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1.
Int J Mol Sci ; 25(11)2024 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-38892290

RESUMEN

Nitric oxide (NO) and reactive nitrogen species (RNS) exert profound biological impacts dictated by their chemistry. Understanding their spatial distribution is essential for deciphering their roles in diverse biological processes. This review establishes a framework for the chemical biology of NO and RNS, exploring their dynamic reactions within the context of cancer. Concentration-dependent signaling reveals distinctive processes in cancer, with three levels of NO influencing oncogenic properties. In this context, NO plays a crucial role in cancer cell proliferation, metastasis, chemotherapy resistance, and immune suppression. Increased NOS2 expression correlates with poor survival across different tumors, including breast cancer. Additionally, NOS2 can crosstalk with the proinflammatory enzyme cyclooxygenase-2 (COX-2) to promote cancer progression. NOS2 and COX-2 co-expression establishes a positive feed-forward loop, driving immunosuppression and metastasis in estrogen receptor-negative (ER-) breast cancer. Spatial evaluation of NOS2 and COX-2 reveals orthogonal expression, suggesting the unique roles of these niches in the tumor microenvironment (TME). NOS2 and COX2 niche formation requires IFN-γ and cytokine-releasing cells. These niches contribute to poor clinical outcomes, emphasizing their role in cancer progression. Strategies to target these markers include direct inhibition, involving pan-inhibitors and selective inhibitors, as well as indirect approaches targeting their induction or downstream effectors. Compounds from cruciferous vegetables are potential candidates for NOS2 and COX-2 inhibition offering therapeutic applications. Thus, understanding the chemical biology of NO and RNS, their spatial distribution, and their implications in cancer progression provides valuable insights for developing targeted therapies and preventive strategies.


Asunto(s)
Neoplasias de la Mama , Ciclooxigenasa 2 , Progresión de la Enfermedad , Óxido Nítrico Sintasa de Tipo II , Humanos , Neoplasias de la Mama/patología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Ciclooxigenasa 2/metabolismo , Femenino , Óxido Nítrico Sintasa de Tipo II/metabolismo , Microambiente Tumoral/efectos de los fármacos , Animales , Óxido Nítrico/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Especies de Nitrógeno Reactivo/metabolismo
2.
Proc Natl Acad Sci U S A ; 117(44): 27423-27434, 2020 11 03.
Artículo en Inglés | MEDLINE | ID: mdl-33060293

RESUMEN

Localization of RNAs at protrusive regions of cells is important for single-cell migration on two-dimensional surfaces. Protrusion-enriched RNAs encode factors linked to cancer progression, such as the RAB13 GTPase and the NET1 guanine nucleotide exchange factor, and are regulated by the tumor-suppressor protein APC. However, tumor cells in vivo often do not move as single cells but rather utilize collective modes of invasion and dissemination. Here, we developed an inducible system of three-dimensional (3D) collective invasion to study the behavior and importance of protrusion-enriched RNAs. We find that, strikingly, both the RAB13 and NET1 RNAs are enriched specifically at the invasive front of leader cells in invasive cell strands. This localization requires microtubules and coincides with sites of high laminin concentration. Indeed, laminin association and integrin engagement are required for RNA accumulation at the invasive front. Importantly, perturbing RNA accumulation reduces collective 3D invasion. Examination of in vivo tumors reveals a similar localization of the RAB13 and NET1 RNAs at potential invasive sites, suggesting that this mechanism could provide a targeting opportunity for interfering with collective cancer cell invasion.


Asunto(s)
Movimiento Celular/genética , Invasividad Neoplásica/genética , Neoplasias/patología , ARN Mensajero/metabolismo , Proteína de la Poliposis Adenomatosa del Colon/genética , Animales , Línea Celular Tumoral , Progresión de la Enfermedad , Femenino , Células HeLa , Humanos , Hibridación Fluorescente in Situ , Microscopía Intravital , Ratones , Microscopía Confocal , Invasividad Neoplásica/prevención & control , Neoplasias/genética , Proteínas Oncogénicas/genética , ARN Interferente Pequeño , Esferoides Celulares , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas de Unión al GTP rab/genética
3.
Br J Cancer ; 125(4): 534-546, 2021 08.
Artículo en Inglés | MEDLINE | ID: mdl-34155340

