Expression Of Active Β-catenin Research Articles

Specificity protein 1/3 regulate T-cell acute lymphoblastic leukemia cell proliferation and apoptosis through β-catenin by acting as targets of miR-495-3p.

T-cell acute lymphoblastic leukemia (T-ALL) is a hematologic heterogeneous disease. This study explored the mechanism of specificity protein 1/3 (Sp1/3) in T-ALL cells through β-catenin by acting as targets of miR-495-3p. Expression levels of miR-495-3p, Sp1, Sp3, and β-catenin in the serum from T-ALL children patients, healthy controls, and the T-ALL cell lines were measured. The cell proliferation ability and apoptosis rate were detected. Levels of proliferation-related proteins proliferating cell nuclear antigen (PCNA)/cyclinD1 and apoptosis-related proteins B-cell lymphoma-2 associated X protein (Bax)/B-cell lymphoma-2 (Bcl-2) were determined. The binding of Sp1/3 and β-catenin promoter and the targeted relationship between miR-495-3p with Sp1/3 were analyzed. Sp1/3 were upregulated in CD4+ T-cells in T-ALL and were linked with leukocyte count and risk classification. Sp1/3 interference prevented proliferation and promoted apoptosis in T-ALL cells. Sp1/3 transcription factors activated β-catenin expression. Sp1/3 enhanced T-ALL cell proliferation by facilitating β-catenin expression. miR-495-3p targeted and repressed Sp1/3 expressions. miR-495-3p overexpression inhibited T-ALL cell proliferation and promoted apoptosis. Conjointly, Sp1/3, as targets of miR-495-3p limit apoptosis and promote proliferation in T-ALL cells by promoting β-catenin expression.

ATP6AP2, a regulator of LRP6/β-catenin protein trafficking, promotes Wnt/β-catenin signaling and bone formation in a cell type dependent manner

Wnt/β-catenin signaling is critical for various cellular processes in multiple cell types, including osteoblast (OB) differentiation and function. Exactly how Wnt/β-catenin signaling is regulated in OBs remain elusive. ATP6AP2, an accessory subunit of V-ATPase, plays important roles in multiple cell types/organs and multiple signaling pathways. However, little is known whether and how ATP6AP2 in OBs regulates Wnt/β-catenin signaling and bone formation. Here we provide evidence for ATP6AP2 in the OB-lineage cells to promote OB-mediated bone formation and bone homeostasis selectively in the trabecular bone regions. Conditionally knocking out (CKO) ATP6AP2 in the OB-lineage cells (Atp6ap2Ocn-Cre) reduced trabecular, but not cortical, bone formation and bone mass. Proteomic and cellular biochemical studies revealed that LRP6 and N-cadherin were reduced in ATP6AP2-KO BMSCs and OBs, but not osteocytes. Additional in vitro and in vivo studies revealed impaired β-catenin signaling in ATP6AP2-KO BMSCs and OBs, but not osteocytes, under both basal and Wnt stimulated conditions, although LRP5 was decreased in ATP6AP2-KO osteocytes, but not BMSCs. Further cell biological studies uncovered that osteoblastic ATP6AP2 is not required for Wnt3a suppression of β-catenin phosphorylation, but necessary for LRP6/β-catenin and N-cadherin/β-catenin protein complex distribution at the cell membrane, thus preventing their degradation. Expression of active β-catenin diminished the OB differentiation deficit in ATP6AP2-KO BMSCs. Taken together, these results support the view for ATP6AP2 as a critical regulator of both LRP6 and N-cadherin protein trafficking and stability, and thus regulating β-catenin levels, demonstrating an un-recognized function of osteoblastic ATP6AP2 in promoting Wnt/LRP6/β-catenin signaling and trabecular bone formation.

Cbfβ regulates Wnt/β-catenin, Hippo/Yap, and Tgfβ signaling pathways in articular cartilage homeostasis and protects from ACLT surgery-induced osteoarthritis.

