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1.
FASEB J ; 37(9): e23108, 2023 09.
Artículo en Inglés | MEDLINE | ID: mdl-37534940

RESUMEN

Recent advances in gene therapy have brought novel treatment options for cancer. However, the full potential of this approach has yet to be unlocked due to the limited payload capacity of commonly utilized viral vectors. Virus-free DNA transposons, including piggyBac, have the potential to obviate these shortcomings. In this study, we improved a previously modified piggyBac system with superior transposition efficiency. We demonstrated that the internal domain sequences (IDS) within the 3' terminal repeat domain of hyperactive piggyBac (hyPB) donor vector contain dominant enhancer elements. Plasmid-free donor vector devoid of IDS was used in conjunction with a helper plasmid expressing Quantum PBase™ v2 to generate an optimal piggyBac system, Quantum pBac™ (qPB), for use in T cells. qPB outperformed hyPB in CD20/CD19 CAR-T production in terms of performance as well as yield of the CAR-T cells produced. Furthermore, qPB also produced CAR-T cells with lower donor-associated variabilities compared to lentiviral vector. Importantly, qPB yielded mainly CD8+ CAR-TSCM cells, and the qPB-produced CAR-T cells effectively eliminated CD20/CD19-expressing tumor cells both in vitro and in vivo. Our findings confirm qPB as a promising virus-free vector system with an enhanced payload capacity to incorporate multiple genes. This highly efficient and potentially safe system will be expected to further advance gene therapy applications.


Asunto(s)
Receptores Quiméricos de Antígenos , Elementos Transponibles de ADN , Plásmidos , Linfocitos T , Vectores Genéticos/genética , Terapia Genética
2.
J Biomed Sci ; 31(1): 72, 2024 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-39010070

RESUMEN

BACKGROUND: Epithelial cell adhesion molecule (EpCAM) has been widely studied as a tumor antigen due to its expression in varieties of solid tumors. Moreover, the glycoprotein contributes to critical cancer-associated cellular functionalities via its extracellular (EpEX) and intracellular (EpICD) domains. In colorectal cancer (CRC), EpCAM has been implicated in the Wnt signaling pathway, as EpICD and ß-Catenin are coordinately translocated to the nucleus. Once in the nucleus, EpICD transcriptionally regulates EpCAM target genes that; however, remains unclear whether Wnt signaling is modulated by EpICD activity. METHODS: Patient-derived organoids (PDOs), patient-derived xenografts (PDXs), and various CRC cell lines were used to study the roles of EpCAM and EpICD in Wnt receptor expression. Fluorescence and confocal microscopy were used to analyze tumors isolated from PDX and other xenograft models as well as CRC cell lines. EpCAM signaling was intervened with our humanized form of EpCAM neutralizing antibody, hEpAb2-6. Wnt receptor promoters under luciferase reporters were constructed to examine the effects of EpICD. Luciferase reporter assays were performed to evaluate promoter, γ-secretase and Wnt activity. Functional assays including in vivo tumor formation, organoid formation, spheroid and colony formation experiments were performed to study Wnt related phenomena. The therapeutic potential of EpCAM suppression by hEpAb2-6 was evaluated in xenograft and orthotopic models of human CRC. RESULTS: EpICD interacted with the promoters of Wnt receptors (FZD6 and LRP5/6) thus upregulated their transcriptional activity inducing Wnt signaling. Furthermore, activation of Wnt-pathway-associated kinases in the ß-Catenin destruction complex (GSK3ß and CK1) induced γ-secretase activity to augment EpICD shedding, establishing a positive-feedback loop. Our hEpAb2-6 antibody blocked EpICD-mediated upregulation of Wnt receptor expressions and conferred therapeutic benefits in both PDX and orthotopic models of human CRC. CONCLUSIONS: This study uncovers relevant functions of EpCAM where Wnt receptors are upregulated via the transcriptional co-factor activity of EpICD. The resultant enhancement of Wnt signaling induces γ-secretase activity further stimulating EpICD cleavage and its nuclear translocation. Our humanized anti-EpCAM antibody hEpAb2-6 blocks these mechanisms and may thereby provide therapeutic benefit in CRC.


