Transformation of mammary epithelial cells into invasive carcinoma results in alterations in their integrin-mediated responses to the extracellular matrix, including a loss of normal epithelial polarization and differentiation, and a switch to a more motile, invasive phenotype. Changes in the actin cytoskeleton associated with this switch suggest that the small GTPases Cdc42 and Rac, which regulate actin organization, might modulate motility and invasion. However, the role of Cdc42 and Rac1 in epithelial cells, especially with respect to integrin-mediated events, has not been well characterized. Here we show that activation of Cdc42 and Rac1 disrupts the normal polarization of mammary epithelial cells in a collagenous matrix, and promotes motility and invasion. This motility does not require the activation of PAK, JNK, p70 S6 kinase, or Rho, but instead requires phosphatidylinositol-3-OH kinase (PI(3)K). Further, direct PI(3)K activation is sufficient to disrupt epithelial polarization and induce cell motility and invasion. PI(3)K inhibition also disrupts actin structures, suggesting that activation of PI(3)K by Cdc42 and Rac1 alters actin organization, leading to increased motility and invasiveness.

Rationale: Rhinoviruses are the major cause of asthma exacerbations; however, its underlying mechanisms are poorly understood. We hypothesized that the epithelial cell-derived cytokine IL-33 plays a central role in exacerbation pathogenesis through augmentation of type 2 inflammation.Objectives: To assess whether rhinovirus induces a type 2 inflammatory response in asthma in vivo and to define a role for IL-33 in this pathway.Methods: We used a human experimental model of rhinovirus infection and novel airway sampling techniques to measure IL-4, IL-5, IL-13, and IL-33 levels in the asthmatic and healthy airways during a rhinovirus infection. Additionally, we cultured human T cells and type 2 innate lymphoid cells (ILC2s) with the supernatants of rhinovirusinfected bronchial epithelial cells (BECs) to assess type 2 cytokine production in the presence or absence of IL-33 receptor blockade.

Rac1 and RhoA are members of the Rho family of Ras-related proteins and function as regulators of actin cytoskeletal organization, gene expression, and cell cycle progression. Constitutive activation of Rac1 and RhoA causes tumorigenic transformation of NIH 3T3 cells, and their functions may be required for full Ras transformation. The effectors by which Rac1 and RhoA mediate these diverse activities, as well as the interrelationship between these events, remain poorly understood. Rac1 is distinct from RhoA in its ability to bind and activate the p65 PAK serine/threonine kinase, to induce lamellipodia and membrane ruffling, and to activate the c-Jun NH 2 -terminal kinase (JNK). To assess the role of PAK in Rac1 function, we identified effector domain mutants of Rac1 and Rac1-RhoA chimeric proteins that no longer bound PAK. Surprisingly, PAK binding was dispensable for Rac1-induced transformation and lamellipodium formation, as well as activation of JNK, p38, and serum response factor (SRF). However, the ability of Rac1 to bind to and activate PAK correlated with its ability to stimulate transcription from the cyclin D1 promoter. Furthermore, Rac1 activation of JNK or SRF, or induction of lamellipodia, was neither necessary nor sufficient for Rac1 transforming activity. Finally, the signaling pathways that mediate Rac1 activation of SRF or JNK were distinct from those that mediate Rac1 induction of lamellipodia. Taken together, these observations suggest that Rac1 regulates at least four distinct effector-mediated functions and that multiple pathways may contribute to Rac1-induced cellular transformation.

IntroductionCellular constituents of the alveolar-capillary wall may be key participants in the recruitment of polymorphonuclear leukocytes to the lung through the generation of the novel neutrophil chemotactic peptide interleukin-8 (IL-8 Invest. 1990Invest. . 86:1945Invest. -1953

