TP53 gene are one of the most common alterations in human cancers. We previously showed that mutant p53 (mutp53) can bind TopBP1, an ATR activator, to attenuate its ATR-activating function. A partially defective ATR function caused by mutp53 makes cancer cells more vulnerable to inhibitors of other TopBP1-independent ATR activators, such as DNA2. DNA2 plays a role in homologous recombination (HR) repair by resecting DNA ends in double-strand breaks and preparing them for invasion of homologous duplex. Here we identify a new DNA2 inhibitor, namely d16, and show that d16 exhibits anticancer activities and overcomes chemotherapy resistance in mutp53-bearing cancers. Similar to DNA2 depletion, d16 treatment results in cell-cycle arrest mainly at S-phase. Moreover, reexpression of mutp53 in a p53-null cancer cell line makes cells more vulnerable to d16-mediated inhibition of ATR activity. As d16 also inhibits HR, a combination of d16 and PARP inhibitors displays synergistic induction of cell death. DNA2 is often overexpressed in cancer, particularly in cancer cells harboring mutp53. Overexpression of DNA2 is associated with poor outcome in ovarian cancer. Overall, our results provide a rationale to target DNA2 as a new synthetic lethality approach in mutp53-bearing cancers, and further extend the benefit of PARP inhibitors beyond BRCA-mutated cancers.The tumor suppressor p53 promotes tumor-suppressive activities including cell-cycle inhibition, apoptosis, senescence, autophagy, and DNA repair. However, somatic mutations in the This study identifies a new DNA2 inhibitor as a synthetic lethal targeted therapy for mutp53-harboring cancers, and provides a new therapeutic strategy by combining DNA2 inhibitors with PARP inhibitors for these cancers. TP53 gene as the most significantly mutated gene in human cancers, resulting in perturbation of replication checkpoint function (TP53 mutations in response to DNA damage. One of the mechanisms caused by TP53 mutations (mutp53) involves the checkpoint activator TopBP1 (topoisomerase IIβ-binding protein), a critical molecule capable of facilitating the complex formation between several hotspot mutp53 proteins and either (nuclear factor Y) NF-Y or p63/p73 , are repaired accurately. One of the major cellular stress sensors triggered by DSBs involves transcription factor p53. The tumor suppressor p53 promotes tumor-suppressive activities including cell-cycle inhibition, apoptosis, senescence, autophagy, and DNA repair, etc. By stopping aberrant DNA replication characterized by oncogenic pressure, p53 function constitutes one of the most powerful shields against tumor growth . In factfunction . Hence,  p63/p73 . ATR act p63/p73 . Previou p63/p73 . As a re p63/p73 . It is n p63/p73  or ETAA1 p63/p73  independ p63/p73 . Loss-of p63/p73 . Therefoin vivo antitumor activities and their anticancer activities remain to be optimized.In addition to the functions mentioned above, DNA2 has been shown to promote homologous recombination (HR) repair by facilitating the recruitment of HR repair factors, such as Rad51, to DSBs . DNA2 poin vivo anticancer activity in an ovarian cancer xenograft model. Moreover, d16 can inhibit HR and synergize with PARPis in several BRCA-proficient ovarian and breast cancer cells, providing a new therapeutic strategy for the treatment of TP53-mutated, BRCA1/2-WT (wild-type), breast and ovarian cancers.Here, we identify a new DNA2 inhibitor, namely d16, and demonstrate the synthetic lethal potential of DNA2 inhibitors for treating mutant p53-harboring cancers. Our data show that d16 exerts https://mcule.com/apps/1-click-docking/).Crystal structure of Dna2 nuclease-helicase was downloaded from the Protein Data Bank (accession no. 5EAW). Molecular docking study was performed using 1-Click Docking of Mcule software (DNA2 inhibitor C5 (NSC-15765) was obtained from NCI Developmental Therapeutics Program . C5 analogs were purchased from MolPort, Inc. or ChemBridge Corporation. Compound d16 was resynthesized in ChemBridge Corporation.2 at 37°C. All cell lines were confirmed negative for Mycoplasma contamination using PlasmoTest Mycoplasma Detection Kit  according to manufacturer's instruction at the time of stock vial preparation. Cells were authenticated by ATCC short tandem repeat profiling and routine examination of morphology and consistent in vitro growth properties. Cell lines were grown for no more than 15 passages after thawing. For transient transfection, WT p53 plasmid and single hotspot mutant p53 plasmid constructs including V143A, R175H, R248W, R249S, and R273H (A2780cis cell line (ECACC 93112517) was purchased from PHE Culture collections through Sigma. Other human cell lines were purchased from ATCC. MDA-MB-468 and BT549 human breast cancer cells, H1299 human lung cancer cells, C33A human cervical cancer cells  and MDAH-2774 ovarian cancer cells  were grown in DMEM, supplemented with 10% FBS, penicillin (50 IU/mL), and streptomycin (50 µg/mL) in a humidified incubator with 5% CORNA was extracted from H1299 lung cancer cells using TRIzol reagent (Invitrogen), and quantitative PCR (QPCR) was performed in triplicates on MX3005P thermal cycler using SYBR Q-PCR Master Mix (GenDEPOT). The primer pairs used for DNA2 amplification are 5′-ATTTCTGGCACCAGCATAGC-3′ and5′-GCTGGATAGCTGGATGAACC-3′, and the primer pairs used for GAPDH amplification are 5′-ATTGGGCGCCTGGTCACCAGGGCTG-3′ and 5′-AAATGAGCCCCAGCCTTCTCCATG-3′. The DNA2 mRNA levels were analyzed with MxPro 4.0 QPCR software  and were normalized relative to GAPDH.g for 20 minutes. Supernatant were boiled in SDS loading buffer, separated by SDS-PAGE and electrotransferred to the polyvinylidene fluoride membrane (Millipore). After blocking with 5% milk, immunoblotting was performed with the appropriate antibodies. Antibodies specific to p53 , Chk1 (G-4), GAPDH (6C5), and Actin (C-2) were from Santa Cruz Biotechnology. Antibodies specific to phospho-Chk1 (S345) and RAD51  were from Cell Signaling Technology. Antibody specific to DNA2  was from Thermo Fisher Scientific. Antibody specific to PARP (Asp 214) was from BD Biosciences.Cells were lysed in 1% SDS buffer. After boiling, the lysates were sonicated and subjected to centrifugation at 20,000 × Xenografts tissues were harvested, and fixed in 10% neutral buffered formalin  for 24–72 hours. Tissues were then placed in 70% ethanol at 4°C. IHC was performed by staining tissues using hematoxylin and eosin (H&E), cleaved PARP1 and Ki67 antibodies under the care of the pathology core and lab of Baylor College of Medicine (BCM) using standard operating protocols.To determine and quantify a dose-effect relationship between DNA2 inhibitor d16 and PARPi [rucaparib (RU) or talazoparib], different doses of d16 or PARPi, or combinations of d16 and PARPi with a fixed constant ratio were added to BT549, MDA-MB-468, MDAH-2774, and C33A cells, respectively. The combinational effect was evaluated using the combination index (CI) method as described previously . The modTP53 mutant and WT TP53 tissues in all types of cancer from TCGA datasets. CSIOVDB  was added to each well, followed by incubation at 37°C for 1 hour. The medium and reagent were washed away and 150 µL of DMSO was added to each well. After incubation at room temperature for 15 minutes, absorbance was read at 490 nm on a plate reader (BioTek Synergy HT). Each experiment was performed at least in triplicates. Apoptosis was determined by the Caspase-Glo 3/7 activity assay (Promega), which measures the caspase-3/7 activity by cleavage of the luminogenic substrate containing the DEVD sequence, and was normalized to protein concentrations. Clonogenic survival assay was performed as described previously (Propidium iodide (PI) staining/flow cytometry and bromodeoxyuridine (BrdU) incorporation assay were performed as describe previously . The datPreparation of DNA substrate was performed following the protocol described previously . BrieflyDownstream: biotin-AGGTCTCGACTAACTCTAGTCGTTGTTCCACCCGTCCACCCGACGCCACCTCCTG; Template: GCAGGAGGTGGCGTCGGGTGGACGGGATTGAAATTTAGGCTGGCACGGTC; Upstream: CGACCGTGCCAGCCTAAATTTCAATA.2, 2 mmol/L DTT, 100 mmol/L NaCl, 0.25 mg/mL BSA with or without recombinant flag-tagged DNA2 (10 nmol/L) in the presence of either DMSO or DNA2 inhibitor d16 (20 µmol/L). All reactions were incubated at 37°C for 10 minutes, followed by the addition of 2X stop solution . The nucleolytic products were boiled for 1 minute and subjected to electrophoresis using 15% denaturing gel containing 7 mol/L urea. The gel was transferred to nitrocellulose membrane by electroblotting, UV cross-linked and subjected to streptavidin/avidin conjugates detection.Nuclease activity assay was performed according to the method described by Masuda-Sasa . Biotin-6 MDAH-2774 cells were suspended in 150 µL PBS and injected subcutaneously into the dorsal flanks of NOD scid IL2 receptor γ chain knockout (NSG) mice. After the tumors had grown to the appropriate size for the experiments, the mice were randomized to receive the treatment with DNA2 inhibitor d16 alone or a combination of d16 and talazoparib. In the experiment investigating the effect of d16 alone, animals received intraperitoneal injection of d16 (30 or 15 mg/kg) twice weekly or vehicle DMSO control. In another study assessing the effect of the combination of d16 and talazoparib, d16 (20 mg/kg) was injected intraperitoneally twice weekly, and talazoparib (0.5 mg/kg) in 10% Dimethylacetamide (DMAc)/4% DMSO/PBS was injected intraperitoneally daily, 5 days per week. The nontreated control group received a mixture of 10% DMAc/PBS and DMSO. Tumor volume was measured every 3 days using caliper and calculated as follows: volume = longest tumor diameter × (shortest tumor diameter)2/2. The evaluator was blinded to the treatments. Animals were euthanized and tumors were harvested on the indicated dates. On the basis of the size variation of the MDAH-2774 xenografts, power analysis estimated 5 mice per group for 80% statistical power to detect 20% differences in tumor size between groups at P < 0.05. All experiments were performed under a BCM IACUC-approved protocol and all experiments confirm to IACUC standards and ethical regulations.All animal care and experimental procedures were performed in accordance with protocols approved by the BCM Institutional Animal Care and Use Committee (IACUC). A total of 2 × 10MDAH-2774 cells were treated with DNA2 inhibitor d16 (10 µmol/L), RU (10 µmol/L), or both for 8 hours. Cells were fixed with 4% paraformaldehyde for 20 minutes, followed by permeabilization in PBS buffer containing 0.5% Triton X-100 and 0.5% NP-40 for 10 minutes. Cells were blocked in 2% BSA-containing PBS at room temperature for 1 hour, followed by incubation with anti-RAD51 rabbit mAb for 1 hour, and Texas Red X-conjugated anti-rabbit secondary antibody for another hour. Nuclei were stained with Hoechst 33258 dye. Images were captured with a Zeiss fluorescence microscope equipped with ApoTome 2 (Axio Observer inverted microscope).The reagents and constructs for traffic light reporter (TLR) system have been kindly provided by Dr. Scharenberg through Addgene. Accession codes: Addgene: 31475 (pCVL SFFV d14GFP