The development of metastases is a decisive step in the course of a cancer disease. The detection of metastases in cancer patients is correlated with a poor prognosis, and over 90% of all deaths from cancer are not due to the primary tumor, which often can be successfully treated, but are due to the metastases. Tumor cell migration, a prerequisite for metastasis development, is not merely genetically determined, but is distinctly regulated by signal substances of the environment including chemokines and neurotransmitters. We have shown previously that the migration of breast, prostate, and colon carcinoma cells is enhanced by the stress-related neurotransmitter norepinephrine in vitro, and that this effect can be inhibited by the b-blocker propranolol. We now provide for the first time evidence for the in vivo relevance of this neurotransmitter-driven regulation using PC-3 prostate carcinoma cells. The development of lumbar lymph node metastases in athymic BALB/c nude mice increased with the application of norepinephrine via microosmotic pumps, while propranolol inhibited this effect. However, the growth of the primary tumor was not affected by either treatment. Additionally, experiments using human tissue microarrays showed that 70-90 percent of breast, colon, and prostate carcinoma tissues express the relevant b2-adrenoceptor. Thus, our work contributes to the understanding of the basic cellular mechanisms of metastasis development, and furthermore delivers a rationale for the chemopreventive use of clinically established b-blockers for the inhibition of metastases. 1 Their theory is supported by the finding that certain mutations, e.g. mutations of the tumor-suppressor gene MADH4, occur more frequently in metastatic tumors.2 On the other hand, the comparative gene expression profiling of primary breast tumors and distant metastases showed striking similarity, suggesting that the metastatic capability in breast cancer is an inherent feature and is not based on clonal selection.3 Recent research has shown that ligands to serpentine receptors play an important role in the regulation of tumor cell migration. These ligands predominantly consist of 2 groups, the chemokines and the neurotransmitters. 4 Among the chemokines, the stromal cell-derived factor-1 (SDF-1) is best investigated for its role in metastasis development, 5 and in the localization of metastases as shown in mice.6 Within the group of neurotransmitters, norepinephrine is one of the most potent known stimulators for the migration of tumor cells. We have shown previously in this journal and elsewhere that norepinephrine induces migration in breast, colon and prostate carcinoma cells, and that this induction can be inhibited by b2-adrenoceptor blocking drugs in vitro. [7][8][9] In this present study, we address the question whether the previously observed increase of tumor cell migration in response to norepinephrine in vitro corresponds to an in vivo increase of metastasis development in mice, and whether this effect can be inhibited by the chemoprev...

The active migration of tumor cells, a crucial requirement for metastasis development and cancer progression, is regulated by signal substances including neurotransmitters. We investigated the migration of tumor cells within a threedimensional collagen matrix using time-lapse videomicroscopy and computer-assisted analysis of the migration path. Tumor cell migration is induced by norepinephrine, dopamine and substance P. We show that this induced migration, using MDA-MB-468 breast and PC-3 prostate carcinoma cells, can be inhibited by using specific, clinically established receptor antagonists to the ␤2-adrenoceptor, the D2 receptor, or the neurokinin-1 receptor, respectively. All of the investigated neurotransmitters significantly activated the cyclic adenosine-monophosphate response element binding protein (CREB). Furthermore, microarray analysis revealed changes of gene expression toward a highly motile tumor cell type, including an upregulation of the ␣2 integrin, which is an essential adhesion receptor for collagen in migration. The gene for the tumor suppressor gelsolin was downregulated. These 2 critical alterations were confirmed on the protein level by flow-cytometry and immunoblotting, respectively. Neurotransmitters thus induce a metastatogenic tumor cell type by directly regulating gene expression and increased migratory activity, which can be prevented by established neurotransmitter antagonists. © 2004 Wiley-Liss, Inc. Key words: metastasis formation; tumor cell migration; neurotransmitters; signal transduction; gene expressionMost patients suffering from cancer do not die due to the primary tumor, but from distant metastases descending from it. The development of these metastases is a multi-step process for which the metastasizing tumor cells need to acquire the ability of active migration. This migratory activity is not solely an intrinsic property of tumor cells, but a cell function which is regulated by signal substances similar to the regulation of migratory activity in leukocytes. 1 Chemokines are the key regulatory signal substances of the immune system, whereas neurotransmitters are also known to regulate the adhesion, migration, and homing of leukocytes. 2 In addition to their function in the immune system, chemokines have been shown to induce the migration of tumor cells, 2,3 and direct the tumor cells to specific organs. 4 The involvement of psychosocial factors in the incidence and progression of cancer has also been discussed for decades and has been reviewed recently by Heffner et al. 5 In further support of this psycho neuro-oncological link we identified neurotransmitters, which induce the migration of MDA-MB-468 breast carcinoma cells. 6 Norepinephrine was the most potent inducer of migration. This induction of migration is not based on a recruitment of formerly non-migrating cells, but is a very subtle composition of changes of migratory parameters such as of the velocity as well as of the frequency and duration of pauses during migratory activity; morphological changes, e.g., with...

