Cytoplasmic Β-catenin Levels Research Articles

Inhibition of DNA methyltransferase 1 by RNA interference reverses epithelial-mesenchymal transition in highly metastatic 95D lung cancer cells by inhibiting the Wnt signaling pathway.

Epigenetic modifications serve important roles in non-small cell lung cancer (NSCLC) tumorigenesis; however, the role of DNA methyltransferase 1 (DNMT1) in lung cancer progression remains unclear. In the present study, the expression of DNMT1 in the human NSCLC cell lines 95D (high invasive ability) and 95C (low invasive ability) was analyzed by western blotting. The results demonstrated that the expression of DNMT1 in 95D cells was significantly higher, compared with in 95C cells and small airway epithelial cells. To further define the role of DNMT1 in tumor migration and invasion in NSCLC cells, RNA interference was used to silence DNMT1 expression. Depletion of DNMT1 significantly inhibited 95D cell invasion and migration. In addition, treatment with DNMT1 small interfering RNA resulted in compact cell morphology and significantly increased epithelial marker E-cadherin expression whilst also decreasing the expression of certain mesenchymal markers, including vimentin and fibronectin. Suppression of DNMT1 increased cytoplasmic β-catenin levels while downregulating nuclear β-catenin and Snail, an important regulator of EMT. The results from the present study suggest that the inhibition of DNMT1 reverses the epithelial-mesenchymal transition partly via the inhibition of the Wnt/β-catenin signaling pathway, and therefore inhibits cell migration and invasion. These results indicate that targeting DNMT1 may inhibit tumor metastasis and that DNMT1 is a promising target for the novel treatment of lung cancer.

Chondroitin sulfate–mediated N-cadherin/β-catenin signaling is associated with basal-like breast cancer cell invasion

Tumor metastasis involves cancer cell invasion across basement membranes and interstitial tissues. The initial invasion step consists of adherence of the tumor cell to the extracellular matrix (ECM), and this binding transduces a variety of signals from the ECM to the tumor cell. Accordingly, it is critical to establish the mechanisms by which extracellular cues influence the intracellular activities that regulate tumor cell invasion. Here, we found that invasion of the basal-like breast cancer cell line BT-549 is enhanced by the ECM component chondroitin sulfates (CSs). CSs interacted with and induced proteolytic cleavage of N-cadherin in the BT-549 cells, yielding a C-terminal intracellular N-cadherin fragment that formed a complex with β-catenin. Of note, the cleavage of N-cadherin increased cytoplasmic and nuclear β-catenin levels; induced the matrix metalloproteinase 9 (MMP9) gene, a target of β-catenin nuclear signaling; and augmented the invasion potential of the cells. We also found that CS-induced N-cadherin proteolysis requires caveolae-mediated endocytosis. An inhibitor of that process, nystatin, blocked both the endocytosis and proteolytic cleavage of N-cadherin induced by CS and also suppressed BT-549 cell invasion. Knock-out of chondroitin 4-O-sulfotransferase-1 (C4ST-1), a key CS biosynthetic enzyme, suppressed activation of the N-cadherin/β-catenin pathway through N-cadherin endocytosis and significantly decreased BT-549 cell invasion. These results suggest that CSs produced by C4ST-1 might be useful therapeutic targets in the management of basal-like breast cancers.

MTA1 promotes epithelial to mesenchymal transition and metastasis in non-small-cell lung cancer.

The present study assessed the role of metastasis-associated protein 1 (MTA1) in epithelial to mesenchymal transition (EMT) and metastasis in non-small-cell lung cancer (NSCLC) cells using a normal lung epithelium cell line, three NSCLC cell lines, a mouse NSCLC model, and 56 clinical NSCLC samples. We observed that MTA1 overexpression decreased cellular adhesion, promoted migration and invasion, and changed cytoskeletal polarity. MTA1 knockdown had the opposite effects. MTA1 overexpression decreased E-cadherin, Claudin-1, and ZO-1 levels and increased Vimentin expression in vitro and in vivo, through activation of AKT/GSK3β/β-catenin signaling. However, treatment with the AKT inhibitor MK2206 did not completely rescue effects associated with MTA1 expression changes, indicating that pathways other than the AKT/GSK3β/β-catenin pathway could be involved in MTA1-induced EMT. Compared with normal lung tissues, MTA1 expression was elevated in NSCLC patient tissues and was correlated with American Joint Committee on Cancer stage, T stage, lymphatic metastasis, and patient overall survival. Additionally, MTA1 expression was positively associated with p-AKT and cytoplasmic β-catenin levels. These findings indicate MTA1 promotes NSCLC cell EMT and metastasis via AKT/GSK3β/β-catenin signaling, which suggests MTA1 may be an effective anti-NSCLC therapeutic target.

