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BACKGROUND: Idiopathic nodular mesangial sclerosis, also called idiopathic nodular glomerulosclerosis (ING), is a rare clinical entity with an unclear pathogenesis. The hallmark of this disease is the presence of nodular mesangial sclerosis on histology without clinical evidence of diabetes mellitus or other predisposing diagnoses. To achieve insights into its pathogenesis, we queried the clinical, histopathologic and transcriptomic features of ING and nodular diabetic nephropathy (DN). METHODS: All renal biopsy reports accessioned at Indiana University Health from 2001 to 2016 were reviewed to identify 48 ING cases. Clinical and histopathologic features were compared between individuals with ING and DN (n = 751). Glomeruli of ING (n = 5), DN (n = 18) and reference (REF) nephrectomy (n = 9) samples were isolated by laser microdissection and RNA was sequenced. Immunohistochemistry of proline-rich 36 (PRR36) protein was performed. RESULTS: ING subjects were frequently hypertensive (95.8%) with a smoking history (66.7%). ING subjects were older, had lower proteinuria and had less hyaline arteriolosclerosis than DN subjects. Butanoate metabolism was an enriched pathway in ING samples compared with either REF or DN samples. The top differentially expressed gene, PRR36, had increased expression in glomeruli 248-fold [false discovery rate (FDR) P = 5.93 × 10-6] compared with the REF and increased 109-fold (FDR P = 1.85 × 10-6) compared with DN samples. Immunohistochemistry revealed a reduced proportion of cells with perinuclear reaction in ING samples as compared to DN. CONCLUSIONS: Despite similar clinical and histopathologic characteristics in ING and DN, the uncovered transcriptomic signature suggests that ING has distinct molecular features from nodular DN. Further study is warranted to understand these relationships.
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Diabetes Mellitus , Nefropatías Diabéticas , Síndrome Nefrótico , Diabetes Mellitus/patología , Nefropatías Diabéticas/genética , Nefropatías Diabéticas/patología , Humanos , Glomérulos Renales/patología , Síndrome Nefrótico/patología , Proteinuria/patología , Esclerosis/patologíaRESUMEN
Coronary transient receptor potential canonical (TRPC) channel expression is elevated in metabolic syndrome (MetS). However, differential contribution of TRPCs to coronary pathology in MetS is not fully elucidated. We investigated the roles of TRPC1 and TRPC6 isoforms in coronary arteries of MetS pigs and determined whether long-term treatment with a mineralocorticoid receptor inhibitor, spironolactone, attenuates coronary TRPC expression and associated dysfunctions. MetS coronary arteries exhibited significant atherosclerosis, endothelial dysfunction, and increased histamine-induced contractions. Immunohistochemical studies revealed that TRPC6 immunostaining was significantly greater in the medial layer of MetS pig coronary arteries compared to that in Lean pigs, whereas little TRPC6 immunostaining was found in atheromas. Conversely, TRPC1 immunostaining was weak in the medial layer but strong in MetS atheromas, where it was predominantly localized to macrophages. Spironolactone treatment significantly decreased coronary TRPC expression and dysfunctions in MetS pigs. In vivo targeted delivery of the dominant-negative (DN)-TRPC6 cDNA to the coronary wall reduced histamine-induced calcium transients in the MetS coronary artery medial layer, implying a role for TRPC6 in mediating calcium influx in MetS coronary smooth muscles. Monocyte adhesion was increased in Lean pig coronary arteries cultured in the presence of aldosterone; and spironolactone antagonized this effect, suggesting that coronary mineralocorticoid receptor activation may regulate macrophage infiltration. TRPC1 expression in atheroma macrophages was associated with advanced atherosclerosis, whereas medial TRPC6 upregulation correlated with increased histamine-induced calcium transients and coronary contractility. We propose that long-term spironolactone treatment may be a therapeutic strategy to decrease TRPC expression and coronary pathology associated with MetS.
