RESUMEN
Immune and epithelial cells express TLR3, a receptor deputed to respond to microbial signals activating the immune response. The prognostic value of TLR3 in cancer is debated and no data are currently available in NSCLC, for which therapeutic approaches that target the immune system are providing encouraging results. Dissecting the lung immune microenvironment could provide new prognostic markers, especially for early stage NSCLC for which surgery is the only treatment option. In this study we investigated the expression and the prognostic value of TLR3 on both tumor and immune compartments of stage I NSCLCs. In a cohort of 194 NSCLC stage I, TLR3 immunohistochemistry expression on tumor cells predicted a favorable outcome of early stage NSCLC, whereas on the immune cells infiltrating the tumor stroma, TLR3 expression associated with a poor overall survival. Patients with TLR3-positive immune infiltrating cells, but not tumor cells showed a worse prognosis compared with all other patients. The majority of TLR3-expressing immune cells resulted to be macrophages and TLR3 expression associates with PD-1 expression. TLR3 has an opposite prognostic significance when expressed on tumor or immune cells in early stage NCSCL. Analysis of TLR3 in tumor and immune cells can help in identifying high risk stage I patients for which adjuvant treatment would be beneficial.
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Carcinoma de Pulmón de Células no Pequeñas/genética , Neoplasias Pulmonares/genética , Receptor Toll-Like 3/genética , Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Carcinoma de Pulmón de Células no Pequeñas/patología , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Estimación de Kaplan-Meier , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Pronóstico , Receptor Toll-Like 3/análisisRESUMEN
Despite different molecular tumor profiles indicate that human epidermal growth factor receptor 2 (HER2) messenger RNA (mRNA) levels mirror HER2 addiction and trastuzumab benefit in HER2-positive breast cancer (BC), the identification of noninvasive clinical predictors of trastuzumab sensitivity remains an unmet clinical need. In the current study, we investigated whether intratumor lactate levels reflect HER2 addiction and, in turn, trastuzumab susceptibility. Accordingly, the gene expression profiles of transgenic murine BC cell lines expressing the human d16HER2 variant (HER2-addicted) or human full-length HER2 (WTHER2; HER2-nonaddicted) revealed a significant enrichment of glycolysis-related gene pathways in HER2-addicted cells. We studied the metabolic content of 22 human HER2-positive BC by quantitative nuclear magnetic resonance spectroscopy and found that those cases with higher lactate levels were characterized by higher HER2 transcript levels. Moreover, gene expression analyses of HER2-positive BC samples from a TCGA data set revealed a significant enrichment in glycolysis-related pathways in high/HER2-addicted tumors. These data were confirmed by metabolic analyses of human HER2-positive BC cell lines with high or low HER2 transcript levels, which revealed significantly more active glycolytic metabolism in high HER2 transcript than in low HER2 transcript cells. Overall, our results provide evidence for noninvasive intratumor lactate detection as a potential metabolic biomarker of HER2 addiction and trastuzumab response suggesting the possibility to use in vivo imaging to assess lactate levels and, in turn, select HER2-positive BC patients who are more likely to benefit from anti-HER2 treatments.
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Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Glucólisis , Ácido Láctico/metabolismo , Dependencia del Oncogén , Receptor ErbB-2/genética , Animales , Antineoplásicos Inmunológicos/uso terapéutico , Biomarcadores de Tumor/antagonistas & inhibidores , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Línea Celular Tumoral , Quimioterapia Adyuvante , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Italia , Lapatinib/farmacología , Espectroscopía de Resonancia Magnética , Ratones , Selección de Paciente , Medicina de Precisión , Inhibidores de Proteínas Quinasas/uso terapéutico , Receptor ErbB-2/antagonistas & inhibidores , Receptor ErbB-2/metabolismo , Estudios Retrospectivos , Trastuzumab/uso terapéutico , Resultado del Tratamiento , Regulación hacia ArribaRESUMEN
Trastuzumab is the standard treatment for HER2+ breast cancer (BC) patients, and even though it significantly improved their clinical outcome, 50% of them do not benefit from this drug and disease recurs, underlining the need of reliable predictive biomarkers and new therapeutic strategies. Strikingly, despite all the molecular analyses performed to identify the escape mechanisms behind this resistance, it still represents a question point. MiRNAs have been correlated with occurrence and progression of human cancer, and their potential as clinical tools has emerged in the last years. We previously reported that oncosuppressive miR-205 targets HER3, thus increasing the responsiveness to TKIs lapatinib and gefitinib in preclinical models. Here we demonstrate that HER3 inhibition by miR-205 ectopic expression or siRNA-mediated silencing improves the responsiveness to Trastuzumab in vitro in HER2+ BC cell lines, and that this effect is exerted through impairment of AKT-mediated pathway. Moreover, evaluating a series of 52 HER2+ BC patients treated with adjuvant Trastuzumab, we observed that higher miR-205 expression is significantly associated with better outcome (disease-free survival). In summary, our data indicate that miR-205 could predict Trastuzumab efficacy and that its modulation might be useful as adjuvant treatment to improve the response to the drug.
