Frizzled7 as an emerging target for cancer therapy

Wnt proteins are secreted glycoproteins that bind to the N-terminal extra-cellular cysteine-rich domain of the Frizzled (Fzd) receptor family. The Fzd receptors can respond to Wnt proteins in the presence of Wnt co-receptors to activate the canonical and non-canonical Wnt pathways. Recent studies indicated that, among the Fzd family, Fzd7 is the Wnt receptor most commonly upregulated in a variety of cancers including colorectal cancer, hepatocellular carcinoma and triple negative breast cancer. Fzd7 plays an important role in stem cell biology and cancer development and progression. In addition, it has been demonstrated that siRNA knockdown of Fzd7, the anti-Fzd7 antibody or the extracellular peptide of Fzd7 (soluble Fzd7 peptide) displayed anti-cancer activity in vitro and in vivo mainly due to the inhibition of the canonical Wnt signaling pathway. Furthermore, pharmacological inhibition of Fzd7 by small interfering peptides or a small molecule inhibitor suppressed β-catenin-dependent tumor cell growth. Therefore, targeted inhibition of Fzd7 represents a rational and promising new approach for cancer therapy.     

The functions and possible significance of Kremen as the gatekeeper of Wnt signalling in development and pathology

Kremen (Krm) was originally discovered as a novel transmembrane protein containing the kringle domain. Both Krm1 (the first identified Krm) and its relative Krm2 were later identified to be the high-affinity receptors for Dickkopf (Dkk), the inhibitor of Wnt/beta-catenin signalling. The formation of a ternary complex composed of Krm, Dkk, and Lrp5/6 (the coreceptor of Wnt) inhibits Wnt/beta-catenin signalling. In Xenopus gastrula embryos, Wnt/beta-catenin signalling regulates anterior-posterior patterning, with low-signalling in anterior regions. Inhibition of Krm1/2 induces embryonic head defects. Together with anterior localization of Krms and Dkks, the inhibition of Wnt signalling by Dkk-Krm action seems to allow anterior embryonic development. During mammalian development, krm1 mRNA expression is low in the early stages, but gradually and continuously increases with developmental progression and differentiation. In contrast with the wide, strong expression of krm1 mRNA in mature tissues, expression of krm1 is diminished in a variety of human tumor cells. Since stem cells and undifferentiated cells rely on Wnt/beta-catenin signalling for maintenance in a low differentiation state, the physiological shutdown of Wnt/beta-catenin signalling by Dkk-Krm is likely to set cells on a divergent path toward differentiation. In tumour cells, a deficit of Krm may increase the susceptibility to tumourigenic transformation. Both positive and negative regulation of Wnt/beta-catenin signalling definitively contributes to diverse developmental and physiological processes, including cell-fate determination, tissue patterning and stem cell regulation. Krm is quite significant in these processes as the gatekeeper of the Wnt/beta-catenin signalling pathway.     

Reconstitution of a Frizzled8��Wnt3a��LRP6 Signaling Complex Reveals Multiple Wnt and Dkk1 Binding Sites on LRP6

Wnt/β-catenin signaling is initiated at the cell surface by association of secreted Wnt with its receptors Frizzled (Fz) and low density lipoprotein receptor-related protein 5/6 (LRP5/6). The study of these molecular interactions has been a significant technical challenge because the proteins have been inaccessible in sufficient purity and quantity. In this report we describe insect cell expression and purification of soluble mouse Fz8 cysteine-rich domain and human LRP6 extracellular domain and show that they inhibit Wnt/β-catenin signaling in cellular assays. We determine the binding affinities of Wnts and Dickkopf 1 (Dkk1) to the relevant co-receptors and reconstitute in vitro the Fz8 CRD·Wnt3a·LRP6 signaling complex. Using purified fragments of LRP6, we further show that Wnt3a binds to a region including only the third and fourth β-propeller domains of LRP6 (E3E4). Surprisingly, we find that Wnt9b binds to a different part of the LRP6 extracellular domain, E1E2, and we demonstrate that Wnt3a and Wnt9b can bind to LRP6 simultaneously. Dkk1 binds to both E1E2 and E3E4 fragments and competes with both Wnt3a and Wnt9b for binding to LRP6. The existence of multiple, independent Wnt binding sites on the LRP6 co-receptor suggests new possibilities for the architecture of Wnt signaling complexes and a model for broad-spectrum inhibition of Wnt/β-catenin signaling by Dkk1.     