Asunto(s)
Neoplasias Colorrectales , Molécula de Adhesión Celular Epitelial , Vía de Señalización Wnt , Humanos , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/genética , Molécula de Adhesión Celular Epitelial/metabolismo , Molécula de Adhesión Celular Epitelial/genética , Ratones , Animales , Línea Celular Tumoral , Progresión de la Enfermedad
3.
Proc Natl Acad Sci U S A ; 118(50)2021 12 14.
Artículo en Inglés | MEDLINE | ID: mdl-34876527

RESUMEN

Pancreatic cancer is usually asymptomatic in the early stages; the 5-y survival rate is around 9%; and there is a lack of effective treatment. Here we show that SSEA-4 is more expressed in all pancreatic cancer cell lines examined but not detectable in normal pancreatic cells; and high expression of SSEA-4 or the key enzymes B3GALT5 + ST3GAL2 associated with SSEA-4 biosynthesis significantly lowers the overall survival rate. To evaluate potential new treatments for pancreatic cancer, homogeneous antibodies with a well-defined Fc glycan for optimal effector functions and CAR-T cells with scFv construct designed to target SSEA-4 were shown highly effective against pancreatic cancer in vitro and in vivo. This was further supported by the finding that a subpopulation of natural killer (NK) cells isolated by the homogeneous antibody exhibited enhancement in cancer-cell killing activity compared to the unseparated NK cells. These results indicate that targeting SSEA-4 by homologous antibodies or CAR-T strategies can effectively inhibit cancer growth, suggesting SSEA-4 as a potential immunotherapy target for treating pancreatic disease.


Asunto(s)
Anticuerpos/inmunología , Neoplasias Pancreáticas/tratamiento farmacológico , Antígenos Embrionarios Específico de Estadio/inmunología , Animales , Línea Celular Tumoral , Tratamiento Basado en Trasplante de Células y Tejidos , Regulación de la Expresión Génica , Humanos , Inmunoterapia , Inmunoterapia Adoptiva , Ratones , Ratones Desnudos , Ensayos Antitumor por Modelo de Xenoinjerto
4.
J Biomed Sci ; 27(1): 92, 2020 Sep 04.
Artículo en Inglés | MEDLINE | ID: mdl-32887585

RESUMEN

BACKGROUND: The Taiwan Human Disease iPSC Service Consortium was established to accelerate Taiwan's growing stem cell research initiatives and provide a platform for researchers interested in utilizing induced pluripotent stem cell (iPSC) technology. The consortium has generated and characterized 83 iPSC lines: 11 normal and 72 disease iPSC lines covering 21 different diseases, several of which are of high incidence in Taiwan. Whether there are any reprogramming-induced recurrent copy number variant (CNV) hotspots in iPSCs is still largely unknown. METHODS: We performed genome-wide copy number variant screening of 83 Han Taiwanese iPSC lines and compared them with 1093 control subjects using an Affymetrix genome-wide human SNP array. RESULTS: In the iPSCs, we identified ten specific CNV loci and seven "polymorphic" CNV regions that are associated with the reprogramming process. Additionally, we established several differentiation protocols for our iPSC lines. We demonstrated that our iPSC-derived cardiomyocytes respond to pharmacological agents and were successfully engrafted into the mouse myocardium demonstrating their potential application in cell therapy. CONCLUSIONS: The CNV hotspots induced by cell reprogramming have successfully been identified in the current study. This finding may be used as a reference index for evaluating iPSC quality for future clinical applications. Our aim was to establish a national iPSC resource center generating iPSCs, made available to researchers, to benefit the stem cell community in Taiwan and throughout the world.


Asunto(s)
Diferenciación Celular , Variaciones en el Número de Copia de ADN , Células Madre Pluripotentes Inducidas/metabolismo , Miocitos Cardíacos/fisiología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Reprogramación Celular , Niño , Preescolar , Femenino , Humanos , Masculino , Persona de Mediana Edad , Taiwán , Adulto Joven
5.
Nucleic Acids Res ; 46(7): 3671-3691, 2018 04 20.
Artículo en Inglés | MEDLINE | ID: mdl-29385530

RESUMEN

Transcriptionally non-co-linear (NCL) transcripts can originate from trans-splicing (trans-spliced RNA; 'tsRNA') or cis-backsplicing (circular RNA; 'circRNA'). While numerous circRNAs have been detected in various species, tsRNAs remain largely uninvestigated. Here, we utilize integrative transcriptome sequencing of poly(A)- and non-poly(A)-selected RNA-seq data from diverse human cell lines to distinguish between tsRNAs and circRNAs. We identified 24,498 NCL events and found that a considerable proportion (20-35%) of them arise from both tsRNAs and circRNAs, representing extensive alternative trans-splicing and cis-backsplicing in human cells. We show that sequence generalities of exon circularization are also observed in tsRNAs. Recapitulation of NCL RNAs further shows that inverted Alu repeats can simultaneously promote the formation of tsRNAs and circRNAs. However, tsRNAs and circRNAs exhibit quite different, or even opposite, expression patterns, in terms of correlation with the expression of their co-linear counterparts, expression breadth/abundance, transcript stability, and subcellular localization preference. These results indicate that tsRNAs and circRNAs may play different regulatory roles and analysis of NCL events should take the joint effects of different NCL-splicing types and joint effects of multiple NCL events into consideration. This study describes the first transcriptome-wide analysis of trans-splicing and cis-backsplicing, expanding our understanding of the complexity of the human transcriptome.