As the most common degenerative joint disease, osteoarthritis (OA) contributes significantly to pain and disability during aging. Several genes of interest involved in articular cartilage damage in OA have been identified. However, the direct causes of OA are poorly understood. Evaluating the public human RNA-seq dataset showed that CBFB (subunit of a heterodimeric Cbfβ/Runx1, Runx2, or Runx3 complex) expression is decreased in the cartilage of patients with OA. Here, we found that the chondrocyte-specific deletion of Cbfb in tamoxifen-induced Cbfbf/f;Col2a1-CreERT mice caused a spontaneous OA phenotype, worn articular cartilage, increased inflammation, and osteophytes. RNA-sequencing analysis showed that Cbfβ deficiency in articular cartilage resulted in reduced cartilage regeneration, increased canonical Wnt signaling and inflammatory response, and decreased Hippo/Yap signaling and Tgfβ signaling. Immunostaining and western blot validated these RNA-seq analysis results. ACLT surgery-induced OA decreased Cbfβ and Yap expression and increased active β-catenin expression in articular cartilage, while local AAV-mediated Cbfb overexpression promoted Yap expression and diminished active β-catenin expression in OA lesions. Remarkably, AAV-mediated Cbfb overexpression in knee joints of mice with OA showed the significant protective effect of Cbfβ on articular cartilage in the ACLT OA mouse model. Overall, this study, using loss-of-function and gain-of-function approaches, uncovered that low expression of Cbfβ may be the cause of OA. Moreover, Local admission of Cbfb may rescue and protect OA through decreasing Wnt/β-catenin signaling, and increasing Hippo/Yap signaling and Tgfβ/Smad2/3 signaling in OA articular cartilage, indicating that local Cbfb overexpression could be an effective strategy for treatment of OA.

CXC chemokine receptor 7 ameliorates renal fibrosis by inhibiting β-catenin signaling and epithelial-to-mesenchymal transition in tubular epithelial cells

Renal fibrosis is a common feature of various chronic kidney diseases. However, the underlying mechanism remains poorly understood. The CXC chemokine receptor (CXCR) family plays a role in renal fibrosis; however, the detailed mechanisms have not been elucidated. In this study, we investigated the potential role of CXCR7 in mediating renal fibrosis. CXCR7 expression is decreased in unilateral ischemia–reperfusion injury (UIRI) and unilateral ureteral obstruction mouse models. Furthermore, CXCR7 was specifically expressed primarily in the Lotus Tetragonolobus Lectin-expressing segment of tubules, was slightly expressed in the peanut agglutinin-expressing segment, and was barely expressed in the Dolichos biflorus agglutinin-expressing segment. Administration of pFlag-CXCR7, an overexpression plasmid for CXCR7, significantly inhibited the activation of β-catenin signaling and protected against the progression of epithelial-to-mesenchymal transition (EMT) and renal fibrosis in a UIRI mouse model. Using cultured HKC-8 cells, we found that CXCR7 significantly downregulated the expression of active β-catenin and fibrosis-related markers, including fibronectin, Collagen I, and α-SMA. Furthermore, CXCR7 significantly attenuated TGF-β1-induced changes in β-catenin signaling, EMT and fibrosis. These results suggest that CXCR7 plays a crucial role in inhibiting the activation of β-catenin signaling and the progression of EMT and renal fibrosis. Thus, CXCR7 could be a novel therapeutic target for renal fibrosis.

Cebpb Regulates Skeletal Stem Cell Osteogenic Differentiation and Fracture Healing via the WNT/β-Catenin Pathway.

