Release of cAMP gating by the alpha6beta4 integrin stimulates lamellae formation and the chemotactic migration of invasive carcinoma cells.

The alpha6beta4 integrin promotes carcinoma in-vasion by its activation of a phosphoinositide 3-OH (PI3-K) signaling pathway (Shaw, L.M., I. Rabinovitz, H.H.-F. Wang, A. Toker, and A.M. Mercurio. Cell. 91: 949-960). We demonstrate here using MDA-MB-435 breast carcinoma cells that alpha6beta4 stimulates chemotactic migration, a key component of invasion, but that it has no influence on haptotaxis. Stimulation of chemotaxis by alpha6beta4 expression was observed in response to either lysophosphatidic acid (LPA) or fibroblast conditioned medium. Moreover, the LPA-dependent formation of lamellae in these cells is dependent upon alpha6beta4 expression. Both lamellae formation and chemotactic migration are inhibited or "gated" by cAMP and our results reveal that a critical function of alpha6beta4 is to suppress the intracellular cAMP concentration by increasing the activity of a rolipram-sensitive, cAMP-specific phosphodiesterase (PDE). This PDE activity is essential for lamellae formation, chemotactic migration and invasion based on data obtained with PDE inhibitors. Although PI3-K and cAMP-specific PDE activities are both required to promote lamellae formation and chemotactic migration, our data indicate that they are components of distinct signaling pathways. The essence of our findings is that alpha6beta4 stimulates the chemotactic migration of carcinoma cells through its ability to influence key signaling events that underlie this critical component of carcinoma invasion.

RhoA function in lamellae formation and migration is regulated by the alpha6beta4 integrin and cAMP metabolism.

Clone A colon carcinoma cells develop fan-shaped lamellae and exhibit random migration when plated on laminin, processes that depend on the ligation of the alpha6beta4 integrin. Here, we report that expression of a dominant negative RhoA (N19RhoA) in clone A cells inhibited alpha6beta4-dependent membrane ruffling, lamellae formation, and migration. In contrast, expression of a dominant negative Rac (N17Rac1) had no effect on these processes. Using the Rhotekin binding assay to assess RhoA activation, we observed that engagement of alpha6beta4 by either antibody-mediated clustering or laminin attachment resulted in a two- to threefold increase in RhoA activation, compared with cells maintained in suspension or plated on collagen. Antibody-mediated clustering of beta1 integrins, however, actually suppressed Rho A activation. The alpha6beta4-mediated interaction of clone A cells with laminin promoted the translocation of RhoA from the cytosol to membrane ruffles at the edges of lamellae and promoted its colocalization with beta1 integrins, as assessed by immunofluorescence microscopy. In addition, RhoA translocation was blocked by inhibiting phosphodiesterase activity and enhanced by inhibiting the activity of cAMP-dependent protein kinase. Together, these results establish a specific integrin-mediated pathway of RhoA activation that is regulated by cAMP and that functions in lamellae formation and migration.

Epileptiform abnormalities predict delayed cerebral ischemia in subarachnoid hemorrhage.

OBJECTIVE: To identify whether abnormal neural activity, in the form of epileptiform discharges and rhythmic or periodic activity, which we term here ictal-interictal continuum abnormalities (IICAs), are associated with delayed cerebral ischemia (DCI). METHODS: Retrospective analysis of continuous electroencephalography (cEEG) reports and medical records from 124 patients with moderate to severe grade subarachnoid hemorrhage (SAH). We identified daily occurrence of seizures and IICAs. Using survival analysis methods, we estimated the cumulative probability of IICA onset time for patients with and without delayed cerebral ischemia (DCI). RESULTS: Our data suggest the presence of IICAs indeed increases the risk of developing DCI, especially when they begin several days after the onset of SAH. We found that all IICA types except generalized rhythmic delta activity occur more commonly in patients who develop DCI. In particular, IICAs that begin later in hospitalization correlate with increased risk of DCI. CONCLUSIONS: IICAs represent a new marker for identifying early patients at increased risk for DCI. Moreover, IICAs might contribute mechanistically to DCI and therefore represent a new potential target for intervention to prevent secondary cerebral injury following SAH. SIGNIFICANCE: These findings imply that IICAs may be a novel marker for predicting those at higher risk for DCI development.

Quantitation of two histochemical markers in the same extract using chemiluminescent substrates.

Factors that influence early events of primary tumor development have been cumbersome to evaluate because of the need to either wait for tumor palpability after experimental manipulation or use of radiolabel to evaluate cell clearance. To facilitate these and similar analyses of cells in vivo, new methods are described that utilize histochemical marker genes to quantitate tumor cell number in a target tissue through the use of luminescent, enzymatic assays for these gene products. 3T3 Cells transfected with either human placental alkaline phosphatase or bacterial lacZ genes were injected subcutaneously into athymic nude mice. Using luminescent substrates designed for marker gene enzymes, extracts from homogenized tumor cell-bearing skins were assayed for the corresponding marker enzyme activities, which were optimized for recovery from skin extracts and correlated to cell number. The homogenization buffer used for these assays was designed to accommodate the optimal and simultaneous recovery of cytosolic beta-galactosidase and membrane-linked alkaline phosphatase from the skin, as well as from cultured cells. These assays provide an inexpensive, sensitive method for quantitatively monitoring the fate of cells genetically tagged with marker genes in various in vivo environments.

Earliest steps in primary tumor formation and micrometastasis resolved with histochemical markers of gene-tagged tumor cells.

