RESUMEN
Expression of the metastasis suppressor NME1 in melanoma is associated with reduced cellular motility, invasion, and metastasis, but mechanisms underlying these activities are not completely understood. Herein we report a novel mechanism through which NME1 drives formation of large, stable focal adhesions (FAs) in melanoma cells via induction of integrin ß3 (ITGß3), and in one cell line, concomitant suppression of integrin ß1 (ITGß1) transcripts. Forced expression of NME1 resulted in a strong activation of the promoter region (-301 to +13) of the ITGB3 gene. Chromatin immunoprecipitation (ChIP) analysis revealed the transcriptional induction was associated with direct recruitment of NME1 and an increase in the epigenetic activation mark, acetylation of histone 3 on lysine 27 (H3K27Ac) to a 1â¯kb stretch of 5'-flanking sequence of the ITGB3 gene. Unexpectedly, NME1 did not affect the amount either ITGß1 or ITGß3 proteins were internalized and recycled, processes commonly associated with regulating expression of integrins at the cell surface. The ability of NME1 to suppress motile and invasive phenotypes of melanoma cells was dependent on its induction of ITGß3. Expression of ITGß3 mRNA was associated with increased disease-free survival time in melanoma patients of the TCGA collection, consistent with its potential role as an effector of the metastasis suppressor function of NME1. Together, these data indicate metastasis suppressor activity of NME1 in melanoma is mediated by induction of ITGB3 gene transcription, with NME1-driven enrichment of ITGß3 protein at the cell membrane resulting in attenuated cell motility through the stabilization of large focal adhesions.
Asunto(s)
Movimiento Celular , Integrina beta3/genética , Melanoma/genética , Melanoma/patología , Nucleósido Difosfato Quinasas NM23/metabolismo , Transcripción Genética , Animales , Línea Celular Tumoral , Movimiento Celular/genética , Fibronectinas/metabolismo , Adhesiones Focales/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Integrina beta3/metabolismo , Ratones Endogámicos C57BL , Invasividad Neoplásica , Metástasis de la Neoplasia , Regiones Promotoras Genéticas/genética , Unión Proteica/genética , Subunidades de Proteína/metabolismo , Transporte de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Análisis de SupervivenciaRESUMEN
Although NME1 is well known for its ability to suppress metastasis of melanoma, the molecular mechanisms underlying this activity are not completely understood. Herein, we utilized a bioinformatics approach to systematically identify genes whose expression is correlated with the metastasis suppressor function of NME1. This was accomplished through a search for genes that were regulated by NME1, but not by NME1 variants lacking metastasis suppressor activity. This approach identified a number of novel genes, such as ALDOC, CXCL11, LRP1b, and XAGE1 as well as known targets such as NETO2, which were collectively designated as an NME1-Regulated Metastasis Suppressor Signature (MSS). The MSS was associated with prolonged overall survival in a large cohort of melanoma patients in The Cancer Genome Atlas (TCGA). The median overall survival of melanoma patients with elevated expression of the MSS genes was >5.6 years longer compared with that of patients with lower expression of the MSS genes. These data demonstrate that NMEl represents a powerful tool for identifying genes whose expression is associated with metastasis and survival of melanoma patients, suggesting their potential applications as prognostic markers and therapeutic targets in advanced forms of this lethal cancer.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Melanoma/metabolismo , Nucleósido Difosfato Quinasas NM23/metabolismo , Animales , Antígenos de Neoplasias/genética , Línea Celular Tumoral , Quimiocina CXCL11/genética , Biología Computacional , Femenino , Fructosa-Bifosfato Aldolasa/genética , Humanos , Melanoma/mortalidad , Ratones Desnudos , Nucleósido Difosfato Quinasas NM23/genética , Metástasis de la Neoplasia , Mutación Puntual , Receptores de LDL/genéticaRESUMEN
Expression of the metastasis suppressor NME1 in melanoma is associated with reduced cellular motility and invasion in vitro and metastasis in vivo, but the underlying molecular mechanisms are not completely understood. Herein, we report a novel mechanism through which NME1 controls melanoma cell morphology via upregulation of the extracellular matrix (ECM) protein fibronectin. Expression of NME1 strongly suppressed cell motility in melanoma cell lines 1205LU and M14. The resulting sedentary phenotype was associated with a more flattened appearance and marked increases in actin stress fibre and focal adhesion formation. NME1-induced focal adhesions were colocalized with dense deposits of fibronectin, which were absent or minimal in the corresponding NME1-deficient parental lines. NME1 was a strong inducer of fibronectin mRNA and protein expression, shown with reciprocal approaches of forced NME1 expression and shRNA-mediated knock-down. Increased synthesis and ECM deposition of fibronectin was necessary for NME1-induced cell spreading, as knock-down of fibronectin opposed the effects of NME1 on cell morphology. Fibronectin knock-down also reversed the ability of NME1 to promote aggregation when cells were plated on a non-adherent substratum. Similarly, inhibiting activation of the fibronectin receptor integrin α4ß1 with an anti-α4 antibody reversed the motility-suppressing effect of NME1. A positive correlation was observed between NME1 and fibronectin mRNA in clinical biopsies of normal skin, benign nevi and primary melanomas, but not in metastatic forms, suggesting the NME1/fibronectin axis represents a barrier to melanoma progression. In summary, these findings indicate fibronectin is an important effector of the motility-suppressing function of NME1 in melanoma cells.
