/farmacología , Neoplasias Ováricas/tratamiento farmacológico , Inhibidores de las Quinasa Fosfoinosítidos-3/farmacología , Piridonas/farmacología , Pirimidinas/farmacología , Triazinas/farmacología , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Humanos , Ratones , Morfolinas/uso terapéutico , Neoplasias Ováricas/patología , Ovario/patología , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3/uso terapéutico , Piridonas/uso terapéutico , Pirimidinas/uso terapéutico , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/metabolismo , Triazinas/uso terapéutico , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Cellular senescence is a stress response program characterized by a robust cell cycle arrest and the induction of a proinflammatory senescence-associated secretory phenotype (SASP) that is triggered through an unknown mechanism. Here, we show that, during oncogene-induced senescence (OIS), the Toll-like receptor 2 (TLR2) and its partner TLR10 are key mediators of senescence in vitro and in murine models. TLR2 promotes cell cycle arrest by regulating the tumor suppressors p53-p21CIP1, p16INK4a, and p15INK4b and regulates the SASP through the induction of the acute-phase serum amyloids A1 and A2 (A-SAAs) that, in turn, function as the damage-associated molecular patterns (DAMPs) signaling through TLR2 in OIS. Last, we found evidence that the cGAS-STING cytosolic DNA sensing pathway primes TLR2 and A-SAAs expression in OIS. In summary, we report that innate immune sensing of senescence-associated DAMPs by TLR2 controls the SASP and reinforces the cell cycle arrest program in OIS.
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Senescencia Celular , Inmunidad Innata , Receptor Toll-Like 2/metabolismo , Alarminas/metabolismo , Animales , Senescencia Celular/efectos de los fármacos , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , FN-kappa B/metabolismo , Nucleotidiltransferasas/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Proteína Amiloide A Sérica/genética , Proteína Amiloide A Sérica/metabolismo , Transducción de Señal , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacología , Receptor Toll-Like 10/antagonistas & inhibidores , Receptor Toll-Like 10/genética , Receptor Toll-Like 10/metabolismo , Receptor Toll-Like 2/antagonistas & inhibidores , Receptor Toll-Like 2/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismoRESUMEN
Angiosarcomas are a rare group of tumours which have poor prognosis and limited treatment options. The development of new therapies has been hampered by a lack of good preclinical models. Here, we describe the development of an autochthonous mouse model of angiosarcoma driven by loss of p53 in VE-cadherin-expressing endothelial cells. Using Cdh5-Cre to drive recombination in adult endothelial cells, mice developed angiosarcomas with 100% penetrance upon homozygous deletion of Trp53 with a median lifespan of 325â days. In contrast, expression of the R172H mutant p53 resulted in formation of thymic lymphomas with a more rapid onset (median lifespan 151â days). We also used Pdgfrb-Cre-expressing mice, allowing us to target predominantly pericytes, as these have been reported as the cell of origin for a number of soft tissue sarcomas. Pdgfrb-Cre also results in low levels of recombination in venous blood endothelial cells in multiple tissues during development. Upon deletion of Trp53 in Pdgfrb-Cre-expressing mice (Pdgfrb-Cre,Trp53fl/fl mice), 65% developed lymphomas and 21% developed pleomorphic undifferentiated soft tissue sarcomas. None developed angiosarcomas. In contrast, 75% of Pdgfrb-Cre,Trp53R172H/R172H mice developed angiosarcomas, with 60% of these mice also developing lymphomas. The median lifespan of the Pdgfrb-Cre,Trp53R172H/R172H mice was 151â days. Re-implantation of angiosarcoma tumour fragments from Cdh5-Cre, Trp53fl/fl mice provided a more consistent and rapid model of angiosarcoma than the two spontaneous models. The ability to passage tumour fragments through the mouse provides a novel model which is amenable to preclinical studies and will help the development of potential new therapies for angiosarcoma.
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Modelos Animales de Enfermedad , Hemangiosarcoma/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Antígenos CD/metabolismo , Cadherinas/metabolismo , Diferenciación Celular , Células Endoteliales/metabolismo , Hemangiosarcoma/patología , Integrasas/genética , Ratones , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/genética , Recombinación Genética , Proteína p53 Supresora de Tumor/genéticaRESUMEN
The observation that BRCA1- and BRCA2-deficient cells are sensitive to inhibitors of poly(ADP-ribose) polymerase (PARP) has spurred the development of cancer therapies that use these inhibitors to target deficiencies in homologous recombination1. The cytotoxicity of PARP inhibitors depends on PARP trapping, the formation of non-covalent protein-DNA adducts composed of inhibited PARP1 bound to DNA lesions of unclear origins1-4. To address the nature of such lesions and the cellular consequences of PARP trapping, we undertook three CRISPR (clustered regularly interspersed palindromic repeats) screens to identify genes and pathways that mediate cellular resistance to olaparib, a clinically approved PARP inhibitor1. Here we present a high-confidence set of 73 genes, which when mutated cause increased sensitivity to PARP inhibitors. In addition to an expected enrichment for genes related to homologous recombination, we discovered that mutations in all three genes encoding ribonuclease H2 sensitized cells to PARP inhibition. We establish that the underlying cause of the PARP-inhibitor hypersensitivity of cells deficient in ribonuclease H2 is impaired ribonucleotide excision repair5. Embedded ribonucleotides, which are abundant in the genome of cells deficient in ribonucleotide excision repair, are substrates for cleavage by topoisomerase 1, resulting in PARP-trapping lesions that impede DNA replication and endanger genome integrity. We conclude that genomic ribonucleotides are a hitherto unappreciated source of PARP-trapping DNA lesions, and that the frequent deletion of RNASEH2B in metastatic prostate cancer and chronic lymphocytic leukaemia could provide an opportunity to exploit these findings therapeutically.
