This study examines the possibilities of applying numerical and physical modeling to the elaboration of technology and design of tools used in the hot forging of needle rails for railroad turnouts. First, a numerical model of a three-stage process for forging a needle from lead was built in order to develop a proper geometry of the tools’ working impressions for physical modeling. Based on preliminary results of the force parameters, a decision was made to verify the numerical modeling at 1:4 scale due to forging force values as well as agreement of the numerical and physical modeling results, which was confirmed by the similar courses of forging forces and a comparison of the 3D scan image of the forged lead rail with the CAD model obtained from FEM. The final stage of our research was modeling an industrial forging process in order to determine the preliminary assumptions of this newly developed method of precision forging using a hydraulic press as well as preparing tools to reforge a needle rail from the target material, i.e., 350HT steel with a 60E1A6 profile to the 60E1 profile used in railroad turnouts. Needle rails are railway infrastructure elements that are fixed in pairs in turnouts so that the train’s track or travel direction can be changed . On one Currently, the processes of needle rail reforging are realized mainly through semi-closed die forging or die forging, as a result of which the obtained reforged parts of the needle rail are characterized by improper geometry, causing the necessity of large-scale mechanical treatment, as well as insufficient hardness and low surface quality. For this reason, the technologies realized so far require improvement and optimization, as they necessitate the production of forgings with big allowances for mechanical treatment on all surfaces of the rail c. This mThe correctness of all die forging technology is dependent on many, often opposing, factors as well as their interaction, which depend mainly on the technology itself and the operation conditions, the type of the forging and its material, and also, often, on the human factor ,10. All The process of forging turnout needle rails consists in preliminary heating of a fragment (about 1 m) of the rail to over 1200 °C, and next reforging the end of the needle rail, on a slightly shorter distance than the heated one, into a track rail. At present, the realized forging processes are conducted usually in a few semi-closed die forging impressions, and sometimes, the last operation is one of die forging ,32. In m-Numerical modeling with the use of the Forge 3.0 Nxt calculation package for the process of reforging a rail section for the model material (lead), not for the target material, i.e., steel R350HT. The simulations were performed for such a material because the following step of research was verification of the obtained results based on physical modeling.The aim of this study is the conceptual development of needle rail reforging tools that will make it possible to realize precision forging technology under industrial conditions with the use of mainly physical and numerical modeling as well as other scientific and measurement methods. In order to reach the assumed objective, the following research studies were conducted, including 3 main stages:The calculations were made at 1:4 scale as the preliminary FEM simulation results for Pb indicated that, in the case of the real size (dimensions) of the tools, the forces would exceed the maximal pressure of a semi-industrial hydraulic press, 200 t;-Physical modeling of the process of forging a needle rail from lead in order to verify the virtual forging process . A justification of performing physical modeling at a reduced scale is the facility and relatively low costs of such an experiment, as well as the fact that the large mass of forging  would cause problems with transport and the manipulation and removal of tools;-Analysis of the obtained results, numerical modeling of the industrial forging process and elaboration of the target tools  used to forge needle rails from R350 HT pearlitic steel.2 K, thermal conductivity with the environment—15 W/m2 K. In addition, with the known parameters of the target forging aggregate, a hydraulic press with a pressure of 3000 t and a maximal movement speed of 30 mm/min, as well as the preliminary assumptions of the elaborated technology, for the target process, it was assumed that the mean deformation rate would be at the level of 1 s−1. In the modeling process, the assumed press speed was at the level of 22 mm/s. The flow curves for lead determined in a trial of upsetting a cylinder  on an Instron testing machine were introduced into the calculation program in a tabular form  to cast a 60E1A6 type needle rail a.The first trials of casting Pb into the mold did not give the desired results b, as theTo verify the physical modeling, we compared a 3D scan of the reforged needle rail made of Pb to the nominal CAD model, which is shown in As we can observe based on the 3D scanning results, the obtained geometry from the laboratorial process is in good agreement with the numerical modeling, and errors of shape, even in extreme areas, do not exceed 1 mm.The following stage of verification of the correctness of the FEM calculations was a comparison of the forging forces determined from the numerical modeling with the forces measured during each of the 3 operations of forming a 60E1A6 rail from lead a–c.We can notice that the results calculated numerically are similar to the real values resulting from the measurements . Additionally, for a selected sample, for each of the 3 operations from physical modeling, an equation was formulated and the error with respect to the forces obtained from FEM was determined. As we can see, for each case, the difference is not more than 8%. This allows us to state that the elaborated numerical model for forging a lead rail is correct. To verify, let us assume yield stresses for Pb at the levels of 28 MPa and 158 MPa for R350HT steel formed in the last operation with a forging temperature of about 900 °C. For them we obtain scale factor C at the level of 158:28 = 5.6. Next, if we assume that the maximal force in the process occurs in the last operation, III, then for the forming of Pb, according to the diagram, it is at the level of 108 t. Therefore, by multiplying 108 × 5.8 and considering the scale factor of ¼, we obtain 108 × 5.6 × 4 = 2419 t, that is, theoretically, such a value of force should be expected in the last operation of needle rail forging in the industrial process for R350HT steel. Therefore, this confirms the maximal load of the selected hydraulic press with a pressure of 3000 t, and the obtained results can be used to develop a proper model for the target forging process for 350HT steel. The correctness of physical modeling using the geometric scale of enlarging/reducing the physical model to the real one was demonstrated in . However2 K, thermal conductivity with the environment—10 W/m2 K. The strength properties of the forging material—the flow curves of R350HT steel  for the process of physical modeling from Pb into the real tool dimensions, which were imported into the FEM program. Next, a numerical model was built with the following assumed initial-boundary conditions: initial temperature of the reforged rail section—1280 °C, tool temperature—280 °C, ambient temperature—30 °C, press speed—22 mm/s, friction coefficient according to the Coulomb model—0.15, thermal conductivity in contact—10 kW/mHT steel a—were inHT steel a.The rail reforging simulations were performed for a process that was ultimately planned to be realized under industrial conditions on a hydraulic press with maximal pressure force of 3000 t. The assumed rail reforging length was at the level of 240 mm, with a 60 mm allowance to be cut off; the total reforging length was 300 mm. It was also assumed that the anvils supporting the die inserts would be 600 mm long b, and soThe constructed numerical model before the forming of the rail and in the last stage of operation I is presented in In turn, For a more thorough analysis, An analysis was also performed of the temperature field distributions in the forging after the consecutive operations . Figure In the case of operation II b, the teWe can notice that the allowances for the trimming treatment are in the correct areas, the shape and dimensions of the rails correlate quite well, and the determined deviations are at the level of 0–1.5 mm, that is, they are within the assumed tolerance field.As we can notice, the maximal force needed to reforge this rail equals about 2400 t and is lower than the maximal pressure force of the press by about 600 t; therefore, we can assume that, in the case of technological trials under industrial conditions on the target hydraulic press with a pressure of 3000 t, there should be no problem with reforging and obtaining a proper forging. The obtained force values simultaneously confirm the results of physical modeling with the use of Pb and the recalculation of the forces from this experiment through the calculated scale factor C. At the same time, we should note that the simulation of forming a needle rail was based on the predetermined initial boundary conditions  and assumptions  that, in the case of a real experiment, can differ. However, an allowance of over 600 t, even in the case of increased friction forces, should be sufficient.On this basis, it was stated that, based on the performed simulation tests confirmed with physical modeling and numerical simulations for the target process, we can make real metal tools.We assumed that the tools, together with the remaining elements of forging instrumentation, would be made from tool steel for hot WLNV operations through milling and electrical machining. Currently, works are being performed connected with assembling the whole tool set and fixing the hydraulic press in the table .This study presents the results of numerical and physical modeling that made it possible to build forging tools as well as develop guidelines for the created technology of hot precision forging needle rails used in railroad turnouts from profile 60E1A6 to 60E1. The first stage of investigations was the construction of a numerical FEM model as well as simulations of the process of forging a needle rail from lead in order to elaborate the preliminary geometry of the working impressions of the tools for physical modeling.-The preliminary numerical simulation results for the process of forging from Pb referring to the force parameters demonstrate that verification of the numerical modeling would be conducted at 1:4 scale due to the obtained high forging force values  as well as the availability of a semi-industrial hydraulic press with a nominal pressure of 200 t. The obtained results of numerical and physical modeling for a Pb rail are in good agreement, which is confirmed by the similar course of forging forces as well as their values in the case of the maximal forging force in numerical modeling ;-A similar comparison of the 3D scan images of the rail forged from lead to the CAD model obtained from FEM shows a good agreement, as the geometrical deviations are within the assumed tolerance of 0–0.15 mm. At the same time, we should emphasize that both in numerical modeling and physical modeling, certain simplifications were made, those being that the main assumed criteria were the mean deformation rate and temperature in the numerical modeling of the target process. Nevertheless, such an approach is justified in order to minimize the costs of the industrial experiment and with undertake attempts at developing the target technology under industrial conditions with the greatest probability of success;-The final stage of research was the construction of a numerical model of the industrial forging process based on the physical modeling results, in particular re-scaling the geometry of the working impressions of the tools used to forge Pb. The obtained numerical simulation results for the target forging process, after recalculation of the forging forces from the physical modeling based on the similarity condition in the plastic scope and consideration of the 1:4 scale, demonstrate a good agreement in terms of forging force values;-At present, works investigating mounting the tools in the holders on the press and selecting the optimal settings and technological parameters are being conducted, after which technological forging trials under industrial conditions will be realized. However, it is assumed that there might be a need for small process modifications and improvement of the technological parameters, e.g., adjusting the working temperature of the dies as well as the parameters and manner of lubrication, which of course is inevitable in the case of activating new forging processes;-The results of the conducted research can serve in the future towards developing similar technologies for precision forging railroad turnout needle rails not only for the commonly used pearlitic steels, but also bainitic steels used for high-speed trains as well as other special applications.