LZTS2 and PTEN collaboratively regulate ß-catenin in prostatic tumorigenesis.

The leucine zipper tumor suppressor 2 (LZTS2) was identified as a tumor susceptibility gene within the 10q24.3 chromosomal region, and is approximately 15Mb from the PTEN locus. This region containing the both loci is frequently deleted in a variety of human malignancies, including prostate cancer. LZTS2 is a ß-catenin-binding protein and a negative regulator of Wnt signaling. Overexpression of PTEN in prostate cancer cell lines reduces ß-catenin-mediated transcriptional activity. In this study, we examined the collaborative effect of PTEN and LZTS2 using multiple in vitro and in vivo approaches. Co-expression of PTEN and LZTS2 in prostate cancer cells shows stronger repressive effect on ß-catenin mediated transcription. Using a newly generated mouse model, we further assessed the effect of simultaneous deletion of Pten and Lzts2 in the murine prostate. We observed that mice with both Lzts2 and Pten deletion have an earlier onset of prostate carcinomas as well as an accelerated tumor progression compared to mice with Pten or Lzts2 deletion alone. Immunohistochemical analyses show that atypical and tumor cells from compound mice with both Pten and Lzts2 deletion are mainly composed of prostate luminal epithelial cells and possess higher levels of cytoplasmic and nuclear β-catenin. These cells also exhibit a higher proliferative capacity than cells isolated from single deletion mice. These data demonstrate the significance of simultaneous Pten and Lzts2 deletion in oncogenic transformation in prostate cells and implicates a new mechanism for the dysregulation of Wnt/β-catenin signaling in prostate tumorigenesis.

2-Methoxyestradiol-Mediated Induction of Frzb Contributes to Cell Death and Autophagy in MG63 Osteosarcoma Cells.

Osteosarcoma is a bone tumor that mainly affects children and adolescents. Although its pathogenesis is still not fully understood, activation of Wnt signaling has been implicated in the development and metastasis of osteosarcoma. In this report, we have investigated the effect of the anti-tumor compound, 2-methoxyestradiol (2-ME) on Wnt antagonist frizzled-related protein b (Frzb), also known as secreted frizzled-related protein (sFRP)3 in human osteosarcoma (MG63) cells. Our results show that 2-ME treatment induces Frzb gene promoter activity, and increases Frzb mRNA and protein levels in osteosarcoma cells. In addition, 2-ME treatment regulates downstream Wnt signaling, increasing the cytoplasmic levels of β-catenin, and blocking β-catenin-mediated Wnt activation in osteosarcoma cells. 2-ME-mediated induction of Frzb protein expression is specific to osteosarcoma cells, as it does not affect Frzb expression in normal primary human osteoblasts. Furthermore, 2-ME-induced apoptosis and autophagy are blocked in osteosarcoma cells transfected with Frzb siRNAs. Taken together, these studies demonstrate that Frzb protein plays an important role in 2-ME-mediated anti-tumor mechanisms in osteosarcoma cells. J. Cell. Biochem. 118: 1497-1504, 2017. © 2016 Wiley Periodicals, Inc.

IMST-22. THERAPEUTIC TARGETING OF THE Wnt-β-CATENIN PATHWAY ALLEVIATES GLIOMA-INDUCED IMMUNOSUPPRESSION AND CAUSES INHIBITION OF GLIOMA PROGRESSION