RESUMEN

BACKGROUND: There is a need to improve the treatment of prostate cancer (PCa) and reduce treatment side effects. Vascular-targeted photodynamic therapy (VTP) is a focal therapy for low-risk low-volume localised PCa, which rapidly disrupts targeted tumour vessels. There is interest in expanding the use of VTP to higher-risk disease. Tumour vasculature is characterised by vessel immaturity, increased permeability, aberrant branching and inefficient flow. FRT alters the tumour microenvironment and promotes transient 'vascular normalisation'. We hypothesised that multimodality therapy combining fractionated radiotherapy (FRT) and VTP could improve PCa tumour control compared against monotherapy with FRT or VTP. METHODS: We investigated whether sequential delivery of FRT followed by VTP 7 days later improves flank TRAMP-C1 PCa tumour allograft control compared to monotherapy with FRT or VTP. RESULTS: FRT induced 'vascular normalisation' changes in PCa flank tumour allografts, improving vascular function as demonstrated using dynamic contrast-enhanced magnetic resonance imaging. FRT followed by VTP significantly delayed tumour growth in flank PCa allograft pre-clinical models, compared with monotherapy with FRT or VTP, and improved overall survival. CONCLUSION: Combining FRT and VTP may be a promising multimodal approach in PCa therapy. This provides proof-of-concept for this multimodality treatment to inform early phase clinical trials.


Asunto(s)
Neovascularización Patológica/terapia , Fotoquimioterapia/métodos , Neoplasias de la Próstata/terapia , Animales , Línea Celular Tumoral , Terapia Combinada , Fraccionamiento de la Dosis de Radiación , Células Endoteliales de la Vena Umbilical Humana , Humanos , Masculino , Ratones , Neoplasias de la Próstata/irrigación sanguínea , Análisis de Supervivencia , Microambiente Tumoral , Ensayos Antitumor por Modelo de Xenoinjerto
4.
Int J Mol Sci ; 22(13)2021 Jun 30.
Artículo en Inglés | MEDLINE | ID: mdl-34209132

RESUMEN

The metabolic requirements and functions of cancer and normal tissues are vastly different. Due to the rapid growth of cancer cells in the tumor microenvironment, distorted vasculature is commonly observed, which creates harsh environments that require rigorous and constantly evolving cellular adaption. A common hallmark of aggressive and therapeutically resistant tumors is hypoxia and hypoxia-induced stress markers. However, recent studies have identified alterations in a wide spectrum of metabolic pathways that dictate tumor behavior and response to therapy. Accordingly, it is becoming clear that metabolic processes are not uniform throughout the tumor microenvironment. Metabolic processes differ and are cell type specific where various factors promote metabolic heterogeneity within the tumor microenvironment. Furthermore, within the tumor, these metabolically distinct cell types can organize to form cellular neighborhoods that serve to establish a pro-tumor milieu in which distant and spatially distinct cellular neighborhoods can communicate via signaling metabolites from stroma, immune and tumor cells. In this review, we will discuss how biochemical interactions of various metabolic pathways influence cancer and immune microenvironments, as well as associated mechanisms that lead to good or poor clinical outcomes.


Asunto(s)
Neoplasias/inmunología , Óxido Nítrico/inmunología , Transducción de Señal/inmunología , Microambiente Tumoral/inmunología , Animales , Humanos , Neoplasias/patología
5.
Br J Cancer ; 123(7): 1089-1100, 2020 09.
Artículo en Inglés | MEDLINE | ID: mdl-32641865

RESUMEN

BACKGROUND: Radiotherapy enhances innate and adaptive anti-tumour immunity. It is unclear whether this effect may be harnessed by combining immunotherapy with radiotherapy fractions used to treat prostate cancer. We investigated tumour immune microenvironment responses of pre-clinical prostate cancer models to radiotherapy. Having defined this landscape, we tested whether radiotherapy-induced tumour growth delay could be enhanced with anti-PD-L1. METHODS: Hypofractionated radiotherapy was delivered to TRAMP-C1 and MyC-CaP flank allografts. Tumour growth delay, tumour immune microenvironment flow-cytometry, and immune gene expression were analysed. TRAMP-C1 allografts were then treated with 3 × 5 Gy ± anti-PD-L1. RESULTS: 3 × 5 Gy caused tumour growth delay in TRAMP-C1 and MyC-CaP. Tumour immune microenvironment changes in TRAMP-C1 at 7 days post-radiotherapy included increased tumour-associated macrophages and dendritic cells and upregulation of PD-1/PD-L1, CD8+ T-cell, dendritic cell, and regulatory T-cell genes. At tumour regrowth post-3 × 5 Gy the tumour immune microenvironment flow-cytometry was similar to control tumours, however CD8+, natural killer and dendritic cell gene transcripts were reduced. PD-L1 inhibition plus 3 × 5 Gy in TRAMP-C1 did not enhance tumour growth delay versus monotherapy. CONCLUSION: 3 × 5 Gy hypofractionated radiotherapy can result in tumour growth delay and immune cell changes in allograft prostate cancer models. Adjuncts beyond immunomodulation may be necessary to improve the radiotherapy-induced anti-tumour response.


