Wnt Signaling Pathway Genes Research Articles

Cribriform morular thyroid carcinoma: Clinicopathological and molecular basis for both a preventive and therapeutic approach for a rare tumor (Review).

Cribriform morular thyroid carcinoma (CMTC) has been included within the group of thyroid tumors of uncertain histogenesis in the recent World Health Organization classification of endocrine tumors. Most CMTCs occur in young euthyroid women with multiple (and bilateral) thyroid nodules in cases associated with familial adenomatous polyposis (FAP) or as single nodules in sporadic cases. CMTC generally behaves indolently, while aggressiveness and mortality are associated with high‑grade CMTC. This tumor histologically displays a distinctive combination of growth patterns with morular structures. Strong diffuse nuclear and cytoplasmic immunostaining for β‑catenin is the hallmark of CMTC. Tumor cells are also positive for thyroid transcription factor‑1 and for estrogen and progesterone receptors, but negative for thyroglobulin and calcitonin. It is possible that the CMTC phenotype could result from blockage in the terminal/follicular differentiation of follicular cells (or their precursor cells) secondary to the permanent activation of the Wnt/β‑catenin pathway. In CMTC, the activation of the Wnt/β‑catenin pathway is the central pathogenetic event, which in FAP‑associated cases results from germline mutations of the APC regulator of WNT signaling pathway (APC) gene, and in sporadic cases from somatic inactivating mutations in the APC, AXIN1 and CTNNB1 genes. Estrogens appear to play a tumor‑promoting role by stimulating both the PI3K/AKT/mTOR and the RAS/RAF/MAPK signaling pathways. Additional somatic mutations (i.e. RET rearrangements, or KRAS, phosphatidylinositol‑4,5‑bisphosphate 3‑kinase catalytic subunit α, telomerase reverse transcriptase or tumor protein 53 mutations) may further potentiate the development and progression of CMTC. While hemithyroidectomy would be the treatment of choice for sporadic cases without high‑risk data, total thyroidectomy would be indicated in FAP‑associated cases. There is insufficient clinical data to propose therapies targeting the Wnt/β‑catenin pathway, but multikinase or selective inhibitors could be used in a manner analogous to that of conventional thyroid tumors. It is also unknown whether adjuvant antiestrogenic therapy could be useful in the subgroup of women undergoing surgery with high‑risk CMTC, as well as when there is tumor recurrence and/or metastasis.

Polystyrene nanoplastics exert cardiotoxicity through the Notch and Wnt pathways in zebrafish (Danio rerio)

The ubiquity of micro(nano)plastics has raised significant concerns among people. Their accumulation in the cardiovascular system necessitates attention to their cardiotoxicity. However, research on the cardiotoxicity of micro(nano)plastics remains scarce. Our study exposed zebrafish embryos to four different concentrations (0, 1, 10, 100 μg/mL) of polystyrene nanoplastics (PSNPs) for a period of 7 days. The results indicated that PSNPs noticeably decreased the hatching and survival rates of zebrafish and also induced cardiac developmental abnormalities. The mRNA level analysis revealed significant upregulations of heart development-related genes nkx2.5, cmlc-2, and myh-7 in response to PSNPs. Additionally, PSNPs significantly up-regulated the mRNA level associated with the Notch signaling pathway (notch-1a, jag-1a, and her-7) while remarkably suppressing the expression of the Wnt signaling pathway gene (wnt-3a). Further research showed that PSNPs significantly increased the expression of endoplasmic reticulum stress genes atf-6 and chop, while noticeably inhibiting mitochondrial copy numbers. Moreover, PSNPs were found to decrease calcium ion level and superoxide dismutase (SOD) activity in zebrafish larvae. Additionally, prolonged exposure to PSNPs for 7 days exacerbated abnormalities in various indicators compared to a 4-day exposure. In conclusion, our study demonstrates that PSNPs induce oxidative stress in zebrafish larvae, thereby activating endoplasmic reticulum stress and inhibiting mitochondrial activity, ultimately disrupting the Notch and Wnt signaling pathways. These disruptions result in abnormalities in cardiac developmental genes, ultimately leading to cardiac developmental abnormalities in zebrafish. The present research contributes to a novel understanding of the cardiotoxicity of PSNPs.

