Erik Sahai

Membrane blebbing during apoptosis results from caspase-mediated activation of ROCK I

The execution phase of apoptosis is characterized by marked changes in cell morphology that include contraction and membrane blebbing. The actin–myosin system has been proposed to be the source of contractile force that drives bleb formation, although the biochemical pathway that promotes actin–myosin contractility during apoptosis has not been identified. Here we show that the Rho effector protein ROCK I, which contributes to phosphorylation of myosin light-chains, myosin ATPase activity and coupling of actin–myosin filaments to the plasma membrane, is cleaved during apoptosis to generate a truncated active form. The activity of ROCK proteins is both necessary and sufficient for formation of membrane blebs and for re-localization of fragmented DNA into blebs and apoptotic bodies.

Fibroblast-led collective invasion of carcinoma cells with differing roles for RhoGTPases in leading and following cells

Imaging of collectively invading cocultures of carcinoma cells and stromal fibroblasts reveals that the leading cell is always a fibroblast and that carcinoma cells move within tracks in the extracellular matrix behind the fibroblast. The generation of these tracks by fibroblasts is sufficient to enable the collective invasion of the squamous cell carcinoma (SCC) cells and requires both protease- and force-mediated matrix remodelling. Force-mediated matrix remodelling depends on integrins α3 and α5, and Rho-mediated regulation of myosin light chain (MLC) activity in fibroblasts, but these factors are not required in carcinoma cells. Instead, carcinoma cells use Cdc42 and MRCK (myotonic dystrophy kinase-related CDC42-binding protein kinases) mediated regulation of MLC to follow the tracks generated by fibroblasts.

Rac Activation and Inactivation Control Plasticity of Tumor Cell Movement

Tumor cells exhibit two different modes of individual cell movement. Mesenchymal-type movement is characterized by an elongated cellular morphology and requires extracellular proteolysis. In amoeboid movement, cells have a rounded morphology, are less dependent on proteases, and require high Rho-kinase signaling to drive elevated levels of actomyosin contractility. These two modes of cell movement are interconvertible. We show that mesenchymal-type movement in melanoma cells is driven by activation of the GTPase Rac through a complex containing NEDD9, a recently identified melanoma metastasis gene, and DOCK3, a Rac guanine nucleotide exchange factor. Rac signals through WAVE2 to direct mesenchymal movement and suppress amoeboid movement through decreasing actomyosin contractility. Conversely, in amoeboid movement, Rho-kinase signaling activates a Rac GAP, ARHGAP22, that suppresses mesenchymal movement by inactivating Rac. We demonstrate tight interplay between Rho and Rac in determining different modes of tumor cell movement, revealing how tumor cells switch between different modes of movement.

Macrophages Promote the Invasion of Breast Carcinoma Cells via a Colony-Stimulating Factor-1/Epidermal Growth Factor Paracrine Loop

Previous studies have shown that macrophages and tumor cells are comigratory in mammary tumors and that these cell types are mutually dependent for invasion. Here we show that macrophages and tumor cells are necessary and sufficient for comigration and invasion into collagen I and that this process involves a paracrine loop. Macrophages express epidermal growth factor (EGF), which promotes the formation of elongated protrusions and cell invasion by carcinoma cells. Colony stimulating factor 1 (CSF-1) produced by carcinoma cells promotes the expression of EGF by macrophages. In addition, EGF promotes the expression of CSF-1 by carcinoma cells thereby generating a positive feedback loop. Disruption of this loop by blockade of either EGF receptor or CSF-1 receptor signaling is sufficient to inhibit both macrophage and tumor cell migration and invasion.

Localized and reversible TGFbeta signalling switches breast cancer cells from cohesive to single cell motility.

Here we use intravital imaging to demonstrate a reversible transition to a motile state as breast cancer cells spread. Imaging primary tumours revealed heterogeneity in cell morphology and motility. Two distinct modes of motility were observed: collective and single-celled. By monitoring the localization of Smad2 and the activity of a TGFβ-dependent reporter gene during breast cancer cell dissemination, we demonstrate that TGFβ signalling is transiently and locally activated in motile single cells. TGFβ1 switches cells from cohesive to single cell motility through a transcriptional program involving Smad4, EGFR, Nedd9, M-RIP, FARP and RhoC. Blockade of TGFβ signalling prevented cells moving singly in vivo but did not inhibit cells moving collectively. Cells restricted to collective invasion were capable of lymphatic invasion but not blood-borne metastasis. Constitutive TGFβ signalling promoted single cell motility and intravasation but reduced subsequent growth in the lungs. Thus, transient TGFβ signalling is essential for blood-borne metastasis.