donor), 31476 [pCVL SFFV d14GFP EF1s HA.NLS.Sce(opt)], [pCVL SFFV-EF1s HA.NLS.Sce(opt)]. The experimental procedure was performed as described by Certo and colleagues  aliquots and heated individually at different temperatures ranging from 40°C to 45°C for 3 minutes followed by cooling for 3 minutes at room temperature. The cell suspensions were freeze-thawed three times using liquid nitrogen. The soluble fraction (lysate) was separated from the cell debris by centrifugation at 20,000 × g for 20 minutes at 4°C. The supernatants were transferred to new microtubes and analyzed by SDS-PAGE followed by Western blot analysis. For xenograft experiments, the frozen tumors were thawed on ice, and homogenized in cold PBS followed by three cycles of freeze-thawing using liquid nitrogen. Tissue lysates were separated from the cellular debris by centrifugation at 20,000 × g for 20 minutes at 4°C. The supernatants were diluted with PBS supplemented with protease inhibitor cocktail, aliquoted, and then heated at different temperatures for 3 minutes, followed by cooling down for 3 minutes at room temperature. Soluble fractions were isolated by centrifugation and analyzed as mentioned above.MDAH-2774 cells were harvested and resuspended in complete DMEM. Cell suspensions were treated with 20 µmol/L d16 in a COt test where P values < 0.05 were considered significant.Data were expressed as mean ± SD from at least three biological replicates. Statistical analyses were conducted using unpaired, two-tailed The data generated in this study are available within the article and its Supplementary Data. All other raw data are available upon request from the corresponding author.TP53 mutations across different cancer types further shows that DNA2 is expressed at a higher level in tumor samples harboring mutant TP53 than those harboring WT TP53 across all of TCGA cancer types  high-grade serous ovarian carcinomas bear TP53 mutations (P = 5.40e-12) and normal fallopian tube (P = 0.00217), respectively  compared with all other subtypes. Notably, ovarian cancers that do not respond to chemotherapy such as platins or taxanes, express higher levels of DNA2 than those that respond in TCGA ovarian cancer cohort  and disease-free survival  and disease-free survival  was identified through a virtual high-throughput screening using a predicted structure of DNA2 helicase domain, which was built through homology modeling from the crystal structures of yeast and human Upf1 . In the ublished . We firsublished . Howeverublished . The lowublished . Figure ublished . Moreoveublished . Other cublished . Compariublished .1-phase  ratio (1/(S+G2)  ratio . Consist2) ratio , d16 als1/(S+G2) . Interes1/(S+G2) . Because1/(S+G2) , it is lTo determine whether d16 targets DNA2 within the cells, we depleted DNA2 in H1299 cells and performed clonogenic survival assay after d16 treatment. Depletion of DNA2 blocked the effect of d16 on the inhibition of clongenic viability in H1299 cells . This reHuman DNA2 can activate the ATR checkpoint under replication stress in S-phase . We previn vivo, we established an ovarian cancer MDAH-2774 xenograft model in NOD scid IL2 receptor γ chain knockout mice (NSG) mice. The MDAH-2774 xenograft-bearing NSG mice were treated with vehicle control or d16 (30 mg/kg) twice a week by intraperitoneal injection for 1 month. As shown in in vivo. We also assessed the proapoptotic effect of d16 by quantifying cleaved PARP1 IHC staining. We found that d16 treatment increased the percentage of cleaved PARP1-positive cells compared with the vehicle control group (in vivo (3). Here we repeated the experiment and started d16 treatment when the xenografts had grown to bigger sizes (150–250 mm3) and reduced the dosage of d16 to 15 mg/kg, twice weekly. Consistently, we observed similar reduction of tumor volumes by d16 treatment , talazoparib , or both. Indeed, combination of d16 and talazoparib showed the best inhibitory effect on tumor growth  in DNA2, and subsequently identified C5 as a DNA2 inhibitor through the Site 1 molecular docking . Here wero assay ; (ii) CEro assay  and I anro assay  provide ro assay  and D; aro assay .Treatment with d16 induces cell-cycle arrest mainly at S-phase and inhibits HR. These activities are consistent with the anticipated outcomes from DNA2 inhibition, regardless of the p53 status of the cancer cells. Thus, d16 not only inhibits proliferation of mutp53-transfected H1299 cells, but also p53-null H1299 cells and WT p53-transfected H1299 cells , althougTP53 are associated with therapy resistance and a poor prognosis in cancer. On the other hand, mutp53-bearing cancer cells appear to be more dependent on DNA2 for survival . Consequently, the majority of DNA2 overexpression arises from alternative mechanisms. In particular, cancers or cancer cell lines harboring one of the hotspot mutp53 variants express higher levels of DNA2 than those harboring WT p53  repair and at stalled replication forks . Inhibitin vitro functional assay, we identify and validate d16 as a novel DNA2 inhibitor. Targeting DNA2 with d16 inhibits cancer cell survival and tumor growth in vivo in cancers harboring mutp53, and this effect is further enhanced when combined with other chemotherapeutic agents. Furthermore, as DNA2 plays a role in DSB repair via HR pathways, the combination of d16 and PARPis can be utilized as a synthetic lethal therapy in BRCA1- and or BRCA2-proficient cancer cells and xenografts. These findings provide opportunities to develop and characterize DNA2 inhibitors for synthetic lethality in mutant p53-bearing cancers.In summary, DNA2 is highly expressed in many types of human cancer, and its expression is associated with mutp53 expression, disease stages, and poor outcomes. Through virtual docking and Supplementary DataFigures S1-S7, Table S1Click here for additional data file.


Enhanced frequency of CD49d+IFNγ+ CTLs in the TBLN and blood of both the Nano11-based SwIAV vaccinates was observed. Animals vaccinated with both Nano11-based vaccines had upregulated cross-reactive secretory IgA in the lungs and serum IgG against heterologous and heterosubtypic viruses. However, in NanoS100–SwIAV vaccinates, a slight early reduction in the H1N1 pandemic virus and a late reduction in the SwIAV H1N1-OH7 load in the nasal passages were detected. Hence, despite vast genetic differences between the vaccine and both the challenge viruses, IN vaccination with NanoS100–SwIAV induced antigen-specific moderate levels of cross-protective immune responses.Swine influenza A viruses (SwIAVs) are pathogens of both veterinary and medical significance. Intranasal (IN) vaccination has the potential to reduce flu infection. We investigated the efficacy of split SwIAV H1N2 antigens adsorbed with a plant origin nanoparticle adjuvant [Nano11–SwIAV] or in combination with a STING agonist ADU-S100 [NanoS100–SwIAV]. Conventional pigs were vaccinated via IN and challenged with a heterologous SwIAV H1N1-OH7 or 2009 H1N1 pandemic virus. Immunologically, in NanoS100–SwIAV vaccinates, we observed enhanced frequencies of activated monocytes in the blood of the pandemic virus challenged animals and in tracheobronchial lymph nodes (TBLN) of H1N1-OH7 challenged animals. In both groups of the virus challenged pigs, increased frequencies of IL-17A Swine influenza A viruses (SwIAVs) cause seasonal respiratory disease and are endemic in pig herds throughout the world, and therefore, they exert severe economic burden on the swine industry. Furthermore, both human and avian influenza viruses infect pigs, and this attribute facilitates reassortment ushering in the potential evolution of virulent virus/es exerting catastrophic effects globally. The predominant SwIAV H1N1, H1N2, and H3N2 subtypes are characterized by significant diversity in the hemagglutinin (HA), neuraminidase (NA), and the remaining six  genes due to antigenic drifts and shifts. Currently, the available commercial SwIAV vaccines in grow-finisher pigs display variable efficacy due to maternal antibody interference, an inability to protect against variants, and inadequate mucosal adaptive immune responses. Hence, SwIAVs pose a challenge, and therefore, novel effective mucosal vaccines are needed for effective control and mitigation strategies  , TBLN cells, and BAL cells were harvested, restimulated, and immunostained as described previously . Five miIntracellular labeling procedure: Cells washing was performed once and permeabilization was performed with 1% saponin for 45 min at room temperature. This was followed by washing with saponin wash buffer (0.1% saponin). Incubation with fluorochrome conjugated mAbs or their corresponding isotype antibodies in 50 µL final volume of saponin wash containing 1% normal rabbit serum for 45 min at 4 °C was carried out subsequently. Cells were washed once in the saponin wash. Labeling with indicated secondary antibodies for 45 min at 4 °C was carried out in the next step. Cell washing was performed once, and the cells were resuspended in 200 µL FACS buffer and transferred to FACS tubes. Acquisition of the samples was performed using a live cell gate in a BD FACS Aria II flow cytometer. For each sample, 100,000 events were collected. The flow cytometry data were investigated using FlowJo software , and data plotting was carried out by employing GraphPad Prism . In all of the flow cytometry labeling panels, isotype and fluorescence minus one (FMO) control was maintained for representative pigs in each of the experimental groups. These controls were used for determining the gating strategy to differentiate positive and negative populations for the indicated markers.SwIAV-specific immunoglobulins IgG and secretory IgA (sIgA) antibodies titers were characterized . High bi4 cells/well) were cultured in 200 μL DMEM complete media. Incubation was carried out in a 37 °C humidified 5% CO2 incubator overnight. Round bottom 96-well plates were used for preparing ten-fold serial dilutions of nasal swab, BAL fluid, and lung lysate samples in serum-free DMEM. The plates with confluent monolayers of MDCK cells were washed with PBS. Cells were inoculated with 100 μL/well of serially diluted test samples for 1.5 h at 37 °C in a 5% CO2 incubator. One hundred microliters of DMEM serum-free medium with 2 µg/mL L-(tosylamido-2-phenyl) ethyl chloromethyl ketone (TPCK)-trypsin  was added to the wells. Plates were incubated at 37 °C in humidified 5% CO2 incubator for 36 h. The cells were fixed using 80% acetone (10 min). Labeled with an influenza A virus (IAV) NP protein specific mAb , and diluted at 1:5000 for 2 h at 37 °C in a 5% CO2 incubator. Plates were washed and incubated for 1.5 h with 1:3000 dilution of (50 μL/well) the secondary antibody Alexa Fluor 488 conjugated with goat anti-mouse IgG (H+L) antibody  at 37 °C in a 5% CO2 incubator. Glycerol was mixed with PBS, pH = 8 at 6:4 proportion, and used as the mounting medium. Infection was documented using an Olympus fluorescent microscope . Infectious titer was calculated by the Reed and Muench method. For live virus detection, by using immunofluorescence assay, the cutoff was 1:5 dilution of the samples, which gives background fluorescence, and anything 1 