RAD21 subunits of cohesin. SGO1 shares this binding interface with CTCF, indicating that these distinct chromosomal regulators control cohesin through a universal principle. This interaction is essential for the localization of SGO1 to centromeres and protects centromeric cohesin against WAPL-mediated cohesin release. SGO1–cohesin binding is maintained until the formation of microtubule–kinetochore attachments and is required for faithful chromosome segregation and the maintenance of a stable karyotype.In the early stages of mitosis, cohesin is released from chromosome arms but not from centromeres. The protection of centromeric cohesin by SGO1 maintains the sister chromatid cohesion that resists the pulling forces of microtubules until all chromosomes are attached in a bipolar manner to the mitotic spindle. Here we present the X-ray crystal structure of a segment of human SGO1 bound to a conserved surface of the cohesin complex. SGO1 binds to a composite interface formed by the SA2 and SCC1 Here, the authors determine the structural basis of how SGO1 protects centromeric cohesin from WAPL-mediated release until the kinetochores and the microtubules have formed functional attachments. In addition, they show that SGO1 and CTCF bind cohesin in a similar manner, suggesting a potential universal mechanism of cohesin control. Cohesin entraps DNA inside its ring-shaped structure4, which at its core consists of SMC1, SMC3 and SCC1 . SCC1 is bound by either of two paralogous HEAT repeat-containing proteins, SA1 or SA2 5.During mitosis, the duplicated genome needs to be accurately distributed over the two daughter cells. The cohesin protein complex holds together the sister DNAs from replication until mitosis6. During mitosis, cohesin is removed from chromosomes in two waves. First, cohesin is removed from chromosome arms in a WAPL-dependent manner through a process known as the prophase pathway10. Cohesion at centromeres is protected by Shugoshin (SGO1)14, giving rise to the typical X-shaped structure of human chromosomes. SGO1 protects centromeric cohesin by recruiting PP2A to counteract cohesin phosphorylation by mitotic kinases, and SGO1 also directly competes with WAPL for cohesin binding18. Centromeric cohesion is maintained until proper attachment of microtubules to the kinetochores, upon which the remaining cohesin is cleaved by separase to trigger anaphase onset19. By protecting centromeric cohesion, SGO1 thus ensures faithful chromosome segregation.Cohesin complexes have a dynamic mode of DNA binding that involves DNA entrapment and release. From DNA replication until mitosis, the cohesin complexes that hold together the sister DNAs are locked on DNA to render cohesin resistant to cohesin’s release factor WAPL20. We showed recently that CTCF binds to cohesin through a conserved YxF motif in the amino terminus of CTCF. This CTCF segment interacts directly with a composite binding interface formed by the SA2 and SCC1 subunits of cohesin21. The SA2 interface is conserved from fungi to mammals and is known as the conserved essential surface (CES)22. For simplicity, we refer to the composite SA2–SCC1 binding pocket as the CES. The direct interaction of the YxF motif of CTCF with the CES is required for formation of CTCF-anchored loops at TAD boundaries21. It has also been suggested that the CES region interacts directly with both SGO1 and WAPL18. The interaction of SGO1 with cohesin is promoted by phosphorylation of SGO1 T346. However, this phosphorylation does not control the direct interaction between the CES and SGO1 (refs. 18). SGO1 contains a YxF motif close to this phosphorylation site that could be key to this interaction. In agreement, previous biochemical experiments have shown that an SGO1 fragment containing the YxF motif can directly compete with CTCF for binding to the CES21. If so, cohesin complexes may be controlled through a shared mechanism, irrespective of whether these complexes build DNA loops or rather hold together the sister DNAs.Cohesin has a dual role, as it not only holds together sister DNAs but also builds the DNA loops that shape the interphase genome. To control this latter function, cohesin is bound by the architectural factor CTCFWe report here the X-ray crystal structure and AlphaFold model of the cohesin subcomplex SA2–SCC1 bound to a fragment of SGO1 Fig. . We demo18. The interacting region contains a YxF motif that is conserved in vertebrate SGO1 proteins , enhances the interaction with SA2–SCC1 but is not essential for binding18. We were able to obtain crystals with a SGO1 peptide spanning amino acids 331–341 containing the YxF motif but not with an extended peptide spanning amino acids 331–349 containing pT346. We determined the X-ray structure by molecular replacement to a minimum Bragg spacing of 3.2 Å  experiments showed that the T346-phosphorylated SGO1 fragment spanning amino acids 331–349 bound SA2–SCC1 with a lower equilibrium dissociation constant (2.3 ± 0.4 μM) compared with a nonphosphorylated SGO1 peptide spanning amino acids 331–341 (13.5 μM ± 1.4) Fig. . Using gY335A F337A as their sole copy of SGO1 . As expected, this SGO1 depletion yielded a massive cohesion defect, but this defect was no stronger than the defect observed in the SGO1Y335A F337A cells. SGO1 depletion in SGO1Y335A F337A cells also barely if at all worsened the cohesion defect of these cells -tagged SGO1 that was either wild type or harbored the SGO1Y335A F337A mutation. We then scored for SGO1 localization by immunofluorescence chromosome spreads, comparing the absence versus the presence of microtubule–kinetochore attachments, using nocodazole- or MG132-treated cells, respectively. In nocodazole-treated cells, wild-type SGO1–GFP localized to the inner centromere as expected. The SGO1Y335A F337A mutant, however, did not localize to the inner centromere and was primarily found at the kinetochores . Here, SGO1 through CES binding protects centromeric cohesin from the WAPL-dependent prophase pathway. Upon establishment of bipolar microtubule attachment, SGO1 then relocates towards kinetochores25 , STAG1W337A (5′-AGTACTGAGACAAACATAACTTCCATCAAAGCTTAGAACAG AGTAACTTACCCTGTCGTGAAGAGTAGCGCCAACATATTTTAGGTAACTGTCATTTAGGAAGGCATCACTATACATTTTCATC-3′) and STAG2W334A (5′-CTTAATGACAGTTATTTAAAATATGTTGGTGCGACTATGCATGATAAGGTAAGATGTGCCCTTCAGACTGCTTCTTTCTATACATCGGCGTGGCTGTCTGCACCTCTCATTCATGAG-3′). We cotransfected pBabePuro at a ratio of 1:10 to the pX330 plasmid. Cells were treated with 2 μg μl−1 puromycin for 2 days for selection. Colonies were picked, genomic DNA of clones was isolated and mutations were validated by Sanger sequencing.HAP1 cells were cultured in Iscove’s modified Dulbecco’s medium (Invitrogen), containing 10% fetal bovine serum (Clontech), 1% UltraGlutamin (Lonza) and 1% penicillin–streptomycin (Invitrogen). Mutant cells were generated by CRISPR–Cas9 technology. Guide RNAs targeting exon 6 of SGO1 , exon 10 of STAG1 (5′- TTGGCTGGACTCTTCATGAC-3′) and exon 11 of STAG2 (5′-GACAGTTATTTAAAATATGT-3′) were annealed into pX330. To mutate the locus of interest, we cotransfected a 100–120 base pair repair oligonucleotide with the desired mutation as well as a silent mutation: for SGO1YAll siRNAs were manufactured by Dharmacon (ON-TARGETplus). For SGO1 and luciferase we used SMARTpools, and for WAPL we used the following sequence: 5′-CAACAGUGAAUCGAGUAAUU-3′. Transfection was performed with 20 μM per siRNA final concentration, using Invitrogen RNAiMAX (Life Technologies), following the manufacturer’s instructions.31. Images were randomized by a homemade ImageJ macro and then visually assigned their corresponding phenotype. A parametric two-tailed t test was used to compare the scoring of cohesion phenotypes.Cells were transfected with the corresponding siRNAs, and after 2 days the cells were treated with nocodazole as described previously31. For immunofluorescence spreads, cells were treated with the corresponding siRNA. After 24 h, cells were transfected using FuGENE transfection reagent (Promega) with 0.8 μg SGO1–GFP plasmid (kindly provided by S. Lens) or a SGO1Y335A F337A–GFP mutant plasmid. One day later, cells were treated with nocodazole for 1.5 h or with MG132 for 2 h, and mitotic cells were collected by shake-off. Cells were washed once in phosphate-buffered saline, followed by a quick spin onto microscope slides with a Shandon Cytospin centrifuge. Cells were extracted with PBS containing 0.3% Triton-X for 5 min and fixed in 4% paraformaldehyde for 15 min. The coverslips were washed three times with PBS containing 0.1% Triton-X before being incubated with antibodies at a 1:1000 dilution in PBS containing 3% BSA and 0.1% Triton-X overnight at 4 °C. Secondary antibody incubations were performed by incubation at room temperature for 1 h with DAPI in PBS containing 3% BSA and 0.1% Triton-X. Coverslips were mounted in Prolong Gold (Invitrogen).For immunofluorescence, cells were treated with nocodazole, fixed and stained as described previouslyImages were obtained using a DeltaVision deconvolution microscope (Applied Precision), and images were acquired using Softworx (Applied Precision) and ImageJ. To establish levels of SGO1 in prometaphase cells, we used an ImageJ macro that allowed us to calculate the level of SGO1 relative to CENPA. To identify the location of SGO1–GFP in mitotic cells, we first blinded the channel corresponding to GFP to prevent bias towards a phenotype. Next, we drew a straight line on four random chromosomes that showed two distinct centromeres and obtained the plot profile of both CENPA and GFP for each location.Cells were grown on glass-bottomed dishes (LabTek). To visualize the DNA, 2 h before imaging, a SiR-DNA probe  was added. Images were taken using a DeltaVision deconvolution microscope (Applied Precision). Cells were imaged every 5 min using a ×40 air objective with 4 × 2.5 μm Z stacks. Images were acquired using Softworx (Applied Precision) and ImageJ.Prometaphase samples cells were obtained as described above. Fixed cells were dropped on cover slides and then dried. We added probes against the centromere of chromosome 8  and shielded the cells with a coverslip and rubber cement. The slides were incubated for 2 min at 75 °C, followed by overnight incubation at 37 °C. The cells were washed with 0.4× SSC at 72 °C for 2 min, followed by washing at room temperature with 2× SSC, 0.05% Tween-20, for 30 s. The slides were washed with water and stained with DAPI, followed by mounting with Prolong Gold (Invitrogen).G2 samples were collected by treating the cells for 18 h with RO-3306. We verified that the cells were synchronized in G2 by incubation in Nicoletti buffer followed by flow cytometry (BD LSRFortessa). Plots were generated with FlowJo (v.10). G2-synchronized cells were spun down and resuspended with fixative solution , followed by the same protocol as described above.32.Images were taken using a DeltaVision deconvolution microscope (Applied Precision), and images were acquired using Softworx (Applied Precision) and ImageJ. The fluorescence signal was categorized as singlet  or doublet , as described previously33.Immunoblot and coimmunoprecipitation were performed as previously describedThe following antibodies were used as primary antibodies for immunofluorescence microscopy: SGO1 , GFP  and CENPA . For immunoblotting, the following primary antibodies were used: SA1 , SA2 , SMC1 , SCC1 , WAPL , Sororin , HSP90 (sc-13119(F-8), Santa Cruz) and α-tubulin . All primary antibodies were used at a 1:1000 dilution with the exception of HSP90 and