Direct measurement of vertical forces shows correlation between mechanical activity and proteolytic ability of invadopodia.

Mechanobiology plays a prominent role in cancer invasion and metastasis. The ability of a cancer to degrade extracellular matrix (ECM) is likely connected to its invasiveness. Many cancer cells form invadopodia-micrometer-sized cellular protrusions that promote invasion through matrix degradation (proteolysis). Although it has been hypothesized that invadopodia exert mechanical force that is implicated in cancer invasion, direct measurements remain elusive. Here, we use a recently developed interferometric force imaging technique that provides piconewton resolution to quantify invadopodial forces in cells of head and neck squamous carcinoma and to monitor their temporal dynamics. We compare the force exerted by individual protrusions to their ability to degrade ECM and investigate the mechanical effects of inhibiting invadopodia through overexpression of microRNA-375. By connecting the biophysical and biochemical characteristics of invadopodia, our study provides a new perspective on cancer invasion that, in the future, may help to identify biomechanical targets for cancer therapy.

Autocrine HBEGF expression promotes breast cancer intravasation, metastasis and macrophage-independent invasion in vivo.

Increased expression of HBEGF in estrogen receptor-negative breast tumors is correlated with enhanced metastasis to distant organ sites and more rapid disease recurrence upon removal of the primary tumor. Our previous work has demonstrated a paracrine loop between breast cancer cells and macrophages in which the tumor cells are capable of stimulating macrophages through the secretion of colony-stimulating factor-1 while the tumor-associated macrophages (TAMs), in turn, aid in tumor cell invasion by secreting epidermal growth factor. To determine how the autocrine expression of epidermal growth factor receptor (EGFR) ligands by carcinoma cells would affect this paracrine loop mechanism, and in particular whether tumor cell invasion depends on spatial ligand gradients generated by TAMs, we generated cell lines with increased HBEGF expression. We found that autocrine HBEGF expression enhanced in vivo intravasation and metastasis and resulted in a novel phenomenon in which macrophages were no longer required for in vivo invasion of breast cancer cells. In vitro studies revealed that expression of HBEGF enhanced invadopodium formation, thus providing a mechanism for cell autonomous invasion. The increased invadopodium formation was directly dependent on EGFR signaling, as demonstrated by a rapid decrease in invadopodia upon inhibition of autocrine HBEGF/EGFR signaling as well as inhibition of signaling downstream of EGFR activation. HBEGF expression also resulted in enhanced invadopodium function via upregulation of matrix metalloprotease 2 (MMP2) and MMP9 expression levels. We conclude that high levels of HBEGF expression can short-circuit the tumor cell/macrophage paracrine invasion loop, resulting in enhanced tumor invasion that is independent of macrophage signaling.

Phosphoinositide 3-kinase signaling is critical for ErbB3-driven breast cancer cell motility and metastasis.

Many malignancies show increased expression of the epidermal growth factor (EGF) receptor family member ErbB3 (HER3). ErbB3 binds heregulin ß-1 (HRGß1) and forms a heterodimer with other ErbB family members, such as ErbB2 (HER2) or EGF receptor (EGFR; HER1), enhancing phosphorylation of specific C-terminal tyrosine residues and activation of downstream signaling pathways. ErbB3 contains six YXXM motifs that bind the p85 subunit of phosphoinositide 3 (PI3)-kinase. Previous studies demonstrated that overexpression of ErbB3 in mammary tumor cells can significantly enhance chemotaxis to HRGß1 and overall metastatic potential. We tested the hypothesis that ErbB3-mediated PI3-kinase signaling is critical for heregulin-induced motility, and therefore crucial for ErbB3-mediated invasion, intravasation and metastasis. The tyrosines in the six YXXM motifs on the ErbB3 C-terminus were replaced with phenylalanine. In contrast to overexpression of the wild-type ErbB3, overexpression of the mutant ErbB3 did not enhance chemotaxis towards HRGß1 in vitro or in vivo. We also observed reduced tumor cell motility in the primary tumor by multiphoton microscopy, as well as a dramatically reduced ability of these cells to cross the endothelium and intravasate into the circulation. Moreover, whereas mutation of the ErbB3 C-terminus had no effect on tumor growth, it had a dramatic effect on spontaneous metastatic potential. Treatment with the PI3-kinase inhibitor PIK-75 similarly inhibited motility and invasion in vitro and in vivo. Our results indicate that stimulation of the early metastatic steps of motility and invasion by ErbB3 requires activation of the PI3-kinase pathway by the ErbB3 receptor.

