This study aims to improve the electrical-mechanical performance of traditional epoxy/graphite composites for engineering applications. The improvement in the properties of these composites depended on the incorporation of different sizes of graphite particles of the same type and controlling their curing process conditions. The thermal properties and microstructural changes were also characterized. A maximum in-plane electrical conductivity value of approximately 23 S/cm was reported for composites containing 80 wt.% G with a particle size of 150 µm. The effect of combining large and small G particles increased this value to approximately 32 S/cm by replacing the large particle size with 10 wt.% smaller particles (75 µm). A further increase in the electrical conductivity to approximately 50 S/cm was achieved due to the increase in curing temperature and time. Increasing the curing temperature or time also had a crucial role in improving the tensile strength of the composites and a tensile strength of ~19 MPa was reported using a system of multiple filler particle sizes processed at the highest curing temperature and time compared to ~9 MPa for epoxy/G150 at 80 wt.%. TGA analysis showed that the composites are thermally stable, and stability was improved by the addition of filler to the resin. A slight difference in the degraded weights and the glass transition temperatures between composites of different multiple filler particle sizes was also observed from the TGA and DSC results. Researchers are still making efforts to achieve better electrical-mechanical performance of conductive polymer composite materials (CPCs) for various engineering applications ,2,3. OthAmong the various conductive fillers used in CPCs, graphite is still of interest because it has high electrical conductivity and various shapes and sizes, and it is easily obtained using conventional processes ,20. The On the other hand, many studies have highlighted the optimization of the processing parameters of fabrication techniques to improve the electrical and mechanical performance of CPCs ,30. The Finally, most of the previous studies in the literature focused on the improvement of the electrical and mechanical properties of polymer/G composites either by using a specified size of G particles incorporated into the composite ,39,40,413 was purchased from Asbury Carbon, New Jersey. The binder (matrix) used was epoxy resin (1.1 g/cm3 density) and hardener (635 thin epoxy resin) of 1.04 g/cm3 density with a 4:1 ratio fast epoxy hardener, which had a low viscosity of 6 poise and was supplied by a US composites company. The SEM images of the different G particle sizes are shown in In this study, three different sizes of the same graphite particle type were used as conductive fillers: particles with an average size of 150 µm (designated G150) were used as the main conductive filler up to 80 wt.%, and smaller average particle sizes of 75 µm (G75) and 44 µm (G44) were used as secondary fillers up to 10 wt.%. Graphite powder with a density of 2.19 g/cm2 square mold and placed in an oven . The process of curing the resin was carried out at different curing temperatures  and different curing times  to obtain the conductive polymer composite materials. Twelve different formulations of epoxy/G composites are shown in The epoxy/G composites were prepared as follows: (i) the epoxy resin and the hardener were initially mixed with a composition ratio of 4:1 according to the manufacturer datasheet, using a mechanical stirrer  at a rotation speed of 250 rpm for 10 min; (ii) the G conductive particles were then added to the epoxy-hardener mixture during stirring based on the predetermined compositions shown in The in-plane electrical conductivity of the composites was measured according to the ASTM C611 method using the conventional four-probe technique at a constant current supply . The tenThe values of the in-plane electrical conductivity of the epoxy/G composites with different G filler loadings at different types of G particle sizes are shown in Epoxy/G150 with 80 wt.% G150 was selected as the master batch formula based on its high in-plane electrical conductivity value. In an attempt to further improve the in-plane electrical conductivity and to determine the effect of the incorporation of different sizes of G particles into the matrix, up to 10 wt.% of G150 was replaced by smaller G particle size, G75 or G44, and the results are shown in The curing conditions were found to have a crucial role in determining the in-plane electrical conductivity, as shown in The results of the tensile test for all types of composites are shown in g) of the epoxy and its composites. It was observed from the DSC curves that there was a slight deviation of Tg from 66 °C for epoxy/G150/G44 to 69 °C for epoxy/G150/G75 composites compared to that of pure epoxy (68 °C). Sun et al. [g compared to composites having micro-sized fillers. They also indicated that the surface conditions of the fillers and their dispersion besides the interaction at the filler–resin interface play a crucial role for determining the Tg of the composites. Morimune-Moriya et al. [g value. In this study, using a narrow range of particle sizes of the secondary filler  might not display much difference on Tg.The thermal stability of pure epoxy and epoxy/70 wt.% G150/10 wt.% (G44 or G75) composites was investigated, and the TGA and DTG curves are shown in n et al.  showed ta et al.  found thAn experimental study to improve the electrical-mechanical behavior of epoxy/G composites based on the effects of G particle size and curing conditions was carried out, and the findings could be summarized as follows: the incorporation of multiple filler sizes of the same type increased the in-plane electrical conductivity of the composites by forming bridges of small G particles between the larger plate-like G particles, increasing the particle-to-particle contact, and consequently improving the electrical conductivity. The curing conditions temperature and time had a significant effect on increasing the electrical conductivity and tensile strength of the epoxy/G150/G75 and epoxy/G150/G44 composites. As a comparison and by optimizing the curing conditions, an increase of about 78% in the in-plane electrical conductivity of epoxy/G150 composites was achieved when combining G150 with a smaller G size compared to that containing only 80 wt.% G150, as reported in the previous work of Suherman et al. , and the


The WNT/β-catenin signaling pathway is involved in fundamental processes for the proliferation and differentiation of mesenchymal stem cells. However, little is known about its relevance for mesenchymal neoplasms, such us soft tissue sarcomas (STS). Chemotherapy based on doxorubicin (DXR) still remains the standard first-line treatment for locally advanced unresectable or metastatic STS, although overall survival could not be improved by combination with other chemotherapeutics. In this sense, the development of new therapeutic approaches continues to be an unmatched goal. This review covers the most important molecular alterations of the WNT signaling pathway in STS, broadening the current knowledge about STS as well as identifying novel drug targets. Furthermore, the current therapeutic options and drug candidates to modulate WNT signaling, which are usually classified by their interaction site upstream or downstream of β-catenin, and their presumable clinical impact on STS are discussed.Soft tissue sarcomas (STS) are a very heterogeneous group of rare tumors, comprising more than 50 different histological subtypes that originate from mesenchymal tissue. Despite their heterogeneity, chemotherapy based on doxorubicin (DXR) has been in use for forty years now and remains the standard first-line treatment for locally advanced unresectable or metastatic STS, although overall survival could not be improved by combination with other chemotherapeutics. In this sense, the development of new therapeutic approaches continues to be a largely unmatched goal. The WNT/β-catenin signaling pathway is involved in various fundamental processes for embryogenic development, including the proliferation and differentiation of mesenchymal stem cells. Although the role of this pathway has been widely researched in neoplasms of epithelial origin, little is known about its relevance for mesenchymal neoplasms. This review covers the most important molecular alterations of the WNT signaling pathway in STS. The detection of these alterations and the understanding of their functional consequences for those pathways controlling sarcomagenesis development and progression are crucial to broaden the current knowledge about STS as well as to identify novel drug targets. In this regard, the current therapeutic options and drug candidates to modulate WNT signaling, which are usually classified by their interaction site upstream or downstream of β-catenin, and their presumable clinical impact on STS are also discussed. Sarcomas are a very heterogeneous group of rare tumors with a very low incidence, accounting for less than 1% of all malignant tumors . The estPDGFR) in GIST; and (c) sarcomas with high-level amplifications of chromosome 12 regions encompassing the MDM2 and CDK4 loci in well-differentiated liposarcomas (WDLPS) and dedifferentiated liposarcomas (DDLPS). The second group, defined by multiple and unspecific complex karyotype abnormalities, includes loss and amplification of numerous genes, chromosome regions, and chromosomes. To this second group belong leiomyosarcomas (LMS), myxofibrosarcomas (MFS), malignant peripheral nerve sheath tumor (MPNST), pleomorphic liposarcomas (P-LPS), pleomorphic rhabdomyosarcomas (P-RMS), and unclassified pleomorphic sarcoma (UPS) [TP53, ATRX, and RB1) are repetitively mutated in all sarcoma types, and secondly, molecular subtypes related to patient outcome could be defined based on genomic driver pathways. In addition, they emphasized that the immune microenvironment may have a different impact on patient outcome depending on the sarcoma subtype and might be useful to provide information about immune checkpoint inhibitors in clinical trials [Histopathological classification traditionally divides sarcomas according to the tissue of apparition. However, sarcomas could also be divided into two main groups according to the type of genetic alterations: (1) sarcomas with near-diploid karyotypes and simple genetic alterations including translocations or specific oncogenic mutations and (2) sarcomas with complex and unbalanced karyotypes. The first group, in turn, could be broken down into three further subgroups: (a) sarcomas carrying translocations, such as SS18-SSX in synovial sarcomas (SS); (b) sarcomas with specific mutated genes, such as c-KIT or platelet-derived growth factor receptor (ma (UPS) ,6. Sincema (UPS) . Furtherl trials . Finallyl trials .Despite the heterogeneity of these tumors, the first treatment option in patients with localized disease is surgery with or without radiotherapy and chemotherapy . UnfortuThe WNT signaling pathway is involved in several fundamental processes of embryogenic development, including the regulation of mesenchymal stem cells, cell migration, and cell turnover to maintain homeostasis in certain adult tissues, such as skin or intestine ,20. TradThe canonical pathway is involved in cell proliferation and survival, while the non-canonical pathway is associated with cell differentiation, migration, and polarity. The non-canonical pathway, in