Phosphatidylinositol 4-kinase IIIα Research Articles

Adaptor proteins mediate CXCR4 and PI4KA crosstalk in prostate cancer cells and the significance of PI4KA in bone tumor growth

The chemokine receptor, CXCR4 signaling regulates cell growth, invasion, and metastasis to the bone-marrow niche in prostate cancer (PCa). Previously, we established that CXCR4 interacts with phosphatidylinositol 4-kinase IIIα (PI4KIIIα encoded by PI4KA) through its adaptor proteins and PI4KA overexpressed in the PCa metastasis. To further characterize how the CXCR4–PI4KIIIα axis promotes PCa metastasis, here we identify CXCR4 binds to PI4KIIIα adaptor proteins TTC7 and this interaction induce plasma membrane PI4P production in prostate cancer cells. Inhibiting PI4KIIIα or TTC7 reduces plasma membrane PI4P production, cellular invasion, and bone tumor growth. Using metastatic biopsy sequencing, we found PI4KA expression in tumors correlated with overall survival and contributes to immunosuppressive bone tumor microenvironment through preferentially enriching non-activated and immunosuppressive macrophage populations. Altogether we have characterized the chemokine signaling axis through CXCR4–PI4KIIIα interaction contributing to the growth of prostate cancer bone metastasis.

CircPI4KA Overexpression Enhances Carcinogenesis and Glycolysis Metabolism in Papillary Thyroid Carcinoma by Causing the miR-1287-5p-Mediated NRP2 Expression Elevation.

Circular RNAs (circRNAs) are implicated in regulating the pathogenesis of papillary thyroid carcinoma (PTC). Herein, we aimed to investigate how circRNA phosphatidylinositol 4-kinase IIIα (circPI4KA, hsa_circ_0062389) functioned as an oncogene in PTC. CircPI4KA, microRNA-1287-5p (miR-1287-5p) and Neuropilin-2 (NRP2) level detection were completed by reverse transcription-quantitative polymerase chain reaction assay. Cell proliferation was assessed through Cell Counting Kit-8 assay, colony formation assay, and EdU assay. Transwell assay was used for detecting migration and invasion abilities. Cell migration was also determined by wound healing assay. Cell apoptosis was assessed using flow cytometry assay. The protein examination was performed using western blot. Glycolysis was evaluated via commercial kits. Dual-luciferase reporter assay and RNA immunoprecipitation assay were conducted for target analysis. The role of circPI4KA in vivo was explored and analyzed via tumor xenograft assay. CircPI4KA was significantly upregulated in PTC tissues and cells. Knockdown of circPI4KA suppressed proliferation, migration, invasion, glycolysis, and induced apoptosis of PTC cells. CircPI4KA interacted with miR-1287-5p in PTC cells. The antitumor function of circPI4KA downregulation was reversed by inhibition of miR-1287-5p. The miR-1287-5p directly targeted NRP2, and circPI4KA elevated the NRP2 expression by sponging miR-1287-5p. PTC progression was impeded by miR-1287-5p via targeting NRP2. Silencing circPI4KA inhibited tumor growth in vivo through the miR-1287-5p/NRP2 axis. The collective results revealed that circPI4KA induced the upregulation of NRP2 via sponging miR-1287-5p, thus acting as a carcinogenic factor in PTC.

A newborn with multiple intestinal atresias found to have variants in tetratricopeptide repeat domain 7A gene

