Human papillomaviruses (HPV) are one of the deadliest viruses causing cancer resulting in over 300,000 deaths annually in the world. HPV16 is the most oncogenic form of HPV and is responsible for over half of all HPV-driven tumors. The viral E7 gene encodes a powerful oncoprotein that can cause infected cells to have uncontrolled growth. A previous study showed that HPV16 isolates with amino acid alterations in the E7 gene were nearly always found in women with HPV infection but no cancer. To study the role of these variants, we expressed them in cultured cells. Most variants produced lower levels of protein, and selected variants had reduced activity in allowing cells to grow in assays that measure tumor cell invasiveness. The data indicate that inhibition of E7 could be a practical approach to treating HPV-driven cancers.The human papillomavirus (HPV) type 16 E7 oncogene is critical to carcinogenesis and highly conserved. Previous studies identified a preponderance of non-synonymous E7 variants amongst HPV16-positive cancer-free controls compared to those with cervical cancer. To investigate the function of E7 variants, we constructed full-length HPV16 E7 genes and tested variants at positions H9R, D21N, N29S, E33K, T56I, D62N, S63F, S63P, T64M, E80K, D81N, P92L, and P92S (found only in controls); D14E, N29H cervical intraepithelial neoplasia (CIN2), and P6L, H51N, R77S (CIN3). We determined the steady-state level of cytoplasmic and nuclear HPV16 E7 protein. All variants from controls showed a reduced level of E7 protein, with 7/13 variants having lower protein levels. In contrast, 2/3 variants from the CIN3 precancer group had near-wild type E7 levels. We assayed the activity of representative variants in stably transfected NIH3T3 cells. The H9R, E33K, P92L, and P92S variants found in control subjects had lower transforming activity than D14E and N29H variants (CIN2), and the R77S (CIN3) had activity only slightly reduced from wild-type E7. In addition, R77S and WT E7 caused increased migration of NIH3T3 cells in a wound-healing assay compared with H9R, E33K, P92L, and P92S (controls) and D14E (CIN2). These data provide evidence that the E7 variants found in HPV16-positive cancer-free women are partially defective for transformation and cell migration, further demonstrating the importance of fully active E7 in cancer development. Cervical cancer is the second most common cancer among women worldwide and remains a clinical problem despite early detection and therapy . HPV16 iHPV16 E7 is a multifunctional protein interacting with multiple cellular factors ,9,10. OnHPV16 E7 is only 98-amino acids, yet it binds to multiple cellular proteins . The E7 In a study of 5570 HPV16 infected cervical precancer/cancer cases and HPV16-positive cancer-free controls, we observed significantly more E7 amino-acid altering variants in controls than in cases . The sub® PCR Cloning Kit according to the manufacturer’s instructions . E7 protein expression was confirmed by Western blotting, with three positive control vectors from Addgene, CMV16 E7 (WT), CMV16 E7 C91S (MT1), CMV16 E7 del DLYC (MT2) in 293T (A synthetic full-length HPV16 A1 sequence (GenBank accession number is NC-001526.4) was used as a template for preparing an HPV16 E7 construct with the primer set: RecNheI_HPV16-A1E7 Forward; RecBamHI_HPV16-A1E7 Reverse . The HPV in 293T . In addi in 293T . We foun® PCR Cloning  and site-directed mutagenesis methods  according to the manufacturer’s instructions. The oligonucleotides used to generate the mutations are listed in The pcDNA3.1(+) IRES GFP-HPV16 E7 WT was used as a template to generate variant constructs using the CloneEZ5 cells per well and transfected after 48 h with 3 µg of plasmid DNA using HilyMax  transfection reagents according to the manufacturer’s instructions. The empty vector and wild-type E7 constructs were used as negative and positive controls, and the transfection efficiency of the mutants was assessed by imaging green fluorescent protein (GFP) signals through fluorescent microscopy with a ZOE Fluorescent Cell Imager  (The cancer cell lines CaSki (RRID: CVCL_1100), SiHa (RRID: CVCL_0032), HeLa (RRID: CVCL_0030), and HEK293T (RRID: CVCL_0063), as well as mouse fibroblast NIH-3T3 (RRID: CVCL_0594) cells, were obtained from ATCC and cultured according to the provider’s instructions and were regularly tested for mycoplasma infection. The identify of CaSki, SiHa, and NIH-3T3 cells have been validated by Identifiler  typing in the last three years. To determine HPV16 E7 protein expression in vitro, HeLa and 293T cells were seeded in 6 well plates at 2.5 × 10 CA, USA) . At 48 hNuclear and cytoplasmic extracts were prepared from transfected HeLa, 293T, and NIH 3T3 cell lines using the CellLytic NuCLEAR extraction kit (Sigma-Aldrich). Protein concentration was measured with a Bio-Rad protein assay. The nuclear and cytoplasmic lysates were resolved on 10% NuPAGE Novex Bis-Tris polyacrylamide gels , transferred to a PVDF membrane, blocked at room temperature for two hours in 5% milk in Tris-buffered saline with 0.1% Tween 20 (TBST), and then blotted. Western blot analysis was performed with the following antibodies: HPV16 E7 (716-325) mouse monoclonal antibody at 1:100 (Santa Cruz). In addition, we selected rabbit monoclonal antibody at 1:1000 of TBP (D5G7Y) and beta-Tubulin (9F3)  as loading controls for nuclear and cytoplasmic proteins for WB, respectively. Densitometric analysis of specific signals using Image Lab software 6 on three independent blots was quantitated and normalized to the loading control TBP and beta-Tubulin, respectively. The nuclear and cytoplasmic proteins of HPV16 E7 wild type were considered as 100% expression. Original Western blot images are found in NIH3T3 cells stably transfected with the empty vector, wild type E7, and the indicated E7 variants were plated in 75 mL flasks at an equal cell density in DMEM medium with G418 500µg/mL. At 60% confluence, cell morphology was examined and imaged under a microscope with 10× and 20× magnification . NIH3T3 cells were used in an HPV16 transformation assay by examining anchorage-independent growth in soft agar. NIH3T3 cells were transfected with pcDNA3.1(+) IRES GFP-HPV16 E7 vectors as described above. At 48 h after transfection, cells were selected in a medium containing G418. After three weeks of selection, the stably transfected cells were seeded into soft agar in 96-well plates.4 cells per well, and duplicate plates were tested using Quantitation of Anchorage-Independent Growth and colony formation. The colony formation was quantified by solubilizing the soft agar, lysing the cells, and incubating cell lysates with the CyQUANT GR Dye (Cell Biolabs) with the GLOMAX Multi Detection System . In addition, the colonies were imaged under a microscope .We used the Cytoselect 96-well Cell Transformation Assay  to assess colony formation according to the manufacturer’s instructions. We counted live cells (variability >95%) using the Countess automated cell counter , and each cell line was counted twice. The cells were seeded in soft agar at a density of 1 × 10 5 live cells per well. Cell monolayers were scratched using an Auto-scratch machine (BioTek). The plate was placed in the Lionheart FX Automated Live Cell Imager in 5% CO2 at 37 °C. The wound area was imaged every 2 h for 20 h using a 4× objective and phase contrast, and bright field illumination. The number of cells migrating into the wound area was counted using percent confluency within the wound area (www.biotek.com (accessed on 4 October 2022)).The migration of NIH3T3 stably transfected cells was monitored by the Lionheart FX Automated Live Cell Imager , and cellular analysis of the kinetic images was performed using Gen5 software. Cells were grown in T75 flasks until they reached 60–75% confluency. The cells were detached from the flask using phenol red-free TrypLE Express Trypsin (Gibco/Thermo Fisher). The method for cell counting was the same as described above, and cells were seeded in 24-well plates with 1.0 × 10t-tests were performed using GraphPad Prism version 8 for Windows 10; p-values of <0.05 were considered significant.Mann–Whitney-U tests and unpaired two-tailed To test E7 protein expression and function of 18 specific E7 variants, we generated expression constructs in the pcDNA3.1(+) IRES GFP vector. We constructed nine variants using the ClonEZ PCR cloning method, nine by site-directed mutagenesis, and all clones were confirmed by DNA sequencing. To determine if the variants affect the level of steady-state E7 protein, we transiently transfected them into HeLa, and 293T cells and nuclear and cytoplasmic proteins were detected by Western blotting (WB). These cell lines were chosen because HeLa cells are derived from cervical cancer and have an intact pRB tumor suppressor pathway ,24,25,26The anti-HPV16 E7 (716-325) antibody was tested for sensitivity and specificity with 293T transfected with three positive control vectors and in cervical cancer cell lines containing HPV16 (CaSki and SiHa) or HPV18 (HeLa) . The posThe HPV16 E7 protein has been previously localized to the cytoplasm and nucleus and observed as a dimer in the nucleus ,31,32,33Similarly, we found a severe to moderate reduction of E7 protein levels for the D14E, N29H variants identified in CIN2 and H9R and E33K in controls, in either HeLa or 293T cells . Two of To test the ability of HPV16 E7 transfected cells in a cellular transformation assay, we chose a representative subset of variants, five from the control group  and three from the CIN2 or CIN3 precancer groups , to assess cell morphology and growth in NIH3T3 cells, an established model of transforming gene function. As shown in We assessed growth and colony size in soft agar to determine the effects of HPV16 E7 variants on cellular transformation. NIH3T3 cells or empty vector-transfected cells display few colonies of small size, whereas WT E7-transfected cells showed an increased number of colonies and of a larger size . We usedTo determine the effect of a representative subset of seven variants  on cell migration in a wound-healing assay, we performed automated scratches of a cell monolayer, monitoring cell migration every two hours over 20 h. HPV16 E7 WT and R77S (from CIN3 precancer) NIH3T3 cells showed increased migration into the wound area as compared with H9R (CIN2), E33K (CIN2), P92L (Control), and P92S (Control) and moderately increased compared with D21N (Control) and D14E CIN2; . The perThe E7 oncogene is one of the two most important genes contributing to the cellular transformation and oncogenicity of high-risk HPV types. E7 is essential for the continued oncogenic growth of cervical cancer cells . As HPV1The study aimed to assess the role of E7 amino acid variants previously identified  in HPV-iThe level of E7 protein does not always reflect the transforming activity of the protein, and we, therefore, used multiple methods to assess E7 activity  in the soft agar assay. Aspartic and glutamic acid are similarly charged amino acids, and this alteration may only slightly affect E7 activity. Likewise, the other alterations identified in CIN2 or CIN3 precancer  appear to be residues that can tolerate change. Gillison et al.  identifiWe also assessed the activity of stably transformed NIH3T3 cells to migrate in culture using the wound-healing (scratch) assay. Using an instrument that automatically generates uniform scratches and continuously monitors cell growth over 20 h we show a dramatic difference between NIH3T3 cells containing an empty vector and those with the wild-type E7 construct. Scratch assays have been used to monitor oncoprotein E6 and E7 function and demonstrate that HPV16 oncoproteins promote cell migration and invasion ,42. StilInterestingly, the previous finding that an alteration of the highly conserved arginine at residue 77 to glutamic acid (R77E)  has a hiNotably, no variants in functional or structural E7 motifs, including the conserved residues of the LxCxE and CXXC motifs, have been found in patients. Mutations in these residues, C24G, E26G for LxCxE, and C58G and C91G for CXXC, dramatically reduce or eliminate E7 transforming activity ,44,45. EThe limitations of this study are that we did not determine the molecular basis for the reduced steady-state level of protein and performed functional analyses with stable clones on a subset of the variants. In addition, our functional assays involve NIH3T3 mouse fibroblast cells, and it would be informative to repeat these results using viral constructs in primary cells or an infectious model of HPV ,44,45. HIn conclusion, cervical cancer development appears to require the continuous activity of a fully functional E7 protein. Our genetic epidemiologic data previously demonstrated that variation in E7 is poorly tolerated . Our res