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Enfermedad de la Arteria Coronaria/prevención & control , Vasos Coronarios/efectos de los fármacos , Síndrome Metabólico/tratamiento farmacológico , Antagonistas de Receptores de Mineralocorticoides/administración & dosificación , Espironolactona/administración & dosificación , Canales Catiónicos TRPC/efectos de los fármacos , Canal Catiónico TRPC6/efectos de los fármacos , Vasoconstricción/efectos de los fármacos , Animales , Señalización del Calcio/efectos de los fármacos , Células Cultivadas , Enfermedad de la Arteria Coronaria/genética , Enfermedad de la Arteria Coronaria/metabolismo , Enfermedad de la Arteria Coronaria/fisiopatología , Vasos Coronarios/metabolismo , Vasos Coronarios/fisiopatología , Modelos Animales de Enfermedad , Regulación hacia Abajo , Esquema de Medicación , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Endotelio Vascular/fisiopatología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Síndrome Metabólico/genética , Síndrome Metabólico/metabolismo , Síndrome Metabólico/fisiopatología , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiopatología , Porcinos , Porcinos Enanos , Canales Catiónicos TRPC/metabolismo , Canal Catiónico TRPC6/genética , Canal Catiónico TRPC6/metabolismo , Factores de Tiempo , Técnicas de Cultivo de TejidosRESUMEN
BACKGROUND: Tamoxifen (TAM) has been widely used for the treatment of estrogen receptor (ER)-positive breast cancer and its combination with other therapies is being actively investigated as a way to increase efficacy and decrease side effects. Here, we evaluate the therapeutic potential of co-treatment with TAM and BreastDefend (BD), a dietary supplement formula, in ER-positive human breast cancer. METHODS: Cell proliferation and apoptosis were determined in ER-positive human breast cancer cells MCF-7 by MTT assay, quantitation of cytoplasmic histone-associated DNA fragments and expression of cleaved PARP, respectively. The molecular mechanism was identified using RNA microarray analysis and western blotting. Tumor tissues from xenograft mouse model were analyzed by immunohistochemistry. RESULTS: Our data clearly demonstrate that a combination of 4-hydroxytamoxifen (4-OHT) with BD lead to profound inhibition of cell proliferation and induction of apoptosis in MCF-7 cells. This effect is consistent with the regulation of apoptotic and TAM resistant genes at the transcription and translation levels. Importantly, TAM and BD co-treatment significantly enhanced apoptosis, suppressed tumor growth and reduced tumor weight in a xenograft model of human ER-positive breast cancer. CONCLUSION: BD sensitized ER-positive human breast cancer cells to 4-OHT/TAM treatment in vitro and in vivo. BreastDefend can be used in an adjuvant therapy to increase the therapeutic effect of tamoxifen in patients with ER-positive breast cancer.
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Productos Biológicos/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Suplementos Dietéticos , Receptores de Estrógenos/metabolismo , Tamoxifeno/uso terapéutico , Adyuvantes Farmacéuticos/farmacología , Adyuvantes Farmacéuticos/uso terapéutico , Animales , Apoptosis , Productos Biológicos/farmacología , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Proliferación Celular , Resistencia a Antineoplásicos , Femenino , Hongos , Genes Relacionados con las Neoplasias , Humanos , Indoles/farmacología , Indoles/uso terapéutico , Células MCF-7 , Magnoliopsida , Ratones , Quercetina/farmacología , Quercetina/uso terapéutico , Tamoxifeno/farmacologíaRESUMEN
Airway epithelial CD55 down-regulation occurs in several hypoxia-associated pulmonary diseases, but the mechanism is unknown. Using in vivo and in vitro assays of pharmacologic inhibition and gene silencing, the current study investigated the role of hypoxia-inducible factor (HIF)-1α in regulating airway epithelial CD55 expression. Hypoxia down-regulated CD55 expression on small-airway epithelial cells in vitro, and in murine lungs in vivo; the latter was associated with local complement activation. Treatment with pharmacologic inhibition or silencing of HIF-1α during hypoxia-recovered CD55 expression in small-airway epithelial cells. HIF-1α overexpression or blockade, in vitro or in vivo, down-regulated CD55 expression. Collectively, these data show a key role for HIF-1α in regulating the expression of CD55 on airway epithelium.
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Antígenos CD55/metabolismo , Epitelio/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Pulmón/metabolismo , Aminoácidos Dicarboxílicos/farmacología , Animales , Hipoxia de la Célula/efectos de los fármacos , Activación de Complemento/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Epitelio/efectos de los fármacos , Silenciador del Gen/efectos de los fármacos , Masculino , Ratones Endogámicos C57BLRESUMEN
We describe a child with dyserythropoietic anemia, thrombocytosis, functional platelet defect, and megakaryocyte dysplasia. We show that (i) this constellation of hematopoietic abnormalities was due to a germline mutation within the 5' untranslated region (5'UTR) of globin transcription factor 1 (GATA1); (ii) the mutation impaired a 5'UTR GATA1 splicing site, with promoted production of the shortened GATA1 isoform lacking the N-terminus; and (iii) expression of the GATA1 N-terminus is restricted to erythroblasts and megakaryocytes in normal marrow, consistent with the patient's abnormal erythropoiesis and megakaryopoiesis. Our findings provide insights into the clinically relevant in vivo function of the N-terminal domain of GATA1 in human hematopoiesis.
