Paracrine Wnt Signaling Research Articles

Tumor Explants Elucidate a Cascade of Paracrine SHH, WNT, and VEGF Signals Driving Pancreatic Cancer Angiosuppression.

We present a key mechanism of tumor angiosuppression, a process that sculpts the physiologic, cellular, and metabolic environment of PDAC. We further present a computational and experimental framework for the dissection of complex signaling cascades that propagate among multiple cell types in the tissue environment. This article is featured in Selected Articles from This Issue, p. 201.

Endothelial FOXC1 and FOXC2 promote intestinal regeneration after ischemia–reperfusion injury

Intestinal ischemia underlies several clinical conditions and can result in the loss of the intestinal mucosal barrier. Ischemia‐induced damage to the intestinal epithelium is repaired by stimulation of intestinal stem cells (ISCs), and paracrine signaling from the vascular niche regulates intestinal regeneration. Here, we identify FOXC1 and FOXC2 as essential regulators of paracrine signaling in intestinal regeneration after ischemia–reperfusion (I/R) injury. Vascular endothelial cell (EC)‐ and lymphatic EC (LEC)‐specific deletions of Foxc1, Foxc2, or both in mice worsen I/R‐induced intestinal damage by causing defects in vascular regrowth, expression of chemokine CXCL12 and Wnt activator R‐spondin 3 (RSPO3) in blood ECs (BECs) and LECs, respectively, and activation of Wnt signaling in ISCs. Both FOXC1 and FOXC2 directly bind to regulatory elements of the CXCL12 and RSPO3 loci in BECs and LECs, respectively. Treatment with CXCL12 and RSPO3 rescues the I/R‐induced intestinal damage in EC‐ and LEC‐Foxc mutant mice, respectively. This study provides evidence that FOXC1 and FOXC2 are required for intestinal regeneration by stimulating paracrine CXCL12 and Wnt signaling.

Pituitary stem cells produce paracrine WNT signals to control the expansion of their descendant progenitor cells

Pituitary stem cells produce paracrine WNT signals to control the expansion of their descendant progenitor cells

Paracrine Wnt signaling is necessary for prostate epithelial proliferation.

The Wnt proteins play key roles in the development, homeostasis, and disease progression of many organs including the prostate. However, the spatiotemporal expression patterns of Wnt proteins in prostate cell lineages at different developmental stages and in prostate cancer remain inadequately characterized. We isolated the epithelial and stromal cells in the developing and mature mouse prostate by flow cytometry and determined the expression levels of Wnt ligands. We used Visium spatial gene expression analysis to determine the spatial distribution of Wnt ligands in the mouse prostatic glands. Using laser-capture microscopy in combination with gene expression analysis, we also determined the expression patterns of Wnt signaling components in stromal and cancer cells in advanced human prostate cancer specimens. To investigate how the stroma-derived Wnt ligands affect prostate development and homeostasis, we used a Col1a2-CreERT2 mouse model to disrupt the Wnt transporter Wntless specifically in prostate stromal cells. We showed that the prostate stromal cells are a major source of several Wnt ligands. Visium spatial gene expression analysis revealed a distinct spatial distribution of Wnt ligands in the prostatic glands. We also showed that Wnt signaling components are highly expressed in the stromal compartment of primary and advanced human prostate cancer. Blocking stromal Wnt secretion attenuated prostate epithelial proliferation and regeneration but did not affect cell survival and lineage maintenance. Our study demonstrates a critical role of stroma-derived Wnt ligands in prostate development and homeostasis.

