Dataset Information

Cleavage of Histone 3 by Cathepsin D in the involuting mammary gland.

ABSTRACT: The post-lactational regression of mammary gland is a complex multi-step process designed to conserve the biological function of the gland for next pregnancy. This developmental stage is a biological intrigue with great relevance to breast cancer research, and thus has been the subject of intensive scrutiny. Multipronged studies (microarray, proteomics profiling, animal knock-out models) have provided a repertoire of genes critical to involution. However, the caveat of these approaches remains in their failure to reveal post-translational modification(s), an emerging and critical aspect of gene regulation in developmental processes and mammary gland remodeling. The massive surge in the lysosomal enzymes concurrent with the onset of involution has been known for decades, and considered essential for "clearance" purposes. However, functional significance of these enzymes in diverse biological processes distinct from their proteolytic activity is just emerging. Studies from our laboratory had indicated specific post-translational modifications of the aspartyl endopeptidase Cathepsin D (CatD) at distinct stages mammary gland development. This study addresses the biological significance of these modifications in the involution process, and reveals that post-translational modifications drive CatD into the nucleus to cleave Histone 3. The cleavage of Histone 3 has been associated with cellular differentiation and could be critical instigator of involution process. From functional perspective, deregulated expression and increased secretion of CatD are associated with aggressive and metastatic phenotype of breast cancer. Thus unraveling CatD's physiological functions in mammary gland development will bridge the present gap in understanding its pro-tumorigenic/metastatic functions, and assist in the generation of tailored therapeutic approaches.

SUBMITTER: Khalkhali-Ellis Z

PROVIDER: S-EPMC4108390 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Cleavage of Histone 3 by Cathepsin D in the involuting mammary gland.

Khalkhali-Ellis Zhila Z Goossens William W Margaryan Naira V NV Hendrix Mary J C MJ

PloS one 20140723 7

The post-lactational regression of mammary gland is a complex multi-step process designed to conserve the biological function of the gland for next pregnancy. This developmental stage is a biological intrigue with great relevance to breast cancer research, and thus has been the subject of intensive scrutiny. Multipronged studies (microarray, proteomics profiling, animal knock-out models) have provided a repertoire of genes critical to involution. However, the caveat of these approaches remains i ...[more]

PMID: 25054204

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Project description:Epithelial cells within the mammary gland undergo developmental programmes of proliferation and apoptosis during the pregnancy cycle. After weaning, secretory epithelial cells are removed by apoptosis. To determine whether members of the Bcl-2 gene family could be involved in regulating this process, we have examined whether changes in their expression occur during this developmental apoptotic program in vivo. Bax and Bcl-x were evenly expressed throughout development. However, expression of Bak and Bad was increased during late pregnancy and lactation, and the proteins were present during the time of maximal apoptotic involution. Thereafter, their levels declined. In contrast, Bcl-w was expressed in pregnancy and lactation but was downregulated at the onset of apoptosis. Bcl-2 was not detected in lactating or early involuting mammary gland. Thus, the pro-apoptotic proteins Bax, Bak and Bad, as well as the death-suppressors Bcl-x, Bcl-2 and Bcl-w, are synthesised in mouse mammary gland, and dynamic changes in the expression profiles of these proteins occurs during development. To determine if changes in Bak and Bcl-w expression could regulate mammary apoptosis, their effect on cultured mouse mammary epithelial cells was examined in transient transfection assays. Enforced expression of Bak induced rapid mammary apoptosis, which could be suppressed by coexpression of Bcl-w. In extracts of mammary tissue in vivo, Bak heterodimerized with Bcl-x whereas Bax associated with Bcl-w, but Bak/Bcl-w heterodimers were not detected. Thus, Bak and Bcl-w may regulate cell death through independent pathways. These results support a model in which mammary epithelial cells are primed for apoptosis during the transition from pregnancy to lactation by de novo expression of the death effectors Bak and Bad. It is suggested that these proteins are prevented from triggering apoptosis by anti-apoptotic Bcl-2 family proteins until involution, when the levels of Bcl-w decline. Our study provides evidence that regulated changes in the expression of cell death genes may contribute to the developmental control of mammary apoptosis.

