Rachelle Kosoff

PAK signalling during the development and progression of cancer

p21-activated kinases (PAKs) are positioned at the nexus of several oncogenic signalling pathways. Overexpression or mutational activation of PAK isoforms frequently occurs in various human tumours, and recent data suggest that excessive PAK activity drives many of the cellular processes that are the hallmarks of cancer. In this Review, we discuss the mechanisms of PAK activation in cancer, the key substrates that mediate the developmental and oncogenic effects of this family of kinases, and how small-molecule inhibitors of these enzymes might be best developed and deployed for the treatment of cancer.

A Rac-Pak signaling pathway is essential for ErbB2-mediated transformation of human breast epithelial cancer cells.

The activation of receptor tyrosine kinases, particularly ErbB2, has an important role in the genesis of breast cancer. ErbB2 kinase activity promotes Ras-mediated stimulation of downstream protein kinase cascades, including the Ras/Raf-1/MAPK/ERK kinase (Mek)/extracellular signal-regulated kinase (Erk) pathway, leading to tumor cell growth and migration. Signaling through the Ras–Erk pathway can be influenced by p21-activated kinase-1 (Pak1), an effector of the Rho family GTPases Rac and Cdc42. In this study, we asked if ErbB2 expression correlates with Pak1 and Erk activity in human breast cancer specimens, and if Pak1 signaling is required for ErbB2 transformation in a three-dimensional (3D) in vitro setting and in xenografts. We found a correlation between ErbB2 expression and activation of Pak in estrogen receptor-positive human breast tumor samples and observed that in 3D cultures, activation of Rac–Pak1 pathway by ErbB2 homodimers induced growth factor-independent proliferation and promoted disruption of 3D mammary acinar-like structures through activation of the Erk and Akt pathways. Further, we found that inhibition of Pak1 by small molecules compromised activation of Erk and Akt, resulting in reversion of the malignant phenotype and restoration of normal acinar architecture. Finally, ErbB2-amplified breast cancer cells expressing a specific Pak inhibitor showed delayed tumor formation and downregulation of Erk and Akt signaling in vivo. These data imply that the Rac–Pak pathway is vital to ErbB2-mediated transformation and that Pak inhibitors represent plausible drug targets in breast cancers in which ErbB2 signaling is activated.

DAI Senses Influenza A Virus Genomic RNA and Activates RIPK3-Dependent Cell Death

Influenza A virus (IAV) is an RNA virus that is cytotoxic to most cell types in which it replicates. IAV activates the host kinase RIPK3, which induces cell death via parallel pathways of necroptosis, driven by the pseudokinase MLKL, and apoptosis, dependent on the adaptor proteins RIPK1 and FADD. How IAV activates RIPK3 remains unknown. We report that DAI (ZBP1/DLM-1), previously implicated as a cytoplasmic DNA sensor, is essential for RIPK3 activation by IAV. Upon infection, DAI recognizes IAV genomic RNA, associates with RIPK3, and is required for recruitment of MLKL and RIPK1 to RIPK3. Cells lacking DAI or containing DAI mutants deficient in nucleic acid binding are resistant to IAV-triggered necroptosis and apoptosis. DAI-deficient mice fail to control IAV replication and succumb to lethal respiratory infection. These results identify DAI as a link between IAV replication and RIPK3 activation and implicate DAI as a sensor of RNA viruses.

Pak2 Kinase Restrains Mast Cell FcϵRI Receptor Signaling through Modulation of Rho Protein Guanine Nucleotide Exchange Factor (GEF) Activity

Background: The protein kinase Pak1 stimulates mast cell degranulation, but the role of the more abundant isoform Pak2 in these cells is unknown. Results: Pak2, unlike Pak1, inhibits mast cell degranulation via a GEF-H1-RhoA pathway. Conclusion: Pak2 mediates signals between FcϵRI and secretion through regulation of the GEF responsible for RhoA activation. Significance: Pak2 has an opposing role to Pak1 in mast cell degranulation. p21-activated kinase-1 (Pak1) is a serine/threonine kinase that plays a key role in mediating antigen-stimulated extracellular calcium influx and degranulation in mast cells. Another isoform in this kinase family, Pak2, is expressed at very high levels in mast cells, but its function is unknown. Here we show that Pak2 loss in murine bone marrow-derived mast cells, unlike loss of Pak1, induces increased antigen-mediated adhesion, degranulation, and cytokine secretion without changes to extracellular calcium influx. This phenotype is associated with an increase in RhoA-GTPase signaling activity to downstream effectors, including myosin light chain and p38MAPK, and is reversed upon treatment with a Rho-specific inhibitor. Pak2, but not Pak1, negatively regulates RhoA via phosphorylation of the guanine nucleotide exchange factor GEF-H1 at an inhibitory site, leading to increased GEF-H1 microtubule binding and loss of RhoA stimulation. These data suggest that Pak2 plays a unique inhibitory role in mast cell degranulation by down-regulating RhoA via GEF-H1.

