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TSG101 - a potential modifier gene for neoplastic transformation in mammary epithelial cells.

It is generally accepted that normal mammary epithelial cells have to acquire a series of genetic changes to transform themselves into breast cancer cells. Genes that contribute to tumor formation are categorized into oncogenes and tumor-suppressor genes. Both classes of genes are tightly interwoven, and they do much more than just stimulate (oncogenes) or prevent (tumor-suppressor genes) the onset of a cancer. They are intimately involved in the regulation of fundamental processes affecting almost every aspect of normal cell growth, differentiation, and cell death. Determining the function(s) of an oncogene or tumor-suppressor gene in normal cells and tissues is essential for our understanding of the role of this gene in abnormal events such as cancer. Like oncogenes, tumor-suppressor genes occupy pivotal positions in regulatory networks that encompass various signal transduction cascades. Therefore, these pathways often involve an interaction of different tumor-suppressor genes. According to the hierarchy in the cascade and the function that genes possess, tumor-suppressor genes are commonly subdivided into 'gatekeepers' and 'caretakers', or sometimes 'modifiers of tumorigenesis'. The latter type of genes have gained more importance in the last couple of years, since their biological function could explain why some patients with inherited mutations of important tumor-suppressor genes such as BRCA1 never develop breast cancer, whereas other patients have an early onset of this disease.

TSG101 is a newly discovered tumor susceptibility gene, which is important for growth restriction of normal cells. Mutations of the TSG101 gene are rare events in human breast cancers, but aberrant products from this gene are observed quite frequently. The occurrence of these abnormal products seems to be tightly correlated with tumor grade and the mutation status of another important tumor-suppressor gene called p53. P53 is affected in more than half of all human breast cancers, and is therefore a key player in the development of this disease. Recent data propose a direct interaction of TSG101 with factors that regulate the function of p53. Scientists have just started to investigate the biological role of TSG101 in normal and cancerous cells. However, the mechanism for the tumor-suppressive function of TSG101 is still elusive.

Scientists frequently modify genes in animal models such as mice to investigate the course of a genetic disease like cancer. The importance of a given gene might vary in a few organs between humans and mice, and this might be a problem for modeling a specific type of human cancer in rodents. For instance, the elimination of an important gene from all cells of the mouse might be deleterious, and it is therefore impossible to study the function of this gene in adult animals. We have developed a technique that allows us to inactivate genes in specific cells of the mammary gland of adult mice. These so-called somatic knockouts are very useful tools for modeling breast cancer in a similar fashion as they occur in human malignancies. The value of these tools has recently been demonstrated as we have generated a mouse model for the hereditary form of mammary cancer by inactivating the BRCA1 gene. We will apply this sophisticated technique to study the biological function of TSG101. We have a unique system that allows us to determine whether TSG101 is a modifier for tumor progression in the mammary gland, and whether there is an interaction of this gene with other important tumor-suppressor genes such as p53. A better understanding of signaling pathways that are subverted in breast cancer cells might provide new clues for the restoration of the function of tumor-suppressor genes and a possible intervention in breast cancer.

The role of the Jak2-Stat5 signaling cascade as an important mediator of the prolactin pathway.

Our objective is to elucidate the mechanisms by which the peptide hormone prolactin regulates cellular growth, differentiation, and contributes to the neoplastic transformation of mammary epithelial cells. It is widely accepted that prolactin (PRL) signals mainly through so-called signal transducers and activators of transcription (STAT), and in particular through Stat5A and Stat5B in the mammary gland. These molecules are tyrosine phosphorylated by a kinase called Jak2 that is temporally part of the receptor complex. Upon phosphorylation, Stat5A and Stat5B form homo- or heterodimers and translocalize into the nucleus where they activate the transcription of downstream genes. The phenotypic analysis of animal models that lack PRL, the PRL receptor, or Stat5a/5b shed light onto the biological function of PRL and its downstream mediators. Most intriguingly, Stat5A and Stat5B have specific and redundant functions depending on the organ of interest. Stat5A seems to be essential for mammary development, whereas Stat5B is necessary for a normal reproductive cycle. There are still many open questions on how the prolactin pathway is modulated in mammary epithelial cells. For instance, it is unclear whether the inhibition of PRL downstream targets affects only the differentiation of alveolar cells or both the proliferation of alveolar precursors and their subsequent differentiation into secretory cell types. Also, the loss of PRL function of differentiated cells might be important to initiate cell death and mammary gland remodeling. This is an important aspect since it is suggested that a constitutively active prolactin pathway has the ability to prevent apoptosis and thereby support neoplastic transformation. To further address some of these issues we have generated transgenic mice that express super-active forms of STAT5a and STAT5b under regulatory elements of the Mouse Mammary Tumor Virus (MMTV-LTR). These genetically engineered mice are currently being analyzed to determine whether the formation of stable homodimers (Stat5a or Stat5b) results in phenotypic abnormalities in organs that express these mutant proteins. By crossing the two mutant mouse strains together we will study the specific role of STAT5a/STAT5b heterodimer formation in vivo. These super-active heterodimers might trigger different phenotypic abnormalities in double transgenic mice compared to single transgenics that express either STAT5a or STAT5b super-active homodimers.

Cloning of a new parity-induced mammary epithelial cell population.

It is a paradigm among many mammary gland researchers that all differentiated cells have to undergo programmed cell death during involution. In the next pregnancy, a new population of mammary epithelial cells with specific functions originates from undifferentiated progenitor cells through proliferation and differentiation. This paradigm might be true for the vast majority of alveolar cells, but we were able to show recently that a significant amount of differentiated cells reside in the mammary gland after complete remodeling. Our hypothesis that self-renewal is critical for the survival of a subset of mammary cells was confirmed recently when we were able to label these residing cells in situ using the Cre-loxP technology. These self-renewed cells gave rise to a new generation of small ducts and alveoli in subsequent pregnancies. The goal of this project is the cloning of these specific cells. The cloning technique is based on the permanent activation of selectable markers in differentiated cells that remain active in those cells that bypass apoptosis and remodeling during involution. By analyzing these cells in vitro, we hope to find unique indicators for self-renewing cells that can be utilized for rapid cell selection. It is likely that the presences of this new epithelial population in parous mice explains some of the physiological differences observed between virgin and parous mammary glands. These cells also might modify the overall response of the mammary epithelium to carcinogenic insults (i.e. the development of breast cancer).