Asunto(s)
Adhesión Celular/fisiología , Movimiento Celular/fisiología , Fibronectinas/fisiología , Melanoma/patología , Nucleósido Difosfato Quinasas NM23/fisiología , Neoplasias Cutáneas/patología , Línea Celular Tumoral , Progresión de la Enfermedad , Matriz Extracelular/patología , Matriz Extracelular/fisiología , Fibronectinas/genética , Regulación Neoplásica de la Expresión Génica/fisiología , Humanos , Técnicas In Vitro , Melanoma/fisiopatología , Nucleósido Difosfato Quinasas NM23/genética , Metástasis de la Neoplasia/patología , Metástasis de la Neoplasia/fisiopatología , ARN Mensajero/genética , ARN Mensajero/fisiología , Transducción de Señal/fisiología , Neoplasias Cutáneas/fisiopatologíaRESUMEN
OBJECTIVE: To credential Stathmin 1 (STMN1) and p16(INK4A) (p16) as adjunct markers for the diagnosis of serous tubal intraepithelial carcinoma (STIC), and to compare STMN1 and p16 expression in p53-positive and p53-negative STIC and invasive high-grade serous carcinoma (HGSC). METHODS: Immunohistochemistry (IHC) was used to examine STMN1 and p16 expression in fallopian tube specimens (n=31) containing p53-positive and p53-negative STICs, invasive HGSCs, and morphologically normal FTE (fallopian tube epithelium). STMN1 and p16 expression was scored semiquantitatively by four individuals. The semiquantitative scores were dichotomized, and reported as positive or negative. Pooled siRNA was used to knockdown p53 in a panel of cell lines derived from immortalized FTE and HGSC. RESULTS: STMN1 and p16 were expressed in the majority of p53-positive and p53-negative STICs and concomitant invasive HGSCs, but only scattered positive cells were present in morphologically normal FTE. Both proteins were expressed consistently across multiple STICs from the same patient and in concomitant invasive HGSC. Knockdown of p53 in immortalized FTE cells and in four HGSC-derived cell lines expressing different missense p53 mutations did not affect STMN1 protein levels. CONCLUSIONS: This study demonstrates that STMN1 and p16 are sensitive and specific adjunct biomarkers that, when used with p53 and Ki-67, improve the diagnostic accuracy of STIC. The addition of STMN1 and p16 helps to compensate for practical limitations of p53 and Ki-67 that complicate the diagnosis in up to one third of STICs.