PIM1–HDAC2 axis modulates intestinal homeostasis through epigenetic modification

The mucosal barrier is crucial for intestinal homeostasis, and goblet cells are essential for maintaining the mucosal barrier integrity. The proviral integration site for Moloney murine leukemia virus-1 (PIM1) kinase regulates multiple cellular functions, but its role in intestinal homeostasis during colitis is unknown. Here, we demonstrate that PIM1 is prominently elevated in the colonic epithelia of both ulcerative colitis patients and murine models, in the presence of intestinal microbiota. Epithelial PIM1 leads to decreased goblet cells, thus impairing resistance to colitis and colitis-associated colorectal cancer (CAC) in mice. Mechanistically, PIM1 modulates goblet cell differentiation through the Wnt and Notch signaling pathways. Interestingly, PIM1 interacts with histone deacetylase 2 (HDAC2) and downregulates its level via phosphorylation, thereby altering the epigenetic profiles of Wnt signaling pathway genes. Collectively, these findings investigate the unknown function of the PIM1–HDAC2 axis in goblet cell differentiation and ulcerative colitis/CAC pathogenesis, which points to the potential for PIM1-targeted therapies of ulcerative colitis and CAC.

Gene Expression Patterns of Colorectal Cancer Stem Cells Following Ibuprofen and Hyperthermia Treatment.

Cancer stem cells (CSCs) substantially influence the development of colorectal cancer (CRC), metastasis, relapse, and resistance to therapy. Ibuprofen and hyperthermia can be effective in the treatment of cancer. Herein, we evaluated the effects of hyperthermia and ibuprofen on the isolated-CSCs of CRC. This experimental study was conducted between Sep 2020 and Jan 2022 at the Department of Pathology, School of Medicine, Shiraz University of Medical Sciences, Iran. A non-adhesive culture system was used to isolate CSCs from HT-29 cells. To confirm the stemness nature of isolated-CSCs, the expression of stemness genes and protein markers was evaluated by quantitative Real-time PCR (qRT-PCR) and flow cytometry assay. The isolated-CSCs were treated with hyperthermia and ibuprofen. The cell viability was determined by MTT assay and trypan blue staining. The expression of stemness, proliferation, Wnt signaling pathway and apoptosis genes was assessed by qRT-PCR. CSCs were isolated within 14 days. The expression of CD-133 marker and OCT3/4, C-MYC, KLF4, and NANOG genes in isolated-CSCs was higher than HT-29 cells (P<0.05). Cell viability of treated-CSCs were considerably reduced (P<0.05). Hperthermia reduced the expression of OCT3/4, NANOG, PCNA, WNT1 and CTNNB1 genes and increased the expression of P53, BAX, and KLF4 genes (P<0.05). Ibuprofen decreased the expression of OCT3/4, BCL2, NANOG, PCNA, WNT1, and CTNNB1 genes and increased the expression of P53, BAX, and KLF4 genes in treated-CSCs (P<0.05). Hyperthermia and ibuprofen treatment demonstrate an inhibitory effect on colorectal CSCs. However, using combination therapy is remaining to be tested.

Novel frameshift variant of WNT10A in a Japanese patient with hypodontia

Congenital tooth agenesis is caused by the impairment of crucial genes related to tooth development, such as Wnt signaling pathway genes. Here, we investigated the genetic causes of sporadic congenital tooth agenesis. Exome sequencing, followed by Sanger sequencing, identified a novel single-nucleotide deletion in WNT10A (NC_000002.12(NM_025216.3):c.802del), which was not found in the healthy parents of the patient. Thus, we concluded that the variant was the genetic cause of the patient’s agenesis.

Integrated analysis strategy of genome-wide functional gene mining reveals DKK2 gene underlying meat quality in Shaziling synthesized pigs

BackgroundShaziling pig is a well-known indigenous breed in China who has superior meat quality traits. However, the genetic mechanism and genomic evidence underlying meat quality characteristics of Shaziling pigs are still unclear. To explore and investigate the germplasm characteristics of Shaziling pigs, we totally analyzed 67 individual’s whole genome sequencing data for the first time (20 Shaziling pigs [S], 20 Dabasha pigs [DBS], 11 Yorkshire pigs [Y], 10 Berkshire pigs [BKX], 5 Basha pigs [BS] and 1 Warthog).ResultsA total of 2,538,577 SNPs with high quality were detected and 9 candidate genes which was specifically selected in S and shared in S to DBS were precisely mined and screened using an integrated analysis strategy of identity-by-descent (IBD) and selective sweep. Of them, dickkopf WNT signaling pathway inhibitor 2 (DKK2), the antagonist of Wnt signaling pathway, was the most promising candidate gene which was not only identified an association of palmitic acid and palmitoleic acid quantitative trait locus in PigQTLdb, but also specifically selected in S compared to other 48 Chinese local pigs of 12 populations and 39 foreign pigs of 4 populations. Subsequently, a mutation at 12,726-bp of DKK2 intron 1 (g.114874954 A > C) was identified associated with intramuscular fat content using method of PCR–RFLP in 21 different pig populations. We observed DKK2 specifically expressed in adipose tissues. Overexpression of DKK2 decreased the content of triglyceride, fatty acid synthase and expression of relevant genes of adipogenic and Wnt signaling pathway, while interference of DKK2 got contrary effect during adipogenesis differentiation of porcine preadipocytes and 3T3-L1 cells.ConclusionsOur findings provide an analysis strategy for mining functional genes of important economic traits and provide fundamental data and molecular evidence for improving pig meat quality traits and molecular breeding.

