PTPN2 regulates the activation of KRAS and plays a critical role in proliferation and survival of KRAS-driven cancer cells.

RAS genes are the most commonly mutated in human cancers and play critical roles in tumor initiation, progression, and drug resistance. Identification of targets that block RAS signaling is pivotal to develop therapies for RAS-related cancer. As RAS translocation to the plasma membrane (PM) is essential for its effective signal transduction, we devised a high-content screening assay to search for genes regulating KRAS membrane association. We found that the tyrosine phosphatase PTPN2 regulates the plasma membrane localization of KRAS. Knockdown of PTPN2 reduced the proliferation and promoted apoptosis in KRAS-dependent cancer cells, but not in KRAS-independent cells. Mechanistically, PTPN2 negatively regulates tyrosine phosphorylation of KRAS, which, in turn, affects the activation KRAS and its downstream signaling. Consistently, analysis of the TCGA database demonstrates that high expression of PTPN2 is significantly associated with poor prognosis of patients with KRAS-mutant pancreatic adenocarcinoma. These results indicate that PTPN2 is a key regulator of KRAS and may serve as a new target for therapy of KRAS-driven cancer.

Evaluation of inhibition effect on microbiologically influenced corrosion of Ti-5Cu alloy against marine Bacillus vietnamensis biofilm.

Titanium is highly susceptible to biofouling due to the poor toxicity and excellent biocompatibility. Microbiologically influenced corrosion of cp-Ti induced by Bacillus vietnamensis and the inhibition effect of Ti-5Cu alloy were evaluated using various electrochemical techniques and surface analyses. Electrochemical results revealed that, cp-Ti and Ti-5Cu alloy exhibited excellent passivation in the sterile medium. In biotic medium, however, attachment of B. vietnamensis biofilm to surface of cp-Ti accelerated the active dissolution and poisons repassivation. In contrast, cells adhered preferentially as a form of discrete colonies on Ti2Cu intermetallic phases. Release of Cu2+ ions induced damage in morphology of B. vietnamensis cells resulting in relative resistance to pitting corrosion estimated by 3.1 ± 0.6 µm and 2.0 ± 0.4 µm for cp-Ti and Ti-5Cu alloy, respectively.

A novel microtubule inhibitor promotes tumor ferroptosis by attenuating SLC7A11/GPX4 signaling.

MP-HJ-1b is a novel microtubule inhibitor that we designed and reported previously. Ferroptosis is a newly identified type of nonapoptotic cell death induced by ferrous catalysis and lipid peroxidation. Here, transcriptomics, proteomics, and molecular docking analyses were combined to explore the novel effects of MP-HJ-1b on tumors. Both omics analyses suggested that MP-HJ-1b affects ribosomes, and we confirmed that it inhibits the ribosomal component proteins RPL35 and MRPL28. Colchicine was used as an analog, and the results showed that MP-HJ-1b and colchicine increased reactive oxygen species and malondialdehyde levels and decreased reduced glutathione levels, suggesting that they promoted ferroptosis in HeLa cells. Specifically, MP-HJ-1b downregulated SLC7A11 and GPX4 to enhance the classical pathway of ferroptosis, while colchicine upregulated LC3A/B-II and enhanced autophagy. Clinically, the serum concentrations of ferrous ions, reduced glutathione, and Hcy were higher in cervical cancer patients than in healthy individuals. ALT, AST, Cho, HDL-C, and LDL-C levels were decreased in the serum of patients. Our study expands understanding of the way MP-HJ-1b promotes cell death and enriches research on microtubule inhibitors in the ferroptosis field.

Microstructure Evolution and Deformation Mechanisms of As-Cast Antibacterial Ti6Al4V-5Cu Alloy for Isothermal Forging Process.

The hot workability behavior of antibacterial Ti6Al4V-5Cu alloy was investigated using a hot compression experiment in the temperature range of 790-1040 °C and strain rate of 10-3-10 s-1 with a strain of 0.4. The deformation behavior of the alloy was characterized by Gleeble 3800 compression experiment, and the relationship among deformed microstructures and deformation parameters was established. The deformations of Ti6Al4V-5Cu alloy were temperature and strain rate-dependent. Higher temperature and lower strain rate made power dissipation efficiency (η) increase and reach 89%. The activation energies (Q) in the dual-phase (α + ß) and single ß phase regions were calculated as 175.43 and 159.03 kJ mol-1, respectively. In the dual (α + ß) phase region, with an increase in strain rate, flow-softening behavior was dominated, however in the single ß phase region such as processing at 940 °C. Flow stress increased slightly in which work-hardening behavior was dominated (especially between strain rates of 10-3-1 s-1). The deformation at various conditions exhibited different stress-strain profiles, providing an insight that work hardening and flow softening coexisted in Ti6Al4V-5Cu alloy. The relative intensity of oscillatory change in flow stress profile decreased as the strain rate decreased. The hot workability of Ti6Al4V-5Cu alloy was also accessed from the viewpoint of the sub-grain structure.