Asunto(s)
Biomarcadores de Tumor/biosíntesis , Cistadenocarcinoma Seroso/metabolismo , Neoplasias de las Trompas Uterinas/metabolismo , Proteínas de Neoplasias/biosíntesis , Neoplasias Ováricas/metabolismo , Estatmina/biosíntesis , Línea Celular Tumoral , Inhibidor p16 de la Quinasa Dependiente de Ciclina , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/patología , Neoplasias de las Trompas Uterinas/genética , Neoplasias de las Trompas Uterinas/patología , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Inmunohistoquímica , Antígeno Ki-67/metabolismo , Mutación , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Proteína p53 Supresora de Tumor/deficiencia , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The metastasis suppressor NM23-H1 possesses 3 enzymatic activities in vitro, a nucleoside diphosphate kinase (NDPK), a protein histidine kinase and a more recently characterized 3'-5' exonuclease. Although the histidine kinase has been implicated in suppression of motility in breast carcinoma cell lines, potential relevance of the NDPK and 3'-5' exonuclease to metastasis suppressor function has not been addressed in detail. To this end, site-directed mutagenesis and biochemical analyses of bacterially expressed mutant NM23-H1 proteins have identified mutations that disrupt the 3'-5' exonuclease alone (Glu(5) to Ala, or E(5) A), the NDPK and histidine kinase activities tandemly (Y(52) A, H(118) F) or all 3 activities simultaneously (K(12) Q). Although forced expression of NM23-H1 potently suppressed spontaneous lung metastasis of subcutaneous tumor explants derived from the human melanoma cell line 1205LU, no significant metastasis suppressor activity was obtained with the exonuclease-deficient variants E(5) A and K(12) Q. The H(118) F mutant, which lacked both the NDPK and histidine kinase while retaining the 3'-5' exonuclease, also exhibited compromised suppressor activity. In contrast, each mutant retained the ability to suppress motility and invasive characteristics of 1205LU cells in culture, indicating that the NM23-H1 molecule possesses an additional activity(s) mediating these suppressor functions. These studies provide the first demonstration that the 3'-5' exonuclease activity of NM23-H1 is necessary for metastasis suppressor function and further indicate cooperativity of the 3 enzymatic activities of the molecule on suppression of the metastatic process.
Asunto(s)
Exonucleasas/metabolismo , Neoplasias Pulmonares/enzimología , Melanoma Experimental/enzimología , Nucleósido Difosfato Quinasas NM23/metabolismo , Secuencia de Aminoácidos , Animales , Western Blotting , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Exonucleasas/química , Exonucleasas/genética , Femenino , Ácido Glutámico/genética , Ácido Glutámico/metabolismo , Histidina/genética , Histidina/metabolismo , Histidina Quinasa , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundario , Lisina/genética , Lisina/metabolismo , Melanoma Experimental/genética , Melanoma Experimental/patología , Ratones , Ratones Desnudos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación Missense , Nucleósido Difosfato Quinasas NM23/química , Nucleósido Difosfato Quinasas NM23/genética , Conformación Proteica , Proteínas Quinasas/química , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Trasplante HeterólogoRESUMEN
The tumor microenvironment evolves during malignant progression, with major changes in nonmalignant cells, cytokine networks, and the extracellular matrix (ECM). In this study, we aimed to understand how the ECM changes during neoplastic transformation of serous tubal intraepithelial carcinoma lesions (STIC) into high-grade serous ovarian cancers (HGSOC). Analysis of the mechanical properties of human fallopian tubes (FT) and ovaries revealed that normal FT and fimbria had a lower tissue modulus, a measure of stiffness, than normal or diseased ovaries. Proteomic analysis of the matrisome fraction between FT, fimbria, and ovaries showed significant differences in the ECM protein TGF beta induced (TGFBI, also known as ßig-h3). STIC lesions in the fimbria expressed high levels of TGFBI, which was predominantly produced by CD163-positive macrophages proximal to STIC epithelial cells. In vitro stimulation of macrophages with TGFß and IL4 induced secretion of TGFBI, whereas IFNγ/LPS downregulated macrophage TGFBI expression. Immortalized FT secretory epithelial cells carrying clinically relevant TP53 mutations stimulated macrophages to secrete TGFBI and upregulated integrin αvß3, a putative TGFBI receptor. Transcriptomic HGSOC datasets showed a significant correlation between TGFBI expression and alternatively activated macrophage signatures. Fibroblasts in HGSOC metastases expressed TGFBI and stimulated macrophage TGFBI production in vitro. Treatment of orthotopic mouse HGSOC tumors with an anti-TGFBI antibody reduced peritoneal tumor size, increased tumor monocytes, and activated ß3-expressing unconventional T cells. In conclusion, TGFBI may favor an immunosuppressive microenvironment in STICs that persists in advanced HGSOC. Furthermore, TGFBI may be an effector of the tumor-promoting actions of TGFß and a potential therapeutic target. SIGNIFICANCE: Analysis of ECM changes during neoplastic transformation reveals a role for TGFBI secreted by macrophages in immunosuppression in early ovarian cancer.