turn, differs in two signaling pathways: the calcium-dependent and the planar cell polarity pathway, and is activated through the binding of WNT ligands to frizzled membrane receptors (FZD) as well as to receptor tyrosine kinase class XII  and class XVII . Despite this theoretical differentiation, both pathways interact to regulate complex processes in a coordinated way, such as embryonic development, maintenance of stem cells, tissue homeostasis, or wound healing, and their aberrant regulation is associated with tumorigenesis, metastasis, and other diseases ,23.Although in the literature, the role of the WNT/β-catenin pathway has been widely described in colorectal carcinogenesis as well as in other neoplasms of epithelial origin, little is known about the involvement of this pathway in mesenchymal neoplasms, such as STS. In this review, we first summarize the most relevant molecular alterations of the components of the WNT signaling pathway found in STS and describe their functional roles. The identification of these biomarkers, together with their functional characterization within pathways controlling sarcomagenesis development and progression, is fundamental to improve the current understanding of STS as well as to design new therapeutic approaches for these tumors. The second part of the review describes current therapeutic options modulating WNT signaling and their clinical impact on STS.The WNT ligand family, which is highly evolutionarily conserved ,25, consActivation of the WNT/β-catenin pathway is induced by the binding of extracellular WNT ligands to FZD receptors, which belong to the family of seven-domain transmembrane receptors and their low-density lipoprotein receptor-related protein 5/6 co-receptors (LRP5/6). After the binding of WNT ligands to these receptors, the protein Disheveled (DVL) is phosphorylated and binds to the protein Axin, causing the inhibition of the β-catenin destruction complex, which is composed of several proteins: a structural protein called Axin, adenomatous polyposis coli (APC) enhancing the affinity of the complex for β-catenin, casein kinase 1α (CK1α), as well as glycogen synthase kinase 3β (GSK-3β) B. As a cIn vertebrates, another mechanism of WNT signaling activation has been described, which involves other secreted proteins, the R-spondins (RSPO1–4) ,53. WhenTLE1, WNT-5A, FZD1, and TLE4, showing the implication of WNT signaling pathway in this subtype of STS. Moreover, WNT-5A and FZD1 were overexpressed in 11 and 9 of the 16 SS analyzed tumors, respectively [AXIN2, LEF1, TCF7, WISP2, FRAG1, DAAM1, FZD8, MYC, PRICKLE1, and SFRP1 in SS, malignant peripheral nerve sheath tumor (MPNST), and myxoid/round-cell liposarcoma [Despite the involvement of the WNT/β-catenin pathway in processes such as self-renewal and differentiation of mesenchymal stem cells, few studies have been published discussing the involvement of the WNT pathway in sarcomagenesis, osteosarcomas (OS), Ewing’s sarcomas (ES), and synovial sarcomas (SS) being the most studied subtypes ,60,61,62osarcoma . Table 2WNT-5A expression was reduced using shRNA in MPNST cells. Rather, WNT-5A knockdown increased the expression of genes involved in extracellular matrix remodeling and communication with inmune cells, suggesting that WNT-5A modulates the MPNST microenvironment thereby inhibiting tumor formation [WNT ligands consist of 350–400 amino acids and have a molecular weight of about 40 kDa. In order to be secreted, their signal sequences at its amino-terminal end contain hydrophobic amino acids of different lengths that are cleaved for maturation . One of ormation .FZD proteins are WNT receptors, which are upregulated in certain tumors. In STS, frizzled homologue 10 (FZD10) is upregulated in SS and plays a role in its cell survival and growth . MoreoveSFRP3 reduced cell growth of human alveolar RMS (A-RMS) cells both in vitro and in vivo. In angiosarcoma , another SFRP protein, SFRP2, has been found to be overexpressed. Specific blocking of SFRP2 with a monoclonal antibody in xenograph angiosarcoma mice models and cell lines demonstrated anti-angiogenic effects and inhibition of the β-catenin pathway [SFRP4 was decreased in both low-grade endometrial stromal sarcoma and undifferentiated endometrial sarcoma, and it was regulated in an opposite manner to that of β-catenin. Altogether, these data suggest a dual role of these SFRP in the sarcomagenesis of different types of sarcoma.The naturally secreted WNT pathway inhibitors, such as the secreted frizzled related protein 3 (SFRP3), have been shown to be altered in rhabdomyosarcoma (RMS) expressing the fusion protein PAX3-FOXO1 . By mean pathway . Thus, i pathway . CompareRSPO2 and RSPO3 fusions in colorectal cancer involve EIF3E exon 1 and RSPO2 exon 2 or exon3, and PTPRK exon 1 or exon 7 and RSPO3 exon 3. Interestingly, these RSPO2 and RSPO3 gene fusions are mutually exclusive and occurred in tumors without APC or CTNNB1 mutations [RSPO2 overexpression was the result of a deletion-mediated gene fusion between exon 1 of EIF3E and exon 2 of RSPO2, the same as described in colorectal cancer. In vitro, they showed that knockdown of RSPO2 was able to reduce WNT signaling activity and consequently cell viability. Altogether, their results suggest that overexpression of RSPO2 could drive a subset of MPNST and that blocking the function of RSPO2 may be a potential therapeutic strategy for MPNST patients with RSPO2 overexpression [RSPO2 and RSPO3 mRNA expression in a set of 86 tumor tissue samples representative of different STS subtypes from the University Hospital Son Espases and the Spanish Group for Research on Sarcoma (GEIS). In these STS tissue samples, mRNA levels of RSPO2 and RSPO3 were increased 42% and 84%, respectively. RSPO3 overexpression was a common event in all analyzed STS subtypes; instead, RSPO2 overexpression was higher in SS compared to LMS and LPS  phenotype in colorectal and endometrial cancer [RNF43 or ZNRF3 mutations in STS, but our group has analyzed mRNA expression of these two ubiquitin ligases in the representative set of STS samples described above. We observed an underexpression of RNF43 and ZNRF3 mRNA levels that could be linked to the truncating mutations already mentioned. Among all analyzed STS subtypes, LMS and SS were those with higher underexpression of RNF43, while underexpression of ZNRF3 was similar for all STS subtypes  were 8.2% of SS cases harboring missense APC mutations were found. The five discovered mutations, A1299T, G1412R, V1414I, S1398N, and M1413I, were all located in the mutation cluster region (MCR) of the APC gene. Moreover, all SS cases with APC mutations showed β-catenin accumulation independently of the presence or absence of β-catenin mutation [The APC protein is another key component of the WNT signaling pathway, which acts as a scaffold protein controlling the intracellular levels of the oncoprotein β-catenin. Mutations in mutation .GSK3β is a signaling mediator of diverse signaling pathways. In the context of WNT signaling, the inhibition of GSK3β-mediated β-catenin phosphorylation is known to be the key event in WNT/β-catenin signaling. In fact, one of the consequences of GSK3β inhibition in cells is the stabilization and nuclear translocation of β-catenin. Some studies have brought to light GSK3β deregulation in STS, mainly in SS. Manish Mani Subramaniam et al. (2010) found expression of phosphorylated GSK3β (active form) in 90 to 100% SS cases studied . In the APC [CTNNB1 mutations have been described. It seems that other mechanisms such as autocrine loop activation [CTNNB1 exon 3 (a C-A transversion at position 37) has been reported as relevant in the oncogenesis of pleomorphic sarcoma. The amino acid change from serine to tyrosine provokes the loss of one of the β-catenin phosphorylation sites, leading to the accumulation of the protein in the cytosol first and then the activation of gene expression by the nuclear β-catenin/TCF4 complex [CTNNB1 mutations have also been detected in SS cell lines and in patient samples. The SYO-1 SS cell line carries a point mutation in CTNNB1 codon 34 (G34L) that does not affect any regulatory phospho-site in the degradation targeting box of β-catenin. Although alterations in this codon do not activate transcription of β-catenin, they cause increased tumor transformation, suggesting that other mechanisms, independently of this mutation, play a crucial role in the activation of canonical WNT signaling [CTNNB1. In addition to SS, many studies have demonstrated a strong nuclear expression of β-catenin in many different STS subtypes in vitro [β-Catenin is the key transcription factor in the regulation of the canonical WNT signaling cascade. While in many cancers, the aberrant activation of the canonical WNT pathway is due to mutations in this gene or other important components of the pathway such as APC ,112, in tivation , fusion tivation ,93,94, otivation ,63 have tivation . These ttivation . Another complex . In addiignaling . Missensignaling ,95. It iignaling . This fain vitro ,83 and iin vitro .APC-mutated LMS cell line the one that showed the highest level of active phospho-β-catenin. Moreover, consistent with β-catenin activation, increased basal TCF reporter activity was found in LMS, FS, and LPS cells, and as consequence, CDC25A, a WNT target gene that has been described to drive proliferation of sarcomas [Martinez et al. (2017) demonstrated that, in several STS cells, including stable cell lines and primary cells from patients, β-catenin and phospho-β-catenin (Ser552) were detected, being an sarcomas , was oveTLE-1 is a transcriptional co-repressor that inhibits the WNT signaling pathway and other cell fate determination signaling pathways. It has no-DNA binding domains but can promote specific gene silencing by downregulating transcriptional activators by enhancing transcriptional repressors or by transforming transcriptional activators into repressors. Generally, TLE-1 inhibits WNT signaling transcription through its binding to TCF/LEF. In SS, the association between the SS18-SSX fusion protein and overexpression of TLE-1 has been described. The concrete mechanism of action of TLE-1 in SS consists of downregulating the activating transcription factor 2 (ATF2). In non-tumoral cells, ATF2 enhances the transcription of early growth response 1 (EGR1) target genes, involving tumor suppressors such as TP53 and PTEN. In SS, the SS18-SSX fusion protein forms a stable complex with TLE-1 and ATF2 in the nucleus that, in turn, represses the transcriptional activity of EGR1 and boosts oncogenesis ,101,102.CREBBP gene mutations have been found in histiocytic sarcomas, which are rare, and aggressive neoplasms of mature histiocytes that can arise de novo or as a secondary malignancy evolving from low-grade B cell lymphoma. The possible common origin between these two neoplasias highlights the need for an accurate differential diagnosis. The detection of somatic mutations I1471T and F1484V in the histone acetyltransferase domain of CREBBP may help with the differential diagnosis [CREBBP mutation at 16p13.3. In fact, mutations of CREBBP gene have been reported in approximately half of Rubinstein–Taybi syndrome patients, which would have the potential to develop E-RMS or LMS. Identification of such correlations, if present, can facilitate appropriate genetic counseling and testing of patients and their relatives, as well as screening, surveillance, and interventional measures, as needed [CBP is one of the transcriptional co-activators of canonical