Localization Of Β-catenin Research Articles

Comparative stemness and differentiation of luminal and basal breast cancer stem cell type under glutamine-deprivation.

Glutamine (gln) metabolism has emerged as a cancer therapeutic target in past few years, however, the effect of gln-deprivation of bCSCs remains elusive in breast cancer. In this study, effect of glutamine on stemness and differentiation potential of bCSCs isolated from MCF-7 and MDAMB-231 were studied. We have shown that bCSCs differentiate into CD24+ epithelial population under gln-deprivation and demonstrated increased expression of epithelial markers such as e-cadherin, claudin-1 and decreased expression of mesenchymal protein n-cadherin. MCF-7-bCSCs showed a decrease in EpCAMhigh population whereas MDAMB-231-bCSCs increased CD44high population in response to gln-deprivation. The expression of intracellular stem cell markers such sox-2, oct-4 and nanog showed a drastic decrease in gene expression under gln-deprived MDAMB-231-bCSCs. Finally, localization of β-catenin in MCF-7 and MDAMB-231 cells showed its accumulation in cytosol or perinuclear space reducing its efficiency to transcribe downstream genes. Conclusively, our study demonstrated that gln-deprivation induces differentiation of bCSCs into epithelial subtypes and also reduces stemness of bCSCs mediated by reduced nuclear localization of β-catenin. It also suggests that basal and luminal bCSCs respond differentially towards changes in extracellular and intracellular gln. This study could significantly affect the gln targeting regimen of breast cancer therapeutics.

Mesenchymal stromal cells can repair radiation-induced pulmonary fibrosis via a DKK-1-mediated Wnt/β-catenin pathway.

Pulmonary injury occurring after thoracic radiotherapy is a main factor limiting the curative effect of radiotherapy. Robust activation of the Wnt signalling pathway induced by ionizing radiation stress plays a critical role in epithelial-mesenchymal transition (EMT) in irradiated type II alveolar epithelial cells and in the proliferation of pulmonary fibroblasts, which contributes to the formation of fibrotic lesions in irradiated lungs. The pathogenesis of radiation-induced pulmonary fibrosis could be restricted by systemic delivery of human adipose-derived mesenchymal stromal cells (Ad-MSCs), as evidenced by the inhibitory effects of Ad-MSCs on EMT in irradiated type II alveolar epithelial cells. The purpose of this study is to observe the effects of mesenchymal stromal cells (MSCs) on repairing fibrosis caused by radiation. We used western blotting and real-time PCR to observe the expression of DKK-1 in MSCs of different origins and passages. After the successful establishment of a radiation-induced lung injury model, we investigated the potency of the supernatant from stromal cells to reduce pro-fibrotic events, including EMT and fibroblast activation. To study the mechanism, we evaluated the levels of active β-catenin, TCF4 and the target genes Snail, Twistand c-Myc. After the injection of Ad-MSCs into mice via the tail vein, proteins related to EMT, fibroblasts and Wnt/β-catenin signalling were investigated. The TGF-β and IL-10 protein concentrations in peripheral blood were measured by ELISA. Ad-MSC-derived supernatant effectively reversed the decrease in E-cadherin expression and inhibited the increase in vimentin expression induced by ionizing radiation in epithelial cells and suppressed the expression of α-SMA, a mediator of fibroblast proliferation. The canonical Wnt pathway may be activated by irradiation but the nuclear localization of active β-catenin was reduced in the presence of the supernatant from Ad-MSCs. In addition, the expression of target genes involved in EMT was downregulated. Additionally, when DKK-1 in the supernatant was neutralized, all these effects were reversed. Changes in the levels of proteins related to EMT and fibroblast activation, as well as those of active β-catenin and TCF4, were similar in vivo and in vitro. The serum level of the immunosuppressive factor IL-10 was increased after radiation and was further enhanced after Ad-MSC interference for one month. In conclusion, Ad-MSCs medium can contain DKK-1 and inhibit the induction of EMT via Wnt/β-catenin signalling in vitro and in vivo.

Identification of Phytochemical-Based β-Catenin Nuclear Localization Inhibitor in NSCLC: Differential Targeting Population from Member of Isothiocyanates.

