Impact of the Helicobacter pylori Oncoprotein CagA in Gastric Carcinogenesis.

Helicobacter pylori CagA is the first and only bacterial oncoprotein etiologically associated with human cancer. Upon delivery into gastric epithelial cells via bacterial type IV secretion, CagA acts as a pathogenic/pro-oncogenic scaffold that interacts with and functionally perturbs multiple host proteins such as pro-oncogenic SHP2 phosphatase and polarity-regulating kinase PAR1b/MARK2. Although H. pylori infection is established during early childhood, gastric cancer generally develops in elderly individuals, indicating that oncogenic CagA activity is effectively counteracted at a younger age. Moreover, the eradication of cagA-positive H. pylori cannot cure established gastric cancer, indicating that H. pylori CagA-triggered gastric carcinogenesis proceeds via a hit-and-run mechanism. In addition to its direct oncogenic action, CagA induces BRCAness, a cellular status characterized by replication fork destabilization and loss of error-free homologous recombination-mediated DNA double-strand breaks (DSBs) by inhibiting cytoplasmic-to-nuclear localization of the BRCA1 tumor suppressor. This causes genomic instability that leads to the accumulation of excess mutations in the host cell genome, which may underlie hit-and-run gastric carcinogenesis. The close connection between CagA and BRCAness was corroborated by a recent large-scale case-control study that revealed that the risk of gastric cancer in individuals carrying pathogenic variants of genes that induce BRCAness (such as BRCA1 and BRCA2) dramatically increases upon infection with cagA-positive H. pylori. Accordingly, CagA-mediated BRCAness plays a crucial role in the development of gastric cancer in conjunction with the direct oncogenic action of CagA.

Kinase Activity of PAR1b, Which Mediates Nuclear Translocation of the BRCA1 Tumor Suppressor, Is Potentiated by Nucleic Acid-Mediated PAR1b Multimerization.

PAR1b is a cytoplasmic serine/threonine kinase that controls cell polarity and cell-cell interaction by regulating microtubule stability while mediating cytoplasmic-to-nuclear translocation of BRCA1. PAR1b is also a cellular target of the CagA protein of Helicobacter pylori, which leads to chronic infection causatively associated with the development of gastric cancer. The CagA-PAR1b interaction inactivates the kinase activity of PAR1b and thereby dampens PAR1b-mediated BRCA1 phosphorylation, which reduces the level of nuclear BRCA1 and thereby leads to BRCAness and BRCAness-associated genome instability underlying gastric carcinogenesis. While PAR1b can multimerize within the cells, little is known about the mechanism and functional role of PAR1b multimerization. We found in the present study that PAR1b was multimerized in vitro by binding with nucleic acids (both single- and double-stranded DNA/RNA) via the spacer region in a manner independent of nucleic-acid sequences, which markedly potentiated the kinase activity of PAR1b. Consistent with these in vitro observations, cytoplasmic introduction of double-stranded DNA or expression of single-stranded RNA increased the PAR1b kinase activity in the cells. These findings indicate that the cytoplasmic DNA/RNA contribute to nuclear accumulation of BRCA1 by constitutively activating/potentiating cytoplasmic PAR1b kinase activity, which is subverted in gastric epithelial cells upon delivery of H. pylori CagA oncoprotein.

Loss of Par1b/MARK2 primes microglia during brain development and enhances their sensitivity to injury.

