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1.
Exp Hematol ; 130: 104134, 2024 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-38052261

RESUMEN

Immunodeficient mice bearing human immune systems, or "humanized" chimeric mice, are widely used in basic research, along with the preclinical stages of drug development. Nonobese diabetic-severe combined immunodeficiency (NOD-SCID) IL2Rγnull (NSG) mice expressing human stem cell factor, granulocyte-macrophage colony stimulating factor, and interleukin-3 (NSG-SGM3) support robust development of human myeloid cells and T cells but have reduced longevity due to the development of fatal hemophagocytic lymphohistiocytosis (HLH). Here, we describe an optimized protocol for development of human immune chimerism in NSG-SGM3 mice. We demonstrate that efficient human CD45+ reconstitution can be achieved and HLH delayed by engraftment of neonatal NSG-SGM3 with low numbers of human umbilical cord-derived CD34+ hematopoietic stem cells in the absence of preconditioning irradiation.


Asunto(s)
Trasplante de Células Madre Hematopoyéticas , Linfohistiocitosis Hemofagocítica , Ratones , Humanos , Animales , Recién Nacido , Linfohistiocitosis Hemofagocítica/terapia , Ratones Endogámicos NOD , Ratones SCID , Células Madre Hematopoyéticas , Antígenos CD34 , Linfocitos T
2.
Res Sq ; 2023 Oct 24.
Artículo en Inglés | MEDLINE | ID: mdl-38076926

RESUMEN

Genome-wide association studies have linked Iroquois-Homeobox 4 (IRX4) as a robust expression quantitative-trait locus associated with prostate cancer (PCa) risk. However, the intricate mechanism and regulatory factors governing IRX4 expression in PCa remain poorly understood. Here, we unveil enrichment of androgen-responsive gene signatures in metastatic prostate tumors exhibiting heightened IRX4 expression. Furthermore, we uncover a novel interaction between IRX4 and the androgen receptor (AR) co-factor, FOXA1, suggesting that IRX4 modulates PCa cell behavior through AR cistrome alteration. Remarkably, we identified a distinctive short insertion-deletion polymorphism (INDEL), upstream of the IRX4 gene that differentially regulates IRX4 expression through the disruption of AR binding. This INDEL emerges as the most significant PCa risk-associated variant within the 5p15 locus, in a genetic analysis involving 82,591 PCa cases and 61,213 controls and was associated with PCa survival in patients undergoing androgen-deprivation therapy. These studies suggest the potential of this INDEL as a prognostic biomarker for androgen therapy in PCa and IRX4 as a potential therapeutic target in combination with current clinical management.

3.
Prostate Cancer Prostatic Dis ; 26(3): 614-624, 2023 09.
Artículo en Inglés | MEDLINE | ID: mdl-37264224

RESUMEN

BACKGROUND: Prostate cancer is a broad-spectrum disease, spanning from indolent to a highly aggressive lethal malignancy. Prostate cancer cell lines are essential tools to understanding the basic features of this malignancy, as well as in identifying novel therapeutic strategies. However, most cell lines routinely used in prostate cancer research are derived from metastatic disease and may not fully elucidate the molecular events underlying the early stages of cancer development and progression. Thus, there is a need for new cell lines derived from localised disease to better span the disease spectrum. METHODS: Prostatic tissue from the primary site, and adjacent non-cancerous tissue was obtained from four patients with localised disease undergoing radical prostatectomy. Epithelial cell outgrowths were immortalised with human papillomavirus type 16 (HPV16) E6 and E7 to establish monoclonal cell lines. Chromosomal ploidy was imaged and STR profiles were determined. Cell morphology, colony formation and cell proliferation characteristics were assessed. Androgen receptor (AR) expression and AR-responsiveness to androgen treatment were analysed by immunofluorescence and RT-qPCR, respectively. RNA-seq analysis was performed to identify prostate lineage markers and expression of prostate cancer tumorigenesis-related genes. RESULTS: Two benign cell lines derived from non-cancer cells (AQ0420 and AQ0396) and two tumour tissue derived cancer cell lines (AQ0411 and AQ0415) were immortalised from four patients with localised prostatic adenocarcinoma. The cell lines presented an epithelial morphology and a slow to moderate proliferative rate. None of the cell lines formed anchorage independent colonies or displayed AR-responsiveness. Comparative RNA-seq expression analysis confirmed the prostatic lineage of the four cell lines, with a distinct gene expression profile from that of the metastatic prostate cancer cell lines, PC-3 and LNCaP. CONCLUSIONS: Comprehensive characterization of these cell lines may provide new in vitro tools that could bridge the current knowledge gap between benign, early-stage and metastatic disease.