Abstract 15521: Wnt Signaling Inhibition Prevents Postnatal Inflammation and Disease Progression in Congenital Myxomatous Valve Disease

Introduction: Myxomatous valve disease (MVD) is the most common cause of mitral regurgitation, leading to cardiac dysfunction and heart failure. MVD in a mouse model of Marfan syndrome (MFS) includes valve leaflet thickening and increased immune cell infiltration. However, the underlying mechanisms by which immune cells are recruited in MFS valves remain undefined. Hypothesis: Inhibition of Wnt signaling prevents MVD progression in MFS valves. Methods: Mice with the Fibrillin 1 variant Fbn1 C1039G/+ recapitulate histopathological features of MFS and Wnt signaling activity was detected in TCF/Lef-lacZ reporter mice. Single-cell RNA sequencing was performed from mitral valves of WT and Fbn1 C1039G/+ mice at 1 month-of-age. Inhibition of Wnt signaling was achieved by conditional induction of Wnt inhibitor Dkk1 in valve interstitial cells of Fbn1 C1039G/+ ; Periostin-Cre; tetO-Dkk1; R26-rtTA mice. Dietary doxycycline was administered for 1 month beginning with MVD initiation (1-month-old) or MVD progression (2-month-old). Histological evaluation and immunofluorescence for immune cells were performed. Results: Wnt signaling pathway gene expression is activated early in mitral valve disease progression, prior to immune cell infiltration in Fbn1 C1039G/+ mice. Single cell transcriptomics revealed similar mitral valve cell heterogeneity between WT and Fbn1 C1039G/+ mice at 1 month-of-age. Wnt ligands and responsive genes were predominantly increased in valve interstitial cells and valve endothelial cells of Fbn1 C1039G/+ mice. Inhibition of Wnt signaling by Dkk1 induction at 1 month-of-age prevented the initiation of MVD as indicated by reduced valve leaflet thickness with decreased CD45+ leukocytes and CCR2+ immune cells in Fbn1 C1039G/+ mice. However, later Wnt inhibition starting at 2 months when inflammation and myxomatous changes are occurring did not prevent the progression of MVD. Conclusions: Wnt signaling is involved in the initiation of mitral valve abnormalities and inflammation, but is not responsible for valve disease progression once it has been initiated. Thus, Wnt signaling inhibition prevents initiation of congenital MVD, which provides a promising therapeutic target for the early treatment of congenital MVD.

Apatinib induces zebrafish hepatotoxicity by inhibiting Wnt signaling and accumulation of oxidative stress.

Apatinib, a small-molecule VEGFR2-tyrosine kinase inhibitor, has shown potent anticancer activity in various clinical cancer treatments, but also different adverse reactions. Therefore, it is necessary to study its potential toxicity and working mechanism. We used zebrafish to investigate the effects of apatinib on the development of embryos. Zebrafish exposed to 2.5, 5, and 10 μM apatinib showed adverse effects such as decreased liver area, pericardial oedema, slow yolk absorption, bladder atrophy, and body length shortening. At the same time, it leads to abnormal liver tissue structure, liver function and related gene expression. Furthermore, after exposure to apatinib, oxidative stress levels were significantly elevated but liver developmental toxicity was effectively ameliorated with oxidative stress inhibitor treatment. Apatinib induces down-regulation of key target genes of Wnt signaling pathway in zebrafish, and it is found that Wnt activator can significantly rescue liver developmental defects. These results suggest that apatinib may induce zebrafish hepatotoxicity by inhibiting the Wnt signaling pathway and up-regulating oxidative stress, helping to strengthen our understanding of rational clinical application of apatinib.

Effect of heterologous platelet-rich plasma on liver and modulation of glucose metabolism and Wnt signalling pathways in diabetic mice.

