Prognostic Significance of O-GlcNAc and PKM2 in Hormone Receptor-Positive and HER2-Nonenriched Breast Cancer.

Predictive metabolic biomarkers for the recurrent luminal breast cancer (BC) with hormone receptor (HR)-positive and human epidermal growth factor receptor type 2 (HER2)-negative are lacking. High levels of O-GlcNAcylation (O-GlcNAc) and pyruvate kinase isoenzyme M2 (PKM2) are associated with malignancy in BC; however, the association with the recurrence risk remains unclear. We first conduct survival analysis by using the METABRIC dataset to assess the correlation of PKM2 expression with BC clinical outcomes. Next, patients with HR+/HER2- luminal BC were recruited for PKM2/O-GlcNAc testing. Logistic regression and receiver operating characteristic curve analysis were performed to evaluate the 10-year DFS predicted outcome. Survival analysis of the METABRIC dataset revealed that high expression of PKM2 was significantly associated with worse overall survival in luminal BC. The high expression of O-GlcNAc or PKM2 was a significant independent marker for poor 10-year DFS using immunohistochemical analysis. The PKM2 or O-GlcNAc status was a significant predictor of DFS, with the combination of PKM2-O-GlcNAc status and T stage greatly enhancing the predictive outcome potential. In summary, O-GlcNAc, PKM2, and T stage serve as good prognostic discriminators in HR+/HER2- luminal BC.

Targeting the histone demethylase PHF8-mediated PKCα-Src-PTEN axis in HER2-negative gastric cancer.

Targeted treatments for advanced gastric cancer (GC) are needed, particularly for HER2-negative GC, which represents the majority of cases (80 to 88%). In this study, in silico analyses of the lysine histone demethylases (KDMs) involved in diverse biological processes and diseases revealed that PHD finger protein 8 (PHF8, KDM7B) was significantly associated with poor clinical outcome in HER2-negative GC. The depletion of PHF8 significantly reduced cancer progression in GC cells and in mouse xenografts. PHF8 regulated genes involved in cell migration/motility based on a microarray analysis. Of note, PHF8 interacted with c-Jun on the promoter of PRKCA which encodes PKCα. The depletion of PHF8 or PKCα greatly up-regulated PTEN expression, which could be rescued by ectopic expression of a PKCα expression vector or an active Src. These suggest that PTEN destabilization occurs mainly via the PKCα-Src axis. GC cells treated with midostaurin or bosutinib significantly suppressed migration in vitro and in zebrafish models. Immunohistochemical analyses of PHF8, PKCα, and PTEN showed a positive correlation between PHF8 and PKCα but negative correlations between PHF8 and PTEN and between PKCα and PTEN. Moreover, high PHF8-PKCα expression was significantly correlated with worse prognosis. Together, our results suggest that the PKCα-Src-PTEN pathway regulated by PHF8/c-Jun is a potential prognostic/therapeutic target in HER2-negative advanced GC.

Mutations in the PKM2 exon-10 region are associated with reduced allostery and increased nuclear translocation.

PKM2 is a key metabolic enzyme central to glucose metabolism and energy expenditure. Multiple stimuli regulate PKM2's activity through allosteric modulation and post-translational modifications. Furthermore, PKM2 can partner with KDM8, an oncogenic demethylase and enter the nucleus to serve as a HIF1α co-activator. Yet, the mechanistic basis of the exon-10 region in allosteric regulation and nuclear translocation remains unclear. Here, we determined the crystal structures and kinetic coupling constants of exon-10 tumor-related mutants (H391Y and R399E), showing altered structural plasticity and reduced allostery. Immunoprecipitation analysis revealed increased interaction with KDM8 for H391Y, R399E, and G415R. We also found a higher degree of HIF1α-mediated transactivation activity, particularly in the presence of KDM8. Furthermore, overexpression of PKM2 mutants significantly elevated cell growth and migration. Together, PKM2 exon-10 mutations lead to structure-allostery alterations and increased nuclear functions mediated by KDM8 in breast cancer cells. Targeting the PKM2-KDM8 complex may provide a potential therapeutic intervention.

