RESUMEN

Identification of conserved co-expression networks is a useful tool for clustering groups of genes enriched for common molecular or cellular functions [1]. The relative importance of genes within networks can frequently be inferred by the degree of connectivity, with those displaying high connectivity being significantly more likely to be associated with specific molecular functions [2]. Previously we utilized cross-species network analysis to identify two network modules that were significantly associated with distant metastasis free survival in breast cancer. Here, we validate one of the highly connected genes as a metastasis associated gene. Tpx2, the most highly connected gene within a proliferation network specifically prognostic for estrogen receptor positive (ER+) breast cancers, enhances metastatic disease, but in a tumor autonomous, proliferation-independent manner. Histologic analysis suggests instead that variation of TPX2 levels within disseminated tumor cells may influence the transition between dormant to actively proliferating cells in the secondary site. These results support the co-expression network approach for identification of new metastasis-associated genes to provide new information regarding the etiology of breast cancer progression and metastatic disease.

Asunto(s)

Neoplasias de la Mama/genética , Redes Reguladoras de Genes , Neoplasias Pulmonares/genética , Animales , Apoptosis , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Transición Epitelial-Mesenquimal , Femenino , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundario , Ratones , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Trasplante de Neoplasias , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Pronóstico , Transcriptoma , Carga Tumoral

RESUMEN

Metastasis is the result of stochastic genomic and epigenetic events leading to gene expression profiles that drive tumor dissemination. Here we exploit the principle that metastatic propensity is modified by the genetic background to generate prognostic gene expression signatures that illuminate regulators of metastasis. We also identify multiple microRNAs whose germline variation is causally linked to tumor progression and metastasis. We employ network analysis of global gene expression profiles in tumors derived from a panel of recombinant inbred mice to identify a network of co-expressed genes centered on Cnot2 that predicts metastasis-free survival. Modulating Cnot2 expression changes tumor cell metastatic potential in vivo, supporting a functional role for Cnot2 in metastasis. Small RNA sequencing of the same tumor set revealed a negative correlation between expression of the Mir216/217 cluster and tumor progression. Expression quantitative trait locus analysis (eQTL) identified cis-eQTLs at the Mir216/217 locus, indicating that differences in expression may be inherited. Ectopic expression of Mir216/217 in tumor cells suppressed metastasis in vivo. Finally, small RNA sequencing and mRNA expression profiling data were integrated to reveal that miR-3470a/b target a high proportion of network transcripts. In vivo analysis of Mir3470a/b demonstrated that both promote metastasis. Moreover, Mir3470b is a likely regulator of the Cnot2 network as its overexpression down-regulated expression of network hub genes and enhanced metastasis in vivo, phenocopying Cnot2 knockdown. The resulting data from this strategy identify Cnot2 as a novel regulator of metastasis and demonstrate the power of our systems-level approach in identifying modifiers of metastasis.

Asunto(s)

Regulación Neoplásica de la Expresión Génica/genética , Metástasis de la Neoplasia/genética , Neoplasias/genética , Proteínas Represoras/genética , Animales , Redes Reguladoras de Genes , Predisposición Genética a la Enfermedad , Humanos , Ratones , MicroARNs/genética , Metástasis de la Neoplasia/patología , Neoplasias/patología , Sitios de Carácter Cuantitativo/genética , ARN Mensajero/genética

RESUMEN

Recent studies suggest that BET inhibitors are effective anti-cancer therapeutics. Here we show that BET inhibitors are effective against murine primary mammary tumors, but not pulmonary metastases. BRD4, a target of BET inhibitors, encodes two isoforms with opposite effects on tumor progression. To gain insights into why BET inhibition was ineffective against metastases the pro-metastatic short isoform of BRD4 was characterized using mass spectrometry and cellular fractionation. Our data show that the pro-metastatic short isoform interacts with the LINC complex and the metastasis-associated proteins RRP1B and SIPA1 at the inner face of the nuclear membrane. Furthermore, histone binding arrays revealed that the short isoform has a broader acetylated histone binding pattern relative to the long isoform. These differential biochemical and nuclear localization properties revealed in our study provide novel insights into the opposing roles of BRD4 isoforms in metastatic breast cancer progression.

