A GAPDH serotonylation system couples CD8+ T cell glycolytic metabolism to antitumor immunity.

Apart from the canonical serotonin (5-hydroxytryptamine [5-HT])-receptor signaling transduction pattern, 5-HT-involved post-translational serotonylation has recently been noted. Here, we report a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) serotonylation system that promotes the glycolytic metabolism and antitumor immune activity of CD8+ T cells. Tissue transglutaminase 2 (TGM2) transfers 5-HT to GAPDH glutamine 262 and catalyzes the serotonylation reaction. Serotonylation supports the cytoplasmic localization of GAPDH, which induces a glycolytic metabolic shift in CD8+ T cells and contributes to antitumor immunity. CD8+ T cells accumulate intracellular 5-HT for serotonylation through both synthesis by tryptophan hydroxylase 1 (TPH1) and uptake from the extracellular compartment via serotonin transporter (SERT). Monoamine oxidase A (MAOA) degrades 5-HT and acts as an intrinsic negative regulator of CD8+ T cells. The adoptive transfer of 5-HT-producing TPH1-overexpressing chimeric antigen receptor T (CAR-T) cells induced a robust antitumor response. Our findings expand the known range of neuroimmune interaction patterns by providing evidence of receptor-independent serotonylation post-translational modification.

Nucleolar HEAT Repeat Containing 1 Up-regulated by the Mechanistic Target of Rapamycin Complex 1 Signaling Promotes Hepatocellular Carcinoma Growth by Dominating Ribosome Biogenesis and Proteome Homeostasis.

BACKGROUND & AIMS: Hyperactivation of ribosome biogenesis leads to hepatocyte transformation and plays pivotal roles in hepatocellular carcinoma (HCC) development. We aimed to identify critical ribosome biogenesis proteins that are overexpressed and crucial in HCC progression. METHODS: HEAT repeat containing 1 (HEATR1) expression and clinical correlations were analyzed using The Cancer Genome Atlas and Gene Expression Omnibus databases and further evaluated by immunohistochemical analysis of an HCC tissue microarray. Gene expression was knocked down by small interfering RNA. HEATR1-knockdown cells were subjected to viability, cell cycle, and apoptosis assays and used to establish subcutaneous and orthotopic tumor models. Chromatin immunoprecipitation and quantitative polymerase chain reaction were performed to detect the association of candidate proteins with specific DNA sequences. Endogenous coimmunoprecipitation combined with mass spectrometry was used to identify protein interactions. We performed immunoblot and immunofluorescence assays to detect and localize proteins in cells. The nucleolus ultrastructure was detected by transmission electron microscopy. Click-iT (Thermo Fisher Scientific) RNA imaging and puromycin incorporation assays were used to measure nascent ribosomal RNA and protein synthesis, respectively. Proteasome activity, 20S proteasome foci formation, and protein stability were evaluated in HEATR1-knockdown HCC cells. RESULTS: HEATR1 was the most up-regulated gene in a set of ribosome biogenesis mediators in HCC samples. High expression of HEATR1 was associated with poor survival and malignant clinicopathologic features in patients with HCC and contributed to HCC growth in vitro and in vivo. HEATR1 expression was regulated by the transcription factor specificity protein 1, which can be activated by insulin-like growth factor 1-mammalian target of rapamycin complex 1 signaling in HCC cells. HEATR1 localized predominantly in the nucleolus, bound to ribosomal DNA, and was associated with RNA polymerase I transcription/processing factors. Knockdown of HEATR1 disrupted ribosomal RNA biogenesis and impaired nascent protein synthesis, leading to reduced cytoplasmic proteasome activity and inhibitory-κB/nuclear factor-κB signaling. Moreover, HEATR1 knockdown induced nucleolar stress with increased nuclear proteasome activity and inactivation of the nucleophosmin 1-MYC axis. CONCLUSIONS: Our study revealed that HEATR1 is up-regulated by insulin-like growth factor 1-mammalian target of rapamycin complex 1-specificity protein 1 signaling in HCC and functions as a crucial regulator of ribosome biogenesis and proteome homeostasis to promote HCC development.

