The m6A writer RBM15 drives the growth of triple-negative breast cancer cells through the stimulation of serine and glycine metabolism.

N6-adenosine methylation (m6A) is critical for controlling cancer cell growth and tumorigenesis. However, the function and detailed mechanism of how m6A methyltransferases modulate m6A levels on specific targets remain unknown. In the current study, we identified significantly elevated levels of RBM15, an m6A writer, in basal-like breast cancer (BC) patients compared to nonbasal-like BC patients and linked this increase to worse clinical outcomes. Gene expression profiling revealed correlations between RBM15 and serine and glycine metabolic genes, including PHGDH, PSAT1, PSPH, and SHMT2. RBM15 influences m6A levels and, specifically, the m6A levels of serine and glycine metabolic genes via direct binding to target RNA. The effects of RBM15 on cell growth were largely dependent on serine and glycine metabolism. Thus, RBM15 coordinates cancer cell growth through altered serine and glycine metabolism, suggesting that RBM15 is a new therapeutic target in BC.

Metformin Suppresses Both PD-L1 Expression in Cancer Cells and Cancer-Induced PD-1 Expression in Immune Cells to Promote Antitumor Immunity.

Background: Metformin, a drug prescribed for patients with type 2 diabetes, has potential efficacy in enhancing antitumor immunity; however, the detailed underlying mechanisms remain to be elucidated. Therefore, we aimed to identify the inhibitory molecular mechanisms of metformin on programmed death ligand 1 (PD-L1) expression in cancer cells and programmed death 1 (PD-1) expression in immune cells. Methods: We employed a luciferase reporter assay, quantitative real-time PCR, immunoblotting analysis, immunoprecipitation and ubiquitylation assays, and a natural killer (NK) cell-mediated tumor cell cytotoxicity assay. A mouse xenograft tumor model was used to evaluate the effect of metformin on tumor growth, followed by flow-cytometric analysis using tumor-derived single-cell suspensions. Results: Metformin decreased AKT-mediated ß-catenin S552 phosphorylation and subsequent ß-catenin transactivation in an adenosine monophosphate-activated protein kinase (AMPK) activation-dependent manner, resulting in reduced CD274 (encoding PD-L1) transcription in cancer cells. Tumor-derived soluble factors enhanced PD-1 protein stability in NK and T cells via dissociation of PD-1 from ubiquitin E3 ligases and reducing PD-1 polyubiquitylation. Metformin inhibited the tumor-derived soluble factor-reduced binding of PD-1 to E3 ligases and PD-1 polyubiquitylation, resulting in PD-1 protein downregulation in an AMPK activation-dependent manner. These inhibitory effects of metformin on both PD-L1 and PD-1 expression ameliorated cancer-reduced cytotoxic activity of immune cells in vitro and decreased tumor immune evasion and growth in vivo. Conclusions: Metformin blocks both PD-L1 and PD-1 within the tumor microenvironment. This study provided a mechanistic insight into the efficacy of metformin in improving immunotherapy in human cancer.

Accuracy of generative deep learning model for macular anatomy prediction from optical coherence tomography images in macular hole surgery.

This study aims to propose a generative deep learning model (GDLM) based on a variational autoencoder that predicts macular optical coherence tomography (OCT) images following full-thickness macular hole (FTMH) surgery and evaluate its clinical accuracy. Preoperative and 6-month postoperative swept-source OCT data were collected from 150 patients with successfully closed FTMH using 6 × 6 mm2 macular volume scan datasets. Randomly selected and augmented 120,000 training and 5000 validation pairs of OCT images were used to train the GDLM. We assessed the accuracy and F1 score of concordance for neurosensory retinal areas, performed Bland-Altman analysis of foveolar height (FH) and mean foveal thickness (MFT), and predicted postoperative external limiting membrane (ELM) and ellipsoid zone (EZ) restoration accuracy between artificial intelligence (AI)-OCT and ground truth (GT)-OCT images. Accuracy and F1 scores were 94.7% and 0.891, respectively. Average FH (228.2 vs. 233.4 µm, P = 0.587) and MFT (271.4 vs. 273.3 µm, P = 0.819) were similar between AI- and GT-OCT images, within 30.0% differences of 95% limits of agreement. ELM and EZ recovery prediction accuracy was 88.0% and 92.0%, respectively. The proposed GDLM accurately predicted macular OCT images following FTMH surgery, aiding patient and surgeon understanding of postoperative macular features.