Project description:Mammary gland involution represents one of the most dramatic examples of programmed cell death/apoptosis and tissue regression during development, yet large gaps still exist in the understanding of the mechanisms involved, and the key factors that trigger involution, are not yet identified. With the focus on identifying “novel” genes associated with mammary gland regression, we used microarray analysis to examine differentially expressed genes during early mammary gland involution in the mouse. We believe that such analysis may facilitate the identification of genes that report on proliferative changes in the breast, particularly with regards to growth arrest and growth inhibition. We further reasoned that genes that inhibit mammary gland proliferation might have relevance to breast tumorigenesis since cancer is generally associated with a defect in apoptosis and cell growth arrest. Gene expression from day 0 (D0) of involution was compared to day 1 (D1, 24 hr post weaning) to identify genes exhibiting differential expression. A total of 5,826 genes were identified as being differentially expressed. Changes in gene expression were confirmed by quantitative real time PCR analysis of 21 randomly selected genes. Following the selection of a number of highly regulated mouse genes within this cohort, database searches were then employed to identify genes associated with human tumorigenesis. With an ultimate goal being the identification of genes, which under normal physiological conditions, inhibit breast proliferation, and therefore have the potential to impact on the clinical management of breast cancer, we further examined their relevance to human tumorigenesis. Among genes identified as having human homologues, and with previously established links to human tumorigenesis, were several known genes among the top 200 differentially expressed genes including EGF, EGFR, TGFß2, PTHLH, IGFBP1, that were down regulated. Upregulated known genes included ANNEXIN A1 and A3, THYMOSIN BETA 10, IGFBP5, LIF, BID, BCL2, JUN, KIT, and CLAUDIN 1. CLAUDIN 1 was the most highly upregulated gene in our dataset. Moreover, we identified a number of genes not previously implicated or not well characterized in human breast cancer including ANGPTL4, SLC34A2, GOS2, STEAP, SOX4, TNFRSF12A and SAA2. The expression levels of these genes in human breast cancer were confirmed in breast cancer cell lines and breast tumor tissues. For some of these genes, expression was consistently down regulated in a small panel of human breast tumor specimens. We believe that these genes now warrant further investigation to determine their relevance to human breast tumorigenesis and their potential utility as therapeutic targets for human breast cancer. This pilot study was used to demonstrate proof of principle that through the analysis of gene expression during mammary gland involution, it may be indeed possible to identify “novel” genes relevant to the growth arrest in the mammary gland and relevant to human breast cancer. Keywords: comparative expression analysis of involuting mouse mammary gland

Project description:Mammary gland involution represents one of the most dramatic examples of programmed cell death/apoptosis and tissue regression during development, yet large gaps still exist in the understanding of the mechanisms involved, and the key factors that trigger involution, are not yet identified. With the focus on identifying ânovelâ genes associated with mammary gland regression, we used microarray analysis to examine differentially expressed genes during early mammary gland involution in the mouse. We believe that such analysis may facilitate the identification of genes that report on proliferative changes in the breast, particularly with regards to growth arrest and growth inhibition. We further reasoned that genes that inhibit mammary gland proliferation might have relevance to breast tumorigenesis since cancer is generally associated with a defect in apoptosis and cell growth arrest. Gene expression from day 0 (D0) of involution was compared to day 1 (D1, 24 hr post weaning) to identify genes exhibiting differential expression. A total of 5,826 genes were identified as being differentially expressed. Changes in gene expression were confirmed by quantitative real time PCR analysis of 21 randomly selected genes. Following the selection of a number of highly regulated mouse genes within this cohort, database searches were then employed to identify genes associated with human tumorigenesis. With an ultimate goal being the identification of genes, which under normal physiological conditions, inhibit breast proliferation, and therefore have the potential to impact on the clinical management of breast cancer, we further examined their relevance to human tumorigenesis. Among genes identified as having human homologues, and with previously established links to human tumorigenesis, were several known genes among the top 200 differentially expressed genes including EGF, EGFR, TGFÃ2, PTHLH, IGFBP1, that were down regulated. Upregulated known genes included ANNEXIN A1 and A3, THYMOSIN BETA 10, IGFBP5, LIF, BID, BCL2, JUN, KIT, and CLAUDIN 1. CLAUDIN 1 was the most highly upregulated gene in our dataset. Moreover, we identified a number of genes not previously implicated or not well characterized in human breast cancer including ANGPTL4, SLC34A2, GOS2, STEAP, SOX4, TNFRSF12A and SAA2. The expression levels of these genes in human breast cancer were confirmed in breast cancer cell lines and breast tumor tissues. For some of these genes, expression was consistently down regulated in a small panel of human breast tumor specimens. We believe that these genes now warrant further investigation to determine their relevance to human breast tumorigenesis and their potential utility as therapeutic targets for human breast cancer. This pilot study was used to demonstrate proof of principle that through the analysis of gene expression during mammary gland involution, it may be indeed possible to identify ânovelâ genes relevant to the growth arrest in the mammary gland and relevant to human breast cancer. Experiment Overall Design: Pooled RNA from D0 mice and from D1 mice, (five mice per time point), was compared for differential gene expression by microarray analysis conducted by Paradigm Array Labs (Research Triangle Park, North Carolina, USA). The Affymetrix GeneChip Mouse Genome 430 Plus 2.0 Array (Affymetrix, Santa Clara CA. USA) was used. The array contained 45,100 probe sets (The Affymetrix probe-set identificator) and represented 39,000 transcripts. The Affymetrix Statistical Algorithms, implemented in the Affymetrix Microarray Suite 5.0 software, were used in the expression analysis of GeneChip probe arrays.

Dataset Information

Cleavage of Histone 3 by Cathepsin D in the involuting mammary gland.

Publications

Cleavage of Histone 3 by Cathepsin D in the involuting mammary gland.

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