Fracture is the most common traumatic organ injury, and fracture nonunion is a critical clinical challenge. The research on the mechanisms of skeletal stem cell (SSC) differentiation and fracture healing may help develop new treatment strategies and improve the prognosis of patients at high risk of nonunion. Bioinformatic analysis of scRNA-seq data of mouse SSCs and mouse osteoprogenitors was applied to discover major transcription factors for the regulation of SSC differentiation. FACS was used to isolate SSCs prospectively. The expression of Cebpb, osteogenesis-related genes (Runx2, Sp7, and Bglap2), and markers for Notch, Hedgehog, MAPK, BMP2/SMAD, and WNT/β-catenin signaling pathways (Hes1, Gli1, p-Erk1/2, p-Smad1/5/9, and β-catenin) were detected in SSCs with qPCR or western blot, respectively. Alkaline phosphatase assay and alizarin red S staining were used to illustrate the osteogenic differentiation ability of SSCs in vitro. A WNT inhibitor, IWR-1, was further used to explore the mechanism of WNT signaling in the differentiation of SSCs. Micro-CT, mechanical testing, and immunohistochemistry of osteogenic and chondrogenic proteins (Sp7 and Col2α1) were used to demonstrate the capacity of Cebpb knockdown in promoting fracture healing in a monocortical defect model. We found that Cebpb was the crucial transcription factor regulating SSC differentiation. Inhibiting Cebpb in SSCs enhanced the expression of active β-catenin to promote the expression of WNT target genes, thus facilitating the osteogenic differentiation of SSCs. Bone mass, mechanical properties, and osteogenic protein expression were also increased in the Cebpb inhibition group compared to the group without Cebpb inhibition. Collectively, our results proved that Cebpb knockdown promotes SSC osteogenic differentiation and fracture healing via the WNT/β-catenin signaling pathway.

Zinc finger protein 501 maintains glioblastoma cell growth through enhancing Frizzled-6 expression

Understanding the regulatory mechanisms of glioblastoma growth is crucial for developing novel therapies. In this study, the expression and function of zinc finger protein 501 (ZNF501) in human glioblastoma cells were characterized. ZNF501 was abundantly expressed in human glioblastoma cell lines U251, U118, U87, and U138. ZNF501 ablation in these cell lines caused inhibition of proliferation, sphere formation, and the expression of SOX2 and OCT4. Besides, ZNF501 ablation increased the sensitivity of these cell lines to Temozolomide but did not influence cell migration. Orthotopic implantation of U251 cells and U118 cells indicated that ZNF501 ablation suppressed glioblastoma cell proliferation and tumor formation in vivo. ZNF501 ablation induced down-regulation of Frizzled-6 (FZD6), a component of the Wnt signaling. Lentivirus-mediated FZD6 overexpression restored the proliferation, sphere formation, drug sensitivity, and SOX2 and OCT4 expression in these cell lines. ZNF501 ablation also incurred down-regulation of JNK phosphorylation which is an indicator of the non-canonical Wnt signaling, but did not change the expression of active β-catenin which is the hallmark of the canonical Wnt signaling. Inhibition of the non-canonical Wnt signaling abrogated the effects of ZNF501 and FZD6. Therefore, for the first time, we showed that ZNF501 is essential for the growth and stemness of glioblastoma cells possibly by maintaining FZD6 expression and the non-canonical Wnt signaling.

GPC1-Targeted Immunotoxins Inhibit Pancreatic Tumor Growth in Mice via Depletion of Short-lived GPC1 and Downregulation of Wnt Signaling.

Glypican-1 (GPC1) is a cell surface proteoglycan that is upregulated in multiple types of human cancers including pancreatic cancer. Here, we investigated whether GPC1 could be a target of antibody-toxin fusion proteins (i.e., immunotoxins) for treating pancreatic cancer. We constructed a panel of GPC1-targeted immunotoxins derived from a functional domain of Pseudomonas exotoxin A. An albumin-binding domain was also introduced into the anti-GPC1 immunotoxin to improve serum half-life. Small-molecule screening was performed to identify irinotecan that shows synergistic efficacy with the immunotoxin. We showed that GPC1 was internalized upon antibody binding. Anti-GPC1 immunotoxins alone inhibited tumor growth in a pancreatic cancer xenograft model. The immunotoxin treatment reduced active β-catenin expression in tumor cells. Furthermore, immunotoxins containing an albumin-binding domain in combination with irinotecan caused pancreatic tumor regression. GPC1 expression was reduced by the immunotoxin treatment due to the degradation of the internalized GPC1 and its short cellular turnover rate. Our data indicate that the GPC1-targeted immunotoxin inhibits pancreatic tumor growth via degradation of internalized GPC1, downregulation of Wnt signaling, and inhibition of protein synthesis. The anti-GPC1 immunotoxin in combination with irinotecan thus provides a potential new treatment strategy for patients with pancreatic tumors.