Asunto(s)
Cistadenocarcinoma Seroso/patología , Matriz Extracelular/patología , Macrófagos/inmunología , Neoplasias Ováricas/patología , Neoplasias Peritoneales/patología , Factor de Crecimiento Transformador beta1/metabolismo , Microambiente Tumoral , Animales , Apoptosis , Proliferación Celular , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/inmunología , Cistadenocarcinoma Seroso/metabolismo , Matriz Extracelular/inmunología , Matriz Extracelular/metabolismo , Femenino , Humanos , Inmunosupresores , Ratones , Ratones Endogámicos C57BL , Neoplasias Ováricas/genética , Neoplasias Ováricas/inmunología , Neoplasias Ováricas/metabolismo , Neoplasias Peritoneales/genética , Neoplasias Peritoneales/inmunología , Neoplasias Peritoneales/metabolismo , Pronóstico , Factor de Crecimiento Transformador beta1/genética , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Hepatocyte growth factor-overexpressing mice that harbor a deletion of the Ink4a/p16 locus (HP mice) form melanomas with low metastatic potential in response to UV irradiation. Here we report that these tumors become highly metastatic following hemizygous deletion of the Nme1 and Nme2 metastasis suppressor genes (HPN mice). Whole-genome sequencing of melanomas from HPN mice revealed a striking increase in lung metastatic activity that is associated with missense mutations in eight signature genes (Arhgap35, Atp8b4, Brca1, Ift172, Kif21b, Nckap5, Pcdha2, and Zfp869). RNA-seq analysis of transcriptomes from HP and HPN primary melanomas identified a 32-gene signature (HPN lung metastasis signature) for which decreased expression is strongly associated with lung metastatic potential. Analysis of transcriptome data from The Cancer Genome Atlas revealed expression profiles of these genes that predict improved survival of patients with cutaneous or uveal melanoma. Silencing of three representative HPN lung metastasis signature genes (ARRDC3, NYNRIN, RND3) in human melanoma cells resulted in increased invasive activity, consistent with roles for these genes as mediators of the metastasis suppressor function of NME1 and NME2. In conclusion, our studies have identified a family of genes that mediate suppression of melanoma lung metastasis, and which may serve as prognostic markers and/or therapeutic targets for clinical management of metastatic melanoma.
Asunto(s)
Biomarcadores de Tumor/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundario , Melanoma/genética , Nucleósido Difosfato Quinasas NM23/genética , Rayos Ultravioleta/efectos adversos , Animales , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Factor de Crecimiento de Hepatocito/genética , Humanos , Melanoma/etiología , Ratones , Mutación Missense , Análisis de Secuencia de ARN , Análisis de Supervivencia , Secuenciación Completa del GenomaRESUMEN
nm23-h1 is a well-documented metastasis suppressor gene whose mechanism(s) of action have yet to be fully elucidated. The purpose of this report is to discuss recent advances in investigating the potential role of a novel 3'-5' exonuclease activity identified recently in our laboratory, a biochemical function associated, in general, with DNA repair and replication. We have employed a site-directed mutagenesis approach to demonstrate that the 3'-5' exonuclease activity of NM23-H1 is required for its metastasis suppressor function. Consistent with a role in DNA repair, we also observe that the single yeast NM23 homolog (YNK1) is required for the maintenance of genomic integrity and normal kinetics of DNA repair in response to exposure to ultraviolet radiation. These results and their implications for understanding the molecular mechanisms underlying NM23-H1 functions in cancer are discussed.
Asunto(s)
Reparación del ADN , Exonucleasas/genética , Genes Supresores de Tumor , Nucleósido Difosfato Quinasas NM23/genética , Metástasis de la Neoplasia/genética , Línea Celular Tumoral , Exonucleasas/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Mutagénesis Sitio-Dirigida , Mutación/genética , Nucleósido Difosfato Quinasas NM23/metabolismo , Metástasis de la Neoplasia/prevención & controlRESUMEN
Recent insights supporting the fallopian tube epithelium (FTE) and serous tubal intraepithelial carcinomas (STIC) as the tissue of origin and the precursor lesion, respectively, for the majority of high-grade serous ovarian carcinomas (HGSOC) provide the necessary context to study the mechanisms that drive the development and progression of HGSOC. Here, we investigate the role of the E3 ubiquitin ligase RNF20 and histone H2B monoubiquitylation (H2Bub1) in serous tumorigenesis and report that heterozygous loss of RNF20 defines the majority of HGSOC tumors. At the protein level, H2Bub1 was lost or downregulated in a large proportion of STIC and invasive HGSOC tumors, implicating RNF20/H2Bub1 loss as an early event in the development of serous ovarian carcinoma. Knockdown of RNF20, with concomitant loss of H2Bub1, was sufficient to enhance cell migration and clonogenic growth of FTE cells. To investigate the mechanisms underlying these effects, we performed ATAC-seq and RNA-seq in RNF20 knockdown FTE cell lines. Loss of RNF20 and H2Bub1 was associated with a more open chromatin conformation, leading to upregulation of immune signaling pathways, including IL6. IL6 was one of the key cytokines significantly upregulated in RNF20- and H2Bub1-depleted FTE cells and imparted upon these cells an enhanced migratory phenotype. These studies provide mechanistic insight into the observed oncogenic phenotypes triggered by the early loss of H2Bub1. SIGNIFICANCE: Loss of RNF20 and H2Bub1 contributes to transformation of the fallopian tube epithelium and plays a role in the initiation and progression of high-grade serous ovarian cancer.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/4/760/F1.large.jpg.
