Alterations in the Wnt signaling pathway are associated with the advancement of cancers; however, the exact mechanisms responsible remain largely unknown. It has recently been established that heightened intratumoral Wnt signaling correlates with tumor immunomodulation and immune suppression, which likely contribute to the decreased efficacy of multiple cancer therapeutics. Here, we review available literature pertaining to connections between Wnt pathway activation in the tumor microenvironment and local immunomodulation. We focus specifically on preclinical and clinical data supporting the hypothesis that strategies targeting Wnt signaling could act as adjuncts for cancer therapy, either in combination with chemotherapy or immunotherapy, in a variety of tumor types.  The Wnt/β-catenin signaling pathway plays a vital role in many cellular functions. Alterations in the pathway are associated with the advancement of cancers. Wnt pathway aberrations have also been correlated with changes in the tumor microenvironment, such as immune evasion, and this has been the topic of two recent reviews ,2. The eWnt pathways act as critical signal transduction cascades that modulate embryonic development, adult homeostasis, stem cell control, and wound repair . The firAs an initial step in this signaling cascade, Wnt ligands must be processed and exported . Wnt proTraditionally, Wnt pathways are associated with intracellular β-catenin stabilization . Cytopla2+ signaling pathways. Most of these pathways overlap to transduce calcium-dependent cell signaling [2+ from the endoplasmic reticulum, leading to activation of protein kinase C (PKC). PKC activates nuclear factor kappa-B (NFκB) and cAMP response element binding protein (CREB). Calcineurin (Cn) and calcium/calmodulin-dependent protein kinase type II (CamKII) are also activated, leading to activation of the nuclear factor of activated T cells (NFAT) and NFκB. NFκB, CREB, and NFAT translocate to the nucleus causing transcription of regulatory genes [Most studies have focused on the role of Wnt in the canonical signaling pathway; however, Wnt can also signal through a number of noncanonical pathways. These pathways include the Wnt-JNK, Wnt-RAP1, Wnt-Ror2, Wnt-PKA, Wnt-GSK3MT, Wnt-aPKC, Wnt-RYK, Wnt-mTOR, and Wnt/Caignaling . It is tignaling . Ror1/2 ry genes . Althougry genes . There are several protein families and genes known to antagonize or modulate Wnt signaling. DKK1, a member of the Dickkopf (DKK) family of proteins, is thought to act as a LRP5/6 ligand antagonist  Figure . There aTCF7L2. This gene encodes for T cell factor-4 (Tcf-4), which forms the β-catenin/Tcf-4 transcription complex. A study by Korinek et al. demonstrated that cessation of Wnt signaling resulted in a loss of intestinal stem cells, leading to a breakdown of the intestinal epithelium [+ tumor infiltration, as related to tumor PD-L1 expression and the effects on cancer progression, are also being investigated [The Wnt signaling pathway is known to be involved in regulating the self-renewal capacity of non-malignant stem cells. For example, an association between Wnt and adult stem cells was established with the gene disruption of mouse ithelium . Wnt sigithelium . In contithelium . Intriguithelium . Overexpithelium . Additioithelium . In addiithelium . There iithelium . Potentistigated . Due to + T cell differentiation into effector cells, promoting self-renewing multipotent CD8+ memory stem cells and maintaining the “stemness” of mature memory CD8+ T cells [With its role in cellular regulation, there is ample evidence that Wnt signaling affects hematopoietic stem cells. Wnt stimulation of hematopoietic stem cells may increase their self-renewal capacity . For exa T cells . This coGSK3B may lead hematopoietic stem cells to progress to acute myeloid leukemia [The associations between Wnt signaling and cancer progression are the topic of intense investigation. This correlation was first established when a WNT factor gene was identified as an oncogene in the mouse mammary cell line RAC311c . Our undleukemia . Mutatioleukemia ,53. It ileukemia . Inactivleukemia ,56. Whil+ T cells, and high CD8+/CD4+ ratios have been correlated with improved overall survival in ovarian cancer [Despite tremendous advancements in traditional chemotherapies, many oncology patients continue to experience rapid progression of disease characterized by chemotherapy resistance, which limits therapeutic options. This has led to the pressing need to investigate alternative methods of treatment, such as immune-directed therapies. Many key aspects of the relationship between the immune system and solid tumors have been elucidated over the last 20 years. There is an appreciable complexity of this system that results in either tumor suppression or tumor progression. It is now established that in order for immune cells to recognize cancer cells and control cancer progression and metastasis, they must first infiltrate the tumor, then remain activated in the TME . The pren cancer . The maiThe Cancer Genome Atlas (TCGA) has provided much insight into the role of the immune system in various types of cancers through combined analysis of genomic and patient outcome data. With this resource, TME may be examined from a transcriptional viewpoint, permitting more nuanced cancer categorizations to be made. For example, Thorsson et al. recently performed an immunogenomic analysis of 10,000 tumors from 33 cancer types to identify underlying immune subtypes that are common to all cancers examined . The six+ T cell differentiation by repressing CD4 genetic networks [Ctnnb1, the gene encoding for β-catenin, or deletions in Tcf7, the gene encoding TCF1, were shown to block thymocyte development [Tcf7 is deleted in CD8+ T cells, functional T cell memory is impaired [The differentiation of multiple leukocyte populations is regulated by Wnt signaling pathways. Alterations in canonical Wnt signaling may have a genomic influence on T cell development. TCF1 and LEF1 genes have been linked to epigenetic changes that may promote CD8networks ,64. Furtelopment ,66. Whenimpaired . Howeverimpaired . Thus, t-/-Tcf7 mice [-/-Lef1 and -/-Fzd9 mice, B cell precursors were diminished in the bone marrow [Ctnnb1 deletion in B cell precursors [Other immune cell lineages are also influenced by canonical Wnt signaling. Innate lymphoid cells, including natural killer (NK) cells, require TCF1 for development, as shown by the earliest linage-specific precursor expressing high levels of TCF1, and defective NK cell survival in -/- mice ,70. In Le marrow . Howeverecursors . Furtherecursors . The obs+ and CD8+ T cell protective immunity [Multiple leukocyte populations within the TME are influenced by both Wnt stimulators and inhibitors. Key Wnt-related immunological alterations are illustrated in immunity . This inH1 responses [H1 cells [+ T cells had sustained β-catenin activation, resulting in TH17 polarization and production of proinflammatory cytokines that favor tumorigenesis [Noncanonical pathway stimulation may support a tumor proliferative environment. WNT5A stimulation has been shown to increase IL-12 production from DCs, causing an increase in Tesponses . AlternaH1 cells . These Wigenesis . It has igenesis . These r+ CTLs for tumor recognition [-/- mice, with a CD11c-specific deletion of β-catenin, vaccination with tumor antigen failed to provide tumor protection. Interestingly, these mice were found to be deficient in CD8+ T cell immunity [Immune tolerance is also impacted by Wnt signaling. Irregularities in antitumor cytotoxic T lymphocyte (CTL) priming are associated with Wnt signaling in DCs. The high levels of Wnt ligands found in the TME condition DCs to a regulatory state  Figure . This suognition ,86. Throimmunity . These fEvidence suggests a strong correlation between Wnt pathway changes and immune exclusion. This was recently evaluated with TCGA data. Multiple cancers were analyzed for gene expression of TILs and categorized into a high, intermediate, or low T cell-inflamed tumor environment, based on their expression for genes associated with T cell infiltration. These tumors were then profiled for Wnt/β-catenin-related gene expression profiles. Up to 90% of tumor types showed an inverse correlation between Wnt/β-catenin pathway activation and a T cell-inflamed gene expression signature , suggestGSK3 inactivation in mouse melanoma resulted in a repression of the PD-1 gene, allowing an improved CD8+ response [Immunoevasion mechanisms may also be represented through alterations in immune checkpoint molecules due to Wnt signaling component changes. response . Howeverresponse . As immuSome immunotherapies are directed at the proteins that regulate T cell function and cytolytic activity. In particular, immune checkpoint inhibitors (ICIs) are monoclonal antibodies against receptors such as CLTA-4 and Programmed Death-1 (PD-1) that act to down-modulate T cell effector function. ICI are now approved for the treatment of malignant melanoma, non-small-cell lung cancer, classical Hodgkin lymphoma, head and neck squamous cell carcinoma, urothelial carcinoma, and renal cell carcinoma . In addiIn a similar fashion, ipilimumab and tremelimumab are monoclonal antibodies to CTLA4, which normally functions to restrain effector T cell activation. Ipilimumab is known for its significant progression-free and overall survival advancement in melanoma . FurtherWith the rising use of mono- and combinatorial immunotherapy in the clinical setting, there is a need to further understand the TME in patients who do not respond. It has been reported that decreased TILs result in resistance to ICIs . Wnt patThere are numerous relationships between Wnt signaling, immune function, and cancer progression; most notable is that of increased Wnt signaling correlating with decreased tumor T cell infiltration, as discussed above. Wnt pathways have a vast number of roles that offer multiple options for pathway modulation in the malignant setting. The United States Patent and Trade Office Patent and Patent Application databases report 103 unique Wnt signaling modulators being investigated, with 34 in clinic trials . ActionsNumerous studies are currently combining Wnt therapies with ICIs, additional Wnt inhibitors, or chemotherapies in an attempt to achieve optimal effects on tumor control. However, it is currently unknown how the combination of therapies will affect tumor progression. In colon cancer, changes in Wnt signaling pathways have shown counter-intuitive results in some studies, such as WNT-TCF blockade actually boosting metastasis . In cont+ T cells. It will be interesting to view future clinical trial results related to effects on the TME that occur following targeted inhibition of a Wnt inhibitor, with and without combination therapy. Therapeutic modulation of Wnt signaling is being investigated through many avenues, several of which are depicted in Alternative therapeutic agents act directly on Wnt ligand secretion. PORCN inhibitors are known to block the extracellular excretion of Wnt by blocking the enzyme responsible for palmitoylation of Wnt ligands . This faArtemisia annua, used as an FDA-approved antimalarial drug. Treatment of colorectal tumor xenografts with this agent correlated with decreased growth of tumors with inhibition of a hyperactive Wnt/β-catenin pathway [Some Wnt altering agents have been previously approved in nonmalignant diseases. Artesunate is a compound extracted from the herb  pathway . The exa pathway . There aAdditional Wnt-inhibiting agents are being tested in clinical trials. Ipafricept, also known as OMP54F28, is a recombinant fusion protein with an extracellular Fzd 8 receptor portion attached to an IgG1 Fc fragment, which acts as a decoy receptor for Wnt ligands . Four phHere, we have reviewed preclinical and clinical data that support the continued investigation of combining Wnt signaling modulators with cancer chemotherapies or immunotherapies, in order to achieve better tumor control in a greater percentage of patients. As discussed, Wnt signaling is involved in regulating a wide variety of complex cellular functions, in both malignant cells and leukocytes. Furthermore, aberrations in Wnt signaling are now well-established in a multitude of malignancies and elevated Wnt signaling shows a strong correlation with overall immune suppression. There is also evidence to suggest that tumor stem cell promotion, via enhanced Wnt signaling, may contribute to immune evasion. Thus, finding ways to alter Wnt signaling specifically in tumor cells and/or tumor stem cells could provide a platform for inducing beneficial changes in the immune-related TME, leading to improved cancer treatment efficacy. Wnt-modulating agents on clinical trial include DKK1 antibodies, PORCN inhibitors, AXIN1 activators, Wnt decoys, WNT5A mimics, β-catenin inhibitors, among others with less clearly-defined modes of action. At this time, additional studies are needed to more fully understand how Wnt-modulating agents are altering the intratumoral immune response and broader TME. However, the synergistic effects seen to date with ICIs or chemotherapies used in combination with Wnt inhibitors lends validity to the idea that targeting the Wnt pathway is a promising therapeutic approach for many tumor types, as doing so may promote protective anti-tumor immunity and convert the tumor milieu to one more susceptible to traditional therapies.


