At the same time, DKD, as a metabolic disease, has many connections related to podocyte autophagy.  Organ fibrosis is a common endpoint of a variety of diseases. Many studies have shown that the pathogenesis of diabetic kidney disease (DKD) is related to the excessive activation of the Wnt/β-catenin signaling pathway, and further investigated the relationship between this effect and autophagy.  We experimented the effects of Mahuang Fuzi and Shenzhuo decoction (MFSD) which is the combination of Mahuang Fuzi decoction and Shenzhuo decoction in traditional Chinese medicine compounds used “The Golden Chamber” in high glucose-induced podocytes, determined whether this effect was related to Wnt/β-catenin signaling pathway inhibitor DKK1 (100 ng/ml) was used to intervene podocytes before high glucose stimulation. Podocyte injury-related proteins, Wnt/β-catenin signaling pathway-related proteins, and autophagy-related proteins were detected by using western blotting and immunofluorescence analysis.  The mice podocytes were stimulated by using 30 mmol/L of high glucose and serum containing MFSD or Wnt/β-catenin protein expression. Furthermore, the protein expressions of beclin1, LC3B, and P62 were also significantly increased in high glucose-induced podocytes.  Our results showed that DKK1 and MFSD treatment significantly upregulated the protein expressions of nephrin, podocin, podocalyxin, and podoplanin in high glucose-induced podocytes and downregulated the β-catenin signaling pathway and the inhibition of autophagy after activation. MFSD treatment can inhibit the activation of Wnt/β-catenin signaling pathway in podocytes stimulated by high glucose and helpful in reducing the podocyte injury. This protective mechanism can be related to the enhancement of podocyte autophagy by MFSD treatment. Our experiments confirmed that the destruction of podocytes in DKD is related to the excessive activation of Wnt/ Diabetic kidney disease (DKD) is one of the major complications of diabetes . The traβ-catenin signaling pathway is a common signaling pathway regulating cell proliferation, β-catenin is the main effector protein [β-catenin signaling pathway is aberrantly activated [β-catenin signaling pathway further amplifies destruction when podocytes are damaged. Many studies have reported abnormal activation of the Wnt/β-catenin signaling pathway in podocytes [β-catenin signaling pathway [Wnt/ protein , 10, and protein , 12. Whectivated . Abnormaodocytes –16. Autoodocytes –19. Exte pathway , 21.β-catenin pathway in CKD [β-catenin and autophagy pathway.Cumulative studies have shown that a variety of traditional Chinese medicines (TCMs) are used to treat various diseases including chronic kidney diseases (CKD) –24. A coy in CKD , 26. Mahy in CKD , 28. Mah40 male Sprague Dawley (SD) mice, aged 6–8 weeks old, weighing between 200 and 220 g, were purchased from Beijing Huakang Biotechnology Co., Ltd. The number of mice was determined according to the experimental needs. Standard rodent chow and water were obtained freely by feeding all the mice in 12-hour/12-hour light/dark conditions without any other specific pathogens. At the time of the experiment, mice were anesthetized according to the body weight of the mice by intraperitoneal injection of 1% sodium pentobarbital at 1 ml/100 g. The blood of the mice was obtained through the abdominal artery under general anesthesia, and finally, the mice died due to excessive blood loss. All of the experimental procedures involving animals were approved by the Experimental Animal Welfare Ethics Committee of the Beijing Institute of Chinese Medicine, Ethics No. 2018090102.The specific information such as the composition and dosage of MFSD is shown in μm filter, and stored at −20°C.20 SD mice were perfused with MFSD for one week to obtain serum containing MFSD (MFSDS), and another 20 SD mice were orally administered with normal saline for one week to obtain control serum (CS). A week later, mice blood was obtained and centrifuged at 3000 rpm for 10 min at 4°C, and the serum was inactivated at 56°C for 30 min, filtered with a 0.22 γ at 33°C in an incubator with 5% CO2, The podocytes passaged until the cells were in good growth state and covered 70% to 80% of the culture dishes. In the interferon-containing 1640 medium, the culture was incubated at 37°C in 5% CO2, and the cells were fully differentiated after 10 to 14 days, which were used for the experiment. These cells were divided into four groups: (1) control group with 10% CS, (2) high glucose group with 10% CS, (3) DKK1 group with 10% CS, and (4) MFSD group with 10% MFSDS.Conditional immortal mouse glomerular podocytes (GPC) were presented by Professor Maria Pia Rastaldi of S. Carlo Hospital, University of Milan, Italy. The podocytes were cultured in RPMI 1640  containing 10% fetal bovine serum and 50 u/ml interferon-The cultured and well-prepared podocytes were fixed with 4% paraformaldehyde for 15 minutes and washed three times with PBS, each time for 5 minutes. The membrane was diafiltered with 0.5% Triton-100 for 30 min at room temperature and washed three times with PBS for 5 minutes each time. 5% BSA was used to block cells at room temperature for 1 hour and then primary antibody was added and incubated overnight at 4°C. After that, the primary antibody was discarded and washed three times with PBS, each time for 5 minutes, and the cytoskeleton was stained with phalloidin . The target protein was stained with Alexa Fluor 594 , and the nucleus was stained with 5%DAPI . At last, the specimen was observed under a confocal microscope .μg of each well was loaded, and the antibody dilution, primary antibody, secondary antibody, chemiluminescence solution, and wash buffer were sequentially added to the sample plate, and experiment was performed on the equipment. The remaining part was completed automatically by the equipment after adjusting the relevant settings.Western blot experiments were performed according to the instructions of the WES kit . The cultured and intervened podocytes were collected in groups, and then, an appropriate amount of RIPA lysate  was added at 4°C, fully lysed for 30 minutes, and centrifuged at 14,000 rpm, for 5 minutes, and the supernatant was collected, and the BCA quantitative assay kit  was used to determine the protein concentration. 1.5 P < 0.05 was considered statistically significant.According to the obtained data, a statistical analysis plan was made and SPSS Statistics 23.0 software was applied for analysis. Data that conformed to the normal distribution were expressed as mean ± standard deviation, and data that did not conform to the normal distribution were expressed as median (quartile spacing). One-way analysis of variance was done to calculate the difference between experimental groups in GraphPad Prism8 . 2 for 10 days to 14 days, the adherent growth area of the cells increases significantly, and a large number of secondary protrusions protrude from the main part of the cell body, making cells to appear “dendritic” or “flower-like.” There is no obvious difference in morphology between the two groups of podocytes. This shows that the MFSD-containing serum has no obvious effect on the morphology of podocytes.We observed the morphology of the MFSD group after a 24-hour intervention under light microscopy . The celBefore the intervention of high glucose (30 mmol/L) in podocytes, first of all, we cultured podocytes with DKK1 (100 ng/ml) or 10% MFSDS for 24 hours. Subsequently, podocytes were incubated with high glucose medium for 24 hours to cause destructive phenomena. After cell culture, the expression of podocyte-related proteins was analyzed by immunofluorescence and western blots. We found out that the expression levels of nephrin and podocin were significantly decreased in high glucose conditions. Western blots showed that the expression levels of nephrin, podocin, podocalyxin, and podoplanin in the DKK1 group and MFSD group also restored compared with the high glucose group . These rβ-catenin signaling pathway inhibitor, showed an inhibitory effect on Wnt/β-catenin signaling pathway which was significant. In order to study the effect of MFSD on Wnt/β-catenin signaling pathway, we further observed the quantity of the key protein of β-catenin and phosphorylated β-catenin expression. The results showed that protein expression levels were significantly increased in the high glucose group, indicating abnormal activation of Wnt/β-catenin signaling pathway. Under the intervention of DKK1 or MFSD, their expressions were significantly inhibited, indicating that the activation of the Wnt/β-catenin signaling pathway was blocked (DKK1, as a Wnt/ blocked .β-catenin signaling pathway and can reduce high glucose-induced podocyte injury. However, the mechanism remains enigmatic.Our experiments confirmed that MFSD treatment can significantly inhibit the abnormal activation of Wnt/In order to explore whether DKK1 or MFSD treatment can regulate the autophagy in podocytes, we experimented the effect of DKK1 and MFSD on autophagy. To observe the status of autophagy after podocytes were intervened, we examined autophagy-related proteins LC3B, beclin1, and p62. These experimental results confirmed that the protein expressions of LC3B, beclin1, and p62 were significantly decreased in the high glucose group, indicating that the autophagy of podocytes was inhibited under high glucose conditions. Interestingly, the protein expressions of LC3B, beclin1, and p62 were significantly increased in the DKK1 and MFSD group, which represented an upregulation of podocyte autophagy levels. At the same time, there was no significant difference in the level of autophagy between the DKK1 group and the MFSD group .in vitro.DKD is the most common cause of end-stage kidney disease worldwide. The pathogenic mechanisms are mostly understood –32. HowePodocytes are unique, highly differentiated glomerular epithelial cells which are important components of the glomerular filtration barrier . Furtherβ-catenin signaling pathway in patients with CKD [β-catenin, thereby activating the Wnt/β-catenin signaling pathway [β-catenin signaling pathway, such as knocking out β-catenin in glomerular podocytes or using the Wnt/β-catenin signaling pathway to inhibit protein DKK1, it can significantly reduce podocyte injury, reduce proteinuria, and also protect the kidney [β-catenin signaling pathway on podocytes.The Wnt protein is a secreted glycoprotein containing a signal peptide and 23 or 24 conserved cysteine residues –40. Wnt with CKD , 45. In  pathway . By inhie kidney –49, at tβ-catenin signaling pathway [β-catenin signaling pathway in podocytes [β-catenin signaling pathway in podocytes. Interestingly, we found out that β-catenin and phosphorylated β-catenin were significantly increased in the high glucose group, indicating abnormal activation of the Wnt/β-catenin signaling pathway, which is consistent with previous studies [β-catenin signaling pathway-related proteins was significantly reduced in the DKK1 and MFSD groups, which indicated that MFSD could inhibit the abnormal activation of Wnt/β-catenin signaling pathway. Combined with the previously