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Regiones no Traducidas 5' , Anemia Diseritropoyética Congénita/genética , Factor de Transcripción GATA1/genética , Megacariocitos , Sitios de Empalme de ARN , Empalme Alternativo , Anemia Diseritropoyética Congénita/metabolismo , Preescolar , Factor de Transcripción GATA1/biosíntesis , Humanos , Masculino , Isoformas de Proteínas/biosíntesis , Isoformas de Proteínas/genéticaRESUMEN
PURPOSE: Plexiform neurofibromas (PNF) are benign peripheral nerve sheath tumors (PNST) associated with neurofibromatosis type 1 (NF1). Despite similar histologic appearance, these neoplasms exhibit diverse evolutionary trajectories, with a subset progressing to malignant peripheral nerve sheath tumor (MPNST), the leading cause of premature death in individuals with NF1. Malignant transformation of PNF often occurs through the development of atypical neurofibroma (ANF) precursor lesions characterized by distinct histopathologic features and CDKN2A copy-number loss. Although genomic studies have uncovered key driver events promoting tumor progression, the transcriptional changes preceding malignant transformation remain poorly defined. EXPERIMENTAL DESIGN: Here we resolve gene-expression profiles in PNST across the neurofibroma-to-MPNST continuum in NF1 patients and mouse models, revealing early molecular features associated with neurofibroma evolution and transformation. RESULTS: Our findings demonstrate that ANF exhibit enhanced signatures of antigen presentation and immune response, which are suppressed as malignant transformation ensues. MPNST further displayed deregulated survival and mitotic fidelity pathways, and targeting key mediators of these pathways, CENPF and BIRC5, disrupted the growth and viability of human MPNST cell lines and primary murine Nf1-Cdkn2a-mutant Schwann cell precursors. Finally, neurofibromas contiguous with MPNST manifested distinct alterations in core oncogenic and immune surveillance programs, suggesting that early molecular events driving disease progression may precede histopathologic evidence of malignancy. CONCLUSIONS: If validated prospectively in future studies, these signatures may serve as molecular diagnostic tools to augment conventional histopathologic diagnosis by identifying neurofibromas at high risk of undergoing malignant transformation, facilitating risk-adapted care.
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Neoplasias de la Vaina del Nervio , Neurofibroma , Neurofibromatosis 1 , Neurofibrosarcoma , Animales , Humanos , Ratones , Perfilación de la Expresión Génica , Neoplasias de la Vaina del Nervio/genética , Neurofibroma/genética , Neurofibromatosis 1/genética , Neurofibrosarcoma/genéticaRESUMEN
The biologic basis of genetic ancestry-dependent variability in disease incidence and outcome is just beginning to be explored. We recently reported enrichment of a population of ZEB1-expressing cells located adjacent to ductal epithelial cells in normal breasts of women of African ancestry compared to those of European ancestry. In this study, we demonstrate that these cells have properties of fibroadipogenic/mesenchymal stromal cells that express PROCR and PDGFRα and transdifferentiate into adipogenic and osteogenic lineages. PROCR + /ZEB1 + /PDGFRα+ (PZP) cells are enriched in normal breast tissues of women of African compared to European ancestry. PZP: epithelial cell communication results in luminal epithelial cells acquiring basal cell characteristics and IL-6-dependent increase in STAT3 phosphorylation. Furthermore, level of phospho-STAT3 is higher in normal and cancerous breast tissues of women of African ancestry. PZP cells transformed with HRasG12V ± SV40-T/t antigens generate metaplastic carcinoma suggesting that these cells are one of the cells-of-origin of metaplastic breast cancers.
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Neoplasias de la Mama , Humanos , Femenino , Incidencia , Neoplasias de la Mama/epidemiología , Neoplasias de la Mama/genética , Receptor de Proteína C Endotelial , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas , Células EpitelialesRESUMEN
Study of genomic aberrations leading to immortalization of epithelial cells has been technically challenging due to the lack of isogenic models. To address this, we used healthy primary breast luminal epithelial cells of different genetic ancestry and their hTERT-immortalized counterparts to identify transcriptomic changes associated with immortalization. Elevated expression of TONSL (Tonsoku-like, DNA repair protein) was identified as one of the earliest events during immortalization. TONSL, which is located on chromosome 8q24.3, was found to be amplified in approximately 20% of breast cancers. TONSL alone immortalized primary breast epithelial cells and increased telomerase activity, but overexpression was insufficient for neoplastic transformation. However, TONSL-immortalized primary cells overexpressing defined oncogenes generated estrogen receptor-positive adenocarcinomas in mice. Analysis of a breast tumor microarray with approximately 600 tumors revealed poor overall and progression-free survival of patients with TONSL-overexpressing tumors. TONSL increased chromatin accessibility to pro-oncogenic transcription factors, including NF-κB and limited access to the tumor-suppressor p53. TONSL overexpression resulted in significant changes in the expression of genes associated with DNA repair hubs, including upregulation of several genes in the homologous recombination (HR) and Fanconi anemia pathways. Consistent with these results, TONSL-overexpressing primary cells exhibited upregulated DNA repair via HR. Moreover, TONSL was essential for growth of TONSL-amplified breast cancer cell lines in vivo, and these cells were sensitive to TONSL-FACT complex inhibitor CBL0137. Together, these findings identify TONSL as a regulator of epithelial cell immortalization to facilitate cancer initiation and as a target for breast cancer therapy. SIGNIFICANCE: The chr.8q24.3 amplicon-resident gene TONSL is upregulated during the initial steps of tumorigenesis to support neoplastic transformation by increasing DNA repair and represents a potential therapeutic target for treating breast cancer.