WNT signaling, and it exerts its effects when interacting with β-catenin in the nucleus. iagnosis . These miagnosis . Some sas needed ,90.SMARCA4 gene and modulates WNT target gene expression through the binding to β-catenin. It is considered a tumor suppressor gene, because many loss-of-function alterations in this subunit are increasingly detected in human malignancies. Cancer-related SMARCA4 mutations alter the open chromatin landscape promoting the expression of pro-oncogenic genes [SMARCA4 that cause loss of nuclear protein expression has emerged as a differential diagnostic marker between some types of sarcoma and carcinomas sharing similar molecular characteristics. For example, in combination with a panel of other molecular features, SMARCA4 has become a novel tool to discern between SMARCA4-deficient undifferentiated uterine sarcoma and undifferentiated and dedifferentiated endometrial carcinomas . Kolin et al. (2020) found a wide variety of SMARCA4 inactivating mutations among the 12 cases of undifferentiated uterine sarcomas studied: one loss-of-function inversion and different frameshift, nonsense, missense, and splice site mutations. One of the cases also presented loss of heterozygosity (LOH) [SMARCA4 mutational studies and copy number analysis, a new molecular entity of aggressive tumors has been described. These studies have provided evidence of consistent alteration at 19p13 encompassing SMARCA4 in thoracic sarcomas, resulting in either LOH or heterozygous deletions. The thoracic sarcomas carrying inactivation mutations in SMARCA4 are enriched in genes involved in proliferation and stemness compared to unclassified sarcomas. Thus, SMARCA4-deficient thoracic sarcomas are at present recognizable in clinical practice, due to simultaneous loss of expression of SMARCA4 and SMARCA2 and concomitant overexpression of SOX2. In this case, the immunodetection of SMARCA4 serves to distinguish between thoracic sarcomas and lung carcinomas, as the latter usually do not display a loss of SMARCA4 nuclear expression [SMARCA4 that has been associated with an increased risk for developing a malignant type of soft tissue tumor called rhabdoid tumor is that germline heterozygous loss-of-function mutations in SMARCA4 (19q13.2) cause rhabdoid tumor predisposition syndrome II. As mentioned before, the identification of carriers involved in the development of malignant STS is an important fact to take into account, because it allows adequate monitoring and management of patients [The protein BRG-1, a subunit of the SWI/SNF chromatin-remodeling protein complex (BAF), is encoded by the ic genes . In the ty (LOH) . Followipression ,113,114.patients .p300 is a member of the histone acetyltransferase family of transcriptional co-activators that can bind to β-catenin when it accumulates in the nucleus due to WNT signaling activation. In a study of 43 SS tumor samples, Liu et al. (2019) found high p300 protein expression in SS, being more prevalent in monophasic SS. Although high p300 expression has been associated with poor overall survival (OS), progression-free survival, and recurrence-free survival in various types of human cancer, such as digestive system malignant neoplasms, the prognostic value of p300 expression has not been proved yet in SS .LEF1 gene encoding for the transcription factor lymphoid enhancer-binding factor 1 participates in the WNT signaling pathway, activating the transcription of target genes in the presence of β-catenin and its co-activators. LEF1 is altered in most SS, which display high mRNA levels and strong nuclear expression, providing evidence that aberrant activation of the WNT/β-catenin pathway is present in SS [nt in SS . On the nt in SS .The molecular alterations described above are potential drug targets for the development of new therapies for STS that aim at key molecular members of the WNT signaling pathway.Specific WNT signaling pathway inhibitors can be classified into functional groups according to their interaction site: upstream of β-catenin or downstream of β-catenin . The molOf the 44 clinical trials focusing on targeting WNT signaling pathway on cancer treatment, 39 trials (89%) have as a target WNT-upstream members, while only 5 of them (11%) focus on pathway inhibition downstream of β-catenin .RNF43 and ZNRF3 loss-of-function mutations, RSPOs translocations, and Notch ligand loss-of-function mutations) are WNT ligand dependent, and those aberrations have a huge potential to be used as biomarkers to predict sensitivity to PORCN inhibitors [Porcupine (PORCN) is considered a highly selective target for the WNT driven cancers , as thishibitors ,121,122.hibitors , XNW7201hibitors  and RXC0hibitors  for advahibitors ,127 alsohibitors ,129,130.RSPO2/3 translocations [ETC-159 has been reported to be highly efficacious in genetically defined colorectal cancer tumors with ocations  and was ocations . Later oRNF43 mutations and RSPOs fusions. Its effects were accompanied by a significant reduction in mRNA expression of WNT targets as AXIN2 and C-MYC [RNF43 mutations) also exhibited a potent inhibition of tumor growth [RXC004 in vitro reduced cell proliferation through cell cycle arrest at G1/S and G2/M, in colorectal and pancreatic cell lines harboring nd C-MYC . Oral trr growth . CurrentRSPO2 fusions after 28 days of treatment. Nowadays, this inhibitor is in phase I trials for treatment of patients with advanced solid tumors or with advanced gastrointestinal tumors as a single agent or in combination with pembrolizumab.CGX1321 is a potent and specific inhibitor developed through several in silico approaches in a comprehensive study from a lead compound series (patent: US9556144) ,171. StuRNF43 or ZNRF3, and fusions in RSPOs are predicted to be sensitive to this inhibitor. Moreover, preliminary clinical data shows that LGK974 has promising potential for antitumor activity in selected patients harboring WNT aberrations [RNF43 and/or ZNRF3 or of fusions that provoke the amplification of RSPO2 and/or RSPO3, these genes were clearly under- and overexpressed in STS cell lines and in patients’ tumors  is an anti-RSPO3 monoclonal antibody that entered clinical trials in 2015 as a single agent for advanced solid tumors and in combination with chemotherapy (FOLFIRI), including metastatic colorectal cancer . The ideAnother molecule that binds WNT ligands is OMP-54F28 (Ipafricept) , a recomClinical trials using WNT receptor inhibitors include Ad5-SGE-REIC/Dkk3 ,140 for OMP18R5 (Vantictumab) ,150,151 Another molecule, UC-961 (Cirmtuzumab) ,157,158,The FZD10 receptor has been reported to be highly expressed on the cell surface of almost all SS tumors, but it is absent in most healthy organs except for the placenta ,181. As CTNNB1 that allow mutated β-catenin to escape from proteasomal degradation, as the mechanism of induction of β-catenin degradation behind CWP232291 is completely different.CWP232291 is a peptidomimetic small molecule that induces degradation of β-catenin through endoplasmic reticulum (ER) stress activation. ER stress leads to the activation of caspase-3-mediated apoptosis, which can induce degradation of β-catenin ,184, andAfter translocation into the nucleus, β-catenin activates transcription of WNT target genes by recruiting a transcriptional co-activator, which can be either cAMP-response-element-binding protein (CREB)-binding protein (CBP) or E1A binding protein 300 kDa (p300). Several high-throughput screenings have been conducted to identify small molecules targeting these interactions, as this has been considered a promising therapeutic approach. Among them, ICG-001, a first-in-class inhibitor of β-catenin/CBP interaction, was developed in 2004. In preclinical studies, this inhibitor has been reported to reduce cell viability and to induce apoptosis in LPS cells  and coloCDC25A, which is highly expressed in STS patient samples, thereby inducing cell death or cycle arrest. Most importantly, combination of PRI-724 with standard STS chemotherapeutic drugs, DXR or trabectedin, enhanced their antitumor activity in a synergistic manner, suggesting that this new strategy with PRI-724 could also have a beneficial clinical effect on patients with STS [In STS, the effect of PRI-724 has been very recently evaluated by our group . The study demonstrated that PRI-724 significantly reduces cell proliferation of different subtypes of STS cell lines in vitro by downregulating the expression of the WNT target gene with STS . These rAXIN2, LEF1, MYC, and TCF7) more than 10-fold more than PRI-724, which also acts downstream of β-catenin [TCF7, MYC, LRP5, DVL2, and BTRC). Currently, patients with advanced solid tumors are being recruited in a phase I clinical trial to evaluate the safety and pharmacokinetics of this inhibitor when administered orally [CDC25A in STS [CCND1, LEF1 and TCF7) in mantle cell lymphoma (MCL) and strongly impaired cell proliferation and induced apoptosis in vitro in MCL and in acute myeloid leukemia (AML) cell lines. In vivo, this compound demonstrated a strong antitumor effect in both neoplasias, and consequently, a phase 1 study assessing safety, tolerability, and pharmacokinetics of SM09419 in subjects with advanced hematologic malignancies has already been initiated [The expression of WNT target genes could also be reduced by alternative mechanisms, such as modulating the alternative splicing. SM08502 is an inhibitor of CDC-like kinase (CLK), which has an important role in precursor-mRNA splicing ,199,200,d orally   of mesenchymal stem cells, which, by its very nature, have unlimited potential to proliferate. It can be hypothesized that STS patients might benefit from both upstream inhibitors of β-catenin, such as PORCN inhibitors or WNT ligand-receptor interaction inhibitors, as well as downstream inhibitors of β-catenin, both of which have shown promising results in clinical treatments of neoplasms of epithelial origin. Although only few clinical trials with WNT/β-catenin pathway inhibitors have been conducted with STS patients, the molecular data suggest that especially those patients with an over-activated WNT signaling pathway could benefit from its inhibition. In this context, determination of the specific deregulation sites in every patient and the development of novel, specifically targeted drugs, administrated as single agents or in combination with other chemotherapeutics, would be an important step towards precision medicine in STS treatment.