The S100A levels were generally increased in CD19+ cells compared to MNCs of CLL. The S100A4 gene expression was significantly stimulated (p < 0.05) by the inhibition of the PI3K/AKT signaling pathway in MNCs. IL-6 stimulated S100A4 and S100A8 protein expression, prevented by the NF-κB and JAK1/2 inhibitors. In contrast, IL-10 reduced S100A8, S100A9, and S100A12 protein expressions in MNCs of CLL. Moreover, IL-10 inhibited activation of NF-κB signaling . In conclusion, inflammation stimulated the S100A protein expression mediated via the proliferation-related signaling and balanced by the cytokines in CLL.The calcium-binding proteins S100A4, S100A8, and S100A9 are upregulated in chronic lymphocytic leukemia (CLL), while the S100A9 promotes NF-κB activity during disease progression. The S100-protein family has been involved in several malignancies as mediators of inflammation and proliferation. The hypothesis of our study is that S100A proteins are mediators in signaling pathways associated with inflammation-induced proliferation, such as NF-κB, PI3K/AKT, and JAK/STAT. The mononuclear cells (MNCs) of CLL were treated with proinflammatory IL-6, anti-inflammatory IL-10 cytokines, inhibitors of JAK1/2, NF-κB, and PI3K signaling pathways, to evaluate S100A4, S100A8, S100A9, and S100A12 expression as well as NF-κB activation by qRT-PCR, immunocytochemistry, and immunoblotting. The quantity of S100A4, S100A8, and S100A9 positive cells ( Chronic lymphocytic leukemia (CLL) is the most common malignancy in the elderly population characterized by increased proliferation and accumulation of B monocytes in the bone marrow . CLL celS100As proteins belong to a group of low-molecular-weight proteins with a significant role in the regulation of inflammation-related processes in a large number of malignancies including CLL ,8. In adA previous study showed that elevated IL-6 levels in plasma were in positive correlation with a poorer medical outcome in CLL patients . IncreasDifferent types of cancer cells increasingly produce and secrete IL-6 because of the constant stimulation of STAT3 or NF-κB . AdditioThe hypothesis of our study was that inflammation induces proliferation and apoptosis related to signaling pathways in CLL. We targeted inflammatory S100A proteins and their regulation by inflammatory cytokines mediated by JAK2-STAT, PI3K-AKT, and NF-κB signaling pathways in patients with CLL.+ cells in CLL patients regardless of genetic abnormalities. S100A4, S100A8, and S100A9 had a similar pattern of expression and showed a higher level of immunopositive cells in the MNCs of healthy controls than in CLL patients, and demonstrated a higher level of expression in CD19+ cells than in MNCs of CLL patients , and Trisomy 12 (10%) . We alsopatients . The perp < 0.05, + cells than in samples of control cells  than in healthy control cells , while Ly294002 significantly stimulated the level of S100A12 gene expression  in comparison to nontreated MNCs of CLL patients (p < 0.01) in comparison to nontreated MNCs (p < 0.001) in combination with IL-6  and Ruxolitinib (p < 0.001) prevented the IL-6 induction of the S100A4 protein expression in MNCs (p < 0.01) and Ruxolitinib (p < 0.05) reduced the S100A9 protein expression (p < 0.001) and S100A12 (p < 0.05) protein expressions  in MNCs of CLL (p < 0.05), S100A9 (p < 0.001), and S100A12 (p < 0.01) protein expressions in MNCs of CLL  protein expressions in MNCs of CLL patients . The S10s of CLL A. IL-10 s of CLL B–D. The + cells of CLL .In our study, we included 60 CLL patients’ samples. All patients’ samples were collected at the time of diagnosis and before treatment. All 60 CLL patients included in our study signed the consent form approved by a local ethical committee in accordance with the Declaration of Helsinki. We collected 30 mL of peripheral blood from all subjects, and it was drawn and collected in disodium EDTA. MNCs, after the process of isolation, were washed in PBS and resuspended in an RPMI-1640 medium . After that, MNCs were pretreated for one hour with 1 μM or 1.2 μM Ruxolitinib , 5 μM or 10 μM Ly294002 , and 0.75 μM or 1.5 μM JSH23 , and treated for one hour with IL-6 or IL-10 . Then MNCs were washed in PBS and treated with RIPA lysis buffer at 4 °C for 30 min. After those treatments, MNCs were centrifuged at 10000× Isolation of proteins from MNCs of CLL was conducted as previously reported  and 30 μ4 cells per slide) were fixed to super frost plates using methanol. Blocking of nonspecific staining derived from endogenous peroxidase was performed by treating the samples with 3% hydrogen peroxide. The samples were then treated and incubated with appropriate primary antibodies overnight at +40 °C. The streptavidin-biotin technique  was used for staining. The DAKO Liquid DAB+ Substrate/Chromogen System (DAKO) was used to visualize the positive immunoreactivity, while the contrast of unstained nuclei was performed using Mayer’s hematoxylin . As a negative control, we used samples treated with PBS without a primary antibody. From each section, five fields with the average number of immunoreactive cells were imaged and processed. Imaging was performed using a light microscope , while immunoreactive cell counting was performed using a special program . All images had an 40x magnification.Immunocytochemical staining was performed on cytospin mononuclear samples isolated from 10 healthy controls and 30 CCL patients. Mononuclear cells . Equal RNA concentrations of all samples were used for transcription into cDNA using the Maxima First Strand cDNA Synthesis kit .Real-time quantitative PCR analysis for S100A4, S100A8, S100A9, and S100A12 were performed using appropriate primers and Maxima SYBR Green/ROX qPCR master mix  at Mastercycler EP RealPlex .The obtained experimental results are presented as mean ± standard deviation. The statistical program Prism 4 was used for the analysis, while one-way ANOVA and Dunnett’s posttest were used as statistical tests.


Intratumoral heterogeneity is the primary challenge in the treatment of glioblastoma (GBM). The presence of glioma stem cells (GSCs) and their conversion between different molecular phenotypes contribute to the complexity of heterogeneity, culminating in preferential resistance to radiotherapy. ARP2/3 (actin-related protein-2/3) complexes (ARPs) are associated with cancer migration, invasion and differentiation, while the implications of ARPs in the phenotype and resistance to radiotherapy of GSCs remain unclear.We screened the expression of ARPs in TCGA-GBM and CGGA-GBM databases. Tumor sphere formation assays and limiting dilution assays were applied to assess the implications of ARPC1B in tumorigenesis. Apoptosis, comet, γ-H2AX immunofluorescence (IF), and cell cycle distribution assays were used to evaluate the effect of ARPC1B on radiotherapy resistance. Immunoprecipitation (IP) and mass spectrometry analysis were used to detect ARPC1B-interacting proteins. Immune blot assays were performed to evaluate protein ubiquitination, and deletion mutant constructs were designed to determine the binding sites of protein interactions. The Spearman correlation algorithm was performed to screen for drugs that indicated cell sensitivity by the expression of ARPC1B. An intracranial xenograft GSC mouse model was used to investigate the role of ARPC1B in vivo.We concluded that ARPC1B was significantly upregulated in MES-GBM/GSCs and was correlated with a poor prognosis. Both in vitro and in vivo assays indicated that knockdown of ARPC1B in MES-GSCs reduced tumorigenicity and resistance to IR treatment, whereas overexpression of ARPC1B in PN-GSCs exhibited the opposite effects. Mechanistically, ARPC1B interacted with IFI16 and HuR to maintain protein stability. In detail, the Pyrin of IFI16 and RRM2 of HuR were implicated in binding to ARPC1B, which counteracted TRIM21-mediated degradation of ubiquitination to IFI16 and HuR. Additionally, the function of ARPC1B was dependent on IFI16-induced activation of NF-κB pathway and HuR-induced activation of STAT3 pathway. Finally, we screened AZD6738, an ataxia telangiectasia mutated and rad3-related (ATR) inhibitor, based on the expression of ARPC1B. In addition to ARPC1B expression reflecting cellular sensitivity to AZD6738, the combination of AZD6738 and radiotherapy exhibited potent antitumor effects both in vitro and in vivo.ARPC1B promoted MES phenotype maintenance and radiotherapy resistance by inhibiting TRIM21-mediated degradation of IFI16 and HuR, thereby activating the NF-κB and STAT3 signaling pathways, respectively. AZD6738, identified based on ARPC1B expression, exhibited excellent anti-GSC activity in combination with radiotherapy.The online version contains supplementary material available at 10.1186/s13046-022-02526-8. Glioblastoma (GBM), characterized by wild-type isocitrate dehydrogenase (IDH), is the most aggressive and fatal primary brain tumor in adults , 2. DespAccording to bulk expression profiles, there are three phenotypes of GBM, termed proneural (PN), classical (CL) and mesenchymal (MES). GBM patients with the MES phenotype exhibit worse survival and enhanced resistance to radiotherapy than patients with the PN phenotype , 7. StudGSCs are characterized by multilineage differentiation and self-renewal ability, exhibiting preferential resistance to radiotherapy and chemotherapy , 12. SimThe actin-related protein-2/3 (ARP2/3) complex was the first molecule identified to initiate new filament polymerization . It is aThe main strategy for treatment of refractory or recurrent gliomas is the combination of standard radiotherapy and chemotherapy with targeted agents. Ataxia telangiectasia and Rad3-related (ATR), serving as a serine/threonine protein kinase, plays an essential role in coordinating the DNA damage response and maintaining genomic stability . A growiIn this study, we found that ARPC1B was upregulated in the MES phenotype and predicted a poor outcome. In vitro and in vivo studies demonstrated that knockdown of ARPC1B suppressed the MES phenotype and promoted radiotherapy sensitivity in GSCs, whereas overexpression of ARPC1B promoted MES phenotype transformation and radiotherapy resistance. Mechanistically, ARPC1B directly binds to IFI16 and HuR, preventing TRIM21-mediated degradation of IFI16 and HuR by ubiquitination, thereby activating the STAT3 and NF-κB signaling pathways, respectively. As discussed previously, activation of STAT3 and