Background: Stent-jailing and stent-jack are used for stent-assisted coil embolism (SCE) in intracranial aneurysm (IA) therapy, and cause different incidences of IA recurrence. Angiogenesis strongly correlates with aneurysm accumulation. Stent-jack causes higher mechanical forces in cerebral vessels than stent-jailing. Mechanical forces, as well as TGF-β/Smad2,3,4 signaling pathway, may play an important factor in IA recurrence by affecting angiogenesis.Methods: We explored the effects of stent-jailing or stent-jack technique on IA recurrence by investigating mechanical forces, TGF-β/Smad2,3,4 signaling pathway and the incidence of angiogenesis in IA patients. One-hundred-eighty-one IA patients were assigned into stent-jailing (n = 93) and stent-jacket groups (n = 88). The clinical outcome was evaluated using Glasgow Outcome Score (GOS) and aneurysm occlusion grades. The percentage of CD34+EPCs (releasing pro-angiogenic cytokines) in peripheral blood was measured by flow cytometer. Endothelial cells were separated from cerebral aneurysm and malformed arteries via immunomagnetic cell sorting. Angiogenesis was measured by microvessel density (MVD) using anti-CD34 monoclonal antibody staining before using the stent, immediately after surgery and 2 years later. Meanwhile, the mechanical forces in cerebral vessels were determined by measuring endothelial shear stress (ESS) via a computational method. TGF-β and Smad2,3,4 were measured by real-time qPCR and Western Blot. Tube formation analysis was performed to test the relationship between angiogenesis and TGF-β, and the effects of different techniques on angiogenesis.Results: After a 2-year follow-up, 85 and 81 patients from stent-jailing and stent-jack groups, respectively, completed the experiment. Stent-jailing technique improved GOS and reduced aneurysm occlusion grades higher than the stent-jack technique (P < 0.05). The counts of CD34+EPCs and MVD values in the stent-jailing group were lower than the stent-jack group (P < 0.05). ESS values in sent-jailing group were lower than the stent-jack group (P < 0.05), and positively correlated with MVD values (P < 0.05). TGF-β and Smad2,3,4 levels in sent-jailing group were also lower than the stent-jack group (P < 0.05). TGF-β was associated with angiogenesis incidence and stent-jack caused angiogenesis incidence more than stent-jailing.Conclusion: Stent-jailing technique reduces IA recurrence more than stent-jack by causing less mechanical forces, angiogenesis and inhibiting TGF-β/Smad2,3,4 signaling in IA patients. Intracranial aneurysm (IA) is a common cerebral disease, which involves various organs and becomes more prevalent with high-level morbidity and mortality . With thStent-jailing technique represents an efficacious adjuvant technique for treating wide-necked persistent trigeminal artery aneurysm , in whicComplicated IAs are often existed in the internal and middle cerebral artery. Embolization of irregular and complicated IAs is still a challenge. The stent-jailing technique  or stentStent-Jailing technique and stent-jacket technique may affect the recurrence and regrowth of IA since stent can induce angiogenesis , which iRabbit anti-Smad2 (ab63576), anti-Smad2 around the phosphorylation site of serine 467 (ab63576), Anti-Smad3 (EP568Y), anti-Smad3 phosphorylated on Serine 423 and 425 (ab52903), anti-Smad4 (EP618Y), anti-CD31 (ab28364), anti-CD34 (ab8536) and goat anti-rabbit HRP (IgG H&L) (ab6721) antibodies were purchased from Abcam trading (Shanghai) co., ltd. . Anti-CD 146-coated dynabeads were purchased from Invitrogen Corporation .Before the present experiment, all steps were approved by Human Research Ethics Committee of the First Hospital of Jilin University , and all patients agreed to sign the informed consent. IAs were measured via digital subtraction angiography (DSA), magnetic resonance angiography (MRA) and computed tomographic angiography (CTA). CTA and MRA can locate the position and show characteristics of IAs. DSA can improve the accuracy of the diagnoses. Severity of IA was evaluated by using Glasgow Outcome Scale (GOS) .The following patients were included:  > 18 yeaThe following excluding criteria were used:  the patiNimodipine was used and act to inhibit cerebral vasospasm. IA subjects were orally administered with 80-mg clopidogrel (daily) and 100-mg aspirin (daily) 3 days before surgery. Before half-an-hour surgery, patients were injected with one-mg atropine sulfate to prevent gland secretion.n = 93) and stent-jack groups (n = 88) based on self-selection after consulting with the brain aneurysm specialists. To eliminate the self-selection bias, a little adjustment was performed to keep no significant difference for IA types and clinical characteristics between two groups after receiving individual agreement  . GOS cat+EPCs are involved with the release of pro-angiogenic cytokines and associated with pathological angiogenesis  was used for biopsy specimens’ retrieval from an occluded IA. Cerebral aneurysm and malformed arteries were isolated from cerebral arties using a cutting plane and separating the surfaces on either side of the plane by using a minimally invasive technique. Endothelial cells were derived from human cerebral aneurysm and malformed arteries, and separated with immunomagnetic cell sorting. Briefly, Anti-CD 146-coated Dynabeads  were prepared according to manufacturer’s instruction and stored at 4°C. Fifty-mg aneurysms were ground with glass pestle and mortar with one-millilter PBS buffer, 0.1% bovine serum albumin, 0.1% sodium azide, and 0.1% a standard broad-spectrum inhibitor cocktail at 4°C. Ten-microliter FcR-blocking agent  and 25-microliter antibody-coated Dynabeads were added and mixed thoroughly. The samples were mixed in a mixer for 1 h at 4°C and washed four times with PBS inside the Big Easy Magnet  at 4°C. Between each washing procedure, the endothelial cells were flushed out with MACS buffer  10 times in a 100-μL pipette.Angiogenesis is associated with MVD and can be evaluated by MVD . For MVDin vivo exactly. However, the mechanical forces mainly produced shear stress due to blood flow . The isolated RNA was digested by DNase I  according to the manufacturer’s instructions. Two-microliter total RNA was used for reverse transcription to cDNA with the reverse transcription kit . The primers for specific genes and β-actin were listed in Table Frozen samples endothelial cells were lysed in RIPA buffer containing 1% (w/v) of protease/phosphatase inhibitor cocktail . Protein concentration was measured by BCA detection kit . The mixed proteins (100 μg) were separated by using SDS-PAGE and transferred to a PVDF membrane for 20 min. Antibodies were incubated overnight at 4°C. The membrane was blocked by using 5% non-fat milk in PBST buffer for 1 h. Secondary antibodies were further incubated and protein bands were visualized chemiluminescence (ECL) system .3 cells in 96 wells overnight. The cells were treated with 2 μM TGF-β inhibitor (SB505124) , or 10 ng/mL TGF-β , the sample supernatants from stent-jailing and stent jack groups for 48 h. A 96-well plate was coated with Matrigel (BD Biosciences) and was cultured at 37°C for half an hour to let Matrigel solidify. HUVECs were plated onto the plate coated by Matrigel. After 20-h culture, the HUVECs were recorded using Olympus IX71 Inverted Compound Microscope .HUVECs were purchased from Shanghai Institutes for Biological Sciences (Cat. No. ECV304) were cultured in RPMI-1640 medium  supplemented with 10% fetal calf serum at a density of 4 × 10The statistical analysis was carried out by using SPSS 20.0. The possible factors associated with IA risks and postoperative symptoms were analyzed by using univariate and multi-factor analysis. The relationship between the levels of MVD values and ESS values was analyzed using Spearman’s correlation coefficient test.P > 0.05).From April 1st, 2016 to March 1st, 2017, 181 IA patients received SCE therapy, including 97 males and 69 females, aged 35.9–81.7 years. Complete coil embolization was 65 cases (76.47%) and 66 cases (81.48%) in stent-jailing and -jack groups, respectively. After a 2-year follow-up period, 6 and 4 patients withdrew from two groups, respectively. Two and 3 patients were died from two groups because of aneurysmal subarachnoid hemorrhage (SAH), respectively. Thus, 85 and 81 patients completed the study, respectively. The statistical differences for all parameters were insignificant between the two groups  or nimodipine (20–30 mg/day) was administrated to prevent the development of vasospasm. There were 4 and 2 cases of cerebral vascular occlusion in the two groups, respectively. Balloon was deployed to dilate blood vessels. There were 2 cases of embolism coil pop-up in stent-jack group and no case of embolism coil pop-up in stent-jailing group.P > 0.05). In contrast, the statistical differences for aneurysm occlusion grade were still insignificant between two groups after immediate SCE surgery (Table P > 0.05) but significant after 2-year follow-up (Table P < 0.05). The results suggested that stent-jailing was more effective than stent-jack in the prevention of aneurysm occlusion.Aneurysm occlusion grades reflect the severity of ischemic stroke . The sta+EPCs was insignificant between stent-jailing and stent-jack groups . After 2-year follow-up, the counts of CD34+EPCs in the stent-jailing group were lower than in the stent-jack group . The results suggest that stent-jack increases the counts of CD34+EPCs, which may increase the incidence of angiogenesis.Before SCE surgery and after immediate SCE surgery, the statistical difference for the counts of CD34P > 0.05, Figures 2. After 2-year follow-up, MVD values in the stent-jailing group were lower than in the stent-jack group . After 2-year follow-up, relative protein levels of TGF-β, Smad2, Smad3, and Smad4 were still comparable between stent-jailing and stent-jack groups .The results suggested that stent-jack promoted phosphorylated TGF-β-sphoryl phosphorylation of Smad2 and Smad3 when compared with stent-jailing technique.Relative protein levels of TGF-β, phospho-TGF-β, Smad2, phospho-Smad2, Smad3, phospho-Smad3 and Smad4 were analyzed by using Western Blot Figure . Before P < 0.05). In contrast, SB505124 potently inhibited the proliferation and tube formation of HUVECs . Comparatively, stent-jailing potently reduced more angiogenesis than stent-jack . The results proved that stent-jailing and stent-jack may cause different angiogenesis by affecting TGF-β.Transforming growth factor-β increased cellular proliferation and promoted angiogenesis when compared with controls Figures  (P < 0.0 Figures  (P < 0.0 Figures –F (P < 0Stent-assisted coil embolism is an important strategy in the therapy of IAs. Compared with craniotomy, endovascular embolization has the advantages of less trauma, fewer complications, lower mortality and short procedure time . AlthougThere were 85 aneurysms treated by stent-jailing technology and 81 aneurysm treated by stent-jack in the present work. When stent-assisted embolism is carried out, the choice between the two methods will depend on the expert’s experiences. Stent-jailing method has more advantages than stent-jack. Firstly, catheter placement into aneurysm dome is more difficult after the deployment of stent, especially for closed and small stents. Secondly, like a balloon, the deployed stent will avoid the kickback of the jailing catheter during coil embolism. Thirdly, the occurrence of coil herniation into the parent artery is low. Finally, unintended untangling of coils around the stent-jack can be avoided in stent-jailing technique. The main shortage of stent-jailing technique is that the catheter tip is forced out from aneurysm dome during stent deployment. More coil loops can be deployed to solve the drawback.Stent-jack can be considered when it is difficult in the application of stent-jailing technique because the stent-jailing technique cannot be adjusted to a satisfactory location. Due to the small opening of aneurysms have been small, scaffold can enter the aneurysm dome via stent-jack. One main drawback for stent-jack is that the detached coil loops may herniate or move, and hang on stent when the stent retrieved.