RESUMEN
BACKGROUND: Follistatin (FST) is an intrinsic inhibitor of activin, a member of the transforming growth factor-ß superfamily of ligands. The prognostic value of FST and its family members, the follistatin-like (FSTL) proteins, have been studied in various cancers. However, these studies, as well as limited functional analyses of the FSTL proteins, have yielded conflicting results on the role of these proteins in disease progression. Furthermore, very few have been focused on FST itself. We assessed whether FST may be a suppressor of tumorigenesis and/or metastatic progression in breast cancer. METHODS: Using publicly available gene expression data, we examined the expression patterns of FST and INHBA, a subunit of activin, in normal and cancerous breast tissue and the prognostic value of FST in breast cancer metastases, recurrence-free survival, and overall survival. The functional effects of activin and FST on in vitro proliferation, migration, and invasion of breast cancer cells were also examined. FST overexpression in an autochthonous mouse model of breast cancer was then used to assess the in vivo impact of FST on metastatic progression. RESULTS: Examination of multiple breast cancer datasets revealed that FST expression is reduced in breast cancers compared with normal tissue and that low FST expression predicts increased metastasis and reduced overall survival. FST expression was also reduced in a mouse model of HER2/Neu-induced metastatic breast cancer. We found that FST blocks activin-induced breast epithelial cell migration in vitro, suggesting that its loss may promote breast cancer aggressiveness. To directly determine if FST restoration could inhibit metastatic progression, we transgenically expressed FST in the HER2/Neu model. Although FST had no impact on tumor initiation or growth, it completely blocked the formation of lung metastases. CONCLUSIONS: These data indicate that FST is a bona fide metastasis suppressor in this mouse model and support future efforts to develop an FST mimetic to suppress metastatic progression.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Folistatina/genética , Receptor ErbB-2/genética , Proteínas Supresoras de Tumor/genética , Animales , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/mortalidad , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular , Supervivencia Celular/genética , Bases de Datos Genéticas , Femenino , Folistatina/metabolismo , Humanos , Estimación de Kaplan-Meier , Ratones , Ratones Transgénicos , Metástasis de la Neoplasia , Estadificación de Neoplasias , Pronóstico , Receptor ErbB-2/metabolismo , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Activin is a well-established modulator of male and female reproduction that stimulates the synthesis and secretion of follicle-stimulating hormone. Nonpituitary effects of activin have also been reported, although the paracrine actions of this growth factor in several reproductive tissues are not well understood. To identify the paracrine functions of activin during mammary gland morphogenesis and tumor progression, we produced transgenic mice that overexpress follistatin (FST), an intrinsic inhibitor of activin, under control of the mouse mammary tumor virus (MMTV) promoter. Although the MMTV-Fst mice were constructed to assess the role of activin in females, expression of the transgene was also observed in the testes and epididymides of males. While all 17 transgenic founder males exhibited copulatory behavior and produced vaginal plugs in females, only one produced live offspring. In contrast, transgenic females were fertile, permitting expansion of transgenic mouse lines. Light and transmission electron microscopic examination of the transgenic testes and epididymides revealed impairment of fluid resorption and sperm transit in the efferent ducts and initial segment of the epididymis, as indicated by accumulation of fluid and sperm stasis. Consequently, a variety of degenerative lesions were observed in the seminiferous epithelium, such as vacuolation and early stages of mineralization and fibrosis. Sperm collected from the caudae epididymidis of MMTV-Fst males had detached heads and were immotile. Together, these data reveal that activin signaling is essential for normal testicular excurrent duct function and that its blockade impairs fertility. These results also suggest that selective inhibitors of activin signaling may provide a useful approach for the development of male contraceptives without compromising androgen synthesis and actions.