RESUMEN
BACKGROUND: CDCP1, a transmembrane protein with tumor pro-metastatic activity, was recently identified as a prognostic marker in TNBC, the most aggressive breast cancer subtype still lacking an effective molecular targeted therapy. The mechanisms driving CDCP1 over-expression are not fully understood, although several stimuli derived from tumor microenvironment, such as factors present in Wound Healing Fluids (WHFs), reportedly increase CDCP1 levels. METHODS: The expression of CDCP1, PDGFRß and ERK1/2cell was tested by Western blot after stimulation of MDA-MB-231 cells with PDGF-BB and, similarly, in presence or not of ERK1/2 inhibitor in a panel of TNBC cell lines. Knock-down of PDGFRß was established in MDA-MB-231 cells to detect CDCP1 upon WHF treatment. Immunohistochemical staining was used to detect the expression of CDCP1 and PDGFRß in TNBC clinical samples. RESULTS: We discovered that PDGF-BB-mediated activation of PDGFRß increases CDCP1 protein expression through the downstream activation of ERK1/2. Inhibition of ERK1/2 activity reduced per se CDCP1 expression, evidence strengthening its role in CDCP1 expression regulation. Knock-down of PDGFRß in TNBC cells impaired CDCP1 increase induced by WHF treatment, highlighting the role if this receptor as a central player of the WHF-mediated CDCP1 induction. A significant association between CDCP1 and PDGFRß immunohistochemical staining was observed in TNBC specimens, independently of CDCP1 gene gain, thus corroborating the relevance of the PDGF-BB/PDGFRß axis in the modulation of CDCP1 expression. CONCLUSION: We have identified PDGF-BB/PDGFRß-mediated pathway as a novel player in the regulation of CDCP1 in TNCBs through ERK1/2 activation. Our results provide the basis for the potential use of PDGFRß and ERK1/2 inhibitors in targeting the aggressive features of CDCP1-positive TNBCs.
Asunto(s)
Antígenos CD/metabolismo , Moléculas de Adhesión Celular/metabolismo , Regulación Neoplásica de la Expresión Génica , Sistema de Señalización de MAP Quinasas/genética , Proteínas de Neoplasias/metabolismo , Neoplasias de la Mama Triple Negativas/genética , Antígenos de Neoplasias , Becaplermina/farmacología , Línea Celular Tumoral , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Persona de Mediana Edad , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , ARN Interferente Pequeño/metabolismo , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/genética , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Regulación hacia ArribaRESUMEN
BACKGROUND: Current insights into the effects of iron deficiency in tumour cells are not commensurate with the importance of iron in cell metabolism. Studies have predominantly focused on the effects of oxygen or glucose scarcity in tumour cells, while attributing insufficient emphasis to the inadequate supply of iron in hypoxic regions. Cellular responses to iron deficiency and hypoxia are interlinked and may strongly affect tumour metabolism. METHODS: We examined the morphological, proteomic, and metabolic effects induced by two iron chelators-deferoxamine (DFO) and di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT)-on MDA-MB-231 and MDA-MB-157 breast cancer cells. RESULTS: These chelators induced a cytoplasmic massive vacuolation and accumulation of lipid droplets (LDs), eventually followed by implosive, non-autophagic, and non-apoptotic death similar to methuosis. Vacuoles and LDs are generated by expansion of the endoplasmic reticulum (ER) based on extracellular fluid import, which includes unsaturated fatty acids that accumulate in LDs. Typical physiological phenomena associated with hypoxia are observed, such as inhibition of translation, mitochondrial dysfunction, and metabolic remodelling. These survival-oriented changes are associated with a greater expression of epithelial/mesenchymal transcription markers. CONCLUSIONS: Iron starvation induces a hypoxia-like program able to scavenge nutrients from the extracellular environment, and cells assume a hypertrophic phenotype. Such survival strategy is accompanied by the ER-dependent massive cytoplasmic vacuolization, mitochondrial dysfunctions, and LD accumulation and then evolves into cell death. LDs containing a greater proportion of unsaturated lipids are released as a consequence of cell death. The consequence of the disruption of iron metabolism in tumour tissue and the effects of LDs on intercellular communication, cancer-inflammation axis, and immunity remain to be explored. Considering the potential benefits, these are crucial subjects for future mechanistic and clinical studies.