Asunto(s)
Cromatina/metabolismo , Cistadenocarcinoma Seroso/patología , Neoplasias de las Trompas Uterinas/patología , Histonas/metabolismo , Neoplasias Ováricas/patología , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Apoptosis , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Carcinoma Epitelial de Ovario/genética , Carcinoma Epitelial de Ovario/metabolismo , Carcinoma Epitelial de Ovario/patología , Proliferación Celular , Cromatina/genética , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/metabolismo , Progresión de la Enfermedad , Neoplasias de las Trompas Uterinas/genética , Neoplasias de las Trompas Uterinas/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Histonas/genética , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Pronóstico , Transducción de Señal , Células Tumorales Cultivadas , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
The degree of genetic aberrations characteristic of high-grade serous ovarian cancer (HGSC) makes identification of the molecular features that drive tumor progression difficult. Here, we perform genome-wide RNAi screens and comprehensive expression analysis of cell-surface markers in a panel of HGSC cell lines to identify genes that are critical to their survival. We report that the tetraspanin CD151 contributes to survival of a subset of HGSC cell lines associated with a ZEB transcriptional program and supports the growth of HGSC tumors. Moreover, we show that high CD151 expression is prognostic of poor clinical outcome. This study reveals cell-surface vulnerabilities associated with HGSC, provides a framework for identifying therapeutic targets, and reports a role for CD151 in HGSC.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Membrana Celular/metabolismo , Cistadenocarcinoma Seroso/metabolismo , Cistadenocarcinoma Seroso/patología , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Tetraspanina 24/metabolismo , Línea Celular Tumoral , Supervivencia Celular , Células Epiteliales/metabolismo , Femenino , Redes Reguladoras de Genes , Humanos , Clasificación del Tumor , Fenotipo , Pronóstico , Ensayos Antitumor por Modelo de Xenoinjerto , Caja Homeótica 2 de Unión a E-Box con Dedos de Zinc/metabolismo , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/metabolismoRESUMEN
Purpose: Ovarian cancer is the leading cause of death from gynecologic malignancy in the United States, with high rates of recurrence and eventual resistance to cytotoxic chemotherapy. Model systems that allow for accurate and reproducible target discovery and validation are needed to support further drug development in this disease.Experimental Design: Clinically annotated patient-derived xenograft (PDX) models were generated from tumor cells isolated from the ascites or pleural fluid of patients undergoing clinical procedures. Models were characterized by IHC and by molecular analyses. Each PDX was luciferized to allow for reproducible in vivo assessment of intraperitoneal tumor burden by bioluminescence imaging (BLI). Plasma assays for CA125 and human LINE-1 were developed as secondary tests of in vivo disease burden.Results: Fourteen clinically annotated and molecularly characterized luciferized ovarian PDX models were generated. Luciferized PDX models retain fidelity to both the nonluciferized PDX and the original patient tumor, as demonstrated by IHC, array CGH, and targeted and whole-exome sequencing analyses. Models demonstrated diversity in specific genetic alterations and activation of PI3K signaling pathway members. Response of luciferized PDX models to standard-of-care therapy could be reproducibly monitored by BLI or plasma markers.Conclusions: We describe the establishment of a collection of 14 clinically annotated and molecularly characterized luciferized ovarian PDX models in which orthotopic tumor burden in the intraperitoneal space can be followed by standard and reproducible methods. This collection is well suited as a platform for proof-of-concept efficacy and biomarker studies and for validation of novel therapeutic strategies in ovarian cancer. Clin Cancer Res; 23(5); 1263-73. ©2016 AACR.
