Β-catenin Activation Research Articles

Abstract 2546: Targeting of AKT/β-catenin signaling pathways driven hepatocellular carcinoma cell lines by combined treatment of HDAC and multi-kinase inhibitors

Abstract Hepatocellular carcinoma (HCC) is the dominant type of liver cancer exhibiting high mortality and poor prognosis with the fastest rate of death due to its aggressiveness and lack of effective therapies. Beta-catenin is a key player transcriptional factor, which orchestrates gene expression in HCC progression. Activation of β-catenin is promoted by its phosphorylation by AKT. Although the studies of metastatic behavior of β-catenin over-expressing HCC cells are not clear, the present study is focusing on the relationship between chromatin remodelling through deacetylation and phosphorylation of β-catenin by AKT. The current study investigated the role of a combination of Lenvatinib, a multi-kinase inhibitor, and Belinostat, a histone deacetylase inhibitor (HDACI), on two HCC cell lines, Hep G2 and Hep 3B, respectively. In-depth, our data showed that combined treatment significantly induced morphological changes in the tested cell lines with activation of caspase-3 and pre-apoptotic signals of Bcl-2, Bcl-XL, and Bax, indicating apoptotic induction after 48 hours of treatment compared to the control or each drug separately. The calculated IC50 was 1µmol/L for Belinostat and 25 n mole/L for Lenvatinib. Western blotting analysis reveals that the combined treatment for 48 hours inhibited the oncogenic β-catenin, AKT, and TGFβ expression in a dose-dependent manner.In addition, the combined treatment inhibited the localization of beta-catenin in the tested cell nuclei after 48 hours as confirmed by IHC staining. Statistical analysis proved that the combination of both drugs significantly decreased the proliferation and invasion of both cell lines (P<0.01, ANOVA) compared to each drug alone. In addition, the percentage of apoptotic cells was significantly increased and showed a cell cycle arrest in the G0/G1 phase of Hep-G2 cells after 48 hours of combined treatment as confirmed by flow cytometry. All in all, our data show that targeting AKT/β-catenin driven HCC progression can be targeted by Lenvatinib and Belinostat combined treatment. Citation Format: Mohamed Mahmoud Elberawi, Mohamed Abdulqadir, Ahmed Samir Sultan. Targeting of AKT/β-catenin signaling pathways driven hepatocellular carcinoma cell lines by combined treatment of HDAC and multi-kinase inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2546.

Abstract 2566: Blocking the functional domain of TIP1 upregulates midkine via the β-catenin/Wnt signaling pathway

Abstract Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related mortalities world-wide. Targeted therapy has been a growing topic of investigation to improve therapeutic efficacy for NSCLC. A few targeted therapies that exploit aberrant protein expression profiles have been approved for NSCLC. However, marginal improvement in efficacy observed with these therapeutic approaches highlights the need for discovery of additional therapeutic targets. Tax interacting protein 1 (TIP1) is a molecular target that is overexpressed on the surface of various cancers and correlates with poor overall patient survival. The functional domain of TIP1 (PDZ domain) caps the C-terminus of many cellular proteins that regulate cell signaling, cancer development, and progression. We found that antibodies against the PDZ domain of TIP1 induced cytotoxicity of lung cancer cells but not normal cells. A quantitative mass spectrometry-based approach identified a ten-fold increase in Midkine protein expression on blockade of TIP1. Midkine is a heparin-binding growth factor that is involved in cancer progression, therapy resistance, and immune suppression. This study tested the hypothesis that Midkine is upregulated by the anti-PDZ/TIP1 antibody via the β-catenin/Wnt signaling pathway, which subsequently modulates downstream signaling mechanisms. In co-immunoprecipitation studies we found a direct interaction of TIP1 with β-catenin on the cell surface. Anti-TIP1 antibody treatment led to nuclear translocation of β-catenin in NSCLC cells. We used a luciferase based TOPFlash/FOPFlash reporter assay to study the transcriptional activity of the canonical β-catenin/Wnt pathway following TIP1 blockade. Reporter assays showed a three to four-fold increase (p<0.0001) in the transcriptional activity of β-catenin following TIP1 blockade in NSCLC cells. Genetic silencing of β-catenin inhibited Midkine mRNA transcription (p<0.005), protein induction and its extracellular release (p<0.0001) following anti-TIP1 antibody treatment. In conclusion, the mechanism of Midkine release following TIP1 blockade involves the β-catenin/Wnt signaling pathway. Ongoing studies are evaluating the dual-targeting of TIP1 and Midkine for enhanced efficacy of NSCLC treatment. Citation Format: Minakshi Saikia, Abhay K. Singh, Nathan Cheung, Dennis E. Hallahan, Vaishali Kapoor. Blocking the functional domain of TIP1 upregulates midkine via the β-catenin/Wnt signaling pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2566.