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FN-kappa B , Oncogenes , Animales , Ratones , Carcinogénesis/genética , Transformación Celular Neoplásica/genética , FN-kappa B/genética , FN-kappa B/metabolismo , Oncogenes/genética , Factores de Transcripción/genéticaRESUMEN
Renal clear cell carcinoma commonly occurs in patients with von HippelLindau disease (VHL). Kidneys of VHL disease patients (VHL kidneys) contain an abundance of independent clear cell proliferation events that have been hypothesized to represent precursor structures of clear cell carcinoma. In the present study, it was tried to identify the site of origin of clear cell proliferation, and the immunophenotype of clear cells. Using 3D histological tracking, the topographic origin of microscopic clear cell proliferation was investigated by identification of informative structures of interest and immunohistochemical staining for cluster of differentiation 10 (CD10) and cytokeratin 7 (CK7) in consecutive serial sections. In addition, the CD10/CK7 immunophenotype of proliferating clear cells was evaluated. Clear cell proliferation uniformly occurred in the distal tubular system. Some clear cell proliferation, however, revealed proximal tubule immunophenotype. It was concluded that early proliferation of VHLdeficient clear cells occurs in the distal tubular system. Despite the association with the distal tubular system, the immunohistochemical profile of early clear cell proliferation may be inconsistent with its distal tubular origin.
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Carcinoma de Células Renales , Neoplasias Renales , Enfermedad de von Hippel-Lindau , Humanos , Neoplasias Renales/genética , Carcinoma de Células Renales/genética , Riñón/patología , Enfermedad de von Hippel-Lindau/complicaciones , Enfermedad de von Hippel-Lindau/genética , Enfermedad de von Hippel-Lindau/patología , Proliferación Celular , Queratina-7 , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genéticaRESUMEN
BACKGROUND: Genome-wide association studies have identified several breast cancer susceptibility loci. However, biomarkers for risk assessment are still missing. Here, we investigated cancer-related molecular changes detected in tissues from women at high risk for breast cancer prior to disease manifestation. Disease-free breast tissue cores donated by healthy women (N = 146, median age = 39 years) were processed for both methylome (MethylCap) and transcriptome (Illumina's HiSeq4000) sequencing. Analysis of tissue microarray and primary breast epithelial cells was used to confirm gene expression dysregulation. RESULTS: Transcriptomic analysis identified 69 differentially expressed genes between women at high and those at average risk of breast cancer (Tyrer-Cuzick model) at FDR < 0.05 and fold change ≥ 2. Majority of the identified genes were involved in DNA damage checkpoint, cell cycle, and cell adhesion. Two genes, FAM83A and NEK2, were overexpressed in tissue sections (FDR < 0.01) and primary epithelial cells (p < 0.05) from high-risk breasts. Moreover, 1698 DNA methylation changes were identified in high-risk breast tissues (FDR < 0.05), partially overlapped with cancer-related signatures, and correlated with transcriptional changes (p < 0.05, r ≤ 0.5). Finally, among the participants, 35 women donated breast biopsies at two time points, and age-related molecular alterations enhanced in high-risk subjects were identified. CONCLUSIONS: Normal breast tissue from women at high risk of breast cancer bears molecular aberrations that may contribute to breast cancer susceptibility. This study is the first molecular characterization of the true normal breast tissues, and provides an opportunity to investigate molecular markers of breast cancer risk, which may lead to new preventive approaches.