Asunto(s)
Activinas/metabolismo , Modelos Animales de Enfermedad , Epidídimo/metabolismo , Folistatina/metabolismo , Infertilidad Masculina/metabolismo , Animales , Receptor alfa de Estrógeno/metabolismo , Femenino , Gonadotropinas Hipofisarias/metabolismo , Infertilidad Masculina/patología , Infertilidad Masculina/fisiopatología , Masculino , Virus del Tumor Mamario del Ratón , Ratones , Ratones Transgénicos , Tamaño de los Órganos , Fenotipo , Hipófisis/metabolismo , Regiones Promotoras Genéticas , Testículo/metabolismo , Testículo/patología , Testículo/fisiopatología , Testosterona/sangreRESUMEN
Krüppel-like factor 4 (KLF4) is a zinc finger transcription factor that functions as an oncogene or tumor suppressor in a highly tissue-specific cell-dependent manner. However, its precise role in breast cancer and metastasis remains unclear. Here, we show that transient adenoviral expression of KLF4 in the 4T1 orthotopic mammary cancer model significantly attenuated primary tumor growth as well as micrometastases to the lungs and liver. These results can be attributed, in part, to decreased proliferation and increased apoptosis. Further supporting a tumor-suppressive role for KLF4 in the breast, we found that KLF4 expression is lost in a mouse model of HER2/NEU/ERBB2-positive breast cancer. To determine whether enforced KLF4 expression could alter tumor latency in these mice, we used a doxycycline-inducible expression model in the context of the MMTV-Neu transgene. Surprisingly, tumors that developed in this model also lost KLF4 expression, suggesting negative selection for sustained expression. We have previously reported that KLF4 inhibits epithelial-to-mesenchymal transition (EMT), a preliminary step in metastatic progression. Overexpression of KLF4 in 4T1 cells led to a significant reduction in the expression of Snail, a key mediator of EMT and metastasis. Conversely, KLF4 silencing increased Snail expression in the nontransformed MCF-10A cell line. Collectively, these data demonstrate the first functional, in vivo evidence for KLF4 as a tumor suppressor in breast cancer cells. Furthermore, our findings suggest an inhibitory role for KLF4 during breast cancer metastases that functions, in part, through repression of Snail.
Asunto(s)
Neoplasias de la Mama/patología , Carcinoma/patología , Factores de Transcripción de Tipo Kruppel/fisiología , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Carcinoma/genética , Carcinoma/metabolismo , Células Cultivadas , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Transición Epitelial-Mesenquimal/genética , Transición Epitelial-Mesenquimal/fisiología , Femenino , Regulación Neoplásica de la Expresión Génica , Genes Supresores de Tumor/fisiología , Humanos , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Metástasis de la Neoplasia , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The LIM-only protein, LMO4, is a transcriptional modulator overexpressed in breast cancer. It is oncogenic in murine mammary epithelium and is required for G2/M progression of ErbB2-dependent cells as well as growth and invasion of other breast cancer cell types. However, the mechanisms underlying the oncogenic activity of LMO4 remain unclear. Herein, we show that LMO4 is expressed in all breast cancer subtypes examined and its expression level correlates with the degree of proliferation of such tumours. In addition, we have determined that LMO4 silencing induces G2/M arrest in cells from various breast cancer subtypes, suggesting that LMO4 action in the cell cycle is not restricted to a single breast cancer subtype. This arrest was accompanied by increased cell death, amplification of centrosomes, and formation of abnormal mitotic spindles. Consistent with its ability to positively and negatively regulate the formation of active transcription complexes, overexpression of LMO4 also resulted in an increase in centrosome number. Centrosome amplification has been shown to prolong the G2/M phase of the cell cycle and induce apoptosis; thus, we conclude that supernumerary centrosomes mediate the G2/M arrest and cell death in LMO4-deficient cells. Furthermore, the correlation of centrosome amplification with genomic instability suggests that the impact of dysregulated LMO4 on the centrosome cycle may promote LMO4-induced tumour formation.
