Charles H. Streuli

Vinculin controls focal adhesion formation by direct interactions with talin and actin.

Focal adhesions (FAs) regulate cell migration. Vinculin, with its many potential binding partners, can interconnect signals in FAs. Despite the well-characterized structure of vinculin, the molecular mechanisms underlying its action have remained unclear. Here, using vinculin mutants, we separate the vinculin head and tail regions into distinct functional domains. We show that the vinculin head regulates integrin dynamics and clustering and the tail regulates the link to the mechanotransduction force machinery. The expression of vinculin constructs with unmasked binding sites in the head and tail regions induces dramatic FA growth, which is mediated by their direct interaction with talin. This interaction leads to clustering of activated integrin and an increase in integrin residency time in FAs. Surprisingly, paxillin recruitment, induced by active vinculin constructs, occurs independently of its potential binding site in the vinculin tail. The vinculin tail, however, is responsible for the functional link of FAs to the actin cytoskeleton. We propose a new model that explains how vinculin orchestrates FAs.

Extracellular matrix remodelling and cellular differentiation

The extracellular matrix is not merely a passive structure. In the past few years, it has emerged that the matrix is a dynamic action zone that functions to instruct cellular phenotype. Extracellular matrix proteins interact directly with cell surface receptors to initiate signal transduction pathways and to modulate those triggered by differentiation and growth factors. The extracellular matrix also controls the activity and presentation of a wide range of growth factors. Thus modulation of the extracellular matrix, by remodelling its structure and activity, has profound effects on its function and the consequent behaviour of cells residing on or within it.

Signal co-operation between integrins and other receptor systems

The multicellular nature of metazoans means that all cellular processes need to be tuned by adhesive interactions between cells and their local microenvironment. The spatial organization of cells within tissues requires sophisticated networks of extracellular signals to control their survival and proliferation, movements and positioning, and differentiated function. These cellular characteristics are mediated by multiple inputs from adhesion systems in combination with soluble and developmental signals. In the present review we explore how one class of adhesion receptor, the integrins, co-operate with other types of receptor to control diverse aspects of cell fate. In particular we discuss: (i) how beta3 and beta1 integrins work together with growth factors to control angiogenesis; (ii) how alpha6beta4 integrin co-operates with receptor tyrosine kinases in normal epithelial function and cancer; (iii) the interplay between beta1 integrins and EGF (epidermal growth factor) receptor; (iv) signal integration connecting integrins and cytokine receptors for interleukins, prolactin and interferons; and (v) how integrins and syndecans co-operate in cell migration.

Desmosomal adhesion regulates epithelial morphogenesis and cell positioning

Desmosomes are intercellular junctions of epithelia and are of widespread importance in the maintenance of tissue architecture. We provide evidence that desmosomal adhesion has a function in epithelial morphogenesis and cell-type-specific positioning. Blocking peptides corresponding to the cell adhesion recognition (CAR) sites of desmosomal cadherins block alveolar morphogenesis by epithelial cells from mammary lumen. Desmosomal CAR-site peptides also disrupt positional sorting of luminal and myoepithelial cells in aggregates formed by the reassociation of isolated cells. We demonstrate that desmosomal cadherins and E-cadherin are comparably involved in epithelial morphoregulation. The results indicate a wider role for desmosomal adhesion in morphogenesis than has previously been considered.

Critical research gaps and translational priorities for the successful prevention and treatment of breast cancer

IntroductionBreast cancer remains a significant scientific, clinical and societal challenge. This gap analysis has reviewed and critically assessed enduring issues and new challenges emerging from recent research, and proposes strategies for translating solutions into practice.MethodsMore than 100 internationally recognised specialist breast cancer scientists, clinicians and healthcare professionals collaborated to address nine thematic areas: genetics, epigenetics and epidemiology; molecular pathology and cell biology; hormonal influences and endocrine therapy; imaging, detection and screening; current/novel therapies and biomarkers; drug resistance; metastasis, angiogenesis, circulating tumour cells, cancer ‘stem’ cells; risk and prevention; living with and managing breast cancer and its treatment. The groups developed summary papers through an iterative process which, following further appraisal from experts and patients, were melded into this summary account.ResultsThe 10 major gaps identified were: (1) understanding the functions and contextual interactions of genetic and epigenetic changes in normal breast development and during malignant transformation; (2) how to implement sustainable lifestyle changes (diet, exercise and weight) and chemopreventive strategies; (3) the need for tailored screening approaches including clinically actionable tests; (4) enhancing knowledge of molecular drivers behind breast cancer subtypes, progression and metastasis; (5) understanding the molecular mechanisms of tumour heterogeneity, dormancy, de novo or acquired resistance and how to target key nodes in these dynamic processes; (6) developing validated markers for chemosensitivity and radiosensitivity; (7) understanding the optimal duration, sequencing and rational combinations of treatment for improved personalised therapy; (8) validating multimodality imaging biomarkers for minimally invasive diagnosis and monitoring of responses in primary and metastatic disease; (9) developing interventions and support to improve the survivorship experience; (10) a continuing need for clinical material for translational research derived from normal breast, blood, primary, relapsed, metastatic and drug-resistant cancers with expert bioinformatics support to maximise its utility. The proposed infrastructural enablers include enhanced resources to support clinically relevant in vitro and in vivo tumour models; improved access to appropriate, fully annotated clinical samples; extended biomarker discovery, validation and standardisation; and facilitated cross-discipline working.ConclusionsWith resources to conduct further high-quality targeted research focusing on the gaps identified, increased knowledge translating into improved clinical care should be achievable within five years.