Abstract 1081: Co-targeting CK1epsilon and PIK3CB/p110beta to treat glioblastoma

Abstract The purpose of this study is to determine whether co-inhibition of CK1epsilon and PIK3CB/p110β is an effective therapeutic treatment for Glioblastoma (GBM). GBM is the most common and malignant primary brain cancer in adults. GBM has a dismal prognosis of death within 6 months if left untreated, thus there is a clinically unmet need for new and effective treatments. CK1epsilon has been identified as a GBM survival kinase gene. CK1epsilon promotes growth of GBM cells by inhibiting β-catenin. However, blocking CK1epsilon alone fails to completely block tumor growth perhaps because β-catenin can be regulated by other kinases. PIK3CB/p110β is another GBM survival kinase gene which also acts upstream of β-catenin. As seen with CK1epsilon, blocking PIK3CB/p110β alone also fails to completely block tumor growth. It is possible that blocking either CK1epsilon or PIK3CB/p110β alone does not substantially activate β-catenin to induce massive GBM cell death, thus limiting their therapeutic efficacy as a single agent. It is therefore imperative to investigate if co-targeting CK1epsilon and PIK3CB/p110β yields a synergistic inhibitory effect on GBM cell growth. GBM cells were treated with either DMSO (control), IC261 (chemical inhibitor of CK1epsilon), AZD6482 (chemical inhibitor of PIK3CB/p110β), or both IC261 and AZD6482. Cell viability was measured using the MTS viability assay. Cell viability for the treatment groups were as follows: AZD6482 (88.81%), IC261 (66.78%), and combination of AZD6482+IC261 (42.59%). Statistical analysis of drug synergy using the Bliss independence model yielded an Excess Over Bliss score greater than 0%, indicating a synergistic effect. To further elucidate the mechanism of GBM cell death, immunoblotting will also be performed to detect β-catenin activity after inhibition of CK1epsilon and PIK3CB/p110β. Overall, preliminary results show that co-inhibition of CK1epsilon and PIK3CB/p110β with chemical inhibitors yields synergistic GBM cell death. Citation Format: Eileen Xu. Co-targeting CK1epsilon and PIK3CB/p110beta to treat glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1081.

Abstract 521: Discovery of novel TNKS inhibitors through structure-based virtual screening

Abstract More than 80% of colorectal cancers (CRCs) have somatic mutations in the adenomatous polyposis coli (APC) gene, which affects abnormal WNT/β-catenin signaling. Since inhibition of tankyrases (TNKS1 and TNKS2) belonging to the poly(ADP-ribose) polymerase family stabilizes AXINs and induces degradation of β-catenin, drug development as a therapeutic target for APC-mutated CRCs is required. To identify selective small molecule inhibitors of TNKS, we performed pharmacophore modeling and structural docking studies targeting nicotinamide, adenosine, and both binding subsites of TNKS. Among the 8 million compounds, 149 of the nicotinamide site binders, 23 of the adenosine site binders, and 15 of the dual-site binders of TNKS were selected through computer-based virtual screening. Using in vitro TNKS1 and TNKS2 enzyme assays, we identified that TI2-N17 and TI3-D11 significantly inhibited TNKS1 and TNKS2 enzyme activities at low nanomolar levels. TI2-N17 and TI3-D11 stabilized AXIN2, reduced active β-catenin, and downregulated β-catenin target genes in COLO320DM, APC-mutated CRC cells. Both compounds exhibited anticancer effects on COLO320DM and SW403 cells. In addition, the combination treatment of 5-Fluorouracil (5-FU), an anticancer agent for CRC patients, and these compounds synergistically inhibited the proliferation of CRC cells compared to single compound treatment. Our findings demonstrated that TI2-N17 and TI3-D11 are therapeutic candidates targeting TNKS for APC-mutated CRCs treatment and suggested that the combined treatment with the compounds and 5-FU chemotherapy may be an effective therapeutic strategy for APC-mutated CRCs. Based on these compounds, further study will develop potent and selective TNKS inhibitors by designing and synthesizing novel derivative compounds. Citation Format: Hwani Ryu, Hye-Ran Seo, Hyo Jeong Kim, Jiyeon Ahn. Discovery of novel TNKS inhibitors through structure-based virtual screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 521.

