Nuclear Β-catenin Research Articles

Inflammatory and Nested Testicular Sex Cord Tumor: A Novel Neoplasm With Aggressive Clinical Behavior and Frequent EWSR1::ATF1 Gene Fusions.

A subset of malignant testicular sex cord tumors (TSCTs), heretofore interpreted as Sertoli cell tumors, not otherwise specified, exhibits distinctive morphologic features that partially overlap with those of seminoma. In this study, we evaluated the clinicopathologic and molecular characteristics of 13 such tumors. The patients were 20 to 73 years old (median, 36y), and all with available data presented with testicular masses (median size, 3cm), with 2 having synchronous retroperitoneal metastases. All 11 patients with available follow-up developed metastases to retroperitoneal lymph nodes, nonretroperitoneal lymph nodes, bone, contralateral testis, and/or lung. Microscopically, the tumors showed solid nests and sheets of epithelioid cells with granular, eosinophilic to clear/vacuolated cytoplasm, admixed in most (12/13) cases with variable proportions of lymphocytes, plasma cells, eosinophils, and neutrophils. Additional features included intracytoplasmic hyaline inclusions and a prominent collagenous, sometimes hyalinized stroma. Mitotic activity was relatively low (median, 1 mitosis/10HPF), but tumor necrosis was frequent (11/13). Local invasion of adjacent structures and lymphovascular invasion were noted in some tumors (4/9 cases with available data for each feature). All were α-inhibin-positive and lacked nuclear reactivity for β-catenin. In addition, all tested cases were positive for epithelial membrane antigen (9/9) and steroidogenic factor-1 (8/8), and 8/10 expressed CD30. Two "index" cases were initially analyzed using a DNA sequencing panel, which identified EWSR1::ATF1 fusions in both. Subsequently, EWSR1::ATF1 fusions were demonstrated in 8 of the remaining 11 cases using fluorescence in situ hybridization or DNA sequencing. One of the 3 cases that were negative for EWSR1::ATF1 harbored ATF1 amplification. This study, therefore, shows that a group of malignant TSCTs resembling seminoma is characterized by α-inhibin and steroidogenic factor-1 positivity, no expression of nuclear β-catenin, frequent CD30 positivity and recurrent EWSR1::ATF1 fusions. We have descriptively termed these neoplasms "inflammatory and nested TSCT." Importantly, inflammatory and nested TSCTs show significant differences in morphology, immunoprofile, molecular biology, and, likely, clinical behavior from Sertoli cell tumors, not otherwise specified and should be classified separately.

Butyrate prevents the migration and invasion, and aerobic glycolysis in gastric cancer via inhibiting Wnt/β-catenin/c-Myc signaling.

Gastric cancer (GC) remains a common cause of cancer death worldwide. Evidence has found that butyrate exhibited antitumor effects on GC cells. However, the mechanism by which butyrate regulate GC cell proliferation, migration, invasion, and aerobic glycolysis remains largely unknown. The proliferation, migration, and invasion of GC cells were tested by EdU staining, transwell assays. Additionally, protein expressions were determined by western blot assay. Next, glucose uptake, lactate production, and cellular ATP levels in GC cells were detected. Furthermore, the antitumor effects of butyrate in tumor-bearing nude mice were evaluated. We found, butyrate significantly prevented GC cell proliferation, migration, and invasion (p < .01). Additionally, butyrate markedly inhibited GC cell aerobic glycolysis, as shown by the reduced expressions of GLUT1, HK2, and LDHA (p < .01). Moreover, butyrate notably decreased nuclear β-catenin and c-Myc levels in GC cells (p < .01). Remarkably, through activating Wnt/β-catenin signaling with LiCl, the inhibitory effects of butyrate on the growth and aerobic glycolysis of GC cells were diminished (p < .01). Moreover, butyrate notably suppressed tumor volume and weight in GC cell xenograft nude mice in vivo (p < .01). Meanwhile, butyrate obviously reduced nuclear β-catenin, c-Myc, GLUT1, HK2 and LDHA levels in tumor tissues in GC cell xenograft mice (p < .01). Collectively, butyrate could suppress the growth and aerobic glycolysis of GC cells in vitro and in vivo via downregulating wnt/β-catenin/c-Myc signaling. These findings are likely to prove useful in better understanding the role of butyrate in GC.

