scholarly journals Rational design of peptides for identification of linear epitopes and generation of neutralizing monoclonal antibodies against DKK2 for cancer therapy

2020 ◽  
Vol 3 (2) ◽  
pp. 63-70 ◽  
Author(s):  
Rongqing Zhao ◽  
Qian Xiao ◽  
Maohua Li ◽  
Wenlin Ren ◽  
Chenxi Xia ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Tumor Growth ◽  
Wnt Signaling ◽  
Rational Design ◽  
Polyclonal Antibodies ◽  
Wnt Signaling Pathway ◽  
Keyhole Limpet Hemocyanin ◽  
Linear Epitopes

Abstract Dickkopf-related protein 2 (DKK2)is a member of the Dickkopf family in Wnt signaling pathway. Recently, we found that antibodies against DKK2 could activate natural killer (NK) and CD8+ T cells in tumors and inhibit tumor growth. In this paper, we report the rational design of peptides for identification of linear epitopes and generation of neutralizing monoclonal anti-DKK2 antibodies. To break the immune tolerance, we designed and chemically synthesized six peptides corresponding to different regions of DKK2 as immunogens and found five of them could generate mouse polyclonal antibodies that can bind to the active recombinant human DKK2 protein. Neutralizing mouse monoclonal antibodies (5F8 and 1A10) against human DKK2 were successfully developed by immunizing the mice with two different peptides (34KLNSIKSSL42 and 240KVWKDATYS248) conjugated to Keyhole limpet hemocyanin (KLH). The monoclonal antibodies not only abolish DKK2’s suppression of Wnt signaling in vitro but also inhibits tumor growth in vivo. Currently, those two mAbs are undergoing humanization as immunotherapy candidates and may offer a new drug for treatment of human cancers.

scholarly journals The endocannabinoid anandamide inhibits cholangiocarcinoma growth via activation of the noncanonical Wnt signaling pathway

2008 ◽  
Vol 295 (6) ◽  
pp. G1150-G1158 ◽  
Author(s):  
Sharon DeMorrow ◽  
Heather Francis ◽  
Eugenio Gaudio ◽  
Julie Venter ◽  
Antonio Franchitto ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Tumor Growth ◽  
Wnt Signaling ◽  
Signaling Pathway ◽  
Calcium Release ◽  
Wnt Signaling Pathway ◽  
Xenograft Model ◽  
Orphan Receptor

Cholangiocarcinomas are cancers that have poor prognosis and limited treatment options. The noncanonical Wnt pathway is mediated predominantly by Wnt 5a, which activates a Ca2+-dependent pathway involving protein kinase C, or a Ca2+-independent pathway involving the orphan receptor Ror2 and subsequent activation of Jun NH2-terminal kinase (JNK). This pathway is associated with growth-suppressing effects in numerous cell types. We have shown that anandamide decreases cholangiocarcinoma growth in vitro. Therefore, we determined the effects of anandamide on cholangiocarcinoma tumor growth in vivo using a xenograft model and evaluated the effects of anandamide on the noncanonical Wnt signaling pathways. Chronic administration of anandamide decreased tumor growth and was associated with increased Wnt 5a expression in vitro and in vivo. Treatment of cholangiocarcinoma cells with recombinant Wnt 5a decreased cell proliferation in vitro. Neither anandamide nor Wnt 5a affected intracellular calcium release, but both increased the JNK phosphorylation. Stable knockdown of Wnt 5a or Ror2 expression in cholangiocarcinoma cells abolished the effects of anandamide on cell proliferation and JNK activation. Modulation of the endocannabinoid system may be important in cholangiocarcinoma treatment. The antiproliferative actions of the noncanonical Wnt signaling pathway warrants further investigation to dissect the mechanism by which this may occur.

scholarly journals Dickkopf Wnt signaling pathway inhibitor 1 regulates the differentiation of mouse embryonic stem cells in vitro and in vivo