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OBJECTIVES: To investigate the risk factors for bronchopulmonary dysplasia (BPD) in twin preterm infants with a gestational age of <34 weeks, and to provide a basis for early identification of BPD in twin preterm infants in clinical practice. METHODS: A retrospective analysis was performed for the twin preterm infants with a gestational age of <34 weeks who were admitted to 22 hospitals nationwide from January 2018 to December 2020. According to their conditions, they were divided into group A (both twins had BPD), group B (only one twin had BPD), and group C (neither twin had BPD). The risk factors for BPD in twin preterm infants were analyzed. Further analysis was conducted on group B to investigate the postnatal risk factors for BPD within twins. RESULTS: A total of 904 pairs of twins with a gestational age of <34 weeks were included in this study. The multivariate logistic regression analysis showed that compared with group C, birth weight discordance of >25% between the twins was an independent risk factor for BPD in one of the twins (OR=3.370, 95%CI: 1.500-7.568, P<0.05), and high gestational age at birth was a protective factor against BPD (P<0.05). The conditional logistic regression analysis of group B showed that small-for-gestational-age (SGA) birth was an independent risk factor for BPD in individual twins (OR=5.017, 95%CI: 1.040-24.190, P<0.05). CONCLUSIONS: The development of BPD in twin preterm infants is associated with gestational age, birth weight discordance between the twins, and SGA birth.

The physiology, pathology and potential therapeutic application of serotonylation.

The classical neurotransmitter serotonin or 5-hydroxytryptamine (5-HT), synthesized from tryptophan, can be produced both centrally and peripherally. Through binding to functionally distinct receptors, serotonin is profoundly implicated in a number of fundamental physiological processes and pathogenic conditions. Recently, serotonin has been found covalently incorporated into proteins, a newly identified post-translational modification termed serotonylation. Transglutaminases (TGMs), especially TGM2, are responsible for catalyzing the transamidation reaction by transferring serotonin to the glutamine residues of target proteins. Small GTPases, extracellular matrix protein fibronectin, cytoskeletal proteins and histones are the most reported substrates for serotonylation, and their functions are triggered by this post-translational modification. This Review highlights the roles of serotonylation in physiology and diseases and provides perspectives for pharmacological interventions to ameliorate serotonylation for disease treatment.

Optical micro-phase-shift dropvolume in a diffractive deep neural network.

To provide a desirable number of parallel subnetworks as required to reach a robust inference in an active modulation diffractive deep neural network, a random micro-phase-shift dropvolume that involves five-layer statistically independent dropconnect arrays is monolithically embedded into the unitary backpropagation, which does not require any mathematical derivations with respect to the multilayer arbitrary phase-only modulation masks, even maintaining the nonlinear nested characteristic of neural networks, and generating an opportunity to realize a structured-phase encoding within the dropvolume. Further, a drop-block strategy is introduced into the structured-phase patterns designed to flexibly configure a credible macro-micro phase dropvolume allowing for convergence. Concretely, macro-phase dropconnects concerning fringe griddles that encapsulate sparse micro-phase are implemented. We numerically validate that macro-micro phase encoding is a good plan to the types of encoding within a dropvolume.

Combination of microbial and chemical synthesis for the sustainable production of ß-elemene, a promising plant-extracted anticancer compound.

Beta-elemene, a class of sesquiterpene derived from the Chinese medicinal herb Curcuma wenyujin, is widely used in clinical medicine due to its broad-spectrum antitumor activity. However, the unsustainable plant extraction prompted the search for environmentally friendly strategies for ß-elemene production. In this study, we designed a Yarrowia lipolytica cell factory that can continuously produce germacrene A, which is further converted into ß-elemene with 100% yield through a Cope rearrangement reaction by shifting the temperature to 250°C. First, the productivity of four plant-derived germacrene A synthases was evaluated. After that, the metabolic flux of the precursor to germacrene A was maximized by optimizing the endogenous mevalonate pathway, inhibiting the competing squalene pathway, and expressing germacrene A synthase gene in multiple copies. Finally, the most promising strain achieved the highest ß-elemene titer reported to date with 5.08 g/L. This sustainable and green method has the potential for industrial ß-elemene production.