Asunto(s)
Neoplasias de la Próstata , Masculino , Humanos , Neoplasias de la Próstata/patología , Próstata/patología , Línea Celular , Antígeno Prostático Específico/metabolismo , Receptores Androgénicos/genética , Receptores Androgénicos/análisis , Andrógenos/metabolismo , Línea Celular Tumoral
4.
J Immunother Cancer ; 9(3)2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33737342

RESUMEN

BACKGROUND: The conventional type 1 dendritic cell subset (cDC1) is indispensable for tumor immune responses and the efficacy of immune checkpoint inhibitor (ICI) therapies in animal models but little is known about the role of the human CD141+ DC cDC1 equivalent in patients with melanoma. METHODS: We developed a flow cytometry assay to quantify and characterize human blood DC subsets in healthy donors and patients with stage 3 and stage 4 metastatic melanoma. To examine whether harnessing CD141+ DCs could improve responses to ICIs in human melanoma, we developed a humanized mouse model by engrafting immunodeficient NSG-SGM3 mice with human CD34+ hematopoietic stem cells (HSCs) from umbilical cord blood followed by transplantation of a human melanoma cell line and treatment with anti-programmed cell death protein-1 (anti-PD-1). RESULTS: Blood CD141+ DC numbers were significantly reduced in patients with stage 4 melanoma compared with healthy controls. Moreover, CD141+ DCs in patients with melanoma were selectively impaired in their ability to upregulate CD83 expression after stimulation with toll-like receptor 3 (TLR3) and TLR7/8 agonists ex vivo. Although DC numbers did not correlate with responses to anti-PD-1 and/or anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) ICIs, their numbers and capacity to upregulate CD83 declined further during treatment in non-responding patients. Treatment with anti-PD-1 was ineffective at controlling tumor growth in humanized mice but efficacy was enhanced by indirectly expanding and activating DCs in vivo with fms-like tyrosine kinase-3 ligand (Flt3L) and a TLR3 agonist. Moreover, intratumoral injections of CD141+ DCs resulted in reduced tumor growth when combined with anti-PD-1 treatment. CONCLUSIONS: These data illustrate quantitative and qualitative impairments in circulating CD141+ DCs in patients with advanced melanoma and that increasing CD141+ DC number and function is an attractive strategy to enhance immunogenicity and response rates to ICIs.


Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacología , Células Dendríticas/trasplante , Trasplante de Células Madre Hematopoyéticas , Inhibidores de Puntos de Control Inmunológico/farmacología , Inmunoterapia Adoptiva , Melanoma/terapia , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Neoplasias Cutáneas/terapia , Trombomodulina/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Animales , Antígenos CD34/metabolismo , Estudios de Casos y Controles , Línea Celular Tumoral , Terapia Combinada , Citocinas/sangre , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Femenino , Células Madre Hematopoyéticas/inmunología , Células Madre Hematopoyéticas/metabolismo , Humanos , Masculino , Melanoma/inmunología , Melanoma/metabolismo , Melanoma/patología , Ratones Endogámicos NOD , Ratones SCID , Persona de Mediana Edad , Receptor de Muerte Celular Programada 1/metabolismo , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto
5.
PeerJ ; 9: e10280, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33585078

RESUMEN

It is now appreciated that long non-coding RNAs (lncRNAs) are important players in orchestrating cancer progression. In this study we characterized GHSROS, a human lncRNA gene on the opposite DNA strand (antisense) to the ghrelin receptor gene, in prostate cancer. The lncRNA was upregulated by prostate tumors from different clinical datasets. Transcriptome data revealed that GHSROS alters the expression of cancer-associated genes. Functional analyses in vitro showed that GHSROS mediates tumor growth, migration and survival, and resistance to the cytotoxic drug docetaxel. Increased cellular proliferation of GHSROS-overexpressing PC3, DU145, and LNCaP prostate cancer cell lines in vitro was recapitulated in a subcutaneous xenograft model. Conversely, in vitro antisense oligonucleotide inhibition of the lncRNA reciprocally regulated cell growth and migration, and gene expression. Notably, GHSROS modulates the expression of PPP2R2C, the loss of which may drive androgen receptor pathway-independent prostate tumor progression in a subset of prostate cancers. Collectively, our findings suggest that GHSROS can reprogram prostate cancer cells toward a more aggressive phenotype and that this lncRNA may represent a potential therapeutic target.

6.
Cancers (Basel) ; 12(12)2020 Nov 24.
Artículo en Inglés | MEDLINE | ID: mdl-33255452

RESUMEN

Recent reports have suggested the role of kallikrein-related peptidase 4 (KLK4) to be that of remodeling the tumor microenvironment in many cancers, including prostate cancer. Notably, these studies have suggested a pro-tumorigenic role for KLK4, especially in prostate cancer. However, these have been primarily in vitro studies, with limited in vivo studies performed to date. Herein, we employed an orthotopic inoculation xenograft model to mimic the growth of primary tumors, and an intracardiac injection to induce metastatic dissemination to determine the in vivo tumorigenic effects of KLK4 overexpressed in PC3 prostate cancer cells. Notably, we found that these KLK4-expressing cells gave rise to smaller localized tumors and decreased metastases than the parent PC-3 cells. To our knowledge, this is the first report of an anti-tumorigenic effect of KLK4, particularly in prostate cancer. These findings also provide a cautionary tale of the need for in vivo analyses to substantiate in vitro experimental data.

