RESUMEN
Cytosolic caspase-8 is a mediator of death receptor signaling. While caspase-8 expression is lost in some tumors, it is increased in others, indicating a conditional pro-survival function of caspase-8 in cancer. Here, we show that tumor cells employ DNA-damage-induced nuclear caspase-8 to override the p53-dependent G2/M cell-cycle checkpoint. Caspase-8 is upregulated and localized to the nucleus in multiple human cancers, correlating with treatment resistance and poor clinical outcome. Depletion of caspase-8 causes G2/M arrest, stabilization of p53, and induction of p53-dependent intrinsic apoptosis in tumor cells. In the nucleus, caspase-8 cleaves and inactivates the ubiquitin-specific peptidase 28 (USP28), preventing USP28 from de-ubiquitinating and stabilizing wild-type p53. This results in de facto p53 protein loss, switching cell fate from apoptosis toward mitosis. In summary, our work identifies a non-canonical role of caspase-8 exploited by cancer cells to override the p53-dependent G2/M cell-cycle checkpoint.
Asunto(s)
Caspasa 8/metabolismo , Núcleo Celular/enzimología , Proliferación Celular , Puntos de Control de la Fase G2 del Ciclo Celular , Neoplasias/enzimología , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitina Tiolesterasa/metabolismo , Antineoplásicos/farmacología , Apoptosis , Caspasa 8/genética , Núcleo Celular/efectos de los fármacos , Núcleo Celular/genética , Núcleo Celular/patología , Proliferación Celular/efectos de los fármacos , Resistencia a Antineoplásicos , Femenino , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Células HCT116 , Células HeLa , Humanos , Células MCF-7 , Masculino , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/patología , Células PC-3 , Estabilidad Proteica , Transducción de Señal , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/genética , Ubiquitina Tiolesterasa/genéticaRESUMEN
Androgen receptor (AR) is a main driver for castration-resistant prostate cancer (CRPC). c-Myc is an oncogene underlying prostate tumorigenesis. Here, we find that the deubiquitinase USP11 targets both AR and c-Myc in prostate cancer (PCa). USP11 expression was up-regulated in metastatic PCa and CRPC. USP11 knockdown (KD) significantly inhibited PCa cell growth. Our RNA-seq studies revealed AR and c-Myc as the top transcription factors altered after USP11 KD. ChIP-seq analysis showed that either USP11 KD or replacement of endogenous USP11 with a catalytic-inactive USP11 mutant significantly decreased chromatin binding by AR and c-Myc. We find that USP11 employs two mechanisms to up-regulate AR and c-Myc levels: namely, deubiquitination of AR and c-Myc proteins to increase their stability and deubiquitination of H2A-K119Ub, a repressive histone mark, on promoters of AR and c-Myc genes to increase their transcription. AR and c-Myc reexpression in USP11-KD PCa cells partly rescued cell growth defects. Thus, our studies reveal a tumor-promoting role for USP11 in aggressive PCa through upregulation of AR and c-Myc activities and support USP11 as a potential target against PCa.
Asunto(s)
Progresión de la Enfermedad , Regulación Neoplásica de la Expresión Génica , Neoplasias de la Próstata , Proteínas Proto-Oncogénicas c-myc , Receptores Androgénicos , Tioléster Hidrolasas , Humanos , Masculino , Línea Celular Tumoral , Proliferación Celular/genética , Histonas/metabolismo , Regiones Promotoras Genéticas/genética , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/genética , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/patología , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Receptores Androgénicos/metabolismo , Receptores Androgénicos/genética , Tioléster Hidrolasas/metabolismo , Tioléster Hidrolasas/genética , Ubiquitinación , Regulación hacia ArribaRESUMEN
Numerous cancers, including prostate cancer (PCa), are addicted to transcription programs driven by specific genomic regions known as super-enhancers (SEs). The robust transcription of genes at such SEs is enabled by the formation of phase-separated condensates by transcription factors and coactivators with intrinsically disordered regions. The androgen receptor (AR), the main oncogenic driver in PCa, contains large disordered regions and is co-recruited with the transcriptional coactivator mediator complex subunit 1 (MED1) to SEs in androgen-dependent PCa cells, thereby promoting oncogenic transcriptional programs. In this work, we reveal that full-length AR forms foci with liquid-like properties in different PCa models. We demonstrate that foci formation correlates with AR transcriptional activity, as this activity can be modulated by changing cellular foci content chemically or by silencing MED1. AR ability to phase separate was also validated in vitro by using recombinant full-length AR protein. We also demonstrate that AR antagonists, which suppress transcriptional activity by targeting key regions for homotypic or heterotypic interactions of this receptor, hinder foci formation in PCa cells and phase separation in vitro. Our results suggest that enhanced compartmentalization of AR and coactivators may play an important role in the activation of oncogenic transcription programs in androgen-dependent PCa.
