RAC1B function is essential for breast cancer stem cell maintenance and chemoresistance of breast tumor cells.

Breast cancer stem cells (BCSC) are presumed to be responsible for treatment resistance, tumor recurrence and metastasis of breast tumors. However, development of BCSC-targeting therapies has been held back by their heterogeneity and the lack of BCSC-selective molecular targets. Here, we demonstrate that RAC1B, the only known alternatively spliced variant of the small GTPase RAC1, is expressed in a subset of BCSCs in vivo and its function is required for the maintenance of BCSCs and their chemoresistance to doxorubicin. In human breast cancer cell line MCF7, RAC1B is required for BCSC plasticity and chemoresistance to doxorubicin in vitro and for tumor-initiating abilities in vivo. Unlike Rac1, Rac1b function is dispensable for normal mammary gland development and mammary epithelial stem cell (MaSC) activity. In contrast, loss of Rac1b function in a mouse model of breast cancer hampers the BCSC activity and increases their chemosensitivity to doxorubicin treatment. Collectively, our data suggest that RAC1B is a clinically relevant molecular target for the development of BCSC-targeting therapies that may improve the effectiveness of doxorubicin-mediated chemotherapy.

Elevated EDAR signalling promotes mammary gland tumourigenesis with squamous metaplasia.

Ectodysplasin A receptor (EDAR) is a death receptor in the Tumour Necrosis Factor Receptor (TNFR) superfamily with roles in the development of hair follicles, teeth and cutaneous glands. Here we report that human Oestrogen Receptor (ER) negative breast carcinomas which display squamous differentiation express EDAR strongly. Using a mouse model with a high Edar copy number, we show that elevated EDAR signalling results in a high incidence of mammary tumours in breeding female mice. These tumours resemble the EDAR-high human tumours in that they are characterised by a lack of oestrogen receptor expression, contain extensive squamous metaplasia, and display strong ß-catenin transcriptional activity. In the mouse model, all of the tumours carry somatic deletions of the third exon of the CTNNB1 gene that encodes ß-catenin. Deletion of this exon yields unconstrained ß-catenin signalling activity. We also demonstrate that ß-catenin activity is required for transformed cell growth, showing that increased EDAR signalling creates an environment in which ß-catenin activity can readily promote tumourigenesis. Together, this work identifies a novel death receptor oncogene in breast cancer, whose mechanism of transformation is based on the interaction between the WNT and Ectodysplasin A (EDA) pathways.

Vinculins interaction with talin is essential for mammary epithelial differentiation.

Vinculin is an essential component of cell adhesion complexes, where it regulates the strength and stability of adhesions. Whilst the role of vinculin in cell motility is well established, it remains unclear how vinculin contributes to other aspects of tissue function. Here we examine the role of vinculin in mammary epithelial cell phenotype. In these cells, correct adhesion to the extracellular matrix is essential for both the formation of polarised secretory acini and for the transcription of tissue-specific milk protein genes. We show that vinculin, through its interaction with talin, controls milk protein gene expression. However, vinculin is not required for the formation of polarised acini. This work reveals new roles for vinculin that are central to cellular differentiation, and for the ability of cells to interpret their extracellular microenvironment.

Influence of the extracellular matrix on cell-intrinsic circadian clocks.

Cell-autonomous circadian clocks coordinate tissue homeostasis with a 24-hourly rhythm. The molecular circadian clock machinery controls tissue- and cell type-specific sets of rhythmic genes. Disruptions of clock mechanisms are linked to an increased risk of acquiring diseases, especially those associated with aging, metabolic dysfunction and cancer. Despite rapid advances in understanding the cyclic outputs of different tissue clocks, less is known about how the clocks adapt to their local niche within tissues. We have discovered that tissue stiffness regulates circadian clocks, and that this occurs in a cell-type-dependent manner. In this Review, we summarise new work linking the extracellular matrix with differential control of circadian clocks. We discuss how the changes in tissue structure and cellular microenvironment that occur throughout life may impact on the molecular control of circadian cycles. We also consider how altered clocks may have downstream impacts on the acquisition of diseases.

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.

Disrupted circadian clocks and altered tissue mechanics in primary human breast tumours.