Asunto(s)
Antineoplásicos/uso terapéutico , Neoplasias Glandulares y Epiteliales/tratamiento farmacológico , Neoplasias Glandulares y Epiteliales/genética , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genética , Ensayos Antitumor por Modelo de Xenoinjerto/métodos , Animales , Ascitis/patología , Antígeno Ca-125/sangre , Carcinoma Epitelial de Ovario , Línea Celular Tumoral , Modelos Animales de Enfermedad , Humanos , Elementos de Nucleótido Esparcido Largo/genética , Proteínas de la Membrana/sangre , Ratones , Neoplasias Glandulares y Epiteliales/sangre , Neoplasias Glandulares y Epiteliales/patología , Neoplasias Ováricas/sangre , Neoplasias Ováricas/patología , Transducción de Señal/efectos de los fármacosRESUMEN
High-grade serous ovarian carcinoma (HGSOC) is the most frequent type of ovarian cancer and has a poor outcome. It has been proposed that fallopian tube cancers may be precursors of HGSOC but evolutionary evidence for this hypothesis has been limited. Here, we perform whole-exome sequence and copy number analyses of laser capture microdissected fallopian tube lesions (p53 signatures, serous tubal intraepithelial carcinomas (STICs), and fallopian tube carcinomas), ovarian cancers, and metastases from nine patients. The majority of tumor-specific alterations in ovarian cancers were present in STICs, including those affecting TP53, BRCA1, BRCA2 or PTEN. Evolutionary analyses reveal that p53 signatures and STICs are precursors of ovarian carcinoma and identify a window of 7 years between development of a STIC and initiation of ovarian carcinoma, with metastases following rapidly thereafter. Our results provide insights into the etiology of ovarian cancer and have implications for prevention, early detection and therapeutic intervention of this disease.
Asunto(s)
Cistadenocarcinoma Seroso/genética , Neoplasias de las Trompas Uterinas/patología , Trompas Uterinas/patología , Neoplasias Quísticas, Mucinosas y Serosas/patología , Neoplasias Ováricas/genética , Alelos , Variaciones en el Número de Copia de ADN/genética , Neoplasias de las Trompas Uterinas/metabolismo , Trompas Uterinas/metabolismo , Femenino , Humanos , Inmunohistoquímica , Captura por Microdisección con Láser , Neoplasias Quísticas, Mucinosas y Serosas/metabolismoRESUMEN
High-grade serous ovarian carcinoma (HGS-OvCa) harbors p53 mutations and can originate from the epithelial cell compartment of the fallopian tube fimbriae. From this site, neoplastic cells detach, survive in the peritoneal cavity, and form cellular clusters that intercalate into the mesothelium to form ovarian and peritoneal masses. To examine the contribution of mutant p53 to phenotypic alterations associated with HGS-OvCA, we developed live-cell microscopy assays that recapitulate these early events in cultured fallopian tube nonciliated epithelial (FNE) cells. Expression of stabilizing mutant variants of p53, but not depletion of endogenous wild-type p53, in FNE cells promoted survival and cell-cell aggregation under conditions of cell detachment, leading to the formation of cell clusters with mesothelium-intercalation capacity. Mutant p53R175H-induced phenotypes were dependent on fibronectin production, α5ß1 fibronectin receptor engagement, and TWIST1 expression. These results indicate that FNE cells expressing stabilizing p53 mutants acquire anchorage independence and subsequent mesothelial intercalation capacity through a mechanism involving mesenchymal transition and matrix production. These findings provide important new insights into activities of mutant p53 in the cells of origin of HGS-OvCa.
RESUMEN
The NME1 gene represents the prototypical metastasis suppressor, whose expression inhibits cell motility and metastasis without impact on primary tumor growth in a number of different human cancers. This report outlines our recent efforts to define the molecular mechanisms through which NME1 both suppresses cell motility and promotes genomic integrity in the setting of human melanoma. Forced NME1 expression in a variety of melanoma-derived cell lines was shown to induce dynamic changes in cell morphology and reorganization of the actin cytoskeleton, with formation of a network of thick stress fibers and assembly of fibronectin fibrils at large focal adhesions. Moreover, NME1 expression results in adhesion reprogramming through an impact on integrin repertoire and focal adhesion dynamics. Having previously demonstrated that NME1 expression promotes repair of DNA damage induced by ultraviolet radiation (UVR) in both yeast and mammalian cells, probably via the nucleotide excision repair pathway, we have more recently demonstrated that NME1 is rapidly recruited to double-strand breaks. This preliminary result represents the first evidence of direct interactions between NME1 and DNA in the context of DNA repair and has set the stage for current efforts to probe its functional interactions with double-strand break repair pathways. Discussed herein are molecular models to explain the interactions of NME1 with such diverse cellular functions as cell motility and DNA repair, potentially through its nucleoside diphosphate kinase and 3'-5' exonuclease activities.