Asunto(s)
Empalme Alternativo/genética , ARN/genética , Trans-Empalme/genética , Transcriptoma/genética , Exones/genética , Perfilación de la Expresión Génica , Humanos , Empalme del ARN/genética , ARN Circular
6.
Exp Cell Res ; 363(2): 299-309, 2018 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-29366807

RESUMEN

Leber's hereditary optic neuropathy (LHON) is the maternally inherited mitochondrial disease caused by homoplasmic mutations in mitochondrial electron transport chain Complex I subunit genes. The mechanism of its incomplete penetrance is still largely unclear. In this study, we created the patient-specific human induced pluripotent stem cells (hiPSCs) from MT-ND4 mutated LHON-affected patient, asymptomatic mutation carrier and healthy control, and differentiated them into retinal ganglion cells (RGCs). We found the defective neurite outgrowth in affected RGCs, but not in the carrier RGCs which had significant expression of SNCG gene. We observed enhanced mitochondrial biogenesis in affected and carrier derived RGCs. Surprisingly, we observed increased NADH dehydrogenase enzymatic activity of Complex I in hiPSC-derived RGCs of asymptomatic carrier, but not of the affected patient. LHON mutation substantially decreased basal respiration in both affected and unaffected carrier hiPSCs, and had the same effect on spare respiratory capacity, which ensures normal function of mitochondria in conditions of increased energy demand or environmental stress. The expression of antioxidant enzyme catalase was decreased in affected and carrier patient hiPSC-derived RGCs as compared to the healthy control, which might indicate to higher oxidative stress-enriched environment in the LHON-specific RGCs. Microarray profiling demonstrated enhanced expression of cell cycle machinery and downregulation of neuronal specific genes.


Asunto(s)
ADN Mitocondrial/genética , Genes Mitocondriales/genética , Células Madre Pluripotentes Inducidas/metabolismo , Atrofia Óptica Hereditaria de Leber/genética , Diferenciación Celular/fisiología , Humanos , Mitocondrias/genética , Mitocondrias/metabolismo , Biogénesis de Organelos , Estrés Oxidativo/efectos de los fármacos , Células Ganglionares de la Retina/metabolismo
7.
Proc Natl Acad Sci U S A ; 113(4): 960-5, 2016 Jan 26.
Artículo en Inglés | MEDLINE | ID: mdl-26677875

RESUMEN

The discovery of cancer stem cells (CSCs), which are responsible for self-renewal and tumor growth in heterogeneous cancer tissues, has stimulated interests in developing new cancer therapies and early diagnosis. However, the markers currently used for isolation of CSCs are often not selective enough to enrich CSCs for the study of this special cell population. Here we show that the breast CSCs isolated with CD44(+)CD24(-/lo)SSEA-3(+) or ESA(hi)PROCR(hi)SSEA-3(+) markers had higher tumorigenicity than those with conventional markers in vitro and in vivo. As few as 10 cells with CD44(+)CD24(-/lo)SSEA-3(+) formed tumor in mice, compared with more than 100 cells with CD44(+)CD24(-/lo). Suppression of SSEA-3 expression by knockdown of the gene encoding ß-1,3-galactosyltransferase 5 (ß3GalT5) in the globo-series pathway, led to apoptosis in cancer cells specifically but had no effect on normal cells. This finding is further supported by the analysis of SSEA-3 and the two related globo-series epitopes SSEA4 and globo-H in stem cells (embryonic stem cells and induced pluripotent stem cells) and various normal and cancer cells, and by the antibody approach to target the globo-series glycans and the late-stage clinical trials of a breast cancer vaccine.


