Mitigating the Effects of Persistent Antipolymer Immune Reactions in Nanomedicine: Evaluating Materials-Based Approaches Using Molecular Imaging.

Poly(ethylene glycol) (PEG) is a hydrophilic polymer ubiquitously used in both medical and nonmedical goods. Recent debate surrounding the observed stimulation of immune responses against PEG has spurred the development of materials that may be suitable replacements for this common polymeric component. The underlying view is that these alternative materials with comparable physicochemical properties can overcome the unfavorable and unpredictable effects of antibody-mediated clearance by being chemically, and therefore antigenically, distinct from PEG. However, this hypothesis has not been thoroughly tested in any defined manner, and the immune response observed against PEG has not been rigorously investigated within the context of these emerging materials. Consequently, it remains unclear whether immunity-mediated discrimination between polymeric entities even occurs in vivo and, if this is the case, how it may be exploited. In this study, we utilize positron emission tomography-computed tomography molecular imaging in mice immunized to develop specific antibody responses to PEG and an alternative polymer in order to visualize and quantify the influence of antipolymer antibodies on the biodistribution of synthetic polymers in vivo as a function of immunization status. Under the conditions of this experiment, mice could be primed to exhibit both innate and adaptive immunity to all of the polymer systems to which they were exposed. We demonstrate that alternating between chemically disparate polymers is a viable approach to extend their efficacy when antipolymer humoral immune responses arise.

Probing the Biocompatibility and Immune Cell Association of Chiral, Water-Soluble, Bottlebrush Poly(2-oxazoline)s.

Poly(2-oxazoline)s (POx) have received substantial attention as poly(ethylene glycol) (PEG) alternatives in the biomedical field due to their biocompatibility, high functionality, and ease of synthesis. While POx have demonstrated strong potential as biomaterial constituents, the larger family of poly(cyclic imino ether)s (PCIE) to which POx belongs remains widely underexplored. One highly interesting sub-class of PCIE is poly(2,4-disubstituted-2-oxazoline)s (PdOx), which bear an additional substituent on the backbone of the polymers' repeating units. This allows fine-tuning of the hydrophilic/hydrophobic balance and renders the PdOx chiral when enantiopure 2-oxazoline monomers are used. Herein, we synthesize new water-soluble (R-/S-/RS-) poly(oligo(2-ethyl-4-methyl-2-oxazoline) methacrylate) (P(OEtMeOxMA)) bottlebrushes and compare them to well-established PEtOx- and PEG-based bottlebrush controls in terms of their physical properties, hydrophilicity, and biological behavior. We reveal that the P(OEtMeOxMA) bottlebrushes show a lower critical solution temperature behavior at a physiologically relevant temperature (∼44 °C) and that the enantiopure (R-/S-) variants display a chiral secondary structure. Importantly, we demonstrate the biocompatibility of the chiral P(OEtMeOxMA) bottlebrushes through cellular association and mouse biodistribution studies and show that these systems display higher immune cell association and organ accumulation than the two control polymers. These novel materials possess properties that hold promise for applications in the field of nanomedicine and may be beneficial carriers for therapeutics that require enhanced cellular association and immune cell interaction.

Modulating Macrophage Clearance of Nanoparticles: Comparison of Small-Molecule and Biologic Drugs as Pharmacokinetic Modifiers of Soft Nanomaterials.

Nanomedicines show benefits in overcoming the limitations of conventional drug delivery systems by reducing side effects, toxicity, and exhibiting enhanced pharmacokinetic (PK) profiles to improve the therapeutic window of small-molecule drugs. However, upon administration, many nanoparticles (NPs) prompt induction of host innate immune responses, which in combination with other clearance pathways such as renal and hepatic, eliminate up to 99% of the administered dose. Here, we explore a drug predosing strategy to transiently suppress the mononuclear phagocyte system (MPS), subsequently improving the PK profile and biological behaviors exhibited by a model NP system [hyperbranched polymers (HBPs)] in an immunocompetent mouse model. In vitro assays allowed the identification of five drug candidates that attenuated cellular association. Predosing of lead compounds chloroquine (CQ) and zoledronic acid (ZA) further showed increased HBP retention within the circulatory system of mice, as shown by both fluorescence imaging and positron emission tomography-computed tomography. Flow cytometric evaluation of spleen and liver tissue cells following intravenous administration further demonstrated that CQ and ZA significantly reduced HBP association with myeloid cells by 23 and 16%, respectively. The results of this study support the use of CQ to pharmacologically suppress the MPS to improve NP PKs.