We report a male infant born with multiple intestinal atresias (MIA) prompting early genetic testing. Bi-allelic, compound heterozygous variants in tetratricopeptide repeat domain 7A (TTC7A) gene were identified. TTC7A is involved in transporting phosphatidylinositol 4-kinase IIIα into the plasma membrane (1). Mutations in this gene disrupt intestinal development and polarity, causing intestinal diseases such as atresia, enteropathy, and very early onset inflammatory bowel disease (VEO-IBD), as well as variable degrees of immunodeficiency (1).The identification of variants in TTC7A gene prompted an immune evaluation since as many as 75% of patients with TTC7A mutations reported in the literature have an underlying immunodeficiency (2). Flow cytometry was relatively unremarkable except for a slightly low memory T-cell count and percentage. NK-cell and B-cell numbers and percentages were appropriate for age. His phytohemagglutinin response was relatively low, but still significant, suggesting a possible component of combined immunodeficiency. To further characterize his T-cell function, we assessed his T-cell proliferation response to anti-CD3, which showed moderately decreased response of CD45+ lymphocytes and CD3+ T-cells to soluble anti-CD3 alone. However, co-stimulation with anti-CD3+anti-CD28 and anti-CD3+IL-2 normalized his T-cell proliferative response. Immunoglobulins were all low. Given his subtle T-cell defect and hypogammaglobulinemia, he was classified as having MIA with combined immunodeficiency (MIA-CID) phenotype of TTC7A.Cobalamin was tried in this patient as it has a low side effect profile and showed benefits in gut motility and intestinal tract luminal narrowing in a TTC7A zebrafish model (3). We considered investigational use of leflunomide, which showed the most promising results in the same zebrafish model (3). Bowel and hematopoietic stem-cell transplantation were considered too. Enterectomy was also offered, as this was shown to be effective in a case report of two patients with similar MIA-CID TTC7A phenotype (4). Unfortunately, the patient did not respond to steroids or cobalamin. His clinical condition worsened, and he passed away at four months of age.Mutations in the TTC7A gene result in various gastrointestinal pathologies with varying degrees of immunodeficiency in approximately 75% of patients (2). All patients with MIA or VEO-IBD should have an immune evaluation and genetic testing.

EFR3 and phosphatidylinositol 4-kinase IIIα regulate insulin-stimulated glucose transport and GLUT4 dispersal in 3T3-L1 adipocytes.

Insulin stimulates glucose transport in muscle and adipocytes. This is achieved by regulated delivery of intracellular glucose transporter (GLUT4)-containing vesicles to the plasma membrane where they dock and fuse, resulting in increased cell surface GLUT4 levels. Recent work identified a potential further regulatory step, in which insulin increases the dispersal of GLUT4 in the plasma membrane away from the sites of vesicle fusion. EFR3 is a scaffold protein that facilitates localization of phosphatidylinositol 4-kinase type IIIα to the cell surface. Here we show that knockdown of EFR3 or phosphatidylinositol 4-kinase type IIIα impairs insulin-stimulated glucose transport in adipocytes. Using direct stochastic reconstruction microscopy, we also show that EFR3 knockdown impairs insulin stimulated GLUT4 dispersal in the plasma membrane. We propose that EFR3 plays a previously unidentified role in controlling insulin-stimulated glucose transport by facilitating dispersal of GLUT4 within the plasma membrane.

Targeting PI4KA sensitizes refractory leukemia to chemotherapy by modulating the ERK/AMPK/OXPHOS axis.

Background: The emergence of chemoresistance in leukemia markedly impedes chemotherapeutic efficacy and dictates poor prognosis. Recent evidence has revealed that phosphatidylinositol 4 kinase-IIIα (PI4KA) plays a critical role in tumorigenesis. However, the molecular mechanisms of PI4KA-regulated chemoresistance and leukemogenesis remain largely unknown.Methods: Liquid chromatography-mass spectrometry (LC-MS), patient samples and leukemia xenograft mouse models were used to investigate whether PI4KA was an effective target to overcome chemoresistance in leukemia. Enzyme-linked immunosorbent assay (ELISA) and molecular mechanics/generalized born surface area (MM/GBSA) method were employed to identify cepharanthine (CEP) as a novel PI4KA inhibitor.Results: High expression of PI4KA was observed in drug-resistant leukemia cells or in relapsed leukemia patients, which was correlated with poor overall survival. Depletion of PI4KA sensitized drug-resistant leukemia cells to chemotherapeutic drugs in vitro and in vivo by regulating ERK/AMPK/OXPHOS axis. We also identified cepharanthine (CEP) as a novel PI4KA inhibitor, which could undermine the stability of the PI4KA/TTC7/FAM126 complex, enhancing the sensitivity of drug-resistant leukemia cells to chemotherapeutic drugs in vitro and in vivo.Conclusions: Our study underscored the potential of therapeutic targeting of PI4KA to overcome chemoresistance in leukemia. A combination of the PI4KA inhibitor with classic chemotherapeutic agents could represent a novel therapeutic strategy for the treatment of refractory leukemia.