Asunto(s)

Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Animales , Proteínas de Ciclo Celular , Línea Celular Tumoral , Núcleo Celular/metabolismo , Modelos Animales de Enfermedad , Femenino , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Histonas/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , Acetiltransferasa E N-Terminal/metabolismo , Acetiltransferasas N-Terminal , Metástasis de la Neoplasia , Neoplasias/metabolismo , Neoplasias/patología , Proteínas Nucleares/genética , Unión Proteica , Isoformas de Proteínas , Transporte de Proteínas , Factores de Transcripción/genética , Carga Tumoral/efectos de los fármacos

RESUMEN

Recent advances in genome wide transcriptional analysis have provided greater insights into the etiology and heterogeneity of breast cancer. Molecular signatures have been developed that stratify the conventional estrogen receptor positive or negative categories into subtypes that are associated with differing clinical outcomes. It is thought that the expression patterns of the molecular subtypes primarily reflect cell-of-origin or tumor driver mutations. In this study however, using a genetically engineered mouse mammary tumor model we demonstrate that the PAM50 subtype signature of tumors driven by a common oncogenic event can be significantly influenced by the genetic background on which the tumor arises. These results have important implications for interpretation of "snapshot" expression profiles, as well as suggesting that incorporation of genetic background effects may allow investigation into phenotypes not initially anticipated in individual mouse models of cancer.

Asunto(s)

Neoplasias Pulmonares/genética , Neoplasias Mamarias Experimentales/genética , Transcriptoma , Animales , Animales no Consanguíneos , Femenino , Estudios de Asociación Genética , Sitios Genéticos , Predisposición Genética a la Enfermedad , Haplotipos , Humanos , Queratinas/metabolismo , Neoplasias Pulmonares/clasificación , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundario , Masculino , Neoplasias Mamarias Experimentales/clasificación , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Transgénicos

RESUMEN

The metastatic cascade is a complex and extremely inefficient process with many potential barriers. Understanding this process is of critical importance because the majority of cancer mortality is associated with metastatic disease. Recently, it has become increasingly clear that microRNAs (miRNA) play important roles in tumorigenesis and metastasis, yet few studies have examined how germline variations may dysregulate miRNAs, in turn affecting metastatic potential. To explore this possibility, the highly metastatic MMTV-PyMT mice were crossed with 25 AKXD (AKR/J × DBA/2J) recombinant inbred strains to produce F1 progeny with varying metastatic indices. When mammary tumors from the F1 progeny were analyzed by miRNA microarray, miR-290 (containing miR-290-3p and miR-290-5p) was identified as a top candidate progression-associated miRNA. The microarray results were validated in vivo when miR-290 upregulation in two independent breast cancer cell lines suppressed both primary tumor and metastatic growth. Computational analysis identified breast cancer progression gene Arid4b as a top target of miR-290-3p, which was confirmed by luciferase reporter assay. Surprisingly, pathway analysis identified estrogen receptor (ER) signaling as the top canonical pathway affected by miR-290 upregulation. Further analysis showed that ER levels were elevated in miR-290-expressing tumors and positively correlated with apoptosis. Taken together, our results suggest miR-290 targets Arid4b while simultaneously enhancing ER signaling and increasing apoptosis, thereby suppressing breast cancer progression. This, to the best of our knowledge, is the first example of inherited differences in miRNA expression playing a role in breast cancer progression.