Asunto(s)
Melanoma , Nucleósido Difosfato Quinasas NM23/metabolismo , Neoplasias Cutáneas , Actinas/metabolismo , Animales , Movimiento Celular , Reparación del ADN , Adhesiones Focales , Inestabilidad Genómica , Humanos , Melanoma/genética , Melanoma/metabolismo , Melanoma/patología , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patologíaRESUMEN
Cutaneous malignant melanoma is the most lethal form of skin cancer, with 5-year survival rates of <5 % for patients presenting with metastatic disease. Mechanisms underlying metastatic spread of UVR-induced melanoma are not well understood, in part due to a paucity of animal models that accurately recapitulate the disease in its advanced forms. We have employed a transgenic mouse strain harboring a tandem deletion of the nm23-m1 and nm23-m2 genes to assess the combined contribution of these genes to suppression of melanoma metastasis. Crossing of the nm23-h1/nm23-h2 knockout in hemizygous-null form ([m1m2](+/-)) to a transgenic mouse strain (hepatocyte growth factor/scatter factor-overexpressing, or HGF(+) strain) vulnerable to poorly-metastatic, UVR-induced melanomas resulted in UVR-induced melanomas with high metastatic potential. Metastasis to draining lymph nodes was seen in almost all cases of back skin melanomas, while aggressive metastasis to lung, thoracic cavity, liver and bone also occurred. Interestingly, no differences were observed in the invasive characteristics of primary melanomas of HGF(+) and HGF(+) × [m1m2](+/-) strains, with both exhibiting invasion into the dermis and subcutis, indicating factors other than simple invasive activity were responsible for metastasis of HGF(+) × [m1m2](+/-) melanomas. Stable cell lines were established from the primary and metastatic melanoma lesions from these mice, with HGF(+) × [m1m2](+/-) lines exhibiting increased single cell migration and genomic instability. These studies demonstrate for the first time in vivo a potent metastasis suppressor activity of NM23 in UVR-induced melanoma, and have provided new tools for identifying molecular mechanisms that underlie melanoma metastasis.
Asunto(s)
Modelos Animales de Enfermedad , Inestabilidad Genómica , Melanoma/etiología , Nucleósido Difosfato Quinasas NM23/fisiología , Neoplasias Cutáneas/etiología , Rayos Ultravioleta/efectos adversos , Animales , Movimiento Celular , Daño del ADN/genética , Reparación del ADN/genética , Femenino , Factor de Crecimiento de Hepatocito/genética , Humanos , Hipoxantina Fosforribosiltransferasa/genética , Masculino , Melanoma/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación/genética , Neoplasias Cutáneas/secundario , Células Tumorales Cultivadas , Cicatrización de HeridasRESUMEN
Reduced expression of the metastasis suppressor NM23-H1 is associated with aggressive forms of multiple cancers. Here, we establish that NM23-H1 (termed H1 isoform in human, M1 in mouse) and two of its attendant enzymatic activities, the 3'-5' exonuclease and nucleoside diphosphate kinase, are novel participants in the cellular response to UV radiation (UVR)-induced DNA damage. NM23-H1 deficiency compromised the kinetics of repair for total DNA polymerase-blocking lesions and nucleotide excision repair of (6-4) photoproducts in vitro. Kinase activity of NM23-H1 was critical for rapid repair of both polychromatic UVB/UVA-induced (290-400 nm) and UVC-induced (254 nm) DNA damage, whereas its 3'-5' exonuclease activity was dominant in the suppression of UVR-induced mutagenesis. Consistent with its role in DNA repair, NM23-H1 rapidly translocated to sites of UVR-induced (6-4) photoproduct DNA damage in the nucleus. In addition, transgenic mice hemizygous-null for nm23-m1 and nm23-m2 exhibited UVR-induced melanoma and follicular infundibular cyst formation, and tumor-associated melanocytes displayed invasion into adjacent dermis, consistent with loss of invasion-suppressing activity of NM23 in vivo. Taken together, our data show a critical role for NM23 isoforms in limiting mutagenesis and suppressing UVR-induced melanomagenesis.
