Hsa_circ_0099198 facilitates the progression of retinoblastoma by regulating miR-1287/LRP6 axis.

Retinoblastoma (RB) is an intraocular malignancy that occurs in children. Circular RNAs (circRNAs) have been confirmed to play an essential role in tumorigenesis and development. This study aimed to ascertain the role and potential mechanism of hsa_circ_0099198 in RB. The levels of circ_0099198, microRNA-1287 (miR-1287) and low-density lipoprotein receptor-related protein 6 (LRP6) were determined by real-time quantitative polymerase chain reaction and Western blot. Cell proliferation was assessed by colony formation assay. Cell cycle arrest and apoptosis were evaluated by flow cytometry. Cell migration and invasion were tested using transwell assay. The activity of caspase-3/caspase-9 was examined with commercial kits. The interaction among circ_0099198, miR-1287 and LRP6 were verified by dual-luciferase reporter assay, RNA immunoprecipitation (RIP) assay or RNA pull-down assay. Xenograft experiment was used to assess tumor growth in vivo. circ_0099198 and LRP6 levels were increased, while miR-1287 level was reduced in RB cells. circ_0099198 silencing suppressed proliferation and metastasis and expedited cell cycle arrest and apoptosis in Y79 and So-RB50 cells. In addition, depletion of circ_0099198 inhibited RB cell progression via regulating miR-1287/LRP6 axis. Moreover, knockdown of circ_0099198 blocked the growth of xenograft tumors. circ_0099198 contributed to RB progression by sponging miR-1287 and up-regulating LRP6, which provided novel biomarkers for RB therapy.

Oncolytic Ad co-expressing decorin and Wnt decoy receptor overcomes chemoresistance of desmoplastic tumor through degradation of ECM and inhibition of EMT.

Pancreatic cancer is a highly lethal disease. Excessive accumulation of tumor extracellular matrix (ECM) and epithelial-to-mesenchymal transition (EMT) phenotype are two main contributors to drug resistance in desmoplastic pancreatic tumors. To overcome desmoplasia and chemoresistance of pancreatic cancer, we utilized an oncolytic adenovirus (Ad) co-expressing decorin and soluble Wnt decoy receptor (HEmT-DCN/sLRP6). An orthotopic pancreatic xenograft tumor model was established in athymic nude mice using Mia PaCa-2 cells, and the antimetastatic and antitumor efficacy of systemically administered HEmT-DCN/sLRP6 was evaluated. Immunohistochemical analysis of tumor tissues was performed to assess ECM degradation, induction of apoptosis, viral dispersion, and inhibition of the Wnt/ß-catenin signaling pathway. HEmT-DCN/sLRP6 effectively degraded tumor ECM and inhibited EMT, leading to enhanced viral distribution, induction of apoptosis, and attenuation of tumor cell proliferation in tumor tissue. HEmT-DCN/sLRP6 prevented metastasis of pancreatic cancer. Importantly, HEmT-DCN/sLRP6 sensitized pancreatic tumor to gemcitabine treatment. Furthermore, HEmT-DCN/sLRP6 augmented drug penetration and dispersion within pancreatic tumor xenografts and patient-derived tumor spheroids. Collectively, these results illustrate that HEmT-DCN/sLRP6 can enhance the dispersion of both oncolytic Ad and a chemotherapeutic agent in chemoresistant and desmoplastic pancreatic tumor, effectively overcoming the preexisting limitations of standard treatments.

Lipid rafts disruption induces apoptosis by attenuating expression of LRP6 and survivin in triple negative breast cancer.

Triple negative breast cancer is a clinically challenging subtype due to lack of biomarker for rational targeted therapy. Lipid rafts are cholesterol enriched rigid platforms, which colocalize signalling molecules of cancer progression. This study explores the effect of lipid rafts disruption by cholesterol depleting agent, MßCD on induction of apoptosis and expression of WNT receptor LRP6, survivin and common apoptotic markers in TNBC cell lines. The in vitro effect of lipid rafts disruption on viability, single cell reproductive ability, proliferation and migration were evaluated by MTT, clonogenic, BrdU incorporation and wound scratch assays, respectively. The morphological changes were assessed by tryphan blue, Wright and Giemsa staining; nuclear changes by Hoechst staining. The induction of apoptosis was evaluated by AO/EtBr staining, DNA damage and DNA fragmentation assays. Expression of Caveolin-1, LRP6, ß-Catenin, Survivin, Bcl2, BAX, Caspase-3, Ki67 and c-myc were analyzed by PCR and Western blotting techniques. The lipid raft disruption resulted in reduction of the proliferation of MDA-MB 231 and MDA-MB 468 cells by 56.3 and 42.0%; survival fraction by 54.7 and 59.4%; migration by 44.3 and 48.4%, respectively. It also induced apoptosis by causing cell shrinkage, membrane blebbing, nuclear condensation, chromatin cleavage, oligonucleotide fragmentation with an apoptotic index of 59.1 and 46.6% in MDA-MB 231 and 468 cells, respectively. Further, it downregulated the expression of caveolin-1, LRP6, ß-catenin, survivin, Bcl2, ki67, c-myc and upregulated BAX, caspase-3. The cholesterol supplementation enhanced the clonogenic potential and upregulated the expression of caveolin-1 and LRP6. The results underline a potential effect of lipid rafts disruption on induction of apoptosis in TNBC cells.