α-tubulin (1:10000). For coimmunoprecipitation, we used 4.5 μg of SMC1  or IgG  per sample. Secondary antibodies were used at a 1:1000 dilution. For immunofluorescence microscopy we used: Alexa Fluor 488 goat anti-mouse , Alexa Fluor 568 goat anti-mouse , Alexa Fluor 488 goat anti-rabbit  and Alexa Fluor 568 goat anti-rabbit . For western blots, we used the following secondary antibodies: anti-goat-PO , anti-rabbit-PO  and anti-mouse-PO .21. Expression and purification were done as described previously21. SGO1 constructs were cloned into the BamHI and NotI sites of pGEX-6P1. Mutagenesis was done using a Q5 Site-Directed Mutagenesis Kit (New England Biolabs). All proteins were expressed in Escherichia coli BL21 (DE3) by autoinduction, and purification was done as described previously21.SA2 amino acid residues 80–1060 were expressed as a GST fusion protein and SCC1 amino acid residues 281–420 as an N-terminally 6×His-tagged fragment as described previously21. Crystals were soaked for 7 days with a 500 μM peptide solution including SGO1 amino acid residues 331–341 SNDAYNFNLEE. Crystals were cryoprotected as described previously21. Diffraction data were collected at 100 K at an X-ray wavelength of 0.9687 Å at beamline ID30A-1/MASSIF-1 (ref. 34) of the European Synchrotron Radiation Facility, with a Pilatus3 2M detector, using automatic protocols for the location and optimal centering of crystals35.Crystallization of the SA2–SCC1 complex was done as described previously36 and imported into CCP4 format using AIMLESS37. The structure was determined by molecular replacement using Phaser (Phenix 1.14-3260)38. A final model was produced by iterative rounds of manual model-building in Coot (COOT 0.8.0-3)39 and refinement using PHENIX (1.14-3260)40. The SA2–SCC1–SGO1 model was refined to a resolution of 3.2 Å with Rwork and Rfree values of 25% and 28%, respectively . The computational model shown in Fig. 42. The computational model shown in Fig. Data were processed with XDSr Phenix .14-3260321 with small modifications. Briefly, 50 μM GST-tagged SGO1 constructs were mixed in 50 μl buffer 1  containing 25 μl of a 50% slurry of GST Sepharose beads (Cytiva) per reaction. GST beads were incubated for 1 h at 4 °C, followed by four washes with 500 μl of buffer 1. Then, 2.5 μM of SA2–SCC1 was added, followed by overnight incubation at 4 °C. A 25-μl volume of the reaction was withdrawn as the reaction input, and the remainder was washed five times with 500 μl of buffer 1. Samples were boiled in 1× sodium dodecyl sulfate (SDS) sample loading buffer (New England Biolabs) for 5 min to obtain the bound fraction, followed by SDS polyacrylamide gel electrophoresis analysis. ITC was performed as described previously21. ITC data were analyzed with Origin 7.0.GST pulldowns were done as described previouslyt test analyses, with a probability threshold of P = 0.05. GST pulldowns were repeated at least three times with consistency.No statistical method was used to predetermine the sample size. No data were excluded from the analyses. All experiments with phenotype calling were randomized, and the SGO1 signal was blinded in all experiments for SGO1 localization with respect to the centromere. Data were visualized with Prism 9. For all pairwise comparisons, we performed Further information on research design is available in the Any methods, additional references, Nature Portfolio reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41594-023-00968-y.Supplementary InformationSupplementary Fig. 1 FACS gating strategy.Reporting Summary


We show that knockout of SAMHD1 enhanced phosphorylation of IKKα, IKKβ, and IKKγ in THP-1 cells treated with the NF-κB activator lipopolysaccharide or infected with Sendai virus and SAMHD1 reconstitution inhibited phosphorylation of IKKα/β/γ in Sendai virus–infected THP-1 cells. We demonstrate that endogenous SAMHD1 interacted with IKKα and IKKβ in THP-1 cells and recombinant SAMHD1 bound to purified IKKα or IKKβ directly in vitro. Mapping of these protein interactions showed that the HD domain of SAMHD1 interacts with both IKKα and IKKβ and that the kinase domain of IKKα and the ubiquitin-like domain of IKKβ are required for their interactions with SAMHD1, respectively. Moreover, we found that SAMHD1 disrupts the interaction between upstream kinase TAK1 and IKKα or IKKβ. Our findings identify a new regulatory mechanism by which SAMHD1 inhibits phosphorylation of IκBα and NF-κB activation.Sterile alpha motif and histidine-aspartate (HD) domain–containing protein 1 (SAMHD1) inhibits HIV-1 replication in nondividing cells by reducing the intracellular dNTP pool. SAMHD1 also suppresses NF-κB activation induced by inflammatory stimuli and viral infections. Specifically, SAMHD1-mediated reduction of NF-κB inhibitory protein (IκBα) phosphorylation is important for the suppression of NF-κB activation. However, while the inhibitors of NF-κB kinase subunit alpha and beta (IKKα and IKKβ) regulate IκBα phosphorylation, the mechanism by which SAMHD1 regulates phosphorylation of IκBα remains unclear. Here, we report that SAMHD1 suppresses phosphorylation of IKKα/β/γ  SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase (dNTPase) that reduces the concentration of intracellular dNTPs . SAMHD1 NF-κB is activated through two distinct signaling pathways defined as canonical and noncanonical . In the The IKK complex consists of two catalytic subunits IKKα/IKKβ and regulatory subunit IKKγ . Both IKKα and IKKβ contain an N-terminal kinase domain (KD), a ubiquitin-like domain (ULD), a scaffold dimerization domain (SDD), and a C-terminal NEMO-binding domain , 20, 21.Our previous study showed that SAMHD1 interacts with NF-κB1/p50 and NF-κB2/p52 and reduces phosphorylation of IκBα, thereby inhibiting NF-κB activation . We alsoIn this study, we found that SAMHD1 inhibited IKKα- and IKKβ-mediated NF-κB activation in cells upon viral infection or inflammatory stimulation. SAMHD1 negatively affected phosphorylation of IKKα, IKKβ, and IKKγ in THP-1 cells treated with lipopolysaccharide (LPS) or infected with Sendai virus (SeV). We demonstrated that SAMHD1 interacted with IKKα and IKKβ in THP-1 cells and HEK293T cells. Furthermore, we identified that the HD domain of SAMHD1, the KD of IKKα, and the ULD of IKKβ were involved in the interaction between SAMHD1 and IKKα or IKKβ. Furthermore, we observed that SAMHD1 overexpression disrupted the interaction between TAK1 and IKKα or IKKβ. Overall, our findings reveal a new regulatory mechanism by which SAMHD1 inhibits NF-κB activation during viral infection and inflammation.A), IKKβ , or both IKKα and IKKβ . When HEK293T cells overexpressed IKKα, IKKβ, or both, a significant increase in luciferase expression from the NF-κB reporter was observed . Treatment of cells with the inflammatory cytokine TNF-α also significantly induces NF-κB activation . SAMHD1 inhibited IKKα- or IKKβ-mediated NF-κB activation in a dose-dependent manner in the absence or presence of TNF-α (A and B). When IKKα and IKKβ were coexpressed in HEK293T cells, we observed synergistic enhancement of the NF-κB activity as expected, which was not further promoted by TNF-α treatment , likely due to saturated NF-κB activation. Interestingly, SAMHD1 inhibited both IKKα- and IKKβ-mediated NF-κB activation in a dose-dependent manner regardless of TNF-α treatment . These results indicate that exogenous SAMHD1 suppresses IKKα- and IKKβ-mediated NF-κB activation in HEK293T cells.We previously reported that SAMHD1 suppresses NF-κB activation by interacting with p50/p52 and by reducing phosphorylation of IκBα . HoweverFig. 1tivation . Indeed,A and B). Because the phosphor-specific antibody detected both IKKα and IKKβ phosphorylation (p-IKKα/β), we labeled and quantified p-IKKα/β together (A–D). After 1 h of LPS treatment, the phosphorylation of IKKα/β and IKKγ gradually decreases to baseline levels. We found that the level of p-IKKα/β and phosphorylation of IKKγ (p-IKKγ) was higher in THP-1 SAMHD1 KO cells compared with the THP-1 control cells (A–D). Other NF-κB activators, TNF-α and interleukin 1 beta (IL-1β), were also tested and showed similar results (We previously reported that LPS-induced phosphorylation of IκBα is increased in THP-1 SAMHD1 knockout (KO) cells compared with THP-1 control cells . To invetogether , A–D. Afol cells , A–D. Ot results . TogetheOur previous studies suggested that the function of SAMHD1 is dependent on cell differentiation , 29. ForC). The total expression levels of IKKα, IKKβ, and IKKγ were not significantly influenced by LPS treatment in the differentiated THP-1 cells. The phosphorylation of IKKα/β and IKKγ was induced by LPS 15 min to 30 min post treatment in PMA-differentiated THP-1 cells, after which levels of p-IKKα/β and p-IKKγ returned to baseline (C). The peak of p-IKKα/β and p-IKKγ in PMA-differentiated cells appeared earlier than that observed in dividing THP-1 cells, suggesting that noncycling THP-1 cells were more sensitive to LPS treatment. The levels of p-IKKα/β and p-IKKγ in the THP-1 control cells were lower than that in THP-1 SAMHD1 KO cells (C and D). These results further corroborate that endogenous SAMHD1 inhibits phosphorylation of IKKα/β/γ induced by LPS treatment.To confirm the effect of SAMHD1 in NF-κB activation in nondividing cells, the phosphorylation levels of IKKα/β/γ were measured in PMA-differentiated THP-1 cells C. The tobaseline C. The peKO cells , C and DA and C). Although the total levels of IKKα, IKKβ, and IKKγ were not significantly influenced by SeV infection, the level of p-IKKα/β and p-IKKγ increased in response to infection and the peak of p-IKKα/β and p-IKKγ appeared at 4 h post infection (hpi) in THP-1 cells (A and B). At 4 hpi, the p-IKKα/β and p-IKKγ in the SAMHD1 KO cells were nearly 4- and 2-fold higher relative to THP-1 control cells (A and B), demonstrating that SAMHD1 suppresses phosphorylation of IKKα/β and IKKγ induced by SeV infection. Similar results were overserved in PMA-differentiated THP-1 cells (C and D), indicating that endogenous SAMHD1 inhibits virus-induced phosphorylation of IKKα/β/γ in both cycling and noncycling cells.We found that endogenous SAMHD1 inhibits phosphorylation of IKKα/β/γ induced by inflammatory stimuli . To exam-1 cells , A and Bol cells , A and B-1 cells , C and DA and B), confirming that SAMHD1 inhibits phosphorylation of IKKα/β and IKKγ in THP-1 cells. To confirm the role of SAMHD1 in inhibiting phosphorylation of IKKα/β/γ in nondividing cells, the cells were differentiated by PMA prior to SeV infection. As shown in C and D, differentiated THP-1 SAMHD1 KO cells and differentiated THP-1 Lvx cells lacking SAMHD1 expression promoted p-IKKα/β and p-IKKγ compared with THP-1 control cells and SAMHD1 KI cells. Thus, SAMHD1 reconstitution in SAMHD1 KO cells inhibits phosphorylation of IKKα/β/γ induced by SeV infection. These results also complement our loss-of-function studies.SAMHD1 knock-in (KI) cells were generated from parental THP-1 SAMHD1 KO cells to reconstitute the expression of SAMHD1 with THP-1 Lvx lentivirus (Lvx) cells serving as a control cell population . THP-1 cA and B). Similar results were obtained after stimulation of THP-1 cells with LPS (C) or HA antibody (D) was used for immunoprecipitation (IP) and IgG was used as a negative control. Coimmunoprecipitation (Co-IP) confirmed that SAMHD1 interacted with exogenous IKKα, IKKβ, and IKKγ in HEK293T cells (C and D). To determine whether the interaction between SAMHD1 and IKKα or IKKβ is direct, we performed an in vitro pull-down assay. As