An Immunoregulatory Role for Complement Receptors in Murine Models of Breast Cancer.

The complement system has demonstrated roles in regulating tumor growth, although these may differ between tumor types. The current study used two murine breast cancer models (EMT6 and 4T1) to investigate whether pharmacological targeting of receptors for complement proteins C3a (C3aR) and C5a (C5aR1) is protective in murine breast cancer models. In contrast to prior studies in other tumor models, treatment with the selective C5aR1 antagonist PMX53 had no effect on tumor growth. However, treatment of mice with a dual C3aR/C5aR1 agonist (YSFKPMPLaR) significantly slowed mammary tumor development and progression. Examination of receptor expression by quantitative polymerase chain reaction (qPCR) analysis showed very low levels of mRNA expression for either C3aR or C5aR1 by EMT6 or 4T1 mammary carcinoma cell lines compared with the J774 macrophage line or bone marrow-derived macrophages. Moreover, flow cytometric analysis found no evidence of C3aR or C5aR1 protein expression by either EMT6 or 4T1 cells, leading us to hypothesize that the tumor inhibitory effects of the dual agonist are indirect, possibly via regulation of the anti-tumor immune response. This hypothesis was supported by flow cytometric analysis of tumor infiltrating leukocyte populations, which demonstrated a significant increase in T lymphocytes in mice treated with the C3aR/C5aR1 agonist. These results support an immunoregulatory role for complement receptors in primary murine mammary carcinoma models. They also suggest that complement activation peptides can influence the anti-tumor response in different ways depending on the cancer type, the host immune response to the tumor and levels of endogenous complement activation within the tumor microenvironment.

Fluorophore Selection and Incorporation Contribute to Permeation and Distribution Behaviors of Hyperbranched Polymers in Multi-Cellular Tumor Spheroids and Xenograft Tumor Models.

Improving our understanding of how design choices in materials synthesis impact biological outcomes is of critical importance in the development of nanomedicines. Here, we show that fluorophore labeling of polymer nanomedicine candidates significantly alters their transport and cell association in multi-cellular tumor spheroids and their penetration in breast cancer xenografts, dependent on the type of the fluorophore and their positioning within the macromolecular structure. These data show the critical importance of the biomaterials structure and architecture in their tissue distribution and intracellular trafficking, which in turn govern their potential therapeutic efficacy. The broader implication of these findings suggests that when developing materials for medical applications, great care should be taken early on in the design process as relatively simple choices may have downstream impacts that could potentially skew preclinical biology data.

Small molecule inhibition of Dynamin-dependent endocytosis targets multiple niche signals and impairs leukemia stem cells.

Intensive chemotherapy for acute leukemia can usually induce complete remission, but fails in many patients to eradicate the leukemia stem cells responsible for relapse. There is accumulating evidence that these relapse-inducing cells are maintained and protected by signals provided by the microenvironment. Thus, inhibition of niche signals is a proposed strategy to target leukemia stem cells but this requires knowledge of the critical signals and may be subject to compensatory mechanisms. Signals from the niche require receptor-mediated endocytosis, a generic process dependent on the Dynamin family of large GTPases. Here, we show that Dynole 34-2, a potent inhibitor of Dynamin GTPase activity, can block transduction of key signalling pathways and overcome chemoresistance of leukemia stem cells. Our results provide a significant conceptual advance in therapeutic strategies for acute leukemia that may be applicable to other malignancies in which signals from the niche are involved in disease progression and chemoresistance.

Controlling the Biological Fate of Micellar Nanoparticles: Balancing Stealth and Targeting.

Integrating nanomaterials with biological entities has led to the development of diagnostic tools and biotechnology-derived therapeutic products. However, to optimize the design of these hybrid bionanomaterials, it is essential to understand how controlling the biological interactions will influence desired outcomes. Ultimately, this knowledge will allow more rapid translation from the bench to the clinic. In this paper, we developed a micellar system that was assembled using modular antibody-polymer amphiphilic materials. The amphiphilic nature was established using either poly(ethylene glycol) (PEG) or a single-chain variable fragment (scFv) from an antibody as the hydrophile and a thermoresponsive polymer (poly(oligoethylene glycol) methyl ether methacrylate) as the hydrophobe. By varying the ratios of these components, a series of nanoparticles with different antibody content was self-assembled, where the surface presentation of targeting ligand was carefully controlled. In vitro and in vivo analysis of these systems identified a mismatch between the optimal targeting ligand density to achieve maximum cell association in vitro compared to tumor accumulation in vivo. For this system, we determined an optimum antibody density for both longer circulation and enhanced targeting to tumors that balanced stealthiness of the particle (to evade immune recognition as determined in both mouse models and in whole human blood) with enhanced accumulation achieved through receptor binding on tumor cells in solid tumors. This approach provides fundamental insights into how different antibody densities affect the interaction of designed nanoparticles with both target cells and immune cells, thereby offering a method to probe the intricate interplay between increased targeting efficiency and the subsequent immune response to nanoparticles.