+ progenitor cells can be stimulated by many cytokines (+EPCs did not increase much after stenting in IA patients between two groups (Figures Peripheral blood CD34ytokines . Surgeryytokines . We prop Figures . After 2 Figures  when com Figures , suggest Figures , hepatoc Figures  and fibr Figures .P < 0.05, Figure We showed that stent-jailing rather than stent-jack reduced angiogenesis partly via reducing mechanical forces in cerebral blood vessels or the TGF-β/Smad2,3,4 pathway. The mechanical forces may stimulate endothelial-cell-inducing angiogenesis . Stent-jin vivo. Some long-term complications existed for the patients that undergo aneurysm treatment. First, in-stent thrombosis may be a source of complications because of the angiogenesis caused by mechanical forces. Second, mechanical forces could be technically problematic and induced a further risk of complications. Thus, in-stent thrombosis may be higher. In the present study, aneurysm neck was mainly covered by stents. Although angiogenesis is associated with aneurysm formation (There were some limitations of the present work. The interaction of stents with the cerebral endothelial cells in a controlled environment has been widely reported . The steormation , angiogeStent-jailing technique reduced aneurysm occlusion grades more than stent-jack technique after 2-year follow-up by controlling angiogenesis in IA patients. Stent-jailing technique was more effective than stent-jack technique in the treatment of IA patients by reducing mechanical forces and activity of TGF-β/Smad2,3,4 signaling pathway. To confirm the present conclusion, further work in highly demanded in a larger population in the future.NX, HM, and TL performed the experiments, enrolled patients, and analyzed the related data. YF and YQ measured MVD and ESS values. DZ and HW contributed to design the techniques. NX, HM, TL, YF, YQ, and DZ performed the surgery. DZ and HW conceived the experimental plan and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Heterotrimeric G proteins are signal transduction proteins involved in regulating numerous signaling events. In particular, previous studies have demonstrated a role for G-proteins in regulating β-catenin signaling. Pasteurella multocida toxin was utilized as a unique tool to activate G-proteins, with LiCl treatment serving as a β-catenin signaling agonist. The results show that Pasteurella multocida toxin (PMT) significantly enhanced LiCl-induced active β-catenin levels in HEK293T cells and mouse embryo fibroblasts. Evaluation of the effect of specific G-alpha proteins on the regulation of β-catenin showed that Gq/11 and G12/13 knockout cells had significantly higher levels of active and total β-catenin than wild-type cells. The stimulation of active β-catenin by PMT and LiCl was lost upon both constitutive and transient knockdown of G12 and G13 but not Gq. Based on our results, we conclude that endogenous G-alpha proteins are negative regulators of active β-catenin; however, PMT-activated G-alpha subunits positively regulate LiCl-induced β-catenin expression in a G12/13-dependent manner. Hence, G-alpha subunit regulation of β-catenin is context dependent.Heterotrimeric G proteins are signal transduction proteins involved in regulating numerous signaling events. In particular, previous studies have demonstrated a role for G-proteins in regulating β-catenin signaling. However, the link between G-proteins and β-catenin signaling is controversial and appears to depend on G-protein specificity. We describe a detailed analysis of a link between specific G-alpha subunits and β-catenin using G-alpha subunit genetic knockout and knockdown approaches. The  PMT is a potent intracellularly acting toxin which activates three families of heterotrimeric G-proteins: Gq/11, G12/13, and Gi/o .Since PMT was found to be a potent activator of G-proteins and enhanced the levels of LiCl-induced active β-catenin, we next investigated the role of specific G-alpha subunits by using MEFs depleted for either Gr G12/13 . Western−/− MEFs . Interespe cells . This survations . The levq/11 or G12/13 subunits had ∼8-fold or 20-fold higher levels of active β-catenin, respectively, than wild-type MEFs  cell cytotoxicity (data not shown). Since both Gq/11−/− and, in particular, G12/13−/− null cells have high endogenous levels of active β-catenin  against G subunit .q, G12, and G13 in the regulation of active β-catenin were assessed individually. First, in contrast to the targeted null cells shown earlier  site for G-alpha proteins, in particular for activated G12 , and that long-term chronic treatment of cells with PMT continuously over months changes their endogenous G-alpha subunit content . Our data also show that cells lacking G-proteins in the long term show potentially deleterious levels of β-catenin. However, short-term inhibition of G-proteins has no direct effect on β-catenin levels but was shown to alter agonist-mediated β-catenin signaling in a specific G-alpha-dependent way. Studying specific activated G-alpha subunits using PMT has revealed a specific role for G12 and G13 in cooperating with β-catenin signaling. Given the established roles of altered G12/13 proteins and β-catenin in tumorigenesis, it is tempting to speculate that PMT-induced G-protein fine-tuning of β-catenin signaling supports the procarcinogenic signature of chronic PMT exposure that we have previously hypothesized  and incubated with radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitor cocktail (1:100 dilution)  and phosphatase inhibitor cocktail (1:100 dilution)  for 5 min on ice. Cells were removed using cell scrapers, and the whole-cell lysates were transferred to a microcentrifuge. The tubes were centrifuged at a relative centrifugal force of 14,000 for 15 min, and the supernatant was aliquoted and stored at −80°C. For anti-QE antibody preparation, hybridoma cells  were cultured in hybridoma-SFM medium supplemented with recombinant human interleukin-6 (IL-6) and purified as described previously (Wild-type mouse embryonic fibroblasts (wild-type MEFs), G Germany ). MEFs aeviously .The proteins were separated on 4 to 12% gradient gels and electrotransferred onto nitrocellulose membrane. The following antibodies were used: anti-active β-catenin antibody  and total anti-β-catenin antibody . All of the G-protein antibodies were purchased from Santa Cruz Biotechnology : anti-Gα12 (sc- 409), anti-Gα13 (sc-410), anti-Gαq (sc-393), anti-Gα11 (sc-394), anti-Gαs (sc-823), anti-Gαi-1 (sc-391), anti-Gαi-2 (sc-7276), anti-Gαi-3 (sc-262), and anti-Gαo (sc-387). Bands were analyzed using the ChemiDoc MP imaging system from Bio-Rad. The band intensity was measured using Image Lab software (version 5.2.1). The band intensity of each protein was normalized against its β-actin housekeeping gene .4 cells/ml) were cultured on 12-mm collagen-coated coverslips under standard conditions as described above and treated with the respective compounds. Cells were then fixed with 4% paraformaldehyde (PFA) for 1 h at room temperature (RT) and permeabilized with 0.2% Triton X-100 in PBS for 15 min at 4°C. They were then incubated with blocking buffer containing 1% bovine serum albumin in PBS for 30 min at RT. The primary anti-active β-catenin antibody and secondary anti-rabbit fluorescent antibodies were diluted in blocking buffer. The cells were incubated with the primary antibody (1:100) at 4°C overnight, washed with PBS, and incubated with secondary antibody (1:500) for 1 h at RT. The cells were washed with PBS three times and mounted using Vectashield mounting medium containing 4′,6-diamidino-2-phenylindole . The imaging was carried out using an ApoTome Zeiss fluorescence microscope, and the images were captured using an AxiCAm MRm Zeiss camera and processed on AxioVision Rel 4.8 software.Cells (5 × 104 cells/well) were plated in a 24-well plate in medium containing no antibiotics and cultured overnight under standard conditions. Specific siRNAs and the transfection reagent were diluted in media without antibiotics in separate tubes. The contents of each tube were gently mixed and incubated for 5 min and then mixed and incubated for a further 20 min. Media were added into the above-described mix to obtain a final siRNA concentration of 25 nM. The cells were incubated with medium containing each siRNA for 72 h. Cells with each respective G-protein knockdown were analyzed by Western blotting for decreased expression and then used for further experiments. Significant knockdown of GNA11 expression was not achieved and was not pursued further.siRNAs developed against GNAQ, GNA12, and GNA13 were purchased from GE Healthcare Dharmacon, Ltd. : ON-TARGET plus human GNAQ (L-008562-00-0005), GNA12 (L-008435-00-0005), GNA13 (L-009948-00-0005), and nontargeting pool (D-001810-10-05). The transfection protocol was performed according to the manufacturer’s instructions. Briefly, HEK293T cells (2 × 10TC method (where TC is threshold cycle) was used for data analysis.For qPCR analysis, cells were cultured until confluence, and RNA was extracted using TRIzol . Reverse transcription was performed using an RTnanoscript2 kit (Primer Design). The following primers were used: β-actin, 3′-GGCTGTATTCCCCTCCATCG-5′ and 5′-CCAGTTGGTAACAATGCCTGT-3′; axin2, 3′-TGACTCTCCTTCCAGATCCCA-5′ and 5′-TGCCCACACTAGGCTGACA-3′. The ΔΔAll experiments were repeated at least three independent times. One-way analysis of variance and Tukey’s test were used to assess the difference between treatments using GraphPad Prism software, version 7.