2015 ◽  
Vol 13 (1) ◽  
pp. 720-730 ◽  
Author(s):  
LIPING OU ◽  
LIAOQIONG FANG ◽  
HEJING TANG ◽  
HAI QIAO ◽  
XIAOMEI ZHANG ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Wnt Signaling ◽  
Signaling Pathway ◽  
Wnt Signaling Pathway ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cells

scholarly journals The Wnt signaling pathway effector TCF7L2 is upregulated by insulin and represses hepatic gluconeogenesis

2012 ◽  
Vol 303 (9) ◽  
pp. E1166-E1176 ◽  
Author(s):  
Wilfred Ip ◽  
Weijuan Shao ◽  
Yu-ting Alex Chiang ◽  
Tianru Jin
Keyword(s):  
Gene Expression ◽  
Type 2 Diabetes ◽  
Transcription Factor ◽  
Wnt Signaling ◽  
Wnt Signaling Pathway ◽  
Glucose Production ◽  
Hepatic Gluconeogenesis

Certain single nucleotide polymorphisms (SNPs) in transcription factor 7-like 2 (TCF7L2) are strongly associated with the risk of type 2 diabetes. TCF7L2 and β-catenin (β-cat) form the bipartite transcription factor cat/TCF in stimulating Wnt target gene expression. cat/TCF may also mediate the effect of other signaling cascades, including that of cAMP and insulin in cell-type specific manners. As carriers of TCF7L2 type 2 diabetes risk SNPs demonstrated increased hepatic glucose production, we aimed to determine whether TCF7L2 expression is regulated by nutrient availability and whether TCF7L2 and Wnt regulate hepatic gluconeogenesis. We examined hepatic Wnt activity in the TOPGAL transgenic mouse, assessed hepatic TCF7L2 expression in mice upon feeding, determined the effect of insulin on TCF7L2 expression and β-cat Ser675 phosphorylation, and investigated the effect of Wnt activation and TCF7L2 knockdown on gluconeogenic gene expression and glucose production in hepatocytes. Wnt activity was observed in pericentral hepatocytes in the TOPGAL mouse, whereas TCF7L2 expression was detected in human and mouse hepatocytes. Insulin and feeding stimulated hepatic TCF7L2 expression in vitro and in vivo, respectively. In addition, insulin activated β-cat Ser675 phosphorylation. Wnt activation by intraperitoneal lithium injection repressed hepatic gluconeogenic gene expression in vivo, whereas lithium or Wnt-3a reduced gluconeogenic gene expression and glucose production in hepatic cells in vitro. Small interfering RNA-mediated TCF7L2 knockdown increased glucose production and gluconeogenic gene expression in cultured hepatocytes. These observations suggest that Wnt signaling and TCF7L2 are negative regulators of hepatic gluconeogenesis, and TCF7L2 is among the downstream effectors of insulin in hepatocytes.

scholarly journals Modulation of Calretinin Expression in Human Mesothelioma Cells Reveals the Implication of the FAK and Wnt Signaling Pathways in Conferring Chemoresistance towards Cisplatin

2019 ◽  
Vol 20 (21) ◽  
pp. 5391 ◽  
Author(s):  
Wörthmüller ◽  
Salicio ◽  
Oberson ◽  
Blum ◽  
Schwaller
Keyword(s):  
Wnt Signaling ◽  
Signaling Pathways ◽  
Signaling Pathway ◽  
Expression System ◽  
Single Agent ◽  
Wnt Signaling Pathway ◽  
Tumor Formation ◽  
Mesenchymal Transition