Hyperbranched Poly(2-oxazoline)s and Poly(ethylene glycol): A Structure-Activity Comparison of Biodistribution.

In light of research reporting abnormal pharmacokinetic behavior for therapeutics and formulations containing poly(ethylene glycol) (PEG), a renewed emphasis has been placed on exploring alternative surrogate materials and tailoring specific materials to distinct nanomedicine applications. Poly(2-oxazolines) (POx) have shown great promise in this regard; however, a comparison of POx and PEG interactions with components of the immune system is needed to inform on their distinct suitability. Herein, the interaction of isolated immune cells following injection of hyperbranched polymers comprised of PEG or hydrophilic POx macromonomers was determined via flow cytometry. All materials showed similar association with all of the splenic immune cells analyzed. Interestingly, splenic CD68hi and CD11bhi macrophages showed similar levels of polymer association, despite CD11bhi being a smaller population, suggesting CD68 is linked to increased recognition and phagocytosis of these nanomaterials. This is of interest given that CD68 is a scavenger receptor and directly facilitates the clearance of cellular debris and promotion of phagocytosis, as opposed to CD11b, which is associated with the mediating inflammation via the production of cytokines as well as complement-mediated uptake of foreign particles. In the liver, PEG and poly(2-methyl oxazoline) hyperbranched polymers showed no discernible differences in their cellular association, while hyperbranched poly(2-ethyl oxazoline) showed increased association with dendrocytes and CD68hi macrophages, suggesting that this material exhibited a greater propensity to interact with components of the immune system. This work highlights the importance of how subtle changes in chemical structure can influence the immune response.

Attenuated Acceleration to Leukemia after Ezh2 Loss in Nup98-HoxD13 (NHD13) Myelodysplastic Syndrome.

Supplemental Digital Content is available in the text.

Restricted cell cycle is essential for clonal evolution and therapeutic resistance of pre-leukemic stem cells.

Pre-leukemic stem cells (pre-LSCs) give rise to leukemic stem cells through acquisition of additional gene mutations and are an important source of relapse following chemotherapy. We postulated that cell-cycle kinetics of pre-LSCs may be an important determinant of clonal evolution and therapeutic resistance. Using a doxycycline-inducible H2B-GFP transgene in a mouse model of T-cell acute lymphoblastic leukemia to study cell cycle in vivo, we show that self-renewal, clonal evolution and therapeutic resistance are limited to a rare population of pre-LSCs with restricted cell cycle. We show that proliferative pre-LSCs are unable to return to a cell cycle-restricted state. Cell cycle-restricted pre-LSCs have activation of p53 and its downstream cell-cycle inhibitor p21. Furthermore, absence of p21 leads to proliferation of pre-LSCs, with clonal extinction through loss of asymmetric cell division and terminal differentiation. Thus, inducing proliferation of pre-LSCs represents a promising strategy to increase cure rates for acute leukemia.

A novel role for Lyl1 in primitive erythropoiesis.

Stem cell leukemia (Scl or Tal1) and lymphoblastic leukemia 1 (Lyl1) encode highly related members of the basic helix-loop-helix family of transcription factors that are co-expressed in the erythroid lineage. Previous studies have suggested that Scl is essential for primitive erythropoiesis. However, analysis of single-cell RNA-seq data of early embryos showed that primitive erythroid cells express both Scl and Lyl1 Therefore, to determine whether Lyl1 can function in primitive erythropoiesis, we crossed conditional Scl knockout mice with mice expressing a Cre recombinase under the control of the Epo receptor, active in erythroid progenitors. Embryos with 20% expression of Scl from E9.5 survived to adulthood. However, mice with reduced expression of Scl and absence of Lyl1 (double knockout; DKO) died at E10.5 because of progressive loss of erythropoiesis. Gene expression profiling of DKO yolk sacs revealed loss of Gata1 and many of the known target genes of the SCL-GATA1 complex. ChIP-seq analyses in a human erythroleukemia cell line showed that LYL1 exclusively bound a small subset of SCL targets including GATA1. Together, these data show for the first time that Lyl1 can maintain primitive erythropoiesis.