log10 and above was considered specific.Virus titers were characterized as per optimized lab protocol . MDCK .The Nano11 surface adsorbed split SwIAV H1N2 Ags and ADU-S100 adjuvant; either together (NanoS100–SwIAV) or only the virus antigen (Nano11–SwIAV) were characterized for their polydispersity index (PDI), adsorption efficiency, size, and zeta potential in our recently published article .We monitored the clinical signs of flu  post-challenge and scored the gross lung lesions of pigs during necropsy. However, neither of the challenge viruses caused appreciable clinical signs of disease, and during necropsy did not observe any difference among the pig groups with respect to lung lesions scores. This is likely because pigs were euthanized at DPC6. To appreciate any differences in the lung lesions, DPC2 to DPC4 is ideal during the active infection. Further, the safeguarding potential of the Nano11–SwIAV and NanoS100–SwIAV IN vaccines was assessed by measuring the titers of the challenge SwIAV H1N1-OH7 and H1N1 pandemic virus in the nasal swabs (NS), BAL fluid, and lung lysate specimens. A remarkable abatement of SwIAV H1N1-OH7 titers (not significant) in the nasal passages of the NanoS100–SwIAV vaccinates compared to the Mock+Ch group animals was observed at DPC6 C but not+ lymphocytes, CD172a+SynCAM+ cells were considered as putative porcine dendritic cells and CD172a+SynCAM− cells as monocytes [+ and CD80/86+ dendritic cells and monocytes in both lung draining TBLN and PBMCs of pigs vaccinated intranasally and challenged with influenza viruses.In order to understand the mechanisms of the immune responses, innate immune cell frequencies in TBLN mononuclear cells (MNCs) and PBMCs were analyzed. The gating strategy employed for the identification of dendritic cells and monocytes is described in onocytes . Differeonocytes . Therefo−CD172a+SynCAM−): In TBLN MNCs challenged with SwIAV H1N1-OH7, the frequencies of CD80/86+ monocytes were decreased significantly, while activated monocytes expressing both CXCL10 and CD80/86 were significantly increased compared to the mock group in all of the animal groups (p < 0.01 to p < 0.001) (p < 0.05 to p < 0.01) (+ monocytes compared to the Mock+Ch group (p < 0.05) in TBLN MNCs challenged with SwIAV-OH7 (Monocytes (CD3< 0.001) A,B. Howe < 0.01) C,D. In awIAV-OH7 A.−CD172+SynCAM+): the frequencies of total CD80/86+ dendritic cells were significantly decreased in both the Nano11–SwIAV+Ch and NanoS100–SwIAV+Ch group of animals compared to the mock group (p < 0.05 to p < 0.01) in TBLN MNCs upon challenge with both SwIAV H1N1-OH7  (p < 0.0001) in all of the SwIAV H1N1-OH7-challenged pigs compared to the mock animals (+ CTLs and CD49d+IL-17A+ CTLs compared to the mock group (p < 0.05) (+ CTLs and CD49d+IFNγ+ CTLs compared to the mock group (p < 0.01 to p < 0.001)  . The exp animals A. The TB < 0.05) B,C, Whil< 0.001) D,E.+ Th/memory cells compared to the mock group animals (p < 0.05) (p < 0.05 to p < 0.0001) in all of the H1N1 pandemic virus-challenged pigs compared to the mock animals (+ CTLs (p < 0.05 to p < 0.01) (+IL-17A+ CTLs (p < 0.01)  F; howeve animals G. A sign < 0.01) H and CD4 < 0.01) I comparep < 0.05) (p < 0.001) (p < 0.05)  A. While < 0.001) B. Howeve < 0.05) B.+CTLs compared to the Mock+Ch group animals (p < 0.05 to p < 0.01) (+IFNγ+ CTLs were enhanced significantly (p < 0.01) in NanoS100–SwIAV vaccinates  C. In addccinates D systemiAnimals vaccinated with NanoS100–SwIAV and Nano11–SwIAV vaccines and challenged with the heterologous SwIAV H1N1-OH7 exhibited significantly augmented SwIAV H1N1-OH7- and H3N2-OH4-specific sIgA antibody titers in lung lysate and BAL fluid compared to the Mock+Ch animals M,P,R, reThe antigen-specific IgG and sIgA titers in serum, lung lysate, and BAL fluid samples of vaccinates were determined at DPC6 A–R. NanoVaccination/immunization is a global health success story that mitigates 3.5–5 million deaths every year from infectious diseases such as pertussis, tetanus, diphtheria, influenza, and measles. Furthermore, human, animal, and plant health are interlinked and interdependent on the health of the ecosystem in which they reside, and this approach is referred to as the One Health approach. Therefore, the development of novel and efficacious vaccines against swine influenza is of critical importance to prevent the emergence of potential pandemics and to secure food security . Previou+ dendritic cells and monocytes co-expressing the activation marker CD80/86 in pigs. NanoS100–SwIAV vaccine upon the SwIAV-H1N1-OH7 and H1N1 pandemic virus challenge infection stimulated the population of CXCL10+CD80/86+-activated monocytes in the draining TBLN and in the systemic compartment . Furthermore, starting from DPI4, antigen-specific CD4+ and CD8+ T lymphocytes generated mainly IFNγ and/or TNF-α and attained maximum peak frequencies at DPI9 in the lungs rather than in the TBLN and blood. These cells also exhibited cross-reactivity to heterologous influenza virus strains [+ T lymphocytes mediated protection against homo- and heterosubtypic IAV infections. Likewise, another study also documented the evidence for the protective significance of cross-protective (heterosubtypic immunity) lung-resident memory CD8+ T cells following intramuscular vaccination with a tandem repeat extracellular domain of M2 (M2e) epitopes on VLP [+ and CD8+ T cells. Furthermore, hemagglutinin (HA) and neuraminidase (NA) epitopes were also targeted by cross-reactive CD4+ T cells. However, CD8+ T cells targeted internal matrix protein (M1), nonstructural protein 2 (NS2), and acid polymerase (PA) epitopes [Our data underline the protective importance of antigen-specific adaptive immunity in influenza infection, and this is consistent with the published literature. Previous work from the lab established the phenotypes of innate, adaptive, and regulatory immune cells during the acute phase of SwIAV H1N1-OH7 infection in pigs . The sig at DPC6 . In addi in pigs ,35. Furtcumented . Consist strains . In a st strains , followis on VLP . Using aepitopes . Our expWith respect to humoral memory responses, intranasal administration of NanoS100–SwIAV upon SwIAV H1N1-OH7 challenge elicited significantly high cross-protective SwIAV H1N1-OH7- and H3N2-OH4-specific sIgA titers in the lungs. However, in the case of the H1N1 pandemic virus challenge, NanoS100–SwIAV stimulated significant titers of H1N1 pandemic virus- and SwIAV-H1N2-OH10-specific serum and lung IgG and lung sIgA and SwIAV H3N2-OH4-specific lung sIgA, compared to their Mock+Ch counterparts. The experimental animals were raised conventionally, reflecting field conditions, with maternal influenza-specific IgG antibodies of various levels as sows were vaccinated. Therefore, we detected high basal/pre-existing immune responses in the animals. The DPC0 data confirm this observation . Hence, Our results demonstrate that NanoS100–SwIAV can efficiently induce the expression of CD49d in both T-helper/memory cells and CTLs in both the lung draining TBLN and systemic compartment. Furthermore, CD49d, due to its ability to act as a signaling receptor, has been shown to augment B cell survivability via the upregulation of the expression of Bcl-2 family members ,41. Henc+ T cells [The above described findings are in agreement with published work on split virus influenza vaccines. In a study in humans, high-dimensional proteomic profiling by mass-cytometry and Luminex was employed to characterize major signaling pathways and cytokine production for all major immune cells in whole-blood stimulated with split virus influenza vaccine (SVIV). SVIV treatment ex vivo stimulated an overlapping but unique proteomic signature profile, different from Toll-like receptor (TLR) activation. The stimulatory activity of SVIV was dependent on the presence of influenza-specific antibodies, suggesting that this stimulatory activity was immune complex-dependent, mediated via Fcγ receptors. Hence, persons with pre-existing immunity mount potent antigen-specific immune responses . Another T cells . In cont T cells .Mycobacterium bovis bacillus Calmette–Guerin (BCG) vaccine resulted in suppression of inflammation, prevented lung immunopathology, and conferred significant protection against M. tuberculosis infection, and this protection was directly linked to IL-17A response [+ alveolar macrophage-mediated killing of opsonized pneumococci [Escherichia coli (ETEC) infection, oral immunization with F4 fimbriae stimulated a robust antigen-specific protective systemic Th17 response [+ CTL responses compared to their non-adjuvant vaccinate counterparts. Furthermore, the dose sparing effect was observed in the case of intradermal vaccination compared to intramuscular vaccination in SPF pigs [Various research groups have documented the therapeutic role/s of antigen-specific Th17 responses. Subcutaneous administration of a trivalent subunit influenza vaccine with a cationic liposome adjuvant system CAF01 augmented the antigen-specific humoral immunity, robust Th1, and Th17 responses in mice. Furthermore, Th1/Th17 cytokines were persistent until 20 weeks after the last vaccination. In addition, CAF01-adjuvanted influenza vaccine conferred complete protection of mice challenged with a drifted H1N1 influenza strain . In anotresponse . In an eumococci . In addiresponse . ContinuAlthough the current pool of serotype-specific influenza vaccines mainly elicits antibody responses against viral surface proteins such as HA and NA, they are deficient in the induction of cell-mediated responses targeting viral conserved internal proteins. Furthermore, antigenic shift and drift contribute toward the constant evolution of field strains and the evasion of host immune responses. Hence, the stimulation of humoral responses against receptor-binding epitopes and broadly neutralizing conserved epitopes, coupled with robust cell-mediated immune responses against highly conserved internal proteins, is a better strategy. The conserved proteins NP, Matrix protein 2 (M2), and relatively conserved HA stem region have been reported to confer broad cross-protection/heterosubtypic immunity in preclinical animal models . AnotherUpcoming research activities are aimed at elucidating the mechanisms of mucosal vaccine efficacy involving multivalent SwIAV vaccine candidates in combination with potent adjuvants. Furthermore, delineation of the interplay of the host and microbiota, mucosal routes of vaccination, and adjuvants by using unbiased systems of biology strategies are essential to combat the threat of constantly evolving SwIAV field strains and potential pandemics.Irrespective of the vast genetic difference (77% HA gene identity) between the vaccine H1N2 and both of the H1N1 challenge influenza viruses, intranasal vaccination of split virus antigens surface adsorbed onto Nano11 particles, along with the STING adjuvant combination, elicited superior cross-protective T cell responses. This was observed both in the mucosal and systemic immune compartments, suggesting the utility of the combination of mucosal adjuvants in mitigating SwIAV infection in pigs and in reducing the transmission of zoonotic SwIAV from pigs to humans.