Optimal conditions for the modification of GR electrodes by conducting polymer-based nanocomposites and GOx were elaborated. The electrodes were investigated by cyclic voltammetry and constant potential amperometry in the presence of the redox mediator phenazine methosulfate (PMS). The improved enzymatic biosensors based on GR/PANI-AuNPs(6 nm)-GOx/GOx and GR/Ppy-AuNPs(6 nm)-GOx/GOx electrodes were characterized by high sensitivity (65.4 and 55.4 μA mM−1 cm−2), low limit of detection (0.070 and 0.071 mmol L−1), wide linear range (up to 16.5 mmol L−1), good repeatability (RSD 4.67 and 5.89%), and appropriate stability  was 22 and 17 days, respectively). The excellent anti-interference ability to ascorbic and uric acids and successful practical application for glucose determination in serum samples was presented for GR/PANI-AuNPs(6 nm)-GOx/GOx electrode.Biosensors for the determination of glucose concentration have a great significance in clinical diagnosis, and in the food and pharmaceutics industries. In this research, short-chain polyaniline (PANI) and polypyrrole (Ppy)-based nanocomposites with glucose oxidase (GOx) and 6 nm diameter AuNPs (AuNPs The design and wide application of electrochemical biosensors in nanoscience, nanotechnology, medicine, environmental, and food monitoring has significantly intensified during the past decade ,3,4,5,6.Recently, enzymes have been used for the design of enzymatic biofuel cells ,16,17,182, SiO2, ZnO, ZrO2, and many others), which are characterized by variable sizes and shapes, have been considered as unique materials for the immobilization of enzymes and have been used for the design of electrochemical biosensors [2O2 and the facilitation of accepted electron transfer towards the electrode [4− ions and form of metallic AuNPs, the size of which depends on the kinetics of the nucleation reaction [Major fundamental and nanotechnological advances have opened new horizons for the application of nanomaterials ,20 and hosensors ,27,28,29osensors ,30. Firsosensors . AuNPs alectrode ,22. GOx reaction .π–π conjugated polymers (including polyaniline (PANI) and polypyrrole (Ppy)), which can be synthesized via chemical or electrochemical oxidation of monomers, are used for the development of rechargeable batteries, smart windows, light emitting diodes, transistors, sensors and etc. [π-boned polymer backbone [π-electron system of the doped polymer [π–π conjugated polymers can be improved by electrochemical doping to induce an insulator-to-conductor transition in these polymers [n-type and p-type charge carriers can be involved in charge transfer within π–π conjugated polymers. PANI is electrically conducting and electroactive in protonated form at low pH values, but at pH 3 or 4 it is insulating and electrochemically inactive [π–π conjugated polymers, the monomer is ‘attacked’ by a radical cation induced by H2O2 and this process is leading towards the polymerization reaction [π–π conjugated structures formed between the redox center of the enzyme and the electrode increase the efficiency of the charge transfer in this structure [and etc. . These cand etc. ,16,17,33backbone ,34. The  polymer . The coninactive . During reaction ,36. The tructure ,16,17,332) of proteins paved the way to the interaction of AuNPs with these proteins [π–π conjugated polymer nanocomposites presented the best sensing performance due to the effective ‘contact’ between the enzyme and electrode. Additionally, such types of biosensors are more resistant to some interfering materials [(300 s)) film, nano Au and GOx and operating in the presence of redox mediator was characterized by the sensitivity of 3.07 μA mM−1 cm−2 [−1 cm−2 in the presence of redox mediator [4− ions was characterized by a sensitivity of 4.31 mA mM−1 cm−2 and narrow linear range (up to 0.70 mmol L−1) in the presence of redox mediator [PANI and Ppy were combined with electroactive AuNPs and GOx, and were integrated as a structure suitable for the development of biosensors dedicated for the monitoring of glucose ,29. The proteins ,37. The aterials ,29,34. Tmediator . Biosensmediator .(6 nm)). For this reason, short chain polymer nanocomposites (PANI-AuNPs(6 nm)-GOx and Ppy-AuNPs(6 nm)-GOx) were prepared, dispersing π–π conjugated polymer-based nanocomposites in the solution by ultrasound. An additional layer of GOx was formed over GR electrode pre-modified with dispersed conducting polymer nanocomposites.The main aim of the presented study was to improve the analytical characteristics of amperometric glucose biosensors based on conducting polymer nanocomposites with embedded GOx and 6 nm diameter AuNPs  and D-(+)-glucose, D(−)-fructose, D(+)-mannose, D(+)-galactose, D(+)-xylose, D(+)-saccharose were obtained from Fluka  and Carl Roth GmbH+Co.KG . Tetrachloroauric acid and tannic acid were purchased from Alfa Aesar GmbH&Co KG and Carl Roth GmbH + Co , sodium citrate—from Penta . 0.05 mol L−1 solution of sodium acetate (SA) buffer with 0.1 mol L−1 potassium chloride (KCl) was prepared from sodium acetate trihydrate and potassium chloride, whose were obtained from Reanal  and Lachema . Aniline and sodium hydroxide were purchased from Merck KGaA , pyrrole—from Acros Organics , graphite rod  and hydrochloric acid—from Sigma-Aldrich , α alumina powder —from Electron Microscopy Sciences . The solution of 25% glutaraldehyde was obtained from Fluka Chemie GmbH , phenazine methosulfate, L-ascorbic acid (AA) and uric acid (UA)—from AppliChem GmbH . 2.3 × 1016 particles L−1 of AuNPs(6 nm) were prepared as was described in previous research [Glucose oxidase -GOx and Ppy-AuNPs(6 nm)-GOx nanocomposites were synthesized for 2 days and separated according to previously described methodology [(6 nm)-GOx and Ppy-AuNPs(6 nm)-GOx were tending to agglomerate according to previously reported results [(6 nm) aniline and pyrrole were polymerized into the spherical particles. In the presence of pyrrole, nanocomposites were formed layered structure.PANI-GOx, Ppy-GOx, PANI-AuNPshodology ,40,41. P results ,42. In t3 g for 8 min using IEC CL31R Multispeed centrifuge . After then, the collected polymer-based nanocomposites were dispersed in 40 μL of SA buffer, pH 6.0, using the ultrasonic bath  to achieve short chain polymers.The separation of polymer nanocomposites from the polymerization solution was performed at 14.6 × 102) was cut, polished using fine emery paper and by humid Al2O3, washed, and dried at +20 ± 2 °C. Then, GR was placed into a silicone tube. 0.43 μL mm−2 of dispersed polymer nanocomposites were deposited on CR electrode. Then 3 μL of 25 mg mL−1 GOx were immobilized on a graphite rod electrode modified by polymer nanocomposite. After the evaporation of water at room temperature, the prepared working electrode was stored for 15 min in a closed vessel over a 25% solution of glutaraldehyde at +20 ± 2 °C, which covalently cross-linked the enzyme adsorbed on the polymer nanocomposites deposited on the GR [Working electrode — graphite rod (surface area 0.071 cm(6 nm)-GOx/GOx electrode was performed according to a methodology presented previously [−1 of glucose in the absence and presence of 1.00 mmol L−1 fructose, mannose, galactose, xylose, and saccharose. To investigate the effect of AA and UA on the GR/PANI-AuNPs(6 nm)-GOx/GOx electrode, serum samples with 10.0 mmol L−1 of glucose; 10.0 mmol L−1 of glucose and 0.01, 0.10 or 0.20 mmol L−1 of AA; 10.0 mmol L−1 of glucose and 0.01, 0.05 or 0.10 mmol L−1 of UA were used.The selectivity tests of glucose biosensor to interfering and electroactive species and the electrochemical determination of glucose concentration in diluted (1:10) serum sample using GR/PANI-AuNPseviously . The inf(6 nm)-GOx/GOx or GR/Ppy-AuNPs(6 nm)-GOx/GOx electrodes were switched into electrochemical cell a working electrodes, 2 cm2 platinum electrode was used as a counter electrode and Ag/AgCl−1(3 mol LKCl) from Metrhom  — as a reference electrode.Electrochemical measurements were performed by the computerized potentiostat PGSTAT 302/Autolab from EcoChemie  with GPES 4.9 software. GR/PANI-GOx/GOx, GR/Ppy-GOx/GOx, GR/PANI-AuNPs−1(3 mol LKCl) using amperometry in stirred (1400 rpm) by a magnetic stirrer IKA from Werke GmbH & Co. KG  solution of 0.05 mol L−1 SA buffer, pH 6.0, containing 0.1 mol L−1 KCl and in the presence of 6 mmol L−1 PMS as soluble redox mediator. Cyclic voltammograms were registered at potential diapason from −0.60 to +0.80 V vs. Ag/AgCl−1(3 mol LKCl) at a potential sweep rate of 0.100 V s−1 in an unstirred solution of 0.05 mol L−1 SA buffer, pH 6.0, containing 0.1 mol L−1 KCl and in the presence 6 mmol L−1 of PMS.The registration of glucose was performed at +0.30 V vs. Ag/AgCl−1 SA, pH 6.0, with 0.1 mol L−1 KCl and centrifuged within 8 min (14.6 × 103 g). All investigations were performed with biosensors based on GR/PANI-AuNPs(6 nm)-GOx/GOx electrode. The electrochemical measurements were applied in 10× diluted serum sample with 10.0 mmol L−1 of glucose before and after the addition of 1.00 mmol L−1 fructose, mannose, galactose, xylose and saccharose. To evaluate the influence of ascorbic and uric acids on the developed biosensor, the analytical signals of glucose were registered in the solutions of 10× diluted serum sample with: 10.0 mmol L−1 of glucose; 10.0 mmol L−1 of glucose and 0.01, 0.10 and 0.20 mmol L−1 of AA; 10.0 mmol L−1 of glucose and 0.01, 0.05 and 0.10 mmol L−1 of UA.The sample of the serum was diluted (1:10) in 0.05 mol LImax) and the apparent Michaelis constant (KM)) and the limit of detection (LOD) were estimated by SigmaPlot software 12.5.The parameters of Michaelis–Menten kinetics -GOx or Ppy-AuNPs(6 nm)-GOx nanocomposites synthesis, their deposition on the surface of GR electrode with the following additional enrichment by GOx and electrochemical investigations are presented in 2) of GOx. Later, electrons from the reduced PMSH2 are transferred directly to the positively charged surface of GR electrode and/or through AuNPs present on the GR electrode [The principles of PANI-AuNPslectrode .(6 nm)-GOx/GOx or Ppy-AuNPs(6 nm)-GOx/GOx nanocomposites, was investigated in 0.05 mol L−1 SA buffer, pH 6.0, with 0.01 mol L−1 KCl in the presence of 6 mmol L−1 PMS by cyclic voltammetry in potential diapason from −0.60 to +0.80 V. To evaluate the influence of additionally on nanocomposite added GOx, the cyclic voltamperograms of GR/PANI-AuNPs(6 nm)-GOx and GR/Ppy-AuNPs(6 nm)-GOx were registered. As it is seen from Epa) of GR electrodes modified by PANI-GOx/GOx, Ppy-GOx/GOx, PANI-AuNPs(6 nm)-GOx/GOx or Ppy-AuNPs(6 nm)-GOx/GOx composites was determined at +0.12 V vs. Ag/AgCl(3 mol L−1KCl). The potential of cathodic peaks (Epc) of GR electrode modified by PANI-GOx/GOx or Ppy-GOx/GOx was −0.14 V, while for PANI-AuNPs(6 nm)-GOx/GOx and Ppy-AuNPs(6 nm)-GOx/GOx it was −0.17 and −0.15 V, respectively. Diffusion-controlled, reversible electrochemical process was observed: the peak separation (ΔEp= Epa-Epc) for PANI-GOx/GOx and Ppy-GOx/GOx was 0.26 V, while for PANI-AuNPs(6 nm)-GOx/GOx and Ppy-AuNPs(6 nm)-GOx/GOx it was 0.29 and 0.27 V, respectively. This fact indicates that the reversibility was of high degree and both (PANI-AuNPs(6 nm)-GOx and Ppy-AuNPs(6 nm)-GOx) polymer nanocomposites increased the efficiency of charge transfer between GOx and GR.The form of cyclic votammograms and peak shifts are providing important information on the action mechanism and charge transfer of the electrochemical species . The ele(6 nm) in polyaniline- and polypyrrole-based nanocomposites (PANI-AuNPs(6 nm)-GOx/GOx and Ppy-AuNPs(6 nm)-GOx/GOx) increased anodic current by 1.12 and 1.16 times, respectively, if compared with that registered for electrodes modified by PANI-GOx/GOx and Ppy-GOx/GOx. This fact indicates that AuNPs show good electrocatalytic activity towards electrochemical oxidation of glucose. The addition of GOx after electrode modification by polymer nanocomposites is recommended to increase the current response by 1.51 and 2.15 times for GR/PANI-AuNPs(6 nm)-GOx/GOx and GR/Ppy-AuNPs(6 nm)-GOx/GOx electrodes, in the comparison with results obtained using GR/PANI-AuNPs(6 nm)-GOx and GR/Ppy-AuNPs(6 nm)-GOx electrodes.It was tentatively determined that glucose biosensors based on GR electrodes modified by GOx-containing nanocomposites are sensitive towards glucose. As is seen from (6 nm) was investigated in this chapter. To determine the advantages of polymer-GOx nanocomposites, the measurements were performed using GR electrodes modified by polymer nanocomposites of different composition: in the presence of GOx without and with AuNPs(6 nm). The investigations were