Wnt Signal Transduction Pathway Research Articles

Comparison of ovarian mRNA expression levels in wild and hatchery-produced greater amberjack Seriola dumerili

The greater amberjack Seriola dumerili is a promising candidate for aquaculture production. This study compares the ovary transcriptome of greater amberjack sampled in the wild (WILD) with hatchery-produced breeders reared in aquaculture sea cages in the Mediterranean Sea. Among the seven sampled cultured fish, three were classified as reproductively dysfunctional (DysF group), while four showed no signs of reproductive alteration (NormalF group). The DysF fish showed 1,166 differentially expressed genes (DEGs) compared to WILD females, and 755 DEGs compared to the NormalF. According to gene ontology (GO) analysis, DysF females exhibited enrichment of genes belonging to the biological categories classified as Secreted, ECM-receptor interaction, and Focal adhesion. Protein–protein interaction analysis revealed proteins involved in the biological categories of ECM-receptor interaction, Enzyme-linked receptor protein signaling, Wnt signal transduction pathways, and Ovulation cycle. KEGG pathway analysis showed DEGs involved in 111 pathways, including Neuroactive ligand-receptor interaction, Steroid hormone biosynthesis, Cell cycle, Oocyte meiosis, Necroptosis, Ferroptosis, Apoptosis, Autophagy, Progesterone-mediated oocyte maturation, Endocytosis and Phagosome, as well as Hedgehog, Apelin, PPAR, Notch, and GnRH signalling pathways. Additionally, DysF females exhibited factors encoded by upregulated genes associated with hypogonadism and polycystic ovary syndrome in mammals. This study -which is part of a broader research effort examining the transcriptome of the entire reproductive axis in greater amberjack of both sexes-, enhances our comprehension of the mechanisms underlying the appearance of reproductive dysfunctions when fish are reared under aquaculture conditions.

A Comprehensive View on the Impact of Chlorogenic Acids on Colorectal Cancer.

Chlorogenic acids are plant secondary metabolites, chemically-polyphenols with similar biological activity, formed through the esterification of quinic acid and hydrocinnamic acid moieties. They are best known for their high concentration in coffee and other dietary sources and the antioxidant properties that they exhibit. Both chlorogenic acids and plant extracts containing significant amounts of the compounds show promising in vitro activity against colorectal cancer. With coffee being the most popular drink in the world, and colorectal cancer at an unfortunate peak in incidence and mortality, the mechanisms through which the anti-tumorigenic effect of chlorogenic acids could be functionalized for CRC prevention seem appealing to study. Therefore, this review aims to enable a better understanding of the modes of action of chlorogenic acids in combating carcinogenesis, with a focus on cell cycle arrest, the induction of apoptosis, and the modulation of Wnt, Pi3K/Akt, and MAPK signal transduction pathways, alongside the reduction in the number of inflammatory cytokines and chemokines and the counterintuitive beneficial elevation of oxidative stress.

Effect of estradiol after bacterial infection on the Wnt/β-catenin pathway in bovine endometrium epithelial cells and organoids