Asunto(s)
Neoplasias de la Próstata , Receptores Androgénicos , Masculino , Humanos , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismo , Andrógenos , Factores de Transcripción/metabolismo , Regulación de la Expresión Génica , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Expresión Génica , Línea Celular Tumoral , Regulación Neoplásica de la Expresión GénicaRESUMEN
Photothermal therapy (PTT) refers to the use of plasmonic nanoparticles to convert electromagnetic radiation in the near infrared region to heat and kill tumor cells. Continuous wave lasers have been used clinically to induce PTT, but the treatment is associated with heat-induced tissue damage that limits usability. Here, the engineering and validation of a novel long-pulsed laser device able to induce selective and localized mild hyperthermia in tumors while reducing the heat affected zone and unwanted damage to surrounding tissue are reported. Long-pulsed PTT induces acute necrotic cell death in heat affected areas and the release of tumor associated antigens. This antigen release triggers maturation and stimulation of CD80/CD86 in dendritic cells in vivo that primes a cytotoxic T cell response. Accordingly, long-pulsed PTT enhances the therapeutic effects of immune checkpoint inhibition and increases survival of mice with bladder cancer. Combined, the data promote long-pulsed PTT as a safe and effective strategy for enhancing therapeutic responses to immune checkpoint inhibitors while minimizing unwanted tissue damage.
Asunto(s)
Inmunoterapia , Terapia Fototérmica , Inmunoterapia/métodos , Animales , Terapia Fototérmica/métodos , Ratones , Neoplasias/terapia , Humanos , Línea Celular TumoralRESUMEN
Chondroitin sulfate (CS) is the placental receptor for the VAR2CSA malaria protein, expressed at the surface of infected erythrocytes during Plasmodium falciparum infection. Infected cells adhere to syncytiotrophoblasts or get trapped within the intervillous space by binding to a determinant in a 4-O-sulfated CS chains. However, the exact structure of these glycan sequences remains unclear. VAR2CSA-reactive CS is also expressed by tumor cells, making it an attractive target for cancer diagnosis and therapeutics. The identities of the proteoglycans carrying these modifications in placental and cancer tissues remain poorly characterized. This information is clinically relevant since presentation of the glycan chains may be mediated by novel core proteins or by a limited subset of established proteoglycans. To address this question, VAR2CSA-binding proteoglycans were affinity-purified from the human placenta, tumor tissues and cancer cells and analyzed through a specialized glycoproteomics workflow. We show that VAR2CSA-reactive CS chains associate with a heterogenous group of proteoglycans, including novel core proteins. Additionally, this work demonstrates how affinity purification in combination with glycoproteomics analysis can facilitate the characterization of CSPGs with distinct CS epitopes. A similar workflow can be applied to investigate the interaction of CSPGs with other CS binding lectins as well.
Asunto(s)
Antígenos de Protozoos/metabolismo , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Placenta/metabolismo , Proteómica , Neoplasias de la Vejiga Urinaria/metabolismo , Antígenos de Protozoos/química , Proteoglicanos Tipo Condroitín Sulfato/química , Cromatografía de Afinidad , Femenino , Humanos , Placenta/química , Embarazo , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
AIMS: Gastric cancer (GC) is one of the leading causes of cancer-related death worldwide. Genes expressed only in cancer tissue may be useful biomarkers for cancer diagnosis and therapeutics. The aims of the present study were to analyse regulator of calcineurin 2 (RCAN2) in a large number of GCs, and to investigate how these expression patterns correlate with clinicopathological parameters and various markers. METHODS AND RESULTS: An immunohistochemical analysis of RCAN2 in 207 GC tissue samples showed that 110 (53%) GCs were positive for RCAN2. RCAN2-positive GCs were more advanced in terms of TNM classification and tumour stage than RCAN2-negative GCs. Furthermore, RCAN2 was an independent prognostic classifier for GC patients. The cell growth and invasiveness of RCAN2 small interfering RNA (siRNA)-transfected GC cell lines were less than those of the negative control siRNA-transfected cell lines, whereas those of RCAN2-transfected cells were significantly increased as compared with those of empty vector-transfected cells. RCAN2 siRNA inhibits the phosphorylation of AKT and p44/p42 (ERK1/2). RCAN2 was colocalised with EGFR, nuclear ß-catenin, MMP7, laminin-γ2, VEGF-A, and VEGF-C. CONCLUSION: These results suggest that RCAN2 is involved in tumour progression and is an independent prognostic classifier in patients with GC.