performed in SA buffer, pH 6.0, with 0.01 mol L−1 KCl and 6.0 mmol L−1 PMS.The composition of polymer nanocomposites has a significant influence on the sensitivity and analytical characteristics of the here developed amperometric biosensors. Thoroughly, the influence of the composition of polymer-GOx nanocomposites with glucose oxidase without and with AuNPs−1, and the influence of these kinds of compounds on the analytical response are presented in I vs. glucose concentration were in agreement with the Michaelis–Menten kinetics. The difference of the maximal current response generated during the enzymatic reaction and the apparent Michaelis constant were correspondingly a and b parameters of the hyperbolic function (y = ax/(b + x)) and were used for the approximation of the obtained results.The hyperbolic dependences of the current response registered by amperometry on the concentration of glucose in the diapason increased from 0.10 to 173 mmol L(6 nm) after additional enrichment by GOx are characterized by significantly higher sensitivity. The maximal current was higher for nanocomposites containing AuNPs(6 nm). Particularly for GR/Ppy/AuNPs(6 nm)-GOx/GOx (ΔImax = 122 μA) and GR/PANI/AuNPs(6 nm)-GOx/GOx (ΔImax = 120 μA) the current response increased by 1.22 and 1.32 times, respectively, if compared with results obtained using electrode modified by Ppy-GOx/GOx (ΔImax = 100 μA) or PANI-GOx/GOx (ΔImax = 91.0 μA). These results are in good agreement with that presented in previous investigations by cyclic voltammetry. Obtained results present the advantage of biosensors based on polymer nanocomposites, which contain AuNPs(6 nm) (Imax for GR electrodes modified by PANI-AuNPs(6 nm)-GOx/GOx or Ppy-AuNPs(6 nm)-GOx/GOx has increased by 1.39 and 1.41 times in the comparison with that registered by GR electrode modified only with AuNPs(6 nm)-GOx (ΔImax = 86.3 μA). The value of ΔImax determined for GR electrodes modified by PANI-GOx/GOx or Ppy-GOx/GOx was 1.65 and 1.81 times higher if compared with that determined for the electrode modified only by GOx (ΔImax = 55.1 μA).As is seen from Ps(6 nm) A. These (6 nm)-GOx/GOx and Ppy-AuNPs(6 nm)-GOx/GOx show an increase linear detection range of glucose, which is the main advantage when the determination is performed directly in real samples. In our investigations, GR/PANI-AuNPs(6 nm)-GOx/GOx, GR/Ppy-AuNPs(6 nm)-GOx/GOx, GR/PANI-GOx/GOx or GR/Ppy-GOx/GOx electrodes were characterized by KM value of 15.1, 11.7, 14.9 and 11.8 mmol L−1, respectively. According to another research, this effect indicates a strong affinity between the enzyme and glucose [(6 nm)-GOx/GOx and Ppy-AuNPs(6 nm)-GOx/GOx were the most efficient and were used in further investigations.High values of the apparent Michaelis constant determined for electrodes based on PANI-AuNPs glucose ,31. Ther(6 nm)-GOx nanocomposites were equally distributed on GR electrode surface and the same amount of GOx was additionally immobilized over formed nanocomposite layer. As it is seen from the diagrams presented in (6 nm)-GOx nanocomposites of 0.43, 0.85 to 1.7 μL mm−2, respectively.The sensitivity of the designed biosensors depend on the composition of the polymer layer deposited on the surface of electrode. The influence of the thickness of polymer nanocomposites, which were deposited on GR electrodes, on the current response of the biosensor was investigated when different amounts of PANI-AuNPsImax registered by GR electrodes modified by 0.43 μL mm−2 polymer nanocomposites was similar (only 1.01 times higher) to 0.85 μL mm−2 and 1.56 times higher than that obtained using 1.7 μL mm−2 polymer nanocomposites. The decrease of ΔImax of the developed biosensor can be explained by the formation of a thick layer of PANI-AuNPs(6 nm)-GOx nanocomposites on GR electrode, which limits the diffusion of glucose to the surface of electrode. The lower amount of polymer nanocomposites than that of 0.43 μL mm−2 was not used in our system through similarity of the difference of maximal current for GR electrode modified by 0.85 or 1.7 μL mm−2 of polymer nanocomposites. In this way, the following investigations were performed in the presence of 0.43 μL mm−2 of polymer nanocomposites.The value of Δ(6 nm)-GOx/GOx, Ppy-AuNPs(6 nm)-GOx/GOx, PANI-GOx/GOx or Ppy-GOx/GOx. As it is seen from the obtained calibration curves (−1. The achieved linear detection range in our study was wider than for glucose biosensors based on carbon-ink/GOx modified stainless steel electrode (linear range up to 1 mmol L−1) [−1) [−1) [−1 was registered using the GR electrode modified by the long-chain Ppy/AuNPs(AuCl4-)-GOx nanocomposite [(6 nm)-GOx/GOx, GR/Ppy-AuNPs(6 nm)-GOx/GOx, GR/PANI-GOx/GOx and GR/Ppy-GOx/GOx were characterized by the correlation coefficient of 0.9968, 0.9961, 0.9927 and 0.9977, respectively  and stability of the developed analytical systems. Firstly, the linear detection range of glucose determination was measured using GR electrode modified by PANI-AuNPsn curves , the linmol L−1) , glassy −1) [−1)  or graph−1) [−1) . It shouomposite ; hence, ectively . As it i(6 nm)-GOx/GOx, Ppy-AuNPs(6 nm)-GOx/GOx, PANI-GOx/GOx or Ppy-GOx/GOx towards 16.5 mmol L−1 of glucose was determined as 65.4, 55.4, 52.0 and 48.0 μA mM−1 cm−2, respectively ((6 nm) in the polymerization solution and in PANI-AuNPs(6 nm)-GOx and Ppy-AuNPs(6 nm)-GOx nanocomposites increased the sensitivity of glucose biosensor based on GR/PANI-AuNPs(6 nm)-GOx/GOx or GR/Ppy-AuNPs(6 nm)-GOx/GOx by 1.26 and 1.15 times, respectively, if compared with results obtained for GR/PANI-GOx/GOx and GR/Ppy-GOx/GOx electrodes. Glucose biosensors based on GR electrodes modified by PANI-AuNPs(6 nm)-GOx/GOx or Ppy-AuNPs(6 nm)-GOx/GOx are 5.61 and 4.76 times more sensitive than glucose biosensor based on GC/PLL/RGO-ZrO2 electrode (11.65 μA mM−1 cm−2) [(6 nm)-GOx/GOx or GR/Ppy-AuNPs(6 nm)-GOx/GOx due to the use of another electrochemical method was very similar or a little bit smaller than that obtained in our previous study in the presence of long-chain Ppy/AuNPs(AuCl4-)-GOx nanocomposite (77.2 μA mM−1 cm−2) [(6 nm)-GOx/GOx-based glucose biosensor developed in recent research is 2.55 times higher than the glucose biosensor based on AuNP(13nm) electrochemically deposited on GR electrode and modified by GOx and Ppy (21.7 μA mM−1 cm−2) [(6 nm)-GOx/GOx-based glucose biosensor is 18 times higher if compared with the glucose biosensor based on GC electrode modified by OOPpy(300 s)-nanoAu and GOx (3.07 μA mM−1 cm−2) [The sensitivity of the developed biosensors based on GR electrodes modified by PANI-AuNPsectively . The pre−1 cm−2) . The sen−1 cm−2) . The sen−1 cm−2) . Moreove−1 cm−2) .(6 nm)-GOx/GOx, Ppy-AuNPs(6 nm)-GOx/GOx, PANI-GOx/GOx or Ppy-GOx/GOx GR was investigated in the presence of 2.99 mmol L−1 of glucose. The relative standard deviations (RSD) calculated for these biosensors were 4.67%, 5.89%, 6.10% and 9.72%, respectively. Developed glucose biosensors based on GR/PANI-AuNPs(6 nm)-GOx/GOx or GR/Ppy-AuNPs(6 nm)-GOx/GOx exhibited better repeatability (up to 3.0 and 2.4 times) than that obtained previously in the presence of long-chain polymer nanocomposites (for Ppy/AuNPs(AuCl4-)-GOx RSD was 13.9%) [(6 nm)-GOx/GOx and GR/Ppy-AuNPs(6 nm)-GOx/GOx electrodes was almost similar — 0.070 and 0.071 mmol L−1, respectively. Higher values of LOD for biosensors were determined using GR/PANI-GOx/GOx (0.084 mmol L−1) or GR/Ppy-GOx/GOx (0.10 mmol L−1) electrodes. Biosensors based on GR/PANI-AuNPs(6 nm)-GOx/GOx and GR/Ppy-AuNPs(6 nm)-GOx/GOx electrodes are characterized by lower LOD if compared with GR/PANI-GOx/GOx and GR/Ppy-GOx/GOx electrodes. The limit of detection determined for the developed biosensor based on short-chain PANI-AuNPs(6 nm)-GOx composites with following immobilization of GOx was 1.4 times lower than that for long-chain Ppy/AuNPs(AuCl4-)-GOx (0.1 mmol L−1) nanocomposite [(6 nm)-GOx/GOx electrode was 2.82 times lower if compared with previously developed biosensor based on electrochemically deposited AuNPs(13nm), immobilized GOx and covered by Ppy (LOD 0.20 mmol L−1) [The repeatability of biosensors based on GR electrodes modified by PANI-AuNPss 13.9%) . The anas 13.9%) . As is somposite . The LODmol L−1) .(6 nm)-GOx/GOx or Ppy-AuNPs(6 nm)-GOx/GOx were stored at +4 °C in a closed vessel hanging over SA buffer solution between electrochemical measurements for 131 days. The changes of analytical signal over time and the apparent Michaelis constant for the developed glucose biosensors are presented in For the stability test, GR electrodes modified by PANI-AuNPs(6 nm)-GOx/GOx and GR/Ppy-AuNPs(6 nm)-GOx/GOx electrodes considerably decreased during first 8 days, and 67.4% and 71.4% of initially current response was registered ((6 nm)-GOx/GOx and GR/Ppy-AuNPs(6 nm)-GOx/GOx electrodes towards glucose decreased from 31.8% down to 11.0% and from 24.4% down to 4.28%, respectively. The τ1/2 values, which represent the time after that 50% of the initial current response is registered for glucose biosensors based on GR/PANI-AuNPs(6 nm)-GOx/GOx or GR/Ppy-AuNPs(6 nm)-GOx/GOx electrodes, were determined as 22 days and 17 days, respectively. These results illustrate that the biosensor based on GR/PANI/AuNPs(6 nm)-GOx/GOx electrode is 1.29 times more stable than that based on the GR/Ppy/AuNPs(6 nm)-GOx/GOx electrode.As is seen from gistered A. The de nm-GOx/G(6 nm)-GOx/GOx was 3.14 times more stable than that of the Pt/PANI/gold nanorod composites/GOx electrode without mediator (τ1/2 was determined as 7.0 days) [(6 nm)-GOx/GOx electrode was 1.73 times more stable than that previously reported for biosensors based on the GR electrode with electrochemical deposited AuNPs(13nm), immobilized GOx and modified by Ppy (9.8 days of τ1/2) [(6 nm)-GOx with a following immobilization of GOx that was by 17, 6.7 and 1.2 times more stable than the biosensor based on long-chain PANI/AuNPs(6 nm)-GOx (τ1/2 = 1.3 days), Ppy/AuNPs(6 nm)-GOx (τ1/2 = 3.3 days) and Ppy/AuNPs(AuCl4-)-GOx (τ1/2 = 19 days) [The developed glucose biosensor based on the GR electrode modified by PANI-AuNPs.0 days) . The gluof τ1/2) . In this19 days) . The higKM constant increased from 15.4 to 92.3 mmol L−1 for the GR/PANI-AuNPs(6 nm)-GOx/GOx electrode and from 12.9 to 42.4 mmol L−1 for the GR/Ppy-AuNPs(6 nm)-GOx/GOx electrode within the time frame ranging from 1 to 131 days (KM for the GR electrode modified by Ppy-AuNPs(6 nm)-GOx/GOx after 131 days of electrode’s storage stability was 2.18 times better than that determined for electrodes modified by PANI-AuNPs(6 nm)-GOx/GOx. However, the higher value of KM correlates with the decreased sensitivity of the biosensor. The glucose biosensor based on GR/PANI-AuNPs(6 nm)-GOx/GOx was selected for further investigation due to their rather good performance  (The 131 days B. It is rements) ). AdditiGlucose is considered as the most important sugar, which is always present in human’s blood serum ,45. Othe(6 nm)-GOx/GOx electrode to interfering and electroactive species were performed in 10× diluted serum samples. To evaluate the influence of sugars on the current response of the developed biosensor, the serum containing 10.0 mmol L−1 of glucose with the following addition of 1.00 mmol L−1 fructose, mannose, galactose, xylose and saccharose was tested ((6 nm)-GOx/GOx electrode was sensitive only to glucose.The selectivity tests of the glucose biosensor based on the GR/PANI-AuNPss tested A. As is −1 for AA [−1 for UA [(6 nm)-GOx/GOx electrode. The addition of 10.0 mmol L−1 glucose with 0.01 and 