Endometrial stromal cells and decidualized stromal cells: Origins,transformation and functions

Decidualization of endometrium, which is characterized by endometrial stromal cell (ESC) decidualization, vascular reconstruction, immune cell recruitment, and plentiful molecule production, is a crucial step for uterus to become receptive for embryo. When implantation takes place, ESCs surround and directly interact with embryo. Decidualized stromal cells (DSCs) are of great importance in endometrial decidualization, having a broad function in regulating immune activity and vascular remodeling of uterus. DSCs are shown to have a higher metabolic level and looser cytoskeleton than ESCs. What's the origin of ESCs and how ESCs successfully transform into DSCs had puzzled scientists in the last decades. Breakthrough had been achieved recently, and many studies had elucidated some of the characters and functions of DSCs. However, several questions still remain unclear. This paper reviews current understanding of where ESCs come from and how ESCs differentiate into DSCs, summarizes some characters and functions of DSCs, analyzes current studies and their limitations and points out research areas that need further investigation.     

Embryonic expression and evolutionary analysis of the amphioxus Dickkopf and Kremen family genes

The secreted Wnt signaling inhibitor Dickkopf1(Dkk1)plays key role in vertebrate head induction.Its receptor Kremen synergizes with Dkkl in Wnt inhibition.Here we have carried out expression and functional studies of the Dkk and Kremen genes in amphioxus(Branchiostoma belcheri).During embryonic and larval development,BbDkk1/2/4 is expressed in the posterior mesoendoderm,anterior somatic mesoderm and the pharyngeal regions.Its expression becomes restricted to the pharyngeal region on the left side at larval stages.In 45 h larvae,BbDkk1/2/4 is expressed specifically in the cerebral vesicle.BbDkk3 was only detected at larval stages in the mid-intestine region.Seven Kremen related genes were identified in the genome of the Florida amphioxus(Branchiostoma floridae),clustered in 4scaffolds,and are designated Kremen1-4 and Kremen-like 1-3,respectively.In B.belcheri,Kremenl is strongly expressed in the mesoendoderm during early development and Kremen3 is expressed asymmetrically in spots in the larval pharyngeal region.In luciferase reporter assays,BbDkk1/2/4 can strongly inhibit Writ signaling,while BbDkk3,BbKremen1 and BbKremen3 can not.No co-operative effect was observed between amphioxus Dkk1/2/4 and Kremens,suggesting that the interaction between Dkk and Kremen likely originated later during evolution.     

EZH2‐mediated repression of Dkk1 promotes hepatic stellate cell activation and hepatic fibrosis

EZH2, a histone H3 lysine‐27‐specific methyltransferase, is involved in diverse physiological and pathological processes including cell proliferation and differentiation. However, the role of EZH2 in liver fibrosis is largely unknown. In this study, it was identified that EZH2 promoted Wnt pathway‐stimulated fibroblasts in vitro and in vivo by repressing Dkk‐1, which is a Wnt pathway antagonist. The expression of EZH2 was increased in CCl₄‐induced rat liver and primary HSCs as well as TGF‐β1‐treated HSC‐T6, whereas the expression of Dkk1 was reduced. Silencing of EZH2 prevented TGF‐β1‐induced proliferation of HSC‐T6 cells and the expression of α‐SMA. In addition, knockdown of Dkk1 promoted TGF‐β1‐induced activation of HSCs. Moreover, silencing of EZH2 could restore the repression of Dkk‐1 through trimethylation of H3K27me3 in TGF‐β1‐treated HSC‐T6 cells. Interestingly, inhibition of EZH2 had almost no effect on the activation of HSC when Dkk1 was silenced. Collectively, EZH2‐mediated repression of Dkk1 promotes the activation of Wnt/β‐catenin pathway, which is an essential event for HSC activation.     