The role of Wnt/β-catenin pathway for skin-derived precursors differentiating into corneal endothelial cell-like cells

Bullous keratopathy is a serious blinding eye disease requiring corneal endothelial transplantation. However, the lack of cornea donors forced us to search for new sources of functional corneal endothelial cells (CECs). In our previous study, we have successfully differentiated the SKPs into CEC-like cells with unclear mechanism. The Wnt/β-catenin signaling pathway is essential for maintaining embryonic eye development and formation. This study aimed to clarify the activity of Wnt/β-catenin pathway in the process of skin-derived precursors (SKPs) differentiating into CEC-like cells. We showed that the expression of active β-Catenin, p-GSK3β, P-LRP6 and LRP6 upregulated, indicating the activation of Wnt/β-catenin pathway during CEC-like cells induction. What’s more, when the pathway was inhibited with a specific inhibitor, the process of induction was obviously suppressed. These findings indicates that Wnt/β-catenin pathway plays an important role in the CEC-like cells induction from SKPs. Our study lays an experimental foundation for providing abundant corneal endothelial cells and promotes CEC-like cells to be clinically applied in cellular replacement therapy or regenerative medicine in the future.

WNT5A Interacts With FZD5 and LRP5 to Regulate Proliferation and Self-Renewal of Endometrial Mesenchymal Stem-Like Cells.

Endometrial mesenchymal stem-like cells (eMSC) reside in the basal layer of the endometrium and are responsible for cyclic regeneration during the reproductive lives of women. Myometrial cells act as a component of the niche and regulate the stem cell fate through the activation of WNT/β-catenin signaling via WNT5A. Since WNT5A-responsive mechanisms on eMSC are still uncertain, we hypothesize that the WNT ligand–WNT5A works to activate WNT/β-catenin signaling through binding to Frizzled receptors (FZDs) and co-receptor low-density lipoprotein receptor-related protein 5 (LRP5). Among the various receptors that have been reported to interact with WNT5A, we found FZD5 abundantly expressed by eMSC when compared to unfractionated stromal cells. Neutralizing the protein expression by using anti-FZD5 antibody suppressed the stimulatory effects on phenotypic expression and the clonogenicity of eMSC in a myometrial cell–eMSC co-culture system as well as in an L-Wnt5a conditioned medium. Gene silencing of FZD5 not only reduced the binding of WNT5A to eMSC but also decreased the TCF/LEF transcriptional activities and expression of active β-catenin. Inhibition of LRP coreceptors with recombinant Dickkopf-1 protein significantly reduced the binding affinity of eMSC to WNT5A as well as the proliferation and self-renewal activity. During postpartum remodeling in mouse endometrium, active β-catenin (ABC) was detected in label-retaining stromal cells (LRSCs), and these ABC+ LRSCs express FZD5 and LRP5, suggesting the activation of WNT/β-catenin signaling. In conclusion, our findings demonstrate the interaction of WNT5A, FZD5, and LRP5 in regulating the proliferation and self-renewal of eMSC through WNT/β-catenin signaling.

Laminin 411 mediates endothelial specification via multiple signaling axes that converge on β-catenin.

SummaryThe extracellular matrix (ECM) provides essential cues to promote endothelial specification during tissue development in vivo; correspondingly, ECM is considered essential for endothelial differentiation outside of the body. However, systematic studies to assess the precise contribution of individual ECM proteins to endothelial differentiation have not been conducted. Further, the multi-component nature of differentiation protocols makes it challenging to study the underlying mechanisms by which the ECM contributes to cell fate. In this study, we determined that Laminin 411 alone increases endothelial differentiation of induced pluripotent stem cells over collagen I or Matrigel. The effect of ECM was shown to be independent of vascular endothelial growth factor (VEGF) binding capacity. We also show that ECM-guided endothelial differentiation is dependent on activation of focal adhesion kinase (FAK), integrin-linked kinase (ILK), Notch, and β-catenin pathways. Our results indicate that ECM contributes to endothelial differentiation through multiple avenues, which converge at the expression of active β-catenin.

Decoding the Role of DVL1 in Intracranial Meningioma.