GOLM1 affects proliferation, invasion, and migration by regulating Wnt pathway in esophageal squamous cell carcinoma cells

AimTo investigate the effect of Golgi membrane protein 1 (GOLM1) on the proliferation, migration, and invasion of esophageal squamous cell carcinoma (ESCC) cells and its underlying oncogenic mechanism. MethodsThe Expression level of GOLM1 in esophageal cancer (EC) tissues were analyzed in the cancer genome database. Fresh ESCC tissues and cancer adjacent tissues of 44 patients with EC who underwent radical esophagectomy were collected. GOLM1 mRNA expression was detected by RT qPCR. ESCC cell lines (EC9706, TE-1, Eca-109, kyse-150) and normal esophageal epithelial cells (HEEC) were cultured in vitro. ResultsThe expression level of GOLM1 in EC tissues was significantly higher (P < 0.05). The mRNA expression level of GOLM1 in ESCC was significantly higher than that in adjacent tissues (P < 0.05). Compared with HEEC cells, the mRNA and protein expression levels of GOLM1 in ESCC cell lines were significantly higher (P < 0.05). Kyse-150 cells showed the highest expression of GOLM1. Kyse-150 cells were divided into control group, lenti-NC group, GOLM1 overexpression group, SH-NC group, GOLM1 silencing group, XAV -939 group, and GOLM1 overexpression + XAV -939 group. After lentivirus transfection, the expression levels of mRNA and protein of GOLM1 were significantly higher (P＜0.05). In the GOLM1 silence group, all these variables were significantly decreased. Overexpression of GOLM1 can increase the expression of Wnt3a and nuclear β-catenin and can reverse the effect of XAV-939 intervention. The effect of GOLM1 silence was consistent with that of the XAV-939 intervention. Compared with the NC group, the tumor volume and tumor index of the GOLM1 knockdown group decreased significantly in vivo (P < 0.05). ConclusionGOLM1 is up-regulated in ESCC tissues and ESCC cells and may function as an oncogene. GOLM1 silencing can reduce the proliferation, migration, and invasion of ESCC cells by inhibiting Wnt/β-catenin signaling pathway.

Stabilization of nuclear β-catenin by inhibiting KDM2A mediates cerebral ischemic tolerance.

Hypoxic postconditioning (HPC) with 8% oxygen increases nuclear accumulation of β-catenin through activating the classical Wnt pathway, thereby alleviating transient global cerebral ischemia (tGCI)-induced neuronal damage in the hippocampal CA1 subregion of adult rats. However, little is understood about the regulatory mechanism of nuclear β-catenin in HPC-mediated cerebral ischemic tolerance. Although lysine(K)-specific demethylase 2A (KDM2A) has been known as a crucial regulator of nuclear β-catenin destabilization, whether it plays an important role through modulating nuclear β-catenin in cerebral ischemic tolerance induced by HPC remains unknown. In this study, we explored the molecular mechanism of stabilizing nuclear β-catenin by inhibiting KDM2A-mediated demethylation in the HPC-offered neuroprotection against tGCI. In addition, we confirmed that nuclear methylated-β-catenin in CA1 decreased and nuclear β-catenin turnover increased after tGCI, which were reversed by HPC. The administration with methyltransferase inhibitor AdOx abrogated HPC-induced methylation and stabilization of nuclear β-catenin in CA1, as well as the neuroprotection against tGCI. Notably, HPC downregulated the expression of KDM2A in CA1 and reduced the interaction between KDM2A and β-catenin in the nucleus after tGCI. The knockdown of KDM2A with small-interfering RNA could upregulate nuclear methylated-β-catenin and stabilize β-catenin, thereby increasing survivin in CA1 and improving the cognitive function of rats after tGCI. Opposite results were observed by the administration of KDM2A-carried adenovirus vector. Furthermore, we demonstrated that KDM2A mediates the demethylation of nuclear β-catenin through jumonji C (JmjC) domain of KDM2A in HEK-293T and SH-SY5Y cells. Our data support that the inhibition of KDM2A-mediated demethylation of nuclear β-catenin contributes to HPC-induced neuroprotection against tGCI.

