Targeting the ATF6-Mediated ER Stress Response and Autophagy Blocks Integrin-Driven Prostate Cancer Progression.

Prostate cancer progression to the lethal metastatic castration-resistant phenotype (mCRPC) is driven by αv integrins and is associated with Golgi disorganization and activation of the ATF6 branch of unfolded protein response (UPR). Overexpression of integrins requires N-acetylglucosaminyltransferase-V (MGAT5)-mediated glycosylation and subsequent cluster formation with Galectin-3 (Gal-3). However, the mechanism underlying this altered glycosylation is missing. For the first time, using HALO analysis of IHC, we found a strong association of integrin αv and Gal-3 at the plasma membrane (PM) in primary prostate cancer and mCRPC samples. We discovered that MGAT5 activation is caused by Golgi fragmentation and mislocalization of its competitor, N-acetylglucosaminyltransferase-III, MGAT3, from Golgi to the endoplasmic reticulum (ER). This was validated in an ethanol-induced model of ER stress, where alcohol treatment in androgen-refractory PC-3 and DU145 cells or alcohol consumption in patient with prostate cancer samples aggravates Golgi scattering, activates MGAT5, and enhances integrin expression at PM. This explains known link between alcohol consumption and prostate cancer mortality. ATF6 depletion significantly blocks UPR and reduces the number of Golgi fragments in both PC-3 and DU145 cells. Inhibition of autophagy by hydroxychloroquine (HCQ) restores compact Golgi, rescues MGAT3 intra-Golgi localization, blocks glycan modification via MGAT5, and abrogates delivery of Gal-3 to the cell surface. Importantly, the loss of Gal-3 leads to reduced integrins at PM and their accelerated internalization. ATF6 depletion and HCQ treatment synergistically decrease integrin αv and Gal-3 expression and temper orthotopic tumor growth and metastasis. IMPLICATIONS: Combined ablation of ATF6 and autophagy can serve as new mCRPC therapeutic.

The non-canonical mechanism of ER stress-mediated progression of prostate cancer.

BACKGROUND: The development of persistent endoplasmic reticulum (ER) stress is one of the cornerstones of prostate carcinogenesis; however, the mechanism is missing. Also, alcohol is a physiological ER stress inducer, and the link between alcoholism and progression of prostate cancer (PCa) is well documented but not well characterized. According to the canonical model, the mediator of ER stress, ATF6, is cleaved sequentially in the Golgi by S1P and S2P proteases; thereafter, the genes responsible for unfolded protein response (UPR) undergo transactivation. METHODS: Cell lines used were non-malignant prostate epithelial RWPE-1 cells, androgen-responsive LNCaP, and 22RV1 cells, as well as androgen-refractory PC-3 cells. We also utilized PCa tissue sections from patients with different Gleason scores and alcohol consumption backgrounds. Several sophisticated approaches were employed, including Structured illumination superresolution microscopy, Proximity ligation assay, Atomic force microscopy, and Nuclear magnetic resonance spectroscopy. RESULTS: Herein, we identified the trans-Golgi matrix dimeric protein GCC185 as a Golgi retention partner for both S1P and S2P, and in cells lacking GCC185, these enzymes lose intra-Golgi situation. Progression of prostate cancer (PCa) is associated with overproduction of S1P and S2P but monomerization of GCC185 and its downregulation. Utilizing different ER stress models, including ethanol administration, we found that PCa cells employ an elegant mechanism that auto-activates ER stress by fragmentation of Golgi, translocation of S1P and S2P from Golgi to ER, followed by intra-ER cleavage of ATF6, accelerated UPR, and cell proliferation. The segregation of S1P and S2P from Golgi and activation of ATF6 are positively correlated with androgen receptor signaling, different disease stages, and alcohol consumption. Finally, depletion of ATF6 significantly retarded the growth of xenograft prostate tumors and blocks production of pro-metastatic metabolites. CONCLUSIONS: We found that progression of PCa associates with translocation of S1P and S2P proteases to the ER and subsequent ATF6 cleavage. This obviates the need for ATF6 transport to the Golgi and enhances UPR and cell proliferation. Thus, we provide the novel mechanistic model of ATF6 activation and ER stress implication in the progression of PCa, suggesting ATF6 is a novel promising target for prostate cancer therapy.

Porcine Reproductive and Respiratory Syndrome Virus Infection Upregulates Negative Immune Regulators and T-Cell Exhaustion Markers.