7.
J Immunother Cancer ; 8(2)2020 07.
Artículo en Inglés | MEDLINE | ID: mdl-32737142

RESUMEN

BACKGROUND: Dendritic cells (DCs) are crucial for the efficacy of cancer vaccines, but current vaccines do not harness the key cDC1 subtype required for effective CD8+ T-cell-mediated tumor immune responses. Vaccine immunogenicity could be enhanced by specific delivery of immunogenic tumor antigens to CD141+ DCs, the human cDC1 equivalent. CD141+ DCs exclusively express the C-type-lectin-like receptor CLEC9A, which is important for the regulation of CD8+ T cell responses. This study developed a new vaccine that harnesses a human anti-CLEC9A antibody to specifically deliver the immunogenic tumor antigen, NY-ESO-1 (New York esophageal squamous cell carcinoma 1), to human CD141+ DCs. The ability of the CLEC9A-NY-ESO-1 antibody to activate NY-ESO-1-specific naïve and memory CD8+ T cells was examined and compared with a vaccine comprised of a human DEC-205-NY-ESO-1 antibody that targets all human DCs. METHODS: Human anti-CLEC9A, anti-DEC-205 and isotype control IgG4 antibodies were genetically fused to NY-ESO-1 polypeptide. Cross-presentation to NY-ESO-1-epitope-specific CD8+ T cells and reactivity of T cell responses in patients with melanoma were assessed by interferon γ (IFNγ) production following incubation of CD141+ DCs and patient peripheral blood mononuclear cells with targeting antibodies. Humanized mice containing human DC subsets and a repertoire of naïve NY-ESO-1-specific CD8+ T cells were used to investigate naïve T cell priming. T cell effector function was measured by expression of IFNγ, MIP-1ß, tumor necrosis factor and CD107a and by lysis of target tumor cells. RESULTS: CLEC9A-NY-ESO-1 antibodies (Abs) were effective at mediating delivery and cross-presentation of multiple NY-ESO-1 epitopes by CD141+ DCs for activation of NY-ESO-1-specific CD8+ T cells. When benchmarked to NY-ESO-1 conjugated to an untargeted control antibody or to anti-human DEC-205, CLEC9A-NY-ESO-1 was superior at ex vivo reactivation of NY-ESO-1-specific T cell responses in patients with melanoma. Moreover, CLEC9A-NY-ESO-1 induced priming of naïve NY-ESO-1-specific CD8+ T cells with polyclonal effector function and potent tumor killing capacity in vitro. CONCLUSIONS: These data advocate human CLEC9A-NY-ESO-1 Ab as an attractive strategy for specific targeting of CD141+ DCs to enhance tumor immunogenicity in NY-ESO-1-expressing malignancies.


Asunto(s)
Antígenos de Neoplasias/metabolismo , Linfocitos T CD8-positivos/metabolismo , Células Dendríticas/inmunología , Lectinas Tipo C/metabolismo , Proteínas de la Membrana/metabolismo , Receptores Mitogénicos/metabolismo , Trombomodulina/metabolismo , Animales , Femenino , Voluntarios Sanos , Humanos , Ratones
8.
Clin Transl Immunology ; 9(6): e1141, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32547743

RESUMEN

OBJECTIVES: Vaccines that prime Wilms' tumor 1 (WT1)-specific CD8+ T cells are attractive cancer immunotherapies. However, immunogenicity and clinical response rates may be enhanced by delivering WT1 to CD141+ dendritic cells (DCs). The C-type lectin-like receptor CLEC9A is expressed exclusively by CD141+ DCs and regulates CD8+ T-cell responses. We developed a new vaccine comprising a human anti-CLEC9A antibody fused to WT1 and investigated its capacity to target human CD141+ DCs and activate naïve and memory WT1-specific CD8+ T cells. METHODS: WT1 was genetically fused to antibodies specific for human CLEC9A, DEC-205 or ß-galactosidase (untargeted control). Activation of WT1-specific CD8+ T-cell lines following cross-presentation by CD141+ DCs was quantified by IFNγ ELISPOT. Humanised mice reconstituted with human immune cell subsets, including a repertoire of naïve WT1-specific CD8+ T cells, were used to investigate naïve WT1-specific CD8+ T-cell priming. RESULTS: The CLEC9A-WT1 vaccine promoted cross-presentation of WT1 epitopes to CD8+ T cells and mediated priming of naïve CD8+ T cells more effectively than the DEC-205-WT1 and untargeted control-WT1 vaccines. CONCLUSIONS: Delivery of WT1 to CD141+ DCs via CLEC9A stimulates CD8+ T cells more potently than either untargeted delivery or widespread delivery to all Ag-presenting cells via DEC-205, suggesting that cross-presentation by CD141+ DCs is sufficient for effective CD8+ T-cell priming in humans. The CLEC9A-WT1 vaccine is a promising candidate immunotherapy for malignancies that express WT1.