NF-κB facilitated conversion of PN-GSCs to MES-GSCs, which exhibited greater resistance to radiotherapy. Moreover, we identified a selective ATR inhibitor, AZD6738, based on the expression of ARPC1B. We validated that AZD6738 in combination with radiotherapy exhibited excellent anti-GSC activity for the first time.The patient-derived PN-GSCs (GSC 8-11 and GSC 11), MES-GSCs  and neural progenitor cell (NPC) were a kind gift from Dr. Frederick F. Lang and Dr. Krishna P.L. Bhat . Phenotypes of GSCs in this study have been identified according to the genetic signature of Philips and Verhaak, and are widely accepted and applied , 17, 41.We performed persistent knockdown of ARPC1B by lentivirus expressing sh-ARPC1B, which were synthesized by GeneChem . The ARPC1B, IFI16 and HuR overexpression and the corresponding control lentiviruses were also synthesized by GeneChem . The RNA-interfering sequences used in this study are listed in The total RNA was extracted by TRIzol  following manufacturer’s protocol. We performed reverse transcription using high-capacity-cDNA Reverse Transcription Kit  according to the protocol. The PCR primer pairs’ sequences were listed in GSCs were inoculated into 6-well plates at a density of 1000 cells per well and cultured for 7 days with 1.5 ml of GSC culture medium. The relative diameters of tumor spheres were recorded by an optical microscope and used for subsequent analysis.http://bioinf.wehi.edu.au/software/elda/).GSCs were inoculated into 96-well plates at a gradient of 0, 2, 4, 8, 16, 32, 64 or 128 cells per well and with 10 replicate wells per set. We observed and recorded the number of wells with tumor spheres formation 7 days later, and analyzed the collecting data using (GSCs were treated with IR (6 Gy) for in vitro radiotherapy assays. The apoptosis, comet assay, cell-cycle analysis and γ-H2AX IF staining assay were performed 96 h later. In term of mouse experiments, we treated mice with 4 doses of IR (2.5 Gy each) within 7 to 14 days after GSCs injection.GSCs were treated with IR (6 Gy) and total GSCs were collected after 96 h to assess apoptotic rate. We leveraged Apoptosis detection kit  for apoptosis detection in accordance with manufacturer’ s protocol. The final results were generated by a BD Accuri C6 flow cytometer  and further processed by FlowJo V10.5 cells/ml density. We then mixed the GSCs with LM agarose in a ratio of 1:10 (V/V) and added 50 µl of the mixture onto a comet slide immediately. We further lysed the cells using alkaline lysis buffer for 12 h at 4℃ and soaked the slides into alkaline electrophoresis solution for 20 min. Then we performed electrophoresis procedure for slides at 25 V for 30 min. Finally, we stained slides with Green-DNA Dye and collected images with a fluorescence microscopy.We performed alkaline comet assay to detect the DNA damage levels following manufacturer’s instruction . In brieTUNEL assay was conducted using TUNEL apoptosis assay kit  in accordance with manufacturer’s instruction. In brief, tissue sections of mice with different interventions were fixed with 4% paraformaldehyde and washed with PBS. Then the sections were permeabilized in PBS with 0.5% Triton X-10. The detection solution and DAPI solution were prepared for staining apoptotic cells and nuclei, respectively. We captured images with a fluorescent microscope and recorded the percentage of apoptotic cells.GSCs were treated with IR (6 Gy), and dissociated and fixed in cold ethanol after 96 h. Then we resuspended and stained the GSCs with propidium iodide (PI) staining solution . Finally, we conducted cell-cycle analysis leveraging a a BD Accuri C6 flow cytometer.For Western blotting assay, we lysed GSCs in different groups using RIPA with 1% protease and phosphate inhibitor. We then separated proteins by electrophoresis using SDS-PAGE gels and transferred the proteins to polyvinylidene fluoride (PVDF) membrane. Primary anti-bodies were incubated with membranes at 4 °C overnight. After that, we removed primary antibodies and incubated the blots with secondary antibodies at room temperature for 1 h. We examined proteins using an Odyssey fluorescence scanner .For IHC assay, tissue sections from GSC xenografted mice were stained with the primary antibodies against CD44. We captured images of sections with an optical microscope and evaluated them quantitatively in accordance with German IHC scoring system .For IF assay, we attached and fixed GSCs in μ-slide 8-well plates . We then permeabilized and blocked GSCs using 0.3% Triton X-100 as well as 5% Goat serum, respectively. After that, we incubated the GSCs with primary antibodies at 4 °C overnight, and then incubated GSCs with fuorescent secondary antibodies and DAPI for 1 h and 15 min, respectively. GSCs were washed three times with PBS after each staining or process. Finally, the representative images were collected leveraging a LeicaSP8 confocal microscope for further analysis.All antibodies used in this research are presented in We conducted co-IP assay leveraging Pierce Classic Magnetic IP/co-IP Kit  in accordance with manufacturer’s protocol. In brief, we incubated antibodies with protein A/G magnetic beads. Then we obtained GSCs lysates and mixed with antibody coupled beads overnight at 4 °C. After washing and denaturation procedures, proteins interacting with the beads are collected for western blotting.CHX is a protein translation inhibitor, widely used to analyze half-life of proteins. We treated different groups of GSCs with 100 μg/ml CHX for 0 h, 3 h, 6 h or 9 h, followed by collecting proteins and performing western blotting assay to detect protein levels.6 GSCs into the right frontal lobe of the mice for the construction of GSCs xenografts. For the IR treatment group, mice were given four doses of IR (2.5 Gy each) between 7 and 14 days postoperatively. We assessed in vivo tumor growth by intraperitoneal injection of 150 mg/kg of fluorescein followed by bioluminescence. Representative images and quantification of bioluminescence were obtained from an IVIS Lumina series III ex vivo imaging system .The animal experiments were approved by the Institutional Animal Care and Use Committee of Qilu Hospital of Shandong University. We purchased 4-week-old male BALB/c nude mice from the Model Animal Research Center of Nanjing University . We randomly grouped similarly situated mice into different groups in preparation for construction of intracranial GSCs xenograft models. We prepared luciferase labeled GSC 267, GSC 20 and GSC 8-11 cells with different interventions of ARPC1B expression. We injected 1 × 10https://portal.gdc.cancer.gov/). The RNA-seq transcriptome data and clinical traits of the CGGA GBM were downloaded from CGGA database (http://www.cgga.org.cn/).Transcript level data and associated clinical information of TCGA GBM were extracted from GDC Data Portal  and analyzed using R package “Seurat 4.1.0”. Method “UMAP” was applied for the visualization of different cell clusters. R package “irGSEA” was used to calculate and visualize the enrichment scores of the Verhaak_GBM_MES signature by method “UCell”.The single-cell RNA-sequencing of gliomas were downloaded from Gene Expression Omnibus  [https://portals.broadinstitute.org/ccle/). The drugs significantly implicated in the expression of ARPC1B were screened according to Spearman correlation analysis.We downloaded the drug sensitivity data of GBM cell lines from Genomics of Drug Sensitivity in Cancer  , whereasWe purchased AZD6738  from MedChemExpress . AZD6738 which was dissolved in dimethyl sulfoxide (DMSO) was stored at -20 °C and used up within one month. GSCs were inoculated into 96-well plates at a density of 5000 cells per well, and treated with different concentrations of AZD6738. The GSCs were cultured at 37 °C for 48 h. After that, we added 10 μl Cell Counting Kit-8 (CCK-8) solution to each well and the absorbance reflecting cell proliferation were measured 1 h later. GSCs were treated with 1 μM AZD6738 prior to IR intervention in vitro experiments. For in vivo experiments, AZD6738 was dissolved in a solution containing 10% DMSO, 40% propylene glycol and 50% deionized water, and administered orally to mice at a dose of 50 mg/kg.P-value < 0.05 were accepted as statistically significant .We leveraged GraphPad Prism 8.0 software and R 4.1.3 to perform all statistical analysis. The normality of distribution and the homogeneity of variance were proved by Shapiro–Wilk normality test and Bartlett test, respectively. The student’s t-Test and one-way ANOVA were performed to compare the certified data (significance > 0.1) between two groups and more than two groups, respectively. We performed Pearson correlation algorithm to evaluate the correlation between different groups. For survival analysis, Kaplan–Meier (KM) curve and log-rank test were performed to visualize and assess survival between different groups, respectively. p value  exhibited an obviously high expression of ARPC1B, whereas the PN GSCs (GSC 8-11 and GSC 11) and NPC barely expressed ARPC1B  sensor that plays an essential role in innate immune response against viral infection . The DNAWe performed qRT–PCR and observed that knockdown of ARPC1B in GSC 20 and GSC 267 cells or overexpression of ARPC1B in GSC 8-11 cells had no statistically significant impact on mRNA expression of either IFI16 or HuR Fig. SB, we hyphttp://ibs.biocuckoo.org/download.php). Collectively, we identified Pyrin and RRM2 as two key domains involved in ARPC1B-IFI16/HuR interactions, which were implicated in the PMT and radiotherapy resistance for GSCs. Our results may ensure more effective precision oncology and provide insights into the development of targeted drugs.Protein domains (PDs) are regions of proteins with specific spatial structures and independent functions, which are the key functional units for proteins to perform their biological functions. In a given protein, identification of the structural domains presented and the detailed functions of the domains provides insight into the function of the protein . To deteThe ubiquitin–proteasome system (UPS) is one of most prevalent types of posttranslational modifications and plays an essential role in various disorders, including cancer . AlthougThe activation of STAT3 and NF-κB signaling pathways driving PMT and radiotherapy resistance has been extensively studied and elucidated. Our fundings demonstrated that knockdown of ARPC1B in GSC 20 and GSC 267 cells reduced protein levels of p-STAT3, STAT3 and p-P65, whereas overexpression of ARPC1B in GSC 8-11 cells elevated protein levels of p-STAT3, STAT3 and p-P65  as well as GSC 267 cells (IC50 = 1.609 μM)   induced by DNA replication-related stress and cell cycle checkpoints . The actIn conclusion, we identified ARPC1B, which is significantly upregulated in MES-GBM/GSCs and is correlated with a poor prognosis. ARPC1B promotes PMT and radiotherapy resistance by inhibiting TRIM21-mediated degradation of IFI16 and HuR, thereby activating the NF-κB and STAT3 signaling pathways, respectively. AZD6738 in combination with radiotherapy exhibited potent anti-GSC effects. Our findings expand the understanding of the heterogeneity and plasticity of GBM and provide a potential therapeutic strategy for GBM treatment.Additional file 1: Figure S1. (A) The expression of ARPs among mesenchymal (MES), proneural (PN), and classical (CL) phenotypes in TCGA GBM. (B) Kaplan–Meier curves visualizing the overall survival of CCGA-GBM patients stratified according to expression of ARPs. (C) The expression of ARPs among MES, PN, and CL phenotypes in CGGA GBM. (D) GSEA exhibited a positive correlation between ARPC1B expression and MES phenotypes, and a negative correlation with PN phenotypes. (E) Single-cell RNA sequencing of GSE138794 visualizing the expression of ARPs other than ARPC1B. Fig. S2. (A) Correlation analysis of ARPC1B with CD44, YKL-40, OLIG2 and SOX2 in TCGA-GBM and CGGA-GBM, respectively. (B) Western blot analysis of ARPC1B and SOX2 protein levels in GSC 8-11 overexpressing ARCP1B.  