Abstract 2293: Role of phosphatidylinositol 4-kinase IIIα in chemokine signaling

Abstract The CXCR4-CXCL12 chemokine signaling axis plays a key role in migration and bone metastasis in prostate cancer (PC). Androgens regulate CXCR4 expression and its receptor activation in lipid-raft micro domains of PC cells, resulting in higher protease expression and invasion. In order to identify some novel CXCR4 co-regulators associated within the lipid-raft, a SILAC (Stable isotope labeling using amino acid in cell culture)-based proteomic analysis was performed with PC3 stable cell-lines over-expressing or knocking-down CXCR4. Phosphatidylinositol 4-kinase III alpha (PI4KIIIα or PI4KA) and SAC1 lipid phosphatase were identified as candidate proteins enriched in CXCR4 expressing cells. PI4KA is an evolutionarily conserved mammalian kinase that converts PI to PI4P; and SAC1 dephosphorylates PI4P to PI. PI4K is required for the maintenance and functioning of the plasma-membrane and vesicular trafficking in the Golgi apparatus. PI4KA is also needed to maintain a steady pool of PI(4,5)P2 in the plasma-membrane, to be utilized in various signaling pathways. We show that CXCR4 interacts with PI4KA through its adaptor proteins EFR3B and TTC7B, recruiting it to the plasma-membrane for PI4P generation. Similarly PI4KA was closely linked to CXCR4 induced PC cell invasion, and knockdown of PI4KA in CXCR4 over-expressing PC3 cell lines reduced cell invasion. Also localized productions of PI4P was evident in invasive projections of CXCR4 over-expressing cell lines through immunofluorescence microscopy. PC tumor microarray data show increased PI4K expression in metastatic PC tissue vs localized or normal adjacent tissue. These data suggest a novel interaction between PI4KA and CXCR4, promoting tumor cell invasion and metastasis. Pharmacological targeting of PI4KA, its adaptor proteins or its signaling-related proteins might prove therapeutically beneficial and enhance survival in PC patients. Citation Format: Barani Govindarajan, Sreenivasa Chinni, Diego Sbrissa. Role of phosphatidylinositol 4-kinase IIIα in chemokine signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2293.

A PI4KIIIα protein complex is required for cell viability during Drosophila wing development

Phosphatidylinositol 4 phosphate (PI4P) and phosphatidylinositol 4,5 bisphosphate [PI(4,5)P2] are enriched on the inner leaflet of the plasma membrane and proposed to be key determinants of its function. PI4P is also the biochemical precursor for the synthesis of PI(4,5)P2 but can itself also bind to and regulate protein function. However, the independent function of PI4P at the plasma membrane in supporting cell function in metazoans during development in vivo remains unclear. We find that conserved components of a multi-protein complex composed of phosphatidylinositol 4-kinase IIIα (PI4KIIIα), TTC7 and Efr3 is required for normal vein patterning and wing development. Depletion of each of these three components of the PI4KIIIα complex in developing wing cells results in altered wing morphology. These effects are associated with an increase in apoptosis and can be rescued by expression of an inhibitor of Drosophila caspase. We find that in contrast to previous reports, PI4KIIIα depletion does not alter key outputs of hedgehog signalling in developing wing discs. Depletion of PI4KIIIα results in reduced PI4P levels at the plasma membrane of developing wing disc cells while levels of PI(4,5)P2, the downstream metabolite of PI4P, are not altered. Thus, PI4P itself generated by the activity of the PI4KIIIα complex plays an essential role in supporting cell viability in the developing Drosophila wing disc.

WNK1 promotes renal tumor progression by activating TRPC6-NFAT pathway.