Endometritis is a disease caused by a postpartum bacterial infection with a poor prognosis that primarily affects dairy cows. Three-dimensional organoids have been used as a model for endometritis, because they exhibit a structure comparable to that of the endometrium, demonstrating both expansibility and hormone responsiveness. These characteristics render them an ideal platform for in vitro investigations of endometrial diseases. Estradiol (E2) is an endogenous steroid hormone with demonstrated anti-inflammatory properties, and the objective of this study was to determine the mechanism by which E2 modulates the inflammatory response and the Wnt signal transduction pathway in bovine endometrial epithelial cells and organoids following E. coli infection. We present the techniques for isolating and culturing primary bovine endometrial epithelial cells (BEECs), and producing endometrial organoids. For the experiments, the endometrial epithelial cells and organoids were infected with E. coli for 1 h, followed by incubation with E2 for 12 h. The mRNA and protein expressions of the inflammation-related genes, IL-1β, IL-6, TLR4, and NF-κB, as well as the Wnt pathway-related genes, Wnt4, β-catenin, c-Myc, and CyclinD1, were assessed using real-time quantitative-PCR and western blotting, respectively. The CCK8 viable cell counting assay was utilized to determine the optimal concentration of the Wnt inhibitor, IWR-1. The mRNA and protein expression of Wnt pathway-related genes was assessed following IWR-1 treatment, while the expression levels of proliferation-associated genes (Ki67, PCNA) and barrier repair genes (occludin, claudin, and Zo-1) in BEECs and organoids were evaluated after E2 treatment. The results of this study show that mRNA expression of the inflammatory genes, IL-1β, TLR4, and NF-κB (P < 0.05) decreased in BEECs following E2 treatment compared to the E. coli group. The protein expression of the IL-1β, IL-6, TLR4 and NF-κB genes was also inhibited (P < 0.05). Similar results were observed in tests on the organoids. Our findings demonstrate that E2 significantly upregulates the expression of Wnt-related genes, including β-catenin and c-Myc, while concurrently downregulating the expression of GSK3β (P < 0.05). Next, we treated E. coli-infected BEECs and organoids with the Wnt inhibitor, IWR-1. Compared with E. coli and E. coli + E2, the expression of mRNA and protein from Wnt 4, β-catenin, and CyclinD1 in E. coli + E2 and E. coli + IWR-1 was down-regulated (P < 0.05). The expression of the proliferation genes, Ki67, PCNA, and the tight junction genes, occludin, claudin1, and Zo-1, in organoids was significantly higher than that in BEECs (P < 0.05). In summary, we found strong potential for E2 mitigation of the E. coli-induced inflammatory response in BEECs and organoids, through activation of the Wnt pathway. In addition, the proliferation and repair capacity of organoids was much higher than that of BEECs.

The origin and evolution of Wnt signalling.

The Wnt signal transduction pathway has essential roles in the formation of the primary body axis during development, cellular differentiation and tissue homeostasis. This animal-specific pathway has been studied extensively in contexts ranging from developmental biology to medicine for more than 40 years. Despite its physiological importance, an understanding of the evolutionary origin and primary function of Wnt signalling has begun to emerge only recently. Recent studies on very basal metazoan species have shown high levels of conservation of components of both canonical and non-canonical Wnt signalling pathways. Furthermore, some pathway proteins have been described also in non-animal species, suggesting that recruitment and functional adaptation of these factors has occurred in metazoans. In this Review, we summarize the current state of research regarding the evolutionary origin of Wnt signalling, its ancestral function and the characteristics of the primal Wnt ligand, with emphasis on the importance of genomic studies in various pre-metazoan and basal metazoan species.

VEGF overexpression in transplanted NSCs promote recovery of neurological function in rats with cerebral ischemia by modulating the Wnt signal transduction pathway

Neural stem cell transplantation is a good method to treat stroke, but the mechanism is still unclear. Therefore, this study aims to explore the regulatory mechanism of VEGF overexpression in transplanted NSCs to promote the recovery of neural function in ischemic rats by regulating Wnt signal transduction pathways. We amplified VEGF gene fragments by PCR and transfected them into NSCs with Ad5 adenovirus. Rat brain IRI model was established by MCAO method, and VEGF transfected NSCs (VEGF-NSCs) were transplanted 24 h after successful IRI model. One week after the transplant, cognitive function was assessed using a neurological deficit score; Brain injury was assessed by histopathology; Photochemical and ELISA methods were used to detect oxidative stress markers and inflammatory factors, respectively. Western blotting has been detected in molecules of the Wnt signaling pathway. The results showed that the transduced NSCs express VEGF at least for 14 days. VEGF-NSCs transplantation (VNT) improved spatial learning and memory in rats, and inhibited oxidative stress injury, inflammatory response, and histopathological injury. VNT also resulted in significant changes in the phosphorylation levels of β-catenin and GSK-3β proteins, ultimately triggering activation of the Wnt signal transduction pathway. These results suggest that the neuroprotective effects of VNT may be related to the regulation of the Wnt signal transduction pathway.