The current study was designed to highlight the effects of heterologous platelet-rich plasma (PRP) on deteriorated hepatic tissues and impaired glucose metabolism of alloxan-induced diabetic mice. 30 male mice were divided into a control (CG), PRP (PG), diabetic (DG), and two treated groups (T1G and T2G). PG was given PRP treatment (0.5 ml/kg body weight) twice a week for four weeks. DG, T1G and T2G were given alloxan (150mg/kg) to induce diabetes. After confirmation, PRP treatment was given to T1G and T2G for two and four weeks respectively while DG was left untreated. Upon completion of the said experimental period, liver samples were taken for histological and gene expression analyses. The study found that the liver tissue of the DG group showed signs of damage, including hepatocyte ballooning, sinusoid dilatation, and collagen deposition. However, these changes were significantly reduced in both T1G and T2G groups. The expression of several genes related to liver function was also affected, with upregulation of Fbp1 and Pklr, and downregulation of Pck1 in the DG group. PRP treatment restored Fbp1 expression and also increased the expression of glycolytic pathway genes Hk1 and Gck, as well as Wnt signalling pathway genes Wnt2, Wnt4, and Wnt9a in both treated groups. Current study revealed that heterologous PRP may partly alleviate high glucose levels in diabetics possibly by mediating glucose metabolism via inhibition of Wnt signalling pathway.

Pexidartinib hydrochloride exposure induces developmental toxicity and immunotoxicity in zebrafish embryos via activation of Wnt signaling.

Pexidartinib, a macrophage colony-stimulating factor receptor (CSF-1R) inhibitor, is indicated for the treatment of tendon sheath giant cell tumor (TGCT). However, few studies on the toxicity mechanisms of pexidartinib for embryonic development. In this study, the effects of pexidartinib on embryonic development and immunotoxicity in zebrafish were investigated. Zebrafish embryos at 6h post fertilization (6 hpf) were exposed to 0, 0.5, 1.0, and 1.5μM concentrations of pexidartinib, respectively. The results showed that different concentrations of pexidartinib induced the shorter body, decreased heart rate, reduced number of immune cells and increase of apoptotic cells. In addition, we also detected the expression of Wnt signaling pathway and inflammation-related genes, and found that these genes expression were significantly upregulated after pexidartinib treatment. To test the effects of embryonic development and immunotoxicity due to hyperactivation of Wnt signaling after pexidartinib treatment, we used IWR-1, Wnt inhibitor, for rescue. Results show that IWR-1 could not only rescue developmental defects and immune cell number, but also downregulate the high expression of Wnt signaling pathway and inflammation-related caused by pexidartinib. Collectively, our results suggest that pexidartinib induces the developmental toxicity and immunotoxicity in zebrafish embryos through hyperactivation of Wnt signaling, providing a certain reference for the new mechanisms of pexidartinib function.

WNT-Conditioned Mechanism of Exit from Postchemotherapy Shock of Differentiated Tumour Cells

Background: the present study aims to prove or disprove the hypothesis that the state of copy number aberration (CNA) activation of WNT signalling pathway genes accounts for the ability of differentiated tumour cells to emerge from postchemotherapy shock. Methods: In the first step, the CNA genetic landscape of breast cancer cell lines BT-474, BT-549, MDA-MB-231, MDA-MD-468, MCF7, SK-BR-3 and T47D, which were obtained from ATCC, was examined to rank cell cultures according to the degree of ectopic activation of the WNT signalling pathway. Then two lines of T47D with ectopic activation and BT-474 without activation were selected. The differentiated EpCAM+CD44-CD24-/+ cells of these lines were subjected to IL6 de-differentiation with formation of mammospheres on the background of cisplatin and WNT signalling inhibitor ICG-001. Results: it was found that T47D cells with ectopic WNT signalling activation after cisplatin exposure were dedifferentiated to form mammospheres while BT-474 cells without ectopic WNT-signalling activation did not form mammospheres. The dedifferentiation of T47D cells after cisplatin exposure was completely suppressed by the WNT signalling inhibitor ICG-001. Separately, ICG-001 reduced, but did not abolish, the ability to dedifferentiate in both cell lines. Conclusions: these data support the hypothesis that the emergence of differentiated tumour cells from postchemotherapy shock after chemotherapy is due to ectopic activation of WNT signalling pathway genes.