Glioblastoma multiforme (GBM) is the most common malignant neoplasm of astrocytic lineage. Despite aggressive treatment, GBM patients have a median survival of 12-15 months. The Wnt-β-catenin pathway has garnered attention in GBM research as it mediates stemness, proliferation and therapeutic resistance in human GBMs. Therapeutic targeting of this pathway could thus serve as an effective therapeutic option for GBM. The Wnt-β-catenin pathway attenuates T-cell activation and enhances survival of regulatory T cells. However, despite extensive studies on the role of this pathway in GBM progression, the significance of this pathway in glioma-induced immunosuppression has never been investigated. Here we hypothesize that targeting the Wnt-β-catenin pathway not only halts glioma progression but also causes alleviation of immunosuppression in GBMs. Immunofluorescence staining showed that in GL261/C57BL6 murine glioma model, huge overexpression of Wnt3a occurs in tumor compared to normal brain. Western blot revealed Wnt3a overexpression in both lysates of adherent GL261 cells as well as GL261 neurospheres. Treatment of GL261 cells with the inhibitor of porcupine, an O-acyl transferase necessary for Wnt secretion, results in decreased Wnt3a secretion and decreased cytoplasmic β-catenin levels, suggesting that the pathway is inhibited in GL261 cells upon treatment with the inhibitor. Graded dosing of GL261 cells with porcupine inhibitor showed cell viability dropping to 50% on 10µM treatment. Flowcytometric studies on T-cells isolated form murine spleen and draining lymph nodes showed significant increases (P<0.001) of both percentage of Tregs (from 4.05 ± 0.36% to 28.36 ± 2.67%) and their proliferation (from 27.73 ± 6.8% to 67.08 ± 3.49%) upon Wnt3a treatment whereas effector CD4 cells were decreased (P<0.001). Through the current study we suggest that overabundance of Wnt in glioma may be an additional means for glioma-induced T-cell immunosuppression and therefore propose a novel angle whereby therapeutic targeting of the Wnt-β-catenin pathway in GBM will alleviate glioma-mediated immunosuppression and inhibit glioma progression.

The isolation and characterization of systemic sclerosis vascular smooth muscle cells: enhanced proliferation and apoptosis resistance

Background Vascular dysfunction is a major pathologic component of systemic sclerosis (SSc). The role of vascular smooth muscle cells (vSMCs) in the development of vascular dysfunction is still unknown. In this study, we describe a method for isolation of dermal vSMCs from skin biopsies, and we outline their functional phenotype. Methods We obtained 4 mm punch-skin biopsies from three SSc patients and matched controls. After trypsin digestion, cells were cultured for 14 days. vSMCs were isolated by first depleting CD31+ cells (endothelial cells), followed by positive selection of CD146+ cells. The CD31- CD146+ cells were then cultured in media optimized for SMCs proliferation. We evaluated cell proliferation, viability and apoptosis in normal and in low serum culture conditions. Cytoplasmic and nuclear expression levels of β-catenin were also investigated. Results The CD31- CD146+ cell population expressed smooth muscle MYH11, Desmin and Vimentin but did not express NG2. Flow cytometry confirmed the high purity of CD31- CD146+ MYHC11+ cell population that was maintained for up to the eleventh passage. SSc-vSMCs exhibited increased cell proliferation and viability compared to control cells. Under serum starvation conditions, SSc-vSMCs exhibited more proliferative capacity, and resistance to apoptosis compared to control-vSMCs. Furthermore, a cytoplasmic to nuclear translocation of β-catenin was seen in SSc-vSMCs but not in control-vSMCs. Conclusions This is the first report of successful isolation and initial characterization of SSc-vSMCs. It is likely that increased proliferation of SSc-vSMCs in association with resistance to apoptosis can adversely impact the vascular lesion in SSc.

The receptor for advanced glycation end products is required for β-catenin stabilization in a chemical-induced asthma model.

Cytoplasmic retention of β-catenin will lead to its nuclear translocation and subsequent interaction with the transcription factor TCF/LEF that regulates target gene expression. We have previously demonstrated aberrant expression of β-catenin in a model of asthma induced by toluene diisocyanate (TDI). The aim of this study was to examine whether the receptor for advanced glycation end products (RAGE) can regulate β-catenin expression in TDI-induced asthma. Male BALB/c mice were sensitized and challenged with TDI to generate a chemically-induced asthma model. Inhibitors of RAGE, FPS-ZM1 and the RAGE antagonist peptide (RAP), were injected i.p. after each challenge. Airway resistance was measured in vivo and bronchoalveolar lavage fluid was analysed. Lungs were examined by histology and immunohistochemistry. Western blotting and quantitative PCR were also used. Expression of RAGE and of its ligands HMGB1, S100A12, S100B, HSP70 was increased in TDI-exposed lungs. These increases were inhibited by FPS-ZM1 or RAP. Either antagonist blunted airway reactivity, airway inflammation and goblet cell metaplasia, and decreased release of Th2 cytokines. TDI exposure decreased level of membrane β-catenin, phosphorylated Akt (Ser(473) ), inactivated GSK3β (Ser(9) ), dephosphorylated β-catenin at Ser(33) /(37) /Thr(41) , which controls its cytoplasmic degradation, increased phosphorylated β-catenin at Ser(552) , raised cytoplasmic and nuclear levels of β-catenin and up-regulated its targeted gene expression (MMP2, MMP7, MMP9, VEGF, cyclin D1, fibronectin), all of which were reversed by RAGE inhibition. RAGE was required for stabilization of β-catenin in TDI-induced asthma, identifying protective effects of RAGE blockade in this model.