Decades of research has convinced us that phytochemical compounds contained within the plant products are the real deal, and they provide benefits such as health maintenance an d cure to illnesses. One of the deadliest noncommunicable diseases today is lung cancer, hence its disease management still deserves attention. Wnt/β-catenin pathway activation conferring cancer stem cell (CSC) activities to non-small cell lung carcinomas (NSCLCs) may explain why the disease is still difficult to cure. In the present study, we assessed several representatives of phytochemical categories consisting of alkaloids, chalcones and isothiocyanates for their inhibitory activity to nuclear localization of β-catenin—an important event for Wnt/β-catenin pathway activation, in lung cancer cell lines. Real-time cell analyzer confirmed that evodiamine (EVO), chelidonine (CHE), isoliquiritigenin (ISO), licochalcone-A (LICO), benzyl isothiocyanate (BI) and phenethylisothiocyanate (PI) exhibited anti-proliferative activities and cytotoxicities to adenocarcinoma cell line SK-LU-1 and human lung CSC primary cell line (HLCSC). Immunofluorescence assay identified that CHE, ISO, LICO, BI and PI were capable of reducing the number of cells harboring β-catenin within the nuclei of these cells. We extended the characterizations of BI and PI in Wnt-dependent squamous cell carcinoma cell line NCI-H1703 on several CSC functions and found that BI was better at inhibiting soft agar colony formation as an output of self-renewal ability, whereas PI was more effective in inhibiting the growth of multicellular tumor spheroid model mimicking micrometastases. Both however were not able to inhibit migration and invasion of NCI-H1703. In conclusion, BI could potentially be used as a safer alternative to target undifferentiated CSCs as adjuvant therapy, whereas PI could be used as chemotherapy to remove bulk tumor.

Immunohistochemical evaluation of potential molecular targets of desmoid-type fibromatosis

Desmoid-type fibromatosis is locally aggressive tumor rare in general population, although commonly present in patients with familial adenomatous polyposis, significantly contributing to the morbidity and mortality of patients. To optimize and individualize the management of patients it is necessary to better understand the biology of these tumors. Immunohistochemical analysis of β-catenin, VEGF, hormone receptors ERβ, ERα and PR, COX-2, APC protein, EGFR, c-kit (CD117), bcl-2 and HER2 expression, potential therapeutic targets, was carried out on 15 archival biopsy samples together with APC gene mutational screening. β-catenin expression was found in all samples, with over 73% showing high range positivity, however with no prognostic significance. Non-specific cytoplasmic localization of β-catenin was observed FAP-associated cases lacking CTNNB1 mutations. Hormone receptor status demonstrated expression of ERβ in 93% of lesions, without detectable ERα or PR. Distinct COX-2 expression of variable intensity was present in all but one desmoid-type fibromatosis case. All lesions demonstrated intense VEGF positivity. Immunoreactivity for the APC protein was found only in 4 cases associated with FAP. No EGFR, HER2, bcl-2 or c-kit expression was detected in any sample. Expression of β-catenin, VEGF, ERβ, COX-2 in high number of cases suggests a potential as future therapeutic targets in desmoid-type fibromatosis.

Transcription factors β-catenin and Hex in postnatal development of the rat adrenal cortex: implication in proliferation control

Transcriptional regulation of growth, maturation, and cell turnover in adrenal cortex during postnatal development has been significantly less studied than in embryonic period, while elucidation of factors mediating its normal postnatal morphogenesis could clarify mechanisms of tumorigenesis in adrenal cortex. Expression of transcription factors Hex, β-catenin, and Wnt signaling in the adrenal cortex of male pubertal and postpubertal Wistar rats were examined. Adrenal cortex morphology and hormone production during postnatal development were also studied. Adrenocortical zones demonstrated similar reduction of Ki-67-expressing cells, but different patterns of morphological and functional changes. Age-dependent decrease in percentage of cells with membrane localization of β-catenin and stable rate of cells with nuclear β-catenin, indicative of Wnt signaling activation, were revealed in each cortical zone. Nuclear β-catenin was not observed in immature areas of zona fasciculata. No association between Wnt signaling activation and rates of proliferation as well as changes in secretion of adrenocortical hormones was observed in postnatal development of rat adrenal cortex. Hex, known as antiproliferative factor, showed up-regulation of expression after puberty. Strong inverse correlations between ratio of Hex-positive cells and proliferating cells were found in zona glomerulosa and zona fasciculata. Zona reticularis demonstrated moderate correlation. Thus, these findings suggest a role for Hex in proliferation control during postnatal development of the rat adrenal cortex and possible implication of Hex down-regulation in adrenocortical dysplasia and neoplasia, which requires further study. Evaluation of Hex expression may also be considered a potent tool in assessment of cell proliferation in rat adrenal cortex.