0.10 mmol L−1 of AA increased the current response by 2.11% and 2.89%, while the addition of 10.0 mmol L−1 glucose with 0.20 mmol L−1 of AA increased the current response by 4.74% if compared with current response after the addition of 10.0 mmol L−1 glucose without any AA. The presence of 0.01, 0.05 and 0.10 mmol L−1 UA in the solution of 10.0 mmol L−1 glucose increased the current response of the developed biosensor by 1.49%, 2.99% and 3.08%, respectively, in comparison with the results obtained after the addition of 10.0 mmol L−1 glucose without any UA. The anti-interference capability of the developed glucose biosensor was higher than that described by other authors for the glucose biosensor based on GC electrode modified by graphene/nano-Au/GOx nanocomposites (the interference of 5.6% for AA and of 3.2% for UA was registered) [(AuCl4-)-GOx nanocomposites [−1 of AA, and 1.47 and 4.48 times more resistant to 0.01 or 0.05 mmol L−1 UA. Just the insignificant interference of electroactive species indicates that the developed glucose biosensor based on GR/PANI-AuNPs(6 nm)-GOx/GOx electrode is characterized by very good anti-interference ability towards the here evaluated interfering materials.The influence of such electroactive species as AA and UA on the registered current response was investigated. The assessed concentrations of AA and UA were higher than their normal physiological concentrations: 0.141 mmol L1 for AA ,50 and 01 for UA ,51. The istered) . If the mposites , it was −1 [−1 [(6 nm)-GOx/GOx for glucose determination in the diluted serum sample was assessed using the addition method. The results of the determined glucose concentration and the recovery are presented in (300 s)-nanoAu/GOx electrode (96%) [(AuCl4-)-GOx nanocomposites (92.7–94.8%) [Glucose concentration of nondiabetic human’s blood serum usually is in the range from 3 to 8 mmol L−1 ,51,52, w−1 [−1 ,29. The 7–94.8%) .−1 cm−2) and stability (τ1/2 = 22 days), (ii) low limit of detection (0.070 mmol L−1), good reproducibility (5.8%) and repeatability (4.67%), (iii) wide linear range (up to 16.5 mmol L−1) and small amount of aliquot required for the analysis, (iv) multiple use and rather low price of single analysis, (v) fast analytical response (10 s), high resistance to interfering species  and suitability for glucose determination (95.0–98.5%). Thus, these characteristics allow the application of the developed biosensor for the determination of glucose in real samples.The analytical characteristics of glucose biosensor based on dispersed enzymatically synthesized PANI nanocomposites with following immobilization of GOx were improved in comparison to polymeric nanocomposites-based nanoparticles . It shou(6 nm) embedded within the conducting polymers PANI or Ppy. Biosensors with shorter polymeric chains are characterized by good reversibility of the soluble redox mediator. Our results show that the glucose biosensors based on GR/PANI-AuNPs(6 nm)-GOx/GOx or GR/Ppy-AuNPs(6 nm)-GOx/GOx electrodes are characterized by high sensitivity, low limit of detection, wide linear range, good repeatability, sufficient storage stability. Additionally, the glucose biosensor based on GR/PANI-AuNPs(6 nm)-GOx/GOx electrode operates well in the serum sample and detects glucose in the presence of interfering species. This glucose biosensor enables the reduction of the amount of aliquot, which is required for the determination of the analyte. The methodology presented in this paper could be used for the design and fabrication of improved enzymatic biosensors for biomedical analysis. Further modification of GR/PANI-AuNPs(6 nm)-GOx/GOx or GR/Ppy-AuNPs(6 nm)-GOx/GOx electrodes by insoluble redox mediator will be the next step in this our research direction.In this research, we demonstrated the improved properties and analytical parameters of a glucose biosensor developed using dispersed nanocomposites based on GOx and AuNPs


AXIN1 mutations are observed in 8–10% of hepatocellular carcinomas (HCCs) and originally were considered to support tumor growth by aberrantly enhancing β-catenin signaling. This view has however been challenged by reports showing neither a clear nuclear β-catenin accumulation nor clearly enhanced expression of β-catenin target genes. Here, using nine HCC lines, we show that AXIN1 mutation or siRNA mediated knockdown contributes to enhanced β-catenin signaling in all AXIN1-mutant and non-mutant lines, also confirmed by reduced signaling in AXIN1-repaired SNU449 cells. Both AXIN1 and AXIN2 work synergistically to control β-catenin signaling. While in the AXIN1-mutant lines, AXIN2 is solely responsible for keeping signaling in check, in the non-mutant lines both AXIN proteins contribute to β-catenin regulation to varying levels. The AXIN proteins have gained substantial interest in cancer research for a second reason. Their activity in the β-catenin destruction complex can be increased by tankyrase inhibitors, which thus may serve as a therapeutic option to reduce the growth of β-catenin-dependent cancers. At concentrations that inhibit tankyrase activity, some lines  were clearly affected in colony formation, but in most cases apparently independent from effects on β-catenin signaling. Overall, our analyses show that AXIN1 inactivation leads to enhanced β-catenin signaling in HCC cell lines, questioning the strong statements that have been made in this regard. Enhancing AXIN activity by tankyrase monotherapy provides however no effective treatment to affect their growth exclusively through reducing β-catenin signaling. Aberrant activation of Wnt/β-catenin signaling is often observed in HCC3. Intracellular β-catenin levels are regulated by a multiprotein complex composed of the APC tumor suppressor, scaffold proteins AXIN1, AXIN2 and the kinases GSK3 and CK1α4. When cells are not exposed to extracellular Wnt ligands, β-catenin is constitutively phosphorylated and degraded to maintain cytoplasmic levels at a minimum. Following Wnt stimulation, the multiprotein complex dissociates resulting in the accumulation of cytosolic and nuclear β-catenin, which in turn triggers the transcription of specific target genes. Aberrant activation of Wnt/β-catenin signaling in HCC has been mainly attributed to activating somatic mutations in the CTNNB1 gene coding for β-catenin (20–25%)8, for which it is well-accepted that they support tumor growth by enhancing β-catenin signaling in a dominant fashion.Hepatocellular carcinoma (HCC) is a prevalent cancer with worldwide around 700,000 new cases diagnosed yearly, and a leading cause of cancer related deathsAXIN1 (8–10%)9. Originally, given its role in the β-catenin destruction complex, mutational inactivation of AXIN1 was considered to support HCC development by aberrantly enhancing β-catenin signaling. This view has however been challenged by several reports showing neither a clear nuclear β-catenin accumulation nor clearly enhanced expression of β-catenin target genes in AXIN1-mutant HCCs12. Others have provided some evidence of increased β-catenin signaling in AXIN1 mutant HCC cells, albeit modest14. Qiao and coworkers showed that HCC induction following AXIN1 deletion in mouse livers was strongly dependent on functional β-catenin, but as argued by others only in the context of simultaneous MET activation17. Hence, whether β-catenin signaling is activated following AXIN1 mutation and its relevance for supporting human HCC growth is still heavily debated.Another component of the Wnt/β-catenin signaling pathway regularly inactivated in HCC is TNKS/TNKS2) associate with AXIN, resulting in their PARsylation and subsequent ubiquitylation and degradation, thereby limiting the activity of the destruction complex21. Blocking tankyrases results in the formation of so-called degradasomes in which all components of the β-catenin destruction complex aggregate, leading to an efficient β-catenin turnover. Application of these tankyrase inhibitors has been investigated for the treatment of various cancer types, with some successful initial results for a subset of tumors30.The AXIN proteins have gained substantial interest in cancer research for a second reason. Their activity in the β-catenin destruction complex can be increased by tankyrase inhibitors, which thus may serve as a therapeutic option to treat β-catenin-dependent cancers. The AXIN proteins, like β-catenin itself, are under tight proteolytic control. Poly-ADP-ribosyltransferases tankyrase-1 and -2  in HCC cell lines both AXIN1 and AXIN2 work synergistically to control β-catenin signaling; (ii) in the AXIN1-mutant lines, AXIN2 is solely responsible for keeping signaling in check, whereas in the non-mutant lines both AXIN proteins contribute to β-catenin regulation to varying levels depending on the cell line under investigation; (iii) in the non-mutant lines 21. Their inhibition can lead to an enhanced β-catenin turnover and growth suppression of a subset of β-catenin-dependent cancers30. All HCC cell lines showed readily detectable expression of TNKS/TNKS2 , and Hep3B and Huh7 (IWR-1). Using the reporter assay, significant reductions with XAV939 were only observed in PLC/PRF/5 and Huh7, whereas this was the case for PLC/PRF/5 and SNU182 using the IWR-1 inhibitor. Importantly, despite the 1.2–3-fold reduction in AXIN2 and/or β-catenin reporter levels observed in some of the lines, the absolute level of nuclear β-catenin signaling remains high in especially the CTNNB1- and AXIN1-mutant lines  polymerases PARP1 and PARP236. Among the AXIN1-mutant cell lines a modest but significant reduction in colony numbers was observed, especially using 5 μM XAV939 and IWR-1. For PLC/PRF/5 this may be attributed to reduced β-catenin signaling, while this is less obvious for Hep3B and SNU449. Lastly, among the non-mutant lines SNU182 was unaffected by tankyrase inhibition. Huh7 was clearly inhibited in colony formation with XAV939, but not using IWR-1, which is in line with the observed effects on β-catenin signaling for both compounds. HepaRG showed reduced numbers, which may be partially explained by reduced β-catenin signaling.Following three days of XAV939 treatment, HCC cell growth was unaltered in a MTT assay, even at higher 5 μM concentration Fig. . Among tCollectively, these findings suggest that tankyrase inhibition can inhibit the growth of several HCC cell lines when plated at low density. Not in all cases can the growth suppressing effects be linked to β-catenin signaling (e.g. HepG2 and SNU398). Tankyrase inhibition is however less effective when initiated with higher cell concentrations, as shown in the MTT-assay.Table 38. We successfully obtained several independent clones with complete repair of AXIN1 9. A second common mechanism originally considered to lead to enhanced β-catenin signaling, is mutational inactivation of AXIN1 observed in about 10% of HCCs, putting it among the most frequently mutated genes in HCC9. Given its activity in the β-catenin destruction complex this was a logic assumption, however several reports have suggested that AXIN1 mutation leads to liver cancer in the absence of increased β-catenin signaling12. Recently, HCC formation in mice following mutation of AXIN1 was however shown to strongly dependent on functional β-catenin15, but as argued by others only in the context of simultaneous MET activation16. Hence, the issue whether AXIN1 mutation leads to activation of β-catenin signaling is still under debate17. In addition, the AXIN proteins have gained substantial interest in cancer research because their