Asunto(s)
Proteínas Musculares/biosíntesis , Neoplasias Gástricas/patología , Adulto , Anciano , Biomarcadores de Tumor/análisis , Femenino , Humanos , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Pronóstico , Modelos de Riesgos Proporcionales , Estudios RetrospectivosRESUMEN
We have recently identified lens epithelium-derived growth factor (LEDGF/p75, also known as PSIP1) as a component of the homologous recombination DNA repair machinery. Through its Pro-Trp-Trp-Pro (PWWP) domain, LEDGF/p75 binds to histone marks associated with active transcription and promotes DNA end resection by recruiting DNA endonuclease retinoblastoma-binding protein 8 (RBBP8/CtIP) to broken DNA ends. Here we show that the structurally related PWWP domain-containing protein, hepatoma-derived growth factor-related protein 2 (HDGFRP2), serves a similar function in homologous recombination repair. Its depletion compromises the survival of human U2OS osteosarcoma and HeLa cervix carcinoma cells and impairs the DNA damage-induced phosphorylation of replication protein A2 (RPA2) and the recruitment of DNA endonuclease RBBP8/CtIP to DNA double strand breaks. In contrast to LEDGF/p75, HDGFRP2 binds preferentially to histone marks characteristic for transcriptionally silent chromatin. Accordingly, HDGFRP2 is found in complex with the heterochromatin-binding chromobox homologue 1 (CBX1) and Pogo transposable element with ZNF domain (POGZ). Supporting the functionality of this complex, POGZ-depleted cells show a similar defect in DNA damage-induced RPA2 phosphorylation as HDGFRP2-depleted cells. These data suggest that HDGFRP2, possibly in complex with POGZ, recruits homologous recombination repair machinery to damaged silent genes or to active genes silenced upon DNA damage.
Asunto(s)
Proteínas Cromosómicas no Histona/genética , Roturas del ADN de Doble Cadena , Histonas/genética , Péptidos y Proteínas de Señalización Intercelular/genética , Reparación del ADN por Recombinación , Transposasas/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Sitios de Unión , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Línea Celular Tumoral , Supervivencia Celular , Cromatina/química , Cromatina/metabolismo , Homólogo de la Proteína Chromobox 5 , Proteínas Cromosómicas no Histona/metabolismo , Endodesoxirribonucleasas , Células HeLa , Histonas/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Osteoblastos/metabolismo , Osteoblastos/patología , Fosforilación , Unión Proteica , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteína de Replicación A/genética , Proteína de Replicación A/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transposasas/antagonistas & inhibidores , Transposasas/metabolismoRESUMEN
Burkitt lymphoma (BL) is a malignant disease, which is frequently found in areas with holoendemic Plasmodium falciparum malaria. We have previously found that the VAR2CSA protein is present on malaria-infected erythrocytes and facilitates a highly specific binding to the placenta. ofCS is absent in other non-malignant tissues and thus VAR2CSA generally facilitates parasite sequestration and accumulation in pregnant women. In this study, we show that the specific receptor for VAR2CSA, the oncofetal chondroitin sulfate (ofCS), is likewise present in BL tissue and cell lines. We therefore explored whether ofCS in BL could act as anchor site for VAR2CSA-expressing infected erythrocytes. In contrast to the placenta, we found no evidence of in vivo sequestering of infected erythrocytes in the BL tissue. Furthermore, we found VAR2CSA-specific antibody titers in children with endemic BL to be lower than in control children from the same malaria endemic region. The abundant presence of ofCS in BL tissue and the absence of ofCS in non-malignant tissue encouraged us to examine whether recombinant VAR2CSA could be used to target BL. We confirmed the binding of VAR2CSA to BL-derived cells and showed that a VAR2CSA drug conjugate efficiently killed the BL-derived cell lines in vitro. These results identify ofCS as a novel therapeutic BL target and highlight how VAR2CSA could be used as a tool for the discovery of novel approaches for directing BL therapy.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Linfoma de Burkitt/metabolismo , Sulfatos de Condroitina/metabolismo , Malaria Falciparum/metabolismo , Placenta/metabolismo , Placenta/parasitología , Adolescente , Anticuerpos Antiprotozoarios/sangre , Antígenos de Protozoos/inmunología , Linfoma de Burkitt/parasitología , Línea Celular Tumoral , Niño , Preescolar , Eritrocitos/parasitología , Femenino , Humanos , Inmunoglobulina G/metabolismo , Malaria Falciparum/complicaciones , Masculino , Plasmodium falciparum/inmunología , Embarazo , Proteoglicanos/metabolismo , Proteínas Recombinantes/metabolismoRESUMEN
