LRP6 Receptor Plays Essential Functions in Development and Human Diseases

LRP6 is a member of the low-density lipoprotein receptor superfamily of cell-surface receptors. It is required for the activation of the Wnt/β-catenin signalling pathway. LRP6 is detected in different tissue types and is involved in numerous biological activities such as cell proliferation, specification, metastatic cancer, and embryonic development. LRP6 is essential for the proper development of different organs in vertebrates, such as Xenopus laevis, chickens, and mice. In human, LRP6 overexpression and mutations have been reported in multiple complex diseases including hypertension, atherosclerosis, and cancers. Clinical studies have shown that LRP6 is involved in various kinds of cancer, such as bladder and breast cancer. Therefore, in this review, we focus on the structure of LRP6 and its interactions with Wnt inhibitors (DKK1, SOST). We also discuss the expression of LRP6 in different model systems, with emphasis on its function in development and human diseases.

DDRE-15. Sema3C SIGNALING CONFERS RESISTANCE TO Wnt INHIBITION IN GLIOBLASTOMA

BACKGROUND Wnt signaling is widely dysregulated in cancer. The therapeutic potential of Wnt inhibitors appears promising in preclinical studies. However, they have uniformly failed clinical trials. How cancer cells develop Wnt inhibitor resistance is poorly understood. Current Wnt inhibitors are designed targeting either ligand or receptor. We hypothesized cancer cells will bypass ligand-receptor interaction through an unknown mechanism. We focused on the neurodevelopmental signaling program of Semaphorin 3C (Sema3C) that is upregulated in 85% of GBM and regulates glioma stem-cell-driven tumor progression. RESULTS Porcupine inhibitor LGK974 reduced TCF1 expression in the GBM tumor mouse models, suggesting successful target engagement in vivo. However, it failed to prolong the overall survival. Sema3C expression strongly correlated with TCF1 expression in human GBM samples by immunohistochemical analysis. Genetic inhibition of Sema3C and TCF1 together prolonged animal survival more than either alone, indicating better control of Wnt pathway signaling with dual pathway blockade. Immunofluorescence and cell fractionation studies revealed that Sema3C signaling drove β-catenin nuclear accumulation. Sema3C regulates transactivation of Wnt target genes including TCF1, c-Myc and c-Met. Sema3C pathway activates Rac1. It is reported that Rac1 activates β-catenin and promotes β-catenin nuclear accumulation. In GSCs, constitutively active Rac1 restored β-catenin nuclear localization and rescued TCF1 and c-Myc down-regulation in the setting of Sema3C silencing. Sema3C can drive canonical Wnt signaling even when Wnt ligand secretion is blocked. Together, the data support that GSCs can escape Wnt inhibition through Sema3C and Rac1. CONCLUSIONS Sema3C signaling drives canonical Wnt signaling, providing an escape mechanism for cancer cells despite Wnt ligand-receptor interruption. Sema3C-β-catenin signaling promotes GSC self-renewal and tumor progression. Upstream Wnt pathway inhibition alone is insufficient to control tumors. Our data provide a therapeutic strategy of dual blockade of Wnt and Sema3C pathways to provide clinically significant tumor control.

Osteoblast/osteocyte-derived interleukin-11 regulates osteogenesis and systemic adipogenesis

Exercise offers mechanical loading to the bone, while it stimulates energy expenditure in the adipose tissue. Thus, bone may secrete a factor to communicate with adipose tissue in response to mechanical loading. Interleukin (IL)-11 is expressed in the bone, upregulated by mechanical loading, enhances osteogenesis and suppresses adipogenesis. Systemic IL-11 deletion (IL-11−/−) exhibited reduced bone mass, suppressed bone formation response to mechanical loading, enhanced expression of Wnt inhibitors, and suppressed Wnt signaling. Enhancement of bone resorption under mechanical unloading was unaffected. Unexpectedly, IL-11−/− mice showed increased systemic adiposity and glucose intolerance. Osteoblast/osteocyte-specific IL-11 deletion in osteocalcin-Cre;IL-11fl/fl mice showed reduced serum IL-11, blunted bone formation under mechanical loading, and increased systemic adiposity similar to IL-11−/− mice. Adipocyte-specific IL-11 deletion in adiponectin-Cre; IL-11fl/fl mice exhibited no abnormality. Thus, IL-11 from osteoblast/osteocyte controls both osteogenesis and systemic adiposity in response to mechanical loading. These findings may bring new therapeutic approaches to osteoporosis and metabolic syndrome.

