RESUMEN
Diamond Blackfan Anemia (DBA) is a rare macrocytic red blood cell aplasia that usually presents within the first year of life. The vast majority of patients carry a mutation in one of approximately 20 genes that results in ribosomal insufficiency with the most significant clinical manifestations being anemia and a predisposition to cancers. Nemo-like Kinase (NLK) is hyperactivated in the erythroid progenitors of DBA patients and inhibition of this kinase improves erythropoiesis, but how NLK contributes to the pathogenesis of the disease is unknown. Here we report that activated NLK suppresses the critical upregulation of mitochondrial biogenesis required in early erythropoiesis. During normal erythropoiesis, mTORC1 facilitates the translational upregulation of Transcription factor A, mitochondrial (TFAM), and Prohibin 2 (PHB2) to increase mitochondrial biogenesis. In our models of DBA, active NLK phosphorylates the regulatory component of mTORC1, thereby suppressing mTORC1 activity and preventing mTORC1-mediated TFAM and PHB2 upregulation and subsequent mitochondrial biogenesis. Improvement of erythropoiesis that accompanies NLK inhibition is negated when TFAM and PHB2 upregulation is prevented. These data demonstrate that a significant contribution of NLK on the pathogenesis of DBA is through loss of mitochondrial biogenesis.
RESUMEN
Ca(2+) is an essential and ubiquitous second messenger. Changes in cytosolic Ca(2+) trigger events critical for tumorigenesis, such as cellular motility, proliferation, and apoptosis. We show that an isoform of Secretory Pathway Ca(2+)-ATPase, SPCA2, is upregulated in breast cancer-derived cells and human breast tumors, and suppression of SPCA2 attenuates basal Ca(2+) levels and tumorigenicity. Contrary to its conventional role in Golgi Ca(2+) sequestration, expression of SPCA2 increased Ca(2+) influx by a mechanism dependent on the store-operated Ca(2+) channel Orai1. Unexpectedly, SPCA2-Orai1 signaling was independent of ER Ca(2+) stores or STIM1 and STIM2 sensors and uncoupled from Ca(2+)-ATPase activity of SPCA2. Binding of the SPCA2 amino terminus to Orai1 enabled access of its carboxyl terminus to Orai1 and activation of Ca(2+) influx. Our findings reveal a signaling pathway in which the Orai1-SPCA2 complex elicits constitutive store-independent Ca(2+) signaling that promotes tumorigenesis.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Canales de Calcio/metabolismo , Señalización del Calcio , ATPasas Transportadoras de Calcio/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Femenino , Perfilación de la Expresión Génica , Humanos , Ratones , Ratones Desnudos , Modelos Moleculares , Datos de Secuencia Molecular , Trasplante de Neoplasias , Proteína ORAI1 , Ratas , Alineación de Secuencia , Trasplante HeterólogoRESUMEN
BACKGROUND: Growth factor receptor-bound 7 (Grb7) is an adaptor protein involved in signal transduction downstream of multiple receptor tyrosine kinases, including ERBB, FGFR, and PDGFR pathways. Experimental studies have implicated Grb7 in regulating cell proliferation, survival, migration, and invasion through its large repertoire of protein-protein interactions. RESULTS: Here, we describe the generation and characterization of a Grb7 knockout mouse. These mice are viable and fertile. A lacZ knock-in reporter was used to visualize Grb7 promoter activity patterns in adult tissues, indicating widespread Grb7 expression in glandular epithelium, the central nervous system, and other tissues. The sole defect observed in these animals was a failure of Grb7 knockout females to successfully raise pups to weaning age, a phenotype that was independent of both paternal and pup genotypes. CONCLUSIONS: These data suggest a regulatory role for Grb7 in mammary lactational physiology.