Terbinafine prevents colorectal cancer growth by inducing dNTP starvation and reducing immune suppression.

Existing evidence indicates that gut fungal dysbiosis might play a key role in the pathogenesis of colorectal cancer (CRC). We sought to explore whether reversing the fungal dysbiosis by terbinafine, an approved antifungal drug, might inhibit the development of CRC. A population-based study from Sweden identified a total of 185 patients who received terbinafine after their CRC diagnosis and found that they had a decreased risk of death (hazard ratio = 0.50) and metastasis (hazard ratio = 0.44) compared with patients without terbinafine administration. In multiple mouse models of CRC, administration of terbinafine decreased the fungal load, the fungus-induced myeloid-derived suppressor cell (MDSC) expansion, and the tumor burden. Fecal microbiota transplantation from mice without terbinafine treatment reversed MDSC infiltration and partially restored tumor proliferation. Mechanistically, terbinafine directly impaired tumor cell proliferation by reducing the ratio of nicotinamide adenine dinucleotide phosphate (NADP+) to reduced form of nicotinamide adenine dinucleotide phosphate (NADPH), suppressing the activity of glucose-6-phosphate dehydrogenase (G6PD), resulting in nucleotide synthesis disruption, deoxyribonucleotide (dNTP) starvation, and cell-cycle arrest. Collectively, terbinafine can inhibit CRC by reversing fungal dysbiosis, suppressing tumor cell proliferation, inhibiting fungus-induced MDSC infiltration, and restoring antitumor immune response.

Characteristics and evaluation of C1 posterior arch variation for transpedicular screw placement between patients with and without basilar invagination.

BACKGROUND: C1 transpedicular screw (C1TS) placement provided satisfactory pullout resistance and 3D stability, but its application might be limited in patients with basilar invagination (BI) due to the high incidences of the atlas anomaly and vertebral artery (VA) variation. However, no study has explored the classifications of C1 posterior arch variations and investigated their indications and ideal insertion trajectories for C1TS in BI. PURPOSE: To investigate the bony and surrounding arterial characteristics of the atlas, classify posterior arch variations, identify indications for C1TS, evaluate ideal insertion trajectories for C1TS in BI patients without atlas occipitalization (AO), and compare them with those without BI and AO as control. METHODS: A total of 130 non-AO patients with and without BI (52 patients and 78 patients, respectively) from two medical centers were included at a 1:1.5 ratio. The posterior arch variations were assessed using a modified C1 morphological classification. Comparisons regarding the bony and surrounding arterial characteristics, morphological classification distributions, and ideal insertion trajectories between BI and control groups were performed. The subgroup analyses based on different morphological classifications were also conducted. In addition, the factors possibly affecting the insertion parameters were investigated using multiple linear regression analyses. RESULTS: The BI group was associated with significantly smaller lateral mass height and width, sagittal length of posterior arch, pedicle height, vertical height of posterior arch, and distance between VA and VA groove (VAG) than control group. Four types of posterior arch variations with indications for different screw placement techniques were classified; Classifications I and II were suitable for C1TS. The BI cohort showed a significantly lower rate of Classification I than the control cohort. In the BI group, the subgroup of Classification I had significantly larger distance between the insertion point (IP) and inferior aspect of the posterior arch. In addition, it had the narrowest width along ideal screw trajectory, but a significantly more lateral ideal mediolateral angle than the subgroup of Classification II. Multiple linear regression indicated that the cephalad angle was significantly associated with the diagnosis of BI (B = 3.708, P < 0.001) and sagittal diameter of C1 (B = 3.417, P = 0.027); the ideal mediolateral angle was significantly associated with BMI (B = 0.264, P = 0.031), sagittal diameter of C1 (B = - 4.559, P = 0.002), and pedicle height (B = - 2.317, P < 0.001); the distance between the IP and inferior aspects of posterior arch was significantly associated with age (B = - 0.002, P = 0.035), BMI (B = - 0.007, P = 0.028), sagittal length of posterior arch (B = - 0.187, P = 0.032), pedicle height (B = - 0.392, P < 0.001), and middle and lower parts of posterior arch (B = 0.862, P < 0.001). CONCLUSION: The incidence of posterior arch variation in BI patients without AO was remarkably higher than that in control patients. The insertion parameters of posterior screws were different between the morphological classification types in BI and control groups. The distance between VA V3 segments and VAG in BI cohort was substantially smaller than that in control cohort. Preoperative individual 3D computed tomography (CT), CT angiography and intraoperative navigation are recommended for BI patients receiving posterior screw placement.