The Prognostic Role of Β-Catenin Subcellular Localization in Hepatocellular Carcinoma of Egyptian Patients

Background: Hepatocellular carcinoma (HCC) is one of the most common cancers in Egypt with fatal outcome. The molecular pathogenesis of this type of cancer is so complex involving interaction of several signaling pathways. Among those is Wingless (WNT)/β-catenin signaling pathway, which is frequently activated in HCC. Objectives: To investigate the relationship of different localization patterns of β-catenin in relation to different histopathologic parameters in Egyptian HCC cases. Patients and methods: One hundred and twenty-one (121) HCC specimens were subjected for β-catenin evaluation by immunohistochemistry. Results: The studied HCC cases showed β-catenin membranous expression in 47/121 (38.8%) of cases, while nucleocytoplasmic localization was observed in 31/121 (25.6%). Most of HCC cases that showed single tumor mass presentation exhibited negative nucleocytoplasmic expression pattern (79.2%, P=0.02). Membranous pool of β-catenin was associated with adverse prognostic factors like increased positivity among high-grade cases (P=0.04), higher H score value in cases having lymphovascular invasion, positive lymph node and higher (advanced) TMN stages (P = 0.01, 0.00, 0.00 &0.03 respectively). Conclusion: HCC cases showed heterogeneous β-catenin expression pattern. Membranous β-catenin localization was associated with aggressive prognostic parameters of HCC.

Pannexin 1 binds β-catenin to modulate melanoma cell growth and metabolism

Melanoma is the most aggressive skin malignancy with increasing incidence worldwide. Pannexin1 (PANX1), a member of the pannexin family of channel-forming glycoproteins, regulates cellular processes in melanoma cells including proliferation, migration, and invasion/metastasis. However, the mechanisms responsible for coordinating and regulating PANX1 function remain unclear. Here, we demonstrated a direct interaction between the C-terminal region of PANX1 and the N-terminal portion of β-catenin, a key transcription factor in the Wnt pathway. At the protein level, β-catenin was significantly decreased when PANX1 was either knocked down or inhibited by two PANX1 blockers, Probenecid and Spironolactone. Immunofluorescence imaging showed a disrupted pattern of β-catenin localization at the cell membrane in PANX1-deficient cells, and transcription of several Wnt target genes, including MITF, was suppressed. In addition, a mitochondrial stress test revealed that the metabolism of PANX1-deficient cells was impaired, indicating a role for PANX1 in the regulation of the melanoma cell metabolic profile. Taken together, our data show that PANX1 directly interacts with β-catenin to modulate growth and metabolism in melanoma cells. These findings provide mechanistic insight into PANX1-mediated melanoma progression and may be applicable to other contexts where PANX1 and β-catenin interact as a potential new component of the Wnt signaling pathway.

Expression of β-catenin in Minor Salivary Glands Adjacent to Oral Squamous Cell Carcinoma

Objective: This study aims to evaluate the expression pattern, localization, and stain intensity of β-catenin in minor salivary glands adjacent to surgically excised oral squamous cell carcinoma (OSCC). 
 Methods: A retrospective study was held with 16 samples of formalin-fixed paraffin-embedded blocks with minor salivary glands adjacent to OSCC. Sections were stained and evaluated immunohistochemically
 with β-catenin. The staining expression was assessed according to cellular localization, stain intensity, and, lastly, the pattern of stain distribution throughout acini.
 Statistical analysis was performed using SPSS version 24.0 software for Windows, and data analyzed by Fisher's exact test. P-value <0.05 was considered as statistically significant.
 Results: All minor salivary glands in the studied sample showed β-catenin staining with different expression in their functional units, as all had ductal and myoepithelial cells staining with a predominant cytoplasmic
 localization. While the mucous acini showed β- catenin expression in 10 cases (62.5%), this marker was significantly less frequently detected in serous acini of two cases of poorly differentiated OSCC (p= .008).
 A highly significant relation was found between the β-catenin cellular localization and stain distribution pattern in mucous and serous acini. 
 Conclusions: β-catenin had altered cytoplasmic expression in all of the minor salivary glands adjacent to OSCC. Furthermore, the diffuse pattern of distribution throughout the acini could identify the multi-patches
 pathological alteration of this area. The current study clarifies that the adjacent clinically normal-appearing salivary glands could harbor genetic aberrations of their subsequent malignant transformation.