available literature, we sort out that MFSD can reduce high glucose-induced podocyte injury by inhibiting the activation of Wnt/β-catenin signaling pathway.Many small molecules from natural products have been demonstrated to target Wnt/ pathway , 50, 51.odocytes , 53. Sin studies . The expβ-catenin signaling pathway to alleviate high glucose-induced podocyte injury. Further studies are required to rule out the whole mechanism. In DKD, podocytes are in a high glucose environment for a long time. Under the stimulation of long-term high glucose, autophagy is inhibited, and accumulated reactive oxygen species, glycosylation end products, etc., cannot be eliminated in time, and podocyte apoptosis is finally induced. Our experimental results showed that the expression levels of autophagy-related proteins LC3B and beclin1 in the high glucose group were reduced, which means that autophagy in podocytes in the high glucose group was suppressed. Under normal circumstances, P62 participates in the process of autophagolysosome degradation of protein and P62 is also degraded by this process. When cell autophagy is impaired, P62 cannot be degraded smoothly and accumulates in the cell [However, it is not enough to explain that MFSD relies on excessive activation of the Wnt/the cell . Strangeβ-catenin signaling pathway plays a key role in autophagy [β-catenin signaling pathway is involved in the regulation of autophagy in prostate cancer cells [β-catenin signaling pathway can promote the expression of autophagy in myocardium P19CL6 cells [β-catenin inhibits autophagy by inhibiting the production of p62 [β-catenin signaling pathway is activated, β-catenin accumulates in the cytoplasm and eventually enters the nucleus affecting the transcription of P62, which results in insufficient amount of P62 in the cytoplasm during the process of autophagy.Studies have shown that activation of the Wnt/utophagy , 57. Forer cells , preventL6 cells , while ββ-catenin signaling pathway. At the level of implementation, specifically, the protective mechanism can be such that long-term high glucose stimulation leads to abnormal activation of the Wnt/β-catenin signaling pathway, resulting in an increase in the expression of β-catenin, thereby inhibiting the production of p62, ultimately leading to inhibition of autophagy results in destruction of podocytes, and intervention by MFSD blocked the abnormal activation of the Wnt/β-catenin signaling pathway and upregulated the process of autophagy in podocytes (Regarding the above experimental results, we found out that the mechanism of high glucose damaging the podocytes involves signaling pathway and autophagy, and there is also a certain relation between signaling pathway and autophagy. We can also clearly see the protective effect of MFSD on podocytes induced by high glucose. This protective effect was likely to upregulate the autophagy of podocytes by inhibiting activation of Wnt/odocytes .β-catenin signaling pathway and the inhibition of autophagy after activation. Inhibition of Wnt/β-catenin signaling pathway can activate autophagy and can significantly reduce the destruction of podocytes in high glucose level. Our findings demonstrated that MFSD treatment can effectively alleviate the destruction of podocytes induced by high glucose and can also protect the podocytes by inhibiting the activation of the Wnt/β-catenin signaling pathway and simultaneously upregulating the autophagy level in podocytes, providing a new way of treating DKD.The destruction of podocytes in DKD is related to the excessive activation of Wnt/


Objective: Osteosarcoma is a common primary highly malignant bone tumor. Kinesin family member 18B (KIF18B) has been identified as a potential oncogene involved in the development and metastasis of several cancer types. While KIF18B overexpression in osteosarcoma tissue is clearly detected, its specific function in the disease process remains to be established.Methods:KIF18B expression was assessed in osteosarcoma tissues and cells. We additionally evaluated the effects of KIF18B on proliferation, migration, and invasion of osteosarcoma cells, both in vitro and in vivo.Results: Our results showed overexpression of KIF18B in osteosarcoma tissues and cells. Knockdown of KIF18B induced G1/S phase arrest and significantly inhibited proliferation, migration, and invasion of osteosarcoma cells, both in vitro and in vivo. KIF18B regulated β-catenin expression at the transcriptional level by controlling nuclear aggregation of ATF2 and at the post-transcriptional level by interacting with the adenomatous polyposis coli (APC) tumor suppressor gene in osteosarcoma cells.Conclusions: KIF18B plays a carcinogenic role in osteosarcoma by regulating expression of β-catenin transcriptionally via decreasing nuclear aggregation of ATF2 or post-transcriptionally through interactions with APC. Our collective findings support the potential utility of KIF18B as a novel prognostic biomarker for osteosarcoma. The majority of osteosarcomas are highly malignant and most patients clinically diagnosed with osteosarcoma have metastatic disease at initial presentation2. Owing to the growth and invasiveness of osteosarcoma, a periosteal reaction occurs in a radial manner. Osteosarcoma is particularly prone to early metastasis to the lung, which accounts for low annual survival rates of 5%–20% post-amputation3, highlighting the urgent requirement for novel molecular biomarkers to facilitate earlier diagnosis, accurately predict clinical outcomes, and promote effective treatments.Osteosarcoma, a malignancy derived from mesenchymal tissue, is the second most prevalent type of primary bone tumor7. Therefore, therapies targeting Wnt/β-catenin signaling present a promising anti-cancer strategy.The classical Wnt/β-catenin signaling pathway is involved in the regulation of multiple biological processes, including embryogenesis and morphogenesis, tissue stability, energy metabolism balance, and stem cell maintenance. Dysregulation of this pathway is often closely associated with major human diseases. β-Catenin protein is highly expressed in almost all tumorigenesis processes, resulting in abnormal activation of Wnt/β-catenin signaling and consequent promotion of cell colonization and migration, effects on the normal cell cycle, and induction of tumorigenesis and deterioration10. The kinesin superfamily comprises a conserved class of microtubule-dependent molecular motor proteins with adenosine triphosphatase activity12. According to the standard nomenclature for kinesin, 45 KIFs have been identified and classified into 14 families (kinesin-1 to kinesin-14)13. These families are subdivided into N-kinesin, M-kinesin, and C-kinesin, based on location of their kinematic domains at the amino, middle, and carboxy terminus, respectively14. During eukaryotic mitosis, KIFs are involved in spindle formation, chromosome recombination and alignment, and cytokinesis15. Abnormal KIF expression and function are implicated in the development of multiple human cancer types. Higher recovery of KIF function may therefore be beneficial in molecular targeted therapies for human cancers16. Assessment of KIF expression patterns may provide clues for biomarkers suitable for early detection and prognosis of human cancers. For instance, KIF5B mRNA is upregulated in several cancer tissues, including bladder, stomach, skin, and breast20. Inactivation of the KAP3 subunit of the KIF3 complex in neural precursor cells promotes embryonic brain tumors21. KIF14, a microtubule motor, is overexpressed in breast and lung cancers and retinoblastoma23. KIF18A is highly expressed in most cancer cells but not paracarcinoma tissue, except testes and lungs, as reported by Shichijo et al.24 Bioinformatic analyses suggest that overexpression of KIF18B is associated with poor prognosis in hepatic carcinoma25. Earlier, Wu and co-workers26 characterized KIF18B as a novel oncogene that promotes tumorigenicity of cervical cancer cells through activating the Wnt/β-catenin signaling pathway. Experiments by our group showed KIF18B overexpression in osteosarcoma tissue, but its specific function in the disease process is currently unclear. Data from the present study suggest that KIF18B is a potential oncogene that promotes osteosarcoma cell proliferation and migration, both in vitro and in vivo. Furthermore, KIF18B exerts its effects through regulation of β-catenin, a key protein in the Wnt/β-catenin pathway, at both transcriptional and post-transcriptional levels.KIF18B is a member of the kinesin superfamily that is closely associated with the pairing and separation of chromosomes in mitosis and controls microtubule length, centering the mitotic spindle at metaphase2 atmosphere at 37 °C.Human osteoblast cells (hFOB1.19) and three sarcoma cell lines  were obtained from American Type Culture Collection . All cell lines were cultured in DMEM supplemented with 10% fetal bovine serum  and 100 mg/mL penicillin/streptomycin . Cultures were additionally supplemented with 200 mM/L glutamine and 2 mg/mL sodium bicarbonate for hFOB1.19 cells, 1.0 g/L glucose and 1 mM sodium pyruvate for HOS and U2OS cells, and 2 mM L-glutamine for Saos-2 cells. Cells were grown in a humidified 5% COKIF18B-targeting small hairpin RNA (shRNA1) sequences were as follows: 5′-GCUCAUCAACGUCCUCAAUTT-3′ (sense) and 5′-AUUGAGGACGUUGAUGAGCTT-3′ (antisense)26. Human KIF18B-targeting small hairpin RNA (shRNA2) was purchased from Sigma  (TRCN0000245670). Recombinant KIF18B or negative control (shRNA-KIF18B or shRNA-NC) lentiviral particles were generated for transfection of cells. ATF2 siRNA (106604), CUX1 siRNA (110461), TCF3 siRNA (114338), RUNX1 siRNA (106565), GATA1 siRNA (157704), APC siRNA (122389) and control siRNA (131905) were purchased from Invitrogen . Plasmids expressing full-length human KIF18B and β-catenin protein were purchased from Genechem . Empty plasmid was used as the negative control. KIF18B and CTNNB1 cDNA were cloned into pcDNA3.1  for constructing overexpression plasmids, with empty vector used as a negative control. Transfection was performed using Oligofectamine , in keeping with the manufacturer’s instructions. Transfection efficiency was detected via quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR).Human KIF18B, CTNNB, WNT5A, MYC, CCND1, ATF2, CUX1, TCF3, RUNX1, GATA1 and β-actin genes are listed in Supplementary Table S1. The QuantStudio™ 6 Flex Real-Time PCR System was employed for collection of qRT-PCR data. The qRT-PCR reaction included an initial denaturation step at 95 °C for 10 min, followed by 40 cycles at 92 °C for 15 s, and 60 °C for 1 min. Experiments were performed in triplicate and relative expression calculated and normalized to β-actin using the 2−ΔΔCt method.Osteoblasts hFOB1.19 and osteosarcoma cells were harvested and collected, and total RNA extracted with TRIzol reagent . The quality and quantity of RNA were assessed using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific Inc.). For qRT-PCR, a reverse transcription kit  was employed. The reaction was performed with 1,000 ng total RNA and SYBR Select Master Mix  in a final volume of 20 μL. The primers