BACKGROUND: Microglia, the resident immune cells of the brain, exhibit various morphologies that correlate with their functions under physiological and pathological conditions. In conditions such as aging and stress, microglia priming occurs, which leads to altered morphology and lower threshold for activation upon further insult. However, the molecular mechanisms that lead to microglia priming are unclear. METHODS: To understand the role of Par1b/MARK2 in microglia, we first expressed shRNA targeting luciferase or Par1b/MARK2 in primary microglial cells and imaged the cells using fluorescent microscopy to analyze for morphological changes. A phagocytosis assay was then used to assess functional changes. We then moved in vivo and used a Par1b/MARK2 knockout mouse model to assess for changes in microglia density, morphology, and phagocytosis using immunohistochemistry, confocal imaging, and 3D image reconstruction. Next, we used two-photon in vivo imaging in live Par1b/MARK2 deficient mice to examine microglia dynamics. In addition, a controlled-cortical impact injury was performed on wild-type and Par1b/MARK2-deficient mice and microglial response was determined by confocal imaging. Finally, to help rule out non-cell autonomous effects, we analyzed apoptosis by confocal imaging, cytokine levels by multiplex ELISA, and blood-brain barrier permeability using Evans Blue assay. RESULTS: Here, we show that loss of the cell polarity protein Par1b/MARK2 facilitates the activation of primary microglia in culture. We next found that microglia in Par1b/MARK2 deficient mice show increased density and a hypertrophic morphology. These morphological changes are accompanied with alterations in microglia functional responses including increased phagocytosis of neuronal particles early in development and decreased surveillance of the brain parenchyma, all reminiscent of a primed phenotype. Consistent with this, we found that microglia in Par1b/MARK2 deficient mice have a significantly lower threshold for activation upon injury. CONCLUSIONS: Together, our studies show that loss of Par1b/MARK2 switches microglia from a surveillant to a primed state during development, resulting in an increased neuroinflammatory response to insults.

Phosphorylation of the E3 ubiquitin ligase RNF41 by the kinase Par-1b is required for epithelial cell polarity.

The establishment and maintenance of cell polarity is an essential property governing organismal homeostasis, and loss of polarity is a common feature of cancer cells. The ability of epithelial cells to establish apical-basal polarity depends on intracellular signals generated from polarity proteins, such as the Par-1 family of proteins, as well as extracellular signals generated through cell contacts with the extracellular matrix (ECM). The Par-1 family has a well-established role in regulating cell-cell contacts in the form of tight junctions by phosphorylating Par-3. In addition, Par-1 has been shown to impact on cell-ECM interactions by regulating laminin receptor localization and laminin deposition on the basal surface of epithelial cells. Laminins are major structural and signaling components of basement membrane (BM), a sheet of specialized ECM underlying epithelia. In this study, we identify RNF41, an E3 ubiquitin ligase, as a novel Par-1b (also known as MARK2) effector in the cell-ECM pathway. Par-1b binds to and phosphorylates RNF41 on serine 254. Phosphorylation of RNF41 by Par-1b is required for epithelial cells to localize laminin-111 receptors to their basolateral surfaces and to properly anchor to laminin-111. In addition, phosphorylation of RNF41 is required for epithelial cells to establish apical-basal polarity. Our data suggests that phosphorylation of RNF41 by Par-1b regulates basolateral membrane targeting of laminin-111 receptors, thereby facilitating cell anchorage to laminin-111 and ultimately forming the cell-ECM contacts required for epithelial cells to establish apical-basal cell polarity.

Natural variant of the Helicobacter pylori CagA oncoprotein that lost the ability to interact with PAR1.

Helicobacter pylori strains carrying the cagA gene are associated with severe disease outcomes, most notably gastric cancer. CagA protein is delivered into gastric epithelial cells by a type IV secretion system. The translocated CagA undergoes tyrosine phosphorylation at the C-terminal EPIYA motifs by host cell kinases. Tyrosine-phosphorylated CagA acquires the ability to interact with and activate SHP2, thereby activating mitogenic signaling and inducing cell morphological transformation (hummingbird phenotype). CagA also interacts with PAR1b via the CM sequence, resulting in induction of junctional and polarity defects. Furthermore, CagA-PAR1b interaction stabilizes the CagA-SHP2 complex. Because transgenic mice systemically expressing CagA develop gastrointestinal and hematological malignancies, CagA is recognized as a bacterium-derived oncoprotein. Interestingly, the C-terminal region of CagA displays a large diversity among H. pylori strains, which influences the ability of CagA to bind to SHP2 and PAR1b. In the present study, we investigated the biological activity of v225d CagA, an Amerindian CagA of H. pylori isolated from a Venezuelan Piaroa Amerindian subject, because the variant CagA does not possess a canonical CM sequence. We found that v225d CagA interacts with SHP2 but not PAR1b. Furthermore, SHP2-binding activity of v225d CagA was much lower than that of CagA of H. pylori isolated from Western countries (Western CagA). v225d CagA also displayed a reduced ability to induce the hummingbird phenotype than that of Western CagA. Given that perturbation of PAR1b and SHP2 by CagA underlies the oncogenic potential of CagA, the v225d strain is considered to be less oncogenic than other well-studied cagA-positive H. pylori strains.

