RESUMEN
Lung diseases develop when telomeres shorten beyond a critical point. We constructed a mouse model in which the catalytic subunit of telomerase (mTert), or its catalytically inactive form (mTertCI), is expressed from the p21Cdkn1a locus. Expression of either TERT or TERTCI reduces global p21 levels in the lungs of aged mice, highlighting TERT non-canonical function. However, only TERT reduces accumulation of very short telomeres, oxidative damage, endothelial cell (ECs) senescence and senile emphysema in aged mice. Single-cell analysis of the lung reveals that p21 (and hence TERT) is expressed mainly in the capillary ECs. We report that a fraction of capillary ECs marked by CD34 and endowed with proliferative capacity declines drastically with age, and this is counteracted by TERT but not TERTCI. Consistently, only TERT counteracts decline of capillary density. Natural aging effects are confirmed using the experimental model of emphysema induced by VEGFR2 inhibition and chronic hypoxia. We conclude that catalytically active TERT prevents exhaustion of the putative CD34 + EC progenitors with age, thus protecting against capillary vessel loss and pulmonary emphysema.
Asunto(s)
Enfisema , Rarefacción Microvascular , Enfisema Pulmonar , Telomerasa , Ratones , Animales , Acortamiento del Telómero , Telomerasa/genéticaRESUMEN
Innovative strategies for enhancing efficacy and overcoming drug resistance in hematologic cancers, such as using antibody-drug conjugates (ADC), have shifted the paradigm of conventional care by delivering promising outcomes in cancer therapies with a significant reduction in the risk of relapse. Transferrin receptor (TfR1), cluster of differentiation 71 (CD71), is known to be overexpressed in malignant cells and considered a potent antitumor target. Therefore, we developed an anti-CD71 ADC, INA03, a humanized antibody conjugated to monomethyl auristatin E through a 3-arylpropiolonitrile-valine-citrulline linker. In this study, we investigated the potency and safety of INA03, in competition with Transferrin (Tf), the CD71's natural ligand, as a novel strategy to specifically target highly proliferative cells. The high expression of CD71 was confirmed on different leukemic cell lines, allowing INA03 to bind efficiently. Subsequently, INA03 rapidly internalizes into lysosomal compartments, in which its cytotoxic drug is released following cathepsin B cleavage. Downregulation of CD71 expression using shRNA highlighted that INA03-induced cell death was dependent on CD71 density at the cell surface. INA03 intravenous treatment in acute leukemia mouse models significantly reduced tumor burden, increased mouse survival, and showed no residual disease compared with conventional chemotherapies. Because INA03 competes with human Tf, a double knock-in (human CD71/human Tf) competent mouse model was generated to mimic human pharmacokinetics and pharmacodynamics. INA03 administration in human CD71/hTf mice did not reveal any improper toxicities, even at high doses. Hence, these data demonstrate the promising preclinical efficacy and safety of INA03 and support its development as a novel acute leukemia treatment. Significance: The Tf receptor is believed to be undruggable because of its ubiquitous expression. By entering into competition with its cognate ligand, the Tf and INA03 ADC can safely achieve potency.
Asunto(s)
Antígenos CD , Inmunoconjugados , Receptores de Transferrina , Transferrina , Humanos , Animales , Ratones , Receptores de Transferrina/metabolismo , Receptores de Transferrina/antagonistas & inhibidores , Inmunoconjugados/farmacología , Inmunoconjugados/uso terapéutico , Antígenos CD/metabolismo , Transferrina/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Línea Celular Tumoral , Leucemia/tratamiento farmacológico , Leucemia/patología , FemeninoRESUMEN
Ectodermal dysplasias form a large disease family with more than 200 members. The combination of hair and tooth abnormalities, alopecia, and cutaneous syndactyly is characteristic of ectodermal dysplasia-syndactyly syndrome (EDSS). We used a homozygosity mapping approach to map the EDSS locus to 1q23 in a consanguineous Algerian family. By candidate gene analysis, we identified a homozygous mutation in the PVRL4 gene that not only evoked an amino acid change but also led to exon skipping. In an Italian family with two siblings affected by EDSS, we further detected a missense and a frameshift mutation. PVRL4 encodes for nectin-4, a cell adhesion molecule mainly implicated in the formation of cadherin-based adherens junctions. We demonstrated high nectin-4 expression in hair follicle structures, as well as in the separating digits of murine embryos, the tissues mainly affected by the EDSS phenotype. In patient keratinocytes, mutated nectin-4 lost its capability to bind nectin-1. Additionally, in discrete structures of the hair follicle, we found alterations of the membrane localization of nectin-afadin and cadherin-catenin complexes, which are essential for adherens junction formation, and we found reorganization of actin cytoskeleton. Together with cleft lip and/or palate ectodermal dysplasia (CLPED1, or Zlotogora-Ogur syndrome) due to an impaired function of nectin-1, EDSS is the second known "nectinopathy" caused by mutations in a nectin adhesion molecule.