KRAS mutations are insensitive to them and do not have an alternative drug to overcome the problem. The levels of β-catenin, EGFR, and RAS, especially mutant KRAS, are increased in CRC patient tissues due to mutations of adenomatous polyposis coli (APC), which occur in 90% of human CRCs. The increases in these proteins by APC loss synergistically promote tumorigenesis. Therefore, we tested KYA1797K, a recently identified small molecule that degrades both β-catenin and Ras via GSK3β activation, and its capability to suppress the cetuximab resistance of KRAS-mutated CRC cells. KYA1797K suppressed the growth of tumor xenografts induced by CRC cells as well as tumor organoids derived from CRC patients having both APC and KRAS mutations. Lowering the levels of both β-catenin and RAS as well as EGFR via targeting the Wnt/β-catenin pathway is a therapeutic strategy for controlling CRC and other types of cancer with aberrantly activated the Wnt/β-catenin and EGFR-RAS pathways, including those with resistance to EGFR-targeting drugs attributed to KRAS mutations.Drugs targeting the epidermal growth factor receptor (EGFR), such as cetuximab and panitumumab, have been prescribed for metastatic colorectal cancer (CRC), but patients harboring  A recently identified small molecule shows promise for tackling resistance to a leading colorectal cancer drug. Three proteins that are over-expressed in colorectal cancer are epidermal growth factor receptor (EGFR), RAS and β-catenin. These proteins and their interconnected signaling pathways are therefore important therapeutic targets. EGFR is the target of the drug cetuximab, but many patients are resistant to this drug attributed to mutations in a gene that influences the signaling pathways of the three key proteins. Kang-Yell Choi at Yonsei University in Seoul, South Korea, and co-workers trialed a novel molecular drug on human colorectal cancer tissues and on mice. They confirmed that the new drug leads to reduced EGFR levels by degrading RAS and β-catenin and therefore suppresses the growth of colorectal cancer cells in samples with or without the resistant mutations. Both APC and KRAS mutations synergistically promote cellular transformation and tumor growth5, which is attributed to the activation of the RAS-ERK pathway via the aberrant activation of the Wnt/β-catenin signaling caused the loss of APC8. One of the key events in the crosstalk between the two pathways is the stabilization of RAS, especially oncogenic mutant KRAS, as well as β-catenin, caused by APC loss. RAS, as well as β-catenin, is degraded via glycogen synthase kinase 3 beta (GSK3β)-mediated phosphorylation and subsequent poly-ubiquitination-dependent proteasomal degradation; however, the inactivation of GSK3β by the loss of APC stabilizes these proteins10.The Wnt/β-catenin and RAS/extracellular signal-regulated kinase (ERK) pathways play important roles in the regulation of various cellular events, including proliferation, differentiation, and transformation. Mutations in genes in both pathways, such as 12, is also overexpressed in human CRC and plays a synergistic role with the Wnt/β-catenin pathway in tumorigenesis15. Therefore, therapies targeting both the Wnt/β-catenin and EGFR-RAS-ERK pathways, especially those lowering the levels of β-catenin, RAS, and EGFR, can be ideal approaches for the treatment of CRC. Based on the identification of the mechanism of RAS destabilization by the Wnt/β-catenin signaling and the large increase in both β-catenin and RAS levels in human CRC, we screened and characterized small molecules that suppress the growth of CRC cells via the destabilization of both β-catenin and RAS17. KYA1797K, a small molecule derived from the chemical synthesis of an initial hit, directly interacts with the N-terminal regulator of G protein signaling (RGS) domain of axin and results in the phosphorylation and subsequent degradation of both β-catenin and RAS via the activation of GSK3β16.The epidermal growth factor receptor (EGFR), a transcriptional target of the Wnt/β-catenin pathwayKRAS mutations20, and have no alternative therapy. Therefore, we tested the effects of KYA1797K on the growth and transformation of KRAS-mutated CRC cells resistant to cetuximab because KYA1797K reduces both β-catenin and RAS protein levels. KYA1797K suppresses the growth and transformation of CRC cells, reduces tumor xenograft growth and decreases the colony formation and growth of tumor organoids derived from CRC patient tissues insensitive to cetuximab due to KRAS mutations via the degradation of oncogenic KRAS and β-catenin with EGFR repression.The drugs targeting the EGFR-RAS-ERK pathway, such as the anti-EGFR antibody drugs including cetuximab and panitumumab, have been prescribed as therapeutic drugs for patients with cancers such as metastatic CRC. However, these drugs have limitations in their usage due to the poor efficacy or the insensitivity in patients harboring APC, KRAS, and EGFR mutations.Together, small molecules that degrade both β-catenin and RAS via the transcriptional repression of EGFR can be a potential therapy for the treatment of CRC patients with aberrantly activated Wnt/β-catenin and EGFR-RAS pathways attributed to the increase in β-catenin, EGFR, and RAS as well as to their activation by pathologically important KRAS 21 were provided by B. Vogelstein . DiFi cells were provided by Won-Ki Kang . All the cell lines were authenticated using short tandem repeat profiling . RKO cells were cultured in DMEM  supplemented with 10% fetal bovine serum . HCT15, DLD1, SW480, DiFi, D-WT, and D-MT cells were maintained in RPMI 1640 medium (Gibco Life Technologies) supplemented with 10% FBS. For the establishment of stable cell lines, D-WT cells were transfected with Myc-KRAS-WT-pcDNA3.1, Myc-KRAS-G12V-pcDNA3.1, or empty-pcDNA3.1 plasmid, and were selected in a medium containing G-418 . For the establishment of the APC knockout (KO) cell lines, RKO cells were transfected with the LentiVRISPRv2 vector containing the APC gene guide sequence and were selected in a medium containing puromycin . Lipofectamine  and Lipofector-EZ  were used for plasmid transfection according to the manufacturer’s instructions. KYA1797K was dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich) for the in vitro studies. Cetuximab  was obtained from Merck .Colorectal cancer (CRC) cells, including HCT15, RKO, SW480, and DLD1, were purchased from the American Type Culture Collection . Isogenic human CRC cells, DLD-1 cells harboring wild-type (WT), or mutant (MT) 9. To construct the APC knockout vector using the CRISPR-CAS system, we designed and synthesized the guide oligos targeting the APC gene (upstream of the NGG PAM sequence) using the following sequences: forward, 5′-CACCGTCGCTCTTCATGGATTTTTA-3′; and reverse, 5′-AAACTAAAAATCCATGAAGAGCGAC-3′ . Two oligos were annealed and ligated into the BsmB1-digested lentiCRISPRv2 vector , and transformed into the JM109 bacterial strain. All the constructs and mutations were confirmed by nucleotide sequencing analyses .The plasmids Myc-APC-pCMV were provided by Eric R. Fearon . The constructs Myc-WT-KRAS-pcDNA3.1 and Myc-G12V-KRAS-pcDNA3.1 were previously generatedThe normal, polyp/adenoma, and adenocarcinoma samples were analyzed using CRC TMA . Immunohistochemical analyses were performed with anti-EGFR, anti-β-catenin, and anti-pan-RAS antibodies. The TMA slides were visualized by microscopy . For quantitative analyses, the H-score of each stained sample was determined by IHC profiler software (Image J) using the following scale: H-score=3×, highly positive population, +2×, positive population, +1×, weak positive population, and +0×, negative population.Apcmin/+ (Apcmin/+) and B6.129S-Krastm3tyj (KrasG12DLA2) mice were obtained from the Jackson Laboratory. To generate Apcmin/+/KrasG12DLA2 mice for the tumor organoid experiment, Apcmin/+ mice were crossed with KrasG12DLA2 mice. For the xenograft study, athymic nu/nu mice were injected subcutaneously in the dorsal flank with D-MT (1 × 107 cells/mouse) in 200 µL of PBS: Matrigel® . When the mean tumor size was between 100–200 mm3, the mice were randomly divided into four groups: vehicle, cetuximab, KYA1797K, or co-treatment with KYA1797K and cetuximab. The tumors were measured using Vernier calipers every 3 days, and the volumes were then calculated according to the following formula: π/6 × length × width × height. Twenty-one days after the drug treatment, the mice were killed, and the tumors were excised and fixed in 4% paraformaldehyde  or snap frozen in liquid nitrogen for further analyses.All animal experiments were performed in accordance with the Korean Food and Drug Administration guidelines. The protocols were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Yonsei University. C57BL/6J-ApcMin/+/KrasG12DLA2 mice were isolated and washed with ice-cold PBS, and single cells isolated from the tumors were collected using 0.25% trypsin containing 10 mM Ly27632 and 100 µg/mL Primocin™ for 30 min. After incubation, 1 × B27 was added, and the mixture was filtered through 100 µm and 40 µm cell strainers  to collect single cells. The cells were mixed with growth factor-reduced Matrigel® (BD Bioscience). After the gel solidified, N2 medium containing 10% R-spondin-1 CM, 100 µg/mL noggin, 1.25 mM N-acetyl cysteine, 10 mM nicotinamide, 50 ng/mL EGF, 10 nM gastrin, 500 nM A83-01, 3 µM SB202190, and 10 nM prostaglandin E2 was added. The growth medium was refreshed every 2 days, and the cells were passaged by mechanical disruption every 10–14 days at a 1:5 split ratio. The measurements of tumor organoid growth were performed by a CellTiter-Glo® assay  according to the manufacturer’s instructions. Luminescence was measured using a FLUOstar Optima instrument .For the human tumor organoid experiments, the use of human tissue was reviewed and approved by the Institutional Review Board of Severance Hospital, Yonsei University College of Medicine. The biopsied tumor samples from CRC patients were cut into 3–5 mm pieces, washed with ice-cold PBS three times, and incubated with collagenase II (2 mg/mL), hyaluronidase (20 µg/mL), and Ly27632 (10 µM) for 1 h at 37 °C. Small intestinal tumors from 16. Immunoblotting was performed with the following antibodies: anti-pan-RAS (sc-4), anti-β-catenin (sc-7199), anti-ERK (sc-514302), anti-APC (sc-896), anti-PCNA (sc-56), anti-KRAS (sc-30), and anti-Myc (sc-40) purchased from Santa Cruz Biotechnology; anti-p-ERK (9101) and anti-α-tubulin (3873) obtained from Cell Signaling Technology; and anti-EGFR (ab52894) and anti-p-EGFR (ab5644) purchased from Abcam .Immunoblot analysis was performed as previously described2] with a 50% bead slurry. Purified GST-Raf1 RBD proteins were incubated with the WCL of D-WT or D-MT cells treated under the indicated conditions, and the beads were washed with GST-FISH buffer at least five times. The pull-down samples were subjected to immunoblot analyses.For the detection of GTP-RAS (active RAS), the GST-Raf1 RAS-binding domain (RBD) fusion protein was purified from cell lysates of transformed BL21 cells using glutathione-agarose beads and then resuspended in GST-FISH buffer [10% glycerol, 50 mM Tris (pH 7.4), 100 mM NaCl, 1% NP-40, and 2 mM MgCl3 cells/well in a 96-well plate. The cells were then treated under the indicated conditions for 96 h. After incubation, 3--2-5-diphenyltetrazolium bromide  reagent was added to each well at a concentration of 0.25 mg/mL for 2 h at 37 °C. Insoluble purple formazan was obtained by removing the medium and extracting it with DMSO. The absorbance at 590 nm was monitored with the FLUOstar Optima microplate reader (BMG Labtech).For the cell proliferation assays, CRC cells were plated at a density of 2–5 × 10CRC cells were seeded in 12-well plates (250–600 cells/well). The cells were treated under the indicated conditions for 7–16 days with changes of the medium every 4 days. At the end of the experiment, the cells were washed, fixed, and stained with 0.5% crystal violet in 20% ethanol for 2 h, and subsequently washed three times with distilled water.