MP20-01 PROTEIN KINASE D1 (PrKD) PROTECTS AGAINST DNA DAMAGE BY INCREASING Α-CATENIN LEVELS THROUGH PUTATIVE SERINE681 AND 685 PHOSPHORYLATION OF CYCLIN DEPENDENT KINASE 12 (CDK12) IN PROSTATE CANCER (PC)

You have accessJournal of UrologyCME1 Apr 2023MP20-01 PROTEIN KINASE D1 (PrKD) PROTECTS AGAINST DNA DAMAGE BY INCREASING Α-CATENIN LEVELS THROUGH PUTATIVE SERINE681 AND 685 PHOSPHORYLATION OF CYCLIN DEPENDENT KINASE 12 (CDK12) IN PROSTATE CANCER (PC) Sanjeev Shukla, Teruko Osumi, Mohammed Al-Toubat, Carlos Riveros, Allison H. Feibus, Ahmed El-shafie, Adam M. Kase, John A. Copland, Joachim L. Petit, Nathalie Meurice, Justyna Gleba, Jonathan Chardon-Robles, and K. C. Balaji Sanjeev ShuklaSanjeev Shukla More articles by this author , Teruko OsumiTeruko Osumi More articles by this author , Mohammed Al-ToubatMohammed Al-Toubat More articles by this author , Carlos RiverosCarlos Riveros More articles by this author , Allison H. FeibusAllison H. Feibus More articles by this author , Ahmed El-shafieAhmed El-shafie More articles by this author , Adam M. KaseAdam M. Kase More articles by this author , John A. CoplandJohn A. Copland More articles by this author , Joachim L. PetitJoachim L. Petit More articles by this author , Nathalie MeuriceNathalie Meurice More articles by this author , Justyna GlebaJustyna Gleba More articles by this author , Jonathan Chardon-RoblesJonathan Chardon-Robles More articles by this author , and K. C. BalajiK. C. Balaji More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003245.01AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: PrKD is protective of DNA damage in PC and loss of PrKD leading to increased genomic instability is a novel potential mechanism. CDK12 mutated PC is now recognized as a distinct aggressive variant associated with genomic instability. We explored whether PrKD influences DNA damage through CDK12. METHODS: The DNA damage and protein levels studied by COMET assay and western blot respectively and validated the findings in human PC samples by querying cBioPortal gene expression. RESULTS: PrKD abrogated etoposide induced DNA damage in castration insensitive C4-2 PC cells. PrKD is a membrane bound protein that phosphorylates proteins in cell adherens junction including β-catenin. As PrKD is the only known kinase to phosphorylate T120 in β-catenin, mutation of T120 leads to nuclear translocation of transcriptionally active β-catenin playing a role in DNA damage, uncouples the binding of β- to α-catenin, which translocates to the nucleus and influences β-catenin. PrKD increased the expression of α-catenin, which was attenuated by CDK12 wild type and double non-phosphorylated mutants of PrKD putative phosphorylation sites; serine (s) 681 and 685 but not as much by s681 mutant. PrKD suppressed the substrate phosphorylation of γH2AX s139 by CDK12 s681 and s681/s685 non-phosphorylated mutant compared to wild type, which links PrKD substrate phosphorylation of CDK12 to DNA damage. Query of cBioPortal for concurrent PrKD1 and CDK12 mutations in PC with characteristic gene signature of DNA damage repair (DDR) revealed the highest concordance of PrKD1 and CDK12 mutations in patients with BRAC2 mutations and the DDR genes expression were not significantly different between patients with PrKD1 and CDK12 mutated patient except BRAC1 expression (rarely mutated in PC) suggesting that PrKD1 and CDK12 could be involved in the same signaling pathway (Figure 1). CONCLUSIONS: The study provides a validated mechanistic basis for the novel protective role of PrKD in DNA damage, which has potential implications in PC therapeutics linked to DNA damage. Source of Funding: none © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e275 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Sanjeev Shukla More articles by this author Teruko Osumi More articles by this author Mohammed Al-Toubat More articles by this author Carlos Riveros More articles by this author Allison H. Feibus More articles by this author Ahmed El-shafie More articles by this author Adam M. Kase More articles by this author John A. Copland More articles by this author Joachim L. Petit More articles by this author Nathalie Meurice More articles by this author Justyna Gleba More articles by this author Jonathan Chardon-Robles More articles by this author K. C. Balaji More articles by this author Expand All Advertisement PDF downloadLoading ...

Formation and function of the meningeal arachnoid barrier around the developing mouse brain.

The arachnoid barrier, a component of the blood-cerebrospinal fluid barrier (B-CSFB) in the meninges, is composed of epithelial-like, tight-junction-expressing cells. Unlike other central nervous system (CNS) barriers, its' developmental mechanisms and timing are largely unknown. Here, we show that mouse arachnoid barrier cell specification requires the repression of Wnt-β-catenin signaling and that constitutively active β-catenin can prevent its formation. We also show that the arachnoid barrier is functional prenatally and, in its absence, a small molecular weight tracer and the bacterium group B Streptococcus can cross into the CNS following peripheral injection. Acquisition of barrier properties prenatally coincides with the junctional localization of Claudin 11, and increased E-cadherin and maturation continues after birth, where postnatal expansion is marked by proliferation and re-organization of junctional domains. This work identifies fundamental mechanisms that drive arachnoid barrier formation, highlights arachnoid barrier fetal functions, and provides novel tools for future studies on CNS barrier development.