Changes in Intraocular Pressure and Anterior Chamber Parameters Following Cataract Surgery, Vitrectomy, and Combined Surgery.

PURPOSE: The aim of this study is to investigate changes in intraocular pressure (IOP) and anterior-segment parameters before and after cataract surgery, vitrectomy, and combined surgery. METHODS: The records of patients who had undergone cataract surgery (cataract group), vitrectomy (vitrectomy group), or combined cataract surgery and vitrectomy (combined group) at our hospital were retrospectively examined. The vitrectomy group consisted of pseudophakic eyes. IOP and anterior-segment measurements, including anterior chamber depth (ACD), angle opening distance (AOD), trabecular-iris angle (TIA), and trabecular-iris space area (TISA), were measured using swept-source anterior-segment optical coherence tomography before and 6 months after surgery in 41, 15, and 40 eyes, respectively. RESULTS: In the cataract and combined groups, there was a decrease in IOP (cataract group: from 15.8 to 13.4 mmHg, p <0.001; combined group: from 15.8 to 14.2 mmHg, p = 0.002) and an increase in the central corneal thickness after surgery (p <0.001). The ACD increased in all groups, with a smaller increase in the vitrectomy group (p <0.03). Postoperative AOD, TIA, and TISA were significantly increased in the cataract and combined groups (p <0.02). Higher preoperative IOP and larger IOP reduction after surgery were correlated with smaller preoperative AOD, TISA, and TIA in cataract and combined groups (p <0.034). A small preoperative ACD was related to smaller preoperative AOD, TISA, TIA (r > 0.649, p <0.001), and postoperative IOP reduction in the cataract and combined groups (r = 0.377, p = 0.018 and r = 0.559, p = 0.001, respectively). CONCLUSIONS: Compared to the vitrectomy group, the cataract and combined groups showed reduced postoperative IOP and increased AOD, TISA, and TIA. In these two groups, patients with shallower preoperative ACDs showed greater changes in IOP after surgery. Changes in IOP after surgery are thought to be related to changes in the anterior segment caused by the removal of the crystalline lens.

Disruption of phosphofructokinase activity and aerobic glycolysis in human bronchial epithelial cells by atmospheric ultrafine particulate matter.

Exposure to ambient ultrafine particulate matter (UPM) causes respiratory disorders; however, the underlying molecular mechanisms remain unclear. In this study, we synthesized simulated UPM (sUPM) with controlled physicochemical properties using the spark-discharge method. Subsequently, we investigated the biological effects of sUPM using BEAS-2B human bronchial epithelial cells (HBECs) and a mouse intratracheal instillation model. High throughput RNA-sequencing and bioinformatics analyses revealed that dysregulation of the glycolytic metabolism is involved in the inhibited proliferation and survival of HBECs by sUPM treatment. Furthermore, signaling pathway and enzymatic analyses showed that the treatment of BEAS-2B cells with sUPM induces the inactivation of extracellular signal-regulated kinase (ERK) and protein kinase B (PKB, also known as AKT), resulting in the downregulation of phosphofructokinase 2 (PFK2) S483 phosphorylation, PFK enzyme activity, and aerobic glycolysis in HBECs in an oxidative stress-independent manner. Additionally, intratracheal instillation of sUPM reduced the phosphorylation of ERK, AKT, and PFK2, decreased proliferation, and increased the apoptosis of bronchial epithelial cells in mice. The findings of this study imply that UPM induces pulmonary toxicity by disrupting aerobic glycolytic metabolism in lung epithelial cells, which can provide novel insights into the toxicity mechanisms of UPM and strategies to prevent their toxic effects.

AMPK-HIF-1α signaling enhances glucose-derived de novo serine biosynthesis to promote glioblastoma growth.