Diallyl trisulfide inhibits proliferation, invasion and angiogenesis of glioma cells by inactivating Wnt/ß-catenin signaling.

Aberrant activation of Wnt/ß-catenin signaling leads to increased cell proliferation and survival and promotes the development of various human tumors, including glioma, one of the most common primary brain tumors. The treatment efficacy of many anticancer drugs remains limited or unsatisfactory and it is urgently necessary to develop effective and low-toxicity anticancer drugs or strategies, especially for glioma. Here, we report that diallyl trisulfide suppresses survival, migration, invasion and angiogenesis in glioma cells. These effects were associated with inhibition of the Wnt/ß-catenin signaling cascade, which was accompanied by decreased expression of LRP6, TRIM29 and Pygo2. A dual-luciferase reporter assay confirmed that DATS treatment decreased TCF/LEF-mediated transcription. Finally, a nude mouse tumorigenicity model was used to examine the biological effect of diallyl trisulfide in vivo. Consistent with the previous results, diallyl trisulfide inhibited proliferation, invasion and angiogenesis in glioma cells by suppressing Wnt/ß-catenin signaling.

Long noncoding RNA papillary thyroid carcinoma susceptibility candidate 3 (PTCSC3) inhibits proliferation and invasion of glioma cells by suppressing the Wnt/ß-catenin signaling pathway.

BACKGROUND: The dysregulation of long noncoding RNAs (lncRNAs) has been identified in a variety of cancers. An increasing number of studies have found the critical role of lncRNAs in the regulation of cellular processes, such as proliferation, invasion and differentiation. Long noncoding RNA papillary thyroid carcinoma susceptibility candidate 3 (PTCSC3) is a novel lncRNA that was primarily detected in papillary thyroid carcinoma. However, the biological function and molecular mechanism of lncRNA PTCSC3 in glioma are still unknown. METHODS: The expression level of lncRNA PTCSC3 in human microglia and glioma cell lines was examined using quantitative real-time polymerase chain reaction (qRT-PCR). The influence of lncRNA PTCSC3 on cell proliferation were studied using the cell counting kit-8, and cell cycle and apoptosis were analyzed by flow cytometry assays. The migration and invasion abilities were investigated by transwell and wound healing assays. The target genes of lncRNA PTCSC3 were explored by qRT-PCR, immunofluorescence and western blot. RESULTS: LncRNA PTCSC3 was significantly downregulated in glioma cell lines. The overexpression of lncRNA PTCSC3 suppressed proliferation and induced apoptosis in U87 and U251 cells. Additionally, the overexpression of lncRNA PTCSC3 inhibited the migration and invasion of U87 and U251 cells. Moreover, lncRNA PTCSC3 inhibited the epithelial-mesenchymal transition of U87 cells. The study also demonstrated that LRP6, as a receptor of the Wnt/ß-catenin pathway, was a target of lncRNA PTCSC3. By evaluating the expression levels of Axin1, active ß-catenin, c-myc, and cyclin D1, the study indicated that lncRNA PTCSC3 inhibited the activation of the Wnt/ß-cateninpathway through targeting LRP6. CONCLUSIONS: LncRNA PTCSC3 inhibits the proliferation and migration of glioma cells and suppresses Wnt/ß-catenin signaling pathway by targeting LRP6. LncRNA PTCSC3 is a potential therapeutic target for treatment of glioma.

Sphingosine-1-phosphate-enhanced Wnt5a promotes osteogenic differentiation in C3H10T1/2 cells.