Myeloma–Bone Interaction: A Vicious Cycle via TAK1–PIM2 Signaling

Multiple myeloma (MM) has a propensity to develop preferentially in bone and form bone-destructive lesions. MM cells enhance osteoclastogenesis and bone resorption through activation of the RANKL–NF-κB signaling pathway while suppressing bone formation by inhibiting osteoblastogenesis from bone marrow stromal cells (BMSCs) by factors elaborated in the bone marrow and bone in MM, including the soluble Wnt inhibitors DKK-1 and sclerostin, activin A, and TGF-β, resulting in systemic bone destruction with loss of bone. Osteocytes have been drawn attention as multifunctional regulators in bone metabolism. MM cells induce apoptosis in osteocytes to trigger the production of factors, including RANKL, sclerostin, and DKK-1, to further exacerbate bone destruction. Bone lesions developed in MM, in turn, provide microenvironments suited for MM cell growth/survival, including niches to foster MM cells and their precursors. Thus, MM cells alter the microenvironments through bone destruction in the bone where they reside, which in turn potentiates tumor growth and survival, thereby generating a vicious loop between tumor progression and bone destruction. The serine/threonine kinases PIM2 and TAK1, an upstream mediator of PIM2, are overexpressed in bone marrow stromal cells and osteoclasts as well in MM cells in bone lesions. Upregulation of the TAK1–PIM2 pathway plays a critical role in tumor expansion and bone destruction, posing the TAK1–PIM2 pathway as a pivotal therapeutic target in MM.

Isolation and Identification of Isocoumarin Derivatives With Specific Inhibitory Activity Against Wnt Pathway and Metabolome Characterization of Lasiodiplodia venezuelensis

The Wnt signaling pathway controls multiple events during embryonic development of multicellular animals and is carcinogenic when aberrantly activated in adults. Breast cancers are dependent on Wnt pathway overactivation mostly through dysregulation of pathway component protein expression, which necessitates the search for therapeutically relevant compounds targeting them. Highly diverse microorganisms as endophytes represent an underexplored field in the therapeutic natural products research. In the present work, the objective was to explore the chemical diversity and presence of selective Wnt inhibitors within a unique collection of fungi isolated as foliar endophytes from the long-lived tropical palm Astrocaryum sciophilum. The fungi were cultured, extracted with ethyl acetate, and screened for their effects on the Wnt pathway and cell proliferation. The endophytic strain Lasiodiplodia venezuelensis was prioritized for scaled-up fractionation based on its selective activity. Application of geometric transfer from analytical HPLC conditions to semi-preparative scale and use of dry load sample introduction enabled the isolation of 15 pure compounds in a single step. Among the molecules identified, five are original natural products described for the first time, and six are new to this species. An active fraction obtained by semi-preparative HPLC was re-purified by UHPLC-PDA using a 1.7 µm phenyl column. 75 injections of 8 µg were necessary to obtain sufficient amounts of each compound for structure elucidation and bioassays. Using this original approach, in addition to the two major compounds, a third minor compound identified as (R)-(-)-5-hydroxymellein (18) was obtained, which was found to be responsible for the significant Wnt inhibition activity recorded. Further studies of this compound and its structural analogs showed that only 18 acts in a highly specific manner, with no acute cytotoxicity. This compound is notably selective for upstream components of the Wnt pathway and is able to inhibit the proliferation of three triple negative breast cancer cell lines. In addition to the discovery of Wnt inhibitors of interest, this study contributes to better characterize the biosynthetic potential of L. venezuelensis.