Abstract 5171: Interleukin-17 acts through MMP7 to promote prostate cancer

Abstract Interleukin-17 (IL-17) is a key cytokine in inflammation, which has been shown to play critical roles in development of prostate cancer, skin cancer, colon cancer, and breast cancer. We have previously demonstrated that IL-17 promotes prostate carcinogenesis, accompanied with increased expression of matrix metalloproteinase 7 (MMP7) in the mouse prostate. The purpose of the present study was to determine whether MMP7 mediates IL-17's action and the underlying mechanisms. Mmp7 knockout (Mmp7−/−) mice were crossbred with phosphatase and tensin homolog (Pten) conditional knockout mice (Ptenpc-/−, prostate-specific through probasin promoter-driven Cre recombinase). The prostate tumor phenotypes were analyzed among Mmp7+/+;Ptenpc-/−, Mmp7+/−;Ptenpc-/−, and Mmp7−/−;Ptenpc-/− mice. MMP7-overexpression or MMP7 knockdown using small interfering RNA (siRNA) was generated in human prostate cancer LNCaP and C4-2B cell lines to explore the molecular mechanisms of MMP7's actions. We found that Mmp7−/−;Ptenpc-/− mice displayed prostates that were smaller than Mmp7+/+;Ptenpc-/− or Mmp7+/−;Ptenpc-/− mice. Mmp7−/−;Ptenpc-/− mice developed a reduced number of invasive prostate adenocarcinomas with lower rate of cellular proliferation and higher rate of apoptosis than Mmp7+/+;Ptenpc-/− or Mmp7+/−;Ptenpc-/− mice. In addition, the invasiveness of prostate tumor cells isolated from Mmp7−/−;Ptenpc-/− mice was significantly reduced compared to those isolated from Mmp7+/+;Ptenpc-/− mice in Matrigel® invasion assays. Expression of the epithelial-to-mesenchymal transition (EMT) markers, such as β-catenin, vimentin, Snail, and Slug, was dramatically reduced in Mmp7−/−;Ptenpc-/− mice compared to Mmp7+/+;Ptenpc-/− or Mmp7+/−;Ptenpc-/− mice. In contrast, expression of the epithelial markers such as E-cadherin and claudin was increased in Mmp7−/−;Ptenpc-/− mice compared to Mmp7+/+;Ptenpc-/− or Mmp7+/−;Ptenpc-/− mice. MMP7 cleaved E-cadherin and disrupted E-cadherin/β-catenin complex to increase cytoplasmic and nuclear β-catenin levels in the prostate cancer cells, thus up-regulating EMT transcription factors. In addition, IL-17 receptor C (Il-17rc) and Pten double knockout (Il-17rc−/−;Ptenpc-/−) mice echoed the reduction of EMT in Mmp7−/−;Ptenpc-/− mice, compared to Il-17rc+/+;Ptenpc-/− mice. Further, IL-17 treatment induced MMP7 expression in LNCaP and C4-2B cells, leading to increased expression of EMT markers. In contrast, MMP7 knockdown inhibited IL-17-induced expression of EMT markers. Taken together, our results demonstrate that MMP7 mediates IL-17's function in promoting prostate carcinogenesis through induction of EMT. This work was partially funded by National Institutes of Health (R01CA174714 and P20GM103518), Department of Defense (W81XWH-14-1-0050, W81XWH-14-1-0149, W81XWH-14-1-0458, and W81XWH-15-1-0444), and Tulane's Institute of Integrated Engineering for Health and Medicine. Citation Format: Qiuyang Zhang, Sen Liu, Keshab Parajuli, Zongbing You. Interleukin-17 acts through MMP7 to promote prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5171.

Suppression of β-catenin Signaling Pathway in Human Prostate Cancer PC3 Cells by Delphinidin.