In this study, we investigated the involvement of Wnt signaling in sphingosine-1-phosphate (S1P)-enhanced osteogenic differentiation of C3H10T1/2 pluripotent stem cells. We found that S1P enhanced the expression of Wnt5a and low-density lipoprotein receptor-related protein 5 or 6 (LRP5/6) during osteogenic differentiation. Wnt5a-neutralizing antibody inhibited S1P-enhanced expression of LRP5/6 and alkaline phosphatase, which are essential for osteogenic differentiation. Conversely, S1P did not affect endogenous canonical Wnt signaling. Taken together, S1P-enhanced Wnt5a promotes LRP5/6 expression, resulting in the trigger of osteogenic differentiation of C3H10T1/2 cells. These findings suggest a potential beneficial role for S1P in bone regeneration.

Systems Genetics Analysis of a Recombinant Inbred Mouse Cell Culture Panel Reveals Wnt Pathway Member Lrp6 as a Regulator of Adult Hippocampal Precursor Cell Proliferation.

In much animal research, genetic variation is rather avoided than used as a powerful tool to identify key regulatory genes in complex phenotypes. Adult hippocampal neurogenesis is one such highly complex polygenic trait, for which the understanding of the molecular basis is fragmented and incomplete, and for which novel genetic approaches are needed. In this study, we aimed at marrying the power of the BXD panel, a mouse genetic reference population, with the flexibility of a cell culture model of adult neural precursor proliferation and differentiation. We established adult-derived hippocampal precursor cell cultures from 20 strains of the BXD panel, including the parental strains C57BL/6J and DBA/2J. The rates of cell proliferation and neuronal differentiation were measured, and transcriptional profiles were obtained from proliferating cultures. Together with the published genotypes of all lines, these data allowed a novel systems genetics analysis combining quantitative trait locus analysis with transcript expression correlation at a cellular level to identify genes linked with the differences in proliferation. In a proof-of-principle analysis, we identified Lrp6, the gene encoding the coreceptor to Frizzled in the Wnt pathway, as a potential negative regulator of precursor proliferation. Overexpression and siRNA silencing confirmed the regulatory role of Lrp6. As well as adding to our knowledge of the pathway surrounding Wnt in adult hippocampal neurogenesis, this finding allows the new appreciation of a negative regulator within this system. In addition, the resource and associated methodology will allow the integration of regulatory mechanisms at a systems level.

Wnt Signaling Inhibits Osteoclast Differentiation by Activating Canonical and Noncanonical cAMP/PKA Pathways.

Although there has been extensive characterization of the Wnt signaling pathway in the osteoblast lineage, the effects of Wnt proteins on the osteoclast lineage are less well studied. We found that osteoclast lineage cells express canonical Wnt receptors. Wnt3a reduced osteoclast formation when applied to early bone-marrow macrophage (BMM) osteoclast differentiation cultures, whereas late addition did not suppress osteoclast formation. Early Wnt3a treatment inactivated the crucial transcription factor NFATc1 in osteoclast progenitors. Wnt3a led to the accumulation of nuclear ß-catenin, confirming activation of canonical Wnt signaling. Reducing low-density lipoprotein receptor-related proteins (Lrp) 5 and Lrp6 protein expression prevented Wnt3a-induced inactivation of NFATc1; however, deletion of ß-catenin did not block Wnt3a inactivation of NFATc1, suggesting that this effect was mediated by a noncanonical pathway. Wnt3a rapidly activated the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway and pharmacological stimulation of cAMP/PKA signaling suppressed osteoclast differentiation; Wnt3a-induced NFATc1 phosphorylation was blocked by inhibiting interactions between PKA and A-kinase anchoring proteins (AKAPs). These data indicate that Wnt3a directly suppresses osteoclast differentiation through both canonical (ß-catenin) and noncanonical (cAMP/PKA) pathways in osteoclast precursors. In vivo reduction of Lrp5 and Lrp6 expressions in the early osteoclast lineage via Rank promoter Cre recombination reduced trabecular bone mass, whereas disruption of Lrp5/6 expression in late osteoclast precursors via cathepsin K (Ctsk) promoter Cre recombination did not alter the skeletal phenotype. Surprisingly, reduction of Lrp5/6 in the early osteoclast lineage decreased osteoclast numbers, as well as osteoblast numbers. Published studies have previously noted that ß-catenin signaling is required for osteoclast progenitor proliferation. Our in vivo data suggest that Rank promoter Cre-mediated deletion of Lrp5/6 may similarly impair osteoclast progenitor proliferation.

microRNA-513c suppresses the proliferation of human glioblastoma cells by repressing low-density lipoprotein receptor-related protein 6.

