PIP3-Independent Activation of TorC2 and PKB at the Cell's Leading Edge Mediates Chemotaxis

SummaryBackgroundStudies show that high phosphotidylinositol 3,4,5-trisphosphate (PIP3) promotes cytoskeletal rearrangements and alters cell motility and chemotaxis, possibly through activation of protein kinase Bs (PKBs). However, chemotaxis can still occur in the absence of PIP3, and the identities of the PIP3-independent pathways remain unknown.ResultsHere, we outline a PIP3-independent pathway linking temporal and spatial activation of PKBs by Tor complex 2 (TorC2) to the chemotactic response. Within seconds of stimulating Dictyostelium cells with chemoattractant, two PKB homologs, PKBA and PKBR1, mediate transient phosphorylation of at least eight proteins, including Talin, PI4P 5-kinase, two Ras GEFs, and a RhoGap. Surprisingly, all of the substrates are phosphorylated with normal kinetics in cells lacking PI 3-kinase activity. Cells deficient in TorC2 or PKB activity show reduced phosphorylation of the endogenous substrates and are impaired in chemotaxis. The PKBs are activated through phosphorylation of their hydrophobic motifs via TorC2 and subsequent phosphorylation of their activation loops. These chemoattractant-inducible events are restricted to the cell's leading edge even in the absence of PIP3. Activation of TorC2 depends on heterotrimeric G protein function and intermediate G proteins, including Ras GTPases.ConclusionsThe data lead to a model where cytosolic TorC2, encountering locally activated small G protein(s) at the leading edge of the cell, becomes activated and phosphorylates PKBs. These in turn phosphorylate a series of signaling and cytoskeletal proteins, thereby regulating directed migration

In vitro Efficacy of a Novel Guanosine-Analog Phosphonate

Actinic keratosis, a frequent carcinoma in situ of non-melanoma skin cancer (NMSC), can transform into life-threatening cutaneous squamous cell carcinoma. Current treatment is limited due to low complete clearance rates and asks for novel therapeutic concepts; the novel purine nucleotide analogue OxBu may be an option. In order to enhance skin penetration, solid lipid nanoparticles (SLN, 136-156 nm) were produced with an OxBu entrapment efficiency of 96.5 ± 0.1%. For improved preclinical evaluation, we combined tissue engineering with clinically used keratin-18 quantification. Three doses of 10-3 mol/l OxBu, dissolved in phosphate-buffered saline as well as loaded to SLN, were effective on reconstructed NMSC. Tumour response and apoptosis induction were evaluated by an increase in caspase-cleaved fragment of keratin-18, caspase-7 activation as well as by reduced expression of matrix metallopeptidase-2 and Ki-67. OxBu efficacy was superior to equimolar 5-fluorouracil solution, and thus the drug should be subjected to the next step in preclinical evaluation

A single high dose of dexamethasone affects the phosphorylation state of glutamate AMPA receptors in the human limbic system

Lopes MW, Leal RB, Guarnieri R, et al. A single high dose of dexamethasone affects the phosphorylation state of glutamate AMPA receptors in the human limbic system. TRANSLATIONAL PSYCHIATRY. 2016;6(12): e986.Glucocorticoids (GC) released during stress response exert feedforward effects in the whole brain, but particularly in the limbic circuits that modulates cognition, emotion and behavior. GC are the most commonly prescribed anti-inflammatory and immunosuppressant medication worldwide and pharmacological GC treatment has been paralleled by the high incidence of acute and chronic neuropsychiatric side effects, which reinforces the brain sensitivity for GC. Synapses can be bi-directionally modifiable via potentiation (long-term potentiation, LTP) or depotentiation (long-term depression, LTD) of synaptic transmission efficacy, and the phosphorylation state of Ser831 and Ser845 sites, in the GluA1 subunit of the glutamate AMPA receptors, are a critical event for these synaptic neuroplasticity events. Through a quasi-randomized controlled study, we show that a single high dexamethasone dose significantly reduces in a dose-dependent manner the levels of GluA1-Ser831 phosphorylation in the amygdala resected during surgery for temporal lobe epilepsy. This is the first report demonstrating GC effects on key markers of synaptic neuroplasticity in the human limbic system. The results contribute to understanding how GC affects the human brain under physiologic and pharmacologic conditions

Chemotaxis: a feedback-based computational model robustly predicts multiple aspects of real cell behaviour