Palmitoylation of GNAQ/11 is critical for tumor cell proliferation and survival in GNAQ/11-mutant uveal melanoma.

More than 85% of patients with uveal melanoma (UM) carry a GNAQ or GNA11 mutation at a hotspot codon (Q209) that encodes G protein α subunit q/11 polypeptides (Gαq/11). GNAQ/11 relies on palmitoylation for membrane association and signal transduction. Despite the palmitoylation of GNAQ/11 was discovered long before, its implication in UM remains unclear. Here, results of palmitoylation-targeted mutagenesis and chemical interference approaches revealed that the loss of GNAQ/11 palmitoylation substantially affected tumor cell proliferation and survival in UM cells. Palmitoylation inhibition through the mutation of palmitoylation sites suppressed GNAQ/11Q209L-induced malignant transformation in NIH3T3 cells. Importantly, the palmitoylation-deficient oncogenic GNAQ/11 failed to rescue the cell death initiated by the knock down of endogenous GNAQ/11 oncogenes in UM cells, which are much more dependent on Gαq/11 signaling for cell survival and proliferation than other melanoma cells without GNAQ/11 mutations. Furthermore, the palmitoylation inhibitor, 2-bromopalmitate, also specifically disrupted Gαq/11 downstream signaling by interfering with the MAPK pathway and BCL2 survival pathway in GNAQ/11-mutant UM cells and showed a notable synergistic effect when applied in combination with the BCL2 inhibitor, ABT-199, in vitro. The findings validate that GNAQ/11 palmitoylation plays a critical role in UM and may serve as a promising therapeutic target for GNAQ/11-driven UM.

DDB1- and CUL4-associated factor 8 plays a critical role in spermatogenesis.

Cullin-RING E3 ubiquitin ligase (CRL)-4 is a member of the large CRL family in eukaryotes. It plays important roles in a wide range of cellular processes, organismal development, and physiological and pathological conditions. DDB1- and CUL4-associated factor 8 (DCAF8) is a WD40 repeat-containing protein, which serves as a substrate receptor for CRL4. The physiological role of DCAF8 is unknown. In this study, we constructed Dcaf8 knockout mice. Homozygous mice were viable with no noticeable abnormalities. However, the fertility of Dcaf8-deficient male mice was markedly impaired, consistent with the high expression of DCAF8 in adult mouse testis. Sperm movement characteristics, including progressive motility, path velocity, progressive velocity, and track speed, were significantly lower in Dcaf8 knockout mice than in wild-type (WT) mice. However, the total motility was similar between WT and Dcaf8 knockout sperm. More than 40% of spermatids in Dcaf8 knockout mice showed pronounced morphological abnormalities with typical bent head malformation. The acrosome and nucleus of Dcaf8 knockout sperm looked similar to those of WT sperm. In vitro tests showed that the fertilization rate of Dcaf8 knockout mice was significantly reduced. The results demonstrated that DCAF8 plays a critical role in spermatogenesis, and DCAF8 is a key component of CRL4 function in the reproductive system.

GNA13 regulates BCL2 expression and the sensitivity of GCB-DLBCL cells to BCL2 inhibitors in a palmitoylation-dependent manner.