Asunto(s)
Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Neoplasias de la Próstata/terapia , Hipofraccionamiento de la Dosis de Radiación , Microambiente Tumoral , Animales , Antígeno B7-H1/análisis , Línea Celular Tumoral , Terapia Combinada , Modelos Animales de Enfermedad , Antígenos de Histocompatibilidad Clase I/análisis , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Trasplante de Neoplasias , Neoplasias de la Próstata/inmunología , Neoplasias de la Próstata/patología
6.
Proc Natl Acad Sci U S A ; 114(49): 13030-13035, 2017 12 05.
Artículo en Inglés | MEDLINE | ID: mdl-29087320

RESUMEN

Proinflammatory signaling pathways are commonly up-regulated in breast cancer. In estrogen receptor-negative (ER-) and triple-negative breast cancer (TNBC), nitric oxide synthase-2 (NOS2) and cyclooxygenase-2 (COX2) have been described as independent predictors of disease outcome. We further explore these findings by investigating the impact of their coexpression on breast cancer survival. Elevated coexpression of NOS2/COX2 proteins is a strong predictor of poor survival among ER- patients (hazard ratio: 21). Furthermore, we found that the key products of NOS2 and COX2, NO and prostaglandin E2 (PGE2), respectively, promote feed-forward NOS2/COX2 crosstalk in both MDA-MB-468 (basal-like) and MDA-MB-231 (mesenchymal-like) TNBC cell lines in which NO induced COX2 and PGE2 induced NOS2 proteins. COX2 induction by NO involved TRAF2 activation that occurred in a TNFα-dependent manner in MDA-MB-468 cells. In contrast, NO-mediated TRAF2 activation in the more aggressive MDA-MB-231 cells was TNFα independent but involved the endoplasmic reticulum stress response. Inhibition of NOS2 and COX2 using amino-guanidine and aspirin/indomethacin yielded an additive reduction in the growth of MDA-MB-231 tumor xenografts. These findings support a role of NOS2/COX2 crosstalk during disease progression of aggressive cancer phenotypes and offer insight into therapeutic applications for better survival of patients with ER- and TNBC disease.


Asunto(s)
Neoplasias de la Mama/genética , Ciclooxigenasa 2/genética , Regulación Neoplásica de la Expresión Génica , Óxido Nítrico Sintasa de Tipo II/genética , Receptores de Estrógenos/genética , Neoplasias de la Mama Triple Negativas/genética , Animales , Aspirina/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/patología , Línea Celular Tumoral , Ciclooxigenasa 2/metabolismo , Dinoprostona/metabolismo , Estrés del Retículo Endoplásmico/efectos de los fármacos , Retroalimentación Fisiológica , Femenino , Guanidinas/farmacología , Humanos , Indometacina/farmacología , Ratones , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Pronóstico , Modelos de Riesgos Proporcionales , Receptores de Estrógenos/deficiencia , Transducción de Señal , Factor 2 Asociado a Receptor de TNF/genética , Factor 2 Asociado a Receptor de TNF/metabolismo , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/mortalidad , Neoplasias de la Mama Triple Negativas/patología , Carga Tumoral/efectos de los fármacos , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto
7.
Chembiochem ; 20(3): 360-365, 2019 02 01.
Artículo en Inglés | MEDLINE | ID: mdl-30358041

RESUMEN

Dysregulated metabolism can fuel cancer by altering the production of bioenergetic building blocks and directly stimulating oncogenic gene-expression programs. However, relatively few optical methods for the direct study of metabolites in cells exist. To address this need and facilitate new approaches to cancer treatment and diagnosis, herein we report an optimized chemical approach to detect the oncometabolite fumarate. Our strategy employs diaryl tetrazoles as cell-permeable photoinducible precursors to nitrileimines. Uncaging these species in cells and cell extracts enables them to undergo 1,3-dipolar cycloadditions with endogenous dipolarophile metabolites such as fumarate to form pyrazoline cycloadducts that can be readily detected by their intrinsic fluorescence. The ability to photolytically uncage diaryl tetrazoles provides greatly improved sensitivity relative to previous methods, and enables the facile detection of dysregulated fumarate metabolism through biochemical activity assays, intracellular imaging, and flow cytometry. Our studies showcase an intersection of bioorthogonal chemistry and metabolite reactivity that can be applied for biological profiling, imaging, and diagnostics.