The mechanism of eukaryotic chemotaxis remains unclear despite intensive study. The most frequently described mechanism acts through attractants causing actin polymerization, in turn leading to pseudopod formation and cell movement. We recently proposed an alternative mechanism, supported by several lines of data, in which pseudopods are made by a self-generated cycle. If chemoattractants are present, they modulate the cycle rather than directly causing actin polymerization. The aim of this work is to test the explanatory and predictive powers of such pseudopod-based models to predict the complex behaviour of cells in chemotaxis. We have now tested the effectiveness of this mechanism using a computational model of cell movement and chemotaxis based on pseudopod autocatalysis. The model reproduces a surprisingly wide range of existing data about cell movement and chemotaxis. It simulates cell polarization and persistence without stimuli and selection of accurate pseudopods when chemoattractant gradients are present. It predicts both bias of pseudopod position in low chemoattractant gradients and-unexpectedly-lateral pseudopod initiation in high gradients. To test the predictive ability of the model, we looked for untested and novel predictions. One prediction from the model is that the angle between successive pseudopods at the front of the cell will increase in proportion to the difference between the cell's direction and the direction of the gradient. We measured the angles between pseudopods in chemotaxing Dictyostelium cells under different conditions and found the results agreed with the model extremely well. Our model and data together suggest that in rapidly moving cells like Dictyostelium and neutrophils an intrinsic pseudopod cycle lies at the heart of cell motility. This implies that the mechanism behind chemotaxis relies on modification of intrinsic pseudopod behaviour, more than generation of new pseudopods or actin polymerization by chemoattractant

Critical Role of VCP/p97 in the Pathogenesis and Progression of Non-Small Cell Lung Carcinoma

Valosin-containing protein (VCP)/p97 is an AAA ATPase molecular chaperone that regulates vital cellular functions and protein-processing. A recent study indicated that VCP expression levels are correlated with prognosis and progression of non-small cell lung carcinoma (NSCLC). We not only verified these findings but also identified the specific role of VCP in NSCLC pathogenesis and progression.Our results show that VCP is significantly overexpressed in non-small cell lung carcinoma (NSCLC) as compared to normal tissues and cell lines (p<0.001). Moreover, we observed the corresponding accumulation of ubiquitinated-proteins in NSCLC cell lines and tissues as compared to the normal controls. VCP inhibition by si/shRNA or small-molecule (Eeyarestatin I, EerI) significantly (p<0.05, p<0.00007) suppressed H1299 proliferation and migration but induced (p<0.00001) apoptosis. Cell cycle analysis by flow cytometry verified this data and shows that VCP inhibition significantly (p<0.001, p<0.003) induced cell cycle arrest in the G0/G1 phases. We also found that VCP directly regulates p53 and NFκB protein levels as a potential mechanism to control tumor cell proliferation and progression. Finally, we evaluated the therapeutic potential of VCP inhibition and observed significantly reduced NSCLC tumor growth in both in vitro and xenograft murine (athymic-nude) models after EerI treatment (p<0.05).Thus, targeting VCP in NSCLC may provide a novel strategy to restore p53 and NFκB levels and ameliorate the growth and tumorigenicity, leading to improved clinical outcomes

Proteasome Particle-Rich Structures Are Widely Present in Human Epithelial Neoplasms: Correlative Light, Confocal and Electron Microscopy Study

A novel cytoplasmic structure has been recently characterized by confocal and electron microscopy in H. pylori-infected human gastric epithelium, as an accumulation of barrel-like proteasome reactive particles colocalized with polyubiquitinated proteins, H. pylori toxins and the NOD1 receptor. This proteasome particle-rich cytoplasmic structure (PaCS), a sort of focal proteasome hyperplasia, was also detected in dysplastic cells and was found to be enriched in SHP2 and ERK proteins, known to play a role in H. pylori-mediated gastric carcinogenesis. However, no information is available on its occurrence in neoplastic growths. In this study, surgical specimens of gastric cancer and various other human epithelial neoplasms have been investigated for PaCSs by light, confocal and electron microscopy including correlative confocal and electron microscopy (CCEM). PaCSs were detected in gastric cohesive, pulmonary large cell and bronchioloalveolar, thyroid papillary, parotid gland, hepatocellular, ovarian serous papillary, uterine cervix and colon adenocarcinomas, as well as in pancreatic serous microcystic adenoma. H. pylori bodies, their virulence factors (VacA, CagA, urease, and outer membrane proteins) and the NOD1 bacterial proteoglycan receptor were selectively concentrated inside gastric cancer PaCSs, but not in PaCSs from other neoplasms which did, however, retain proteasome and polyubiquitinated proteins reactivity. No evidence of actual microbial infection was obtained in most PaCS-positive neoplasms, except for H. pylori in gastric cancer and capsulated bacteria in a colon cancer case. Particle lysis and loss of proteasome distinctive immunoreactivities were seen in some tumour cell PaCSs, possibly ending in sequestosomes or autophagic bodies. It is concluded that PaCSs are widely represented in human neoplasms and that both non-infectious and infectious factors activating the ubiquitin-proteasome system are likely to be involved in their origin. PaCS detection might help clarify the role of the ubiquitin-proteasome system in carcinogenesis