Adoptive transfer of T cells genetically modified to express chimeric antigen receptors (CARs) targeting the CD19 B cell-associated protein have demonstrated potent activity against relapsed/refractory B-lineage acute lymphoblastic leukemia (B-ALL). Not all patients respond, and CD19-negative relapses have been observed. Overexpression of the thymic stromal lymphopoietin receptor (TSLPR; encoded by CRLF2) occurs in a subset of adults and children with B-ALL and confers a high risk of relapse. Recent data suggest the TSLPR signaling axis is functionally important, suggesting that TSLPR would be an ideal immunotherapeutic target. We constructed short and long CARs targeting TSLPR and tested efficacy against CRLF2-overexpressing B-ALL. Both CARs demonstrated activity in vitro, but only short TSLPR CAR T cells mediated leukemia regression. In vivo activity of the short CAR was also associated with long-term persistence of CAR-expressing T cells. Short TSLPR CAR treatment of mice engrafted with a TSLPR-expressing ALL cell line induced leukemia cytotoxicity with efficacy comparable with that of CD19 CAR T cells. Short TSLPR CAR T cells also eradicated leukemia in 4 xenograft models of human CRLF2-overexpressing ALL. Finally, TSLPR has limited surface expression on normal tissues. TSLPR-targeted CAR T cells thus represent a potent oncoprotein-targeted immunotherapy for high-risk ALL.
Asunto(s)
Inmunoterapia Adoptiva/métodos , Leucemia-Linfoma Linfoblástico de Células Precursoras B/terapia , Receptores de Citocinas/antagonistas & inhibidores , Linfocitos T/inmunología , Animales , Antígenos CD19/metabolismo , Línea Celular Tumoral , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Leucemia-Linfoma Linfoblástico de Células Precursoras B/inmunología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/uso terapéutico , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Gastric cancer (GC) is one of the most common human cancers. Genes expressed only in cancer tissue, especially on the cell membrane, will be useful biomarkers for cancer diagnosis and therapeutics. METHODS: To identify novel genes encoding transmembrane protein specifically expressed in GC, we generated an Escherichia coli ampicillin secretion trap (CAST) library from diffuse-type GC cell line MKN-45. CAST is a survival-based signal sequence trap method that exploits the ability of mammalian signal sequences to confer ampicillin resistance to a mutant ß-lactamase lacking the endogenous signal sequence. RESULTS: By sequencing 1,536 colonies, we identified 23 genes encoding the transmembrane protein present in GC. Among these genes, TSPAN8 (also known as CO-029 and TM4SF3) gene, which encodes transmembrane protein tetraspanin 8, was emphasized as a candidate. Immunohistochemical analysis of tetraspanin 8 in human GC tissues revealed that 72 (34 %) of 210 GC cases were positive for tetraspanin 8, and microvessel density was significantly higher in tetraspanin 8-positive GC than in tetraspanin 8-negative GC. Furthermore, univariate and multivariate analyses revealed that tetraspanin 8 expression is an independent prognostic classifier of patients with GC. TSPAN8 knockdown by siRNA reduced the invasion of GC cell line. The reduction of invasiveness was retrieved by the tetraspanin 8-containing exosome. CONCLUSION: These results suggest that tetraspanin 8 is involved in tumor progression and is an independent prognostic classifier in patients with GC.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , Tetraspaninas/genética , Anciano , Ampicilina/farmacología , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Línea Celular Tumoral , Escherichia coli/genética , Exosomas/metabolismo , Femenino , Biblioteca de Genes , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Neoplasias Gástricas/metabolismo , Tetraspaninas/metabolismoRESUMEN