used for via SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes. Next, membranes were blocked in 5% skimmed milk powder in Tris-buffered saline/Tween 20 (TBS-T) for 1 h and incubated overnight at 4 °C with primary antibodies against KIF18B , β-catenin , Wnt5a , Myc , cyclin D1 , ATF2 , DDK , β-tubulin , Lamin B1 , β-actin , APC  or GAPDH . After washing with TBS-T, membranes were incubated with goat anti-rabbit secondary antibody  at room temperature for 2 h. The primary and secondary antibodies were purchased from Abcam . Blots were visualized via enhanced chemiluminescence detection . All experiments were independently repeated at least three times.Cells were harvested and processed in lysis buffer , β-mercaptoethanol, and glycerol) on ice. A BCA Kit  was utilized for protein quantitation. Equal amounts of protein were separated Cell proliferation was examined using Cell Counting Kit-8 (CCK-8) (KeyGEN). Transfected cells and controls were seeded at densities of 5,000 and 10,000 cells per well, and absorbance measured at 450 nm using an ELx-800 universal microplate reader . Cells were grown for one week, with changing of medium every second day. Cells were subsequently fixed with 4% paraformaldehyde and stained with 0.1% crystal violet for analysis. The visible colonies were counted. Each experiment was performed in triplicate.4) in 100 μL serum-free medium were added to the upper chamber and the lower chamber filled with complete medium containing 10% serum. After 48 h of incubation at 37 °C and 5% CO2, non-migrating cells present on the upper surface were removed. Membranes were fixed in 4% paraformaldehyde and stained with 0.1% crystal violet. The number of migrating cells from four random fields was evaluated under a microscope. Each sample was assayed in triplicate. A similar system with Matrigel-coated membranes was employed for assessing invasion. In this case, Transwells were first coated with Matrigel  and cells were allowed to invade for 72 h.Cell migration and invasion assays were performed in 6.5 mm Transwells . Cells . The percentages of cells in G0–G1, S, and G2–M phases were compared. Cells were washed and resuspended in PBS buffer, and the Annexin V-FITC cell apoptosis detection kit used for analysis of apoptosis according to the manufacturer’s instructions (BD Biosciences). Following incubation at room temperature in the dark for 20 min, cells were immediately analyzed 4 cells/well. Upon reaching 70% confluency, cells were transfected with 50 ng pGL3-Basic-CTNNB1 promoter and 0.5 ng pRL-TK , along with 50 ng pGL3-Basic-CTNNB1 (mutant) promoter using the Lipofectamine™ 2000 Transfection Reagent (Invitrogen Life Technologies), in keeping with the manufacturer’s instructions. The total amount of plasmid DNA was maintained as 200 ng in each well. Cells were harvested and lysed at 24 h post-transfection. Firefly and renilla luciferase activities were determined with the Dual-Luciferase Reporter Assay System in a GloMax96 luminescence reader  according to the manufacturer’s protocol. Relative luciferase activity was expressed as the ratio of firefly luciferase to renilla luciferase activity in each sample. All values were obtained from at least three independent repeat transfections, with six wells for each transfection mixture/sample.The day prior to transfection, U2OS cells were suspended in fresh medium and plated in 96-well plates at a density of 2 × 10via centrifugation at 1,000 × g for 1 min. ATF2, DDK, APC or β-catenin antibodies bound to protein A/G agarose using the Protein G immunoprecipitation kit . The agarose slurry (20 μL) was washed twice with 200 μL PBS buffer solution and incubated with 100 μL antibody (10 μL antibody + 85 μL H2O + 5 μL 20 × PBS) in PBS for 30 min at 25 °C on a mixer. In parallel, 100 μL rabbit serum with the same IgG concentration or anti-rabbit IgG peroxidase secondary antibody was prepared as a negative control. Agar glycoprotein and 10 μL antibody were incubated in a mixer for 1 h at 25 °C, followed by overnight incubation with 600 μL pre-clarified lysates. Immunoprecipitation products were washed five times with washing buffer and eluted with 2× Laemmli buffer at 100 °C for 10 min. Three independent experiments were conducted.Cells were harvested with 1% NP40 lysis buffer containing protease and phosphatase inhibitors . The supernatant was removed 7) grown in 150 mm dishes were fixed with 1/10 volume of cross-linking solution [11% formaldehyde/0.1 mol/L NaCl/1 mmol/L Na-EDTA/0.5 mmol/L Na-EGTA/50 mmol/L HEPES (pH 8.0)] for 10 min at 37 °C. Chromatin immunoprecipitation (ChIP) analysis was performed as reported previously27. qPCR primers for the CTNNB1 promoter regions are listed in Supplementary Table S1.Cells (1 × 106) transfected with shKIF18B or shCtrl. Tumor volumes were measured every 7 days using calipers. Approximately six weeks later, mice were anesthetized with 1% sodium pentobarbital and intraperitoneally injected with luciferin (10 μL/g). Tumor volumes were measured and tumors collected for immunohistochemical (IHC) examination.Ten nude mice  purchased from the Baylor College of Medicine Institutional Animal Care and Use Committee were subcutaneously injected in the axilla region with exponentially growing U2OS cells with fluorescent tag (1.0 × 102O2 (30 min) to inhibit endogenous peroxidase activity, washed with PBS for 5 min, and blocked with normal rabbit serum for 20 min at room temperature (RT). Incubation with anti-PTEN , anti-β-catenin , anti-Ki67  and anti-KIF18B  was performed overnight at 4 °C. Sections were subsequently incubated with the biotinylated secondary antibody  and avidin-conjugated peroxidase . Between each step, sections were washed three times with PBS. The peroxidase staining reaction was conducted with diaminobenzidine (1 mg/mL) and H2O2 for 5 min and terminated by immersion in tap water for 10 min. Sections were counterstained with hematoxylin for 1 min. In control groups, the primary antibody was replaced with pre-immune mouse serum. Both positive and negative controls were included for all assays.Paraffin sections (7 μm) were incubated in methanol/HP values < 0.05 were considered statistically significant.All statistical analyses were conducted using the SPSS 18.0 statistical software package . Values are expressed as means ± standard deviation. The Mann-Whitney U test was applied for comparison of two groups of independent data. The Kolmogorov-Smirnov test was used for statistical analysis of the normality of parameter distribution. KIF18B in immortalized human osteoblasts, hFOB1.19, and three sarcoma cell lines, HOS, U2OS, and Saos-2, was detected with qRT-PCR and Western blot, respectively. KIF18B mRNA expression was 2–3 times higher in osteosarcoma cells than hFOB1.19 cells . Consistently, KIF18B protein was overexpressed in all three osteosarcoma cell lines, in particular, HOS and U2OS . Further qRT-PCR analysis of KIF18B in 20 paired osteosarcoma tissue samples  revealed overexpression in malignant tissue in 75% (15/20) cases . The results of immunohistochemical staining for KIF18B and Ki67 of cancer tissue and adjacent tissue of osteosarcoma patients were analyzed, reveled higher expression of Ki67 and KIF18B in tissue of osteosarcoma compared to that of adjacent tissue .Differential mRNA and protein expression of KIF18B were constructed for infection of U2OS and HOS cells after viral packaging. The KIF18B mRNA level in the shKIF18B  group was 18% that in the U2OS shCtrl group , and the protein level was < 10% that in the shCtrl group , according to qRT-PCR and Western blot analyses, respectively. The KIF18B mRNA level in the shKIF18B (shKIF18B#2) group was 21% relative to the U2OS shCtrl group  and the protein level was < 13% that in the shCtrl group . Consistent results were obtained with the HOS cell line. KIF18B mRNA levels in the shKIF18B#3 and shKIF18B#4 groups were 26% and 27%  and the corresponding protein levels were < 15% and 17% relative in the HOS shCtrl group , respectively.Two shRNA lentiviral vectors specifically targeting Figure 2C and 2D). To gain further insights into the effects of KIF18B suppression in U2SO cells, cell cycle distribution was analyzed. KIF18B knockdown led to alterations in the periodic distribution of U2OS cells. Compared to shCtrl cells, cells depleted of KIF18B were significantly increased in the G1 phase and decreased in the S and G2/M phases , indicating that downregulation of KIF18B induces G1/S phase arrest in U2OS cells. Furthermore, KIF18B knockdown promoted apoptosis of U2OS cells .With prolonged culture, the differences between the number of cells in the shKIF18B and control groups gradually became clear. By day 5, the shKIF18B cell number was only a third of the control group , suggesting that knockdown of KIF18B attenuates the U2OS migration and invasion. Further examination of EMT-associated proteins revealed that compared with shCtrl cells, the shKIF18B cells contained lower E-cadherin and higher vimentin levels, supporting a positive correlation between expression of KIF18B and EMT . Similar results were obtained with the HOS cell line .Transwell assays were additionally performed to investigate the effects of KIF18B on U2OS cell migration and invasion. As shown in via RNA-seq. Among the top 15 genes downregulated in the two cell lines (Supplementary Table S3), we identified CTNNB1 and WNT5A, two key genes participating in the Wnt classical and non-canonical pathways, respectively. Accordingly, we speculated that KIF18B may affect the tumorigenicity of osteosarcoma cells by influencing the Wnt signaling pathway. To examine this hypothesis, expression of CTNNB1 and WNT5A in shKIF18B cells was examined via qRT-PCR and Western blot. In agreement with results of enrichment analyses, KIF18B knockdown resulted in lower mRNA  and protein  levels of CTNNB1 than shCtrl treatment whereas expression of WNT5A was not affected. Examination of c-MYC and cyclin D1 genes downstream of CTNNB1 (β-catenin) additionally revealed significant downregulation of both mRNA  and protein  levels. Notably, CTNNB1 overexpression in shKIF18B cell lines  resulted in recovery of proliferation , migration, invasion, proportion of cells in each cell cycle phase, and proportion of apoptotic cells to control levels . These results clearly suggest that the function of KIF18B in osteosarcoma cells is closely associated with activation of β-catenin, a key member of the Wnt/β-catenin pathway.To establish the specific role of KIF18B in progression of osteosarcoma, differentially expressed genes between shCtrl and shKIF18B cells were detected KIF18B additionally affected the relative β-catenin mRNA content in the current study. The promoter region of β-catenin was amplified using PCR and used to construct a luciferase reporter vector. Upon KIF18B knockdown, luciferase activity was significantly decreased, suggesting that KIF18B acts on the promoter region and positively regulates transcription of β-catenin . Data from ChIP experiments further validated binding of KIF18B to β-catenin promoter . No previous studies have described a DNA binding domain although an ATP-binding domain of KIF18B has