Modeling Paracrine Noncanonical Wnt Signaling <em>In Vitro</em>

Noncanonical Wnt signaling regulates intracellular actin filament organization and polarized migration of progenitor cells during embryogenesis. This process requires complex and coordinated paracrine interactions between signal-sending and signal-receiving cells. Given that these interactions can occur between various types of cells from different lineages, in vivo evaluation of cell-specific defects can be challenging. The present study describes a highly reproducible method to evaluate paracrine noncanonical Wnt signaling in vitro. This protocol was designed with the ability to (1) conduct functional and molecular assessments of noncanonical Wnt signaling between any two cell types of interest; (2) dissect the role of signal-sending versus signal-receiving molecules in the noncanonical Wnt signaling pathway; and (3) perform phenotypic rescue experiments with standard molecular or pharmacologic approaches. This protocol was used to evaluate neural crest cell (NCC)-mediated noncanonical Wnt signaling in myoblasts. The presence of NCCs is associated with an increased number of phalloidin-positive cytoplasmic filopodia and lamellipodia in myoblasts and improved myoblast migration in a wound-healing assay. The Wnt5a-ROR2 axis was identified as a crucial noncanonical Wnt signaling pathway between NCC and second heart field (SHF) cardiomyoblast progenitors. In conclusion, this is a highly tractable protocol to study paracrine noncanonical Wnt signaling mechanisms in vitro.

Pituitary stem cells produce paracrine WNT signals to control the expansion of their descendant progenitor cells.

In response to physiological demand, the pituitary gland generates new hormone-secreting cells from committed progenitor cells throughout life. It remains unclear to what extent pituitary stem cells (PSCs), which uniquely express SOX2, contribute to pituitary growth and renewal. Moreover, neither the signals that drive proliferation nor their sources have been elucidated. We have used genetic approaches in the mouse, showing that the WNT pathway is essential for proliferation of all lineages in the gland. We reveal that SOX2+ stem cells are a key source of WNT ligands. By blocking secretion of WNTs from SOX2+ PSCs in vivo, we demonstrate that proliferation of neighbouring committed progenitor cells declines, demonstrating that progenitor multiplication depends on the paracrine WNT secretion from SOX2+ PSCs. Our results indicate that stem cells can hold additional roles in tissue expansion and homeostasis, acting as paracrine signalling centres to coordinate the proliferation of neighbouring cells.

Dally-like protein sequesters multiple Wnt ligands in the Drosophila germarium

Cells in multicellular organisms rely on secreted ligands for development and morphogenesis. Several mechanisms modulate the availability and distribution of secreted ligands, determining their ability to signal locally and at long range from their source. One of these mechanisms is Dally-like protein (Dlp), a cell-surface glypican that exhibits biphasic functions in Drosophila wing discs, promoting Wg signaling at long-range from Wg source cells and inhibiting Wg signaling near source cells. In the germarium at the tip of the ovary, Dlp promotes long-range distribution of Wg from cap cells to follicle stem cells. However, the germarium also expresses other Wnts - Wnt2, Wnt4, and Wnt6 - that function locally in escort cells to promote oogenesis. Whether and how local functions of these Wnts are regulated remains unknown. Here we show that the dlp overexpression phenotype is multifaceted and phenocopies multiple Wnt loss-of-function phenotypes. Each aspect of dlp overexpression phenotype is suppressed by co-expression of individual Wnts, and the suppression pattern exhibited by each Wnt suggests that Wnts have functional specificity in the germarium. Further, dlp knockdown phenocopies Wnt gain-of-function phenotypes. Together these data show that Dlp inhibits the functions of each Wnt. All four Wnts co-immunoprecipitate with Dlp in S2R+ ​cells, suggesting that in the germarium, Dlp sequesters Wnts to inhibit local paracrine Wnt signaling. Our results indicate that Dlp modulates the availability of multiple extracellular Wnts for local paracrine Wnt signaling in the germarium.