Asunto(s)
Antígenos de Carbohidratos Asociados a Tumores/análisis , Biomarcadores de Tumor/análisis , Neoplasias de la Mama/patología , Galactosiltransferasas/análisis , Células Madre Neoplásicas/química , Antígenos Embrionarios Específico de Estadio/análisis , Animales , Apoptosis , Secuencia de Bases , Línea Celular Tumoral , Femenino , Humanos , Ratones , Datos de Secuencia Molecular
8.
Diabetologia ; 61(1): 158-167, 2018 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-28864913

RESUMEN

AIMS/HYPOTHESIS: It has been proposed that the neuro-insular network enables rapid, synchronised insulin secretion. However, to date, acquiring the pancreatic tissue map to study the neural network remains a challenging task as there is a lack of feasible approaches for large-scale tissue analysis at the organ level. Here, we have developed 3-dimensional (3D) panoramic histology to characterise the pancreatic neuro-insular network in young mice. METHODS: Pancreases harvested from young wild-type B6 mice (3 and 8 weeks old) and db/db mice (3 weeks old; db/db vs db/+) were used to develop 3D panoramic histology. Transparent pancreases were prepared by optical clearing to enable deep-tissue, tile-scanning microscopy for qualitative and quantitative analyses of islets and the pancreatic tissue network in space. RESULTS: 3D panoramic histology reveals the pancreatic neurovascular network and the coupling of ganglionic and islet populations via the network. This integration is identified in both 3- and 8-week-old mice, featuring the peri-arteriolar neuro-insular network and islet-ganglionic aggregation. In weaning hyperphagic db/db mice, the 3D image data identifies the associated increases in weight, adipose tissue attached to the pancreas, density of large islets (major axis > 150 µm) and pancreatic sympathetic innervation compared with db/+ mice. CONCLUSIONS/INTERPRETATION: Our work provides insight into the neuro-insular integration at the organ level and demonstrates a new approach for investigating previously unknown details of the pancreatic tissue network in health and disease.


Asunto(s)
Insulina/metabolismo , Páncreas/metabolismo , Animales , Peso Corporal/fisiología , Ganglión/metabolismo , Ratones , Red Nerviosa/metabolismo , Obesidad/metabolismo
9.
Diabetologia ; 61(1): 168-181, 2018 01.
Artículo en Inglés | MEDLINE | ID: mdl-28852792

RESUMEN

AIMS/HYPOTHESIS: Identification of a pancreatic neuro-insular network in mice suggests that a similar integration of islets and nerves may be present in the human pancreas. To characterise the neuro-insular network and the intra-pancreatic ganglia in a clinically related setting, we examined human pancreases in health and with fatty infiltration via 3-dimensional (3D) histology and compared the human pancreatic microenvironment with its counterpart in mice. METHODS: Human pancreatic specimens from individuals with normal BMI, high BMI (≥ 25) and type 2 diabetes were used to investigate the neuro-insular network. Transparent specimens were prepared by tissue clearing for transmitted light and deep-tissue fluorescence imaging to simultaneously visualise infiltrated adipocytes, islets and neurovascular networks. RESULTS: High-definition images of human islets reveal that both the sympathetic and parasympathetic nerves enter the islet core and reside in the immediate microenvironment of islet cells. Around the islets, the neuro-insular network is visualised with 3D histology to identify the intra-pancreatic ganglia (peri-lobular and intra-parenchymal ganglia) and the islet-ganglionic association. In humans, but not in mice, pancreatic fatty infiltration (BMI dependent) features adipocytes infiltrating into the parenchyma and accumulating in the peri-lobular space, in which the peri-lobular ganglia also reside. We identified the formation of adipose-ganglionic complexes in the peri-lobular space and enlargement of ganglia around adipocytes. In the specimen from the individual with type 2 diabetes, an increase in the number of nerve projections from the intra-parenchymal ganglia is associated with severe fatty infiltration. CONCLUSIONS/INTERPRETATION: We present new perspectives of human pancreas and islet innervation via 3D histology. Our results strongly suggest that fatty infiltration in the human pancreas creates a neurotrophic microenvironment and promotes remodelling of pancreatic innervation.


Asunto(s)
Páncreas/metabolismo , Adipocitos/metabolismo , Animales , Índice de Masa Corporal , Diabetes Mellitus Tipo 2/metabolismo , Humanos , Islotes Pancreáticos/metabolismo , Ratones , Obesidad/metabolismo , Sistema Nervioso Simpático/metabolismo
10.
Am J Physiol Gastrointest Liver Physiol ; 311(3): G412-22, 2016 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-27340125