Cortactin-dependent control of Par1b-regulated epithelial cell polarity in Helicobacter infection.

Cell polarity is crucial for gastric mucosal barrier integrity and mainly regulated by polarity-regulating kinase partitioning-defective 1b (Par1b). During infection, the carcinogen Helicobacter pylori hijacks Par1b via the bacterial oncoprotein CagA leading to loss of cell polarity, but the precise molecular mechanism is not fully clear. Here we discovered a novel function of the actin-binding protein cortactin in regulating Par1b, which forms a complex with cortactin and the tight junction protein zona occludens-1 (ZO-1). We found that serine phosphorylation at S405/418 and the SH3 domain of cortactin are important for its interaction with both Par1b and ZO-1. Cortactin knockout cells displayed disturbed Par1b cellular localization and exhibited morphological abnormalities that largely compromised transepithelial electrical resistance, epithelial cell polarity, and apical microvilli. H. pylori infection promoted cortactin/Par1b/ZO-1 abnormal interactions in the tight junctions in a CagA-dependent manner. Infection of human gastric organoid-derived mucosoids supported these observations. We therefore hypothesize that CagA disrupts gastric epithelial cell polarity by hijacking cortactin, and thus Par1b and ZO-1, suggesting a new signaling pathway for the development of gastric cancer by Helicobacter.

MARK2 regulates directed cell migration through modulation of myosin II contractility and focal adhesion organization.

Cancer cell migration during metastasis is mediated by a highly polarized cytoskeleton. MARK2 and its invertebrate homolog Par1B are kinases that regulate the microtubule cytoskeleton to mediate polarization of neurons in mammals and embryos in invertebrates. However, the role of MARK2 in cancer cell migration is unclear. Using osteosarcoma cells, we found that in addition to its known localizations on microtubules and the plasma membrane, MARK2 also associates with the actomyosin cytoskeleton and focal adhesions. Cells depleted of MARK proteins demonstrated that MARK2 promotes phosphorylation of both myosin II and the myosin phosphatase targeting subunit MYPT1 to synergistically drive myosin II contractility and stress fiber formation in cells. Studies with isolated proteins showed that MARK2 directly phosphorylates myosin II regulatory light chain, while its effects on MYPT1 phosphorylation are indirect. Using a mutant lacking the membrane-binding domain, we found that membrane association is required for focal adhesion targeting of MARK2, where it specifically enhances cell protrusion by promoting FAK phosphorylation and formation of focal adhesions oriented in the direction of migration to mediate directionally persistent cell motility. Together, our results define MARK2 as a master regulator of the actomyosin and microtubule cytoskeletal systems and focal adhesions to mediate directional cancer cell migration.

Helicobacter pylori: a stealth assassin.

Helicobacter pylori is a pathogen that confers the highest known risk for gastric cancer. Research directed at understanding the pathogenesis of H. pylori is crucial to identify colonized persons that may subsequently develop neoplasia. Imai et al. describe how H. pylori elicits BRCAness and endows epithelial cells with the ability to evade apoptosis.

Helicobacter pylori CagA elicits BRCAness to induce genome instability that may underlie bacterial gastric carcinogenesis.