Delphinidin possesses strong anti-oxidant, anti-inflammatory, and anti-cancer properties. Suppression of the Wnt/β-catenin signaling pathway is a potential strategy for chemoprevention and therapy. As aberrant activation of the β-catenin signaling pathway contributes to prostate cancer progression, we evaluated the effect of delphinidin on this pathway in human PC3 prostate cancer cells. An MTT assay showed that treatment with delphinidin (15–180 μM, 72 hours) resulted in a dose-dependent growth inhibition of cells. Treatment with delphinidin increased the phosphorylation of serine or threonine residues on β-catenin and decreased the levels of cytoplasmic β-catenin. Moreover, treatment with delphinidin inhibited the nuclear translocation of β-catenin and the expression of β-catenin target genes such as cyclin D1, c-myc, Axin-2, and T cell factor-1. Delphinidin also induced the phosphorylation of glycogen synthase kinase 3β and the expression of adenomatous polyposis coli and Axin proteins. Our results indicate that inhibition of cell growth by delphinidin is mediated, at least in part, through modulation of the β-catenin signaling pathway. We suggest that delphinidin is a potent inhibitor of Wnt/β-catenin signaling in prostate cancer cells.

Caught in a Wnt storm: Complexities of Wnt signaling in hematopoiesis.

The Wnt signaling pathway is an evolutionary conserved pathway that is involved in the development of almost every organ system in the body and provides self-renewal signals for most, if not all, adult stem cell systems. In recent years, this pathway has been studied by various research groups working on hematopoietic stem cells, resulting in contradicting conclusions. Here, we discuss and interpret the results of these studies and propose that Wnt dosage, the source of hematopoietic stem cells, and interactions with other pathways explain these disparate results.

Delphinidin, a major anthocyanin, inhibits 3T3-L1 pre-adipocyte differentiation through activation of Wnt/β-catenin signaling.

Delphinidin (Del) is a major anthocyanin that is widely found in pigmented fruits and vegetables. Herein, we investigated the antiadipogenic effect and the molecular mechanism by which Del affects 3T3-L1 preadipocyte differentiation. We found that Del effectively reduced intracellular lipid accumulation and promoted lipolysis while regulating the expression of adipogenic transcription factors and their target genes. This lipid lowering effect of Del was largely limited to the early phase of adipogenesis, which is governed by the delayed cell cycle progression due to G1 cell cycle arrest. Subsequently, Del suppressed the expression of early adipogenic transcription factors, such as C/EBPβ and C/EBPδ, as well as that of intermediate markers, C/EBPα, PPARγ, and the PPARγ-target adipocyte markers adiponectin and aP2 (FABP4). Furthermore, Del treatment activated Wnt1, Wnt10b, the Wnt receptor Fzd2, and the coreceptors Lrp5/6, while it inactivated Gsk3β, a member of the β-catenin destruction complex. Moreover, Del treatment stabilized cytoplasmic β-catenin levels and promoted its nuclear translocation, with subsequent activation of the expression of its downstream target genes, c-Myc and cyclin D1. Our findings therefore suggest that Del can effectively inhibit adipogenesis followed by the stabilization of β-catenin during early 3T3-L1 differentiation via inhibition of its destructive complexes and activation of Wnt signaling, and may thus be a promising candidate for the prevention of metabolic diseases, including obesity.

Mouse Crumbs3 sustains epithelial tissue morphogenesis in vivo.

The human apical protein CRB3 (Crb3 in mouse) organizes epithelial cell polarity. Loss of CRB3 expression increases the tumorogenic potential of cultured epithelial cells and favors metastasis formation in nude mice. These data emphasize the need of in vivo models to study CRB3 functions. Here, we report the phenotypic analysis of a novel Crb3 knockout mouse model. Crb3-deficient newborn mice show improper clearance of airways, suffer from respiratory distress and display perinatal lethality. Crb3 is also essential to maintain apical membrane identity in kidney epithelial cells. Numerous kidney cysts accompany these polarity defects. Impaired differentiation of the apical membrane is also observed in a subset of cells of the intestinal epithelium. This results in improper remodeling of adhesive contacts in the developing intestinal epithelium, thereby leading to villus fusion. We also noted a strong increase in cytoplasmic β-catenin levels in intestinal epithelial cells. β-catenin is a mediator of the Wnt signaling pathway, which is overactivated in the majority of colon cancers. In addition to clarifying the physiologic roles of Crb3, our study highlights that further functional analysis of this protein is likely to provide insights into the etiology of diverse pathologies, including respiratory distress syndrome, polycystic kidney disease and cancer.

Tuning WNT-β-catenin signaling via BCL9 proteins for targeting colorectal cancer cells

Tuning WNT-β-catenin signaling via BCL9 proteins for targeting colorectal cancer cells

β-Catenin is required for intrinsic but not extrinsic BCR-ABL1 kinase-independent resistance to tyrosine kinase inhibitors in chronic myeloid leukemia.