Abstract 1124: Wnt-producing niche cells support cancer stem-like cells in pancreatic ductal adenocarcinoma

Abstract Despite modern advances in cancer therapy, the prognosis for pancreatic ductal adenocarcinoma (PDAC) remains dismal, highlighting the need for better understanding of the biologic underpinnings of the disease. Well established as a pathway for growth and regeneration, Wnt signaling maintains subpopulations of cancer cells in a stem-like state, contributing to the heterogeneous composition of tumors. Our previous work demonstrated that Porcupine, an essential enzyme for secretion of paracrine Wnt signals, can be used to mark Wnt-producing cells in normal and tumor tissues. We detected the presence of nuclear beta-catenin, a biomarker of active Wnt-signaling, in a subpopulation of cancer cells adjacent to Porcupine+ niche cells in murine PDAC tissue. Furthermore, genetic inactivation of Porcupine translated to a 25% increase in survival in PDAC-bearing mice. In vitro, we see a marked increase in spheroid formation and proliferation of the stem-like subpopulation over the rest of the primary tumor cells. Ongoing lineage-tracing experiments in an autochthonous murine model of PDAC will determine whether these cancer stem cells are able to produce their Wnt-secreting niche in vivo. Our current efforts are focused on the preclinical evaluation of Wnt inhibitors in the treatment of mouse and human PDAC as single agents or in combination with chemotherapy. Such findings open the door for interrogation of the Wnt pathway as a possible target for improving the clinical outcome of pancreatic cancer. Citation Format: Katherine Wu, Olivera Grbovic-Huezo, Griffin Hartmann, Tyler Jacks, Tuomas Tammela. Wnt-producing niche cells support cancer stem-like cells in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1124.

Direct visualization of the Wntless-induced redistribution of WNT1 in developing chick embryos

Paracrine Wnt signals are critical regulators of cell proliferation, specification, and differentiation during embryogenesis. Consistent with the discovery that Wnt ligands are post-translationally modified with palmitoleate (a 16 carbon mono-unsaturated fatty acid), our studies show that the vast majority of bioavailable chick WNT1 (cWNT1) produced in stably transfected L cells is cell-associated. Thus, it seems unlikely that the WNT1 signal is propagated by diffusion alone. Unfortunately, the production and transport of vertebrate Wnt proteins has been exceedingly difficult to study as few antibodies are able to detect endogenous Wnt proteins and fixation is known to disrupt the architecture of cells and tissues. Furthermore, vertebrate Wnts have been extraordinarily refractory to tagging. To help overcome these obstacles, we have generated a number of tools that permit the detection of WNT1 in palmitoylation assays and the visualization of chick and zebrafish WNT1 in live cells and tissues. Consistent with previous studies in fixed cells, live imaging of cells and tissues with overexpressed cWNT1-moxGFP shows predominant localization of the protein to a reticulated network that is likely to be the endoplasmic reticulum. As PORCN and WLS are important upstream regulators of Wnt gradient formation, we also undertook the generation of mCherry-tagged variants of both proteins. While co-expression of PORCN-mCherry had no discernible effect on the localization of WNT1-moxGFP, co-expression of WLS-mCherry caused a marked redistribution of WNT1-moxGFP to the cell surface and cellular projections in cultured cells as well as in neural crest and surface ectoderm cells in developing chick embryos. Our studies further establish that the levels of WLS, and not PORCN, are rate limiting with respect to WNT1 trafficking.

Abstract PR06: Reciprocal prostate cancer signaling with its microenvironment mediates castrate-resistant disease progression