BACKGROUND: Gastric cancer (GC) is the fifth commonest malignancy worldwide and still one of the leading causes of cancer-related death. The aim of this study was to identify a novel prognostic marker or therapeutic target for GC. METHODS: We analyzed candidate genes from our previous Escherichia coli ampicillin secretion trap (CAST) libraries in detail, and focused on the FKTN gene because it was overexpressed in both GC cell line CAST libraries, MKN-1 and MKN-45. RESULTS: Quantitative reverse transcriptase PCR analysis of FKTN revealed that FKTN messenger RNA was overexpressed in nine of 28 (32.1 %) GC tissue samples compared with nonneoplastic gastric mucosa. Immunostaining of fukutin showed that 297 of 695 cases (42.7 %) were positive for fukutin. Fukutin-positive GC cases were significantly associated with differentiated histological features, and advanced T grade and N grade. In addition, fukutin expression was observed more frequently in the intestinal phenotype (51 %) of GC than in other phenotypes (37 %) when defined by the expression patterns of mucin 5AC, mucin 6, mucin 2, and CD10. FKTN small interfering RNA treatment decreased GC cell proliferation. CONCLUSIONS: These results indicate that the expression of fukutin may be a key regulator for progression of GC with the intestinal mucin phenotype.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Proteínas de la Membrana/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , Anciano , Ampicilina/farmacología , Factor de Transcripción CDX2/metabolismo , Línea Celular Tumoral , Proliferación Celular , Escherichia coli/genética , Femenino , Biblioteca de Genes , Humanos , Inmunoquímica , Masculino , Proteínas de la Membrana/metabolismo , Persona de Mediana Edad , Mucina 5AC/metabolismo , Mucina 2/metabolismo , Mucina 6/metabolismoRESUMEN
PURPOSE: Systemic therapy for advanced bladder cancer has not changed substantially in more than 2 decades and mortality rates remain high. The recognition of HER2 over expression in bladder cancer has made HER2 a promising therapeutic target. T-DM1, a new drug consisting of the HER2 antibody trastuzumab conjugated with a cytotoxic agent, has been shown in breast cancer to be superior to trastuzumab. We tested T-DM1 in preclinical models of bladder cancer. MATERIALS AND METHODS: We evaluated the effect of T-DM1 compared to trastuzumab in different in vitro and in vivo models of HER2 over expressing bladder cancer. RESULTS: RT4V6 was the highest HER2 expressing bladder cancer cell line and it showed higher growth inhibition with T-DM1 compared to trastuzumab. T-DM1 but not trastuzumab induced apoptosis of RT4V6 cells after G2/M arrest on cell cycle analysis. HER2 expression was higher in cell lines with acquired cisplatin resistance compared to the corresponding parental cell lines. Resistant cells showed higher sensitivity to T-DM1 by the induction of apoptosis. In addition, cells cultured in anchorage independent conditions increased HER2 expression compared to cells cultured in adherent conditions and T-DM1 significantly inhibited colony formation in soft agar compared to trastuzumab. In an orthotopic bladder cancer xenograft model tumor growth of cisplatin resistant RT112 was significantly inhibited by T-DM1 via the induction of apoptosis compared to treatment with control IgG or trastuzumab. CONCLUSIONS: T-DM1 has promising antitumor effects in preclinical models of HER2 over expressing bladder cancer.
Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacología , Antineoplásicos/farmacología , Maitansina/análogos & derivados , Receptor ErbB-2/biosíntesis , Receptor ErbB-2/efectos de los fármacos , Trastuzumab/farmacología , Neoplasias de la Vejiga Urinaria/metabolismo , Ado-Trastuzumab Emtansina , Animales , Apoptosis , Modelos Animales de Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Maitansina/farmacología , Ratones , Células Tumorales CultivadasRESUMEN
OBJECTIVE: Colorectal cancer (CRC) develops through the deregulation of gene expression and the accumulation of epigenetic abnormalities, leading to tumor cell acquisition of malignant features. MicroRNAs (miRNAs) play a critical role in cancer development, where they can act as oncogenes or oncosuppressors. METHODS: miR-148a expression was measured by qRT-PCR in patients with colorectal adenoma (n = 21) and CRC (stage I-IV, n = 159) using formalin-fixed paraffin-embedded tissue samples. In situ hybridization (ISH) using an miR-148a-specific probe was also performed. To further confirm the direct effect of miR-148a on matrix metalloproteinase (MMP)7 expression in CRC, MTT and cell invasion assays using HT29 and WiDr cells were performed. RESULTS: miR-148a expression was found to be clearly downregulated in high-grade adenoma compared to low-grade adenoma on both qRT-PCR and ISH analysis. Downregulation of miR-148a expression was significantly correlated with advanced clinicopathological features and was an independent prognostic classifier in patients with stage III CRC. In CRC cells and tissues, miR-148a expression was inversely correlated with the expression of MMP7. CONCLUSION: We showed the collaborative participation of miR-148a and MMP7 in CRC cell invasion. These results also demonstrate that the downregulation of miR-148a expression promotes CRC progression, especially carcinogenesis and cancer cell invasion.