Porcine alveolar macrophage (PAM) is one of the primary cellular targets for porcine reproductive and respiratory syndrome virus (PRRSV), but less than 2% of PAMs are infected with the virus during the acute stage of infection. To comparatively analyze the host transcriptional response between PRRSV-infected PAMs and bystander PAMs that remained uninfected but were exposed to the inflammatory milieu of an infected lung, pigs were infected with a PRRSV strain expressing green fluorescent protein (PRRSV-GFP), and GFP+ (PRRSV infected) and GFP- (bystander) cells were sorted for RNA sequencing (RNA-seq). Approximately 4.2% of RNA reads from GFP+ and 0.06% reads from GFP- PAMs mapped to the PRRSV genome, indicating that PRRSV-infected PAMs were effectively separated from bystander PAMs. Further analysis revealed that inflammatory cytokines, interferon-stimulated genes, and antiviral genes were highly upregulated in GFP+ compared to GFP- PAMs. Importantly, negative immune regulators, including NF-κB inhibitors (NFKBIA, NFKBID, NFKBIZ, and TNFAIP3) and T-cell exhaustion markers (programmed death ligand-1 [PD-L1], PD-L2, interleukin-10 [IL-10], IDO1, and transforming growth factor ß2 [TGFB2]) were highly upregulated in GFP+ cells compared to GFP- cells. By using an in situ hybridization assay, RNA transcripts of tumor necrosis factor (TNF) and NF-κB inhibitors were detected in PRRSV-infected PAMs cultured ex vivo and lung sections of PRRSV-infected pigs during the acute stage of infection. Collectively, the results suggest that PRRSV infection upregulates expression of negative immune regulators and T-cell exhaustion markers in PAMs to modulate the host immune response. Our findings provide further insight into PRRSV immunopathogenesis. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) is widespread in many swine-producing countries, causing substantial economic losses to the swine industry. Porcine alveolar macrophage (PAM) is considered the primary target for PRRSV replication in pigs. However, less than 2% of PAMs from acutely infected pigs are infected with the virus. In the present study, we utilized a PRRSV strain expressing green fluorescent protein to infect pigs and sorted infected and bystander PAMs from the pigs during the acute stage of infection for transcriptome analysis. PRRSV-infected PAMs showed a distinctive gene expression profile and contained many uniquely activated pathways compared to bystander PAMs. Interestingly, upregulated expression of NF-κB signaling inhibitors and T-cell exhaustion molecules were observed in PRRSV-infected PAMs. Our findings provide additional knowledge on the mechanisms that PRRSV employs to modulate the host immune system.

Post-ER Stress Biogenesis of Golgi Is Governed by Giantin.

BACKGROUND: The Golgi apparatus undergoes disorganization in response to stress, but it is able to restore compact and perinuclear structure under recovery. This self-organization mechanism is significant for cellular homeostasis, but remains mostly elusive, as does the role of giantin, the largest Golgi matrix dimeric protein. METHODS: In HeLa and different prostate cancer cells, we used the model of cellular stress induced by Brefeldin A (BFA). The conformational structure of giantin was assessed by proximity ligation assay and atomic force microscopy. The post-BFA distribution of Golgi resident enzymes was examined by 3D SIM high-resolution microscopy. RESULTS: We detected that giantin is rather flexible than an extended coiled-coil dimer and BFA-induced Golgi disassembly was associated with giantin monomerization. A fusion of the nascent Golgi membranes after BFA washout is forced by giantin re-dimerization via disulfide bond in its luminal domain and assisted by Rab6a GTPase. GM130-GRASP65-dependent enzymes are able to reach the nascent Golgi membranes, while giantin-sensitive enzymes appeared at the Golgi after its complete recovery via direct interaction of their cytoplasmic tail with N-terminus of giantin. CONCLUSION: Post-stress recovery of Golgi is conducted by giantin dimer and Golgi proteins refill membranes according to their docking affiliation rather than their intra-Golgi location.

The Role of Alcohol-Induced Golgi Fragmentation for Androgen Receptor Signaling in Prostate Cancer.

Multiple epidemiologic observations and meta-analysis clearly indicate the link between alcohol abuse and the incidence and progression of prostate cancer; however, the mechanism remains enigmatic. Recently, it was found that ethanol (EtOH) induces disorganization of the Golgi complex caused by impaired function of the largest Golgi matrix protein, giantin (GOLGB1), which, in turn, alters the Golgi docking of resident Golgi proteins. Here, it is determined that in normal prostate cells, histone deacetylase 6 (HDAC6), the known regulator of androgen receptor (AR) signaling, localizes in the cytoplasm and nucleus, while its kinase, glycogen synthase kinase ß (GSK3ß), primarily resides in the Golgi. Progression of prostate cancer is accompanied by Golgi scattering, translocation of GSK3ß from the Golgi to the cytoplasm, and the cytoplasmic shift in HDAC6 localization. Alcohol dehydrogenase-generated metabolites induces Golgi disorganization in androgen-responsive LNCaP and 22Rv1 cells, facilitates tumor growth in a mouse xenograft model and activates anchorage-independent proliferation, migration, and cell adhesion. EtOH-treated cells demonstrate reduced giantin and subsequent cytoplasmic GSK3ß; this phenomenon was validated in giantin-depleted cells. Redistribution of GSK3ß to the cytoplasm results in phosphorylation of HDAC6 and its retention in the cytoplasm, which, in turn, stimulates deacetylation of HSP90, AR import into the nucleus, and secretion of prostate-specific antigen (PSA). Finally, the relationship between Golgi morphology, HDAC6 cytoplasmic content, and clinicopathologic features was assessed in human prostate cancer patient specimens with and without a history of alcohol dependence. IMPLICATIONS: This study demonstrates the importance of alcohol-induced Golgi fragmentation in the activation of AR-mediated proliferation.