® reagent  according to the manufacturer’s instructions. The total RNA (2 µg) was reverse transcribed using 200 units of M-MLV reverse transcriptase (Invitrogen) in a 20 µL reaction mixture at 42 °C for 1 h. For quantitative real-time PCR analyses, the resulting cDNA (1 µL) was amplified in 10 µL of‘ Rotor-gene SYBR® green . The comparative cycle threshold (CT) method was used, and β-actin served as an endogenous control. The primers are listed in the supplementary table The total RNA was isolated using TRIzol17. For peroxidase IHC analysis, sections were incubated with the primary antibody overnight at 4 °C, followed by incubation with biotinylated anti-mouse  or biotinylated anti-rabbit (Vector Laboratories) secondary antibodies at RT for 1 h. The samples were incubated in the avidin-biotin complex solution (Vector Laboratories), they were then stained with 3, 3′ diaminobenzidine  for 5 min and counterstained with Mayer’s hematoxylin . The signals were analyzed using a bright field microscope (Nikon TE-2000U). The fluorescence signal was visualized using a confocal microscope  at excitation wavelengths of 488 nm (Alexa Fluor® 488), 543 nm (Alexa Fluor® 555), and 405 nm (DAPI). At least three fields of view per section were analyzed. The IHC images were quantified from the representing images using an IHC profiler in the ImageJ software.Immunohistochemistry (IHC) was performed as previously described® 488), 543 nm (Alexa Fluor® 555), and 405 nm (DAPI).Cells were seeded on collagen-coated (500 µg/mL) coverslips. The cells were then fixed with 5% formalin for 30 min, permeabilized with 0.1% Triton X-100 for 20 min, and preblocked with PBS containing 5% bovine serum albumin  and 1% normal goat serum  for 1 h. The cells were then incubated with the indicated primary antibody overnight at 4 °C, followed by Alexa Fluor 488  or Alexa Fluor 555 (Life Technologies) secondary antibodies for 4 h at 4 °C and counterstained with DAPI for 10 min at room temperature. After incubation, the cells were mounted in Gel/Mount media . The fluorescence signal was visualized using a confocal microscope  at excitation wavelengths of 488 nm  and KRAS  in the CRC patient samples, total genomic DNA was isolated from the tumor organoids and analyzed by whole exome sequencing (Macrogen).To analyze the mutational status of both t-test. The significance was denoted as *P<0.05, **P<0.01, and ***P<0.001.All the data are represented as the means ± standard deviations of at least three independent experiments. The statistical significance was assessed using the Student’s APC mutations and that EGFR is a transcriptional target of the Wnt/β-catenin pathway, we tested whether expression levels of β-catenin, RAS, and EGFR are correlated in CRC patient tissues. The levels of the EGFR were higher along with β-catenin and pan-RAS, especially in the polyp/adenoma and adenocarcinoma regions of the CRC patient tissues, compare with paired normal tissues -mutated CRC cell lines  of APC. The levels of β-catenin, pan-RAS, and EGFR were simultaneously increased  genes, both well-known Wnt/β-catenin signaling response genes, were increased in the APC-KO RKO cells  KRAS mutations18–20. Similarly, the growth of KRAS-mutated D-MT, HCT15, and SW480 cells was not significantly inhibited, while the growth of DiFi and D-WT cells harboring WT KRAS was effectively inhibited by cetuximab -KRAS  injected with cetuximab did not show an effect in tumor growth, whereas mice treated with KYA1797K alone or co-treated with KYA1797K and cetuximab showed a significantly reduced tumor volume and weight  and KRAS (Gly12Ser) mutations, which were identified by whole exome sequencing (Fig. 22. However, KYA1797K effectively inhibited gland formation in tumor organoids (Supplementary Figure KRAS mutations by reducing the protein levels via degradation.To test the potential application of KYA1797K in humans, we established tumor organoids derived from CRC patient tissues harboring both ing Fig. . The hum24. In CRC, mutations of genes in the Wnt/β-catenin and RAS/ERK pathways, such as APC and KRAS, synergistically promote transformation25. Aberrant activation of the Wnt/β-catenin and RAS/ERK pathways by the co-stabilization of β-catenin and RAS as well as by genetic mutations in these pathway components contribute to colorectal tumorigenesis. EGFR is also overexpressed in human CRC and plays a role in synergistic tumorigenesis14.Cancer development is a dynamic and multistep process involving multiple genetic changes and molecular alterationsKRAS mutations. The effectiveness of KYA1797K on the inhibition of cetuximab-resistant KRAS-mutated CRC cells is shown by both in vitro and in vivo studies using the mouse xenograft tumor model generated from CRC cells harboring KRAS mutations. The effectiveness and potential application of this compound in patients are demonstrated by the reduction in these proteins and the suppression of growth in tumor organoids derived from CRC patient tissues harboring both APC and KRAS mutations.Therefore, the inhibition of both the Wnt/β-catenin and EGFR-RAS-ERK pathways, especially by reducing the levels of the proteins elevated in CRC, can be an ideal approach for the treatment of human CRC. However, a clinically applicable drug that inhibits both the Wnt/β-catenin and EGFR-RAS-ERK pathways or even the Wnt/β-catenin pathway alone is not available. Small molecules such as KYA1797K reduce the levels of β-catenin, RAS, and EGFR and are potential drug candidates to resolve the problem of resistance to EGFR-targeting therapy attributed to APC, which is observed in ~90% of human CRCs. The approach to controlling CRC by the specific degradation of the β-catenin and RAS proteins can be further supported by a recent study suggesting that the degradation of specific oncogenic proteins is an ideal approach for cancer therapy26. Our approach to controlling RAS, especially oncogenic mutant KRAS, via its degradation provides an alternative approach for the development of a clinically applicable drug targeting the undruggable RAS30. Although β-catenin- and RAS-destabilizing compounds induced the degradation of HRAS and NRAS, these could be beneficial for the treatment of cancer, because the overexpression of WT-HRAS or -NRAS is known to play roles in the promotion of MT-KRAS-driven tumorigenesis31. The importance of the additional KRAS mutations in colorectal tumorigenesis with APC mutations is shown by the role of MT KRAS in the strong secondary activation of Wnt/β-catenin signaling, which involves cancer stem cell activation by a positive feedback loop via the activation of ERK after the initial activation of the pathway caused by the loss of APC25.The small molecule drugs that reduce the levels of β-catenin, RAS, and EGFR are highly effective for the treatment of CRC because of the elevated protein levels caused by the loss of APC, and increases in these proteins critically promote colorectal tumorigenesis. Therefore, small molecules that degrade both β-catenin and RAS with the reduction of EGFR transcription can be potential drug candidates for the treatment of CRC, including CRCs that are resistant to the EGFR-targeting therapies because of KRAS mutations.In summary, we show that the levels of β-catenin, RAS, and EGFR are upregulated in a correlated manner by the loss of Supplementary Information


Evolving systems, be it an antibody repertoire in the face of mutating pathogens or a microbial population exposed to varied antibiotics, constantly search for adaptive solutions in time-varying fitness landscapes. Generalists refer to genotypes that remain fit across diverse selective pressures; while multi-drug resistant microbes are undesired yet prevalent, broadly-neutralizing antibodies are much wanted but rare. However, little is known about under what conditions such generalists with a high capacity to adapt can be efficiently discovered by evolution. In addition, can epistasis—the source of landscape ruggedness and path constraints—play a different role, if the environment varies in a non-random way? We present a generative model to estimate the propensity of evolving generalists in rugged landscapes that are tunably related and alternating relatively slowly. We find that environmental cycling can substantially facilitate the search for fit generalists by dynamically enlarging their effective basins of attraction. Importantly, these high performers are most likely to emerge at intermediate levels of ruggedness and environmental relatedness. Our approach allows one to estimate correlations across environments from the topography of experimental fitness landscapes. Our work provides a conceptual framework to study evolution in time-correlated complex environments, and offers statistical understanding that suggests general strategies for eliciting broadly neutralizing antibodies or preventing microbes from evolving multi-drug resistance. Generalists are robust performers under varied environmental conditions, even though they are less fit than specialists in any particular environment. For better  or worse (e.g. emergence of multi-drug resistance in microbes), it is important to be able to evolve generalists efficiently. Yet, whether and when environmental changes select generalists over specialists remain largely unknown. Here we develop a dynamic landscape model to study the evolutionary discovery of generalists in time-varying correlated environments. We demonstrate that cycling rugged fitness landscapes can enhance the propensity of evolving fit generalists, via dynamic augmentation of their attractors. We find that high performers are most reliably evolved under intermediate environmental correlations, reflecting a tension between diversity and accessibility. Our approach offers design principles for choosing correlated environments in dynamic protocols to speed or slow generalist evolution in diverse contexts. Temporally varying environments profoundly influence various properties of evolving systems, including their structure –4, robusTheoretical studies have examined the adaptive utility of survival strategies at different timescales of environmental fluctuations –21. Whilgenotypes can be recognized as local fitness optima shared by distinct landscapes representing varied environments. Remarkable examples of generalists in adaptive evolution complement each other in an inspiring way: It is desirable for the immune system to evolve broadly neutralizing antibodies [Generalists can reuse partial solutions in new contexts and hence rapidly adapt to previously unseen environmental conditions –33. In otibodies  that tartibodies  and virutibodies . Attempttibodies –41, whictibodies ).tunably related and cycling relatively slowly , irrespective of the specific structure of the evolving system NK model  to repreNK model –67.N is defined as the average over the fitness contribution of each bit:In an NK landscape, the fitness i, ϵ depends on K + 1 coupled sites influencing the fitness contribution of site i. An additive landscape (K = 0) has a single global optimum reachable from an arbitrary starting genotype, whereas in a completely random landscape (K = N − 1) statistical independence of nearby states leads to an extraordinarily rough surface in which on average 2N/(N + 1) local maxima can be surrounded by fitness valleys. Natural populations are likely to be guided by fitness landscapes in between these extremes.Here the fitness contribution of bit N binary antibody genotypes. To link the level of fitness conservation (the likelihood that a fitness contribution is preserved across environments) to topographical relatedness between landscapes, we consider two environments A and B in whichIn the case of antibody-antigen binding affinity, each distinct antigen defines a unique hypersurface spanning over a hypercube of 2B from landscape A; the latter is generated according to ai of correlation between fitness contributions of site i in two environments is given byThis constructs landscape ggedness , this faai = 1) or subject to tradeoff (ai = −1). While np = N corresponds to identical landscapes, np = 0 characterizes completely inverted