Wnts, bone and cancer

While the role of Wnt signaling is well established in colorectal carcinogenesis, its function in gynecologic cancers has not been elucidated. Here, we describe the current state of knowledge of canonical Wnt signaling in endometrial cancer (EC), and its implications for future therapeutic targets. Deregulation of the Wnt/β-catenin signaling pathway in EC occurs by inactivating β-catenin mutations in approximately 10–45% of ECs, and via downregulation of Wnt antagonists by epigenetic silencing. The Wnt pathway is intimately involved with estrogen and progesterone, and emerging data implicate it in other important signaling pathways, such as mTOR and Hedgehog. While no therapeutic agents targeting the Wnt signaling pathway are currently in clinical trials, the preclinical data presented suggest a role for Wnt signaling in uterine carcinogenesis, with further research warranted to elucidate the mechanism of action and to proceed towards targeted cancer drug development.

This Month in Gastroenterology

Parathyroid hormone-related protein (PTHrP) is an important regulator of bone destruction in bone metastatic tumors. Transforming growth factor-beta (TGF-β) stimulates PTHrP production in part through the transcription factor Gli2, which is regulated independent of the Hedgehog signaling pathway in osteolytic cancer cells. However, inhibition of TGF-β in vivo does not fully inhibit tumor growth in bone or tumor-induced bone destruction, suggesting other pathways are involved. While Wnt signaling regulates Gli2 in development, the role of Wnt signaling in bone metastasis is unknown. Therefore, we investigated whether Wnt signaling regulates Gli2 expression in tumor cells that induce bone destruction. We report here that Wnt activation by β-catenin/T cell factor 4 (TCF4) over-expression or lithium chloride (LiCl) treatment increased Gli2 and PTHrP expression in osteolytic cancer cells. This was mediated through the TCF and Smad binding sites within the Gli2 promoter as determined by promoter mutation studies, suggesting cross-talk between TGF-β and Wnt signaling. Culture of tumor cells on substrates with bone-like rigidity increased Gli2 and PTHrP production, enhanced autocrine Wnt activity and led to an increase in the TCF/Wnt signaling reporter (TOPFlash), enriched β-catenin nuclear accumulation, and elevated Wnt-related genes by PCR-array. Stromal cells serve as an additional paracrine source of Wnt ligands and enhanced Gli2 and PTHrP mRNA levels in MDA-MB-231 and RWGT2 cells in vitro and promoted tumor-induced bone destruction in vivo in a β-catenin/Wnt3a-dependent mechanism. These data indicate that a combination of matrix rigidity and stromal-secreted factors stimulate Gli2 and PTHrP through Wnt signaling in osteolytic breast cancer cells, and there is significant cross-talk between the Wnt and TGF-β signaling pathways. This suggests that the Wnt signaling pathway may be a potential therapeutic target for inhibiting tumor cell response to the bone microenvironment and at the very least should be considered in clinical regimens targeting TGF-β signaling.

Researchers studying the Wnt pathway frequently like to joke that `Wnts do everything', and that this fact keeps us all in business. Over time, this claim has become less facetious, as the number of known roles for Wnt signaling has grown rapidly. Following on from the first identified functions of

The Wnt signaling network, which is composed of Wnt ligands, receptors, antagonists, and intracellular signaling molecules, has emerged as a powerful regulator of cell fate, proliferation, and function in multicellular organisms. Over the past two decades, the critical role of Wnt signaling in embryonic cartilage and bone development has been well established, and much has been learnt regarding the role of Wnt signaling in chondrogenesis and cartilage development. However, relatively little is known about the role of Wnt signaling in adult articular cartilage and degenerative cartilage tissue. This review will briefly summarize recent advances in Wnt regulation of chondrogenesis and hypertrophic maturation of chondrocytes, and review data concerning the role of Wnt signaling in the maintenance and degeneration of articular chondrocytes and cartilage.