New Anti-Prelog Stereospecific Whole-Cell Biocatalyst for Asymmetric Reduction of Prochiral Ketones.

The biocatalytic asymmetric reduction of prochiral ketones for the production of enantiopure alcohols is highly desirable due to its inherent advantages over chemical methods. In this study, a new bacterial strain capable of transforming ketones to corresponding alcohols with high activity and excellent enantioselectivity was discovered in a soil sample. The strain was subsequently identified as Bacillus cereus TQ-2 based on its physiological characteristics and 16S rDNA sequence analysis. Under optimized reaction conditions, the resting cells of B. cereus TQ-2 converted acetophenone to enantioenriched (R)-1-phenylethanol with 99% enantiometric excess following anti-Prelog's rule, which is scarce in biocatalytic ketone reduction. The optimum temperature for the cells was 30 °C, and considerable catalytic activity was observed over a broad pH range from 5.0 to 9.0. The cells showed enhanced catalytic activity in the presence of 15% (v/v) glycerol as a co-substrate. The catalytic activity can also be substantially improved by adding Ca2+ or K+ ions. Moreover, the B. cereus TQ-2 cell was highly active in reducing several structurally diverse ketones and aldehydes to form corresponding alcohols with good to excellent conversion. Our study provides a versatile whole-cell biocatalyst that can be used in the asymmetric reduction of ketones for the production of chiral alcohol, thereby expanding the biocatalytic toolbox for potential practical applications.

Multivalent Polypeptide and Tannic Acid Cooperatively Iron-Coordinated Nanohybrids for Synergistic Cancer Photothermal Ferroptosis Therapy.

Owing to having a unique mechanism to kill cancer cells via the membrane accumulation of lipid peroxide (LPO) and the downregulation of glutathione peroxidase-4 (GPX-4), the ferroptosis therapy (FT) of tumors based on the Fenton reaction of iron nanoparticles has been receiving much attention in the past decade; however, there are some hurdles including the uncontrollable release of iron ions, slower kinetics of the intracellular Fenton reaction, and poor efficacy of FT that need to be overcome. Considering cooperative coordination of a multivalent thiol-pendant polypeptide ligand with iron ions, we put forward a facile strategy for constructing the iron-coordinated nanohybrid of methacryloyloxyethyl phosphorylcholine-grafted polycysteine/iron ions/tannic acid (i.e., PCFT), which could deliver a higher concentration of iron ions into cells. The dynamic and unsaturated coordination in PCFT is favorable for the intracellular stimuli-triggered release and fast Fenton reaction to realize efficient FT, while its intrinsic photothermia would boost the Fenton reaction to induce a synergistic effect between FT and photothermal therapy (PTT). Both immunofluorescence analyses of reactive oxygen species (ROS) and LPO confirmed that the intracellular Fenton reaction resulted in efficient FT, during which process the photothermia greatly boosted ferroptosis, and the Western blot assay corroborated that the expression level of GPX-4 was downregulated by FT and highly degraded by the photothermia to induce synergistic PTT-FT in vitro. Excitingly, by a single intravenous dose of PCFT plus one NIR irradiation, in vivo PTT-FT treatment completely eradicated 4T1 tumors without skin scar and tumor recurrence for 16 days, demonstrating prominent antitumor efficacy, as evidenced by the GPX-4, H&E, and TUNEL assays.