The F-actin side binding activity of the Arp2/3 complex is essential for actin nucleation and lamellipod extension.

Most eukaryotic cells rely on localized actin polymerization to generate and sustain the protrusion activity necessary for cell movement [1, 2]. Such protrusions are often in the form of a flat lamellipod with a leading edge composed of a dense network of actin filaments [3, 4]. The Arp2/3 complex localizes within that network in vivo [3, 4] and nucleates actin polymerization and generates a branched network of actin filaments in vitro [5-7]. The complex has thus been proposed to generate the actin network at the leading edge of crawling cells in vivo [3, 4, 8]. However, the relative contributions of nucleation and branching to protrusive force are still unknown. We prepared antibodies to the p34 subunit of the Arp2/3 complex that selectively inhibit side binding of the complex to F-actin. We demonstrate that side binding is required for efficient nucleation and branching by the Arp2/3 complex in vitro. However, microinjection of these antibodies into cells specifically inhibits lamellipod extension without affecting the EGF-stimulated appearance of free barbed ends in situ. These results indicate that while the side binding activity of the Arp2/3 complex is required for nucleation in vitro and for protrusive force in vivo, it is not required for EGF-stimulated increases in free barbed ends in vivo. This suggests that the branching activity of the Arp2/3 complex is essential for lamellipod extension, while the generation of nucleation sites for actin polymerization is not sufficient.

Lamellipodia in invasion.

In vivo imaging of GFP-labeled metastatic tumor cells reveals cell orientation towards blood vessels. Orientation of tumor cells during chemotactic responses to ligands such as EGF begins with lamellipod extension. Evaluation of some of the downstream events in lamellipod extension indicates: (1) plasma membrane distribution of the EGF receptor is uniform but internalized receptor accumulates on the side of the cell closest to the source of EGF; (2) the alpha p110 isoform of PI-3 kinase is required; and (3) protrusion of the lamellipod relies upon the combined actions of the Arp2/3 complex and cofilin for generation of filamentous actin.

N-terminal domains of the class ia phosphoinositide 3-kinase regulatory subunit play a role in cytoskeletal but not mitogenic signaling.

Phosphoinositide (PI) 3-kinases are required for the acute regulation of the cytoskeleton by growth factors. We have shown previously that in the MTLn3 rat adenocarcinoma cells line, the p85/p110alpha PI 3-kinase is required for epidermal growth factor (EGF)-stimulated lamellipod extension and formation of new actin barbed ends at the leading edge of the cell. We have now examined the role of the p85alpha regulatory subunit in greater detail. Microinjection of recombinant p85alpha into MTLn3 cells blocked both EGF-stimulated mitogenic signaling and lamellipod extension. In contrast, a truncated p85(1-333), which lacks the SH2 and iSH2 domains and does not bind p110, had no effect on EGF-stimulated mitogenesis but still blocked EGF-stimulated lamellipod extension. Additional deletional analysis showed that the SH3 domain was not required for inhibition of lamellipod extension, as a construct containing only the proline-rich and breakpoint cluster region (BCR) homology domains was sufficient for inhibition. Although the BCR domain of p85 binds Rac, the effects of the p85 constructs were not because of a general inhibition of Rac signaling, because sorbitol-induced JNK activation in MTLn3 cells was not inhibited. These data show that the proline-rich and BCR homology domains of p85 are involved in the coupling of p85/p110 PI 3-kinases to regulation of the actin cytoskeleton. These data provide evidence of a distinct cellular function for the N-terminal domains of p85.

Chemotaxis assays for eukaryotic cells.

Chemotaxis is a complex response of a cell to an external stimulus. It involves detecting and measuring the concentration of the chemoattractant, biochemical transmission of the information, and the motility and adhesive changes associated with the response. This unit describes a number of chemotaxis assays that can be used to identify chemoattractants individually and in large-scale screenings, to distinguish chemotaxis from chemokinesis, and to analyze cellular behavioral and biochemical responses. Some of these assays such as the filter, under agarose, and small population assays, can be used to monitor the behavior of large groups of cells; the bridge, pipet, and upshift assays can be used to analyze the responses of single cells.