Shigella flexneri Disruption of Cellular Tension Promotes Intercellular Spread

SUMMARYDuring infection, some bacterial pathogens invade the eukaryotic cytosol and spread between cells of an epithelial monolayer. Intercellular spread occurs when these pathogens push against the plasma membrane, forming protrusions that are engulfed by adjacent cells. Here, we show that IpaC, a Shigella flexneri type 3 secretion system protein, binds the host cell-adhesion protein β-catenin and facilitates efficient protrusion formation. S. flexneri producing a point mutant of IpaC that cannot interact with β-catenin is defective in protrusion formation and spread. Spread is restored by chemical reduction of intercellular tension or genetic depletion of β-catenin, and the magnitude of the protrusion defect correlates with membrane tension, indicating that IpaC reduces membrane tension, which facilitates protrusion formation. IpaC stabilizes adherens junctions and does not alter β-catenin localization at the membrane. Thus, Shigella, like other bacterial pathogens, reduces intercellular tension to efficiently spread between cells.

Blockers of Wnt3a, Wnt10a, or β-Catenin Prevent Chemotherapy-Induced Neuropathic Pain In Vivo.

Although chemotherapy is a key cancer treatment, many chemotherapeutic drugs produce chronic neuropathic pain, called chemotherapy-induced neuropathic pain (CINP), which is a dose-limiting adverse effect. To date, there is no medicine that prevents CINP in cancer patients and survivors. We determined whether blockers of the canonical Wnt signaling pathway prevent CINP. Neuropathic pain was induced by intraperitoneal injection of paclitaxel (PAC) on four alternate days in male Sprague-Dawley rats or male Axin2-LacZ knock-in mice. XAV-939, LGK-974, and iCRT14, Wnt/β-catenin blockers, were administered intraperitoneally as a single or multiple doses before or after injury. Mechanical allodynia, phosphoproteome profiling, Wnt ligands, and inflammatory mediators were measured by von Frey filament, phosphoproteomics, reverse transcription-polymerase chain reaction, and Western blot analysis. Localization of β-catenin was determined by immunohistochemical analysis in the dorsal root ganglia (DRGs) in rats and human. Our phosphoproteome profiling of CINP rats revealed significant phosphorylation changes in Wnt signaling components. Importantly, repeated systemic injections of XAV-939 or LGK-974 prevented the development of CINP in rats. In addition, XAV-939, LGK-974, and iCRT14 ameliorated CINP. PAC increased Wnt3a and Wnt10a, activated β-catenin in DRG, and increased monocyte chemoattractant protein-1 and interleukin-1β in DRG. PAC also upregulated rAxin2 in mice. Furthermore, β-catenin was expressed in neurons, including calcitonin gene-related protein-expressing neurons and satellite cells in rat and human DRG. In conclusion, chemotherapy increases Wnt3a, Wnt10a, and β-catenin in DRG and their pharmacological blockers prevent and ameliorate CINP, suggesting a target for the prevention and treatment of CINP.

Role of Wnt signaling in dermatofibroma induction phenomenon.

Dermatofibroma (DF) is a common benign skin neoplasm. Induction above DF lesions, including follicular unit induction, is a frequently observed phenomenon. Wnt signaling is known to be critical in hair follicle morphogenesis. Our study assesses the role of Wnt signaling in DF induction by evaluating intracellular localization of β-catenin in various types of DF induction. Archived tissue collected between 1 October 1980 and 1 October 2013 was stained per protocol using hematoxylin and eosin and anti-β-catenin monoclonal antibody. Specimens were grouped into categories based on the presence or absence and type of induction. All specimens were scored for nuclear β-catenin localization. Of 62 specimens, 42 (68%) showed induction while 20 (32%) showed none. Nuclear β-catenin staining was detected in 23 (55%) of the induction and in none of the no-induction specimens (P-value < 0.001). Types of induction included: 15 (24%) follicular induction, 31 (50%) acanthosis, and 4 (6%) sebaceous induction. For follicular induction, 13 (87%) showed positive nuclear β-catenin staining compared to 11 (35%) for acanthosis and 1 (25%) for sebaceous induction (P-value = 0.002). Our findings support the hypothesis that DFs promote an ectopic activation of Wnt pathway signaling in follicular induction phenomenon.