The mechanics of tumor cells dictate malignancy

Tumor cells are generally softer than healthy cells and tumor cells with high self-renewal or malignancy exhibit even lower stiffness, suggesting the inverse correlation between cellular stiffness and malignant potential. However, it remains mysterious whether tumor cell softening is a by-product of or a driving force for tumor progression. In this study, hepatocellular carcinoma (HCC) was chosen as a model of disease. The stiffness of various HCC cells was modulated by targeting actomyosin activity through both genetic and pharmaceutical approaches. Softening and stiffening tumor cells promotes and suppresses their self-renewal in vitro and tumor formation/growth in vivo, suggesting the regulatory role of cell mechanics in self-renewal and tumorigenicity. These findings are also observed in multiple other cancer types, including breast, lung, colon, and cervical cancer. Mechanistically, softening/stiffening cytoskeleton weakens/strengthens the interaction between APC and β-catenin, which inhibits/enhances β-catenin degradation and promotes/suppresses its nuclear translocation. Nuclear β-catenin binds the promoter of the stemness gene Oct4 and facilitates its transcription, which enhances tumor cell self-renewal. Further, cell softening facilitates the de-differentiation of non-CSCs into cancer stem cells (CSCs), while cell stiffening promotes the differentiation of CSCs into non-CSCs, both of which depend on Wnt/β-catenin signaling. The cytoskeleton-APC-Wnt/β-catenin-Oct4 signaling are correlated in clinical samples and associated with tumor differentiation and patient survival. In summary, our findings demonstrate that actomyosin-mediated mechanical properties of tumor cells dictate their self-renewal and tumorigenicity through cytoskeleton-APC-Wnt/β-catenin-Oct4 signaling, which may identify cell mechanics not only as a marker for diagnosis but also as a new mechanical target for cancer therapeutics.

KLF3 Transcription Activates WNT1 and Promotes the Growth and Metastasis of Gastric Cancer via Activation of the WNT/β-Catenin Signaling Pathway

The transcription factor Krüppel-like factor (KLF) 3 is one of the members of the KLF family, which plays an important role in tumor progression. Nevertheless, the role of KLF3 in the growth and metastasis of gastric cancer (GC) still needs to be elucidated. Bioinformatics analysis showed that KLF3 was overexpressed in patients with GC, and the high expression of KLF3 was correlated with poor survival. KLF3 was also overexpressed in GC clinical samples and cell lines. In vitro functional role of KLF3 in GC cells was explored by a gain-of-function and loss-of-function assay. Overexpressed KLF3 promoted the cell proliferation, migration, invasion, and epithelial-mesenchymal transition of GC cells, whereas suppressed KLF3 inhibited these biological behaviors. The clinical samples and bioinformatics analysis showed that WNT1 was also highly expressed in GC tumor tissues and positively correlated with KLF3 expression. The luciferase reporter assay and chromatin immunoprecipitation result confirmed that KLF3 could directly bind to the WNT1 promoter to increase the transcriptional activity of WNT1, thus regulating its expression. Overexpressed KLF3 enhanced the protein expression level of p-GSK3β(Ser9) and β-catenin, the key elements in the WNT/β-catenin signaling pathway. Repression of KLF3 decreased the level of p-GSK3β(Ser9) and β-catenin. Immunofluorescence images showed that KLF3 promoted nuclear β-catenin accumulation. Inhibition of WNT1 attenuated the proliferation, migration, and invasiveness of KLF3-overexpressing GC cells. Moreover, the xenograft mouse model confirmed that KLF3 promotes GC tumor growth and metastasis in vivo. Our results demonstrated that KLF3 activates the WNT/β-catenin signaling pathway via WNT1 to promote GC tumor growth and metastasis, indicating that repression of KLF3 may act as a potential therapeutic target for patients with GC.

Developmental Exposure to DDT Disrupts Transcriptional Regulation of Postnatal Growth and Cell Renewal of Adrenal Medulla.

Dichlorodiphenyltrichloroethane (DDT) is the most widespread persistent pollutant with endocrine-disrupting properties. DDT has been shown to disrupt secretory and morphogenetic processes in the adrenal cortex. The present investigation aimed to evaluate transcriptional regulation of postnatal growth of the adrenal medulla and formation of the pools necessary for self-renewal of medullary cells in rats that developed under low-dose exposure to DDT. The study was performed using male Wistar rats exposed to low doses of o,p'-DDT during prenatal and postnatal development. Light microscopy and histomorphometry revealed diminished medulla growth in the DDT-exposed rats. Evaluation of Ki-67 expression in chromaffin cells found later activation of proliferation indicative of retarded growth of the adrenal medulla. All DDT-exposed rats exhibited a gradual decrease in tyrosine hydroxylase production by adrenal chromaffin cells. Immunohistochemical evaluation of nuclear β-catenin, transcription factor Oct4, and ligand of sonic hedgehog revealed increased expression of all factors after termination of growth in the control rats. The DDT-exposed rats demonstrated diminished increases in Oct4 and sonic hedgehog expression and lower levels of canonical Wnt signaling activation. Thus, developmental exposure to the endocrine disruptor o,p'-DDT alters the transcriptional regulation of morphogenetic processes in the adrenal medulla and evokes a slowdown in its growth and in the formation of a reserve pool of cells capable of dedifferentiation and proliferation that maintain cellular homeostasis in adult adrenals.