KDM4B is a coactivator of c-Jun and involved in gastric carcinogenesis.

KDM4/JMJD2 Jumonji C-containing histone lysine demethylases (KDM4A-D) constitute an important class of epigenetic modulators in the transcriptional activation of cellular processes and genome stability. Interleukin-8 (IL-8) is overexpressed in gastric cancer, but the mechanisms and particularly the role of the epigenetic regulation of IL-8, are unclear. Here, we report that KDM4B, but not KDM4A/4C, upregulated IL-8 production in the absence or presence of Helicobacter pylori. Moreover, KDM4B physically interacts with c-Jun on IL-8, MMP1, and ITGAV promoters via its demethylation activity. The depletion of KDM4B leads to the decreased expression of integrin αV, which is exploited by H. pylori carrying the type IV secretion system, reducing IL-8 production and cell migration. Elevated KDM4B expression is significantly associated with the abundance of p-c-Jun in gastric cancer and is linked to a poor clinical outcome. Together, our results suggest that KDM4B is a key regulator of JNK/c-Jun-induced processes and is a valuable therapeutic target.

Helicobacter pylori cholesterol glucosylation modulates autophagy for increasing intracellular survival in macrophages.

Cholesterol-α-glucosyltransferase (CGT) encoded by the type 1 capsular polysaccharide biosynthesis protein J (capJ) gene of Helicobacter pylori converts cellular cholesterol into cholesteryl glucosides. H. pylori infection induces autophagy that may increase bacterial survival in epithelial cells. However, the role of H. pylori CGT that exploits lipid rafts in interfering with autophagy for bacterial survival in macrophages has not been investigated. Here, we show that wild-type H. pylori carrying CGT modulates cholesterol to trigger autophagy and restrain autophagosome fusion with lysosomes, permitting a significantly higher bacterial burden in macrophages than that in a capJ-knockout (∆CapJ) mutant. Knockdown of autophagy-related protein 12 impairs autophagosome maturation and decreases the survival of internalised H. pylori in macrophages. These results demonstrate that CGT plays a crucial role in the manipulation of the autophagy process to impair macrophage clearance of H. pylori.

Cholesterol glucosylation by Helicobacter pylori delays internalization and arrests phagosome maturation in macrophages.

BACKGROUND/PURPOSE: Helicobacter pylori colonizes the human stomach and contributes to chronic inflammation of the gastric mucosa. H. pylori persistence occurs because of insufficient eradication by phagocytic cells. A key factor of H. pylori, cholesterol-α-glucosyltransferase encoded by capJ that extracts host cholesterol and converts it to cholesteryl glucosides, is important to evade host immunity. Here, we examined whether phagocytic trafficking in macrophages was perturbed by capJ-carrying H. pylori. METHODS: J774A.1 cells were infected with H. pylori at a multiplicity of infection of 50. Live-cell imaging and confocal microscopic analysis were applied to monitor the phagocytic trafficking events. The viability of H. pylori inside macrophages was determined by using gentamicin colony-forming unit assay. The phagocytic routes were characterized by using trafficking-intervention compounds. RESULTS: Wild type (WT) H. pylori exhibited more delayed entry into macrophages and also arrested phagosome maturation more than did capJ knockout mutant. Pretreatment of genistein and LY294002 prior to H. pylori infection reduced the internalization of WT but not capJ-knockout H. pylori in macrophages. CONCLUSION: Cholesterol glucosylation by H. pylori interferes with phagosome trafficking via a lipid-raft and PI3K-dependent manner, which retards engulfment of bacteria for prolonged intracellular survival of H. pylori.

The CrdRS two-component system in Helicobacter pylori responds to nitrosative stress.