Asunto(s)
Carcinogénesis/genética , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Regulación hacia Abajo , MicroARNs/genética , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias Colorrectales/diagnóstico , Femenino , Regulación Neoplásica de la Expresión Génica , Células HT29 , Humanos , Masculino , Metaloproteinasa 7 de la Matriz/genética , Persona de Mediana Edad , Invasividad Neoplásica/genética , Pronóstico , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Esophageal squamous cell carcinoma (ESCC) is one of the most common malignancies worldwide. In the present study, to identify novel prognostic markers or therapeutic targets for ESCC, we reviewed a list of genes with upregulated expression in ESCC compared with normal esophagus, as identified by our serial analysis of gene expression (SAGE) analysis. We focused on the NRD1 gene, which encodes the nardilysin protein. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) in 34 ESCC tissue samples revealed that mRNA expression of NRD1 was upregulated in 56% of ESCC tissue samples. Immunohistochemical analysis of nardilysin in 109 ESCC tissue samples demonstrated that 43 (39%) ESCC cases were positive for nardilysin. Nardilysin-positive ESCC cases were more advanced in terms of T classification (P = 0.0007), N classification (P = 0.0164), and tumor stage (P < 0.0001) than nardilysin-negative ESCC cases. Furthermore, nardilysin expression was significantly associated with poorer prognosis (P = 0.0258). Univariate and multivariate analyses revealed that nardilysin expression is an independent prognostic classifier of patients with ESCC. The invasiveness of NRD1-knockdown TE1 and TE5 esophageal cancer cell lines was less than that of the negative control siRNA-transfected cell lines. Expression of MMP2 and MMP3 mRNA was significantly lower in NRD1-knockdown TE5 cells than in negative control siRNA-transfected cells. These results suggest that nardilysin is involved in tumor progression, and is an independent prognostic classifier in patients with ESCC.
Asunto(s)
Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patología , Metaloproteinasa 2 de la Matriz/biosíntesis , Metaloproteinasa 3 de la Matriz/biosíntesis , Metaloendopeptidasas/metabolismo , Anciano , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Línea Celular Tumoral , Inducción Enzimática , Neoplasias Esofágicas/enzimología , Neoplasias Esofágicas/genética , Femenino , Humanos , Masculino , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 3 de la Matriz/genética , Metaloproteinasa 3 de la Matriz/metabolismo , Metaloendopeptidasas/genética , Invasividad Neoplásica , Pronóstico , Regulación hacia ArribaRESUMEN
Gastric cancer (GC) develops through deregulation of gene expression and accumulation of epigenetic abnormalities, leading to tumor cell acquisition of malignant features. MicroRNAs (miRNAs) play a critical role in cancer development where they can act as oncogenes or oncosuppressors. To identify miRNAs that are associated with some clinicopathologic features of GC and/or participate in tumor progression, miRNA expression in 20 GC tissues and five corresponding non-neoplastic gastric mucosa was examined by miRNA microarray. Oligonucleotide array analysis was carried out for miRNA target prediction. The functions of candidate miRNAs and their target genes were also analyzed by quantitative RT-PCR, Western blotting, reporter gene assay, and cell invasion assay. Comparison of miRNA expression profiles revealed that downregulation of miR-148a was identified in most of the GC tissues. Downregulation of miR-148a was significantly correlated with an advanced clinical stage, lymph node metastasis, and poor clinical outcome. Custom oligonucleotide array analysis revealed that MMP7 expression was markedly downregulated in miR-148a-overexpressing GC cells; MMP7 was found to be a direct and functional target of miR-148a, participating in cell invasion. These results suggest that miR-148a contributes to the maintenance of homeostasis in normal stomach tissue and plays an important role in GC invasion by regulating MMP7 expression.