GNA13, encoding one of the G protein alpha subunits of heterotrimeric G proteins that transduce signals of G protein-coupled receptors (GPCR), is frequently mutated in germinal center B-cell-like diffuse large B-cell lymphoma (GCB-DLBCL) with poor prognostic outcomes. Due to the "undruggable" nature of GNA13, targeted therapy for these patients is not available. In this study, we found that palmitoylation of GNA13 not only regulates its plasma membrane localization, but also regulates GNA13's stability. It is essential for the tumor suppressor function of GNA13 in GCB-DLBCL cells. Interestingly, GNA13 negatively regulates BCL2 expression in GCB-DLBCL cells in a palmitoylation-dependent manner. Consistently, BCL2 inhibitors were found to be effective in killing GNA13-deficient GCB-DLBCL cells in a cell-based chemical screen. Furthermore, we demonstrate that inactivating GNA13 by targeting its palmitoylation enhanced the sensitivity of GCB-DLBCL to the BCL2 inhibitor. These studies indicate that the loss-of-function mutation of GNA13 is a biomarker for BCL2 inhibitor therapy of GCB-DLBCL and that GNA13 palmitoylation is a potential target for combination therapy with BCL2 inhibitors to treat GCB-DLBCL with wild-type GNA13.

IRF8 Impacts Self-Renewal of Hematopoietic Stem Cells by Regulating TLR9 Signaling Pathway of Innate Immune Cells.

IRF8 is a key regulator of innate immunity receptor signaling and plays diverse functions in the development of hematopoietic cells. The effects of IRF8 on hematopoietic stem cells (HSCs) are still unknown. Here, it is demonstrated that IRF8 deficiency results in a decreased number of long-term HSCs (LT-HSCs) in mice. However, the repopulation capacity of individual HSCs is significantly increased. Transcriptomic analysis shows that IFN-γ and IFN-α signaling is downregulated in IRF8-deficient HSCs, while their response to proinflammatory cytokines is unchanged ex vivo. Further tests show that Irf8-/- HSCs can not respond to CpG, an agonist of Toll-like receptor 9 (TLR9) in mice, while long-term CpG stimulation increases wild-type HSC abundance and decreases their bone marrow colony-forming capacity. Mechanistically, as the primary producer of proinflammatory cytokines in response to CpG stimulation, dendritic cells has a blocked TLR9 signaling due to developmental defect in Irf8-/- mice. Macrophages remain functionally intact but severely reduce in Irf8-/- mice. In NK cells, IRF8 directly regulates the expression of Tlr9 and its deficiency leads to no increased IFNγ production upon CpG stimulation. These results indicate that IRF8 regulates HSCs, at least in part, through controlling TLR9 signaling in diverse innate immune cells.

Combination therapy of BCR-ABL-positive B cell acute lymphoblastic leukemia by tyrosine kinase inhibitor dasatinib and c-JUN N-terminal kinase inhibition.

BACKGROUND: The Philadelphia chromosome (Ph), which leads to the creation and expression of the fusion gene product BCR-ABL, underlines the pathogenesis of chronic myelogenous leukemia (CML) and a fraction of adult and pediatric acute B-lymphoblastic leukemia (B-ALL). The BCR-ABL tyrosine kinase inhibitors (TKIs) have shown a remarkable clinical activity in patients with CML, but their efficacy in treating Ph+ B-ALL is limited. Identifying additional therapeutic targets is important for the effective treatment of Ph+ B-ALL. METHODS: Activation of the JNK signaling pathway in human and mouse BCR-ABL+ B-ALL cells with or without dasatinib treatment was analyzed by Western blotting. JNK was inhibited either by RNA interference or chemical inhibitors, such as JNK-IN-8. The effect of JNK inhibition with or without BCR-ABL TKI dasatinib on BCR-ABL+ B-ALL cells was analyzed by the CellTiter-Glo® Luminescent Cell Viability Assay. The in vivo effects of JNK-IN-8 and dasatinib alone or in combination were tested using a BCR-ABL induced B-ALL mouse model. RESULTS: We found that the c-JUN N-terminal kinase (JNK) signaling pathway is abnormally activated in both human and mouse BCR-ABL+ B-ALL cells, but the BCR-ABL TKI does not inhibit JNK activation in these cells. Inhibition of JNK, either by RNAi-mediated downregulation or by JNK inhibitors, could significantly reduce viability of Ph+ B-ALL cells. JNK inhibition by RNAi-mediated downregulation or JNK inhibitors also showed a synergistic effect with the BCR-ABL TKI, dasatinib, in killing Ph+ B-ALL cells in vitro. Furthermore, a potent JNK inhibitor, JNK-IN-8, in combination with dasatinib markedly improved the survival of mice with BCR-ABL induced B-ALL, as compared to the treatment with dasatinib alone. CONCLUSIONS: Our findings indicate that simultaneously targeting both BCR-ABL and JNK kinase might serve as a promising therapeutic strategy for Ph+ B-ALL.