Aberrant activation of the Wnt/ß-catenin signaling pathway is frequently observed in glioblastoma (GBM) cells. Therefore, it was hypothesized that low­density lipoprotein receptor­related protein 6 (LRP6) may be involved in activating the Wnt/ß­catenin pathway in the progression of GBM. The present study reported that the expression of microRNA (miR)­513c was markedly downregulated in GBM cells and GBM tissues compared with that in normal human astrocytes and normal brain tissues. Previous studies have demonstrated that miR­513c is critical in a variety of biological processes in various human cancer cells. The role of this miR in GBM cells was therefore investigated in the present study. Ectopic expression of miR­513c reduced the proliferation and anchorage­independent growth of GBM cells, whereas inhibition of miR­513c promoted this effect. Bioinformatic analysis further identified LRP6, a putative tumor suppressor, as a potential target of miR­513c. Luciferase reporter assays revealed that miR­513c directly bound to the 3'­untranslated region of LRP6 mRNA and repressed the expression at the transcriptional as well as the translational level. In functional assays, miR­513c suppressed GBM cell proliferation, which was reversed by an inhibitor of miR­513c. In conclusion, the present study provided compelling evidence that miR­513c functions as a tumor suppressor miRNA, which may be important in the inhibition of cell proliferation in GBM. In addition, the tumor suppressive effects were mediated predominantly through the direct suppression of the expression of LRP6.

Salinomycin suppresses LRP6 expression and inhibits both Wnt/ß-catenin and mTORC1 signaling in breast and prostate cancer cells.

Emerging evidence indicates that activation of Wnt/ß-catenin signaling at the cell surface results in inhibition of glycogen synthase kinase 3ß (GSK3ß), leading to activation of mTORC1 signaling in cancer cells. The low density lipoprotein receptor-related protein-6 (LRP6) is an essential Wnt co-receptor for Wnt/ß-catenin signaling. Salinomycin is a novel small molecule inhibitor of LRP6. In the present study, we found that LRP6 overexpression induced mTORC1 signaling activation in cancer cells, and that salinomycin was not only a potent Wnt/ß-catenin signaling inhibitor, but also a strong mTORC1 signaling antagonist in breast and prostate cancer cells. Mechanistically, salinomycin activated GSK3ß in cancer cells. Moreover, salinomycin was able to suppress the expression of cyclin D1 and survivin, two targets of both Wnt/ß-catenin and mTORC1 signaling, in prostate and breast cancer cells, and displayed remarkable anticancer activity. Our results present novel mechanisms underlying salinomycin-mediated cancer cell death.

Fiber type-specific expression of low-density lipoprotein receptor-related protein 6 in human skeletal muscles.

OBJECTIVE: Gene expression patterns differ in the two types of skeletal muscle fiber. The Wnt signaling pathway, which includes low-density lipoprotein receptor-related protein 6 (LRP6), has been associated with cell differentiation and glucose metabolism in skeletal muscles. We examined the relationships between muscle fiber types and LRP6 expression. METHODS: Adenosine triphosphatase was assayed histochemically, and the levels of expression of LRP6 and myosin were analyzed immunohistochemically, in frozen sections of muscle fiber obtained from 16 muscle biopsy samples. The expression pattern of LRP6 in C2C12 cells was assayed by immunocytochemistry. RESULTS: LRP6 was expressed only in type II fibers. Type IIc fibers showed variations in LRP6 expression. Expression of LRP6 was observed at the stage of myoblast differentiation. CONCLUSION: Antibody to LRP6 may be useful for identifying type II skeletal muscle fibers. LRP6 may influence glucose metabolism in type II fibers of human skeletal muscles.

Molecular chaperone gp96 is a novel therapeutic target of multiple myeloma.

PURPOSE: gp96 (grp94) is a key downstream chaperone in the endoplasmic reticulum (ER) to mediate unfolded protein response (UPR) and the pathogenesis of multiple myeloma is closely linked to dysregulated UPR. In this study, we aimed to determine the roles of gp96 in the initiation and progression of multiple myeloma in vivo and in vitro. EXPERIMENTAL DESIGN: We generated a mouse model with overexpression of XBP1s and conditional deletion of gp96 in B-cell compartment simultaneously to identify the roles of gp96 in the development of multiple myeloma in vivo. Using a short hairpin RNA (shRNA) system, we silenced gp96 in multiple human multiple myeloma cells and examined the effect of gp96 knockdown on multiple myeloma cells by cell proliferation, cell-cycle analysis, apoptosis assay, immunohistochemistry, and human myeloma xenograft model. The anticancer activity of gp96 selective inhibitor, WS13, was evaluated by apoptosis assay and MTT assay. RESULTS: Genetic deletion of gp96 in XBP1s-Tg mice attenuates multiple myeloma. Silencing of gp96 causes severe compromise in human multiple myeloma cell growth through inhibiting Wnt-LRP-survivin pathway. We also confirmed that knockdown of gp96 decreased human multiple myeloma growth in a murine xenograft model. The targeted gp96 inhibitor induced apoptosis and blocked multiple myeloma cell growth, but did not induce apoptosis in pre-B leukemic cells. We have demonstrated that myeloma growth is dependent on gp96 both genetically and pharmacologically. CONCLUSIONS: gp96 is essential for multiple myeloma cell proliferation and survival, suggesting that gp96 is a novel therapeutic target for multiple myeloma. Clin Cancer Res; 19(22); 6242-51. ©2013 AACR.