Abstract A017: Deciphering the role of integrated stress response (ISR) in the developmental stages of mutant KRAS lung cancer

Abstract Lung cancer is the leading cause of cancer death worldwide. Non-small cell lung cancer (NSCLC) constitutes 80% of all lung malignancies with 15-25% attributed to mutations in the KRAS gene. KRAS mutations expose cells to stress-inducing conditions, e.g., genotoxic, proteotoxic and metabolic stress, which disrupt normal proliferation and tissue homeostasis. Successful proliferation of tumors is dependent on stress-adaptive pathways, which promote growth and contribute to resistance to chemotherapeutic drugs. Thus, inhibition of the adaptation process is considered a suitable anti-tumor approach for the treatment of KRAS cancers including lung cancer. An important mechanism of adaptation to stress involves the phosphorylation of the translation initiation factor eIF2α at serine 52 (p-eIF2α). Increased p-eIF2α is the nodal point of the integrated stress response (ISR), a master regulator of translational and transcriptional reprogramming that determines survival and adaptation to stress. We demonstrated the tumorigenic function of p-eIF2α in a mouse model of mutant KRAS cancer (Nature Communications 2021). We delineate the role of the ISR in lineage diversity and tumor heterogeneity during KRAS LUAD progression by single-cell RNA sequencing (sc-RNA seq). As such, the ISR was found to drive high stemness and epithelial to mesenchymal transition (EMT) programs in KRAS LUAD cells. The ISR also employs a high-plasticity cell-state mechanism for successful lung tumor progression and evolution. The involvement of the ISR in the mentioned processes further emphasizes its role as master regulator of pro-tumorigenic pathways and highlights the therapeutic potential of ISR inhibitors as novel targets in KRAS lung cancer. Our recent findings demonstrate that increased p-eIF2α stimulates the activity of the gene trans-activator proteins YAP and TAZ in mutant KRAS lung tumors. Genetic approaches identify a stimulatory effect of p-eIF2 on the expression of YAP/TAZ-dependent genes with roles in proliferation and epithelial to mesenchymal transition (EMT) including genes of WNT signaling pathway. We provide strong evidence that stimulation of YAP/TAZ activity by p-eIF2α occurs, at least partially, through the upregulation of the transcription factor ATF4 in tumor cells. Our findings demonstrate that p-eIF2α is a focal point of different tumorigenic pathways, intimately implicated in mutant KRAS-mediated transformation and tumor resistance to mutant KRAS inactivation. Citation Format: Shiqi Diao, Nour Ghaddar, Jia Yi Zou, Shuo Wang, Antonis E. Koromilas. Deciphering the role of integrated stress response (ISR) in the developmental stages of mutant KRAS lung cancer [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A017.

The non-canonical Wnt receptor Ror2 is required for cartilage cell polarity and morphogenesis of the craniofacial skeleton in zebrafish.

Non-canonical/β-catenin-independent Wnt signaling plays crucial roles in tissue/cell polarity in epithelia, but its functions have been less well studied in mesenchymal tissues, such as the skeleton. Mutations in non-canonical Wnt signaling pathway genes cause human skeletal diseases such as Robinow syndrome and Brachydactyly Type B1, which disrupt bone growth throughout the endochondral skeleton. Ror2 is one of several non-canonical Wnt receptor/co-receptors. Here, we show that ror2-/- mutant zebrafish have craniofacial skeletal defects, including disruptions of chondrocyte polarity. ror1-/- mutants appear to be phenotypically wild type, but loss of both ror1 and ror2 leads to more severe cartilage defects, indicating partial redundancy. Skeletal defects in ror1/2 double mutants resemble those of wnt5b-/- mutants, suggesting that Wnt5b is the primary Ror ligand in zebrafish. Surprisingly, the proline-rich domain of Ror2, but not its kinase domain, is required to rescue its function in mosaic transgenic experiments in ror2-/- mutants. These results suggest that endochondral bone defects in ROR-related human syndromes reflect defects in cartilage polarity and morphogenesis.

Mechanistic studies in Drosophila and chicken give new insights into functions of DVL1 in dominant Robinow syndrome.