Asunto(s)
Fluorescencia , Fumaratos/análisis , Fumaratos/efectos de la radiación , Línea Celular , Colorantes Fluorescentes/análisis , Colorantes Fluorescentes/química , Colorantes Fluorescentes/efectos de la radiación , Fumaratos/metabolismo , Humanos , Microscopía Confocal , Estructura Molecular , Imagen Óptica , Tetrazoles/química
8.
Blood ; 128(17): 2135-2143, 2016 10 27.
Artículo en Inglés | MEDLINE | ID: mdl-27557945

RESUMEN

Cell motility, division, and structural integrity depend on dynamic remodeling of the cellular cytoskeleton, which is regulated in part by actin polymerization and depolymerization. In 3 families, we identified 4 children with recurrent infections and varying clinical manifestations including mild neutropenia, impaired wound healing, severe stomatitis with oral stenosis, and death. All patients studied had similar distinctive neutrophil herniation of the nuclear lobes and agranular regions within the cytosol. Chemotaxis and chemokinesis were markedly impaired, but staphylococcal killing was normal, and neutrophil oxidative burst was increased both basally and on stimulation. Neutrophil spreading on glass and cell polarization were also impaired. Neutrophil F-actin was elevated fourfold, suggesting an abnormality in F-actin regulation. Two-dimensional differential in-gel electrophoresis identified abnormal actin-interacting protein 1 (Aip1), encoded by WDR1, in patient samples. Biallelic mutations in WDR1 affecting distinct antiparallel ß-strands of Aip1 were identified in all patients. It has been previously reported that Aip1 regulates cofilin-mediated actin depolymerization, which is required for normal neutrophil function. Heterozygous mutations in clinically normal relatives confirmed that WDR1 deficiency is autosomal recessive. Allogeneic stem cell transplantation corrected the immunologic defect in 1 patient. Mutations in WDR1 affect neutrophil morphology, motility, and function, causing a novel primary immunodeficiency.


Asunto(s)
Citoesqueleto de Actina/patología , Síndromes de Inmunodeficiencia/patología , Trastornos Leucocíticos/genética , Proteínas de Microfilamentos/genética , Neutrófilos/patología , Niño , Electroforesis en Gel Bidimensional , Femenino , Predisposición Genética a la Enfermedad , Humanos , Immunoblotting , Síndromes de Inmunodeficiencia/inmunología , Trastornos Leucocíticos/inmunología , Trastornos Leucocíticos/patología , Masculino , Espectrometría de Masas , Proteínas de Microfilamentos/deficiencia , Proteínas de Microfilamentos/inmunología , Microscopía Confocal , Mutación , Neutrófilos/inmunología , Linaje
9.
Infect Immun ; 85(1)2017 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-27799332

RESUMEN

Burkholderia mallei and B. pseudomallei cause glanders and melioidosis, respectively, in humans and animals. A hallmark of pathogenesis is the formation of granulomas containing multinucleated giant cells (MNGCs) and cell death. These processes depend on type 6 secretion system 1 (T6SS-1), which is required for virulence in animals. We examined the cell biology of MNGC formation and cell death. We found that chloroquine diphosphate (CLQ), an antimalarial drug, inhibits Burkholderia growth, phagosomal escape, and subsequent MNGC formation. This depends on CLQ's ability to neutralize the acid pH because other alkalinizing compounds similarly inhibit escape and MNGC formation. CLQ inhibits bacterial virulence protein expression because T6SS-1 and some effectors of type 3 secretion system 3 (T3SS-3), which is also required for virulence, are expressed at acid pH. We show that acid pH upregulates the expression of Hcp1 of T6SS-1 and TssM, a protein coregulated with T6SS-1. Finally, we demonstrate that CLQ treatment of Burkholderia-infected Madagascar hissing cockroaches (HCs) increases their survival. This study highlights the multiple mechanisms by which CLQ inhibits growth and virulence and suggests that CLQ be further tested and considered, in conjunction with antibiotic use, for the treatment of diseases caused by Burkholderia.