Hordenine Activated Dermal Papilla Cells and Promoted Hair Regrowth by Activating Wnt Signaling Pathway.

Hordenine is effective in treating hyperpigmentation, fighting diabetes and resisting fibrosis and acute inflammation. However, the role of Hordenine on hair growth has not been elucidated. Here, we found that Hordenine treatments significantly enhance proliferation of primary mouse dermal-papilla cells (DPCs) and increase the activity of DPCs in a dose-dependent manner. Additionally, Hordenine markedly promoted the elongation of the hair shaft in the model of in vitro-cultured mouse vibrissa follicle and accelerated hair regrowth in a mouse model of depilation-induced hair regeneration. Real-time PCR, Western Blot and immunofluorescent assays showed that nuclear β-catenin and its downstream gene expression such as Lef1, Axin2, Cyclin D1 and ALP were greatly upregulated in DPCs and mouse hair follicles after Hordenine treatments. Moreover, the increased DPCs' proliferation and hair shaft elongation of cultured mouse vibrissa follicles induced by Hordenine treatments were rescued by a Wnt/β-catenin signaling inhibitor, FH535. These data indicate that Hordenine can effectively enhance DPCs' activity and accelerate hair regrowth through activating the Wnt/β-catenin signaling pathway. Therefore, these findings suggest Hordenine/its derivatives may be potentially used for preventing and treating alopecia in the future.

Immunity-related long noncoding RNA WDFY3-AS2 inhibited cell proliferation and metastasis through Wnt/β-catenin signaling in oral squamous cell carcinoma

ObjectiveLong noncoding RNA WDFY3-AS2 has been shown to play dual roles in the modulation of cancer progression. This study aimed at clarifying the biological role of WDFY3-AS2 as well as the association between WDFY3-AS2 expression, β-catenin expression, and OSCC immunity in oral squamous cell carcinoma (OSCC). DesignBioinformatics analyses, CCK8, EdU, wound healing, transwell, RT-qPCR, western blot, immunofluorescence, in situ hybridization, and immunohistochemistry assays were adopted for exploring the role of WDFY3-AS2 in OSCC. ResultsBioinformatics analyses showed that WDFY3-AS2 conferred a poor prognosis for OSCC patients. Further analyses identified WDFY3-AS2 as an independent prognostic indicator for OSCC. Moreover, silencing WDFY3-AS2 inhibits OSCC cell proliferation, migration and invasion. Gene set enrichment analysis indicated that WDFY3-AS2 participated in the regulation of Wnt signaling. In addition, WDFY3-AS2 expression was positively associated with β-catenin mRNA levels, the key component of Wnt signaling. Interestingly, WDFY3-AS2 knockdown inhibited β-catenin expression and nuclear translocation, thus suppressing OSCC progression through Wnt signaling. Furthermore, WDFY3-AS2 expression correlated with an immunosuppressive phenotype in the tumor immune microenvironment. In situ hybridization and immunohistochemistry verified that WDFY3-AS2 was positively associated with total and nuclear β-catenin protein levels and negatively associated with CD4 expression. ConclusionsThis study demonstrates that the immunity-associated WDFY3-AS2 augments OSCC proliferation and metastasis through Wnt/β-catenin signaling and may serve as a novel treatment target and a new prognostic factor for OSCC.

β-catenin/TCF4 inhibitors ICG-001 and LF3 alleviate BDL-induced liver fibrosis by suppressing LECT2 signaling

