RESUMO
The expression of Wingless and Int-related protein (Wnt) ligands is aberrantly high in human breast cancer. We report here that WNT4 is significantly upregulated at the mRNA and protein level in mammary carcinoma cells expressing autocrine human growth hormone (hGH). Depletion of WNT4 using small interfering (si) RNA markedly decreased the rate of human breast cancer cell proliferation induced by autocrine hGH. Forced expression of WNT4 in the nonmalignant human mammary epithelial cell line MCF-12A stimulated cell proliferation in low and normal serum conditions, enhanced cell survival and promoted anchorage-independent growth and colony formation in soft agar. The effects of sustained production of WNT4 were concomitant with upregulation of proliferative markers (c-Myc, Cyclin D1), the survival marker BCL-XL, the putative WNT4 receptor FZD6 and activation of ERK1 and STAT3. Forced expression of WNT4 resulted in phenotypic conversion of MCF-12A cells, such that they exhibited the molecular and morphological characteristics of mesenchymal cells with increased cell motility. WNT4 production resulted in increased mesenchymal and cytoskeletal remodeling markers, promoted actin cytoskeleton reorganization and led to dissolution of cell-cell contacts. In xenograft studies, tumors with autocrine hGH expressed higher levels of WNT4 and FZD6 when compared with control tumors. In addition, Oncomine data indicated that WNT4 expression is increased in neoplastic compared with normal human breast tissue. Accordingly, immunohistochemical detection of WNT4 in human breast cancer biopsies revealed higher expression in tumor tissue vs normal breast epithelium. WNT4 is thus an autocrine hGH-regulated gene involved in the growth and development of the tumorigenic phenotype.
Assuntos
Neoplasias da Mama/metabolismo , Hormônio do Crescimento Humano/metabolismo , Proteína Wnt4/metabolismo , Apoptose , Mama/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular , Movimento Celular , Proliferação de Células , Feminino , Humanos , Células MCF-7 , RNA Mensageiro/metabolismo , Proteína Wnt4/genéticaRESUMO
CONTEXT: Empirical evidence suggests that autocrine human GH (hGH) may possess a proliferative and oncogenic role in human mammary carcinoma. However, this concept is largely derived from studies using cultured human mammary carcinoma cell (HMCC) lines. OBJECTIVE: We investigated the expression and functionality of hGH and the hGH receptor in isolated cultures of primary HMCC. DESIGN: Epithelial cell adhesion molecule-positive primary HMCC were isolated from surgical biopsies of patients with mammary carcinoma and cultured in vitro. Expression of hGH and hGH receptor was determined by RT-PCR, immunofluorescence microscopy, and ELISA. The proliferative response of the cultured primary HMCC to hGH stimulation or hGH inhibition with a hGH antagonist was determined. RESULTS: One hundred percent of cultured primary HMCC expressed the hGH receptor, and 52% expressed hGH at the mRNA level. hGH-positive primary HMCC produced hGH protein within the cell and secreted hGH to the media. Both hGH-negative and hGH-positive HMCC responded to hGH stimulation with large increases in cell number. hGH-positive HMCC responded to inhibition of hGH by a hGH antagonist with a decrease in cell number, whereas hGH-negative HMCC did not. CONCLUSION: Primary HMCC proliferate in response to hGH, and the proliferation of hGH-positive HMCC is inhibited by hGH antagonism. Inhibition of hGH in patients with mammary carcinoma may therefore limit tumor growth.