Regulation of Wnt Signaling by Heterotrimeric G-Proteins

Wnt signal transduction pathways play crucial roles in animal development, and after birth, they are involved in maintaining cellular and tissue homeostasis. Deregulation of the Wnt-mediated pathways occurs in many human diseases, including a wide range of human cancers. Many years ago, due to the topological similarity between Frizzled receptors and G-protein-coupled receptors, it was proposed that heterotrimeric G-proteins might be involved in the regulation of Wnt/Frizzled-mediated signaling pathways. Currently, there is a wealth of evidence indicating that heterotrimeric G-proteins regulate both canonical and non-canonical Wnt signaling pathways. This review article will discuss some of this evidence and the significance of this interaction.

Myeloid-derived suppressor cells and tolerogenic dendritic cells are distinctively induced by PI3K and Wnt signaling pathways

Imbalanced immune responses are a prominent hallmark of cancer and autoimmunity. Myeloid cells can be overly suppressive, inhibiting protective immune responses or inactive not controlling autoreactive immune cells. Understanding the mechanisms that induce suppressive myeloid cells, such as myeloid-derived suppressor cells (MDSCs) and tolerogenic dendritic cells (TolDCs), can facilitate the development of immune-restoring therapeutic approaches. MDSCs are a major barrier for effective cancer immunotherapy by suppressing antitumor immune responses in cancer patients. TolDCs are administered to patients to promote immune tolerance with the intent to control autoimmune disease. Here, we investigated the development and suppressive/tolerogenic activity of human MDSCs and TolDCs to gain insight into signaling pathways that drive immunosuppression in these different myeloid subsets. Moreover, monocyte-derived MDSCs (M-MDSCs) generated invitro were compared to M-MDSCs isolated from head-and-neck squamous cell carcinoma patients. PI3K-AKT signaling was identified as being crucial for the induction of human M-MDSCs. PI3K inhibition prevented the downregulation of HLA-DR and the upregulation of reactive oxygen species and MerTK. In addition, we show that the suppressive activity of dexamethasone-induced TolDCs is induced by β-catenin-dependent Wnt signaling. The identification of PI3K-AKT and Wnt signal transduction pathways as respective inducers of the immunomodulatory capacity of M-MDSCs and TolDCs provides opportunities to overcome suppressive myeloid cells in cancer patients and optimize therapeutic application of TolDCs. Lastly, the observed similarities between generated- and patient-derived M-MDSCs support the use of invitro-generated M-MDSCs as powerful model to investigate the functionality of human MDSCs.

CNSC-02. INVESTIGATING THE ROLE OF THE WNT SIGNALING TRANSDUCTION PATHWAY IN GLIOMA BLOOD-BRAIN-BARRIER INTEGRITY

Abstract The WNT signaling transduction pathway has been linked to cancer stem cell self-renewal, differentiation, and blood brain barrier (BBB) development. Previous studies have shown that WNT pathway inhibition increased BBB permeability, specifically in WNT medulloblastoma. Glioblastoma, an aggressive malignant tumor with poor prognosis, has also been shown to have high WNT expression and variable BBB permeability. We hypothesize that activating WNT pathway inhibition in glioblastoma stem cells (GSCs) will inhibit glioma progression, increase BBB permeability within the tumor microenvironment, and enhance overall treatment responsiveness. We transduced primary pediatric derived glioma stem cells with overexpressing vectors for WNT inhibitors (DKK1 or WIF1). To characterize effects of DKK1 or WIF1 overexpression, we evaluated cell migration patterns via RTCA xCelligence, cell cycle progression via Annexin 5 assays, and cell viability via cell-titer glo. Brain endothelial cell integrity was evaluated with the treatment of CHIR99021, WNT pathway activator, or GSC condition media with immunoblotting for junctional protein expression (Claudin-5, Claudin-3, Occludin, ZO-1, VE-Cadherin). Verification of WNT inhibitory overexpressing GSCs for DKK1 and WIF1 was performed using RNAseq, proteomics, and immunoblotting. DKK1-GSCs demonstrated a statistically significant impairment in the migratory slope compared to empty vector GSCs. Cell cycle analysis also revealed that DKK1-GSCs were arrested in G0/1 phase while WIF1-GSCs were arrested in S/G2M phase. CHIR99021 endothelial treatment resulted in increased secretion of WNT inhibitors DKK1 and SFRP1 and resultantly, there was a decrease in junctional protein expression. Overall, WNT inhibitor overexpressing GSCs exhibited unique cellular patterns and indirectly disrupted endothelial cell integrity. Future studies will evaluate orthotopic WNT inhibitor overexpressing transplant rodent models to determine BBB integrity, drug permeability, model survival, and chemotherapeutic treatment response. By enhancing our understanding of the WNT pathway in GSCs, we anticipate understanding the use of future therapeutic WNT pathway targeting to increase chemotherapeutic responsiveness and improve glioblastoma prognosis.