Abstract Our lab has previously reported that the mouse model knocked out for TGF-beta receptor type II in fibroblasts results in castrate resistance of prostate tissues, mediated by paracrine Wnt signaling. We found that antagonizing androgen signaling leads to Wnt ligand expression by prostatic fibroblasts, interestingly dependent on cell-cell contact. This led us to hypothesize that a juxtacrine action within the stromal compartment was important to the development of castrate resistance. Building upon this observation, we found that Notch signaling was upregulated in prostate cancer associated fibroblasts (CAFs) compared with normal tissue associated fibroblasts (NAFs). Further, inhibition of Notch signaling in CAFs, using DAPT, resulted in decrease in Wnt2b and decreased expression of Notch target genes. Similarly, castration or androgen receptor antagonism (i.e. bicalutamide or enzalutamide) resulted in increased Notch signaling and downstream Wnts 2 and 2b up regulation. The further generation of chimeric tissue recombinant xenografts with human RV1 prostate cancer epithelia with mouse prostate stromal cells with activated Notch signaling, derived from NICD transgenic mice resulted in increased tumor size compared with wild type control stromal recombinants. Conversely, recombinants derived from prostatic fibroblasts having a loss of Notch signaling, from RBPJ-knockout mice, had diminished tumorigenesis, compared to xenografts with the wild type fibroblasts counterpart. In trying to determine the mediator of Wnt-associated paracrine prostate epithelial expansion, we found downstream YAP activation in the RV1 cells. In a reciprocal epithelial-to-fibroblast communication we found that the RV1 cells treated with conditioned media from NICD fibroblasts showed increased IL-6 expression. Conversely, conditioned media from prostate cancer cells increased Notch target gene expression in CAFs, which was blocked upon addition of IL-6 neutralizing antibody. Taken together there is a stromal-derived signaling cascade involving Notch and Wnt ligands, that activates YAP in the epithelia for paracrine IL-6-dependent Notch activity back in the stromal fibroblasts. Critically, disruption of this cross-talk in the fibroblasts can limit the gain of stem features and castrate resistance of the adjacent epithelia in halting lethal progression of prostate cancer. This abstract is also presented as Poster A44. Citation Format: Manisha Tripathi, Srinivas Nandana, Jen-Ming Huang, Manabu Kato, Leland W. K. Chung, Rajeev Mishra, Li Xin, Neil Bhowmick. Reciprocal prostate cancer signaling with its microenvironment mediates castrate-resistant disease progression. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr PR06.

Control of Wnt Receptor Turnover by R-spondin-ZNRF3/RNF43 Signaling Module and Its Dysregulation in Cancer.

Aberrant activation of the Wnt/β-catenin pathway is frequently found in various cancers, often through mutations of downstream components. Inhibiting β-catenin signaling in tumors with downstream pathway mutations remains challenging, due to a lack of favorable targets. On the other hand, targeting upstream components of the Wnt pathway is rather straightforward. However, it is difficult to identify tumors addicted to autocrine or paracrine Wnt signaling. Discovery of the R-spondin-ZNRF3/RNF43 signaling module and its genetic alterations in cancers represents a breakthrough in this area. Membrane E3 ligase ZNRF3 and RNF43 are critical negative feedback regulators of the Wnt pathway, which function through promoting ubiquitination and degradation of Wnt receptors. R-spondin proteins (RSPO1-4) serve as natural antagonists of ZNRF3/RNF43. To maintain strong and sustained Wnt/β-catenin signaling, cancers need to overcome ZNRF3/RNF43-mediated feedback inhibition. Indeed, mutations of RNF43/ZNRF3 and recurrent translocations of RSPO2/RSPO3 have recently been identified in various cancers. Significantly, genetic alterations in RNF43/ZNRF3/RSPO2/RSPO3 have shown promise as predictive biomarkers in pre-clinical models for the efficacy of upstream Wnt inhibitors. In this review, we will discuss the biology of the R-spondin-ZNRF3/RNF43 signaling module, cancer-associated alterations of this signaling module, and their value as biomarkers to identify Wnt-addicted tumors.

To Control Site-Specific Skin Gene Expression, Autocrine Mimics Paracrine Canonical Wnt Signaling and Is Activated Ectopically in Skin Disease

Despite similar components, the heterogeneity of skin characteristics across the human body is enormous. It is classically believed that site-specific fibroblasts in the dermis control postnatal skin identity by modulating the behavior of the surface-overlying keratinocytes in the epidermis. To begin testing this hypothesis, we characterized the gene expression differences between volar (ventral; palmoplantar) and nonvolar (dorsal) human skin. We show that KERATIN 9 (KRT9) is the most uniquely enriched transcript in volar skin, consistent with its etiology in genetic diseases of the palms and soles. In addition, ectopic KRT9 expression is selectively activated by volar fibroblasts. However, KRT9 expression occurs in the absence of all fibroblasts, although not to the maximal levels induced by fibroblasts. Through gain-of-function and loss-of-function experiments, we demonstrate that the mechanism is through overlapping paracrine or autocrine canonical WNT-β-catenin signaling in each respective context. Finally, as an invivo example of ectopic expression of KRT9 independent of volar fibroblasts, we demonstrate that in the human skin disease lichen simplex chronicus, WNT5a and KRT9 are robustly activated outside of volar sites. These results highlight the complexities of site-specific gene expression and its disruption in skin disease.