Asunto(s)
Proteína Adaptadora GRB7 , Ratones Noqueados , Animales , Femenino , Ratones , Proteína Adaptadora GRB7/metabolismo , Proteína Adaptadora GRB7/genética , Masculino , Lactancia/genética , Insuficiencia de Crecimiento/genética , Insuficiencia de Crecimiento/metabolismo , Glándulas Mamarias Animales/metabolismo , Glándulas Mamarias Animales/crecimiento & desarrolloRESUMEN
PURPOSE: The epidermal growth factor receptor ligand, Amphiregulin, is a transcriptional target of estrogen receptor alpha and is required for pubertal mammary gland development. Previous studies using immortalized human breast cancer cell line xenografts have suggested that Amphiregulin may be an important effector of estrogen receptor alpha during breast cancer development, at least in immune-compromised animals. Here, we evaluate the requirement for Amphiregulin in an immune-competent mouse model which is prone to developing estrogen receptor-positive tumors. METHODS: We have intercrossed mice with mammary-specific mutation of p53 with mice deficient in Amphiregulin in order to assess the requirement for Amphiregulin in the initiation and progression of both estrogen receptor-positive and estrogen receptor-negative mammary tumors. RESULTS: Deletion of Amphiregulin significantly delayed the onset of palpable mammary tumors and also strongly reduced the proportion of estrogen receptor alpha-positive tumors formed. Upon necropsy, no substantial differences in the prevalence of non-palpable lesions were observed between cohorts, suggesting that the importance of Amphiregulin in mammary tumorigenesis is limited to the post-initiation phase. CONCLUSIONS: This study underlines the importance of the EGFR ligand, Amphiregulin, as a key mediator of estrogen receptor action in breast cancer.
Asunto(s)
Anfirregulina/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Eliminación de Gen , Mutación , Receptores de Estrógenos/metabolismo , Proteína p53 Supresora de Tumor/genética , Alelos , Animales , Biomarcadores de Tumor , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/patología , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Modelos Animales de Enfermedad , Femenino , Inmunohistoquímica , Ratones , Pronóstico , Proteína p53 Supresora de Tumor/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Although the treatment of metastatic melanoma has been significantly improved by both anti-BRAF/MEK and checkpoint immunotherapies, resistance to these treatment modalities remains a substantial clinical problem. Multiple clinical studies are addressing the optimal sequencing of these agents in larger patient cohorts, but successful long-term individualized treatment will likely require the elucidation of resistance mechanisms from post-progression samples. Here, we describe a patient with BRAF-V600E-positive metastatic melanoma who was sequentially treated with BRAF/MEK inhibitors (dabrafenib/trametinib) and checkpoint inhibitor immunotherapy (nivolumab, followed by pembrolizumab). After the emergence of resistance, whole exome sequencing was performed, implicating MAP2K2 and B2M mutations in loss of response to anti-BRAF/MEK and anti-PD1 therapies, respectively.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , MAP Quinasa Quinasa 2/antagonistas & inhibidores , Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Melanoma/tratamiento farmacológico , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Neoplasias Cutáneas/tratamiento farmacológico , Microglobulina beta-2/antagonistas & inhibidores , Anticuerpos Monoclonales Humanizados/administración & dosificación , Humanos , Imidazoles/administración & dosificación , MAP Quinasa Quinasa 2/genética , Quinasas Quinasa Quinasa PAM/metabolismo , Masculino , Melanoma/genética , Persona de Mediana Edad , Mutación , Nivolumab/administración & dosificación , Oximas/administración & dosificación , Inhibidores de Proteínas Quinasas/administración & dosificación , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Piridonas/administración & dosificación , Pirimidinonas/administración & dosificación , Neoplasias Cutáneas/genética , Insuficiencia del Tratamiento , Microglobulina beta-2/genéticaRESUMEN
BACKGROUND: Amphiregulin (AREG), a ligand of the epidermal growth factor receptor, is not only essential for proper mammary ductal development, but also associated with breast cancer proliferation and growth. In the absence of AREG, mammary ductal growth is stunted and fails to expand. Furthermore, suppression of AREG expression in estrogen receptor-positive breast tumor cells inhibits in-vitro and in-vivo growth. METHODS: We crossed AREG-null (AREG-/-) mice with the murine luminal B breast cancer model, MMTV-PyMT (PyMT), to generate spontaneous breast tumors that lack AREG (AREG-/- PyMT). We evaluated tumor growth, cytokeratin-8 (K8)-positive luminal cells, cytokeratin-14 (K14)-positive myoepithelial cells, and expression of AREG, Ki67, and PyMT. Primary myoepithelial cells from nontumor-bearing AREG+/+ mice underwent fluorescence-activated cell sorting and were adapted to culture for in-vitro coculture studies with AT-3 cells, a cell line derived from C57Bl/6 PyMT mammary tumors. RESULTS: Intriguingly, PyMT-induced lesions progress more rapidly in AREG-/- mice than in AREG+/+ mice. Quantification of K8+ luminal and K14+ myoepithelial cells in non-PyMT AREG-/- mammary glands showed fewer K14+ cells and a thinner myoepithelial layer. Study of AT-3 cells indicated that coculture with myoepithelial cells or exposure to AREG, epidermal growth factor, or basic fibroblast growth factor can suppress PyMT expression. Late-stage AREG-/- PyMT tumors are significantly less solid in structure, with more areas of papillary and cystic growth. Papillary areas appear to be both less proliferative and less necrotic. In The Cancer Genome Atlas database, luminal-B invasive papillary carcinomas have lower AREG expression than luminal B invasive ductal carcinomas. CONCLUSIONS: Our study has revealed a previously unknown role of AREG in myoepithelial cell development and PyMT expression. AREG expression is essential for proper myoepithelial coverage of mammary ducts. Both AREG and myoepithelial cells can suppress PyMT expression. We find that lower AREG expression is associated with invasive papillary breast cancer in both the MMTV-PyMT model and human breast cancer.