Malignant mesothelioma (MM) is an aggressive asbestos-linked neoplasm, characterized by dysregulation of signaling pathways. Due to intrinsic or acquired chemoresistance, MM treatment options remain limited. Calretinin is a Ca2+-binding protein expressed during MM tumorigenesis that activates the FAK signaling pathway, promoting invasion and epithelial-to-mesenchymal transition. Constitutive calretinin downregulation decreases MM cells’ growth and survival, and impairs tumor formation in vivo. In order to evaluate early molecular events occurring during calretinin downregulation, we generated a tightly controlled IPTG-inducible expression system to modulate calretinin levels in vitro. Calretinin downregulation significantly reduced viability and proliferation of MM cells, attenuated FAK signaling and reduced the invasive phenotype of surviving cells. Importantly, surviving cells showed a higher resistance to cisplatin due to increased Wnt signaling. This resistance was abrogated by the Wnt signaling pathway inhibitor 3289-8625. In various MM cell lines and regardless of calretinin expression levels, blocking of FAK signaling activated the Wnt signaling pathway and vice versa. Thus, blocking both pathways had the strongest impact on MM cell proliferation and survival. Chemoresistance mechanisms in MM cells have resulted in a failure of single-agent therapies. Targeting of multiple components of key signaling pathways, including Wnt signaling, might be the future method-of-choice to treat MM.

scholarly journals TRIM33 Overexpression Inhibits the Progression of Clear Cell Renal Cell Carcinoma In Vivo and In Vitro

2020 ◽  
Vol 2020 ◽  
pp. 1-18
Author(s):  
Yingkun Xu ◽  
Guangzhen Wu ◽  
Jiayao Zhang ◽  
Jianyi Li ◽  
Ningke Ruan ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Mrna Expression ◽  
Wnt Signaling Pathway ◽  
Xenograft Model ◽  
The Cancer Genome Atlas ◽  
Migration And Invasion ◽  
Cell Renal Cell Carcinoma ◽  
Expression Levels

Purpose. To evaluate the expression of tripartite motif-containing 33 (TRIM33) in ccRCC tissues and explore the biological effect of TRIM33 on the progress of ccRCC. Method. The Cancer Genome Atlas (TCGA) database was used to examine the mRNA expression levels of TRIM33 in ccRCC tissues and its clinical relevance. Immunohistochemistry (IHC) was performed to evaluate its expression in ccRCC tissues obtained from our hospital. The correlation between TRIM33 expression and clinicopathological features of the patients was also investigated. The effects of TRIM33 on the proliferation of ccRCC cells were examined using the CCK-8 and colony formation assays. The effects of TRIM33 on the migration and invasion of ccRCC cells were explored through wound healing and transwell assays, along with the use of Wnt signaling pathway agonists in rescue experiments. Western blotting was used to explore the potential mechanism of TRIM33 in renal cancer cells. A xenograft model was used to explore the effect of TRIM33 on tumor growth. Result. Bioinformatics analysis showed that TRIM33 mRNA expression in ccRCC tissues was downregulated, and low TRIM33 expression was related to poor prognosis in ccRCC patients. In agreement with this, low TRIM33 expression was detected in human ccRCC tissues. TRIM33 expression levels were correlated with clinical characteristics, including tumor size and Furman’s grade. Furthermore, TRIM33 overexpression inhibited proliferation, migration, and invasion of 786-O and ACHN cell lines. The rescue experiment showed that the originally inhibited migration and invasion capabilities were restored. TRIM33 overexpression reduced the expression levels of β-catenin, cyclin D1, and c-myc, and inhibited tumor growth in ccRCC cells in vivo. Conclusion. TRIM33 exhibits an abnormally low expression in human ccRCC tissues. TRIM33 may serve as a potential therapeutic target and prognostic marker for ccRCC.

scholarly journals Amygdala-Based Altered miRNome and Epigenetic Contribution of miR-128-3p in Conferring Susceptibility to Depression-Like Behavior via Wnt Signaling

2020 ◽  
Vol 23 (3) ◽  
pp. 165-177 ◽  
Author(s):  
Bhaskar Roy ◽  
Michael Dunbar ◽  
Juhee Agrawal ◽  
Lauren Allen ◽  
Yogesh Dwivedi
Keyword(s):  
Wnt Signaling ◽  
Target Genes ◽  
Rna Binding ◽  
Statistical Significance ◽  
Neuroblastoma Cell ◽  
Wnt Signaling Pathway ◽  
Target Prediction ◽  
In Vivo Model