RESUMEN

Pericytes and glial cells are accessory cells of neurovascular networks, which have been reported to participate in scar formation after tissue injury. However, it remains unclear whether similar reactive cellular responses occur in pancreatic intraepithelial neoplasia (PanIN). In this study we developed three-dimensional (3D) duct lesion histology to investigate PanIN and the associated pericyte, glial, and islet remodeling. Transparent mouse pancreata with a Kras(G12D) mutation were used to develop 3D duct lesion histology. Deep-tissue, tile-scanning microscopy was performed to generate panoramic views of the diseased pancreas for global examination of early stage and advanced duct lesion formation. Fluorescence signals of ductal and neurovascular networks were simultaneously detected to reveal associated remodeling. Significantly, in Kras(G12D)-mutant mice, when the low-grade PanINs emerge, duct lesions appear as epithelial buds with perilesional pericyte and glial activation. When PanINs occur in large scale (induced by cerulein injections to the mutant mice), the 3D image data identifies 1) aggregation of PanINs in clusters in space; 2) overexpression of the pericyte marker NG2 in the PanIN microenvironment; and 3) epithelial in-growth to islets, forming the PanIN-islet complexes. Particularly, the PanIN-islet complexes associate with proliferating epithelial and stromal cells and receive substantial neurovascular supplies, making them landmarks in the atrophic lobe. Overall, perilesional pericyte and glial activation and formation of the PanIN-islet complex underline cellular heterogeneity in the duct lesion microenvironment. The results also illustrate the advantage of using 3D histology to reveal previously unknown details of neurovascular and endocrine links to the disease.


Asunto(s)
Carcinoma in Situ/patología , Carcinoma Ductal Pancreático/patología , Islotes Pancreáticos/fisiología , Neuroglía/fisiología , Páncreas/patología , Pericitos/fisiología , Animales , Regulación Neoplásica de la Expresión Génica , Ratones , Ratones Transgénicos
11.
Biochem Biophys Res Commun ; 477(4): 874-880, 2016 09 02.
Artículo en Inglés | MEDLINE | ID: mdl-27381866

RESUMEN

Patients with type 1 diabetes mellitus are associated with impairment in vitamin A metabolism. This study evaluated whether treatment with retinoic acid, the biologically active metabolite of vitamin A, can ameliorate diabetes. All-trans retinoic acid (atRA) was used to treat streptozotocin (STZ)-induced diabetic mice which revealed atRA administration ameliorated blood glucose levels of diabetic mice. This hyperglycemic amelioration was accompanied by an increase in the amount of ß cells co-expressed Pdx1 and insulin and by restoration of the vascular laminin expression. The atRA-induced production of vascular endothelial growth factor-A from the pancreatic islets was possibly the key factor that mediated the restoration of islet vascularity and recovery of ß-cell mass. Furthermore, the combination of islet transplantation and atRA administration significantly rescued hyperglycemia in diabetic mice. These findings suggest that vitamin A derivatives can potentially be used as a supplementary treatment to improve diabetes management and glycemic control.


Asunto(s)
Diabetes Mellitus Tipo 1/diagnóstico , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Diabetes Mellitus Tipo 1/metabolismo , Islotes Pancreáticos/metabolismo , Tretinoina/administración & dosificación , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Glucemia/metabolismo , Relación Dosis-Respuesta a Droga , Hipoglucemiantes/administración & dosificación , Insulina/sangre , Islotes Pancreáticos/efectos de los fármacos , Masculino , Ratones , Estreptozocina , Resultado del Tratamiento
12.
Stem Cells ; 33(12): 3468-80, 2015 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-26303070

RESUMEN

Leukemia inhibitory factor (LIF) regulates mouse embryonic stem cell (mESC) pluripotency through STAT3 activation, but the downstream signaling remains largely unelucidated. Using cDNA microarrays, we verified B cell leukemia/lymphoma 3 (Bcl3) as the most significantly downregulated factor following LIF withdrawal in mESCs. Bcl3 knockdown altered mESC morphology, reduced expression of pluripotency genes including Oct4, Sox2, and Nanog, and downregulated DNA binding of acetylated histone 3 and RNA polymerase II on the Oct4 promoter. Conversely, Bcl3 overexpression partially prevented cell differentiation and promoted Oct4 and Nanog promoter activities. Furthermore, coimmunoprecipitation and chromatin immunoprecipitation experiments demonstrated that Bcl3 regulation of mESC pluripotency may be through its association with Oct4 and ß-catenin and its promoter binding capability. These results establish that Bcl3 positively regulates pluripotency genes and thus shed light on the mechanism of Bcl3 as a downstream molecule of LIF/STAT3 signaling in pluripotency maintenance.


