Aegilops biuncialis has high β-glucan content, which makes it an attractive gene source to develop wheat lines with modified fiber composition. In order to support introgression breeding, this work examined genetic variability in grain β-glucan, pentosan, and protein content in a collection of Ae. biuncialis. A large variation in grain protein and edible fiber content was revealed, reflecting the origin of Ae. biuncialis accessions from different eco-geographical habitats. Association analysis using DArTseq-derived SNPs identified 34 QTLs associated with β-glucan, pentosan, water-extractable pentosan, and protein content. Mapping the markers to draft chromosome assemblies of diploid progenitors of Ae. biuncialis underlined the role of genes on chromosomes 1Mb, 4Mb, and 5Mb in the formation of grain β-glucan content, while other QTLs on chromosome groups 3, 6, and 1 identified genes responsible for total- and water-extractable pentosan content. Functional annotation of the associated marker sequences identified fourteen genes, nine of which were identified in other monocots. The QTLs and genes identified in the present work are attractive targets for chromosome-mediated gene transfer to improve the health-promoting properties of wheat-derived foods.Grain dietary fiber content is an important health-promoting trait of bread wheat. A dominant dietary fiber component of wheat is the cell wall polysaccharide arabinoxylan and the goatgrass  Triticum aestivum L., 2n = 6x = 42; AABBDD) is grown on more than 220 million hectares worldwide and is one of the most important components of the human diet. Due to its major role in human nutrition, wheat is a major source of dietary fiber (DF). DF has numerous health-promoting effects, including lowering the risk of coronary heart disease, colorectal cancer, inflammatory bowel disease, breast cancer, tumor formation, and mineral-related abnormalities [Bread wheat  is closely related to Triticum and includes eleven diploid and twelve polyploid species with divergent U, M, N, C, S, D, and T genomes. Numerous reports pointed to wild goatgrasses as useful and untapped sources of new gene variants for wheat improvement [Ae. biuncialis Vis.  evolved after natural hybridization between Ae. umbellulata Zhuk.  and Ae. comosa Sm. in Sibth. et Sm. , which served as U- and M genome donors. Ae. biuncialis is native from the Iberian Peninsula to Central Asia and includes the entire Mediterranean region, Near East, Cis- and Transcaucasia, and the southern part of Ukraine [Ae. biuncialis exhibits high genetic diversity [Ae. biuncialis grains have also been found to contain a significantly larger amount of micronutrients [Aegilops, some Ub- and Mb-genome chromosomes were transferred into wheat by the development of wheat-Ae. biuncialis amphiploids, disomic addition lines carrying chromosomes 1Ub, 2Ub, 3Ub, 2Mb, 3Mb and 7Mb, a double disomic addition line 1Ub and 6Ub, 3Mb(4B) substitution, and 3Mb.4BS translocation lines [The genus rovement ,14,15,16 Ukraine ,17. In piversity . Some geiversity , powderyiversity , yellow iversity , brown riversity , leaf ruiversity , and steiversity , while oiversity , salt [2iversity ,27, and iversity . Ae. biuutrients , edible utrients ,32,33 coon lines ,36,37,38Ae. biuncialis remains unexploited in wheat breeding [Ae. biuncialis. For example, biochemical analysis of wheat-Aegilops addition lines showed that chromosomes 5Ug, 7Ug, 7Mg, and 7Mb of Ae. geniculata or Ae. biuncialis have a positive effect on DF content of bread wheat [Aegilops parents (3-500% β-glucan content compared to wheat). These results suggested the presence of additional QTLs in the U and M genome chromosomes, which are needed to further increase the β-glucan content of wheat. Examination of β-glucan content in a diverse collection of Ae. biuncialis accessions would facilitate the selection of the most suitable crossing partners for introgression breeding programs and allow mapping of the additional QTL regions and candidate chromosomes for targeted gene transfer into wheat.Although numerous wheat-alien addition and translocation lines have been developed, the genetic potential of breeding . Chromosad wheat . HoweverAegilops species, including Ae. taushii [Ae. sharonensis [Diversity Array Technology (DArT), originally developed as a hybridization-based microarray platform, is a sequence-independent, cost-effective, high-throughput genotyping-by-sequencing approach combining the genome complexity reduction method and next-generation sequencing ,40,41. D taushii  and Ae. ronensis .Ae. biuncialis accessions with geographically diverse origins using Silico-DArT markers. More recently, the complete chromosome set of Ae. umbellulata and Ae. comosa [Aegilops chromosomes [Ivanizs et al.  characte. comosa  was flow. comosa . The chromosomes .Ae. biuncialis. Using the allelic composition of DArTSeq derived SNP markers in combination with a sequence similarity search on the U- and M-chromosome assemblies, we characterized the Aegilops genotypes to find markers and genomic regions closely associated with the desired quality traits.Motivated by the need to discover accessions suitable for interspecific hybridization programs and to identify QTLs for targeted transfer of high fiber and/or protein content, this work focused on the evaluation of phenotypic variability of β-glucan, total- and water-extractable pentosan, and protein content in a collection of 86 genotypes of Ae. biuncialis accessions and the control wheat line Mv9kr1. As three Aegilops accessions produced a low quantity of grains, the quality traits were only determined in 83 genotypes. The accessions were chosen to represent a wide geographical distribution of Ae. biuncialis, extending from the Balkans to the Caspian Sea. As a result, a large variation was found for all traits that were analyzed.Grain samples from single plots were analyzed to determine the variation in dietary fiber components, β-glucan (BG) and arabinoxylan, measured as total- (TP) and water-extractable pentosans (WEP) as well as the protein content of Aegilops collection was significantly higher than that of the Mv9kr1 wheat .The BG content of the r1 wheat , with thAegilops accessions (40.11 mg/g) and wheat (40.17 mg/g), the Aegilops collection showed a wide phenotypic variation (29.60 mg/g–50.77 mg/g). While the average content of water-extractable pentosan in Aegilops genotypes (10.82 mg/g) did not differ considerably from that of Mv9kr1 wheat (10.79 mg/g), a wide range (6.83 mg/g–15.42 mg/g) was observed in Ae. biuncialis. In terms of protein content, Mv9kr1 wheat line (12.91%) was significantly outperformed by all Ae. biuncialis accessions . In summary, all Ae. biuncialis genotypes had significantly higher grain BG and protein content than Mv9kr1 wheat, as well as a high variation in the pentosan fractions.Although there was no difference in total pentosan content between the Ae. biuncialis accessions were investigated. Each year, the frequency distribution of the traits was studied separately. Because all of the analyzed traits had a normal distribution, their polygenic nature was confirmed  values and the fact that G accounted for only 16% of the total variance. Nonetheless, for the genome-wide association analysis, all of the quality traits were used.The analysis of protein content revealed that the G × E interaction determined 83% of the total variance , indicatAe. biuncialis accessions.After excluding accessions with failed DNA amplification, monomorphism, missing data, and allele frequencies that differed from what was described in the methods section, a total of 2602 DArTSeq derived SNP tags were used for whole-genome profiling  in the set of 86 Aegilops accessions was studied using the Bayesian approach implemented in STRUCTURE, which provided the ΔK plotted against the K numbers of the subgroups. The optimal number of subpopulations was determined using the STRUCTURE Harvester software. The maximum ΔK (686.70) occurred at K = 2  were assigned to subpopulation 1, with a Q1 mean membership of 0.85. The remaining 23 accessions were assigned to subpopulation 2, with a Q2 mean of 0.96. Based on the genetic data of the SNP-DArT markers, an unrooted phylogenetic tree was constructed using the maximum likelihood method to group accessions into clades    was perfAe. biuncialis population, in which the Aegilops genotypes were grouped into two subpopulations based on their geographic distribution. The accessions of Ae. biuncialis in subpopulation 1 come from the Peloponnese, Asia Minor, Azerbaijan, and the Near East, while the 23 members of the other subpopulation originate from the Balkans and North Africa , and based on genome-wide DArTSeq derived SNP markers was low with a mean r2 = 0.049. The information about the LD of DArTSeq derived SNP markers has been presented in Genome-wide association study (GWAS) was conducted during two seasons using the MLM (Q+K) model, allowing the identification of 34 QTLs representing regions associated with β-glucan, total-pentosan, water-extractable pentosan, and protein content . As threAe. umbellulata and Ae. comosa, respectively [Aegilops U or M genomes and hexaploid wheat A, B, and D genomes. Twenty-five (78.1%) of the 32 markers were found on the same homoeologous group chromosomes in Aegilops as in wheat  and to the assemblies of chromosomes 1U-7U and 1M-7M, which were obtained by sequencing chromosomes flow-sorted from diploid ectively . Using tin wheat .https://wheat.pw.usda.gov, accessed on 30 October 2021) and used for the NCBI BLAST search. The analysis identified fourteen genes, nine of which encode putative ripening-related protein 6 associated with the QTL for WEP and protein; 1-deoxy-D-xylulose-5-phosphate synthase 1, glutamate receptor 2.8-like, endoglucanase 5-like, and flavonol synthase/flavanone 3-hydroxylase-like, all co-located with WEP QTL; glutathione S-transferase 3-like linked to β-glucan, WEP and protein loci , which correlated with their geographic origin: A (North Africa), B , C , D  and E (Near East and Azerbaijan) [Ae. biuncialis population showed high similarity. Based on co-dominant SNP markers, the accessions assigned previously to clusters D and E were grouped into subpopulation 1, while the genotypes belonging to clusters A and B were classified as subpopulation 2 in the present study  were clustered into one group (subpopulation 2) in the present study, suggesting that these genotypes share a common phylogenetic origin. Subpopulation 1 included genotypes previously classified into clusters D  and E (Near East and Azerbaijan) also indicating a closer phylogenetic relationship. A majority of Aegilops accessions from former cluster D have a mixed genome shared in different proportions by subpopulations 1 and 2, indicating a significant admixture of their ancestral genotypes. Because genotypes in subpopulation 1 are mostly found in southern Greece and Turkey, while most of the accessions in subpopulation 2 are originating from the Balkans and the western part of Turkey, their natural distribution areas may overlap in the Aegean region and Asia Minor, allowing intraspecific hybridizations to occur. The analysis of chloroplast DNA could provide more information about the genealogical relationship of subpopulations and the intraspecific evolution of Ae. biuncialis [Using the Bayesian approach and PCoA for the SNP-DArT marker data, the present study identified two subpopulations with different geographic distributions in the rbaijan) . Althougnt study . Genotypuncialis .2 based on DArTSeq derived SNP markers was low (mean r2 = 0.049). This is expected as in self-pollinated species, such Arabidospis or barley, LD can extend up to 10–30 kb and 212 kb, respectively [Pairwise LD measured by rectively ,55. LD, ectively . LD estiAe. biuncialis, 34 marker-trait associations were found, identifying