Liver fibrosis can be characterized by the over-deposition of extracellular matrix (ECM). It has been reported that β-catenin/TCF4 interaction was enhanced in bile duct ligation (BDL) model, which implicated the critical role of β-catenin/TCF4 interaction during the progression of fibrosis. However, whether inhibiting β-catenin/TCF4 signaling attenuates liver fibrosis remains unknown. In the current study, we used ICG-001, an inhibitor that disrupts the interaction between CREB binding protein (CBP) and β-catenin, to inhibit β-catenin/TCF4 transcriptional activity. We also used LF3, a small molecule antagonist, to inhibit β-catenin/TCF4 interaction. The antifibrotic effect of ICG-001 and LF3 was assessed on BDL-induced liver fibrosis model. The results indicated both ICG-001 and LF3 significantly reduced the positive staining area of Sirius Red and α-SMA. The protein expression levels of α-SMA, Collagen Ⅰ and CD31 were also significantly downregulated in BDL + ICG-001 and BDL + LF3 groups. Besides, ICG-001 and LF3 promoted portal angiogenesis and inhibited sinusoids capillarization in fibrotic livers. For mechanistic study, we measured the level of leukocyte cell-derived chemotaxin 2 (LECT2), a direct target of β-catenin/TCF4, which was recently reported to participate in hepatic fibrosis by regulating angiogenesis. The results showed that both ICG-001 and LF3 reduced LECT2 expression in BDL mice. LF3 also downregulated pSer 675 β-catenin and nuclear β-catenin. In conclusion, this study demonstrated that inhibiting β-catenin/TCF4 signaling by ICG-001 or LF3 mitigated liver fibrosis by downregulating LECT2, promoting portal angiogenesis and inhibiting sinusoids capillarization, which provided new evidence that β-catenin/TCF4 signaling might be a target for the treatment of liver fibrosis.

Significance of Wnt/β-Catenin Signal Activation for Resistance to Neoadjuvant Chemoradiotherapy in Rectal Cancer.

Although a therapeutic response to neoadjuvant chemoradiotherapy (NACRT) is important to improve oncological outcomes after surgery in patients with locally advanced rectal cancer, there is no reliable predictor for this. The Wnt/β-catenin signal is known to be crucial for the tumorigenesis of colorectal cancer. This study aimed to investigate the association of Wnt/β-catenin signal activation with a pathological response to NACRT. The immunohistochemical expression of nuclear and membranous β-catenin was analyzed in biopsy samples obtained from 60 patients with locally advanced rectal cancer who received curative surgery following NACRT. The association of Wnt/β-catenin signal activation with their clinical outcomes was investigated. Notably, the body mass index of these patients was significantly higher in the low nuclear β-catenin expression group. Moreover, patients in the high nuclear β-catenin expression group tended to have more advanced disease and a higher rate of positive vascular invasion than those in the low expression group. Furthermore, the rate of good histological responses was significantly higher in the low nuclear β-catenin expression group (72% vs. 37.1%, p &lt; 0.01). Overall, relapse-free survival tended to be better in patients with low nuclear/high membranous β-catenin expression (n = 9) than in other individuals (n = 51) (p = 0.093 and p = 0.214, respectively). Activation of the Wnt/β-catenin signal pathway represented by nuclear β-catenin accumulation was significantly associated with a poor response to NACRT in patients with rectal cancer. Analysis of nuclear β-catenin accumulation before starting treatment might help predict the therapeutic response to NACRT.

A Wnt/β-catenin signaling inhibitor, IMU1003, suppresses the emergence of osimertinib-resistant colonies from gefitinib-resistant non-small cell lung cancer cells

Drug resistance has become a challenge in effective longterm molecular targeted therapy. Longterm non-small cell lung cancer (NSCLC) treatments with the first-generation epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) shorten the effective duration of the third-generation EGFR-TKI, osimertinib, via genetic or epigenetic mechanisms in addition to the gatekeeper mutation T790M. This study reproduced this persistence in vitro using gefitinib-resistant NSCLC PC-9 cells (GR cells) and revealed that pharmacological nuclear localization inhibition of β-catenin suppressed the osimertinib resistance. Osimertinib effectively reduced GR cell survival but left significantly more resistant colonies than parental PC-9 cells. The nuclear fraction of β-catenin was enriched in GR cells during acquisition of osimertinib resistance. A chemical nuclear localization inhibitor of β-catenin, IMU1003, dramatically decreased the emergence of osimertinib-resistant colonies. Forced nuclear localization of β-catenin reduced IMU1003 efficacy. Thus, suppression of the nuclear β-catenin function may overcome the transgenerational EGFR-TKI-resistance.