activity in the β-catenin destruction complex can be increased by tankyrase inhibitors, which thus may serve as a therapeutic option to reduce the growth of β-catenin-dependent cancers. Here, using a panel of 9 HCC cell lines with specific mutations in components of the β-catenin signaling pathway, we have investigated both aspects of AXIN biology.Inappropriate activation of Wnt/β-catenin signaling has been reported frequently in HCC. This has been mainly attributed to somatic mutations in the AXIN1-mutant HCCs develop without β-catenin activation is largely based on expression profiling using a 23-β-catenin target gene signature12. Most AXIN1-mutated HCCs clustered in a group with no evident β-catenin activation program, and only about 20% in a group with weak activation. Interestingly, these same groups also show an enrichment in CTNNB1 mutations within armadillo repeats 5 and 6 , for which we have recently unequivocally shown that they are efficient inducers of HCC development in mice, associated with a weak but significant activation of β-catenin signaling8. Expression profiling of tumor tissues has the inherent shortcoming that average expression is determined of a heterogeneous group of cells, including contaminating normal cells. Small but biologically relevant increases of β-catenin signaling, like the ones imposed by armadillo repeat mutant β-catenin, in a subset of cells can easily be missed. Likewise we feel that it is difficult to use expression profiling to fully exclude increased β-catenin signaling in AXIN1-mutated cancers.The conclusion that 15, indeed suggesting that there is no nuclear signaling. However, this is also observed in tumors carrying oncogenic CTNNB1 mutations within the armadillo repeats and a subset of S45-mutant cancers43, while these clearly lead to enhanced signaling. Thus, although nuclear accumulation of β-catenin is a reliable predictor of active signaling, its absence does not exclude that a low level of biologically relevant signaling is active44. It shows that both genetic profiling and IHC have their shortcomings to unambiguously determine the absence of nuclear β-catenin signaling.The same holds true for immunohistochemical demonstration of nuclear β-catenin. AXIN1-mutant HCCs predominantly show an exclusive membranous β-catenin staining14. Furthermore, we show that AXIN1 knockdown in three non-mutant lines leads to enhanced signaling, in accordance with other reports15. In the reverse experiment in which we restore AXIN1 expression in SNU449 cells, we consistently observe reduced expression of the β-catenin target gene AXIN2. Taken together, this shows that AXIN1 mutation leads to a modest increase of β-catenin signaling that may be relevant for hepatocellular tumorigenesis.Our analyses show that all three AXIN1-mutant HCC cell lines have increased β-catenin reporter activity maximum accumulation of the tankyrase enzymes. Overall our study suggests that tankyrase inhibition at such concentrations is unlikely to contribute to HCC treatment as monotherapy exclusively through reduction of β-catenin signaling. A potential exception could be the rare subset of HCCs carrying APC mutations leading to high level signaling of wild-type β-catenin49.Using a colony formation assay we observe significant reductions in colony numbers in 7 out of 9 lines, albeit modest in some . Not in all cases can this be easily linked to effects on β-catenin signaling, like the strong inhibitory effect on colony formation we observe for HepG2 and SNU398, despite retaining high levels of signaling. For other lines like Huh7, reduced β-catenin signaling clearly associates with reduced colony formation, while this may be somewhat the case for PLC/PRF/5 and HepaRG. Tankyrases have however also been linked to several other cellular processes such as telomere maintenance, Hippo signaling, mitosis and DNA strand break repairAXIN2. In the three non-mutant lines reducing AXIN1 levels also in all cases leads to enhanced signaling. Overall, these analyses show that AXIN1 mutation or inactivation leads to enhanced β-catenin signaling in HCC, questioning the strong statements that have been made in this regard. We also show that AXIN1 and AXIN2 work synergistically to control β-catenin signaling. While in the AXIN1-mutant lines, AXIN2 is solely responsible for keeping signaling in check, in the non-mutant lines both AXIN proteins contribute to β-catenin regulation to varying levels. Lastly, at concentrations that inhibit tankyrase activity some HCC lines were clearly affected in colony formation, but in most cases apparently independent from effects on β-catenin signaling.In conclusion, using a panel of nine HCC cell lines we observe that all three AXIN1-mutant lines display a clearly increased activity in a β-catenin reporter assay. Repair of the AXIN1 mutation in SNU449 confirms this observation as it results in reduced activation of the β-catenin target gene 14. The term “non-mutant” is used throughout the paper to indicate that these lines do not contain mutations in genes known to be linked to β-catenin signaling. Identity of all cell lines and clones thereof, was confirmed by the Erasmus Molecular Diagnostics Department, using Promega Powerplex-16 STR genotyping in October 2018. All cell lines tested negative for mycoplasma. Supplemental Table CTNNB1-mutant HepG2, Huh6, SNU398, AXIN1-mutant Hep3B, PLC/PRF/5, SNU449 and non-mutant HepaRG, Huh7, SNU182 HCC and CRC cell line SW480 were cultured as reported previouslyXAV939 and IWR-1 were purchased from Sigma-Aldrich. Antibodies used: β-catenin , phospho-β-catenin (Ser33/37) , AXIN1 , AXIN2 , Tankyrase-1/2 , β-actin . Anti-rabbit or anti-mouse IRDye-conjugated secondary antibodies  and anti-rabbit-HRP  were used for western blot analysis.46. In short, we plated 5 × 104 cells per well on 24-well plates, which were transfected with 250 ng Wnt Responsive Element (WRE) vector and 10 ng CMV-Renilla using FuGENE HD or Lipofectamin 2000 Transfection Reagent. We measured luciferase activities and normalized the data for the transfection efficiency by using the Dual Luciferase Reporter Assay system. Following normalization, WRE/CMV-Renilla ratios are obtained. For the data presented in Supplemental Figs. The β-catenin reporter assays were performed basically as previously described14. The mean and standard error were calculated for each condition.After incubation with XAV939 for 72 h, cells were analyzed by MTT assay as previously reported14. Results were visualized with Odyssey 3.0 software. For AXIN1, AXIN2 and pS33/37-β-catenin western blot analysis we used Immobilon ECL Ultra HRP substrate (MerckMillipore). Membranes for ECL detection were blocked and incubated using Immobilon Block-FL reagent (MerckMillipore). Original western blot images are presented in Supplemental Fig. Cells were lysed and run for fluorescent western blotting analysis as previously reportedCells were seeded on Nunc Lab-TekII CC2 Chamber Slides (Thermo-Fisher Scientific). After 16 h treatment with 1 µM XAV939 or DMSO, cells were washed with PBS, fixed in PBS-buffered 4% paraformaldehyde for 10 min, permeabilized with PBS-0.2% Triton X-100 solution for 5 min and blocked with PBS containing 3% BSA for 30 min. Samples were incubated with primary antibodies at room temperature for 1 h, followed by PBS-Tween 20 0.05% washes and incubation with appropriate secondary antibodies for 1 h. Primary antibodies were diluted as follows: AXIN1 ; AXIN2 ; Tankyrase-1/2 . The following secondary reagents were used: Donkey anti-Goat-Alexa 647 (#A-21447), Donkey anti-Rabbit-Alexa 488 (#A-21206), Donkey anti-Mouse-Alexa 594 (#A-21203); all from Invitrogen, at 1:500 dilution. Slides were mounted with Vectashield mounting medium with DAPI . Images were generated using a Zeiss LSM510META confocal electroscope.50. In short, RNA was isolated with a Machery-NucleoSpin RNA II kit  according to the manufacturer’s instructions, and quantified using a Nanodrop ND-1000 . CDNA was prepared from total RNA using a cDNA Synthesis Kit (TAKARA BIO INC). Quantitative PCR was performed using Sensimix SYBRGreen (Applied Biosystems) or TaqMan  Gene Expression Assays (Applied Biosystems). Analyses were performed using the StepOne Real-Time PCR System and the StepOnev2.0 software . All expression levels are depicted relative to the expression of GAPDH. Primer sequences are provided in Supplemental Table RNA isolation and qRT-PCR analysis were performed as described previously50. Smartpool ON-TARGETplus siRNAs targeting AXIN1, AXIN2 or APC were purchased from Dharmacon 8. A single guide RNA (sgRNA) encompassing the homozygous c.2134C>T AXIN1 mutation present in SNU449 cells was designed and cloned into pSpCas9(BB)-2A-GFP (PX458), a gift from Feng Zhang (Addgene plasmid # 48138) using standard procedures. To repair this mutation a single-stranded oligodeoxynucleotide (ssODN) was ordered as Ultramer (Integrated DNA Technologies). Both the sgRNA and ssODN are described in Supplemental Table 6 SNU449 cells were cotransfected with 2 µg PX458 and 2 µg ssODN. After nucleofection, complete DMEM medium with 7.5 µM RAD51-stimulatory compound-1  was used for cell culture. After 48 h, GFP positive cells were sorted by FACS and seeded as single cells in 96-well plates. DNA from clones grown successfully from single cells was isolated using the QuickExtract DNA Extraction Solution (Epicentre). For each clone, a PCR product encompassing the mutation was subjected to sequencing.CRISPR/Cas9 repair of the AXIN1 mutations was performed as reported previously52.Total RNA was isolated with the Machery-NucleoSpin RNA II kit  and quantified using a Nanodrop ND-1000 . RNA quality was checked using a RNA Pico chip on the Agilent Bioanalyzer. Library was constructed and sequenced with an Illumina HiSeqTM2000 . Briefly, the mRNA was enriched using oligo-dT magnetic beads, followed by fragmentation (about 200 bp). Then first strand cDNA was synthesized using random hexamer-primers and the second strand was further synthesized in a reaction buffer including dNTPs, RNase H and DNA polymerase I. Double stranded cDNA was purified with magnetic beads. Then, the 3′-end single nucleotide A (adenine) was added and adapters were ligated to the fragments which were enriched by PCR amplification.The RNA sequencing procedure was performed as previously describedAXIN1-repaired SNU449 samples (n = 3 each) was analyzed using UCSC human genome build hg38 and GENCODE annotation release 26 (GRCh38). FASTQC (v0.11.5)53 was applied on the single-end FASTQ files for quality control, both before and after running trimmomatic (v0.36)54, which removed TrueSeq adapter sequences. STAR (v2.5.3a) was used as aligner, with 2-pass mapping for each sample separately55. Mapping quality plot was generated and checked based on sambamba Flagstat (v0.6.7) statistics56. Count files, with the number of reads for each gene were created with subread FeatureCounts (1.5.2)57. Settings of different tools can be seen in Supplemental Table 58 and the Wald-test. A significance cut-off of 0.05 on the adjusted P-value was utilized, using the Benjamini–Hochberg procedure. The RNA-sequencing data from this study have been submitted to the Gene Expression Omnibus (GEO)-database under the accession number GSE119001.RNA-seq data of control and P value less than 0.05 .All results are presented as mean ± SD. Statistical analyses were carried out using software GraphPad Prism version 8.4.1 . Differences were considered significant at a Supplementary Tables.Supplementary Figures.