been identified, highlighting the possibility that KIF18B interacts indirectly with the promoter of β-catenin. Next, we used the online tools PROMO  and TRANSFAC (http://gene-regulation.com/pub/databases.html) to predict transcription factors that potentially bind the β-catenin promoter. ATF-2 (ATF2), CUTL1 (CUX1), E47 (TCF3), Evi-1 (RUNX1), and GATA-1 (GATA1) were the top ranked hits. Upon utilization of siRNAs to suppress the levels of these transcription factors, mRNA expression of β-catenin and luciferase activity of the promoter-reporter vector in the ATF2 and RUNX1 siRNA groups showed different degrees of downregulation . ChIP experiments confirmed that KIF18B binding at the promoter region was significantly decreased, along with a specific decrease in ATF2 expression . After deletion of the potential binding site of ATF2 at the promoter region of β-catenin in the luciferase reporter vector , KIF18B induced significant downregulation of the transcriptional activation of luciferase . Our results suggest that KIF18B interacts with the promoter region of β-catenin via ATF2. In osteosarcoma specimens, qPCR analysis showed positive co-expression of β-catenin with KIF18B and ATF2 relative to that in adjacent tissues . Immunoprecipitation assays (Co-IP) performed on U2OS cells overexpressing KIF18B with a DDK tag revealed interactions between KIF18B and ATF2 . Knockdown of KIF18B did not affect the mRNA and protein expression levels of ATF2  but altered nuclear distribution and decreased nuclear aggregation of ATF2 to a significant extent . Kinesin is based on cytoskeleton components, such as microtubules, and transports different molecular cargo proteins to designated intracellular locations. As a member of the kinesin family, KIF18B may be contribute to regulation of β-catenin transcription by transporting ATF2 into the nucleus.Studies to date have shown β-catenin regulation at the protein rather than transcript level. However, knockdown and overexpression of Figure 6A). Protein stability testing was subsequently performed using cycloheximide to inhibit protein translation. The protein stability of β-catenin in the shKIF18B group was significantly lower than that in the shCtrl group . After cycloheximide treatment for 8 h, the β-catenin content of the shKIF18B group was approximately half that of the shCtrl group. We detected interactions between KIF18B and APC . APC binds β-catenin and promotes its degradation. Notably, overexpression of KIF18B significantly blocked interactions between APC and β-catenin . The protein stability of β-catenin in the shKIF18B-siAPC group (with knockdown of APC in the shKIF18B group) was recovered to a similar level as that in the shCtrl group . Our collective results suggest that KIF18B competitively binds APC and indirectly regulates β-catenin protein levels.Co-IP experiments revealed no interactions between KIF18B and β-catenin directly . Tumor volume and weight were lower in the shKIF18B than control group . IHC staining revealed attenuated expression of Ki67, PTEN, KIF18B, and β-catenin in the sh-KIF18B group , indicating that silencing of KIF18B inhibits tumor growth in vivo.A nude mouse xenograft model using U2OS cells was further established. The luciferase activity detected in mice injected with shKIF18B was significantly lower than that in mice injected with shCtrl cells , were significantly downregulated. CTNNB1 overexpression led to recovery of the phenotype of shKIF18B cells, such as inhibition of proliferation, migration, and invasion. These results suggest that the function of KIF18B in osteosarcoma cells is closely associated with activation of the Wnt/β-catenin pathway. Several diseases, including malignant tumors, are associated with imbalances in the intracellular Wnt/β-catenin signaling pathway34. A host of important transcription and signal transduction factors are involved in regulation of the Wnt/β-catenin signaling pathway. β-Catenin, a marker of Wnt activity and a core factor transmitting Wnt signals, is highly expressed in almost all types of tumors. Consequently, the Wnt signaling pathway is abnormally activated, thus promoting cell colonization and migration, affecting the normal cell cycle and inducing tumor deterioration 35.To clarify the specific mechanisms underlying the tumor-promoting effect of KIF18B in U2OS cells, microarray analysis was applied to detect the differences in transcription before and after KIF18B induced a decrease in β-catenin mRNA expression. Generally, β-catenin is thought to be regulated at the protein level, with limited reports of transcriptional regulation. To ascertain whether KIF18B regulates β-catenin transcription, we incorporated the promoter region of β-catenin into a luciferase reporter vector. Notably, luciferase activity was significantly decreased with knockdown of KIF18B. Data from ChIP assays additionally supported KIF18B activity on the promoter region of β-catenin. However, no studies to date have identified a DNA binding domain in KIF18B, indicating that interactions between KIF18B and the β-catenin promoter may be indirect. Further analyses revealed interactions between KIF18B and ATF2. With decreased KIF18B expression, distribution in the cytoplasm and nucleus was altered and the degree of nuclear aggregation of ATF2 was significantly decreased. Accordingly, we propose that KIF18B, a member of the driver protein family, regulates β-catenin at the transcriptional level by transporting the transcription factor, ATF2, into the nucleus. We additionally observed significantly lower β-catenin protein stability in the KIF18B knockout relative to the control group. The mechanism by which KIF18B affects the stability of β-catenin protein is yet to be established. Interestingly, we observed interactions between KIF18B and APC. In mammals, APC is expressed in most fetal tissues and adult epithelial cells. The APC gene encodes a multi-domain protein of 2843 amino acids. Mammalian APC contains an oligomerization domain, armadillo repeat domain, and 15 or 20 residue repeat domain, which is critical for binding to β-catenin37. Interestingly, an earlier report by Behrens et al.38 showed that β-catenin is downregulated via interactions with APC. Consequently, activation of the typical Wnt pathway triggered by APC inactivation could result in intestinal tumor occurrence. The protein stability of β-catenin in the shKIF18B-siAPC group was restored to that of the control group, compared with that in the shKIF18B group. APC inactivation is proposed to promote tumorigenesis through loss of cell adhesion40. Overexpression of KIF18B significantly hindered the interactions between APC and β-catenin, indicating that KIF18B competitively binds APC and protects β-catenin stability. The data support an indirect regulatory effect of KIF18B on β-catenin at the translational level.Knockout of in vitro. Downregulation of KIF18B leads to arrest of U2OS osteosarcoma cells in the G1/S phase, resulting in decreased proliferation, migration, and invasion. In addition, KIF18B knockdown suppresses expression of β-catenin, a key protein in the Wnt/β-catenin pathway, at the transcriptional level by decreasing the degree of nuclear aggregation. Simultaneously, KIF18B regulates β-catenin at the post-transcriptional level through interactions with the tumor suppressor gene APC. Based on the collective findings, we propose that KIF18B plays a carcinogenic role and may effectively serve as a novel prognostic biomarker of osteosarcoma.KIF18B is overexpressed in osteosarcoma cells and tissues and its expression is positively correlated with the degree of malignancy of U2OS cells. KIF18B promotes osteosarcoma cell proliferation Click here for additional data file.


A disintegrin and metalloproteinase (ADAM) family of proteins play versatile roles in cancer development and progression. In the present study, we investigated the role of ADAM proteins in colorectal cancer (CRC) cell migration and invasion focusing on the epigenetic mechanism whereby ADAM transcription is regulated. We report that higher levels of ADAM10, ADAM17, and ADAM19 were detected in SW480 cells than in HCT116 cells. Expression levels of the same set of ADAMs were higher in human CRC biopsy specimens of advanced stages than in those of a less aggressive phenotype. Overexpression of ADAM10/17/19 in HCT116 cells enhanced, whereas depletion of ADAM10/17/19 in SW480 cells weakened, migration and invasion. ADAM expression was activated by the Wnt signaling pathway, which could be attributed to direct binding of β-catenin on the ADAM promoters. Mechanistically, β-catenin recruited the chromatin remodeling protein BRG1, which in turn enlisted histone demethylase KDM4 to alter the chromatin structure, thereby leading to ADAM transactivation. In conclusion, our data suggest that the Wnt signaling may promote CRC metastasis, at least in part, by recruiting an epigenetic complex to activate ADAM transcription. Colorectal cancer (CRC) represents one of the deadliest forms of cancers worldwide with approximately 2 million projected new diagnoses each year . ImplemeA complicated network of signaling pathways, of which the Wnt signaling pathway is the best characterized, orchestrates the pathogenesis of CRC . In normA disintegrin and metalloproteinases (ADAMs) belong to a family of transmembrane proteolytic enzymes that play keys roles in cell proliferation, adhesion, migration, and invasion. ADAMs exert their wide-ranging effects by separating membrane-anchored proteins from their extracellular domains in a process termed “ectodomain shedding.” Previous investigations have implicated ADAMs in the development and progression of CRC . Park anWe first profiled the expression of 12 human ADAM isoforms that have been confirmed to possess enzymatic activities  in HCT11trans-well assay  situated on each of three ADAM promoters . ChromatWe have previously shown that the chromatin remodeling protein BRG1 activates Wnt signaling pathway to regulate liver regeneration by recruiting the histone demethylase KDM4 to modulate β-catenin activity . We askeRe-ChIP assay revealed that a much stronger BRG1–β-catenin interaction could be detected across the WRE region, but not the transcription start site (TSS) region, of the ADAM promoters in SW480 cells than in HCT116 cells . DepletiWe next performed ChIP assays to examine whether BRG1 may recruit KDM4 to the ADAM promoters. As shown in Frizzled) and Wnt ligand genes (Wnt). A follow-up study by Weng and colleagues have found that the long non-coding RNA LncFZD6 recruits BRG1 to the Frizzled6 promoter and the Wnt5a promoter to activate transcription, thereby driving liver cancer cell self-renewal (Villin-Cre) or in the intestinal stem cells (driven by the Lgr5-Cre) attenuates colorectal tumorigenesis and improves survival in Apc-deficient mice, which is attributable to the suppression of key β-catenin target genes involved in the initiation and progression of CRCs. As tantalizing as the model appears, it is not without a caveat. A recent investigation by RosaBrg1/+; Villin-Cre) protects, whereas BRG1 deletion in epithelial cells  exacerbates, colorectal carcinogenesis