Epidermal growth factor receptor distribution during chemotactic responses.

To determine the distribution of the epidermal growth factor (EGF) receptor (EGFR) on the surface of cells responding to EGF as a chemoattractant, an EGFR-green fluorescent protein chimera was expressed in the MTLn3 mammary carcinoma cell line. The chimera was functional and easily visualized on the cell surface. In contrast to other studies indicating that the EGFR might be localized to certain regions of the plasma membrane, we found that the chimera is homogeneously distributed on the plasma membrane and becomes most concentrated in vesicles after endocytosis. In spatial gradients of EGF, endocytosed receptor accumulates on the upgradient side of the cell. Visualization of the binding of fluorescent EGF to cells reveals that the affinity properties of the receptor, together with its expression level on cells, can provide an initial amplification step in spatial gradient sensing.

The collection of the motile population of cells from a living tumor.

In this study, we report that needles containing chemoattractants can be used to collect the subpopulation of motile and chemotactic tumor cells from a primary tumor in a live rat as a pure population suitable for further analysis. The most efficient cell collection requires the presence of chemotactic cytokines, such as epidermal growth factor and serum components, and occurs with 15-fold higher efficiency in metastatic tumors compared with nonmetastatic tumors. Although tumor cells of the nonmetastatic tumors show a motility response to serum, they were not collected with high efficiency into needles in vivo in response to serum, indicating that additional factors besides motility are required to explain differences in cell collection efficiencies between metastatic and nonmetastatic tumors. The results reported here indicate that needles filled with growth factors and matrigel, when inserted into the primary tumor, can faithfully mimic the environment that supports invasion and intravasation in vivo. Furthermore, the results indicate that the same cell behaviors that contribute to chemotaxis in vitro also contribute to invasion in vivo.

Imaging of cancer invasion and metastasis using green fluorescent protein.

The use of green fluorescent protein to fluorescently tag tumour cells has allowed investigators to open the "black box" of metastasis in order to visualise the behaviour of tumour cells in living tissues. Analysis of cells leaving the primary tumour indicates that highly metastatic cells are able to polarise more effectively towards blood vessels while poorly metastatic cells fragment more often when interacting with blood. In addition, there appear to be greater numbers of host immune system cells interacting with metastatic tumours. After arresting in target organs such as the lungs or liver, most tumour cells become dormant or apoptose. A small fraction of the arrested cells form metastases. In some target organs, migration of tumour cells may enhance the ability to form metastases.

A critical step in metastasis: in vivo analysis of intravasation at the primary tumor.

Detailed evaluation of all steps in tumor cell metastasis is critical for evaluating the cell mechanisms controlling metastasis. Using green fluorescent protein transfectants of metastatic (MTLn3) and nonmetastatic (MTC) cell lines derived from the rat mammary adenocarcinoma 13762 NF, we have measured tumor cell density in the blood, individual tumor cells in the lungs, and lung metastases. Correlation of blood burden with lung metastases indicates that entry into the circulation is a critical step for metastasis. To examine cell behavior during intravasation, we have used green fluorescent protein technology to view these cells in time lapse images within a single optical section using a confocal microscope. In vivo imaging of the primary tumors of MTLn3 and MTC cells indicates that both metastatic and nonmetastatic cells are motile and show protrusive activity. However, metastatic cells show greater orientation toward blood vessels and larger numbers of host cells within the primary tumor, whereas nonmetastatic cells fragment when interacting with vessels. These results demonstrate that a major difference in intravasation between metastatic and nonmetastatic cells is detected in the primary tumor and illustrate the value of a direct visualization of cell properties in vivo for dissection of the metastatic process.

Role of cofilin in epidermal growth factor-stimulated actin polymerization and lamellipod protrusion.