In the search for molecular candidates for targeted meningioma therapies, increasing attention has been paid to the role of signaling pathways in the development and progression of intracranial meningiomas. Although it is well known that the Wnt signaling pathway is involved in meningioma progression, the role of its central mediator, DVL1, is still unclear. In order to investigate the influence of DVL1 gene alterations on the progression of human intracranial meningioma, we focused on its central PDZ domain, which is responsible for DVL interaction with the Fzd receptor and the phosphorylation of DVL mediated through the casein kinases CK1 and CK2. A genetic analysis of genomic instability revealed the existence of microsatellite instability in 9.09% and the loss of heterozygosity in 6.06% of the samples. The sequencing of the PDZ gene region showed repetitive deletions of two bases located in intron 7 and exon 8, and a duplication in intron 8 in most samples, with different outcomes on the biological function of the DVL1 protein. Immunohistochemistry revealed that the nuclear expression of DVL1 was significantly correlated with a higher expression of active β-catenin (p = 0.029) and a higher meningioma grade (p = 0.030), which leads to the conclusion that it could be used as biomarker for meningioma progression and the activation of the Wnt signaling pathway.

Differences in Fracture Healing Between Female and Male C57BL/6J Mice.

BackgroundMice are increasingly used in fracture healing research because of the opportunity to use transgenic animals. While both, male and female mice are employed, there is no consensus in the literature whether fracture healing differs between both sexes. Therefore, the aim of the present study was to analyze diaphyseal fracture healing in female and male C57BL/6J mice, a commonly used mouse strain in bone research.MethodsFor that purpose, 12-week-old Female (17–20 g) and Male mice (22–26 g) received a standardized femur midshaft osteotomy stabilized by an external fixator. Mice were euthanized 10 and 21 days after fracture and bone healing was analyzed by biomechanical testing, μCT, histology, immunohistochemistry and qPCR.ResultsTen days after fracture, Male mice displayed significantly more cartilage but less fibrous tissue in the fracture callus compared to Female mice, whereas the amount of bone did not differ. At day 21, Male mice showed a significantly larger fracture callus compared to Female mice. The relative amount of bone in the fracture callus did not significantly differ between both sexes, whereas its tissue mineral density was significantly higher in Male mice on day 21, indicating more mature bone and slightly more rapid fracture healing. These results were confirmed by a significantly greater absolute bending stiffness of the fractured femurs of Male mice on day 21. On the molecular level, Male mice displayed increased active β-catenin expression in the fracture callus, whereas estrogen receptor α (ERα) expression was lower.ConclusionThese results suggest that Male mice display more rapid fracture healing with more prominent cartilaginous callus formation. This might be due to the higher weight of Male mice, resulting in increased mechanical loading of the fracture. Furthermore, Male mice displayed significantly greater activation of osteoanabolic Wnt/β-catenin signaling, which might also contribute to more rapid bone regeneration.

KLK11 acts as a tumor-inhibitor in laryngeal squamous cell carcinoma through the inactivation of Akt/Wnt/β-catenin signaling.

Kallikrein-associated peptidase 11 (KLK11) has emerged as a key tumor-associated protein that is implicated in a wide spectrum of tumor types. However, the detailed involvement of KLK11 in laryngeal squamous cell carcinoma (LSCC) has not been well studied. The aims of our work were to evaluate whether KLK11 plays a role in LSCC. We found that both the mRNA and protein expression of KLK11 were significantly lower in LSCC tissues than in normal tissues. Low expression of KLK11 was also observed in LSCC cell lines, and the up-regulation of KLK11 caused a significant inhibitory effect on the proliferation, colony formation and invasion of LSCC cells. On the contrary, the knockdown of KLK11 markedly accelerated the proliferative and invasive abilities of LSCC cells. Molecular mechanism research revealed that KLK11 overexpression decreased the phosphorylation of glycogen synthase kinase-3β (GSK-3β) and down-regulated the expression of active β-catenin, leading to the inactivation of Wnt/β-catenin signaling in LSCC cells. Furthermore, GSK-3β inhibition markedly abrogated the KLK11-mediated suppressive effect on Wnt/β-catenin signaling. Notably, the reactivation of Wnt/β-catenin partially reversed KLK11-mediated tumor-inhibition effect in LSCC. In addition, the xenograft tumor assay demonstrated that the up-regulation of KLK11 retarded tumor formation and the growth of LSCC cells in vivo. Taken together, the findings of our work demonstrate that KLK11 exerts a tumor-inhibition role in LSCC by down-regulating Wnt/β-catenin signaling. Our work highlights a pivotal role of KLK11 in LSCC progression and suggests it as an attractive anticancer target for LSCC treatment.