Asunto(s)
Moléculas de Adhesión Celular/genética , Displasia Ectodérmica/complicaciones , Displasia Ectodérmica/genética , Mutación/genética , Sindactilia/complicaciones , Sindactilia/genética , Anomalías Múltiples/genética , Adulto , Secuencia de Aminoácidos , Animales , Moléculas de Adhesión Celular/química , Moléculas de Adhesión Celular/metabolismo , Niño , Extremidades/embriología , Familia , Femenino , Regulación del Desarrollo de la Expresión Génica , Cabello/patología , Humanos , Masculino , Ratones , Datos de Secuencia Molecular , Linaje , Transporte de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Piel/patología , SíndromeRESUMEN
There is increasing evidence that breast tumors are organized in a hierarchy, with a subpopulation of tumorigenic cancer cells, the cancer stem cells (CSCs), which sustain tumor growth. The characterization of protein networks that govern CSC behavior is paramount to design new therapeutic strategies targeting this subpopulation of cells. We have sought to identify specific molecular pathways of CSCs isolated from 13 different breast cancer cell lines of luminal or basal/mesenchymal subtypes. We compared the gene expression profiling of cancer cells grown in adherent conditions to those of matched tumorsphere cultures. No specific pathway was identified to be commonly regulated in luminal tumorspheres, resulting from a minor CSC enrichment in tumorsphere passages from luminal cell lines. However, in basal/mesenchymal tumorspheres, the enzymes of the mevalonate metabolic pathway were overexpressed compared to those in cognate adherent cells. Inhibition of this pathway with hydroxy-3-methylglutaryl CoA reductase blockers resulted in a reduction of breast CSC independent of inhibition of cholesterol biosynthesis and of protein farnesylation. Further modulation of this metabolic pathway demonstrated that protein geranylgeranylation (GG) is critical to breast CSC maintenance. A small molecule inhibitor of the geranylgeranyl transferase I (GGTI) enzyme reduced the breast CSC subpopulation both in vitro and in primary breast cancer xenografts. We found that the GGTI effect on the CSC subpopulation is mediated by inactivation of Ras homolog family member A (RHOA) and increased accumulation of P27(kip1) in the nucleus. The identification of protein GG as a major contributor to CSC maintenance opens promising perspectives for CSC targeted therapy in basal breast cancer.
Asunto(s)
Neoplasias de la Mama/metabolismo , Ácido Mevalónico/metabolismo , Neoplasias Basocelulares/metabolismo , Células Madre Neoplásicas/metabolismo , Animales , Antineoplásicos/uso terapéutico , Benzamidas , Western Blotting , Neoplasias de la Mama/tratamiento farmacológico , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Docetaxel , Femenino , Perfilación de la Expresión Génica , Humanos , Ratones , Ratones SCID , Neoplasias Basocelulares/tratamiento farmacológico , Células Madre Neoplásicas/citología , Células Madre Neoplásicas/efectos de los fármacos , Taxoides/uso terapéuticoRESUMEN
Replication stress (RS) has a pivotal role in tumor initiation, progression, or therapeutic resistance. In this study, we depicted the mechanism of breast cancer stem cells' (bCSCs) response to RS and its clinical implication. We demonstrated that bCSCs present a limited level of RS compared with non-bCSCs in patient samples. We described for the first time that the spatial nuclear location of BMI1 protein triggers RS response in breast cancers. Hence, in bCSCs, BMI1 is rapidly located to stalled replication forks to recruit RAD51 and activate homologous-recombination machinery, whereas in non-bCSCs BMI1 is trapped on demethylated 1q12 megasatellites precluding effective RS response. We further demonstrated that BMI1/RAD51 axis activation is necessary to prevent cisplatin-induced DNA damage and that treatment of patient-derived xenografts with a RAD51 inhibitor sensitizes tumor-initiating cells to cisplatin. The comprehensive view of replicative-stress response in bCSC has profound implications for understanding and improving therapeutic resistance.