Background: S100 Calcium Binding Protein A16 (S100A16), a novel member of S100 protein family, is linked to tumorigenic processes and abundantly expressed in CNS tissues. Our study aimed to explore the biological function and possible mechanism of S100A16 in the progression of glioma.Methods: Sequence data of S100A16 and survival prognosis of glioma patients were initially analyzed using public databases. Glioma tissues were collected to examine S100A16 expression levels. Glioma cell lines and nude mice were subjected to in vitro and in vivo functional experiments. Western blot, immunofluorescence (IF), immunoprecipitation (IP) and ubiquitination assays were done to further elucidate the underlying mechanism.Results: This study firstly revealed that S100A16 was markedly up-regulated in glioma, and patients with higher S100A16 levels have a shorter survival time. S100A16 overexpression promoted the proliferation, invasion and migration of glioma cells, and the tumor formation of nude mice. Importantly, we identified S100A16 as a negative regulator of the Hippo pathway which could decrease LATS1 expression levels, promote the YAP nuclear import and initiate the downstream target genes CYR61 and CTGF. Moreover, our data showed that S100A16 destabilized LATS1 protein by inducing the CUL4A-mediated LATS1 ubiquitination degradation.Conclusions: This study demonstrated a vital biological role of S100A16 in glioma progression mechanism by promoting CUL4A-mediated LATS1 ubiquitination to inhibit Hippo signaling pathway. S100A16 could be a novel biomarker and treatment option for glioma patients. Glioma is a primary intracranial tumor that originates from glial cells, which account for approximately 24.5% of all brain tumors, as well as 80.9% of malignant tumors The S100 family is a class of small acidic calcium-binding proteins that act as calcium sensors or extracellular factors to regulate a variety of biological processes. S100 proteins are involved in numerous tumor behavior, such as cell proliferation, invasion, angiogenesis and immune escape The Hippo signaling pathway, initially discovered in Drosophila, is a highly conserved growth control pathway regulating tissue development and regeneration, as well as cell proliferation and apoptosis in vitro and in vivo functional tests. The results indicated that S100A16 predicted poor prognosis in glioma by promoting cell proliferation, migration and invasive abilities via inhibiting LATS1 expression in the Hippo pathway. Our study established a critical role of S100A16 in glioma development, and offered a new direction for glioma diagnosis, therapy and prognosis.In this study, we aimed to gain a better understanding of S100A16's biological function and molecular mechanism in glioma progression by using bioinformatics analysis, http://gepia.cancer-pku.cn/detail.php), CGGA (http://www.cgga.org.cn/), TCGA  and GEO database (http://www.ncbi.nlm.nih.gov/geo).Bioinformatics analyses were carried out using gene expression profiles and survival information from public databases, including GEPIA  between high- and low-S100A16 expression groups. R package “DESeq2” was used to distinguish DEGs, which were presented with heatmaps via R package “heatmap” and volcano maps via R package “ggplot”. Glioma patient tissues and clinical characteristics were collected from the First Affiliated Hospital of Nanjing Medical University. Normal brain tissues (NBTs) were taken from patients receiving decompressive craniotomy for traumatic brain injury (TBI) as the control group. The post-surgical specimens were confirmed with histopathologic diagnosis based on WHO criteria. All patients gave their written informed consent before the procedure.2. Logarithmic-phase cells with superior performance were chosen for the present study.The glioma cell lines U87, U251, T98G, LN229, A172, SW1738, N3 were purchased from the Chinese Academy of Sciences and grown in DMEM with 10% FBS and 0.5% Penicillin/Streptomycin. Human astrocyte cell NHA was obtained from University of Bergen, and cultured following the manufacturer's instruction. Cells were propagated in a humidified chamber at 37°C with 5% COTable S1.Plasmids sh-S100A16, sh-NC, sh-LATS1, si-CUL4A and S100A16 cDNA, LATS1 cDNA, CUL4A cDNA were transfected into glioma cells by utilizing Lipofectamine® 3000 transfection reagent . Lentivirus was constructed by Genechem , and used to infect cells following the manufacturer's guidelines. After 48h of infection, we cultured cells in DMEM with 1ug/ml puromycin  to obtain stable clones. The target sequences applied in this study were presented in 2O. Real‐time quantitative PCR (qRT-PCR) was finally performed on the StepOnePlus Real-Time PCR system  according to pre-set amplification parameters. The qRT-PCR primers were listed in Table S2. The relative expression value of each gene was computed with the 2-ΔΔCT method.Total RNA was isolated from cells and tissues utilizing Trizol reagent , then reverse-transcribed to cDNAs utilizing HiScript Synthesis kit  according to the manual. PCR mixtures were prepared with SYBR ® Green Master Mix , RNA primer, cDNA and ddHTotal protein was lysed from cells and tissues utilizing RIPA buffer, and its concentrations were measured using a BCA kit . Equal amounts of protein were separated by SDS-PAGE, then electro-transferred onto nitrocellulose (NC) membrane . The membrane was blocked with 5% skimmed milk for 2h at room temperature (RT). Blots were incubated with primary antibodies overnight at 4℃, washed with 1×TBST (3×10 min), and subsequently incubated with secondary antibodies for 2h at RT. Protein bands were exposed with an enhanced chemiluminescence reagent , and visualized by gel analysis imaging system . The following antibodies were used: S100A16, pLATS1 and pMST1/2 , β-Actin , LATS1, MST1, MST2, SAV, pMOB, MOB, pYAP, YAP/TAZ, Ub, CUL4A , and DCAF1, DDB1 .After cell lysis, equal amounts of protein were taken to incubate with primary antibody  or IgG  overnight at 4°C. Then, the protein-antibody immunoprecipitates were collected by protein A/G magnetic beads  for 2h at RT. Finally, the complexes were washed with PBS, boiled in loading buffer, and subjected to western blot. For the ubiquitination assay, cells were pre-treated with 10μM MG132 for 8h before IP analysis.To identify YAP intracellular distribution, nuclear and cytoplasmic sections were isolated with Nuclear and Cytoplasmic Extraction Reagents consisting of CER I, CER II and NER , following the manufacturer's guidelines. Histone H3  and β-Tubulin  were used as loading controls for cytoplasmic and nuclear proteins, respectively.2O2 for 10 min to prevent endogenous peroxidase activity. The slides were incubated with 5% goat serum for 30 min at RT, then incubated overnight at 4°C with S100A16 , LATS1 antibody , or stained with H&E. After being washed with PBS, tissues were added with secondary antibody binding to primary antibody for 1 h at RT. Streptavidin-biotin complex (SABC) and Diaminobenzidine (DAB) were used to visualize the immunostaining. Finally, sections were counterstained with hematoxylin, dehydrated with alcohol, dried and covered in resin. The stained samples were reviewed and analyzed under a microscope .Tissues were fixed in 4% paraformaldehyde and embedded in paraffin, then cut into 5 um slices for IHC. Paraffin slices were deparaffinized in xylene and hydrated in gradient alcohol. After antigen repair, tissues were immersed in 3% HTissue fixation, deparaffinization, hydration, and antigen repair were the same as the IHC procedure. Cells were fixed with 4% paraformaldehyde for 30 min. Next, tissues or cells were permeabilized with 0.1% Triton-X 100, and blocked with 5% BSA for 30 min. Then, they were incubated overnight with primary antibodies. A TRITC-labelled goat anti-Rabbit IgG  was used as secondary antibody for 1 h at RT in the dark. DAPI was employed to stain the nuclei for another 10 min. IF images were observed under a light microscope  or Confocal microscope .GBM cells were seeded in 96-well plates  in triplicate overnight. Next, the cell growth was assayed at different time points with a CCK-8 kit  following the manufacturer's protocol. Absorbance at 450 nm was measured by a microplate reader .2 incubator for 2 weeks. Colonies were subsequently fixed and stained with 4% paraformaldehyde and Crystal violet. The colony-forming capacity was compared by counting the number of colonies.GBM cells were plated in 6-well plates (500 cells / well) and cultured in a 37 °C 5% CO5 cells/well) in triplicate overnight. Cells were stained with 50 μM EdU  for 2 h, fixed with 4% paraformaldehyde and permeabilizated with 0.5% Triton X‐100. After three PBS washes, they were incubated with 100 µL of 1 × Apollo® reaction for 30 min, followed by 1×Hoechst33342 staining for another 30 min. The stained cells were photographed by fluorescence microscopy .GBM cells were seeded in 96‐well plates (1×105 cells/well) pre-coated with Matrigel  in serum-free DMEM. The bottom chambers were filled with DMEM containing 10% FBS. After incubation for 24 h, the penetrated cells were fixed with 4% methanol, then stained with 0.1% crystal violet. Each well was randomly photographed and counted by a light microscope .GBM cells were added into the top of 24-well Transwell chambers . The scratch healing ratio was determined using the formula: (0h scratch width - 48h scratch width) / 0h scratch width × 100%.6cells / 100μL) were implanted subcutaneously into the right shoulder of nude mice. Tumor volume was monitored with a vernier caliper weekly. At 30 days after inoculation, xenografts were excised, weighed and photographed. For the intracranial orthotopic model, luciferase-expressing U87 cells (1×106 cells / 10 μL) were injected into the frontal lobes of nude mice brains under a stereotaxic apparatus. Tumor growth was visualized weekly by a bioluminescence imaging system. Mice were monitored for clinical symptoms, moribund animals were euthanized, and brains were removed for the next experiments. Animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Nanjing Medical University (Number: IACUC-2106046).To establish a subcutaneous xenograft model, stable-transfected glioma cells . The result suggested that S100A16 was up-regulated in GBM and LGG, as well as bladder urothelial carcinoma (BLCA), cervical squamous cell carcinoma (CESC), colon adenocarcinoma (COAD) et.al, whereas down-regulated in adrenocortical carcinoma (ACC), esophageal carcinoma (ESCA), and kidney Chromophobe (KICH) et.al. The overexpression of S100A16 in GBM was further examined in TCGA database . Furthermore, we analyzed S100A16 expression and clinical information of 325 glioma patients based on the CGGA database and showed that the level of S100A16 expression increased with the grade of malignancy, with the highest expression in GBM patients . Survival analysis showed that high S100A16 expression significantly shortened the survival time of glioma patients, especially for GBM patients . and survival differences were also validated in GEO database .S100A16 expression in tumor was first screened in GEPIA database   were screened and displayed on the hot map Fig. A-B and vFig. , qRT-PCR indicated that S100A16 mRNA levels were significantly up-regulated in HGG (WHOgrade 3-4) and LGG (WHO grade 1-2) compared to NBT. In addition, western blot suggested that S100A16 protein expression was evaluated with increasing glioma grade . IHC assay also confirmed S100A16 overexpression in glioma, and its positivity was observed in the cytoplasm and nuclei of glioma cells . IF staining revealed the strong cytosolic expression of S100A16 in GBM relative to NBT . Next, we analyzed S100A16 expression in glioma U87, U251, T98G, LN229, A172, SW1738, N3 cells and control NHA cells by western blot, and showed different levels of S100A16 proteins . U251 and T98G cells, presenting the relatively higher expression of S100A16, were chosen for sh-S100A16, while U87 and A172 cells for S100A16 cDNA transfection. These findings revealed that S100A16 was up-regulated in both glioma tissues and cells, which may play a vital role in tumorigenesis and malignancy.To validate the bioinformatic data, we analyzed S100A16 expression in clinical samples from 5 NBTs and 20 glioma tissues of different grades. As seen in Fig. ), and S100A16 overexpression in both U87 and A172 cells by using western blot . CCK-8 growth curves showed that silencing S100A16 markedly reduced T98G and U251 cells' growth , while over-expressing S100A16 increased U87 and A172 cells' growth . The findings were corroborated in EdU and cloning assays. The EdU-positive cells or colonies were decreased by sh-S100A16 in T98G and U251 cells , but increased by S100A16 overexpressing in U87 and A172 cells . Moreover, we also performed a transwell assay and a wound-healing assay, the results demonstrated that silencing S100A16 by shRNA markedly decreased T98G and U251 invasion cells  and scratch healing