shown in A and B, recombinant SAMHD1 interacted directly with purified IKKα and IKKβ, suggesting a potential mechanism of SAMHD1-mediated inhibition of p-IκBα through the SAMHD1-IKKs complex.We previously found that endogenous SAMHD1 interacts with key proteins like p50 and IκBα and inhibits NF-κB activation . To explwith LPS . Next, Hwith LPS C or HA aantibody D was use3T cells , C and DA). Co-IP results showed that SAMHD1 M1 and M7 lacking the HD domain did not interact with IKKα or IKKβ, suggesting that the HD domain of SAMHD1 is required for its interaction with IKKα and IKKβ (B and C). Because of lower expression of the HD domain alone (M3) compared with other mutants  and FLAG-IKKβ and p-IKKβ (active) . Thus, the HD domain of SAMHD1 is necessary and sufficient for its interaction with IKKα and IKKβ.We reported that the HD domain of SAMHD1 interacts with IFN regulatory factor 7 (IRF7) and mediates suppression of IFN-I activation . To map Fig. 7 mutants , HEK293TThe above data indicate an important role of the HD domain of SAMHD1 in suppressing NF-κB activation . ConsistA and C) (A and B), suggesting that the KD of IKKα is required for its interaction with SAMHD1. Interestingly, IKKβ M1 interacted only weakly with SAMHD1, while IKKβ WT, M2, M3, and M4 containing the ULD strongly interacted with SAMHD1 (D), suggesting that the interaction is mostly dependent on the ULD of IKKβ. Thus, the KD of IKKα and ULD of IKKβ mediate their respective interactions with SAMHD1.To map the specific domains of IKKα and IKKβ required for interaction with SAMHD1, a Co-IP assay was performed in HEK293T cells coexpressing WT SAMHD1 and WT IKKα, WT IKKβ, or a series of truncated IKKα or IKKβ mutants (M1-M5) , A and CA and C) . IKKα M4A and C) , A and Bh SAMHD1 D, suggesA) or IKKβ (B) in a dose-dependent manner. These data suggest that SAMHD1 blocks the TAK1-binding site of IKKα and IKKβ and thereby inhibits phosphorylation of IKKα and IKKβ.To explore the exact mechanism of SAMHD1-mediated inhibition of p-IKKα and p-IKKβ, HEK293T cells were cotransfected with constructs expressing FLAG-TAK1, increasing amounts of HA-SAMHD1 and Myc-IKKα or Myc-IKKβ. Co-IP and immunoblot analysis showed that exogenous SAMHD1 decreases the interaction between TAK1 and IKKα A or IKKβ or IKKβ B in a doC), indicating that IKKα and IKKβ could synergistically activate the NF-κB pathway. Moreover, we revealed that SAMHD1 suppressed the phosphorylation of IKK complex proteins, which play an important role in NF-κB activation. Upon inflammatory stimuli treatment or SeV infection, the phosphorylation of IKKα/β reaches a peak and diminishes quickly , Tubulin , IKKα , IKKα , IKKβ , Phospho-IKKα/β (Ser176/180) , IKKγ , Phospho-IKKγ (Ser376) , IκBα , Phospho-IκBα (Ser32/36) , GAPDH , HA , FLAG , and SeV . Secondary antibodies used were goat anti-mouse IgG (H + L) HRP (W4021) and goat anti-rabbit lgG (H + L) HRP (W401B) (Promega); mouse anti-rabbit IgG (L) (211-032-171) and goat anti-mouse IgG (L) (115-035-174) (Jackson ImmunoResearch Inc).LPS (L6529), puromycin (P8833), and PMA (P8139) were purchased from Sigma-Aldrich. TNF-α (300-01A) and IL-1β (200-01B) were from Peprotech. Cell lysis buffer (9803) was from Cell Signaling. Phosphatase Inhibitor Cocktail 3 (P0044) and Protease inhibitor cocktail (P8340) were from Sigma-Aldrich. Dynabeads Protein G (10004D), Dynabeads Protein A (10001D), SuperSignal West Femto Maximum Sensitivity Substrate (34096), and Pierce BCA Protein Assay (23225) were from Thermo Fisher Scientific. Dual-Luciferase Reporter Assay System (E1910) was from Promega. Nitrocellulose membrane (1620115) was from Bio-Rad. Polyethyleneimine (PEI) (24313-2) was purchased from Polysciences. Purified recombinant IKKα (TP761707) and IKKβ (TP750220) were purchased from Origene.2 and confirmed free from mycoplasma contamination using the universal mycoplasma detection kit (ATCC 30-101-2K). Trypsin-EDTA (0.25%) (25200072), Dulbecco’s modified Eagle’s medium (11965092), Opti-MEM (31985070), and Penicillin-Streptomycin (15140122) were bought from Gibco. Fetal Bovine Serum (S1150H) was bought from R&D Systems.THP-1 control, THP-1 SAMHD1 KO, THP-1 Lvx, and THP-1 SAMHD1 KI cell lines were described previously . The celA) were reported or constructed as described (Plasmids (pRK based) encoding HA-tagged SAMHD1 WT and truncated mutants   and titered on LLCMK2 cells. SeV infection of THP-1 control, THP-1 SAMHD1 KO, THP-1 Lvx, and THP-1 SAMHD1 KI cell lines was conducted as described . THP-1 cHEK293T cells were transfected with pNF-κB-luciferase, pRL-TK-renilla, and indicated plasmids for 48 h using PEI. For dose response experiments, an empty vector was used to maintain the same amount of the total plasmid in each transfection. The cells were lysed by passive lysis buffer, and luciferase was measured by VICTOR Nivo Multimode Microplate Reader using the method described .The cells were lysed in cell lysis buffer as described . ProteinThe THP-1 control cells or PMA-differentiated THP-1 control cells were harvested and lysed in the cell lysis buffer. SAMHD1 antibody (4 μg) and Dynabeads protein G were used for IP. The same amounts of mouse IgG were used as the negative control. The bound beads were washed with PBS and 0.1% Tween three times and then boiled in protein loading buffer . HEK293TRecombinant SAMHD1 was purified as described  and the t tests were performed for statistical comparison between groups. Statistical differences were considered significant at a value of p < 0.05.The results were presented as mean ± SD. Data were analyzed using the GraphPad Prism software and nonparametric All data are contained within the article.This article contains The authors declare that they have no conflicts of interest with the contents of this article.


The growing interest in materials derived from biomass has generated a multitude of solutions for the development of new sustainable materials with low environmental impact. We report here, for the first time, a strategy to obtain bio-based nanocomposites from epoxidized linseed oil (ELO), itaconic acid (IA), and surface-treated nanofibrillated cellulose (NC). The effect of nanofibrillated cellulose functionalized with silane (NC/S) and then grafted with methacrylic acid (NC/SM) on the properties of the resulted bio-based epoxy systems was thoroughly investigated. The differential scanning calorimetry (DSC) results showed that the addition of NCs did not influence the curing process and had a slight impact on the maximum peak temperature. Moreover, the NCs improved the onset degradation temperature of the epoxy-based nanocomposites by more than 30 °C, regardless of their treatment. The most important effect on the mechanical properties of bio-based epoxy nanocomposites, i.e., an increase in the storage modulus by more than 60% at room temperature was observed in the case of NC/SM addition. Therefore, NC’s treatment with silane and methacrylic acid improved the epoxy–nanofiber interface and led to a very good dispersion of the NC/SM in the epoxy network, as observed by the SEM investigation. The dielectric results proved the suitability of the obtained bio-based epoxy/NCs materials as substitutes for petroleum-based thermosets in the fabrication of flexible electronic devices. Epoxy resins are one of the most widely used thermosetting polymers, which become irreversibly hardened when cured by heating and/or by the addition of crosslinking agents ,2. TodayTo achieve solid, dimensionally, and thermally stable three-dimensional networks, epoxy resins need to be cured in the presence of crosslinking agents, either at room temperature or at elevated temperatures . HoweverAspergillus family . Co. Co38]. Nevertheless, a distinct morphology was noticed in the case of E/NC/SM nanocomposite, probably due to the presence of poly(metacrylic acid) on the NCs surface which led to an increased interface volume and compatibility with the epoxy matrix . AdditioThe higher magnification SEM images of the fractured samples confirm the previous observations. More than that, a homogenous distribution of the grafted NCs was noticed in the epoxy matrix due to the good miscibility between the surface-treated nanofibrillated cellulose and the epoxy matrix .Generally, epoxy resins-based materials have widespread use in many applications because of their favorable mechanical properties, excellent chemical resistance, and especially good electrical insulation. Considering this last characteristic, the obtained bio-based nanocomposites were investigated through BDS in order to assess their suitability as potential candidates for replacing the petroleum-based thermosets in electronic applications.r′ decreased with frequency for all the samples in the studied range. The εr′ values are mainly determined by the dielectric response of bio-based epoxy. The presence of NC, the epoxy–NC interface, and the silane treatment seem to have a small influence on the dielectric activity of the bio-epoxy material as previously observed for common epoxy nanocomposites [r′ value was observed for the E/NC/S compared with the E reference and the other nanocomposites, regardless of the frequency. The lower permittivity of this nanocomposite as compared to the unfilled resin is unexpected and may be explained by the silane treatment of NC which prevents the absorption of water and reduces the number of hydrophilic OH on the surface of nanocellulose. A similar behavior was not observed in the case of E/NC/SM due to the polymethacrylic acid lateral chains and their hydrophilic carboxyl groups. It is worth remarking that the εr’s values obtained in the case of bio-based epoxy and its nanocomposites are very close to those reported for both common and bio-based epoxy resins in a similar frequency range [The dielectric spectra of the neat bio-based epoxy system and epoxy nanocomposites containing differently treated NCs are shown in mposites . The deccy range ,66,67.r″) values are higher for all the samples at low frequencies due to electrode polarization , and it may be attributed to the β-relaxation of the dipoles associated with the polar groups of the bio-epoxy resin [The dielectric loss , while IA (≥99.9%), and THF  were acquired from Sigma-Aldrich  and used as received. Microcrystalline cellulose  from Sigma-Aldrich  was used as raw material for NC’s isolation.