Spindle and kinetochore‑associated complex subunit 3 accelerates breast cancer cell proliferation and invasion through the regulation of Akt/Wnt/β-catenin signaling.

Spindle and kinetochore‑associated complex subunit 3 (SKA3) has recently been identified as a novel regulator of carcinogenesis in multiple types of cancers. However, the function and potential regulatory mechanisms of SKA3 in breast cancer remain poorly understood. The present study was designed to gain a detailed relevance of SKA3 in breast cancer. Expression of SKA3 in breast cancer was examined via real-time quantitative PCR, western blotting and immunohistochemistry analysis. Malignant behaviors of breast cancer cells were investigated via cell counting kit-8, cell apoptosis, and transwell invasion assays. The activity of Wnt/β-catenin signaling was monitored via luciferase reporter assay. The tumorigenicity of breast cancer cells in vivo was assessed via xenograft tumor assay. SKA3 expression was elevated in breast cancer tissue and was correlated with shorter survival rates in breast cancer patients. Knockdown of SKA3 caused marked reductions in cellular proliferation and invasion in breast cancer cells, whereas SKA3 overexpression accelerated proliferation and invasion. Knockdown of SKA3 resulted in decreased Akt and glycogen synthase kinase-3β phosphorylation, and decreased expression of active β-catenin, which lead to the inactivation of Wnt/β-catenin signaling. Inhibition of Akt significantly reversed the SKA3 overexpression-induced activation of Wnt/β-catenin signaling. Inhibition of Wnt/β-catenin signaling markedly abrogated SKA3 overexpression-induced tumor-promotion effects, while re-activation of Wnt/β-catenin signaling significantly reversed SKA3 knockdown-mediated tumor-inhibition effects. Knockdown of SKA3 resulted in a significant decrease in breast cancer tumor formation in vivo. SKA3 accelerates proliferation and invasion in breast cancer through the modulation of Akt/Wnt/β-catenin signaling.

Heat-stable enterotoxin inhibits intestinal stem cell expansion to disrupt the intestinal integrity by downregulating the Wnt/β-catenin pathway.

Enterotoxigenic Escherichia coli causes severe infectious diarrhea with high morbidity and mortality in newborn and weanling pigs mainly through the production of heat-stable enterotoxins (STs). However, the precise regulatory mechanisms involved in ST-induced intestinal epithelium injury remain unclear. Consequently, we conducted the experiments in vivo (mice), ex vivo (mouse and porcine enteroids), and in vitro (MODE-K and IPEC-J2 cells) to explore the effect of STp (one type of STa) on the integrity of the intestinal epithelium. The results showed that acute STp exposure led to small intestinal edema, disrupted intestinal integrity, induced crypt cell expansion into spheroids, and downregulated Wnt/β-catenin activity in the mice. Following a similar trend, the enteroid-budding efficiency and the expression of Active β-catenin, β-catenin, Lgr5, PCNA, and KRT20 were significantly decreased after STp treatment, as determined ex vivo. In addition, STp inhibited cell proliferation, induced cell apoptosis, destroyed cell barriers, and reduced Wnt/β-catenin activity by downregulating its membrane receptor Frizzled7 (FZD7). In contrast, Wnt/β-catenin reactivation protected the IPEC-J2 cells from STp-induced injury. Taking these findings together, we conclude that STp inhibits intestinal stem cell expansion to disrupt the integrity of the intestinal mucosa through the downregulation of the Wnt/β-catenin signaling pathway.