Abstract B019: APC and P53 mutations synergize to create a therapeutic vulnerability to NOTUM inhibition in advanced colorectal cancer

Abstract Colorectal cancer (CRC) is a leading cause of cancer-related deaths globally, with the majority of cases initiated by inactivation of the APC tumor suppressor. This results in the constitutive transcriptional activation of the canonical WNT signal transduction pathway effector b-Catenin, along with induction of WNT feedback inhibitors, including the extracellular palmitoleoyl-protein carboxylesterase NOTUM. Here, we show that NOTUM retains cell-autonomous tumor suppressive activity in APC-null adenomatous lesions despite constitutive b-Catenin activation. Strikingly, we find that NOTUM becomes an obligate oncogene upon subsequent P53 inactivation during the adenoma-adenocarcinoma transition, and that these phenotypes are WNT-independent, resulting from differential activity of NOTUM upon its enzymatic targets Glypican 1 and 4 in early vs. late-stage disease, respectively. Ultimately, preclinical mouse models of CRC and human tumoroid cultures demonstrate that pharmacological inhibition of NOTUM is highly effective in arresting primary adenocarcinoma growth and inhibiting metastatic colonization of distal organs. The finding that a single agent targeting an extracellular enzyme is effective in treating highly aggressive tumors make NOTUM a novel therapeutic vulnerability in advanced colorectal adenocarcinomas. Citation Format: Christopher Lengner. APC and P53 mutations synergize to create a therapeutic vulnerability to NOTUM inhibition in advanced colorectal cancer [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr B019.

Three Effects of Β-Catenin Factors in Liver Cancer and Its Treatment

Liver cancer is a typical malignant tumor and the fourth most typical cancer in the Earth. Clinically, more than 90% of patients with primary liver cancer are caused by hepatocellular carcinoma. Epidemiological and experimental data show that human infection with HBV and HCV hepatitis virus is firmly connected with the occurrence of liver cancer. In the past 20 years, the level of diagnosis and cure of liver cancer in China has been greatly improved, but little progress has been made in the study of liver cancer markers. For then, with the understanding of Wnt signal transduction pathway, it is found that β-catenin, as a key molecule in Wnt signal transduction pathway, is closely associated to the incidence of HCC. Recently, more and more studies have shown that the abnormal activation of classical Wnt signal pathway plays a compelling part in the occurrence and advancement of hepatocellular carcinoma. Hepatocytes because of hidden onset, early rise can be no clinical symptoms, so the clinical discovery is mostly late, the mortality rate is high. At present, the clinical methods for the treatment of hepatocellular carcinoma are liver transplantation, radiotherapy and chemotherapy. However, hepatocellular carcinoma is easy to metastasize and has a high recurrence rate, so the mortality rate of hepatocellular carcinoma is still high. Wnt/β-catenin signal pathway has become major topic of debate in cancer research. In this paper, we begin with the classification of liver cancer and some pathogenic mechanisms. The effects of β-catenin protein on the occurrence, metastasis and immune regulation of hepatocellular carcinoma were also discussed. Under the background of the popular "signal transduction therapy" in recent years, to explore the therapeutic effect of targeted drugs targeting Wnt/β-catenin signal pathway in hepatocellular carcinoma. Because of the high degree of malignancy and limited treatment of hepatocellular carcinoma, we will mainly discuss the effect of Wnt signal pathway on the metastasis of hepatocellular carcinoma and its effect on the differentiation of immune cells. Abnormal activation of Wnt signaling pathway in hepatocellular carcinoma.