−1, when a nanofibrillated cellulose (NC) powder was obtained. The resulting NC was firstly treated with a 5 wt% γ-methacryloyloxypropyltrimethoxysilane (S) water solution at 50 °C, under strong magnetic stirring, for 4 h for generating active sites on its surface and then subjected to graft polymerization with methacrylic acid (M). The graft polymerization reaction was carried out using acetone as solvent and AIBN as initiator by mixing all components together and allowing them to react under reflux for 4 h. The silane modified (NC/S) and methacrylic acid grafted (NC/SM) nanocellulose were further washed with distilled water, freeze-dried, and milled  via a high-pressure mechanical treatment . Brieflyd milled a. The reAll formulations were prepared by maintaining a stoichiometric ratio of 1:1 between the epoxy groups of ELO and the acid groups of IA, while the weight ratio between THF and IA was set at 3:1. The content of NC, NC/S, and NC/SM, respectively, was maintained constant at 0.75 wt% in all the epoxy nanocomposites. For the preparation of the ELO-based nanocomposites, the IA was first solubilized in THF under magnetic stirring at 50 °C until a transparent solution was obtained. Simultaneously, the desired amount of unmodified or surface-modified NCs was dispersed in the ELO resin and homogenized at 80 °C for 30 min under vigorous stirring. Afterwards, the THF/IA solution was poured over the ELO/NC mixture, and the stirring continued for another 30 min. Subsequently, the resulting nanocomposites were poured into silicon molds, degassed under vacuum at ambient temperature, and cured in an oven at 90 °C for 90 min, 120 °C for 60 min, and 140 °C for another 60 min. A neat epoxy system, without NCs, was prepared following the same protocol and served as a control. The schematic illustration of the nanocomposites’ preparation protocol is displayed in −1. The resolution and number of scanning cycles were 4 cm−1 and 32, respectively.The FT-IR absorbance spectra of pristine IA and ELO, the neat epoxy system and epoxy nanocomposites obtained following the curing protocol were recorded using a Jasco FTIR 6300 instrument  in attenuated total reflectance (ATR) mode over a wavenumber range from 4000 to 400 cmTMDSC analysis was employed for monitoring the efficiency of the curing protocol used in the preparation of the neat epoxy system and epoxy nanocomposites and was conducted by using a TA Instruments DSC Q2000 equipment  under helium flow (25 mL/min), with the underlying rate of 10 °C/min, amplitude of 0.8 °C/min, and a period of 30 s as follows: equilibrating at −85 °C; isothermal for 5 min; heating from −85 °C to 305 °C.−1 during heating from room temperature to 750 °C at 10 °C min−1. All samples were run in duplicate to verify the reproducibility, and the average values, along with the standard deviation, were reported.The thermal stability of the cured neat epoxy matrix and epoxy nanocomposites was evaluated using TA Q5000 equipment . The samples were analyzed in nitrogen at a flow rate of 50 mL min3 (length × width × thickness) from −30 to 90 °C at a heating rate of 3 °C min−1 and an oscillation frequency of 1.0 Hz.The viscoelastic behavior of cured epoxy nanocomposites was assessed using a TA Instruments DMA Q800 . The tests were performed in multi-frequency strain mode (tension clamp) by heating the bar samples with dimensions of 13 × 7 × 1.2 mmg + 50) in Kelvin, and R is the ideal gas constant.The crosslink density (ν) was determined in accordance with the Flory theory of rubber elasticity , using EThe transmittance spectra of cured bio-based epoxy matrix and epoxy/NCs nanocomposites were recorded in the visible region (300–800 nm) on a JASCO V570 UV-VIS-NIR spectrophotometer , equipped with a JASCO ILN-472 (150 mm) integrating sphere.The cured epoxy nanocomposites were fractured in liquid nitrogen and then sputter-coated  with a 5 nm layer of gold. The surface of fractured blends was visualized with a Hitachi TM4000Plus II Benchtop microscope working in a low vacuum at 10 kV accelerating voltage and a working distance of about 7 mm. The SEM images were taken using backscattered electron detector (BSE).−2 to 1 × 106 Hz. The real (εr′) and imaginary (εr″) parts of the complex relative permittivity were determined for each sample at the temperature of 30 °C. The samples were maintained for 30 min at this temperature before measurements.BDS measurements were carried out with a Novocontrol Alpha-A Analyzer equipped with an Active Sample Cell ZGS over a frequency range from 5 × 10Bio-based epoxy nanocomposites consisting of ELO, surface-modified NCs as fillers, and IA as cross-linking agent were developed for the first time. The overall results showed that NCs can be used for tailoring the mechanical properties of ELO-based systems. DSC results revealed that the addition of NCs into the bio-based epoxy network did not alter the curing mechanism and had a slight impact on the maximum peak temperature. Even at low content, NCs exhibited a favorable effect in terms of both thermal stability and mechanical behavior. As compared with the neat epoxy, the onset degradation temperature of the epoxy-based nanocomposites increased by more than 30 °C, regardless of their treatment. The nanocomposite containing NC/SM showed the best mechanical performance, exhibiting an increase in the storage modulus by more than 60% at room temperature. This indicates a better dispersion of the nanofibers and a higher adhesion between the epoxy system and the poly(methacrylic acid) grafts, which resulted in a more homogenous structure, as supported by the SEM results. A small influence of NCs on the dielectric activity of the bio-epoxy nanocomposites was observed from the dielectric measurements, thus showing the suitability of the obtained bio-based epoxy/NCs for replacing the petroleum-based thermosets in electronic applications. Therefore, the developed bio-based nanocomposites may provide a new perspective on using them as coatings or flexible films for different engineering applications.


In a continuous search for the improvement of antitumor therapies, the inhibition of the Wnt signaling pathway has been recognized as a promising target. The altered functioning of the Wnt signaling in human tumors points to the strategy of the inhibition of its activity that would impact the clinical outcomes and survival of patients. Because the Wnt pathway is often mutated or epigenetically altered in tumors, which promotes its activation, inhibitors of Wnt signaling are being intensively investigated. It has been shown that knocking down specific components of the Wnt pathway has inhibitory effects on tumor growth in vivo and in vitro. Thus, similar effects are expected from the application of Wnt inhibitors. In the last decades, molecules acting as inhibitors on the pathway’s specific molecular levels have been identified and characterized. This review will discuss the inhibitors of the canonical Wnt pathway, summarize knowledge on their effectiveness as therapeutics, and debate their side effects. The role of the components frequently mutated in various tumors that are principal targets for Wnt inhibitors is also going to be brought to the reader’s attention. Some of the molecules identified as Wnt pathway inhibitors have reached early stages of clinical trials, and some have only just been discovered. All things considered, inhibition of the Wnt signaling pathway shows potential for the development of future therapies. In the last thirty years, inhibitors of the Wnt signaling pathway have been identified and characterized along with the functional explanation of the pathway’s molecular targets ,2,3. Herint-1 and Drosophila’s wingless (wg). The discovery of a novel cellular proto-oncogene int-1, which was later on mapped to the chromosomal position of Drosophila gene wg, launched the marvelous research on this essential pathway and its many important components.Wnt signaling is a conserved cellular pathway in all multicellular organisms that has been studied for more than four decades. The name was coined from the names of two genes, mouse 2+ [2+ pathway regulates cytoplasmic concentration of calcium ions through their exit from the endoplasmic reticulum [It is generally accepted that Wnt signaling consists of canonical β-catenin and two non-canonical beta-catenin independent pathways—the planar cell polarity (PCP) and the Wnt/Ca2+ ,5. Canon2+ ,7. In ad2+ ,9. Activeticulum ,10,11.2+ pathways are thought to be important in the acquisition of metastatic properties, primarily because of their role in cytoskeletal reorganization [The aberrant canonical Wnt pathway is involved in the formation and evolution of various types of tumors ,13. In anization ,15. Thernization  in mammanization . Thus, sc-myc, N-myc, c-jun, cyclin D1, Sox9) [MMP-7, VEGF), and the TERT gene encodes a component of the telomerase enzyme whose activity is an important feature of cellular immortality [The canonical Wnt signaling pathway has been extensively characterized both in development and disease. When active, it contributes to the stabilization of cytoplasmic β-catenin, a molecule that in combination with transcription factors and coactivators TCF, LEF, PYGO, BCL9, p300, or CBP activates the transcription of Wnt target genes . The fir1, Sox9) , some arortality .When the Wnt pathway is inactive, the β-catenin degradation complex, sometimes referred as degradosome, phosphorylates free cytoplasmic β-catenin, making it susceptible to ubiquitination and proteasome degradation ,28,29. Uβ-catenin transcriptional activity does not necessarily have the same consequences under all conditions nor in every cell type. To generate a transcriptionally active complex, β-catenin–TCF recruits the transcriptional co-activator CREB-binding protein (CBP) or its closely related homologue E1A-associated protein p300. The preference to the specific coactivator is the first step in deciding between programs of differentiation and proliferation. Thus, when considering the optimum therapeutic effect, inhibitors acting specifically on each of these two programs should be taken into consideration .Because β-catenin does not contain nuclear localization signal sequences (NLS), the mechanism of its nuclear transfer is still debated. There are conflicting reports from several studies ,39,40 onIt is important to understand that there is another pool of β-catenin in the cell. β-catenin is part of the protein complex that forms intercellular adherens junctions, along with E-cadherin on the outer side of the junction and α-catenin bound to the inner cytoskeletal proteins. Adherens junctions between epithelial cells preserve cell-to-cell adhesion but also prevent the dissociation of individual tumor cells from the tumor mass . The strIt is also important to know that there are six families of proteins that act as extracellular inhibitors of the Wnt pathway. These are Dickkopf, WIF, SOST/Sclerostin, Cerberus, SFRP, and IGFBP4 ,53,54. DThe Wnt signaling pathway malfunctions in a number of tumors, including colorectal, breast, glioma, melanoma, pancreatic, and many others ,55,56,57APC and the CTNNB1 genes [CTNNB1 gene, which encodes β-catenin, accounts for 90% of mutations, leading to a change in the N-terminal domain whereby β-catenin loses its key sites for degradation complex enzyme activity [Changes in the molecules of the Wnt pathway in tumors can be mutational or non-mutational. Non-mutational changes include all levels of epigenetic changes—DNA methylation, histone modification, and RNA interference. Both type of changes promote tumor formation and invasiveness, which usually happens because of the loss of function of negative regulators or the overexpression of activators of the Wnt pathway. Generally, the most common Wnt pathway mutations in tumors are mutations of the B1 genes . The mutactivity .APC gene are present in about 85%, whereas in 50% of tumors that do not contain the APC mutation, an activating β-catenin mutation is present [RNF43 gene have been identified in up to 18% of colorectal cancers and are mutually exclusive with the APC mutations. RNF43 encodes the E3 ubiquitin ligase needed for Frizzled receptor ubiquitination and inhibition of the Wnt pathway [Historically, the first Wnt mutations were detected in sporadic as well as hereditary forms of colorectal cancer. Mutations in at least one regulatory component of the Wnt pathway are present in over 93% of colorectal cancers . Mutatio present ,68. The  present . Similar pathway ,67,70.DKK1, DKK3, WIF1, SFRP5, APC, GSK3B, MCC, and CTNNBIP1 were also found in 32–44% of tested samples and methylation of their promoters in 40–68%. miR-221/222 interferes with the transcription of Wnt pathway