Asunto(s)
Neoplasias de la Mama/patología , Núcleo Celular/metabolismo , Resistencia a Antineoplásicos/efectos de los fármacos , Células Madre Neoplásicas/efectos de los fármacos , Complejo Represivo Polycomb 1/metabolismo , Recombinasa Rad51/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Cisplatino/farmacología , Daño del ADN/efectos de los fármacos , Replicación del ADN/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Femenino , Recombinación Homóloga , Humanos , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Complejo Represivo Polycomb 1/antagonistas & inhibidores , Recombinasa Rad51/antagonistas & inhibidoresRESUMEN
BACKGROUND: The success and limitations of current immunotherapies have pushed research toward the development of alternative approaches and the possibility to manipulate other cytotoxic immune cells such as natural killer (NK) cells. Here, we targeted an intracellular inhibiting protein 'cytokine inducible SH2-containing protein' (CISH) in NK cells to evaluate the impact on their functions and antitumor properties. METHODS: To further understand CISH functions in NK cells, we developed a conditional Cish-deficient mouse model in NK cells (Cishfl/flNcr1Ki/+ ). NK cells cytokine expression, signaling and cytotoxicity has been evaluated in vitro. Using intravenous injection of B16F10 melanoma cell line and EO711 triple negative breast cancer cell line, metastasis evaluation was performed. Then, orthotopic implantation of breast tumors was performed and tumor growth was followed using bioluminescence. Infiltration and phenotype of NK cells in the tumor was evaluated. Finally, we targeted CISH in human NK-92 or primary NK cells, using a technology combining the CRISPR(i)-dCas9 tool with a new lentiviral pseudotype. We then tested human NK cells functions. RESULTS: In Cishfl/flNcr1Ki/+ mice, we detected no developmental or homeostatic difference in NK cells. Global gene expression of Cishfl/flNcr1Ki/+ NK cells compared with Cish+/+Ncr1Ki/+ NK cells revealed upregulation of pathways and genes associated with NK cell cycling and activation. We show that CISH does not only regulate interleukin-15 (IL-15) signaling pathways but also natural cytotoxicity receptors (NCR) pathways, triggering CISH protein expression. Primed Cishfl/flNcr1Ki/+ NK cells display increased activation upon NCR stimulation. Cishfl/flNcr1Ki/+ NK cells display lower activation thresholds and Cishfl/flNcr1Ki/+ mice are more resistant to tumor metastasis and to primary breast cancer growth. CISH deletion favors NK cell accumulation to the primary tumor, optimizes NK cell killing properties and decreases TIGIT immune checkpoint receptor expression, limiting NK cell exhaustion. Finally, using CRISPRi, we then targeted CISH in human NK-92 or primary NK cells. In human NK cells, CISH deletion also favors NCR signaling and antitumor functions. CONCLUSION: This study represents a crucial step in the mechanistic understanding and safety of Cish targeting to unleash NK cell antitumor function in solid tumors. Our results validate CISH as an emerging therapeutic target to enhance NK cell immunotherapy.