Muramyl dipeptide alleviates estrogen deficiency-induced osteoporosis through canonical Wnt signaling.

Wnt signaling is a positive regulator of bone formation through the induction of osteoblast differentiation and down-regulation of osteoclast differentiation. We previously reported that muramyl dipeptide (MDP) increases bone volume by increasing osteoblast activity and attenuating osteoclast activity in receptor activator of nuclear factor-κBligand(RANKL)-induced osteoporotic model mice. In this study, we investigated whether MDP could alleviate post-menopausal osteoporosis through Wnt signaling regulation inan ovariectomy (OVX)-induced mouse osteoporosis model. MDP-administered OVX mice exhibited higher bone volume and bone mineral density than mice of the control group. MDP significantly increased P1NP in the serum of OVX mice, implying increased bone formation. The expression of pGSK3β and β-catenin in the distal femur of OVX mice was lower than that in the distal femur of sham-operated mice. Yet, the expression of pGSK3β and β-catenin was increased in MDP-administered OVX mice compared with OVX mice. In addition, MDP increased the expression and transcriptional activity of β-catenin in osteoblasts. MDP inhibited the proteasomal degradation of β-catenin via the down-regulation of its ubiquitination by GSK3β inactivation. When osteoblasts were pretreated with Wnt signaling inhibitors, DKK1 or IWP-2, the induction of pAKT, pGSK3β, and β-catenin was not observed. In addition, nucleotide oligomerization domain-containing protein 2-deficient osteoblasts were not sensitive to MDP. MDP-administered OVX mice exhibited fewer tartrate-resistant acid phosphatase (TRAP)-positive cells than did OVX mice, attributed to a decrease in the RANKL/OPG ratio. In conclusion, MDP alleviates estrogen deficiency-induced osteoporosis through canonical Wnt signaling and could be an effective therapeutic for the treatment of post-menopausal bone loss. © 2023 The Pathological Society of Great Britain and Ireland.

Osteocalcin modulates parathyroid cell function in human parathyroid tumors.

The bone matrix protein osteocalcin (OC), secreted by osteoblasts, displays endocrine effects. We tested the hypothesis that OC modulates parathyroid tumor cell function. Primary cell cultures derived from parathyroid adenomas (PAds) and HEK293 cells transiently transfected with the putative OC receptor GPRC6A or the calcium sensing receptor (CASR) were used as experimental models to investigate γ-carboxylated OC (GlaOC) or uncarboxylated OC (GluOC) modulation of intracellular signaling. In primary cell cultures derived from PAds, incubation with GlaOC or GluOC modulated intracellular signaling, inhibiting pERK/ERK and increasing active β-catenin levels. GlaOC increased the expression of PTH, CCND1 and CASR, and reduced CDKN1B/p27 and TP73. GluOC stimulated transcription of PTH, and inhibited MEN1 expression. Moreover, GlaOC and GluOC reduced staurosporin-induced caspase 3/7 activity. The putative OC receptor GPRC6A was detected in normal and tumor parathyroids at membrane or cytoplasmic level in cells scattered throughout the parenchyma. In PAds, the membrane expression levels of GPRC6A and its closest homolog CASR positively correlated; GPRC6A protein levels positively correlated with circulating ionized and total calcium, and PTH levels of the patients harboring the analyzed PAds. Using HEK293A transiently transfected with either GPRC6A or CASR, and PAds-derived cells silenced for CASR, we showed that GlaOC and GluOC modulated pERK/ERK and active β-catenin mainly through CASR activation. Parathyroid gland emerges as a novel target of the bone secreted hormone osteocalcin, which may modulate tumor parathyroid CASR sensitivity and parathyroid cell apoptosis.

The WNT/β-catenin system in chronic kidney disease-mineral bone disorder syndrome.