Asunto(s)

Neoplasias de la Mama/genética , Proteínas de Unión al ADN/genética , Regulación Neoplásica de la Expresión Génica , Variación Genética , MicroARNs/genética , Animales , Apoptosis/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Progresión de la Enfermedad , Femenino , Humanos , Ratones , Ratones Transgénicos , Metástasis de la Neoplasia/genética , Fenotipo , Receptores de Estrógenos/genética , Receptores de Estrógenos/metabolismo , Transducción de Señal

RESUMEN

Metastasis is a complex process utilizing both tumor-cell-autonomous properties and host-derived factors, including cellular immunity. We have previously shown that germline polymorphisms can modify tumor cell metastatic capabilities through cell-autonomous mechanisms. However, how metastasis susceptibility genes interact with the tumor stroma is incompletely understood. Here, we employ a complex genetic screen to identify Cadm1 as a novel modifier of metastasis. We demonstrate that Cadm1 can specifically suppress metastasis without affecting primary tumor growth. Unexpectedly, Cadm1 did not alter tumor-cell-autonomous properties such as proliferation or invasion, but required the host's adaptive immune system to affect metastasis. The metastasis-suppressing effect of Cadm1 was lost in mice lacking T cell-mediated immunity, which was partially phenocopied by depleting CD8(+) T cells in immune-competent mice. Our data show a novel function for Cadm1 in suppressing metastasis by sensitizing tumor cells to immune surveillance mechanisms, and this is the first report of a heritable metastasis susceptibility gene engaging tumor non-autonomous factors.

Asunto(s)

Moléculas de Adhesión Celular/genética , Genes Supresores de Tumor , Inmunidad Celular/genética , Inmunoglobulinas/genética , Metástasis de la Neoplasia/genética , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/terapia , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Molécula 1 de Adhesión Celular , Moléculas de Adhesión Celular/metabolismo , Proliferación Celular , Femenino , Regulación Neoplásica de la Expresión Génica , Predisposición Genética a la Enfermedad , Humanos , Inmunoglobulinas/metabolismo , Estimación de Kaplan-Meier , Ratones , Ratones Transgénicos , Invasividad Neoplásica/genética , Metástasis de la Neoplasia/inmunología , Pronóstico

RESUMEN

Accumulating evidence suggests that breast cancer metastatic progression is modified by germline polymorphism, although specific modifier genes have remained largely undefined. In the current study, we employ the MMTV-PyMT transgenic mouse model and the AKXD panel of recombinant inbred mice to identify AT-rich interactive domain 4B (Arid4b; NM_194262) as a breast cancer progression modifier gene. Ectopic expression of Arid4b promoted primary tumor growth in vivo as well as increased migration and invasion in vitro, and the phenotype was associated with polymorphisms identified between the AKR/J and DBA/2J alleles as predicted by our genetic analyses. Stable shRNA-mediated knockdown of Arid4b caused a significant reduction in pulmonary metastases, validating a role for Arid4b as a metastasis modifier gene. ARID4B physically interacts with the breast cancer metastasis suppressor BRMS1, and we detected differential binding of the Arid4b alleles to histone deacetylase complex members mSIN3A and mSDS3, suggesting that the mechanism of Arid4b action likely involves interactions with chromatin modifying complexes. Downregulation of the conserved Tpx2 gene network, which is comprised of many factors regulating cell cycle and mitotic spindle biology, was observed concomitant with loss of metastatic efficiency in Arid4b knockdown cells. Consistent with our genetic analysis and in vivo experiments in our mouse model system, ARID4B expression was also an independent predictor of distant metastasis-free survival in breast cancer patients with ER+ tumors. These studies support a causative role of ARID4B in metastatic progression of breast cancer.

Asunto(s)

Movimiento Celular/genética , Proteínas de Unión al ADN/genética , Neoplasias Mamarias Animales/genética , Proteínas Represoras/genética , Alelos , Animales , Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Neoplasias Mamarias Animales/metabolismo , Neoplasias Mamarias Animales/patología , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , Metástasis de la Neoplasia , Proteínas Nucleares/metabolismo , Proteínas Represoras/metabolismo , Transducción de Señal/genética , Complejo Correpresor Histona Desacetilasa y Sin3