Abstract Background Recent studies suggest that microRNAs (miRNAs) can participate in depression pathogenesis by altering a host of genes that are critical in corticolimbic functioning. The present study focuses on examining whether alterations in the miRNA network in the amygdala are associated with susceptibility or resiliency to develop depression-like behavior in rats. Methods Amygdala-specific altered miRNA transcriptomics were determined in a rat depression model following next-generation sequencing method. Target prediction analyses (cis- and trans) and qPCR-based assays were performed to decipher the functional role of altered miRNAs. miRNA-specific target interaction was determined using in vitro transfection assay in neuroblastoma cell line. miRNA-specific findings from the rat in vivo model were further replicated in postmortem amygdala of major depressive disorder (MDD) subjects. Results Changes in miRNome identified 17 significantly upregulated and 8 significantly downregulated miRNAs in amygdala of learned helpless (LH) compared with nonlearned helpless rats. Prediction analysis showed that the majority of the upregulated miRNAs had target genes enriched for the Wnt signaling pathway. Among altered miRNAs, upregulated miR-128-3p was identified as a top hit based on statistical significance and magnitude of change in LH rats. Target validation showed significant downregulation of Wnt signaling genes in amygdala of LH rats. A discernable increase in expression of amygdalar miR-128-3p along with significant downregulation of key target genes from Wnt signaling (WNT5B, DVL, and LEF1) was noted in MDD subjects. Overexpression of miR-128-3p in a cellular model lead to a marked decrease in the expression of Dvl1 and Lef1 genes, confirming them as validated targets of miR-128-3p. Additional evidence suggested that the amygdala-specific diminished expression of transcriptional repressor Snai1 could be potentially linked to induced miR-128-2 expression in LH rats. Furthermore, an amygdala-specific posttranscriptional switching mechanism could be active between miR-128-3p and RNA binding protein Arpp21 to gain control over their target genes such as Lef1. Conclusion Our study suggests that in amygdala a specific set of miRNAs may play an important role in depression susceptibility, which could potentially be mediated through Wnt signaling.

scholarly journals Monoclonal Antibodies Targeting the HR2 Domain and the Region Immediately Upstream of the HR2 of the S Protein Neutralize In Vitro Infection of Severe Acute Respiratory Syndrome Coronavirus

2006 ◽  
Vol 80 (2) ◽  
pp. 941-950 ◽  
Author(s):  
Kuo-Ming Lip ◽  
Shuo Shen ◽  
Xiaoming Yang ◽  
Choong-Tat Keng ◽  
Aihua Zhang ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Polyclonal Antibodies ◽  
Heptad Repeat ◽  
Functional Domain ◽  
Protein Fragment ◽  
S Protein ◽  
Linear Epitopes ◽  
Respiratory Coronavirus ◽  
Neutralizing Epitopes

ABSTRACT We have previously shown that an Escherichia coli-expressed, denatured spike (S) protein fragment of the severe acute respiratory coronavirus, containing residues 1029 to 1192 which include the heptad repeat 2 (HR2) domain, was able to induce neutralizing polyclonal antibodies (C. T. Keng, A. Zhang, S. Shen, K. M. Lip, B. C. Fielding, T. H. Tan, C. F. Chou, C. B. Loh, S. Wang, J. Fu, X. Yang, S. G. Lim, W. Hong, and Y. J. Tan, J. Virol. 79:3289-3296, 2005). In this study, monoclonal antibodies (MAbs) were raised against this fragment to identify the linear neutralizing epitopes in the functional domain and to investigate the mechanisms involved in neutralization. Eighteen hybridomas secreting the S protein-specific MAbs were obtained. Binding sites of these MAbs were mapped to four linear epitopes. Two of them were located within the HR2 region and two immediately upstream of the HR2 domain. MAbs targeting these epitopes showed in vitro neutralizing activities and were able to inhibit cell-cell membrane fusion. These results provide evidence of novel neutralizing epitopes that are located in the HR2 domain and the spacer region immediately upstream of the HR2 of the S protein.

scholarly journals Replicating Adenoviruses That Target Tumors with Constitutive Activation of the wnt Signaling Pathway