Asunto(s)
Neoplasias de la Mama/metabolismo , Quelantes del Hierro/farmacología , Hierro/metabolismo , Metabolismo de los Lípidos/efectos de los fármacos , Neoplasias de la Mama/patología , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Deferoxamina/farmacología , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/patología , Femenino , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Proteoma , Proteómica/métodos , Vacuolas/metabolismoRESUMEN
Immunotherapy, including the use of checkpoint inhibitors, is a potent therapeutic approach for some cancers, but has limited success with prostate tumors, in which immune suppression is instigated by the tumor. The immunosuppressive capacity of mast cells, which promote adenocarcinoma development in the prostate, prompted our investigation on whether mast cells promote tolerance to SV40 Large-T antigen, the transforming oncogene in transgenic adenocarcinoma of the mouse prostate (TRAMP) mice. The incidence of adenocarcinoma was reduced in the offspring of a cross between TRAMP mice and mast cell-deficient KitWsh mice. TRAMP mice are tolerant to the SV40 Large T antigen, which is otherwise immunogenic in normal syngeneic B6 mice. Genetic ablation of mast cells in TRAMP mice restored their ability to mount a tumor-specific cytotoxic T-cell response. In KitWsh-TRAMP mice, the restored T-cell immunity correlated with the reduced activity of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC), along with their reduced expression of Arg1, Nos2, and Stat3 Having found that CD40L-expressing mast cells can interact in vivo with CD40-expressing PMN-MDSC, we then determined that only KitWsh-TRAMP mice reconstituted with mast cells expressing CD40L could restore PMN-MDSCs suppressive functions, T-cell unresponsiveness and adenocarcinoma development. Thus, mast cells have an immunoregulatory effect on PMN-MDSCs activity through CD40L-CD40 interaction, favoring immunosuppression and tumor onset. In prostate cancer patients, in silico analyses correlated poor clinical outcomes with high expression of genes related to mast cells and PMN-MDSCs. Cancer Immunol Res; 6(5); 552-65. ©2018 AACR.
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Adenocarcinoma/terapia , Comunicación Celular/inmunología , Terapia de Inmunosupresión , Mastocitos/fisiología , Células Supresoras de Origen Mieloide/fisiología , Neoplasias de la Próstata/terapia , Adenocarcinoma/inmunología , Adenocarcinoma/patología , Animales , Células Cultivadas , Humanos , Terapia de Inmunosupresión/métodos , Inmunoterapia , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neoplasias de la Próstata/inmunología , Neoplasias de la Próstata/patologíaRESUMEN
Exosomes-secreted microRNAs play an important role in metastatic spread. During this process breast cancer cells acquire the ability to transmigrate through blood vessels by inducing changes in the endothelial barrier. We focused on miR-939 that is predicted to target VE-cadherin, a component of adherens junction involved in vessel permeability. By in silico analysis miR-939 was found highly expressed in the basal-like tumor subtypes and in our cohort of 63 triple-negative breast cancers (TNBCs) its expression significantly interacted with lymph node status in predicting disease-free survival probability. We demonstrated, in vitro, that miR-939 directly targets VE-cadherin leading to an increase in HUVECs monolayer permeability. MDA-MB-231 cells transfected with a miR-939 mimic, released miR-939 in exosomes that, once internalized in endothelial cells, favored trans-endothelial migration of MDA-MB-231-GFP cells by the disruption of the endothelial barrier. Notably, when up taken in endothelial cells exosomes caused VE-cadherin down-regulation specifically through miR-939 as we demonstrated by inhibiting miR-939 expression in exosomes-releasing TNBC cells. Together, our data indentify an extracellular pro-tumorigenic role for tumor-derived, exosome-associated miR-939 that can explain its association with worse prognosis in TNBCs.
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Antígenos CD/metabolismo , Cadherinas/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , MicroARNs/metabolismo , Migración Transendotelial y Transepitelial , Neoplasias de la Mama Triple Negativas/metabolismo , Antígenos CD/genética , Cadherinas/genética , Línea Celular Tumoral , Supervivencia sin Enfermedad , Regulación hacia Abajo , Exosomas/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Células Endoteliales de la Vena Umbilical Humana/patología , Humanos , MicroARNs/genética , Metástasis de la Neoplasia , Comunicación Paracrina , Permeabilidad , Modelos de Riesgos Proporcionales , Transducción de Señal , Factores de Tiempo , Transfección , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
CDCP1, a transmembrane noncatalytic receptor, the expression of which has been associated with a poor prognosis in certain epithelial cancers, was found to be expressed in highly aggressive triple-negative breast cancer (TNBC) cell models, in which it promoted aggressive activities-ie, migration, invasion, anchorage-independent tumor growth, and the formation of vascular-like structures in vitro. By immunohistochemical (IHC) analysis of 100 human TNBC specimens, CDCP1 was overexpressed in 57% of samples, 38% of which exhibited a gain in CDCP1 copy number by fluorescence in situ hybridization (FISH). CDCP1 positivity was significantly associated between FISH and IHC. CDCP1 expression and gains in CDCP1 copy number synergized with nodal (N) status in determining disease-free and distant disease-free survival. The hazard ratios (HRs) of the synergies between CDCP1 positivity by IHC and FISH and lymph node positivity in predicting relapse did not differ significantly, indicating that CDCP1 overexpression in human primary TNBCs, regardless of being driven by gains in CDCP1, is for a critical factor in the progression of N-positive TNBCs. Thus, CDCP1 is a novel marker of the most aggressive N-positive TNBCs and a potential therapeutic target.