Inhibition of CML Stem Cell Growth By Targeting WNT Signaling Using a Porcupine Inhibitor

BCR-ABL tyrosine kinase inhibitors (TKIs) have revolutionized the treatment of chronic myeloid leukemia (CML). However, TKIs are unable to eradicate the leukemia stem cells (LSCs) from which CML arises, despite effective inhibition of BCR-ABL kinase activity in this population. There is evidence that extrinsic signals from the bone marrow (BM) microenvironment play an important role in the resistance of CML LSC to TKI treatment. Our previous studies have shown a role for Wnt signaling in protecting CML LSC from TKI treatment (Blood. 2013; 121(10):1824-38). Palmitoylation of Wnts by the Porcupine acyltransferase (PORCN) is required for their secretion and activity. LGK974, a potent PORCN inhibitor, has recently been shown to inhibit Wnt signaling and shows in vivo efficacy against several Wnt-dependent tumors (PNAS. 2013; 110(50):20224-9). In this study, we investigated whether inhibition of Wnt secretion using LGK974 could sensitize CML stem/progenitor cells to TKI treatment. CML and normal CD34+ cells and purified CD34+CD38- stem cells and CD34+CD38+ committed progenitors were treated with LGK974 with or without addition of the BCR-ABL TKI (nilotinib), and in the presence and absence of human BM mesenchymal stromal cells (MSC). CML CD34+ cells showed enhanced Wnt signaling following exposure to recombinant Wnt3a (200ng/ml) compared to normal CD34+ cells as evidenced by increased phospho-LRP6 (ser1490) and β-catenin protein levels. Analysis of all 10 frizzled receptors expression by qRT-PCR revealed a significant upregulation of FZD4 (p=0.026, n=10), and FZD5 (p=0.003, n=10), but downregulation of FZD8 (p=0.009, n=10), in CML compared to normal CD34+ cells. Co-culture with MSC was associated with increased Wnt signaling in CML CD34+ cells as detected by increased expression and nuclear translocation of β-catenin protein, and increased expression of Wnt target genes including c-Myc, Cyclin-D1, PPARδ, and Axin2. Treatment with LGK974 reduced Wnt secretion from MSC measured in a TCF/LEF reporter assay (LGK 0.5μM, 0.74 fold reduction compared to control, p=0.016, n=5, LGK 1μM, 0.62 fold reduction, p=0.007, n=5) and potently inhibited Wnt signaling in CML CD34+ cells both in the absence and presence of MSC. Treatment with LGK974 (1μM) in combination with nilotinib (1μM) resulted in significantly increased inhibition of CML CFC growth compared to nilotinib alone, both in the presence and absence of MSC (34+38- cells LGK alone 82.75±6.38% inhibition of control, Nil alone 43.12±15.32%, combination 23.17±6.48%, p<0.05, n=4; 34+38+ cells LGK alone 87.65±6.13% inhibition of control, Nil alone 26.99±4.54%, combination 16.04±0.48%, p<0.05, n=4) Importantly, LGK974 inhibited CML CFC growth to a significantly greater extent than the normal CFC growth ( normal 34+38- cells LGK alone 88.09±6.85% inhibition of control, Nil alone 89.79±10.09%, combination 70.95±9.39%, p=ns, n=4; 34+38+ cells LGK alone 86.42±8.41% inhibition of control, Nil alone 87.43±4.66%, combination 73.39±6.91%, p=ns, n=4). However, LGK974 treatment did not induce apoptosis in CML or normal progenitors. These results suggest that the effects of LGK974 on clonogenic growth could be related to the long-term inhibition of LSC self-renewal. Therefore, studies designed to evaluate the effect of in vivo administration of LGK974 in a transgenic BCR-ABL mouse model of CML are in progress. Finally, our preliminary studies indicate that in vivo treatment of control healthy mice with LGK974 (5mg/kg bid p.o.), nilotinib (50mg/kg qd p.o.), or the combination for 4 weeks (n=7 per cohort) did not result in clinical toxicity or alter the short-term and long-term hematopoietic stem cell populations. In conclusion, our results indicate that treatment with the PORCN inhibitor LGK974 effectively inhibits both autocrine and paracrine WNT signaling in CML stem cells and inhibits their clonogenic capacity in vitro, and support further evaluation of this approach to selectively target CML stem cells. DisclosuresWang:Novartis Institute for Biomedical Research: Employment. McLaughlin:Novartis Institute for Biomedical Research: Employment.