BACKGROUND: Cancer cells undergo cellular adaptation through metabolic reprogramming to sustain survival and rapid growth under various stress conditions. However, how brain tumors modulate their metabolic flexibility in the naturally serine/glycine (S/G)-deficient brain microenvironment remain unknown. METHODS: We used a range of primary/stem-like and established glioblastoma (GBM) cell models in vitro and in vivo. To identify the regulatory mechanisms of S/G deprivation-induced metabolic flexibility, we employed high-throughput RNA-sequencing, transcriptomic analysis, metabolic flux analysis, metabolites analysis, chromatin immunoprecipitation (ChIP), luciferase reporter, nuclear fractionation, cycloheximide-chase, and glucose consumption. The clinical significances were analyzed in the genomic database (GSE4290) and in human GBM specimens. RESULTS: The high-throughput RNA-sequencing and transcriptomic analysis demonstrate that the de novo serine synthesis pathway (SSP) and glycolysis are highly activated in GBM cells under S/G deprivation conditions. Mechanistically, S/G deprivation rapidly induces reactive oxygen species (ROS)-mediated AMP-activated protein kinase (AMPK) activation and AMPK-dependent hypoxia-inducible factor (HIF)-1α stabilization and transactivation. Activated HIF-1α in turn promotes the expression of SSP enzymes phosphoglycerate dehydrogenase (PHGDH), phosphoserine aminotransferase 1 (PSAT1), and phosphoserine phosphatase (PSPH). In addition, the HIF-1α-induced expression of glycolytic genes (GLUT1, GLUT3, HK2, and PFKFB2) promotes glucose uptake, glycolysis, and glycolytic flux to fuel SSP, leading to elevated de novo serine and glycine biosynthesis, NADPH/NADP+ ratio, and the proliferation and survival of GBM cells. Analyses of human GBM specimens reveal that the levels of overexpressed PHGDH, PSAT1, and PSPH are positively correlated with levels of AMPK T172 phosphorylation and HIF-1α expression and the poor prognosis of GBM patients. CONCLUSION: Our findings reveal that metabolic stress-enhanced glucose-derived de novo serine biosynthesis is a critical metabolic feature of GBM cells, and highlight the potential to target SSP for treating human GBM.

Mutual regulation between phosphofructokinase 1 platelet isoform and VEGF promotes glioblastoma tumor growth.

Glioblastoma (GBM) is a highly vascular malignant brain tumor that overexpresses vascular endothelial growth factor (VEGF) and phosphofructokinase 1 platelet isoform (PFKP), which catalyzes a rate-limiting reaction in glycolysis. However, whether PFKP and VEGF are reciprocally regulated during GBM tumor growth remains unknown. Here, we show that PFKP can promote EGFR activation-induced VEGF expression in HIF-1α-dependent and -independent manners in GBM cells. Importantly, we demonstrate that EGFR-phosphorylated PFKP Y64 has critical roles in both AKT/SP1-mediated transcriptional expression of HIF-1α and in the AKT-mediated ß-catenin S552 phosphorylation, to fully enhance VEGF transcription, subsequently promoting blood vessel formation and brain tumor growth. Levels of PFKP Y64 phosphorylation in human GBM specimens are positively correlated with HIF-1α expression, ß-catenin S552 phosphorylation, and VEGF expression. Conversely, VEGF upregulates PFKP expression in a PFKP S386 phosphorylation-dependent manner, leading to increased PFK enzyme activity, aerobic glycolysis, and proliferation in GBM cells. These findings highlight a novel mechanism underlying the mutual regulation that occurs between PFKP and VEGF for promoting GBM tumor growth and also suggest that targeting the PFKP/VEGF regulatory loop might show therapeutic potential for treating GBM patients.

Phosphofructokinase 1 platelet isoform induces PD-L1 expression to promote glioblastoma immune evasion.