Asunto(s)
Daño del ADN , Melanoma Experimental/prevención & control , Nucleósido Difosfato Quinasas NM23/fisiología , Neoplasias Inducidas por Radiación/prevención & control , Rayos Ultravioleta , Animales , Línea Celular Tumoral , Hipoxantina Fosforribosiltransferasa/genética , Melanoma Experimental/etiología , Ratones , Ratones Endogámicos C57BL , Mutación , Nucleósido Difosfato Quinasas NM23/genéticaRESUMEN
ErbB2+ human breast cancer is a major clinical problem. Prior results have suggested that tetraspanin CD151 might contribute to ErbB2-driven breast cancer growth, survival, and metastasis. In other cancer types, CD151 sometimes supports tumor growth and metastasis. However, a definitive test of CD151 effects on de novo breast cancer initiation, growth, and metastasis has not previously been done. We used CD151 gene-deleted mice expressing the MMTV-ErbB2 transgene to show that CD151 strongly supports ErbB2+ mammary tumor initiation and metastasis. Delayed tumor onset (by 70-100 days) in the absence of CD151 was accompanied by reduced survival of mammary epithelial cells and impaired activation of FAK- and MAPK-dependent pathways. Both primary tumors and metastatic nodules showed smooth, regular borders, consistent with a less invasive phenotype. Furthermore, consistent with impaired oncogenesis and decreased metastasis, CD151-targeted MCF-10A/ErbB2 cells showed substantial decreases in three-dimensional colony formation, EGF-stimulated tumor cell motility, invasion, and transendothelial migration. These CD151-dependent functions were largely mediated through α6ß4 integrin. Moreover, CD151 ablation substantially prevented PKC- and EGFR/ERK-dependent α6ß4 integrin phosphorylation, consistent with retention of epithelial cell polarity and intermediate filament cytoskeletal connections, which helps to explain diminished metastasis. Finally, clinical data analyses revealed a strong correlation between CD151 and ErbB2 expression and metastasis-free survival of breast cancer patients. In conclusion, we provide strong evidence that CD151 collaborates with LB integrins (particularly α6ß4 and ErbB2 (and EGFR) receptors to regulate multiple signaling pathways, thereby driving mammary tumor onset, survival, and metastasis. Consequently, CD151 is a useful therapeutic target in malignant ErbB2+ breast cancer.
Asunto(s)
Neoplasias Mamarias Animales/metabolismo , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Metástasis de la Neoplasia , Receptor ErbB-2/metabolismo , Tetraspanina 24/metabolismo , Animales , Butadienos/farmacología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Factor de Crecimiento Epidérmico/farmacología , Femenino , Quinasa 1 de Adhesión Focal/metabolismo , Humanos , Integrina alfa6beta4/metabolismo , Lapatinib , Glándulas Mamarias Animales/metabolismo , Neoplasias Mamarias Animales/mortalidad , Neoplasias Mamarias Animales/patología , Ratones , Ratones Noqueados , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Invasividad Neoplásica , Nitrilos/farmacología , Fosforilación/genética , Quinazolinas/farmacología , Receptor ErbB-2/antagonistas & inhibidores , Tetraspanina 24/genética , Migración Transendotelial y Transepitelial/efectos de los fármacosRESUMEN
nm23-h1 was the first metastasis suppressor gene to be identified in humans, with early studies demonstrating its ability to inhibit the metastatic potential of breast carcinoma and melanoma cell lines. This report outlines recent findings from our laboratory indicating that the metastasis suppressor function of NM23-H1 in human melanoma involves a spectrum of molecular mechanisms. Analysis of NM23-H1-dependent profiles of gene expression in human melanoma cell lines has identified a host of target genes that appear to mediate suppression of directional motility. Of particular interest is a subset of motility-suppressing genes whose regulation by NM23-H1 is independent of its known kinase and 3'-5' exonuclease activities. In parallel, we have recently observed that NM23-H1 expression appears to be required for genomic stability and for optimal repair of DNA damage produced by ultraviolet radiation and other agents. Thus, NM23-H1 might oppose not only the motile and invasive characteristics of metastatic cells but also the acquisition of mutations that drive malignant progression to the metastatic phenotype itself.