Adenoid Ameloblastoma Shares Clinical, Histologic, and Molecular Features With Dentinogenic Ghost Cell Tumor: The Histologic Spectrum of WNT Pathway–Altered Benign Odontogenic Tumors

An epithelial odontogenic tumor called adenoid ameloblastoma (AA) has recently been included in the new WHO classification. However, AA has considerable overlapping features with a preexisting entity, dentinogenic ghost cell tumor (DGCT). This study compared the clinical, histologic, and molecular characteristics of AA and DGCT. Eight cases of odontogenic tumors initially diagnosed as AA or DGCT were included in this study. Quantitative histologic analysis, β-catenin immunohistochemistry, and molecular profiling using next generation sequencing were performed. Additionally, accumulated clinical data of AA and DGCT were statistically analyzed. Nuclear β-catenin accumulation was detected in all cases in common, although the tumors studied histologically consisted of varying combinations of the AA-like phenotype, ghost cells, and dentinoid. However, CTNNB1 hotspot mutations were not found in any case. Instead, loss-of-function mutations in tumor suppressor genes involved in the WNT pathway, including the APC, SMURF1, and NEDD4L genes, were found regardless of histologic type. In addition, KRT13 mutations were detected in 2 cases with a high proportion of ghost cells. Finally, a literature analysis revealed clinical similarities between the previously reported cases of AA and DGCT. These findings suggest that from a clinical and molecular point of view, AA and DGCT represent a histologic spectrum of WNT pathway–altered benign odontogenic tumors rather than 2 distinct tumors. Moreover, previously unidentified keratin mutations may be associated with ghost cell formation found in specific types of odontogenic lesions.

Ovarian Clear Cell Carcinoma and Markers of Epithelial-Mesenchymal Transition (EMT): Immunohistochemical Characterization of Tumor Budding.

Tumor budding, largely considered a manifestation of epithelial-mesenchymal transition (EMT) is an established prognostic marker for several cancers. In a recent study, tumor budding was associated with poor clinical outcomes in early-stage ovarian clear cell carcinoma. Here, we evaluated the immune expression of 3 proteins shown to be associated with EMT (E-cadherin, β-catenin, and glypican-3) in 72 primary tumors of ovarian clear cell carcinoma with median follow-up of 39.47mo. E-cadherin and β-catenin expression was further evaluated in tumor buds in 29 (40%) cases. In the tumor mass, diffuse membranous expression of E-cadherin and β-catenin was seen in 83% (60/72) and 81% (58/72) cases, respectively. Nuclear accumulation of E-cadherin was seen in 7 (10%) cases, while none of the cases showed nuclear β-catenin expression. Glypican-3 expression was diffuse in 33.3% (24/72), patchy in 29.2% (21/72), and absent in 37.5% (27/72) cases. Evaluation of tumor buds showed aberrant patterns of expression (complete loss/cytoplasmic accumulation/diminished, discontinuous incomplete membranous staining) of E-cadherin in 29/29 (100%) and of β-catenin in 26/29 (90%) cases. E-cadherin, β-catenin, and glypican-3 expression in the main tumor mass had no association with stage, lymph node status, recurrent/progressive disease, status at last follow-up, survival and histopathologic features ( P >0.05). Our finding of aberrant expression of both E-cadherin and β-catenin in tumor buds indicates involvement of Wnt signaling pathway/EMT in tumor budding and outlines its significance as a prognostic marker especially for early-stage ovarian clear cell carcinoma.

Pharmacological inhibition of FOXO1 promotes lymphatic valve growth in a congenital lymphedema mouse model.

Mutations in many genes that regulate lymphatic valve development are associated with congenital lymphedema. Oscillatory shear stress (OSS) from lymph provides constant signals for the growth and maintenance of valve cells throughout life. The expression of valve-forming genes in lymphatic endothelial cells (LECs) is upregulated by OSS. The transcription factor FOXO1 represses lymphatic valve formation by inhibiting the expression of these genes, which makes FOXO1 a potential target for treating lymphedema. Here, we tested the ability of a FOXO1 inhibitor, AS1842856, to induce the formation of new lymphatic valves. Our quantitative RT-PCR and Western blot data showed that treatment of cultured human LECs with AS1842856 for 48h significantly increased the expression levels of valve-forming genes. To investigate the function of AS1842856 in vivo, Foxc2 +/- mice, the mouse model for lymphedema-distichiasis, were injected with AS1842856 for 2 weeks. The valve number in AS-treated Foxc2+/- mice was significantly higher than that of the vehicle-treated Foxc2+/- mice. Furthermore, since β-catenin upregulates the expression of Foxc2 and Prox1 during lymphatic valve formation, and AS1842856 treatment increased the level of active β-catenin in both cultured human LECs and in mouse mesenteric LECs in vivo, we used the mouse model with constitutive active β-catenin to rescue loss of lymphatic valves in Foxc2 +/- mice. Foxc2 +/- mice have 50% fewer lymphatic valves than control, and rescue experiments showed that the valve number was completely restored to the control level upon nuclear β-catenin activation. These findings indicate that pharmacological inhibition of FOXO1 can be explored as a viable strategy to resolve valve defects in congenital lymphedema.