Activation of nuclear β-catenin and expression of its transcriptional targets promotes chronic myeloid leukemia (CML) progression, tyrosine kinase inhibitor (TKI) resistance, and leukemic stem cell self-renewal. We report that nuclear β-catenin plays a role in leukemia cell-intrinsic but not -extrinsic BCR-ABL1 kinase-independent TKI resistance. Upon imatinib inhibition of BCR-ABL1 kinase activity, β-catenin expression was maintained in intrinsically resistant cells grown in suspension culture and sensitive cells cultured in direct contact (DC) with bone marrow (BM) stromal cells. Thus, TKI resistance uncouples β-catenin expression from BCR-ABL1 kinase activity. In β-catenin reporter assays, intrinsically resistant cells showed increased transcriptional activity versus parental TKI-sensitive controls, and this was associated with restored expression of β-catenin target genes. In contrast, DC with BM stromal cells promoted TKI resistance, but had little effects on Lef/Tcf reporter activity and no consistent effects on cytoplasmic β-catenin levels, arguing against a role for β-catenin in extrinsic TKI resistance. N-cadherin or H-cadherin blocking antibodies abrogated DC-based resistance despite increasing Lef/Tcf reporter activity, suggesting that factors other than β-catenin contribute to extrinsic, BM-derived TKI resistance. Our data indicate that, while nuclear β-catenin enhances survival of intrinsically TKI-resistant CML progenitors, it is not required for extrinsic resistance mediated by the BM microenvironment.

Abstract 3791: The inhibition of protein kinase CK2 activity by the CIGB-300 synthetic peptide impairs the three-dimensional cell growth, Wnt and nuclear factor қB signaling pathways in lung cancer cells

Abstract CK2 is a serine/threonine kinase involved in cell growth, survival and apoptosis. The CIGB-300 is a synthetic peptide capable of binding to CK2 substrates thus preventing the enzyme activity. Previously we have determined that CIGB-300 presented an inhibitory concentration 50 dose (IC50) of 119±2.4 µM in NCI-H125 human lung cancer cells. Throughout an Annexin V-FITC assay we could determine that CIGB-300 is a potent apoptosis inductor since the treatment with this drug for 60 minutes induced apoptosis at a level comparable with that observed with etoposide (10 µM). Concomitantly with cell death induction, we could observe caspase-3 activation and the decreased expression of c-myc and cyclin D1 and D2. In this work we analyzed whether CIGB-300 is able to alter the ability of cells to grow in 3D and to modulate signaling pathways involved in tumor progression. First, we developed stable spheroids of NCI-H125 cells, which were able to grow in culture for at least 14 days. After 5 days of treatment, CIGB-300 induced a significant growth inhibition (median volume: 4.07x107±0.70x107 µm3 in control spheroids vs 2.13x107±0.35x107 µm3 in spheroids treated with the IC50 dose p&amp;lt;0.05). From the sixth day of treatment the size of the spheroids was dramatically reduced, returning to the initial volume on day eight. To analyze the effect of CIGB-300 on canonical Wnt signaling, this pathway was initially activated by incubating NCI-H125 cells with a conditioned media containing Wnt3a factor. This incubation led to a significant increase in the levels of cytoplasmic β-catenin. By Western blot we could observe that treatment with CIGB-300 blocked this increase, suggesting its role in the modulation of the Wnt pathway. In order to analyze the nuclear factor κB (NF-κB) dependent pathway, this transcription factor was activated by a phorbol ester treatment (PMA, 15 nM). Short treatment (15 min) with CIGB-300 induced an important reduction in NF-κB nuclear levels. However, this inhibition was reverted 24 h later as determined by Western blot and reporter gene expression assays. Our results indicate that the treatment with CIGB-300 induces a significant anti-proliferative response in a three-dimensional model, which resembles in vivo tumor growth conditions, also affecting key signaling pathways involved in tumor progression. Altogether, our data suggest that this peptide may become a new strategy for the treatment of lung cancer malignancies. Citation Format: Stéfano M. Cirigliano, María Inés Díaz Bessone, Carolina Flumian, Damián Emilio Berardi, Silvio E. Perea, Elisa Bal De Kier Joffé, Hernán Farina, Laura Todaro, Alejandro Urtreger. The inhibition of protein kinase CK2 activity by the CIGB-300 synthetic peptide impairs the three-dimensional cell growth, Wnt and nuclear factor қB signaling pathways in lung cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3791. doi:10.1158/1538-7445.AM2014-3791

Galectin-4 expression is associated with reduced lymph node metastasis and modulation of Wnt/β-catenin signalling in pancreatic adenocarcinoma.