Beta-Catenin in Pseudoexfoliation Syndrome

To find whether it is possible that beta-catenin, associated with the development of serious systemic diseases, as well as the neoplastic process, plays a role in the development of pseudoexfoliation syndrome (PEX). If so, identifying PEX, an age-related, vision-threatening disorder of elastic fibers, which is manifested in eyes by the accumulation of an abnormal fibrillar material on the tissues of the anterior segment, with its poorly understood pathogenesis, may be an early indicator of other systemic diseases. The specimens of anterior lens capsules were obtained during routine cataract surgeries from patients with PEX (study group) and those without it (control group). Patients with previously diagnosed renal, cardiac or neoplasm diseases were excluded. In order to determine the localization of β-catenin at the ultrastructural level, the post-embedding colloidal gold (AU) method was used. For the analysis of the presence of proteins involved in cell–cell junctions, including β-catenin, fluorescence staining was performed. An enhanced accumulation of AU in the area of cell junctions in the PEX group was observed in comparison to control patients. A statistically significant increase in the level of β-catenin expression in lens epithelial cells (LECs) for the PEX group (MFI = 808.98) in comparison to the control patients (MFI = 731.6) was also noted. Our study presented the increase in the β-catenin in LECs of PEX group in comparison to control patients. It might be possible for PEX, due to it being easily recognizable, to be the first indicator of serious kidney or cardiac diseases, as well as cancer metastases. Further studies are needed in order to confirm this hypothesis.

Abstract 2605: Cytokeratin 5 promotes endocytosis to remodel cell adhesions in breast cancer

Abstract Breast cancer (BC) is clinically classified according to expression of estrogen receptor, progesterone receptor (ER+ BC), HER2 (HER2+), or absence of these markers (triple negative BC). While TNBC has the inferior prognosis in the short term, ER+ breast cancer has an extended risk of recurrence that lasts up to 20 years and accounts for the most deaths overall. Cytokeratin 5 (CK5) is an intermediate filament protein that is expressed in the majority of TNBC cases and 10-50% of ER+ BC. In CK5+/ER+ BC, there is widespread heterogeneity in the amount of CK5+ tumor cells (~1-50%). The mere presence of CK5+ cells is an indicator of poor prognosis across both subtypes of BC; the reasons for this are poorly understood. We have shown that CK5+ cells within ER+ and TNBC cell lines, and heterogeneous PDX tumor models lose membrane localization of adherens junction proteins β-catenin and E-cadherin. Loss of membrane β-catenin and E-cadherin is associated with poor prognosis in breast cancer and is a precursor to cell invasion. CK5+ cells have been reported to have increased invasive potential by us and others. Thus, identifying the mechanism of CK5-dependent loss of adherens proteins at the membrane could lead to development of therapies targeting this process. β-catenin and E-cadherin localization have been reported to be regulated through endocytosis which is mediated through the small GTPase family of Rab proteins. We performed a screen to identify potential CK5 interacting proteins in breast cancer cells and identified several Rab proteins. Thus, we hypothesize that CK5 acts as a scaffold for Rabs to promote endocytosis of β-catenin and E-cadherin to increase invasive potential.To investigate this, we treated CK5 overexpressing ER+ and TNBC cells with the endocytosis inhibitor Dyngo4a and found membrane β-catenin and E-cadherin localization were restored, suggesting that CK5 may be mediating endocytosis of β-catenin and E-cadherin. Rab5 is responsible for trafficking vesicles from the plasma membrane to the early endosomes. CK5 was confirmed to interact with Rab5 by co-IP in TNBC and CK5 overexpressing ER+ cell lines. In ongoing studies, we are testing whether Rab5 knockdown prevents loss of membrane β-catenin and E-cadherin and decreases cell invasiveness. These experiments will collectively determine whether CK5 regulated endocytosis is a targetable feature in breast cancer cells. Citation Format: Olivia Frances McGinn, Ashley V. Ward, Jessica Finlay-Schultz, Kiran Vinod Paul, Peter Kabos, Carol Sartorius. Cytokeratin 5 promotes endocytosis to remodel cell adhesions in breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2605.