Asunto(s)
Receptor 1 Gatillante de la Citotoxidad Natural , Neoplasias , Animales , Humanos , Células Asesinas Naturales , Ratones , Receptor 1 Gatillante de la Citotoxidad Natural/genética , Receptor 1 Gatillante de la Citotoxidad Natural/metabolismo , Proteínas Supresoras de la Señalización de Citocinas/metabolismoRESUMEN
Triple-negative breast cancer (TNBC) is a highly aggressive breast cancer subtype characterized by a remarkable molecular heterogeneity. Currently, there are no effective druggable targets and advanced preclinical models of the human disease. Here, a unique mouse model (MMTV-R26Met mice) of mammary tumors driven by a subtle increase in the expression of the wild-type MET receptor is generated. MMTV-R26Met mice develop spontaneous, exclusive TNBC tumors, recapitulating primary resistance to treatment of patients. Proteomic profiling of MMTV-R26Met tumors and machine learning approach show that the model faithfully recapitulates intertumoral heterogeneity of human TNBC. Further signaling network analysis highlights potential druggable targets, of which cotargeting of WEE1 and BCL-XL synergistically kills TNBC cells and efficiently induces tumor regression. Mechanistically, BCL-XL inhibition exacerbates the dependency of TNBC cells on WEE1 function, leading to Histone H3 and phosphoS33RPA32 upregulation, RRM2 downregulation, cell cycle perturbation, mitotic catastrophe, and apoptosis. This study introduces a unique, powerful mouse model for studying TNBC formation and evolution, its heterogeneity, and for identifying efficient therapeutic targets.
RESUMEN
Analysis of mycobacterial strains that have lost their ability to cause disease is a powerful approach to identify yet unknown virulence determinants and pathways involved in tuberculosis pathogenesis. Two of the most widely used attenuated strains in the history of tuberculosis research are Mycobacterium bovis BCG (BCG) and Mycobacterium tuberculosis H37Ra (H37Ra), which both lost their virulence during in vitro serial passage. Whereas the attenuation of BCG is due mainly to loss of the ESAT-6 secretion system, ESX-1, the reason why H37Ra is attenuated remained unknown. However, here we show that a point mutation (S219L) in the predicted DNA binding region of the regulator PhoP is involved in the attenuation of H37Ra via a mechanism that impacts on the secretion of the major T cell antigen ESAT-6. Only H37Ra "knock-ins" that carried an integrated cosmid with the wild-type phoP gene from M. tuberculosis H37Rv showed changes in colony morphology, increased virulence, ESAT-6 secretion, and induction of specific T cell responses, whereas other H37Ra constructs did not. This finding established a link between the PhoP regulator and ESAT-6 secretion that opens exciting new perspectives for elucidating virulence regulation in M. tuberculosis.
Asunto(s)
Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/metabolismo , Mycobacterium tuberculosis/metabolismo , Linfocitos T/inmunología , Animales , Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Células Cultivadas , Análisis Mutacional de ADN , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Activación de Linfocitos , Macrófagos/microbiología , Masculino , Ratones , Ratones SCID , Mutación , Mycobacterium tuberculosis/patogenicidad , Análisis de Secuencia por Matrices de Oligonucleótidos , Bazo/citología , Bazo/inmunología , VirulenciaRESUMEN
Triple negative breast cancers (TNBC) remain a major medical challenge due to poor prognosis and limited treatment options. Mesothelin is a glycosyl-phosphatidyl inositol-linked membrane protein with restricted normal expression and high level expression in a large proportion of TNBC, thus qualifying as an attractive target. Its overexpression in breast tumors has been recently correlated with a decreased disease-free survival and an increase of distant metastases. The objective of the study was to investigate the relevance of a bispecific antibody-based immunotherapy approach through mesothelin targeting and CD16 engagement using a Fab-like bispecific format (MesobsFab). Using two TNBC cell lines with different level of surface mesothelin and epithelial/mesenchymal phenotypes, we showed that, in vitro, MesobsFab promotes the recruitment and penetration of NK cells into tumor spheroids, induces potent dose-dependent cell-mediated cytotoxicity of mesothelin-positive tumor cells, cytokine secretion, and decreases cell invasiveness. MesobsFab was able to induce cytotoxicity in resting human peripheral blood mononuclear cells (PBMC), mainly through its NK cells-mediated antibody dependent cell cytotoxicity (ADCC) activity. In vivo, the anti-tumor effect of MesobsFab depends upon a threshold of MSLN density on target cells. Collectively our data support mesothelin as a relevant therapeutic target for the subset of TNBC that overexpresses mesothelin characterized by a low overall and disease-free survival as well as the potential of MesobsFab as antibody-based immunotherapeutics.