Role of whole grains versus fruits and vegetables in reducing subclinical inflammation and promoting gastrointestinal health in individuals affected by overweight and obesity: a randomized controlled trial.

BACKGROUND: Whole grains (WG) and fruits and vegetables (FV) have been shown to reduce the risk of metabolic disease, possibly via modulation of the gut microbiota. The purpose of this study was to determine the impact of increasing intake of either WG or FV on inflammatory markers and gut microbiota composition. METHODS: A randomized parallel arm feeding trial was completed on forty-nine subjects with overweight or obesity and low intakes of FV and WG. Individuals were randomized into three groups (3 servings/d provided): WG, FV, and a control (refined grains). Stool and blood samples were collected at the beginning of the study and after 6 weeks. Inflammatory markers [tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), lipopolysaccharide binding protein (LBP), and high sensitivity C-reactive protein (hs-CRP)] were measured. Stool sample analysis included short/branched chain fatty acids (S/BCFA) and microbiota composition. RESULTS: There was a significant decrease in LBP for participants on the WG (- 0.2 µg/mL, p = 0.02) and FV (- 0.2 µg/mL, p = 0.005) diets, with no change in those on the control diet (0.1 µg/mL, p = 0.08). The FV diet induced a significant change in IL-6 (- 1.5 pg/mL, p = 0.006), but no significant change was observed for the other treatments (control, - 0.009 pg/mL, p = 0.99; WG, - 0.29, p = 0.68). The WG diet resulted in a significant decrease in TNF-α (- 3.7 pg/mL; p < 0.001), whereas no significant effects were found for those on the other diets (control, - 0.6 pg/mL, p = 0.6; FV, - 1.4 pg/mL, p = 0.2). The treatments induced individualized changes in microbiota composition such that treatment group differences were not identified, except for a significant increase in α-diversity in the FV group. The proportions of Clostridiales (Firmicutes phylum) at baseline were correlated with the magnitude of change in LBP during the study. CONCLUSIONS: These data demonstrate that WG and FV intake can have positive effects on metabolic health; however, different markers of inflammation were reduced on each diet suggesting that the anti-inflammatory effects were facilitated via different mechanisms. The anti-inflammatory effects were not related to changes in gut microbiota composition during the intervention, but were correlated with microbiota composition at baseline. TRIAL REGISTRATION: ClinicalTrials.gov , NCT02602496 , Nov 4, 2017.

Overaccumulation of γ-Glutamylcysteine in a Jasmonate-Hypersensitive Arabidopsis Mutant Causes Jasmonate-Dependent Growth Inhibition.

Glutathione (GSH) is essential for many aspects of plant biology and is associated with jasmonate signaling in stress responses. We characterized an Arabidopsis (Arabidopsis thaliana) jasmonate-hypersensitive mutant (jah2) with seedling root growth 100-fold more sensitive to inhibition by the hormone jasmonyl-isoleucine than the wild type. Genetic mapping and genome sequencing determined that the mutation is in intron 6 of GLUTATHIONE SYNTHETASE2, encoding the enzyme that converts γ-glutamylcysteine (γ-EC) to GSH. The level of GSH in jah2 was 71% of the wild type, while the phytoalexin-deficient2-1 (pad2-1) mutant, defective in GSH1 and having only 27% of wild-type GSH level, was not jasmonate hypersensitive. Growth defects for jah2, but not pad2, were also seen in plants grown to maturity. Surprisingly, all phenotypes in the jah2 pad2-1 double mutant were weaker than in jah2. Quantification of γ-EC indicated these defects result from hyperaccumulation of this GSH precursor by 294- and 65-fold in jah2 and the double mutant, respectively. γ-EC reportedly partially substitutes for loss of GSH, but growth inhibition seen here was likely not due to an excess of total glutathione plus γ-EC because their sum in jah2 pad2-1 was only 16% greater than in the wild type. Further, the jah2 phenotypes were lost in a jasmonic acid biosynthesis mutant background, indicating the effect of γ-EC is mediated through jasmonate signaling and not as a direct result of perturbed redox status.