Asunto(s)
Moléculas de Adhesión Celular/análisis , Proteínas de Neoplasias/análisis , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Antígenos CD/análisis , Antígenos CD/genética , Antígenos de Neoplasias , Biomarcadores de Tumor/análisis , Moléculas de Adhesión Celular/genética , Línea Celular Tumoral , Femenino , Humanos , Inmunohistoquímica , Hibridación Fluorescente in Situ , Metástasis de la Neoplasia , Proteínas de Neoplasias/genética , Microambiente TumoralRESUMEN
Organization of cancer cells into endothelial-like cell-lined structures to support neovascularization and to fuel solid tumors is a hallmark of progression and poor outcome. In triple-negative breast cancer (TNBC), PDGFRß has been identified as a key player of this process and is considered a promising target for breast cancer therapy. Thus, we aimed at investigating the role of miRNAs as a therapeutic approach to inhibit PDGFRß-mediated vasculogenic properties of TNBC, focusing on miR-9 and miR-200. In MDA-MB-231 and MDA-MB-157 TNBC cell lines, miR-9 and miR-200 promoted and inhibited, respectively, the formation of vascular-like structures in vitro Induction of endogenous miR-9 expression, upon ligand-dependent stimulation of PDGFRß signaling, promoted significant vascular sprouting of TNBC cells, in part, by direct repression of STARD13. Conversely, ectopic expression of miR-200 inhibited this sprouting by indirectly reducing the protein levels of PDGFRß through the direct suppression of ZEB1. Notably, in vivo miR-9 inhibition or miR-200c restoration, through either the generation of MDA-MB-231-stable clones or peritumoral delivery in MDA-MB-231 xenografted mice, strongly decreased the number of vascular lacunae. Finally, IHC and immunofluorescence analyses in TNBC specimens indicated that PDGFRß expression marked tumor cells engaged in vascular lacunae. In conclusion, our results demonstrate that miR-9 and miR-200 play opposite roles in the regulation of the vasculogenic ability of TNBC, acting as facilitator and suppressor of PDGFRß, respectively. Moreover, our data support the possibility to therapeutically exploit miR-9 and miR-200 to inhibit the process of vascular lacunae formation in TNBC. Cancer Res; 76(18); 5562-72. ©2016 AACR.
Asunto(s)
MicroARNs/genética , Neovascularización Patológica/genética , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/biosíntesis , Neoplasias de la Mama Triple Negativas/patología , Animales , Western Blotting , Diferenciación Celular , Células Endoteliales/patología , Femenino , Técnica del Anticuerpo Fluorescente , Regulación Neoplásica de la Expresión Génica/genética , Técnicas de Silenciamiento del Gen , Xenoinjertos , Humanos , Inmunohistoquímica , Hibridación in Situ , Ratones , Ratones SCID , Neovascularización Patológica/patología , Reacción en Cadena de la Polimerasa , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/genética , Neoplasias de la Mama Triple Negativas/genéticaRESUMEN
Cancer cells within a tumor are functionally heterogeneous and specific subpopulations, defined as cancer initiating cells (CICs), are endowed with higher tumor forming potential. The CIC state, however, is not hierarchically stable and conversion of non-CICs to CICs under microenvironment signals might represent a determinant of tumor aggressiveness. How plasticity is regulated at the cellular level is however poorly understood. To identify determinants of plasticity in lung cancer we exposed eight different cell lines to TGFß1 to induce EMT and stimulate modulation of CD133(+) CICs. We show that response to TGFß1 treatment is heterogeneous with some cells readily switching to stem cell state (1.5-2 fold CICs increase) and others being unresponsive to stimulation. This response is unrelated to original CICs content or extent of EMT engagement but is tightly dependent on balance between epithelial and mesenchymal features as measured by the ratio of expression of CDH1 (E-cadherin) to SNAI2. Epigenetic modulation of this balance can restore sensitivity of unresponsive models to microenvironmental stimuli, including those elicited by cancer-associated fibroblasts both in vitro and in vivo. In particular, tumors with increased prevalence of cells with features of partial EMT (hybrid epithelial/mesenchymal phenotype) are endowed with the highest plasticity and specific patterns of expression of SNAI2 and CDH1 markers identify a subset of tumors with worse prognosis. In conclusion, here we describe a connection between a hybrid epithelial/mesenchymal phenotype and conversion to stem-cell state in response to external stimuli. These findings have implications for current endeavors to identify tumors with increased plasticity.