Human breast progenitor cell numbers are regulated by WNT and TBX3.

BackgroundAlthough human breast development is mediated by hormonal and non-hormonal means, the mechanisms that regulate breast progenitor cell activity remain to be clarified. This limited understanding of breast progenitor cells has been due in part to the lack of appropriate model systems to detect and characterize their properties.MethodsTo examine the effects of WNT signaling and TBX3 expression on progenitor activity in the breast, primary human mammary epithelial cells (MEC) were isolated from reduction mammoplasty tissues and transduced with lentivirus to overexpress WNT1 or TBX3 or reduce expression of their cognate receptors using shRNA. Changes in progenitor activity were quantified using characterized assays. We identified WNT family members expressed by cell populations within the epithelium and assessed alterations in expression of WNT family ligands by MECs in response to TBX3 overexpression and treatment with estrogen and progesterone.ResultsGrowth of MECs on collagen gels resulted in the formation of distinct luminal acinar and basal ductal colonies. Overexpression of TBX3 in MECs resulted in increased ductal colonies, while shTBX3 expression diminished both colony types. Increased WNT1 expression led to enhanced acinar colony formation, shLRP6 decreased both types of colonies. Estrogen stimulated the formation of acinar colonies in control MEC, but not shLRP6 MEC. Formation of ductal colonies was enhanced in response to progesterone. However, while shLRP6 decreased MEC responsiveness to progesterone, shTBX3 expression did not alter this response.ConclusionsWe identified two phenotypically distinguishable lineage-committed progenitor cells that contribute to different structural elements and are regulated via hormonal and non-hormonal mechanisms. WNT signaling regulates both types of progenitor activity. Progesterone favors the expansion of ductal progenitor cells, while estrogen stimulates the expansion of acinar progenitor cells. Paracrine WNT signaling is stimulated by estrogen and progesterone, while autocrine WNT signaling is induced by the embryonic T-box transcription factor TBX3.

The DEK oncogene promotes cellular proliferation through paracrine Wnt signaling in Ron receptor-positive breast cancers.

Disease progression and recurrence are major barriers to surviving breast cancer. Understanding the etiology of recurrent or metastatic breast cancer and underlying mechanisms is critical for the development of new treatments and improved survival. Here, we report that two commonly over-expressed breast cancer oncogenes, Ron and DEK, cooperate to promote advanced disease through multi-pronged effects on β-catenin signaling. The Ron receptor is commonly activated in breast cancers, and Ron over-expression in human disease stimulates β-catenin nuclear translocation and is an independent predictor of metastatic dissemination. Dek is a chromatin-associated oncogene whose expression has been linked to cancer through multiple mechanisms, including β-catenin activity. We demonstrate here that Dek is a downstream target of Ron receptor activation in murine and human models. The absence of Dek in the MMTV-Ron mouse model led to a significant delay in tumor development, characterized by decreased cell proliferation, diminished metastasis, and fewer cells expressing cancer stem cell markers. Dek complementation of cell lines established from this model was sufficient to promote cellular growth and invasion in vitro and in vivo. Mechanistically, Dek expression stimulated the production and secretion of Wnt ligands to sustain an autocrine/paracrine canonical β-catenin signaling loop. Finally, we show that Dek over-expression promotes tumorigenic phenotypes in immortalized human mammary epithelial MCF10A cells and, in the context of Ron receptor activation, correlates with disease recurrence and metastasis in patients. Overall, our studies demonstrate that DEK over-expression, due in part to Ron receptor activation, drives breast cancer progression through the induction of Wnt/β-catenin signaling.