Expression, purification and characterization of murine Dkk1 protein

Dickkopf-1 (Dkk1) protein is a secreted inhibitor of canonical Wnt signaling and modulates that pathway during embryonic development. It is also implicated in several diseases and hence Dkk1 is a potential target for therapeutic intervention. In the present study 6His-tagged Dkk1 expression and secretion was assessed in five mammalian cell types. Only FreeStyle 293-F cells showed significant Dkk1 protein expression in culture medium. High and stable expression of the Dkk1 protein was obtained from a selected stable FreeStyle 293-F clone 3F8, that grows in suspension in serum-free medium. The 3F8 clone showed a high Dkk1 production level (10 mg/L) for up to 2 months of culture. A one step purification procedure resulting in large amounts of highly pure and active Dkk1 protein was developed. Purified Dkk1 binds its receptors LRP5 and LRP6, and is able to dose dependently inhibit canonical Wnt signaling. Recombinant Dkk1 is glycosylated, but this modification is not essential for its biological activity. In summary, an abundant source of pure and functionally active Dkk1 protein is developed that will support the identification of inhibitors such as neutralizing antibodies that could find therapeutic use.     

Hypoblast controls mesoderm generation and axial patterning in the gastrulating rabbit embryo

Gastrulation in higher vertebrate species classically commences with the generation of mesoderm cells in the primitive streak by epithelio-mesenchymal transformation of epiblast cells. However, the primitive streak also marks, with its longitudinal orientation in the posterior part of the conceptus, the anterior-posterior (or head-tail) axis of the embryo. Results obtained in chick and mouse suggest that signals secreted by the hypoblast (or visceral endoderm), the extraembryonic tissue covering the epiblast ventrally, antagonise the mesoderm induction cascade in the anterior part of the epiblast and thereby restrict streak development to the posterior pole (and possibly initiate head development anteriorly). In this paper we took advantage of the disc-shape morphology of the rabbit gastrula for defining the expression compartments of the signalling molecules Cerberus and Dickkopf at pre-gastrulation and early gastrulation stages in a mammal other than the mouse. The two molecules are expressed in novel expression compartments in a complementary fashion both in the hypoblast and in the emerging primitive streak. In loss-of-function experiments, carried out in a New-type culturing system, hypoblast was removed prior to culture at defined stages before and at the beginning of gastrulation. The epiblast shows a stage-dependent and topographically restricted susceptibility to express Brachyury, a T-box gene pivotal for mesoderm formation, and to transform into (histologically proven) mesoderm. These results confirm for the mammalian embryo that the anterior-posterior axis of the conceptus is formed first as a molecular prepattern in the hypoblast and then irrevocably fixed, under the control of signals secreted from the hypoblast, by epithelio-mesenchymal transformation (primitive streak formation) in the epiblast.Edited by D. Tautz     

IDKK1 inhibits canonical Wnt signaling in human papillomavirus-positive penile cancer cells

Penile squamous cell cancer (PSCC) is the most frequent penile malignant disease. Infections with human papillomaviruses (HPV) are a major etiologic driver of PSCC. However, the molecular details of the underlying carcinogenesis are understudied because of rare clinical specimens and missing cell lines. Here, we investigated if the expression of high-risk HPV16 oncogenes causes an augmentation of the Wnt pathway using unique HPV-positive penile cancer (PeCa) cell lines in monolayer and organotypic 3D raft cultures as well as tissue micro arrays containing clinical tissue specimens. The HPV oncoproteins enhanced the expression of Leucine-rich repeat-containing G-protein coupled receptor 6 (LGR6) and the HPV-positive PeCa cells expressed a signature of Wnt target and stemness-associated genes. However, the notable lack of nuclear β-catenin in vitro and in situ raised the question if the enhanced expression of Wnt pathway factors is tantamount to an active Wnt signaling. Subsequent TOP-flash reporter assays revealed Wnt signaling as absent and not inducible by respective Wnt ligands in PeCa cell lines. The HPV-positive PeCa cells and especially HPV-positive PeCa specimens of the tumor core expressed the Wnt antagonist and negative feedback-regulator Dickkopf1 (DKK1). Subsequent neutralization experiments using PeCa cell line-conditioned media demonstrated that DKK1 is capable to impair ligand-induced Wnt signaling. While gene expression analyses suggested an augmented and active canonical Wnt pathway, the respective signaling was inhibited due to the endogenous expression of the antagonist DKK1. Subsequent TMA stainings indicated Dkk1 as linked with HPV-positivity and metastatic disease progression in PeCa suggesting potential as a prognostic marker.