Antiproliferative Effects of Monoclonal Antibodies against (Pro)Renin Receptor in Pancreatic Ductal Adenocarcinoma

We previously reported that silencing of the PRR gene, which encodes the (pro)renin receptor [(P)RR], significantly reduced Wnt/β-catenin-dependent development of pancreatic ductal adenocarcinoma (PDAC). Here, we examined the effects of a panel of blocking mAbs directed against the (P)RR extracellular domain on proliferation of the human PDAC cell lines PK-1 and PANC-1 in vitro and in vivo We observed that four rat anti-(P)RR mAbs induced accumulation of cells in the G0-G1-phase of the cell cycle and significantly reduced proliferation in vitro concomitant with an attenuation of Wnt/β-catenin signaling. Systemic administration of the anti-(P)RR mAbs to nude mice bearing subcutaneous PK-1 xenografts significantly decreased tumor expression of active β-catenin and the proliferation marker Ki-67, and reduced tumor growth. In contrast, treatment with the handle region peptide of (pro)renin did not inhibit tumor growth in vitro or in vivo, indicating that the effects of the anti-(P)RR mAbs were independent of the renin-angiotensin system. These data indicate that mAbs against human (P)RR can suppress PDAC cell proliferation by hindering activation of the Wnt/β-catenin signaling pathway. Thus, mAb-mediated (P)RR blockade could be an attractive therapeutic strategy for PDAC.

The mTOR/ULK1 signaling pathway mediates the autophagy-promoting and osteogenic effects of dicalcium silicate nanoparticles

A novel bioactive inorganic material containing silicon, calcium and oxygen, calcium silicate (Ca2SiO4, C2S) with a CaO-SiO2 ingredient, has been identified as a potential candidate for artificial bone. Autophagy has an essential function in adult tissue homoeostasis and tumorigenesis. However, little is known about whether silicate nanoparticles (C2S NPs) promote osteoblastic differentiation by inducing autophagy. Here we investigated the effects of C2S NPs on bone marrow mesenchymal stem cell differentiation (BMSCs) in osteoblasts. Furthermore, we identified the osteogenic gene and protein expression in BMSCs treated with C2S NPs. We found that autophagy is important for the ability of C2S NPs to induce osteoblastic differentiation of BMSCs. Our results showed that treatment with C2S NPs upregulated the expression of BMP2, UNX2, and OSX in BMSCs, and significantly promoted the expression of LC3 and Beclin, while P62 (an autophagy substrate) was downregulated. C2S NP treatment could also enhance Alizarin red S dye (ARS), although alkaline phosphatase (ALP) activity was not significantly changed. However, all these effects could be partially reversed by 3-MA. We then detected potential signaling pathways involved in this biological effect and found that C2S NPs could activate autophagy by suppressing mTOR and facilitating ULK1 expression. Autophagy further activated β-catenin expression and promoted osteogenic differentiation. In conclusion, C2S NPs promote bone formation and osteogenic differentiation in BMSCs by activating autophagy. They achieve this effect by activating mTOR/ULK1, inducing autophagy, and subsequently triggering the WNT/β-catenin pathway to boost the differentiation and biomineralization of osteoblasts.

Wnt/β-Catenin and Hippo Pathway Deregulation in Mammary Tumors of Humans, Dogs, and Cats.

Mammary cancer is a common neoplasm in women, dogs, and cats that still represents a therapeutic challenge. Wnt/β-catenin and Hippo pathways are involved in tumor progression, cell differentiation, and metastasis. The aim of this study was to evaluate mRNA and protein expression of molecules involved in these pathways in human (HBC), canine (CMT), and feline mammary tumors (FMT). Real-time quantitative polymerase chain reaction (qPCR) for β-catenin, CCND1, YAP, TAZ, CTGF, and ANKRD1, western blotting for YAP, TAZ, and β-catenin, and immunohistochemistry for estrogen receptor (ER), progesterone receptor (PR), ERBB2, β-catenin, and YAP/TAZ were performed on mammary tumor tissues. The protein expression of active β-catenin was higher in tumors than in healthy tissues in all 3 species. The mRNA expression of the downstream gene CCND1 was increased in HBC ER+ and CMTs compared to healthy tissues. Membranous and cytoplasmic protein expression of β-catenin were strongly negatively correlated in all 3 species. Tumors showed an increased protein expression of YAP/TAZ when compared to healthy tissues. Notably, YAP/TAZ expression was higher in triple negative breast cancers when compared to HBC ER+ and in FMTs when compared to CMTs. The mRNA expression of β-catenin, YAP, TAZ, CTGF, and ANKRD1 was not different between tumors and healthy mammary gland in the 3 species. This study demonstrates deregulation of Wnt/β-catenin and Hippo pathways in mammary tumors, which was more evident at the protein rather than the mRNA level. Wnt/β-catenin and Hippo pathways seem to be involved in mammary carcinogenesis and therefore represent interesting therapeutic targets that should be further investigated.