Asunto(s)
Anticuerpos Biespecíficos/uso terapéutico , Neoplasias de la Mama/terapia , Proteínas Ligadas a GPI/inmunología , Inmunoterapia/métodos , Células Asesinas Naturales/inmunología , Receptores de IgG/inmunología , Neoplasias de la Mama Triple Negativas/terapia , Citotoxicidad Celular Dependiente de Anticuerpos , Neoplasias de la Mama/inmunología , Línea Celular Tumoral , Epítopos , Femenino , Humanos , Mesotelina , Neoplasias de la Mama Triple Negativas/inmunologíaRESUMEN
Women with low levels of vitamin D have a higher risk of developing breast cancer. Numerous studies associated the presence of a CD8+ T cell infiltration with a good prognosis. As vitamin D may play a key role in the modulation of the immune system, the objective of this work was to evaluate the impact of vitamin D on the breast cancer progression and mammary tumor microenvironment. We show that vitamin D decreases breast cancer tumor growth. Immunomonitoring of the different immune subsets in dissociated tumors revealed an increase in tumor infiltrating CD8+ T cells in the vitamin D-treated group. Interestingly, these CD8+ T cells exhibited a more active T cell (TEM/CM) phenotype. However, in high-fat diet conditions, we observed an opposite effect of vitamin D on breast cancer tumor growth, associated with a reduction of CD8+ T cell infiltration. Our data show that vitamin D is able to modulate breast cancer tumor growth and inflammation in the tumor microenvironment in vivo. Unexpectedly, this effect is reversed in high-fat diet conditions, revealing the importance of diet on tumor growth. We believe that supplementation with vitamin D can in certain conditions represent a new adjuvant in the treatment of breast cancers.
Asunto(s)
Neoplasias de la Mama/inmunología , Linfocitos T CD8-positivos/inmunología , Vitamina D/inmunología , Animales , Neoplasias de la Mama/patología , Linfocitos T CD8-positivos/patología , Línea Celular Tumoral , Proliferación Celular/fisiología , Progresión de la Enfermedad , Femenino , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/patología , Ratones , Ratones Endogámicos C57BL , Pronóstico , Microambiente Tumoral/inmunologíaRESUMEN
Therapeutic resistance is a major clinical challenge in oncology. Evidence identifies cancer stem cells (CSCs) as a driver of tumor evolution. Accordingly, the key stemness property unique to CSCs may represent a reservoir of therapeutic target to improve cancer treatment. Here, we carried out a genome-wide RNA interference screen to identify genes that regulate breast CSCs-fate (bCSC). Using an interactome/regulome analysis, we integrated screen results in a functional mapping of the CSC-related processes. This network analysis uncovered potential therapeutic targets controlling bCSC-fate. We tested a panel of 15 compounds targeting these regulators. We showed that mifepristone, salinomycin, and JQ1 represent the best anti-bCSC activity. A combination assay revealed a synergistic interaction of salinomycin/JQ1 association to deplete the bCSC population. Treatment of primary breast cancer xenografts with this combination reduced the tumor-initiating cell population and limited metastatic development. The clinical relevance of our findings was reinforced by an association between the expression of the bCSC-related networks and patient prognosis. Targeting bCSCs with salinomycin/JQ1 combination provides the basis for a new therapeutic approach in the treatment of breast cancer.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/fisiopatología , Descubrimiento de Drogas/métodos , Pruebas Genéticas/métodos , Estudio de Asociación del Genoma Completo/métodos , Células Madre Neoplásicas/fisiología , Interferencia de ARN , Antineoplásicos/farmacología , Femenino , Redes Reguladoras de Genes , Humanos , Mapas de Interacción de Proteínas , Células Tumorales CultivadasRESUMEN
Tumor initiation, progression, and therapeutic resistance have been proposed to originate from a subset of tumor cells, cancer stem cells (CSCs). However, the current understanding of the mechanisms involved in their self-renewal and tumor initiation capacity remains limited. Here, we report that expression of LANO/LRRC1, the vertebrate paralog of SCRIB tumor suppressor, is associated with a stem cell signature in normal and tumoral mammary epithelia. Through in vitro and in vivo experiments including a Lano/Lrrc1 knockout mouse model, we demonstrate its involvement in the regulation of breast CSC (bCSC) fate. Mechanistically, we demonstrate that Lano/LRRC1-depleted cells secrete increased levels of WNT ligands, which act in a paracrine manner to positively deregulate the WNT/ß-catenin pathway in bCSCs. In addition to describing the first function of LANO/LRRC1, our results suggest that its expression level could be used as a biomarker to stratify breast cancer patients who could benefit from WNT/ß-catenin signaling inhibitors.