Representative images and quantification of tumor sphere formation of GSC 8-11 (C) and GSC 11 (D) transduced with vector or ARPC1B. Scale bar, 100μm. (E) The protein expression of ARPC1B in GSC 20 and GSC 267 under different IR dose treatments. Fig. S3. (A) Flow cytometric analysis showing the effect of ARPC1B overexpression on the apoptosis in IR-treated (6Gy) GSC 8-11 cells. The right panels showing the quantification of apoptosis rate. (B) Representative images and quantification of comet assay showing the effect of ARPC1B overexpression on DNA damage of GSC 8-11 with IR treatment (6 Gy). Scale bar, 20μm. (C) Representative images and quantification of γ-H2AX IF staining showing the effect of ARPC1B knockdown on DNA damage of GSC 267 and GSC 20 with IR treatment (6 Gy). Scale bar, 40μm. (D) Representative images and quantification of γ-H2AX IF staining in GSC 8-11. Scale bar, 40μm. (E) Cell-cycle analysis of GSC 267, GSC 20 and GSC 8-11 in different treatment groups. The proportions of cells arrested in G2/M phase were quantified (right panel). Fig. S4.  Bioluminescence imaging of tumor size on day 7 in sh-control, sh-ARPC1B#1 and sh-ARPC1B#2 GSC 267 (A) or GSC 20 (B) xenograft nude mice in indicated groups. The right panel shows the quantification of photon counts of GSC 267 and GSC 20 xenografts.  Bioluminescence imaging of tumor size on day 7 (C) and day 30 (D) in Vector or ARPC1B-transfected GSC 8-11 xenograft nude mice receiving or exempt from IR treatment. The right panel showing the quantification of photon counts of GSC 8-11 xenografts. Fig. S5. (A) Representative images and quantification of IHC staining for CD44 in sections of non-IR GSC 267 xenografts , and TUNEL staining in sections of IR treated GSC 267 xenografts . (B) Representative images and quantification of IHC staining for CD44 in sections of non-IR GSC 8-11 xenografts , and TUNEL staining in sections of IR treated GSC 8-11 xenografts . (C) Representative images of H&E staining in sections from indicated xenografts. Scale bar, 400μm. (D) Western blotting analysis of protein levels of DBN1, ACTN4, FLNA, and CORO1C upon knockdown of ARPC1B in GSC 20 and GSC 267, or overexpression of ARPC1B in GSC 8-11. Fig. S6. (A) Co-IF staining exhibiting the distribution of ARPC1B with IFI16 or HuR in GSC 20. Scale bar, 5μm. (B) The protein levels of IFI16 and HuR after overexpression of ARPC1B in GSC 8-11. (C) The mRNA expression of IFI16 and HuR assessed by qRT-PCR assay. (D) The protein levels of IFI16 and HuR in sh-control or sh-ARPC1B-GSCs treated with 100μg/ml CHX for indicated times. (E) Representative images and quantification of IHC staining for ARPC1B, HuR and IFI16 in different groups of GSC 267 xenograft sections . Fig. S7. (A) Western blotting analysis showing the effect of TRIM21 knockdown on the protein levels of IFI16 and HuR in sh-ARPC1B GSCs. (B) Western blotting analysis showing the effect of ARPC1B knockdown in GSC 20 and GSC 267, or ARPC1B overexpression in GSC 8-11 on the protein levels of STAT3, p-STAT3, P65, and p-P65. (C) Western blotting analysis of protein levels of ARPC1B, IFI16, HuR, P65, p-P65, STAT3, p-STAT3 and, CD44 in GSC 20 treated with indicated interventions. (D) Western blotting analysis showing that knockdown of TRIM21 could reverse the effect of ARPC1B inhibition on IFI16 and HuR ubiquitination. GSCs were pretreated with MG132 (10 µM) for 6 hours before cell lysates were collected. Fig. S8. (A) Representative images of tumor sphere formation of GSC 267 and GSC 20 treated with indicated interventions. Scale bar, 100μm. (B) Representative images of flow cytometry assays showing apoptosis of GSC 267 and GSC 20 treated with indicated interventions. (C) Representative images of comet assays showing DNA damage of GSC 267 and GSC 20 treated with indicated interventions. Scale bar, 20μm. Fig. S9. (A) The correlation between the ARPC1B expression and drug sensitivity assessed by Spearman algorithm. (B) CCK-8 assay in GSC 267 and GSC 20 treated with different concentrations of AZD6738 for 48 h. (C) Representative images and quantification of apoptosis assays (upper panel) and comet assays  for GSC 20 and GSC 267 upon treatment with the indicated interventions. (D) The representative images and quantification of apoptosis assays (upper panel) and comet assays  for GSC 8-11 upon treatment with the indicated interventions. The right panels are the quantification of apoptosis rate and DNA damage, respectively. (E) Representative images and quantification of TUNEL staining in sections of GSC 267 xenografts for different groups. Scale bar, 200μm. (F) Bioluminescence imaging of tumor size on day 7 in GSC 267 xenograft nude mice treated with the indicated interventions. (G) The quantification of photon counts on day 7 of the GSC 267 xenografts. Supplementary Materials and Methods.Additional file 2: Table S1. Correlation analysis of ARPC1B with PN and MES marker genes (Correlation). Table S2. Mass spectrometry result of proteins related with ARPC1B. Table S3. The correlation between the ARPC1B expression and drug sensitivity assessed by Spearman algorithm (correlation).


Mfn1/Mfn2 double null cells and Mfn2 knock out (KO) fibroblasts, we measured the ability of this variant protein to change conformations and hydrolyze GTP. We found that a mutation in the HR1 domain (M376A) of MFN2 results in conformational change dysfunction while maintaining GTPase ability. Prolonged exposure to mitofusin agonist MiM 111 reverses mitochondrial fusion dysfunction in the HR1 mutant through encouraging an open conformation, resulting in a potential therapeutic model in this variant. Herein, we describe a novel mechanism of dysfunction in MFN2 variants through exploring domain-specific mitochondrial characteristics leading to CMT2A. Phenotypic variations in Charcot-Marie-Tooth disease type 2A (CMT2A) result from the many mutations in the mitochondrial fusion protein, mitofusin 2 (MFN2). While the GTPase domain mutations of MFN2 lack the ability to hydrolyze GTP and complete mitochondrial fusion, the mechanism of dysfunction in HR1 domain mutations has yet to be explored. Using  Mitochondria are double-membraned organelles that regulate cellular integrity, biological process and embryonic development. Notably, they possess highly dynamic morphology regulated by mitochondrial fusion and fission . MitochoIn mammalian cells, mitochondrial fusion is mediated by mitofusin 1 (MFN1), mitofusin 2 (MFN2), and optic protein atrophy-1 (OPA1). While OPA1 is an inner membrane protein, mitofusins are GTPase ubiquitinated proteins on the outer mitochondrial membrane . RegulatMFN1 and MFN2 share 80% of their sequences, with the N-terminus and C-terminus domains external to the cytosol, the transmembrane domain inside of the mitochondrial membranes and two coiled-coil heptad-repeat regions (HR1 and HR2) linked by hydrophobic bonds. Besides mitochondrial fusion, MFN2 is involved in tethering mitochondria to the endoplasmic reticulum and releasing calcium ,8. MFN2 MFN2 [MFN2 dysfunction, accounting for the progressive distal weakness and sensory loss associated with CMT2A [Charcot-Marie-Tooth disease type 2A (CMT2A), an untreatable neurodegenerative condition, can result from varying dominant hereditary mutations in MFN2 . Moreoveth CMT2A . We’ve pth CMT2A .MFN2 are related to CMT2A, with varying mild, moderate or severe phenotypes. These mutations take place in several mitofusin 2 domains , suggesting a necessary investigation into the effect of the location of the domain mutation on the properties of mitochondrial fusion. Loss of function mutations in the MFN2 GTPase domain, like The105Met and Arg94Glu, have an impaired ability to hydrolyze GTP to GDP, the last step in mitochondrial fusion. This results in shortened and depolarized “clumped mitochondria” that tether together but cannot fuse due to the absence of GTPase activity [MFN2 M376A (Met 376 to Ala) in the HR1 domain and MFN2 T105M (Thr 105 Met) and R94Q (Arg 94 Gln), GTPase mutations. Approximately 100 different dominant missense mutations in activity . It is cThe conformation for a closed/inactive mitofusin involves antiparallel intramolecular binding between the coiled-coil heptad-repeat regions HR1 and HR2 . The opeMFN2 T105M mutation, the first therapeutic approach to cure CMT2A in the mouse model. In CMT2A disease, only one copy of MFN2 is mutated. Mitofusin agonists interact with the two endogenous allele copies for MFN1 and the one functional copy of MFN2 to tether and fuse mitochondria together [Promoting mitochondrial elongation and fusion, mitofusin agonists can modulate the interactions between HR1 and HR2 domains ,14,15. Mtogether . 376 and His380 in the HR1 domain and Asp725 and Leu727 in the HR2 domain. We focused on Met376 because MFN2 M376V corresponds to a moderate phenyotype of human CMT2A. MFN2 M376 to Alanine in the HR1 domain induces a mitochondrial fusion defect, just like MFN2 R94Q and MFN2 T105M in the GTPase domain [Rocha AG et al.  demonstre domain .MFN2 M376A results in a dysfunctional conformation unable to complete mitochondrial fusion. GTPase mutations favor the protein’s closed/inactive conformation, resulting in short mitochondria that lack tethering and fusion abilities . Similar(1)MFN2 M376A mutation in the HR1 domain stems from mitofusin in the inactive conformation, notably maintaining its GTPase abilities.The source of dysfunction for the (2)MFN2 M376A lacks perinuclear mitochondrial clumping.(3)MFN2 M376A mutant protein and reverse its dysfunctional mitochondrial fusion abilities.Mitofusin agonists MiM 111 and Chimera C open the Here, we demonstrated: Mfn1/Mfn2 double null MEFs and Mfn2 KO cell fibroblasts were purchased from American Type Culture Collection . MEFs were cultured at 37 °C, 5% CO2—95% air in Dulbecco’s minimal essential medium (DMEM) containing glucose (4.5 g/L)  with 10% (v/v) fetal bovine serum , 1× nonessential amino acids (Gibco Cat: # 11130051), 2 mM L-glutamine , and 1% (v/v) penicillin/streptomycin (Gibco Cat: # 15140-122). MFN2 mutants and FRET probes were generated using PCR mutagenesis, sequence verified, and sent to Vector BioLabs. Ad-CMV-β-Gal was purchased from Vector BioLab (Cat: #1080).Hypothetical structures of human MFN2 were generated using I-TASSER and Chimera UCSF. The putative closed and open conformation were downloaded (PDB: 2J69 and AF-095140-F1). Domain coloring is as follows: Green GTPase (AA 94-265); Red HR1 (AA 338-421).2PO4, 15 nM Glucose, 20 nM HEPES pH: 7.2–7.5, and 1 mM CaCl2). Laser excitation was at 488 nm with emission at 510 nm for MitoTracker GREEN and laser excitation was 549 nm with emission at 590 nm for TMRE. Mitochondrial clumping and depolarization were measured as the percentage of cells with clumped mitochondria or depolarized mitochondria, respectively. Live cell studies assessing mitochondrial morphology were performed as described ,18. BrieMfn1/Mfn2 double-null MEFs were transduced with wild type ad-MFN2, ad-MFN2 R94Q, ad-MFN2 T105M or ad-MFN2 M376A at multiplicity of infection (M.O.I.) 50. Mitochondria were isolated as described [escribed , prepareMfn1/Mfn2 double-null MEFs were transduced with FRET ad-MFNs. Seventy-two hours later, mitochondria were isolated, and FRET was measured