QTL regions for grain BG, TP, WEP, and protein content. The present study also demonstrated the utility of chromosome genomics in facilitating genome analysis of wild gene source species with poorly studied genomes [Ae. biuncialis was not available, we used a sequence similarity search against the assemblies of individual chromosomes from the U and M genomes of Ae. umbellulata and Ae. comosa, respectively [Aegilops. However, the chromosomal position of the QTLs established could be affected by the purity of the flow-sorted M-genome chromosome fractions that were sequenced [Using the allelic distribution of SNP markers in the collection of  genomes . Becauseectively , to locaequenced ,45.Aegilops chromosomes 1M and 4M, while the same markers were found on wheat group 1 chromosomes. Previous comparative studies of wheat and diploid Aegilops species based on the mapping of conserved orthologous genes using single-gene FISH [Ae. comosa (4M) and wheat (group 1) could be due to cross-contamination of chromosome 4M fraction by 1M chromosomes during flow-sorting. This explanation is supported by the fact that the 4M and 1M chromosome populations were detected close to each other on the bivariate flow karyotype of Ae. comosa [The present study identified five QTLs  on ene FISH  and COS ene FISH ,58 revea. comosa ,45. ThusAe. umbellulata synteny should also be considered when determining the chromosomal position of marker-trait associations. The U genome of Ae. umbellulata has been reported to have undergone multiple genome rearrangements during the evolution, resulting in synteny breaks in some chromosomes relative to wheat [Aegilops chromosome addition lines [Wheat-to wheat ,59,60,61on lines , and phyon lines  revealedon lines ,59,60,61Ae. geniculata and wheat-Ae. biuncialis addition lines [We identified 30 QTLs for traits related to the cell wall polysaccharide  synthesis that were evenly distributed across all M genome chromosomes . These findings are partially consistent with the previous biochemical analysis of wheat-on lines .Aegilops group 5 chromosomes significantly affects cell wall polysaccharide synthesis and can modify the grain BG, TP, and WEP content in wheat. Our results suggest that genes on chromosomes 1M and 4M may also be required for BG synthesis in Ae. biuncialis, and that a transfer of chromosome 5M alone is insufficient to increase the wheat BG content to that of Aegilops.We mapped three QTLs related to grain BG content on chromosomes 1M, 4M, and 5M. Although chromosome 4M was not represented in the set of addition lines previously investigated by Rakszegi et al. , their rAegilops chromosomes of 1M and 5M, which is consistent with previous studies made on durum wheat [CslF9 and CslF7) involved in β-glucan biosynthesis were assigned to the same homoeologous group chromosomes (groups 1 and 5) in barley and hexaploid wheat [Csl gene family members have not been identified on group 4 chromosomes in barley or wheat, it is less likely that QTL 1 on the structurally similar chromosome 4M contains a Csl gene variant in Ae. biuncialis. On the other hand, functional annotation revealed that the marker associated with QTL 1 co-located with a glutathione S-transferase 3-like gene (GTS3L), which is involved in various metabolic pathways with a wide range of substrates, including α,β-unsaturated carbonyls [Two (QTLs 2 and 3) of the three QTLs associated with BG content were identified on the um wheat  indicatiid wheat ,63,64. Tarbonyls . As the The water-extractable pentosan content was associated with the highest number of QTLs (24 of the 30) related to the cell wall polysaccharide synthesis. Among them, 11 QTLs affected positively the edible fiber content, with group 1 (2 QTLs), group 3 (4 QTLs), group 6 (4 QTLs), and group 4 (1 QTLs) chromosomes being over-represented. This is consistent with earlier findings in durum and bread wheat. Thus, a QTL strongly affecting the WEP content was found on chromosome 1B of hexaploid wheat , which rGT) play an important role in the regulation of AX biosynthesis [GT47 gene encoding a subunit of β-1,4 xylan synthase located on the group three chromosomes in hexaploid wheat [GT61 encoding α-1,3-arabinosyltransferase located on chromosomes 1B and 6A [GT47 and GT61) on the 1B, 3ABD, and 6A chromosomes correlated well with the current result that the majority of QTLs associated with WEP content were identified on the same homoeologous groups of Aegilops chromosomes.Glycosyltransferase gene families -beta-glucosyl linkages, such as xyloglucan ; solubleAe. biuncialis.The association of QTLs related to WEP content and candidate genes influencing polysaccharide metabolism emphasizes the important role of genes on chromosomes 1M, 3M, and 6M in the biosynthesis of cell wall components in Aegilops biuncialis Vis.  accessions, originating from a wide range of ecological habitats and representing the geographic distribution of the species, was previously genotyped using the DArTSeq platform [Aegilops accessions were maintained and propagated by the Cereal Genebank in Martonvásár. Hexaploid winter wheat (Triticum aestivum L.) line Mv9kr1, which contains a recessive crossability gene kr1 [A set of 86 platform . The accplatform . The Aeggene kr1 , was usegene kr1  and was Ae. biuncialis was grown during two growing seasons (2015–16 and 2016–17) in an experimental area  owned by the Agricultural Institute, Centre for Agricultural Research. Each genotype was sown in 6 × 3 m rows  in three replicates  using randomized complete blocks design in chernozem soil as previously described by Ivanizs et al. [Ae. biuncialis accessions were harvested manually in each replicate, where the grain mixture of replicates 1 and 2 or replicates 2 and 3 were considered as two biological samples for each genotype.The diverse collection of Ae. biuncialis accessions were characterized for quality traits  together with the wheat genotype Mv9kr1. For each genotype, measurements were performed on two biological and two technical replications. Whole grain flour was prepared from 4 grams of grain per sample using a Retsch Mixer Mill MM 400 ball mill . Wholemeal samples were immediately refrigerated and stored at −20 °C before use. Crude protein content was determined by the Dumas method in accordance with ICC Standard Method No. 167 [Grain composition analysis was performed on grain samples from two growing seasons (2015–16 and 2016–17). Since three genotypes could not be maintained in field trials, 83  No. 167  using th No. 167  with min No. 167 , as repo No. 167 . Total m No. 167 ; AACC Me No. 167  as descr No. 167 . Raw datAe. biuncialis using a QuickGene-Mini80 device  and a QuickGene DNA tissue kit (FujiFilm) according to the manufacturer’s instructions. DNA samples of the Ae. biuncialis accessions were genotyped utilizing the DArTseq™ 1.0 array platform by Diversity Arrays Technologies Pty. Ltd. . A total of 77,376 SNP-DArT markers were obtained after genome complexity reduction and subsequent SNP calling. Before performing GWAS, markers with a minimum allele frequency of less than 10% and those with more than 5% missing data points were removed from the data matrix using GenAlEx [Genomic DNA was extracted from young leaves of 86 accessions of  GenAlEx ,84. AfteTo determine the population structure, three different approaches were used: a principal coordinate analysis (PCoA) was performed using GenAlEx ,84; BayeAe. comosa MvGB1039  and Ae. umbellulata AE740/03 , respectively [ftp://ftp.ncbi.nlm.nih.gov/, accessed on 21 May 2021). Alignments that met certain criteria , were considered significant. The two best alignment hits were determined for each SNP marker and summarized in In order to obtain the chromosomal location of the thirty-four SNP markers closely associated with the quality traits, the SNP marker (35–69 bp) sequences were used as queries for the BLASTn search on 1M-7M and 1U-7U chromosomal scaffolds of ectively , and agaectively ) using Bftp://ftp.uniprot.org, accessed on 21 May 2021; UniProt release 2019_11) database, and protein domains were identified using the Hidden Markov Model (HMM)-based HMMER 3.0 software package  [ftp://ftp.ebi.ac.uk, accessed on 21 May 2021) [Ae. comosa, and Ae. umbellulata, the protein collection extracted from the database (PFAM), and gene ontology information (GO) are summarized in Aegilops sequences, each “gene” or “gene family” name was searched for in the CAZy database [http://www.plantgdb.org/cgi-bin/prj/PLEXdb/ProbeMatch.pl, accessed on 21 May 2021).Functional annotations were performed using translated sequences from known wheat genes. Gene Ontology information was obtained from the Universal Protein Resource (ay 2021)  in Pfam ay 2021) . The resdatabase . To charGrain quality trait parameters were measured with two replications in two parallel samples per accession. The measurement was repeated two more times when the difference between the results of duplicated analyses exceeded 10%. For statistical evaluation, Linear Mixed Model analysis  was applied for each compositional quality trait using SPSS 16.0 software  as described by Virk et al. . For allAe. Biuncialis, and revealed large genetic variation in grain edible fiber and protein content. BG, TP, and WEP content showed a high level of inheritance, making them promising traits to study a relationship between genotype and phenotype. Our GWAS analysis suggested that loci on chromosomes 1Mb, 4Mb, and 5Mb affect grain BG content, whereas chromosome groups 3, 6, and 1 contain a majority of QTLs responsible for TP and WEP content. Aegilops orthologs for genes found in these QTLs may be transferred from selected accessions and pyramided in bread wheat to achieve improved grain quality. The present study will facilitate more efficient use of Ae. biuncialis in pre-breeding programs to develop wheat varieties with specially modified fiber content and improved health benefits.A genome-wide association approach was employed for the first time in


Schizosaccharomyces pombe, showed that the TORC2 signaling pathway, which is essential for cell proliferation under glucose-limited conditions, ensures cell-surface localization of a high-affinity hexose transporter, Ght5, by downregulating its endocytosis. The TORC2 signaling pathway retains Ght5 on the cell surface, depending on the presence of nitrogen sources in medium. Ght5 is transported to vacuoles upon nitrogen starvation. In this review, we discuss the molecular mechanisms underlying this regulation to cope with nutritional stress, a response which may be conserved from yeasts to mammals.Target of rapamycin (TOR) kinases form two distinct complexes, TORC1 and TORC2, which are evolutionarily conserved among eukaryotes. These complexes control intracellular biochemical processes in response to changes in extracellular nutrient conditions. Previous studies using the fission yeast,  Schizosaccharomyces pombe, TORC2 contains a TOR kinase encoded by the tor1+ gene as the catalytic subunit as well as TORC2-specific RICTOR and SIN1 proteins, which are encoded by the ste20+ and sin1+ genes, respectively [S. pombe TORC1 contains a catalytic subunit, encoded by the tor2+ gene, and the TORC1-specific subunit RAPTOR, which is encoded by the mip1+ gene.Eukaryotic cells modulate intracellular biochemical processes in response to changes in availability of nutrients for proper growth, proliferation, and survival under a given extracellular microenvironment. While cells require nutrients as energy and substrates for growth and proliferation, nutritional conditions in the environment are not always favorable. Thus, cells need mechanisms to cope with nutritional stresses, such as starvation, for adaptation and survival. The TOR (Target of Rapamycin) kinases are critical for cellular responses to changes in nutritional conditions. As reviewed in the previous article