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.

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.

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.

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 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.

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, 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.

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.

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.

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.