The mammary gland contains a subset of cells with regenerative capacity that is able to generate both luminal and myoepithelial mammary epithelial lineages. Those cells are described as mammary epithelial stem cells. The fate of stem cells is tightly controlled by their microenvironment and adhesion receptors on the stem cells play a vital role in the microenvironment-stem cell communication. They facilitate the interaction of stem cells with the extracellular matrix as well as adjacent cells, and they regulate stem cell homing to their niches, as well as stem cell proliferation, self-renewal, and differentiation. Stem cells express high levels of ECM binding adhesion receptors such as β1 and alpha6-integrins. Those integrins were used to isolate stem cells from the rest of the differentiated epithelial cells within the mammary gland. However, little is known about the role of those integrins in stem cell self-renewal and differentiation. This project aimed to understand how β1-integrin receptors contribute to stem cell behavior. To achieve this, FACS sorting method of stem cells, the organoid assay, and lentivirus knockdown of β1-integrin using shRNA were optimised. The organoid assay was used as an in-vitro test to assess for the frequency of bi-lineage and luminal progenitor cells in a given mammary epithelial population. It is known that bi-lineage cells produce solid organoids in culture while luminal progenitors produce hollow organoids. The frequency of solid and hollow organoids might therefore be an indication of the stem cells and luminal progenitor frequency respectively. My results showed that cells with the highest solid organoid forming ability were within the basal population, which is high for β1- and alpha6-integrin. The β1-integrin signaling pathway was shown to be important for maintaining the organoid-forming population in basal and luminal populations. Knocking out β1-integrin in MECs resulted in abolishing their solid and hollow organoid-forming activity. Downstream of β1-integrin, I found that Rac1 but not ILK is important in β1-integrin maintenance of solid organoid-forming cells. Active Rac1 was able to rescue solid organoid formation but was not able to rescue hollow organoids in the β1-integrin knockdown cells. β1-integrin and Rac1 deletion resulted in the down regulation of Wnt/β-catenin signaling, which is important for stem cells. This down regulation was rescued using active Rac1. Activating Wnt/ β1-catenin signaling in primary cells (using Wnt3a ligand or GSK3β inhibitor) resulted in an increase in solid organoid and a decrease in hollow organoid formation. When activating Wnt signaling using GSK3I in β1-integrin knockdown cells, the solid organoid activity was rescued. However, Wnt3a did not rescue solid organoid formation in the β1-integrin knockdown cells. When active Rac1 was overexpressed in β1-integrin null cells, Wnt3a was able to activate solid organoid formation. When inhibiting Rac1 in primary MECs, solid but not hollow organoid activity was significantly decreased. Wnt3a or GSK3I addition did not rescue this reduction. Taken these results together, it can be concluded that β1integrin-Rac1 signaling play a role in controlling stem cells and this is might be achieved through controlling Wnt/β-catenin signaling. These studies are important in understanding the role of integrins in mammary stem cells. They will also provide new insight on how integrins might be controlling breast cancer and thereby, help in providing new targets for cancer therapy.
|Date of Award||31 Dec 2016|
- The University of Manchester
|Supervisor||Charles Streuli (Supervisor) & Keith Brennan (Supervisor)|
- Mammary stem cells