Asunto(s)
Anfirregulina/metabolismo , Células Epiteliales/patología , Glándulas Mamarias Animales/patología , Neoplasias Mamarias Experimentales/patología , Anfirregulina/genética , Animales , Antígenos Transformadores de Poliomavirus/genética , Antígenos Transformadores de Poliomavirus/metabolismo , Línea Celular Tumoral , Proliferación Celular , Células Epiteliales/virología , Femenino , Humanos , Glándulas Mamarias Animales/citología , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/virología , Virus del Tumor Mamario del Ratón/genética , Virus del Tumor Mamario del Ratón/patogenicidad , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Invasividad Neoplásica/patología , Poliomavirus/genética , Poliomavirus/inmunologíaRESUMEN
Unlike humans, inbred genetically engineered mice have minimal inter-individual variation and, consequently, offer substantially increased statistical power for robust definition of recurrent cooperating cancer mutations. While technically feasible, whole exome sequencing is expensive and extremely data-intensive. Somatic mutation analysis using panels of 25-75 genes now provides detailed insight into the biology of human tumors. Here we report an adaptation for mouse tumors of a human PCR amplicon-based panel (Ion Torrent Cancer Hotspot Panel v2) allowing analysis of 18 cancer genes, including Kras, Nras, Hras, Pten, Pik3ca and Smad4, and encompassing regions homologous to more than 2000 known human cancer mutations.
Asunto(s)
Análisis Mutacional de ADN/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Neoplasias Mamarias Experimentales/genética , Oncogenes , Quinasas de la Proteína-Quinasa Activada por el AMP , Animales , Fosfatidilinositol 3-Quinasa Clase I/genética , ADN de Neoplasias/genética , Femenino , Genes ras , Humanos , Ratones , Ratones Transgénicos , Técnicas de Amplificación de Ácido Nucleico/métodos , Reacción en Cadena de la Polimerasa/métodos , Proteínas Serina-Treonina Quinasas/genética , Especificidad de la EspecieRESUMEN
Ultra-late melanoma recurrence is infrequent, poorly understood and, in most cases, difficult to unambiguously distinguish from a new primary melanoma. We identified a patient with a second melanoma diagnosed after a 30-year disease-free interval, and sought to determine if this new lesion was a recurrence of the original melanoma. Here we report the genomic sequence analysis of the exomes of 2 melanoma lesions isolated from the same individual in 1985 and 2015, and their comparison to each other and to the germline DNA of the patient. Identification of many shared somatic mutations between these lesions proves a lineal relationship spanning 30 years. Unlike prior reports of ultra-late melanoma recurrence, the availability of the original tumor and the use of comprehensive genomic analysis allowed us to confirm that the second lesion is truly a recurrence. We demonstrate the acquisition of numerous additional mutations during the 3 decade asymptomatic period. These data highlight the low but very long-lasting risk of recurrence in this patient population.