in a colitis-induced mouse model. The underlying mechanism, according to the authors, can be ascribed to the ability of BRG1 to regulate autophagy, which helps sequester reactive oxygen species and rein in aberrant inflammation, thus shielding the intestines from oncogenic attacks; it is not clear whether Wnt/β-catenin signaling pathway was altered by BRG1 gain of function or loss of function in this model. Therefore, BRG1 may act as both a promoter and suppressor of CRC pathogenesis, depending on the genetic background and the etiology, which advises against non-discriminate targeting of BRG1 in devising preventive and interventional approaches.CRCs have been one of the leading causes of cancer-related deaths for the last decade and remain a deadly pathology in the era of personalized medicine. Malignant forms of CRC are characterized by aggressive migration and invasion that enable cancer cells to spread to secondary sites. Here we describe a novel pathway in which the chromatin remodeling protein BRG1 promotes CRC cell migration and invasion by activating the transcription of ADAM proteinases . We show-renewal . More reOur data suggest that BRG1 contributes to transcriptional activation of ADAM genes in part by recruiting H3K9 demethylases. In keeping with our observation, In summary, our data suggest that a β-catenin–BRG1–KDM4 transcriptional complex regulates ADAM10/17/19 expression. There are a few limitations regarding this study. First, although we have provided ample evidence to show that the β-catenin–BRG1–KDM4 transcriptional complex contributes to ADAM10/17/19 transcription, the underlying pathophysiological implication in CRC cell migration/invasion remains unclear. Additional experiments should be performed in additional cancer cells, authenticated in animal models of CRC metastasis, and verified in different, larger cohorts of patients. Second, the effects of KDM4 knockdown on ADAM expression in SW480 cells were rather modest pointing to alternative epigenetic mechanisms that might facilitate the regulation of ADAM transcription by β-catenin/BRG1. Third, the requirement of different KDM4s for global β-catenin–dependent transcription awaits further investigation. Clearly, further studies are needed before targeting this newly identified axis can be considered as a viable approach in personalized treatment of CRC patients.TM) were purchased from ATCC. BRG1 expression constructs  with freshly added protease inhibitor (Roche) as previously described . SpecifiADAM10, 5′-GCTGTGATTGCCCAGAT ATCCA-3′ and 5′-CACCATGAAACTGATGTTACGGA-3′; ADAM17, 5′-CTTATGTTGGCTCTCCCAGA-3′ and 5′-CCAG GTCAGCTTCCTTTGTA-3′; ADAM19, 5′-AGAGTGTGGGT CCTGTGGTA-3′ and 5′-AACTGAACTGTTGCCTCAGC-3′. Ct values of target genes were normalized to the Ct values of a housekeeping control gene  using theDDCt method (RNA was extracted with the RNeasy RNA isolation kit (Qiagen) as previously described . Reverset method  and exprWound healing assay was performed as previously described . Cells wTranswell assay was performed as previously described . Twenty-Cell proliferation was measured using an MTT kit (Abcam) as previously described . Briefly3), diluted with the re-ChIP buffer , and subject to immunoprecipitation with a second antibody of interest. Precipitated genomic DNA was amplified by real-time PCR with the following primers:ADAM10 WRE, 5′-AAAAGTTCTAGTTCATGAC-3′ and 5′-AAATTTATGTCACAGACTAAGGC-3′; ADAM10 TSS, 5′-AAAGGAGGAAGGAAACGAAC-3′ and 5′-AAGCACCTCC CTCTCGCTCC-3′; ADAM17 WRE, 5′-AAAGAGGCTCAGA AAAAGGAAC-3′ and 5′-AACACACCTTATTTTCC-3′; ADA M17 TSS, 5′-AGCACTTAGTTCTTTGTGCC-3′ and 5′-AAAC TGGACCCCTCTCCGCAC-3′; ADAM19 WRE, 5′-AAACCGG GAGATTGGAGTCC-3′ and 5′-ATTGACTACTCTTGGAGTC-3′; ADAM19 TSS, 5′-AGAAGCCCTGAAGGGCTAG-3′ and 5′-AGGTTTTCAAGCAAAC-3′. A total of 10% of the starting material is also included as the input. Data are then normalized to the input and expressed as% recovery relative the input. All experiments were performed in triplicate wells and repeated three times.ChIP assays were performed essentially as described before . In briet test was performed using an SPSS package. Unless otherwise specified, p values smaller than.05 were considered statistically significant.Sample sizes reflected the minimal number needed for statistical significance based on power analysis and prior experience. Two-tailed Student The original contributions presented in the study are included in the article/All human studies were reviewed and approved by the Nanjing University of Chinese Medicine Committee on Ethical Conduct of Studies with Human Subjects. Colorectal cancer tissues were collected, under informed consent, from surgical resection specimens of patients who had not undergone radiotherapy or chemotherapy in the Jiangsu Province Hospital of Chinese Medicine.YF and LS conceived the project. LS, BC, JH Wu, CJ, ZW Fan, YF, and YX designed the experiments. LS, BC, JH Wu, CJ, and ZW Fan performed the experiments and collected and analyzed the data. YX wrote the manuscript. LS provided the funding. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The role of the WNT signaling pathway in key cellular processes, such as cell proliferation, differentiation and migration is well documented. WNT signaling cascade is initiated by the interaction of WNT ligands with receptors belonging to the Frizzled family, and/or the ROR1/ROR2 and RYK families. The downstream signaling cascade results in the activation of the canonical β-catenin dependent pathway, ultimately leading to transcriptional control of cell proliferation, or the non-canonical pathway, mainly acting on cell migration and cell polarity. The high level of expression of both WNT ligands and WNT receptors in cancer cells and in the surrounding microenvironment suggests that WNT may represent a central conduit of interactions between tumor cells and microenviroment. In this review we will focus on WNT pathways deregulation in hematological cancers, both at the ligand and receptor levels. We will review available literature regarding both the classical β-catenin dependent pathway as well as the non-canonical pathway, with particular emphasis on the possible exploitation of WNT aberrant activation as a therapeutic target, a notion supported by preclinical data. The WNT signaling pathway is central for development and homeostasis within tissues. The WNT signaling cascade is initiated by the interaction of WNT proteins (lipid-modified secreted glycoproteins) with various receptors and co-receptors whose activation elicit several processes, such as cell proliferation, differentiation, apoptosis, polarity, migration and invasion , 2.WNT proteins are evolutionary conserved and in mammals WNT family genes comprise 19 members. These secreted, cystein-rich proteins exert their effects through the interaction with the Frizzled family of proteins (consisting of 10 members). Frizzled (FZD) are seven-pass transmembrane, and each FZD protein contains a cysteine-rich domain (CRD) with approximately 10 cysteine residues, essential for the binding to WNT proteins , 4.Apart from FZD, other proteins are involved in WNT recognition: the co-receptor low-density lipoprotein receptor-related proteins (LRP 5 and 6), the receptor Tyr kinase-like orphan receptor family (ROR1 and ROR2), the Receptor Tyrosine Kinase (RYK) and the protein Tyr kinase 7 (PTK7).The WNT pathway is broadly divided into canonical (β-catenin-dependent) and non-canonical (β-catenin-independent), depending on the agonists, the receptors and the intracellular players involved. Given the high number of both WNT ligands and receptors, the possible combinations are numerous, making its regulation exceedingly complex. The activation of either signaling pathway is receptor and ligand dependent and yet is also influenced by the cellular context. As an approximation, certain WNTs  activate the canonical β-catenin-dependent WNT signaling pathway by preferentially binding to FZD receptor coupled to LRP5/6 co-receptors. Conversely, WNT5A and WNT11 preferentially bind to ROR1, ROR2 and RYK receptors and are predominantly involved in the β-catenin-independent, non-canonical pathway.Irrespectively from the signaling branch activated upon binding of WNT ligands to FZD or other receptors, the signal is commonly transduced to a protein called Disheveled (DVL) that represents the molecular hub executing both the canonical and non-canonical pathways . How canAs mentioned earlier, FZD proteins act as main receptors for WNT ligands, together with the co-receptor LRP5/6.In the absence of exogenous WNT, the pathway is maintained in the “off” state by the so-called “β-catenin destruction complex”. This multiprotein complex, composed by Axin, β-catenin, APC, GSK3 and CK1, keeps β-catenin at low levels by phosphorylating it, and thus marking it for recognition by β-TRCP ubiquitin ligase, leading to its subsequent ubitiquination and degradation , 8. When2+ pathway, characterized by an increase in the intracellular calcium level as a result of WNT binding. This pathway subsequently activates heterotrimeric G proteins and Phospholipase-C (PLC) which hydrolyses membrane phospholipids to di-acyl glycerol and inositol 1,4,5-triphosphate (IP3). This neo-generated IP3 causes the release of calcium from the endoplasmic reticulum which activates Protein kinase C (PKC) and ultimately activating calcineurin and NFAT transcription factor, thus regulating transcriptional programs involved in cell fate and cell migration (The interactions of other WNT ligands (such as WNT5A and WNT11) and receptors (including ROR1 and ROR2) results in the activation of alternative signaling routes different from the canonical β-catenin dependent pathway, known as WNT non-canonical pathways. The first example of a non-canonical pathway is the Planar Cell Polarity (PCP)/Jun N-terminal kinase (JNK) signaling cascade that involves the activation of JNK, Rho, Rac and Rho kinase (ROCK) proteins . In thisigration  . As for the antagonists they can be broadly divided in two categories: the one interfering with WNT ligand  or other binding to the receptor complex (Dickkopf-related proteins- DKKs) .WNT agonists: R-Spondin family of growth factors consists of 4 members (RSPO1 to 4), acting on both canonical (β-catenin dependent) and non-canonical signaling. Their mechanism of action is still controversial but it is suggested that RSPO proteins bind to leuchin-rich G protein 4 and 5 (LRG-4 and 5) to sequester ZNRF3, an E3 ubiquitin ligase involved in the turn-over of Fz-LRP6 receptor complex. By sequestering ZNRF3, RSPO-LRG interaction induces the accumulation of the receptor complex on the cell surface, thus enhancing WNT signaling , all sharing a conserved CRD at the N-terminus. They act binding WNT ligands and preventing their interaction with the receptor, although mouse sFRP3 has been shown to interact with FZD8  is another secreted factor that acts binding to WNT ligands and preventing their interaction with the receptors. In this way, WIF-1 can inhibit both canonical and non-canonical signaling.Lastly, the DKK family includes four members (DKK1 to 4), with DDK1, 2 and 4 binding to LRP5/6 and inhibiting the