RESUMEN

The bromodomain-containing chromatin-modifying factor BRD4 is an inherited susceptibility gene for breast cancer progression and metastasis, but its functionality in these settings has yet to be explored. Here we show that deletion of either of the BRD4 bromodomains had modest effects on the metastatic suppression ability of BRD4. In contrast, expression of the natural short isoform of BRD4 that truncates the protein after the SEED domain restored progression and metastatic capacity. Unexpectedly, deletion of the proline-rich region induced mesenchymal-like conversion and acquisition of cancer stem cell-like properties, which are mediated by the carboxy-terminal P-TEFb binding domain. Deletion of this proline-rich region also induced a gene expression signature that predicted poor outcome in human breast cancer data sets and that overlapped G3 grade human breast tumors. Thus our findings suggest that BRD4 may be altering the predisposition of tumors to undergo conversion to a more de-differentiated or primitive state during metastatic progression.

Asunto(s)

Eliminación de Gen , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/patología , Células Madre Neoplásicas/patología , Proteínas Nucleares/genética , Dominios Proteicos Ricos en Prolina/genética , Factores de Transcripción/genética , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Procesos de Crecimiento Celular/genética , Transición Epitelial-Mesenquimal/genética , Femenino , Predisposición Genética a la Enfermedad , Humanos , Ratones , Metástasis de la Neoplasia , Transcripción Genética

RESUMEN

In cell lines from advanced lung cancer, breast cancer, and melanoma, endogenous tensin-3 contributes to cell migration, anchorage-independent growth, and tumorigenesis. Although SH2 domains have not been reported previously to be phosphorylated, the tensin-3 SH2 domain is a physiologic substrate for Src. Tyrosines in the SH2 domain contribute to the biological activity of tensin-3, and phosphorylation of these tyrosines can regulate ligand binding. In a mouse breast cancer model, tensin-3 tyrosines are phosphorylated in a Src-associated manner in primary tumors, and experimental metastases induced by tumor-derived cell lines depend on endogenous tensin-3. Thus, tensin-3 is implicated as an oncoprotein regulated by Src and possessing an SH2 domain with a previously undescribed mechanism for the regulation of ligand binding.

Asunto(s)

Metástasis de la Neoplasia , Neoplasias/etiología , Fosfoproteínas Fosfatasas/química , Fosfoproteínas Fosfatasas/metabolismo , Animales , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Femenino , Humanos , Ratones , Ratones Endogámicos NOD , Ratones Endogámicos , Ratones SCID , Fosfoproteínas Fosfatasas/genética , Fosforilación , ARN Interferente Pequeño/metabolismo , Tensinas , Transfección , Dominios Homologos src

RESUMEN

Previous work identified the Rap1 GTPase-activating protein Sipa1 as a germ-line-encoded metastasis modifier. The bromodomain protein Brd4 physically interacts with and modulates the enzymatic activity of Sipa1. In vitro analysis of a highly metastatic mouse mammary tumor cell line ectopically expressing Brd4 demonstrates significant reduction of invasiveness without altering intrinsic growth rate. However, a dramatic reduction of tumor growth and pulmonary metastasis was observed after s.c. implantation into mice, implying that activation of Brd4 may somehow be manipulating response to tumor microenvironment in the in vivo setting. Further in vitro analysis shows that Brd4 modulates extracellular matrix gene expression, a class of genes frequently present in metastasis-predictive gene signatures. Microarray analysis of the mammary tumor cell lines identified a Brd4 activation signature that robustly predicted progression and/or survival in multiple human breast cancer datasets analyzed on different microarray platforms. Intriguingly, the Brd4 signature also almost perfectly matches a molecular classifier of low-grade tumors. Taken together, these data suggest that dysregulation of Brd4-associated pathways may play an important role in breast cancer progression and underlies multiple common prognostic signatures.