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Neoplasias de la Mama/diagnóstico , Epigénesis Genética/genética , Medición de Riesgo/métodos , Activación Transcripcional/genética , Adulto , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/fisiopatología , Estudios de Cohortes , Metilación de ADN/genética , Metilación de ADN/fisiología , Femenino , Estudio de Asociación del Genoma Completo/métodos , Estudio de Asociación del Genoma Completo/estadística & datos numéricos , Humanos , Persona de Mediana Edad , Medición de Riesgo/estadística & datos numéricos , Activación Transcripcional/fisiologíaRESUMEN
BACKGROUND: Family with sequence similarity 83 member A (FAM83A) presents oncogenic properties in several cancers including breast cancer. Recently, we reported FAM83A overexpression in normal breast tissues from women at high risk of breast cancer. We now hypothesize that FAM83A is a key factor in breast cancer initiation. METHODS: Immunohistochemical staining was used to evaluate FAM83A protein levels in both a normal breast tissue microarray (TMA, N = 411) and a breast tumor TMA (N = 349). EGFR staining and its correlation with FAM83A expression were also assessed. Lentivirus-mediated manipulation of FAM83A expression in primary and hTERT-immortalized breast epithelial cells was employed. Biological and molecular alterations upon FAM83A overexpression/downregulation and FAM83A's interaction partners were investigated. RESULTS: TMA analysis revealed a 1.5-fold increase in FAM83A expression level in breast cancer cases as compared with normal breast tissues (p < 0.0001). FAM83A protein expression was directly correlated with EGFR level in both normal and breast cancer tissues. In in vitro assays, exogenous expression of FAM83A in either primary or immortalized breast epithelial cells promoted cell viability and proliferation. Additionally, Ingenuity Pathway Analysis (IPA) revealed that FAM83A overexpression in primary cells affected the expression of genes involved in cellular morphology and metabolism. Mass spectrometry analysis identified DDX3X and LAMB3 as potential FAM83A interaction partners in primary cells, while we detected FAM83A interaction with cytoskeleton reorganization factors, including LIMA1, MYH10, PLEC, MYL6 in the immortalized cells. CONCLUSIONS: This study shows that FAM83A promotes metabolic activation in primary breast epithelial cells and cell proliferation in both primary and immortalized cells. These findings support its role in early breast oncogenesis.
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Preclinical studies of primary cancer cells are typically done after tumors are removed from patients or animals at ambient atmospheric oxygen (O2, ~21%). However, O2 concentrations in organs are in the ~3 to 10% range, with most tumors in a hypoxic or 1 to 2% O2 environment in vivo. Although effects of O2 tension on tumor cell characteristics in vitro have been studied, these studies are done only after tumors are first collected and processed in ambient air. Similarly, sensitivity of primary cancer cells to anticancer agents is routinely examined at ambient O2. Here, we demonstrate that tumors collected, processed, and propagated at physiologic O2 compared to ambient air display distinct differences in key signaling networks including LGR5/WNT, YAP, and NRF2/KEAP1, nuclear reactive oxygen species, alternative splicing, and sensitivity to targeted therapies. Therefore, evaluating cancer cells under physioxia could more closely recapitulate their physiopathologic status in the in vivo microenvironment.
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TP63, a member of the TP53 gene family, is a nuclear marker of myoepithelial cells. Antibody against p63 is frequently used to aid in the diagnosis of prostate carcinoma, as well as in the identification of myoepithelial cells in other tissues including the breast. p63 is also a marker for squamous cell carcinoma. Recently, it was found that all p53 family members are involved in regulating the process of muscle differentiation through the retinoblastoma (RB) protein. Ablation of these p53 family functions blocks the differentiation program and promotes malignant transformation by enabling cooperating oncogenes to transform myoblasts. We therefore studied p63 expression in a number of neoplasms with myogenic differentiation. Immunohistochemical staining for p63 was performed on paraffin sections from 38 rhabdomyosarcomas, five leiomyomas, five leiomyosarcomas, five rhabdomyomas, five rhabdomyomatous Wilms tumors, three normal cardiac muscles, one medullomyoblastoma, one pleuropulmonary blastoma with rhabdomyomatous differentiation, and one teratoma with prominent rhabdomyoblasts. Each case was also stained with desmin. Unlike the nuclear staining scored in myoepithelial cells, only cytoplasmic staining for p63 was considered positive. Of 38 cases of rhabdomyosarcoma, 36 showed cytoplasmic p63 staining; 24 of these showed highlighting of cross-striations superior to that of desmin. In addition, 5/5 rhabdomyomas, 5/5 rhabdomyomatous Wilms tumors, 1/1 pleuropulmonary blastoma with rhabdomyomatous differentiation, 1/1 teratoma with atypical rhabdoblasts, and 1/1 medullomyoblastoma exhibited cytoplasmic p63 staining. Normal cardiac muscle samples (3/3) also demonstrated positive cytoplasmic staining and distinct cross-striations. Smooth muscle tumors exhibited only very focal and faint cytoplasmic staining in 5/5 leiomyomas and 4/5 leiomyosarcomas. Immunoelectron microscopic study of skeletal muscle showed p63 localization to the Z bands of sarcomeres. We conclude that p63 immunostain is a sensitive marker for skeletal muscle differentiation and highlights the cross-striations of strap cells with exceptional definition.