antagonists, including WIF1, SFRP2, DKK2, and AXIN2. It has also been shown that the Wnt pathway is most active in breast cancer stem cells relative to the rest of the tumor mass [FZD 6, 7, and 8 genes in triple-negative breast cancers, which has been associated with poorer survival, invasiveness, and tumor stem cell characteristics.Another cancer in which Wnt signaling plays a major role is breast cancer. Studies have shown that Wnt signaling is active in over 50% of examined breast cancers subtypes, and this activity, higher in comparison to wild type cells, has been associated with poorer survival. It is most active in triple-negative breast cancers, where Wnt ligands and receptors are commonly overexpressed, whereas secreted Wnt antagonists are hypoexpressed. However, β-catenin itself is rarely mutated in breast carcinomas . Deletiomor mass . PreclinAPC or AXIN1 mutations are present in another 5% of cases [However, changes in the Wnt pathway need not be associated with a poorer prognosis in all tumors. Several studies report a less invasive phenotype and increased survival rate in melanoma patients with increased levels of nuclear β-catenin ,72,73,74of cases ,76. Of tof cases .AXIN1 or CTNNB1 genes are frequently altered, whereas APC gene alterations are uncommon [https://www.cbioportal.org/, accessed on 7 March 2023), of 740 patients with hepatocellular carcinoma, 31% had a mutation in the β-catenin gene .Studies investigating the role of Wnt signaling in hepatocellular carcinoma (HCC) have shown the activation of the pathway in about 25–50% of HCC as well as a tumor-specific mutational profile that differs to alterations found in CRC. In HCC, uncommon ,78,79. ASFRP1 gene was found to be hypermethylated in 32% of samples, and the SFRP1 protein expression was reduced in 45.8% of samples. SFRP1 hypermethylation as well as enhanced LEF1 expression were associated with a higher tumor grade [WIF1, SFRP1/2, and NKD1/2 (Naked) [DKK1 promoter was found in 60% of glioblastomas [Several studies have shown that the excessive activation of the Wnt pathway is responsible for glioma formation and is associated with a poor prognosis ,80,81,82or grade . There i (Naked) ,85 by thlastomas . All thelastomas ,87,88. IThe previously described research shows the important role of the Wnt signaling pathway in a large number of human tumors. Therefore, further research and development of inhibitors could prove a useful therapeutic strategy.APC mutation, significant growth inhibition, differentiation, and reduction of proliferation occurred. However, after the cessation of beta-catenin silencing, tumor growth rapidly recovered, suggesting that Wnt inhibitor therapy would require continuous administration [The reason for searching for inhibitors of the Wnt pathway is the hope that the inhibition would have a therapeutic effect on tumors ,89. For stration . Anotherstration . Moreovestration . Althougstration . A similFZD7 gene. A reduced ability of tumor formation in mice was found after the transplantation of triple-negative breast cancer cell lines with knocked down FZD7 [Parallel studies have also focused on examining the effect of growth inhibition of other Wnt pathway components, for example, the own FZD7 . A similown FZD7 . Inhibitown FZD7 .Based on all these findings, molecular targets to which Wnt inhibitors are directed have been recognized, and the inhibitors are usually divided according to their site of action .In addition, inhibitors which enhance the activity of the so-called negative Wnt pathway regulators have also been developed. Most of these molecules have been considered as inhibitors based on screening, i.e., testing thousands of molecules, both synthetic and natural, to identify the most potent ones. Various tests were used to evaluate the inhibition, most commonly the TOPFlash assay, which shows how much the examined molecules interfere with β-catenin-dependent transcription of dTF12 (top flash-like luciferase reporter) . In othePorcupine (PORCN) is a member of the membrane-bound O-acyltransferases (MBOAT) that acylates  Wnt ligands in the endoplasmic reticulum before their secretion into the intercellular space. Increased expression of this enzyme is associated with a poorer prognosis in squamous cell carcinomas of the head and neck . PORCN-iLGK974 (WNT974)  is a small molecule able to decrease the expression of Wnt target genes. In renal carcinoma cell lines, LGK974 inhibits cellular proliferation and migration and increases the proportion of G1 cells . In prechttps://clinicaltrials.gov/ct2/show/NCT01351103, accessed on 19 February 2023). In patients with metastatic colorectal cancer containing WNT and BRAF mutations, LGK974 is on trial in combination with BRAF inhibitor LGX818 and cetuximab [A similar effect of LGK974 was observed in a mouse model of the Wnt-dependent human head and neck squamous cell carcinoma (HNSCC) cell line in which a dose of 3.0 mg/kg caused substantial tumor regression of T/C% = −50% was achieved. In both studies, LGK974 did not cause significant weight loss in mice . It has etuximab ,102.R-spondin 2 and 3 genes. This indicates that R-spondin, which is a secreted agonist of the pathway, can safely and efficiently be inhibited with ETC-159 [Another porcupine oral inhibitor with high bioavailability is ETC-153 (ETC-1922159)  , also specifically targets PORCN. It affects the production of Wnt ligands by inhibiting the post-translational palmitoylation and secretion of Wnt ligands in the endoplasmic reticulum. In such a manner, it can block downstream Wnt signaling. Thus, CGX1321 is effective in patients with an active canonical Wnt pathway. Currently, CGX1321 is being tested in clinical trials on colon cancer patients through oral administration .Suppression of Wnt signaling in the extracellular space is another potential territory for cancer therapy. Ipafricept OMP54F28; IPA)  is a recombinant fusion antibody consisting of an extracellular portion of FZD8 receptor and Fc fragment of human IgG1. The binding of Wnt ligands to Ipafricept prevents their interaction with FZD receptors ,11,105. 4F28; IPAA phase Ib clinical trial for the combination of ipafricept with paclitaxel and carboplatin in recurrent ovarian cancers was also conducted. After the revision of the first protocol, doses per cohort were increased from 2 to 6 mg/kg resulting in the overall response rate of 75.7%, the median survival of 33 months, and the median progression-free survival of 10.3 months. Adverse reactions were observed in ≥15% of patients of which neutropenia was the most common. Bone toxicities at efficacy doses prevented further testing of this treatment regimen . AnotherVantictumab (OMP-18R5)  is a human IgG2 monoclonal antibody that inhibits FZD receptors, namely FZD1, FZD2, FZD5, FZD7, and FZD8. It binds to their extracellular domains and probably sterically inhibits the binding of Wnt ligands. Thereby, the phosphorylation of LRP6 co-receptors is blocked, β-catenin concentration is reduced, and Wnt signaling is stopped . AccordiVantictumab in monotherapy significantly slowed tumor growth in xenograft models of squamous cell carcinoma of the head and neck. Furthermore, a very potent effect of the combination of taxol and vantictumab on xenografts of breast and lung cancer was reported . A furthA phase Ia clinical trial has been conducted for vantictumab on different solid tumors, including colon, breast, sarcoma, and neuroendocrine tumors. In most patients, vantictumab therapy was discontinued within 60 days due to disease progression, and in three elderly patients with neuroendocrine tumors, a prolonged phase of disease stabilization occurred . HoweverDue to the important role of the Wnt pathway in bone metabolism, bone-related side effects and their frequency were one of the main concerns in all clinical trials. Vantictumab increased the concentration of the biomarker of bone degradation, βCTX, even at low doses; moreover, at doses greater than 5 mg/kg, it decreased the biomarkers of new bone formation, P1NP and osteocalcin . The occA chimeric monoclonal antibody OTSA101 , Kawasaki City, Japan) is yet another FZD receptor antagonist directed specifically to FZD10. This antibody was developed after it was demonstrated that synovial sarcoma cells express significantly more FZD10 than normal tissues. Additionally, when it was radiolabeled with Yttrium-90, this radioactive form, OTSA101-DTPA-90Y, showed even stronger antitumor activity ,5,119. IDrugs that act antagonistically to the LRP co-receptors were originally registered for the treatment of parasite infections, but their potential for tumor treatment is currently being intensively investigated. Salinomycin  is a monocarboxylic polyether ionophore previously known for its antibiotic and coccidiostatic effects. Ionophores are small molecules that help specific ions cross the membrane. Numerous studies have shown that salinomycin also shows antitumor activity, especially against tumor stem cells. Although its exact mechanism of action has not been fully elucidated, it has been proposed that it is through the inactivation of the Wnt pathway. In addition to inducing LRP co-receptor degradation, salinomycin activates the transcription factor FOXO3a, which prevents β-catenin from binding to TCF ,121. SalAdditional studies indicated a potential benefit from the combination of salinomycin with resveratrol, which led to the decreased expression of the EMT marker vimentin and the induction of apoptosis in breast cancer cells . AlthougMallotus philippinensis, which has historically been used as an antihelmintic drug. It affects a number of signaling pathways in tumors [Rottlerin  is a natural polyphenol isolated from the plant n tumors . The effn tumors . In anotn tumors .Streptomyces cinnamonensis [Another antagonist to the LRP co-receptor is monensin , an antibiotic and antiparasitic ionophore derived from the bacterium monensis . It showmonensis  and redumonensis  and showmonensis .Another oral antihelmintic drug that has recently been credited with effects on a wide range of diseases including tumors is niclosamide . The antiproliferative effect of niclosamide in colorectal, prostate, lung, ovary, and breast cancer by the inhibition of Wnt, mTOR, STAT3, Notch, and NF-kB signaling pathways has been reported . It prevTankyrase is an enzyme that regulates the stability of scaffolding proteins AXIN1 and AXIN2 by a reversible post-translational modification—poly-ADP-ribosylation. Such poly-ADP-ribosylated AXINs (PAR-ilylated AXINs) are recognized by ubiquitin ligase RNF146, which labels them for proteasome degradation. Due to the consequent decrease in the AXIN concentration, the formation of the β-catenin degradation complex is prevented . Thus, AAPC mutations, the application of JW-74 resulted in the reduction of tumor growth and cell cycle arrest in the G1/S phase [Further tankyrase inhibitors include JW-55, JW-74, and G007-LK . Generally, they all increase cytoplasmic AXIN levels, β-catenin degradation, and downregulate the expression of Wnt target genes. In colorectal cancer cell lines with /S phase . Tankyra/S phase . In glio/S phase . The LGRAPC. The concentration which reached 50% growth inhibition was 0.449 μmol/L. However, it showed no significant effect on the growth of cell lines with gain-of-function CTNNB1 mutations, lines with longer forms of APC mutant proteins, or lines with wild-type APC [APC mutation is an important factor for sensitivity to tankyrase inhibitors in colorectal cancer. Immunohistochemistry showed that both