pairs. Therefore, the fraction of conserved fitness contributions, np/N, naturally measures the level of conservation. Furthermore, by making ai independent of the state of the K + 1 epistatically interacting sites, we assume that fitness correlations are preserved in all backgrounds, which decouples the effect of inter-landscape correlations (fitness conservation characterized by np) from that of intra-landscape correlations (epistasis measured by K). This decoupling in turn implies that np tunes the topographical similarity without affecting the degree of ruggedness. As a consequence, landscapes thus constructed are tunably related yet statistically equivalent  and mean fitness (panel B) of local optima, while shifting their locations and modifying topography in their neighborhood.In this model, the fitness effect of single mutations under different environments is either conserved  control the relative dominance of two attractor types and govern their distribution in sequence space.Natural environments are often partially related over the course of systems adaptation; the very existence of generalists demands a minimal commonality. An immediate consequence of environmental correlation is that different parts of the sequence space may experience different levels of fitness variations as environments cycle: genotypes undergoing large fitness swings correspond to specialists that are fit in a particular environment, whereas genotypes facing small fitness fluctuations represent generalists. Thus, in a landscape description  is close to that between independent landscapes, so that np > N/2 (np < N/2) corresponds to overall positively (negatively) correlated landscapes.Local fitness optima that remain in the same location as the environment changes—shared optima across landscapes—represent generalists. Intuitively, as the number expected . Note thK of the epistatic groups, we plot the fraction of local optima being shared between landscapes  than exponential for K ≤ (≥)N/2. Therefore, stronger ruggedness appears to promote optimum sharing, both by boosting the prevalence of generalists at a given conservation level (K increasing in the direction of the arrow) and by extending their presence to a lower level of fitness conservation  specialists are on average more fit, at sufficiently high levels of environmental relatedness (large np), generalists become selectively favorable . Note that stronger ruggedness enlarges the generalist-favored regime toward a lower conservation level, at the expense of a modest reduction in average fitness.When are generalists favored over specialists? As known from ecology, generalist birds with intermediate bill lengths may evolve when prey types are alike, whereas specialization develops when more diverse prey types require highly dissimilar beaks . This alA that belongs to basin B). In this way, landscape cycling dynamically enlarges the attractor size of generalists by connecting basins segregated in static environments , the frequency of basin linking declines with increasing np; too similar landscape topography makes it unlikely that a generalist basin in one landscape contains a specialist peak in the other landscape. Increasing epistasis also monotonically diminishes the chance of basin linking; stronger decorrelation in fitness (larger K) results in fewer (K ≥ 9), the likelihood of basin linking exhibits a relatively weak dependence on np. Intermediate values of np result in, on average, longer chains  to phase II where generalists constitute stationary attractors in changing environments (colored region). While selectively accessible  trajectories are rarely circular on a static landscape, closed paths may prevail under environmental cycling in phase I—either due to oscillations between nearby specialists each present in only one landscape . Notably, the chance of evolving fit generalists is worst in the strong-conservation high-epistasis corner (blue color), where the sequence space divides into many small and rarely linked basins surrounding generalist peaks of which the majority are unfit .Starting from diverse sequences, how likely will a population discover a generalist under environmental cycling? To estimate the mutational accessibility of generalists (phase II), we first computed the fraction of genotypes that would follow an adaptive path to a shared optimum as landscapes alternate . This qu large K  or when rge K  oscillations or cycles among specialists in the absence of generalists, and (II) convergence to a generalist after multiple environmental switches. We find that an intermediate amount of epistasis, reflective of evolved functional constraints in biological systems, appears to balance the diversity  and accesimultaneously considering the role of epistasis and topographical similarity between landscapes in guiding the evolutionary discovery.In the context of adaptive immunity, recent work has suggested that temporal correlations in antigenic environments crucially regulate evolution of generalist antibodies –74. HereThe NK model was originally developed for studying antibody affinity maturation in a constant environment. Our extended NK model helps to solve the problems of evolving generalists in time varying correlated environments. By combining laboratory selection experiments with next-generation sequencing, it is now feasible to map sizable fitness landscapes under different environments, e.g. binding of antibodies to different mutants of influenza haemagglutinin  and growK can be determined by comparing the contribution of epistasis in the empirical landscape to that in simulated NK landscapes at varied K values [np can be estimated by comparing fitness contributions of single mutations across different environments. For example, we performed Hadamard-Walsh transform on fitness landscapes of β-lactamase under different β-lactam antibiotics [ai ∼ 1) or subject to tradeoff (ai ∼ −1), with more weight on the conserved side K values . Meanwhiibiotics . We founved side .np qualitatively. For example, single amino acid substitutions can affect protein stability and function (e.g. binding affinity to antigens or drugs) [np than those with dissimilar profile.If we know more about the mechanism underlying the mutational effects, we can also estimate r drugs) , 81. ForK. Our finding suggests that evolving antibodies that recognize simple epitopes  require switching between similar antigens (large np), whereas antibodies that bind well to complex epitopes (large K) emerge when cycled antigens are sufficiently dissimilar . Hence, intermediate np adjusted to K would do best. For the latter, dissimilar antibiotics  could avoid generalist microbes and confine the population to a small region of the sequence space under landscape switching (i.e. tight limit cycles), whereas very similar drugs with strong epistasis (large np and large K) may trap the microbial population to unfit generalist genotypes. In fact, experiment has shown that alternating environments can constrain the evolution of multi-drug resistance, in the regime where np is small between different antibiotics [np is relatively large and hence multi-drug resistance is easily achieved [Given that we can empirically estimate ruggedness within a landscape and correlations between landscapes, our results . As the variety of distinct environments increases, the possibilities of switching schemes expand quickly, since it permits combinatorial ways of arranging successive environments and wide choices for the timing of switches. We expect, within our model, that generalists will likely become rarer given more diverse environments. And yet, guiding principles derived from this work still hold in choosing sistance  and antisistance , which asistance . While aFinally, our results based on temporally varying environments can be extended to understand adaptation in spatially heterogeneous environments, where landscape switching arises from migration between distinct yet connected habitats or microenvironments , 91.ϵ labels the environment and j runs through in total ϵ. Generalists correspond to local optima common to different environments and are represented by K) and the level of fitness conservation (np) vary, both the number and the distribution of generalists and specialists in sequence space will change. Below we introduce the measures to quantify the prevalence and accessibility of generalists in static and switching environments.Consider two environments A and B. Each environment defines a fitness landscape in the sequence space, characterized by a set of local optima (fitness peaks), θg, as the ratio of the number of generalists  a linked basin. We then search through this linked basin for fitness peaks in environment A; if identified, we link the fittest peak and its basin (in environment A). Iterate until the newly linked basin no longer contains any peak in the other environment. Repeating this process starting from environment B, we obtain a full chain of linked basins hinged at the generalist ; these generalists gain linkage to additional specialist basins under cycling.Here the numerator counts the number of generalists that have enlarged basin size in switching landscapes  = m(XϵXϵ) − m(Xϵ)m(Xϵ) to compute mean and variance respectively. The correlation strength is then averaged over environments.In a static environment In cycling environments,A0, is defined as the total coverage of generalists’ effective basins in switching landscapes:The absolute accessibility of all generalists , denotedAc, is measured by the total effective basin size of generalists within the top 30% of maximum fitness, normalized by the total basin size of all generalists combined:The conditional accessibility of fit generalists , denotedS1 Text(PDF)Click here for additional data file.S1 Fignp (different symbols) indicates that changing np does not alter the number and average fitness of local optima for a given number K of interacting sites. Each data point is an average over 1000 landscapes.(A) Total number of local optima. (B) Average fitness of local optima. Almost complete overlap of data points at different values of (PDF)Click here for additional data file.S2 FigK = 9 (upper row) and np = 8 (lower row). The number of linked basins excludes the one associated with the shared optimum. For each combination of np and K, around 200 generalists are collected. (A and B) With relatively strong epistasis (K = 9), an intermediate level of fitness conservation  leads to longer chains of linked basins (A) of larger average size (B), compared to weaker or stronger conservation. Inset of A: the weight of the np = 8 term for various numbers of linked basins; the grey line marks 1/3. Inset of B: the average size of linked basins at different values of np. (C and D) At an intermediate level of fitness conservation (np = 8), stronger epistasis (larger K) results in shorter chains of linked basins (C) of significantly smaller average size (D).Histograms of the number (left column) and average size (right column) of linked basins under landscape switching for (PDF)Click here for additional data file.S3 Fignp and K. While the number of generalists rapidly grows with np and K (A), the number of fit ones saturates (B). Average basin sizes of generalists decrease as K increases and are largest at intermediate np for large K, consistent with the trend of the probability of basin linking (The number (left column) and average basin size (right column) of all generalists  and fit generalists . All color-coded values are in logarithmic scales and averaged over 1000 landscape pairs for each combination of (PDF)Click here for additional data file.S4 Figβ-lactam antibiotics are taken from Table in Ref. [L = 4, 16 genotypes).(A) Fitness effect of single drug-resistance mutations, i.e., first-order Fourier coefficients, is found to be predominantly conserved (Pearson correlation close to 1) or anticorrelated (Pearson correlation close to −1) under pairs of different drugs. The data of empirical fitness landscapes under 15 different  in Ref. . (B) Cor(PDF)Click here for additional data file.