To facilitate detection of tumor cells at the highest resolution in any organ in athymic nude mouse model systems, a histochemical marker gene [bacterial lacZ or human placental alkaline phosphatase (ALP)] was transfected into specified transformed/tumor cells (fibrosarcoma or neuroblastoma). The fates of tumor cells were followed qualitatively and quantitatively by histochemical staining of whole organs or organ sections. Primary tumors developed initially via formation of "curly-haired" complexes of cells in the subcutis or dermis, followed by division of a large fraction of cells. When two tumor classes were mixed before injection, outgrowth occurred in regional concentrations of the primary tumor. Blood microvessels were detectable within 72 hr of injection, growing into tumor regions. iv injection routinely yielded multicellular foci in the lungs within minutes as precursors of experimental metastases. Micrometastasis was further resolved with cells "inactivated" by different treatments and by co-injection of two different tagged cell types. These approaches using different histochemical marker genes to "tag" different tumor cell classes, along with more advanced molecular biological approaches, permit us to characterize gene expression and its reversibility during the earliest stages of primary tumor formation and micrometastasis to virtually any organ in the recipient animal.

The metastatic odyssey: the integrin connection.

This article explores the mechanistic basis of carcinoma progression by focusing on the contribution of integrins. Integrins are essential for progression because of their ability to mediate physical interactions with extracellular matrices and their ability to regulate signaling pathways that control actin dynamics and cell movement, and for growth and survival. This article centers on a6 integrins (a6B1 and a6B4), which are receptors for the laminin family of basement membrane components. Numerous studies have implicated these integrins in cancer progression and have provided a rationale for studying the mechanistic basis of their contribution to aggressive disease.

Coupling S100A4 to Rhotekin alters Rho signaling output in breast cancer cells.

Rho signaling is increasingly recognized to contribute to invasion and metastasis. In this study, we discovered that metastasis-associated protein S100A4 interacts with the Rho-binding domain (RBD) of Rhotekin, thus connecting S100A4 to the Rho pathway. Glutathione S-transferase pull-down and immunoprecipitation assays demonstrated that S100A4 specifically and directly binds to Rhotekin RBD, but not the other Rho effector RBDs. S100A4 binding to Rhotekin is calcium-dependent and uses residues distinct from those bound by active Rho. Interestingly, we found that S100A4 and Rhotekin can form a complex with active RhoA. Using RNA interference, we determined that suppression of both S100A4 and Rhotekin leads to loss of Rho-dependent membrane ruffling in response to epidermal growth factor, an increase in contractile F-actin 'stress' fibers and blocks invasive growth in three-dimensional culture. Accordingly, our data suggest that interaction of S100A4 and Rhotekin permits S100A4 to complex with RhoA and switch Rho function from stress fiber formation to membrane ruffling to confer an invasive phenotype.

Protein kinase A regulates Rac and is required for the growth factor-stimulated migration of carcinoma cells.

Members of the Rho family of small GTPases, such as Rho and Rac, are required for actin cytoskeletal reorganization during the migration of carcinoma cells. Phosphodiesterases are necessary for this migration because they alleviate cAMP-dependent protein kinase (PKA)-mediated inhibition of RhoA (O'Connor, K. L., Shaw, L. M., and Mercurio, A. M. (1998) J. Cell Biol. 143, 1749-1760; O'Connor K. L., Nguyen, B.-K., and Mercurio, A. M. (2000), J. Cell Biol. 148, 253-258). In this study, we report that the migration of breast and squamous carcinoma cells toward either lysophosphatidic acid or epidermal growth factor involves not only phosphodiesterase activity but also cooperative signaling from PKA. Furthermore, we demonstrate that Rac1 activation in response to chemoattractant or beta(1) integrin clustering is regulated by PKA and that Rac1 is required for this migration. Also, we find that beta(1) integrin signaling stimulates the rapid and transient activation of PKA. A novel implication of these findings is that carcinoma cell migration is controlled by cAMP-dependent as well as cAMP inhibitory signaling mechanisms.

Complementation of two related tumour cell classes during experimental metastasis tagged with different histochemical marker genes.

Intercellular complementation during tumour development and metastasis was analysed for two different oncogene (ras or sis) transformants of Balb/c 3T3 cells, tagged with different histochemical marker genes (lacZ or ALP to generate LZEJ or APSI cells, respectively), by localising them after their co-injection with specific double-staining protocols. This model evaluates whether limited progression of each tumour class can be facilitated reciprocally during co-localisation and co-growth in nude mice by taking advantage of the sensitivity of the histochemical marker genes for localising them. After intravenous co-injection of equal numbers of both cells to analyse experimental metastasis, most foci transiently established in the lung for several hours were comprised of only one cell class. However, a significant fraction of foci contained both cell types, as identified in double-stained whole-lung tissues and in lung sections. Evidence was obtained that LZEJ cells increase the survivability and subsequent growth of APSI-containing micrometastases during co-localisation in lung, when compared to APSI cells injected alone. Conversely, APSI cells facilitate expansion of LZEJ cells from micrometastatic foci into overt-metastatic nodules in the lung. These analyses reveal reciprocity during experimental metastasis by two related tumour cell classes derived from the same parental cell.

Integrin laminin receptors and breast carcinoma progression.

This review explores the mechanistic basis of breast carcinoma progression by focusing on the contribution of integrins. Integrins are essential for progression not only for their ability to mediate physical interactions with extracellular matrices but also for their ability to regulate signaling pathways that control actin dynamics and cell movement, as well as for growth and survival. Our comments center on the alpha6 integrins (alpha6beta1 and alpha6beta4), which are receptors for the laminin family of basement membrane components. Numerous studies have implicated these integrins in breast cancer progression and have provided a rationale for studying the mechanistic basis of their contribution to aggressive disease. Recent work by our group and others on mechanisms of breast carcinoma invasion and survival that are influenced by the alpha6 integrins are discussed.