by Tatebe and Shiozaki , the TORectively . S. pombgad8+ gene is proposed to be equivalent to mammalian AKT [To perform their physiological functions, TORC1 and TORC2 phosphorylate and activate downstream effector kinases belonging to the AGC-family . An AGC-lian AKT . Like AKlian AKT . As fisslian AKT ,5,6, GadS. pombe TORC2 signaling pathway inhibits endocytic vesicle trafficking of a high-affinity hexose transporter, Ght5, from the plasma membrane to vacuoles to ensure cell surface abundance of Ght5 and efficient glucose uptake under glucose-limited conditions [Several groups, including ours, have reported that as in other organisms, the TORC2 signaling pathway in fission yeast regulates glucose metabolism and enables proper cell proliferation under glucose-limited conditions. Recently, we found that the nditions . Curiousnditions , indicatS. pombe cells require TORC2 as well as AMP-activated protein kinase (AMPK) signaling pathways [S. pombe mutant cells defective for a component of the TORC2 signaling pathway  or the AMPK pathway  fail to form colonies on glucose-limited solid media, while they proliferate and form colonies on glucose-rich (>2%) media. The high-affinity hexose transporter, Ght5, which is encoded by the ght5+ gene, is indispensable for cell proliferation under glucose-limited conditions. In wild-type cells, Ght5 is localized on the plasma membrane and mediates transport of glucose from the medium to the cytoplasm. While the AMPK signaling pathway is required for transcriptional upregulation of ght5+ upon glucose limitation, the TORC2 pathway is necessary for Ght5 localization on the plasma membrane [While laboratory media for yeasts are normally supplied with high concentrations 2–3%, 111–168 mM) of glucose, fission yeast cells can grow and proliferate in medium containing only 0.08% (4.4 mM) glucose, which is equivalent to concentrations in normal human blood . To prolpathways . AMPK, w1–168 mM membrane ,12. Defemembrane . ConsistS. pombe cells to respond to nitrogen starvation. Consistent with this, TORC2 is involved in transcriptional regulation of a gene encoding an amino acid transporter protein upon nitrogen starvation [Fission yeast genes for the TORC2-AKT signaling were originally identified as those required for cell cycle arrest at G1 phase upon nitrogen starvation ,13,14,15arvation .S. pombe TORC2-AKT is central to cellular responses in both nitrogen and glucose starvation, to which fission yeast cells respond differently. Further studies are required for a full understanding of molecular mechanisms regulating the activities and/or substrate specificities of TORC2 and AKT depending on concentrations of amino acids and glucose.Collectively, S. pombe gad8 mutant cells in glucose-limited media [As mentioned in the previous section, fission yeast TORC2-Gad8/AKT is required for proper localization of the Ght5 hexose transporter on the cell surface. To reveal the mechanism underlying TORC2-dependent localization of Ght5 on the cell surface, Toyoda et al. screened for genomic suppressor mutations that rescue the growth deficiency of ed media . In thisaly3 gene mutation was the strongest suppressor. Loss-of-function mutations in the +aly3 gene greatly rescue growth deficiency under glucose-limited conditions and fully restore cell surface localization of the Ght5 transporter in gad8 mutant cells. The aly3+ gene encodes a novel α-arrestin protein belonging to the arrestin superfamily. Arrestin superfamily proteins are evolutionarily conserved among eukaryotes and classified into four subfamilies: α-arrestins, β-arrestins, visual-arrestins, and Vps26-related proteins [S. pombe. Arrestin was originally identified as a protein terminating the signal from the light-activated photoreceptor, rhodopsin, in bovine eyes [S. pombe Aly3 supposedly promotes association of Ght5 with a ubiquitin ligase, which has been proposed by Toyoda et al., but which remains to be identified, as well as its ubiquitylation [Among mutations identified in the screening, proteins . Aly3 isine eyes ,19. Arretylation   hexose transporters, such as Hxt1 and Hxt3, are expressed on the cell surface for glucose uptake, whereas high-affinity transporters, including Hxt6, are ubiquitylated and transported to vacuoles in a manner dependent on the arrestin, Rod1, and the E3 ubiquitin ligase, Rsp5  triggers Aly3-dependent transport of Ght5 to vacuoles, regardless of glucose concentrations. As amino acid starvation due to an auxotroph mutation also causes vacuolar transport of Ght5, reduction of intracellular amino acid levels is supposedly the direct trigger of Ght5 transport to vacuoles. Upon amino acid starvation, TORC2 and Gad8/AKT, which inhibit Aly3-dependent endocytosis of Ght5, may be down-regulated by an unknown mechanism, and consequently, Ght5 is transported to vacuoles and degraded  may replenish intracellular amino acids essential to survive nitrogen starvation at the cost of glucose uptake ability. Reducing glucose uptake ability under nitrogen-starved conditions may be part of reestablishing metabolic homeostasis, in which fission yeast Aly3 serves an important function. It is noteworthy that the budding yeast TORC1 subunit, RAPTOR/Kog1, reportedly regulates metabolic homeostasis under glucose and amino acid limitation independently of TORC1 activity [Similarly, fission yeast cells use different types of hexose transporters depending on the glucose concentration in medium . S. pomb to Ght8 ,27. Unde to Ght8 . Howeverdegraded . It remaactivity . Therefoactivity ,30. The The above-mentioned results of Toyoda et al. imply that activity of the TORC2-Gad8/AKT pathway is regulated by abundance of nitrogen sources . HoweverS. pombe, may be responsible for ubiquitylation of Ght5. Once endocytosed, ubiquitylated Ght5 is recognized and internalized within MVB in intraluminal vesicles by the ESCRT, followed by the transport of such a species of Ght5 to vacuoles, in which the protein is degraded into amino acids. The regenerated amino acids may contribute to increased activity of the TORC2-Gad8/AKT pathway and/or cell survival under low-glucose, nitrogen-starved conditions.A schematic model showing how TORC2-AKT ensures cell-surface localization of the high affinity hexose transporter Ght5 and reflects our recent results . In the As discussed above, arrestin-mediated transport of hexose transporters to vacuoles is regulated by the Gad8/AKT kinase in fission yeast, while it is regulated by the AMPK pathway in budding yeast. Similar to fission yeast, arrestin-mediated internalization of hexose transporters is reportedly regulated by AKT in mammalian cells. Mammalian thioredoxin-interacting protein (TXNIP) belongs to the α-arrestin family ,33,34. AS. pombe TORC2-Gad8/AKT ensures glucose uptake and cell proliferation under glucose-limited conditions. As long as nitrogen sources are present in the medium, S. pombe TORC2-Gad8/AKT inactivates the α-arrestin, Aly3, which is essential for vesicle transport of the Ght5 hexose transporter from the plasma membrane to cytoplasmic vacuoles. Similarly, in cells of mammals, including humans, internalization of hexose transporters from the cell surface to endocytic vesicles is controlled by AKT-dependent phosphorylation of the α-arrestin, TXNIP. Thus, inactivation of an α-arrestin by phosphorylation appears to be an evolutionarily conserved function of TORC2-AKT, although it remains to be determined whether α-arrestins are regulated via the TORC2-AKT pathway in other model organisms. Further studies are required to uncover molecular mechanisms regulating AKT-dependent phosphorylation of arrestins in response to nutritional/growth hormone stimuli.In this review, we have discussed how


Proteinopathy and excessive production of reactive oxygen species (ROS), which are the principal features observed in the Alzheimer’s disease (AD) brain, contribute to neuronal toxicity. β-amyloid and tau are the primary proteins responsible for the proteinopathy  in AD, which depends on ROS production; these aggregates can also generate ROS. These mechanisms work in concert and reinforce each other to drive the pathology observed in the aging brain, which primarily involves oxidative stress (OS). This, in turn, triggers neurodegeneration due to the subsequent loss of synapses and neurons. Understanding these interactions may thus aid in the identification of potential neuroprotective therapies that could be clinically useful. Here, we review the role of β-amyloid and tau in the activation of ROS production. We then further discuss how free radicals can influence structural changes in key toxic intermediates and describe the putative mechanisms by which OS and oligomers cause neuronal death. Moreover, ROS generation occurs at elevated rates in normal aging. It is an inevitable process in both acute and chronic pathophysiological conditions [Reactive oxygen species (ROS) result from normal daily cellular metabolism. Research conducted in the last two decades has clarified the role of ROS as secondary signaling molecules that regulate various biological and physiological processes, including proliferation, host defense, and gene expression ,2. Furth and DNA A. A highnditions . Thus, O2 and H2O2, whereas CAT and GPx convert H2O2 into H2O and O2. Along with the primary antioxidant defense against ROS, secondary antioxidant and cellular detoxification programs are mainly regulated by NF-E2-related factor 2 (Nrf2) and Kelch-like ECH-associated protein 1 (Keap1). Under normal conditions, Nrf2 is retained in the cytoplasm by the actin-binding protein Keap1; a substrate adaptor protein for the Cullin3-containing E3–ligase complex, which targets Nrf2 for ubiquitination and degradation by the proteasome [ROS are predominantly produced in mitochondria via mitochondrial enzymes. The electron transport chain (ETC) of mitochondria produces superoxide radicals at respiratory complexes I and III of the oxidative phosphorylation (OXPHOS) pathway through the single-electron leak ,6. Neveroteasome . Keap1 ioteasome . OS abrooteasome . It heteoteasome ,11,12. Ioteasome ,14,15. Aoteasome . 2O2 leads to enhanced Aβ production in different cell models [Deposition and spreading of aggregated proteins are the main characteristics of sporadic (s) and familial (f) forms of various neurodegenerative disorders, such as AD. This, in turn, results in excessive ROS production leading to OS, chronic neuroinflammation, and mitochondrial dysfunction, which altogether cause neuronal loss  and protl models . In addil models . Moreovel models . Productl models ,27. Bothl models ,29,30. H2 and, therefore, can damage cellular macromolecules. For example, ROS can react with nucleic acids (NA) by attacking nitrogenous bases and the sugar–phosphate backbone. Further, these can evoke single- and double-stranded DNA breaks, affecting the protein-coding region of mtDNA and influencing OXPHOS [ROS are oxygen-containing molecules that are more chemically reactive than Og OXPHOS ,32. mtDNg OXPHOS . In addig OXPHOS . Thus, pg OXPHOS ,32,33. Mg OXPHOS . Due to Furthermore, AGEs have been found to accumulate in numerous tissues throughout physiological aging, which leads to OS since the ability to respond to OS reduces with age. Due to this, many proteins lose their function, including those involved in gene transcription regulation ,33. ThusThe molecular associations of proteinopathy and proteotoxicity with OS are varied and complicated. Indeed, considerable evidence suggests that OS occurs before the appearance of symptoms in AD and that oxidative damage is detected not only in the vulnerable brain regions  but alsoAPP is a type I membrane protein that is synthesized and modified post-translationally in the endoplasmic reticulum (ER) and Golgi apparatus (GA) ,52,53. Ad-aspartate receptors (NMDARs); this, in turn, triggers a series of toxic synaptic events [The expression, processing, and intracellular protein trafficking of APP and Aβ peptides reportedly occur in the trans-Golgi network, endosomes, and PM and are well-defined phenomena ,58. Impoc events ,60,61. Tc events ,60,61. Tc events ,60,61,62c events ,61,62. Tc events ,60,61,624 and H2O2/FeSO4, resulted in a significant generation of 4-HNE. They also reported that an increase in the levels of 4-HNE was well correlated with an increase in BACE1 protein levels and was accompanied by a proportional increase in carboxy-terminal fragments of APP [Aβ toxicity has been demonstrated in vitro . When pls of APP ,22. Theys of APP ,22. 