Effect of pantoprazole on human gastric adenocarcinoma SGC7901 cells through regulation of phospho­LRP6 expression in Wnt/ß-catenin signaling.

Recent studies have found that an acidic tumor microenvironment is the key to managing cancer progression and metastasis. Our previous study found that proton pump inhibitors (PPIs) inhibit the expression of vacuolar-ATPases (V-ATPases) and reverse the transmembrane pH gradient. The present study was conducted to explore the effect of pantoprazole on gastric adenocarcinoma through the regulation of Wnt/ß-catenin signaling. We used SGC7901 human gastric cancer cells as an in vitro model to study the effect of pantoprazole. The antiproliferative, pro-apoptotic and anti­invasive effects of pantoprazole were examined. The effects of pantoprazole on the expression of the Wnt/ß-catenin signaling pathway were also studied by western blotting. Our study found that pantoprazole inhibited the proliferation and induced the apoptosis of SGC7901 human gastric cancer cells. The expression of V-ATPases was decreased following treatment with pantoprazole. Further study found that pantoprazole treatment caused a decrease in phospho-LRP6, but not in LRP6. ß-catenin in Wnt/ß-catenin signaling and its target genes c-Myc and cyclin D1 were also decreased upon the inhibition of V-ATPases. Therefore, pantoprazole could be characterized as a V-ATPase inhibitor for treating gastric cancer by inhibiting the phosphorylation of LRP6 in Wnt/ß-catenin signaling.

SOX9 regulates low density lipoprotein receptor-related protein 6 (LRP6) and T-cell factor 4 (TCF4) expression and Wnt/ß-catenin activation in breast cancer.

Gene expression profiling has identified breast cancer (BCa) subtypes, including an aggressive basal-like (BL) subtype. The molecular signals underlying the behavior observed in BL-BCa group are largely unknown, although recent results indicate a prevalent increase in Wnt/ß-catenin activity. Our immunohistochemistry study confirmed that SOX9, one of the BL-BCa signature genes, was expressed by most BL-BCa, and its expression correlated with indicators of poor prognosis. Importantly, BCa gene expression profiling strongly associated SOX9 with the expression of Wnt/ß-catenin pathway components, LRP6 and TCF4. In cancer cell lines, SOX9 silencing reduced cell proliferation and invasion, LRP6 and TCF4 transcription, and decreased Wnt/ß-catenin activation. SOX9 expression was also increased by Wnt, indicating that SOX9 is at the center of a positive feedback loop that enhances Wnt/ß-catenin signaling. Consistently, SOX9 overexpression in BCa cell lines and transgenic SOX9 expression in breast epithelium caused increased LRP6 and TCF4 expression and Wnt/ß-catenin activation. These results identify SOX9-mediated Wnt/ß-catenin activation as one of the molecular mechanisms underlying aberrant Wnt/ß-catenin activity in BCa, especially in the BL-BCa subgroup.

Antagonists of LRP6 regulate PTH-induced cAMP generation.

LRP6 is a common coreceoptor for different G protein-coupled seven-transmembrane receptors in production of cAMP. Extracelluar proteins sclerostin and DKK1, initially identified as antagonists for Wnt signaling by binding to LRP6, are negative regulators for bone formation. Here, we show that both sclerostin and DKK1 inhibit PTH-stimulated cAMP production. In addition, PTH suppresses expression of sclerostin in osteocytes in mice. We also found that sclerostin and DKK1 binds to LRP6 as antagonists to increase the availability of LRP6 to facilitate PTH signaling in a positive-feedback fashion. These studies reveal a previously unrecognized function of sclerostin and DKK1, which provides an alternative explanation for the application of sclerostin and DKK1 neutralization on enhancing bone formation as a potential therapy for skeletal diseases.