Galectin-4 (Gal-4) has been recently identified as a pivotal factor in the migratory capabilities of a set of defined pancreatic ductal adenocarcinoma (PDAC) cell lines using zebrafish as a model system. Here we evaluated the expression of Gal-4 in PDAC tissues selected according to their lymph node metastatic status (N0 vs. N1), and investigated the therapeutic potential of targeting the cross-link with the Wnt signaling pathway in primary PDAC cells. Analysis of Gal-4 expression in PDACs showed high expression of Gal-4 in 80% of patients without lymph node metastasis, whereas 70% of patients with lymph node metastases had low Gal-4 expression. Accordingly, in primary PDAC cells high Gal-4 expression was negatively associated with migratory and invasive ability in vitro and in vivo. Knockdown of Gal-4 in primary PDAC cells with high Gal-4 expression resulted in significant increase of invasion (40%) and migration (50%, P<0.05), whereas enforced expression of Gal-4 in primary cells with low Gal-4 expression reduced the migratory and invasive behavior compared to the control cells. Gal-4 markedly reduces β-catenin levels in the cell, counteracting the function of Wnt signaling, as was assessed by down-regulation of survivin and cyclin D1. Furthermore, Gal-4 sensitizes PDAC cells to the Wnt inhibitor ICG-001, which interferes with the interaction between CREB binding protein (CBP) and β-catenin. Collectively, our data suggest that Gal-4 lowers the levels of cytoplasmic β-catenin, which may lead to lowered availability of nuclear β-catenin, and consequently diminished levels of nuclear CBP-β-catenin complex and reduced activation of the Wnt target genes. Our findings provide novel insights into the role of Gal-4 in PDAC migration and invasion, and support the analysis of Gal-4 for rational targeting of Wnt/β-catenin signaling in the treatment of PDAC.

β-catenin mediates the inflammatory cytokine expression induced by the Der p 1 house dust mite allergen

The modulations of β-catenin were analyzed during the inflammatory response induced by the Der p 1 house dust mite allergen. Der p 1 induced the dose-dependent expression of inflammatory cytokines, including interleukin (IL)-6, IL-8, monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-α (TNF-α) in THP-1 human monocytic cells. The mRNA expression levels of β-catenin were not altered, however protein levels increased following Der p 1 treatment, demonstrating that β-catenin was modulated by post-transcriptional processes. It was also revealed that nuclear β-catenin levels were significantly increased while cytoplasmic β-catenin levels were reduced, which demonstrated the nuclear translocation of β-catenin by the Der p 1 allergen. Glycogen synthase kinase 3β (GSK3β), a regulator of β-catenin stability, was demonstrated to be phosphorylated following Der p 1 treatment. When β-catenin was knocked down by the transfection of its small interfering RNA (siRNA), inflammatory cytokine expression as well as nuclear factor-κB (NF-κB) activity, which were induced by Der p 1 treatment, were all significantly reduced. The results demonstrated that Der p 1-induced inflammatory responses were mediated by β-catenin.

PI3K/Akt Signaling Interacts With Wnt/β-Catenin Signaling But Does Not Induce An Accumulation Of β-Catenin In The Nucleus Of Acute Lymphoblastic Leukemia Cell Lines

