ABSTRACT
Maspin is a unique serpin involved in the suppression of tumor growth and metastasis. To investigate whether increased levels of maspin protect against tumor progression in vivo, we established a transgenic model in which maspin is targeted to mammary epithelial cells by the Whey Acidic Protein (WAP) promoter for overexpression. We crossed these WAP-maspin transgenic mice with the WAP-TAg mouse model of tumor progression. Maspin overexpression increased the rate of apoptosis of both preneoplastic and carcinomatous mammary epithelial cells. Maspin reduced tumor growth through a combination of reduced angiogenesis and increased apoptosis. The number of pulmonary metastases was reduced in the presence of maspin overexpression. These data demonstrate that targeted overexpression of maspin can inhibit tumor progression in vivo, likely through a combination of increased apoptosis, decreased angiogenesis, and inhibition of tumor cell migration.
Subject(s)
Adenocarcinoma/pathology , Antigens, Viral, Tumor/metabolism , Disease Progression , Mammary Neoplasms, Animal/pathology , Milk Proteins/genetics , Proteins/metabolism , Serpins/metabolism , Adenocarcinoma/blood supply , Adenocarcinoma/genetics , Animals , Antigens, Viral, Tumor/genetics , Apoptosis , Cell Division , Cell Movement , Disease-Free Survival , Epithelial Cells/metabolism , Epithelial Cells/pathology , Female , Gene Expression , Genes, Tumor Suppressor , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Lung Neoplasms/secondary , Mammary Neoplasms, Animal/blood supply , Mammary Neoplasms, Animal/genetics , Mice , Mice, Inbred C57BL , Mice, Transgenic , Neovascularization, Pathologic , Nuclease Protection Assays , Precancerous Conditions/blood supply , Precancerous Conditions/genetics , Precancerous Conditions/pathology , Promoter Regions, Genetic/genetics , Proteins/genetics , RNA, Messenger/analysis , RNA, Messenger/genetics , Serpins/genetics , Transgenes/geneticsABSTRACT
Maspin is a unique member of the serpin family, which functions as a class II tumor suppressor gene. Despite its known activity against tumor invasion and motility, little is known about maspin's functions in normal mammary gland development. In this paper, we show that maspin does not act as a tPA inhibitor in the mammary gland. However, targeted expression of maspin by the whey acidic protein gene promoter inhibits the development of lobular-alveolar structures during pregnancy and disrupts mammary gland differentiation. Apoptosis was increased in alveolar cells from transgenic mammary glands at midpregnancy. However, the rate of proliferation was increased in early lactating glands to compensate for the retarded development during pregnancy. These findings demonstrate that maspin plays an important role in mammary development and that its effect is stage dependent.
Subject(s)
Mammary Glands, Animal/embryology , Proteins/physiology , Serine Proteinase Inhibitors/physiology , Serpins/physiology , Animals , Apoptosis , Cell Division , Female , Genes, Tumor Suppressor , Lactation , Mice , Mice, Transgenic , Milk Proteins/genetics , Pregnancy , Proteins/genetics , Serpins/genetics , Tissue Plasminogen Activator/antagonists & inhibitors , TransgenesABSTRACT
elafin is a unique elastase inhibitor. It is differentially expressed at the transcriptional level in human normal mammary epithelial cells and carcinomas. The elafin gene is induced by PMA in 21MT2 breast tumor cells. By deletion analysis and mutagenesis, we have identified the Ap1 site in the promoter as the cis element mediating transcriptional activation of elafin in 70N normal breast cells and its induction by phorbol 12-myristate 13-acetate (PMA) in 21MT2 breast tumors. PMA treatment induces AP1 factor expression, which binds to the Ap1 site of the elafin promoter. Mutation of this Ap1 abolishes the capability of induction by PMA. Furthermore, our data provide a basis for therapeutic manipulation of proteinase inhibitors such as elafin.
Subject(s)
Breast Neoplasms/metabolism , Breast/metabolism , Promoter Regions, Genetic , Protein Biosynthesis , Proteins , Serine Proteinase Inhibitors/biosynthesis , Tetradecanoylphorbol Acetate/pharmacology , Transcription Factor AP-1/metabolism , Transcription, Genetic/drug effects , Binding Sites , Breast/cytology , Cells, Cultured , Chloramphenicol O-Acetyltransferase/biosynthesis , Epithelial Cells/metabolism , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , Kinetics , Promoter Regions, Genetic/drug effects , Proteinase Inhibitory Proteins, Secretory , Recombinant Fusion Proteins/biosynthesis , Transfection , Tumor Cells, CulturedABSTRACT
Prostate cancer is the most common cancer in men. The molecular mechanisms leading to its development are poorly understood. Maspin is a tumor-suppressing serpin expressed in normal breast and prostate epithelium. We have found that expression of maspin in normal and carcinoma-derived prostate epithelial cells is differentially regulated at the transcriptional level. We have identified two different kinds of cis elements, Ets and hormonal responsive element (HRE), in the maspin promoter. The Ets element is active in regulating maspin expression in normal prostate epithelial cells but inactive in tumor cells. The HRE site is a negative element that is active in both cell types. This negative DNA sequence can repress a heterologous promoter recognized by the androgen receptor. We conclude that expression of maspin is under the influence of both a positive Ets and a negative HRE element. Loss of maspin expression during tumor progression apparently results from both the absence of transactivation through the Ets element and the presence of transcription repression through the negative HRE element recognized by androgen receptor.
Subject(s)
DNA-Binding Proteins/metabolism , Promoter Regions, Genetic , Protein Biosynthesis , Receptors, Androgen/metabolism , Serpins/biosynthesis , Transcription, Genetic , Base Sequence , Binding Sites , Cell Line , Chloramphenicol O-Acetyltransferase/biosynthesis , Epithelium , Gene Expression Regulation, Neoplastic , Genes, Tumor Suppressor , Humans , Male , Oligodeoxyribonucleotides , Prostate/metabolism , Prostatic Neoplasms/metabolism , Proteins/genetics , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-ets , Recombinant Fusion Proteins/biosynthesis , Serine Proteinase Inhibitors/biosynthesis , Serpins/genetics , Transcription Factors/metabolism , Tumor Cells, CulturedABSTRACT
Tumor invasion and metastasis are processes poorly understood at the molecular level. Maspin is a serine protease inhibitor (serpin) with tumor-suppressing function in the mammary gland. Maspin gene expression is decreased with malignancy and is lost in metastatic cells. We show in this report that differential expression of maspin in normal and carcinoma-derived mammary epithelial cells is regulated at the transcriptional level. We have identified the Ets and Ap1 sites in the maspin promoter that are active in regulating maspin expression in normal mammary epithelial cells but inactive in tumor cells. The Ets site alone is sufficient to activate transcription in a heterologous promoter, whereas the Ap1 site cooperates with Ets in activation. The enhancing function by Ets and Ap1 elements is decreased in primary tumor cells (21NT) and is abolished in invasive tumor cells (MDA-231). Thus, loss of maspin expression during tumor progression results at least in part from the absence of transactivation through the Ets and Ap1 sites.