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Biomarcadores de Tumor/análisis , Diferenciación Celular , Citoplasma/química , Citoplasma/patología , Inmunohistoquímica , Microscopía Inmunoelectrónica , Músculo Esquelético/química , Músculo Esquelético/patología , Neoplasias/química , Neoplasias/patología , Factores de Transcripción/análisis , Proteínas Supresoras de Tumor/análisis , Neoplasias Cerebelosas/química , Neoplasias Cerebelosas/patología , Citoplasma/ultraestructura , Humanos , Neoplasias Renales/química , Neoplasias Renales/patología , Leiomioma/química , Leiomioma/patología , Leiomiosarcoma/química , Leiomiosarcoma/patología , Meduloblastoma/química , Meduloblastoma/patología , Músculo Esquelético/ultraestructura , Músculo Liso/química , Músculo Liso/patología , Miocardio/química , Miocardio/patología , Neoplasias/ultraestructura , Blastoma Pulmonar/química , Blastoma Pulmonar/patología , Rabdomiosarcoma/química , Rabdomiosarcoma/patología , Teratoma/química , Teratoma/patología , Tumor de Wilms/química , Tumor de Wilms/patologíaRESUMEN
Breast cancers are classified into five intrinsic subtypes and 10 integrative clusters based on gene expression patterns and genomic aberrations, respectively. Although the cell-of-origin, adaptive plasticity, and genomic aberrations shape dynamic transcriptomic landscape during cancer progression, how interplay between these three core elements governs obligatory steps for a productive cancer progression is unknown. Here, we used genetic ancestry-mapped immortalized breast epithelial cell lines generated from breast biopsies of healthy women that share gene expression profiles of luminal A, normal-like, and basal-like intrinsic subtypes of breast cancers and breast cancer relevant oncogenes to develop breast cancer progression model. Using flow cytometry, mammosphere growth, signaling pathway, DNA damage response, and in vivo tumorigenicity assays, we provide evidence that establishes cell context-dependent effects of oncogenes in conferring plasticity, self-renewal/differentiation, intratumor heterogeneity, and metastatic properties. In contrast, oncogenic aberrations, independent of cell context, shaped response to DNA damage-inducing agents. Collectively, this study reveals how the same set of genomic aberration can have distinct effects on tumor characteristics based on cell-of-origin of tumor and highlights the need to utilize multiple "normal" epithelial cell types to decipher oncogenic properties of a gene of interest. In addition, by creating multiple isogenic cell lines ranging from primary cells to metastatic variants, we provide resources to elucidate cell-intrinsic properties and cell-oncogene interactions at various stages of cancer progression. IMPLICATIONS: Our findings demonstrate that how an interplay between the normal cell type that encountered genomic aberrations and type of genomic aberration influences heterogeneity, self-renewal/differentiation, and tumor properties including propensity for metastasis.
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Neoplasias de la Mama/genética , Transformación Celular Neoplásica/genética , Genómica/métodos , Animales , Carcinogénesis , Diferenciación Celular , Femenino , Humanos , Ratones Endogámicos NODRESUMEN
OBJECTIVE: To evaluate the effects of surgical weight loss on hepatic lipid peroxidation levels and cytochrome P-450 protein expression in patients with nonalcoholic fatty liver disease (NAFLD). SUMMARY BACKGROUND DATA: NAFLD and nonalcoholic steatohepatitis (NASH) affect hepatic cytochrome P-450 (CYP) protein expression and activity, and CYP2E1 may play a role in the pathogenesis of NAFLD and NASH through induction of oxidative stress and lipid peroxidation. NAFLD and NASH are associated with increased systemic lipid peroxidation levels and elevated hepatic CYP2E1 activity, but hepatic CYP3A4/5 activity is decreased. METHODS: Liver biopsies from 20 patients with NAFLD who underwent bariatric surgery were obtained intraoperatively and at 15 +/- 7 months following surgery. Hepatic malondialdehyde (MDA) levels (a marker of lipid peroxidation), CYP2E1 and CYP3A4/5 protein expression, and steatosis, as a percent of total area, were measured by immunohistochemistry followed by digital image quantitation. RESULTS: Following weight loss, as reflected by reduced BMI (54 +/- 9 vs. 37 +/- 9 kg/m2; P < 0.001), features of the metabolic syndrome, grade and stage of liver disease, and liver histology were all significantly improved (P < 0.01). Hepatic MDA staining (35 +/- 18% vs. 23 +/- 14%; P = 0.02), CYP2E1 protein content (68 +/- 9% vs. 56 +/- 11%; P < 0.001), and steatosis (17 +/- 7% vs. 2 +/- 3%; P < 0.001) were significantly reduced following weight loss. CYP3A4/5 protein content was unchanged (57 +/- 13% vs. 55 +/- 13%; P = 0.433). The reduction in lipid peroxidation was independently associated with changes in CYP2E1 protein expression after bariatric surgery (r = 0.477; P = 0.033). CONCLUSION: Elevations in hepatic lipid peroxidation and CYP2E1 expression that are seen in NAFLD improve significantly with weight loss induced by bariatric surgery.