MafB, WDR77, and ß-catenin interact with each other and have similar genome association profiles.

MafB (a bZIP transcription factor), ß-catenin (the ultimate target of the Wnt signal transduction pathway that acts as a transcriptional co-activator of LEF/TCF proteins), and WDR77 (a transcriptional co-activator of multiple hormone receptors) are important for breast cellular transformation. Unexpectedly, these proteins interact directly with each other, and they have similar genomic binding profiles. Furthermore, while some of these common target sites coincide with those bound by LEF/TCF, the majority are located just downstream of transcription initiation sites at a position near paused RNA polymerase (Pol II) and the +1 nucleosome. Occupancy levels of these factors at these promoter-proximal sites are strongly correlated with the level of paused Pol II and transcriptional activity.

The effect of TLR-4 on the proliferation and differentiation of bone mesenchymal stem cells and its relationship with the Wnt signal transduction pathway during bone nonunion.

BackgroundBone nonunion is a special fracture complication that occurs in about 5% to 10% of cases. This type of fracture is difficult to heal, and causes great pain to patients and affects their quality of life. The mechanism of bone nonunion is not clear. In our study, we investigated the influence of Toll-like receptor (TLR)-3, TLR-4, and Wnt signaling pathways on the occurrence of bone nonunion.MethodsFirstly, we established a Sprague Dawley (SD) rat model of femoral nonunion, and detected the expression levels of TLR-3, TLR-4, β-catenin, nemo-like kinase (NLK), c-Jun N-terminal kinase (JNK), and other proteins during model construction. For in vitro experiments, primary cultured bone mesenchymal stem cells (BMSCs) were divided into 4 groups: lipopolysaccharide (LPS, agonist of TLR-4) group, LPS + CLI095 (inhibitor of TLR-4) group, control group, and LPS + substance P (SP) group. The expression of β-catenin, NLK, JNK, and ALP and the osteogenic differentiation ability of cells were detected during culture.ResultsX-ray and hematoxylin and eosin (HE) staining results confirmed the successful modeling of bone nonunion. During the formation of the bone nonunion model, the expression of TLR-4 showed an upward trend. In vitro experiment results showed that inhibition of TLR-4 expression could enhance the proliferation and differentiation ability of BMSCs. The expression of β-catenin, the core protein of the canonical Wnt signaling pathway, increased rapidly in the first 2 weeks of bone nonunion construction, and decreased after 2 weeks. Non-canonical Wnt signaling pathway proteins NLK and JNK had no change in the first 2 weeks, and showed an upward trend after 2 weeks. In vitro experiment results showed that the expression of β-catenin was dominant in BMSCs with strong proliferation and differentiation ability, while the expression of NLK and JNK was dominant in BMSCs with weak proliferation and differentiation ability. These results suggest that the Wnt signaling pathway may regulate the occurrence of bone nonunion.ConclusionsTLR-4 inhibits the proliferation and differentiation of BMSCs, and the transformation of the canonical Wnt signaling pathway to the non-canonical Wnt signaling pathway may lead to bone nonunion. Our study may provide new insights into the treatment of bone nonunion.