Roles of Beclin1 protein expression in cervical cancer: a meta-analysis and bioinformatics analysis.

Beclin1 is a key regulator of a family of autophagy-related proteins. The aim of our study was to elucidate the clinicopathological and prognostic significance of Beclin1 expression which is a positive regulator of autophagy in cervical cancer. The results showed that a total of 2682 patients were enrolled in 21 case-control studies. The results showed that, as for Beclin1 expression, significant differences were found in cervical cancer vs. normal cervical tissues (p<.00001) and cancer tissues with vs. no lymph node metastasis (p<.00001); tumour diameter no less than vs. less than 4 cm (p=.001), myometrial invasion depth no less than vs. less than 1/2 and FIGO I vs. II (p=.02); relationship between Beclin1 expression and prognosis of cervical cancer (p=.03). Kaplan-Meier's plotter showed that Beclin1 expression was negative. It was associated with overall, post-progressive and distant metastatic survival. According to the Oncomine database, Beclin1 mRNA expression in cervical cancer tissues was higher than that in normal tissues. Cox multivariate showed that lymph node metastasis and TNM stage were important factors affecting the survival time of patients. Beclin1 expression can be used as an indicator of prognosis in patients, and provide methods and ideas for prevention and treatment.

Percutaneous Prodding Reduction and K-Wire Fixation Via Sinus Tarsi Approach Versus ORIF for Sanders Type III Calcaneal Fractures: A Prospective Case-Controlled Trial.

We conducted a prospective randomized controlled trial to compare the radiological and clinical outcomes of Sanders type III calcaneal fractures treated with percutaneous prodding reduction and K-wire fixation via a sinus tarsi approach (PPRKF) versus open reduction and internal fixation (ORIF). Fifty-one patients with closed, unilateral, Sanders type III calcaneal fractures were randomly assigned to the PPRKF group (n = 26) or the ORIF group (n = 25). The clinical outcomes evaluated were time to surgery, blood loss, operative time, hospital stay, wound healing time, wound complications, and Maryland foot score. Radiological results were evaluated on lateral and axial X-rays and computed tomography images and included Böhler's angle, Gissane's angle, and calcaneal width. Compared with the ORIF group, the PPRKF group had shorter time to surgery, shorter operative time, less blood loss, shorter hospital stay, shorter wound healing time, and fewer wound complications (p < .001). The postoperative Böhler's angle, Gissane's angle, and calcaneal width in both groups were significantly better than those measured preoperatively (p < .001) and did not differ between the PPRKF group and ORIF group (p> .05). Regarding clinical results, there was no significant difference in Maryland foot score between the two groups at 12 months after surgery (p > .05). Both PPRKF and ORIF can result in satisfactory clinical function. PPRKF is superior to ORIF in reducing the time to surgery, operative time, blood loss, hospital stay, wound healing time, and wound complications.

Combined Use of Aspirin and Selective Serotonin Reuptake Inhibitors Is Associated With Lower Risk of Colorectal Cancer: A Nested Case-Control Study.