Assuntos
Comunicação Autócrina/fisiologia , Neoplasias da Mama/metabolismo , Carcinoma/metabolismo , Proliferação de Células , Hormônio do Crescimento Humano/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Proteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Células Tumorais CultivadasRESUMO
Deregulated PAX5 expression has been associated with metastatic mammary carcinoma, although the precise role of PAX5 in cancer progression is unclear. Stable forced expression of PAX5alpha in the mammary carcinoma cell lines MCF-7 and MDA-MB-231 reduced cell cycle progression, cell survival, and anchorage-independent cell growth. In xenograft studies, forced expression of PAX5alpha was associated with a significant reduction in tumor volume. Furthermore, forced expression of PAX5alpha in mammary carcinoma cells resulted in altered cell morphology with resultant enhancement of epithelial cell characteristics. Morphologic changes were associated with localization of beta-CATENIN at cell-cell junctions and with altered mRNA expression of mesenchymal markers in mammary carcinoma cells. In addition, forced expression of PAX5alpha in MCF-7 and MDA-MB-231 cells significantly reduced cell migration and invasion. Concomitantly, small interfering RNA-mediated depletion of PAX5alpha increased MCF-7 total cell number, cell motility, migration, and invasion. These studies show that PAX5alpha enhances the epithelial characteristics of mammary carcinoma cells, reminiscent of mesenchymal to epithelial transition.
Assuntos
Neoplasias da Mama/metabolismo , Carcinoma/metabolismo , Desdiferenciação Celular/fisiologia , Células Epiteliais/metabolismo , Fator de Transcrição PAX5/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Biomarcadores/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Carcinoma/genética , Carcinoma/patologia , Contagem de Células , Diferenciação Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Regulação para Baixo/genética , Células Epiteliais/citologia , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Junções Intercelulares/metabolismo , Junções Intercelulares/ultraestrutura , Mesoderma/citologia , Mesoderma/metabolismo , Invasividade Neoplásica/genética , Invasividade Neoplásica/fisiopatologia , Fator de Transcrição PAX5/genética , Interferência de RNA/fisiologia , RNA Mensageiro/metabolismo , Proteínas Supressoras de Tumor/genética , beta Catenina/metabolismoRESUMO
The functional role of autocrine trefoil factor-1 (TFF1) in mammary carcinoma has not been previously elucidated. Herein, we demonstrate that forced expression of TFF1 in mammary carcinoma cells resulted in increased total cell number as a consequence of increased cell proliferation and survival. Forced expression of TFF1 enhanced anchorage-independent growth and promoted scattered cell morphology with increased cell migration and invasion. Moreover, forced expression of TFF1 increased tumor size in xenograft models. Conversely, RNA interference-mediated depletion of TFF1 in mammary carcinoma cells significantly reduced anchorage-independent growth and migration. Furthermore, neutralization of secreted TFF1 protein by polyclonal antibody decreased mammary carcinoma cell viability in vitro and resulted in regression of mammary carcinoma xenografts. We have therefore demonstrated that TFF1 possesses oncogenic functions in mammary carcinoma cells. Functional antagonism of TFF1 can therefore be considered as a novel therapeutic strategy for mammary carcinoma.
Assuntos
Neoplasias da Mama/metabolismo , Carcinoma/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Neoplasias da Mama/patologia , Carcinoma/patologia , Ciclo Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Progressão da Doença , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica , Interferência de RNA , RNA Mensageiro/metabolismo , Fator Trefoil-1 , Proteínas Supressoras de Tumor/imunologiaRESUMO
Human growth hormone (hGH) is expressed by mammary epithelial cells and associated with proliferative disorders of the human breast. Our goal is to characterize the paracrine effects of hGH on morphological and functional changes of mammary carcinoma cells using MCF7 cells stably transfected with the hGH gene (MCFhGH). To identify the molecular actors involved in autocrine hGH-induced cell proliferation, we have used a protein chip technology using a commercial antibody microarray. The results enabled us to qualitatively characterize MCF-hGH cell's proteome from a panel of 500 proteins. Statistical analysis of variations in protein levels between the two cell lines did not highlight any significant differences. Thus, we concluded that variations in MCF-hGH proteome are more likely to reside in the activation status rather than drastic variations in the expression level of the 500 spotted proteins. To test this hypothesis, we confronted the protein chip result to the study of the regulation of the transcriptional factor Pax (Paired-box)-5 whose expression was not found to be altered on the protein chip. Surprisingly, we found that autocrine production of hGH in MCF7 cells was associated with a strong nuclear accumulation of Pax5 in a JAK2-dependent manner associated with an increase in Pax5-DNA binding activity. Our work indicates that subtle changes mediated by Pax5 are responsible for autocrine hGH-induced cell proliferation.