Intestinal crypts are responsible for the total cell renewal of the lining of the intestines; this turnover is governed by the interplay between signalling pathways and the cell cycle. The role of Wnt signalling in cell proliferation and differentiation in the intestinal crypt has been extensively studied, with increased signalling found towards the lower regions of the crypt. Recent studies have shown that the Wnt signalling gradient found within the crypt may arise as a result of division-based spreading from a Wnt ‘reservoir’ at the crypt base. The discovery of the Hippo pathway’s involvement in maintaining crypt homeostasis is more recent; a mechanistic understanding of Hippo pathway dynamics, and its possible cross-talk with the Wnt pathway, remains lacking. To explore how the interplay between these pathways may control crypt homeostasis, we extended an ordinary differential equation model of the Wnt signalling pathway to include a phenomenological description of Hippo signalling in single cells, and then coupled it to a cell-based description of cell movement, proliferation and contact inhibition in agent-based simulations. Furthermore, we compared an imposed Wnt gradient with a division-based Wnt gradient model. Our results suggest that Hippo signalling affects the Wnt pathway by reducing the presence of free cytoplasmic β-catenin, causing cell cycle arrest. We also show that a division-based spreading of Wnt can form a Wnt gradient, resulting in proliferative dynamics comparable to imposed-gradient models. Finally, a simulated APC double mutant, with misregulated Wnt and Hippo signalling activity, is predicted to cause monoclonal conversion of the crypt.

Chapter 12 - Wnt signaling and dementia

Intestinal crypts constitute a niche in which epithelial progenitors respond to Wnt signals, replicate, and prepare to differentiate. Because mutations in Wnt pathway genes lead to intestinal cancer, the role of Wnt signaling in gut epithelial homeostasis is a subject of intense investigation. We studied how Wnt signaling is established during intestine development. We studied spatiotemporal features of Wnt signaling at formative stages in mouse embryos, when villous projections appear and crypt precursors occupy intervillus regions. We used TOP-GAL transgenic and Axin2(LacZ) mice, which report faithfully on canonical Wnt activity, relevant molecular markers, and embryos with aberrant beta-catenin activation. Developing intestines first display evidence for Wnt signaling after appearance of villi. During villus morphogenesis, intervillus cells proliferate actively but lack signs of canonical Wnt signaling. Surprisingly, in late gestation and briefly thereafter, conspicuous Wnt activity is evident in differentiated, postmitotic villus epithelium. Neither Tcf4, a principal transcriptional effector of intestinal Wnt signals, nor candidate Wnt targets CD44 and cyclinD1 are expressed in late fetal villus cells that show high Wnt activity. Instead, those cells express the related factor Tcf3 and a different Wnt target, c-Myc. Premature and deregulated beta-catenin activation causes severe villus dysmorphogenesis in transgenic mice. Relationships among Wnt signaling, epithelial proliferation, and tissue differentiation are reversed in the developing and adult gut. The canonical Wnt pathway has independent, albeit possibly overlapping, functions in early intestinal villi and adult crypts. These observations advance understanding of Wnt functions in intestinal development and disease.

Chapter 7 - WNT Signaling in the Intestine: Development, Homeostasis, Disease

Keratoacanthoma (KA) has a unique life cycle of rapid growth and spontaneous regression that shows similarities to the hair follicle cycle, which involves an active Wnt signaling during physiological regeneration. We analyzed the expression of the Wnt signaling proteins β-catenin, Lef1, Sox9, and Cyclin D1 in young and old human KAs to investigate a possible role for Wnt signaling in KAs. To investigate the role of the Wnt/β-catenin signaling pathway in human KAs. Formalin-fixed, paraffin-embedded tissue samples of 67 KAs were analyzed for protein expression using immunohistochemistry. The majority of KAs were positive for Sox9 and Cyclin D1 but not for nuclear-localized β-catenin or Lef-1. No significant differences in protein expressions were seen between young and old KAs. However, we found a significant association between Ki67 and Cyclin D1 proteins (P= .008). The Wnt signaling pathway does not appear to play a significant role in the biogenesis of human KA. Sox9 overexpression may be indicative of inhibition of Wnt signaling. Sox-9 and Cyclin D1 are proliferation markers that are most likely transactivated by alternate signaling pathways.