Asunto(s)
Metaloproteinasa 7 de la Matriz/genética , MicroARNs/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , Procesos de Crecimiento Celular/fisiología , Línea Celular Tumoral , Metilación de ADN , Regulación hacia Abajo , Femenino , Humanos , Metástasis Linfática , Masculino , Metaloproteinasa 7 de la Matriz/metabolismo , MicroARNs/metabolismo , Persona de Mediana Edad , Invasividad Neoplásica , Pronóstico , Neoplasias Gástricas/metabolismoRESUMEN
OBJECTIVE: Scirrhous-type gastric cancer (GC) is highly aggressive and has a poor prognosis due to rapid cancer cell infiltration accompanied by extensive stromal fibrosis. The aim of this study is to identify genes that encode transmembrane proteins frequently expressed in scirrhous-type GC. METHODS: We compared Escherichia coli ampicillin secretion trap (CAST) libraries from 2 human scirrhous-type GC tissues with a normal stomach CAST library. By sequencing 2,880 colonies from scirrhous CAST libraries, we identified a list of candidate genes. RESULTS: We focused on the TM9SF3 gene because it has the highest clone count, and immunohistochemical analysis demonstrated that 46 (50%) of 91 GC cases were positive for TM9SF3, which was observed frequently in scirrhous-type GC. TM9SF3 expression showed a significant correlation with the depth of invasion, tumor stage and undifferentiated GC. There was a strong correlation between TM9SF3 expression and poor patient outcome, which was validated in two separate cohorts by immunostaining and quantitative RT-PCR, respectively. Transient knockdown of the TM9SF3 gene by siRNA showed decreased tumor cell-invasive capacity. CONCLUSION: Our results indicate that TM9SF3 might be a potential diagnostic and therapeutic target for scirrhous-type GC.
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Adenocarcinoma Escirroso/fisiopatología , Ampicilina , Escherichia coli , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Análisis de Secuencia de ADN/métodos , Neoplasias Gástricas/fisiopatología , Adenocarcinoma Escirroso/diagnóstico , Adenocarcinoma Escirroso/genética , Anciano , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/fisiología , Proteínas Portadoras , Línea Celular Tumoral , Movimiento Celular/genética , Movimiento Celular/fisiología , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Estimación de Kaplan-Meier , Masculino , Invasividad Neoplásica/genética , Invasividad Neoplásica/fisiopatología , Pronóstico , Señales de Clasificación de Proteína , ARN Interferente Pequeño/farmacología , Neoplasias Gástricas/diagnóstico , Neoplasias Gástricas/genéticaRESUMEN
RNF185 is a RING finger domain-containing ubiquitin ligase implicated in ER-associated degradation. Prostate tumor patient data analysis revealed a negative correlation between RNF185 expression and prostate cancer progression and metastasis. Likewise, several prostate cancer cell lines exhibited greater migration and invasion capabilities in culture upon RNF185 depletion. Subcutaneous inoculation of mouse prostate cancer MPC3 cells stably expressing short hairpin RNA against RNF185 into mice resulted in larger tumors and more frequent lung metastases. RNA-sequencing and Ingenuity Pathway Analysis identified wound-healing and cellular movement among the most significant pathways upregulated in RNF185-depleted lines, compared with control prostate cancer cells. Gene Set Enrichment Analyses performed in samples from patients harboring low RNF185 expression and in RNF185-depleted lines confirmed the deregulation of genes implicated in epithelial-to-mesenchymal transition. Among those, COL3A1 was identified as the primary mediator of RNF185's ability to impact migration phenotypes. Correspondingly, enhanced migration and metastasis of RNF185 knockdown (KD) prostate cancer cells were attenuated upon co-inhibition of COL3A1. Our results identify RNF185 as a gatekeeper of prostate cancer metastasis, partly via its control of COL3A1 availability. IMPLICATIONS: RNF185 is identified as an important regulator of prostate cancer migration and metastasis, in part due to its regulation of COL3A1. Both RNF185 and COL3A1 may serve as novel markers for prostate tumors.