BACKGROUND: Overexpression of PD-L1 is observed in many types of human cancer, including glioblastoma (GBM) and contributes to tumor immune evasion. In addition, GBM shows highly-activated aerobic glycolysis due to overexpression of phosphofructokinase 1 platelet isoform (PFKP), which the key enzyme in the glycolysis. However, it remains unclear whether the metabolic enzyme PFKP plays a role in the regulation of PD-L1 expression and GBM immune evasion. OBJECTIVE: We aimed to investigate the non-metabolic role of PFKP in PD-L1 expression-induced GBM immune evasion. METHODS: The mechanisms of PFKP-induced PD-L1 expression were studied by several experiments, including real-time PCR, immunoblot analysis, and ATP production. The coculture experiments using GBM cell and T cells were performed to evaluate the effect of PFKP on T cell activation. The clinical relationship between PFKP and PD-L1 was analyzed in The Cancer Genome Atlas (TCGA) database and in human GBM specimens. RESULTS: We showed that PFKP promotes EGFR activation-induced PD-L1 expression in human GBM cells. Importantly, we demonstrated that EGFR-phosphorylated PFKP Y64 plays an important role in AKT-mediated ß-catenin transactivation and subsequent PD-L1 transcriptional expression, thereby enhancing the GBM immune evasion. In addition, based on our findings, the levels of PFKP Y64 phosphorylation are positively correlated with PD-L1 expression in human GBM specimens, highlighting the clinical significance of PFKP Y64 phosphorylation in the GBM immune evasion. CONCLUSION: These findings provide new mechanistic insight into the regulation of PD-L1 expression by a non-metabolic function of PFKP on tumor cells.

Cytotoxicities and wound healing effects of contact lens multipurpose solution on human corneal epithelial cell.

CLINICAL RELEVANCE: Contact lens multipurpose solutions (MPSs) contain several components that have the potential to cause corneal epithelial cell toxicity. Evaluating the components and the toxic effect of MPS should be considered for effective eye care. BACKGROUND: The cytotoxic and wound healing effects of five commercially available MPSs on human corneal epithelial cells (HCECs) are is investigated. METHODS: The following commercially available MPSs were used: Queen's PLURISOL®, Frenz®, Boston SIMPLUS®, DL+PLUS EYE® (DL), and NEW YORK DEFINE® (NY). The proliferation of HCECs exposed to each MPS for 1, 6, and 24 h and the cytotoxicity of these solutions were analyzed using methyl thiazolyl tetrazolium-based colorimetric and lactate dehydrogenase leakage assays, respectively. The cellular morphology was evaluated by inverted phase-contrast and electron microscopy. A scratch-wound assay was performed to measure wound widths 24 h after confluent HCEC monolayers were scratch-wounded. RESULTS: The tested MPS had a time-dependent inhibitory effect on HCEC proliferation and cytotoxicity, significantly at 24 h after exposure (p< 0.05 in all MPSs). HCECs exposed to MPS detached from the bottom of the culture dishes, showed degenerative changes such as loss of microvilli, cytoplasmic vacuole formation and nuclear condensation, and decreased wound healing, compared to the controls (p< 0.001 in Boston, DL and NY). Among the tested MPS, DL and NY were more cytotoxic and showed less wound healing. CONCLUSION: MPS has a toxic effect on HCECs, which is dependent on the concentration of the disinfecting component. Since the components that constitute the MPS are absorbed and retained in the lens, cautious scrutiny of the concentration and attention to lens cleaning are warranted to mitigate the related cytotoxicity.

Mechanosensitive turnover of phosphoribosyl pyrophosphate synthetases regulates nucleotide metabolism.

Cells coordinate their behaviors with the mechanical properties of the extracellular matrix (ECM). Tumor cells frequently harbor an enhanced nucleotide synthesis, presumably to meet the increased demands for rapid proliferation. Nevertheless, how ECM rigidity regulates nucleotide metabolism remains elusive. Here we show that shift from stiff to soft matrix blunts glycolysis-derived nucleotide synthesis in tumor cells. Soft ECM results in TNF receptor-associated factor 2 (TRAF2)-dependent K29 ubiquitination and degradation of phosphoribosyl pyrophosphate synthetase (PRPS)1/2. Recruitment of TRAF2 to PRPS1/2 requires phosphorylation of PRPS1 S285 or PRPS2 T285, which is mediated by low stiffness-activated large tumor suppressor (LATS)1/2 kinases. Further, non-phosphoryable or non-ubiquitinatable PRPS1/2 mutations maintain PRPS1/2 expression and nucleotide synthesis at low stiffness, and promote tumor growth and metastasis. Our findings demonstrate that PRPS1/2 stability and nucleotide metabolism is ECM rigidity-sensitive, and thereby highlight a regulatory cascade underlying mechanics-guided tumor metabolism reprogramming.