INTRODUCTION: Chemoprevention against colorectal cancer (CRC) is greatly needed. As the development of CRC involves multiple dysfunctional pathways, it is thus reasonable to combine some agents that address several pathways to achieve better chemoprotection. We aimed to explore whether the use of aspirin and selective serotonin reuptake inhibitors (SSRIs)-either as monotherapy or combined-can have a clinical benefit against CRC. METHODS: We performed a nested case-control study using nationwide Swedish registers. We recruited 24,786 CRC cases and randomly matched to 74,358 controls conditional on birth year and sex using incidence-density sampling. The conditional logistic regression model was used to calculate odds ratios (ORs) and 95% confidence intervals (CIs). Additive interaction was calculated as the relative excess risk for interaction, and multiplicative interaction was calculated by including a product term in the regression model. RESULTS: Both aspirin and SSRIs monotherapy were negatively associated with CRC risk, but the combined use of aspirin and SSRIs was associated with an even lower CRC risk (adjusted OR, 0.77, 95% CI, 0.67-0.89) than aspirin monotherapy (adjusted OR, 0.91, 95% CI, 0.87-0.97) or SSRI monotherapy (adjusted OR, 0.93, 95% CI, 0.86-1.00). A significant interaction was observed at the additive scale with a relative excess risk for interaction of -0.07 (P < 0.001), whereas no interaction was noted on the interactive scale. The inverse associations of CRC with aspirin and SSRIs showed a dose-dependent pattern. DISCUSSION: This study suggests that the use of aspirin and SSRIs-either as monotherapy or combined-was associated with a reduced risk of CRC. The stronger chemoprevention of combined use of aspirin and SSRIs is innovative and calls for further studies to confirm the underlying mechanisms and the plausibility of clinical recommendation.

Ikarugamycin inhibits pancreatic cancer cell glycolysis by targeting hexokinase 2.

Mangrove-derived actinobacteria strains are well-known for producing novel secondary metabolites. The polycyclic tetramate macrolactam (PTM), ikarugamycin (IKA) isolated from Streptomyces xiamenensis 318, exhibits antiproliferative activities against pancreatic ductal adenocarcinoma (PDAC) in vitro. However, the protein target for bioactive IKA is unclear. In this study, whole transcriptome-based profiling revealed that the glycolysis pathway is significantly affected by IKA. Metabolomic studies demonstrated that IKA treatment induces a significant drop in glucose-6-phosphate and a slight increase in intracellular glucose level. Analysis of glucose consumption, lactate production, and the extracellular acidification rate confirmed the inhibitory role of IKA on the glycolytic flux in PDAC cells. Surface plasmon resonance (SPR) experiments and docking studies identified the key enzyme of glycolysis, hexokinase 2 (HK2), as a molecular target of IKA. Moreover, IKA reduced tumor size without overt cytotoxicity in mice with PDAC xenografts and increased chemotherapy response to gemcitabine in PDAC cells in vitro. Taken together, IKA can block glycolysis in pancreatic cancer by targeting HK2, which may be a potential drug candidate for PDAC treatment.

Phase-shift determination for a 4 × 4 intelligent photonic neural network with compatible learning.

Intelligent photonic circuits (IPCs) tuned with an appropriate phase-shift vector could enable a photonic intelligent matrix possibly implemented in multiple neural layers for a task-oriented topologies. A photonic Mach-Zehnder Interferometer (MZI) is a fundamental photonic component in IPCs, whose matrix representation could be broadcasted into an arbitrary matrix that is equipped with an optimized phase-shift vector. The initialized MZIs' phases are tentatively probed between analytical elements and a digital weight matrix that is learned from samples with efficient compatible learning for complex-valued neural networks. Nonlinear least squares is utilized to formulate a phase determination system to refine the optimal phase-shift solutions. The robustness of phase determination system for photonic neural networks is discussed in detail. For a preliminary implementation, a basic 4×4 intelligent photonic neural network is utilized to verify the proof of concept on phase-shift determination in IPC through numerical experiments.

Loss of Setd2 promotes Kras-induced acinar-to-ductal metaplasia and epithelia-mesenchymal transition during pancreatic carcinogenesis.