Most patients suffering from breast carcinoma do not die due to the primary tumor but from the development of metastases. Active migration of cancer cells is a prerequisite for development of these metastases. We used time-lapse videomicroscopy and computer-assisted cell tracking of MDA-MB-468 human breast carcinoma cells, which were incorporated into a three-dimensional collagen matrix, in order to analyze the migratory activity of these cells in response to different neurotransmitters. Our results show that met-enkephalin, substance P, bombesin, dopamine, and norepinephrine have a stimulatory effect on the migration of the breast cancer cells; moreover, these cells show positive chemotaxis towards norepinephrine as was analyzed by the directionality and persistence on a single-cell basis. Gamma-aminobutyric acid (GABA) however has an inhibitory effect. Endorphin and leu-enkephalin, as well as histamin and acetylcholine, had no influence on the migratory activity of the cells. In summary, we provide evidence for a strong regulatory involvement of neurotransmitters in the regulation of breast cancer cell migration, which might provide the basis for the use of the pharmacological agonists and antagonists for the chemopreventive inhibition of metastasis development.

Cell migration may depend on integrin-mediated adhesion to and deadhesion from extracellular matrix ligands. This concept, however, has not yet been confirmed for T lymphocytes migrating in three-dimensional extracellular matrices. We investigated receptor involvement in T cell migration combining a three-dimensional collagen matrix model with time-lapse videomicroscopy, computer-assisted cell tracking and confocal microscopy. In collagen lattices, the migration of CD4+ T cells (1) involved interactions with collagen fibers at the leading edge and uropod likewise, (2) occurred independently of the co-clustering of beta1, beta2, or beta3 integrins with F-actin, focal adhesion kinase, and phosphotyrosine at interactions with collagen fibers, (3) was counteracted by high-affinity beta1 integrin binding induced by antibody TS2/16; however, (4) the migration could not be blocked by a combination of adhesion-perturbing anti-beta1, -beta2, -beta3, and alpha v integrin antibodies. Integrin blocking neither affected cell polarization, interaction with fibers, beta1 integrin distribution, migration velocity, path structure, nor the number of locomoting cells in spontaneously migrating or concanavalin A-activated cells. Hence, T lymphocytes migrating in three-dimensional collagen matrices may utilize highly transient interactions with collagen fibers of low adhesivity, thereby differing from focal adhesion-dependent migration strategies employed by other cells.

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Induction of tumor cell migration is a key step in invasion and metastasis. Here we report that the epidermal growth factor (EGF)-induced cell migration of breast cancer cells is attributed to a transient, rather than a sustained, activation of phospholipase C (PLC)-gamma1 due to c-erbB-2 signaling. EGF stimulation of EGF receptor (EGFR) overexpressing cells resulted in long-term PLC-gamma1 tyrosine phosphorylation and sustained levels of inositol-1,4,5-triphosphate (IP3) and diacylglycerol (DAG) producing sinusoidal calcium oscillations. In contrast, c-erbB-2/EGFR expressing cells displayed baseline transient calcium oscillations after EGF treatment due to short-term PLC-gamma1 tyrosine phosphorylation and short-term IP3 and DAG turnover. A third cell line expressing a point-mutated c-erbB-2 receptor that lacks the autophosphorylation Y1248 was generated to investigate whether the different PLC-gamma1 activation was attributed to this structure. Neither PLC-gamma1 tyrosine phosphorylation nor IP3 and DAG turnover and calcium oscillations were observed in this cell line, indicating the modulation of the PLC-g1 activation time course by c-erbB-2 signaling. Induction of cell migration was solely observable in the c-erbB-2-positive cell line as proved by the mode of actin reorganization and a cell migration assay, using a 3D-collagen lattice. In summary, c-erbB-2 up-regulation switches on the cell migration program by modulating the time course of PLC-gamma1 activation.