Stimulation of metastatic MTLn3 cells with epidermal growth factor (EGF) causes a rapid and transient increase in actin nucleation activity resulting from the appearance of free barbed ends at the extreme leading edge of extending lamellipods. To investigate the role of cofilin in EGF-stimulated actin polymerization and lamellipod extension in MTLn3 cells, we examined in detail the temporal and spatial distribution of cofilin relative to free barbed ends and characterized the actin dynamics by measuring the changes in the number of actin filaments. EGF stimulation triggers a transient increase in cofilin in the leading edge near the membrane, which is precisely cotemporal with the appearance of free barbed ends there. A deoxyribonuclease I binding assay shows that the number of filaments per cell increases by 1.5-fold after EGF stimulation. Detection of pointed ends in situ using deoxyribonuclease I binding demonstrates that this increase in the number of pointed ends is confined to the leading edge compartment, and does not occur within stress fibers or in the general cytoplasm. Using a light microscope severing assay, cofilin's severing activity was observed directly in cell extracts and shown to be activated after stimulation of the cells with EGF. Microinjection of function-blocking antibodies against cofilin inhibits the appearance of free barbed ends at the leading edge and lamellipod protrusion after EGF stimulation. These results support a model in which EGF stimulation recruits cofilin to the leading edge where its severing activity is activated, leading to the generation of short actin filaments with free barbed ends that participate in the nucleation of actin polymerization.

Specific requirement for the p85-p110alpha phosphatidylinositol 3-kinase during epidermal growth factor-stimulated actin nucleation in breast cancer cells.

We have studied the role of phosphatidylinositol 3-kinases (PI 3-kinases) in the regulation of the actin cytoskeleton in MTLn3 rat adenocarcinoma cells. Stimulation of MTLn3 cells with epidermal growth factor (EGF) induced a rapid increase in actin polymerization, with production of lamellipodia within 3 min. EGF-stimulated lamellipodia were blocked by 100 nM wortmannin, suggesting the involvement of a class Ia PI 3-kinase. MTLn3 cells contain equal amounts of p110alpha and p110beta, and do not contain p110delta. Injection of specific inhibitory antibodies to p110alpha induced cell rounding and blocked EGF-stimulated lamellipod extension, whereas control or anti-p110beta antibodies had no effect. In contrast, both antibodies inhibited EGF-stimulated DNA synthesis. An in situ assay for actin nucleation showed that EGF-stimulated formation of new barbed ends was blocked by injection of anti-p110alpha antibodies. In summary, the p110alpha isoform of PI 3-kinase is specifically required for EGF-stimulated actin nucleation during lamellipod extension in breast cancer cells.

Integration of Rac-dependent regulation of cyclin D1 transcription through a nuclear factor-kappaB-dependent pathway.

The small GTP-binding protein Rac1, a member of the Ras superfamily, plays a fundamental role in cytoskeleton reorganization, cellular transformation, the induction of DNA synthesis, and superoxide production. Cyclin D1 abundance is rate-limiting in normal G(1) phase progression, and the abundance of cyclin D1 is induced by activating mutations of both Ras and Rac1. Nuclear factor-kappaB (NF-kappaB) proteins consist of cytoplasmic hetero- or homodimeric Rel-related proteins complexed to a member of the IkappaB family of inhibitor proteins. In the current studies, activating mutants of Rac1 (Rac(Leu-61), Rac(Val-12)) induced cyclin D1 expression and the cyclin D1 promoter in NIH 3T3 cells. Induction of cyclin D1 by Rac1 required both an NF-kappaB and an ATF-2 binding site. Inhibiting NF-kappaB by overexpression of an NF-kappaB trans-dominant inhibitor (nonphosphorylatable IkappaBalpha) reduced cyclin D1 promoter activation by the Rac1 mutants, placing NF-kappaB in a pathway of Rac1 activation of cyclin D1. Specific amino acid mutations in the amino-terminal effector domain of Rac(Leu-61) had comparable effects on NF-kappaB transcriptional activity and activation of the cyclin D1 promoter. The NF-kappaB factors Rel A (p65) and NF-kappaB(1) (p50) induced the cyclin D1 promoter, requiring both the NF-kappaB binding site and the ATF-2 site. Stable overexpression of Rac(Leu-61) increased binding of Rel A and NF-kappaB(1) to the cyclin D1 promoter NF-kappaB site. Activation of Rac1 in NIH 3T3 cells induces both NF-kappaB binding and activity and enhances expression of cyclin D1 through an NF-kappaB and ATF-2 site in the proximal promoter, suggesting a critical role for NF-kappaB in cell cycle regulation through cyclin D1 and Rac1.