rate Fig. F, and up-regulating S100A16 exhibited elevated A172 and U87 invasion cells  and healing rate . Taken together, these findings indicated that S100A16 promoted glioma cells' proliferation, migration, and invasion.We further conducted functional experiments to investigate the significance of S100A16 in glioma progression. We first confirmed the knockdown efficiency of two sh-S100A16 knockdown in both T98G and U251 cells . As the downstream transcriptional regulators of YAP/TAZ, CTGF and CYR61, we used qRT-PCR to examine their mRNA levels. It was revealed that YAP/TAZ target genes were declined in T98G- and U251-sh-S100A16 cells , and raised in U87- and A172-S100A16 cells . To observe whether S100A16 affects YAP/TAZ activity by boosting its nuclear localization, YAP/TAZ protein levels in cytoplasmic and nuclear components were separately measured. In U251-shS100A16 cells , YAP protein was significantly reduced in nucleus compared to controls, but increased in U87-S100A16 nucleus . In addition, IF showed sh-S100A16 increased YAP expression and accumulation in the cytoplasm of U251 cells, as well as the mobility of YAP from the nucleus to the cytoplasm . Conversely, nuclear localization of YAP was enhanced in U87-S100A16 cells . In total, these findings suggest that S100A16 inhibited Hippo signaling pathway, thus regulating YAP behavior and cellular distribution in glioma cells.Hippo signaling pathway has been previously reported to play a vital role in tumor initiation and progression. Bioinformatics in our study predicted that S100A16 may regulate Hippo signaling pathway in glioma. Therefore, we examined the impact of S100A16 on this pathway's core members in glioma cells by Western blot. As shown in Fig. , S100A16 expression was not influenced by LATS1, whereas knockdown of LATS1 in U251-shS100A16 cells reduced the upregulated LATS1 expression due to S100A16 knockdown, ectopic expression of LATS1 in U87-S100A16 cells restored the poor LATS1 expression due to S100A16 overexpression. We also conducted a series of functional experiments to demonstrate the effect of LATS1 in S100A16-independent proliferation, migration and invasion. CCK-8  showed that down-regulation of LATS1 restored the proliferation rate of U251 cells inhibited by shS100A16, while upregulation of LATS1 reduced the growth ability of U87 cells transfected by S100A16. This was consistent with the results of EdU  and colony-forming assays . Moreover, transwell  and wound healing assays  suggested that sh-LATS1 reversed the inhibitory impact of sh-S100A16 on glioma migration and invasion in U251 cells. In contrast, over-expression of LATS1 in U87-S100A16 cells obtained the inverse effects. These data support that LATS1 could be a potential downstream and active target of S100A16 in the development of glioma.We have identified that S100A16 induced a cascade response in the Hippo pathway. Among them, the upstream MST1/2 protein and its phosphorylation were not altered obviously, while LATS1 protein was significantly different. Despite changes in its phosphorylation, it may be due to obvious changes in total LATS1 protein. These allowed us to consider that the effects of S100A16 on tumorigenesis were dependent on LATS1 protein. Therefore, we co-transfected shRNA targeting LATS1 into U251-shS100A16, and co-transfected LATS1 cDNA into U87-S100A16. Overexpression or knockdown efficiency was assessed by western blot. As shown in Fig. ). This led to the possibility that S100A16 modulates LATS1 protein stability. We next determined LATS1 protein levels in GBM cells pretreated with the proteasome inhibitor MG132. As shown in Fig. , MG132 restored LATS1 levels after overexpressing S100A16 in U87 cells. In addition, a protein synthesis inhibitor, CHX, was added to GBM cells to detect the half-life of LATS1 protein. The result showed that LATS1 half-life was lengthened in U251-shS100A16 cells , but was lessened in U87-S100A16 cells . Thus, S100A16 could promote proteasome-mediated degradation of LATS1.To elucidate the mechanism by which S100A16 controls LATS1 activity, we tested whether S100A16 affects LATS1 transcriptionally. Interestingly, alteration of S100A16 had no obvious effect on mRNA levels of LATS1 , while increased in U87-S100A16 cells . In total, the data above indicated that S100A16 mediated the stability of LATS1 in a ubiquitin-independent way.Ubiquitin (Ub) is a classical post-translational modification involved in proteasomal degradation. Thus, we investigated whether S100A16 regulates LATS1 proteolysis through the ubiquitin pathway in GBM cells pre-treated with MG132. The results suggested that endogenous LATS1 ubiquitination was decreased in U251-shS100A16 cells , up-regulating CUL4A reversed the reduced LATS1 ubiquitination by sh-S100A16. In the U87-S100A16 cells , silencing CUL4A attenuated the increased LATS1 ubiquitination caused by S100A16 overexpression. We also performed CO-IP experiments to confirm the existence of the interaction of S100A16 and CUL4A . Overall, these results support our hypothesis that S100A16 destabilizes LATS1 by facilitating CUL4A-mediated ubiquitination for proteasomal degradation.Ubiquitination regulation is usually catalyzed by in vivo, we infected glioma cells with S100A16 shRNA or cDNA lentivirus, and constructed the subcutaneous or orthotopic animal model. Representative images of subcutaneous tumors were shown in Fig. . The oncogenic efficiency of S100A16 was confirmed and evaluated by tumor volume  and tumor weight . Compared with controls, tumor growth was decreased in U251-shS100A16 cells, whereas increased in U87-S100A16 cells. IHC showed that S100A16 was inversely linked with LATS1 expression, indicating possible interaction of these two proteins . We have previously validated the LATS1 antibody specificity using LATS1 KD glioma cells . In an orthotopic xenograft model, the tumor growth was monitored by bioluminescence signals, and representative images were shown in Fig. . Quantitative analysis demonstrated that S100A16 significantly enlarged the tumor volume in vivo . In survival curve, overexpression of S100A16 significantly shortened the survival time of nude mice bearing orthotopic tumors . The HE-stained orthotopic tumors are presented in Fig. , with an expanded tumor infiltration in the S100A16-transfected group. To confirm the relationship between S100A16 and LATS1, we chose four pairs of resected tumors at random from shCtrl- and S100A16-transfected groups, respectively. Western blot showed that overexpression of S100A16 lead to a reduction of LATS1 expression . These results are consistent with the previously proposed mechanism that S100A16 could down-regulate the downstream protein LATS1, and play an oncogenic role in glioma. A schematic diagram of S100A16 promoted glioma progression was concluded in Fig. 12.To prove the promoting effect of S100A16 Glioma is the most frequent and lethal type of primary CNS malignancy, with a high surgical risk, easy recurrence after surgery and poor survival prognosis in vitro and vivo. It was first established that S100A16 upregulation greatly enhanced glioma proliferation, migration and invasion. These suggested that S100A16 might act as an oncogene promoter in glioma.In this study, we first found that S100A16 expression differed between normal brain and glioma tissues, which varied across tumor types, based on GEPIA database. This indicated that S100A16 possesses a dual role, acting as a tumor suppressor or tumor promoter in different malignancies. In human brain tissues, S100A16 is highly expressed in glioma relative to non-neoplastic brain tissues, which was also verified in the TCGA, CGGA and GEO databases. In addition, S100A16 was significantly associated with glioma patients' prognosis, meaning that the survival time of patients with high-S100A16 expression was greatly shorter than those with low S100A16. To further confirm the role of S100A16, we determined S100A16 expression in real clinical specimens, and conducted functional experiments The potential mechanisms of S100A16-mediated glioma development require deep exploration. Our bioinformatic analysis implied that the Hippo signaling pathway, MTOR pathway, WNT pathway et.al might be implicated in glioma malignant progression regulated by S100A16. Previous studies have reported that MTOR and WNT signaling play important roles in cellular growth, migration and survival in glioma progression LATS1 encodes a serine/threonine kinase that negatively regulates YAP, and exhibits anti-tumor activity by restricting cell proliferation or promoting apoptosis However, our study still has several limitations. The specific mechanism of interaction among S100A16, CUL4A and LATS1, as well as the binding sites warrants more investigation in our future work. Moreover, there are more than one E3 ubiquitin ligase for LATS1, further investigations are needed to characterize other CUL4A adaptors or additional E3 ligases in the regulation of tumor progression by S100A16.In conclusion, we discovered that S100A16 was highly expressed in glioma and related to prognostic survival. Furthermore, S100A16 acted as a negative regulator of the Hippo signaling pathway to assist in glioma proliferation, invasion and migration, the mechanism by which was probably to interact with and promote CUL4A, an E3 ubiquitin ligase binding to LATS1 and induce its ubiquitination degradation, which resulted in YAP/TAZ entry into the nucleus, targeting the downstream genes CTGF and CYR61. Consequently, these data revealed a new insight into glioma pathogenesis and identified S100A16 as a prospective molecular target for glioma patients' diagnosis, treatment and prognosis.Supplementary figures and tables.Click here for additional data file.


S. cerevisiae LYS2 half-locus experimental system and a novel method for detecting rare translocation events, they provide mechanistic insight into the kinetics and dependencies of MIRs during chromosomal segregation and the formation of structural variations.In this study, Reitz et al. delineate two molecular models for multi-invasion-induced rearrangements (MIRs) involved in homologous recombination. Using the  δ-independent manner, unlike recombinational DNA synthesis. This work provides a refined mechanistic understanding of these HR-based SV formation pathways and shows that complex repeat-mediated SVs can occur without displacement DNA synthesis. Sequence signatures for inferring MIR1 from long-read data are proposed.Punctuated bursts of structural genomic variations (SVs) have been described in various organisms, but their etiology remains incompletely understood. Homologous recombination (HR) is a template-guided mechanism of repair of DNA double-strand breaks and stalled or collapsed replication forks. We recently identified a DNA break amplification and genome rearrangement pathway originating from the endonucleolytic processing of a multi-invasion (MI) DNA joint molecule formed during HR. Genome-wide approaches confirmed that multi-invasion-induced rearrangement (MIR) frequently leads to several repeat-mediated SVs and aneuploidies. Using molecular and genetic analysis and a novel, highly sensitive proximity ligation-based assay for chromosomal rearrangement quantification, we further delineate two MIR subpathways. MIR1 is a universal pathway occurring in any sequence context, which generates secondary breaks and frequently leads to additional SVs. MIR2 occurs only if recombining donors exhibit substantial homology and results in sequence insertion without additional breaks or SVs. The most detrimental MIR1 pathway occurs late on a subset of persisting DNA joint molecules in a PCNA/Pol Structural variants (SVs) of the genome are the predominant class of driver mutations in most cancer types and are more numerous than the more widely studied single-nucleotide variants and indels . These SThe maintenance of genome stability involves independent molecular mechanisms that inhibit the occurrence of DNA damage and achieve their accurate repair . HomologThe tenet of BIR is a displacement DNA synthesis step that goes unabated until stabilization of the DSB end by either telomere capture, merging with a converging replication fork, D-loop disruption and annealing of the extended ssDNA to a complementary DNA end, or end joining . BIR depThe role of structure-selective endonucleases (SSEs) in the generation of HR-mediated chromosomal rearrangements has long been recognized as part of the canonical double-strand break repair (DSBR) model. Endonucleolytic resolution of an allelic interhomolog double-Holliday junction (dHJ) intermediate can lead to the formation of either a crossover or a noncrossover with equal likelihood. Resolution into a crossover can cause loss of heterozygosity of the centromere-devoid chromosomal region at the next cell generation . If DNA Saccharomyces cerevisiae cells (We previously identified a genomic instability mechanism originating from “multi-invasions” (MIs), a JM in which a single DSB end has invaded two independent dsDNA donors . MIs areae cells , 2021b. ae cells . This trae cells . MIR insae cells . Finallyδ-PCNA, unlike D-loop extension. These results elucidate a novel HR-based mechanism leading to tripartite SVs in the absence of displacement DNA synthesis.Here we investigated the existence of two MIR subpathways postulated on the basis of past molecular and genetic evidence: MIR1 that generates a translocation and additional DSB ends, applicable in any sequence context, and MIR2 that generates an insertion without forming an additional DSB but has specific sequence requirements . The system has three components: a site-specific DSB-inducible construct and two donors. The heterozygous DSB-inducible construct on chromosome V consists of the HO cut site (HOcs) and, on one side, the central part (YS) of the LYS2 gene. This YS sequence has nonoverlapping homology with two donors: the LY and S2 halves of the LYS2 gene . These latter configurations resulted in higher frequencies of secondary rearrangements  analysis of MIR products confirmed that all recombinants resulted in the formation of the mosome V . In ∼15% product . The prembinants A. Most  cases, as expected  of the analyzed small Lysmosome V D. The mamosome V D. Howevesome XII E. Coloni by PFGE C. The SV by PFGE E. Finall by PFGE C. Conseq by PFGE E. In all by PFGE C. Recomb by PFGE D, which  by PFGE D, but th by PFGE . This baIn conclusion, detailed characterization of a subset of the ∼10% of small MIR recombinants confirmed that MIR is frequently associated with secondary rearrangements in addition to the primary selected translocation event . These rLY and S2 donors together with the MIR translocation and (2) the presence of additional chromosomal rearrangements , depending on the template used (LY donor) will segregate with the primary MIR translocation at the next cell division. If the donors are in an ectopic configuration, accurate repair is not possible, and additional rearrangements are observed at high frequencies  the segregation pattern of the ngements . In the ngements A. This pS2 donor A. In an ate used . This reS2 donor. No secondary rearrangement is expected.In contrast, MIR2 resolves the terminal invasion by synthesis-dependent strand annealing (SDSA), in which the extended end of the terminal D-loop pairs with the single-ended DSB generated upon cleavage of the internal D-loop B. This pThe MIR1 and MIR2 mechanisms thus make clear predictions as to which donor segregates with the MIR translocation product C. The obIn the following sections, we address the existence of the MIR1 and MIR2 pathways by challenging these predictions and better define their relative contributions to the formation of chromosomal rearrangements in different sequence contexts.pol32Δ mutant defective for long-range recombination-associated DNA synthesis. POL32 deletion caused a significant 2.5-fold decrease in MIR frequency (LYS2 + S2) decreased from 55 out of 93 to eight out of 24 , with a compensatory increase in MIR1 signatures (LYS2 + LY and 2× LYS2). The proportion of LYS2 + LY recombinants reached 45.8%, approaching the minimal proportion expected for MIR1-only events (50%)  C. The sh)  donor to an ectopic location on chromosome V  , unlike in the initial allelic configuration. Only MIR1 is expected in this configuration, whose outcome is a chromosome II:V translocation yielding an ∼895-kb chromosome with LYS2 present in place of the S2 donor at CAN1. We then restored substantial flanking homologies  at the 3′ side of the donors by modifying the S2 donor  see . In thisS2 donor A. RestorS2 donor led to a modest, nonsignificant increase in MIR frequency . Of the 48 remaining colonies, 38 had LYS2 in place of the S2 donor, typical of MIR1, and one was a mixed colony   D. These S2 70 bp and S2 1000 bp did not exhibit significant differences in their class distribution  fulfills the last prediction that MIR1, but not MIR2, is associated with frequent secondary rearrangements. In conclusion, the three predictions of our two MIR models were satisfied: (1) MIR2 specifically requires flanking homology on the 3′ side of the donors, but MIR1 does not (MIR recombinants were subclassified based on their category (MIR1 or MIR2), the segregating donor(s) (A–D), and the secondary rearrangements detected (0–3) to yield classes in the form of, for example, 1-C1 A. First,ribution B. Half tribution B. In totormality . In contormality . This sidoes not . (2) MIRdoes not C. (3) Addoes not .S2 70 bp and S2 1000 bp pooled)  was never associated with a V:II(Y) SV  (LY donors (B0) with the II:V(Y) SV (C1), in which five out of 84 B1 events would be expected , and indicates that the two are mutually exclusive. This observation allows mechanistic inferences as to the repair of the primary and secondary DSBs generated at donor sites following MIR1  A,B. Highctively) . This diing MIR1 D.Y region of the LY donor (e in LY-containing chromatid will result in 50% of MIR1 recombinants bearing the LY donor (B + D class) and 50% lacking it (A + C class), not significantly different from the observed donor class distributions   BInor e in D followinor e in D,E. Repanor e in E. Howevectively) E. Conseqmosome V D,E. In cS2) donor is retained in the majority (56 out of 84) of MIR1 recombinants (C and D classes)   of the C1 clones analyzed by aCGH  B,C. Becaegregate D. Cosegrary DSBs D. For in1 class) B. The re by aCGH . Consist by aCGH . Thus, tmologies , likely YS-containing initiating DSB (a or b) and the S2 donor. The co-occurrence of V:II(YS2) and V:II(Y) SVs (class 3: three out of 84) is not significantly lower than that expected for independent occurrence of class 1 and class 2 SVs (approximately five out of 84). Consequently, the formation and segregation of these two SVs occur without detectable interference. This finding implies that cells undergoing MIR1 experience a failure to faithfully repair both initiating DSBs using the homolog as a template.Finally, V:II(YS2) SVs are infrequently observed  C. This SLY) and terminal (S2) donors largely recapitulates the observed donor and SV class distributions  in the DNA of a MIR1-devoid strain (induced Lys−) showed that CR-C can reliably detect translocation frequencies down to ∼5 × 10−5 (+) frequency and MIR1 levels per haploid genome equivalent 24 h after DSB induction were in good quantitative agreement , a highly sensitive molecular assay for detection of rare chromosomal translocation in a cell population   and CR-C−4) amounted to only a fraction of the MIs detected at any given time point (from 8.6 × 10−4 at 4 h to 2.6 × 10−4 at 8 h)  H. Conseq at 8 h) I. This r at 8 h) . We veri at 8 h) , as prev at 8 h) . Consistd mutant , with nod mutant . These rδ-PCNA  events have recently been reported , revealid mutant J. Cdc5 td mutant , an SSE d mutant , MIR  model, which entails the endonucleolytic resolution of a dHJ formed between two ectopic repeats . This mo only 7% . Likewis only 7% , which wated SVs A, the moated SVs . This isated SVs A. Comparand mice , may helormation , and MIRormation . This reNonetheless, rearrangement patterns in yeast consistent with MIR have been reported. For instance, a break outside a Ty element is more prone to generating rearrangements than a DSB induced within . Also, tOf note, and depending on the repair of the initiating and secondary DSBs, MIR can elicit reciprocal translocations in 100% of daughter cells B. ConseqLINE and Alu repeats, appear to be one of the primary sources of these de novo SVs, which are surprisingly common even in healthy individuals  used in this study are listed in HO endonuclease gene under the control of the GAL1/10 promoter at the TRP1 locus on chromosme IV. The DSB-inducible construct contained the 117-bp HOcs  on chromosome V. The HOcs at the mating type loci (MAT) on chromosome III was inactivated by a point mutation to prevent HO cleavage (MATa-inc/MATα-inc) . S2 was oriented so as to avoid generating a dicentric II:V chromosome upon translocation with the LY donor left on chromsome II.The genotype of the diploid -bp HOcs , variousATα-inc) . In the RFC1-AID-9Myc::hphMX construct was a gift from Neil Hunter and has been described previously (POL3-iAID construct was a gift from Lorraine Symington and has been described previously (TIR1 and TetR expression integrated at SSN6 have been described previously (cdc5-ad mutation (T751G)  and providing the dsDNA repair template (top strand): 5′-GTGAACGTAAATACACAATATGCGGAACACCTAATTACATCGCACCTGAAGTGTGGATGGGTAAGCATTCTGGACATTCATTTGAAGTAG-3′ (nucleotide substitutions are underlined). The template contained a PAM-inactivating silent mutation in addition to the T751G mutation.The eviously . The POLeviously . The coneviously . IntroduSynthetic dropout (SD) and rich YPD  solid and liquid media were prepared according to standard protocols . Liquid pol3-iAID RFC1 or pol3-iAID rfc1-AID-9Myc strains, a single colony of the appropriate strain was used to inoculate a 5-mL YPD culture, grown to saturation overnight, and diluted in 250 mL of YEP-lactate. Following ∼13–16 h of growth overnight at 30°C, the OD600 of the culture was determined, and the culture was split equally into two smaller flasks. Protein depletion was conducted as in For time courses involving + frequency was determined by plating yeast cells exponentially growing in YEP-lactate liquid culture on SD-LYS and YPD plates (control plating). The expression of the HO endonuclease was triggered in the remaining liquid culture upon addition of 2% galactose. Two hours after galactose addition, when HO cutting was >99%  and two unrepairable (homozygous) DSBs was determined upon plating exponentially growing YP-lactate cultures on galactose-containing plates, incubating them for 16 h at 30°C, and counting the number of microcolonies (defined as more than six cell bodies) under the microscope, similar to 600 of the cultures was determined, and the cultures were used to inoculate fresh 5-mL YPD cultures at an equivalent OD. The 5-mL cultures were grown on a rotator for ∼6 h at 30°C, the OD600 of the cultures was determined again, and all cultures were diluted to an OD600 of 0.19. A series of 1:10 dilutions was prepared for each of the strains from these starting dilutions and spotted onto YPD + solvent or YPD + 1.5 mM IAA and 50 µg/mL doxycycline plates in parallel using a multichannel pipette. Plates were imaged following 2 d of growth at 30°C.Cell viability was assessed on YPD + solvent or YPD + 1.5 mM IAA and 50 µg/mL doxycycline as follows: Five-milliliter YPD cultures in 15-mL glass tubes were inoculated with a single colony corresponding to the appropriate strain and grown on a rotator overnight at 30°C. After ∼16 h of growth, the OD+ colonies were patched on SD-LYS plates, and their DNA was extracted from a 5-mL SD-LYS liquid culture saturated overnight. DNA was digested by HindIII , PstI (for the ectopic S2 70-bp donor construct), or PstI + EcoRI (for the ectopic S2 1000-bp donor construct) for 4 h at 37°C and migrated overnight in 0.8% agarose-LE (Affymetrix) in 1× TBE at 50 V. The DNA was transferred from the gel onto an Amersham Hybond-XL membrane  following the manufacturer's instructions . The membrane was blocked with Church buffer  for 2–3 h at 65°C. The LY, S2, or LYS2 probes , together with phage λ DNA (molecular ladder), were radiolabeled by random priming with 6000 Ci/mmol P32-αdCTP (Perkin-Elmer) using the Decaprime II kit  and incubated with the membrane overnight at 65°C. After three to five washes for 10 min at 65°C , membranes were exposed for 8–24 h, and the storage phosphor screen  was scanned on a Storm phosphorimager (Molecular Dynamics).Independent Lys+ recombinants were performed as described previously (S. cerevisiae S288c R64-2-1 genome with Bowtie 2 (Preparation of DNA plugs and pulse-field gel electrophoresis (PFGE) of Lyseviously . Array ceviously . Whole-gBowtie 2  with defThe DLC and DLE assays have been described previously . A psora8 cells collected at various times after DSB induction by spheroplasting cells with zymolyase, protein digestion, phenol–chloroform–isoamyl alcohol (25:24:1) extraction, isopropanol precipitation, and RNase A treatment according to standard protocols. DNA was quantified on a Life Technologies Qubit 2.0 fluorometer using the dsDNA HS assay kit (Invitrogen Q32854) according to the manufacturer's instructions. Five-hundred nanogreams of DNA was digested for 1 h at 37°C with 50 U of EcoRI-HF (NEB R3101), and the enzyme was inactivated for 20 min at 65°C. Eighty nanograms of digested DNA was ligated in 800 µL of ligation buffer  with 20 U of DNA ligase T4 (NEB M0202) for 1.5 h at 16°C. DNA was subsequently purified by phenol:chloroform:isoamyl alcohol  extraction followed by isopropanol precipitation. DNA was resuspended in 40 µL of TE , and 2 µL was used per 20 µL of qPCR reaction, representing ∼105 haploid genomes per