Deregulation of Ca2+ signaling has been regarded as one of the key features of cancer progression. Lysine-deficient protein kinase 1 (WNK1), a major regulator of renal ion transport, regulates Ca2+ signaling through stimulating the phosphatidylinositol 4-kinase IIIα (PI4KIIIα) to activate Gαq-coupled receptor/PLC-β signaling. However, the contribution of WNK1-mediated Ca2+ signaling in the development of clear-cell renal-cell carcinoma (ccRCC) is yet unknown. We found that the canonical transient receptor potential channel (TRPC)6 was widely expressed in ccRCC tissues and functioned as a primary Ca2+ influx mechanism. We further identified that the expressions of WNK1, PI4KIIIα, TRPC6, and the nuclear factor of activated T cells cytoplasmic 1 (NFATc1) were elevated in the tumor tissues compared with the adjacent normal tissues. WNK1 expression was directly associated with the nuclear grade of ccRCC tissues. Functional experiments showed that WNK1 activated TRPC6-mediated Ca2+ influx and current by stimulating PI4KIIIα. Notably, the inhibition of WNK1-mediated TRPC6 activation and its downstream substrate calcineurin attenuated NFATc1 activation and the subsequent migration and proliferation of ccRCC. These findings revealed a novel perspective of WNK1 signaling in targeting the TRPC6-NFATc1 pathway as a therapeutic potential for renal-cell carcinoma.-Kim, J.-H., Hwang, K.-H., Eom, M., Kim, M., Park, E. Y., Jeong, Y., Park, K.-S., Cha, S.-K. WNK1 promotes renal tumor progression by activating TRPC6-NFAT pathway.

A novel cross-talk between CXCR4 and PI4KIIIα in prostate cancer cells.

Chemokine signaling regulates cell migration and tumor metastasis. CXCL12, a member of the chemokine family, and its receptor, CXCR4, a G protein coupled receptor (GPCR), are key mediators of prostate-cancer (PC) bone metastasis. In PC cells androgens activate CXCR4 gene expression and receptor signaling on lipid rafts, which induces protease expression and cancer cell invasion. To identify novel lipid-raft-associated CXCR4 regulators supporting invasion/metastasis, we performed a SILAC-based quantitative proteomic analysis of lipid-rafts derived from PC3 stable cell lines with overexpression or knockdown of CXCR4. This analysis identified the evolutionarily conserved phosphatidylinositol 4-kinase IIIα (PI4KIIIα), and SAC1 phosphatase that dephosphorylates phosphatidylinositol-4-phosphate as potential candidate CXCR4 regulators. CXCR4 interacted with PI4KIIIα membrane targeting machinery recruiting them to the plasma membrane for PI4P production. Consistent with this interaction, PI4KIIIα was found tightly linked to the CXCR4 induced PC cell invasion. Thus, ablation of PI4KIIIα in CXCR4-expressing PC3 cells reduced cellular invasion in response to a variety of chemokines. Immunofluorescence microscopy in CXCR4 expressing cells revealed localized production of PI4P on the invasive projections. Human tumor studies documented increased PI4KIIIα expression in metastatic tumors vs. the primary tumor counterparts, further supporting the PI4KIIIα role in tumor metastasis. Furthermore, we also identified an unexpected function of PI4KIIIα in GPCR signaling where CXCR4 regulates PI4KIIIα activity and mediate tumor metastasis. Together, our study identifies a novel cross-talk between PI4KIIIα and CXCR4 in promoting tumor metastasis and suggests that PI4KIIIα pharmacological targeting may have therapeutic benefit for advanced prostate cancer patients.