2001 ◽  
Vol 75 (6) ◽  
pp. 2857-2865 ◽  
Author(s):  
Michele Brunori ◽  
Maddalena Malerba ◽  
Haruhiko Kashiwazaki ◽  
Richard Iggo
Keyword(s):  
Wnt Signaling ◽  
Cancer Cells ◽  
Wnt Pathway ◽  
Wnt Signaling Pathway ◽  
Lung Model ◽  
Colorectal Tumors ◽  
Colon Tumors ◽  
Treat All

ABSTRACT Despite important advances in understanding the molecular basis of cancer, few treatments have been devised which rationally target known causal oncogenic defects. Selectively replicating viruses have a major advantage over nonreplicating viruses to target these defects because the therapeutic effect of the injected virus is augmented by virus produced within the tumor. To permit rational targeting of colon tumors, we have developed replicating adenoviruses that express the viral E1B and E2 genes from promoters controlled by the Tcf4 transcription factor. Tcf4 is constitutively activated by mutations in the adenomatous polyposis coli and β-catenin genes in virtually all colon tumors and is constitutively repressed by Groucho and CtBP in normal tissue. The Tcf-E2 and Tcf-E1B promoters are active in many, but not all, cell lines with activation of the wnt pathway. Viruses with Tcf regulation of E2 expression replicate normally in SW480 colon cancer cells but show a 50- to 100-fold decrease in replication in H1299 lung cancer cells and WI38 normal fibroblasts. Activation of wnt signaling by transduction of a stable β-catenin mutant into normal fibroblasts renders the cells permissive for virus replication. Insertion of Tcf4 sites in the E1B promoter has only small effects on replication in vitro but significantly reduces the inflammatory response in a rodent lung model in vivo. Replicating adenoviruses with Tcf regulation of both E1B and E2 transcription are potentially useful for the treatment of liver metastases from colorectal tumors, but additional changes will be required to produce a virus that can be used to treat all colon tumors.

Ellagic Acid Attenuates BLM-Induced Pulmonary Fibrosis via Inhibiting Wnt Signaling Pathway

2020 ◽  
Author(s):  
Xiaohe Li ◽  
kai huang ◽  
Xiaowei Liu ◽  
Hao Ruan ◽  
Ling Ma ◽  
...  
Keyword(s):  
Pulmonary Fibrosis ◽  
Wnt Signaling ◽  
Signaling Pathway ◽  
Ellagic Acid ◽  
Cell Damage ◽  
Wnt Signaling Pathway ◽  
Pharmacological Activities ◽  
Natural Polyphenol

Abstract Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with high mortality, which characterized by epithelial cell damage and fibroblasts activation. Ellagic acid (EA) is a natural polyphenol compound widely found in fruits and nuts which has demonstrated multiple pharmacological activities. Herein we showed that Ellagic acid significantly alleviated bleomycin(BLM)-induced pulmonary fibrosis in mice, and also inhibited the Wnt/β-catenin signal in primary pulmonary fibroblasts. In vitro experiments indicated that Ellagic acid apparently suppressed Wnt3a-induced myofibroblasts activation and ECM accumulation mainly via inhibiting the phosphorylation of Erk2 and Akt. Further studies showed that Ellagic acid could induce autophagy formation of myofibroblasts mainly by suppressing mTOR signaling and promote apoptosis of myofibroblasts. In vivo experiments reveled that Ellagic acid significantly inhibited myofibroblasts activation and promoted autophagy formation. Taken together, our results showed that Ellagic acid effectively attenuated BLM-induced pulmonary fibrosis in mice by suppressing myofibroblasts activation, promoting autophagy and apoptosis of myofibroblasts mainly via inhibiting Wnt signaling pathway.