Pak2 restrains endomitosis during megakaryopoiesis and alters cytoskeleton organization

Megakaryocyte maturation and polyploidization are critical for platelet production; abnormalities in these processes are associated with myeloproliferative disorders, including thrombocytopenia. Megakaryocyte maturation signals through cascades that involve p21-activated kinase (Pak) function; however, the specific role for Pak kinases in megakaryocyte biology remains elusive. Here, we identify Pak2 as an essential effector of megakaryocyte maturation, polyploidization, and proplatelet formation. Genetic deletion of Pak2 in murine bone marrow is associated with macrothrombocytopenia, altered megakaryocyte ultrastructure, increased bone marrow megakaryocyte precursors, and an elevation of mature CD41(+) megakaryocytes, as well as an increased number of polyploid cells. In Pak2(-/-) mice, platelet clearance rate was increased, as was production of newly synthesized, reticulated platelets. In vitro, Pak2(-/-) megakaryocytes demonstrate increased polyploidization associated with alterations in β1-tubulin expression and organization, decreased proplatelet extensions, and reduced phosphorylation of the endomitosis regulators LIM domain kinase 1, cofilin, and Aurora A/B/C. Together, these data establish a novel role for Pak2 as an important regulator of megakaryopoiesis, polyploidization, and cytoskeletal dynamics in developing megakaryocytes.

Development and characterization of an organotypic model of Barrett's esophagus

Understanding the molecular and cellular processes underlying the development, maintenance, and progression of Barretts esophagus (BE) presents an empirical challenge because there are no simple animal models and standard 2D cell culture can distort cellular processes. Here we describe a three‐dimensional (3D) cell culture system to study BE. BE cell lines (CP‐A, CP‐B, CP‐C, and CP‐D) and esophageal squamous keratinocytes (EPC2) were cultured on a matrix consisting of esophageal fibroblasts and collagen. Comparison of growth and cytokeratin expression in the presence of all‐trans retinoic acid or hydrochloric acid was made by immunohistochemistry and Alcian Blue staining to determine which treatments produced a BE phenotype of columnar cytokeratin expression in 3D culture. All‐trans retinoic acid differentially affected the growth of BE cell lines in 3D culture. Notably, the non‐dyplastic metaplasia‐derived cell line (CP‐A) expressed reduced squamous cytokeratins and enhanced columnar cytokeratins upon ATRA treatment. ATRA altered the EPC2 squamous cytokeratin profile towards a more columnar expression pattern. Cell lines derived from patients with high‐grade dysplasia already expressed columnar cytokeratins and therefore did not show a systematic shift toward a more columnar phenotype with ATRA treatment. ATRA treatment, however, did reduce the squamoid‐like multilayer stratification observed in all cell lines. As the first study to demonstrate long‐term 3D growth of BE cell lines, we have determined that BE cells can be cultured for at least 3 weeks on a fibroblast/collagen matrix and that the use of ATRA causes a general reduction in squamous‐like multilayered growth and an increase in columnar phenotype with the specific effects cell‐line dependent. J. Cell. Physiol. 227: 2654–2659, 2012.

Pak2 is required for actin cytoskeleton remodeling, TCR signaling, and normal thymocyte development and maturation

The molecular mechanisms that govern thymocyte development and maturation are incompletely understood. The P21-activated kinase 2 (Pak2) is an effector for the Rho family GTPases Rac and Cdc42 that regulate actin cytoskeletal remodeling, but its role in the immune system remains poorly understood. In this study, we show that T-cell specific deletion of Pak2 gene in mice resulted in severe T cell lymphopenia accompanied by marked defects in development, maturation, and egress of thymocytes. Pak2 was required for pre-TCR β-selection and positive selection. Surprisingly, Pak2 deficiency in CD4 single positive thymocytes prevented functional maturation and reduced expression of S1P1 and KLF2. Mechanistically, Pak2 is required for actin cytoskeletal remodeling triggered by TCR. Failure to induce proper actin cytoskeletal remodeling impaired PLCγ1 and Erk1/2 signaling in the absence of Pak2, uncovering the critical function of Pak2 as an essential regulator that governs the actin cytoskeleton-dependent signaling to ensure normal thymocyte development and maturation. DOI: http://dx.doi.org/10.7554/eLife.02270.001

Sequential phosphorylation of SLP-76 at tyrosine 173 is required for activation of T and mast cells