Probing the Architecture, Dynamics, and Inhibition of the PI4KIIIα/TTC7/FAM126 Complex

Phosphatidylinositol 4-kinase IIIα (PI4KIIIα) is the lipid kinase primarily responsible for generating the lipid phosphatidylinositol 4-phosphate (PI4P) at the plasma membrane, which acts as the substrate for generation of the signaling lipids PIP2 and PIP3. PI4KIIIα forms a large heterotrimeric complex with two regulatory partners, TTC7 and FAM126. We describe using an integrated electron microscopy and hydrogen–deuterium exchange mass spectrometry (HDX-MS) approach to probe the architecture and dynamics of the complex of PI4KIIIα/TTC7/FAM126. HDX-MS reveals that the majority of the PI4KIIIα sequence was protected from exchange in short deuterium pulse experiments, suggesting presence of secondary structure, even in putative unstructured regions. Negative stain electron microscopy reveals the shape and architecture of the full-length complex, revealing an overall dimer of PI4KIIIα/TTC7/FAM126 trimers. HDX-MS reveals conformational changes in the TTC7/FAM126 complex upon binding PI4KIIIα, including both at the direct TTC7-PI4KIIIα interface and at the putative membrane binding surface. Finally, HDX-MS experiments of PI4KIIIα bound to the highly potent and selective inhibitor GSK-A1 compared to that bound to the non-specific inhibitor PIK93 revealed substantial conformational changes throughout an extended region of the kinase domain. Many of these changes were distant from the putative inhibitor binding site, showing a large degree of allosteric conformational changes that occur upon inhibitor binding. Overall, our results reveal novel insight into the regulation of PI4KIIIα by its regulatory proteins TTC7/FAM126, as well as additional dynamic information on how selective inhibition of PI4KIIIα is achieved.

Inhibition of PI4K IIIα radiosensitizes in human tumor xenograft and immune-competent syngeneic murine tumor model.

Phosphatidylinositol (PI) 4-kinase (PI4K) has emerged as a potential target for anti-cancer treatment. We recently reported that simeprevir, an anti-hepatitis C viral (HCV) agent, radiosensitized diverse human cancer cells by inhibiting PI4K IIIα in vitro. In this study, we investigated the radiosensitizing effect of simeprevir in an in vivo tumor xenograft model and the mechanism of its interaction. The immune modulatory effect of PI4K IIIα was evaluated in an immune-competent syngeneic murine tumor model.In in vivo xenograft models using BT474 breast cancer and U251 brain tumor cells, inhibition of PI4K IIIα induced by simeprevir combined with radiation significantly delayed tumor growth compared to either treatment alone. PI4K IIIα inhibition led to eversion of the epithelial-mesenchymal transition as suggested by decreased invasion/migration and vascular tube formation. Simeprevir down-regulated PI3Kδ expression and PI3Kδ inhibition using RNA interference radiosensitized breast cancer cells. PI4K IIIα inhibition enhanced the radiosensitizing effect of anti-programmed death-ligand 1 (PD-L1) and decreased the expression of PI3Kδ, phosphorylated-Akt, and PD-L1 in breast cancer cells co-cultured with human T-lymphocytes. The immune modulatory effect in vivo was evaluated in immune-competent syngeneic 4T1 murine tumor models. Simeprevir showed significant radiosensitizing effect and immune modulatory function by affecting the CD4(+)/CD8(+) ratio of tumor infiltrating lymphocytes.These findings suggest that targeting PI4K IIIα with an anti-HCV agent is a viable drug repositioning approach for enhancing the therapeutic efficacy of radiation therapy. The immune regulatory function of PI4K IIIα via modulation of PI3Kδ suggests a strategy for enhancing the radiosensitizing effect of immune checkpoint blockades.

Tuning a cellular lipid kinase activity adapts hepatitis C virus to replication in cell culture.

With a single exception, all isolates of hepatitis C virus (HCV) require adaptive mutations to replicate efficiently in cell culture. Here, we show that a major class of adaptive mutations regulates the activity of a cellular lipid kinase, phosphatidylinositol 4-kinase IIIα (PI4KA). HCV needs to stimulate PI4KA to create a permissive phosphatidylinositol 4-phosphate-enriched membrane microenvironment in the liver and in primary human hepatocytes (PHHs). In contrast, in Huh7 hepatoma cells, the virus must acquire loss-of-function mutations that prevent PI4KA overactivation. This adaptive mechanism is necessitated by increased PI4KA levels in Huh7 cells compared with PHHs, and is conserved across HCV genotypes. PI4KA-specific inhibitors promote replication of unadapted viral isolates and allow efficient replication of patient-derived virus in cell culture. In summary, this study has uncovered a long-sought mechanism of HCV cell-culture adaptation and demonstrates how a virus can adapt to changes in a cellular environment associated with tumorigenesis.