Asunto(s)
Antiácidos/farmacología , Burkholderia mallei/efectos de los fármacos , Burkholderia pseudomallei/efectos de los fármacos , Cloroquina/farmacología , Células Gigantes/efectos de los fármacos , Sistemas de Secreción Tipo VI/efectos de los fármacos , Virulencia/efectos de los fármacos , Animales , Proteínas Bacterianas/metabolismo , Burkholderia mallei/metabolismo , Burkholderia pseudomallei/metabolismo , Línea Celular , Muermo/tratamiento farmacológico , Muermo/microbiología , Concentración de Iones de Hidrógeno , Melioidosis/tratamiento farmacológico , Melioidosis/microbiología , Ratones , Sistemas de Secreción Tipo III/efectos de los fármacos , Factores de Virulencia/metabolismo
10.
Biochim Biophys Acta ; 1853(1): 222-32, 2015 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-25447673

RESUMEN

Valosin-containing protein (VCP or p97), a member of the AAA family (ATPases associated with diverse cellular activities), plays a key role in many important cellular activities. A genetic deficiency of VCP can cause inclusion body myopathy associated with Paget's disease of bone and frontotemporal dementia (IBMPFD). Previous studies showed that the VCP N domain is essential for the regulation of nuclear entry of VCP. Here we report that IBMPFD mutations, which are mainly located in the N domain, suppress the nuclear entry of VCP. Moreover, the peptide sequence G780AGPSQ in the C-terminal region regulates the retention of VCP in the nucleus. A mutant lacking this sequence can increase the nuclear distribution of IBMPFD VCP, suggesting that this sequence is a potential molecular target for correcting the deficient nucleocytoplasmic shuttling of IBMPFD VCP proteins.


Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Transporte Activo de Núcleo Celular , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/fisiología , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Demencia Frontotemporal/genética , Células HEK293 , Humanos , Distrofia Muscular de Cinturas/genética , Miositis por Cuerpos de Inclusión/genética , Osteítis Deformante/genética , Estructura Terciaria de Proteína , Proteína que Contiene Valosina
11.
J Immunol ; 193(1): 56-67, 2014 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-24860189

RESUMEN

TCR-dependent signaling events have been observed to occur in TCR microclusters. We found that some TCR microclusters are present in unstimulated murine T cells, indicating that the mechanisms leading to microcluster formation do not require ligand binding. These pre-existing microclusters increase in absolute number following engagement by low-potency ligands. This increase is accompanied by an increase in cell spreading, with the result that the density of TCR microclusters on the surface of the T cell is not a strong function of ligand potency. In characterizing their composition, we observed a constant number of TCRs in a microcluster, constitutive exclusion of the phosphatase CD45, and preassociation with the signaling adapters linker for activation of T cells and growth factor receptor-bound protein 2. The existence of TCR microclusters prior to ligand binding in a state that is conducive for the initiation of downstream signaling could explain, in part, the rapid kinetics with which TCR signal transduction occurs.


Asunto(s)
Antígenos Comunes de Leucocito/inmunología , Microdominios de Membrana/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Transducción de Señal/inmunología , Linfocitos T/inmunología , Animales , Antígenos Comunes de Leucocito/genética , Microdominios de Membrana/genética , Ratones , Ratones Noqueados , Receptores de Antígenos de Linfocitos T/genética , Transducción de Señal/genética
12.
Traffic ; 14(1): 57-69, 2013 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-22998189

RESUMEN

Retrotransposon and retroviral RNA delivery to particle assembly sites is essential for their replication. mRNA and Gag from the Ty1 retrotransposon colocalize in cytoplasmic foci, which are required for transposition and may be the sites for virus-like particle (VLP) assembly. To determine which Ty1 components are required to form mRNA/Gag foci, localization studies were performed in a Ty1-less strain expressing galactose-inducible Ty1 plasmids (pGTy1) containing mutations in GAG or POL. Ty1 mRNA/Gag foci remained unaltered in mutants defective in Ty1 protease (PR) or deleted for POL. However, Ty1 mRNA containing a frameshift mutation (Ty1fs) that prevents the synthesis of all proteins accumulated in the nucleus. Ty1fs RNA showed a decrease in stability that was mediated by the cytoplasmic exosome, nonsense-mediated decay (NMD) and the processing body. Localization of Ty1fs RNA remained unchanged in an nmd2Δ mutant. When Gag and Ty1fs mRNA were expressed independently, Gag provided in trans increased Ty1fs RNA level and restored localization of Ty1fs RNA in cytoplasmic foci. Endogenously expressed Gag also localized to the nuclear periphery independent of RNA export. These results suggest that Gag is required for Ty1 mRNA stability, efficient nuclear export and localization into cytoplasmic foci.