Asunto(s)
Melanoma/genética , Melanoma/patología , Recurrencia Local de Neoplasia/genética , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Anciano , Humanos , Masculino , Neoplasias Primarias Múltiples/patología , Neoplasias de la Próstata/patologíaRESUMEN
Myelodysplastic syndromes (MDS) are characterized by ineffective hematopoiesis that leads to peripheral cytopenias. We observed that SMAD7, a negative regulator of transforming growth factor-beta (TGF-ß) receptor-I kinase, is markedly reduced in MDS and leads to ineffective hematopoiesis by overactivation of TGF-ß signaling. To determine the cause of SMAD7 reduction in MDS, we analyzed the 3'UTR of the gene and determined that it contains a highly conserved putative binding site for microRNA-21. We observed significantly elevated levels of miR-21 in MDS marrow samples when compared with age-matched controls. miR-21 was shown to directly bind to the 3'UTR of SMAD7 and reduce its expression in hematopoietic cells. Next, we tested the role of miR-21 in regulating TGF-ß signaling in a TGF-ß-overexpressing transgenic mouse model that develops progressive anemia and dysplasia and thus serves as a model of human bone marrow failure. Treatment with a chemically modified miR-21 inhibitor led to significant increases in hematocrit and led to an increase in SMAD7 expression in vivo. Inhibition of miR-21 also led to an increase in erythroid colony formation from primary MDS bone marrow progenitors, demonstrating its ability in stimulating hematopoiesis in vitro. Taken together, these studies demonstrate the role of miR-21 in regulating overactivated TGF-ß signaling in MDS.
Asunto(s)
Hematopoyesis/genética , MicroARNs/genética , Síndromes Mielodisplásicos/genética , Transducción de Señal/genética , Factor de Crecimiento Transformador beta1/genética , Regiones no Traducidas 3'/genética , Anciano , Anciano de 80 o más Años , Animales , Sitios de Unión/genética , Células de la Médula Ósea/metabolismo , Línea Celular , Células Cultivadas , Femenino , Expresión Génica , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Células K562 , Masculino , Ratones , Ratones Transgénicos , Microscopía Fluorescente , Persona de Mediana Edad , Mutación , Síndromes Mielodisplásicos/metabolismo , Proteína smad7/genéticaRESUMEN
Plasma membrane calcium ATPases (PMCAs) actively extrude Ca(2+) from the cell and are essential components in maintaining intracellular Ca(2+) homeostasis. There are four PMCA isoforms (PMCA1-4), and alternative splicing of the PMCA genes creates a suite of calcium efflux pumps. The role of these different PMCA isoforms in the control of calcium-regulated cell death pathways and the significance of the expression of multiple isoforms of PMCA in the same cell type are not well understood. In these studies, we assessed the impact of PMCA1 and PMCA4 silencing on cytoplasmic free Ca(2+) signals and cell viability in MDA-MB-231 breast cancer cells. The PMCA1 isoform was the predominant regulator of global Ca(2+) signals in MDA-MB-231 cells. PMCA4 played only a minor role in the regulation of bulk cytosolic Ca(2+), which was more evident at higher Ca(2+) loads. Although PMCA1 or PMCA4 knockdown alone had no effect on MDA-MB-231 cell viability, silencing of these isoforms had distinct consequences on caspase-independent (ionomycin) and -dependent (ABT-263) cell death. PMCA1 knockdown augmented necrosis mediated by the Ca(2+) ionophore ionomycin, whereas apoptosis mediated by the Bcl-2 inhibitor ABT-263 was enhanced by PMCA4 silencing. PMCA4 silencing was also associated with an inhibition of NFκB nuclear translocation, and an NFκB inhibitor phenocopied the effects of PMCA4 silencing in promoting ABT-263-induced cell death. This study demonstrates distinct roles for PMCA1 and PMCA4 in the regulation of calcium signaling and cell death pathways despite the widespread distribution of these two isoforms. The targeting of some PMCA isoforms may enhance the effectiveness of therapies that act through the promotion of cell death pathways in cancer cells.