Since the first Wnt gene was identified in 1982, the functions and mechanisms of Wnt signaling have been extensively studied. Wnt signaling is conserved from invertebrates to vertebrates and regulates early embryonic development as well as the homeostasis of adult tissues. In addition, both embryonic stem cells and adult stem cells are regulated by Wnt signaling. Deregulation of Wnt signaling is associated with many human diseases, particularly cancers. In this review, we will discuss in detail the functions of many components involved in the Wnt signal transduction pathway. Then, we will explore what is known about the role of Wnt signaling in stem cells and cancers.

Wnt/β-catenin signaling regulates follicular development by modulating the expression of Foxo3a signaling components

897-F Figure 1 (A) 005GSC-GFP cells were orthotopically implanted and randomized into control, WNT974, aPD1 and WNT974+aPD1 and survival was monitored. The median survival for each arm. *p<0.05 using Log...

β-catenin-dependent Wnt signaling is initiated as Wnt binds to both the receptor FZD and coreceptor LRP5/6, which then assembles a multimeric complex at the cytoplasmic membrane face to recruit and inactivate the kinase GSK3. The large number and sequence diversity of Wnt isoforms suggest the possibility of domain-specific ligand-coreceptor interactions, and distinct binding sites on LRP6 for Wnt3a and Wnt9b have recently been identified in vitro. Whether mechanistically different interactions between Wnts and coreceptors might mediate signaling remains to be determined. It is also not clear whether coreceptor homodimerization induced extracellularly can activate Wnt signaling, as is the case for receptor tyrosine kinases. We generated monoclonal antibodies against LRP6 with the unexpected ability to inhibit signaling by some Wnt isoforms and potentiate signaling by other isoforms. In cell culture, two antibodies characterized further show reciprocal activities on most Wnts, with one antibody antagonizing and the other potentiating. We demonstrate that these antibodies bind to different regions of LRP6 protein, and inhibition of signaling results from blocking Wnt binding. Antibody-mediated dimerization of LRP6 can potentiate signaling only when a Wnt isoform is also able to bind the complex, presumably recruiting FZD. Endogenous autocrine Wnt signaling in different tumor cell lines can be either antagonized or enhanced by the LRP6 antibodies, indicating expression of different Wnt isoforms. As anticipated from the roles of Wnt signaling in cancer and bone development, antibody activities can also be observed in mice for inhibition of tumor growth and in organ culture for enhancement of bone mineral density. Collectively, our results indicate that separate binding sites for different subsets of Wnt isoforms determine the inhibition or potentiation of signaling conferred by LRP6 antibodies. This complexity of coreceptor-ligand interactions may allow for differential regulation of signaling by Wnt isoforms during development, and can be exploited with antibodies to differentially manipulate Wnt signaling in specific tissues or disease states.

Transcription factors of the T cell factor/lymphoid enhancing factor (Tcf/Lef) family are key regulators in the development of T cell precursors to the CD4+8+ stage. These factors are known targets of the canonical Wnt signalling pathway, and regulate transcription of Wnt target genes following interaction with the armadillo repeat-containing protein beta-catenin. However, as recent studies show normal thymocyte maturation in the absence of either beta-catenin or its homologue gamma-catenin, the role of Wnt signalling in Tcf/Lef activation during T cell development is controversial. To directly investigate the importance of catenin-mediated Wnt signalling in early thymocytes, we have compared the expression of beta- and gamma-catenin and analysed distinct stages of T cell precursor maturation following overexpression of inhibitor of beta-catenin and Tcf (ICAT), which inhibits Wnt signalling by preventing binding of armadillo repeat-containing proteins to Tcf/Lef. By direct retroviral gene targeting of CD4-8- and CD4+8+ precursors, we show that ICAT overexpression inhibits the CD4-8--to-CD4+8+ transition, but not the CD4+8+-to-CD4+8- or -CD4-8+ transition. Collectively, our data support a model in which canonical Wnt signalling influences T cell development in the thymus by playing an essential role in the maturation of CD4-8- but not CD4+8+ thymocytes.