Abstract 2453: Inhibition of β-catenin/CBP signaling with E7386 targets epigenetic changes associated with cancer stem cells in head and neck cancer

Abstract Head and neck squamous cell carcinoma (HNSCC) is a pernicious malignancy that arises from populations of cancer stem cells (CSCs). We and others have shown that the Wnt/β-catenin signaling pathway drives CSC gene expression mediated, in part, by epigenetic alterations directed by interactions between nuclear β-catenin and the cAMP-responsive element binding (CREB)-binding protein (CBP). In HNSCC, the β-catenin/CBP complex recruits the histone methyltransferase, MLL1, to drive trimethylation of H3K4me3 to induce an open chromatin structure and expression of CSC genes. Further, β-catenin/CBP signaling is highly correlated with the activity of the paralogous transcriptional regulators YAP and TAZ (YAP/TAZ), which are pro-tumorigenic factors in HNSCC. We reported that a small molecule inhibitor of the β-catenin-CBP interaction, ICG-001, blocks oncogenic phenotypes in cellular, zebrafish, and murine models of HNSCC, concomitant with the reduction of CSC traits. Recently, a novel β-catenin/CBP modulator, E7386, has been shown to be effective against a number of neoplasms in preclinical studies. Here, we compared anti-cancer properties of E7386 with ICG-001 to define its molecular mechanisms and validate the β-catenin/CBP axis as a bona fide therapeutic target in HNSCC. Anti-HNSCC activity of E7386 was evaluated in four human HNSCC cell lines using genomic, molecular, biochemical and functional approaches, including global ChIP-seq for H3K4me3. The set of transcripts significantly down-regulated by E7386 in HNSCC cells was projected onto a TCGA RNA-seq data (n=318) using ASSIGN, where samples were scored based on the coordinated expression of the gene signature which, in turn, reflected the level of E7386 inhibition per sample. The E7386 inhibition score was then tested for its association with survival by stratifying TCGA patients (n=318) into high- and low-score groups. Results showed that E7386 had highly overlapping activity signatures with ICG-001 (R = 0.997) with ~50 - 100-fold lower EC50. Similar to ICG-001, treatment with E7386 blocked association between β-catenin and CBP with a concomitant reduction in CBP and MLL1 abundance and global H3K4 trimethylation. E7386 repressed an oncogenic gene expression signature regulated by YAP1/TAZ and impeded HNSCC cell proliferation, promoting E-cadherin adhesion and junctional localization of β-catenin. Importantly, E7386 inhibition-associated transcriptional signatures tracked with tumor grade and poor human HNSCC patient survival. In conclusion, inhibiting β-catenin/CBP activity with E7386 represents a novel approach aimed at targeting epigenetically driven changes in the chromatin structure in HNSCC. Citation Format: Huamei Yang, Vinay Kartha, Khalid A. Alamoud, Anthony Federico, Andrew Tilston-Lunel, Bach-Cuc Nguyen, Takashi Owa, Kenichi Nomoto, Xaralabos Varelas, Stefano Monti, Maria A. Kukuruzinska. Inhibition of β-catenin/CBP signaling with E7386 targets epigenetic changes associated with cancer stem cells in head and neck cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2453.

R-Spondin 2 Induces Odontogenic Differentiation of Dental Pulp Stem/Progenitor Cells via Regulation of Wnt/β-Catenin Signaling.

Odontoblast cells generated from human dental pulp stem/progenitor cells (hDPSCs) secrete reparative dentin in responds to an injury. Endogenous Wnt signaling is also activated during this process, and these Wnt-activated cells are responsible for the following repair response. R-spondin 2 (Rspo2) is a potent stem cell growth factor, which strongly potentiates Wnt/β-catenin signaling and plays a vital role in cell differentiation and regeneration. However, the role of Rspo2 during odontoblast differentiation in hDPSCs has not yet been completely understood. This study investigated the effects of Rspo2 on hDPSCs to provide therapeutic insight into dentin regeneration and reparative dentin formation. HDPSCs were extracted from human molars or premolars. Immunofluorescence staining and flow cytometric analysis were used to detect the mesenchymal stem cell markers in hDPSCs. EdU assay and Cell Counting Kit-8 (CCK-8) were performed to explore cell proliferation. The odontogenic differentiation levels were determined by measuring the mRNA and protein expression of DSPP, DMP-1, ALP, and BSP. Immunofluorescence staining was performed to detect the localization of β-catenin. The biological effects of Rspo2 on hDPSCs was investigated using the Lentivirus-based Rspo2 shRNA and recombined human Rspo2 (rhRspo2). Recombined human DKK-1 (rhDKK-1) and recombined human Wnt3a (rhWnt3a) were used for further investigation. The cells generated from human dental pulp expressed mesenchymal stem cell markers Vimentin, Stro-1, Nestin, C-kit, CD90, and CD73, while were negative for CD3, CD31, and CD34. The mRNA expression levels of the odontogenic-related genes DSPP, DMP-1, ALP, and BSP were upregulated in the rhRspo2 treated cells. Silencing Rspo2 suppressed the proliferation and differentiation of the hDPSCs. Blockade of Wnt signaling with DKK-1 inhibited Rspo2-induced activation of Wnt/β-catenin signaling and cell differentiation. The combined use of rhWnt3a and rhRspo2 created a synergistic effect to improve the activation of Wnt/β-catenin signaling. Rspo2 promoted the proliferation and odontogenic differentiation of hDPSCs by regulating the Wnt/β-catenin signaling pathway.