Asunto(s)
Neoplasias de la Mama/enzimología , Señalización del Calcio , Proteínas de Neoplasias/metabolismo , ATPasas Transportadoras de Calcio de la Membrana Plasmática/metabolismo , Transporte Activo de Núcleo Celular/efectos de los fármacos , Transporte Activo de Núcleo Celular/genética , Neoplasias de la Mama/dietoterapia , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Ionóforos de Calcio/farmacología , Línea Celular Tumoral , Núcleo Celular/genética , Núcleo Celular/metabolismo , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Ionomicina/farmacología , Isoenzimas/genética , Isoenzimas/metabolismo , FN-kappa B/genética , FN-kappa B/metabolismo , Necrosis , Proteínas de Neoplasias/genética , ATPasas Transportadoras de Calcio de la Membrana Plasmática/genéticaRESUMEN
Bisected, complex N-glycans on glycoproteins are generated by the glycosyltransferase MGAT3 and cause reduced cell surface binding of galectins. Previously, we showed that MGAT3 reduces growth factor signaling and retards mammary tumor progression driven by the Polyoma middle T antigen (PyMT) expressed in mammary epithelium under the mouse mammary tumor virus (MMTV) promoter. However, the penetrance of the tumor phenotype became variable in mixed FVB/N and C57BL/6 female mice and we therefore investigated a congenic C57BL/6 Mgat3(-/-)/MMTV-PyMT model. In the absence of MGAT3, C57BL/6 Mgat3(-/-)/MMTV-PyMT females exhibited accelerated tumor appearance and increased tumor burden, glucose uptake in tumors and lung metastasis. Nevertheless, activation of extracellular signal-regulated kinase (ERK)1/2 or protein kinase B (AKT) was reduced in â¼20-week C57BL/6 MMTV-PyMT tumors lacking MGAT3. Activation of focal adhesion kinase (FAK), protein tyrosine kinase Src, and p38 mitogen-activated protein kinase were similar to that of controls. All the eight mouse galectin genes were expressed in mammary tumors and tumor epithelial cells (TECs), but galectin-2 and -12 were not detected by western analysis in tumors, and galectin-7 was not detected in 60% of the TEC lines. From microarray data reported for human breast cancers, at least 10 galectin and 7 N-glycan N-acetylglucosaminyl (GlcNAc)-transferase (MGAT) genes are expressed in tumor tissue, and expression often varies significantly between different breast cancer subtypes. Thus, in summary, while MGAT3 and bisected complex N-glycans retard mouse mammary tumor progression, genetic background may modify this effect; identification of key galectins that promote mammary tumor progression in mice is not straightforward because all the eight galectin genes are expressed; and high levels of MGAT3, galectin-4, -8, -10, -13 and -14 transcripts correlate with better relapse-free survival in human breast cancer.
Asunto(s)
Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Progresión de la Enfermedad , Galectinas/metabolismo , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Polisacáridos/metabolismo , Animales , Antígenos Transformadores de Poliomavirus/metabolismo , Neoplasias de la Mama/genética , Modelos Animales de Enfermedad , Femenino , Humanos , Neoplasias Mamarias Experimentales/genética , Virus del Tumor Mamario del Ratón/metabolismo , Ratones , Ratones Endogámicos , N-Acetilglucosaminiltransferasas/deficiencia , N-Acetilglucosaminiltransferasas/genética , N-Acetilglucosaminiltransferasas/metabolismoRESUMEN
The local microenvironment influences tumor progression in several important ways. A recent study by Nguyen-Ngoc and colleagues used explants of primary human and mouse mammary tumors to examine how the composition of the extracellular matrix modulates tumor cell invasion. Culture in the presence of a three-dimensional laminin-rich basement membrane, similar to the mammary basement membrane in vivo, resulted in minimal invasion. In contrast, identical tumor fragments in a collagen I matrix which resembles the interstitial breast stroma exhibited a pronounced protrusive migration and local dissemination. These data emphasize the importance of the laminin-rich basement membrane in constraining tumor cell invasion.
Asunto(s)
Neoplasias de la Mama/genética , Laminina/metabolismo , Neoplasias Mamarias Animales/genética , Invasividad Neoplásica/genética , Animales , Membrana Basal/metabolismo , Membrana Basal/patología , Neoplasias de la Mama/patología , Movimiento Celular , Femenino , Humanos , Neoplasias Mamarias Animales/patología , Ratones , Invasividad Neoplásica/patología , Microambiente Tumoral/genéticaRESUMEN
The epidermal growth factor receptor (EGFR) is frequently expressed in triple-negative breast cancer (TNBC) and is a marker of poor prognosis in this patient population. Because activating mutations in this kinase are very rare events in breast cancer, we screened breast tumor gene expression profiles to examine the distribution of EGFR ligand expression. Of the six known EGFR ligands, transforming growth factor alpha (TGFα) was expressed more highly in triple-negative breast tumors than in tumors of other subtypes. TGFα is synthesized as a transmembrane precursor requiring tumor necrosis factor alpha converting enzyme (TACE)/ADAM17-dependent proteolytic release to activate its receptor. In our study, we show that an inhibitor of this proteolytic release blocks invasion, migration and colony formation by several TNBC cell lines. Each of the effects of the drug was reversed upon expression of a soluble TGFα mutant that does not require TACE activity, implicating this growth factor as a key metalloproteinase substrate for these phenotypes. Together, these data demonstrate that TACE-dependent TGFα shedding is a key process driving EGFR activation and subsequent proliferation and invasion in TNBC cell lines.