Ablation of β1 integrin in mammary epithelium reveals a key role for integrin in glandular morphogenesis and differentiation

Integrin-mediated adhesion regulates the development and function of a range of tissues; however, little is known about its role in glandular epithelium. To assess the contribution of β1 integrin, we conditionally deleted its gene in luminal epithelia during different stages of mouse mammary gland development and in cultured primary mammary epithelia. Loss of β1 integrin in vivo resulted in impaired alveologenesis and lactation. Cultured β1 integrin–null cells displayed abnormal focal adhesion function and signal transduction and could not form or maintain polarized acini. In vivo, epithelial cells became detached from the extracellular matrix but remained associated with each other and did not undergo overt apoptosis. β1 integrin–null mammary epithelial cells did not differentiate in response to prolactin stimulation because of defective Stat5 activation. In mice where β1 integrin was deleted after the initiation of differentiation, fewer defects in alveolar morphology occurred, yet major deficiencies were also observed in milk protein and milk fat production and Stat5 activation, indicating a permissive role for β1 integrins in prolactin signaling. This study demonstrates that β1 integrin is critical for the alveolar morphogenesis of a glandular epithelium and for maintenance of its differentiated function. Moreover, it provides genetic evidence for the cooperation between integrin and cytokine signaling pathways.

Integrins and cell-fate determination.

All cellular processes are determined by adhesive interactions between cells and their local microenvironment. Integrins, which constitute one class of cell-adhesion receptor, are multifunctional proteins that link cells to the extracellular matrix and organise integrin adhesion complexes at the cell periphery. Integrin-based adhesions provide anchor points for assembling and organising the cytoskeleton and cell shape, and for orchestrating migration. Integrins also control the fate and function of cells by influencing their proliferation, apoptosis and differentiation. Moreover, new literature demonstrates that integrins control the cell-division axis at mitosis. This extends the influence of integrins over cell-fate decisions, as daughter cells are frequently located in new microenvironments that determine their behaviour following cell division. In this Commentary, I describe how integrins influence cell-fate determination, placing particular emphasis on their role in influencing the direction of cell division and the orientation of the mitotic spindle.

Control of Integrin Expression by Extracellular Matrix

Integrin-mediated interactions between cells and the extracellular matrix play a fundamental role in the development and function of a variety of tissues by triggering intracellular signals that regulate gene expression. In this study, mouse mammary epithelial cells plated on tissue culture plastic were shown to dramatically up-regulate the steady state levels of mRNA encoding the α1, α2, α3, α5, α6, α7, αv, and β1 integrin subunits, in contrast to cells cultured on a basement membrane matrix or cells in vivo. This pattern of expression was also observed in a mouse mammary epithelial strain, CID-9 and in other mouse cell lines such as MMTE cells and K1735-M2 melanoma cells. The control of integrin expression was mediated at different levels in different cell types. In K1735-M2 cells, transcription of the β1 integrin gene was influenced by the substratum, although the levels of integrin protein remained similar. In mammary epithelial cells, the rates of β1 integrin gene transcription were similar, but mRNA and protein levels were higher in cells cultured on plastic than those on basement membrane. For both cell types, the rate of integrin protein turnover was nearly identical in cells cultured on either substratum. Our results demonstrate that extracellular matrix controls the expression of β1 integrin subunits and that this regulation is exerted at both transcriptional and post-transcriptional levels.

Stat5 as a Target for Regulation by Extracellular Matrix

Transcription of tissue-specific genes in mammary gland requires signals from both prolactin and basement membrane. Here we address the mechanism by which this specialized extracellular matrix regulates transcription. Using mammary cell cultures derived from transgenic mice harboring the ovine β-lactoglobulin gene, we show that either a basement membrane extract, or purified laminin-1, induced high levels of β-lactoglobulin synthesis. It is known that prolactin signals through Stat5 (signal transducer and activator of transcription). This transcription factor interacts with -interferon activation site-related motifs within the β-lactoglobulin promoter, which we show are required for matrix dependence of β-lactoglobulin expression. The DNA binding activity of Stat5 was present only in extracts of mammary cells cultured on basement membrane, indicating that the activation state of Stat5 is regulated by the type of substratum the cell encounters. Thus, basement membrane controls transcription of milk protein genes through the Stat5-mediated prolactin signaling pathway, providing a molecular explanation for previous studies implicating extracellular matrix in the control of mammary differentiation.

β1 integrins regulate mammary gland proliferation and maintain the integrity of mammary alveoli

Integrin–extracellular matrix interactions play important roles in the coordinated integration of external and internal cues that are essential for proper development. To study the role of β1 integrin in the mammary gland, Itgβ1flox/flox mice were crossed with WAPiCre transgenic mice, which led to specific ablation of β1 integrin in luminal alveolar epithelial cells. In the β1 integrin mutant mammary gland, individual alveoli were disorganized resulting from alterations in cell–basement membrane associations. Activity of focal adhesion kinase (FAK) was also decreased in mutant mammary glands. Luminal cell proliferation was strongly inhibited in β1 integrin mutant glands, which correlated with a specific increase of p21Cip1 expression. In a p21Cip1 null background, there was a partial rescue of BrdU incorporation, providing in vivo evidence linking p21Cip1 to the proliferative defect observed in β1 integrin mutant glands. A connection between p21Cip1 and β1 integrin as well as FAK was also established in primary mammary cells. These results point to the essential role of β1 integrin signaling in mammary epithelial cell proliferation.