Oncogenic ZEB2 activation drives sensitivity toward KDM1A inhibition in T-cell acute lymphoblastic leukemia.

Elevated expression of the Zinc finger E-box binding homeobox transcription factor-2 (ZEB2) is correlated with poor prognosis and patient outcome in a variety of human cancer subtypes. Using a conditional gain-of-function mouse model, we recently demonstrated that ZEB2 is an oncogenic driver of immature T-cell acute lymphoblastic leukemia (T-ALL), a heterogenic subgroup of human leukemia characterized by a high incidence of remission failure or hematological relapse after conventional chemotherapy. Here, we identified the lysine-specific demethylase KDM1A as a novel interaction partner of ZEB2 and demonstrated that mouse and human T-ALLs with increased ZEB2 levels critically depend on KDM1A activity for survival. Therefore, targeting the ZEB2 protein complex through direct disruption of the ZEB2-KDM1A interaction or pharmacological inhibition of the KDM1A demethylase activity itself could serve as a novel therapeutic strategy for this aggressive subtype of human leukemia and possibly other ZEB2-driven malignancies.

AKT isoforms 1 and 3 regulate basal and epidermal growth factor-stimulated SGHPL-5 trophoblast cell migration in humans.

Protein kinase B/AKT is critically involved in murine placental development and migration of human placental trophoblasts into maternal uterine tissue. However, localization of the three AKT isoforms within human placenta and their roles in extravillous trophoblasts have not been elucidated. Therefore, we analyzed the expression pattern and function of AKT1, AKT2, and AKT3 in migratory human trophoblasts using SGHPL-5 cell pools stably expressing small-hairpin microRNA (shRNAmir) against AKT1, AKT2, or AKT3 as a model. Western blot analyses using isoform-specific antibodies revealed ubiquitous expression of AKT1, AKT2, and AKT3 in primary villous and extravillous trophoblasts and the trophoblastic cell lines JEG-3, HTR-8/SVneo, and SGHPL-5. Immunofluorescence of first-trimester placentae localized AKT2 and AKT3 to the cytoplasm and nucleus, respectively, in all subtypes of cytotrophoblasts, whereas AKT1 was detected in both cellular compartments. A similar distribution of AKT isoforms was detectable in SGHPL-5 cells. Gene silencing using shRNAmir decreased protein expression of AKT1, AKT2, and AKT3 to 16%, 8%, and 11%, respectively, in SGHPL-5 cells. Compared with shRNAmir controls, proliferation and camptothecin-induced apoptosis were not affected in the different AKT knockdown cells. However, basal and epidermal growth factor (EGF)-induced trophoblast migration was significantly reduced in AKT1 and AKT3 gene-silenced cells, whereas downregulation of AKT2 was not effective. Accordingly, a decrease in EGF-stimulated phosphorylation of AKT (Ser473 and Thr308) and its downstream target mTORC1 (Ser2448) was noticed in AKT1 and AKT3 shRNAmir cell pools. In summary, the results suggest that the AKT isoforms 1 and 3 promote basal as well as EGF-induced trophoblast migration.

Interleukin 11 regulates endometrial cancer cell adhesion and migration via STAT3.

Endometrial carcinoma is the most common gynaecological malignancy. There is however a lack of curative therapies, especially for patients diagnosed with late stage, recurrent or aggressive disease, who have a poor prognosis. Interleukin (IL) 11 is a pleiotropic cytokine that has a role in a number of cancers including colon and breast cancer. IL11 was recently found to be upregulated in endometrial cancers, however the function of IL11 in endometrial cancer is not known. This study aimed to determine the effects of IL11 on endometrial cancer cell proliferation, adhesion and migration. Three endometrial cancer cell lines, Ishikawa, HEC-1A and AN3CA (derived from endometrial cancers grade I, II and III, respectively), were used to determine the effect of IL11 on endometrial cancer cell function. Cell proliferation and viability were assessed by BrdU and Wst-1 assays. Cell adhesion to the extracellular matrix proteins fibronectin, collagen I and IV, vitronectin and laminin was assessed. Modified boyden chambers were utilized to access IL11 action on migration and invasion, respectively. The specific effect of IL11 action on these processes was determined using a unique IL11 inhibitor. IL11 phosphorylated (p)-STAT3 protein abundance in all 3 cell lines but had no effect on pERK and pAKT abundance. Similarly, IL11 had no effect on cell proliferation and viability but increased adhesion of ANC3A cells to fibronectin while having no effect on the other extracellular matrix proteins. IL11 did not alter the adhesive properties of the Ishikawa and HEC-1A cells. In the AN3CA cells, IL11 treatment resulted in a 50% increase in migration and co-treatment with the specific IL11 inhibitor or a STAT3 inhibitor abolished the effect. This study shows a role for IL11 in endometrial cancer and suggests IL11 may be involved in endometrial cancer development and thus may be useful as a therapeutic target.