Transmembrane protein 88 exerts a tumor-inhibitory role in thyroid cancer through restriction of Wnt/β-catenin signaling

Transmembrane protein 88 (TMEM88) has emerged as a newly discovered cancer-related protein that acts as a cancer-promoting or cancer-inhibiting regulator in multiple tumor types. However, the exact role of TMEM88 in thyroid cancer is undetermined. The current study was designed to determine the expression, function, and potential underlying mechanism of TMEM88 in thyroid cancer. Our data demonstrated low TMEM88 expression in thyroid cancer tissues. Decreased TMEM88 expression was also found in several thyroid cancer cell lines, and restoration of TMEM88 markedly suppressed the proliferation, colony formation, and invasive potential of thyroid cancer cells. On the contrary, TMEM88 depletion significantly accelerated the proliferation, colony formation, and invasion of thyroid cancer cells. Further experiments documented that TMEM88 overexpression markedly decreased the expression of the active form of β-catenin and inhibited the expression of Wnt/β-catenin signaling targets genes, such as c-myc and cyclin D1. Notably, reactivation of Wnt/β-catenin signaling by transfecting a vector that expressed constitutively active β-catenin partially reversed the TMEM88-mediated suppressive effect on thyroid cancer cell proliferation and invasion. In addition, TMEM88 upregulation markedly retarded the tumor growth of thyroid cancer cells in vivo using xenograft models associated with downregulation of active β-catenin expression. Taken together, our findings demonstrated that TMEM88 overexpression impeded the proliferation and invasion of thyroid cancer cells through downregulation of Wnt/β-catenin signaling. These data indicate a potential tumor-suppressive function of TMEM88 in thyroid cancer. Our study highlights a key role for TMEM88 in regulating Wnt/β-catenin signaling during the progression of thyroid cancer and suggests that TMEM88 is an attractive anticancer target for thyroid cancer.

Secreted frizzled-related protein 2 prevents pressure-overload-induced cardiac hypertrophy by targeting the Wnt/β-catenin pathway.

Background and aimSecreted frizzled-related protein 2 (sFRP2) has been reported to be involved in cardiovascular diseases. However, its role in cardiac hypertrophy induced by pressure overload is still elusive. We aimed to examine the role of sFRP2 in the development of cardiac hypertrophy in vivo and in vitro.Methods and resultsFollowing cardiac hypertrophy stimulated by aortic banding (AB), the expression of sFRP2 was downregulated in the hypertrophic ventricle. Adeno-associated virus 9 (AAV9) was injected through the tail vein to overexpress sFRP2 in the mouse myocardium. Overexpression of sFRP2 alleviated cardiomyocyte hypertrophy and interstitial fibrosis, as identified by the reduced cardiomyocyte cross-sectional area, heart weight/body weight ratio, and left ventricular (LV) collagen ratio. Additionally, sFRP2 decreased cardiomyocyte apoptosis induced by pressure overload. Western blot showed that sFRP2 prevented the expression of active β-catenin. The Wnt/β-catenin agonist LiCl (1 mmol/kg) abolished the inhibitory effects of sFRP2 on cardiac hypertrophy and apoptosis, as evidenced by the increased cross-sectional area and LV collagen ratio and the deterioration of echocardiographic data.ConclusionOur study indicated that decreased sFRP2 levels were observed in failing mouse hearts. Overexpression of sFRP2 attenuated myocyte hypertrophy and interstitial fibrosis induced by hypertrophic stimuli by inhibiting the Wnt/β-catenin pathway. We revealed that sFRP2 may be a promising therapeutic target for the development of cardiac remodeling.Electronic supplementary materialThe online version of this article (10.1007/s11010-020-03802-x) contains supplementary material, which is available to authorized users.