Cooperatively assembled signalling complexes, nucleated by adaptor proteins, integrate information from surface receptors to determine cellular outcomes. In T and mast cells, antigen receptor signalling is nucleated by three adaptors: SLP‐76, Gads and LAT. Three well‐characterized SLP‐76 tyrosine phosphorylation sites recruit key components, including a Tec‐family tyrosine kinase, Itk. We identified a fourth, evolutionarily conserved SLP‐76 phosphorylation site, Y173, which was phosphorylated upon T‐cell receptor stimulation in primary murine and Jurkat T cells. Y173 was required for antigen receptor‐induced phosphorylation of phospholipase C‐γ1 (PLC‐γ1) in both T and mast cells, and for consequent downstream events, including activation of the IL‐2 promoter in T cells, and degranulation and IL‐6 production in mast cells. In intact cells, Y173 phosphorylation depended on three, ZAP‐70‐targeted tyrosines at the N‐terminus of SLP‐76 that recruit and activate Itk, a kinase that selectively phosphorylated Y173 in vitro. These data suggest a sequential mechanism whereby ZAP‐70‐dependent priming of SLP‐76 at three N‐terminal sites triggers reciprocal regulatory interactions between Itk and SLP‐76, which are ultimately required to couple active Itk to its substrate, PLC‐γ1.

Pak2 regulates hematopoietic progenitor cell proliferation, survival, and differentiation.

p21‐Activated kinase 2 (Pak2), a serine/threonine kinase, has been previously shown to be essential for hematopoietic stem cell (HSC) engraftment. However, Pak2 modulation of long‐term hematopoiesis and lineage commitment remain unreported. Using a conditional Pak2 knockout mouse model, we found that disruption of Pak2 in HSCs induced profound leukopenia and a mild macrocytic anemia. Although loss of Pak2 in HSCs leads to less efficient short‐ and long‐term competitive hematopoiesis than wild‐type cells, it does not affect HSC self‐renewal per se. Pak2 disruption decreased the survival and proliferation of multicytokine stimulated immature progenitors. Loss of Pak2 skewed lineage differentiation toward granulocytopoiesis and monocytopoiesis in mice as evidenced by (a) a three‐ to sixfold increase in the percentage of peripheral blood granulocytes and a significant increase in the percentage of granulocyte‐monocyte progenitors in mice transplanted with Pak2‐disrupted bone marrow (BM); (b)Pak2‐disrupted BM and c‐kit+ cells yielded higher numbers of more mature subsets of granulocyte‐monocyte colonies and polymorphonuclear neutrophils, respectively, when cultured in the presence of granulocyte‐macrophage colony‐stimulating factor. Pak2 disruption resulted, respectively, in decreased and increased gene expression of transcription factors JunB and c‐Myc, which may suggest underlying mechanisms by which Pak2 regulates granulocyte‐monocyte lineage commitment. Furthermore, Pak2 disruption led to (a) higher percentage of CD4+CD8+ double positive T cells and lower percentages of CD4+CD8− or CD4−CD8+ single positive T cells in thymus and (b) decreased numbers of mature B cells and increased numbers of Pre‐Pro B cells in BM, suggesting defects in lymphopoiesis. Stem Cells 2015;33:1630–1641

An in vitro co-culture model of esophageal cells identifies ascorbic acid as a modulator of cell competition

BackgroundThe evolutionary dynamics between interacting heterogeneous cell types are fundamental properties of neoplastic progression but can be difficult to measure and quantify. Cancers are heterogeneous mixtures of mutant clones but the direct effect of interactions between these clones is rarely documented. The implicit goal of most preventive interventions is to bias competition in favor of normal cells over neoplastic cells. However, this is rarely explicitly tested. Here we have developed a cell culture competition model to allow for direct observation of the effect of chemopreventive or therapeutic agents on two interacting cell types. We have examined competition between normal and Barretts esophagus cell lines, in the hopes of identifying a system that could screen for potential chemopreventive agents.MethodsOne fluorescently-labeled normal squamous esophageal cell line (EPC2-hTERT) was grown in competition with one of four Barretts esophagus cell lines (CP-A, CP-B, CP-C, CP-D) under varying conditions and the outcome of competition measured over 14 days by flow cytometry.ResultsWe demonstrate that ascorbic acid (vitamin C) can help squamous cells outcompete Barretts cells in this system. We are also able to show that ascorbic acids boost to the relative fitness of squamous cells was increased in most cases by mimicking the pH conditions of gastrointestinal reflux in the lower esophagus.ConclusionsThis model is able to integrate differential fitness effects on various cell types, allowing us to simultaneously capture effects on interacting cell types without having to perform separate experiments. This model system may be used to screen for new classes of cancer prevention agents designed to modulate the competition between normal and neoplastic cells.