Targeting Phosphatidylinositol 4-Kinase IIIα for Radiosensitization: A Potential Model of Drug Repositioning Using an Anti-Hepatitis C Viral Agent

To investigate which isotype of phosphatidylinositol 4-kinase (PI4K) may affect radiosensitivity and examine whether anti-hepatitis C viral (HCV) agents, some of which have been shown to inhibit PI4K IIIα activity, could be repositioned as a radiosensitizer in human cancer cells. U251, BT474, and HepG2 cell lines and normal human astrocyte were used. Ribonucleic acid interference, clonogenic assays, Western blotting, immunofluorescence, annexin V assay, lysotracker staining, and β-galactosidase assay were performed. Of the 4 PI4K isotypes, specific inhibition of IIIα increased radiosensitivity. For pharmacologic inhibition of PI4K IIIα, we screened 9 anti-HCV agents by half-maximal inhibitory concentration assay. Simeprevir was selected, and its inhibition of PI4K IIIα activity was confirmed. Combination of simeprevir treatment and radiation significantly attenuated expression of phospho-phospho-PKC and phospho-Akt and increased radiation-induced cell death in tested cell lines. Pretreatment with simeprevir prolonged γH2AX foci formation and down-regulation of phospho-DNA-PKcs, indicating impairment of nonhomologous end-joining repair. Cells pretreated with simeprevir exhibited mixed modes of cell death, including apoptosis and autophagy. These data demonstrate that targeting PI4K IIIα using an anti-HCV agent is a viable approach to enhance the therapeutic efficacy of radiation therapy in various human cancers, such as glioma, breast, and hepatocellular carcinoma.

Tyrphostin AG1478 Inhibits Encephalomyocarditis Virus and Hepatitis C Virus by Targeting Phosphatidylinositol 4-Kinase IIIα.

Encephalomyocarditis virus (EMCV), like hepatitis C virus (HCV), requires phosphatidylinositol 4-kinase IIIα (PI4KA) for genome replication. Here, we demonstrate that tyrphostin AG1478, a known epidermal growth factor receptor (EGFR) inhibitor, also inhibits PI4KA activity, both in vitro and in cells. AG1478 impaired replication of EMCV and HCV but not that of an EMCV mutant previously shown to escape PI4KA inhibition. This work uncovers novel cellular and antiviral properties of AG1478, a compound previously regarded only as a cancer chemotherapy agent.