in a 96 well format as described previously [eviously . t-test; multiple group comparisons used one-way ANOVA with Tukey’s post hoc test for individual statistical comparisons. Data are reported as means ± SEM. Sample number (n) indicates the number of independent biological samples. Two-group comparisons used Student’s unpaired Most of the severe CMT2A disorders involve mutations in the mitofusin 2 GTPase domain (T105M and R94Q), compromising GTP hydrolysis and impairing mitochondrial fusion. MFN2 and MFN1 are dynamin family GTPases; catalytic GTPase activity is required for mitochondrial fusion, controlling the last/final step in fusing mitochondrial membranes. MFN2 Met376Val, mostly results in a moderate phenotype for CMT2A  break the intramolecular bond in the molecular core of mitofusins, promoting an active conformation capable of tethering. Using FRET mitofusin assay, GTPase defective CMT2A mutants R94Q and T105M retained the ability to change conformations under basal conditions; however, HR1 domain mutant MFN2 M376A failed to open and remained inactive  in mitofusins null mouse embryonic fibroblasts were compared to MFN2 WT. All three variants T105M, R94Q and M376A had a reduction in mitochondrial length/width (aspect ratio) A,B, showGTPase mutants R94Q and T105M contain depolarized and aggregated “clumped mitochondria” A,C,D. ThMitofusin 2 KO cells in the presence of functional endogenous mitofusin 1 retain the dominant mitochondrial fusion defects of the HR1 and GTPase domain mutants. Short mitochondria were observed in T105M, R94Q and M376A B, validaMFN2 WT. Furthermore, MiM 111 reduced perinuclear mitochondria clump formation in MFN2 R94Q and MFN2 T105M in mitofusin 2 null cells with endogenous mitofusin 1.We can modulate conformational changes in MFN2 using the mitofusin agonist MiM 111 (1 μM overnight). MiM 111 with endogenous mitofusin 1 in mitofusin 2 KO cells, can recover mitochondrial fusion defects and depolarization in cells with the recombinant expression variants  compared to MFN2 KO , but are unable to hydrolyze GTP to fuse mitochondria (second step). In T015M and R94Q, mitochondrial clumping was prevalent, consistent with the domain mutation interrupting GTP-dependent mitochondrial fusion, while not interfering with GTPase-independent mitochondrial tethering. Applying 1 µM of mitofusin activator MiM 111 for 24 h in the presence of functional GTPase protein (endogenous mitofusin 1) largely reversed these abnormalities. MFN2 R94Q and T105M (GTPase mutants), inducing mitochondrial depolarization and perinuclear aggregates. Based on our evidence of perinuclear mitochondrial clumps, GTPase mutants are able to tether together, but fail to complete mitochondrial fusion in the absence of GTPase activity  yielded no conformational change in the MFN2 M376A recovered its mitochondrial fusion abilities, allowing for allosteric modulation. Mitofusin agonists encourage an open conformation in mitofusin null cells expressing recombinant mutants, allowing the functional GTPase to complete mitochondrial fusion in this HR1 domain mutation. This demonstrates that GTPase activity is not compromised by dysfunctional mitofusin conformations. These delayed mitofusin conformational changes with mitofusin agonists represent the first direct evidence to reverse an HR1 domain mutation resulting in CMT2A.After introducing mitofusin agonist MiM 111 overnight, MFN2 T105M, R94Q and M376A result in short mitochondria unable to participate in fusion. MFN2 T105M and R94Q mutations result in depolarized clumped perinuclear mitochondria, able to tether but unable to complete fusion with a lack of GTPase activity. Treatment with the mitofusin activator in a mouse model carrying human MFN2 T105M can reverse the neuromuscular diseased phenotype [It has been demonstrated previously and here that MFN2 KO cell with recombinant mitofusin 1 expression after MiM 111 treatment. This suggests that mitofusin 1 GTPase activity was sufficient to fuse mitochondria. In the absence of mitofusins, GTPase domain mutants with perinuclear mitochondrial clumps cannot regain its normal mitochondrial fusion abilities through treatment with a mitofusin activator MiM 111. Rather, the number of perinuclear mitochondrial aggregates was reduced and fusion was seen in 376 instead of the human CMT2A mutation which substitutes valine. The presence of valine can compromise the hydrophobic mitofusin core. Because Met376 is crucial for mitofusin allosteric activation, studying the domain effects on mitochondrial function at this position was imperative.A limitation of this study is that we substituted alanine onto MetMore experiments are required to understand the relationship between mitochondrial fusion and tethering. Moreover, these results need to be validated in neuronal cells carrying human CMT2A mutants. Confirming these results may lead to promising mitofusin activator treatment in HR1 domain mutations in CMT2A disease.


Peptidergic systems play an important role in cancer progression. The galaninergic system  is involved in tumorigenesis, the invasion and migration of tumor cells and angiogenesis and it has been correlated with tumor stage/subtypes, metastasis and recurrence rate in many types of cancer. Galanin exerts a dual action in tumor cells: a proliferative or an antiproliferative effect depending on the galanin receptor involved in these mechanisms. Galanin receptors could be used in certain tumors as therapeutic targets and diagnostic markers for treatment, prognosis and surgical outcome. This review shows the importance of the galaninergic system in the development of tumors and suggests future promising clinical antitumor applications using galanin agonists or antagonists.1R promoted an antiproliferative effect, whereas the activation of GAL2R induced antiproliferative or proliferative actions. GALRs could be used in certain tumors as therapeutic targets and diagnostic markers for treatment, prognosis and surgical outcome. The current data show the importance of the galaninergic system in the development of certain tumors and suggest future potential clinical antitumor applications using GAL agonists or antagonists.The aim of this review is to show the involvement of the galaninergic system in neuroendocrine  and non-neuroendocrine  tumors. The galaninergic system is involved in tumorigenesis, invasion/migration of tumor cells and angiogenesis, and this system has been correlated with tumor size/stage/subtypes, metastasis and recurrence rate. In the galaninergic system, epigenetic mechanisms have been related with carcinogenesis and recurrence rate. Galanin (GAL) exerts both proliferative and antiproliferative actions in tumor cells. GAL receptors (GALRs) mediate different signal transduction pathways and actions, depending on the particular G protein involved and the tumor cell type. In general, the activation of GAL The GLOBOCAN 2020 database ) states that of the 7,794,798,844 inhabitants of our planet, 19,292,789 of them were diagnosed with some type of cancer and 9,958,133 died, with prevalence cases at 5 years of 50,550,287. Female breast cancer is the most diagnosed cancer and the leading cause of cancer death is lung cancer (1.8 million deaths) [Neuropeptides such as galanin (GAL), angiotensin II, apelin, adrenomedullin, endothelin-1, bombesin, orexin, substance P, neuropeptide Y, calcitonin gene-related peptide, vasoactive intestinal peptide and neurotensin are involved in cancer ,6,7,8,9.GAL was discovered in porcine intestinal extracts and contains 29 amino acids ; howeverThe galaninergic system (GAL and GAL receptors (GALRs)) is widely distributed by the mammalian gastrointestinal tract, testis, ovary, uterus, kidney and heart, and by the immune, endocrine, peripheral and central nervous systems  ,34,35,361R), GAL 2 receptor (GAL2R), GAL 3 receptor (GAL3R)) belong to the rhodopsin-like (class A) G protein-couple receptor family (seven transmembrane receptors or 7TM) [1R-GAL3R (33%) and GAL2R-GAL3R (54%) [3R and GAL2R respectively show 89% and 92% sequence homology with their receptor homologs present in the rat [3R, subnanomolar to nanomolar affinity at GAL2R and subnanomolar affinity at GAL1R [1R, 37 residues; GAL2R, 30; GAL3R, 13; N-terminus: GAL1R, 47 residues; GAL2R, 80; GAL3R, 62) [1R, GAL2R and GAL3R; several signaling pathways are activated after the binding of GAL to these receptors: the stimulation of phospholipase C  or the inhibition of cyclic adenosine monophosphate (cAMP)/PKA (mediated by GAL1R/GAL3R) [2R mediates the inhibition of adenylate cyclase (AC) via coupling to Gi type G protein [3R binding cavity is narrower than that observed in GAL1R or GAL2R) [2R and GAL3R genes have respectively been localized in chromosomes 17q25 and 22q12.2-13.1 [GALRs (GAL 1 receptor (GAL or 7TM) . They co or 7TM) ,61. The  or 7TM) . GALRs h3R (54%) , whereas the rat . Human Gat GAL1R . Althougat GAL1R ,61. ThusL3R, 62) . The phyR/GAL3R) . Moreove protein ,66. GALRr GAL2R) . Human G1R was isolated from a human melanoma cell line [1R inhibited AC activity via an interaction with G-proteins (Gαi/αo), leading to G protein-coupled inwardly-rectifying potassium (GIRK) channels opening [1R activation can also inhibit the transcription factor cAMP regulatory element binding protein (CREB)-dependent signaling pathway [1R (but not that of GAL2R or GAL3R) was controlled by cAMP via CREB [1R gene (located in chromosome 18q23) in humans shows three exons that are translated into a long protein containing 349 amino acids; GAL1R homology is high between species  [1R has been located in the central  and peripheral  nervous systems [GALell line . It is cell line ,68. More opening ,67,69. G pathway , and thevia CREB ,72. The  humans) . GAL1R h systems ,34 and i systems . 2R was first identified in the rat central nervous system [2R contains His252/His253 (transmembrane domain 6) and Phe264/Tyr271 (extracellular loop 3) residues, which play a crucial role in the binding of ligands and in the activation of the receptor [2R shows a high homology with that observed in the rat (85–92%) and it was 39% identical to human GAL1R [2R shows 38% amino acid identity with GAL1R [1R, the distribution of GAL2R is more widespread since it has been observed in the nervous system , skeletal muscle, liver, testis, ovary, uterus, spleen, heart, kidney, lung, gastrointestinal tract and pituitary gland [2R mRNA expression has been reported in the neocortex, dentate gyrus, hypothalamus, cerebellar cortex, substantia nigra, vestibular complex and dorsal root ganglion [2R expression was modified in the thalamus and cerebral cortex during brain development; this suggests that the receptor is involved in important mechanisms during the establishment/maturation of synaptic circuits and during neural damage/repair in the mature nervous system [2R activates the G protein (Gαq/11) pathway by triggering the intracellular phosphoinositol turnover, the activity of PLC and the release of Ca2+ into the cytoplasm [2R, via PKC and G protein (Gαo), activated MAPKs, favoring the downstream phosphatidylinositol 3-kinase (PI3K)-dependent phosphorylation of PKB and blocked the activity of caspases 3 and 9 [2R, induced the nuclear factor of activated T-cells and the cytoplasmic 2 (NFATC2)-mediated transcription of cyclooxygenase 2 and GAL, leading to the secretion of prostaglandin E2 and GAL, which favored cell invasion and neuritogenesis, respectively [2R can block forskolin-stimulated cAMP production; this suggests the activation of Gαi/αo [2R, activated extracellular-regulated protein kinase (ERK) and the phosphorylation of the serine/threonine