canonical β-catenin pathway. DDK3 does not seem to bind to LRP5/6 and is likely acting on a different pathway such as the TGF-β one .WNTS are secreted proteins exerting their function on a short- or long-range gradient in the neighboring cells. Their proper secretion and release in the extracellular space represents an additional level of signaling modulation. Indeed, WNT proteins need indeed to be posttranslational modified in order to be exported and to be biologically active. One of the most crucial modification is acylation, critical for the transportation from the Golgi to the cell surface  and for The high level of expression of both WNT ligands and WNT receptors in cancer suggests that WNT may represent a central conduit of interactions between tumor cells and the microenvironment. Although most of the studies of WNT involvement in cancer come from solid tumors, WNT signaling deregulation has also been observed in hematological cancers.WNT ligands are normally expressed in the hematopoietic stem cells (HSC) compartment and the surrounding microenvironment, and WNT signaling is crucial for HSC self-renewal and homeostasis , as wellAberrant WNT signaling has been reported for example in acute myeloid leukemia (AML). Common chromosomal translocations found in AML  result in the stimulation of WNT signaling in AML cells , 29, indin vitro (In chronic myelogenous leukaemia (CML), increased levels of nuclear β- catenin were detected, thus enhancing the self-renewal capacity of CML cells both in vitro  and in ain vitro . Both cain vitro , 38.WNT pathway deregulation is suggested to exert a crucial role in acute lymphoblastic leukemias (ALL) as well. In T-ALL, that is strictly dependent on Notch pathway alterations, a pivotal role for canonical WNT signaling has been postulated, based on the high expression of β-catenin, its cofactor LEF1  and on tAberrant WNT signaling is not only involved in the development of leukemias originating from the stem cell compartments, but it is also observed in disorders originating from mature cells.in vivo results in detachment of myeloma cells from their niche and delays disease progression. We also demonstrated, using in vitro and ex vivo 3D-culture systems, that ROR2 exerts a pivotal role in the adhesion of cancer cells to the microenvironment, mainly through the PI3K-AKT pathway, and that genetic and pharmacological inhibition of the AKT pathway is able to reduce ROR2-induced adhesion of malignant cells to bone marrow components . Indeed, β- catenin is often overexpressed and constitutively activated in MM cells, impacting on cell proliferation . Moreovemponents . Deregulmponents , 50.In chronic lymphocytic leukemia (CLL) several components of the WNT pathway have been found deregulated: WNT3, WNT5B, WNT6, WNT10A, WNT14, and WNT16, as well as the WNT receptor FZD3, are highly expressed in CLL when compared with normal B cells –53. MoreAberrant WNT signaling can also be found also in lymphomas. Indeed, elevated levels of nuclear β-catenin have been detected in primary samples of different lymphoma sub-types, even in the absence of mutations in APC or β-catenin –60, sugghttps://clinicaltrials.gov/, summarized in Many players of both the canonical and non-canonical WNT signaling are deregulated in hematological cancers, making the WNT pathway an interesting therapeutic opportunity for blood cancers. In this context, candidate drugs can act on WNT signaling pathway at different levels. The ideal druggable targets are both WNT ligands and the receptors or co-receptors, as they are easily accessible for a small compound. However, also intracellular mediators at different signaling levels or additional regulators of WNT pathways (extracellular regulators like DKKs or sFRPs or regulators of WNT secretion like PORCN) have been proposed as possible targets. In this section we report the main therapeutic agents targeting the WNT pathway and currently under clinical trials for the treatment of hematological malignancies  also in the context of drug resistant cells , 65. A pAnother interesting molecule that impacts on WNT-β-catenin signaling is the CWP232291 inhibitor. This molecule targets β -catenin for degradation, thus inhibiting its signaling and exerting anti-apoptotic and anti-proliferative activities. CWP232291 is currently in use in different clinical trials in AMLs and MM, and it was recently reported that this compound is well tolerated and shows encouraging clinical activity .in vivo  or with the anti-DKK1 monoclonal antibody BHQ880, which has been included in an already completed phase 1 (NCT01302886) and phase 2 trials (NCT01337752 and NCT00741377) showing potential clinical activity in myeloma patients . DKK1 haIn this review we tried to focus on the specific aberrations affecting the WNT pathway in different hematological cancer and how these aberrations could be exploited as therapeutic targets, developing drugs and compounds with specific effects on that particular biological process. If on the one hand the presence of many WNT components (19 WNT ligands and more than 15 receptors) offers several possibilities of tackling the pathway (though the problem of redundancy still remains), on the other hand the fact that WNT signaling is essential for many processes in development and homeostasis poses several challenges on the side of drug specificity and safety. The considerable bulk of knowledge accrued so far and the initial testing of several compounds impinging on this pathway suggest that the WNT network is both a crucial mediator of cancer cell survival and proliferation as well as an enticing target for new drugs. Nevertheless, much effort is still active on the identification of peculiar abnormalities and on the way to specifically target them, with the final aim of exploiting the WNT pathway alterations to stratify patients and provide novel tools to be exploited in a personalized medicine setting.MF wrote the manuscript. GT reviewed and wrote the manuscript. All authors contributed to the article and approved the submitted version.Funding for this research was provided by Fondazione Cariplo, Association for International Cancer Research (AICR no. 09-0713 to GT), Associazione Italiana per la Ricerca sul Cancro , Multiple Myeloma Research Foundation (MMRF Research Fellow Award to MF), and Ministero della salute (GR-2011-02351686 to MF).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.


The Wnt activator LiCl induced upregulation of CXCR4 and Wnt target gene expression. Treatment with the Wnt inhibitors had opposite effects. LiCl significantly increased [68Ga] Pentixafor uptake, while treatment with Wnt inhibitors decreased radiopeptide uptake. Wnt pathway modulation influences CXCR4 expression and function in NET cell lines. Wnt modulation might be a tool to enhance the efficacy of CXCR4-directed therapies in NET or to inhibit CXCR4-dependent proliferative signaling. The underlying mechanisms for the interaction of the Wnt pathway with CXCR4 expression and function have yet to be clarified.Loss of Somatostatin Receptor 2 (SSTR2) expression and rising CXC Chemokine Receptor Type 4 (CXCR4) expression are associated with dedifferentiation in neuroendocrine tumors (NET). In NET, CXCR4 expression is associated with enhanced metastatic and invasive potential and worse prognosis but might be a theragnostic target. Likewise, activation of Wnt/β-catenin signaling may promote a more aggressive phenotype in NET. We hypothesized an interaction of the Wnt/β-catenin pathway with CXCR4 expression and function in NET. The NET cell lines BON-1, QGP-1, and MS-18 were exposed to Wnt inhibitors  or the Wnt activator LiCl. The expressions of Wnt pathway genes and of CXCR4 were studied by qRT-PCR, Western blot, and immunohistochemistry. The effects of Wnt modulators on uptake of the CXCR4 ligand [ Neuroendocrine neoplasia (NEN) is a heterogeneous group of tumors with increasing incidence originating from cells of the neuroendocrine system. Two thirds of all neuroendocrine tumors develop in the gastrointestinal tract or the pancreas ,2.Neuroendocrine carcinomas (NEC) are undifferentiated tumors with high proliferation rates (Ki-67 > 80%), whereas the histologically differentiated neuroendocrine tumors (NET) typically show proliferation rates between 1% and 50%. NET retain histological characteristics and functional properties of the original neuroendocrine tissue. This may result in tissue-specific expression of somatostatin receptors .68Ga]Pentixafor and [177Lu]/[90Y]Pentixather were developed [68Ga]Pentixafor PET CT, [68Ga]DOTATOC PET CT, and [18F]FDG PET CT  Pentixafor) were incubated for 1 h/2 h at 37 °C. After incubation, the cells were washed twice with ice-cold washing medium. The cells were extracted with 0.1 M NaOH to obtain the internalized fraction. By Wizard2 2480 , internalized counts per minute were determined in each treatment condition. The values were corrected for differences in cell number as measured by the protein content per well .BON-1, QGP-1, or MS-18 cells were seeded in 24 well plates (Greiner bio-one) .Statistical evaluations were performed using Student’s paired In the three cell lines, the effects of different Wnt-modulators on canonical Wnt signal transduction were characterized by qRT-PCR of the Wnt effector β-catenin and the Wnt target genes MYC, TCF7, and CCND1 .We observed marked differences between the cell lines concerning the effects of Wnt modulators. In general, the three Wnt inhibitors 5-aza-CdR, quercetin, and niclosamide decreased β-catenin mRNA expression in BON-1 and QGP-1 cells, while the Wnt activator LiCl increased the expression of β-catenin. The latter effect was more pronounced in QGP-1 cells. In MS-18 cells, β-catenin mRNA expression was not affected by the Wnt modulators.Depending on the cell line, the Wnt target genes were either downregulated or unchanged after treatment with the Wnt inhibitors. In BON-1 cells, all three inhibitors reduced Wnt target gene expression. This was most pronounced with 5-aza-CdR and quercetin, where TCF7 and MYC were markedly downregulated. In QGP-1 cells, 5-aza-CdR significantly downregulated TCF7 and CCND1 but did not change MYC expression. Quercetin did not affect the expression of these Wnt target genes in QGP-1 cells. In MS-18 cells, 5-aza-CdR did not affect TCF7, MYC, and CCND1 mRNA expression, while quercetin and niclosamide exposure led to a downregulation of those genes. Niclosamide was effective in reducing Wnt target genes in all three cell lines.Treatment with the Wnt activator LiCl resulted in an upregulation of Wnt target genes  in all three cell lines. The efficacy of LiCl was strongest in QGP-1 cells and weakest in MS-18 cells.The effects of the Wnt modulators on CXCR4 expression were analyzed by qRT-PCR, Western blot, and immunohistochemistry (IHC). When standardized for the housekeeper Glycerinaldehyd-3-phosphat-Dehydrogenase (GAPDH), basal CXCR4 mRNA expression was considerably higher in MS-18 cells as compared to the other cell lines .p < 0.01). The smallest effect on CXCR4 mRNA expression was observed in BON-1 cells after 5-aza-CdR exposure . Stimulation with the Wnt activator LiCl significantly increased CXCR4 mRNA expression in BON-1 and MS-18 cells, with no influence on CXCR4 mRNA in QGP-1 cells.All Wnt inhibitors reduced CXCR4 mRNA expression in BON-1, QGP-1, and MS-18 cells . This waImmunohistochemical staining of CXCR4 was performed in all cell lines after incubation with the Wnt modulators . The str68Ga] Pentixafor with and without prior exposure to the Wnt modulators. 