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Carcinoma de Pulmón de Células no Pequeñas/patología , Transición Epitelial-Mesenquimal , Neoplasias Pulmonares/patología , Células Madre Neoplásicas/patología , Factores de Transcripción/metabolismo , Microambiente Tumoral , Antígeno AC133 , Animales , Antígenos CD/metabolismo , Cadherinas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Línea Celular Tumoral , Plasticidad de la Célula , Epigénesis Genética , Transición Epitelial-Mesenquimal/genética , Femenino , Glicoproteínas/metabolismo , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Ratones , Ratones Desnudos , Células Madre Neoplásicas/metabolismo , Péptidos/metabolismo , Fenotipo , Cultivo Primario de Células , Factores de Transcripción de la Familia Snail , Factor de Crecimiento Transformador beta1/metabolismo , Microambiente Tumoral/genéticaRESUMEN
The tumor-suppressor protein fragile histidine triad (Fhit) exerts its functions in the cytoplasm, although some reports suggest that it may also act in the nucleus. We previously showed that cytosolic Fhit protein levels in cancer cell lines stimulated to proliferate were reduced by proteasomal degradation. Here, we demonstrate that Fhit is physiologically present in the nucleus of breast cancer cell lines and tissues at a low level and that proliferative stimulation increases nuclear levels. Breast cancer cells expressing the FhitY114F mutant, which do not undergo proteasomal degradation, contained mutated Fhit in the nucleus, while cells treated with a proteasome inhibitor accumulated nuclear Fhit during proliferation. Thus, Fhit nuclear shuttling and proteasome degradation phenomena occur independently. When Fhit was coupled to a nuclear localization sequence, the proliferation rate of the transfected cells increased together with levels of proliferation pathway mediators cyclin D1, phospho-MAPK, and phospho-STAT3. Fhit nuclear translocation upon mitogenic stimulation may represent a new regulatory mechanism that allows rapid restoration of Fhit cytoplasmic levels and promotes the proliferation cascade activated by mitogenic stimulation.
Asunto(s)
Ácido Anhídrido Hidrolasas/genética , Neoplasias de la Mama/genética , Núcleo Celular/metabolismo , Proliferación Celular/genética , Proteínas de Neoplasias/genética , Ácido Anhídrido Hidrolasas/biosíntesis , Apoptosis/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Núcleo Celular/genética , Ciclina D1/biosíntesis , Citoplasma/genética , Citoplasma/metabolismo , Factor de Crecimiento Epidérmico/administración & dosificación , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Quinasas de Proteína Quinasa Activadas por Mitógenos/biosíntesis , Proteínas de Neoplasias/biosíntesis , Complejo de la Endopetidasa Proteasomal/genética , Factor de Transcripción STAT3/biosíntesisRESUMEN
Neoplastic B-cell clones commonly arise within secondary lymphoid organs (SLO). However, during disease progression, lymphomatous cells may also colonize the bone marrow (BM), where they localize within specialized stromal niches, namely the osteoblastic and the vascular niche, according to their germinal center- or extra-follicular-derivation, respectively. We hypothesized the existence of common stromal motifs in BM and SLO B-cell lymphoid niches involved in licensing normal B-cell development as well as in fostering transformed B lymphoid cells. Thus, we tested the expression of prototypical mesenchymal stromal cell (MSC) markers and regulatory matricellular proteins in human BM and SLO under physiologically unperturbed conditions and during B-cell lymphoma occurrence. We identified common stromal features in the BM osteoblastic niche and SLO germinal center (GC) microenvironments, traits that were also enriched within BM infiltrates of GC-associated B-cell lymphomas, suggesting that stromal programs involved in central and peripheral B-cell lymphopoiesis are also involved in malignant B-cell nurturing. Among factors co-expressed by stromal elements within these different specialized niches, we identified the pleiotropic matricellular protein secreted protein acidic and rich in cysteine (SPARC). The actual role of stromal SPARC in normal B-cell lymphopoiesis, investigated in Sparc-/- mice and BM chimeras retaining the Sparc-/- genotype in host stroma, demonstrated defective BM and splenic B-cell lymphopoiesis. Moreover, in the Trp53 knockout (KO) lymphoma model, p53-/-/Sparc-/- double-KO mice displayed impaired spontaneous splenic B-cell lymphomagenesis and reduced neoplastic clone BM infiltration in comparison with their p53-/-/Sparc+/+ counterparts. Our results are among the first to demonstrate the existence of common stromal programs regulating both the BM osteoblastic niche and the SLO GC lymphopoietic functions potentially fostering the genesis and progression of B-cell malignancies.