Machine learning assisted analysis of breast cancer gene expression profiles reveals novel potential prognostic biomarkers for triple-negative breast cancer

Tumor heterogeneity and the unclear metastasis mechanisms are the leading cause for the unavailability of effective targeted therapy for Triple-negative breast cancer (TNBC), a breast cancer (BrCa) subtype characterized by high mortality and high frequency of distant metastasis cases. The identification of prognostic biomarker can improve prognosis and personalized treatment regimes. Herein, we collected gene expression datasets representing TNBC and Non-TNBC BrCa. From the complete dataset, a subset reflecting solely known cancer driver genes was also constructed. Recursive Feature Elimination (RFE) was employed to identify top 20, 25, 30, 35, 40, 45, and 50 gene signatures that differentiate TNBC from the other BrCa subtypes. Five machine learning algorithms were employed on these selected features and on the basis of model performance evaluation, it was found that for the complete and driver dataset, XGBoost performs the best for a subset of 25 and 20 genes, respectively. Out of these 45 genes from the two datasets, 34 genes were found to be differentially regulated. The Kaplan-Meier (KM) analysis for Distant Metastasis Free Survival (DMFS) of these 34 differentially regulated genes revealed four genes, out of which two are novel that could be potential prognostic genes (POU2AF1 and S100B). Finally, interactome and pathway enrichment analyses were carried out to investigate the functional role of the identified potential prognostic genes in TNBC. These genes are associated with MAPK, PI3-AkT, Wnt, TGF-β, and other signal transduction pathways, pivotal in metastasis cascade. These gene signatures can provide novel molecular-level insights into metastasis.

Studies on Screening of MicroRNA Related to Irradiation Response and the Regulation Mechanism of miRNA-96-5p on Irradiation Resistance in Rectal Cancer Cells

<h3>Purpose/Objective(s)</h3> To screen microRNAs that are positively correlated with radiation resistance in rectal cancer, and to investigate their regulatory mechanism. <h3>Materials/Methods</h3> Fresh rectal cancer tissues were obtained intraoperatively and divided into two equal parts, one of which was pre-stored in a refrigerator at -150℃, and the other was constructed with Patients Derived Xenograft (PDX) models for rectal cancer and irradiated to transplantation tumor screen out radiation-sensitive tumor tissues and radiation-resistant tumor tissues. According to the screening results, the pre-stored samples were screened for differential microRNAs in radiation reactivity, and the microRNAs that were positively correlated with irradiation resistance were obtained. On the base of screening results, the pre-stored samples were screened for differential microRNAs in irradiation reactivity by miRNAs microarrays, and the microRNAs that were positively correlated with irradiation resistance were obtained. Biological prediction software was used to predict the target genes regulated by microRNA, and the prediction results were verified in radiation-resistant rectal cancer cell lines. Lentivirus plasmid transfection technique was used to construct rectal cancer cell lines with down-regulated target microRNA expression, in order to further verify the effects of the changes in the expression of the microRNA on the radiation reactivity, proliferation, invasion and migration of rectal cancer cells. The target genes regulated by microRNAs were screened and verified, and the expression of the key genes in the signal transduction pathways involved in the regulation were detected. <h3>Results</h3> The results of microRNA screening and validation showed that there were 14 microRNAs with fold change > 1.5. Among them, miRNA-552-3p, miRNA-96-5p, miRNA-182-5p, and miRNA-183-5p were up-regulated in irradiation-resistant tumor tissues. The enrichment signaling pathway analysis showed that the Wnt signal transduction pathway was highly active in irradiation-resistant tumor tissues. The clone formation experiment under radiation interference showed that HR-8348 was the most radiation-resistant cell line among the five cell lines, and the expression level of miRNA-96-5p was positively correlated with the radiation-resistant ability of rectal cancer cell lines. The results of prediction and validation showed that the expression of GPC3 gene was directly regulated by miRNA-96-5p, and the down-regulated expression of miRNA-96-5p inhibited the activity of Wnt signal transduction pathway. <h3>Conclusion</h3> The high expression of miRNA-96-5p enhanced the radiation resistance of rectal cancer cells, and its effect may be related to the down-regulation of GPC3 expression and the abnormal activation of Wnt signal transduction pathway.

Wnt ligands 3a and 5a regulate proliferation and migration in human fetal liver progenitor cells.