The study of rare genetic diseases provides valuable insights into human gene function. The autosomal dominant or autosomal recessive forms of Robinow syndrome are genetically heterogeneous, and the common theme is that all the mutations lie in genes in Wnt signaling pathways. Cases diagnosed with Robinow syndrome do survive to adulthood with distinct skeletal phenotypes, including limb shortening and craniofacial abnormalities. Here, we focus on mutations in dishevelled 1 (DVL1), an intracellular adaptor protein that is required for both canonical (β-catenin-dependent) or non-canonical (requiring small GTPases and JNK) Wnt signaling. We expressed human wild-type DVL1 or DVL1 variants alongside the endogenous genome of chicken and Drosophila. This design is strategically suited to test for functional differences between mutant and wild-type human proteins in relevant developmental contexts. The expression of variant forms of DVL1 produced a major disorganization of cartilage and Drosophila wing morphology compared to expression of wild-type DVL1. Moreover, the variants caused a loss of canonical and gain of non-canonical Wnt signaling in several assays. Our data point to future therapies that might correct the levels of Wnt signaling, thus improving skeletal growth.

Clinical relevance of genetic polymorphisms in WNT signaling pathway (SFRP1, WNT3A, CTNNB1, WIF-1, DKK-1, LRP5, LRP6) on pulmonary tuberculosis in a Chinese population.

The present study was performed to evaluate the association of WNT signaling pathway genes variants with pulmonary tuberculosis (PTB) risk in Chinese Han population. Our study subjects were composed of 452 PTB patients and 465 normal controls, and seventeen SNPs of seven genes in WNT signaling pathway (SFRP1, WNT3A, CTNNB1, WIF-1, DKK-1, LRP5, LRP6) were genotyped by SNPscan technique. We found no significant relationship of SFRP1 rs10088390, rs4736958, rs3242, WNT3A rs752107, rs3121310, CTNNB1 rs2293303, rs1798802, rs4135385, WIF-1 rs1026024, rs3782499, DKK-1 rs2241529, rs1569198, LRP5 rs3736228, rs556442, LRP6 rs2302685, rs11054697, rs10743980 polymorphisms with PTB susceptibility. While, WIF-1 rs3782499 variant was associated with susceptibility to PTB under recessive model, and haplotype analysis showed that DKK-1 GA haplotype frequency was significantly increased in PTB patients. The WNT3A rs3121310, CTNNB1 rs2293303 polymorphisms were respectively associated with drug-induced liver injury (DILI), sputum smear-positive in PTB patients. The rs3782499 in WIF-1 gene was related to fever, leukopenia, and the rs1569198 in DKK-1 was linked to sputum smear-positive in PTB patients. In LRP5 gene, rs3736228, rs556442 variants respectively affected the occurrence of DILI, fever, and LRP6 gene rs2302685, rs10743980 variants respectively influenced the development of hypoproteinemia, sputum smear-positive in PTB patients. Our results revealed that WNT signaling pathway genes variation were not associated with the susceptibility to PTB, while WNT3A, CTNNB1, WIF-1, DKK-1, LRP5, LRP6 genetic variations might be closely related to the occurrence of several clinical characteristics of PTB patients.

Ruxolitinib enhances cytotoxic and apoptotic effects of temozolomide on glioblastoma cells by regulating WNT signaling pathway-related genes.

Although temozolomide is the primary chemotherapeutic agent in glioblastoma, current studies have focused on its combinational applications to overcome resistance by targeting multiple pathways. JAK/STAT and WNT are among the essential cancer-related signaling pathways. Ruxolitinib, the first approved JAK1/2 inhibitor, has promise in glioblastoma with its blood-brain barrier (BBB) crossing ability. The mentioned study aims to evaluate the anti-cancer potential of ruxolitinib individually and in combination with temozolomide on glioblastoma cells, brain cancer stem cells (BCSCs), and BBB-forming healthy cells. It also intends to determine the effects of JAK inhibitor treatment in combination with temozolomide on WNT signaling, which is known to cross-talk with the JAK/STAT pathway. The U87MG, BCSC, and HBMEC cell lines were the in vitro models. The cytotoxic and apoptotic effects of ruxolitinib and the combination were determined by the WST-1 test and Annexin V assay, respectively. The expression level changes of WNT signaling pathway genes caused by ruxolitinib and the combination treatments were defined by the qRT-PCR method. Network analysis of significantly upregulated and downregulated genes was performed via the GO KEGG pathway enrichment module of the String V11.5 database. The IC50 value of the ruxolitinib on U87MG glioblastoma cells was determined as 94.07µM at 24th h. The combination of temozolomide and ruxolitinib had a synergistic effect on U87MG cells at 24th h. The combination index (CI) was determined as 0.796, and ED60 values of ruxolitinib and temozolomide were determined as 89.75 and 391.48µM, respectively. Ruxolitinib improves the apoptotic effect of temozolomide on glioblastoma cells and brain cancer stem cells. Ruxolitinib regulates the WNT signaling pathway both individually and in combination with temozolomide. Our study indicates the potential of ruxolitinib to increase the cytotoxic and apoptotic activity of temozolomide in glioblastoma cells, also considering CSCs and healthy BBB-forming cells. As supported by gene expression and network analyses, the BBB-crossing agent ruxolitinib promises the potential to increase the efficacy of temozolomide in glioblastoma by affecting multiple signaling pathways in both cancer cells and CSCs.