MP84-16 PHOSPHATIDYLINOSITOL 4-KINASE TYPE III? (PI4KA) AND CXCR4 CROSSTALK IN PROSTATE CANCER CELLS

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology III1 Apr 2016MP84-16 PHOSPHATIDYLINOSITOL 4-KINASE TYPE III? (PI4KA) AND CXCR4 CROSSTALK IN PROSTATE CANCER CELLS Michael Cher, Diego Sbrissa, Louie Semaan, Yanfeng Li, Assia Shisheva, and Srinivasa Chinni Michael CherMichael Cher More articles by this author , Diego SbrissaDiego Sbrissa More articles by this author , Louie SemaanLouie Semaan More articles by this author , Yanfeng LiYanfeng Li More articles by this author , Assia ShishevaAssia Shisheva More articles by this author , and Srinivasa ChinniSrinivasa Chinni More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.2240AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The CXCR4 receptor directs homing of hematopoietic stem cells to CXCL12 ligand-rich bone marrow. Previous studies demonstrated that the CXCL12/CXCR4 axis promotes prostate cancer (PC) invasion and bone metastasis. We showed that CXCR4 expression is transcriptionally upregulated by TMPRSS2-ERG gene fusions in PC tumors, and its localization in lipid raft membrane microdomains promotes expression of invasive proteases. To identify novel lipid raft-associated CXCR4 regulators supporting invasion and metastasis, we performed proteomics analysis of lipid raft microdomains from stable CXCR4-overexpressing (CXCR4) and CXCR4-knockdown (shCXCR4) PC3 cell lines generated by lentiviral infection. METHODS We used stable isotope labeling by amino acids in cell culture (SILAC) proteomics, sucrose gradient buoyant density ultracentrifugation, fluorescence activated cell sorting (FACS), knockdown by siRNA silencing, cell invasion/growth assays, [2-3H]inositol cell labeling, SDS PAGE, Western blotting (WB), and inositol-lipid HPLC profiling. RESULTS SILAC identified phosphatidylinositol 4-kinase IIIα (PI4KA) to be highly associated with CXCR4 cells, along with slightly elevated SAC1 phosphatase. WB confirmed that expression of these phosphoinositide-regulating enzymes is associated with CXCR4 overexpression, thus anticipating their promotion of invasion and metastasis. Several lines of data demonstrated a link between PI4KA and CXCR4 signaling: PI4KA knockdown (i) reduced surface CXCR4 expression and CXCL12-stimulated invasion of CXCR4 cells; (ii) blocked CXCR4/AKT signaling lowering pAKT(Ser473) and pCXCR4(Ser339); and (iii) dropped global cellular PI4P levels. Unexpectedly, PI4KA knockdown resulted in a rise in PI4,5P2, suggesting a homeostatic response. Conversely, CXCR4 knockdown showed an expected increase in PI4,5P2 but an unexpected decrease in PI4P. Importantly, human PC tumor microarray data showed higher PI4KA expression in metastatic vs. localized PC or normal adjacent tissue. CONCLUSIONS These data confirm that PI4KA upregulates CXCR4 expression by altering inositol lipid levels thereby promoting CXCR4/AKT signaling, and that inhibition of CXCR4 signaling via PI4KA knockdown can suppress PC growth/invasion. Thus, PI4KA inhibitors may beneficial in patients with prostate cancer. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e1095 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Michael Cher More articles by this author Diego Sbrissa More articles by this author Louie Semaan More articles by this author Yanfeng Li More articles by this author Assia Shisheva More articles by this author Srinivasa Chinni More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Hepatitis C virus NS5A protein cooperates with phosphatidylinositol 4-kinase IIIα to induce mitochondrial fragmentation

Hepatitis C virus (HCV) has long been observed to take advantage of the host mitochondria to support viral replication and assembly. The HCV core protein has been implicated to fragment host mitochondria. In this report, we have discovered that the non-structural protein 5A (NS5A) plays an instructive role in attaching ER with mitochondria, causing mitochondrial fragmentation. Dynamin-related protein 1(Drp1), a host protein essential to mitochondrial membrane fission, does not play a role in NS5A-induced mitochondrial fragmentation. Instead, phosphatidylinositol 4-kinase IIIα (PI4KA), which has been demonstrated to bind to NS5A and is required to support HCV life cycle, is required for NS5A to induce mitochondrial fragmentation. Both NS5A and core are required by HCV to fragment the mitochondria, as inhibiting either of their respective downstream proteins, PI4KA or Drp1, resulted in lengthening of mitochondria tubules in HCVcc-infected cells. By fragmenting the mitochondria, NS5A renders the cells more resistant to mitochondria mediated apoptosis. This finding indicates previously-ignored contribution of NS5A in HCV-induced mitochondria dysfunction.

Novel perspectives for hepatitis A virus therapy revealed by comparative analysis of hepatitis C virus and hepatitis A virus RNA replication