The WNT/β-catenin system is an evolutionarily conserved signaling pathway that plays a crucial role in morphogenesis and cell tissue formation during embryogenesis. Although usually suppressed in adulthood, it can be reactivated during organ damage and regeneration. Transient activation of the WNT/β-catenin pathway stimulates tissue regeneration after acute kidney injury, while persistent (uncontrolled) activation can promote the development of chronic kidney disease (CKD). CKD-MBD is a clinical syndrome that develops with systemic mineral and bone metabolism disorders caused by CKD, characterized by abnormal bone mineral metabolism and/or extraosseous calcification, as well as cardiovascular disease associated with CKD, including vascular stiffness and calcification. This paper aims to comprehensively review the WNT/β-catenin signaling pathway in relation to CKD-MBD, focusing on its components, regulatory molecules, and regulatory mechanisms. Additionally, this review highlights the challenges and opportunities for using small molecular compounds to target the WNT/β-catenin signaling pathway in CKD-MBD therapy. We conducted a comprehensive literature review using various scientific databases, including PubMed, Scopus, and Web of Science, to identify relevant articles. We searched for articles that discussed the WNT/β-catenin signaling pathway, CKD-MBD, and their relationship. We also reviewed articles that discussed the components of the WNT/β-catenin signaling pathway, its regulatory molecules, and regulatory mechanisms. The WNT/β-catenin signaling pathway plays a crucial role in CKD-MBD by promoting vascular calcification and bone mineral metabolism disorders. The pathway's components include WNT ligands, Frizzled receptors, and LRP5/6 co-receptors, which initiate downstream signaling cascades leading to the activation of β-catenin. Several regulatory molecules, including GSK-3β, APC, and Axin, modulate β-catenin activation. The WNT/β-catenin signaling pathway also interacts with other signaling pathways, such as the BMP pathway, to regulate CKD-MBD. The WNT/β-catenin signaling pathway is a potential therapeutic target for CKD-MBD. Small molecular compounds that target the components or regulatory molecules of the pathway may provide a promising approach to treat CKD-MBD. However, more research is needed to identify safe and effective compounds and to determine the optimal dosages and treatment regimens.

Basic Fibroblast Growth Factor Promotes Mesenchymal Stem Cell Migration by Regulating Glycolysis-Dependent β-Catenin Signaling.

Migration of mesenchymal stem cells (MSCs) to the site of injury is crucial in transplantation therapy. Studies have shown that cell migration is regulated by the cellular microenvironment and accompanied by changes in cellular metabolism. However, limited information is available about the relationship between MSC migration and cellular metabolism. Here, we show that basic fibroblast growth factor (bFGF) promotes the migration of MSCs with high levels of glycolysis and high expression of hexokinase 2 (HK2), a rate-limiting enzyme in glycolysis. The enhancement of glycolysis via the activation of HK2 expression promoted the migration of MSCs, whereas the inhibition of glycolysis, but not of oxidative phosphorylation, inhibited the bFGF-induced migration of these cells. Furthermore, bFGF enhanced glycolysis by increasing HK2 expression, which consequently promoted β-catenin accumulation, and the inhibition of glycolysis inhibited the bFGF-induced accumulation of β-catenin. When the accumulation of glycolytic intermediates was altered, phosphoenolpyruvate was found to be directly involved in the regulation of β-catenin expression and activation, suggesting that bFGF regulates β-catenin signaling through glycolytic intermediates. Moreover, transplantation with HK2-overexpressing MSCs significantly improved the effect of cell therapy on skull injury in rats. In conclusion, we propose a novel glycolysis-dependent β-catenin signaling regulatory mechanism and provide an experimental and theoretical basis for the clinical application of MSCs.

Disturbed flow increases endothelial inflammation and permeability via a Frizzled-4-β-catenin-dependent pathway.

Multidirectional or disturbed flow promotes endothelial dysfunction and is associated with early atherogenesis. Here we investigated the role of Wnt signalling in flow-mediated endothelial dysfunction. The expression of Frizzled-4 was higher in cultured human aortic endothelial cells (ECs) exposed to disturbed flow compared to that seen for undisturbed flow, obtained using an orbital shaker. Increased expression was also detected in regions of the porcine aortic arch exposed to disturbed flow. The increased Frizzled-4 expression in cultured ECs was abrogated following knockdown of R-spondin-3. Disturbed flow also increased the nuclear localisation and activation of β-catenin, an effect that was dependent on Frizzled-4 and R-spondin-3. Inhibition of β-catenin using the small-molecule inhibitor iCRT5 or knockdown of Frizzled-4 or R-spondin-3 resulted in reduced expression of pro-inflammatory genes in ECs exposed to disturbed flow, as did inhibition of WNT5A signalling. Inhibition of the canonical Wnt pathway had no effect. Inhibition of β-catenin also reduced endothelial paracellular permeability; this was associated with altered junctional and focal adhesion organisation and cytoskeletal remodelling. These data suggest the presence of an atypical Frizzled-4-β-catenin pathway that promotes endothelial dysfunction in response to disturbed flow.

Interdependence of SS18-SSX-driven YAP1 and β-Catenin Activation in Synovial Sarcoma.

