Lactobacillus plantarum alleviates irradiation-induced intestinal injury by activation of FXR-FGF15 signaling in intestinal epithelia.

Abdominal irradiation (IR) may destroy the intestinal mucosal barrier, leading to severe intestinal infection and multiple organ dysfunction syndromes. The role of intestinal microbiota in the development of IR-induced intestinal injury remains largely unknown. Herein, we reported that abdominal IR altered the composition of the microbiota and reduced the abundance and diversity of the gut microbiome. Alterations of bacteria, in particular reduction of Lactobacillus, played a critical role in IR-induced intestinal injury. Fecal microbiota transplant (FMT) from normal mice or administration of Lactobacillus plantarum to intestinal microbiota-eliminated mice substantially reduced IR-induced intestinal damage and prevented mice from IR-induced death. We further characterized that L. plantarum activated the farnesoid X receptor (FXR) - fibroblast growth factor 15 (FGF15) signaling in intestinal epithelial cells and hence promoted DNA-damage repair. Application of GW4064, an activator of FXR, to microbiota eliminated mice markedly mitigated IR-induced intestinal damage, reduced intestinal epithelial cell death and promoted the survival of IR mice. In contrast, suppression of FXR with Gly-ß-MCA, a bile acid and an intestine-selective and high-affinity FXR inhibitor, abrogated L. Plantarum-mediated protection on the ileum of IR mice. Taken together, our findings not only provide new insights into the role of intestinal flora in radiation-induced intestinal injury but also shed new light on the application of probiotics for the protection of radiation-damaged individuals.

Gain-of-function mutant p53 promotes cell growth and cancer cell metabolism via inhibition of AMPK activation.

Many mutant p53 proteins (mutp53s) exert oncogenic gain-of-function (GOF) properties, but the mechanisms mediating these functions remain poorly defined. We show here that GOF mutp53s inhibit AMP-activated protein kinase (AMPK) signaling in head and neck cancer cells. Conversely, downregulation of GOF mutp53s enhances AMPK activation under energy stress, decreasing the activity of the anabolic factors acetyl-CoA carboxylase and ribosomal protein S6 and inhibiting aerobic glycolytic potential and invasive cell growth. Under conditions of energy stress, GOF mutp53s, but not wild-type p53, preferentially bind to the AMPKα subunit and inhibit AMPK activation. Given the importance of AMPK as an energy sensor and tumor suppressor that inhibits anabolic metabolism, our findings reveal that direct inhibition of AMPK activation is an important mechanism through which mutp53s can gain oncogenic function.

Shortened ex vivo manufacturing time of EGFRvIII-specific chimeric antigen receptor (CAR) T cells reduces immune exhaustion and enhances antiglioma therapeutic function.

BACKGROUND: Non-viral manufacturing of CAR T cells via the Sleeping Beauty transposon is cost effective and reduces the risk of insertional mutagenesis from viral transduction. However, the current gold standard methodology requires ex vivo numerical expansion of these cells on artificial antigen-presenting cells (AaPCs) for 4 weeks to generate CAR T cells of presumed sufficient quantity and function for clinical applications. METHOD: We engineered EGFRvIII-specific CAR T cells and monitored phenotypic changes throughout their ex vivo manufacturing. To reduce the culture time required to generate the CAR T-cell population, we selected for T cells in peripheral blood mononuclear cells prior to CAR modification (to eliminate the competing NK cell population). RESULTS: While we found increased expression of exhaustion markers (such as PD-1, PD-L1, TIM-3, and LAG-3) after 2 weeks in culture, whose levels continued to rise over time, we were able to generate a CAR+ T-cell population with comparable CAR expression and cell numbers in 2 weeks, thereby reducing manufacturing time by 50%, with lower expression of immune exhaustion markers. The CAR T cells manufactured at 2 weeks showed superior therapeutic efficacy in mice bearing established orthotopic EGFRvIII+ U87 gliomas. CONCLUSION: These findings demonstrate a novel, rapid method to generate CAR T cells by non-viral modification that results in CAR T cells superior in phenotype and function and further emphasizes that careful monitoring of CAR T-cell phenotype prior to infusion is critical for generating an optimal CAR T-cell product with full antitumor potential.

O-linked GlcNAcylation elevated by HPV E6 mediates viral oncogenesis.