2+-mediated modification of γ-secretase activity [2+ enhanced γ-secretase activity results in an increase in the Aβ42/Aβ40 ratio [2+ [In AD, the presence of elevated extracellular Aβ levels at potential sites of lipid peroxidation only serves to elevate the risk of oxidative damage. Compellingly, compared with age-matched controls, in areas such as the hippocampus, where AD pathology is concentrated, higher levels of 4-HNE in AD patients are observed . Reportsactivity . Using c40 ratio ,73. They40 ratio ,73. Howe40 ratio ,73. Altoatio [2+ ,73.2O2, a key ROS, from O2 through electron transfer interactions involving bound redox-active Cu2+ and Fe3+ [2O2 is readily converted into an aggressive OH radical by Fenton chemistry (4). These two types of ROS have been reported to be responsible for the early oxidative damage seen in AD. Some studies have shown that the levels of H2O2 generated by Aβ can be enhanced by co-incubation of the peptide with a reducing substrate, which becomes oxidized in the process [Aβ has been reported to generate Hand Fe3+ ,74. H2O2 process ,74,75. FAGEs are regarded as chemical molecules that can be cross-linked to long-lived proteins ,77. In A2+ is reportedly found bound to several enzymes, such as SOD, cytochrome c oxidase, ceruloplasmin, and tyrosinase, which are involved in critical neuronal and non-neuronal cellular biochemical pathways. For instance, astrocytes can store and export Cu2+ to neurons. However, excess Cu2+ in astrocytes results in binding of Cu2+ to Aβ to form a cuproenzyme-like complex, which can transfer an electron to Cu2+ to convert Cu2+ to Cu+, thus forming the Aβ radical (Aβ+•) [+ can donate two electrons to O2 to generate H2O2 and produce OH radicals (Fenton-type reaction) [2+ is highly reactive, and excess of this metal ion often overproduces reactive chemical species (OH•) [3+ to Fe2+ and the generation of H2O2 [• formation further damages biomolecules, such as lipids, proteins, and NA, due to the ability of OH• to catalyze specific reactions, including hydrogen abstraction, addition reactions, and oxidation reactions in AD. These findings demonstrate that the interactions between Aβ and metals also produce ROS.Abnormal Cu, Zn, and Fe levels have been reported in the hippocampus and amygdala, along with severe histopathological changes in patients with AD ,85,86. Al (Aβ+•) ,88,89,90eaction) ,88,89. Fes (OH•) ,91. Fe aes (OH•) . For inses (OH•) . The bin of H2O2 . It has Besides Aβ proteinopathy, another prominent feature of AD pathogenesis is the accumulation of phosphorylated tau protein within neurons, known as neurofibrillary tangles (NFTs). These tau neurites also contribute to synaptic dysfunction and axonal degeneration . Tau usu2O2 production, lipid peroxidation, and eventually neuronal loss [According to the published experiments and reports, OS is well associated with tau pathology. Moreover, cells with overexpressing tau protein are more vulnerable to the OS, likely caused by peroxisome depletion ,104. Taunal loss ,106. Mornal loss  increasenal loss . Interestingly, P301S transgenic mouse brains showed enhanced OS and higher protein carbonyl levels in the cortical mitochondria. The relationship between tau pathology and OS was confirmed in P301L and P301S transgenic mouse models carrying the human tau gene with either the P301L or P301S mutation; these mice display accumulation of hyperphosphorylated tau, which causes neurodegeneration and the development of NFTs . Tau, boApplication of extracellular tau at different stages of aggregation to cortical co-cultures of neurons and astrocytes showed that only insoluble aggregates of tau could induce ROS production by activating nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in a calcium-dependent manner . The essRecently, several pieces of evidence link the role of microbiota in brain biology and aging, being an essential factor involved in various physiological processes via interactive symbiotic network system with host ,122,123.Physiological levels of OS have been generated in the microbiota, which can interfere with its composition and functionality . Furtherl-alanine (BMAA), an OS-inducing neurotoxin [However, the production of amyloid proteins helps in the formation of bacterial biofilms by promoting the binding of bacterial cells with each other, thus providing resistance from physical or immune factor-mediated destruction . Howeverurotoxin ,140, in urotoxin ,140,141.urotoxin ,143,144.urotoxin , which rurotoxin . In addiurotoxin ,147. C/Eurotoxin . Furthermore, reduction in the relative abundance of Proteobacteria and the low levels of Bifidobacteria can reduce beneficial short-chain fatty acids, leading to lipid peroxidation . This, i2O2. This chemical reaction serves a vital role in cell biology as H2O2 can produce OH radicals in the presence of transition metals such as Fe2+, for which there is no detoxification system [It is now evident that Aβ and tau pathologies are modulated by ROS and are also self-perpetuating concerning ROS formation . Hence, n system .Several antioxidant studies in AD models have also been reported, demonstrating that antioxidants consistently positively affect the animals’ behavioral and amyloidotic phenotypes . Vitamin1 and NRK-52E cells, Balogun and colleagues reported that curcumin upregulates Aβ-induced SOD and catalase and can further activate Nrf2 by selectively binding to Keap1 [Furthermore, curcumin is a potent inhibitor of Aβ fibrillization  and oligto Keap1 . Luteolito Keap1 . Melatonto Keap1 . Similarto Keap1 ,169,170.2O2, which can prepare the body for subsequent pro-oxidant exposure, which is similar to the formation of antibodies in vaccines. The significant outcomes of these studies are reductions in Aβ levels, phosphorylated tau, mitochondrial dysfunction, microglial activation, enhanced synaptic activity, and amelioration of cognitive decline. These results indicate that antioxidant treatment is beneficial in reducing and/or preventing AD progression. The findings also show that combination therapy positively impacts cognitive behavior and lowers AD pathology. The positive findings of these studies are promising. However, they warrant prospective studies  and clinical trials (antioxidant treatment for patients with AD). Recent work has also highlighted the importance of a healthy and detoxified innate response by consuming diet precursors and enhancing responsiveness . For insROS are the byproducts of normal cell metabolism and are therefore unavoidable. However, an imbalance between pro-oxidative and antioxidative cellular mechanisms leads to a vicious cycle since OS reciprocally aggravates ROS production, which results in the oxidation of lipids, proteins, and NA in neurons. This oxidation is a frequently encountered pathological marker in the case of AD. It contributes to the disease’s progression by increasing Aβ deposition, hyperphosphorylation of tau, and synaptic and neuronal loss. Altogether, the relationship between OS and AD suggests that OS is an essential part of the pathological process and that antioxidants may be helpful in treating AD. However, AD demands a precisely targeted treatment. Furthermore, non-antioxidant, targeted protection against OS, including transition metal chelators, compounds that modify the oligomeric structure, and inhibitors of enzymatic ROS production (such an NADPH oxidase), may potentially exert a strong therapeutic effect against AD. Additionally, consumption of precursors in the diet and mild exposure to pro-oxidants can benefit future exposure to the same stressor. All of these approaches are currently being rapidly developed.


Systemic treatment options for advanced neuroendocrine tumors have significantly been improved in the last decade. However efficacy of systemic therapy is limited by tumor resistance and therefore there is a need for further treatment options. Inhibition of the Ras-Raf-Mek-Erk signaling cascade might be a promising new treatment strategy in neuroendocrine neoplasms. In this study we investigated the effects of the MEK inhibitor trametinib, the ERK inhibitor SCH772984 and the CDK4/6 inhibitor ribociclib in human neuroendocrine tumor cell lines BON1, QGP1 and NCI-H727 in vitro. Trametinib alone and in synergism with ribociclib demonstrated antiproliferative effects. Combination therapy of MEK inhibitors and CDK4/6 inhibitors might be a potential strategy to overcome CDK4/6 inhibitor resistance in neuroendocrine tumors.Objectives: This study assessed the antitumoral activity of the MEK inhibitor trametinib (TMT212) and the ERK1/2 inhibitor SCH772984, alone and in combination with the CDK4/6 inhibitor ribociclib (LEE011) in human neuroendocrine tumor (NET) cell lines in vitro. Methods: Human NET cell lines BON1, QGP-1, and NCI-H727 were treated with trametinib or SCH772984, alone and in combination with ribociclib, to assess cell proliferation, cell cycle distribution, and protein signaling using cell proliferation, flow cytometry, and Western blot assays, respectively. Results: Trametinib and SCH772984, alone and in combination with ribociclib, significantly reduced NET cell viability and arrested NET cells at the G1 phase of the cell cycle in all three cell lines tested. In addition, trametinib also caused subG1 events and apoptotic PARP cleavage in QGP1 and NCI-H727 cells. A western blot analysis demonstrated the use of trametinib alone and trametinib in combination with ribociclib to decrease the expression of pERK, cMyc, Chk1, pChk2, pCDK1, CyclinD1, and c-myc in a time-dependent manner in NCI-H727 and QGP-1 cells. Conclusions: MEK and ERK inhibition causes antiproliferative effects in human NET cell lines in vitro. The combination of the MEK inhibitor trametinib (TMT212) with the CDK4/6 inhibitor ribociclib (LEE011) causes additive antiproliferative effects. Future preclinical and clinical studies of MEK inhibition in NETs should be performed. Neuroendocrine neoplasms (NENs) are a heterogeneous group of neuroendocrine tumors (NETs) and neuroendocrine carcinomas (NECs) that can occur in most parts of the human body, most often in the intestine, pancreas, and lungs ,2. TreatAlthough over the past decade, NET treatment strategies and treatment outcomes have improved , their eDuring human carcinogenesis and cancer progression, tumor cells possess at least six essential hallmarks, i.e., tumor cell self-sufficiency in growth signals, insensitivity to growth–inhibitory signals, evasion of apoptosis, limitless replicative potential, sustained angiogenesis, and tissue invasion and metastasis . KRAS, BRAF, PI3KCA, PTEN, CDKN2A, CDKN2B, or AKT, can be found in NENs [NENs of the pancreas, small intestines, and lungs harbor various somatic mutations, as has been recently described ,11,12. F in NENs ,11,12. The CDK4/6 inhibitors ribociclib, palbociclib, and abemaciclib have been approved in the treatment of breast cancer in combination with endocrine therapy . Abemacin = 20 patients with pancreatic NETs, palbociclib failed to demonstrate clinically significant antitumoral effects, with best-response stable disease and a median progression-free survival of 2.6 months [n = 20 patients with foregut NETs, ribociclib caused best-response stable