Asunto(s)
Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proteínas Portadoras/metabolismo , Linaje de la Célula , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Células Madre Neoplásicas/metabolismo , Homología de Secuencia de Aminoácido , Proteínas Supresoras de Tumor/química , Vía de Señalización Wnt , Animales , Proteínas Portadoras/genética , Línea Celular Tumoral , Regulación hacia Abajo/genética , Células Epiteliales/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ligandos , Proteínas de la Membrana/genética , Ratones , Metástasis de la Neoplasia , Células Madre Neoplásicas/patología , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Adrenocortical carcinoma is a rare neoplasm with a poor prognosis. Very important advances have been made in the identification of the genetic determinants of adrenocortical carcinoma pathogenesis but our understanding is still limited about the mechanisms that determine cancer spread and metastasis. One major problem hindering preclinical experimentation for new therapies for adrenocortical carcinoma is represented by the lack of suitable animal models for metastatic disease. With the aim to overcome these limitations, in this study we tested several protocols in order to establish a mouse xenograft model of metastatic adrenocortical carcinoma. The most efficient method, based upon intrasplenic injection followed by splenectomy, produced metastases with high efficiency, whose development could be followed over time by bioluminescence measurements. We expect that the availability of this model will greatly improve the possibilities for preclinical testing of new treatments for advanced-stage disease.
RESUMEN
Components of the evolutionarily conserved developmental planar cell polarity (PCP) pathway were recently described to play a prominent role in cancer cell dissemination. However, the molecular mechanisms by which PCP molecules drive the spread of cancer cells remain largely unknown. PRICKLE1 encodes a PCP protein bound to the promigratory serine/threonine kinase MINK1. We identify RICTOR, a member of the mTORC2 complex, as a PRICKLE1-binding partner and show that the integrity of the PRICKLE1-MINK1-RICTOR complex is required for activation of AKT, regulation of focal adhesions, and cancer cell migration. Disruption of the PRICKLE1-RICTOR interaction results in a strong impairment of breast cancer cell dissemination in xenograft assays. Finally, we show that upregulation of PRICKLE1 in basal breast cancers, a subtype characterized by high metastatic potential, is associated with poor metastasis-free survival.
Asunto(s)
Neoplasias de la Mama/patología , Proteínas con Dominio LIM/metabolismo , Complejos Multiproteicos/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteínas Portadoras/metabolismo , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Femenino , Adhesiones Focales/metabolismo , Genes Dominantes , Humanos , Proteínas con Dominio LIM/química , Diana Mecanicista del Complejo 2 de la Rapamicina , Metástasis de la Neoplasia , Fosforilación , Pronóstico , Unión Proteica , Dominios Proteicos , Mapeo de Interacción de Proteínas , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteína Asociada al mTOR Insensible a la Rapamicina , Proteínas Supresoras de Tumor/química , Regulación hacia ArribaRESUMEN
Constitutive activation of the PI3K/mTOR signaling pathway contributes to carcinogenesis and metastasis in most, if not all, breast cancers. From a chromene backbone reported to inhibit class I PI3K catalytic subunits, several rounds of chemical syntheses led to the generation of a new collection of chromologues that showed enhanced ability to kill PI3K-addicted cancer cells and to inhibit Akt phosphorylation at serine 473, a hallmark of PI3K/mTOR activation. This initial screen uncovered a chromene designated DHM25 that exerted potent antitumor activity against breast tumor cell lines. Strikingly, DHM25 was shown to be a selective and covalent inhibitor of mTOR using biochemical and cellular analyses, modeling, and a large panel of kinase activity assays spanning the human kinome (243 kinases). Finally, in vivo, this novel drug was an efficient inhibitor of growth and metastasis of triple-negative breast cancer cells, paving the way for its clinical application in oncology.