Signaling pathways play essential roles in biological processes as development, cell proliferation and homeostasis. The accurate modulation of signaling pathways, their adapted interaction and their time- and tissue-specific adjusted regulation are required for normal cell development. PI3K/Akt and Wnt/β-Catenin signaling pathways act as key regulators in cell proliferation, differentiation and growth. Both signaling pathways include GSK3β as a common protein, which may mediate an interaction and cross-talk between the pathways. Aberrant activation of PI3K/Akt signaling has been linked to different types of leukemia while Wnt/β-Catenin signaling is known to be deregulated in some solid tumors. However, a potential role of Wnt/β-Catenin signaling for pathogenesis of acute lymphoblastic leukemia (ALL) has not yet been analyzed.In our study we analyzed both signaling pathways in different B- and T-ALL cell lines (RS4;11, SEM, REH, CEM, Jurkat, MOLT-4), thereby focusing mainly on their potential interaction via the protein GSK3β. Western Blot experiments were performed to evaluate the expression of specific PI3K/Akt and Wnt/β-Catenin key proteins. To evaluate the activation status of Wnt signaling immunofluorescence and protein fractionation experiments were performed, analyzing the activation linked nucleic localization of β-Catenin. The effect of pathway activation and inhibition on cell proliferation via chemical compounds was analyzed by WST-1 test.High pAkt levels were detected in B-ALL cell line SEM and T-ALL cell line CEM, indicating a hyperactive PI3K/Akt signaling, whereas other analyzed cell lines diplayed lower pAkt status. Among all cell lines analyzed SEM and CEM also showed the highest cytoplasmic β-Catenin levels, indicating a direct interaction of both signaling pathways. However, immunofluorescence and fractionation experiments revealed that a translocation of β-Catenin into the nucleus did not occur. To further investigate the role and interaction of PI3K/Akt and Wnt/β-Catenin signaling, pathway inhibiting and stimulating experiments were performed. Treatment of cells with Wnt3a led to activation of the Wnt/β-Catenin signaling cascade, characterized by nuclear β-Catenin accumulation. Inhibition of cell proliferation was detected after treatment with high concentrations Wnt3a (≥ 500 ng/ml). PI3K inhibition by LY294002 led to decreased phosphorylation of GSK3β at Ser9 and an increased decay of β-Catenin. Stimulation of PI3K/Akt signaling using activating ligand FLT3L induced GSK3β phosphorylation at Ser9 and accumulation of cytoplasmic β-Catenin. However a translocation of β-Catenin into the nucleus seems not to occur.In summary our results indicate that PI3K/Akt and Wnt/β-Catenin signaling can interact through their common protein GSK3β, but stimulation of the PI3K/Akt signaling pathway by addition of PI3K/Akt specific activators does not fully activate Wnt/β-Catenin signaling in ALL cells. Complete activation of the Wnt cascade characterized by translocation of β-Catenin into the nucleus can only be induced by use of specific Wnt effectors. Disclosures:No relevant conflicts of interest to declare.

405 The Critical Role of Heparan Sulfate in Intestinal Crypt Regeneration of Small Intestine via Wnt/β-Catenin Signaling After Radiation Injury

[Background & Aim] Heparan sulfate (HS) is a linear polysaccharide present on the cell surface. HS proteoglycans modulate functions of several growth factors and signaling molecules. Despite many evidences demonstrating that HSPGs play pivotal roles in embryogenesis, it remains unclear how HSPGs are involved intestinal epithelial homeostasis of small intestine. The aim of this study is to examined the role of epithelial HS in crypt regeneration of small intestine after radiation injury. [Methods](1)We used Villin-Cre mice and conditional Ext1 allele (Ext1flox) . To examine whether HS on IEC of the small intestine affects crypt homeostasis, we generatedmice with intestine-specific conditional Ext1 knockout by crossing Ext1flox/flox mice with Villin-Cre mice as reported previously. (2) Total body irradiation (TBI) was performed in wild type (WT) mice and conditional KO(cKO) to examine the effects of intestinal epithelial HS on radiation damage to the small intestine in vivo. (3) We investigated the Wnt/beta-catenin pathways in intestinal epithelial cells (IECs) after TBI by using primay mouse IECs isolated from small intestine of cKO and WT mice. [Results] (1) Both macroscopic and microscopic findings were normal in cKO mice, suggesting that HS on IECs are not critically involved in crypt homeostasis in the steady state. (2) All cKO mice with 12Gy TBI died within 6 days, while 71% of WT mice were alive more than 30 days after TBI (p,0.01) Histologic evaluation 4 days after 12Gy TBI revealed the small intestinal mucosa with regenerative crypts covering most of the inner intestinal surface in WT mice, while severe intestinal damage with almost no villi or crypts in the small intestinal mucosa of cKO mice was observed. Intestine-specific deletion of Ext1 significantly decreased the number of surviving crypts, which were labeled with BrdU 3.5 days after 10Gy TBI. (3) Immunoblot analysis showed the protein levels of total β-catenin were lower in IECs isolated from the small intestine of cKO mice 4 days after 10Gy TBI than those of WT mice. Non-phosphorylated β-catenin (active β-catenin) was also lower in cKO mice, suggesting the decreased protein levels of cytoplasmic β-catenin with attenuated canonical Wnt signaling in IECs of cKO mice. HS-deficient IECs reduced expression of Wnt target genes including Lgr5 during crypt regeneration. Epithelial HS increased Wnt binding affinity of IECs and promotedWnt/β-catenin signaling following ex vivo stimulation withWnt3a, whereas activation of canonical Wnt signaling following direct inhibition of glycogen synthase kinase-3 β by lithium chloride was similar between HS-deficient and wild-type mice. [Conclusion] HS influences the binding affinity of IECs to Wnt, thereby promoting activation of canonical Wnt signaling and facilitating regeneration of small intestinal crypts after epithelial injury.