RK-287107 and G007-LK inhibitors reduced unphosphorylated active β-catenin levels. RK-287107 also induced the accumulation of AXIN2 and the downregulation of MYC. The next experiment aimed to determine whether RK-287107 is available for oral dosage. Intraperitoneal (150 mg/kg twice daily) and oral (300 mg/kg twice daily) administration of this drug in mouse xenografts with APC mutations that produce shorter protein forms resulted in 47.2% and 51.9% tumor growth inhibition, respectively. Pharmacokinetic analysis revealed higher plasma concentrations after oral than after intraperitoneal administration [Another tankyrase inhibitor is the spirindoline derivative RK-287107  . When adtype APC . Of notestration , and itsstration .Currently, there are two other tankyrase inhibitors in preclinical phases: LZZ-02  and NVP-TNKS656 . A combination of NVP-TNKS656 and PI3K and AKT inhibitors has shown to be a promising tool for decreased beta-catenin translocation to the nucleus and Wnt silencing in PI3K or AKT inhibitor-resistant cells of colorectal carcinoma. A similar result on the restraint of active β-catenin was noted when the novel tankyrase inhibitor LZZ-02 was used.APC mutations were sensitive to pyrvinium treatment, showing a decrease in cell proliferation. Furthermore, pyrvinium targets ovarian cancer cells through suppressing Wnt signaling [One of the novel strategies for the inhibition of the aberrant Wnt signaling pathway is through the restoration and stabilization of the activity of the molecules responsible for the degradation of β-catenin. One such molecule is pyrvinium , an anthelmintic drug already approved by the FDA that exerts its antitumor effects by restoring the activity of several different kinases that are directly or indirectly involved in β-catenin degradation. It is important to highlight that pyrvinium pamoate is approved as antihelmintic drug, but at present the FDA approval does not extend to its antitumor effect. A recent study showed that pyrvinium directly binds to CK1α as an activator, stabilizing its protein ,151,152.ignaling .A new potent and selective inhibitor of Wnt signaling that acts by promoting β-catenin proteosomal degradation is MSAB , or methyl-3 (4-methylphenyl) sulfonyl amino-benzoate. This was recognized by the screening of 22,000 molecules in a colorectal cancer cell line harboring a deletion of β-catenin’s phosphorylation site of CK-1 . It has The selectivity of MSAB for tumor cells was demonstrated in epithelial cells or skin fibroblasts. MSAB also caused a reduction in tumor mass in mouse xenografts in Wnt-dependent tumors, causing apoptosis of tumor cells. It is important to note that MSAB also inhibited the proliferation of LS174T human colon adenocarcinoma cell line carrying β-catenin mutations . The futAPC, these compounds could prove very useful [The next section will focus on the inhibitors that target the downstream protein–protein interactions of β-catenin with transcription factors and coactivators. Such inhibition is primarily by blocking the β-catenin/TCF complex. Several compounds, such as PKF115-584, CGP049090 , and PKF222-815 , acting in a dose-dependent manner were identified using high-throughput ELISA screening. However, these molecules proved to be insufficiently selective because they also inhibited the APC/β-catenin interaction . Neverthy useful .In a study performed by Huang et al. , a selec2 in continuous infusion for 7 days, and the inhibitor proceeded to phase II trial [2/day. The response was the stabilization of disease in 40% of patients. Grade 3 and 4 adverse events were observed in seven patients, but none of the side effects were dose-limiting [In addition to TCF, the coactivators LEF, CBP, p300, and PYGO also form complexes with β-catenin. The co-activator CREB binding protein (CBP) is a particularly interesting target. Several CBP inhibitors have been developed in recent years, including PRI-724 (Prism Pharma Co Ltd. (Prism Pharma), Yokohama, Japan), a small molecule that inhibits the interaction of β-catenin with CBP by competitively binding to CBP. Preclinical studies have shown that PRI-724 displays antitumor effects, promotes the differentiation of tumor stem cells, and makes them more sensitive to chemotherapy ,101,160.II trial . In a phlimiting .2. Two dose-limiting toxicities occurred at the highest dose: grade 3 and 4, hypoxia and thrombocytopenia. The continuation of phase Ib is planned to evaluate the combination therapy of CWP232291 with lenalidomide and dexamethasone [2 [The peptide CWP232291  is another potent inhibitor of β-catenin and CBP complex formation . Two phaethasone . In a seasone [2 ,165.BCL9 (B-cell lymphoma 9) is another coactivator for β-catenin-mediated transcription that is highly expressed in tumors. Carnosic acid represents a pharmacologic strategy for inhibiting oncogenic BCL9 and β-catenin interaction by blocking the binding site for BCL9 on β-catenin ,166. CarVL2, TCF7, ERBB2, and LRP5 genes and exon skipping in LEF1 and TCF7L2 genes, making the resulting mRNA unstable. SM08502 was 10 times more potent than PRI-724, the inhibitor of β-catenin transcriptional activity. Moreover, SM08502 inhibited spheroid formation in pancreatic cancer cell lines. SM08502 induced apoptosis and a significant reduction in tumor growth in all gastrointestinal tumor cell lines in vitro and in mouse colorectal cancer xenografts [CDC-like kinases (CLKs) are evolutionary conserved kinases that are able to phosphorylate serine, threonine, and tyrosine residues. CLKs have primarily been involved in precursor-mRNA splicing, where they catalyze the phosphorylation of splicing factors 1–12 (SRSF1-12). In spite of the fact that the knowledge about their biological roles is still rudimentary, the therapeutic potential of CLK inhibitors has been extensively explored, and the compound SM08502 (Cirtuvivint)  has recently entered clinical trials . It is anografts . The effnografts . Similarnografts . SM08502nografts . BecauseOther compounds have been employed to targeted CLKs. TG003  has shown potent antitumor properties in prostate cancer cells in vitro and in a xenograf model and caused significant changes in the alternative splicing of cancer-associated genes. The use of compounds CC-671  and T-025  also resulted in significant antitumor effects .Because an optimally regulated Wnt pathway ensures cellular differentiation as well as the regeneration of many tissues, especially those with rapid cell turnover such as hematopoietic and gastrointestinal tissues, the inhibition of this pathway carries serious risks of side effects. Side effects can be expected when inhibiting such an essential cellular pathway. Nevertheless, early clinical trials have shown that the severity of side effects is not significant compared to the therapeutic benefits. They are usually associated with the malfunction of tissue regeneration  and coulThe most common side effects upon inhibition of Wnt signaling are gastrointestinal problems, hair loss, immunosuppression, fatigue, vitiligo, anemia, neutropenia, thrombocytopenia, bone fractures, and neurodegeneration. In addition to these adverse reactions, elevations in bilirubin and alkaline phosphatase have been observed, as well as hypophosphatemia. This could be explained by the change in bone remodeling, but also by the fact that proper Wnt signaling is important in the regeneration of liver and kidney tissues after injury . HoweverBecause the Wnt pathway regulates bone remodeling in a complex way, one of the side effects reported in the early clinical stages is an increase in bone remodeling. However, it has been shown that this can be prevented with bisphosphonates, specifically zolendronic acid. It is known that decreased LRP5 expression in mouse osteoblasts leads to an osteopenic phenotype, whereas increased LRP5 expression results in increased bone mass . The actHowever, novel research indicated  that a cNeedless to say, all Wnt pathway inhibitors are contraindicated in pregnancy. Wnt signaling is extremely important in development. For example, Wnt signaling gradients control the establishment of the anterior–posterior axis in the central nervous system and also play a role in dendritic and axon guidance and synaptogenesis ,178.In all phases of clinical trials, the question remains as to how the inhibition of the Wnt pathway would affect the cognitive abilities of patients, especially the younger ones. In the adult brain, the proper activation of the Wnt pathway is crucial for adult neurogenesis and the survival of neurons in the supraventricular zone and hippocampus, but also for the maintenance of higher cognitive functions and dopaminergic pathways, and it appears to affect synapse formation ,178. MurAll in all, the side effects caused by Wnt inhibitors are similar to those of chemotherapeutics that are already known and well established. However, it is important to distinguish whether the reported side effects are strictly due to Wnt inhibitors or whether they are the consequence of chemotherapeutics in combinational therapy. Interactions with other molecules which can lead to the so-called off-target side effects have also been insufficiently investigated . To overThe beneficial effect of Wnt inhibitors on other modalities of antitumor therapy was also investigated. The first interesting possibility was alleviating the resistance to checkpoint inhibitors by using Wnt inhibitors . It has In line with these findings are the results of a phase 1 clinical trial using the porcupine inhibitor LGK974 in combination with spartalizumab, a monoclonal antibody to PD-1, that reported impressive results in patients with several types of solid tumors, including the stabilization of disease in 53% of urothelial carcinoma previously resistant to checkpoint inhibitors .Besides influencing immunotherapy, the combination of Wnt inhibitors with taxanes also demonstrated superior clinical response. Taxanes, including paclitaxel, nab-paclitaxel, and docetaxel, block the M-phase of cell division by acting on microtubules. Wnt pathway components are involved in the cell cycle, too. β-catenin is necessary for the separation of centrosomes in the formation of the mitotic spindle, whereas APC and Dishevelled participate in the regulation of kinetochores binding and, together with FZD and LRP, affect the orientation of the spindle . Thus, tNumerous studies have yielded molecules that are capable of inhibiting abnormal Wnt signaling activity in tumor cells. The preclinical and clinical studies for many of these molecules show promising results. However, the mutational status of Wnt components has not been adequately addressed, so the question remains whether a particular inhibitor would have an effect on their mutated forms. The majority of reported studies did not monitor disease progression or recurrences, but instead only measured the inhibitory effect on tumor size or mass. For future investigations, it would be necessary to distinguish the specific tumor in which a particular Wnt inhibitor is successful. The most likely candidate for future antitumor therapies is the PRI-724 inhibitor, which is currently in phase II clinical trials. Given the many serious side effects caused by Wnt inhibitors, as well as the insufficient knowledge of the additional adverse effects, further research is recommended on the safety, efficacy, and drug delivery modes. Drugging the Wnt signaling pathway continues to be one of the promising approaches for future tumor treatment, both alone and in combination therapy.