Asunto(s)
Antineoplásicos/farmacología , Benzopiranos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Animales , Antineoplásicos/síntesis química , Benzopiranos/síntesis química , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Ratones , Ratones Endogámicos NOD , Modelos Moleculares , Proteína Oncogénica v-akt/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
Defective nectin-1 and -4 have been implicated in ectodermal dysplasia (ED) syndromes with variably associated features including orofacial and limb defects. In particular, nectin-1 mutations cause cleft lip/palate ED (CLPED1; OMIM#225060), whereas defective nectin-4 is associated with ED-syndactyly syndrome (EDSS1; OMIM#613573). Although the broad phenotypic overlap suggests a common mode of action of nectin-1 and -4, little is known about the pathogenic mechanisms involved. We report the identification of, to our knowledge, a previously undescribed nectin-4 homozygous p.Val242Met missense mutation in a patient with EDSS1. We used patient skin biopsy and primary keratinocytes, as well as nectin-4 ectopic expression in epithelial cell lines, to characterize functional consequences of p.Val242Met and p.Thr185Met mutations, the latter previously identified in compound heterozygosity with a truncating mutation. We show that nectin-4-altered expression perturbs nectin-1 clustering at keratinocyte contact sites and delays, but does not impede cell-cell aggregation and cadherin recruitment at adherens junctions (AJs). Moreover, trans-interaction of nectin-1 and -4 induces the activation of Rac1, a member of the Rho family of small GTPases, and regulates E-cadherin-mediated cell-cell adhesion. These data outline a synergistic action of nectin-1 and -4 in the early steps of AJ formation and implicate this interaction in modulating the Rac1 signaling pathway.
Asunto(s)
Uniones Adherentes/fisiología , Moléculas de Adhesión Celular/fisiología , Displasia Ectodérmica/fisiopatología , Mutación , Transducción de Señal/fisiología , Sindactilia/genética , Proteína de Unión al GTP rac1/fisiología , Animales , Adhesión Celular , Moléculas de Adhesión Celular/análisis , Moléculas de Adhesión Celular/química , Moléculas de Adhesión Celular/genética , Agregación Celular , Células Cultivadas , Perros , Displasia Ectodérmica/genética , Humanos , Cinética , NectinasRESUMEN
PURPOSE: Cancer stem cells (CSC) are the tumorigenic cell population that has been shown to sustain tumor growth and to resist conventional therapies. The purpose of this study was to evaluate the potential of histone deacetylase inhibitors (HDACi) as anti-CSC therapies. EXPERIMENTAL DESIGN: We evaluated the effect of the HDACi compound abexinostat on CSCs from 16 breast cancer cell lines (BCL) using ALDEFLUOR assay and tumorsphere formation. We performed gene expression profiling to identify biomarkers predicting drug response to abexinostat. Then, we used patient-derived xenograft (PDX) to confirm, in vivo, abexinostat treatment effect on breast CSCs according to the identified biomarkers. RESULTS: We identified two drug-response profiles to abexinostat in BCLs. Abexinostat induced CSC differentiation in low-dose sensitive BCLs, whereas it did not have any effect on the CSC population from high-dose sensitive BCLs. Using gene expression profiling, we identified the long noncoding RNA Xist (X-inactive specific transcript) as a biomarker predicting BCL response to HDACi. We validated that low Xist expression predicts drug response in PDXs associated with a significant reduction of the breast CSC population. CONCLUSIONS: Our study opens promising perspectives for the use of HDACi as a differentiation therapy targeting the breast CSCs and identified a biomarker to select patients with breast cancer susceptible to responding to this treatment.