kinase Akt signaling pathway [2R promoted, via the Akt (PKB) pathway, cell survival and proliferation; both processes were MAPK1/MAPK3-dependent [2R mediated the neuroprotective effect promoted by GAL after injury and also activated PKC, PLC and ERK via Gq/11 [2R, GAL agonists could be used to treat neurodegenerative diseases  [2R-specific agonists.GALs system ,75,76 ans system . GAL2R creceptor . The seqan GAL1R ,63,78. Ith GAL1R . In compry gland ,63,79,80ganglion . GAL2R es system . GAL2R aytoplasm ,63,68. G 3 and 9 . GAL, viectively . GAL2R cf Gαi/αo ,85, and f Gαi/αo . GAL, vi pathway . The actependent . GAL2R mia Gq/11 ,68,88; tlerosis) . This is3R was first isolated from rat hypothalamic cDNA libraries [3R (368 amino acids long) shows 36% amino acids identity with human GAL1R, 58% with human GAL2R and 90% with rat GAL3R [3R  is more restricted than that reported in the brain for GAL1R or GAL2R [3R mRNA has been located in the amygdala, periaqueductal gray, locus coeruleus, brainstem reticular formation, spinal cord, pancreas, adrenal gland and testis [3R promotes the activation of Gαi/αo, blocking AC activity and opening GIRK channels [2/3Rs (not to GAL1R), exerting a higher potency toward GAL3R than GAL [GALibraries . Human Gat GAL3R . The disor GAL2R ,89,90,91d testis ,91. GAL3channels ,90. Spexthan GAL ,92.1R lead to a higher affinity of this receptor for GAL1-15 than for GAL, increasing the signaling (mediated by Gi/o) and decreasing AC activity and CREB level [1R/GAL2R heteroreceptor complex [1R-GAL2R-5-HT1A receptor complex could explain why GAL1-15, but not GAL1-29, antagonistically moderated the serotonin receptor [GAL agonists or antagonists  have been used for the treatment of several disorders: GAL antagonists have been administered for the treatment of food intake disorders and Alzheimer’s disease, whereas GAL agonists have been used for the treatment of chronic pain ,93. SomeEB level . GALRs mEB level . Thus, t complex  and hete complex . The forreceptor . In addireceptor ,100. Thu1R was involved in tumor suppression and GAL2R favored tumor development and decreased survival [1R was generally antiproliferative, whereas the activation of GAL2R showed antiproliferative or proliferative effects [Peptides and their receptors are one of the molecular bases for the therapeutic targeting of tumors . The galsurvival ,123. GALsurvival . Thus, Gsurvival  and the  effects . The sta effects ,106. In Neuroendocrine tumors (NETs) are a very heterogeneous tumor group including: (1) carcinoid gastroenteropancreatic tumors; (2) non-carcinoid gastroenteropancreatic tumors oma, gastrinoma, insulinoma); (3) catecholamine-secreting tumors ; (4) chromophobe pituitary tumors; (5) medullary carcinoma of the thyroid; (6) Merkel cell tumors; and (7) small-cell lung cancer. NETs originate from neuroendocrine cells, which release peptides  and express their corresponding receptors ,125,126.Regarding the galaninergic system, many data demonstrated its involvement in NETs pathophysiology and carcinogenesis; for example, high doses of estrogens or dopamine agonists reversed rat pituitary hyperplasia and decreased the expression of GAL, suggesting that the peptide acted as a proliferative agent ,131,132.2R mRNA expression was observed in human phaeochromocytomas [GAL and GALRs have been observed in human phaeochromocytomas . Compareocytomas  and GAL ocytomas . GAL bloocytomas .1R have respectively been reported in RINm5F [2+ level [Insulinomas appear sporadically or can be related with multiple endocrine neoplasia type 1  . This syn RINm5F  and Rin1n RINm5F  insulinon RINm5F   in neuroblastoma, whereas the presence of GAL2R mRNA is less common than that of GAL1R mRNA [1R and GAL3R decreased in ganglioneuromas [2R and GAL3R mRNAs were detected in the rat neuroblastoma B104 cell line, but not GAL1R mRNA [2R in the human neuroblastoma SH-SY5Y cell line [2R mediated apoptosis in the latter cell line; however, the GAL antiproliferative potency was 100-fold higher in SH-SY5Y neuroblastoma cells overexpressing GAL2R than in SH-SY5Y neuroblastoma cells overexpressing GAL1R, suggesting that a high level of GAL2R is able to block tumor cell proliferation [2R transfection into neuroblastoma SH-SY5Y cells inhibited cell proliferation and promoted a caspase-dependent apoptotic mechanism [Neuroblastoma is the result of an aberrant sympathetic nervous system development, usually arising from the paraspinal ganglia or adrenal medulla ,164. ThuL1R mRNA ,137. By L1R mRNA . It is iL1R mRNA . Thus, GL1R mRNA . By contell line . GAL2R mferation . In thisechanism . Finallyechanism ,112,142 echanism .1R was the most abundant receptor observed in normal anterior pituitaries, followed by GAL3R, whereas GAL2R was not found [1R and GAL2R mRNAs were found in human pituitaries, but not GAL3R mRNA [GAL and the three GALRs have been observed in normal pituitary glands ,36,145. ot found . In anotL3R mRNA .1R [3R have been reported in some patients who relapsed shortly after surgical intervention [3R could be a marker for relapsing pituitary tumors and that GAL3R antagonists could be a therapeutic approach for the treatment of pituitary tumors [GAL was detected in some, but not all, pituitary tumors  Table 1Table 1. 1R , while hrvention . This suy tumors . GAL mayy tumors .GAL and prolactin coexist in lactotrophs ,167. In The coexistence of growth hormone (GH) and GAL has been reported in somatotrophs ,167. GALMost of the corticotroph adenomas express GAL . In normThe expression of the GAL gene was blocked in thyrotroph adenomas; this means that GAL did not exert a stimulatory proliferative action on thyrotrophs . AnotherSmall-cell lung cancer (SCLC) is a poorly differentiated neuroendocrine carcinoma . Approxi2R, the proliferation of SCLC cells [2+-mobilizing peptides  promoted the growth of SCLC cells through autocrine and paracrine mechanisms [2+-mobilizing receptors could exert a therapeutic antitumor action and, in fact, these antagonists inhibited SCLC cell growth [2+, and the peptide also promoted the growth of both cell lines, which was dependent on the concentration of GAL [2R, a mitogenic action on these cells by activating Gq, Gi and G12 G proteins [2+ mobilization [GAL mediated, via GALLC cells ,151,173 chanisms . This fil growth . In H69 n of GAL ,154. SBCn of GAL . This men of GAL ,156. GALproteins . Thus, Glization .1R were lower in gastric cancer tissues compared with those found in adjacent regions; however, the GAL2R/GAL3R levels did not change [In nerve cells, the galaninergic system plays an important role in tumor development. In human stomach samples, obtained from the vicinity of invasive cancer cells, neurons located in the myenteric plexus showed a high expression of both caspases 3 and 8, but a low expression of GAL ,174 Tab. In carct change . The lowt change .A prolonged administration of GAL (4 µg/kg) blocked gastric carcinogenesis by inhibiting the proliferation of antral epithelial cells . Human gColorectal cancer (CRC), the third most prevalent cancer worldwide, is an invasive tumor process due to the proliferation of epithelial cells that acquire a neoplastic phenotype . This pr1R/GAL3R was observed in CRC cells and it has been reported that the high expression of GAL3R in CRC tissue was associated with a better prognosis and longer survival of CRC patients; this means that GAL3R is a prognostic factor for these patients [CRC tissues showed higher GAL levels than the corresponding non-tumor tissues ,179,180,patients .The number of neurons containing GAL was higher in CRC patients than in those showing a healthy intestine, and an increased GAL gene/protein expression was observed in CRC tissues ,179. Com1R is mainly expressed in the human colon. The silencing of this receptor or GAL promoted apoptosis in drug-sensitive/resistant cell lines and enhanced the effects mediated by chemotherapy; thus, GAL1R regulates drug resistance [2R gene has been suggested in CRC as a chemosensitive methylation candidate to bevacizumab, since HCT116 CRC cells overexpressing GAL2R were more chemosensitive to the monoclonal antibody than control cells [1R silencing promoted a downregulation of the FLIPL-like inhibitory protein long form , meaning this inhibitor is a key downstream effector of the anti-apoptotic signaling mediated by GAL/GAL1R [GALsistance . The GALol cells . GAL1R sAL/GAL1R . Thus, tAL/GAL1R .2R expressed in tumor cells and by inducing NFATC2-mediated transcription of cyclooxygenase-2 and GAL; then, GAL released from tumor cells promoted neuritogenesis, favoring PNI [Head and neck squamous cell carcinoma arises from mucosal surfaces of the head and neck  Table 3Table 3. ring PNI .1R genes, and a hypermethylation promoted the inactivation of GAL/GAL1R/GAL2R genes [1R gene promoter is widely hypermethylated in HNSCC ; this is related to reduced GAL1R expression, which can be restored by treating with a histone deacetylase inhibitor (trichostatin A) or with a methyltransferase inhibitor (5-azacytodine) [1R gene promoter has been related to HNSCC carcinogenesis [1R/GAL2R hypermethylation has been associated with a higher recurrence rate and reduced disease-free survival [1R methylation status could be a biomarker for predicting HNSCC clinical outcomes. Importantly, because methylation suppresses GAL/GALRs expression in some tumors and because GAL/GAL1R act as tumor suppressors (see below) [The promoter methylation status of the peptide-encoding gene GAL was studied in HNSCC samples; methylation was observed in 20% of them . The aut2R genes . The GALytodine) ,198. Thiogenesis . GAL1R/Gsurvival ,200. GALsurvival ,194; thue below) , these f1R blocked human oral tumor cell proliferation [1R inhibited the proliferation of keratinocytes  by blocking the MAPK pathway [1R acts as a tumor suppressor gene, which is frequently silenced in HNSCC [1R is absent [1R blocked the proliferation of tumor cells through the activation of ERK1/2 and cyclin-dependent kinase inhibitors, leading to cell-cycle arrest  [1R in GAL1R/GAL2R-negative HNSCC cells also suppressed tumor cell proliferation through ERK1/2-mediated actions on cyclin-dependent kinase inhibitors and cyclin D1 [2R in HNSCC cell lines favored the survival and proliferation of these cells by activating respectively the PI3K/Akt and MAPK/ERK-dependent pathways [2R activated rap1B  favoring a p38-mediated inactivation of the mRNA binding protein tristetraprolin, which inhibited the production of many pro-inflammatory cytokines. This means that GAL2R-p38-mediated cytokine production could be a therapeutic target against HNSCC, since p-38 inhibitors are currently used in clinical practice. In HNSCC, GAL2R promotes tumor angiogenesis by enhancing the secretion of cytokines  via the p38-MAPK-mediated inhibition of tristetraprolin [2R exerted an antitumor effect by inducing cell-cycle arrest and apoptotic mechanisms (caspase 3-dependent) [2R in HNSCC could be explained by the signaling pathways activated depending on the coupled G protein type. Moreover, GAL2R transfection into human HNSCC cells suppressed cell proliferation [2R blocked HNSCC cell proliferation (showing mutant p53) [1R by GAL have not been reported, and in HNSCC cells, GAL1R/GAL2R are suppressor tumors in a p53-independent manner [2R mediated apoptotic mechanisms (caspase-independent) in HEp-2 cells by downregulating ERK1/2 and inducing Bim (a pro-apoptotic Bcl-2 protein) [A high level of GAL has been detected in HNSCC ; GAL/GALferation , and GAL pathway . Thus, Gin HNSCC ,198; in 57, p27) ,198,200.yclin D1 . The ovepathways . Rap1   and thisferation ,197 and ant p53) . Importat manner . GAL2R mprotein) . Althougprotein) .1R, followed by GAL3R; GAL2R was not found (astrocytic/oligodendroglia tumors) [3R, no correlation with oligodendroglial, astrocytic and mixed neural–glial tumors was reported [3R [1R, followed by GAL3R and GAL2R [3R, but GAL1R/GAL2R were not observed around the blood vessels [3R, and this means that GAL could regulate the activity of GAMs [The GAL/GALR system has been described in glioma ,118 in w tumors)  (Table 4 tumors) , and regreported . Moreoverted [3R . GAL has vessels . This me of GAMs ,204.1R, the proliferation of human glioma cell lines  and tumor growth in nude mice [GAL blocked, through GALude mice . The aut1R DNA methylation is among the most epigenetic molecular alterations in endometrial cancer; this methylation indicates malignancy with a high degree of sensitivity and specificity [1R gene in bladder cancer has been involved in the prognosis of the disease, but the role played by the galaninergic system in this cancer is currently unknown [Although the expressions of GAL and pre-pro-GAL mRNA have been reported in breast cancer, it has been suggested that the GALN gene (which encodes the pre-pro-GAL protein) is an unlikely candidate oncogene in breast tumors because an increase in pre-pro-GAL mRNA expression with GALN amplification was not observed ,206 Tab. Many necificity . The met unknown .1R/GAL2R are therapeutic targets and prognostic factors in salivary duct carcinoma [1R/GAL2R methylation rates were higher in salivary duct carcinomas than in normal tissues, and these rates were correlated with a decrease in overall survival. The expression of GAL has been reported in melanoma, and human Bowes melanoma cells expressed GAL1R [It has been suggested that GALarcinoma . GAL1R/Ged GAL1R ,213. In ed GAL1R . The lated GAL1R . These ced GAL1R .2+ concentration [1R/GAL3R, mainly coupled to Gi/o, decrease the cAMP level and inactivate PKA, whereas GAL2R, preferably coupled to Gq/11 mobilizing intracellular Ca2+, promotes (via PKC) the activation of cell survival (via Akt or PKB) and MAPK1/MAPK3-dependent cell proliferation pathways [1R can also be coupled to Gβγ- and/or Gi-signaling pathways and then the activation of MAPKs occurs in a Ras/Raf-dependent manner [1R activation also favors the Akt/Akt substrate of the 160 kDa (AS160) cascade [1R induces cell-cycle control proteins  and suppresses cyclin D1 in cancer cells [2R, mainly coupled to Gq/11, mediated the activation of PLC and small GTPase proteins in the Rho family [2+ [2R activated the small GTPase protein Rho A in SCLC cells, suggesting the coupling to G12/13 [2R inhibited the production of cAMP, meaning that the receptor was coupled to Gi protein [2R decreased cofilin activation and Rho and Cdc42 GTPase activity [2R activated the MAPK/ERK pathway in a PKC manner, meaning that GAL2R was coupled to a Go protein [2R regulated cell-cycle control proteins  and cyclin D1 and promoted apoptosis (caspase 3-dependent) in HNSCC cells [2R decreased the expression of p21cip1, phosphorylated BAD forms (pBad) and phosphorylated Akt (pAkt), downstream of the Gq11/PI3K pathway [2R-mediated apoptosis in tumor cells was induced by the activation of the pro-apoptotic Bcl-2 protein Bim, through a mechanism independent of caspase [3R, involved in inward potassium ion (K+) currents, is coupled to the Gi/o signaling pathway and its activation favored the inhibition of cAMP and AC altering CREB phosphorylation [+ channels and hyperpolarized cell membranes in the rat RINm5F insulinoma cell line [ntration . The galpathways ,30,87. Gt manner ,25,87. G cascade ,77 and a cascade . GAL1R ier cells . GAL2R, o G12/13 . GAL2R i protein . GAL2R dactivity . In tumo protein . GAL2R rCC cells . GAL2R d pathway . The GAL caspase . GAL3R, rylation ,87,90. Gell line , and theell line ,215. C7 ell line . C7 inhiell line .1R/GAL2R-5-hydroxytryptamine 1A receptor heteromer (a macromolecular complex formed by at least two different receptor units) promoted conformational changes in GAL recognition sites, altering the binding affinity of GAL [1R/GAL2R complex favored a higher affinity of GAL1R for GAL1-15 than for GAL, increasing Gi/o-mediated signaling and decreasing AC activity and CREB levels [3R heteromerization with other peptide receptors or other GALRs has been suggested [The interaction between GALy of GAL . In thisB levels . In addiuggested .3R antagonist, promoted apoptosis in promyelocytic leukemia cells expressing GAL2R [Peptides play an important role in cancer; the in-depth knowledge of the functions mediated by these substances is an emerging and promising line of research that could lead to new clinical applications in oncology. One line of research could be the use of peptides coupled to cytotoxic agents to exert an antitumor action, and another, the use of peptide receptor antagonists or agonists. In the case of GAL, GALR antagonists or agonists could be used as antitumor treatments according to the different signaling pathways and actions mediated by GALRs. GALR antagonists have been administered for the treatment of food intake disorders, anxiety, depression and Alzheimer’s disease, whereas GALR agonists have been used for the treatment of chronic pain ,93. It hng GAL2R .In vitro and in vivo experiments using human gastric cancer cell lines have been performed to study the antitumor action of a triple treatment with GAL, serotonin and octreotide (an octapeptide that mimics the actions mediated by somatostatin) . TreatmePeptide analogs have been used as an antiproliferative strategy and promising results targeting peptidergic systems have been reported; accordingly, GAL analogs could be tested in tumors expressing certain GALRs. The half-life of GAL in plasma is about 5 min, but the half-life of synthetic GAL  is 60–120 min. GAL1-16 was synthesized as a free carboxylic acid, whereas GAL1-29 was synthesized with a C-terminal amide corresponding to the endogenous peptide; the data mean that analogs showing an increased half-life are required for a therapeutic application of the peptide ; this isThe potential antitumor clinical application of GALR ligands (GALR antagonists or agonists) has unfortunately been neglected by the scientific community and the pharmaceutical industry. However, the expression of the galaninergic system could be used for the diagnosis, treatment and prognosis of tumors ,111,112,1R has been demonstrated in insulinoma cells [2R-expressing cells than in those expressing GAL1R, meaning that a high level of GAL2R could block cancer cell proliferation [3R could be used as a marker for relapsing pituitary tumors and GAL3R antagonists could also be used to treat these tumors [2R, promoted the proliferation of SCLC cells through an autocrine manner [2R antagonists exert an antitumor action against SCLC cells. GAL inhibited the proliferation of glioma cells and tumor growth via GAL1R [3R expression has been related to high-grade glioma [1R/GAL2R are therapeutic targets and prognostic factors in salivary duct carcinoma, as well as whether GAL1R DNA methylation indicates malignancy or not in endometrial cancer. Finally, the dual role of GAL as a proliferative and antiproliferative agent must be clarified in pancreatic carcinogenesis.Some peptidergic systems  exclusively promote the proliferation of tumor cells; however, GAL, via different GALRs, exerts a tumor cell proliferative action, but also the peptide suppresses the development of tumors ,122,157.ma cells ,162; howe tumors ; this mue tumors ,160; thee tumors . GAL, vie manner ,151,173;ia GAL1R ; a reducia GAL1R , and GALe glioma : the lin1R genes in HNSCC and it has been reported that hypermethylation promoted the inactivation of GAL/GAL1R/GAL2R genes [1R/GAL2R hypermethylation has been associated with higher recurrence rates and reduced disease-free survival [1R methylation status could be a biomarker for predicting HNSCC clinical outcomes. However, more studies must be performed to confirm whether GAL1R/GAL2R are potential therapeutic targets and prognostic factors.Epigenetic mechanisms regulate the galaninergic system and play a crucial role in tumor development . GAL dow2R genes . Thus, m2R genes , and GALsurvival ,200. TheMany of the proliferative and antiproliferative actions mediated by GAL on tumor cells could be explained by the signal transduction pathways depending on the coupled G protein type ,87,196. The galaninergic system is involved in tumorigenesis, invasion and migration and has been correlated with tumor stage/subtypes and metastasis and, in this system, epigenetic mechanisms have been related with carcinogenesis and recurrence rates. GALRs play a crucial role in cancer and their specific actions must be clearly understood in many tumor types because GALRs mediate different signal transduction pathways and actions depending on the tumor cell type and the particular G protein involved. GALRs could be used as a therapeutic target and diagnostic marker for the treatment, prognosis and surgical outcome in certain tumors. Different from other peptidergic systems, the galaninergic system exerts a proliferative action on tumor cells, but GAL also suppresses the development of tumors . Thus, i


Male germ cells experience a drastic chromatin remodeling through the nucleo-histone to nucleo-protamine (NH-NP) transition necessary for proper sperm functionality. Post-translational modifications (PTMs) of H4 Lys5, such as acetylation (H4K5ac), play a crucial role in epigenetic control of nucleosome disassembly facilitating protamine incorporation into paternal DNA. It has been shown that butyrylation on the same residue (H4K5bu) participates in temporal regulation of NH-NP transition in mice, delaying the bromodomain testis specific protein (BRDT)-dependent nucleosome disassembly and potentially marking retained nucleosomes. However, no information was available so far on this modification in human sperm. Here, we report a dual behavior of H4K5bu and H4K5ac in human normal spermatogenesis, suggesting a specific role of H4K5bu during spermatid elongation, coexisting with H4K5ac although with different starting points. This pattern is stable under different testicular pathologies, suggesting a highly conserved function of these modifications. Despite a drastic decrease of both PTMs in condensed spermatids, they are retained in ejaculated sperm, with 30% of non-colocalizing nucleosome clusters, which could reflect differential paternal genome retention. Whereas no apparent effect of these PTMs was observed associated with sperm quality, their presence in mature sperm could entail a potential role in the zygote. Histone post-translational modifications (PTMs) play an essential role in epigenetic regulation of spermiogenesis, the last phase of spermatogenesis, marked by a unique process of chromatin remodeling of male germ cell’s nucleus that is necessary for proper sperm function ,5,6,7,8.Acetylation of H4 has been widely studied in normal and pathogenic testes within the context of NH-NP transition and has been recently found altered in the testis of patients affected by testicular tumors ,30,31,32The importance of histone PTMs in male germ cells is not limited to their regulatory role during chromatin remodeling. Sperm cells contain many layers of epigenetic information, with modified histones contributing to the non-random distribution of the nucleo-histone genomic domain along specific regions of the paternal genome and marking differential sperm DNA accessibility ,38,39,40One of the current challenges of assisted reproduction techniques (ART) relies on selecting the best spermatozoa for intracytoplasmic sperm injection (ICSI), overcoming the intrinsic high heterogeneity of the human sperm sample. Current methods for sperm selection are based on multilayer density gradient centrifugation, often followed by swim-up. However, these techniques mainly rely on the quality of sperm motility and morphology not accounting on the rest of sperm molecular features that could affect the ART success rate (around 30–35% per cycle in Europe) . In termDespite the relevance of the H4K5 residue in the regulation of mouse NH-NP transition, there is a lack of information about the significance and distribution of acetylated and butyrylated forms in normal human spermatogenesis and testicular pathologies, as well as about their possible retention in the mature human spermatozoon. Therefore, the aim of our work was to increase the current knowledge about these specific-residue PTMs in human male germ cells, as well as to try to decipher whether they could be markers of human sperm quality. We report a different behavior of H4K5bu and H4K5ac during human spermatogenesis, which would indicate a highly specific role of H4K5bu during round spermatid (rSPD) elongation, coexisting with H4K5ac during spermiogenesis. Results in patients with testicular cancer evidence that H4K5ac and H4K5bu remain stable under pathological conditions, supporting the important role of these residue-specific modifications towards spermatogenic development. Of note, both H4K5bu and H4K5ac are retained in ejaculated sperm showing differential location in 30% of nucleosome clusters. Their role as sperm chromatin maturity markers after sperm selection through density gradient centrifugation has also been explored.p < 0.001) to finally recover intermediate levels as spermiogenesis started. Specifically, early-stage (I to V) round spermatids (rSPD I–V) restored the H-Score to 282.7 ± 66.2 (p < 0.0001), which increased to 415.8 ± 80.8 (p < 0.05) in late-stage rSPD (rSPD VI–VIII) maintaining similar levels in elongated spermatids (eSPD). H4K5ac levels finally decreased to the minimum in condensed spermatids . Somatic Sertoli-cells (SC) displayed low levels of H4K5ac (p < 0.01), reaching maximum levels in eSPD . Finally, H4K5bu levels decreased in cSPD . SC showed an H4K5bu H-Score close to zero (2.5 ± 6.1)  were subjected to immunohistochemistry (IHC) analysis to detect H4K5 butyrylation and acetylation . The H4Kf H4K5ac . In turn5 ± 6.1) . IndividAfter observing the predominant detection of H4K5bu in the late stages of spermatogenesis, we explored the characteristic behavior of this modification in patients displaying different primary testicular failures, such as hypospermatogenesis (HP), spermatogenic arrest (SA), and Sertoli-cell only syndrome (SCOS), aiming to identify potential atypical H4K5bu signals in meiotic or mitotic phases, or in supporting somatic SC. H4K5bu H-Scores were analyzed and are shown in p < 0.05) (p < 0.05)  and teratoma (TER), to identify potential epigenetic alterations. TER patients showed a slight increase of the H4K5bu H-score in SPG  A. In tur < 0.05) A. Despit < 0.05) A. Data cp < 0.05)  Representative IHC images are shown in Considering the relevance of global spermatogenic H4 acetylation in testicular cancer patients, which is completely disturbed in the seminiferous tubules adjacent to the tumor, we proceeded to evaluate specific acetylation of H4K5 in SEM and TER patients. Of note, significant changes to the normal pattern were only identified in SPC from TER patients, evidencing a discreet increase of H4K5ac H-score  A. Despit < 0.05) A. SomatiTo further investigate H4K5 butyrylation in male germ cells, we examined its presence and that of the acetylation on the same residue in mature human sperm from patients with normal semen parameters. Both H4K5bu and H4K5ac were detected in all ejaculated sperm cells evaluated from the patients included in the analysis A. To deep > 0.05), which might be due to the high dispersion of the results observed after the multi-layer gradient preparation, compared to the 50% gradients.A 50% density gradient centrifugation, which allows obtaining purified sperm representing the total sperm population of a semen sample without the presence of non-sperm contaminating cells (G50), was performed in order to define the relative abundance of both marks in normozoospermic samples (represented as H4K5bu/H4K5ac ratio). Moreover, a 3-layered (50–70–90%) density gradient centrifugation was performed to compare cell populations according to sperm quality . The presence of H4K5bu and H4K5ac was detected in all cell populations evaluated in our experimental set up. When considering the whole semen sample (G50), the relation H4K5bu/H4K5ac was established as 1.02 ± 0.1 . This raDespite that H4 acetylation has been largely described during mammalian spermatogenesis and has shown to be directly involved in chromatin remodeling during spermiogenesis ,29,31,32The spermatogenic pattern of H4K5ac found in this study was consistent to that of global H4ac previously described ,30,31,32Due to the apparent highly specific role of H4K5bu during spermiogenesis, we explored whether butyrylation levels were altered under different primary testicular failures. No relevant changes were observed in patients with hypospermatogenesis, SCOS, or spermatogenic arrest, as previously reported for H4ac . DespiteGrowing evidence highlights the impact of sperm chromatin state in sperm functionality, fertilization events, and beyond, including sperm modified histones as epigenetic marks inducing differential DNA accessibilities ,43,44,45While colocalization results might suggest a role of H4K5ac and H4K5bu in sperm function, no variations were observed associated with sperm quality. The study of different sperm populations isolated from neat sample following procedures routinely used in the reproductive clinics to select the best spermatozoon for ICSI sheds light on the molecular cargo of the fertile sperm giving rise to a new individual. In that case, the observed results would discard an impact of H4K5bu and H4K5ac relative levels in sperm fertilizing ability. Further work should focus on determining quantitative H4K5bu and H4K5ac levels in selected sperm populations by complementary techniques, which would help disentangle their individual potential as an indicator of chromatin maturity. Due to the known role of sperm histone marks as epigenetic regulators in early embryo, it will be worthy to know whether they could have an impact beyond fertilization, such as in the formation of preimplantation embryo and transmission of epigenetic information.n = 23, mean age 37 ± 7, ranging from 21 to 52 years old) were provided by the Departments of Pathology from the Hospital Clínic de Barcelona  and the Hospital Universitari de Vic . The samples were grouped into three main groups: control group, spermatogenic alterations, and testicular cancer. The control group (N) comprised azoospermic infertile patients corresponding to male partners of couples undergoing assisted reproduction studies, displaying normal spermatogenesis at the histological level, and showing all germ cell types and spermatogenic stages. The origin of the azoospermia was classified as post-vasectomy (n = 1), obstructive azoospermia due to CFTR genotypes (n = 1), and azoospermia of unknown cause when obstructive azoospermia, karyotype alterations, CTCF pathogenic variants, and chromosome Y microdeletions were discarded (n = 4). H4K5ac and H4K5bu levels were assessed in 6 N samples, from which 5 were common to both evaluations, and 1 patient was exclusively subjected to either H4K5bu or H4K5ac assessment. The group of infertile patients with spermatogenic alterations (n = 8) included patients stratified according to the type of spermatogenic impairment into patients with Sertoli-cell only syndrome , hypospermatogenesis , and spermatogenic arrest . Among the SA patients, two of them presented spermatocyte arrest , and the third one evidenced a partial arrest at round spermatid level. The last group of patients with testicular cancer (n = 7) included patients affected by seminoma  and teratoma . In addition to the groups above, one sample corresponding to a patient with normal spermatogenesis was used as technical internal control in all evaluations. Karyotype, AZF status, and CFTR status per individual patient are indicated in Testicular biopsies (n = 9) from patients undergoing routine semen analysis were obtained at the Assisted Reproduction Unit from the Clinic Institute of Gynecology, Obstetrics and Neonatology, at the Hospital Clínic de Barcelona, Spain. The ejaculates were collected by masturbation into sterile containers after 3–5 days of sexual abstinence. Evaluation of the seminal parameters was performed using the automatic semen analysis system CASA  and samples were classified according to the World Health Organization guidelines , presenting a dynamic range from 0 to 600. Since the intensity of the signal within cells belonging to the same specific cell type per spermatogenic stage was homogeneous along the seminiferous tubule section, the mean intensity of each cell type was considered. Thus, H-score for each testicular cell type analyzed per seminiferous tubule section was provided for each individual patient.Classification of testicular cells and spermatogenic stages of the seminiferous tubules sections was based on current classification of human spermatogenesis described by Muciaccia et al. 2013 . For eac2O before permeabilization for 20 min in 0.05% Triton X-100 in PBS. After washings in PBS, slides were incubated for 1h at RT in blocking solution . Primary antibody incubation . Images were acquired by confocal microscopy  at the Advanced Optical Microscopy Unit (Campus Clínic) from Scientific and Technological Centers from University of Barcelona.Extensions consisting in 1 million sperm (Mz) per slide were incubated in decondensing solution  to facilitate antibody accessibility, monitoring decondensation in optic microscope. Sperm cells were fixed 10 min in 2% paraformaldehyde and washed with PBS and Hcubation  at dil 1Colocalization was quantified using ImageJ software distribution FIJI . A custog for 10 min at 4 °C. Quantification was done using Pierce™ BCA Protein Assay Kit , following the manufacturers’ recommendation. Replicates with Ct < 35 for the leukocyte marker PTPRC were discarded. A total of 10 µg of soluble protein lysate were loaded into 16% acrylamide gels by SDS-PAGE [® (TBS) blocking buffer  for 1 h at RT to incubate O/N with primary antibodies at 4 °C . Signal intensities were determined on the raw scanned data using Image Studio Lite v5.2 software (LI-COR Inc.). Intensities below the minimum linear cutoff were discarded from the analysis. Therefore, after exclusion criteria, from the 6 patients analyzed, n = 5 in G50, P70, and P90 and n = 3 in P70 were considered for subsequent statistical analysis.Sperm soluble protein extraction was conducted by incubating purified sperm cells in lysis buffer  for 15 min on ice followed by centrifugation 17,000×  1:1000, . After w 1:1000,  at 1:12,www.graphpad.com). Normal distribution was assessed by Shapiro–Wilk normality test followed by Levene’s homoscedasticity test. According to the distribution, either Student t-test or Mann–Whitney U test was performed for the analysis of H-Score levels. Only groups formed by at least three patients were included in the statistical analysis. Paired Student t-test was applied for the analysis of H4K5bu/ac ratios. p-values < 0.05 were considered statistically significant.Data analysis was conducted in RStudio software version 2022.02.2  and the