Nonsalivary Primary Adenocarcinomas of the Base of the Tongue: A Single Tertiary-Care Oncology Center Series of 6 Cases.

Nonsalivary primary adenocarcinomas of the base of the tongue (PABOTs) are extremely rare and worth reporting. To study the detailed clinicopathologic features of PABOT. Cases of PABOT diagnosed on pathology material were retrieved from the archived electronic surgical pathology records. Six cases in 4 men and 2 women (M:F ratio, 2:1), with an age range of 31 to 76 years, satisfied the criteria. The tumor epicenter was the base of the tongue in all (6 of 6; 100%), with extension to the epiglottis in 50% (3 of 6), nodal metastasis in 66.7% (4 of 6), and distant metastasis in 33.3% (2 of 6). On histology, all but one were pure adenocarcinoma. Five of 6 cases (83.3%) had a gastrointestinal (GI) phenotype, of which 2 (40%) had a colonic/lower-GI-type (small groups of cells floating in mucin, CK20+, SATB2+, and CDX2+) and 3 (60%) had an upper-GI-like adenocarcinoma (UGI-LA; malignant glands with intracellular mucin, CK7+) histology. Cystic structure suggestive of teratomatous origin was identified in 2 of 5 cases (40%), both with UGI-LA phenotype. The non-GI-type case had a unique histology with squamous differentiation in addition to adenocarcinoma areas, diffuse nuclear β-catenin on immunohistochemistry, and a corresponding exon 3 CTNNB1 mutation. One patient succumbed to disease, and 4 are alive with disease (follow-up of 1-9 months after completion of therapy). We suggest using the broad term primary adenocarcinomas of the base of tongue (PABOTs), which can be further subdivided into colonic-type adenocarcinoma of the tongue and oral cavity, UGI-LA, and not otherwise specified categories, and reiterate a need for recognition and distinction of PABOT from salivary gland tumors. A subset originates from teratoid/duplication cysts, necessitating extensive sampling. Multicentric studies are essential to clinically and biologically prognosticate each of these categories.

Açai Berry Administration Promotes Wound Healing through Wnt/β-Catenin Pathway

Recently, wound healing has received increased attention from both a scientific and clinical point of view. It is characterized by an organized series of processes: angiogenesis, cell migration and proliferation, extracellular matrix production, and remodeling. Many of these processes are controlled by the Wnt pathway, which activates them. The aim of the study was to evaluate the molecular mechanism of açai berry administration in a mouse model of wound healing. CD1 male mice were used in this research. Two full-thickness excisional wounds (5 mm) were performed with a sterile biopsy punch on the dorsum to create two circular, full-thickness skin wounds on either side of the median line on the dorsum. Açai berry was administered by oral administration (500 mg/kg dissolved in saline) for 6 days after induction of the wound. Our study demonstrated that açai berry can modulate the Wnt pathway, reducing the expression of Wnt3a, the cysteine-rich domain of frizzled (FZ)8, and the accumulation of cytosolic and nuclear β-catenin. Moreover, açai berry reduced the levels of TNF-α and IL-18, which are target genes strictly downstream of the Wnt/β-catenin pathway. It also showed important anti-inflammatory activities by reducing the activation of the NF-κB pathway. Furthermore, Wnt can modulate the activity of growth factors, such as TGF-β, and VEGF, which are the basis of the wound-healing process. In conclusion, we can confirm that açai berry can modulate the activity of the Wnt/β-catenin pathway, as it is involved in the inflammatory process and in the activity of the growth factor implicated in wound healing.

AMPK activator decelerates osteoarthritis development by inhibition of β-catenin signaling in chondrocytes.

Osteoarthritis (OA) is a common degenerative joint disease with significant negative impact on the quality of life. It has been reported that abnormal upregulation of β-catenin signaling could lead to OA development; however, the upstream regulatory mechanisms of β-catenin signaling have not been determined. Primary rat chondrocytes and ATDC5 chondrocyte cell line were stimulated with AKT2 and treated with or without metformin, an adenosine 5'-monophosphate-activated protein kinase (AMPK) activator. Westerrn blot analysis, luciferase reporter assay and immunofluorescent (IF) staining were performed to examine changes in β-cateninS552 phosphorylation and β-catenin nuclear translocation in ATDC5 cells and in primary chondrocytes. We found that metformin inhibited β-cateninS552 phosphorylation in ATDC5 cells and in primary chondrocytes in a time-dependent manner. Metformin inhibited β-catenin nuclear translocation and β-catenin reporter activity. In addition, metformin also attenuated the expression of β-catenin downstream target genes. We also demonstrated that metformin inhibited β-cateninS552 phosphorylation in articular cartilage in mice. These findings suggest that metformin may exert its chondro-protective effect at least in part through the inhibition of β-catenin signaling in chondrocytes. This study demonstrated the interaction between AMPK and β-catenin signaling in chondrocytes and defined novel molecular targets for the treatment of OA disease.