Asunto(s)
Factor Inhibidor de Leucemia/metabolismo , Células Madre Embrionarias de Ratones/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismo , Animales , Proteínas del Linfoma 3 de Células B , Regulación de la Expresión Génica , Factor Inhibidor de Leucemia/genética , Ratones , Factor 3 de Transcripción de Unión a Octámeros/genética , Proteínas Proto-Oncogénicas/genética , Factor de Transcripción STAT3/genética , Factores de Transcripción/genética
13.
Exp Dermatol ; 25(5): 355-61, 2016 05.
Artículo en Inglés | MEDLINE | ID: mdl-26739701

RESUMEN

The ATP-binding cassette transporter ABCG2 is expressed in the interfollicular epidermis and mediates the side-population phenotype in skin cells. However, the role of ABCG2 in skin is unclear. Increased expression levels of ABCG2 were found at the basal layer of transitional epidermis adjacent to cutaneous wounds in human patients, indicating that ABCG2 may be involved in regulating the wound healing process. To investigate the role of ABCG2 in cutaneous wound healing, full-thickness skin wounds were created in ABCG2 knockout (ABCG2-KO) and wild-type mice. The healing process was analysed and revealed that ABCG2 deficiency in skin results in delays in wound closure and impairments in re-epithelialization, as evidenced by reductions in both suprabasal differentiation and in p63-expressing keratinocytes migrating from transitional epidermis to epithelial tongues. The reduction in p63-expressing cells may be due to elevated levels of reactive oxygen species in ABCG2-KO epidermis, which can cause DNA damage and lead to proliferation arrest. To determine whether ABCG2 deficiency affects the potency of epidermal stem/progenitor cells (EPCs), transplantation studies were carried out, which demonstrated that ABCG2-KO EPCs display higher levels of γH2AX and lose the capacity to differentiate into suprabasal keratinocytes. A competitive repopulation assay confirmed that ABCG2 expression is critical for the proper expansion and differentiation of EPCs in cutaneous wounds. As EPCs are known to contribute to the healing of larger wounds, the current findings imply a functional role for ABCG2 in the expansion and differentiation of p63-expressing EPCs. Thus, ABCG2 deficiency in skin impairs re-epithelialization in cutaneous wound healing.


Asunto(s)
Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/deficiencia , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Células Madre Adultas/fisiología , Epidermis/fisiología , Proteínas de Neoplasias/metabolismo , Repitelización , Adulto , Animales , Daño del ADN , Células Epidérmicas , Femenino , Humanos , Masculino , Ratones Noqueados , Especies Reactivas de Oxígeno/metabolismo
14.
Int J Urol ; 23(9): 734-44, 2016 09.
Artículo en Inglés | MEDLINE | ID: mdl-27397852

RESUMEN

Distant organ metastasis of prostate cancer is a puzzle, and various theories have successively arisen to explain the mechanism of lethal cancer progression. While perhaps agreeable to many cancer biologists, the very statement of "seed and soil" proposed by Stephan Paget in 1881 is arguably still the major statement for organ-specific cancer metastasis. Since recent studies showed important correlations of regulation of cancer cells and the microenvironment, exosomes from cancer and stromal cells seem to create another important niche for metastasis. Stromal cells pretreated with exosomes from metastatic cancer cells increase the potential of change stromal cells. The poorly metastatic cancer cells could also enhance malignancy through transfer of proteins, microribonucleic acid and messenger ribonucleic acid to recipient cancer cells. Herein, we reviewed extracellular exosomes as a factor involved in cross-talk between stromal and prostate cancer epithelial cells.


Asunto(s)
Exosomas , Neoplasias de la Próstata/patología , Microambiente Tumoral , Progresión de la Enfermedad , Humanos , Masculino , Metástasis de la Neoplasia
15.
Am J Respir Crit Care Med ; 190(6): 675-87, 2014 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-25152164