68Ga] Pentixafor uptake in BON-1 and MS-18 cells with the exception of 5-aza-CdR in MS-18 cells, where no significant effect was observed. This is in accordance with the IHC results but different from the observations made with qRT-PCR. In QGP-1 cells, [68Ga] Pentixafor uptake was mainly unaffected by the Wnt inhibitors. Exposure to the Wnt activator LiCl resulted in a marked increase in [68Ga] Pentixafor uptake in all three cell lines, which was most pronounced in QGP-1 cells .To study the effects of Wnt modulation on CXCR4 function, the cells were incubated with the radioactively labeled CXCR4 preferring ligand  Pentixafor uptake in BON-1, QGP-1, or MS-8 cells is available. As [68Ga] Pentixafor has a high affinity to CXCR4, the specific uptake reflects the amount of radiopeptide internalized by endocytosis after binding to functionally intact membrane-bound CXCR4 [68Ga] Pentixafor uptake in all cell lines, suggesting a direct effect of Wnt activation or inhibition on CXCR4 function. In BON-1 and MS-18 cells, the Wnt inhibitors 5-aza-CdR, quercetin, and niclosamide decreased radiopeptide uptake, while in QGP-1 cells [68Ga] Pentixafor, uptake was not affected by Wnt inhibitor exposure. This is inconsistent with the qRT-PCR results, where we observed the strongest CXCR4 mRNA downregulation upon Wnt inhibitor exposure in QGP-1 cells. These differences between CXCR4 mRNA expression and the respective radiopeptide uptake might be due to the fact that receptor endocytosis and redirection are required for modulating CXCR4 signaling [68Ga] Pentixafor uptake does not necessarily need to be the result of an increased number of CXCR4 molecules on the cellular surface but may also be caused by an increase in turnover and redirection of the receptor to the membrane.So far, no information on [nd CXCR4 ,11,12. Tignaling . TherefoSince different Wnt inhibitors that interfere with the Wnt pathway at different levels exerted comparable direct effects on CXCR4 expression and since a Wnt activator had the opposite effect, the results strongly support a direct interaction of Wnt signaling with CXCR4 expression.Because there is a general paucity of pancreatic NEN cell lines and most studies are limited to BON-1 and QGP-1 cells, we added the primary NEC cell line MS-18, which was recently established by our group and displays a higher basal CXCR4 expression. A limitation of the present study is that this cell line is not fully characterized at this time. Furthermore, this study only takes into account the canonical pathway while the effects of the used Wnt modulators might extend their effects on the noncanonical Wnt pathway or other intracellular signal transduction pathways.Wnt pathway modulation affects CXCR4 expression and function in NET cell lines. In the dedifferentiated tumor cell lines used in our experiments, Wnt inhibitors inhibited CXCR4 expression and function while Wnt activation had the opposite effect. The exact underlying mechanisms need further investigation, but the notion that all cell lines responded indicates a more general applicability of Wnt modulators. Whether these results can be translated and reproduced in vivo in patients with dedifferentiated neuroendocrine tumors requires further investigation. The clinical goal would be to reduce CXCR4 receptor density by Wnt inhibition and therefore to decrease pro-proliferative and antiapoptotic signaling without unwanted hematopoietic stem cell mobilization. Moreover, short-time CXCR4 upregulation by simultaneous Wnt activation during ERT might result in higher radiopeptide uptake without increased radiation-related side effects. However, these perspectives must be validated in animal models and clinical studies.


The Wnt signaling pathway is evolutionarily conserved, regulating both embryonic development and maintaining adult tissue homeostasis. Wnt signaling controls several fundamental cell functions, including proliferation, differentiation, migration, and stemness. It therefore plays an important role in the epithelial homeostasis and regeneration of the gastrointestinal tract. Often, both hypo- or hyper-activation of the pathway due to genetic, epigenetic, or receptor/ligand alterations are seen in many solid cancers, such as breast, colorectal, gastric, and prostate. Gastric cancer (GC) is the fourth commonest cause of cancer worldwide and is the second leading cause of cancer-related death annually. Although the number of new diagnoses has declined over recent decades, prognosis remains poor, with only 15% surviving to five years. Geographical differences in clinicopathological features are also apparent, with epidemiological and genetic studies revealing GC to be a highly heterogeneous disease with phenotypic diversity as a result of etiological factors. The molecular heterogeneity associated with GC dictates that a single ‘one size fits all’ approach to management is unlikely to be successful. Wnt pathway dysregulation has been observed in approximately 50% of GC tumors and may offer a novel therapeutic target for patients who would otherwise have a poor outcome. This mini review will highlight some recent discoveries involving Wnt signaling in GC. Gastric cancer (GC) is the sixth commonest cause of cancer worldwide and the second leading cause of cancer-related death , and fivH. pylori infection [GC can be histologically characterized using the Lauren classification, with tumors described as either gland-forming adenocarcinoma  or poorly differentiated with infiltrative single cell morphology (diffuse type) . These tnfection . A varianfection . Dependinfection . The Sounfection . Furthernfection . UnfortuThe prognosis for patients with metastatic GC is very poor, with a median survival varying from 4 to 9 months depending on the degree of metastatic disease and whether patients receive palliative chemotherapy . AlthougThere are 19 highly conserved Wnt ligands, described as secreted morphogens that carry out their function from medium to long distance ranges that elicit several signaling pathways. Whether Wnt ligands act locally or distantly is dependent on how the Wnt ligands are released. Wnt ligands can be released from the plasma membrane directly, as part of an exosome or lipid protein particles, or can be tethered to the plasma membrane . The varIn the absence of Wnt signaling, cytoplasmic β-catenin is targeted by ubiquitin-directed degradation by the intracellular regulator: The β-catenin destruction complex . This de2+) pathway [Drosophila and is determined by the asymmetrical location of core PCP components . The proximal subset includes the atypical cadherin flamingo (FMI), the LIM domain protein prickle (PK), and the Van Gogh transmembrane protein (VANG1/2). The distal subset includes Fmi, Fzd receptors, DVL, and ankyrin repeat protein Diego (DGO), and it is the intercellular interactions between these subsets that allow for the asymmetrical localization of these core PCP proteins [The non-canonical Wnt signaling pathway occurs via a transcriptional β-catenin-independent signal transduction and can be broadly categorized into the planar cell polarity (PCP) and the Wnt/calcium  and R-SPO to Fzd7, transmitting Wnt5a -induced internalization of the ligand/receptor complex [Rac1 and RhoA (Rho GTPases) are effectors of cytoskeletal rearrangement. Activation of PCP signaling causes both RhoA and Daam1 to interact with DVL, leading to ROCK1 activation, mediating cytoskeletal rearrangement . The dis complex . 2+-dependent events through the release of intracellular Ca2+. The Wnt/Ca2+ was first identified in zebrafish, where overexpression of Wnt5a induced an increase in Ca2+ signaling, and in Xenopus caused the activation of protein kinase C (PKC) [2+ signaling through Fzd/ROR1/2 interaction activates inositol 1,4,5-triphosphate (IP3), 1,2-diacylglycerol (DAG) and Ca2+ by phospholipase C (PLC). PLC is responsible for the modification of IP3 and DAG, allowing IP3 to interact with Ca2+ channels on the endoplasmic reticulum, thus causing the release of Ca2+ ions. DAG can then work with Ca2+ ions to activate PKC. Further, Ca2+ activates calmodulin-dependent protein kinase II (CaMKII), which works with PKC to activate both CREB and NFκB to regulate gene transcription [In vertebrates, Wnt ligands can activate Ca C (PKC) ,19. InitRecently, a novel arm of non-canonical Wnt signaling known as the Wnt/STOP pathway (Wnt-dependent stabilization of proteins) was discovered. Canonical Wnt signaling peaks during the G2/M phase of the cell cycle as LRP6 is primed by Cyclin Y/CDK14, and at this point, activation of β-catenin-independent stabilization of proteins is seen, leading to inhibition of GSK-3β activity. This prevents degradation of multiple proteins, resulting in an increased cell protein content, essential for cellular division .Swim [Frzb, and Cres [We do not fully understand how Wnt ligands travel between cells to exert their long-range signaling activity, with both secreted- and membrane-associated mechanisms observed. Extracellular factors, such as Swim , Frzb, aand Cres , can proand Cres  and cytoand Cres . Interesand Cres .WNT1 [WNT2b [WNT5a [WNT6 [WNT10a [F/F mice [Genomic analysis identified 46% (range 43%–48%) of gastric tumors exhibit deregulation of the Wnt/β-catenin pathway . SeveralWNT1 , WNT2b [1 [WNT2b , WNT5a [b [WNT5a , WNT6 [25a [WNT6 , and WNT [WNT10a  , and these samples were positively associated with the depth of invasion and the degree of lymph node metastasis . FurtherRecently, deregulation of Wnt components at the level of the ligand/receptor has been identified, which in several cancers, including gastric, lung, glioblastoma, breast, melanoma, and prostate, are often more frequent than the deregulation of cytoplasmic components .  