Effect of azomethine group containing compounds on gene profiles in Wnt and MAPK signal patterns in lung cancer cell line: In silico and in vitro analyses

The main aims of anticancer drug development studies is to reduce the toxicity of the developed compound and maximize the effectiveness, as well as the discovery of artificial and natural compounds. In recent years, scientists have accelerated their research on new molecules with anticancer activity. In recent years, new drugs containing the azomethine group are thought to be promising in the treatment of cancer. In this study, firstly, the synthesis of azomethine group-containing compounds, i.e. Schiff bases, which was designed theoretically, was carried out. Secondly, the application of the newly synthesized compounds 1, 2, 3 and 4 to the lung cancer cell line (A-549), followed by the determination of their anticancer activities, and finally the Wnt signaling pathway (CSNK1A1, CTNNB1), MAPK signaling pathway (DUSP1, DUSP2, DUSP4 and DUSP10) genes on expression levels was investigated. The compounds synthesized in our study were characterized by 1H and 13C NMR spectroscopy methods. The anticancer activities of the new synthesized molecules were determined in the A-549 lung cancer cell line using the MTT method. Expression levels of Wnt signaling pathway (CSNK1A1, CTNNB1) and MAPK signaling pathway (DUSP1, DUSP2, DUSP4 and DUSP10) genes were determined by RT-PCR method. In addition, A-549 cells were evaluated in terms of biochemical parameters. In addition to experimental studies, theoretical studies were carried out. Molecular docking results were found to be compatible with the experiments. Compounds 1, 2, 3 and 4 applied to cell line A-549 showed the highest activity after 72 h of incubation. As a result, it was determined that compounds 2 and 4 increased the expression of CTNNB1 and DUSP10 genes compared to the control group. It was determined that compound 4 increased the expression level of CSNK1A1, CTNNB1, DUSP1, DUSP2, DUSP4 and DUSP10 genes compared to other groups. A-549 lung cancer cells showed a 70% reduction in GST levels in compound 1, while a 96% reduction in CAT levels in compound 1 compared to the control group. Molecular docking calculations supported the Experimental observations. Calculated binding energies provided important clues about drug efficiencies of molecules studied.

CSIG-40. STEAROYL-COA DESATURASE 1 (SCD1) IS REQUIRED FOR WNT SIGNALING TO INDUCE AN APOPTOSIS IN IDH MUTANT GLIOMA

Abstract BACKGROUND AND HYPOTHESES SCD1, a major enzyme of saturated fatty acids, has been implicated to be important for tumor metabolic reprograming. Our previous study show that a high level of SCD1 mRNA is associated with IDH1mut lower grade gliomas. IDH1mut glioma cells are more sensitive to SFA induced apoptosis. However, the underlying mechanism remains unclear. In this study, we investigate the functions of SCD1 in IDHmut glioma and the potential contribution of SCD1 for cancer therapy of glioma. STUDY DESIGN AND METHODS The genetically engineered IDH wild-type, IDHmut and patient derived glioma cell lines were used to evaluate SCD1 functions. The expression of proteins were checked by Western-blotting assay. SCD1 was silenced by CRISPR or siRNA. The transcriptome change after SCD1 knockdown was profiled by RNA-seq or single cell RNA-seq (scRNA-seq). RESULTS AND CONCLUSIONS SCD1 transient silencing slowed down the cell growth, suggesting that SCD1 may possess an oncogenic property. RNA-seq analysis revealed that SCD1 inhibition decreased the expression of wnt-signaling pathway genes in IDH-1mut cells. scRNA confirmed that CRISPR SCD1 significantly decreased wnt signaling in the patient cell line Ts603. Therefore, we activated wnt pathway using a small chemical compound, BML-2838. Consistent with recent studies, wnt pathway induction led to a dramatically suppression of glioma cells growth. However, SCD1 silencing reversed this inhibitory effect. Further investigation revealed that SCD1 inhibition reduced the nucleus translocation of phosphorylated beta-catenin. Overall, the results suggest that SCD1 is vital for the onset of wnt pathway in glioma cells. High level of SCD1 expression may render the IDHmut glioma cells more sensitive to wnt pathway induced apoptosis. RELEVANCE AND IMPORTANCE In clinical, the 5-years survival rate of glioma remains low. SCD1 have been considered as a target for glioma therapy, recently. Our data provides a new insight on the strategy to target SCD1 in clinical.