Since human fetal liver progenitor cells (hFLPC) can differentiate into multiple liver cell types in vitro and in vivo, hFLPC may be a suitable source for cell therapy and regeneration strategies. Imperative for effective clinical applications of hFLPC is the enhanced knowledge of growth factors that mediate and improve migration and proliferation. The canonical wingless/int-1 (Wnt) signal transduction pathway is known to play a key role in proliferation and migration of stem cells. So, we investigated a role for Wnt3a and Wnt5a ligands in regulating the proliferation and migration of hFLPC. We used alamarBlue assay and transwell migration assay and examined proliferation and migration of hFLPC to Wnt3a and Wnt5a. In addition, the target genes of Wnt signal transduction pathway was identified using microarray analysis and validated by quantitative real-time polymerase chain reaction (qPCR). We found that Wnt3a or Wnt5a independently significantly increased migration and proliferation in a dose-dependent manner which was significantly inhibited by Wnt inhibitors Wnt-C59 or KN-62. Addition of Wnt3a to hFLPC resulted in increased mRNA expression of the known Wnt target genes Axin-2, DKK2, while Wnt5a increased CXCR7, all of which are closely associated with an enhanced proliferation capacity of stem cells. Thus, we report that Wnt3a and Wnt5a may play an important role in the proliferation and migration of hFLPC by possibly regulating key target genes-involved in these processes. Incorporating recombinant human Wnt3a and Wnt5a in regenerative strategies using liver stem/progenitor cells might improve the process of liver regeneration.

A CRISPR knockout screen reveals new regulators of canonical Wnt signaling

The Wnt signaling pathways play fundamental roles during both development and adult homeostasis. Aberrant activation of the canonical Wnt signal transduction pathway is involved in many diseases including cancer, and is especially implicated in the development and progression of colorectal cancer. Although extensively studied, new genes, mechanisms and regulatory modulators involved in Wnt signaling activation or silencing are still being discovered. Here we applied a genome-scale CRISPR-Cas9 knockout (KO) screen based on Wnt signaling induced cell survival to reveal new inhibitors of the oncogenic, canonical Wnt pathway. We have identified several potential Wnt signaling inhibitors and have characterized the effects of the initiation factor DExH-box protein 29 (DHX29) on the Wnt cascade. We show that KO of DHX29 activates the Wnt pathway leading to upregulation of the Wnt target gene cyclin-D1, while overexpression of DHX29 inhibits the pathway. Together, our data indicate that DHX29 may function as a new canonical Wnt signaling tumor suppressor and demonstrates that this screening approach can be used as a strategy for rapid identification of novel Wnt signaling modulators.

Screening of MicroRNA Related to Irradiation Response and the Regulation Mechanism of miRNA-96-5p in Rectal Cancer Cells.

Neoadjuvant chemoradiotherapy has been widely used in the treatment of locally advanced rectal cancer due to the excellent advantages of irradiation in cancer therapy. Unfortunately, not every patient can benefit from this treatment, therefore, it is of great significance to explore biomarkers that can predict irradiation sensitivity. In this study, we screened microRNAs (miRNAs) which were positively correlated with irradiation resistance and found that miRNA-552 and miRNA-183 families were positively correlated with the irradiation resistance of rectal cancer, and found that high expression of miRNA-96-5p enhanced the irradiation resistance of rectal cancer cells through direct regulation of the GPC3 gene and abnormal activation of the canonical Wnt signal transduction pathway. Based on the radioreactivity results of patient-derived xenograft models, this is the first screening report for radio-resistant biomarkers in rectal cancer. Our results suggest that miRNA-96-5p expression is an important factor affecting the radiation response of colorectal cancer cells.

Abstract 533: Breast cancer IHC subtypes display heterogenous and independent targetable signaling pathway activity profiles