BACKGROUND: Circadian rhythms maintain tissue homeostasis during the 24-h day-night cycle. Cell-autonomous circadian clocks play fundamental roles in cell division, DNA damage responses and metabolism. Circadian disruptions have been proposed as a contributing factor for cancer initiation and progression, although definitive evidence for altered molecular circadian clocks in cancer is still lacking. In this study, we looked at circadian clocks in breast cancer. METHODS: We isolated primary tumours and normal tissues from the same individuals who had developed breast cancer with no metastases. We assessed circadian clocks within primary cells of the patients by lentiviral expression of circadian reporters, and the levels of clock genes in tissues by qPCR. We histologically examined collagen organisation within the normal and tumour tissue areas, and probed the stiffness of the stroma adjacent to normal and tumour epithelium using atomic force microscopy. RESULTS: Epithelial ducts were disorganised within the tumour areas. Circadian clocks were altered in cultured tumour cells. Tumour regions were surrounded by stroma with an altered collagen organisation and increased stiffness. Levels of Bmal1 messenger RNA (mRNA) were significantly altered in the tumours in comparison to normal epithelia. CONCLUSION: Circadian rhythms are suppressed in breast tumour epithelia in comparison to the normal epithelia in paired patient samples. This correlates with increased tissue stiffness around the tumour region. We suggest possible involvement of altered circadian clocks in the development and progression of breast cancer.

Epithelial and stromal circadian clocks are inversely regulated by their mechano-matrix environment.

The circadian clock is an autonomous molecular feedback loop inside almost every cell in the body. We have shown that the mammary epithelial circadian clock is regulated by the cellular microenvironment. Moreover, a stiff extracellular matrix dampens the oscillations of the epithelial molecular clock. Here, we extend this analysis to other tissues and cell types, and identify an inverse relationship between circadian clocks in epithelia and fibroblasts. Epithelial cells from mammary gland, lung and skin have significantly stronger oscillations of clock genes in soft 3D microenvironments, compared to stiff 2D environments. Fibroblasts isolated from the same tissues show the opposite response, exhibiting stronger oscillations and more prolonged rhythmicity in stiff microenvironments. RNA analysis identified that a subset of mammary epithelial clock genes, and their regulators, are upregulated in 3D microenvironments in soft compared to stiff gels. Furthermore, the same genes are inversely regulated in fibroblasts isolated from the same tissues. Thus, our data reveal for the first time an intrinsic difference in the regulation of circadian genes in epithelia and fibroblasts.

Extracellular matrix promotes clathrin-dependent endocytosis of prolactin and STAT5 activation in differentiating mammary epithelial cells.

The hormone prolactin promotes lactational differentiation of mammary epithelial cells (MECs) via its cognate receptor and the downstream JAK2-STAT5a signalling pathway. In turn this regulates transcription of milk protein genes. Prolactin signalling depends on a cross-talk with basement membrane extracellular matrix (ECM) via ß1 integrins which activate both ILK and Rac1 and are required for STAT5a activation and lactational differentiation. Endocytosis is an important regulator of signalling. It can both enhance and suppress cytokine signalling, although the role of endocytosis for prolactin signalling is not known. Here we show that clathrin-mediated endocytosis is required for ECM-dependent STAT5 activation. In the presence of ECM, prolactin is internalised via a clathrin-dependent, but caveolin-independent, route. This occurs independently from JAK2 and Rac signalling, but is required for full phosphorylation and activation of STAT5. Prolactin is internalised into early endosomes, where the master early endosome regulator Rab5b promotes STAT5 phosphorylation. These data reveal a novel role for ECM-driven endocytosis in the positive regulation of cytokine signalling.

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.

Integrins as architects of cell behavior.

Integrins are cell surface receptors that bind cells to their physical external environment, linking the extracellular matrix to cell function. They are essential in the biology of all animals. In the late 1980s, we discovered that integrins are required for the ability of breast epithelia to do what they are programmed to do, which is to differentiate and make milk. Since then, integrins have been shown to control most other aspects of phenotype: to stay alive, to divide, and to move about. Integrins also provide part of the mechanism that allows cells to form tissues. Here I discuss how we discovered that integrins control mammary gland differentiation and explore the role of integrins as central architects of other aspects of cell behavior.

Circadian clocks and breast cancer.

Circadian clocks respond to environmental time cues to coordinate 24-hour oscillations in almost every tissue of the body. In the breast, circadian clocks regulate the rhythmic expression of numerous genes. Disrupted expression of circadian genes can alter breast biology and may promote cancer. Here we overview circadian mechanisms, and the connection between the molecular clock and breast biology. We describe how disruption of circadian genes contributes to cancer via multiple mechanisms, and link this to increased tumour risk in women who work irregular shift patterns. Understanding the influence of circadian rhythms on breast cancer could lead to more efficacious therapies, reformed public health policy and improved patient outcome.