Infection with CagA-producing Helicobacter pylori plays a causative role in the development of gastric cancer. Upon delivery into gastric epithelial cells, CagA deregulates prooncogenic phosphatase SHP2 while inhibiting polarity-regulating kinase PAR1b through complex formation. Here, we show that CagA/PAR1b interaction subverts nuclear translocation of BRCA1 by inhibiting PAR1b-mediated BRCA1 phosphorylation. It hereby induces BRCAness that promotes DNA double-strand breaks (DSBs) while disabling error-free homologous recombination-mediated DNA repair. The CagA/PAR1b interaction also stimulates Hippo signaling that circumvents apoptosis of DNA-damaged cells, giving cells time to repair DSBs through error-prone mechanisms. The DSB-activated p53-p21Cip1 axis inhibits proliferation of CagA-delivered cells, but the inhibition can be overcome by p53 inactivation. Indeed, sequential pulses of CagA in TP53-mutant cells drove somatic mutation with BRCAness-associated genetic signatures. Expansion of CagA-delivered cells with BRCAness-mediated genome instability, from which CagA-independent cancer-predisposing cells arise, provides a plausible "hit-and-run mechanism" of H. pylori CagA for gastric carcinogenesis.

High-Molecular-Weight Hyaluronan Is a Hippo Pathway Ligand Directing Cell Density-Dependent Growth Inhibition via PAR1b.

High-molecular-weight hyaluronan, a major component of the extracellular matrix, is anti-oncogenic, whereas low-molecular-weight hyaluronan is pro-oncogenic, though the mechanisms underlying the size-dependent opposite bioactivities of hyaluronan remain uncertain. We show here that treatment with high-molecular-weight hyaluronan stimulates tumor-suppressive Hippo signaling in breast epithelial cells. Mechanistically, clustering of the CD44 extracellular domain by high-molecular-weight hyaluronan leads to recruitment of the polarity-regulating kinase PAR1b by the CD44 intracellular domain, which results in disruption of the Hippo signaling-inhibitory PAR1b-MST complex. Once liberated from PAR1b, MST activates Hippo signaling. Conversely, low-molecular-weight hyaluronan, which is produced by hyaluronidase-mediated degradation of high-molecular-weight hyaluronan, inhibits Hippo signaling by competing with high-molecular-weight hyaluronan for CD44 binding. Triple-negative breast cancers with higher hyaluronidase-2 expression show poorer prognosis than those with lower hyaluronidase-2 expression. Consistently, decreased hyaluronidase-2 is associated with reduced tumorigenicity in a tumor xenograft model. Hence, perturbation of high-molecular-weight hyaluronan-mediated Hippo signaling activation contributes to cancer aggressiveness.

The Spatiotemporal Construction of the Axon Initial Segment via KIF3/KAP3/TRIM46 Transport under MARK2 Signaling.

The axon initial segment (AIS) is a compartment that serves as a molecular barrier to achieve axon-dendrite differentiation. Distribution of specific proteins during early neuronal development has been proposed to be critical for AIS construction. However, it remains unknown how these proteins are specifically targeted to the proximal axon within this limited time period. Here, we reveal spatiotemporal regulation driven by the microtubule (MT)-based motor KIF3A/B/KAP3 that transports TRIM46, influenced by a specific MARK2 phosphorylation cascade. In the proximal part of the future axon under low MARK2 activity, the KIF3/KAP3 motor recognizes TRIM46 as cargo and transports it to the future AIS. In contrast, in the somatodendritic area under high MARK2 activity, KAP3 phosphorylated at serine 60 by MARK2 cannot bind with TRIM46 and be transported. This spatiotemporal regulation between KIF3/KAP3 and TRIM46 under specific MARK2 activity underlies the specific transport needed for axonal differentiation.

MARK2/Par1b Insufficiency Attenuates DVL Gene Transcription via Histone Deacetylation in Lumbosacral Spina Bifida.