OBJECTIVE: SETD2, the sole histone H3K36 trimethyltransferase, is frequently mutated or deleted in human cancer, including pancreatic ductal adenocarcinoma (PDAC). However, whether SETD2/H3K36me3 alteration results in PDAC remains largely unknown. DESIGN: TCGA(PAAD) public database and PDAC tissue array with SETD2/H3K36me3 staining were used to investigate the clinical relevance of SETD2 in PDAC. Furthermore, to define the role of SETD2 in the carcinogenesis of PDAC, we crossed conditional Setd2 knockout mice (PdxcreSetd2flox/flox) together with KrasG12D mice. Moreover, to examine the role of SETD2 after ductal metaplasia, Crisp/cas9 was used to deplete Setd2 in PDAC cells. RNA-seq and H3K36me3 ChIP-seq were performed to uncover the mechanism. RESULTS: SETD2 mutant/low expression was correlated with poor prognosis in patients with PDAC. Next, we found that Setd2 acted as a putative tumour suppressor in Kras-driven pancreatic carcinogenesis. Mechanistically, Setd2 loss in acinar cells facilitated Kras-induced acinar-to-ductal reprogramming, mainly through epigenetic dysregulation of Fbxw7. Moreover, Setd2 ablation in pancreatic cancer cells enhanced epithelia-mesenchymal transition (EMT) through impaired epigenetic regulation of Ctnna1. In addition, Setd2 deficiency led to sustained Akt activation via inherent extracellular matrix (ECM) production, which would favour their metastasis. CONCLUSION: Together, our findings highlight the function of SETD2 during pancreatic carcinogenesis, which would advance our understanding of epigenetic dysregulation in PDAC. Moreover, it may also pave the way for development of targeted, patients-tailored therapies for PDAC patients with SETD2 deficiency.

Correction to: Platelets promote breast cancer cell MCF-7 metastasis by direct interaction: surface integrin α2ß1-contacting-mediated activation of Wnt-ß-catenin pathway.

An amendment to this paper has been published and can be accessed via the original article.

GABRP regulates chemokine signalling, macrophage recruitment and tumour progression in pancreatic cancer through tuning KCNN4-mediated Ca2+ signalling in a GABA-independent manner.

BACKGROUND AND AIMS: Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer-related death worldwide. Neurotransmitter-initiated signalling pathway is profoundly implicated in tumour initiation and progression. Here, we investigated whether dysregulated neurotransmitter receptors play a role during pancreatic tumourigenesis. METHODS: The Cancer Genome Atlas and Gene Expression Omnibus datasets were used to identify differentially expressed neurotransmitter receptors. The expression pattern of gamma-aminobutyric acid type A receptor pi subunit (GABRP) in human and mouse PDAC tissues and cells was studied by immunohistochemistry and western blot analysis. The in vivo implications of GABRP in PDAC were tested by subcutaneous xenograft model and lung metastasis model. Bioinformatics analysis, transwell experiment and orthotopic xenograft model were used to identify the in vitro and in vivo effects of GABRP on macrophages in PDAC. ELISA, co-immunoprecipitation, proximity ligation assay, electrophysiology, promoter luciferase activity and quantitative real-time PCR analyses were used to identify molecular mechanism. RESULTS: GABRP expression was remarkably increased in PDAC tissues and associated with poor prognosis, contributed to tumour growth and metastasis. GABRP was correlated with macrophage infiltration in PDAC and pharmacological deletion of macrophages largely abrogated the oncogenic functions of GABRP in PDAC. Mechanistically, GABRP interacted with KCNN4 to induce Ca2+ entry, which leads to activation of nuclear factor κB signalling and ultimately facilitates macrophage infiltration by inducing CXCL5 and CCL20 expression. CONCLUSIONS: Overexpressed GABRP exhibits an immunomodulatory role in PDAC in a neurotransmitter-independent manner. Targeting GABRP or its interaction partner KCNN4 may be an effective therapeutic strategy for PDAC.

Overexpression of Rac GTPase Activating Protein 1 Contributes to Proliferation of Cancer Cells by Reducing Hippo Signaling to Promote Cytokinesis.