Asunto(s)
Núcleo Celular/metabolismo , ARN Mensajero/metabolismo , Retroelementos/genética , Retroviridae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Activo de Núcleo Celular , Proteínas Adaptadoras Transductoras de Señales/genética , Citoplasma/metabolismo , Exosomas/metabolismo , Mutación del Sistema de Lectura , Proteínas de Fusión gag-pol/genética , Proteínas de Fusión gag-pol/metabolismo , Eliminación de Gen , Estabilidad del ARN , Transporte de ARN , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
13.
bioRxiv ; 2024 Jul 23.
Artículo en Inglés | MEDLINE | ID: mdl-39091795

RESUMEN

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potential cancer therapeutic that induces apoptosis in cancer cells while sparing the non-malignant cells in preclinical models. However, its efficacy in clinical trials has been limited, suggesting unknown modulatory mechanisms responsible for the lack of TRAIL activity in patients. Here, we hypothesized that TRAIL treatment elicits transcriptional changes in triple negative breast cancer (TNBC) cells that alter the immune milieu. To test this, we performed an RNAseq analysis of MDA-MB-231 cells treated with TRAIL, followed by validation in additional TNBC cell lines. TRAIL significantly induces expression of multiple cytokines such as CXCLs 1, 2, 3, 8,11 and IL-6, which are known to modify neutrophil function. Mechanistically, the induction of these cytokines was predominantly mediated by death receptor 5, caspase 8 (but not caspase 8 enzymatic activity), and the non-canonical NFKB2 pathway. The cytokines produced by the TRAIL-treated TNBC cells enhanced chemotaxis of healthy human donor isolated neutrophils. In vivo , TRAIL treated TNBC murine xenograft tumors showed activation of the NFKB2 pathway, elevated production of CXCLs and IL-6, and increased neutrophil recruitment into the tumors. Moreover, donor isolated neutrophils preincubated in supernatants from TRAIL-treated TNBC cells exhibited impaired cytotoxic effect against TNBC cells. Transcriptomic analysis of neutrophils incubated with either TRAIL alone or supernatant of TRAIL-treated TNBC cells revealed increased expression of inflammatory cytokines, immune modulatory genes, immune checkpoint genes, and genes implicated in delayed neutrophil apoptosis. Functional studies with these neutrophils confirmed their suppressive effect on T cell proliferation and an increase in Treg suppressive phenotype. Collectively, our study demonstrates a novel role of TRAIL-induced NFKB2-dependent cytokine production that promotes neutrophil chemotaxis and immune suppression.

14.
JCI Insight ; 9(12)2024 May 21.
Artículo en Inglés | MEDLINE | ID: mdl-38912586

RESUMEN

Immune therapy is the new frontier of cancer treatment. Therapeutic radiation is a known inducer of immune response and can be limited by immunosuppressive mediators including cyclooxygenase-2 (COX2) that is highly expressed in aggressive triple negative breast cancer (TNBC). A clinical cohort of TNBC tumors revealed poor radiation therapeutic efficacy in tumors expressing high COX2. Herein, we show that radiation combined with adjuvant NSAID (indomethacin) treatment provides a powerful combination to reduce both primary tumor growth and lung metastasis in aggressive 4T1 TNBC tumors, which occurs in part through increased antitumor immune response. Spatial immunological changes including augmented lymphoid infiltration into the tumor epithelium and locally increased cGAS/STING1 and type I IFN gene expression were observed in radiation-indomethacin-treated 4T1 tumors. Thus, radiation and adjuvant NSAID treatment shifts "immune desert phenotypes" toward antitumor M1/TH1 immune mediators in these immunologically challenging tumors. Importantly, radiation-indomethacin combination treatment improved local control of the primary lesion, reduced metastatic burden, and increased median survival when compared with radiation treatment alone. These results show that clinically available NSAIDs can improve radiation therapeutic efficacy through increased antitumor immune response and augmented local generation of cGAS/STING1 and type I IFNs.


Asunto(s)
Proteínas de la Membrana , Transducción de Señal , Linfocitos T Citotóxicos , Animales , Proteínas de la Membrana/metabolismo , Ratones , Femenino , Transducción de Señal/efectos de los fármacos , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/efectos de los fármacos , Neoplasias de la Mama Triple Negativas/patología , Neoplasias de la Mama Triple Negativas/inmunología , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/radioterapia , Indometacina/farmacología , Indometacina/uso terapéutico , Línea Celular Tumoral , Humanos , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/tratamiento farmacológico , Inhibidores de la Ciclooxigenasa/farmacología , Inhibidores de la Ciclooxigenasa/uso terapéutico , Nucleotidiltransferasas/metabolismo , Interferón Tipo I/metabolismo , Ciclooxigenasa 2/metabolismo , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Ratones Endogámicos BALB C
15.
Cancer Res Commun ; 4(10): 2766-2782, 2024 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-39356141
16.
Nat Genet ; 36(9): 984-8, 2004 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-15300252