Asunto(s)
Antineoplásicos/farmacología , Benzofuranos/farmacología , Neoplasias de la Mama/patología , Diferenciación Celular/efectos de los fármacos , Ácidos Hidroxámicos/farmacología , Células Madre Neoplásicas/fisiología , ARN Largo no Codificante/metabolismo , Animales , Antineoplásicos/uso terapéutico , Benzofuranos/uso terapéutico , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Resistencia a Antineoplásicos , Femenino , Expresión Génica , Regulación Neoplásica de la Expresión Génica , Inhibidores de Histona Desacetilasas/farmacología , Inhibidores de Histona Desacetilasas/uso terapéutico , Humanos , Ácidos Hidroxámicos/uso terapéutico , Concentración 50 Inhibidora , Ratones , Ratones Endogámicos NOD , Ratones SCID , Células Madre Neoplásicas/efectos de los fármacos , ARN Largo no Codificante/genética , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Cancer stem-like cells (CSC) have been widely studied, but their clinical relevance has yet to be established in breast cancer. Here, we report the establishment of primary breast tumor-derived xenografts (PDX) that encompass the main diversity of human breast cancer and retain the major clinicopathologic features of primary tumors. Successful engraftment was correlated with the presence of ALDH1-positive CSCs, which predicted prognosis in patients. The xenografts we developed showed a hierarchical cell organization of breast cancer with the ALDH1-positive CSCs constituting the tumorigenic cell population. Analysis of gene expression from functionally validated CSCs yielded a breast CSC signature and identified a core transcriptional program of 19 genes shared with murine embryonic, hematopoietic, and neural stem cells. This generalized stem cell program allowed the identification of potential CSC regulators, which were related mainly to metabolic processes. Using an siRNA genetic screen designed to target the 19 genes, we validated the functional role of this stem cell program in the regulation of breast CSC biology. Our work offers a proof of the functional importance of CSCs in breast cancer, and it establishes the reliability of PDXs for use in developing personalized CSC therapies for patients with breast cancer.
Asunto(s)
Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , Isoenzimas/metabolismo , Células Madre Neoplásicas/enzimología , Retinal-Deshidrogenasa/metabolismo , Familia de Aldehído Deshidrogenasa 1 , Animales , Biomarcadores de Tumor/genética , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Diferenciación Celular/genética , Femenino , Perfilación de la Expresión Génica , Xenoinjertos , Humanos , Isoenzimas/genética , Ratones , Ratones Endogámicos NOD , Ratones SCID , Células Madre Neoplásicas/patología , Hibridación de Ácido Nucleico , Pronóstico , Estudios Prospectivos , Retinal-Deshidrogenasa/genética , TransfecciónRESUMEN
BACKGROUND: Targeted therapies, associated with standard chemotherapies, have improved breast cancer care. However, primary and acquired resistances are frequently observed and the development of new concepts is needed. High-throughput approaches to identify new active and safe molecules with or without an "a priori" are currently developed. Also, repositioning already-approved drugs in cancer therapy is of growing interest. The thiomorpholine hydroxamate compound TMI-1 has been previously designed to inhibit metalloproteinase activity for the treatment of rheumatoid arthritis. We present here the repositioning of TMI-1 drug in breast cancer. METHODOLOGY/PRINCIPAL FINDINGS: We tested the effect of TMI-1 on luminal, basal and ERBB2-overexpressing breast tumor cell lines and on MMTV-ERBB2/neu tumor evolution. We measured the effects on i) cell survival, ii) cell cycle, iii) extrinsic and intrinsic apoptotic pathways, iv) association with doxorubicin, docetaxel and lapatinib, v) cancer stem cells compartment. In contrast with conventional cytotoxic drugs, TMI-1 was highly selective for tumor cells and cancer stem cells at submicromolar range. All non-malignant cells tested were resistant even at high concentration. TMI-1 was active on triple negative (TN) and ERBB2-overexpressing breast tumor cell lines, and was also highly efficient on human and murine "primary" ERBB2-overexpressing cells. Treatment of transgenic MMTV-ERBB2/neu mice with 100 mg/kg/day TMI-1 alone induced tumor apoptosis, inhibiting mammary gland tumor occurrence and development. No adverse effects were noticed during the treatment. This compound had a strong synergistic effect in association with docetaxel, doxorubicin and lapatinib. We showed that TMI-1 mediates its selective effects by caspase-dependent apoptosis. TMI-1 was efficient in 34/40 tumor cell lines of various origins (ED50: 0.6 µM to 12.5 µM). CONCLUSIONS/SIGNIFICANCE: This is the first demonstration of the tumor selective cytotoxic action of a thiomorpholin hydroxamate compound. TMI-1 is a novel repositionable drug not only for the treatment of adverse prognosis breast cancers but also for other neoplasms.