Oncogenic activation of FAK drives apoptosis suppression in a 3D-culture model of breast cancer initiation.

A key hallmark of cancer cells is the loss of positional control over growth and survival. Focal adhesion kinase (FAK) is a tyrosine kinase localised at sites of integrin-mediated cell adhesion to the extracellular matrix. FAK controls a number of adhesion-dependent cellular functions, including migration, proliferation and survival. Although FAK is overexpressed and activated in metastatic tumours, where it promotes invasion, it can also be elevated in cancers that have yet to become invasive. The contribution of FAK to the early stages of tumourigenesis is not known. We have examined the effect of activating FAK in non-transformed mammary epithelial cells (MECs) to understand its role in tumour initiation. In agreement with previous studies, we find FAK activation in 2D-culture promotes proliferation, migration, and epithelial-to-mesenchymal transition. However in 3D-cultures that better resemble normal tissue morphology, mammary cells largely respond to FAK activation via suppression of apoptosis, promoting aberrant acinar morphogenesis. This is an acquired function of FAK, because endogenous FAK signalling is not required for normal morphogenesis in 3D-culture or in vivo. Thus, FAK activation may facilitate tumour initiation by causing resistance to apoptosis. We suggest that aberrant FAK activation in breast epithelia is dependent upon the tissue context in which it occurs.

Rac1 Controls Both the Secretory Function of the Mammary Gland and Its Remodeling for Successive Gestations.

An important feature of the mammary gland is its ability to undergo repeated morphological changes during each reproductive cycle with profound tissue expansion in pregnancy and regression in involution. However, the mechanisms that determine the tissue's cyclic regenerative capacity remain elusive. We have now discovered that Cre-Lox ablation of Rac1 in mammary epithelia causes gross enlargement of the epithelial tree and defective alveolar regeneration in a second pregnancy. Architectural defects arise because loss of Rac1 disrupts clearance in involution following the first lactation. We show that Rac1 is crucial for mammary alveolar epithelia to switch from secretion to a phagocytic mode and rapidly remove dying neighbors. Moreover, Rac1 restricts the extrusion of dying cells into the lumen, thus promoting their eradication by live phagocytic neighbors while within the epithelium. Without Rac1, residual milk and cell corpses flood the ductal network, causing gross dilation, chronic inflammation, and defective future regeneration.

Raised mammographic density: causative mechanisms and biological consequences.

High mammographic density is the most important risk factor for breast cancer, after ageing. However, the composition, architecture, and mechanical properties of high X-ray density soft tissues, and the causative mechanisms resulting in different mammographic densities, are not well described. Moreover, it is not known how high breast density leads to increased susceptibility for cancer, or the extent to which it causes the genomic changes that characterise the disease. An understanding of these principals may lead to new diagnostic tools and therapeutic interventions.

The Integrin-Mediated ILK-Parvin-αPix Signaling Axis Controls Differentiation in Mammary Epithelial Cells.

Epithelial cell adhesion to the surrounding extracellular matrix is necessary for their proper behavior and function. During pregnancy and lactation, mammary epithelial cells (MECs) receive signals from their interaction with laminin via ß1-integrin (ß1-itg) to establish apico-basal polarity and to differentiate in response to prolactin. Downstream of ß1-itg, the scaffold protein Integrin Linked Kinase (ILK) has been identified as the key signal transducer that is required for both lactational differentiation and the establishment of apico-basal polarity. ILK is an adaptor protein that forms the IPP complex with PINCH and Parvins, which are central to its adaptor functions. However, it is not known how ILK and its interacting partners control tissue-specific gene expression. Expression of ILK mutants, which weaken the interaction between ILK and Parvin, revealed that Parvins have a role in mammary epithelial differentiation. This conclusion was supported by shRNA-mediated knockdown of the Parvins. In addition, shRNA knockdown of the Parvin-binding guanine nucleotide exchange factor αPix prevented prolactin-induced differentiation. αPix depletion did not disrupt focal adhesions, MEC proliferation, or polarity. This suggests that αPix represents a differentiation-specific bifurcation point in ß1-itg-ILK adhesive signaling. In summary, this study has identified a new role for Parvin and αPix downstream of the integrin-ILK signaling axis for MEC differentiation. J. Cell. Physiol. 231: 2408-2417, 2016. © 2016 Wiley Periodicals, Inc.

Cellular microenvironment controls the nuclear architecture of breast epithelia through ß1-integrin.