High-risk human papillomaviruses (HPVs) are causative agents of anogenital cancers and a fraction of head and neck cancers. The mechanisms involved in the progression of HPV neoplasias to cancers remain largely unknown. Here, we report that O-linked GlcNAcylation (O-GlcNAc) and O-GlcNAc transferase (OGT) were markedly increased in HPV-caused cervical neoplasms relative to normal cervix, whereas O-GlcNAcase (OGA) levels were not altered. Transduction of HPV16 oncogene E6 or E6/E7 into mouse embryonic fibroblasts (MEFs) up-regulated OGT mRNA and protein, elevated the level of O-GlcNAc, and promoted cell proliferation while reducing cellular senescence. Conversely, in HPV-18-transformed HeLa cervical carcinoma cells, inhibition of O-GlcNAc with a low concentration of a chemical inhibitor impaired the transformed phenotypes in vitro. We showed that E6 elevated c-MYC via increased protein stability attributable to O-GlcNAcylation on Thr58. Reduction of HPV-mediated cell viability by a high concentration of O-GlcNAc inhibitor was partially rescued by elevated c-MYC. Finally, knockdown of OGT or O-GlcNAc inhibition in HeLa cells or in TC-1 cells, a mouse cell line transformed by HPV16 E6/E7 and activated K-RAS, reduced c-MYC and suppressed tumorigenesis and metastasis. Thus, we have uncovered a mechanism for HPV oncoprotein-mediated transformation. These findings may eventually aid in the development of effective therapeutics for HPV-associated malignancies by targeting aberrant O-GlcNAc.

AKT isoform-specific expression and activation across cancer lineages.

BACKGROUND: Aberrant AKT activation is prevalent across human cancer lineages, providing an important therapeutic target. AKT comprises three isoforms that mediate critical non-redundant, even opposing functions in cancer pathophysiology. Therefore, targeting specific AKT isoforms in particular cancers may be more effective than pan-AKT inhibition while avoiding disadvantages of pan-AKT inhibition. Currently, AKT isoform-specific expression and activation in cancer are not clearly characterized. METHODS: We systematically characterized AKT isoform-specific expression and activation in 211 cancer cell lines derived from different lineages and genetic backgrounds using a reverse-phase protein array platform. RESULTS: We found that phosphorylation, but not expression, of AKT1 and AKT2 was coordinated in most but not all cells. Different cancer lineages displayed differential AKT1 and AKT2 expression and phosphorylation. A PIK3CA hotspot mutation H1047R but not E545K was associated with selective activation of AKT2 but not AKT1. CONCLUSIONS: Our study identified and validated AKT isoform-specific expression and phosphorylation in certain cell lines and demonstrated that genetic changes can affect AKT isoform-specific activation. These results provide a more precise understanding of AKT isoform-specific signaling and, in addition, facilitate AKT isoform targeting for personalized cancer therapies.

Densification of chlorine-doped continuous CNT sheet/polyvinylidene fluoride sandwich film and improvement of the mechanical and dielectric properties.

The compact structure of a chlorine-doped continuous CNT sheet/polyvinylidene fluoride (Cl-CNT sheet/PVDF) was successfully optimized by means of a hot-press treatment to improve the mechanical and dielectric properties with a high densification degree. Then, the densified Cl-CNT sheet/PVDF dielectric layer was inserted between two PVDF insulating layers to fabricate a sandwich composite. It was found that the dielectric and mechanical properties were effectively enhanced, with a dielectric permittivity of 40.4 (@102 Hz), a dielectric loss of 0.16 (@102 Hz), a tensile strength of 139 MPa, and a tensile modulus of 4.4 GPa under a hot-pressing pressure of 20 MPa. Furthermore, the densified Cl-CNT sheet/PVDF was used as an electrode in a multilayer sandwich composite film, and good performance was obtained. The improvement mechanism was discussed and the studied CNT composite and other dielectric composites were compared. It demonstrates great potential for applications in dielectric and electrode materials to achieve structural and functional integration.

MIR106B and MIR93 prevent removal of bacteria from epithelial cells by disrupting ATG16L1-mediated autophagy.

