Integrin-Rac signalling for mammary epithelial stem cell self-renewal.

BACKGROUND: Stem cells are precursors for all mammary epithelia, including ductal and alveolar epithelia, and myoepithelial cells. In vivo mammary epithelia reside in a tissue context and interact with their milieu via receptors such as integrins. Extracellular matrix receptors coordinate important cellular signalling platforms, of which integrins are the central architects. We have previously shown that integrins are required for mammary epithelial development and function, including survival, cell cycle, and polarity, as well as for the expression of mammary-specific genes. In the present study we looked at the role of integrins in mammary epithelial stem cell self-renewal. METHODS: We used an in vitro stem cell assay with primary mouse mammary epithelial cells isolated from genetically altered mice. This involved a 3D organoid assay, providing an opportunity to distinguish the stem cell- or luminal progenitor-driven organoids as structures with solid or hollow appearances, respectively. RESULTS: We demonstrate that integrins are essential for the maintenance and self-renewal of mammary epithelial stem cells. Moreover integrins activate the Rac1 signalling pathway in stem cells, which leads to the stimulation of a Wnt pathway, resulting in expression of ß-catenin target genes such as Axin2 and Lef1. CONCLUSIONS: Integrin/Rac signalling has a role in specifying the activation of a canonical Wnt pathway that is required for mammary epithelial stem cell self-renewal.

The requirement of integrins for breast epithelial proliferation.

Epithelial cells forming mammary gland ducts and alveoli require adhesion to the extracellular matrix for their function. Mammary epithelial cells need ß1-integrins for normal cell cycle regulation. However, the role of ß1-integrins in tumorigenesis has not been fully resolved. ß1-integrin is necessary for tumour formation in transgenic mice expressing the Polyomavirus Middle T antigen, but it is dispensable in those overexpressing ErbB2. This suggests that some oncogenes can manage without ß1-integrin to proliferate and form tumours, while others still require it. Here we have developed a model to test whether expression of an oncogene can surpass the need for ß1-integrin to drive proliferation. We co-expressed the ErbB2 or Akt oncogenes with shRNA to target ß1-integrin in mammary epithelial cells, and found that they show a differential dependence on ß1-integrin for cell division. Moreover, we identified a key proliferative role of the Rac1-Pak axis downstream of ß1-integrin signalling. Our data suggest that, in mammary epithelial cells, oncogenes with the ability to signal to Pak surpass the requirement of integrins for malignant transformation. This highlights the importance of using the correct combination therapy for breast cancer, depending on the oncogenes expressed in the tumour.

Cellular mechano-environment regulates the mammary circadian clock.

Circadian clocks drive ∼24 h rhythms in tissue physiology. They rely on transcriptional/translational feedback loops driven by interacting networks of clock complexes. However, little is known about how cell-intrinsic circadian clocks sense and respond to their microenvironment. Here, we reveal that the breast epithelial clock is regulated by the mechano-chemical stiffness of the cellular microenvironment in primary cell culture. Moreover, the mammary clock is controlled by the periductal extracellular matrix in vivo, which contributes to a dampened circadian rhythm during ageing. Mechanistically, the tension sensing cell-matrix adhesion molecule, vinculin, and the Rho/ROCK pathway, which transduces signals provided by extracellular stiffness into cells, regulate the activity of the core circadian clock complex. We also show that genetic perturbation, or age-associated disruption of self-sustained clocks, compromises the self-renewal capacity of mammary epithelia. Thus, circadian clocks are mechano-sensitive, providing a potential mechanism to explain how ageing influences their amplitude and function.

Fascin is a key regulator of breast cancer invasion that acts via the modification of metastasis-associated molecules.

The actin-bundling protein, fascin, is a member of the cytoskeletal protein family that has restricted expression in specialized normal cells. However, many studies have reported the induction of this protein in various transformed cells including breast cancer cells. While the role of fascin in the regulation of breast cancer cell migration has been previously shown, the underlying molecular mechanism remained poorly defined. We have used variety of immunological and functional assays to study whether fascin regulates breast cancer metastasis-associated molecules. In this report we found a direct relationship between fascin expression in breast cancer patients and; metastasis and shorter disease-free survival. Most importantly, in vitro interference with fascin expression by loss or gain of function demonstrates a central role for this protein in regulating the cell morphology, migration and invasion potential. Our results show that fascin regulation of invasion is mediated via modulating several metastasis-associated genes. We show for the first time that fascin down-regulates the expression and nuclear translocation of a key metastasis suppressor protein known as breast cancer metastasis suppressor-1 (BRMS1). In addition, fascin up-regulates NF-kappa B activity, which is essential for metastasis. Importantly, fascin up-regulates other proteins that are known to be critical for the execution of metastasis such as urokinase-type plasminogen activator (uPA) and the matrix metalloproteases (MMP)-2 and MMP-9. This study demonstrates that fascin expression in breast cancer cells establishes a gene expression profile consistent with metastatic tumors and offers a potential therapeutic intervention in metastatic breast cancer treatment through fascin targeting.