Substantial evidence supports a critical role for the activation of the Raf-1/MEK/mitogen-activated protein kinase pathway in oncogenic Ras-mediated transformation. For example, dominant negative mutants of Raf-1, MEK, and mitogen-activated protein kinase all inhibit Ras transformation. Furthermore, the observation that plasma membrane-localized Raf-1 exhibits the same transforming potency as oncogenic Ras suggests that Raf-1 activation alone is sufficient to mediate full Ras transforming activity. However, the recent identification of other candidate Ras effectors (e.g., RalGDS and phosphatidylinositol-3 kinase) suggests that activation of other downstream effector-mediated signaling pathways may also mediate Ras transforming activity. In support of this, two H-Ras effector domain mutants, H-Ras(12V, 37G) and H-Ras(12V, 40C), which are defective for Raf binding and activation, induced potent tumorigenic transformation of some strains of NIH 3T3 fibroblasts. These Raf-binding defective mutants of H-Ras induced a transformed morphology that was indistinguishable from that induced by activated members of Rho family proteins. Furthermore, the transforming activities of both of these mutants were synergistically enhanced by activated Raf-1 and inhibited by the dominant negative RhoA (19N) mutant, indicating that Ras may cause transformation that occurs via coordinate activation of Raf-dependent and -independent pathways that involves Rho family proteins. Finally, cotransfection of H-Ras(12V, 37G) and H-Ras(12V, 40C) resulted in synergistic cooperation of their focus-forming activities, indicating that Ras activates at least two Raf-independent, Ras effector-mediated signaling events.Ras proteins act as molecular switches that cycle between active GTP-and inactive GDP-bound forms (reviewed in references 6 and 9) and function as essential components of signal transduction pathways that regulate cell growth and differentiation (reviewed in references 21, 36, and 51). Upon activation by ligand-stimulated receptors, activated Ras proteins transmit their signals to a cascade of serine/threonine kinases (reviewed in references 59, 64, 68, and 86). Activated Ras complexes with and promotes activation of the Raf-1 serine/threonine kinase (50,76,78,79,87). Raf-1 in turn activates mitogen-activated protein kinase (MAPK) kinases (MEK1 and MEK2), which in turn activate p42 and p44 MAPKs/extracellular signal-regulated kinases (ERKs) (16,34,41,42,61,88). Activated MAPKs then translocate into the nucleus, where they phosphorylate and activate nuclear transcription factors such as Elk-1 (30

The signaling pathways linking integrins to nuclear events are incompletely understood. We have examined intracellular signaling by the α6β4 integrin, a laminin receptor expressed in basal keratinocytes and other cells. Ligation of α6β4 in primary human keratinocytes caused tyrosine phosphorylation of Shc, recruitment of Grb2, activation of Ras and stimulation of the MAP kinases Erk and Jnk. In contrast, ligation of the laminin‐ and collagen‐binding integrins α3β1 and α2β1 did not cause these events. While the stimulation of Erk by α6β4 was suppressed by dominant‐negative Shc, Ras and RhoA, the activation of Jnk was inhibited by dominant‐negative Ras and Rac1 and by the phosphoinositide 3‐kinase inhibitor Wortmannin. Adhesion mediated by α6β4induced transcription from the Fos serum response element and promoted cell cycle progression in response to mitogens. In contrast, α3β1‐ and α2β1‐dependent adhesion did not induce these events. These findings suggest that the coupling of α6β4 integrin to the control of cell cycle progression mediated by Shc regulates the proliferation of basal keratinocytes and possibly other cells which are in contact with the basement membrane in vivo.

No abstract

Despite abundant evidence that aberrant Rho-family GTPase activation contributes to most steps of cancer initiation and progression, there is a dearth of inhibitors of their effectors (e.g., p21-activated kinases). Through high-throughput screening and structure-based design, we identify PF-3758309, a potent (K d = 2.7 nM), ATP-competitive, pyrrolopyrazole inhibitor of PAK4. In cells, PF-3758309 inhibits phosphorylation of the PAK4 substrate GEF-H1 (IC 50 = 1.3 nM) and anchorage-independent growth of a panel of tumor cell lines (IC 50 = 4.7 ± 3 nM). The molecular underpinnings of PF-3758309 biological effects were characterized using an integration of traditional and emerging technologies. Crystallographic characterization of the PF-3758309/PAK4 complex defined determinants of potency and kinase selectivity. Global high-content cellular analysis confirms that PF-3758309 modulates known PAK4-dependent signaling nodes and identifies unexpected links to additional pathways (e.g., p53). In tumor models, PF-3758309 inhibits PAK4-dependent pathways in proteomic studies and regulates functional activities related to cell proliferation and survival. PF-3758309 blocks the growth of multiple human tumor xenografts, with a plasma EC 50 value of 0.4 nM in the most sensitive model. This study defines PAK4-related pathways, provides additional support for PAK4 as a therapeutic target with a unique combination of functions (apoptotic, cytoskeletal, cell-cycle), and identifies a potent, orally available small-molecule PAK inhibitor with significant promise for the treatment of human cancers.