Wnt signaling was initially recognized to be vital for tissue development and homeostasis maintenance. Further studies revealed that this pathway is also important for tumorigenesis and progression. Abnormal expression of signaling components through gene mutation or epigenetic regulation is closely associated with tumor progression and poor prognosis in several tissues. Additionally, Wnt signaling also influences the tumor microenvironment and immune response. Some strategies and drugs have been proposed to target this pathway, such as blocking receptors/ligands, targeting intracellular molecules, beta-catenin/TCF4 complex and its downstream target genes, or tumor microenvironment and immune response. Here we discuss the roles of these components in Wnt signaling pathway in tumorigenesis and cancer progression, the underlying mechanisms that is responsible for the activation of Wnt signaling, and a series of drugs targeting the Wnt pathway provide multiple therapeutic values. Although some of these drugs exhibit exciting anti-cancer effect, clinical trials and systematic evaluation should be strictly performed along with multiple-omics technology. Wnt signaling activation can go through the canonical or non-canonical pathway depending on the association with the transcription activator beta-catenin. Following the stimulation of the canonical pathway, beta-catenin is translocated from the cytoplasm to the nucleus . By contignaling . Studiesignaling .Table 1Wnt signaling also plays significant roles in tumorigenesis and progression. Dysregulated Wnt signaling activity promotes malignant transformation of stem/progenitor cell, leading to increased cell proliferation and abnormal differentiation. Wnt signaling also crosstalks with other signaling pathways (e.g. Hedgehog (Hh), Notch) to synergistically regulate tumor progression –7. AbnorAbnormal activation of Wnt signaling cascade is associated with tumorigenesis in several tissues including the esophagus and liver. Canonical Wnt signaling has been shown to promote the self-renewal of cancer stem cells (CSCs) , 12. EsoWnt signaling influences tumor growth at multiple levels. In squamous cell carcinoma (SCC) cell lines, beta-catenin is a key regulator for the expression of DeltaNp63 , a majorIn addition, inactivation of Wnt/beta-catenin and PI3K/AKT through depletion of Ras GTPase-activating protein SH3 domain-binding protein 1 (G3BP1) attenuates the proliferative, invasive and migration potential of esophageal carcinoma cells . Wnt sigAbnormal Wnt signaling is also associated with tumorigenesis of EAC. For example, the mRNA levels of several members of the pathway are frequently altered during the progression of Barrett’s esophagus (BE) towards EAC. Changes in the methylation levels of the APC promoter were found in BE samples and 95% of EAC cases . MethylaMutations of CTNNB1 encoding beta-catenin are closely correlated with multiple cancers including hepatocarcinoma, pancreatic cancer, colorectal cancer (CRC), gastroesophageal junction carcinomas and gastric adenocarcinoma. The mutation of p.S45F in beta-catenin is a common mutation, leading to constitutively activated Wnt/beta-catenin signaling. This mutation has been found in a variety of solid tumors as a potential driver mutation, accounting for 3.3–10.4% of the total identified beta-catenin mutations . MoreoveTP53mut/APCmut/AMER1wt/TCF7L2wt/FBXW7wt and TP53mut/APCmut/AMER1wt/TCF7L2wt/FBXW7wt/SOX9wt/CTNNB1wt genotypes had shorter progression-free survival. These two signatures are negatively correlated with the overall survival of CRC [Mutation of APC is another important driver for tumor formation. Sixteen recurrently mutated genes were analyzed in 98 advanced CRC patients by next-generation sequencing. Multiple correspondence analysis (MCA) suggested that APC and TP53 mutations are close to the negative outlier group, whereas mutations in other members of WNT signaling are in proximity to the positive outliers. Moreover, patients with tumors harboring l of CRC . In anotl of CRC .Normal human intestinal epithelium-derived organoids have been used to study mutations of Wnt components and other genes associated with tumor. For example, organoids containing mutations of the five genes including SMAD4, APC, TP53, KRAS and/or PIK3CA, can grow independently of niche factors. These organoids also form tumors after implantation under the kidney capsule. Moreover, these organoids give rise to micrometastases post-injection into the mouse spleen, but fail to colonize in the liver . Mouse mThe Wnt antagonists SFRP1, SFRP2, DKK2 and Wnt inhibitory factor-1 (WIF-1) are hypermethylated during the transition from colorectal adenoma to carcinoma, whereas mutations in BRAF, APC, and KRAS occur at the transition from normal to adenoma phases, these events may drive the colorectal cancer formation . Among 1RNF43 is a negative regulator of Wnt signaling, and mutation of RNF43 is frequently found in CRC and endometrial cancers , 44, gasMutations of other Wnt family members including ZNRF3 and AXIN. Mutation of ZNRF3, a Wnt signaling suppressor, is often found in adrenocortical carcinoma , while AActivation of the Wnt/beta-catenin cascade can also be obtained through suppressing its inhibitor. For example, expression of HOTAIR and secreted Wnt antagonist WIF-1 is inversely correlated in ESCC cell lines and tumor tissues. HOTAIR lowers the expression of WIF-1 by enhancing the methylation of H3K27 (one type of histone of WIF-1) at its promoter site, thereby triggering the activation of Wnt/beta-catenin signaling pathway . FurtherEpigenetic regulation of the WIF-1 is also involved in gastro-intestinal tumorigenesis. Downregulation of WIF-1 caused by CpG island hypermethylation in the promoter region has been found in multiple cancer cell lines and esophageal, gastric, colorectal and pancreatic cancer tissues , 61. ComEpigenetic inactivation of WIF-1 with promoter methylation also existing in ESCC cell lines and tissues. WIF-1 promoter methylation is commonly found in 46% ESCC tissues and 50% cell lines. Removal of promoter methylation with a demethylating agent such as 5-aza-2′-deoxycytidine results in the attenuation of cellular proliferation and migration, along with decreased activity of beta-catenin/T-cell factor-dependent transcription . ConsistActivation of Wnt signaling is correlated with tumorigenesis in multiple tissues. Therefore, a number of strategies targeting Wnt signaling as therapies have been proposed , 65, as Blockage of Wnt signaling can be realized through ligands and receptors. While Frizzled (Fz)-related proteins (FRPs), Cerberus and WIF act by binding and sequestering Wnts, inhibitors against the receptors like LRP and Frizzled (FZD) also provide useful insights for cancer therapy . For exaAnother approach at attenuating Wnt signaling is through blockade of the receptor FZD , since FOther strategies targeting Wnt ligands or receptors have also been developed. For example, RNAi compounds, monoclonal antibodies and small molecule inhibitors were proposed to target WNT2B  and the Multiple intracellular components of Wnt signaling have been targeted for downregulation of the pathway. As a pivotal member of the Wnt signal pathway, reduction of intracellular beta-catenin or inhibition of the translocation of cytoplasmic beta-catenin to the nucleus, can effectively inhibit tumorigenesis and development, representing a pivotal therapeutic target for cancer treatment. Non-steroidal anti-inflammatory drugs (NSAIDs), such as aspirin and indomethacin, can down-regulate beta-catenin/TCF activity and their target genes cyclin D1 . An impoDishevelled (Dvl) is a key scaffold protein that transmits Wnt signals from the cell membrane to the cell. It consists of three conserved functional domains: DIX, PDZ and DEP. Among these domains, the PDZ domain directly binds to the FZD intracellular segment, thereby inhibiting GSK-3β activity, blocking beta-catenin phosphorylation, and causing the accumulation of beta-catenin in the nucleus. Finally, the Wnt signal is transmitted from the cell membrane into the cell . The actAXIN is a negative regulator of Wnt signal and a scaffold protein involved in Wnt pathway signaling. It has multiple interaction sites with APC, GSK-3β, and CK1 to form beta-catenin destruction complexes, and it can also interact with other components of Wnt signals including Dvl and PP2A , 113. TaAnother study revealed that Delta isoform of the CK1 family of serine/threonine kinases (CK1δ), an important mediator of intracellular Wnt signaling, is always amplified, and overexpressed in a subset of breast cancer. Inhibition of CK1δ with knockdown or inhibitors leads to apoptosis, tumor regression and blocking nuclear accumulation of beta-catenin, thus it provides strategy for effective therapy for breast cancer expressing CK1δ . when coIn another study, significantly higher levels of beta-catenin and lower levels of miR-214 were detected in esophageal carcinoma specimens than paired adjacent tissue. The levels of beta-catenin and miR-214 were inversely correlated in esophageal cancer specimens. Further study revealed that miR-214 regulates the translation of beta-catenin. Overexpression and depletion of miR-214 inhibits and promotes cell growth and invasion in esophageal cancer cell lines, respectively . Further(−/−)/APC (min/+) mutant mice form fewer intestinal tumors. The TNIK inhibitor NCB-0846 downregulates the levels of LRP5 and LRP6 and reduces the expression of the Wnt downstream targets AXIN2 and cMYC in HCT116 and DLD-1 colorectal cancer cells [Beta-catenin regulates the expression of downstream target genes via interaction with TCF/LEF family members. Therefore, targeting the formation of beta-catenin-TCF/LEF complex or reduction in the levels of relevant proteins provides potential means to block the transcription of downstream target genes during cancer progression. Traf2- and Nck-interacting kinase (TNIK) is an important regulator of beta-catenin/TCF4 transcription complexes and is also required for the tumor-initiating capability of colorectal cancer stem cells. TNIK-deficient mice showed resistance to colon tumorigenesis induced by azoxymethane, and TNIK er cells . TherefoThe 4-thioureido-benzenesulfonamide derivative LF3 also inhibits canonical Wnt signaling by directly interfering with beta-catenin/TCF-4 interactions. LF3 application reduced cell motility and self-renewal of colon CSCs; it also blocked tumor growth and induced differentiation in mouse xenografts . In addiThe Wnt downstream target S100A4 is involved in metastasis, promoting cancer cell migration and invasion. High levels of S100A4 predict metastasis and reduced survival in CRC patients. Therefore, S100A4 shRNA or small molecule inhibitors have been tested to treat CRC. In addition, a repurposed anti-helminthic drug niclosamide can inhibit the expression of S100A4 and a prospective phase II clinical trial was carried out for treating CRC patients , 120. Se+ or FOXP3+ TIL subsets are linked with beta-catenin overexpression in breast carcinoma [Oncogenic cascade activation in tumor cells affects the local anti-tumor immune reaction. Wnt signaling cross-talks with various immune cells, and thereby exhibits effects on tumor progression. It not only helps the maintenance and renewal of leucocytes, but also promotes immune tolerance, thereby limiting the anti-tumor response . For exaarcinoma . Correlaarcinoma .In addition to beta-catenin, other members such as WNT5a and DKK1 in the canonical Wnt signaling pathway were also explored for cancer immunotherapy. For example, nanoparticle-mediated trapping of WNT5a remodeled the immunosuppressive tumor microenvironment and enhanced the impact of immunotherapy, especially when combined with low-dose doxorubicin . In addi+, K+, and Pb2+ ionophore, psoralen, ethacrynic acid and pyrvinium pamoate are used to treat vitiligo, hypertension and oedema, and helminth, respectively.Although much progress in tumor suppress has been achieved upon targeting the member of Wnt signaling, no drug targeting this pathway has been approved in clinical applications due to potential side effects. However, some drugs which have been widely used for treating other diseases have been validated to suppress cancer progress, the strategy of analyzing their anti-cancer effect induced by current clinical drugs will save the time and cost. For example, niclosamide is an anthelminthic drug used for treating tapeworms in human, whereas accumulating studies also found that it can also target and inhibit the Wnt signaling in multiple types of cancer, such as ovarian cancer , colorechttps://clinicaltrials.gov/ct2/results. As shown in Table Another strategy of drug development against Wnt signaling is to screen novel inhibitors. So far, many potential drugs targeting Wnt signaling for cancer therapy have been studied in clinical trials at different stages, and representative drugs in specific phases are summarized in Table The Wnt signaling cascade not only controls the development and homeostasis of diverse organs, but also mediates the malignant transformation. Abnormal Wnt activity detected in tumor tissues is associated with tumor initiation, invasion, metastasis, and drug resistance. Further in vitro and in vivo modeling indicates that Wnt signaling can be abnormally activated through genetic and epigenetic modifications. Therefore, several strategies have been proposed for targeting this pathway, such as blocking receptors/ligands, targeting multiple molecules including intracellular molecules, beta-catenin/TCF4 complex and its downstream target genes, or tumor microenvironment and immune response mediated by Wnt signaling. Based on previous attempts, a series of potential drugs have been screened and developed, these drugs comprise small molecules, monoclonal antibody, and other molecules. In addition, some of these drugs also exhibit synergistic effect when combined with chemotherapy. Immunotherapy is a novel therapeutic method, and it also provides strong survival hopes for patients with cancer, especially for those with chemotherapy resistance, thus the combination of Wnt inhibitors with immunotherapy reagents are worth attempting.Although a series of drugs against the components of Wnt signaling have been obtained, many more drugs targeting this pathway should also be explored with approved drug pool to save time and cost. Moreover, novel pharmacological molecules such as peptide can also be screened from current peptide pool. As described in our previous study, P42, a peptide targeting Sox2 protein, was successfully screened from a peptide library with BiFc method and immunoprecipitation, more importantly, this peptide has inhibitive roles on ESCC progression in vitro and in vivo . TherefoWe have just begun to understand how mutations of the critical Wnt signaling genes  cooperate with other tumor-associated genes like p53 and PTEN etc. during initial tumor formation, growth, and metastasis. Similarly, findings of Wnt signaling activation through epigenetic modifications (e.g. methylation or acetylation) are also relatively new. We therefore need more comprehensive models to address the functional relevance of these new findings. In the future, new techniques including CRISPR/Cas9 gene editing, single cell analysis and high-resolution molecule imaging are key to facilitate the establishment of new models, allowing us for deep appreciation of the roles played by Wnt signaling in tumorigenesis. Accordingly, new therapeutic targets and therapies will likely emerge.https://clinicaltrials.gov/ct2/home. However, since Wnt signal also acts in tissue development and homeostasis maintenance, it may also bring some side-effect when use these drugs, thus it is helpful for us to integrate these drugs with nanomaterials to avoid the side-effect on normal tissue development, thereby enhancing their exclusive anti-cancer effects on malignant tissues. Additionally, systematic evaluation on their anti-cancer effects should be strictly performed along with multiple-omics data integration, and the strict clinical studies should be accelerated.Currently, some clinical trials have also been performed with some drugs including niclosamide and LGK974 after searching at website:


In this paper, we investigate the CUSUM statistic of change point under the negatively associated (NA) sequences. By establishing the consistency estimators for mean and covariance functions respectively, the limit distribution of the CUSUM statistic is proved to be a standard Brownian bridge, which extends the results obtained under the case of an independent normal sample and the moving average processes. Finally, the finite sample properties of the CUSUM statistic are given to show the efficiency of the method by simulation studies and an application on a real data analysis.