DO ORIENTED DIVISIONS DICTATE FATE DECISIONS IN MAMMARY STEM CELLS?

Breast cancer is the most common cancer in women worldwide. In the UK alone, the number of new cases of breast cancer is estimated at 55,200, and around 11,400 patients die every year. Throughout the world, the disease causes 522,000 deaths every year. It is believed that breast cancer arises from the normal self-renewing mammary (breast) stem cell that drives tissue growth and homeostasis, which is subject to genetic mutations that alter its ability to balance proliferation and differentiation programs. Yet, the mechanisms that regulate the normal functions of breast stem cells and how their dysregulation leads to tumour growth remain ill defined. Thus, a better understanding of the normal biology of breast stem cells is urgently needed to enhance our knowledge of breast cancer, and develop more efficient therapeutic strategies that eradicate their breast cancer stem cell counterparts in an effort to limit the mortality the disease causes.

Here we propose a multi-disciplinary research program, bringing together expertise from Stem Cell research and Physics and Mathematical research to uncover the role a cellular process called oriented cell division plays in the control of normal breast development to work out what goes wrong in breast cancer. Execution of this critical process allows stem cells in many tissues to balance proliferation and differentiation during development in order to generate the myriad of cell types that comprise our body. Oriented cell division is often defective in breast cancer stem cells, so understanding how it normally works in breast stem cells will provide important insight into the biological changes that allow tumours to grow. To achieve this, we plan to use transgenic reporter mouse models and advanced microscopic techniques in order to track the fate of dividing breast stem cells in their native environment and determine their dynamic contribution to the development of the normal breast tissue at a high spatiotemporal resolution. By the same approaches in combination with a gene invalidation strategy, we will investigate the consequences of loss-of-function of kinesin-1 - a key protein that controls the positioning of the mitotic spindle - on the execution of oriented cell division, and determine how this influences breast tissue differentiation and architecture. Ultimately, we aim to develop and implement a mathematical/computational framework involving the use of stochastic models and Bayesian analyses, which will provide the means to validate with high confidence our hypotheses and draw a model explaining how oriented cell division rules the binary choices between proliferation and differentiation made by breast stem cells.

It is our expectation that this work will provide a rigorous evaluation of the precise role oriented cell division plays in regulating breast stem cell ability to build and maintain the normal breast tissue, which will help resolve their identity, dynamics and differentiation potential. The outputs of this project will enhance our understanding of breast cancer, and will give rationale for designing tailored therapeutic strategies that control and restore the normal number and mode of division in breast stem cells. Thus our work will help bring breast cancer stem cell therapy one step closer to reality.

Adult mammary stem cells drive postnatal gland organogenesis and remodelling, and their self-renewal and fate have direct implications for breast cancer. Their true differentiation potential, however, remains unclear. We recently identified a developmental window during pregnancy where basal cells execute asymmetric cell divisions (ACDs) to achieve unequal partitioning of basal and luminal fate determinants, suggesting that ACDs allow a subset of basal stem cells to manifest bipotency. This project will test this hypothesis by assessing the dynamic relationship between spindle orientation and cell fate decisions in dividing basal cells, and how this translates into alveologenesis. Building on our recent findings establishing KIF5/kinesin-1 as the first plus-end microtubule processive motor controlling spindle orientation and apical polarity, we will determine the consequences of its knockdown in basal cells during pregnancy on the execution of ACDs, and how this impacts on epithelial differentiation and architecture. To this end, we developed a genetic lineage tracing strategy to indelibly mark and follow the fate and dynamics of single mitotic basal cells and their progeny at distinct stages of pregnancy, combined with whole-mount 3D confocal imaging. By this approach, clones arising from a mitotic basal cell can be visualized in their entirety. We will use mathematical modelling to compare the clonal data with predictions from stochastic models for cell fate dynamics, and test the potential causal relationship between mitotic spindle dynamic orientation and cell fate outcomes via Bayesian inference. This unique framework is expected to resolve bipotency in a subset of basal stem cells by establishing oriented divisions as a critical mechanism that rules their fate decisions. Our findings will be important for elucidating the mechanisms controlling the dynamic shifts within the mammary epithelial cell hierarchy, and for understanding how breast cancer arises.

Our research will result in a significant impact on the following areas:

Academic Research: Our work will help resolve with high confidence the differentiation potential of basal stem cells, by providing quantitative data establishing KIF5/kinesin-1-mediated asymmetric cell divisions (ACDs) as a critical cellular mechanism that allows basal stem cells to manifest bipotency. This will have considerable implications for investigators interested in deciphering the mechanisms that rule the dynamics and fates of mammary stem cells during development, homeostasis and transformation. Furthermore, the cutting-edge "wet science" approaches and mathematical models used in this work offer a rigorous framework for the wider stem cell research community assessing the lineage relationship and stem cell fate in different organs and tissues. Our findings will be disseminated to a wider academic audience through publications in high impact peer-reviewed journals and by presenting the work at national and international conferences.

Healthcare: Although not imminently translatable, our work will provide a rationale for designing future therapeutic strategies that target effectively mitotic breast cancer stem cells to correct their unbalanced cell divisions and fate outcomes. Building on our expertise in 3D organoid cultures we will collaborate with clinical scientists from the closely located Cancer Sciences of the University of Southampton (UoS) to generate patient specific cell cultures for therapeutic purposes and drug discovery by targeting KIF5/kinesin-1 and other mitotic proteins. These will represent cost-effective methods, which will ultimately help increase anti-cancer treatment efficiency, enhance the quality of life and decrease the burden on the National Health Service.

Biotechnology and Pharmaceutical Industry: Our work will enhance our understanding of how unbalance in cell division and fate choices in mammary stem cells can lead to the abnormal cell behaviour and tissue architecture observed in cancer, with considerable implications on drug discovery. Those in biotechnology and pharmaceutical industries looking to develop novel and improved approaches for the treatment of breast cancer will greatly benefit from the outcomes of this study. Knowledge transfer activities with these partners will be actively implemented (e.g. through industry-funded studentships) - this is likely to positively influence these industries and create new jobs to increase UK's competitiveness.

Education: Our work will contribute to building a workforce for the future of stem cell research with unique and highly desirable/marketable skill-sets. This project will train one PDRA and will contribute to the training of a PhD student, as well as undergraduate students who pass through the lab. The results generated during this project will serve as an invaluable tool for research-led teaching activities in the relevant higher education courses for basic scientists and health professionals in the UK and abroad.

The General Public: We are committed to engaging the public with our research to educate and raise awareness of the importance of our current research for understanding breast cancer and for the development of novel therapeutic approaches to combat the disease. We will also be actively involved in various UoS outreach events and implement partnerships with local schools to illustrate the broader impact of our research, to inspire the next generation of young scientists. The UoS has a Public Engagement with Research unit "PERu", with a dedicated team who will assist in identifying activities and events for promoting our research. Through this program, our work will also have an ongoing impact on public awareness, engagement, and debates around the importance of stem cell research for understanding human diseases. We will also use UoS press office and appropriate social media tools to reach the wider public.