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Cirugía Bariátrica , Citocromo P-450 CYP2E1/metabolismo , Citocromo P-450 CYP3A/metabolismo , Hígado Graso/metabolismo , Peroxidación de Lípido , Hígado/metabolismo , Pérdida de Peso , Adulto , Hígado Graso/patología , Femenino , Humanos , Hígado/patología , MasculinoRESUMEN
Although the roles of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and hepatocyte growth factor (HGF) in pathologic neovascularization have been well characterized in certain tissues, their particular functions and expression patterns in choroidal neovascularization (CNV) have not been clearly established. After localized laser trauma to Bruch's membrane to induce CNV development, the temporal changes in mRNA and protein expression of these 3 cytokines were documented and compared histologically to areas of immunofluorescence, the proliferation of endothelial cells, neovascular development, and temporal changes in vascular permeability. Changes in mRNA and protein levels of bFGF and HGF occurred quickly and reached peak expression within hours. This activity corresponded in time to intense and localized immunofluorescence for these cytokines within the choriocapillaris within laser lesion sites. During this same initial time period, mRNA upregulation of VEGF occurred, primarily within the neural retina and this expression corresponded to intense immunolabeling of Müller cells immediately adjacent to the lesion sites. By 3 days after lasering, increased VEGF(164) protein expression was measurable, whereas early neovascular development histologically corresponded to HGF and bFGF mRNA expansion into the developing choroidal neovascular membrane (CNVM). At 7 days, CNV expansion, maturation, and increased vascular permeability corresponded to peak VEGF mRNA and protein expression and to immunofluorescence of the CNVM. Differences also occurred in the expression of precursor and activated isoforms of these cytokines in the retinal pigment epithelium/choroid as compared to those in the retina. These molecular and immunocytochemical results suggest that bFGF and HGF may be important as initial regulators neovascularization in this CNV model; whereas VEGF may be important during later phases of angiogenesis and neovascular hyperpermeability.
Asunto(s)
Neovascularización Coroidal/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Animales , Western Blotting/métodos , Permeabilidad Capilar , Neovascularización Coroidal/etiología , Neovascularización Coroidal/patología , Neovascularización Coroidal/fisiopatología , Progresión de la Enfermedad , Factor 2 de Crecimiento de Fibroblastos/genética , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Expresión Génica , Factor de Crecimiento de Hepatocito/genética , Factor de Crecimiento de Hepatocito/metabolismo , Péptidos y Proteínas de Señalización Intercelular/genética , Coagulación con Láser , Masculino , Microscopía Fluorescente/métodos , ARN Mensajero/genética , Ratas , Ratas Endogámicas BN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Regulación hacia Arriba , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Recombinant adeno-associated virus (rAAV)-mediated gene delivery shows promise to transduce the pancreas, but safety/efficacy in a neoplastic context is not well established. To identify an ideal AAV serotype, route, and vector dose and assess safety, we have investigated the use of three AAV serotypes (6, 8, and 9) expressing GFP in a self-complementary (sc) AAV vector under an EF1α promoter (scAAV.GFP) following systemic or retrograde pancreatic intraductal delivery. Systemic delivery of scAAV9.GFP transduced the pancreas with high efficiency, but gene expression did not exceed >45% with the highest dose, 5 × 1012 viral genomes (vg). Intraductal delivery of 1 × 1011 vg scAAV6.GFP transduced acini, ductal cells, and islet cells with >50%, â¼48%, and >80% efficiency, respectively, and >80% pancreatic transduction was achieved with 5 × 1011 vg. In a KrasG12D-driven pancreatic cancer mouse model, intraductal delivery of scAAV6.GFP targeted acini, epithelial, and stromal cells and exhibited persistent gene expression 5 months post-delivery. In normal mice, intraductal delivery induced a transient increase in serum amylase/lipase that resolved within a day of infusion with no sustained pancreatic inflammation or fibrosis. Similarly, in PDAC mice, intraductal delivery did not increase pancreatic intraepithelial neoplasia progression/fibrosis. Our study demonstrates that scAAV6 targets the pancreas/neoplasm efficiently and safely via retrograde pancreatic intraductal delivery.