Dishevelled (DVL/Dvl) genes play roles in canonical and noncanonical Wnt signaling, both of which are essential in neural tube closing and are involved in balancing neural progenitor growth and differentiation, or neuroepithelial cell polarity, respectively. In mouse Dvl haploinsufficiency leads to neural tube defects (NTDs), which represent the second most common birth defects. However, DVL genes' genetic contributions in human NTDs are modest. We sought to explore the molecular impact on such genes in human NTDs in a Han Chinese cohort. In 47 cases with NTDs and 61 matched controls, in brain tissues, the DVL1/2 mRNA levels were correlated with the levels of a serine/threonine protein kinase MARK2, and in 20 cases with lumbosacral spina bifida, the mRNA levels of DVL1 and MARK2 were significantly decreased; by contrast, only an intronic rare variant was found. Moreover, in an extended population, we found merely three novel rare missense variants in 1 % of individuals with NTDs. In cell-based assays, Mark2 depletion indeed reduces Dvl gene expression and interrupts neural stem cell (NSCs) growth and differentiation, which are likely to be mediated through a decrease in class IIa HDAC phosphorylation and reduced H3K4ac and H3K27ac occupancies at the Dvl1/2 promoters. Finally, the detections of folate concentration in human brain tissue and NSCs and MEF cells indicates that folate deficiency contributes to the observed decreases in Mark2 and Dvl1 expression. Our present study raises a potential common pathogenicity mechanism in human lumbosacral spina bifida about DVL genes rather than their genetic pathogenic role.

Type IV Secretion and Signal Transduction of Helicobacter pylori CagA through Interactions with Host Cell Receptors.

Helicobacter pylori is a highly successful human bacterium, which is exceptionally equipped to persistently inhabit the human stomach. Colonization by this pathogen is associated with gastric disorders ranging from chronic gastritis and peptic ulcers to cancer. Highly virulent H. pylori strains express the well-established adhesins BabA/B, SabA, AlpA/B, OipA, and HopQ, and a type IV secretion system (T4SS) encoded by the cag pathogenicity island (PAI). The adhesins ascertain intimate bacterial contact to gastric epithelial cells, while the T4SS represents an extracellular pilus-like structure for the translocation of the effector protein CagA. Numerous T4SS components including CagI, CagL, CagY, and CagA have been shown to target the integrin-ß1 receptor followed by translocation of CagA across the host cell membrane. The interaction of CagA with membrane-anchored phosphatidylserine and CagA-containing outer membrane vesicles may also play a role in the delivery process. Translocated CagA undergoes tyrosine phosphorylation in C-terminal EPIYA-repeat motifs by oncogenic Src and Abl kinases. CagA then interacts with an array of host signaling proteins followed by their activation or inactivation in phosphorylation-dependent and phosphorylation-independent fashions. We now count about 25 host cell binding partners of intracellular CagA, which represent the highest quantity of all currently known virulence-associated effector proteins in the microbial world. Here we review the research progress in characterizing interactions of CagA with multiple host cell receptors in the gastric epithelium, including integrin-ß1, EGFR, c-Met, CD44, E-cadherin, and gp130. The contribution of these interactions to H. pylori colonization, signal transduction, and gastric pathogenesis is discussed.

Satellite Cells in Muscular Dystrophy - Lost in Polarity.

Recent findings employing the mdx mouse model for Duchenne muscular dystrophy (DMD) have revealed that muscle satellite stem cells play a direct role in contributing to disease etiology and progression of DMD, the most common and severe form of muscular dystrophy. Lack of dystrophin expression in DMD has critical consequences in satellite cells including an inability to establish cell polarity, abrogation of asymmetric satellite stem-cell divisions, and failure to enter the myogenic program. Thus, muscle wasting in dystrophic mice is not only caused by myofiber fragility but is exacerbated by intrinsic satellite cell dysfunction leading to impaired regeneration. Despite intense research and clinical efforts, there is still no effective cure for DMD. In this review we highlight recent research advances in DMD and discuss the current state of treatment and, importantly, how we can incorporate satellite cell-targeted therapeutic strategies to correct satellite cell dysfunction in DMD.