Relationship between Arp2/3 complex and the barbed ends of actin filaments at the leading edge of carcinoma cells after epidermal growth factor stimulation.

Using both light and high resolution electron microscopy, we analyzed the spatial and temporal relationships between the Arp2/3 complex and the nucleation activity that is required for lamellipod extension in mammary carcinoma cells after epidermal growth factor stimulation. A rapid two- to fourfold increase in filament barbed end number occurs transiently after stimulation and remains confined almost exclusively to the extreme outer edge of the extending lamellipod (within 100-200 nm of the plasma membrane). This is accompanied by an increase in filament density at the leading edge and a general decrease in filament length, with a specific loss of long filaments. Concomitantly, the Arp2/3 complex is recruited with a 1.5-fold increase throughout the entire cortical filament network extending 1-1.5 microm in depth from the membrane at the leading edge. The recruitment of the Arp2/3 complex at the membrane of the extending lamellipod indicates that Arp2/3 may be involved in initial generation of growing filaments. However, only a small subset of the complex present in the cortical network colocalizes near free barbed ends. This suggests that the 100-200-nm submembraneous compartment at the leading edge of the extending lamellipod constitutes a special biochemical microenvironment that favors the generation and maintenance of free barbed ends, possibly through the locally active Arp2/3 complex, severing or decreasing the on-rate of capping protein. Our results are inconsistent with the hypothesis suggesting uncapping is the dominant mechanism responsible for the generation of nucleation activity. However, they support the hypothesis of an Arp2/3-mediated capture of actin oligomers that formed close to the membrane by other mechanisms such as severing. They also support pointed-end capping by the Arp2/3 complex, accounting for its wide distribution at the leading edge.

Cell polarization: chemotaxis gets CRACKing.

An early stage in the establishment of cell polarity during chemotaxis of Dictyostelium dicoideum has been identified by a recent study; the new results also show that the development of cell polarity does not rely upon cytoskeletal rearrangement, and may use a spatial sensing mechanism.

Chemoattractant-induced lamellipod extension.

The mammary adenocarcinoma cell line MTLn3 is chemotactic towards epidermal growth factor (EGF), and this induced motility is thought to promote breast cancer invasion and metastasis. Stimulation of MTLn3 cells with EGF results in the extension of a flat, thin structure filled with filamentous actin and termed a lamellipod. Lamellipod extension is dependent on actin polymerization and is localized to the border of adherent cells. The structure of EGF-stimulated lamellipods in MTLn3 cells is well suited to analysis of chemoattractant-stimulated protrusion. Actin polymerization occurs within 200 nm of the extending edge of the lamellipod. Although extension of the lamellipod is not dependent upon interaction with the substratum, stabilization of the extended lamellipod is dependent on an adhesive substratum. Dorsal ruffling is suppressed during lamellipod extension. Tyrosine phosphorylation is reduced in preexisting focal contacts compared to new contacts induced by EGF stimulation. The coordination of turnover of focal contacts with lamellipod extension is proposed to result in polarized cell motility in response to gradients of chemoattractants.

Suppression of ruffling by the EGF receptor in chemotactic cells.

To clarify the relationship between ruffling and lamellipod extension in growth factor-stimulated chemotactic responses, we utilized cell lines derived from the rat 13762 NF mammary adenocarcinoma. Nonmetastatic MTC cells expressing the human EGF receptor (termed MTC HER cells) demonstrated chemotactic responses to TGF-alpha, an EGF receptor ligand typically present in mammary tissue. In microchemotaxis chambers, peak chemotactic responses occurred in response to 5 nM TGF-alpha. MTC HER cells showed dramatic ruffling edges in the absence of external stimuli, and addition of 5 nM TGF-alpha led to a transient reduction in ruffling concomitant with lamellipod extension. Lamellipod extension correlated with an overall increase in actin polymerization. These responses were blocked by the PI 3 kinase inhibitor wortmannin but not by the MAP kinase inhibitors PD98059 and SB203580. We conclude that the initial chemotactic response to TGF-alpha involves lamellipod extension and that ruffling reflects a dynamic turnover of lamellipodia that is arrested during lamellipod extension. By regulating the dissolution of ruffles and extension of lamellipods, a chemotactic response can be achieved, which may contribute to the metastatic process.