Asunto(s)
Proteínas ADAM/metabolismo , Neoplasias de la Mama/genética , Invasividad Neoplásica/genética , Factor de Crecimiento Transformador alfa/metabolismo , Proteínas ADAM/antagonistas & inhibidores , Proteínas ADAM/genética , Proteína ADAM17 , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Receptores ErbB/genética , Receptores ErbB/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Mutación , Invasividad Neoplásica/patología , Neoplasias , Fosforilación , Transducción de Señal , Factor de Crecimiento Transformador alfa/genéticaRESUMEN
The mitochondrial calcium uniporter (MCU) transports free ionic Ca(2+) into the mitochondrial matrix. We assessed MCU expression in clinical breast cancer samples using microarray analysis and the consequences of MCU silencing in a breast cancer cell line. Our results indicate that estrogen receptor negative and basal-like breast cancers are characterized by elevated levels of MCU. Silencing of MCU expression in the basal-like MDA-MB-231 breast cancer cell line produced no change in proliferation or cell viability. However, distinct consequences of MCU silencing were seen on cell death pathways. Caspase-dependent cell death initiated by the Bcl-2 inhibitor ABT-263 was not altered by MCU silencing; whereas caspase-independent cell death induced by the calcium ionophore ionomycin was potentiated by MCU silencing. Measurement of cytosolic Ca(2+) levels showed that the promotion of ionomycin-induced cell death by MCU silencing occurs independently of changes in bulk cytosolic Ca(2+) levels. This study demonstrates that MCU overexpression is a feature of some breast cancers and that MCU overexpression may offer a survival advantage against some cell death pathways. MCU inhibitors may be a strategy to increase the effectiveness of therapies that act through the induction of caspase-independent cell death pathways in estrogen receptor negative and basal-like breast cancers.
Asunto(s)
Neoplasias de la Mama/patología , Canales de Calcio/metabolismo , Caspasas/metabolismo , Silenciador del Gen , Mitocondrias/metabolismo , Compuestos de Anilina/farmacología , Antineoplásicos/farmacología , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/metabolismo , Canales de Calcio/genética , Línea Celular Tumoral , Proliferación Celular , Femenino , Humanos , Ionomicina/farmacología , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Sulfonamidas/farmacologíaRESUMEN
Invasive lobular carcinoma (ILC) of the breast, characterized by loss of E-cadherin expression, accounts for 5-15% of invasive breast cancers and it is believed to arise via a linear histological progression. Genomic studies have identified a clonal relationship between ILC and concurrent lobular carcinoma in situ (LCIS) lesions, suggesting that LCIS may be a precursor lesion. It has been shown that an LCIS diagnosis confers a 15-20% risk of progression to ILC over a lifetime. Currently no molecular test or markers can identify LCIS lesions likely to progress to ILC. Since microRNA (miRNA) expression changes have been detected in a number of other cancer types, we explored whether their dysregulation might be detected during progression from LCIS to ILC. Using the Illumina miRNA profiling platform, designed for simultaneous analysis of 470 mature miRNAs, we analysed the profiles of archived normal breast epithelium, LCIS lesions found alone, LCIS lesions concurrent with ILC, and the concurrent ILCs as a model of linear histological progression towards ILC. We identified two sets of differentially expressed miRNAs, the first set highly expressed in normal epithelium, including hsa-miR-224, -139, -10b, -450, 140, and -365, and the second set up-regulated during lobular neoplasia progression, including hsa-miR-375, -203, -425-5p, -183, -565, and -182. Using quantitative RT-PCR, we validated a trend of increasing expression for hsa-miR-375, hsa-miR-182, and hsa-miR-183 correlating with ILC progression. As we detected increased expression of hsa-miR-375 in LCIS lesions synchronous with ILC, we sought to determine whether hsa-miR-375 might induce phenotypes reminiscent of lobular neoplasia by expressing it in the MCF-10A 3D culture model of mammary acinar morphogenesis. Increased expression of hsa-miR-375 resulted in loss of cellular organization and acquisition of a hyperplastic phenotype. These data suggest that dysregulated miRNA expression contributes to lobular neoplastic progression.