Asunto(s)

Neoplasias de la Mama/patología , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Animales , Neoplasias de la Mama/genética , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Bases de Datos Genéticas , Matriz Extracelular/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Ganglios Linfáticos/patología , Neoplasias Mamarias Animales/genética , Neoplasias Mamarias Animales/patología , Invasividad Neoplásica , Metástasis de la Neoplasia , Análisis de Secuencia por Matrices de Oligonucleótidos , Receptores de Estrógenos/metabolismo , Análisis de Supervivencia

RESUMEN

Microarray expression signature analyses have suggested that extracellular matrix (ECM) gene dysregulation is predictive of metastasis in both mouse mammary tumorigenesis and human breast cancer. We have previously demonstrated that such ECM dysregulation is influenced by hereditary germline-encoded variation. To identify novel metastasis efficiency modifiers, we performed expression QTL (eQTL) mapping in recombinant inbred mice by characterizing genetic loci modulating metastasis-predictive ECM gene expression. Three reproducible eQTLs were observed on chromosomes 7, 17 and 18. Candidate genes were identified by correlation analyses and known associations with metastasis. Seven candidates were identified (Ndn, Pi16, Luc7l, Rrp1b, Brd4, Centd3 and Csf1r). Stable transfection of the highly metastatic Mvt-1 mouse mammary tumor cell line with expression vectors encoding each candidate modulated metastasis-predictive ECM gene expression. Implantation of these cells into mice demonstrated that candidate gene ectopic expression impacts tumor progression. Gene expression analyses facilitated the construction of a transcriptional network that we have termed the 'Diasporin Pathway'. This pathway contains the seven candidates, as well as metastasis-predictive ECM genes and metastasis suppressors. Brd4 and Rrp1b appear to form a central node within this network, which likely is a consequence of their physical interaction with the metastasis efficiency modifier Sipa1. Furthermore, we demonstrate that the microarray gene expression signatures induced by activation of ECM eQTL genes in the Mvt-1 cell line can be used to accurately predict survival in a human breast cancer cohort. These data imply that the Diasporin Pathway may be an important nexus in tumor progression in both mice and humans.

Asunto(s)

Neoplasias de la Mama/mortalidad , Redes Reguladoras de Genes , Sitios de Carácter Cuantitativo , Transducción de Señal , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Progresión de la Enfermedad , Matriz Extracelular/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Metástasis de la Neoplasia

RESUMEN

A novel candidate metastasis modifier, ribosomal RNA processing 1 homolog B (Rrp1b), was identified through two independent approaches. First, yeast two-hybrid, immunoprecipitation, and functional assays demonstrated a physical and functional interaction between Rrp1b and the previous identified metastasis modifier Sipa1. In parallel, using mouse and human metastasis gene expression data it was observed that extracellular matrix (ECM) genes are common components of metastasis predictive signatures, suggesting that ECM genes are either important markers or causal factors in metastasis. To investigate the relationship between ECM genes and poor prognosis in breast cancer, expression quantitative trait locus analysis of polyoma middle-T transgene-induced mammary tumor was performed. ECM gene expression was found to be consistently associated with Rrp1b expression. In vitro expression of Rrp1b significantly altered ECM gene expression, tumor growth, and dissemination in metastasis assays. Furthermore, a gene signature induced by ectopic expression of Rrp1b in tumor cells predicted survival in a human breast cancer gene expression dataset. Finally, constitutional polymorphism within RRP1B was found to be significantly associated with tumor progression in two independent breast cancer cohorts. These data suggest that RRP1B may be a novel susceptibility gene for breast cancer progression and metastasis.

Asunto(s)

Proteínas Reguladoras de la Apoptosis/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Proteínas Cromosómicas no Histona/genética , Proteínas Nucleares/genética , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Baltimore , Proteínas Cromosómicas no Histona/metabolismo , Estudios de Cohortes , Progresión de la Enfermedad , Susceptibilidad a Enfermedades , Matriz Extracelular/genética , Femenino , Proteínas Activadoras de GTPasa/metabolismo , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Haplotipos , Humanos , Ratones , Ratones Endogámicos , Proteínas Mutantes/metabolismo , Metástasis de la Neoplasia , Proteínas Nucleares/metabolismo , Polimorfismo Genético , Regiones Promotoras Genéticas , Unión Proteica , Sitios de Carácter Cuantitativo , Análisis de Supervivencia , Resultado del Tratamiento