disease and a median progression-free survival of 10.4 months [In neuroendocrine tumors, the CDK4/6 inhibitor palbociclib (PD-0332991) demonstrated significant antitumoral activity in a preclinical in vitro and in vivo model . By cont6 months . Recentl6 months . In a cl4 months .CDK4/6 inhibitor resistance  might beThe MEK inhibitor trametinib (TMT212), a selective allosteric inhibitor of MEK1/2, is able to control cancer cells with an overactive MEK-ERK pathway . For theIn neuroendocrine tumor cell lines, different MEK inhibitors have been reported to demonstrate antitumoral effects in vitro ,35,36,37In this study, we further assess the effects of the MEK inhibitor (MEKi) trametinib (TMT212), the ERK1/2 inhibitor (ERKi) SCH772984, and their combination with the CDK4/6 inhibitor (CDK4/6i) ribociclib (LEE011) on NETs in vitro and explore the underlying molecular events. We provide insightful information regarding antitumoral efficacy of MEK inhibition alone and in combination with CDK4/6 inhibition in NETs. 2 at 37 °C. Human pancreatic NET BON1 cell line ,38,39 waRibociclib (LEE011) and trametinib (TMT212) were provided by Novartis , while SCH772984 was purchased from Selleckchem (Germany). These reagents were dissolved in dimethyl sulfoxide (DMSO) as stock solutions and stored at –20 °C. Their working solutions were used to treat NETs cells as single therapies and as combinations for different durations of time for various assays. ® cell viability assay kit from Promega . In brief, NET cells were seeded into 96-well plates, grown overnight, and then treated with different doses of ribociclib (LEE011), trametinib (TMT212), SCH772984, and or their combinations for up to 144 h. Our group previously calculated the population doubling time (PDT) to be 0.895 ± 0.066 d for BON1, 1.536 ± 0.051 d for QGP-1, and 1.781 ± 0.295 d for NCI-H727 cells, respectively [To assess the effects of ribociclib (LEE011), trametinib (TMT212), and SCH772984 alone or in appropriate combinations, we utilized the Cell Titer Blueectively . In addiectively . Therefoectively .50 values were calculated for each drug and cell line. At the end of each experiment, the cells were subjected to the kit procedures according to the manufacturer’s protocol. The optical density of each cell line was measured using a spectrophotometer  and inhibitory concentration ICNET cells were seeded and grown in 6-well plates overnight and then treated with various doses of trametinib (TMT212), SCH772984, or trametinib (TMT212) plus ribociclib (LEE011) or SCH772984 plus ribociclib (LEE011) for 72 h. Next, the cells were harvested using 0.05% trypsin, centrifuged and washed in phosphate-buffered saline (PBS), then resuspended in the propidium iodide solution  for flow cytometric analysis and quantification of the cell cycle distribution with BD Accuri C6 Analysis software .After NET cells were grown and treated with ribociclib (LEE011), trametinib (TMT212), or SCH772984, alone or in appropriate combinations, the total cellular protein was lysed in a lysis buffer containing the M-PER  and the HALT protease and phosphatase inhibitor cocktail . The supernatants of the cell lysis were then quantified using the RotiQuant Universal protein assay kit . The protein samples containing 50 µg of each were separated in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred onto the polyvinylidene fluoride membranes . For Western blotting, the membranes were blocked in the Clear Milk Blocking Buffer  for 30 min and incubated at 4 °C overnight with different primary antibodies. The primary antibodies were anti-CyclinD1, CyclinD3, CyclinB1, CDK1, CDK4, CDK6, Chk1, pChk2 T68, Chk2, c-Myc, pEGFRY1068, EGFR, pIGF1RY1135, IGF1R, pAktS473, Akt, pp70S6K T389, p70S6K, p4EBP1 S65, 4EBP1, pERK T202/Y204, ERK, pMEK S217/221, MEK, pRB S780, pRSK1 S380, RSK, pJNK T183/Y185, JNK, and pp38 T180/Y182, p38, Puma, Mcl-1, Bcl-2, BclxL, Caspase 3, cleaved Caspase 3, PARP, pS6 S240/4,S6 , p21 Waf1/Cip1 , RB , p53, AIF, and ß-Actin .On the next day, the membranes were washed with Tris-based saline-Tween 20 (TBS-T) briefly three times and then incubated with a horseradish-peroxidase-conjugated secondary antibody  at a dilution of 1:25,000 for 2 h. After washing, the positive-bound protein bands were visualized with a chemiluminescence Western blotting detection system  and detected with an ECL imaging system . The level of each protein sample was quantified using the ImageJ software .p < 0.05 was considered statistically significant.All experiments were performed in triplicate and repeated at least once. The data were summarized as the mean ± SD of the triplicate samples and statistically analyzed using a one-way analysis of variance test (ANOVA) followed by Dunnett’s t test for multiple comparisons. Biosoft CalcuSyn 2.1 software  was used to assess the synergistic effects of the combination treatments. Here, 50 for ribociclib (LEE011) has been previously reported by our group [50 = 2.6 μM, QGp-1 cells IC50 = 1.2 μM, H727 cells IC50 = 10.9 μM.We have previously reported effects of ribociclib (LEE011) in neuroendocrine tumor cells for up to 144 h . The IC5ur group  as follo50 values were 0.44 nM, 6.359 nM, and 84.12 nM for BON1, QGP-1, and NCI-H727 cells, respectively , trametinib (TMT212), and SCH772984 on regulation of NET cell proliferation when used in a panel of NET cell lines (ectively . The medectively . These dWe then examined the effects of the MEKi trametinib or the ERKi SCH772984 in combination with the CDK4/6i LEE011 on NET cell proliferation . After tp < 0.05) stronger cell cycle G1 arrest in BON1 cells  and in QGP-1 cells , and a corresponding decrease in cells in S and G2-M phases. Similarly, the combination of SCH772984 with LEE011 versus SCH772984 alone caused a significantly (p < 0.05) stronger cell cycle G1 arrest in BON1 cells , QGP-1 cells , and NCH-H727 cells , and a corresponding decrease in cells in the S-phase in all three cell lines  , and c-MHowever, LEE011 combined with SCH772984 maintained the high levels of CDK4/6 and slightly decreased cyclinD1/D3 levels . The timThus, the MEK and ERK inhibitors induced the effects of LEE011 on NET cells, suggesting that the inhibition of the MAPK signaling could at least partially confer the sensitivity of tumor cells to LEE011.We next determined the effects of the combination of trametinib or SCH772984 with LEE011 on regulation of the MEK/ERK signaling in NETs cells. Trametinib alone and in combination with LEE011 treatment significantly decreased pERK1/2 and cMyc expression in BON1, QGP-1, and NCI-H727 cells .Moreover, SCH772984 in combination with LEE011 synergistically upregulated the level of pERK1/2 in NCI-H727 but downregulated it in BON1 and QGP-1 cells . SCH7729Furthermore, trametinib alone or in combination with LEE011 upregulated pMEK1/2 levels in NCI-H727 and QGP-1 cells in a time-dependent manner . The graTreatment of NET cells with trametinib or SCH772984 was able to upregulate levels of pp38 and pJNK in NCI-H727, whereas it decreased them in BON1 and QGP-1 . SCH7729Trametinib plus LEE011 was able to significantly reduce levels of pAKT in BON1 and NCI-H727 but not in QGP-1 as compared to each of the single agents, while reductions of p70S6K, pS6, and p4EBP1 were more pronounced in combination with LEE011 . MoreoveTrametinib or SCH772984 in combination with LEE011 caused significant reductions of pEGFR/EGFR levels vs. the controls .The levels of pIGFR after SCH772984 or trametinib treatment did not show any significant changes, although there was a decrease in BON1 cells after treatment with SCH772984 or trametinib plus LEE011 . InteresIn the current study, we assessed the antitumor activities of the MEKi trametinib (TMT212) and the ERKi SCH772984 and evaluated their effects in combination with the CDK4/6i ribociclib (LEE011) in three different human NET cell lines in vitro and explored the underlying molecular events. We demonstrated that trametinib or SCH772984 in combination with LEE011 dose- and time-dependently inhibited NET cell growth  and arreHRAS, KRAS, NRAS, and BRAF are rare events that have been reported only in 1%, 8%, 0.7%, and 1% of patients, respectively [KRAS and BRAF are frequent events, which have been reported in 8% to 60% and 9% to 60% of tumors, respectively [BRAF-mutated NECs with the BRAF inhibitor dabrafenib and the MEK inhibitor trametinib prevented tumor growth in a xenograft model [NET somatic mutations in ectively . In contectively ,51. Treaft model  and alsoBRAF or RAS mutations, but our current study did not analyze such an association. However, the human pancreatic neuroendocrine tumor cell line BON1 harbors a mutation in NRAS, while the human pancreatic neuroendocrine tumor cell line QGP-1 harbors a mutation in KRAS [Our current in vitro study does have some limitations; for example, a previous study reported that the most efficient antitumor effects of MEKi and ERKi occurred on tumors harboring  in KRAS . Thus, oRAS, BRAF, MEK1, and MEK2 could cooperate with CDKN2A loss in different cancers [Dysregulation of the CDK4/6-Rb pathway, as with the activation of CCND1/CDK4 or CDKN2A loss, contributes to cancer development and confers resistance of cancer cells to treatment with the MAPK/ERK inhibitors . Convers cancers ,55. Thus cancers . Indeed,Furthermore, our current study revealed that trametinib and SCH772984 caused potent inhibitory effects on NET cells; both increased levels of pMEK in these three NET cell lines, while trametinib or SCH772984 in combination with LEE011 also upregulated the expression of pMEK. In contrast, trametinib alone or in combination with LEE011 induced durable suppression of pERK1/2 level. Moreover, SCH772984 alone or in combination with LEE011 reversed the upregulation of pERK by LEE011 alone. Although the inhibitory activity of trametinib and SCH772984 was not identical, trametinib seemed more potent in the suppression of MAPK and CDK4/6 than SCH772984. This phenomenon may be due to the inhibition of feedback mechanisms involving Sprouty and dual-specificity phosphatases . MoreoveAgain, the PI3K/Akt pathway is crucial in promoting cyclinD/CDK4/6-induced cell proliferation via regulation of the cell cycle progression . In our A previous study reported that trametinib blocked p38 MAPK phosphorylation, which was mainly due to a unique off-target effect of trametinib . HoweverEGFR expression has been reported in 80–100% of NETs . A previRAS/BRAF signaling cascade [In conclusion, our current study revealed potent antiproliferative effects of the MEKi trametinib in NETs. MEKi might be especially interesting as an antitumoral strategy in NECs, as NECs harbor a high rate of mutations in the  cascade ,51. In a cascade , neuroen cascade , melanom cascade ,44,45, c cascade ,25,46, p cascade , and NSC cascade .A major limitation of our in vitro study is the fact that the human neuroendocrine tumor cell lines BON1, QGP-1, and NCI-H727 are distinct from neuroendocrine tumors in patients regarding genome, tumor biology, and growth rate factors ,38,39,59Clinical trials with MEKi in combination with CDK4/6i are ongoing in other solid tumors. The current preclinical results in neuroendocrine tumors still need to be evaluated in future preclinical and clinical studies. Future investigations in patient-derived primary tumor cultures, spheroids, and organoids ,61,62 shInhibition of the MAPK/ERK signaling cascade by MEK inhibitors and ERK inhibitors in human neuroendocrine tumor cell lines in vitro demonstrates potent antiproliferative effects. Combination of the MEK inhibitor trametinib and the CDK4/6 inibitor ribociclib demonstrated synergistic antiproliferative effects in vitro. Further preclinical in vivo studies and clinical studies should investigate the therapeutic efficacy of MEK inhibitors alone and in combination with CDK4/6 inhibitors in neuroendocrine neoplasms.