This study provides deeper insights into synovial sarcoma tumor biology demonstrating a mutual dependence between YAP1/TAZ and β-catenin transcriptional activity and a complex interplay with the SS18-SSX fusion protein within the BAF complex.

Topographic Cues of a PLGA Scaffold Promote Odontogenic Differentiation of Dental Pulp Stem Cells through the YAP/β-Catenin Signaling Axis.

The underlying mechanism of how topographic cues of artificial scaffolds regulate cell function remains poorly understood. Yes-associated protein (YAP) and β-catenin signaling have both been reported to play important roles in mechano-transduction and dental pulp stem cells (DPSCs) differentiation. We investigated the effects of YAP and β-catenin in spontaneous odontogenic differentiation of DPSCs induced by topographic cues of a poly(lactic-co-glycolic acid) (PLGA) membrane. The topographic cues and function of a fabricated PLGA scaffold were explored via scanning electron microscopy (SEM), alizarin red staining (ARS), reverse transcription-polymerase chain reaction (RT-PCR), and pulp capping. Immunohistochemistry (IF), RT-PCR, and western blotting (WB) were used to observe the activation of YAP and β-catenin when DPSCs were cultured on the scaffolds. Further, YAP was inhibited or overexpressed on either side of the PLGA membrane, and YAP, β-catenin, and odontogenic marker expression were analyzed using IF, ARS, and WB. The closed side of the PLGA scaffold promoted spontaneous odontogenic differentiation and nuclear translocation of YAP and β-catenin in vitro and in vivo compared to the open side. The YAP antagonist verteporfin inhibited β-catenin expression, nuclear translocation, and odontogenic differentiation on the closed side, but the effects were rescued by LiCl. YAP overexpressing DPSCs on the open side activated β-catenin signaling and promoted odontogenic differentiation. The topographic cue of our PLGA scaffold promotes odontogenic differentiation of DPSCs and pulp tissue through the YAP/β-catenin signaling axis.

Activation of β-catenin in mesenchymal progenitors leads to muscle mass loss.

Loss of muscle mass is a common manifestation of chronic disease. We find the canonical Wnt pathway to be activated in mesenchymal progenitors (MPs) from cancer-induced cachectic mouse muscle. Next, we induce β-catenin transcriptional activity in murine MPs. As a result, we observe expansion of MPs in the absence of tissue damage, as well as rapid loss of muscle mass. Because MPs are present throughout the organism, we use spatially restricted CRE activation and show that the induction of tissue-resident MP activation is sufficient to induce muscle atrophy. We further identify increased expression of stromal NOGGIN and ACTIVIN-A as key drivers of atrophic processes in myofibers, and we verify their expression by MPs in cachectic muscle. Finally, we show that blocking ACTIVIN-A rescues the mass loss phenotype triggered by β-catenin activation in MPs, confirming its key functional role and strengthening the rationale for targeting this pathway in chronic disease.

Cyclic Tensile Stress Induces Skeletal Muscle Hypertrophy and Myonuclear Accretion in a 3D Model.

Skeletal muscle is highly adaptive to mechanical stress due to its resident stem cells and the pronounced level of myotube plasticity. Herein, we study the adaptation to mechanical stress and its underlying molecular mechanisms in a tissue-engineered skeletal muscle model. We subjected differentiated 3D skeletal muscle-like constructs to cyclic tensile stress using a custom-made bioreactor system, which resulted in immediate activation of stress-related signal transducers (Erk1/2, p38). Cell cycle re-entry, increased proliferation, and onset of myogenesis indicated subsequent myoblast activation. Furthermore, elevated focal adhesion kinase and β-catenin activity in mechanically stressed constructs suggested increased cell adhesion and migration. After 3 days of mechanical stress, gene expression of the fusogenic markers MyoMaker and MyoMixer, myotube diameter, myonuclear accretion, as well as S6 activation, were significantly increased. Our results highlight that we established a promising tool to study sustained adaptation to mechanical stress in healthy, hypertrophic, or regenerating skeletal muscle. Impact statement Sustained adaption to mechanical stress presents a key feature for skeletal muscle functionality and growth. Knowledge of these processes, however, is mostly based on in vivo or 2D cell culture models, both of which entail significant shortcomings. Herein, we generated highly hypertrophic tissue-engineered skeletal muscle-like constructs that are comparable to the results of successful in vivo models of adaption to mechanical stimuli, achieving an outcome that only few in vitro approaches have reached. Second, we aimed at studying the underlying molecular mechanisms, which is of interest since there is little knowledge of the intracellular events during hypertrophy upon mechanical stimulation.