Wnt1 is anti-lymphangiogenic in a melanoma mouse model.

Wnt signals contribute to melanoma progression by boosting their proliferation and survival. Initially, we expected that activated Wnt signaling also improves their proficiency to recruit blood and lymph vessels. To assess this, we added cell culture supernatants (SNs) of Wnt1(+) and Wnt1(-) melanoma to endothelial spheroids. Whereas SNs of Wnt1(-) melanoma cells induced lymphatic sprouts, those of Wnt1(+) cells were unable to do so and this was restored by vascular endothelial growth factor C (VEGF-C). Subsequent testing of several human melanoma lines revealed that Wnt1 suppressed their VEGF-C expression. This Wnt1 effect did not depend on glycogen synthase kinase-3ß (GSK3ß), ß-catenin, or activator protein-1, but was blocked by cyclosporine A (CsA). To analyze Wnt1 effects in melanoma in vivo, we selected Wnt1(-) melanoma cell lines, overexpressed Wnt1, and injected them subepidermally into severe combined immunodeficient (SCID) mice. We found reduced VEGF-C expression, reduced lymphangiogenesis, and delayed metastasis to sentinel nodes in Wnt1(+) as compared with Wnt1(-) melanoma (P<0.05). Concomitant overexpression of VEGF-C or feeding of animals with CsA restored lymphangiogenesis and metastasis in Wnt1(+) melanoma. In conclusion, Wnt1 is anti-lymphangiogenic by suppressing melanoma-derived VEGF-C expression.

Interleukin (IL)11 mediates protein secretion and modification in human extravillous trophoblasts.

BACKGROUND: Human trophoblast invasion and differentiation are essential for a successful pregnancy outcome. Dysregulation of these processes can lead to placental pathologies such as pre-eclampsia. The molecular mechanisms; however, are poorly understood. Interleukin (IL)11--a cytokine that regulates endometrial epithelial cell adhesion, trophoblast motility and invasion during implantation--may be involved in some of these processes. METHODS AND RESULTS: The effect of IL11 on protein expression was investigated in trophoblastic HTR8/SVneo cells and primary extravillous trophoblasts (EVTs) purified from first- trimester placentas. Two-dimension (2D)-differential in-gel electrophoresis analyses revealed that 731 spots were significantly differentially regulated by IL11 in HTR8/SVneo cells: seven spots were analyzed by liquid chromatography-tandem mass spectrometry and 14 unique proteins identified. Protein disulfide isomerase family A, member 3 (PDIA3; endoplasmic reticulum p57) and glucose-regulated protein 78 (GRP78) were further validated to be regulated by IL11 in HTR8/SVneo and primary EVT. One dimension western blot analysis confirmed that PDIA3 was down-regulated in EVT. 2D western blot analysis revealed that GRP78 was post-translationally modified following IL11 treatment. Moreover, IL11 stimulated the secretion of GRP78 in EVT. CONCLUSIONS: Data suggest that IL11, possibly via signal transducers and activators of transcription 3 signaling pathway, regulates PDIA3 protein expression and modification/secretion of GRP78. This is the first study to identify PDIA3 and GRP78 as IL11 targets in invasive trophoblasts and identifies a possible mechanism by which IL11 regulates trophoblast function.

Transcription factor AP-2α promotes EGF-dependent invasion of human trophoblast.