BACKGROUND & AIMS: Variants in genes that regulate autophagy have been associated with Crohn's disease (CD). Defects in autophagy-mediated removal of pathogenic microbes could contribute to the pathogenesis of CD. We investigated the role of the microRNAs (miRs) MIR106B and MIR93 in induction of autophagy and bacterial clearance in human cell lines and the correlation between MIR106B and autophagy-related gene 16L1 (ATG16L1) expression in tissues from patients with CD. METHODS: We studied the ability of MIR106B and MIR93 to regulate ATG transcripts in human cancer cell lines (HCT116, SW480, HeLa, and U2OS) using luciferase report assays and bioinformatics analyses; MIR106B and MIR93 mimics and antagonists were transfected into cells to modify levels of miRs. Cells were infected with LF82, a CD-associated adherent-invasive strain of Escherichia coli, and monitored by confocal microscopy and for colony-forming units. Colon tissues from 41 healthy subjects (controls), 22 patients with active CD, 16 patients with inactive CD, and 7 patients with chronic inflammation were assessed for levels of MIR106B and ATG16L1 by in situ hybridization and immunohistochemistry. RESULTS: Silencing Dicer1, an essential processor of miRs, increased levels of ATG protein and formation of autophagosomes in cells, indicating that miRs regulate autophagy. Luciferase reporter assays indicated that MIR106B and MIR93 targeted ATG16L1 messenger RNA. MIR106B and MIR93 reduced levels of ATG16L1 and autophagy; these increased after expression of ectopic ATG16L1. In contrast, MIR106B and MIR93 antagonists increased formation of autophagosomes. Levels of MIR106B were increased in intestinal epithelia from patients with active CD, whereas levels of ATG16L1 were reduced compared with controls. Levels of c-Myc were also increased in intestinal epithelia of patients with active CD compared with controls. These alterations could impair removal of CD-associated bacteria by autophagy. CONCLUSIONS: In human cell lines, MIR106B and MIR93 reduce levels of ATG16L1 and autophagy and prevent autophagy-dependent eradication of intracellular bacteria. This process also appears to be altered in colon tissues from patients with active CD.

Tat-activating regulatory DNA-binding protein regulates glycolysis in hepatocellular carcinoma by regulating the platelet isoform of phosphofructokinase through microRNA 520.

UNLABELLED: Metabolic changes are common features of many cancer cells and are frequently associated with the clinical outcome of patients with various cancers, including hepatocellular carcinoma (HCC). Thus, aberrant metabolic pathways in cancer cells are attractive targets for cancer therapy. However, our understanding of cancer-specific regulatory mechanisms of cell metabolism is still very limited. We found that Tat-activating regulatory DNA-binding protein (TARDBP) is a novel regulator of glycolysis in HCC cells. TARDBP regulates expression of the platelet isoform of phosphofructokinase (PFKP), the rate-limiting enzyme of glycolysis that catalyzes the irreversible conversion of fructose-6-phosphate to fructose-1,6-bisphosphate. Silencing of TARDBP expression in multiple HCC cell lines leads to impaired glucose metabolism and inhibition of in vitro and in vivo growth of HCC cells. Notably, the microRNA 520 (miR-520) family is an intermediate regulator of TARDBP-mediated regulation of glycolysis. Mechanistically, TARDBP suppressed expression of the miR-520 family, which, in turn, inhibited expression of PFKP. We further showed that expression of TARDBP is significantly associated with the overall survival of patients with HCC. CONCLUSION: Our study provides new mechanistic insights into the regulation of glycolysis in HCC cells and reveals TARDBP as a potential therapeutic target for HCC.

The energy sensing LKB1-AMPK pathway regulates p27(kip1) phosphorylation mediating the decision to enter autophagy or apoptosis.

Nutrients and bioenergetics are prerequisites for proliferation and survival of mammalian cells. We present evidence that the cyclin-dependent kinase inhibitor p27(Kip1), is phosphorylated at Thr 198 downstream of the Peutz-Jeghers syndrome protein-AMP-activated protein kinase (LKB1-AMPK) energy-sensing pathway, thereby increasing p27 stability and directly linking sensing of nutrient concentration and bioenergetics to cell-cycle progression. Ectopic expression of wild-type and phosphomimetic Thr 198 to Asp 198 (T198D), but not unstable Thr 198 to Ala 198 (p27(T198A)) is sufficient to induce autophagy. Under stress conditions that activate the LKB1-AMPK pathway with subsequent induction of autophagy, p27 knockdown results in apoptosis. Thus LKB1-AMPK pathway-dependent phosphorylation of p27 at Thr 198 stabilizes p27 and permits cells to survive growth factor withdrawal and metabolic stress through autophagy. This may contribute to tumour-cell survival under conditions of growth factor deprivation, disrupted nutrient and energy metabolism, or during stress of chemotherapy.