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Sistemas CRISPR-Cas , Daño del ADN , Edición Génica , Neoplasias/genética , Neoplasias/patología , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Ribonucleótidos/genética , Animales , Proteína BRCA1/deficiencia , Proteína BRCA1/genética , Línea Celular , Daño del ADN/efectos de los fármacos , Reparación del ADN/genética , Replicación del ADN , ADN-Topoisomerasas de Tipo I/metabolismo , Femenino , Genes BRCA1 , Genoma/genética , Células HeLa , Humanos , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/enzimología , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/patología , Masculino , Ratones , Neoplasias/tratamiento farmacológico , Neoplasias/enzimología , Ftalazinas/farmacología , Piperazinas/farmacología , Poli(ADP-Ribosa) Polimerasa-1/deficiencia , Poli(ADP-Ribosa) Polimerasa-1/genética , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/enzimología , Neoplasias de la Próstata/patología , Ribonucleasa H/deficiencia , Ribonucleasa H/genética , Ribonucleasa H/metabolismo , Mutaciones Letales Sintéticas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
In breast cancer, increased expression of the cytoskeletal adaptor protein Kindlin-1 has been linked to increased risks of lung metastasis, but the functional basis is unknown. Here, we show that in a mouse model of polyomavirus middle T antigen-induced mammary tumorigenesis, loss of Kindlin-1 reduced early pulmonary arrest and later development of lung metastasis. This phenotype relied on the ability of Kindlin-1 to bind and activate ß integrin heterodimers. Kindlin-1 loss reduced α4 integrin-mediated adhesion of mammary tumor cells to the adhesion molecule VCAM-1 on endothelial cells. Treating mice with an anti-VCAM-1 blocking antibody prevented early pulmonary arrest. Kindlin-1 loss also resulted in reduced secretion of several factors linked to metastatic spread, including the lung metastasis regulator tenascin-C, showing that Kindlin-1 regulated metastatic dissemination by an additional mechanism in the tumor microenvironment. Overall, our results show that Kindlin-1 contributes functionally to early pulmonary metastasis of breast cancer.Significance: These findings provide a mechanistic proof in mice that Kindin-1, an integrin-binding adaptor protein, is a critical mediator of early lung metastasis of breast cancer. Cancer Res; 78(6); 1484-96. ©2018 AACR.
Asunto(s)
Neoplasias de la Mama/patología , Proteínas Portadoras/metabolismo , Neoplasias Pulmonares/secundario , Animales , Anticuerpos Monoclonales/farmacología , Antígenos Transformadores de Poliomavirus/toxicidad , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Proteínas Portadoras/genética , Adhesión Celular/efectos de los fármacos , Células Endoteliales/patología , Femenino , Regulación Neoplásica de la Expresión Génica , Integrinas/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Neoplasias Mamarias Experimentales/patología , Neoplasias Mamarias Experimentales/virología , Ratones Transgénicos , Molécula 1 de Adhesión Celular Vascular/inmunologíaRESUMEN
WT1 is a transcription factor which regulates the epithelial-mesenchymal balance during embryonic development and, if mutated, can lead to the formation of Wilms' tumour, the most common paediatric kidney cancer. Its expression has also been reported in several adult tumour types, including breast cancer, and usually correlates with poor outcome. However, published data is inconsistent and the role of WT1 in this malignancy remains unclear. Here we provide a complete study of WT1 expression across different breast cancer subtypes as well as isoform specific expression analysis. Using in vitro cell lines, clinical samples and publicly available gene expression datasets, we demonstrate that WT1 plays a role in regulating the epithelial-mesenchymal balance of breast cancer cells and that WT1-expressing tumours are mainly associated with a mesenchymal phenotype. WT1 gene expression also correlates with CYP3A4 levels and is associated with poorer response to taxane treatment. Our work is the first to demonstrate that the known association between WT1 expression in breast cancer and poor prognosis is potentially due to cancer-related epithelial-to-mesenchymal transition (EMT) and poor chemotherapy response.
Asunto(s)
Antineoplásicos/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Citocromo P-450 CYP3A/metabolismo , Taxoides/uso terapéutico , Proteínas WT1/genética , Proteínas WT1/metabolismo , Antineoplásicos/farmacología , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Docetaxel , Transición Epitelial-Mesenquimal/efectos de los fármacos , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Humanos , Células MCF-7 , Mutación , Pronóstico , Taxoides/farmacología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Novel pyrazolopyrimidines displaying high potency and selectivity toward SRC family kinases have been developed by combining ligand-based design and phenotypic screening in an iterative manner. Compounds were derived from the promiscuous kinase inhibitor PP1 to search for analogs that could potentially target a broad spectrum of kinases involved in cancer. Phenotypic screening against MCF7 mammary adenocarcinoma cells generated target-agnostic structure-activity relationships that biased subsequent designs toward breast cancer treatment rather than to a particular target. This strategy led to the discovery of two potent antiproliferative leads with phenotypically distinct anticancer mode of actions. Kinase profiling and further optimization resulted in eCF506, the first small molecule with subnanomolar IC50 for SRC that requires 3 orders of magnitude greater concentration to inhibit ABL. eCF506 exhibits excellent water solubility, an optimal DMPK profile and oral bioavailability, halts SRC-associated neuromast migration in zebrafish embryos without inducing life-threatening heart defects, and inhibits SRC phosphorylation in tumor xenografts in mice.