Abstract Background: Primary breast cancer is routinely subtyped using immunohistochemistry (IHC) staining and treatment choice is guided by the subtype. Targeted treatment requires the targeted signaling pathway to be active and tumor driving. IHC staining does not provide reliable information on active signaling pathways, and we reported before that clinical response of ER positive breast cancer patients to neoadjuvant aromatase inhibitor therapy appears to be more closely related to ER pathway activity rather than ER/PR IHC staining. To better understand the role of tumor driving signaling pathways within breast cancer subtypes and potentially improve precision medicine we evaluated signaling pathway activity profiles per IHC subtype, using novel assays to quantitatively measure activity of multiple main tumor driving signal transduction pathways, within tissue samples. Methods: mRNA-based estrogen (ER) and androgen (AR) receptor, MAPK, PI3K, Hedgehog (HH), TGFβ, Notch, Wnt signal transduction pathway assays have been previously described. Pathway activity scores (PAS) were measured on primary untreated breast cancer samples, and compared to normal breast epithelium, using Affymetrix expression dataset GSE65194 (Institut Marie Curie), excluding the duplicate measures, containing 29 Luminal A, 30 Luminal B, 30 HER2, 41 triple negative (TN) breast cancer and 6 normal breast tissue sample data. Pathway activity scores exceeding the 95th percentile of normal were considered highly active. Results: PAS thresholds for defining highly active pathway activity were for MAPK 23, AR 29, ER 34, PI3K 42, HH 34, Notch 65, TGFβ 48, Wnt 29. Luminal A was characterized by high ER pathway activity (n=25, 86%, mean PAS 45, SD 10); luminal B by high ER (n=10, 33%; mean PAS 32, SD 11) increased PI3K pathway activity (n=27, 90%; mean PAS 53, SD 10) and increased HH pathway activity (n=23, 77% mean PAS 29, SD 7); HER2 by high AR (n=18, 60%; mean PAS 30, SD 6) increased PI3K pathway activity (n=19, 63%; mean PAS 48, SD8) and increased HH pathway activity (n=27, 90% mean PAS 30 SD 7) ; TNBC by high PI3K (n=37, 90%; mean PAS 53, SD 7) plus high developmental pathway activity (Notch, n=6, 15%; mean PAS 53, SD 12; Wnt, n=28, 68%; mean PAS 31, SD 6). All subtypes had elevated MAPK pathway activity. Discussion: Each breast cancer subtype based on IHC, had in the overall population a typical pathway activity profile. However within each IHC subtype, a substantial range of pathway activities between patients is observed, suggesting that within same IHC subtypes, clear differences in signal pathway activation can be measured that may provide a novel approach to more effectively select patients for specific precision oncology targeted therapy treatments. Citation Format: Anthony M. Magliocco, Anja van de Stolpe, Martijn Akse, Eveline den Biezen, Laurent Holtzer. Breast cancer IHC subtypes display heterogenous and independent targetable signaling pathway activity profiles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 533.

WNT signaling in atrial fibrillation.

The Wnt signaling pathway regulates physiological processes such as cell proliferation and differentiation, cell fate decisions, and stem cell maintenance and, thus, plays essential roles in embryonic development, but also in adult tissue homeostasis and repair. The Wnt signaling pathway has been associated with heart development and repair and has been shown to be crucially involved in proliferation and differentiation of progenitor cells into cardiomyocytes. The investigation of the role of the Wnt signaling pathway and the regulation of its expression/activity in atrial fibrillation has only just begun. The present minireview (I) provides original data regarding the expression of Wnt signaling components in atrial tissue of patients with atrial fibrillation or sinus rhythm and (II) summarizes the current state of knowledge of the regulation of Wnt signaling components' expression/activity and the contribution of the various levels of the Wnt signal transduction pathway to the processes of the development, maintenance, and progression of atrial fibrillation.

Research progress of hair follicle and related stem cells in scar-free wound healing

To explore the research progress of hair follicle and related stem cells in scar-free skin healing in recent years. The literature related to hair follicle and related stem cells, wound healing, and scar formation in recent years was extensively reviewed and summarized from the aspects of cell function and molecular mechanism. Scar tissue after wound healing treated with hair follicle transplantation and related stem cell therapy is more mild or even without scar formation. The cell types and molecular mechanisms of the above phenomena are complex, and the bone morphogenetic protein signal transduction pathway and Wnt signal transduction pathway are strongly correlated. The research of hair follicle and related stem cells in scar-free skin healing is at the initial stage at present. Strengthening the mechanism research may provide new ideas for the treatment of wound and scar.