BRIT1 regulates early DNA damage response, chromosomal integrity, and cancer.

BRIT1, initially identified as an hTERT repressor, has additional functions at DNA damage checkpoints. Here, we demonstrate that BRIT1 formed nuclear foci minutes after irradiation. The foci of BRIT1 colocalized with 53BP1, MDC1, NBS1, ATM, RPA, and ATR. BRIT1 was required for activation of these elements, indicating that BRIT1 is a proximal factor in the DNA damage response pathway. Depletion of BRIT1 increased the accumulation of chromosomal aberrations. In addition, decreased levels of BRIT1 were detected in several types of human cancer, with BRIT1 expression being inversely correlated with genomic instability and metastasis. These results identify BRIT1 as a crucial DNA damage regulator in the ATM/ATR pathways and suggest that it functions as a tumor suppressor gene.

Tumor microenvironment in glioblastoma: Current and emerging concepts.

Glioblastoma (GBM) tumor microenvironment (TME) is a highly heterogeneous and complex system, which in addition to cancer cells, consists of various resident brain and immune cells as well as cells in transit through the tumor such as marrow-derived immune cells. The TME is a dynamic environment which is heavily influenced by alterations in cellular composition, cell-to-cell contact and cellular metabolic products as well as other chemical factors, such as pH and oxygen levels. Emerging evidence suggests that GBM cells appear to reprogram their the TME, and hijack microenvironmental elements to facilitate rapid proliferation, invasion, migration, and survival thus generating treatment resistance. GBM cells interact with their microenvironment directly through cell-to-cell by interaction mediated by cell-surface molecules, or indirectly through apocrine or paracrine signaling via cytokines, growth factors, and extracellular vehicles. The recent discovery of neuron-glioma interfaces and neurotransmitter-based interactions has uncovered novel mechanisms that favor tumor cell survival and growth. Here, we review the known and emerging evidence related to the communication between GBM cells and various components of its TME, discuss models for studying the TME and outline current studies targeting components of the TME for therapeutic purposes.

A Case Study of Chimeric Antigen Receptor T Cell Function: Donor Therapeutic Differences in Activity and Modulation with Verteporfin.

BACKGROUND: Chimeric antigen receptor (CAR) T cells have recently been demonstrated to extract and express cognate tumor antigens through trogocytosis. This process may contribute to tumor antigen escape, T cell exhaustion, and fratricide, which plays a central role in CAR dysfunction. We sought to evaluate the importance of this effect in epidermal growth factor receptor variant III (EGFRvIII) specific CAR T cells targeting glioma. METHODS: EGFRvIII-specific CAR T cells were generated from various donors and analyzed for cytotoxicity, trogocytosis, and in vivo therapeutic activity against intracranial glioma. Tumor autophagy resulting from CAR T cell activity was evaluated in combination with an autophagy inducer (verteporfin) or inhibitor (bafilomycin A1). RESULTS: CAR T cell products derived from different donors induced markedly divergent levels of trogocytosis of tumor antigen as well as PD-L1 upon engaging target tumor cells correlating with variability in efficacy in mice. Pharmacological facilitation of CAR induced-autophagy with verteporfin inhibits trogocytic expression of tumor antigen on CARs and increases CAR persistence and efficacy in mice. CONCLUSION: These data propose CAR-induced autophagy as a mechanism counteracting CAR-induced trogocytosis and provide a new strategy to innovate high-performance CARs through pharmacological facilitation of T cell-induced tumor death.

Metformin impairs the growth of liver kinase B1-intact cervical cancer cells.