BACKGROUND & AIMS: Agents designed to block or alter cytokinesis can kill or stop proliferation of cancer cells. We aimed to identify cytokinesis-related proteins that are overexpressed in hepatocellular carcinoma (HCC) cells and might be targeted to slow liver tumor growth. METHODS: Using the Oncomine database, we compared the gene expression patterns in 16 cancer microarray datasets and assessed gene enrichment sets using gene ontology. We performed immunohistochemical analysis of an HCC tissue microarray and identified changes in protein levels that are associated with patient survival times. Candidate genes were overexpressed or knocked down with small hairpin RNAs in SMMC7721, MHCC97H, or HCCLM3 cell lines; we analyzed their proliferation, viability, and clone-formation ability and their growth as subcutaneous or orthotopic xenograft tumors in mice. We performed microarray analyses to identify alterations in signaling pathways and immunoblot and immunofluorescence assays to detect and localize proteins in tissues. Yeast 2-hybrid screens and mass spectrometry combined with co-immunoprecipitation experiments were used to identify binding proteins. Protein interactions were validated with co-immunoprecipitation and proximity ligation assays. Chromatin immunoprecipitation, promoter luciferase activity, and quantitative real-time polymerase chain reaction analyses were used to identify factors that regulate transcription of specific genes. RESULTS: The genes that were most frequently overexpressed in different types of cancer cells were involved in cell division processes. We identified 3 cytokinesis-regulatory proteins among the 10 genes most frequently overexpressed by all cancer cell types. Rac GTPase activating protein 1 (RACGAP1) was the cytokinesis-regulatory protein that was most highly overexpressed in multiple cancers. Increased expression of RACGAP1 in tumor tissues was associated with shorter survival times of patients with cancer. Knockdown of RACGAP1 in HCC cells induced cytokinesis failure and cell apoptosis. In microarray analyses, we found knockdown of RACGAP1 in SMMC7721 cells to reduce expression of genes regulated by yes-associated protein (YAP) and WW domain containing transcription regulator 1 (WWTR1 or TAZ). RACGAP1 reduced activation of the Hippo pathway in HCC cells by increasing activity of RhoA and polymerization of filamentous actin. Knockdown of YAP reduced phosphorylation of RACGAP1 and redistribution at the anaphase central spindle. We found transcription of the translocated promoter region, nuclear basket protein (TPR) to be regulated by YAP and coordinately expressed with RACGAP1 to promote proliferation of HCC cells. TPR redistributed upon nuclear envelope breakdown and formed complexes with RACGAP1 during mitosis. Knockdown of TPR in HCC cells reduced phosphorylation of RACGAP1 by aurora kinase B and impaired their redistribution at the central spindle during cytokinesis. STAT3 activated transcription of RACGAP in HCC cells. CONCLUSIONS: In an analysis of gene expression patterns of multiple tumor types, we found RACGAP1 to be frequently overexpressed, which is associated with shorter survival times of patients. RACGAP1 promotes proliferation of HCC cells by reducing activation of the Hippo and YAP pathways and promoting cytokinesis in coordination with TPR.

Alternative transcription start site selection in ACSS2 controls its nuclear localization and promotes ribosome biosynthesis in hepatocellular carcinoma.

Acetyl-CoA synthetase 2 (ACSS2) generates acetyl-CoA from acetate is important for histone acetylation and gene expression. ACSS2 fulfills distinct functions depending on its cellular location in tumor cells. The role and cellular localization of ACSS2 in hepatocellular carcinoma (HCC) remains to be studied. Herein, we identified that the alternative transcription start site selection of ACSS2 was significantly different between HCC and corresponding adjacent tissues. Alternative transcription start site selection produced two different ACSS2 transcripts, ACSS2-S1 and ACSS2-S2. The two isoforms of ACSS2 had different subcellular localization and different functions. Overexpression of ACSS2-S2 promoted cell proliferation and invasion, but ACSS2-S1 did not. The ACSS2-S1 was mainly present in cytoplasm, and ACSS2-S2 was distributed in both nucleus and cytoplasm. Finally, we demonstrated that alternative transcription start site selection of ACSS2 correlates ribosome biogenesis in HCC. Our findings reveal an oncogenic role of ACSS2-S2 in HCC progression via increase of ribosome biogenesis, and suggest ACSS2-S2 might be a potential therapeutic target against the HCC.