Excess hydrogen peroxide inhibits head and foot regeneration in hydra by affecting DNA repair and expression of essential genes.

Reactive oxygen species (ROS) are necessary for various cellular processes. However, excess ROS cause damage to many biological molecules and therefore must be tightly regulated in time and space. Hydrogen peroxide (H2 O2 ) is the most commonly used ROS as second messenger in the cell. It is a relatively long-lived freely diffusible signaling molecule during early events of injury. In the Cnidarian hydra, injury-induced ROS production is essential for regeneration to proceed. In the present study, we have examined influence of varying exposure to H2 O2 on head and foot regeneration in the middlepieces of trisected hydra. We find that longer (4 hours) exposure to 1 mM H2 O2 inhibits both head and foot regeneration while shorter exposure (2 hours) does not. Longer exposure to H2 O2 resulted in extensive damage to DNA that could not be repaired, probably due to suboptimal induction of APE1, an enzyme necessary for base excision repair (BER). Concomitantly, genes involved in activation of Wnt pathway, necessary for head regeneration, were significantly downregulated. This appeared to be due to failure of both stabilization and transient nuclear localization of β-catenin. Similarly, genes involved in foot regeneration were also downregulated on longer exposure to H2 O2 . Thus, exposure to excess ROS inhibits regenerative processes in hydra through reduced expression of genes involved in regeneration and diminished DNA repair.

Improved β-catenin detection in spinal cord tissue sections: autofluorescence quenching

Experimental studies on spinal cord regeneration are focusing on the windows of opportunity to improve spinal cord microenvironment via spinal-centric repair pathways. One pathway of particular interest is the Wnt/β-catenin signalling pathway which plays a vital role in axonal guidance, synaptic assembly and function, neuronal survival and connectivity after spinal cord trauma to induce repair. Upregulation of β-catenin expression is often taken as evidence of regeneration mechanisms through the Wnt/ β-catenin pathway. However, these studies may not have optimised the staining protocol for β-catenin to enable accurate detection of the protein. Given possible issues with the background or endogenous tissue autofluorescence, there is a need to optimise the protocol further to allow better visualisation of β-catenin. So far, there are no studies which report optimising spinal cord tissues for β-catenin to reduce autofluorescence, and as β-catenin is widely used in spinal cord injury (SCI) and other spinal cord tissue studies, thus it is an important issue to address. To achieve reliable detection and localisation of β-catenin, we utilised sequential quenching techniques using 1% NaBH4 and 1mM CuSO4 in 50mM ammonium acetate buffer to reduce both background and fixative-induced autofluorescence. Our results showed that sequential autofluorescence quenching is crucial in β-catenin detection, and this improved technique indicates that β-catenin is localised in the spinal cord white matter regions. Objective approach for the β-catenin localisation is highly significant as it unravelled an objective identification and illuminate the pattern of distribution of β-catenin for researcher focusing on spinal cord repair studies via the Wnt/β-catenin pathway following SCI.

Hypoxia-Induced miR-675-5p Supports β-Catenin Nuclear Localization by Regulating GSK3-β Activity in Colorectal Cancer Cell Lines.