RIP1 (receptor-interacting protein kinase 1) is an important component of TNF-α signaling that contributes to various pathological effects. Here, we revealed new potential roles of RIP1 in controlling WNT/β-catenin canonical signaling to enhance metastasis of colorectal cancer (CRC). First, we showed that WNT3A treatment sequentially increased the expression of RIP1 and β-catenin. Immunohistochemical analyses of human CRC tissue arrays consisting of normal, primary, and metastatic cancers indicated that elevated RIP1 expression might be related to β-catenin expression, carcinogenesis, and metastasis. Intravenous injection of RIP1 over-expressed CRC cells into mice has demonstrated that RIP1 may promote metastasis. Immunoprecipitation (IP) results indicated that WNT3A treatment induces direct binding between RIP1 and β-catenin, and that this stabilizes the β-catenin protein in a manner that depends on the regulation of RIP1 ubiquitination via downregulation of the E3 ligase, cIAP1/2. Elimination of cIAP1/2 expression and inhibition of its ubiquitinase activity enhance WNT3A-induced RIP1 and β-catenin protein expression and binding, which stimulates endothelial-mesenchymal transition (EMT) induction to enhance the migration and invasion of CRC cells in vitro. The results of the in vitro binding assay and IP of exogenous RIP1-containing CRC cells additionally verified the direct binding of RIP1 and β-catenin. RIP1 expression can destroy the β-catenin–β-TrCP complex. Taken together, these results suggest a novel EMT-enhancing role of RIP1 in the WNT pathway and suggest a new canonical WNT3A–RIP1–β-catenin pathway that contributes to CRC malignancy by promoting EMT. Colorectal cancer (CRC) is one of the most common human malignancies, and its incidence is steadily rising . DespiteReceptor interaction protein kinase 1 (RIP1) is an important upstream kinase of various inflammation signals, including those mediated by TLR3, TNF-α, IL-6, etc –12. RIP1Based on these previous reports, we hypothesized that RIP1 contributes to CRC metastasis regulated by WNT/β-catenin signaling . Here, w2. Mouse embryonic fibroblasts  were obtained from Dr. Zheng-Gang Liu (NIH/NCI) and maintained using Dulbecco’s modified Eagle’s medium  supplemented with 10% FBS in an incubator at 37 °C with 5% CO2. Transfection was performed as described previously [Ctr and HT29-Luc-Flag-RIP1 stable cells were generated from HT29-Luciferase containing cells . The cells were transfected with a Flag-pcDNA3.1 or Flag-RIP1 plasmids  and selected using G418 selection. DLD-1shcon and DLD-1shRIP1 were constructed from DLD-1 cells by transfection with the non-silencing pGIPZ lentiviral shRNA control vector (Addgene_121488) and pGIPZ lentiviral shRNA RIP1, respectively. After repeated selection with puromycin , IB revealed stable inhibition of RIP1 expression in the transduced cell lines, which were designated DLD-1shcon, DLD-1shRIP1-#1, DLD-1shRIP1-#2 and DLD-1shRIP1-#3 (data not shown). We used DLD-1shcon and DLD-1shRIP1-#1 for migration and invasion assays.The human colorectal cancer cells cell lines, HCT116 and DLD-1, were purchased from the American Type Culture Collection  and maintained in an incubator using RPMI-1640 medium  supplemented with 10% fetal bovine serum  at 37 °C with 5% COeviously . HT29-Luubiquitin plasmids (pRK5-HA-Ubiquitin) were provided by Dr. Seongman Kang (Korea University). The siRNA constructs for RIP1, cIAP1, cIAP2, and non-targeted control were obtained from Santa Cruz Biotechnology, Inc. . The non-silencing pGIPZ lentiviral shRNA control vector and GIPZ lentiviral RIP1 shRNAs were purchased from Dharmacon . Human WNT3A (hWNT3A) was purchased from R&D Systems . Cycloheximide (CHX) and the proteasome inhibitor MG132 were purchased from Sigma Aldrich  and EMD Millipore Corp Chemical , respectively. MG132 was dissolved in dimethyl sulfoxide (DMSO) as a 10mM stock solution and stored at −20 °C.HA-tagged IB analyses were conducted as previously described . Anti-RIMock (pcDNA3.1) or Flag-RIP1. After 48 h, cells were harvested and lysed on ice for 60 min in 0.7% NP-40 lysis buffer supplemented with protease inhibitors . The resulting lysates were centrifuged at 13,000 × g for 20 min at 4 °C, and the supernatants were incubated with the anti- β-catenin antibody at 4 °C overnight. Protein G-Sepharose beads  were then added, and bead-bound proteins were analyzed using sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis.Immunoprecipitation (IP) experiments were performed as described previously . BrieflyHCT116 and DLD-1 cells were serum-starved for 16 h and then treated with or without WNT3A (100 ng/ml) for 2 h. After 24 h, the cells were treated with 100 μg/mL cycloheximide  to inhibit protein synthesis. Cycloheximide-treated cells were collected at the indicated time points  and processed for IB using antibodies against RIP1, β-catenin, and β-actin.Ubiquitin for 24 h, followed by incubation with the proteasome inhibitor MG132 (10 µM) for 7 h. Cells were lysed for 60 min at 4 °C in a RIPA buffer  containing the indicated protease inhibitor. Protein concentrations were determined using the Bio-Rad Protein Assay Kit . Cell lysates were immunoprecipitated using an anti-RIP1 antibody, after which the precipitated proteins were subjected to IB analysis and the blots were probed using an anti-HA antibody.In vivo ubiquitylation assays were performed as previously described . HCT116 g for 5 min. The beads were then washed five times with five bead volumes of the Ni-NTA resin wash buffer. The bead-bound proteins were analyzed by IB using anti-GST and anti-HIS antibodies, according to standard protocols.For the in vitro binding assays, 200 ng of GST-tagged β-catenin  was incubated with 200 ng of HIS-tagged RIP1 , in different protein combinations, in 150 μl of cold buffer A  containing 0.1% Triton X-100 for 1.5 h at 4 °C. Ni-NTA  was added, the mixtures were incubated with the proteins for 3 h at 4 °C, and centrifugation was performed at 1000×4) in 200 μL serum-free medium were added to 0.1% bovine serum albumin and seeded into the upper chamber of a transwell system.Migration and invasion assays were performed as previously described . WNT3A-tHuman CRC TMA (CDA3) was purchased from SUPER BIO CHIPS  and CO952a TMA was obtained from US Biomax . Immunohistochemistry (IHC) was performed as previously described .Ctr) or HT29-Luciferase-RIP1 (HT29-Luc-RIP1) cells stably expressing pcDNA3.1 or RIP1 were injected into 20 female NOD/SCID mice through the tail vein (1 × 107 cells/mouse). Mice injected with these cells were separated into two groups (10 mice/group): control (HT29-LucCtr-) and RIP1 (HT29-Luc-RIP1). Tumor metastasis was assessed using an In-Vivo Xtreme bioluminescence system  starting on day 1 and at 2-week intervals for 10 weeks. d-Luciferin  was injected 15 min prior to each bioluminescence scan.The protocols of all animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC No. Kirams 2020-0052). Female NOD/SCID mice (5–6-week old) were purchased from GemPharmatech . HT29-Luciferase-pcDNA3.1  and CO952a TMAs from US Biomax  were performed as follows: The stained TMAs were evaluated with an Olympus BX50 light microscope . For semi-quantitative evaluation of the slides, multi-score of staining frequency and intensity was applied. The percentage of positive cells was divided into five grades (percentage scores), as follows: 0 (<10%); 1 (10–25%); 2 (26–50%); 3 (51–75%); and 4 (>75%). The staining intensity was rated as follows: 0 (no staining); 1 (light brown); 2 (brown); and 3 (dark brown). Positive staining was determined using the following formula: overall score = percentage score × intensity score. Expression was classified into two groups (positive and negative) with a cut-off value based on the median value of the respective overall score.t-test. Error bars indicate standard deviations (SD). Individual p-values are denoted by asterisks .Data were analyzed using the GraphPad Prism software . The significance of differences between experimental groups was determined using Student’s RIP1-/- mouse embryonic fibroblasts (MEFs) for 2 h increased the level of β-catenin in wtMEFs, but not RIP1-/- MEFs  and normal colorectal tissues (n = 9) to determine the expression patterns of RIP1 and β-catenin  and metastatic tissues (n = 10), such as lymph nodes (LN), ovaries, and lungs, indicated that the expression levels of RIP1 and β-catenin were elevated in metastatic tissues  to wild-type (wt) or EFs Fig. . RIP1-/-ols Fig. . To begiion Fig. . These r00 ng/mL nin Fig. . Quantitues Fig. . In thisnts Fig. . Among NRIP1 to evaluate the effect of RIP1 on β-catenin ubiquitination in HCT116 and DLD1 cells. Previous studies have found that, in the absence of WNT stimulation, β-catenin is phosphorylated by a destruction complex that includes Axin, CK1, GSK3, and APC. Phosphorylated β-catenin is ubiquitinated by E3 ligase β-TrCP and degraded by the 26S proteasome. In contrast, under WNT stimulation, the destruction complex binds to the activated Wnt receptor, which leads to inhibition of the phosphorylation and ubiquitination of β-catenin and its subsequent translocation into the nucleus [RIP1  for RIP1 and β-catenin. Knockdown of cIAP1/2 upregulated the expression of RIP1 and β-catenin in WNT3A cells  revealed that the half-lives of RIP1 and β-catenin increased in the presence of WNT3A Fig. . We addi nucleus , 26. IntIP1 Fig. . These dIP1 Fig. . To furtlls Fig. . As prevlls Fig. . FurtherT3A Fig. . TogethecIAP1/2 siRNAs and performed IB. Indeed, the co-application of WNT3A and cIAP1/2 siRNAs clearly induced EMT  exhibited a critical decrease in migration and invasion, to <2-fold that of DLD-1shcon. WNT3A treatment of DLD-1shRIP1 cells intensified this decrease by more than 3-fold  to increase mitochondrial Ca2+ uptake and energy metabolism [RIP1 siRNA. This treatment significantly decreased the expression of endogenous β-catenin in our system (Fig. RIP1 expression levels are associated with the type of cancer. In CRC, in particular, RIP1 is upregulated and promotes cell proliferation via interacting with mitochondrial Catabolism . Our pretabolism , 35, we tabolism . Moreovetabolism . We usedtabolism . Consisttem Fig. , implyintem Fig. . In vitrtem Fig. . These rcIAP1/2 siRNA treatment also decreases the ubiquitination and upregulates the expression of RIP1 and β-catenin. These results are consistent with previous reports that cIAP1/2 can directly conjugate diverse ubiquitin chains to RIP1 to recruit other proteins [We found that RIP1 inhibited the ubiquitination of β-catenin to decrease its proteasomal degradation Fig. . β-catenproteins , the othproteins , 26. TheTaken together, our results suggest that RIP1 may be a novel EMT inducer that promotes metastasis in CRC by decreasing the ubiquitination and increasing the stability of β-catenin in WNT/β-catenin signaling. This novel role of RIP1 in WNT signaling may suggest that crosstalk between TNF and WNT signaling may modulate CRC malignancy. Also, given that many researchers are seeking to develop novel drugs targeting RIP1 and WNT signaling, our study could support the development of new combinatorial therapeutic strategies for regulating CRC by controlling the two major inflammatory pathways of TNF and Wnt/β-catenin signaling.Supplementary FiguresSupplementary Figure legendssupplementary western image