RESUMEN

Transition through telomere crisis is thought to be a crucial event in the development of most breast carcinomas. Our goal in this study was to determine where this occurs in the context of histologically defined breast cancer progression. To this end, we assessed genome instability (using fluorescence in situ hybridization) and other features associated with telomere crisis in normal ductal epithelium, usual ductal hyperplasia, ductal carcinoma in situ and invasive cancer. We modeled this process in vitro by measuring these same features in human mammary epithelial cell cultures during ZNF217-mediated transition through telomere crisis and immortalization. Taken together, the data suggest that transition through telomere crisis and immortalization in breast cancer occurs during progression from usual ductal hyperplasia to ductal carcinoma in situ.


Asunto(s)
Neoplasias de la Mama/genética , Inestabilidad Cromosómica , Telómero/diagnóstico por imagen , Neoplasias de la Mama/patología , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/patología , Carcinoma Intraductal no Infiltrante/genética , Carcinoma Intraductal no Infiltrante/patología , Progresión de la Enfermedad , Humanos , Hiperplasia/genética , Hiperplasia/patología , Hibridación in Situ , Células Tumorales Cultivadas , Ultrasonografía
17.
Methods Mol Biol ; 2608: 263-280, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-36653713

RESUMEN

Localization of mRNAs at the front of migrating cells is a widely used mechanism that functionally supports efficient cell movement. It is observed in single cells on two-dimensional surfaces, as well as in multicellular three-dimensional (3D) structures and in tissue in vivo. 3D multicellular cultures can reveal how the topology of the extracellular matrix and cell-cell contacts influence subcellular mRNA distributions. Here we describe a method for mRNA imaging in an inducible system of collective cancer cell invasion. MDA-MB-231 cancer cell spheroids are embedded in Matrigel, induced to invade, and processed to image mRNAs with single-molecule sensitivity. An analysis algorithm is used to quantify and compare mRNA distributions at the front of invasive leader cells. The approach can be easily adapted and applied to analyze RNA distributions in additional settings where cells polarize along a linear axis.


Asunto(s)
Neoplasias , Esferoides Celulares , Línea Celular Tumoral , ARN Mensajero/genética , ARN Mensajero/metabolismo , Matriz Extracelular/metabolismo , Movimiento Celular , Neoplasias/genética , Neoplasias/metabolismo
18.
bioRxiv ; 2023 Apr 06.
Artículo en Inglés | MEDLINE | ID: mdl-37066331

RESUMEN

A strong correlation between NOS2 and COX2 tumor expression and poor clinical outcomes in ER-breast cancer has been established. However, mechanisms of tumor induction of these enzymes are unclear. Analysis of The Cancer Genome Atlas (TCGA) revealed correlations between NOS2 and COX2 expression and Th1 cytokines. Herein, single cell RNAseq analysis of TNBC cells shows potent NOS2 and COX2 induction by IFNγ combined with IL1ß or TNFα. Given that IFNγ is secreted by cytolytic lymphocytes, which improve clinical outcomes, this role of IFNγpresents a dichotomy. To explore this conundrum, tumor NOS2, COX2, and CD8 + T cells were spatially analyzed in aggressive ER-, TNBC, and HER2+ breast tumors. High expression and clustering of NOS2-expressing tumor cells occurred at the tumor/stroma interface in the presence of stroma-restricted CD8 + T cells. High expression and clustering of COX2-expressing tumor cells extended into immune desert regions in the tumor core where CD8 + T cell penetration was limited or absent. Moreover, high NOS2-expressing tumor cells were proximal to areas with increased satellitosis suggestive of cell clusters with a higher metastatic potential. Further in vitro experiments revealed that IFNγ+IL1ß/TNFα increased elongation and migration of treated tumor cells. This spatial analysis of the tumor microenvironment provides important insight of distinct neighborhoods where stroma-restricted CD8 + T cells exist proximal to NOS2-expressing tumor niches that could have increased metastatic potential.