The basic helix-span-helix transcription factor activating protein (AP)-2α is critically involved in cell-specific hormone expression of syncytializing human trophoblasts. Its role in invasive trophoblast differentiation, however, remains largely elusive. Using RT-PCR, Western blotting, and immunofluorescence of first-trimester placentae, we here show that AP-2α is expressed in extravillous trophoblasts (EVTs) both in situ and in vitro as well as in invasive trophoblast cell lines. Its protein expression was increased upon supplementation of epidermal growth factor (EGF) both in primary EVTs and trophoblastic SGHPL-5 cells. Gene silencing of AP-2α using small hairpin microRNA (shRNAmir) did not affect basal invasion of SGHPL-5 cells through Matrigel-coated filters but reduced EGF-stimulated invasion. Similarly, treatment of primary EVTs with AP-2α small interfering RNA decreased EGF-dependent invasion. Proliferation of SGHPL-5 cells and primary EVTs, measured by cumulative cell numbers and 5-bromo-2'-deoxyuridine labeling, respectively, were not affected on loss of AP-2α. EGF-dependent induction of matrix metalloproteinase (MMP)-2, pro- and active form of urokinase plasminogen activator, and chorionic gonadotropin (CG)-ß was noticed in shRNAmir-control cells, whereas these genes were suppressed in EGF-treated shRNAmir-AP-2α cells. Similarly, EGF-stimulated MMP-2 and CGß protein expression was reduced in AP-2α small interfering RNA-treated primary EVTs. Knockdown of AP-2α also decreased luciferase activity of the CGß5 promoter in SGHPL-5 cells, which was compensated upon transient overexpression of AP-2α cDNA. In conclusion, we show that AP-2α expression positively affects human trophoblast invasion under EGF-stimulated conditions, likely by inducing critical invasion-promoting genes such MMP-2, urokinase plasminogen activator, and CG.

Governing the invasive trophoblast: current aspects on intra- and extracellular regulation.

This review summarizes several aspects especially of regulating factors governing trophoblast invasion. Those include the composition of the extracellular matrix containing a variety of matrix metalloproeinases and their inhibitors, but also intracellular signals. Furthermore, a newly described trophoblast subtype, the endoglandular trophoblast, is presented. Its presence may provide a possible mechanism for opening and connecting uterine glands into the intervillous space. Amongst others, two intracellular signalling pathways are crucial for regulation of trophoblast functions and development: Wnt- and signal transducer and activator of transcription (STAT)3 signalling. Wnt signalling promotes implantation, placentation and trophoblast differentiation. Several Wnt-dependent cascades and regulatory mechanisms display different functions in trophoblast cells. The STAT3 signalling system is fundamental for induction and regulation of invasiveness in physiological trophoblastic cells, but also in tumours. The role of galectins (Gal) in trophoblast regulation and placenta development comes increasingly into focus. The Gal- 1-4, 7-10 and 12-14 have been detected in humans. Detailed information is only available for Gal-1, -2, -3, -4, -9 and -12 in endometrium and decidua. Gal-1, -3 and -13 (-14) have been detected and studied in trophoblast cells.

Wingless (Wnt)-3A induces trophoblast migration and matrix metalloproteinase-2 secretion through canonical Wnt signaling and protein kinase B/AKT activation.

Invasion of human trophoblasts is promoted through activation of wingless (Wnt) signaling, suggesting a role of the pathway in placental development and morphogenesis. However, details on the process such as involvement of canonical and/or noncanonical Wnt signaling cascades as well as their target genes are largely unknown. Hence, signal transduction via canonical Wnt signaling or phosphatidylinositide 3-kinase (PI3K)/AKT and their cross talk as well as trophoblast-specific protease expression were investigated in trophoblastic SGHPL-5 cells and primary extravillous trophoblasts purified from first-trimester placentas. Western blot analyses revealed that the recombinant Wnt ligand Wnt-3A increased phosphorylation of AKT and the downstream kinase glycogen synthase kinase (GSK)-3beta as well as accumulation of activated, nuclear beta-catenin. In accordance, luciferase expression of a canonical Wnt/TCF reporter and cell migration in first-trimester villous explant cultures and of SGHPL-5 cells were stimulated. Chemical inhibition of PI3K abolished Wnt-dependent phosphorylation of AKT and GSK-3beta and trophoblast motility but did not affect appearance of activated beta-catenin or Wnt/TCF reporter activity. In contrast, inhibition of the canonical pathway through soluble Dickkopf-1 did not influence AKT and GSK-3beta phosphorylation but reduced Wnt reporter activity, accumulation of active beta-catenin, and cell migration. Both inhibitors decreased Wnt-3A-induced secretion of pro- and active matrix metalloproteinase-2 from SGHPL-5 cells and pure EVT. The data suggest that Wnt-3A may activate canonical Wnt signaling and PI3K/AKT through distinct receptors. The two signaling cascades act independently in trophoblasts; however, both pathways promote Wnt-dependent migration and the release of matrix metalloproteinase-2, which has been identified as novel Wnt target in invasive trophoblasts.