9.
Int J Oncol ; 55(6): 1223-1236, 2019 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-31638176

RESUMEN

Recent evidence suggests that numerous long non­coding RNAs (lncRNAs) are dysregulated in cancer, and have critical roles in tumour development and progression. The present study investigated the ghrelin receptor antisense lncRNA growth hormone secretagogue receptor opposite strand (GHSROS) in breast cancer. Reverse transcription­quantitative polymerase chain reaction revealed that GHSROS expression was significantly upregulated in breast tumour tissues compared with normal breast tissue. Induced overexpression of GHSROS in the MDA­MB­231 breast cancer cell line significantly increased cell migration in vitro, without affecting cell proliferation, a finding similar to our previous study on lung cancer cell lines. Microarray analysis revealed a significant repression of a small cluster of major histocompatibility class II genes and enrichment of immune response pathways; this phenomenon may allow tumour cells to better evade the immune system. Ectopic overexpression of GHSROS in the MDA­MB­231 cell line significantly increased orthotopic xenograft growth in mice, suggesting that in vitro culture does not fully capture the function of this lncRNA. This study demonstrated that GHSROS may serve a relevant role in breast cancer. Further studies are warranted to explore the function and therapeutic potential of this lncRNA in breast cancer progression.


Asunto(s)
Neoplasias de la Mama/genética , Movimiento Celular/genética , Regulación Neoplásica de la Expresión Génica , ARN Largo no Codificante/metabolismo , Animales , Apoptosis/genética , Mama/patología , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/patología , Progresión de la Enfermedad , Regulación hacia Abajo , Femenino , Perfilación de la Expresión Génica , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase II/inmunología , Humanos , Células MCF-7 , Ratones , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Receptores de Ghrelina/genética , Escape del Tumor/genética , Ensayos Antitumor por Modelo de Xenoinjerto
10.
Front Pharmacol ; 9: 1319, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30505274

RESUMEN

Alternative therapies against cancer cells with minimal or no effect on healthy tissues are highly sought after. Prostate cancer (PCa) is the second most frequently diagnosed malignancy in males. The Carica papaya L. leaf extract has been traditionally used by Australian aboriginal people for anticancer properties. In this study, medium polar fraction of papaya leaf extract that had shown anti-proliferative activity in PCa cell lines in vitro, in earlier studies, was further fractionated to 28 fractions by semi-preparative HPLC. Nine of these fractions were identified to possess selective anti-proliferative responses on PCa cells in comparison to non-cancerous cells of prostate gland origin. When these nine sub-fractions were mixed in various combinations, a combination containing six of the specific fractions (FC-3) showed the best potency. FC3 inhibited the growth of BPH-1, PC-3, and LNCaP cells in a concentration-dependent manner with an IC50 value <20 µg/mL, while (unlike paclitaxel, the positive control) minimal effect was observed on the proliferation of non-cancerous, WPMY-1 and RWPE-1cells. Furthermore, synergistic interaction of FC-3 with paclitaxel was observed with combination index values in the range of 0.89-0.98 and 0.85-1.10 on PC-3 and LNCaP cells, respectively. Untargeted qualitative analysis using UHPLC (Ultra High-Performance Liquid Chromatography)-QToF (Quadrupole Time of-Flight) mass spectrometry and screening against the METLIN database indicated presence of multiple known anticancer compounds in the FC-3 extract. These outcomes show that the potent and selective anti-proliferative effects are due to a range of bio-active compounds within the medium polar fraction of papaya leaf juice.