Therapeutic window for Wnt-driven cancers: Role of Porcupine inhibitor

Wnt signaling plays a critical role in carcinogenesis; many studies over the last two decades have identified numerous signaling components that have helped to build a molecular framework for the many branches of the Wnt signal transduction pathway. However, the diverse function, integration and specificity of the Wnt signaling are still unclear. The success of Wnt pathway inhibitors has been limited for long-time by the narrow therapeutic window afforded by the requirement for Wnt signaling in normal tissue homeostasis and the lack of predictive biomarkers of response. Porcupine is a membrane bound O-acyltransferase enzyme that is required for and dedicated to palmitoylating Wnt ligands, a necessary step in the process of Wnt ligand secretion. Inhibition of Porcupine blocks Wnt dependent activities, including LRP6 phosphorylation and the expression of Wnt target genes, such as Axin2, which in turn reduces the growth of cancer cells dependent on autocrine or paracrine Wnt signaling. LGK974 is a highly potent, selective and orally bioavailable Porcupine inhibitor and efficacious in multiple tumor models at well-tolerated doses in vivo, including murine and rat mechanistic breast cancer models driven by MMTV–Wnt1, a human head and neck squamous cell carcinoma model (HN30) and RNF43-mutant pancreatic xenograft models. In this review, we will summarize the most recent advances in our understanding of these Wnt signaling pathways and the role of Porcupine in inhibition of Wnt activity.

Stearoyl CoA Desaturase Is Required to Produce Active, Lipid-Modified Wnt Proteins

Wnt proteins contain palmitoleic acid, an unusual lipid modification. Production of an active Wnt signal requires the acyltransferase Porcupine and depends on the attachment of palmitoleic acid to Wnt. The source of this monounsaturated fatty acid has not been identified, and it is not known how Porcupine recognizes its substrate and whether desaturation occurs before or after fatty acid transfer to Wnt. Here, we show that stearoyl desaturase (SCD) generates a monounsaturated fatty acid substrate that is then transferred by Porcupine to Wnt. Treatment of cells with SCD inhibitors blocked incorporation of palmitate analogs into Wnt3a and Wnt5a and reduced Wnt secretion as well as autocrine and paracrine Wnt signaling. The SCD inhibitor effects were rescued by exogenous addition of monounsaturated fatty acids. We propose that SCD is a key molecular player responsible for Wnt biogenesis and processing and that SCD inhibition provides an alternative mechanism for blocking Wnt pathway activation.

Paracrine Wnt signaling both promotes and inhibits human breast tumor growth

Wnt signaling in mouse mammary development and tumorigenesis has been heavily studied and characterized, but its role in human breast cancer remains elusive. Although Wnt inhibitors are in early clinical development, it is unclear whether they will be of therapeutic benefit to breast cancer patients, and subsequently, to which ones. To address this, we generated a panel of Wnt reporting human breast cancer cell lines and identified a previously unrecognized enrichment for the ability to respond to Wnt in the basal B or claudin-low subtype, which has a poor prognosis and no available targeted therapies. By co-injecting Wnt3A expressing human mammary fibroblasts with human breast cancer cell lines into mouse mammary fat pads, we showed that elevated paracrine Wnt signaling was correlated with accelerated tumor growth. Using this heterotypic system and a dual lentiviral reporter system that enables simultaneous real-time measurement of both Wnt-responsive cells and bulk tumor cells, we analyzed the outcome of elevated Wnt signaling in patient-derived xenograft (PDX) models. Interestingly, the PDX models exhibited responses not observed in the cell lines analyzed. Exogenous WNT3A promoted tumor growth in one human epidermal growth factor receptor 2-overexpressing PDX line but inhibited growth in a second PDX line obtained from a patient with triple-negative breast cancer. Tumor suppression was associated with squamous differentiation in the latter. Thus, our work suggests that paracrine Wnt signaling can either fuel or repress the growth of human breast cancers depending on yet to be determined aspects of the molecular pathways they express.