Helicobacter pylori inhabits the gastric mucosa where it senses and responds to various stresses via a two-component systems (TCSs) that enable its persistent colonization. The aim of this study was to investigate whether any of the three paired TCSs (ArsRS, FleRS and CrdRS) in H. pylori respond to nitrosative stress. The results showed that the expression of crdS was significantly increased upon exposure to nitric oxide (NO). crdS-knockout (ΔcrdS) and crdR/crdS-knockout (ΔcrdRS) H. pylori, but not arsS-knockout (ΔarsS) or fleS-knockout (ΔfleS) H. pylori, showed a significant loss of viability upon exposure to NO compared with wild-type strain. Knockin crdS (ΔcrdS-in) significantly restored viability in the presence of NO. Global transcriptional profiling analysis of wild-type and ΔcrdS H. pylori in the presence or absence of NO showed that 101 genes were differentially expressed, including copper resistance determinant A (crdA), transport, binding and envelope proteins. The CrdR binding motifs were investigated by competitive electrophoretic mobility shift assay, which revealed that the two AC-rich regions in the crdA promoter region are required for binding. These results demonstrate that CrdR-crdA interaction enables H. pylori to survive under nitrosative stress.

Increased numbers of Foxp3-positive regulatory T cells in gastritis, peptic ulcer and gastric adenocarcinoma.

AIM: To determine the number of regulatory T cells (Tregs) in gastric mucosa of patients with gastritis, peptic ulcers and gastric cancer. METHODS: This study was a retrospective analysis of gastric antrum biopsy specimens from healthy controls (n = 22) and patients with gastritis (n = 30), peptic ulcer (n = 83), or gastric cancer (n = 32). Expression of CD4, CD25 and Foxp3 was determined by immunohistochemistry in three consecutive sections per sample. RESULTS: Compared with healthy controls, there was an increased number of CD25(+) and Foxp3(+) cells in patients with gastritis (P = 0.004 and P = 0.008), peptic ulcer (P < 0.001 and P < 0.001), and gastric cancer (P < 0.001 and P < 0.001). The ratio of CD25(+)/CD4(+) or Foxp3(+)/CD4(+) cells was also significantly higher in all disease groups (P < 0.001, respectively). The number of CD4(+), CD25(+), and Foxp3(+) cells, and the ratio of CD25(+)/CD4(+) and Foxp3(+)/CD4(+) cells, were associated with the histological grade of the specimens, including acute inflammation, chronic inflammation, lymphoid follicle number, and Helicobacter pylori infection. The number of CD4(+), CD25(+) and Foxp3(+) cells, and the ratio of CD25(+)/CD4(+) and Foxp3(+)/CD4(+) cells, were negatively associated with intestinal metaplasia among gastritis (P < 0.001, P < 0.001, P < 0.001, P = 0.002 and P = 0.002) and peptic ulcer groups (P = 0.013, P = 0.004, P < 0.001, P = 0.040 and P = 0.003). CONCLUSION: Tregs are positively associated with endoscopic findings of gastroduodenal diseases and histological grade but negatively associated with intestinal metaplasia in gastritis and peptic ulcer groups.

Helicobacter pylori cholesteryl glucosides interfere with host membrane phase and affect type IV secretion system function during infection in AGS cells.

Helicobacter pylori infection is an aetiological cause of gastric disorders worldwide. H. pylori has been shown to assimilate and convert host cholesterol into cholesteryl glucosides (CGs) by cholesterol-α-glucosyltransferase encoded by capJ. Here, we show that CapJ-deficient (ΔcapJ) H. pylori resulted in greatly reduced type IV secretion system (TFSS)-associated activities, including the hummingbird phenotype of AGS cells, IL-8 production, CagA translocation/phosphorylation and CagA-mediated signalling events. Complementation of the ΔcapJ mutation with wild type cagJ or by adding CGs-containing lysates or exogenous fluorophore-tagged CGs reversed the mutant phenotypes. We also show that the wild-type but not ΔcapJ H. pylori recruited raft-associated components to sites of bacterial attachment. Fluorescence recovery after photobleaching (FRAP) analysis of AGS cells treated with fluorescence-tagged cholesterol/CGs revealed that there was a higher proportion of CGs associated with immobile fractions. CGs-associated membranes were also more resistant to a cold detergent extraction. Thus, we propose that CGs synthesized by H. pylori around host-pathogen contact sites partition in detergent-resistant membranes (DRMs), alters lateral-phase segregation in membrane and reorganizes membrane architecture. These processes together promote the formation of a functional TFSS and H. pylori infection.