Asunto(s)
Neoplasias de la Mama/metabolismo , Centrosoma/patología , Proteínas de Homeodominio/biosíntesis , Huso Acromático/patología , Factores de Transcripción/biosíntesis , Proteínas Adaptadoras Transductoras de Señales , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Ciclo Celular/fisiología , Centrosoma/metabolismo , Femenino , Genes BRCA1 , Proteínas de Homeodominio/genética , Humanos , Proteínas con Dominio LIM , Índice Mitótico , Mutación , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , ARN Mensajero/genética , ARN Neoplásico/genética , Receptores de Estrógenos/deficiencia , Huso Acromático/metabolismo , Factores de Transcripción/genéticaRESUMEN
Breast cancers that overexpress the receptor tyrosine kinase ErbB2/HER2/Neu result in poor patient outcome because of extensive metastatic progression. Herein, we delineate a molecular mechanism that may govern this malignant phenotype. ErbB2 induction of migration requires activation of the small GTPases Rac1 and Cdc42. The ability of ErbB2 to activate these small GTPases necessitated expression of p120 catenin, which is itself up-regulated by signaling through ErbB2 and the tyrosine kinase Src. Silencing p120 in ErbB2-dependent breast cancer cell lines dramatically inhibited migration and invasion as well as activation of Rac1 and Cdc42. In contrast, overexpression of constitutively active mutants of these GTPases reversed the effects of p120 silencing. Lastly, ectopic expression of p120 promoted migration and invasion and potentiated metastatic progression of a weakly metastatic, ErbB2-dependent breast cancer cell line. These results suggest that p120 acts as an obligate intermediate between ErbB2 and Rac1/Cdc42 to modulate the metastatic potential of breast cancer cells.
Asunto(s)
Neoplasias de la Mama/metabolismo , Cateninas/metabolismo , Receptor ErbB-2/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Animales , Western Blotting , Neoplasias de la Mama/genética , Cateninas/genética , Línea Celular , Línea Celular Tumoral , Movimiento Celular/genética , Movimiento Celular/fisiología , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Inmunohistoquímica , Técnicas In Vitro , Ratones , Ratones Desnudos , Receptor ErbB-2/genética , Cicatrización de Heridas/genética , Cicatrización de Heridas/fisiología , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP rac1/genética , Catenina deltaRESUMEN
The Krüppel-like factor 4 (KLF4) is a transcriptional regulator of proliferation and differentiation in epithelial cells, both during development and tumorigenesis. Although KLF4 functions as a tumor suppressor in several tissues, including the colon, the role of KLF4 in breast cancer is less clear. Here, we show that KLF4 is necessary for maintenance of the epithelial phenotype in non-transformed MCF-10A mammary epithelial cells. KLF4 silencing led to alterations in epithelial cell morphology and migration, indicative of an epithelial-to-mesenchymal transition. Consistent with these changes, decreased levels of KLF4 also resulted in the loss of E-cadherin protein and mRNA. Promoter/reporter analyses revealed decreased E-cadherin promoter activity with KLF4 silencing, while chromatin immunoprecipitation identified endogenous KLF4 binding to the GC-rich/E-box region of this promoter. Furthermore, forced expression of KLF4 in the highly metastatic MDA-MB-231 breast tumor cell line was sufficient to restore E-cadherin expression and suppress migration and invasion. These findings identify E-cadherin as a novel transcriptional target of KLF4. The clear requirement for KLF4 to maintain E-cadherin expression and prevent epithelial-to-mesenchymal transition in mammary epithelial cells supports a metastasis suppressive role for KLF4 in breast cancer.
Asunto(s)
Neoplasias de la Mama/metabolismo , Cadherinas/biosíntesis , Epitelio/metabolismo , Regulación Neoplásica de la Expresión Génica , Factores de Transcripción de Tipo Kruppel/fisiología , Mesodermo/metabolismo , Apoptosis , Cadherinas/metabolismo , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Humanos , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/metabolismo , Modelos Biológicos , Metástasis de la Neoplasia , Regiones Promotoras Genéticas , ARN Mensajero/metabolismoRESUMEN
Classical cadherins are the transmembrane proteins of the adherens junction and mediate cell-cell adhesion via homotypic interactions in the extracellular space. In addition, they mediate connections to the cytoskeleton by means of their association with catenins. Decreased cadherin-mediated adhesion has been implicated as an important component of tumorigenesis. Cadherin switching is central to the epithelial to mesenchymal transitions that drive normal developmental processes. Cadherin switching has also been implicated in tumorigenesis, particularly in metastasis. Recently, cadherins have been shown to be engaged in cellular activities other than adhesion, including motility, invasion, and signaling. In this study, we show that inappropriate expression of R-cadherin in tumor cells results in decreased expression of endogenous cadherins (cadherin switching) and sustained signaling through Rho GTPases. In addition, we show that R-cadherin induces cell motility when expressed in epithelial cells and that this increased motility is dependent upon Rho GTPase activity.