Haptokinetic cell migration across surfaces is mediated by adhesion receptors including ␤1 integrins and CD44 providing adhesion to extracellular matrix (ECM) ligands such as collagen and hyaluronan (HA), respectively. Little is known, however, about how such different receptor systems synergize for cell migration through three-dimensionally (3-D) interconnected ECM ligands. In highly motile human MV3 melanoma cells, both ␤1 integrins and CD44 are abundantly expressed, support migration across collagen and HA, respectively, and are deposited upon migration, whereas only ␤1 integrins but not CD44 redistribute to focal adhesions. In 3-D collagen lattices in the presence or absence of HA and cross-linking chondroitin sulfate, MV3 cell migration and associated functions such as polarization and matrix reorganization were blocked by anti-␤1 and anti-␣2 integrin mAbs, whereas mAbs blocking CD44, ␣3, ␣5, ␣6, or ␣v integrins showed no effect. With use of highly sensitive time-lapse videomicroscopy and computer-assisted cell tracking techniques, promigratory functions of CD44 were excluded. 1) Addition of HA did not increase the migratory cell population or its migration velocity, 2) blocking of the HA-binding Hermes-1 epitope did not affect migration, and 3) impaired migration after blocking or activation of ␤1 integrins was not restored via CD44. Because ␣2␤1-mediated migration was neither synergized nor replaced by CD44 -HA interactions, we conclude that the biophysical properties of 3-D multicomponent ECM impose more restricted molecular functions of adhesion receptors, thereby differing from haptokinetic migration across surfaces. INTRODUCTIONTumor cell invasion and migration are supported by different adhesion receptor systems, including integrins and CD44 (Stetler-Stevenson et al., 1993;Sherman et al., 1994; Friedl and Brö cker, 1999). Integrins and CD44 interact with an array of extracellular matrix (ECM) components, including collagen, fibronectin, laminin, vitronectin, and hyaluronan (HA), respectively (Etoh et al., 1992;Danen et al., 1993;Aznavoorian et al., 1996;Goebeler et al., 1996;Klominek et al., 1997), simultaneously acting as structural links from the cytoskeleton to the ECM as well as signaling molecules (Lokeshwar et al., 1994;Clark and Brugge, 1995;Entwistle et al., 1996;Cox and Huttenlocher, 1998).In human melanoma, the expression of ␣2␤1 integrin is correlated with metastatic behavior (Klein et al., 1991b) and is involved in melanoma cell migration on collagen surfaces (Etoh et al., 1992) and in the reorganization of collagen matrices (Klein et al., 1991b;Schiro et al., 1991;Riikonen et al., 1995). Likewise, ␣3␤1 and ␣v␤3 integrins mediate chemotactic and haptotactic motility toward their respective ligands collagen (Lauer et al., 1998) and vitronectin □ V Online version of this article contains video material. Online version available at www.molbiolcell.org. ‡ Corresponding author. E-mail address: peter.fr@mail.uniwuerzburg.de. Abbreviations used: CS, chondroitin sulfate; ECM, extracellular matri...

Background:During the last decade, epidemiological studies uncovered the tremendous impact of metabolic syndrome/diabetes mellitus type 2 (DM T2) as risk factors of the progression of cancer. Therefore, we studied the impact of diabetogenic glucose and insulin concentrations on the activities of tumour cells, because little is known about how high glucose and insulin levels are influencing gene activities causing changes in the signal cascade activities with respect to kinases involved in the proliferation and migration of cancer cells.Methods:To address this question we analysed the activity of more than 400 gene signatures related to (i) cell cycle, (ii) cell movement as well as (iii) signal transduction. We examined transcriptoms of kinases (PKCα, PI3K), cadherins (E-, N- VE-), integrins and cyclins by comparing physiological (5.5 m) vs diabetogenic (11 m) glucose concentrations (without and with insulin).Results:Proliferation assays revealed that high levels of glucose (11 m) and insulin (100 ng ml−1) did promote the proliferation of the tumour cell lines HT29, SW480, MCF-7, MDA MB468, PC3 and T24. Using a 3D-migration assay, we have shown that high glucose concentrations caused increased motility rates of the tumour cells. The increase in migratory activity at high glucose and insulin concentrations was mediated by an activation of PI3K, PKCα and MLCK, as figured out by the pharmacological inhibitors wortmannin, Go6976 and ML-7.Conclusion:We present molecular and functional data, which could help to understand how hyperglycaemia and hyperinsulinemia might trigger tumour cell proliferation and motility in patients, too.