RESUMEN
Thyroid cancer incidence is rapidly increasing. Papillary Thyroid Carcinoma (PTC), the most frequent hystotype, usually displays good prognosis, but no effective therapeutic options are available for the fraction of progressive PTC patients. BRAF and RET/PTC are the most frequent driving genetic lesions identified in PTC. We developed two complementary in vitro models based on RET/PTC1 oncogene, starting from the hypothesis that miRNAs modulated by a driving PTC-oncogene are likely to have a role in thyroid neoplastic processes. Through this strategy, we identified a panel of deregulated miRNAs. Among these we focused on miR-199a-3p and showed its under-expression in PTC specimens and cell lines. We demonstrated that miR-199a-3p restoration in PTC cells reduces MET and mTOR protein levels, impairs migration and proliferation and, more interesting, induces lethality through an unusual form of cell death similar to methuosis, caused by macropinocytosis dysregulation. Silencing MET or mTOR, both involved in survival pathways, does not recapitulate miR-199a-3p-induced cell lethality, thus suggesting that the cooperative regulation of multiple gene targets is necessary. Integrated analysis of miR-199a-3p targets unveils interesting networks including HGF and macropinocytosis pathways. Overall our results indicate miR-199a-3p as a tumor suppressor miRNA in PTC.
Asunto(s)
Apoptosis , Carcinoma Papilar/genética , Movimiento Celular , Genes Supresores de Tumor , MicroARNs/genética , Neoplasias de la Tiroides/genética , Western Blotting , Carcinoma Papilar/metabolismo , Carcinoma Papilar/patología , Proliferación Celular , Células Cultivadas , Técnica del Anticuerpo Fluorescente , Humanos , Técnicas para Inmunoenzimas , Pinocitosis , Proteínas Proto-Oncogénicas c-met/genética , Proteínas Proto-Oncogénicas c-met/metabolismo , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Glándula Tiroides/metabolismo , Neoplasias de la Tiroides/metabolismo , Neoplasias de la Tiroides/patologíaRESUMEN
Triple negative breast cancer (TNBC) is a very aggressive subgroup of breast carcinoma, still lacking specific markers for an effective targeted therapy and with a poorer prognosis compared to other breast cancer subtypes. In this study we investigated the possibility that TNBC cells contribute to the establishment of tumor vascular network by the process known as vasculogenic mimicry, through endothelial cell differentiation. Vascular-like functional properties of breast cancer cell lines were investigated in vitro by tube formation assay and in vivo by confocal microscopy, immunofluorescence or immunohistochemistry on frozen tumor sections. TNBCs express endothelial markers and acquire the ability to form vascular-like channels in vitro and in vivo, both in xenograft models and in human specimens, generating blood lacunae surrounded by tumor cells. Notably this feature is significantly associated with reduced disease free survival. The impairment of the main pathways involved in vessel formation, by treatment with inhibitors (i.e. Sunitinib and Bevacizumab) or by siRNA-mediating silencing, allowed the identification of PDGFRß and FGFR2 as relevant players in this phenomenon. Inhibition of these tyrosine kinase receptors negatively affects vascular lacunae formation and significantly inhibits TNBC growth in vivo. In summary, we demonstrated that TNBCs have the ability to form vascular-like channels in vitro and to generate blood lacunae lined by tumor cells in vivo. Moreover, this feature is associated with poor outcome, probably contributing to the aggressiveness of this breast cancer subgroup. Finally, PDGFRß and FGFR2-mediated pathways, identified as relevant in mediating this characteristic, potentially represent valid targets for a specific therapy of this breast cancer subgroup.
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Mama/patología , Células Endoteliales/patología , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/metabolismo , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Animales , Mama/irrigación sanguínea , Mama/metabolismo , Diferenciación Celular , Línea Celular Tumoral , Células Endoteliales/citología , Células Endoteliales/metabolismo , Femenino , Humanos , Inmunohistoquímica , Ratones SCID , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Interferencia de ARN , ARN Interferente Pequeño/genética , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genética , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/genética , Neoplasias de la Mama Triple Negativas/irrigación sanguínea , Neoplasias de la Mama Triple Negativas/genéticaRESUMEN
Altered degradation and deposition of extracellular matrix are hallmarks of tumor progression and response to therapy. From a microarray supervised analysis on a dataset of chemotherapy-treated breast carcinoma patients, maspin, a member of the serpin protease inhibitor family, has been the foremost variable identified in non-responsive versus responsive tumors. Accordingly, in a series of 52 human breast carcinomas, we detected high maspin expression in tumors that progressed under doxorubicin (DXR)-based chemotherapy. Our analysis of the role of maspin in response to chemotherapy in human MCF7 and MDAMB231 breast and SKOV3 ovarian carcinoma cells transfected to overexpress maspin and injected into mice showed that maspin overexpression led to DXR resistance through the maspin-induced collagen-enriched microenvironment and that an anti-maspin neutralizing monoclonal antibody reversed the collagen-dependent DXR resistance. Impaired diffusion and decreased DXR activity were also found in tumors derived from Matrigel-embedded cells, where abundant collagen fibers characterize the tumor matrix. Conversely, liposome-based DXR reached maspin-overexpressing tumor cells despite the abundant extracellular matrix and was more efficient in reducing tumor growth. Our results identify maspin-induced accumulation of collagen fibers as a cause of disease progression under DXR chemotherapy for breast cancer. Use of a more hydrophilic DXR formulation or of a maspin inhibitor in combination with chemotherapy holds the promise of more consistent responses to maspin-overexpressing tumors and dense-matrix tumors in general.