Effects of the tip shape on the localized field enhancement and far field radiation pattern of the plasmonic inverted pyramidal nanostructures with the tips for surface-enhanced Raman scattering.

The plasmonic 2D W-shape and 3D inverted pyramidal nanostructures with and without the tips are studied. The effects of the tip height and tip tilt angle on the near field enhancement and far field radiation pattern are discussed in this paper. The localized hot spots are found around the pits and the radiation pattern can be affected by the tip structures. The inverted pyramidal nanostructures with and without the tips were fabricated and their reflection spectra and surface-enhanced Raman scattering (SERS) signals for the chemical molecules thiophenol were measured. The simulation according to the geometry parameters of the fabricated structures is demonstrated. We found that the SERS of our proposed structures with the tips can have stronger light field enhancements than the inverted pyramidal nanostructures without the tips, and the far field radiation pattern can be varied by changing the tip height and tip tilt angle. The study of surface plasmon modes and charge distributions can help the understanding of how to arrange the plasmonic structures to achieve high field enhancement and preferred far field radiation pattern. Our study can be useful for the design of the strong field enhancement SERS substrate with specific far field radiation properties. It can be also applied to the portable Raman detectors for in situ and remote measurements in specific applications.

Interleukin-1beta and -10 polymorphisms influence erosive reflux esophagitis and gastritis in Taiwanese patients.

BACKGROUND AND AIMS: Helicobacter pylori (H. pylori) infection induces cytokine production and is associated with gastrointestinal diseases. This study examined the relationship of gene polymorphisms, including interleukin (IL)-1beta, -10, -8, and tumor necrosis factor-alpha (TNF-alpha), H. pylori infection, and susceptibility to gastrointestinal disorders in Taiwanese patients. METHODS: IL-1beta-511/-31/+3953, -10-1082/-819/-592, -8-251, and TNF-alpha-308 polymorphisms were assessed in 628 gastrointestinal disease patients, and 176 healthy controls were analyzed using the polymerase chain reaction-restriction fragment length polymorphism method. RESULTS: IL-1beta-511 T/T and -31 C/C genotypes, and IL-1beta-511 T and -31 C alleles were associated with an increased risk of reflux esophagitis (P = 0.034, odds ratio [OR] = 1.384, 95% confidence interval [CI]: 1.023-1.871; P = 0.031, OR = 1.388, 95% CI: 1.028-1.873; P = 0.044, OR = 1.342, 95% CI: 1.008-1.786; and P = 0.040, OR = 1.349, 95% CI: 1.014-1.796, respectively). No relationship was found between H. pylori infection and the risk of reflux esophagitis. IL-10-819 C/T and -10-592 A/C genotypes and IL-10-1082/-819/-592 ATA/ACC and ATA/GCC haplotypes were associated with an increased risk of gastritis (P = 0.021, OR = 1.721, 95% CI: 1.084-2.733; P = 0.016, OR = 1.766, 95% CI: 1.112-2.805; P = 0.039, OR = 1.662, 95% CI: 1.024-2.697; and P = 0.035, OR = 1.600, 95% CI: 1.024-2.499, respectively). CONCLUSION: Among Taiwanese patients, IL-1beta and -10 polymorphisms were associated with an increased risk of erosive reflux esophagitis and gastritis, respectively.