Illuminating the metastatic process

Until recently most studies of metastasis only measured the end point of the process — macroscopic metastases. Although these studies have provided much useful information, the details of the metastatic process remain somewhat mysterious owing to difficulties in studying cell behaviour with high spatial and temporal resolution in vivo. The use of luminescent and fluorescent proteins and developments in optical imaging technology have enabled the direct observation of cancer cells spreading from their site of origin and arriving at secondary sites. This Review will describe recent advances in our understanding of the different steps of metastasis gained from cellular resolution imaging, and how these techniques can be used in preclinical drug evaluation.

Identification and Testing of a Gene Expression Signature of Invasive Carcinoma Cells within Primary Mammary Tumors

We subjected cells collected using an in vivo invasion assay to cDNA microarray analysis to identify the gene expression profile of invasive carcinoma cells in primary mammary tumors. Expression of genes involved in cell division, survival, and cell motility were most dramatically changed in invasive cells indicating a population that is neither dividing nor apoptotic but intensely motile. In particular, the genes coding for the minimum motility machine that regulates β-actin polymerization at the leading edge and, therefore, the motility and chemotaxis of carcinoma cells, were dramatically up-regulated. However, ZBP1, which restricts the localization of β-actin, the substrate for the minimum motility machine, was down-regulated. This pattern of expression implicated ZBP1 as a suppressor of invasion. Reexpression of ZBP1 in metastatic cells with otherwise low levels of ZBP1 reestablished normal patterns of β-actin mRNA targeting and suppressed chemotaxis and invasion in primary tumors. ZBP1 reexpression also inhibited metastasis from tumors. These experiments support the involvement in metastasis of the pathways identified in invasive cells, which are regulated by ZBP1.

Classifying collective cancer cell invasion

Most invasive solid tumours display predominantly collective invasion, in which groups of cells invade the peritumoral stroma while maintaining cell-cell contacts. As the concepts and experimental models for functional analysis of collective cancer cell invasion are rapidly developing, we propose a framework for addressing potential mechanisms, experimental strategies and technical challenges to study this process.

ROCK- and Myosin-Dependent Matrix Deformation Enables Protease-Independent Tumor-Cell Invasion In Vivo

Tumor cells invading three-dimensional matrices need to remodel the extracellular matrix (ECM) in their path. Many studies have focused on the role of extracellular proteases; however, cells with amoeboid or rounded morphologies are able to invade even when these enzymes are inhibited. Here, we describe the mechanism by which cells move through a dense ECM without proteolysis. Amoeboid tumor cells generate sufficient actomyosin force to deform collagen fibers and are able to push through the ECM. Force generation is elevated in metastatic MTLn3E cells, and this correlates with increased invasion and altered myosin light chain (MLC) organization. In metastatic cells, MLC is organized perpendicularly to the direction of movement behind the invading edge. Both the organization of MLC and force generation are dependent upon ROCK function. We demonstrate that ROCK regulates the phosphorylation of MLC just behind the invading margin of the cell. Imaging of live tumors shows that MLC is organized in a similar ROCK-dependent fashion in vivo and that inhibition of ROCK but not matrix-metalloproteases reduces cancer cell motility in vivo.

Cross-talk between Ras and Rho signalling pathways in transformation favours proliferation and increased motility.

Transformation by oncogenic Ras requires the function of the Rho family GTPases. We find that Ras‐transformed cells have elevated levels of RhoA‐GTP, which functions to inhibit the expression of the cell cycle inhibitor p21/Waf1. These high levels of Rho‐GTP are not a direct consequence of Ras signalling but are selected for in response to sustained ERK–MAP kinase signalling. While the elevated levels of Rho‐GTP control the level of p21/Waf, they no longer regulate the formation of actin stress fibres in transformed cells. We show that the sustained ERK–MAP kinase signalling resulting from transformation by oncogenic Ras down‐regulates ROCK1 and Rho‐kinase, two Rho effectors required for actin stress fibre formation. The repression of Rho‐ dependent stress fibre formation by ERK–MAP kinase signalling contributes to the increased motility of Ras‐transformed fibroblasts. Overexpression of the ROCK target LIM kinase restores actin stress fibres and inhibits the motility of Ras‐transformed fibroblasts. We propose a model in which Ras and Rho signalling pathways cross‐talk to promote signalling pathways favouring transformation.