Increasing Wnt3a Signaling in Myeloma Cells Inhibits the Osteolytic Phenotype and Tumor Growth In Vivo through Regulation of the RANKL/OPG Axis in Osteoblasts and Indirectly Regulates Osteoclastogenesis.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3506-3506
Author(s):  
Ya-Wei Qiang ◽  
Nathan Brown ◽  
Yu Chen ◽  
Shmuel Yaccoby ◽  
Bart Barlogie ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Wnt Signaling ◽  
Cell Line ◽  
Osteoclast Formation ◽  
C2c12 Cells ◽  
Myeloma Cells ◽  
Protein Levels ◽  
Canonical Wnt

Abstract We have demonstrated that canonical and non-canonical Wnt signaling occurs in myeloma cells (Qiang et al., 2005) and overexpression of Wnt3a in myeloma cells inhibits the osteolytic phenotype and also tumor growth in vivo (Qiang et al Blood, Abstract #3420, 2006). To further investigate the mechanisms that contribute to this process we have expanded our in vivo data by showing that while H929 cells stably expressing Wnt3a (H929/W3a) leads to reduced tumor growth in the in-vivo SCID-hu bone graft model compared with H929 vector alone transfected control cells (H929/EV), there was no significant difference in the subcutaneous growth of the two cell lines in SCID mice. Taken together these data suggests that alteration of the human bone marrow microenvironment is central to Wnt-mediated reduction in tumor growth in bone. We next employed an in-vitro co-culture model in which the mouse osteoprogenitor cell line, C2C12, and human osteoblast cell line, Saos-2 were co-cultured with either H929/Wnt3 or H929/EV cells. QPCR analysis demonstrated that osteoprotegerin (OPG) mRNA expression (relative OPG mRNA to GAPDH) in C2C12 cells co-cultured with H929/W3a was significantly elevated compared with H929/EV (mean±SD: 14.34±0.97 vs 8.43±0.16; P<0.001). ELISA analysis showed that OPG protein levels in the cell culture supernatant were also significantly higher (71.02 ± 6.178 vs 0 pg/ml; P<0.001). Similar results in OPG mRNA and protein levels were observed in Saos-2 cells co-cultured with H929/W3a relative to H929/EV. Furthermore, treatment of C2C12 cells with recombinant Wnt3a protein induced both OPG mRNA (48.1 ±1.2 vs 1.0±0.5; P<0.001) and protein levels (1767.03 ± 44.8 vs 1.11 ± 0.03 p< 0.0001) compared with vehicle alone. These results suggest that forced expression of a canonical Wnt ligand by MM cells might promote OPG transcription in osteoblast progenitors in-vivo. To further confirm the role of Wnt signaling in regulation of OPG and RANKL transcription, we produced C2C12 cells that stably express Dkk1. These clones showed a significant inhibition of Wnt3a induced OPG mRNA (22.2± 2.3 vs 1.7±0.35; p<0.001) and protein (73.3 ± 18.0 vs. 0 pg/ml; p<0.01) compared with vector control. In contrast, RANKL mRNA (5.1±0.9 vs 1.0± 0.5, p<0.01) and protein (9.3±3.8 vs. 0 pg/ml; p<0.01) were increased in Dkk1 expressing clones compared with control. Moreover, supernatant from C2C12 clones stably expressing a DN-beta-catenin (DNBC/C2C12) contained a significantly higher level of RANKL (17.3± 3.5 pg/ml vs. 0±0; P<0.001) and a dramatically lower level of OPG protein (0±0 vs. 431.186 pg/ml; P<0.001) compared with control. Finally, the numbers of multinuclear TRAP-positive osteoclasts were significantly more abundant in culture containing supernatant from DNBC/C2C12 than that from vector control, while Wnt3a exposure had no effect on osteoclast formation in-vitro. Taken together, these data suggest that Wnt ligand-mediated inhibition of myeloma cell growth, and inhibition of osteolytic lesions, in-vivo may result from upregulation of OPG and loss of RANKL in osteoblast progenitors, which subsequently diminishes osteoclast formation. Results of these studies provide new insights into mechanism by which Wnts may serve as an important indirect regulator of myeloma growth and osteoclast formation, and as such, targeting Wnt signaling may be an new therapeutic strategy for controlling myeloma growth and associated bone disease.

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