Effect of VD3 on cell proliferation and the Wnt signaling pathway in bovine endometrial epithelial cells treated with lipopolysaccharide

Vitamin D (VD) deficiency plays an important role in the occurrence and development of various uterine diseases. At present, most studies on the mechanism of VD in the Wnt signaling pathway focus on cancer, while there are no relevant reports on its mechanism in endometritis. This study investigated the effect of vitamin D3 (VD3) on the Wnt signaling pathway in endometrial epithelial cells (BEECs) induced by lipopolysaccharide (LPS). BEECs obtained from bovine uteri were treated with VD3 (0, 50 ng/mL) and LPS (0, 10, 100 ng/mL) separately or in combination, and treated with the Wnt signaling pathway inhibitor IWR-1 to study the mechanism of action. The proliferation of BEECs was evaluated by a CCK-8 assay. qRT-PCR was used to assess the gene expression of Wnt pathway-related factors, including MYC, PCNA, LGR5, GREM1, β-catenin, FZD7, FZD2, Wnt4 and VDR. The results showed that VD3 had no significant effect on cell proliferation (P > 0.05); LPS inhibited BEEC proliferation in a time- and dose-dependent manner, and cells treated with LPS at different concentrations for 24–48 h in combination with VD3 promoted cell proliferation to varying degrees. IWR-1 inhibited cell proliferation in a time- and concentration-dependent manner, while LPS + IWR-1 treatment also significantly promoted cell proliferation after VD3 treatment (P < 0.01). The qRT-PCR results showed that the expression of Wnt4 and PCNA genes showed different trends with different LPS concentrations for stimulation, and the expression of the MYC and GREM1 genes was only stimulated by high-dose (100 ng/mL) LPS stimulation. The expression of FZD7, LGR5, FZD2 and β-catenin was upregulated by LPS at both concentrations. LPS + VD3 significantly downregulated the expression of the Wnt pathway-related genes MYC, PCNA, LGR5, GREM1 and β-catenin (P < 0.001), Wnt4 and FZD2 (P < 0.01), and significantly upregulated the expression of VDR (P < 0.05). After LPS + IWR-1 treatment, the expression of the β-catenin (P < 0.01) and LGR5 (P < 0.05) genes was significantly downregulated, while the Wnt4 (P < 0.01) and VDR (P < 0.001) genes were significantly upregulated, MYC was downregulated but without a significant difference (P > 0.05). In conclusion, VD3 treatment can mitigate the LPS-induced abnormal expression of Wnt signaling pathway genes in BEECs, showing that the Wnt pathway may be a protective pathway of VD3 against LPS-induced gene overexpression in BEECs. The results suggest that VD3 may play a regulatory role in pathways other than the Wnt signaling pathway. Whether VD3 affects the Wnt signaling pathway by affecting Wnt4 gene expression requires further study.

Genome of the sea anemone Exaiptasia pallida and transcriptome profiles during tentacle regeneration.

Cnidarians including sea anemones, corals, hydra, and jellyfishes are a group of animals well known for their regeneration capacity. However, how non-coding RNAs such as microRNAs (also known as miRNAs) contribute to cnidarian tissue regeneration is poorly understood. Here, we sequenced and assembled the genome of the sea anemone Exaiptasia pallida collected in Hong Kong waters. The assembled genome size of E. pallida is 229.21 Mb with a scaffold N50 of 10.58 Mb and BUSCO completeness of 91.1%, representing a significantly improved genome assembly of this species. The organization of ANTP-class homeobox genes in this anthozoan further supported the previous findings in jellyfishes, where most of these genes are mainly located on three scaffolds. Tentacles of E. pallida were excised, and both mRNA and miRNA were sequenced at 9 time points (0 h, 6 h, 12 h, 18 h, 1 day, 2, 3, 6, and 8 days) from regenerating tentacles. In addition to the Wnt signaling pathway and homeobox genes that are shown to be likely involved in tissue regeneration as in other cnidarians, we have shown that GLWamide neuropeptides, and for the first time sesquiterpenoid pathway genes could potentially be involved in the late phase of cnidarian tissue regeneration. The established sea anemone model will be useful for further investigation of biology and evolution in, and the effect of climate change on this important group of animals.