VDAC2, GLS2, FLT3, TLR4, PHKG2, phosphogluconate dehydrogenase (PGD), PANX1, KRAS, PEBP1, ALOX15, and ALOX12B, and suppressor genes ACSL3, CISD1, FANCD2, and SLC3A2) in The Cancer Genome Atlas (TCGA)-LUAD cohort. The signature’s predictive ability was validated in the GSE68465 and GSE72094 cohorts by survival analysis and independent prognostic analysis with clinical features. Nomograms were provided for clinical reference. Functional analysis revealed that ferroptosis was closely related to cell cycle, cell metabolism, and immune pathways. Pan-cancer analysis comprehensively analyzed these 15 genes in 33 cancer types, indicating that the heterogeneity of 15 genes was evident across different cancer types. Besides, these genes were critical regulators modulating drug resistance, tumor microenvironment infiltration, and cancer stemness. Then, we screened 10 genes  as potential targets for further research because their biological functions in ferroptosis were consistent with their prognostic significance. Somatic mutation and copy number variation analysis revealed that the alteration rates of KRAS, PGD, and ALOX15 were more than 1% and significantly associated with overall survival in LUAD. Moreover, the expression of KRAS and PGD was positively related to tumor mutation burden, indicating that KRAS and PGD could serve as novel biomarkers for predicting immunotherapy response rate. Our study identified and validated a ferroptosis-related gene signature for LUAD, provided a 10-gene set for future research, and screened KRAS and PGD as potential novel immunotherapy biomarkers.It is reported that ferroptosis has close relation with tumorigenesis and drug resistance. However, the clinical significance of ferroptosis in lung adenocarcinoma (LUAD) remains elusive, and the potential targets for ferroptosis-based treatment are limited. In this study, we constructed a 15-gene prognostic signature predicting overall survival based on ferroptosis-related genes (ferroptosis driver genes  Ferroptosis genes in lung adenocarcinoma. Lung cancer would cause 2.1 million new cases and 1.8 million deaths in 2018, as estimated . Lung adNFS1 (NFS1 cysteine desulfurase) (CISD1 (CDGSH iron–sulfur domain 1)  accumulation by glutathione (GSH) consumption and glutathione peroxidase 4 (GPX4) inactivation, which results in subsequent cell death . Latest lfurase)  and CISDomain 1) , may becomain 1) . Lei et omain 1) .HO-1) expression  as potential novel immunotherapy biomarkers.We established a novel ferroptosis-related 15-gene prognostic signature in The Cancer Genome Atlas (TCGA)-LUAD cohort and validated it in the GSE68465 and GSE72094. Survival analysis and independent prognostic analysis acted mutually to examine the reliability of our signature. Then, we constructed our prognostic signature nomogram to provide a clinical reference for clinicians. Furthermore, functional analysis revealed the relationship between ferroptosis and cell cycle, cell metabolism, and immune response. The pan-cancer analysis provided a comprehensive and systematic characterization of these 15 genes in all 33 cancer types. The results showed that these genes had a close relationship with tumor immunity, drug resistance, tumor microenvironment (TME) infiltration, and cancer stemness, providing new insights for ferroptosis in tumor behavior. Among these 15 genes, 10 genes could be potential treatment targets for basic researches in LUAD because their biological functions in the process of ferroptosis were consistent with their prognostic significance. We performed mutation analysis to explore the alteration and function of these 15 genes in tumor development at the level of genomics. To conclude, our study identified and validated a ferroptosis-related gene (FRG) signature for LUAD, provided a 10-gene set for future research, and screened 1 LUAD project, including 535 tumor samples and 59 normal samples. The corresponding clinical data were also retrieved from the LUAD project, including 500 patients [13 excluded due to lack of overall survival (OS) and 2 excluded due to lack of RNA-seq] as train cohort. The normalized series matrixes of two total-RNA microarrays, GSE68465 2 and GSE72094 (442 tumor samples),3 were retrieved from the Gene Expression Omnibus (GEO) database (Preprocessed RNA-sequencing (RNA-seq) by fragments per kilobase of exon model per million mapped fragments (FPKM) normalization was downloaded from the TCGA databasedatabase . The cor4 to download the FRGs  between tumor and normal tissues by Mann–Whitney test. Then, we executed the univariate Cox regression to detect the prognostic significance of all FRGs based on OS in the TCGA cohort. Then, the FRDEGs and prognostic FRGs were intersected. The overlapped genes were named prognostic FRDEGs. An online protein–protein interaction (PPI) database, STRING  (version 11.0), was used to plot the PPI network based on prognostic FRDEGs . The setWe also constructed our prognostic gene signature nomogram using the R language. All the prognostic factors included in the multivariate Cox regression of the independent prognosis analysis were included in the nomograms. The calibration curve was plotted to evaluate the fitting and predictive ability of our prognostic model.P  < 0.05 were set as cutoff value. In the GSE68465 cohort, functions “lmFit” and “eBayes” were utilized, and | log2FC| ≥ 0.5 and adj.P < 0.05 were set as cutoff value. In the GSE72094 cohort, functions “lmFit” and “eBayes” were utilized, and | log2FC| ≥ 0.9 and adj.P < 0.05 were set as cutoff value. Gene Ontology  and | log2FC| ≥ 1 and adj.logy GO;  and Kyotlogy GO;  enrichme5 including RNA-seq, clinical data, and stemness scores  after drug treatment. We calculated the Pearson correlation between each FRPSG expression and the GI50 to explore the association between FRPSGs and drug sensitivity. We selected 262 Food and Drug Administration-approved drugs or drugs that are currently in clinical trials in this drug sensitivity analysis (7 (P < 0.05). Furthermore, we utilized the online bioinformatics tool, GEPIA, to explore the correlation between the ferroptosis suppressor genes in FRPSGs and two previously discovered cancer stemness cell biomarkers  were adjusted by Benjamini–Hochberg (BH). Adj.P < 0.05 was considered as statistically significant.The descriptive analysis of each cohort was performed by Chi-square test, Somer’s d test, and independent-samples n 4.0.3) . All theP-value was 8.09e-07, indicating the network has significantly more interactions than a random protein set. The PPI network  and negatively related to DPP4. These findings revealed many interactions between the gene pairs, and the regulatory mechanisms of ferroptosis in LUAD were complex.The research flowchart and corresponding online resources were presented in Graphical Abstract. The three cohorts’ basic characteristics are summarized in  network  selectedse genes  ]}. The median value of risk score (−1.58) divided the 500 patients into two groups (250 in the high-risk group/250 in the low-risk group) (P < 0.001). The ROC curve showed the area under the curve (AUC) reached 0.718, 0.730, and 0.721, respectively, which was acceptable  were increased, and two ferroptosis driver genes (FLT3 and PHKG2) were decreased in higher-stage LUAD. However, some ferroptosis driver genes were increased in higher-stage LUAD. We hypothesized that these genes might involve other regulatory pathways of tumor behavior. For example, PGD is highly expressed in stage IV LUAD. In addition, many previous studies have demonstrated that overexpression of PGD could lead to cancer metastasis and poor prognosis through diverse mechanisms  . The park group) . Survivaceptable . The surdatabase . A briefdatabase . The expD stages . We founchanisms .P < 0.001) (P < 0.001)  . The bas< 0.001) . AUCs re< 0.001) , which w< 0.001) . OS in t< 0.001) . AUCs re< 0.001) . We attrKRAS, EGFR, and TP53 mutation were included. The uni- and multivariate Cox regression showed that gender, stage, and risk score were statistically significant independent prognostic factors , all of the four ferroptosis suppressor genes were significantly upregulated in the high-risk groups, and most ferroptosis driver genes  were significantly downregulated in the high-risk groups (TCGA cohort). In addition, only three ferroptosis driver genes  were significantly upregulated in the high-risk groups. Furthermore, the ferroptosis suppressor genes were also upregulated in the high-risk groups in the GSE68465  and GSE72094  cohorts , activated DCs (aDCs), B cells, CD8 + T cells, iDCs, neutrophils, pDCs, T helper cells, Tfh, mast cells, and TIL were significantly reduced in the high-risk group (TCGA) . As for ALOX12B, ALOX15, FLT3, and GLS2 was deficient, and the expression of PEBP1 was the highest, indicating PEBP1 could be a promising treatment target. Spearman correlation showed that PEBP1 and FANCD2 had the highest correlation. There were some potential interactions between these two genes. Gene differential expression analysis showed that the 15 FRPSGs were differently expressed in tumor tissues compared with corresponding normal tissues. It is noteworthy that the ferroptosis suppressor genes, SLC3A2, FANCD2, CISD1, and ACSL3, were upregulated in most cancer types, indicating that ferroptosis was generally inhibited in cancers (VDAC2 was negatively associated with OS in low-grade glioma (LGG) and positively in pheochromocytoma and paraganglioma (PCPG)  kinase inhibitor for renal cell cancer and some other tumors, and nelfinavir is a protease inhibitor for HIV infection. These findings demonstrated that ferroptosis was involved in some tumor-targeted therapy.The expression of 15 FRPSGs in pan-cancer was different . The exp cancers . As for a (PCPG) . It is rP < 0.001) for each cancer type. Moreover, TME analysis showed the 15 FRPSGs were related to immune and stromal cell infiltration in most cancer types , especially FANCD2 (FANCD2 expression and RNAss reached 0.56 (P < 2.2e-16), indicating FANCD2 plays a critical role in promoting tumor heterogeneity. We further validated these findings by exploring the association between the four ferroptosis suppressor genes with two previously discovered LUAD stemness cell biomarkers, CD133 and CD44. As shown in P < 0.05), which were consistent with the results of cancer stemness feature analysis. In summary, we identified that the four ferroptosis suppressor genes, ACSL3, CISD1, FANCD2, and SLC3A2, could increase the cancer stemness, indicating that the lower the ferroptosis level in lung cancer cells, the higher the tumor heterogeneity.Immune analysis of the 15 FRPSGs was performed. Six immune subtypes were defined as C1 , C2 (INF-r dominant), C3 (inflammatory), C4 (lymphocyte depleted), C5 , and C6 (TGFβ dominant). As shown in er types . These fcer type . Specifiy FANCD2 . The SpeFLT3, ALOX12B, ALOX15, and VDAC2, had a low CNV, and four ferroptosis suppressor genes ACSL3, CISD1, FANCD2, and SLC3A2, had a high CNV, indicating that ferroptosis was inhibited in tumor development. Besides, the generally accepted oncogene KRAS had a high CNV. Among the 15 FRPSGs, the alteration status of three genes, ALOX15, KRAS, and PGD, was significantly related to OS analysis , with an alteration rate of 1.2, 27, and 1.2%  with correlations of 0.61 and 0.16 , and the Spearman correlation reached around 0.2, indicating that KRAS and PGD played a crucial in maintaining genomic integrity. Given that TMB is positively related to immune therapy response rate, we suppose that KRAS and PGD could serve as novel biomarkers for predicting immunotherapy response rate.To fundamentally understand the ferroptosis process during tumor development, we performed mutation analysis of the 