Par-1b is required for morphogenesis and differentiation of myoepithelial cells during salivary gland development.

The salivary epithelium initiates as a solid mass of epithelial cells that are organized into a primary bud that undergoes morphogenesis and differentiation to yield bilayered acini consisting of interior secretory acinar cells that are surrounded by contractile myoepithelial cells in mature salivary glands. How the primary bud transitions into acini has not been previously documented. We document here that the outer epithelial cells subsequently undergo a vertical compression as they express smooth muscle α-actin and differentiate into myoepithelial cells. The outermost layer of polarized epithelial cells assemble and organize the basal deposition of basement membrane, which requires basal positioning of the polarity protein, Par-1b. Whether Par-1b is required for the vertical compression and differentiation of the myoepithelial cells is unknown. Following manipulation of Par-1b in salivary gland organ explants, Par-1b-inhibited explants showed both a reduced vertical compression of differentiating myoepithelial cells and reduced levels of smooth muscle α-actin. Rac1 knockdown and inhibition of Rac GTPase function also inhibited branching morphogenesis. Since Rac regulates cellular morphology, we investigated a contribution for Rac in myoepithelial cell differentiation. Inhibition of Rac GTPase activity showed a similar reduction in vertical compression and smooth muscle α-actin levels while decreasing the levels of Par-1b protein and altering its basal localization in the outer cells. Inhibition of ROCK, which is required for basal positioning of Par-1b, resulted in mislocalization of Par-1b and loss of vertical cellular compression, but did not significantly alter levels of smooth muscle α-actin in these cells. Overexpression of Par-1b in the presence of Rac inhibition restored basement membrane protein levels and localization. Our results indicate that the basal localization of Par-1b in the outer epithelial cells is required for myoepithelial cell compression, and Par-1b is required for myoepithelial differentiation, regardless of its localization.

Composition, structure and function of the Helicobacter pylori cag pathogenicity island encoded type IV secretion system.

Many Gram-negative pathogens harbor type IV secretion systems (T4SS) that translocate bacterial virulence factors into host cells to hijack cellular processes. The pathology of the gastric pathogen Helicobacter pylori strongly depends on a T4SS encoded by the cag pathogenicity island. This T4SS forms a needle-like pilus, and its assembly is accomplished by multiple protein-protein interactions and various pilus-associated factors that bind to integrins followed by delivery of the CagA oncoprotein into gastric epithelial cells. Recent studies revealed the crystal structures of six T4SS proteins and pilus formation is modulated by iron and zinc availability. All these T4SS interactions are crucial for deregulating host signaling events and disease progression. New developments in T4SS functions and their importance for pathogenesis are discussed.

The special case of hepatocytes: unique tissue architecture calls for a distinct mode of cell division.

Columnar epithelia (e.g., kidney, intestine) and hepatocytes embody the two major organizational phenotypes of non-stratified epithelial cells. Columnar epithelia establish their apical and basal domains at opposing poles and organize in monolayered cysts and tubules, in which their apical surfaces form a single continuous lumen whereas hepatocytes establish their apical domains in the midst of their basolateral domains and organize a highly branched capillary luminal network, the bile canaliculi, in which a single hepatocyte can engage in lumen formation with multiple neighbors. To maintain their distinct tissue architectures, columnar epithelial cells bisect their luminal domains during symmetric cell divisions, while the cleavage furrow in dividing hepatocytes avoids bisecting the bile canalicular domains. We discuss recently discovered molecular mechanisms that underlie the different cell division phenotypes in columnar and hepatocytic model cell lines. The serine/threonine kinase Par1b determines both the epithelial lumen polarity and cell division phenotype via cell adhesion signaling that converges on the small GTPase RhoA.