IMRT in oral cavity cancer

BACKGROUND: Except for early T1,2 N0 stages, the prognosis for patients with oral cavity cancer (OCC) is reported to be worse than for carcinoma in other sites of the head and neck (HNC). The aim of this work was to assess disease outcome in OCC following IMRT. Between January 2002 and January 2007, 346 HNC patients have been treated with curative intensity modulated radiation therapy (IMRT) at the Department of Radiation Oncology, University Hospital Zurich. Fifty eight of these (16%) were referred for postoperative (28) or definitive (30) radiation therapy of OCC. 40 of the 58 OCC patients (69%) presented with locally advanced T3/4 or recurred lesions. Doses between 60 and 70 Gy were applied, combined with simultaneous cisplatin based chemotherapy in 78%. Outcome analyses were performed using Kaplan Meier curves. In addition, comparisons were performed between this IMRT OCC cohort and historic in-house cohorts of 33 conventionally irradiated (3DCRT) and 30 surgery only patients treated over the last 10 years. RESULTS: OCC patients treated with postoperative IMRT showed the highest local control (LC) rate of all assessed treatment sequence subgroups (92% LC at 2 years). Historic postoperative 3DCRT patients and patients treated with surgery alone reached LC rates of ~70–80%. Definitively irradiated patients revealed poorest LC rates with ~30 and 40% following 3DCRT and IMRT, respectively. T1 stage resulted in an expectedly significantly higher LC rate (95%, n = 19, p < 0.05) than T2-4 and recurred stages (LC ~50–60%, n = 102). Analyses according to the diagnosis revealed significantly lower LC in OCC following definitive IMRT than that in pharyngeal tumors treated with definitive IMRT in the same time period (43% vs 82% at 2 years, p < 0.0001), while the LC rate of OCC following postoperative IMRT was as high as in pharyngeal tumors treated with postoperative IMRT (>90% at 2 years). CONCLUSION: Postoperative IMRT of OCC resulted in the highest local control rate of the assessed treatment subgroups. In conclusion, generous indication for IMRT following surgical treatment is recommended in OCC cases with unfavourable features like tight surgical margin, nodal involvement, primary tumor stage >T1N0, or already recurred disease, respectively. Loco-regional outcome of OCC following definitive IMRT remained unsatisfactory, comparable to that following definitive 3DCRT

Different modes of state transitions determine pattern in the Phosphatidylinositide-Actin system

<p>Abstract</p> <p>Background</p> <p>In a motile polarized cell the actin system is differentiated to allow protrusion at the front and retraction at the tail. This differentiation is linked to the phosphoinositide pattern in the plasma membrane. In the highly motile <it>Dictyostelium </it>cells studied here, the front is dominated by PI3-kinases producing PI(3,4,5)tris-phosphate (PIP3), the tail by the PI3-phosphatase PTEN that hydrolyses PIP3 to PI(4,5)bis-phosphate. To study de-novo cell polarization, we first depolymerized actin and subsequently recorded the spontaneous reorganization of actin patterns in relation to PTEN.</p> <p>Results</p> <p>In a transient stage of recovery from depolymerization, symmetric actin patterns alternate periodically with asymmetric ones. The switches to asymmetry coincide with the unilateral membrane-binding of PTEN. The modes of state transitions in the actin and PTEN systems differ. Transitions in the actin system propagate as waves that are initiated at single sites by the amplification of spontaneous fluctuations. In PTEN-null cells, these waves still propagate with normal speed but loose their regular periodicity. Membrane-binding of PTEN is induced at the border of a coherent PTEN-rich area in the form of expanding and regressing gradients.</p> <p>Conclusions</p> <p>The state transitions in actin organization and the reversible transition from cytoplasmic to membrane-bound PTEN are synchronized but their patterns differ. The transitions in actin organization are independent of PTEN, but when PTEN is present, they are coupled to periodic changes in the membrane-binding of this PIP3-degrading phosphatase. The PTEN oscillations are related to motility patterns of chemotaxing cells.</p

Two Nuclear Localization Signals in USP1 Mediate Nuclear Import of the USP1/UAF1 Complex

The human deubiquitinase USP1 plays important roles in cancer-related processes, such as the DNA damage response, and the maintenance of the undifferentiated state of osteosarcoma cells. USP1 deubiquitinase activity is critically regulated by its interaction with the WD40 repeat-containing protein UAF1. Inhibiting the function of the USP1/UAF1 complex sensitizes cancer cells to chemotherapy, suggesting that this complex is a relevant anticancer target. Intriguingly, whereas UAF1 has been reported to locate in the cytoplasm, USP1 is a nuclear protein, although the sequence motifs that mediate its nuclear import have not been functionally characterized. Here, we identify two nuclear localization signals (NLSs) in USP1 and show that these NLSs mediate the nuclear import of the USP1/UAF1 complex. Using a cellular relocation assay based on these results, we map the UAF1-binding site to a highly conserved 100 amino acid motif in USP1. Our data support a model in which USP1 and UAF1 form a complex in the cytoplasm that subsequently translocates to the nucleus through import mediated by USP1 NLSs. Importantly, our findings have practical implications for the development of USP1-directed therapies. First, the UAF1-interacting region of USP1 identified here might be targeted to disrupt the USP1/UAF1 interaction with therapeutic purposes. On the other hand, we describe a cellular relocation assay that can be easily implemented in a high throughput setting to search for drugs that may dissociate the USP1/UAF1 complex