showed that both DKK1/2 were hyper-methylated in GC patient samples when compared to adjacent normal tissue. Further, hierarchical clustering of GC samples revealed DKK2 and secreted Frizzled-related protein 2 (sFRP2) to be concurrently hypermethylated [DKK2 and sFRP2, mRNA expression of both genes was analyzed, and showed that gene transcripts were lower in GC samples when compared to normal adjacent tissue, suggesting the hypermethylation was responsible for the silencing of these genes [Epigenetic silencing of Wnt antagonists that regulate the pathway at the level of the ligand/receptor have been identified in gastric tumors. Dickkopf 1/2 (DKK1/2) are both antagonists of canonical Wnt signaling that bind to LRP5/6, preventing their interaction with Wnt-Fzd complexes. Wang et al. thylated . sFRPs ase genes . sFRP-1,se genes  whilst ose genes . Conversse genes , and thuDKK1 overexpression was seen to decrease viability, anchorage-independent colony formation, and invasion of CD44+ GC cells through inhibition of canonical Wnt signaling [DKK1 overexpression significantly impeded the tumorigenesis of CD44+ GC cells in vivo [DKK1 overexpression could be a suitable gene therapy for targeting CD44+ cancer stem cells in GC. Further, Hong et al. reported that DKK1 expression was higher in tumors with lymph node metastasis, and patients with high DKK1 expression had a shorter OS and disease-free survival [Adenovirus (chimeric Ad5/35 vector)-mediated ignaling . Further in vivo . These dsurvival . High expression of the RYK co-receptor  was found to be correlated with poor differentiation, high TNM stage, and liver metastasis in GC patients (cohort of 250 patients) . SubcutaRNF43 gene, of which 62.5% were a truncating mutation [RNF43 was significantly lower in GC cell lines when compared to normal gastric epithelial cells. This result was corroborated by the same phenotype seen in primary gastric carcinoma tissue when compared to matched normal mucosal tissue [RNF43 expression that was seen in the gastric carcinomas was highest in the well-differentiated tumors and lowest in poorly differentiated tumors. A decrease in RNF43 expression was also seen with the progression of the pTNM stage. Overexpression of RNF43 in GC cell lines elicited a decrease in proliferation with an increase in apoptotic cells, due to upregulation of p53 and cleaved caspase 3 [Whole genome sequencing of 100 tumor-normal pairs identified that 54.6% of MSI tumors had mutations in the E3 ubiquitin ligase mutation  demonstrl tissue . Furtheraspase 3 . Rnf43 together with closely related Znrf3 triggered tumorigenesis in the intestine that is dependent on a Wnt-secreting niche. Blocking of this Wnt-secreting niche via porcupine inhibition attenuated the hyperplasia without affecting normal crypts [Functional analysis demonstrated that deletion of l crypts ; howeverThese data demonstrate that deregulation of Wnt signaling at the level of the ligand/receptor can modulate the initiation, growth, and progression of gastric tumors, highlighting that this part of the Wnt pathway is an attractive target for therapeutic intervention in GC. Fzd7 inhibited the initiation and growth of gastric tumors in vitro and in vivo [To establish whether targeting of the Wnt signaling pathway at the level of the ligand/receptor would be therapeutically beneficial for GC, we recently published a paper identifying that pharmacologically targeting Fzd receptors or specific genetic deletion of  in vivo . Here, wOf the 10 Fzd receptors, Fzd2, 5, 7, 8, and 9 have been shown to be upregulated in GC tissue , with reF/Fgp130 mouse model of GC [APC mutant and APC wild-type (wt) GC cells from forming anchorage-independent colonies [APC mutant colorectal cancer cell lines (CRC). However, IWP-2 had no effect on Wnt signaling in the same APC mutant CRC cell lines, suggesting that further research is needed to determine which Wnt-driven cancers are sensitive to porcupine inhibitors and what are the molecular mechanisms behind its function. Fzd7 is abundantly expressed in GC cell lines, and in the tumors of the Stat3-driven el of GC . Pharmaccolonies . This emcolonies  recentlyFzd7 in vivo or shRNA-targeted knockdown of Fzd7 in vitro showed that Fzd7 was essential for the growth and initiation of GC cells [The molecular targets of OMP-18R5/Vantictumab are Fzd1, 2, 5, 7, and 8. Both Fzd2/7 were shown to be highly expressed in GC cell lines, thus indicating that either Fzd2/7 was responsible for transmitting Wnt signaling in GC cells . PreviouGC cells . Fzd7 in the normal gastric epithelium is deleterious, resulting in rapid repopulation with Fzd7-proficient cells [+ gp130F/F Fzd7fl/flCre mice, Fzd7-deleted cells were not repopulated but rather survived 20 days post-tamoxifen induction [Fzd7 deletion and reflects the aberrant biology of tumors compared to the normal epithelium. Deletion of nt cells . In the nduction  and are H. pylori; however, the mechanism of how infection causes GC is currently unknown. Interestingly, it has been found that H. pylori can activate the Wnt/β-catenin pathway through upregulation of Fzd7, which was associated with H. pylori infection-induced cell proliferation. Knockdown of Fzd7 in H. pylori-infected GC cells suppressed both cell proliferation and colony formation [H. pylori-infected gastric carcinogenesis, miRNAs involved in GC were explored. Through bioinformatic analysis and functional assays, it was found that miR-27b harbored a putative binding site for Fzd7 3-UTR [H. pylori infection and the Wnt signaling pathway through inhibition of Fzd7 [ showed that H. pylori caused an upregulation of TRPC6  expression, by regulating the Wnt/-βcatenin pathway, thus promoting GC progression. Thus, targeting Fzd7 with specific miRNAs could be a therapeutic strategy for GC. A pivotal risk factor for developing GC is infection with ormation . To furtd7 3-UTR . Further of Fzd7 . A studyc-Myc is a well characterized β-catenin/TCF target gene within the gastrointestinal tract. Both GC cells and mouse gastric adenomas show upregulation of c-Myc in an Fzd7-dependent manner [c-Myc in +;Apcfl/fl;Mycfl/flCre mice showed a dramatic reduction of gastric adenoma initiation and Wnt activation compared to the respective controls, identifying c-Myc as a key modulator of gastric tumor growth downstream of Fzd7 [c-Myc in the intestinal epithelium is deleterious and triggers rapid repopulation [c-Myc leading to no changes in stem cell activity or homeostasis in vivo [c-Myc itself for the treatment of GC, would be well tolerated in the stomach.t manner . Conditi of Fzd7 . Deletiopulation . We rece in vivo . Thus, aThere are very few functional experiments that decipher the role of other Fzd receptors, besides Fzd7 in GC. Interestingly, Fzd6 is downregulated in GC tissue samples and cell lines, and overexpression of Fzd6 was able to suppress both the proliferation and migration of GC , thus FzAPCKO CRC cells induced the formation of the signalosome (Wnt receptor complex), resulting in activated Wnt signaling. However, CRC cells with APC mutations treated with IWP-2 did not inhibit the activation of the signalosome, suggesting that Wnt ligands are dispensable in Wnt pathway activation resulting from APC truncation. Interestingly, LRP6 deletion in SW480 and DLD1 cells (both APC mutant) did inhibit canonical Wnt signaling and decreased cytoplasmic levels of β-catenin. These data suggest the Wnt receptor signalosome is activated by mutant APC and can induce Wnt signaling independent of Wnt ligands. Saito-Diaz et al. [APC mutant cells was due to internalization of the complex via clathrin-dependent endocytosis. Similarly, recent work from Owen Sansom’s group showed that GTPases RalA and RalB were required for efficient internalization of Fzd7 to activate Wnt signaling in intestinal stem cells [APC mutant cells. Wnt signaling is frequently deregulated in several cancers by mutations of the cytoplasmic components of the pathway, including APC or β-catenin. However, APC mutant cells do not simply switch on Wnt signaling, but rather, regulation is permissible at other levels of the pathway. This is well illustrated by the observation that APC mutant tumors display a variable intensity of nuclear β-catenin, suggesting an environmental factor can regulate APC mutant cells ,61. Thisz et al.  also shoem cells , illustrAPC can respond to Tankyrase inhibition, suppressing oncogenic signaling in response to AXIN1/2 stabilization. However, CRC cells containing an early truncating mutation (minAPC) were unresponsive to tankyrase inhibition, suggesting repression of Wnt signaling through tankyrase inhibition is highly dependent on specific APC disruption. We recently demonstrated that GC organoids derived from ERT2/+;Apcfl/flTff1Cre mice treated with the porcupine inhibitor IWP-2, prevented upregulation of the Wnt pathway and was associated with reduced organoid proliferation [APC mutant GC and CRC cells respond to different Wnt inhibitors depending on the location of the mutation in the APC gene.Schatoff et al.  demonstrferation . These dA recently published study from the Surveillance, Epidemiology and End Results database (SEER) showed that 7792 out of 19,022 (41%) patients presented with metastatic GC, predominantly in the liver (3218 patients) . AnotherIt is well established that Wnt signaling not only drives the initiation of solid cancers but also contributes to the metastatic progression of the primary tumor. The reactivation of Wnt signaling in the cancer stroma favors cancer stem cell survival. Further, the reactivation of Wnt signaling in the primary tumor aids the epithelial-mesenchymal transition (EMT) of tumor cells, the migration and invasion of tumor cells, and escaping dormancy at metastatic secondary sites . Thus, tThough there are limited studies on Wnt signaling driving metastatic GC, Li et al.  discoverFurther, the microRNA miR-544a was found to induce EMT through the activation of Wnt signaling in GC. Specifically, overexpression of miR-544a induced the translocation of β-catenin from the cytoplasm to the nucleus, increasing canonical Wnt signaling in GC cells (MKN28s) . AlongsiAXIN2 and TCF1 [More so, the stem cell marker LGR5 was found to promote proliferation, invasion, and migration of GC cells through the regulation of canonical Wnt signaling  as GC ceand TCF1 .LGR5 overexpression increased GC cell motility by inducing a morphological change, as cells became elongated with a fibroblast-like appearance and this phenotype was reversed when the GC cells were treated with C-59, suggesting that LGR5 regulates cell migration through Wnt signaling . This isWnt5a-targeted knockdown in GC cell lines reduced cell migration both in vitro and in vivo through inhibition of Rac1 and laminin ϒ2, both key drivers of GC cell invasion. Further, suppression of Wnt5a using an anti-Wnt5a antibody prevented the clathrin-mediated rapid internalization of the Wnt5a-Fzd2 receptor complex [A study carried out by Hanaki et al.  discover complex . TogetheSurgery remains the primary modality of cure in GC. Unfortunately, in the UK, approximately 35% of patients present with unresectable or metastatic disease . The surIt is possible to pharmacologically target Wnt signaling at several places in the pathway , which iApc mutant gastric organoids [Very few of the available pharmacological agents that target the Wnt pathway have been tested in preclinical human GC platforms. This may explain why there are no GC-exclusive Wnt inhibitor trials in the pipeline. Nevertheless, there is evidence to support a program of Wnt inhibitor research in GC. In MKN28 (APC mutant) cells and rganoids , IWP-2, rganoids . In a phrganoids . Given tWnt inhibitors are largely very well tolerated in phase 1 clinical trials. The principle adverse event of administering Wnt inhibitors is iatrogenic osteopenia and pathological fractures. Therefore, many therapeutic regimens now incorporate a supplementary bisphosphonate inhibitor, such as zolendronic acid . This haGC represents a heterogenous disease with very variable outcomes, independent of the tumor stage. Deregulation of the Wnt pathway is seen in approximately 50% of tumors  and ther