reaction. Single-step qPCR amplification was performed on a Roche LightCycler 480 or a Bio-Rad CFX96 thermocycler using the respective manufacturer's SYBR Green kit and instructions. The amplification of the rearranged molecule produced upon circularization of the 4628-bp MIR1-containing DNA fragment was normalized on the average circularization efficiency of three DNA fragments of similar size . Primers used are listed in Genomic DNA was purified from 5 × 10Protein extracts for Western blot were prepared using a standard protocol for tricholoroacetic acid (TCA) extraction. An ∼25-µL sample was run out on a 7.5% SDS-PA at 150 V for ∼1 h alongside a protein standard . Samples were transferred to a PVDF membrane (Bio-Rad TransBlot Turbo midsize LF PVDF membrane) using the Bio-Rad Trans-Blot Turbo transfer system. The membrane was then blotted using mouse antimini-AID tag (1:1000) primary antibody (MBL M214-3), mouse anti-c-Myc 9E11 (1:1000) primary antibody (Santa Cruz Biotechnology sc-47694), mouse anti-GAPDH  primary antibody (Thermo Fisher MA5-15738), and antimouse HRP-conjugated (1:1000) secondary antibody (Agilent P0447).Alu elements were retained and sorted with bedtools sort, and overlapping elements were merged with the bedtools merge function, providing the proportion of the genome made of Alu elements (7%). Intervals were extended by 1, 2, and 4 kb and merged, and the genome fraction at risk of Alu-mediated SVs through MIR was computed. Alternatively, initial intervals were shortened by 0.1 kb on each side to determine the genome fraction at risk of Alu-mediated SVs through DSBR, which required the DSB to fall away from the repeat element edges.The RepeatMasker BED track for the human genome assembly hg38 T2T CHM13v2.0 was obtained from the UCSC table browser on October 19, 2022. Lines corresponding to S. cerevisiae genome, raw aCGH data, and SV-supporting nanopore reads have been deposited at GEO projects GSE232732 and GSE233144. A summary of data availability for MIR samples is provided in Raw sequencing data have been deposited at SRA BioProject PRJNA967211. Processed data (coverage of Illumina reads) aligned on the reference R64-2-1 Supplement 1Supplement 2Supplement 3Supplement 4Supplement 5Supplement 6Supplement 7Supplement 8Supplement 9Supplement 10Supplement 11Supplement 12Supplement 13


Background: Solute Carrier Family 3 Member 2 (SLC3A2) is a member of the solute carrier family that plays pivotal roles in regulation of intracellular calcium levels and transports L-type amino acids. However, there are insufficient scientific researches on the prognostic and immunological roles of SLC3A2 in breast cancer (BC) and whether everolimus regulates novel SLC3A2 related molecular mechanism in the immuno-oncology context of the tumor microenvironment (TME), therefore, we see a necessity to conduct the current in silico and biological experimental study.Methods: Using diverse online databases, we investigated the role of SLC3A2 in therapy response, clinicopathological characteristics, tumor immune infiltration, genetic alteration, methylation and single cell sequencing in BC. WB, Co-IP, cell proliferation assay, Edu staining, ROS and GSH assay and in vivo tumor xenograft assays were performed to verify FKBP1A/SLC3A2 axis in everolimus inducing ferroptosis of breast cancer. Co-cultures and IL-9 ELISA were performed to demonstrate the T lymphocyte function.Results: We demonstrated that SLC3A2 was aberrantly expressed among various BC cohorts. Our results also suggested that SLC3A2 expression was associated with chemotherapeutic outcome in BC patients. Our results further indicated that SLC3A2 was associated with tumor infiltration of cytotoxic T cell but not other immune cells among BC TME. The alterations in SLC3A2 gene had a significant correlation to relapse free survival and contributed a significant impact on BC tumor mutational burden. Finally, SLC3A2 was illustrated to be expressed in diverse BC cellular populations at single cell level, and negatively linked to angiogenesis, inflammation and quiescence, but positively correlated with other functional phenotypes. Noteworthily, everolimus (a targeted therapy drug for BC) related protein, FK506-binding protein 1A (FKBP1A) was found to bind with SLC3A2, and negatively regulated SLC3A2 expression during the processes of everolimus inducing ferroptosis of BC cells and promoting anti-proliferation of Th9 lymphocytes.Conclusions: Altogether, our study strongly implies that SLC3A2 is an immuno-oncogenic factor and FKBP1A/SLC3A2 axis would provide insights for a novel immunotherapy approach for the treatment of BC in the context of TME. Further results suggested that SLC3A2 and FKBP1A were associated with chemotherapeutic outcome in BC patients. Our results also indicated that SLC3A2 was associated with tumor infiltration of cytotoxic T cell among BC TME. The alterations in SLC3A2 and FKBP1A genes both contributed a significant impact on BC tumor mutational burden. Moreover, SLC3A2 was illustrated expressed in diverse BC cellular populations at single cell level, and negatively linked to angiogenesis, inflammation and quiescence, but positively correlated with other functional phenotypes. In terms of mechanism, FKBP1A was found to bind with SLC3A2, and negatively regulated SLC3A2 expression during the processes of everolimus inducing ferroptosis of BC cells and promoting anti-proliferation of Th9 cells. Identification of FKBP1A/SLC3A2 axis would provide in-depth insights for TME-targeted strategies for BC treatments.The SLC3A2 protein was only detected in plasma membrane and nucleoplasm Fig. A, and toSince BC therapeutic backbones include endocrine therapy, anti-HER2 therapy, and chemotherapy In UALCAN database, we found SLC3A2 expression in male BC patient was the top A. With tImmune cells among TME are associated with the hallmarks of BC In COSMIC database, the pie chart included various mutations information including nonsense substitution, missense substitution, synonymous substitution, etc. The proportion of missense substitution was the highest 35.87%)  utilizing the CancerSEA database A. In EXPe.g., total ROS and GSH level. Our results showed that in MCF7 cells everolimus significantly increased intracellular total ROS level . MCF7 and 293T were cultured in Dulbecco's modified Eagle's medium. T47D and Th9 were cultured in Roswell Park Memorial Institute (RPMI) medium. Growth media were supplemented with 10% fetal calf serum and penicillin/streptomycin. All human cell lines were cultured at 37 °C in a humidified incubator supplied with 5% COAntibodies were used in the following dilutions: SLC3A2 , FKBP1A , SLC7A11 , GPX4 , IL-9R , GAPDH , HA , Flag . Secondary antibody goat anti-mouse , secondary antibody goat anti-rabbit . Everolimus was purchased from APExBIO (#A8169), and resolved in DMSO at 10mM. Erastin was purchased from meilunbio (#MB4114), and resolved in DMSO at 10mM. IL-9 was purchased from MCE (#HY-P7046), and resolved in PBS at 100μg/mL.Knockdown and overexpression efficiencies and biochemical responses were analyzed by western blotting. Cells were lysed in RIPA lysis buffer (EMD Millipore Corp.). Separated proteins were transferred to PVDF membranes and blocked in 5% milk in Tris-buffered saline, with 0.05% Tween-20. Immunodetection was done with various primary antibodies. Appropriate horseradish peroxidase-conjugated secondary antibodies were used and signals were visualized with Bio-Rad chemiluminescence by Bio-Rad ChemiDoc™ MP Imaging System.Cells for co-IP were lysed in lysis buffer  at 4 °C for 30 min. After sonication and centrifugation, cell lysates were incubated with HA beads (Sigma) at 4 °C overnight on a rotator. After six washes with wash buffer , 50 μL of elution buffer  was added to resuspend the beads, and the eluted proteins were obtained by centrifugation, followed by SDS-PAGE and immunoblotting analysis.FKBP1A-HA, SLC3A2-Flag and the control plasmid were constructed by Tsingke Biotechnology Co., Ltd.® 2000 Reagent (Invitrogen) or siRNAs using Lipofectamine® RNAiMAX Reagent (Invitrogen) as instructed by the manufacturer. The siRNA targeting SLC3A2 or FKBP1A, as well as negative control siRNA were purchased from RIBBIO . 72 h later, the whole-cell extract was prepared for western blot analysis.MCF7, T47D or 293T cells cultured in 12-well tissue culture plates were transiently transfected with plasmids using LipofectamineThe shRNA against SLC3A2 was purchased from RIBBIO . The shRNA targeting sequences of SLC3A2 were as follows: CCGGCAATTCACAAGAACCAGAAGGCTCGAGTTCTGGTTCTTGTGAATTGGATTTTT. In brief, 293T packaging cells were transfected with 800 ng shRNA in combination with the packaging plasmids 200 ng lenti-VSV-G, 400 ng lenti-RRE, and 140 ng lenti-REV. Virus containing supernatant was harvested at 48 h after transfection and filtered through a 0.45 mM syringe filter with the addition of 10 mM DEAE. Supernatants were used to infect target cells in another 12 h period.2 for 24, 48, 72 h, followed by the addition of 10 μl of CCK-8 solution. The absorbance in each well was measured after 1 h incubation using a microculture plate reader at a test wavelength of 450 nm. Three replicate wells were set up in each group, and three independent experiments were performed.Cell proliferation was analyzed using a Cell Counting Kit-8 (CCK-8) (DOJINDO). All cells were seeded into 96-well plates at a density of 5000 cells/well in a 100 µl volume and incubated at 37°C under 5% COThe Edu-488 cell proliferation detection kit was used for cell proliferation detection, in which the thymidine analog Edu was incorporated and labeled in the process of DNA synthesis. 100 μL of Edu working solution (10 μM) was added into MCF7 or T47D cells in the 96-well plate for incubation for 2 h. Subsequently, the cells were fixed in 4% paraformaldehyde for 15 min, sealed in PBS solution containing 3% bovine serum albumin, permeabilized in 0.3% Triton X-100 solution for 15 min, and washed with PBS (5 min/time). Finally, the staining was observed at the wavelength of 495 nm under a microscope.MCF7 cells were co-cultured with Th9 cells by using a Transwell culture system with 0.4 μm pore polyester membrane inserts  for 3 days. Specifically, MCF7 cells were seeded on plates, while Th9 cells were seeded in Transwell chambers.The total ROS level in cells was assessed using a Reactive oxygen species detection kit . Cells were treated as indicated, and then, 10 μmol/L DCFH-DA diluted in serum-free medium was added and incubated with the cells at 37 °C for 20 min. Excess DCFH-DA was removed by washing the cells three times with serum-free cell culture medium. DCFH-DA can be hydrolyzed by intracellular esterases to produce DCFH. Labeled cells were trypsinized and resuspended in serum-free medium. Intracellular reactive oxygen species can oxidize nonfluorescent DCFH to produce DCF, which is fluorescent. The fluorescence of DCF was analyzed using flow cytometry.The relative GSH concentration in cell lysates was assessed using a total Glutathione Assay Kit  according to the manufacturer's instructions. Briefly, Glutathione reductase reduces oxidized glutathione (GSSG) to reduced glutathione (GSH), which reacts with the chromatin substrate DTNB to produce yellow TNB and GSSG. The combination of the two reactions reveals the total glutathione (GSSG + GSH), and the amount of TNB (yellow) formation represents the amount of total glutathione. Thus, the total glutathione content can be calculated by measuring A412.2 with/without everolimus, the supernatant and the cells were separated via 5-minute centrifugation at 1000 g. IL-9 ELISA was performed on the supernatant using Human IL-9 ELISA Kit (CUSABIO).After incubation for 72 h under 37 °C der 5% CO6 cells/mice) were implanted subcutaneously into the left underarm of nude mice. After tumor formation, everolimus (5 mg/kg) was administered orally to its respective animal treatment groups for 8 days. Tumor volume (mm3) was measured every two days and calculated by the formula (length × width × width)/2. After the tumors were surgically removed, a portion of the tumors was immediately fixed in 10% buffered formalin for immunohistochemistry.Four-week-old female BALB/c nude mice were purchased from Vital River Laboratory Animal Technology Co, Ltd . Animal experimental procedures were approved by the Medical Ethics Committee of Zhejiang Provincial People's Hospital. The four-week-old female mice were randomized into different groups. T47D shControl and T47D shSLC3A2 cells (1 ×10The localization and expression of SLC3A2 and FKBP1A protein were generated by PROTTER database Two-tailed student's t-test was used in this study. Data shown was mean±SD from at least three independent experiments. Statistical probability was expressed as *p < 0.05, **p < 0.01, ***p < 0.001. p values generated by different databases were automatically calculated by each database.Supplementary figures.Click here for additional data file.