11.
Sci Rep ; 7(1): 16862, 2017 12 04.
Artículo en Inglés | MEDLINE | ID: mdl-29203868

RESUMEN

Short tandem repeats (STRs) are repetitive sequences of a polymorphic stretch of two to six nucleotides. We hypothesized that STRs are associated with prostate cancer development and/or progression. We undertook RNA sequencing analysis of prostate tumors and adjacent non-malignant cells to identify polymorphic STRs that are readily expressed in these cells. Most of the expressed STRs in the clinical samples mapped to intronic and intergenic DNA. Our analysis indicated that three of these STRs (TAAA-ACTG2, TTTTG-TRIB1, and TG-PCA3) are polymorphic and differentially expressed in prostate tumors compared to adjacent non-malignant cells. TG-PCA3 STR expression was repressed by the anti-androgen drug enzalutamide in prostate cancer cells. Genetic analysis of prostate cancer patients and healthy controls (N > 2,000) showed a significant association of the most common 11 repeat allele of TG-PCA3 STR with prostate cancer risk (OR = 1.49; 95% CI 1.11-1.99; P = 0.008). A significant association was also observed with aggressive disease (OR = 2.00; 95% CI 1.06-3.76; P = 0.031) and high mortality rates (HR = 3.0; 95% CI 1.03-8.77; P = 0.045). We propose that TG-PCA3 STR has both diagnostic and prognostic potential for prostate cancer. We provided a proof of concept to be applied to other RNA sequencing datasets to identify disease-associated STRs for future clinical exploratory studies.


Asunto(s)
Antígenos de Neoplasias/genética , Repeticiones de Microsatélite/genética , Neoplasias de la Próstata/patología , ARN Largo no Codificante/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Alelos , Secuencia de Bases , Estudios de Casos y Controles , Genotipo , Humanos , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Oportunidad Relativa , Pronóstico , Modelos de Riesgos Proporcionales , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/mortalidad , Factores de Riesgo
12.
Biomed Pharmacother ; 89: 515-523, 2017 May.
Artículo en Inglés | MEDLINE | ID: mdl-28249253

RESUMEN

BACKGROUND: Prostate cancer (PCa) is the leading cause of cancer related deaths in men. Carica papaya is a popular tropical plant that has been traditionally used for its nutritional and medicinal properties. METHODS: We investigated the anti-proliferative responses of papaya leaf juice (LJP) and its various extracts ("biological"- in vitro digested, "physical"- size exclusion, and "chemical"-solvent extraction) on a range of cell lines representing benign hyperplasia, tumorigenic and normal cells of prostate origin. RESULTS: Time course analysis (by 24h, 48h and 72h) of LJP (1-0.1mg/mL) before and after in vitro digestion, and of molecular weight based fractions of LJP showed anti-proliferative responses. The medium polarity fraction of LJP (0.03-0.003mg/mL) after 72h exposure showed potent growth inhibitory (IC50=0.02-0.07mg/mL) and cytotoxic activities on all prostate cells, with the exception of the normal (RWPE-1 and WPMY-1) cells. Flow cytometry analysis showed S phase cell cycle arrest and apoptosis as a possible mechanism for these activities. Medium polar fraction of LJP also inhibited migration and adhesion of metastatic PC-3 cells. CONCLUSION: This is the first report suggesting selective anti-proliferative and anti-metastatic attributes of LJP extract against prostatic diseases, including PCa.


Asunto(s)
Carica/química , Extractos Vegetales/farmacología , Hojas de la Planta/química , Neoplasias de la Próstata/tratamiento farmacológico , Puntos de Control del Ciclo Celular , Línea Celular Tumoral , Supervivencia Celular , Humanos , Masculino , Extractos Vegetales/química
13.
Endocrine ; 52(3): 609-17, 2016 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-26792793

RESUMEN

The peptide hormone ghrelin is a potent orexigen produced predominantly in the stomach. It has a number of other biological actions, including roles in appetite stimulation, energy balance, the stimulation of growth hormone release and the regulation of cell proliferation. Recently, several ghrelin gene splice variants have been described. Here, we attempted to identify conserved alternative splicing of the ghrelin gene by cross-species sequence comparisons. We identified a novel human exon 2-deleted variant and provide preliminary evidence that this splice variant and in1-ghrelin encode a C-terminally truncated form of the ghrelin peptide, termed minighrelin. These variants are expressed in humans and mice, demonstrating conservation of alternative splicing spanning 90 million years. Minighrelin appears to have similar actions to full-length ghrelin, as treatment with exogenous minighrelin peptide stimulates appetite and feeding in mice. Forced expression of the exon 2-deleted preproghrelin variant mirrors the effect of the canonical preproghrelin, stimulating cell proliferation and migration in the PC3 prostate cancer cell line. This is the first study to characterise an exon 2-deleted preproghrelin variant and to demonstrate sequence conservation of ghrelin gene-derived splice variants that encode a truncated ghrelin peptide. This adds further impetus for studies into the alternative splicing of the ghrelin gene and the function of novel ghrelin peptides in vertebrates.