RESUMEN

RATIONALE: Metabolic alterations contribute to cancer development and progression. However, the molecular mechanisms relating metabolism to cancer metastasis remain largely unknown. OBJECTIVES: To identify a key metabolic enzyme that is aberrantly overexpressed in invasive lung cancer cells and to investigate its functional role and prognostic value in lung cancer. METHODS: The differential expression of metabolic enzymes in noninvasive CL1-0 cells and invasive CL1-5 cells was analyzed by a gene expression microarray. The expression of target genes in clinical specimens from patients with lung cancer was examined by immunohistochemistry. Pharmacologic and gene knockdown/overexpression approaches were used to investigate the function of the target gene during invasion and metastasis in vitro and in vivo. The association between the target gene expression and clinicopathologic parameters was further analyzed. Bioinformatic analyses were used to discover the signaling pathways involved in target gene-regulated invasion and migration. MEASUREMENTS AND MAIN RESULTS: Squalene synthase (SQS) was up-regulated in CL1-5 cells and in the tumor regions of the lung cancer specimens. Loss of function or knockdown of SQS significantly inhibited invasion/migration and metastasis in cell and animal models and vice versa. High expression of SQS was significantly associated with poor prognosis among patients with lung cancer. Mechanistically, SQS contributed to a lipid-raft-localized enrichment of tumor necrosis factor receptor 1 in a cholesterol-dependent manner, which resulted in the enhancement of nuclear factor-κB activation leading to matrix metallopeptidase 1 up-regulation. CONCLUSIONS: Up-regulation of SQS promotes metastasis of lung cancer by enhancing tumor necrosis factor-α receptor 1 and nuclear factor-κB activation and matrix metallopeptidase 1 expression. Targeting SQS may have considerable potential as a novel therapeutic strategy to treat metastatic lung cancer.


Asunto(s)
Farnesil Difosfato Farnesil Transferasa/metabolismo , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/secundario , Microdominios de Membrana/metabolismo , Invasividad Neoplásica/fisiopatología , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Animales , Línea Celular Tumoral , Colesterol/biosíntesis , Modelos Animales de Enfermedad , Farnesil Difosfato Farnesil Transferasa/genética , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Pulmonares/patología , Metaloproteinasa 1 de la Matriz/metabolismo , Pronóstico , Regulación hacia Arriba
16.
Nat Cancer ; 5(3): 400-419, 2024 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-38267627

RESUMEN

Kirsten rat sarcoma virus (KRAS) signaling drives pancreatic ductal adenocarcinoma (PDAC) malignancy, which is an unmet clinical need. Here, we identify a disintegrin and metalloproteinase domain (ADAM)9 as a modulator of PDAC progression via stabilization of wild-type and mutant KRAS proteins. Mechanistically, ADAM9 loss increases the interaction of KRAS with plasminogen activator inhibitor 1 (PAI-1), which functions as a selective autophagy receptor in conjunction with light chain 3 (LC3), triggering lysosomal degradation of KRAS. Suppression of ADAM9 by a small-molecule inhibitor restricts disease progression in spontaneous models, and combination with gemcitabine elicits dramatic regression of patient-derived tumors. Our findings provide a promising strategy to target the KRAS signaling cascade and demonstrate a potential modality to enhance sensitivity to chemotherapy in PDAC.


Asunto(s)
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Proteínas Proto-Oncogénicas p21(ras) , Proliferación Celular , Neoplasias Pancreáticas/tratamiento farmacológico , Carcinoma Ductal Pancreático/tratamiento farmacológico , Gemcitabina , Proteínas de la Membrana/metabolismo , Proteínas ADAM/metabolismo , Proteínas ADAM/uso terapéutico
17.
Adv Sci (Weinh) ; 10(36): e2301240, 2023 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-37964407

RESUMEN

Over 90% of patients with pancreatic ductal adenocarcinoma (PDAC) have oncogenic KRAS mutations. Nevertheless, mutated KRAS alone is insufficient to initiate pancreatic intraepithelial neoplasia (PanIN), the precursor of PDAC. The identities of the other factors/events required to drive PanIN formation remain elusive. Here, optic-clear 3D histology is used to analyze entire pancreases of 2-week-old Pdx1-Cre; LSL-KrasG12D/+ (KC) mice to detect the earliest emergence of PanIN and observed that the occurrence is independent of physical location. Instead, it is found that the earliest PanINs overexpress Muc4 and associate with αSMA+ fibroblasts in both transgenic mice and human specimens. Mechanistically, KrasG12D/+ pancreatic cells upregulate Muc4 through genetic alterations to increase proliferation and fibroblast recruitments via Activin A secretion and consequently enhance cell transformation for PanIN formation. Inhibition of Activin A signaling using Follistatin (FST) diminishes early PanIN-associated fibroblast recruitment, effectively curtailing PanIN initiation and growth in KC mice. These findings emphasize the vital role of interactions between oncogenic KrasG12D/+ -driven genetic alterations and induced microenvironmental changes in PanIN initiation, suggesting potential avenues for early PDAC diagnostic and management approaches.