Defects in nuclear architecture occur in a variety of diseases, however the fundamental mechanisms that control the internal structure of nuclei are poorly defined. Here we reveal that the cellular microenvironment has a profound influence on the global internal organization of nuclei in breast epithelia. A 3D microenvironment induces a prolonged but reversible form of cell cycle arrest that features many of the classical markers of cell senescence. This unique form of arrest is dependent on signaling from the external microenvironment through ß1-integrins. It is concomitant with alterations in nuclear architecture that characterize the withdrawal from cell proliferation. Unexpectedly, following prolonged cell cycle arrest in 3D, the senescence-like state and associated reprogramming of nuclear architecture are freely reversible on altering the dimensionality of the cellular microenvironment. Breast epithelia can therefore maintain a proliferative plasticity that correlates with nuclear remodelling. However, the changes in nuclear architecture are cell lineage-specific and do not occur in fibroblasts, and moreover they are overcome in breast cancer cells.

Increased peri-ductal collagen micro-organization may contribute to raised mammographic density.

BACKGROUND: High mammographic density is a therapeutically modifiable risk factor for breast cancer. Although mammographic density is correlated with the relative abundance of collagen-rich fibroglandular tissue, the causative mechanisms, associated structural remodelling and mechanical consequences remain poorly defined. In this study we have developed a new collaborative bedside-to-bench workflow to determine the relationship between mammographic density, collagen abundance and alignment, tissue stiffness and the expression of extracellular matrix organising proteins. METHODS: Mammographic density was assessed in 22 post-menopausal women (aged 54-66 y). A radiologist and a pathologist identified and excised regions of elevated non-cancerous X-ray density prior to laboratory characterization. Collagen abundance was determined by both Masson's trichrome and Picrosirius red staining (which enhances collagen birefringence when viewed under polarised light). The structural specificity of these collagen visualisation methods was determined by comparing the relative birefringence and ultrastructure (visualised by atomic force microscopy) of unaligned collagen I fibrils in reconstituted gels with the highly aligned collagen fibrils in rat tail tendon. Localised collagen fibril organisation and stiffness was also evaluated in tissue sections by atomic force microscopy/spectroscopy and the abundance of key extracellular proteins was assessed using mass spectrometry. RESULTS: Mammographic density was positively correlated with the abundance of aligned periductal fibrils rather than with the abundance of amorphous collagen. Compared with matched tissue resected from the breasts of low mammographic density patients, the highly birefringent tissue in mammographically dense breasts was both significantly stiffer and characterised by large (>80 µm long) fibrillar collagen bundles. Subsequent proteomic analyses not only confirmed the absence of collagen fibrosis in high mammographic density tissue, but additionally identified the up-regulation of periostin and collagen XVI (regulators of collagen fibril structure and architecture) as potential mediators of localised mechanical stiffness. CONCLUSIONS: These preliminary data suggest that remodelling, and hence stiffening, of the existing stromal collagen microarchitecture promotes high mammographic density within the breast. In turn, this aberrant mechanical environment may trigger neoplasia-associated mechanotransduction pathways within the epithelial cell population.

Integrin α4ß1 controls G9a activity that regulates epigenetic changes and nuclear properties required for lymphocyte migration.

The mechanical properties of the cell nucleus change to allow cells to migrate, but how chromatin modifications contribute to nuclear deformability has not been defined. Here, we demonstrate that a major factor in this process involves epigenetic changes that underpin nuclear structure. We investigated the link between cell adhesion and epigenetic changes in T-cells, and demonstrate that T-cell adhesion to VCAM1 via α4ß1 integrin drives histone H3 methylation (H3K9me2/3) through the methyltransferase G9a. In this process, active G9a is recruited to the nuclear envelope and interacts with lamin B1 during T-cell adhesion through α4ß1 integrin. G9a activity not only reorganises the chromatin structure in T-cells, but also affects the stiffness and viscoelastic properties of the nucleus. Moreover, we further demonstrated that these epigenetic changes were linked to lymphocyte movement, as depletion or inhibition of G9a blocks T-cell migration in both 2D and 3D environments. Thus, our results identify a novel mechanism in T-cells by which α4ß1 integrin signaling drives specific chromatin modifications, which alter the physical properties of the nucleus and thereby enable T-cell migration.

The Immunology of Breast Development.

The immune system is not normally viewed as a regulator of breast development. However, in this issue of Developmental Cell, Plaks et al. (2015) reveal that antigen-presenting cells and T cells have a key role in controlling the development of the mammary gland's epithelial ductal network.