RESUMEN
OBJECTIVES: We investigated whether a single episode of exercise could acutely increase the numbers of endothelial progenitor cells (EPCs) and cultured/circulating angiogenic cells (CACs) in human subjects. BACKGROUND: Endothelial progenitor cells and CACs can be isolated from peripheral blood and have been shown to participate in vascular repair and angiogenesis. We hypothesized that exercise may acutely increase either circulating EPCs or CACs. METHODS: Volunteer subjects (n = 22) underwent exhaustive dynamic exercise. Blood was drawn before and after exercise, and circulating EPC numbers as well as plasma levels of angiogenic growth factors were assessed. The CACs were obtained by culturing mononuclear cells and the secretion of multiple angiogenic growth factors by CACs was determined. RESULTS: Circulating EPCs (AC133+/VE-Cadherin+ cells) increased nearly four-fold in peripheral blood from 66 +/- 27 cells/ml to 236 +/- 34 cells/ml (p < 0.05). The number of isolated CACs increased 2.5-fold from 8,754 +/- 2,048 cells/ml of peripheral blood to 20,759 +/- 4,676 cells/ml (p < 0.005). Cultured angiogenic cells isolated before and after exercise showed similar secretion patterns of angiogenic growth factors. CONCLUSIONS: Our study demonstrates that exercise can acutely increase EPCs and CACs. Given the ability of these cell populations to promote angiogenesis and vascular regeneration, the exercise-induced cell mobilization may serve as a physiologic repair or compensation mechanism.
Asunto(s)
Circulación Colateral/fisiología , Endotelio Vascular/citología , Ejercicio Físico/fisiología , Células Madre Hematopoyéticas/metabolismo , Macrófagos/metabolismo , Miocitos Cardíacos/metabolismo , Neovascularización Fisiológica , Adulto , Biomarcadores/sangre , Femenino , Factor Estimulante de Colonias de Granulocitos/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Factor de Crecimiento de Hepatocito/metabolismo , Humanos , Leucocitos Mononucleares/metabolismo , Masculino , Persona de Mediana Edad , Factores de Tiempo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Y-box binding protein 1 [YBX1] is a multifunctional protein known to facilitate many of the hallmarks of cancer. Elevated levels of YBX1 protein are highly correlated with cancer progression, making it an excellent marker in cancer. The connection between YBX1 and the important nuclear factor κB [NF-κB] has never been reported. Here, we show that overexpression of wild type YBX1 [WT-YBX1] activates NF-κB, suggesting that YBX1 is a potential NF-κB activator. Furthermore, using mass spectrometry analysis we identified novel phosphorylation of serine 165 [S165] on YBX1. Overexpression of the S165A-YBX1 mutant in either HEK293 cells or colon cancer HT29 cells showed dramatically reduced NF-κB activating ability as compared with that of WT-YBX1, confirming that S165 phosphorylation is critical for the activation of NF-κB by YBX1. We also show that expression of the S165A-YBX1 mutant dramatically decreased the expression of NF-κB-inducible genes, reduced cell growth, and compromised tumorigenic ability as compared with WT-YBX1. Taken together, we provide the first evidence that YBX1 functions as a tumor promoter via NF-κB activation, and phosphorylation of S165 of YBX1 is critical for this function. Therefore, our important discovery may lead to blocking S165 phosphorylation as a potential therapeutic strategy to treat colon cancer.
Asunto(s)
Neoplasias del Colon/metabolismo , Factor de Transcripción ReIA/metabolismo , Proteína 1 de Unión a la Caja Y/metabolismo , Proliferación Celular , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Células HEK293 , Células HT29 , Humanos , Proteínas I-kappa B/metabolismo , Mutación , Inhibidor NF-kappaB alfa , Fosforilación , Serina , Transducción de Señal , Espectrometría de Masas en Tándem , Factores de Tiempo , Factor de Transcripción ReIA/genética , Transfección , Proteína 1 de Unión a la Caja Y/genéticaRESUMEN
Decreased renal blood flow following an ischemic insult contributes to a reduction in glomerular filtration. However, little is known about the underlying cellular or subcellular mechanisms mediating reduced renal blood flow in human ischemic acute kidney injury (AKI) or acute renal failure (ARF). To examine renal vascular injury following ischemia, intraoperative graft biopsies were performed after reperfusion in 21 cadaveric renal allografts. Confocal fluorescence microscopy was utilized to examine vascular smooth muscle and endothelial cell integrity as well as peritubular interstitial pericytes in the biopsies. The reperfused, transplanted kidneys exhibited postischemic injury to the renal vasculature, as demonstrated by disorganization/disarray of the actin cytoskeleton in vascular smooth muscle cells and disappearance of von Willebrand factor from vascular endothelial cells. Damage to peritubular capillary endothelial cells was more severe in subjects destined to have sustained ARF than in those with rapid recovery of their graft function. In addition, peritubular pericytes/myofibroblasts were more pronounced in recipients destined to recover than those with sustained ARF. Taken together, these data suggest damage to the renal vasculature occurs after ischemia-reperfusion in human kidneys. Preservation of peritubular capillary endothelial integrity and increasing pericytes may be critical to recovery from postischemic AKI.