Asunto(s)
Carcinoma in Situ , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Ratones , Humanos , Animales , Proteínas Proto-Oncogénicas p21(ras)/genética , Mucina 4 , Neoplasias Pancreáticas/genética , Carcinoma Ductal Pancreático/genética , Ratones Transgénicos , Carcinoma in Situ/genética , Carcinoma in Situ/patología
18.
Nat Commun ; 14(1): 3395, 2023 06 09.
Artículo en Inglés | MEDLINE | ID: mdl-37296117

RESUMEN

Optical clearing with high-refractive-index (high-n) reagents is essential for 3D tissue imaging. However, the current liquid-based clearing condition and dye environment suffer from solvent evaporation and photobleaching, causing difficulties in maintaining the tissue optical and fluorescent features. Here, using the Gladstone-Dale equation [(n-1)/density=constant] as a design concept, we develop a solid (solvent-free) high-n acrylamide-based copolymer to embed mouse and human tissues for clearing and imaging. In the solid state, the fluorescent dye-labeled tissue matrices are filled and packed with the high-n copolymer, minimizing scattering in in-depth imaging and dye fading. This transparent, liquid-free condition provides a friendly tissue and cellular environment to facilitate high/super-resolution 3D imaging, preservation, transfer, and sharing among laboratories to investigate the morphologies of interest in experimental and clinical conditions.


Asunto(s)
Colorantes Fluorescentes , Imagenología Tridimensional , Ratones , Humanos , Animales , Imagenología Tridimensional/métodos , Solventes , Acrilamida , Imagen Óptica
19.
Stem Cells ; 29(12): 1995-2004, 2011 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-21956927

RESUMEN

Glycosphingolipids (GSLs) are ubiquitous components of cell membranes that can act as mediators of cell adhesion and signal transduction and can possibly be used as cell type-specific markers. Our previous study indicated that there was a striking switch in the core structures of GSLs during differentiation of human embryonic stem cells (hESCs) into embryoid body (EB), suggesting a close association of GSLs with cell differentiation. In this study, to further clarify if alterations in GSL patterns are correlated with lineage-specific differentiation of hESCs, we analyzed changes in GSLs as hESCs were differentiated into neural progenitors or endodermal cells by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) and tandem mass spectrometry (MS/MS) analyses. During hESC differentiation into neural progenitor cells, we found that the core structures of GSLs switched from globo- and lacto- to mostly ganglio-series dominated by GD3. On the other hand, when hESCs were differentiated into endodermal cells, patterns of GSLs totally differed from those observed in EB outgrowth and neural progenitors. The most prominent GSL identified by the MALDI-MS and MS/MS analysis was Gb(4) Ceramide, with no appreciable amount of stage-specific embryonic antigens 3 or 4, or GD3, in endodermal cells. These changes in GSL profiling were accompanied by alterations in the biosynthetic pathways of expressions of key glycosyltransferases. Our findings suggest that changes in GSLs are closely associated with lineage specificity and differentiation of hESCs.


Asunto(s)
Diferenciación Celular , Ectodermo/citología , Células Madre Embrionarias/citología , Endodermo/citología , Glicoesfingolípidos/metabolismo , Linaje de la Célula , Ectodermo/metabolismo , Células Madre Embrionarias/metabolismo , Endodermo/metabolismo , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Gangliósidos/metabolismo , Globósidos/metabolismo , Glicosiltransferasas/metabolismo , Humanos , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Espectrometría de Masas en Tándem
20.
Proteome Sci ; 10(1): 69, 2012 Nov 21.
Artículo en Inglés | MEDLINE | ID: mdl-23170877

RESUMEN

BACKGROUND: CD133-positive liver cancer stem cells, which are characterized by their resistance to conventional chemotherapy and their tumor initiation ability at limited dilutions, have been recognized as a critical target in liver cancer therapeutics. In the current work, we developed a label-free quantitative method to investigate the proteome of CD133-positive liver cancer stem cells for the purpose of identifying unique biomarkers that can be utilized for targeting liver cancer stem cells. Label-free quantitation was performed in combination with ID-based Elution time Alignment by Linear regression Quantitation (IDEAL-Q) and MaxQuant. RESULTS: Initially, IDEAL-Q analysis revealed that 151 proteins were differentially expressed in the CD133-positive hepatoma cells when compared with CD133-negative cells. We then analyzed these 151 differentially expressed proteins by MaxQuant software and identified 10 significantly up-regulated proteins. The results were further validated by RT-PCR, western blot, flow cytometry or immunofluorescent staining which revealed that prominin-1, annexin A1, annexin A3, transgelin, creatine kinase B, vimentin, and EpCAM were indeed highly expressed in the CD133-positive hepatoma cells. CONCLUSIONS: These findings confirmed that mass spectrometry-based label-free quantitative proteomics can be used to gain insights into liver cancer stem cells.

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