15 FRPSGs. The location on chromosome and CNV status of the 15 FRPSGs are presented in and 1.2% . Besidesgression . The altand 0.16 . Comparipression . The mosTLR4, PHKG2, PEBP1, GLS2, FLT3, ALOX15, ACSL3, CISD1, FANCD2, and SLC3A2) could be potential targets for further research because their biological functions in ferroptosis were consistent with their prognostic significance.Among the 15 FRPSGs, most ferroptosis driver genes were associated with a better prognosis , and all the ferroptosis suppressor genes  were associated with a worse prognosis, which indicates that the ferroptosis process in LUAD inhibits the tumor development as previously reported . We suggACSL3, ALOX15, ALOX12B, and PEBP1. ACSL3 is a principal modulator of ferroptosis by activating the exogenous monounsaturated fatty acids, which subsequently replace polyunsaturated fatty acids (PUFAs) and block lipid ROS accumulation oxidative metabolism, and energy metabolism are the main regulatory pathways of ferroptosis. In our signature, lipid metabolism involves mulation . Lipoxygmulation . In partmulation . ALOX12Bmulation . Jiang emulation .SLC3A2, VDAC2, GLS2, and FLT3, within which SLC3A2 is the heavy chain subunit of system xc-cystine/glutamate antiporter, maintaining the steady state of redox (GLS2 in mouse embryonic fibroblasts and found that the ferroptosis is reduced. (Anti)oxidant metabolism involves of redox . It is rof redox . VDAC2 cof redox . GLS2 plPGD was more than 1.2% and was related to a significantly poor prognosis. It is noteworthy that the CNV was relatively common in PGD alteration. Shallow deletion, amplification, and deep deletion occurred, and the gene expression was significantly related to CNV. More research is needed to explore the relationship between PGD’s CNV and ferroptosis.PGD involves energy metabolism such that it functions in the pentose phosphate pathway (PPP). Although the association between energy metabolism and ferroptosis remains puzzling, TLR4, FANCD2, and KRAS, serve as specific mediators. TLR4 is a toll-like receptor responsible for activating the innate immune system and has been confirmed to be associated with inflammation, autophagy, cell migration, and adhesion/metastasis in NSCLC . Besides, the data quality and preprocessing of the two data resources (RNA-seq and microarray) were different, which lead to different data processing methods and different cutoff values. However, we could not find an appropriate RNA-seq validation cohort currently. Second, the clinical significance of our model needs to be further evaluated through prospective data in the real world. Third, experimental evidence was not acquired to prove our conclusion, including drug sensitivity analysis and cancer stemness analysis.The original contributions presented in the study are included in the article/ZR and MH performed the data analysis and wrote the first draft. ZW and JG revised the manuscript and carried out data collection. XZ and GZ revised the manuscript and tables. HZ designed and supervised the study. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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Hereditary cancer syndromes, which are characterized by onset at an early age and an increased risk of developing certain tumors, are caused by germline pathogenic variants in tumor suppressor genes and are mostly inherited in an autosomal dominant manner. Therefore, hereditary cancer syndromes have been used as powerful models to identify and characterize susceptibility genes associated with cancer. Furthermore, clarification of the association between genotypes and phenotypes in one disease has provided insights into the etiology of other seemingly different diseases. Molecular genetic discoveries from the study of hereditary cancer syndrome have not only changed the methods of diagnosis and management, but have also shed light on the molecular regulatory pathways that are important in the development and treatment of sporadic tumors. The main cancer susceptibility syndromes that involve gynecologic cancers include hereditary breast and ovarian cancer syndrome as well as Lynch syndrome. However, in addition to these two hereditary cancer syndromes, there are several other hereditary syndromes associated with gynecologic cancers. In the present review, we provide an overview of the clinical features, and discuss the molecular genetics, of four rare hereditary gynecological cancer syndromes; Cowden syndrome, Peutz-Jeghers syndrome, DICER1 syndrome and rhabdoid tumor predisposition syndrome 2. BRCA1 and BRCA2 germline pathogenic variants (PVs) have been identified. HBOC has been reported to occur in approximately one in 300 to one in 800 in the general population [BRCA-related HBOC, and have allowed more detailed estimates of ovarian cancer risk for several homologous recombination (HR)-related genes with moderate-penetrance such as BRIP1, RAD51C/D, PALB2, and ATM [MLH1, MSH2, MSH6 and PMS2, cause Lynch syndrome. Immune checkpoint inhibitor (ICI) has been proven to be a highly effective treatment strategy for microsatellite instability high (MSI-H) tumors associated with MMR-deficiency, such as Lynch syndrome [Approximately 5–10% of all malignancies are caused by hereditary cancer predisposition syndromes , and gyn and ATM ,7. Poly, Peutz-Jeghers syndrome (PJS), DICER1 syndrome and rhabdoid tumor predisposition syndrome (RTPS) 2, provides a summary of clinical features and relevant gynecologic cancers, and discusses molecular genetics. PTEN PVs occur in up to 85% of CS/CS-like individuals who meet International Cowden Consortium operational diagnostic criteria, in up to 60% of BRRS cases, 7–20% of PS cases and 50–67% of Proteus-like syndrome cases [The reported frequency of CS is estimated to be approximately one in 200,000 ; howeverme cases .Although endometrial cancer (EC) has been recognized as a key component of CS, its prevalence and clinical features are not well characterized. Strong clinical predictors for the presence of germline PTEN variants in EC in CS/CS-like patients suggest an age of under 50 years, macrocephaly, high phenotypic burden, and/or coexistence with renal cell carcinoma . PatientPTEN is located at chromosome 10q23.3, encoding for a 403-amino acid protein that possesses both lipid and protein phosphatase activities. Since the PI3K/Akt/mTOR signaling pathway results in the regulation of signal transduction and biological processes such as cell proliferation, apoptosis, metabolism and angiogenesis, an increased in this pathway activity is a major contributor to human cancer. The PI3K/Akt/mTOR signaling pathway is tightly regulated by PTEN, which acts as a negative regulator of the pathways by converting phosphatidylinositol 3,4,5-trisphosphate back to phosphatidylinositol 4,5-bisphosphate. Inactivation of PTEN by loss-of-function mutations leads to elevated levels of phosphatidylinositol 3,4,5-trisphosphate and accelerates tumor initiation and progression. Various genetic alterations, including point mutations, large chromosomal deletions, and epigenetic mechanisms frequently cause PTEN inactivation in a large array of cancers [PTEN, approximately two-thirds of germline PTEN PVs occur in exons 5, 7, and 8, encoding the phosphatase domain [PTEN variants [PTEN promoter variants in the germline, in addition to intragenic mutations [AKT1 (2.2%) and PIK3CA (8.8%) associated with the PI3K/AKT/mTOR pathway was reported in PTEN wild-type CS/CS-like patients [e domain . In partvariants ,43. Apprutations . Recentlutations . Large dutations ,44,46. Opatients .PTEN-mutated disorders. Recently, promising studies have been reported, in which a pan-AKT inhibitor (ARQ 092) was used to treat primary fibroblasts in vitro in patients with PIK3CA-associated overgrowth spectrum (PROS), and the PIK3CA inhibitor BYL719 (Alperisib) was used to treat a preclinical mouse model of PROS, followed by 19 patients with severe PROS disorders. [Since PTEN negatively regulates the PI3K/AKT/mTOR pathway, inhibition of mTOR is a reasonable therapeutic strategy, but its use is still limited to clinical trials. A phase II open-label clinical trial involving 18 CS patients showed that administration of oral mTOR-inhibitor (sirolimus) was tolerable in terms of its side effect profile, and was sufficient to down-regulate mTOR signaling in skin and gastrointestinal tissue . A doublsorders. ,12. BYL7PTEN, several germline PVs have been reported to exhibit the CS phenotype. The KLLN gene localizes to 10q23 and shares a transcription start site with PTEN. KLLN, a target gene of the tumor suppressor p53, is involved in cell cycle arrest and apoptosis. Germline hypermethylation in the promoter accounts for up to 35% of PTEN mutation-negative CS/CS-like patients, and is associated with increased prevalence of breast and renal cell carcinomas [Beyond rcinomas ,49.SDHA, SDHB, SDHC and SDHD genes, and is assembled in the mitochondria to form the mitochondrial complex 2, a key respiratory enzyme which links the Krebs cycle and the electron transport chain. Mutations in the SDHx genes are associated with a predisposition for developing hereditary pheochromocytoma and/or paraganglioma, GIST and Renal cell carcinoma [PTEN wild-type CS/CS-like patients had germline SDHB/D PVs, which were associated with an increased prevalence of thyroid cancer compared with germline PTEN PVs alone [The succinate dehydrogenase complex (SDHx) is a multipart enzyme made of four subunits encoded by arcinoma . ApproxiVs alone .SEC23B gene provides instructions for making one component of a large group of interacting proteins called coat protein complex II and regulates the transportation of proteins and lipids from the endoplasmic reticulum to the Golgi apparatus in cells [SEC23B variants were observed in three patients (3%) [The in cells . Previouin cells ,54,55. Ints (3%) .WWP1 variants were observed in five of 126 patients (4%) from a study population consisting of CS and CS-like affected patients with wild-type PTEN who had gastrointestinal oligopolyposis as a predominant phenotype [WWP1 (WW domain containing E3 ubiquitin protein ligase 1) is a member of the NEDD4-like family of HECT ubiquitin ligases, and plays important roles in a diverse variety of biochemical and cellular processes, such as protein degradation, transcriptional regulation, cell proliferation and differentiation, apoptosis, and senescence. Overexpression of WWP1 has been identified in a variety of cancers, and is associated with poor prognosis . A recenhenotype .PTEN mutations in 7% of patients, germline SDHx variants in 9.8%, and KLLN promoter hypermethylation in 10.5% of patients [SDHx, KLLN, AKT1, PIK3CA, SEC23B, and WWP1 as candidate genes for predisposition to CS, these genes have not yet been validated for use in the clinical setting. Identifying and accumulating data on susceptibility genes are important for patients to receive gene-specific genetic counseling, predictive testing of family members, and accurate risk assessment.A study evaluating 371 EC patients with CS or CS-like clinical picture found germline patients . AlthougPJS is a relatively rare disorder with an estimated incidence of about one in 25,000 to one in 280,000 births and is an autosomal dominant disorder characterized by melanocytic macules of the lips, buccal mucosa, and digits; multiple gastrointestinal hamartomatous polyps . The cliOvarian sex cord tumor with annular tubules (SCTATs) is one of the gynecological cancers associated with PJS. Although SCTATs are very rare neoplasms comprising less than 1% of sex cord ovarian tumors, it has been reported that 36% (27/74) of SCTAT is complicated by PJS . CompareAnother PJS-related gynecological cancer is minimal deviation adenocarcinoma (MDA), which occurs in the cervix. Although MDA is a very rare malignancy, accounting for less than 1% of uterine cervical cancer, the incidence of MDA in PJS patients is estimated to be 15–30%, while up to 10% of MDA cases are complicated by PJS . The meaSTK11 gene, also known as the LKB1, which is located on 19p13.3 and comprises nine coding exons and one noncoding exon, coding a member of the serine/threonine kinase family with 433 amino acids. STK11 is known as a tumor suppressor gene and is an essential serine/threonine kinase that regulates various cellular processes, including cell metabolism, cell polarity, and apoptosis. The catalytic kinase domain is highly conserved in STK11 protein, which is comprised of amino acid residues in 49–309 positions. Although 80–85% of individuals with PJS have a mutation detected by sequencing, the remaining 15–20% have large rearrangements, deletions and duplications [STK11 PVs have been identified in up to 80% of familial PJS cases; for the remaining patients, PJS likely resulted from de novo mutations [PJS is a rare autosomal dominant inherited disorder caused by a germline PV in the ications . Hence, utations . A smallutations ,74. LossSTK11 have been identified in many sporadic malignancies [STK11 gene , followed by amplification (as in sarcomas) [In the past few decades, somatic mutations of gnancies . The prearcomas) . STK11/Larcomas) , an effeSTK11-mutated pancreatic cancer experienced partial responses to mTOR inhibitor (everolimus) [STK11 mutations are also associated with T cell excluded tumors, which are characterized by low or no PD-L1, low T-cell densities, high levels of granulocyte colony stimulating factor and IL-8 family cytokines, high density of neutrophil-like cells, and production of myeloid cell-recruiting chemokines such as IL-6 [STK11 alteration is associated with poor response to immune checkpoint inhibitors (ICIs) in patients with non-small cell lung cancer, including those with tumors harboring co-occurring KRAS mutation [Several approaches have been proposed to target STK11-deficient tumors based on preclinical observations. A case study reported that a PJS patient with rolimus) . Howeverrolimus) . Tumors  as IL-6 ,81,82. Smutation ,84.STK11 to date, several ongoing clinical trials are being conducted. STK11 is involved in DNA damage response, promoting homologous recombination and fostering genomic stability by interacting with BRCA1 [Although there have been few effective treatments targeting th BRCA1 . Two clith BRCA1 . STK11 hth BRCA1 . A cliniDICER1 PV, an important component of the microRNA processing pathway [DICER1 have been reported to be found in about 70% of PPB cases [DICER1 mutation carriers, including cystic nephroma, rhabdomyosarcoma, multinodular goiter and thyroid carcinoma, which often develops in childhood [DICER1 PVs  than in family controls  [DICER1 syndrome is an autosomal dominant manner caused by a germline  pathway . The hal pathway ,88,89. APB cases ,91. Asidhildhood ,88,89. E43, 12%) .DICER1 [DICER1 variants were more likely to exhibit clinical relapse, although this tendency was not statistically significant [Sertoli-Leydig cell tumor (SLCT), a rare ovarian tumor that belongs to the group of sex cord stromal tumors, accounts for less than 0.5% of ovarian tumors, and is the second most frequent DICER1-related tumor after PPB. Approximately 60% of SLCTs occur against a background of PVs in the germline of DICER1 . CompareDICER1 ,95. De PDICER1 , which iDICER1 ,98. ProgDICER1 . A studynificant .DICER1 somatic alterations are identified in 65–95% of uterine embryonal rhabdomyosarcomas, with up to 50% of these patients harboring germline alterations [DICER1 hot-spot mutation is the key-driving event in a subset of gynandroblastomas containing components of SLCT and juvenile granulosa cell tumor [DICER1 germline PVs has been reported to be 16 years old [Embryonal rhabdomyosarcoma of the uterine cervix incidence is a very rare form of cancer that develops in childhood, and is reported to account for about 0.4–1% of all cervical malignancies . DICER1 erations ,103. Gynll tumor ,105. Theears old .DICER1 PVs, approximately 80% of the germline PVs were inherited from a parent, with the remaining 20% were de novo [DICER1 include deletions, duplications, insertions, transitions, or transversions, as well as point mutations [DICER1 variants were also reported to be associated with DICER1 syndrome in patients with severe phenotypes [DICER1 PV in 2% of the allelic reads in the patient’s blood sample [DICER1 PV is estimated to occur in the general population in approximately one in 10,600 [The DICER1 gene, located on chromosome 14q32.13, has 27 exons and encodes the endoribonuclease Dicer protein of the ribonuclease III family. DICER1, as RNase III, is known to be involved in the regulation of post-transcriptional gene expression through the production of microRNAs, as well as in the generation of siRNAs that regulate gene expression. In individuals with PPB with a detectable germline  de novo ,107. Gerutations . The majutations . In a prenotypes . Germlind sample . Despiten 10,600 . A recenn 10,600 .Tumors that develop as DICER1 syndrome typically have a germline nonsense or frameshift PV in one allele, and a somatic missense mutation in the other allele. Since RNase IIIa cleaves the 3p hairpin arm and RNase IIIb cleaves the 5p hairpin arm, the DICER1 cancer hotspot mutant has a selective defect in processing the miRNA-5p strand, resulting in an overall decrease in the 5p/3p ratios. Somatic mutations found in tumors of DICER1 syndrome affect five “hot spot” codons, encoding critical amino acids in the metal-binding catalytic cleft of the RNase IIIb domain: E1705, D1709, G1809, D1810, and E1813 . These mDICER1 mutations, those with a single functional DICER1 allele may benefit from metformin or similar compounds [Some studies have demonstrated that metformin can upregulate DICER1 in mouse and human cells ,16,17. Aompounds . A DICERompounds . Recentlompounds .SMARCB1 gene have RTPS1, whereas those with a germline variant in SMARCA4 have RTPS2. The majority of RTs show alterations that result in loss of SMARCB1 function, and a small proportion of RTs carry inactivated SMARCA4 [SMARCB1 and 5–15% for SMARCA4, respectively [RTPS are rare, aggressive malignancies, typically diagnosed in infants and children aged younger than 3 years. The origins of Rhabdoid tumors (RTs) are the central nervous system (65%), kidneys (9%), and, in the remaining 26% of cases, the soft tissues of the head and neck, paravertebral muscles, liver, bladder, mediastinum, retroperitoneum and pelvis . RTPS in SMARCA4 . The perectively .SMARCA4 deficient (RTPS2). One of the most common cancers in RTPS2 is small-cell carcinoma of the ovary, hypercalcemic type (SCCOHT), which is a rare and aggressive cancer that mainly occurs in adolescent and young women. This tumor has a poor prognosis, with overall survival of less than two years in most cases [SMARCA4 PVs were diagnosed at a significantly younger median age (21.5 years) than the 34 non-carriers  [SMARCA4 [Patients with RTPS associated with gynecologic cancers are typically st cases . Sequencst cases ,124. To st cases . In a st = 0.02) . Familia = 0.02) ,128. It [SMARCA4 , so the [SMARCA4 .SMARCA4 gene is located in the chromosome 19p13.2, and encodes 1647 amino acids, a 185kDa catalytic subunit of switch/sucrose-non-fermenting (SWI/SNF). The SWI/SNF complex is an evolutionarily conserved ATP-dependent chromatin remodeling complex that plays important roles in DNA repair, transcriptional activation of genes normally repressed by chromatin, differentiation, and organ development, and has been shown to behave as a tumor suppressing complex [The  complex . Each SW complex ,133.SMARCA4 and SMARCB genes is known as Coffin-Siris syndrome (CSS). It is a congenital multi-systemic genetic disorder characterized by intellectual disability, coarse facial features, hypoplasia or absence of the fifth fingernail or toenail, and multiple other abnormalities, including ocular features [SMARCB1 and SMARCA4 were involved in the CSS phenotype [SMARCB1  and SMARCA4  were also reported to be identified among 208 CSS patients [Germline variants in subunits of the BRG1/BRM-associated factor (BAF) or SWI/SNF chromatin reassembly complex have been revealed in benign and malignant tumors and neurodevelopmental disorders . A rare features . Mutatiofeatures . Some sthenotype ,138. Gerpatients .SMARCA4 genomic alterations and loss of SMARCA4 expression have been observed in some tumors [Recent sequencing studies have identified somatic mutations in subunits of the SWI/SNF chromatin remodeling complexes in over 20% of human cancers . In parte tumors . Some ree tumors ,141,142.Although little is known about the efficacy of ICIs for SMARCA4-alterated cancers, some case reports for patients with relapsed SCCOHT, SMARCA4-deficient thoracic sarcoma and SMACA4-altered non-small cell lung cancer have been published ,20,21. FDysfunction of the SWI/SNF complex, due to loss of SMARCA4, leads to oncogenic dependence on EZH2 through transcriptional repression caused by aberrant trimethylated lysine 27 of histone H3. A subset of SMARCA4-deficient tumors was found to be sensitive to EZH2 inhibition, which serves as the catalytic subunit of polycomb repressive complex 2 . The onlLysine-specific histone demethylase 1 (LSD1) regulates the chromatin landscape and gene expression by demethylating proteins such as histone H3, and is highly expressed in SWI/SNF-mutated tumors in the TCGA database. Soldi et al. showed that SWI/SNF-deficient ovarian cancer appears to be dependent on LSD1 activity, because SCCOHT and ovarian clear cell carcinoma cell lines are sensitive to SP-2577 (Seclidemstat), a reversible LSD1 inhibitor . A phaseXue et al. found that inactivation of SMARCA4 causes significant downregulation of cyclin D1, limits CDK4/6 kinase activity in SCCOHT cells, and results in sensitivity to CDK4/6 inhibitors in vitro and in vivo . They alBET inhibitors bind to the bromodomains of the bromodomain and extra-terminal motif (BET) proteins BRD2, BRD3, BRD4, and BRDT, and act to prevent protein–protein interaction between BET proteins and acetylated histones and transcription factors. SCCOHT cells were highly sensitive to BET inhibitors such as JQ1 and OTX-015, and the latter in particular showed strong antitumor activity in an orthotopic xenograft model of SCCOHT . A recenHistone deacetylases (HDACs) are expressed at increased levels in cells of various malignancies, and HDACs inhibitors have shown clinical benefits in hematological malignancies but failed in solid tumors due to the lack of biomarker-driven stratification . AlthougSWI/SNF components, including SMARCA4, have also been implicated in DNA-damage repair . SMARCA4Aurora kinase A (AURKA) is a serine/threonine kinase involved in a number of central biological processes, such as the G2/M transition, mitotic spindle assembly, and DNA replication. Since AURKA activity has been identified as essential for survival and proliferation in non-small cell lung cancer cells lacking SMARCA4, AURKA inhibitors may provide a therapeutic strategy for biomarker-driven clinical studies to treat the non-small cell lung cancers harboring SMARCA4-inactivating mutations .Four rare hereditary gynecological cancer syndromes have been described in the review. The challenges faced by people at high risk of certain cancers include the development and validation of better methods for cancer detection and prevention. An understanding of the genetic causes and molecular pathways of hereditary cancer syndromes can inform our knowledge and potential new treatment of various types of cancers.