Furthermore, related experiments were performed to detect cellar phenotype‐associated signalling pathway, including epithelial‐mesenchymal transition (EMT) and Wnt/β‐catenin. Cell assays indicated that RE could increase trophoblasts migration and invasion. In addition, hypertension and proteinuria were markedly ameliorated by RE compared with the controls in PE mice model. Moreover, treatment by RE in trophoblasts or in PE model, we found that RE activated EMT progress through the regulation of E‐cadherin, β‐catenin, N‐cadherin, vimentin expression, and further altered the WNT‐related gene expression, including WNT1, WNT3 and WNT5B. Our findings demonstrated that RE might stimulate the invasive capability of human trophoblasts by promoting EMT and mediating the Wnt/β‐catenin pathway in PE.Impairment spiral arteries remodelling was considered to be the underlying cause of pathogenesis of pre‐eclampsia (PE). Resveratrol (RE) was reported that it could modulate cellar phenotype to ameliorate diverse human diseases. However, the biological function of RE in PE remains poorly understood. In this report, we investigated the effect of RE on trophoblast phenotype both in vivo and in vitro. We conducted MTT and transwell assays to explore cell proliferation and invasion events in HTR‐8/SVneo. In mice model, the clinical characteristics of PE were established through the injection of NG‐nitro‐ It is characterized by the breakdown of cell‐cell adhesion, loss of epithelial phenotypes and cell depolarization, which accelerate progression in multiple diseases, especially cancer.Resveratrol , a traditional Chinese medicine, is known as a naturally occurring compound with potent biological properties.l‐arginine methyl ester (L‐NAME), an inhibitor of nitric oxide (NO) synthase.Hence, PE mice model was first established through the intraperitoneal injection of NG‐nitro‐22.1The research involving humans was performed with prior approval from the Ethics Committee of the First Affiliated Hospital of Nanjing Medical University. All participants provided a written informed consent prior to the study. All animal experiments were performed with approval from the Institutional Animal Ethics Committee and performed in accordance with the guidelines of the Committee for the Purpose of Control and approved by the Care of Experimental Animals Committee of Nanjing Medical University Ethics Committee. All efforts were made to minimize suffering in experimental animals.2.2am every morning during pregnancy. Urine samples were collected on GD 0.5, 4.5, 9, 13.5 and 18.5 and stored at −80°C. Urinary levels of albumin were detected on the same GDs .A mouse model of PE was established through the intraperitoneal injection of the eNOS inhibitor L‐NAME (125 mg/kg body weight). Pregnant adult female albino Wistar mice (weighing 200‐250 g) were divided immediately and randomly into three groups (n = 16 per group): control, L‐NAME, and L‐NAME + RE groups. RE was administered intragastrically at a dose of 20 mg/kg/day. On the gestational day (GD) 18.5, the mice were killed and urine, blood, placenta and uterine samples were collected. Blood pressure was detected using a programmed electronic sphygmomanometer  by the tail‐cuff method at 8.30 2.32. HTR‐8/SVneo cells were seeded in six‐well culture plates overnight and exposed to the following treatments for 24 hours: no treatment , 20 mmol/L DOX  (DOX group),and 20 mmol/L DOX + 100 µmol/L RE  (DOX+RE group).In this study, HTR‐8/SVneo Cells were provided by Dr Charles Graham, Queen's University, Canada. Human umbilical vein endothelial cells (HUVEC)‐C were obtained from the Type Culture Collection of the Chinese Academy of Sciences . HTR‐8/SVneo and HUVEC cells were cultured in RPMI 1640 and ECM , respectively, which were supplemented with 10% foetal bovine serum , 100 U/mL penicillin and 100 mg/mL streptomycin (Invitrogen). The cells were maintained at 37°C in a humidified atmosphere with 5% CO2.4Total RNA was extracted from approximately 0.1 g of placenta tissues or from trophoblasts using TRIzol reagent (Invitrogen Life Technologies). A reverse transcription kit (Takara) and Power SYBR Green (Takara) were used for cDNA synthesis and amplification respectively. The expression of genes regulating migration, invasion, angiogenesis and EMT‐related factors were detected by SYBR Green qRT‐PCR. The expression of glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) was used for normalization. The primers used were as follows: E‐cadherin ; N‐cadherin ; Snail ; β‐catenin ; Vimentin ; MMP‐2 ; MMP‐9 ; VEGF ; sFlt‐1 ; Ang‐I ; Ang‐II ; GAPDH . An ABI 7500 system was used to carry out the qPCR and data collection.2.5Proteins were extracted from HTR‐8/SVneo cells or from mice placentas, treated with RIPA Protein lysis buffer (Beyotime), and supplemented with protease inhibitor cocktail (Roche) and phenylmethylsulfonyl‐fluoride (Roche). Protein concentration was measured using the Bradford assay. Next, samples containing 50‐100 µg of protein were separated by 10% SDS‐PAGE. The protein on the gel was then transferred onto 0.22‐mm polyvinylidene difluoride membranes (Sigma). Specific antibodies against E‐cadherin, N‐cadherin, snail, vimentin, β‐catenin, MMP‐2, MMP‐9, WNT1, WNT3 and WNT5B were all purchased from Cell Signaling Technology  and used as primary antibodies (1:1000). The secondary antibody used was horseradish peroxidase‐conjugated goat anti‐rabbit IgG or goat anti‐mouse IgG . GAPDH  was used as a control. We used the Quantity One software (Bio‐Rad) to quantify the intensity of the protein bands.2.6The placental tissues were removed from the rats and immediately fixed in 4% paraformaldehyde according to a standard protocol. HTR‐8/SVneo cells were also fixed through the same procedure. Specific polyclonal antibodies against E‐cadherin , N‐cadherin (BD), β‐catenin (BD), vimentin (BD) and snail  at a concentration of 1:100 were used as primary antibodies. TRITC‐labelled anti‐Rabbit IgG  was used as a secondary antibody. An Olympus BX51 microscope  was used to capture the immunofluorescence images.2.75 cells/well to the upper well of a transwell chamber  with a membrane pore diameter of 8 µm. Medium containing 10% FBS, as a chemo‐attractant, was immediately placed in the lower chamber. After 24 hours of incubation, the cells on the upper surface were removed, whereas the cells on the lower surface were fixed and stained with 0.1% crystal violet. Finally, the number of migrated cells was calculated under a digital microscope in five random fields for each sample.Cell invasion assays were performed as previously reported by Xu et al.2.84 cells was seeded on the upper layer of the chamber and incubated at 37°C with 5% CO2 for 24 hours. Then, the inserts were washed three times to remove scattered or the bridge‐like cells that did not form networks. Next, the cells were fixed by methanol for 30 minutes and stained with crystal violet. Finally, images from each well were captured under a light microscope. The number of cell‐cell protracted contacts and the length of tubes were counted as the number of capillary‐like networks present in each field.Tube formation assay was performed as previously described by Xu et al.2.9t tests were used to compare the results of two groups and ANOVA was used to compare the data of more than two groups. P < 0.05 was considered statistically significant.All data are expressed as the mean ± SD. All statistical analyses were performed with the SPSS statistical software package . Paired and unpaired Student's 33.1To explore the role of RE in PE, we first used L‐NAME to simulate a PE mice model. After treatment with RE, the related clinical indicators, including systolic blood pressure, proteinuria, foetus number, foetus birth weight, placental weight and external malformations, were measured and observed in all groups (n = 16 per group) .In conjunction with the results of in vivo study, we choose trophoblast cells to further invalidate the signalling pathway in vitro. Here, to unravel whether RE regulates the EMT in trophoblast cells, we first selected RE and doxorubicin . In addition, the number of branch point was significantly higher in trophoblasts treated with RE than that of the control, indicating a significant increase network formation ability . Taken together, these findings suggested that RE could ameliorate cell invasion and tube formation by promoting the EMT process.Our previous work has reported that the EMT was involved in trophoblast cell invasion and migration.y Figure D and E. 3.5Previous studies have reported that β‐catenin is the key transcriptional activator in canonical Wnt signalling in the nucleus, and L‐NAME represses Wnt/β‐catenin‐mediated transcriptional activity by promoting the cytoplasmic localization of β‐catenin.4Based on its great harm to pregnant women and perinatal infants, PE has attracted much attention in the clinical field. PE may be caused by placental ischaemia, vascular endothelial injury, poor immune adaptation, oxidative stress and genetic susceptibility,Epithelial‐mesenchymal transition process is generally associated with the pathogenesis and progression of various humans disorders, included PE.In our report we explored the role of RE in trophoblast both in vivo and in vitro. Hypertension and proteinuria were markedly improved after injection of RE compared with the controls in PE mice model. MTT and transwell assays were performed to further establish the various regulatory roles of RE in trophoblasts. And we revealed that RE improved cell migration and invasion but mildly and insignificantly improved cell viability. Moreover, RE could significantly increase the expression of MMP‐2 and MMP‐9 at mRNA and protein level, consistent with the results of the transwell assay. Also, RE could promote EMT process through the regulation of E‐cadherin, β‐catenin, N‐cadherin, vimentin expression by qPCR and Western blot assays. RE was involved in controlling the remodelling of spiral arteries by evaluating the angiogenesis‐promoting ability of RE in HUVEC and showed that RE ameliorated the effects of DOX and improved the network formation ability of HUVEC. RE considerably increased the number of capillary‐like networks and the expression of VEGF, sFlt1, AngI and AngII in vitro. Therefore, RE may promote angiogenesis and play an essential role in the remodelling of spiral arteries in PE mice model.β‐catenin is the key transcriptional activator in the canonical Wnt signalling pathway in the nucleus, and RE increases Wnt/β‐catenin‐mediated transcriptional activity by promoting the nucleus localization of β‐catenin.Thus, our study provides new insights into the biological mechanisms of RE in PE, in which RE might stimulate the invasive capability of human trophoblasts by promoting EMT process, partially by mediating Wnt/β‐catenin signalling pathway, to further impede the occurrence and development of PE.The authors declare no conflict of interest.