Microtubule-associated protein 4 promotes epithelial mesenchymal transition in hepatocellular cancer cells via regulating GSK3β/β-catenin pathway

Metastasis is a major obstacle in the treatment of hepatocellular carcinoma (HCC). Microtubule-associated protein 4 (MAP4) plays an important role as a coordinator between microtubules and microfilaments. However, the role of MAP4 in HCC migration and epithelial mesenchymal transition (EMT) is unclear. We compared the protein and mRNA levels of MAP4 in human HCC and adjacent normal tissues using western blotting, immunohistochemistry and RT-qPCR. The migration and invasion abilities and the levels of EMT markers (E-Cadherin, N-Cadherin, Vimentin, and Snail) were compared between MAP4-knockdown and MAP4-overexpressed HCC cells. Finally, we examined whether β-catenin and glycogen synthase kinase 3β (GSK3β) are involved in the stimulatory effects of MAP4 on HCC migration, invasion and EMT. The results revealed that MAP4 levels were higher in the HCC tissues than in the normal hepatic tissues. More importantly, MAP4 knockdown suppressed migration and invasion abilities and EMT processes in HCC cells, which were confirmed by the stimulatory effects of MAP4 overexpression on EMT processes in HCC cells. Further evidence demonstrated that the up-regulation of β-catenin activity induced by the interaction between MAP4 and GSK3β possibly accounted for the pro-migration and pro-EMT effects of MAP4 on HCC cells. Taken together, these results suggest that MAP4 promotes migration, invasion, and EMT in HCC cells by regulating the GSK3β/β-catenin pathway.

Abstract PD5-06: PD5-06 CCN6 suppresses spindle metaplastic breast carcinoma in part via antagonizing Wnt/β-catenin signaling

Abstract Background: Metaplastic breast carcinomas (mBrCAs) are a rare and highly aggressive subtype of triple negative breast cancer, with histological evidence of non-glandular differentiation and frequent activation of the canonical (-catenin-dependent) Wnt pathway. Our laboratory has reported that CCN6 is expressed in normal mammary epithelium, but CCN6 expression is lost in 68% of spindle mBrCAs. We found mice with mammary epithelial cell-specific conditional deletion of Ccn6 (MMTV-Cre; Ccn6fl/fl mice) develop mammary tumors that recapitulate human spindle mBrCAs, including upregulation of Wnt pathway genes. We investigated if and how secreted CCN6 protein functions in tumor suppression in spindle mBrCA via effects on the canonical Wnt pathway. Methods: To investigate CCN6 binding to the Wnt co-receptors LRP6 and FZD8 proteins, we performed Flag-IPs on MDA-MB231 mesenchymal-like breast cancer cells expressing Flag-CCN6 or vector. Effects of CCN6 on b-catenin subcellular localization and gene and protein expression were studied by IHC, IF, qRT-PCR and immunoblot in human cell lines and MMTV-Cre;Ccn6fl/fl tumors. To test effects of recombinant CCN6 on canonical Wnt signaling, we used the Leading-Light Wnt Reporter Assay and also tested CCN6 effects in WNT3A- and WNT10B-mediated Wnt signaling activation and on MDA-MB231 cell invasion. To study b-catenin/TCF function in invasive growth of CCN6-deficient cancer cells, we employed two independent approaches: i) expression of a dominant-negative Tcf4 (dnTcf4) versus control vector in MMTV-Cre; Ccn6fl/fl tumor-derived cells; and ii) expression of a constitutively active mutant (S33Y) b-catenin in concert with treatment with recombinant human CCN6 (rhCCN6; 500 ug/ml) versus BSA control. Syngeneic orthotopic mammary tumor transplants of MMTV-Cre;Ccn6fl/fl were used in vivo for rescue experiments with i.p. injections of rhCCN6 or BSA. We monitored tumor growth and morphology, and performed IHC to determine b-catenin localization and expression. Results: We found in co-IPs that CCN6 interacts with LRP6 and FZD8 to form a complex that antagonizes canonical Wnt signaling. CCN6 ectopic expression in MDA-MB231 cells led to reduced nuclear and increased membrane localization of b-catenin and decreased invasive growth in vitro. In vivo, CCN6 protein administration to MMTV-Cre; Ccn6fl/fl mice reduced tumor growth and was linked to decreased nuclear b-catenin in the tumors. Conclusion: CCN6 antagonizes canonical Wnt/b-catenin in part by binding Wnt ligands, leading to reduced active b-catenin in the cytoplasm and nucleus. Our data indicate a critical role for b-catenin activation for CCN6-deficient mBrCA tumor phenotypes. In vivo, rhCCN6 protein reduces tumorigenesis in MMTV-Cre; Ccn6fl/fl mBrCA tumors, highlighting how CCN6 restoration or b-catenin inhibition could be new therapeutic approaches for mBrCAs. Citation Format: Maria E. Gonzalez, Eric R. Fearon, Celina Kleer. PD5-06 CCN6 suppresses spindle metaplastic breast carcinoma in part via antagonizing Wnt/β-catenin signaling [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD5-06.