OBJECTIVE: Metformin is one of the most widely used drugs for the treatment of type 2 diabetes. Recent investigations demonstrated that application of metformin reduces cancer risk. The present study aimed to determine the role of liver kinase B1 (LKB1) in the response of cervical cancer cells to metformin. METHODS: LKB1 expression and the integrity of LKB1-AMPK signaling were determined with immunoblot in 6 cervical cancer cell lines. Cellular sensitivity to metformin was analyzed with MTT assay. RESULTS: Metformin inhibited growth of cervical cancer cells, C33A, Me180, and CaSki, but was less effective against HeLa, HT-3, and MS751 cells. Analyzing the expression status and the integrity of LKB1-AMPK-mTOR signaling, we found that cervical cancer cells sensitive to metformin were LKB1 intact and exerted an integral AMPK-mTOR signaling response after the treatment. Ectopic expression of LKB1 with stable transduction system or inducible expression construct in endogenous LKB1 deficient cells improved the activation of AMPK, promoted the inhibition of mTOR, and prompted the sensitivity of cells to metformin. In contrast, knock-down of LKB1 compromised cellular response to metformin. Our further investigation demonstrated that metformin could induce both apoptosis and autophagy in cervical cancer cells when LKB1 is expressed. CONCLUSIONS: Metformin is a potential drug for the treatment of cervical cancers, in particular to those with intact LKB1 expression. Administration of cell metabolism agonists may enhance LKB1 tumor suppression, inhibit cell growth, and reduce tumor cell viability via the activation of LKB1-AMPK signaling.

Lactobacillus Suppresses Tumorigenesis of Oropharyngeal Cancer via Enhancing Anti-Tumor Immune Response.

Deficiency in T cell-mediated adaptive immunity, such as low CD8+ T cell infiltration, inhibits the immune surveillance, promotes malignant transformation, and facilitates tumor growth. Microbiota dysbiosis diminishes the immune system and contributes to the occurrence of cancer. However, the impact of oral dysbiosis on the occurrence and molecular mechanisms of oropharyngeal cancer (OPC) remains largely unknown. In the current study, we used 4-nitroquinoline-1-oxide (4NQO) to mimic tobacco-related carcinogenesis to generate a murine OPC model and determine the role of microbiota changes in OPC tumorigenesis. Our results showed that the oral flora composition of mice was deregulated during the tumorigenesis of OPC. The abundance of Streptococcus, Veillonella, Muribacter, Rodentibacter, and Gemella was increased, whereas the dominant genus Lactobacillus was gradually decreased with disease progression. We further demonstrated that infiltration of CD8+ T lymphocytes was markedly reduced due to the reduction of Lactobacillus. Supplementation of Lactobacillus increased the infiltration of CD8+ T cells, promoted the expression of IFN-γ and granzyme B, and lessened the OPC progression. Analyzing the metabolites of the Lactobacillus, we demonstrated that Lactobacillus enhanced the anti-tumor immune response by producing acetate in OPC development. Administration of acetate to mice could increase the expression of IFN-γ and IFN-γ-inducible chemokines in tumor tissues by activating GPR43 to promote the infiltration of CD8+ T lymphocytes and substantially delay the development of OPC. Together, our data suggest that dysbiosis of oral microbiota promotes the tumorigenesis of OPC through downregulation of cytotoxic T lymphocytes. Lactobacillus and its metabolite acetate improve the tumor microenvironment, which could be applied in the treatment of OPC.

PARP1 inhibitors attenuate AKT phosphorylation via the upregulation of PHLPP1.

Poly(ADP-ribose) polymerase-1 (PARP1) inhibitors are emerging as an important class of drugs for treating BRCA-deficient cancers. Recent discoveries have shown that PARP1 inhibitors may treat other cancer patients in addition to the relatively small proportion of patients carrying BRCA mutations. However, the additional targets by which PARP1 inhibitor-mediated tumor suppression remain poorly understood. In this study, we show that two PARP1 inhibitors, PJ-34 and 3-AB, attenuate AKT phosphorylation at serine 473 (S473) independent of DNA repair impairment. These inhibitors decrease the AKT-associated phosphorylation of FOXO3A, enhance the nuclear retention of FOXO3A, and activate its transcriptional activity. We further demonstrate that treatment with PJ-34 or 3-AB dramatically increases the level of PHLPP1. Overexpression of PHLPP1 enhances the PARP1 inhibitor-induced downregulation of AKT phosphorylation and increases tumor cell death. In contrast, knockdown of PHLPP1 abrogates the PARP1 inhibitor-mediated AKT inhibition and desensitizes cells to its treatment. Therefore, our findings not only show the robust role of PARP1 inhibitors in AKT inhibition but also develop a novel strategy to increase the effectiveness of cancer treatment via PARP1 inhibitor-induced PHLPP1 upregulation.