The reduction of oxygen partial pressure in growing tumors triggers numerous survival strategies driven by the transcription factor complex HIF1 (Hypoxia Inducible Factor-1). Recent evidence revealed that HIF1 promotes rapid and effective phenotypic changes through the induction of non-coding RNAs, whose contribution has not yet been fully described. Here we investigated the role of the hypoxia-induced, long non-coding RNA H19 (lncH19) and its intragenic miRNA (miR-675-5p) into HIF1-Wnt crosstalk. During hypoxic stimulation, colorectal cancer cell lines up-regulated the levels of both the lncH19 and its intragenic miR-675-5p. Loss of expression experiments revealed that miR-675-5p inhibition, in hypoxic cells, hampered β-catenin nuclear localization and its transcriptional activity, while lncH19 silencing did not induce the same effects. Interestingly, our data revealed that miRNA inhibition in hypoxic cells restored the activity of Glycogen Synthase Kinase 3β (GSK-3β) reducing the amount of P-Ser9 kinase, thus unveiling a role of the miR-675-5p in controlling GSK-3β activity. Bioinformatics analyses highlighted the serine/threonine-protein phosphatases PPP2CA, responsible for GSK-3β activation, among the miR-675-5p targets, thus indicating the molecular mediator through which miR-675-5p may control β-catenin nuclear localization. In conclusion, here we demonstrated that the inhibition of the hypoxia-induced non-coding RNA miR-675-5p hampered the nuclear localization of β-catenin by regulating GSK-3β activity, thus proposing the miR-675-5p as a new therapeutic target for the treatment of colorectal cancer.

Bisphenol A triggers the malignancy of nasopharyngeal carcinoma cells via activation of Wnt/β-catenin pathway

It is critical to understand the risk factors responsible for the tumorigenesis and progression of nasopharyngeal carcinoma (NPC). Bisphenol A (BPA) can regulate the estrogenic signals to modulate cancer progression, while its roles in NC were not investigated. Our present study revealed that the BPA can increase proliferation and migration of NPC cells while decrease the chemosensitivity to doxorubicin (Dox). The inhibitor of GSK-3β/β-catenin (LiCl) can restore BPA-induced cell proliferation of NPC cells, which is due to that BPA can decrease phosphorylation while increase expression and nucleus localization of β-catenin. Mechanistically, BPA can increase the mRNA stability of β-catenin (encoded by CTNNB1) via suppressing the expression of miR-214-3p, which can direct target the 3’UTR of β-catenin mRNA. Further, BPA can decrease phosphorylation of β-catenin via repressing the expression of CK1α. Collectively, our data showed that BPA can trigger the proliferation and malignancy of NPC cells via activation of Wnt/β-catenin pathway. It indicated that body accumulation and inhalation exposure of BPA might be a risk factor for NPC development.

HOXA10 inhibit the osteogenic differentiation of periodontal ligament stem cells by regulating β-catenin localization and DKK1 expression

ABSTRACT Introduction: Human periodontal ligament stem cells (hPDLSCs) are stem cells found near the tooth periodontal ligament. These cels are involved in the regeneration of the periodontal ligament and alveolar bone during orthodontic treatment and chronic periodontitis. Objectives: The Homeobox gene HOXA10 regulates the osteogenic differentiation of stem cells. However, the role of HOXA10 in hPDLSCs remains unclear. Therefore, we studied the effects of HOXA10 on human PDLSC osteogenic differentiation in vitro. Methods: First, hPDLSCs were isolated and characterized. Second, we assessed the effects of overexpression and knockdown of HOXA10 on PDLSC osteogenic differentiation. Finally, the specific Wnt signaling pathway activator lithium chloride (LiCl) and inhibitor ICG-001 were used to investigate the involvement of the Wnt signaling pathway in HOXA10-induced regulation of osteogenic differentiation. Results: Overexpressing HOXA10 inhibited PDLSC osteogenic differentiation in vitro, shown by ALP and Alizarin Red staining, while HOXA10 knockdown demonstrated the opposite effects. HOXA10 negatively regulated nuclear β-catenin and osteogenic differentiation markers including alkaline phosphatase (ALPL) and integrin-binding sialoprotein (IBSP). Upregulating HOXA10 reduced nuclear β-catenin and increased DKK1 expression. However, HOXA10 knockdown enhanced nuclear β-catenin accumulation and reduced DKK1 expression. These negative effects on osteogenic differentiation by HOXA10 overexpression were restored by the Wnt/β-catenin pathway activator LiCl. The increased osteogenic differentiation effects of HOXA10 knockdown were antagonized by ICG-001, a Wnt pathway inhibitor. Conclusion: These data demonstrate that HOXA10 inhibits the osteogenic differentiation of periodontal ligament stem cells by regulating β-catenin localization and DKK1.