The quasistatic fracture toughness of modified epoxy resin increased with the particle content. However, the fracture toughness of epoxy resin modified with high content fillers decreased for particle agglomeration that existed in epoxy resin. The crack propagation velocity can be decreased for epoxy resin modified with particles under dynamic loading. The dynamic initiation fracture toughness of modified epoxy resin increases with both particle size and content, but when the fillers have a high content, the particle size effects are weak. For the composite under dynamic loading conditions, the toughening mechanism is also affected by particle size.Epoxy resin, modified with different particle sizes  and contents  was manufactured. The mechanical behaviors of tensile, quasistatic fracture and dynamic fracture under SHPB (split Hopkinson pressure bar) loading were investigated. The dynamic fracture behaviors of the composites were evaluated by 2D-DIC  and the strain gauge technique, and the fracture surface was examined by SEM (scanning electron microscope). According to the results, the tensile modulus and strength significantly increased for epoxy resin modified with 5 wt % Al Particulate-filled epoxy resin composites have been widely used in the engineering field due to their excellent mechanical properties and strong designability, which are useful for different applications. With the development of nanotechnology, nanoparticle-filled composites are attracting increasing attention, to further enhance the mechanical performance of composites. However, the fracture behavior of composites under different loading conditions must be taken into consideration in designing and evaluating mechanical properties.2 (with an average diameter of 36 nm) on nanocomposite bulk mechanical properties and dynamic fracture toughness, where an increase in dynamic fracture toughness relative to quasistatic fracture toughness was observed. Zhang et al. [3 into isotactic polypropylene (PP), and found that the composite tensile properties did not significantly change but that the notched Izod impact energy of the composites increased significantly. Marur et al. [2 and nano-Al2O3, where the quasistatic fracture toughness and flexural strength were improved with increasing nanoparticle contents, and where the fracture toughness was higher for composites modified with small-sized particles. Zamanian [Epoxy resin is normally modified by adding rigid and soft particles such as rubber, glass, and silica, to improve fracture toughness under impact loading ,2,3. Guog et al.  added 44r et al.  added dir et al.  showed tr et al.  found thr et al.  studied Zamanian  modifiedMany studies have discussed epoxy resin modified with different particle sizes and amounts. While 100 nm was the critical dimension, we usually used to discriminate the nanoparticles, and most researchers have not studied the particle size near the critical dimension. Therefore, particles of 50 nm, 100 nm, and 200 nm will be a better choice for evaluating the particle size effect on the fracture behavior, as well as the particle size effect on the toughening mechanisms of the composite under dynamic loading conditions, both of which need to be studied further. Regarding the analysis methods for the fracture behavior of nonhomogeneous composites, the interaction energy integral method ,18,19,20In this work, we investigated the mechanical properties of epoxy resin modified with different nanoparticle sizes and contents under quasistatic and SHPB loading conditions. A 2D-DIC  method and strain gauge technique were used to study the composites dynamic fracture behavior. The fracture morphology was examined by SEM (scanning electron microscope), and the toughening mechanism has been given.o-Phthalic anhydride and tris(2-hydroxyethyl)amine  were used as the curing agent and accelerating agent, respectively. Nano-alumina particles with an average diameter of 50 nm, 100 nm, and 200 nm were provided by Shenzhen Crystal Chemical Co., Shenzhen, China.Bisphenol-A epoxy resin E51  was used as the matrix. The nano-alumina particles  were dried for 24 h at 100 °C in order to remove surface water. The epoxy resin was first heated at 80 °C, to reduce the viscosity of the resin and improve the fluidity of the epoxy resin. Then, three sizes of nanoparticles with 1 wt %, 3 wt %, 5 wt %, and 7 wt % were added to the epoxy resin, and the particles were dispersed via a mechanical agitator and magnetic stirring apparatus for 3 h at 60 °C. After that, a curing agent was added into the mixture and stirred until it dissolved completely. In order to remove the air bladder, we put the mixture in a vacuum drying oven, with a vacuum degree of 0.2 KPa for 1 h at 60 °C. After that, the accelerating agent was added, and the mixture was poured into a preheated mold. The curing temperature was set to 120 °C for 30 min, and to 135 °C for 4 h. The whole manufacturing process is shown in ICK, which is defined by Equation (1):P is the load when cracks begin to propagate, S is the span length (S = 80), B is the specimen thickness, W is the specimen width, and a is the initial crack length of the specimen.The tensile test was carried out on a Zwick/Roell Z010 Universal Testing Machine  with a capacity of 10 kN at room temperature and with a crosshead speed of 2 mm/min. For the fracture toughness testing, specimens were prepared as a single edge notched bending (SENB) configuration, with dimensions of 106 mm × 20 mm × 10 mm (length × width × thickness). The average length of the pre-crack was 10 mm, which was determined by tapping a new razor blade in the specimens’ notch. Quasistatic mode-I fracture toughness was estimated though a three-point bending test at room temperature with a crosshead speed of 1 mm/min. The fracture toughness of materials is usually expressed by the critical stress intensity factor The SHPB test system is shown in For the dynamic fracture toughness test, single edge notched bending specimens with pre-cracks were used. Meanwhile, speckle was sprayed onto the specimen surface and the strain gage was pasted onto the specimen surface, without speckle, near the crack tip. Information on specimen size, strain gage position, and the test span is shown in maxt was obtained from the strain gauge that was pasted near the crack tip of specimens . The velocity of the wave in epoxy resin is 2690 m/s, and l and h are the horizontal and vertical distances between the strain gauge center and the crack tip, respectively. The dynamic fracture-initiation toughness IdK (the stress intensity factor (SIF) when the crack begins to grow) is an important parameter to characterize the fracture properties of the composite. Thus, IdK can be obtained from the value of SIF at the crack growth initiation time, ft, i.e.,The strain gauge was stuck near the crack tip of every test specimen, and the crack initiation time could be evaluated using Equation (3).p(t)u [p(t)u may be evaluated using Equations (4)–(6) [The dynamic SIF history can be calculated via the load point displacement, up(t) , and canby Rubio ), and kβa et al. . During n is the numbers of images recorded by the high-speed camera during crack growth. L is the crack propagation length for the duration of bta-t. By calculating the change of time-adjacent images of gray values, we were able to obtain the length of crack growth for a given period. Then, we were able to add up the time-adjacent crack length and the crack growth length L from at to bt.The 2D-DIC method was used to study the dynamic fracture behavior by measuring the deformations of the specimens. The crack propagation velocity was estimated from the crack length variation with time, as shown in xu at t = 0 µs (crack begins to grow), t = 24.98 µs, t = 49.96 µs, and t = 74.94 µs are shown in p(t)u from the xu displacement field. The load-point displacement p(t)u obtained by the DIC method and Equation (6), as shown in The DIC method was also used to evaluate the load-point (the position where the incident bar contacted the specimen) displacement. A sub-image size of 60 pixels × 60 pixels was chosen to calculate the displacement fields. The displacement fields contours of 2O3 at a high content, the trend was reversed, and the modulus decreased. This variation trend for nanoparticle-modified epoxy resin at a high content was also reported in [The physical properties of all the particles that modified the epoxy resin are shown in orted in . FurtherICK, for both unmodified and modified epoxy resin. In the case of the neat epoxy resin, the fracture toughness ICK was determined to be 0.78 MPa·m1/2, while a maximum ICK of 1.56 MPa·m1/2 was measured for epoxy resin with 7 wt % of 50 nm. When comparing the toughness of modified epoxy resin with different particle sizes, epoxy resin modified with 50 nm particles showed higher values. The fracture toughness of 50 nm particle-modified resin is higher than when 200 nm nano-Al2O3 particles were incorporated into the matrix. Meanwhile, the toughness of ICK decreased for epoxy resin modified with 5 wt % of 50 nm and 100 nm particles. For epoxy resin modified with high particle contents, the increasing trend reversed, which suggests the occurrence of nano-Al2O3 agglomeration. The fracture surface morphology of epoxy resin modified with 7 wt % of 50 nm particles, examined by TEM, shows the occurrence of agglomeration  and contents  under dynamic loading are shown in 2O3, the crack velocity did not obviously decrease, and the maximum velocity reduction rate (compared with the neat epoxy resin) was 14.29%. For epoxy resin modified with 100 nm and 200 nm particles, the maximum velocity reduction rates were 36.95% and 37.96%, respectively. Thus, for modified epoxy resin under dynamic loading, the crack velocity could be reduced more effectively by larger particle sizes. The results in ref [The crack velocity (steady state crack velocity) was estimated from Equation (10). The velocities of the neat epoxy resin modified with nano-Als in ref  showed tI(t)K history curve was similar. With the particle content increasing, the slope of the I(t)K history curve also increased, as can be seen in I(t)K history curve increased with the particle content, while for particle content over 5 wt %, the slope decreased.The dynamic stress intensity factor (DSIF) histories of the epoxy resin modified with different particle sizes and contents were evaluated by Equation (5), as shown in 1–d1 represents the crack propagation of the neat epoxy resin, and the epoxy resin modified with 50 nm (1 wt %), 100 nm (1 wt %), and 200 nm (1 wt %) at the same time during the SHPB loading process). 1,b1, shows that the crack propagated almost along a straight line; however, the dynamic initiation fracture toughness was higher than that of neat epoxy resin, as shown in 2,b2. During the crack growth, for the epoxy resin modified with 50 nm particles, the crack will be hampered by particles, and a tiny deflection path will be formed, increasing the dynamic fracture toughness. With the particle size increasing as shown in 1,d1, the angle of crack deflection became larger. 2,d2 gives the crack path deflection mechanisms of epoxy resin modified with 100 nm particles. With a particle content at 7 wt %, the crack initiation toughness (IdK) of the composite tended to be equal. The reason for this is that the number of particles was higher for 50 nm than for either 100 nm or 200 nm; particle agglomeration of 50 nm particles may lead to the energy necessarily consumed for crack initiation being the same for the epoxy modified with different-sized particles at 7 wt %.When comparing the crack propagation velocity, the large-sized particles (at the same content) could effectively reduce the velocity. The energy dissipation mechanisms of the particle size effect on the composite are shown in 2O3 explained by the crack deflection theory is not enough. The detailed damage information of the fracture surface observed by visual inspection was not effective, so the fracture surfaces were examined via scanning electron microscopy (SEM) . 2O3 at 3 wt %, respectively. For the neat epoxy resin, river-like lines were formed after specimen fracture, as shown in 2O3, a plastic void growth could be observed after particle debonding, and this results in matrix plastic deformation [2O3. The crack pinning mechanism has been explained for epoxy resin modified with microparticles [2O3-modified epoxy resin, dynamic fracture toughness can be enhanced, and particle size and content could affect the toughening mechanisms differently.It has been suggested that in epoxy resin, crack deflection processes play an important role in toughening, and a theory has been proposed by Faber and Evans . It is aormation ,29. As sarticles , but alsarticles . In shor2O3 sizes  and contents  under SHPB loading. The tensile and quasistatic mode-I fracture toughness properties of particle-modified epoxy resin have also been given. The influence of particle size and content on dynamic fracture toughness, loading rate, crack propagation velocity (at a steady state), and particle toughness mechanisms were discussed. From the experimental results, we can conclude the following:◆2O3 particles of different size increased with particle content, with the highest increase of 29.2% for epoxy resin modified with 5 wt % of 50 nm particles. The tensile strength significantly increased for the epoxy resin modified with 5 wt % and 7 wt % of 50 nm particles, while there were almost no changes for the epoxy resin modified with 100 nm and 200 nm particles with different contents.The tensile modulus of epoxy resin modified with three Al◆ICK had a maximum value of 1.56 MPa·m1/2 for epoxy resin modified with 7 wt % of 50 nm particles, and the ICK increased with particle content. While the toughness did not increase with particle content for the fillers at high content, the agglomeration reduced the effective number of Al2O3 particles in the epoxy resin, thus reversing the trend.The quasistatic fracture toughness ◆Crack propagation velocity was evaluated by the DIC method for the composites under dynamic loading condition; the crack path was more easily deflected for large-sized particles than for small-sized particles. Meanwhile, the crack propagation velocity could be reduced with an increase in particle content.◆IdK increased with particle content and size. The dynamic fracture toughness was almost equal for epoxy resin modified with three particle sizes at 7 wt %. For high content fillers, the effect of the particle size could be neglected, and more energy would be expended during the fracture process. The loading rate could be affected by the particle content, while for the epoxy resin modified with low content particles, the effect was obvious; there was no significant size effect for epoxy resin modified with high content particles.The dynamic initiation fracture toughness ◆It has been suggested that in particle-modified epoxy resin, the crack deflection processes play an important role in toughening; under dynamic loading and with an increase in particle size, the crack deflection angle became larger. Toughening mechanisms are affected by particle size: for the epoxy resin modified with 50 nm particles, crazed shear band and shear plastic deformation improved the fracture toughness; for the resin modified with 100 nm particles, debonding and plastic void growth were observed; finally, particle debonding and crack pinning were shown to occur in epoxy resin modified with 200 nm particles.In this study, the strain gauge method and DIC method were used to calculate the dynamic fracture behavior of epoxy resin modified with different nano-Al