19.
Cell Death Dis ; 14(5): 319, 2023 05 11.
Artículo en Inglés | MEDLINE | ID: mdl-37169743

RESUMEN

A strong correlation between NOS2 and COX2 tumor expression and poor clinical outcomes in ER breast cancer has been established. However, the mechanisms of tumor induction of these enzymes are unclear. Analysis of The Cancer Genome Atlas (TCGA) revealed correlations between NOS2 and COX2 expression and Th1 cytokines. Herein, single-cell RNAseq analysis of TNBC cells shows potent NOS2 and COX2 induction by IFNγ combined with IL1ß or TNFα. Given that IFNγ is secreted by cytolytic lymphocytes, which improve clinical outcomes, this role of IFNγ presents a dichotomy. To explore this conundrum, tumor NOS2, COX2, and CD8+ T cells were spatially analyzed in aggressive ER-, TNBC, and HER2 + breast tumors. High expression and clustering of NOS2-expressing tumor cells occurred at the tumor/stroma interface in the presence of stroma-restricted CD8+ T cells. High expression and clustering of COX2-expressing tumor cells extended into immune desert regions in the tumor core where CD8+ T cell penetration was limited or absent. Moreover, high NOS2-expressing tumor cells were proximal to areas with increased satellitosis, suggestive of cell clusters with a higher metastatic potential. Further in vitro experiments revealed that IFNγ + IL1ß/TNFα increased the elongation and migration of treated tumor cells. This spatial analysis of the tumor microenvironment provides important insight into distinct neighborhoods where stroma-restricted CD8+ T cells exist proximal to NOS2-expressing tumor niches that could have increased metastatic potential.


Asunto(s)
Interferón gamma , Neoplasias de la Mama Triple Negativas , Microambiente Tumoral , Femenino , Humanos , Linfocitos T CD8-positivos , Línea Celular Tumoral , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Interferón gamma/genética , Interferón gamma/metabolismo , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Microambiente Tumoral/genética , Microambiente Tumoral/inmunología , Factor de Necrosis Tumoral alfa/metabolismo
20.
bioRxiv ; 2023 Dec 23.
Artículo en Inglés | MEDLINE | ID: mdl-38187532

RESUMEN

Estrogen receptor-negative (ER-) breast cancer is an aggressive breast cancer subtype with limited therapeutic options. Upregulated expression of both inducible nitric oxide synthase (NOS2) and cyclo-oxygenase (COX2) in breast tumors predicts poor clinical outcomes. Signaling molecules released by these enzymes activate oncogenic pathways, driving cancer stemness, metastasis, and immune suppression. The influence of tumor NOS2/COX2 expression on the landscape of immune markers using multiplex fluorescence imaging of 21 ER- breast tumors were stratified for survival. A powerful relationship between tumor NOS2/COX2 expression and distinct CD8+ T cell phenotypes was observed at 5 years post-diagnosis. These results were confirmed in a validation cohort using gene expression data showing that ratios of NOS2 to CD8 and COX2 to CD8 are strongly associated with poor outcomes in high NOS2/COX2-expressing tumors. Importantly, multiplex imaging identified distinct CD8+ T cell phenotypes relative to tumor NOS2/COX2 expression in Deceased vs Alive patient tumors at 5-year survival. CD8+NOS2-COX2- phenotypes defined fully inflamed tumors with significantly elevated CD8+ T cell infiltration in Alive tumors expressing low NOS2/COX2. In contrast, two distinct phenotypes including inflamed CD8+NOS2+COX2+ regions with stroma-restricted CD8+ T cells and CD8-NOS2-COX2+ immune desert regions with abated CD8+ T cell penetration, were significantly elevated in Deceased tumors with high NOS2/COX2 expression. These results were supported by applying an unsupervised nonlinear dimensionality-reduction technique, UMAP, correlating specific spatial CD8/NOS2/COX2 expression patterns with patient survival. Moreover, spatial analysis of the CD44v6 and EpCAM cancer stem cell (CSC) markers within the CD8/NOS2/COX2 expression landscape revealed positive correlations between EpCAM and inflamed stroma-restricted CD8+NOS2+COX2+ phenotypes at the tumor/stroma interface in deceased patients. Also, positive correlations between CD44v6 and COX2 were identified in immune desert regions in deceased patients. Furthermore, migrating tumor cells were shown to occur only in the CD8-NOS2+COX2+ regions, identifying a metastatic hot spot. Taken together, this study shows the strength of spatial localization analyses of the CD8/NOS2/COX2 landscape, how it shapes the tumor immune microenvironment and the selection of aggressive tumor phenotypes in distinct regions that lead to poor clinical outcomes. This technique could be beneficial for describing tumor niches with increased aggressiveness that may respond to clinically available NOS2/COX2 inhibitors or immune-modulatory agents.

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