Asunto(s)
Empalme Alternativo , Ghrelina/genética , Secuencia de Aminoácidos , Animales , Regulación del Apetito/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Secuencia Conservada , Ghrelina/farmacología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Isoformas de Proteínas/genética , Isoformas de Proteínas/farmacología , Especificidad de la Especie
14.
BMC Genomics ; 16: 1021, 2015 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-26626734

RESUMEN

BACKGROUND: Fusion transcripts are found in many tissues and have the potential to create novel functional products. Here, we investigate the genomic sequences around fusion junctions to better understand the transcriptional mechanisms mediating fusion transcription/splicing. We analyzed data from prostate (cancer) cells as previous studies have shown extensively that these cells readily undergo fusion transcription. RESULTS: We used the FusionMap program to identify high-confidence fusion transcripts from RNAseq data. The RNAseq datasets were from our (N = 8) and other (N = 14) clinical prostate tumors with adjacent non-cancer cells, and from the LNCaP prostate cancer cell line that were mock-, androgen- (DHT), and anti-androgen- (bicalutamide, enzalutamide) treated. In total, 185 fusion transcripts were identified from all RNAseq datasets. The majority (76%) of these fusion transcripts were 'read-through chimeras' derived from adjacent genes in the genome. Characterization of sequences at fusion loci were carried out using a combination of the FusionMap program, custom Perl scripts, and the RNAfold program. Our computational analysis indicated that most fusion junctions (76%) use the consensus GT-AG intron donor-acceptor splice site, and most fusion transcripts (85%) maintained the open reading frame. We assessed whether parental genes of fusion transcripts have the potential to form complementary base pairing between parental genes which might bring them into physical proximity. Our computational analysis of sequences flanking fusion junctions at parental loci indicate that these loci have a similar propensity as non-fusion loci to hybridize. The abundance of repetitive sequences at fusion and non-fusion loci was also investigated given that SINE repeats are involved in aberrant gene transcription. We found few instances of repetitive sequences at both fusion and non-fusion junctions. Finally, RT-qPCR was performed on RNA from both clinical prostate tumors and adjacent non-cancer cells (N = 7), and LNCaP cells treated as above to validate the expression of seven fusion transcripts and their respective parental genes. We reveal that fusion transcript expression is similar to the expression of parental genes. CONCLUSIONS: Fusion transcripts maintain the open reading frame, and likely use the same transcriptional machinery as non-fusion transcripts as they share many genomic features at splice/fusion junctions.


Asunto(s)
Regulación Neoplásica de la Expresión Génica , Neoplasias de la Próstata/genética , Sitios de Carácter Cuantitativo , Empalme del ARN , Transcripción Genética , Andrógenos/farmacología , Antineoplásicos Hormonales/farmacología , Biología Computacional/métodos , Secuencia Conservada , Conjuntos de Datos como Asunto , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Motivos de Nucleótidos , Sitios de Empalme de ARN , Secuencias Repetitivas de Ácidos Nucleicos
15.
Hum Mol Genet ; 24(5): 1478-92, 2015 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-25378557

RESUMEN

Common variants in the hepatocyte nuclear factor 1 homeobox B (HNF1B) gene are associated with the risk of Type II diabetes and multiple cancers. Evidence to date indicates that cancer risk may be mediated via genetic or epigenetic effects on HNF1B gene expression. We previously found single-nucleotide polymorphisms (SNPs) at the HNF1B locus to be associated with endometrial cancer, and now report extensive fine-mapping and in silico and laboratory analyses of this locus. Analysis of 1184 genotyped and imputed SNPs in 6608 Caucasian cases and 37 925 controls, and 895 Asian cases and 1968 controls, revealed the best signal of association for SNP rs11263763 (P = 8.4 × 10(-14), odds ratio = 0.86, 95% confidence interval = 0.82-0.89), located within HNF1B intron 1. Haplotype analysis and conditional analyses provide no evidence of further independent endometrial cancer risk variants at this locus. SNP rs11263763 genotype was associated with HNF1B mRNA expression but not with HNF1B methylation in endometrial tumor samples from The Cancer Genome Atlas. Genetic analyses prioritized rs11263763 and four other SNPs in high-to-moderate linkage disequilibrium as the most likely causal SNPs. Three of these SNPs map to the extended HNF1B promoter based on chromatin marks extending from the minimal promoter region. Reporter assays demonstrated that this extended region reduces activity in combination with the minimal HNF1B promoter, and that the minor alleles of rs11263763 or rs8064454 are associated with decreased HNF1B promoter activity. Our findings provide evidence for a single signal associated with endometrial cancer risk at the HNF1B locus, and that risk is likely mediated via altered HNF1B gene expression.