The complex relationship of Wnt-signaling pathways and cilia.

Wnt family proteins are secreted glycolipoproteins that signal through multitude of signal transduction pathways. The Wnt-pathways are conserved and critical in all metazoans. They are essential for embryonic development, organogenesis and homeostasis, and associated with many diseases when defective or deregulated. Wnt signaling pathways comprise the canonical Wnt pathway, best known for its stabilization of β-catenin and associated nuclear β-catenin activity in gene regulation, and several non-canonical signaling branches. Wnt-Planar Cell Polarity (PCP) signaling has received the most attention among the non-canonical Wnt pathways. The relationship of cilia to Wnt-signaling is complex. While it was suggested that canonical Wnt signaling requires cilia this notion was always challenged by results suggesting the opposite. Recent developments provide insight and clarification to the relationship of Wnt signaling pathways and cilia. First, it has been now demonstrated that while ciliary proteins, in particular the IFT-A complex, are required for canonical Wnt/β-catenin signaling, the cilium as a structure is not. In contrast, recent work has defined a diverged canonical signaling branch (not affecting β-catenin) to be required for ciliary biogenesis and cilia function. Furthermore, the non-canonical Wnt-PCP pathway does not affect cilia biogenesis per se, but it regulates the position of cilia within cells in many cell types, possibly in all cells where it is active, with cilia being placed near the side of the cell that has the Frizzled-Dishevelled complex. This Wnt/PCP feature is conserved with both centrioles and basal bodies/cilia being positioned accordingly, and it is also used to align mitotic spindles within the Wnt-PCP polarization axis. It also coordinates the alignment of cilia in multiciliated cells. This article addresses these new insights and different links and relationships between cilia and Wnt signaling.

E3 ubiquitin ligase TRIM21 targets TIF1γ to regulate β-catenin signaling in glioblastoma.

Background: Elucidation of the mechanism of ubiquitation has led to novel ways to treat glioblastoma (GBM). A tripartite motif (TRIM) protein mediates a reversible, stringent ubiquitation which is closely related to glioma malignancy. This study intends to screen the most vital and abnormal regulating component of the tripartite motif protein and to explore its underlying mechanisms. Methods: TRIM21 is identified as an important oncogene that accelerates the progression of glioma cell through database in a multidimensional way and this is confirmed in human samples and cells. Tandem Mass Tags (TMT) and MS analysis are performed to discover the substrates of TRIM21.The underlying mechanisms are further investigated by CO-IP, luciferase reporter assays and gain and loss of function assays. In vivo treatment with siRNA is applied to evaluate the therapeutic significance of TRIM21. Result: We screened a panel of TRIM proteins and identified TRIM21, a E3 ubiquitin-protein ligase and autoantigen, as well as a prognostic biomarker for GBM. Functionally, high expression of wild-type TRIM21 accelerates tumor progression in vitro and in vivo, whereas TRIM21 mutants, including one with a critical RING-finger deletion, do not. Mechanistically, TRIM21 stimulates K63-linked ubiquitination and subcellular translocation of active β-catenin from the cytoplasm to the nucleus. Moreover, TRIM21 forms a complex with the β-catenin upstream regulator, TIF1γ, in the nucleus and accelerated its degradation by inducing K48-linked ubiquitination at K5 site, consequently increasing further nuclear β-catenin presence. Endogenous TRIM21 levels are found to be inversely correlated with TIF1γ but positively correlated with β-catenin in glioma tissue microarray experiments. Furthermore, direct injection of TRIM21 small interfering RNA (siRNA) into U87 cell-derived tumors (in vivo treatment with siRNA) is proved to inhibit tumor growth in nude mice. Conclusion: This work suggests that TRIM21/TIF1γ/β-catenin axis is involved in the progression of human GBM. TRIM21 is a promising therapeutic and prognostic biomarker for glioma with hyperactive β-catenin.