PKB/Akt phosphorylates p27, impairs nuclear import of p27 and opposes p27-mediated G1 arrest.

Mechanisms linking mitogenic and growth inhibitory cytokine signaling and the cell cycle have not been fully elucidated in either cancer or in normal cells. Here we show that activation of protein kinase B (PKB)/Akt, contributes to resistance to antiproliferative signals and breast cancer progression in part by impairing the nuclear import and action of p27. Akt transfection caused cytoplasmic p27 accumulation and resistance to cytokine-mediated G1 arrest. The nuclear localization signal of p27 contains an Akt consensus site at threonine 157, and p27 phosphorylation by Akt impaired its nuclear import in vitro. Akt phosphorylated wild-type p27 but not p27T157A. In cells transfected with constitutively active Akt(T308DS473D)(PKB(DD)), p27WT mislocalized to the cytoplasm, but p27T157A was nuclear. In cells with activated Akt, p27WT failed to cause G1 arrest, while the antiproliferative effect of p27T157A was not impaired. Cytoplasmic p27 was seen in 41% (52 of 128) of primary human breast cancers in conjunction with Akt activation and was correlated with a poor patient prognosis. Thus, we show a novel mechanism whereby Akt impairs p27 function that is associated with an aggressive phenotype in human breast cancer.

AMPK-mediated phosphorylation of murine p27 at T197 promotes binding of 14-3-3 proteins and increases p27 stability.

The tuberous sclerosis complex 2 (Tsc2) gene product, tuberin, acts as a negative regulator of mTOR signaling, and loss of tuberin function leads to tumors of the brain, skin, kidney, heart, and lungs. Previous studies have shown that loss of tuberin function affects the stability and subcellular localization of the cyclin-dependent kinase inhibitor (CKI) p27, although the mechanism(s) by which tuberin modulates p27 stability has/have not been elucidated. Previous studies have also shown that AMP-activated protein kinase (AMPK), which functions in an energy-sensing pathway in the cell, becomes activated in the absence of tuberin. Here we show that in Tsc2-null tumors and cell lines, AMPK activation correlates with an increase in p27 levels, and inhibition of AMPK signaling decreases p27 levels in these cells. In addition, activation of AMPK led to phosphorylation of p27 at the conserved terminal threonine residue of murine p27 (T197) in both in vitro kinase assays and in cells. Phosphorylation of p27 at T197 led to increased interaction between p27 and 14-3-3 proteins and increased the protein stability of p27. Furthermore, activation of AMPK signaling promoted the interaction between p27 and 14-3-3 proteins and increased the stability of the p27 protein in a manner that was dependent on T197. These data identify a conserved mechanism for the regulation of p27 stability via phosphorylation at the terminal threonine (mT197/hT198) and binding of 14-3-3 proteins, which when AMPK is activated results in stabilization of the p27 protein.

Upregulation of programmed death ligand 1 by liver kinase B1 and its implication in programmed death 1 blockade therapy in non-small cell lung cancer.

AIMS: Liver kinase B1 (LKB1) deficiency is associated with reduced expression of programmed death ligand 1 (PD-L1) and inferior clinical outcomes of PD-1/PD-L1 blockade in non-small cell lung cancer (NSCLC). This study aimed to investigate the mechanism by which LKB1 regulates PD-L1 expression and its role in programmed death 1 (PD-1) blockade therapy in NSCLC. MAIN METHODS: The impact of LKB1 on PD-L1 was assessed by western blot, qRT-PCR and immunohistochemistry in NSCLC. Activators/inhibitors of AMPK and NRF2 were applied to explore the mechanisms underlying the regulation of PD-L1 by LKB1. Efficiency of combined application of metformin and PD-1 blockade was evaluated in immunocompetent C57BL/6 mice. KEY FINDINGS: A remarkable positive correlation between LKB1 and PD-L1 expression was demonstrated in NSCLC tissues. Knockdown of LKB1 decreased PD-L1 in TC-1 cells, whereas overexpression of LKB1 increased PD-L1 in A549 cells. We further characterized that AMPK mediated the upregulation of PD-L1 by LKB1. Inhibition of AMPK or NRF2 markedly reduced PD-L1 in LKB1-intact NSCLC cells. In contrast, activation of AMPK or NRF2 reversed PD-L1 expression in LKB1-deficient NSCLC cells. Combined administration of metformin and anti-PD-1 antibody efficiently inhibited the growth of LKB1-intact tumors, whereas no obvious suppression was observed in LKB1-deficient tumors. SIGNIFICANCE: These findings demonstrated that LKB1 upregulates PD-L1 expression in NSCLC by activating the AMPK and KEAP1/NRF2 signaling. Activation of LKB1-AMPK with metformin improves the therapeutic effect of PD-1 blockade in NSCLC with wild-type LKB1.