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Neoplasias de la Mama/metabolismo , Doxorrubicina/uso terapéutico , Resistencia a Antineoplásicos , Neoplasias Ováricas/metabolismo , Serpinas/metabolismo , Microambiente Tumoral/efectos de los fármacos , Animales , Antibióticos Antineoplásicos/uso terapéutico , Anticuerpos Monoclonales/inmunología , Neoplasias de la Mama/tratamiento farmacológico , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Colágeno/metabolismo , Progresión de la Enfermedad , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Femenino , Humanos , Células MCF-7 , Ratones , Ratones Desnudos , Neoplasias Ováricas/tratamiento farmacológico , Serpinas/biosíntesis , Serpinas/inmunologíaRESUMEN
Altered expression of matricellular proteins can become pathogenic in the presence of persistent perturbations in tissue homeostasis. Here, we show that autoimmunity associated with Fas mutation was exacerbated and transitioned to lymphomagenesis in the absence of SPARC (secreted protein acidic rich in cysteine). The absence of SPARC resulted in defective collagen assembly, with uneven compartmentalization of lymphoid and myeloid populations within secondary lymphoid organs (SLO), and faulty delivery of inhibitory signals from the extracellular matrix. These conditions promoted aberrant interactions between neutrophil extracellular traps and CD5(+) B cells, which underwent malignant transformation due to defective apoptosis under the pressure of neutrophil-derived trophic factors and NF-κB activation. Furthermore, this model of defective stromal remodeling during lymphomagenesis correlates with human lymphomas arising in a SPARC-defective environment, which is prototypical of CD5(+) B-cell chronic lymphocytic leukemia (CLL).
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Autoinmunidad , Linfocitos B/inmunología , Tejido Linfoide/inmunología , Linfoma/inmunología , Neutrófilos/inmunología , Animales , Antígenos CD5/inmunología , Células Cultivadas , Matriz Extracelular/inmunología , Humanos , Tejido Linfoide/citología , Linfoma/genética , Ratones , Ratones Mutantes , FN-kappa B/inmunología , Osteonectina/genética , Osteonectina/inmunología , Receptor fas/genéticaRESUMEN
We recently showed that differential expression of extracellular matrix (ECM) genes delineates four subgroups of breast carcinomas (ECM1, -2, -3- and -4) with different clinical outcome. To further investigate the characteristics of ECM signature and its impact on tumor progression, we conducted unsupervised clustering analyses in 6 additional independent datasets of invasive breast tumors from different platforms for a total of 643 samples. Use of four different clustering algorithms identified ECM3 tumors as an independent group in all datasets tested. ECM3 showed a homogeneous gene pattern, consisting of 58 genes encoding 43 structural ECM proteins. From 26 to 41% of the cases were ECM3-enriched, and analysis of datasets relevant to gene expression in neoplastic or corresponding stromal cells showed that both stromal and breast carcinoma cells can coordinately express ECM3 genes. In in vitro experiments, ß-estradiol induced ECM3 gene production in ER-positive breast carcinoma cell lines, whereas TGFß induced upregulation of the genes leading to ECM3 gene classification, especially in ER-negative breast carcinoma cells and in fibroblasts. Multivariate analysis of distant metastasis-free survival in untreated breast tumor patients revealed a significant interaction between ECM3 and histological grade (pâ=â0.001). Cox models, estimated separately in grade I-II and grade III tumors, indicated a highly significant association between ECM3 and worse survival probability only in grade III tumors (HRâ=â3.0, 95% CIâ=â1.3-7.0, pâ=â0.0098). Gene Set Enrichment analysis of ECM3 compared to non-ECM3 tumors revealed significant enrichment of epithelial-mesenchymal transition (EMT) genes in both grade I-II and grade III subsets of ECM3 tumors. Thus, ECM3 is a robust cluster that identifies breast carcinomas with EMT features but with accelerated metastatic potential only in the undifferentiated (grade III) phenotype. These findings support the key relevance of neoplastic and stroma interaction in breast cancer progression.