LncRNA MALAT1 promotes growth and metastasis of head and neck squamous cell carcinoma by repressing VHL through a non-canonical function of EZH2

Long non-coding RNAs (LncRNAs) are implicated in malignant progression of human cancers. Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a well-known lncRNA, has been reported to play crucial roles in multiple malignancies including head and neck squamous cell carcinoma (HNSCC). However, the underlying mechanisms of MALAT1 in HNSCC progression remain to be further investigated. Here, we elucidated that compared with normal squamous epithelium, MALAT1 was notably upregulated in HNSCC tissues, especially in which was poorly differentiated or with lymph nodes metastasis. Moreover, elevated MALAT1 predicted unfavorable prognosis of HNSCC patients. The results of in vitro and in vivo assays showed that targeting MALAT1 could significantly weaken the capacities of proliferation and metastasis in HNSCC. Mechanistically, MALAT1 inhibited von Hippel–Lindau tumor suppressor (VHL) by activating EZH2/STAT3/Akt axis, then promoted the stabilization and activation of β-catenin and NF-κB which could play crucial roles in HNSCC growth and metastasis. In conclusion, our findings reveal a novel mechanism for malignant progression of HNSCC and suggest that MALAT1 might be a promising therapeutic target for HNSCC treatment.

Rare Genetic Variants in Human APC Are Implicated in Mesiodens and Isolated Supernumerary Teeth

The activation of Wnt/β-catenin signalling is a prerequisite for odontogenesis. APC, a member of the AXIN-CK1-GSK3β-APC β-catenin destruction complex, functions to modulate Wnt/β-catenin signalling to establish regular teeth number and positions. APC loss-of-function mutations are associated with the over-activation of WNT/β-catenin signalling and subsequent familial adenomatous polyposis (FAP; MIM 175100) with or without multiple supernumerary teeth. The ablation of Apc function in mice also results in the constitutive activation of β-catenin in embryonic mouse epithelium and causes supernumerary tooth formation. The objective of this study was to investigate if genetic variants in the APC gene were associated with supernumerary tooth phenotypes. We clinically, radiographically, and molecularly investigated 120 Thai patients with mesiodentes or isolated supernumerary teeth. Whole exome and Sanger sequencing identified three extremely rare heterozygous variants (c.3374T>C, p.Val1125Ala; c.6127A>G, p.Ile2043Val; and c.8383G>A, p.Ala2795Thr) in APC in four patients with mesiodentes or a supernumerary premolar. An additional patient with mesiodens was compound as heterozygous for two APC variants (c.2740T>G, p.Cys914Gly, and c.5722A>T, p.Asn1908Tyr). Rare variants in APC in our patients are likely to contribute to isolated supernumerary dental phenotypes including isolated mesiodens and an isolated supernumerary tooth.

YTHDF1 Enhances Chondrogenic Differentiation by Activating the Wnt/β-Catenin Signaling Pathway.

Cartilage is derived from the chondrogenic differentiation of stem cells, for which the regulatory mechanism has not been fully elucidated. N6-methyladenosine (m6A) messenger RNA (mRNA) methylation is the most common posttranscriptional modification in eukaryotic mRNAs and is mediated by m6A regulators. However, whether m6A regulators play roles in chondrogenic differentiation is unknown. Herein, we aim to determine the role of a main m6A reader protein, YTH N6-methyladenosine RNA binding protein 1 (YTHDF1), in chondrogenic differentiation regulation. Western blotting (WB) assays found that the expression of YTHDF1 increased during chondrogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs). The results of quantitative polymerase chain reaction, WB, immunohistochemistry, and Alcian blue staining revealed that overexpression of YTHDF1 increased cartilage matrix synthesis and the expression of chondrogenic markers when hBMSCs, ATDC5 cells, or C3H10T1/2 cells were induced to undergo chondrogenesis. Conversely, chondrogenesis was clearly inhibited when YTHDF1 was knocked down in hBMSCs, ATDC5 cells, or C3H10T1/2 cells. Further RNA sequencing and molecular biology experiments found that YTHDF1 activated the Wnt/β-catenin signaling pathway during chondrogenic differentiation. Finally, the effects of overexpression and knockdown of YTHDF1 on chondrogenic differentiation were reversed by inhibiting or activating β-catenin activity. Therefore, we demonstrated that YTDHF1 promoted chondrogenic differentiation through activation of the Wnt/β-catenin signaling pathway.