Foxf2 in Intestinal Fibroblasts Reduces Numbers of Lgr5+ Stem Cells and Adenoma Formation by Inhibiting Wnt Signaling

The stem cell niche at the base of the intestinal crypts, as well as stemness and high clonogenicity in colon cancer cells, depend on Wnt signaling to β-catenin. Fibroblasts modulate the Wnt pathway in normal and neoplastic epithelial cells via unclear mechanisms. We investigated how in intestinal fibroblasts the forkhead transcription factor Foxf2 controls Wnt signaling to affect numbers of stem cells and formation and growth of adenomas in mice. We created mice with different copy numbers of Foxf2 by generating Foxf2(-/+) mice and a transgenic strain, Tg(FOXF2). Adenoma formation was investigated in Apc(Min/+) mice, stem cells were counted in mice with the Lgr5-enhanced green fluorescent protein knock-in allele, proliferation was measured by incorporation of bromodeoxyuridine, Foxf2 and Sfrp1 were localized by immunohistochemistry, and signaling pathways were analyzed by quantitative polymerase chain reaction and immunoblot assays. Epithelial β-catenin was stabilized in Foxf2(-/+) mice, resulting in increased number and size of adenomas. Tg(FOXF2) mice, however, were partially resistant to intestinal neoplasia and developed fewer and smaller adenomas; Foxf2(-/+) mice developed 24-fold more tumors than Tg(FOXF2) mice. Epithelial cells of Foxf2(-/+) mice also had higher numbers of Lgr5(+) stem cells and greater amounts of crypt cell proliferation and expression of Myc (a target of Wnt signaling) than Tg(FOXF2) mice. Expression of Sfrp1, which encodes an extracellular inhibitor of Wnt, in fibroblasts increased with copy number of Foxf2. Foxf2 is a fibroblast factor that inhibits paracrine Wnt signaling and restricts the crypt stem cell niche in intestines of mice. Loss of Foxf2 promotes adenoma formation and growth.

Gpr177, a novel locus for bone mineral density and osteoporosis, regulates osteogenesis and chondrogenesis in skeletal development

Human genetic analysis has recently identified Gpr177 as a susceptibility locus for bone mineral density and osteoporosis. Determining the unknown function of this gene is therefore extremely important to furthering our knowledge base of skeletal development and disease. The protein encoded by Gpr177 exhibits an ability to modulate the trafficking of Wnt, similar to the Drosophila Wls/Evi/Srt. Because it plays a critical role in Wnt regulation, Gpr177 might be required for several key steps of skeletogenesis. To overcome the early lethality associated with the inactivation of Gpr177 in mice, conditional gene deletion is used to assess its functionality. Here we report the generation of four different mouse models with Gpr177 deficiency in various skeletogenic cell types. The loss of Gpr177 severely impairs development of the craniofacial and body skeletons, demonstrating its requirement for intramembranous and endochondral ossifications, respectively. Defects in the expansion of skeletal precursors and their differentiation into osteoblasts and chondrocytes suggest that Wnt production and signaling mediated by Gpr177 cannot be substituted. Because the Gpr177 ablation impairs Wnt secretion, we therefore identify the sources of Wnt proteins essential for osteogenesis and chondrogenesis. The intercross of Wnt signaling between distinct cell types is carefully orchestrated and necessary for skeletogenesis. Our findings lead to a proposed mechanism by which Gpr177 controls skeletal development through modulation of autocrine and paracrine Wnt signals in a lineage-specific fashion.