C3AR1 was reported in driving tumor immunity in multiple cancers. However, its roles in ovarian cancer remain unclear. This study aims to determine role of C3AR1 in prognosis and regulating tumor infiltrating immune cells of ovarian cancer (OC).The expression, prognosis and clinical data related to C3AR1 were collected from public databases such as The Cancer Genome Atlas (TCGA), Human Protein Atlas (HPA) and Clinical Proteomics Tumor Analysis Alliance (CPTAC), and further analyze their relationship with immune infiltration. Immunohistochemistry verified the expression of C3AR1 in ovarian cancer and control tissues. C3AR1 was forced expressed in SKOV3 cells by plasmid transfection, and verified by qRT-PCR and Western blot. Cell proliferation were evaluated by EdU assay.Bioinformatics analysis  and immunohistochemical staining of clinical samples confirmed higher C3AR1 expression in ovarian cancer than that in normal tissues. High C3AR1 expression predicted adverse clinical outcomes. KEGG and GO analysis showed that the biological processes of C3AR1 in ovarian cancer are mainly involved in T cell activation, cytokine and chemokine activation. C3AR1 expression was positively correlated with chemokines and their receptors in the tumor microenvironment, such as CCR1(R = 0.83), IL10RA (R = 0.92), and INFG (R = 0.74). In addition, increased C3AR1 expression predicted more infiltration of tumor-associated macrophages, dendritic cell and CD8 + T cell. Some important m6A regulators, such as IGF2BP2, ALKBH5, IGFBP3 and METL14, are significantly positively or negatively correlated with C3AR1. Finally, overexpression of C3AR1 significantly increased proliferation of SKOV3 cells.In summary, our study suggested that C3AR1 is associated with the prognosis and immune cell infiltration of ovarian cancer, and is a promising immunotherapeutic target.The online version contains supplementary material available at 10.1186/s13048-023-01140-2. Ovarian cancer (OC) is a highly aggressive malignant tumor, ranking fifth in the cancer-related death in . PatientOvarian cancer cells evade immune surveillance and achieve tumor proliferation by shaping a highly immunosuppressive tumor microenvironment (TME). AccordiExistence of cancer is often accompanied by changes in the complement system. On the one hand, due to its water-soluble properties, complement can often infiltrate into deeper tumors faster than immune cells. It recruits leukocytes through anaphylactoxin, opsonin and lysis, promotes tumor lysis and induces the release of pro-inflammatory mediators to enhance the anti-cancer . On the http://gepia.cancer-pku.cn/) database. Protein expression level of C3AR1 were investigated in the Clinical Proteomics Tumor Analysis Alliance (CPTAC) dataset and the HPA database (https://www.proteinatlas.org/). C3AR1 expression in different ovarian cancer cells were detected in CCLE . In addition, correlations between C3AR1 and the clinicopathological characteristics of OC patients is explored in TCGA database. Prognostic value of C3AR1 was analyzed in the Kaplan Meier plotter (http://kmplot.com/).The transcription levels of C3AR1 in pan-cancers were explored in the GEPIA  were analyzed using the DESeq2.0 package, and criteria was |Log2FC|>2 and p < 0.05. Functional pathway analysis of GO and KEGG was conducted on DEGs and co-expressed genes individually, and visualized by the ggplot2 software package.www.cistrome.shinyapps.io/timer). We also compiled the associations of 22 immune cell marker genes with C3AR1 mRNA levels in the TIMER, GEPIA and TCGA databases. The matrix score, immune score and ESTIMATE score between different C3AR1 expression groups are evaluated by the ESTIMATE algorithm. Infiltration level of immune cells in two C3AR1 groups was compared by the MCP counter algorithm. We further assessed the correlation between expression of C3AR1 and immune checkpoint.To explore the role of C3AR1 in regulating immune microenvironment of OC, we analyzed the relationship between the expression of C3AR1 and B cells, CD4 + T cells, CD8 + T cells, macrophages, neutrophil cell and dendritic cell infiltration in the TIMER database (Enhancing or inhibiting m6A methyltransferase (writer) or demethylase (eraser) alters tumor development, and function status of m6A RNA methylation is highly dependent on the cell microenvironment. We analyzed gene expression of m6A mediators in two C3AR1 groups in the R software package. The prognostic value of C3AR1 related m6A genes in OC were explored. The ggplot2 is used for data visualization.Tissues of 5 OC patients and 5 healthy controls were stained by immunohistochemistry (IHC) after formalin fixation and paraffin-embedding. C3AR1 specific antibody (Proteintech) was diluted at 1:100.Human ovarian cancer cell line SKOV3 was purchased from NEWGAINBIO (China). We maintained cells at 37 °C in a humidified atmosphere containing 5% carbon dioxide in RPMI 1640 medium  supplemented with 10% fetal bovine serum  and 1% penicillin/streptomycin solution . According to the manufacturer’s protocol, the plasmids  with different concentrations were transfected into SKOV3 cells using jetPRIME transfection reagent . After 48 h of transfection, Total RNA Extraction mini kit  was used to extract total RNA from cultured cells for qRT-PCR. C3AR1 forward primer: CCCTACGGCAGGTTCCTATG; C3AR1 reverse primer: GACAGCGATCCAGGCTAATGG-3,‘ β-actin forward primer: GTGGGGCGCCCCAGGCACCAGGGC; β-actin reverse primer: CTCCTTAATGTCACGCACGATTTC. RIPA buffer (Solarbio) was used to lyse the cells and extract total proteins. Western blotting assay were conducted as described , the antC3AR1 overexpression and control cells were treated with EdU reagent  for 3 h. The cells were then immobilized with 4% paraformaldehyde and stained with fluorescent dye and Hoechst. Fluorescence detection was performed under an inverted fluorescence microscope.All data were statistically analyzed using GraphPad Prism (version 9.1.0). The student t test, one-way ANOVA, and Chi-square test were used to assess differences in variables between groups. The log-rank test evaluated the statistical significance of Kaplan-Meier survival curves. Spearman and statistical significance were used to analyze gene expression correlations. |R| > 0.3 is considered to be relevant, P values < 0.05 was considered statistically significant.First, we compared the difference of C3AR1 between ovarian cancer and other human tumors and normal tissues. Increased expression of C3AR1 is higher in esophageal carcinoma (ESCA), glioblastoma multiforme (GBM), kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), acute myeloid leukemia (LAML), brain lower grade glioma (LGG), ovarian serous cystadenocarcinoma (OC), pancreatic adenocarcinoma (PAAD), skin cutaneous melanoma (SKCM), stomach adenocarcinoma (STAD), and testicular germ cell tumors (TGCT) than normal tissues. However, C3AR1 mRNA expression levels in adrenocortical carcinoma (ACC), lymphoid neoplasm diffuse large B-cell lymphoma (DLBC), lung squamous cell carcinoma (LUSC), and thymoma (THYM) are lower than normal tissues ). Top 9 genes with the strongest correlation with C3AR1 are presented  were presented in the volcano plot Fig. . The enrCorrelation between C3AR1 expression in OC and immune cell infiltration were evaluated and found that C3AR1 is positively correlated with the six types of immune cells. According to correlation index, they are neotrophil , dendritic cell , macrophage , CD8 + T cells , CD4 + T cells  cells and B cells  Fig. , includiWe further evaluated the association between C3AR1 and various immune cell markers in OC cohorts of TIMER, GEPIA and TCGA database Table . ResultsEnrichment analysis of co-expressed genes and DEGs indicated that C3AR1 is involved in the densely mediated signaling pathways of chemokines, interleukins, and interferons. Therefore, we evaluated the correlation between C3AR1 and chemokines, interleukins and interferons in the ovarian cancer microenvironment in the TCGA database. As shown in Fig. M6A-related genes are widely involved in multiple processes such as ovarian cancer metastasis, chemotherapy resistance, and tumor microenvironment regulation. Analysis of TCGA found that C3AR1 co-expressed with multiple m6A-related genes, including HNRNPC, IGF2BP2, RBMX and ZC3H13, which were significantly positively correlated Figure . Kaplan-The expression level of C3AR1 in SKOV3 cells was low, therefore we overexpressed C3AR1 to conduct subsequent functional experiments. mRNA and protein levels of C3AR1 were significantly increased after plasmid transfection Fig. B-D, and In this study, the expression of C3AR1 in OC was analyzed and verified from both mRNA and protein levels by bioinformatics and experimental methods. Correlation analysis between the methylation and the expression of C3AR1 showed that low methylation levels partly contribute to the high C3AR1 mRNA level. Previous studies have found that GBM, LGG, and COAD patients with high C3AR1 expression tend to have a poor .We also Current researches on C3AR1 in tumors mainly focuses on stomach adenocarcinomas (STAD),  and kidnWe analyzed the related genes of C3AR1 and found that the expression of LAPTM5, CD53, CYBB, LAIR1, MS4A6A, HAVCR2, CD86, FCCR2A and FCCR2C in OC has the strongest correlation with C3AR1. Chen et al. found that down-regulation of LAPTM5 induces cell cycle arrest in G0/G1 phase to inhibit the proliferation and viability of bladder cancer . HAVCR2 Immune cell infiltration affects tumor metastasis, chemotherapy resistance, and prognosis of OC patients. We analyzed data from multiple databases and confirmed that C3AR1 is closely related to immune cell infiltration, including macrophages. In addition, immune cell markers from 3 data sets also confirmed these results. Among them, C3AR1 has the strongest correlation with TAM, M2 macrophage and monocyte markers, suggesting that C3AR1 may affect the immune microenvironment of OC mainly by regulating macrophage infiltration. The role of M2 and TAM in promoting tumor progression in ovarian cancer has been widely recognized. In ovarian cancer, M2 macrophages promote the formation of ascites by reducing the expression of VLA4 in their cell membranes and reducing the level of VCAM1 in endothelial . MechaniSeveral studies have confirmed m6A mediators in promoting ovarian cancer proliferation, angiogenesis, chemotherapy resistance and tumor microenvironment regulation. Wang et al. found that knocking down YTHDF1 in cisplatin-resistant ovarian cancer cells can inhibit cancer stem cell-like characteristics by overexpression of TRIM29 . Liu et This study demonstrates that C3AR1 is up-regulated and promotes cancer cell proliferation in ovarian cancer, as well as associated with various pathological features. In particular, C3AR1 overexpression is associated with immune cell infiltration, mediating the tumor immune microenvironment, and is associated with poor prognosis of ovarian cancer patients. Patients with high C3AR1 are more likely to benefit from immunotherapy as a result of elevated immune checkpoints expression levels. Taken together, these results indicate that C3AR1 may be a potential new immunotherapeutic target for ovarian cancer.Authors’ contributions.Kaixian Deng conceptualized the study design and supervised the analysis. Jinfa Huang collected the data, performed the statistical analysis, and wrote the paper. Lei Zhou conducted the experiment and polished the draft. All authors read and approved the final manuscript.Below is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2Supplementary Material 3Supplementary Material 4Supplementary Material 5Supplementary Material 6