Wnt signaling promotes tumor development in part through phosphofructokinase 1 platelet isoform upregulation.

The activation of Wnt signaling has been detected in various types of human cancer and has been shown to be associated with cancer development. In the present study, it was revealed that Wnt signaling induced the expression of phosphofructokinase 1 platelet isoform (PFKP), which has been reported to catalyze a rate­limiting reaction in glycolysis and is important for the Warburg effect, proliferation, colony formation and cancer cell migration. Moreover, it was demonstrated that Wnt3A induced PFKP expression in a ß­catenin­independent manner, resulting in increased PFK enzyme activity. Wnt3A­induced epidermal growth factor receptor transactivation activated PI3K/AKT, which stabilized PFKP through PFKP S386 phosphorylation and subsequent PFKP upregulation. Wnt3A­induced PFKP S386 phosphorylation increased PFKP expression and promoted the Warburg effect, cell proliferation, colony formation and the migratory ability of cancer cells. On the whole, the findings of the present study underscore the potential role of PFKP in Wnt signaling­induced tumor development.

Analysis of factors affecting visual field recovery following surgery for pituitary adenoma.

PURPOSE: To analyze the factors influencing visual field recovery in patients with pituitary adenoma following surgical treatment. METHODS: We retrospectively reviewed 144 eyes of 72 patients with pituitary adenoma who had been followed up for more than 6 months following surgery between January 2016 and December 2019. Pre and postoperative visual acuity, visual field test and retinal nerve fiber layer (RNFL) thickness were investigated. We defined recovery of visual field defects as being an improvement in mean deviation (MD) of 2 dB or more. RESULT: The average age of the 72 patients (144 eyes) was 51.94 ± 14.69 years, making for 37 patients in the recovery group and 35 patients in the non-recovery group. Preoperative MD, pattern standard deviation (PSD), and visual field indexes (VFI) were negatively correlated to postoperative MD, PSD and VFI changes and positively correlated to postoperative MD, PSD, and VFI values. Using multiple regression analysis, a shorter duration of symptoms (Odds ratio [OR], 0.990; p = 0.033), higher preoperative MD values (OR, 0.871; p = 0.025), and thicker temporal RNFL (OR, 1.068; p = 0.048) were associated with a visual field recovery following surgery. CONCLUSIONS: The prognosis for visual field recovery is favorable for patients who have a short period from symptom onset to surgery, a higher MD value of preoperative VF, and a thicker peripapillary temporal RNFL thickness. Therefore, the preoperative MD, temporal RNFL thickness, and the symptom period can be predictive variables affecting postoperative visual field recovery.

Immune cell composition in normal human kidneys.

An understanding of immunological mechanisms in kidney diseases has advanced using mouse kidneys. However, the profiling of immune cell subsets in human kidneys remains undetermined, particularly compared with mouse kidneys. Normal human kidneys were obtained from radically nephrectomised patients with urogenital malignancy (n = 15). Subsequently, human kidney immune cell subsets were analysed using multicolor flow cytometry and compared with subsets from C57BL/6 or BALB/c mice under specific pathogen-free conditions. Twenty kidney sections from healthy kidney donors or subjects without specific renal lesions were additionally analysed by immunohistochemistry. In human kidneys, 47% ± 12% (maximum 63%) of immune cells were CD3+ T cells. Kidney CD4+ and CD8+ T cells comprised 44% and 56% of total T cells. Of these, 47% ± 15% of T cells displayed an effector memory phenotype (CCR7- CD45RA- CD69-), and 48% ± 19% were kidney-resident cells (CCR7- CD45RA- CD69+). However, the proportions of human CD14+ and CD16+ myeloid cells were approximately 10% of total immune cells. A predominance of CD3+ T cells and a low proportion of CD14+ or CD68+ myeloid cells were also identified in healthy human kidney sections. In mouse kidneys, kidney-resident macrophages (CD11blow F4/80high) were the most predominant subset (up to 50%) but the proportion of CD3+ T cells was less than 20%. These results will be of use in studies in which mouse results are translated into human cases under homeostatic conditions or with disease.