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Neoplasias de la Próstata , Masculino , Humanos , Ratones , Animales , Neoplasias de la Próstata/patología , Próstata/patología , Movimiento Celular/genética , Transición Epitelial-Mesenquimal , ARN Interferente Pequeño , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Colágeno Tipo III/genética , Colágeno Tipo III/metabolismo , Proteínas Mitocondriales/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
RNF185 is a RING finger domain-containing ubiquitin ligase implicated in ER-associated degradation. Prostate tumor patient data analysis revealed a negative correlation between RNF185 expression and prostate cancer progression and metastasis. Likewise, several prostate cancer cell lines exhibited greater migration and invasion capabilities in culture upon RNF185 depletion. Subcutaneous inoculation of mouse prostate cancer MPC3 cells stably expressing shRNA against RNF185 into mice resulted in larger tumors and more frequent lung metastases. RNA-sequencing and Ingenuity Pathway Analysis identified wound healing and cellular movement among the most significant pathways upregulated in RNF185-depleted, compared to control prostate cancer cells. Gene Set Enrichment Analyses performed in samples from patients harboring low RNF185 expression and in RNF185-depleted lines confirmed the deregulation of genes implicated in EMT. Among those, COL3A1 was identified as the primary mediator of RNF185's ability to impact migration phenotypes. Correspondingly, enhanced migration and metastasis of RNF185 KD prostate cancer cells were attenuated upon co-inhibition of COL3A1. Our results identify RNF185 as a gatekeeper of prostate cancer metastasis, partly via its control of COL3A1 availability.
RESUMEN
PURPOSE: Histone deacetylase (HDAC) inhibition has been shown to induce pharmacologic "BRCAness" in cancer cells with proficient DNA repair activity. This provides a rationale for exploring combination treatments with HDAC and PARP inhibition in cancer types that are insensitive to single-agent PARP inhibitors (PARPi). Here, we report the concept and characterization of a novel bifunctional PARPi (kt-3283) with dual activity toward PARP1/2 and HDAC enzymes in Ewing sarcoma cells. EXPERIMENTAL DESIGN: Inhibition of PARP1/2 and HDAC was measured using PARP1/2, HDAC activity, and PAR formation assays. Cytotoxicity was assessed by IncuCyte live cell imaging, CellTiter-Glo, and spheroid assays. Cell-cycle profiles were determined using propidium iodide staining and flow cytometry. DNA damage was examined by γH2AX expression and comet assay. Inhibition of metastatic potential by kt-3283 was evaluated via ex vivo pulmonary metastasis assay (PuMA). RESULTS: Compared with FDA-approved PARP (olaparib) and HDAC (vorinostat) inhibitors, kt-3283 displayed enhanced cytotoxicity in Ewing sarcoma models. The kt-3283-induced cytotoxicity was associated with strong S and G2-M cell-cycle arrest in nanomolar concentration range and elevated DNA damage as assessed by γH2AX tracking and comet assays. In three-dimensional spheroid models of Ewing sarcoma, kt-3283 showed efficacy in lower concentrations than olaparib and vorinostat, and kt-3283 inhibited colonization of Ewing sarcoma cells in the ex vivo PuMA model. CONCLUSIONS: Our data demonstrate the preclinical justification for studying the benefit of dual PARP and HDAC inhibition in the treatment of Ewing sarcoma in a clinical trial and provides proof-of-concept for a bifunctional single-molecule therapeutic strategy.
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Puma , Sarcoma de Ewing , Animales , Humanos , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Sarcoma de Ewing/patología , Inhibidores de Histona Desacetilasas/farmacología , Inhibidores de Histona Desacetilasas/uso terapéutico , Vorinostat/uso terapéuticoRESUMEN
Prostate cancer is the most common cancer in men and it is estimated that over 350,000 men worldwide die of prostate cancer every year. There remains an unmet clinical need to improve how clinically significant prostate cancer is diagnosed and develop new treatments for advanced disease. Aberrant glycosylation is a hallmark of cancer implicated in tumour growth, metastasis, and immune evasion. One of the key drivers of aberrant glycosylation is the dysregulated expression of glycosylation enzymes within the cancer cell. Here, we demonstrate using multiple independent clinical cohorts that the glycosyltransferase enzyme GALNT7 is upregulated in prostate cancer tissue. We show GALNT7 can identify men with prostate cancer, using urine and blood samples, with improved diagnostic accuracy than serum PSA alone. We also show that GALNT7 levels remain high in progression to castrate-resistant disease, and using in vitro and in vivo models, reveal that GALNT7 promotes prostate tumour growth. Mechanistically, GALNT7 can modify O-glycosylation in prostate cancer cells and correlates with cell cycle and immune signalling pathways. Our study provides a new biomarker to aid the diagnosis of clinically significant disease and cements GALNT7-mediated O-glycosylation as an important driver of prostate cancer progression.