Asunto(s)
Células Acinares/patología , Neoplasias de la Mama/genética , Carcinoma in Situ/genética , Carcinoma Lobular/genética , Polaridad Celular/genética , MicroARNs/genética , Neoplasias de la Mama/patología , Carcinoma in Situ/patología , Carcinoma Lobular/patología , Progresión de la Enfermedad , Femenino , Técnica del Anticuerpo Fluorescente , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Inmunohistoquímica , Microdisección , Microscopía Confocal , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ARNRESUMEN
INTRODUCTION: Metastasis of breast cancer is the main cause of death in patients. Previous genome-wide studies have identified gene-expression patterns correlated with cancer patient outcome. However, these were derived mostly from whole tissue without respect to cell heterogeneity. In reality, only a small subpopulation of invasive cells inside the primary tumor is responsible for escaping and initiating dissemination and metastasis. When whole tissue is used for molecular profiling, the expression pattern of these cells is masked by the majority of the noninvasive tumor cells. Therefore, little information is available about the crucial early steps of the metastatic cascade: migration, invasion, and entry of tumor cells into the systemic circulation. METHODS: In the past, we developed an in vivo invasion assay that can capture specifically the highly motile tumor cells in the act of migrating inside living tumors. Here, we used this assay in orthotopic xenografts of human MDA-MB-231 breast cancer cells to isolate selectively the migratory cell subpopulation of the primary tumor for gene-expression profiling. In this way, we derived a gene signature specific to breast cancer migration and invasion, which we call the Human Invasion Signature (HIS). RESULTS: Unsupervised analysis of the HIS shows that the most significant upregulated gene networks in the migratory breast tumor cells include genes regulating embryonic and tissue development, cellular movement, and DNA replication and repair. We confirmed that genes involved in these functions are upregulated in the migratory tumor cells with independent biological repeats. We also demonstrate that specific genes are functionally required for in vivo invasion and hematogenous dissemination in MDA-MB-231, as well as in patient-derived breast tumors. Finally, we used statistical analysis to show that the signature can significantly predict risk of breast cancer metastasis in large patient cohorts, independent of well-established prognostic parameters. CONCLUSIONS: Our data provide novel insights into, and reveal previously unknown mediators of, the metastatic steps of invasion and dissemination in human breast tumors in vivo. Because migration and invasion are the early steps of metastatic progression, the novel markers that we identified here might become valuable prognostic tools or therapeutic targets in breast cancer.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Animales , Neoplasias de la Mama/mortalidad , Línea Celular Tumoral , Movimiento Celular/genética , Estudios de Cohortes , Modelos Animales de Enfermedad , Femenino , Redes Reguladoras de Genes , Xenoinjertos , Humanos , Invasividad Neoplásica , Evaluación del Resultado de la Atención al Paciente , Pronóstico , Reproducibilidad de los Resultados , TranscriptomaRESUMEN
Triple-negative breast cancer (TNBC) is a heterogeneous disease that is usually associated with poor prognosis, and frequently associated with the basal-like breast cancer gene expression profile. There are no targeted therapeutic modalities for this disease, and no useful biomarkers. High GRB7 RNA expression levels are associated with an elevated risk of recurrence in patients with operable TNBC treated with standard adjuvant anthracycline and taxane therapy. To determine whether GRB7 is involved in the pathobiology of TNBC, we evaluated the biological effects of GRB7 inhibition in a panel of triple-negative cell lines-MDA-MB-468, MDA-MB-231, HCC70, and T4-2. We found GRB7 inhibition reduced cell motility and invasion of these cell lines and promoted cell death by apoptosis in 3D culture. These data suggest that GRB7 itself, or GRB7-dependent pathways, may prove to be important therapeutic targets in this disease.