BIOL-01. THE RELATIONSHIP OF BRAIN ENDOTHELIAL WNT SIGNAL INHIBITION ON BLOOD-TUMOR BARRIER INTEGRITY

The blood-tumor barrier (BTB) is the primary site of nutrient and drug transport to tumor cells such as malignant gliomas. Yet, signaling pathways and factors influencing BTB permeability are poorly understood. Previous studies demonstrate the role of WNT/β-catenin signaling in establishing and fortifying blood-brain barrier integrity in a non-diseased state. Additionally, WNT proteins are highly expressed in gliomas and their surrounding vasculature. Thus, we propose inhibition of WNT/β-catenin signaling at the brain endothelium of malignant glioma can impair BTB integrity to enhance permeability for select cytotoxic agents. We used immortalized mouse brain endothelial cells (bEnd.3), akin to brain tumor endothelium, treated for 24 hours with WNT inhibitors (ICG-001, IWR-1, and LGK974). Inhibition of WNT/β-catenin signaling was confirmed by gene expression of transcription factors (Tcf4 and Birc5). Cell viability was confirmed by CellTiter Glo®. Brain endothelial cell-cell interaction was evaluated by cell impedance and resistance via the Agilent xCELLigence and ABP TEER24 systems. Using qPCR and flow cytometry, we observed changes in expression and function of Abcb1 and Abcg2 transporters. Using an in vitro BTB (bEnd.3 cells and mouse H3.3WT/K27 glioma cells) we evaluated the effect of WNT inhibition on permeability and glioma viability. We found that all the inhibitors downregulated Tcf4 and Birc5 in brain endothelium dose-dependently. Viability with inhibitors demonstrated an IC50 of 28μM for ICG-001, and 42μM for both IWR-1 and LGK974. Endothelial cell-cell interaction was transiently decreased by approximately 50% with all inhibitors at 30 minutes; increasing closer to baseline after 2-4hrs. All WNT inhibitors dose-dependently decreased Abcg2 transporter expression and function. While In vitro BTB studies are ongoing, preliminary findings demonstrate increasing permeability of BTB amongst H3.3K27 glioma cells. Our results demonstrate potential of WNT inhibitors to modulate BTB integrity and drug efflux function. More studies are warranted to explore WNT/β-catenin signaling inhibition on BTB in vivo.

sFRP1 Expression Regulates Wnt signaling in Chronic Myeloid Leukemia K562 Cells

Background: Wnt signaling cascades play important roles in cell fate decisions and their deregulation has been documented in many diseases, including malignant tumors and leukemia. One mechanism of aberrant Wnt signaling is the silencing of Wnt inhibitors through epigenetic mechanisms. The sFRPs are one of the most studied Wnt inhibitors; and the sFRP1 loss is known in many hematological malignancies. Therefore, we aimed to compare the expression of Wnt related genes in the presence and absence of sFRP1 in chronic myeloid leukemia (CML) cell line. Objective: It is important to understand how sFRP1 and sFRP1 perform on CML to design new agents and strategies for resistant and advanced forms of CML. Materials and Methods: We used K562 cells, which normally do not express sFRP1 and its sFRP1 expressing subclone K562s. Total RNA was isolated from K562 and K562s cell lines end converted cDNA. PCR Array experiments performed using Human Wnt Signaling Pathway Plus RT2 Profiler™ kit. Wnt signaling pathway activation was studied by western blot for downstream signaling targets. Results: The WNT3, LRP6, PRICKLE1 and BTRC expressions were significantly decreased in the presence of sFRP1; while WNT5B increased. The sFRP1 expression inhibited stabilization of total β-catenin protein and downstream effector phosphorylation of noncanonical Wnt/PCP signaling; whereas Ca2+/PKC signaling remained active. Conclusion: The results suggest that sFRP1 could be a promising therapeutic anticancer agent. Defining these pathway interactions is crucial to design new agents resistant and advanced forms of CML.

Impaired bone microarchitecture in premenopausal women with acromegaly: the possible role of Wnt signaling

Context Acromegaly can impair bone integrity, increasing the risk of vertebral fractures (VF). Objective To evaluate the impact of isolated GH/IGF-I hypersecretion on bone turnover markers,Wnt inhibitors, BMD, microarchitecture, bone strength and vertebral fractures in female patients with acromegaly (Acro), compared to healthy control group (HC). Design, setting, and patients Cross-sectional study including 83 premenopausal women without any pituitary deficiency:18 acromegaly in remission (AcroR), 12 in group with active acromegaly (AcroA) and 53 HC. Serum P1NP, β-CTX, osteocalcin, sclerostin and DKK1 were measured in blood samples. DXA, HR-pQCT and vertebral fractures evaluation were also assessed simultaneously. Main outcome and Results AcroA showed significantly lower sclerostin and higher DKK1 as compared to HC. On HR-pQCT of tibia and radius, Acro showed impairment of trabecular (area and trabecular number), increased cortical porosity and increased cortical area and cortical thickness compared to HC. The only significant correlation found with HR-pQCT parameters was a positive correlation between cortical porosity and serum DKK1 (R=0.45, P=0.044). Mild VF were present in approximately thirty percent of patients. Conclusions Eugonadal women with acromegaly without any pituitary deficiency showed increased cortical BMD, impairment of trabecular bone microstructure and increased VF. Sclerostin was not correlated with any HR-pQCT parameters, however DKK1 was correlated with cortical porosity in tibia (P=0.027). Additional studies are needed to clarify, the role of Wnt inhibitors on bone microarchitecture impairment in acromegaly.