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Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Proteínas de la Matriz Extracelular/genética , Células del Estroma/metabolismo , Células del Estroma/patología , Transcriptoma , Anciano , Neoplasias de la Mama/mortalidad , Línea Celular Tumoral , Análisis por Conglomerados , Progresión de la Enfermedad , Proteínas de la Matriz Extracelular/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Persona de Mediana Edad , Clasificación del Tumor , Pronóstico , Carga TumoralRESUMEN
FOXP3 is a member of the forkhead/winged-helix family of transcriptional regulators. Defects in the FOXP3 gene cause an X-linked autoimmune/immunodeficiency syndrome in humans and the Scurfy phenotype in mice. FOXP3 acts mainly in regulating the development and function of CD4+CD25+ regulatory T cells. Although initially thought to be specific for these cells, FOXP3 expression has recently been described in non-hematopoietic cells, including epithelial cells of multiple lineages and of different tissue origins. Moreover, FOXP3 expression has been detected in tumor cells of both epithelial and non-epithelial tissues. The role of FOXP3 expression by tumor cells remains controversial, with in vitro studies pointing to an onco-suppressive action, whereas studies conducted on human samples associate FOXP3 expression by tumor cells with metastatic spread. Here, we review evidence for the multi-faceted role of FOXP3 in cancer cells.
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Factores de Transcripción Forkhead/metabolismo , Regulación Neoplásica de la Expresión Génica/fisiología , Animales , Factores de Transcripción Forkhead/genética , Humanos , Metástasis de la Neoplasia , Subgrupos de Linfocitos T/metabolismoRESUMEN
PURPOSE: To assess the occurrence of breast cancer (BC) after exposure to ionizing radiation for pediatric cancer, by means of a multimodal screening program. PATIENTS AND METHODS: We identified 86 patients who had received chest wall radiation therapy for pediatric cancer. Clinical breast examination (CBE), ultrasound (US), and mammography (MX) were performed yearly. Magnetic resonance imaging (MRI) was added as of October 2007. We calculated the risk of developing BC by radiation therapy dose, patient age, and menarche before or after primary treatment. RESULTS: Eleven women developed a BC from July 2002-February 2010. The sensitivity of the screening methods was 36% for CBE, 73% for MX, 55% for US, and 100% for MRI; the specificity was 91%, 99%, 95%, and 80% for CBE, MX, US, and MRI, respectively. The annual BC detection rate was 2.9%. The median age at BC diagnosis was 33 years. Although age had no influence, menarche before as opposed to after radiation therapy correlated significantly with BC (P=.027): the annual BC detection rate in the former subgroup was 5.3%. CONCLUSIONS: Mammography proved more sensitive and specific in our cohort of young women than CBE or US. Magnetic resonance imaging proved 100% sensitive (but this preliminary finding needs to be confirmed). Our cohort of patients carries a 10-fold BC risk at an age more than 20 years younger than in the general population.
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Neoplasias de la Mama/diagnóstico , Neoplasias Inducidas por Radiación/diagnóstico , Neoplasias Primarias Secundarias/diagnóstico , Adolescente , Adulto , Factores de Edad , Neoplasias de la Mama/etiología , Niño , Detección Precoz del Cáncer/métodos , Femenino , Humanos , Imagen por Resonancia Magnética , Mamografía , Menarquia , Persona de Mediana Edad , Neoplasias Primarias Secundarias/etiología , Palpación , Sensibilidad y Especificidad , Tórax/efectos de la radiación , Ultrasonografía Mamaria , Adulto JovenRESUMEN
Antineutrophil cytoplasmic antibodies (ANCAs) target proteins normally retained within neutrophils, indicating that cell death is involved in the autoimmunity process. Still, ANCA pathogenesis remains obscure. ANCAs activate neutrophils inducing their respiratory burst and a peculiar form of cell death, named NETosis, characterized by formation of neutrophil extracellular traps (NETs), decondensed chromatin threads decorated with cytoplasmic proteins endorsed with antimicrobial activity. NETs have been consistently detected in ANCA-associated small-vessel vasculitis, and this association prompted us to test whether the peculiar structure of NET favors neutrophil proteins uploading into myeloid dendritic cells and the induction of ANCAs and associated autoimmunity. Here we show that myeloid DCs uploaded with and activated by NET components induce ANCA and autoimmunity when injected into naive mice. DC uploading and autoimmunity induction are prevented by NET treatment with DNAse, indicating that NET structural integrity is needed to maintain the antigenicity of cytoplasmic proteins. We found NET intermingling with myeloid dendritic cells also positive for neutrophil myeloperoxidase in myeloperoxidase-ANCA-associated microscopic poliangiitis providing a potential correlative picture in human pathology. These data provide the first demonstration that NET structures are highly immunogenic such to trigger adaptive immune response relevant for autoimmunity.