The role of ROMA a novel transcriptional regulator in mammary lineage determination.

Stem cells are required in adult tissues to provide new cells to replace those that are damaged or destroyed. For example, skin and gut cells are replaced on a daily basis and liver and bone cells can be replaced following injury. Stem cells provide a reservoir of cells that last for the lifetime of the animal or person and are used to provide the progenitor cells that divide rapidly to replace these lost or damaged cells. One feature of adult stem cells is that they divide very infrequently and are usually found in specific parts of the tissue. In the breast, stem cells are known to exist and may be the cells that become breast tumours. However, it is not known where the breast stem cells are located although new techniques have allowed these cells to be highly enriched, thus allowing their study. We have recently shown that specific secreted factors called cytokines can influence how stem and progenitor cells grow during pregnancy to provide the large increase in secretory milk-producing alveolar cells that are required for lactation. During our studies, we identified a novel protein that we have named ROMA. We hypothesise that ROMA may be a master regulator of mammary stem cells. The main objective of this project is to characterise ROMA further and investigate its role in the development of the mammary gland during pregnancy. We will also identify genes that are regulated by ROMA. This project should provide essential information on how stem cells are maintained and induced to become more differentiated cells. This will have major implications for stem cell therapy and regenerative medicine and for understanding diseases that arise from stem cell failure.

Adult stem cells are required for tissue homeostasis and for the development of organs. In the mammary gland, stem cells are required during embryonic development and in the pubertal female for the development of the branching ductal network. Stem cells are required during each pregnancy to provide the progenitor cells that differentiate into the milk-producing lobuloalveolar units. There are at least four mammary epithelial lineages although the mammary epithelial hierarchy is not well defined and there are no definitive stem or progenitor cell markers. Essential regulators of the luminal alveolar lineage have recently been identified and include GATA-3, a zinc finger transcription factor, and Elf5. We have shown recently that Stat6, a regulator of GATA-3, is required for unhindered alveologenesis as mice deficient for either Stat6 or its activating cytokines IL-4 and IL-13 display up to 70% reduction in the number of alveoli during early gestation. We have identified a novel target of Stat6 that we hypothesise is a master regulator of alveologenesis and named this gene Roma. Bioinformatic analysis reveadled that ROMA is a KRAB domain zinc finger protein and a putative transcriptional repressor. The aim of this project is to characterise ROMA further and define its role in lineage commitment in mammary gland using mice with loss of function of ROMA and stem and progenitor cell analysis using FACS analysis and fat pad transfer. In addition, we will identify transcriptional targets of ROMA, the pathways that interact with ROMA, and the epigenetic status of the Roma promoter as other lineage regulators such as Elf5 are epigentically regulated.

Commercial impact: There are a number of possibilities for a commercial impact from this project. We will be generating an antibody as part of the characterisation of ROMA. Should this antibody prove to be specific and effective, we will make it available to a very limited number of collaborators. Since this is likely to be a very interesting protein and a useful lineage marker, there would be considerable commercial interest in marketing this antibody. We would initially approach the Cambridge-based antibody company AbCam as we have a good rapport with them and they would be able to market the antibody effectively. Following preliminary discussions with AbCam, negotiations through Cambridge Enterprise would be essential. The global gene expression analysis is likely to identify novel genes, progenitor cell markers and possibly a stem cell marker. Patenting opportunities, depending on the function of the markers and their likely use in therapeutics, will be discussed with Cambridge Enterprise. Should we identify a gene that has a role in stem cell self-renewal, there will be opportunities to investigate the value of this as a marker of breast cancer or the targeting of this as a possible treatment for breast cancer which will be of interest to big pharmaceutical companies. Finally, milk production in farm animals is of high commercial significance. Our work may provide insights into how the lactation period in cows can be extended without reduction in milk production (due to loss of function of the alveolar cells which undergo apoptosis) and thus will benefit the agricultural community and ultimately the consumer. Public impact: Public interest in stem cells and regenerative medicine is currently high. This project should clarify the mammary epithelial hierarchy and will be of interest to the public in terms of understanding how cells that produce milk in cows and other dairy animals are generated. It will also be of interest in elucidating which cells may become cancerous. The PI has presented to lay audiences and members of the public. This includes the annual Cambridge Science festival where an interactive display on stem cells, the huge variety of mammals (from rats to whales) and milk production and composition is presented. Children are able to use microscopes and play with a mock FACS analyser giving them (and their parents) the opportunity to discuss methods that are used in scientific research and the kinds of experiments that we carry out. We have been interviewed on the radio, had our work highlighted on the University and BBSRC web sites, and presented talks to fund raisers and university alumni. We will inform the university media office of interesting discoveries and breakthroughs. Enhancing public understanding of stem cell biology could impact government policy on the use of stem cells.