Hepatitis A virus (HAV) and hepatitis C virus (HCV) are two positive‐strand RNA viruses sharing a similar biology, but causing opposing infection outcomes, with HAV always being cleared and HCV establishing persistence in the majority of infections. To gain deeper insight into determinants of replication, persistence, and treatment, we established a homogenous cell‐culture model allowing a thorough comparison of RNA replication of both viruses. By screening different human liver‐derived cell lines with subgenomic reporter replicons of HAV as well as of different HCV genotypes, we found that Huh7‐Lunet cells supported HAV‐ and HCV‐RNA replication with similar efficiency and limited interference between both replicases. HAV and HCV replicons were similarly sensitive to interferon (IFN), but differed in their ability to establish persistent replication in cell culture. In contrast to HCV, HAV replicated independently from microRNA‐122 and phosphatidylinositol 4‐kinase IIIα and β (PI4KIII). Both viruses were efficiently inhibited by cyclosporin A and NIM811, a nonimmunosuppressive analog thereof, suggesting an overlapping dependency on cyclophilins for replication. However, analysis of a broader set of inhibitors revealed that, in contrast to HCV, HAV does not depend on cyclophilin A, but rather on adenosine‐triphosphate–binding cassette transporters and FK506‐binding proteins. Finally, silibinin, but not its modified intravenous formulation, efficiently inhibited HAV genome replication in vitro, suggesting oral silibinin as a potential therapeutic option for HAV infections. Conclusion: We established a cell‐culture model enabling comparative studies on RNA replication of HAV and HCV in a homogenous cellular background with comparable replication efficiency. We thereby identified new host cell targets and potential treatment options for HAV and set the ground for future studies to unravel determinants of clearance and persistence. (Hepatology 2015;62:397–408

Cyclophilin and NS5A inhibitors, but not other anti-hepatitis C virus (HCV) agents, preclude HCV-mediated formation of double-membrane-vesicle viral factories.

Although the mechanisms of action (MoA) of nonstructural protein 3 inhibitors (NS3i) and NS5B inhibitors (NS5Bi) are well understood, the MoA of cyclophilin inhibitors (CypI) and NS5A inhibitors (NS5Ai) are not fully defined. In this study, we examined whether CypI and NS5Ai interfere with hepatitis C virus (HCV) RNA synthesis of replication complexes (RCs) or with an earlier step of HCV RNA replication, the creation of double-membrane vesicles (DMVs) essential for HCV RNA replication. In contrast to NS5Bi, both CypI and NS5Ai do not block HCV RNA synthesis by way of RCs, suggesting that they exert their antiviral activity prior to the establishment of enzymatically active RCs. We found that viral replication is not a precondition for DMV formation, since the NS3-NS5B polyprotein or NS5A suffices to create DMVs. Importantly, only CypI and NS5Ai, but not NS5Bi, mir-122, or phosphatidylinositol-4 kinase IIIα (PI4KIIIα) inhibitors, prevent NS3-NS5B-mediated DMV formation. NS3-NS5B was unable to create DMVs in cyclophilin A (CypA) knockdown (KD) cells. We also found that the isomerase activity of CypA is absolutely required for DMV formation. This not only suggests that NS5A and CypA act in concert to build membranous viral factories but that CypI and NS5Ai mediate their early anti-HCV effects by preventing the formation of organelles, where HCV replication is normally initiated. This is the first investigation to examine the effect of a large panel of anti-HCV agents on DMV formation, and the results reveal that CypI and NS5Ai act at the same membranous web biogenesis step of HCV RNA replication, thus indicating a new therapeutic target of chronic hepatitis C.

Multiple intestinal atresia with combined immune deficiency

To update on the molecular and cellular basis of multiple intestinal atresia (MIA). Mutations of the tetratricopeptide repeat domain 7A gene have been identified in patients with MIA and other related disorders, including MIA associated with combined immunodeficiency and very early onset inflammatory bowel disease with apoptotic enterocolitis. Pathological findings in patients with MIA and MIA associated with combined immunodeficiency include abnormalities of enterocyte apicobasal polarity, increased apoptosis of intestinal cells, decreased proliferation of intestinal crypts, and defects of thymic architecture associated with lymphoid depletion. Dysregulated RhoA signaling and defective expression of phosphatidylinositol 4-kinase IIIα represent biochemical cellular hallmarks of the disease. The study of patients with MIA and related disorders has established that tetratricopeptide repeat domain 7A plays a critical role in intestinal and immune homeostasis. Identification of biochemical defects may pave the way to novel pharmacological interventions for this group of severe congenital disorders.

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