Asunto(s)
Neoplasias de la Mama/patología , Proteína Adaptadora GRB7/fisiología , Apoptosis/efectos de los fármacos , Apoptosis/genética , Neoplasias de la Mama/genética , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Supervivencia Celular/genética , Femenino , Proteína Adaptadora GRB7/antagonistas & inhibidores , Proteína Adaptadora GRB7/genética , Humanos , Invasividad Neoplásica/genética , Neoplasias Basocelulares/genética , Neoplasias Basocelulares/patología , Péptidos Cíclicos/farmacología , Receptor ErbB-2/deficiencia , Receptores de Estrógenos/deficiencia , Receptores de Progesterona/deficiencia , Reproducibilidad de los ResultadosRESUMEN
The implementation of monoclonal antibody therapeutics during the COVID-19 pandemic altered the selective pressures encountered by SARS-CoV-2, raising the possibility of selection for resistant variants. Within-host viral evolution was reported in treated immunocompromised individuals but whether this signifies a real risk of onward transmission is unclear. We used a regional SARS-CoV-2 sequencing program to monitor lineages with clinically relevant variants in identified patients, which facilitated analysis of parameters potentially relevant to new variant emergence. Here we describe a newly acquired spike E484K mutation detected within the B.1.311 lineage. Multiple individuals in 2 households of the same extended family were infected. The timing and patterns of spread were consistent with de novo emergence of this E484K variant in the bamlanivimab-treated index patient. Our study suggests that the selective pressures introduced by the widespread administration of these antibodies may warrant increased genomic surveillance to identify and mitigate spread of therapy-induced variants.
Asunto(s)
COVID-19 , SARS-CoV-2 , Anticuerpos Monoclonales Humanizados , Anticuerpos Neutralizantes , Humanos , Mutación , Pandemias , SARS-CoV-2/genética , Glicoproteína de la Espiga del CoronavirusRESUMEN
Amphiregulin is a transmembrane protein which, when cleaved by the TACE/ADAM17 protease, releases a soluble epidermal growth factor receptor ligand domain that promotes proliferation of normal and malignant cells. We previously described a rabbit monoclonal antibody, GMF-1A3, that selectively recognizes the cell-associated cleaved amphiregulin epitope. Antibody-drug conjugates had anti-tumor activity against human breast cancer xenografts. Several tumor types express amphiregulin, but evidence for a functional requirement for amphiregulin in these malignancies is limited. By directly evaluating amphiregulin cleavage with immunohistochemistry, GMF-1A3 provides a more direct measure of amphiregulin activity. Using 370 specimens from 10 tumor types (as well as normal controls), we demonstrate that cleaved amphiregulin is widely expressed in solid tumors and is especially common (> 50% of cases) in breast, prostate, liver and lung cancer. As a potential companion diagnostic for this antibody-drug conjugate, this assay allows identification of tumors with high levels of the cleaved amphiregulin target.
RESUMEN
BACKGROUND: While dialysis patients are at greater risk of serious SARS-CoV-2 complications, stringent infection prevention measures can help mitigate infection and transmission risks within dialysis facilities. We describe an outbreak of 14 cases diagnosed in a hospital-based outpatient ESRD facility over 13 days in the second quarter of 2021, and our coordinated use of epidemiology, viral genome sequencing, and infection control practices to quickly end the transmission cycle. METHODS: Symptomatic patients and staff members were diagnosed by RT-PCR. Facility-wide screening utilized SARS-CoV-2 antigen tests. SARS-CoV-2 genome sequences were obtained from residual diagnostic specimens. RESULTS: Of the 106 patients receiving dialysis in the facility, 10 were diagnosed with SARS-CoV-2 infection, as was 1 patient support person. Of 3 positive staff members, 2 were unvaccinated and had provided care for 6 and 4 of the affected patients, respectively. Sequencing demonstrated that all cases in the cluster shared an identical B.1.1.7./Alpha substrain. Attack rates were greatest among unvaccinated patients and staff. Vaccine effectiveness was 88% among patients. CONCLUSIONS: Prompt recognition of an infection cluster and rapid intervention efforts successfully ended the outbreak. Alongside consistent adherence to core infection prevention measures, vaccination was highly effective in reducing disease incidence and morbidity in this vulnerable population.