Enhanced O-linked Glcnacylation in Crohn's disease promotes intestinal inflammation.

BACKGROUND: Treatment of Crohn's disease (CD) remains to be a challenge due to limited insights for its pathogenesis. We aimed to determine the role of O-Linked ß-N-acetylglucosamine (O-GlcNAc) in the development of CD and evaluate therapeutic effects of O-GlcNAc inhibitors on CD. METHODS: O-GlcNAc in intestinal epithelial tissues of CD, adherent-invasive Escherichia coli (AIEC) LF82-infected cells and mice was determined by immunoblot and immunohistochemistry. AIEC LF82 and dextran sulfate sodium were administrated into C57BL/6 mice for estabolishing inflammatory bowel disease model and for therapeutic study. FINDINGS: O-GlcNAc was increased in intestinal epithelial tissues of CD patients and AIEC LF82-infected mice. Infection of AIEC LF82 up-regulated the level of UDP-GlcNAc and increased O-GlcNAc in human colon epithelial HCT116 and HT-29 cells. We identified that IKKß and NF-κB were O-Glycosylated in AIEC LF82-treated cells. Mutations of IKKß (S733A) and p65 (T352A) abrogated the O-GlcNAc in IKKß and NF-κB and inhibited AIEC LF82-induced activation of NF-κB. Application of 6-diazO-5-oxO-L-norleucine, an agent that blocks the production of UDP-GlcNAc and inhibits O-GlcNAc, inactivated NF-κB in AIEC LF82-infected cells, enhanced the formation of autophagy, promoted the removal of cell-associated AIEC LF82, alleviated intestinal epithelial inflammation, and improved the survival of the colitis mice. INTERPRETATION: Intestinal inflammation in CD is associated with increased O-GlcNAc modification, which is required for NF-κB activation and suppression of autophagy. Targeting O-GlcNAc could be an effective therapy for inflammatory bowel disease. FUNDING: National Natural Science Foundation of China (Nos. 81573087 and 81772924) and International Cooperation Foundation of Jilin Province (20190701006GH).

Verteporfin Inhibits PD-L1 through Autophagy and the STAT1-IRF1-TRIM28 Signaling Axis, Exerting Antitumor Efficacy.

Programmed cell death 1 ligand 1 (PD-L1) is a key driver of tumor-mediated immune suppression, and targeting it with antibodies can induce therapeutic responses. Given the costs and associated toxicity of PD-L1 blockade, alternative therapeutic strategies are needed. Using reverse-phase protein arrays to assess drugs in use or likely to enter trials, we performed a candidate drug screen for inhibitors of PD-L1 expression and identified verteporfin as a possible small-molecule inhibitor. Verteporfin suppressed basal and IFN-induced PD-L1 expression in vitro and in vivo through Golgi-related autophagy and disruption of the STAT1-IRF1-TRIM28 signaling cascade, but did not affect the proinflammatory CIITA-MHC II cascade. Within the tumor microenvironment, verteporfin inhibited PD-L1 expression, which associated with enhanced T-lymphocyte infiltration. Inhibition of chromatin-associated enzyme PARP1 induced PD-L1 expression in high endothelial venules (HEV) in tumors and, when combined with verteporfin, enhanced therapeutic efficacy. Thus, verteporfin effectively targets PD-L1 through transcriptional and posttranslational mechanisms, representing an alternative therapeutic strategy for targeting PD-L1.