Asunto(s)
Mapeo Cromosómico , Neoplasias Endometriales/genética , Sitios Genéticos , Factor Nuclear 1-beta del Hepatocito/genética , Alelos , Estudios de Casos y Controles , Línea Celular Tumoral , Biología Computacional , Bases de Datos Genéticas , Epigénesis Genética , Femenino , Variación Genética , Estudio de Asociación del Genoma Completo , Genotipo , Haplotipos , Factor Nuclear 1-beta del Hepatocito/metabolismo , Humanos , Desequilibrio de Ligamiento , Polimorfismo de Nucleótido Simple , Regiones Promotoras Genéticas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Riesgo , Población Blanca/genética
16.
Reprod Biol Endocrinol ; 11: 70, 2013 Jul 23.
Artículo en Inglés | MEDLINE | ID: mdl-23879975

RESUMEN

BACKGROUND: Ghrelin is a 28 amino acid peptide hormone that is expressed in the stomach and a range of peripheral tissues, where it frequently acts as an autocrine/paracrine growth factor. Ghrelin is modified by a unique acylation required for it to activate its cognate receptor, the growth hormone secretagogue receptor (GHSR), which mediates many of the actions of ghrelin. Recently, the enzyme responsible for adding the fatty acid residue (octanoyl/acyl group) to the third amino acid of ghrelin, GOAT (ghrelin O-acyltransferase), was identified. METHODS: We used cell culture, quantitative real-time reverse transcription (RT)-PCR and immunohistochemistry to demonstrate the expression of GOAT in prostate cancer cell lines and tissues from patients. Real-time RT-PCR was used to demonstrate the expression of prohormone convertase (PC)1/3, PC2 and furin in prostate cancer cell lines. Prostate-derived cell lines were treated with ghrelin and desacyl ghrelin and the effect on GOAT expression was measured using quantitative RT-PCR. RESULTS: We have demonstrated that GOAT mRNA and protein are expressed in the normal prostate and human prostate cancer tissue samples. The RWPE-1 and RWPE-2 normal prostate-derived cell lines and the LNCaP, DU145, and PC3 prostate cancer cell lines express GOAT and at least one other enzyme that is necessary to produce mature, acylated ghrelin from proghrelin (PC1/3, PC2 or furin). Finally, ghrelin, but not desacyl ghrelin (unacylated ghrelin), can directly regulate the expression of GOAT in the RWPE-1 normal prostate derived cell line and the PC3 prostate cancer cell line. Ghrelin treatment (100nM) for 6 hours significantly decreased GOAT mRNA expression two-fold (P < 0.05) in the PC3 prostate cancer cell line, however, ghrelin did not regulate GOAT expression in the DU145 and LNCaP prostate cancer cell lines. CONCLUSIONS: This study demonstrates that GOAT is expressed in prostate cancer specimens and cell lines. Ghrelin regulates GOAT expression, however, this is likely to be cell-type specific. The expression of GOAT in prostate cancer supports the hypothesis that the ghrelin axis has autocrine/paracrine roles. We propose that the RWPE-1 prostate cell line and the PC3 prostate cancer cell line may be useful for investigating GOAT regulation and function.


Asunto(s)
Aciltransferasas/genética , Regulación Neoplásica de la Expresión Génica , Ghrelina/farmacología , Neoplasias de la Próstata/genética , Aciltransferasas/metabolismo , Línea Celular , Línea Celular Tumoral , Furina/genética , Furina/metabolismo , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Inmunohistoquímica , Masculino , Proproteína Convertasa 1/genética , Proproteína Convertasa 1/metabolismo , Proproteína Convertasa 2/genética , Proproteína Convertasa 2/metabolismo , Próstata/enzimología , Próstata/metabolismo , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
17.
Int J Oncol ; 43(2): 566-74, 2013 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-23722988

RESUMEN

The molecular mechanisms involved in non­small cell lung cancer tumourigenesis are largely unknown; however, recent studies have suggested that long non-coding RNAs (lncRNAs) are likely to play a role. In this study, we used public databases to identify an mRNA-like, candidate long non-coding RNA, GHSROS (GHSR opposite strand), transcribed from the antisense strand of the ghrelin receptor gene, growth hormone secretagogue receptor (GHSR). Quantitative real-time RT-PCR revealed higher expression of GHSROS in lung cancer tissue compared to adjacent, non-tumour lung tissue. In common with many long non-coding RNAs, GHSROS is 5' capped and 3' polyadenylated (mRNA-like), lacks an extensive open reading frame and harbours a transposable element. Engineered overexpression of GHSROS stimulated cell migration in the A549 and NCI-H1299 non-small cell lung cancer cell lines, but suppressed cell migration in the Beas-2B normal lung-derived bronchoepithelial cell line. This suggests that GHSROS function may be dependent on the oncogenic context. The identification of GHSROS, which is expressed in lung cancer and stimulates cell migration in lung cancer cell lines, contributes to the growing number of non-coding RNAs that play a role in the regulation of tumourigenesis and metastatic cancer progression.


Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/patología , Neoplasias Pulmonares/patología , ARN Largo no Codificante/genética , Receptores de Ghrelina/genética , Secuencia de Bases , Carcinogénesis/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Pulmonares/genética , Metástasis de la Neoplasia , Análisis de Secuencia de ADN , Transfección
18.
PLoS One ; 8(2): e57056, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23451143

RESUMEN

High tumor kallikrein-related-peptidase 4 (KLK4) levels are associated with a poor outcome for women with serous epithelial ovarian cancer (EOC), for which peritoneal dissemination and chemoresistance are key events. To determine the role of KLK4 in these events, we examined KLK4-transfected SKOV-3 and endogenous KLK4 expressing OVCA432 cells in 3-dimensional (3D) suspension culture to mimic the ascites microenvironment. KLK4-SKOV-3 cells formed multicellular aggregates (MCAs) as seen in ascites, as did SKOV-3 cells treated with active KLK4. MCA formation was reduced by treatment with a KLK4 blocking antibody or the selective active site KLK4 sunflower trypsin inhibitor (SFTI-FCQR). KLK4-MCAs formed larger cancer cell foci in mesothelial cell monolayers than those formed by vector and native SKOV-3 cells, suggesting KLK4-MCAs are highly invasive in the peritoneal microenvironment. A high level of KLK4 is expressed by ascitic EOC cells compared to matched primary tumor cells, further supporting its role in the ascitic microenvironment. Interestingly, KLK4 transfected SKOV-3 cells expressed high levels of the KLK4 substrate, urokinase plasminogen activator (uPA), particularly in 3D-suspension, and high levels of both KLK4 and uPA were observed in patient cells taken from ascites. Importantly, the KLK4-MCAs were paclitaxel resistant which was reversed by SFTI-FCQR and to a lesser degree by the general serine protease inhibitor, Aprotinin, suggesting that in addition to uPA, other as yet unidentified substrates of KLK4 must be involved. Nonetheless, these data suggest that KLK4 inhibition, in conjunction with paclitaxel, may improve the outcome for women with serous epithelial ovarian cancer and high KLK4 levels in their tumors.


Asunto(s)
Antineoplásicos/farmacología , Ascitis/patología , Calicreínas/metabolismo , Neoplasias Ováricas/tratamiento farmacológico , Paclitaxel/farmacología , Esferoides Celulares , Microambiente Tumoral , Antineoplásicos/uso terapéutico , Ascitis/enzimología , Línea Celular Tumoral , Cartilla de ADN , Resistencia a Antineoplásicos , Femenino , Humanos , Calicreínas/antagonistas & inhibidores , Neoplasias Ováricas/enzimología , Neoplasias Ováricas/patología , Paclitaxel/uso terapéutico , Reacción en Cadena de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
19.
Endocr Rev ; 33(6): 849-91, 2012 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-22826465

RESUMEN

Ghrelin, the endogenous ligand for the GH secretagogue receptor (GHSR), is a peptide hormone with diverse physiological roles. Ghrelin regulates GH release, appetite and feeding, gut motility, and energy balance and also has roles in the cardiovascular, immune, and reproductive systems. Ghrelin and the GHSR are expressed in a wide range of normal and tumor tissues, and a fluorescein-labeled, truncated form of ghrelin is showing promise as a biomarker for prostate cancer. Plasma ghrelin levels are generally inversely related to body mass index and are unlikely to be useful as a biomarker for cancer, but may be useful as a marker for cancer cachexia. Some single nucleotide polymorphisms in the ghrelin and GHSR genes have shown associations with cancer risk; however, larger studies are required. Ghrelin regulates processes associated with cancer, including cell proliferation, apoptosis, cell migration, cell invasion, inflammation, and angiogenesis; however, the role of ghrelin in cancer is currently unclear. Ghrelin has predominantly antiinflammatory effects and may play a role in protecting against cancer-related inflammation. Ghrelin and its analogs show promise as treatments for cancer-related cachexia. Further studies using in vivo models are required to determine whether ghrelin has a role in cancer progression.


Asunto(s)
Ghrelina/metabolismo , Neoplasias/metabolismo , Receptores de Ghrelina/metabolismo , Animales , Caquexia/tratamiento farmacológico , Línea Celular Tumoral , Progresión de la Enfermedad , Ghrelina/uso terapéutico , Humanos
20.
Mol Cell Endocrinol ; 340(1): 65-9, 2011 Jun 20.
Artículo en Inglés | MEDLINE | ID: mdl-21616120

RESUMEN

Ghrelin is a peptide hormone that was originally isolated from the stomach as the endogenous ligand for the growth hormone secretagogue receptor (GHSR). Ghrelin has many functions, including the regulation of appetite and gut motility, growth hormone release from the anterior pituitary and roles in the cardiovascular and immune systems. Ghrelin and its receptor are expressed in a number of cancers and cancer cell lines and may play a role in processes associated with cancer progression, including cell proliferation, apoptosis, and cell invasion and migration.


Asunto(s)
Ghrelina/metabolismo , Neoplasias/metabolismo , Animales , Comunicación Autocrina , Humanos , Modelos Biológicos , Neoplasias/patología , Comunicación Paracrina
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