Human Pluripotency Is Initiated and Preserved by a Unique Subset of Founder Cells.

The assembly of organized colonies is the earliest manifestation in the derivation or induction of pluripotency in vitro. However, the necessity and origin of this assemblance is unknown. Here, we identify human pluripotent founder cells (hPFCs) that initiate, as well as preserve and establish, pluripotent stem cell (PSC) cultures. PFCs are marked by N-cadherin expression (NCAD+) and reside exclusively at the colony boundary of primate PSCs. As demonstrated by functional analysis, hPFCs harbor the clonogenic capacity of PSC cultures and emerge prior to commitment events or phenotypes associated with pluripotent reprogramming. Comparative single-cell analysis with pre- and post-implantation primate embryos revealed hPFCs share hallmark properties with primitive endoderm (PrE) and can be regulated by non-canonical Wnt signaling. Uniquely informed by primate embryo organization in vivo, our study defines a subset of founder cells critical to the establishment pluripotent state.

Mural Wnt/ß-catenin signaling regulates Lama2 expression to promote neurovascular unit maturation.

Neurovascular unit and barrier maturation rely on vascular basement membrane (vBM) composition. Laminins, a major vBM component, are crucial for these processes, yet the signaling pathway(s) that regulate their expression remain unknown. Here, we show that mural cells have active Wnt/ß-catenin signaling during central nervous system development in mice. Bulk RNA sequencing and validation using postnatal day 10 and 14 wild-type versus adenomatosis polyposis coli downregulated 1 (Apcdd1-/-) mouse retinas revealed that Lama2 mRNA and protein levels are increased in mutant vasculature with higher Wnt/ß-catenin signaling. Mural cells are the main source of Lama2, and Wnt/ß-catenin activation induces Lama2 expression in mural cells in vitro. Markers of mature astrocytes, including aquaporin 4 (a water channel in astrocyte endfeet) and integrin-α6 (a laminin receptor), are upregulated in Apcdd1-/- retinas with higher Lama2 vBM deposition. Thus, the Wnt/ß-catenin pathway regulates Lama2 expression in mural cells to promote neurovascular unit and barrier maturation.

Formal proof of the requirement of MESP1 and MESP2 in mesoderm specification and their transcriptional control via specific enhancers in mice.

MESP1 and MESP2 are transcriptional factors involved in mesoderm specification, somite boundary formation and somite polarity regulation. However, Mesp quadruple mutant zebrafish displayed only abnormal somite polarity without mesoderm specification defects. In order to re-evaluate Mesp1/Mesp2 mutants in mice, Mesp1 and Mesp2 single knockouts (KOs), and a Mesp1/Mesp2 double KO were established using genome-editing techniques without introducing selection markers commonly used before. The Mesp1/Mesp2 double KO embryos exhibited markedly severe mesoderm formation defects that were similar to the previously reported Mesp1/Mesp2 double KO embryos, indicating species differences in the function of MESP family proteins. However, the Mesp1 KO did not display any phenotype, including heart formation defects, which have been reported previously. We noted upregulation of Mesp2 in the Mesp1 KO embryos, suggesting that MESP2 rescues the loss of MESP1 in mesoderm specification. We also found that Mesp1 and Mesp2 expression in the early mesoderm is regulated by the cooperation of two independent enhancers containing T-box- and TCF/Lef-binding sites. Deletion of both enhancers caused the downregulation of both genes, resulting in heart formation defects. This study suggests dose-dependent roles of MESP1 and MESP2 in early mesoderm formation.

Sclerostin blockade promotes bone metastases of Wnt-responsive breast cancer cells.

The secreted protein sclerostin is primarily produced by osteocytes and suppresses osteoblast differentiation and function by inhibiting the canonical Wnt signaling pathway. Genetic and pharmacological inhibition of sclerostin has been shown to increase bone formation and an anti-sclerostin antibody has been clinically approved for the treatment of osteoporosis. Canonical Wnt signaling is also involved in the progression of several types of cancers including breast cancer. Here, we studied the effects of sclerostin inhibition on the development of bone metastases of breast cancer using mouse models. TOPFLASH assay and real-time PCR analysis of AXIN2, a target of canonical Wnt signaling, revealed that, among four cell lines tested, MDA-MB-231 human breast cancer cells responded highly to the canonical Wnt ligand Wnt3a, whereas other cell lines exhibited marginal responses. Consistent with these results, treatment with an anti-sclerostin antibody significantly increased the bone metastases of MDA-MB-231 but not those of other breast cancer cells. Immunohistochemical studies demonstrated that an anti-sclerostin antibody induced intracellular accumulation of ß-catenin in bone-colonized MDA-MB-231 cells. Suspension culture assays showed that Wnt3a accelerated the tumorsphere formation of MDA-MB-231 cells, whereas monolayer cell proliferation and migration were not affected. Furthermore, the numbers of osteoclasts and their precursor cells in bone metastases of MDA-MB-231 were significantly increased in mice treated with an anti-sclerostin antibody. These results collectively suggest that sclerostin blockade activates canonical Wnt signaling in ligand-responsive breast cancer cells metastasized to bone, thereby increasing bone metastases, likely to have been mediated at least in part by enhancing stem cell-like properties of cancer cells and osteoclastogenesis.

Inhibition of Canonical Wnt Signaling Promotes Ex Vivo Maintenance and Proliferation of Hematopoietic Stem Cells in Zebrafish.

The maintenance and proliferation of hematopoietic stem cells (HSCs) are tightly regulated by their niches in the bone marrow. The analysis of niche cells or stromal cell lines that can support HSCs has facilitated the finding of novel supporting factors for HSCs. Despite large efforts in the murine bone marrow; however, HSC expansion is still difficult ex vivo, highlighting the need for new approaches to elucidate the molecular elements that regulate HSCs. The zebrafish provides a unique model to study hematopoietic niches as HSCs are maintained in the kidney, allowing for a parallel view of hematopoietic niches over evolution. Here, using a stromal cell line from the zebrafish kidney, zebrafish kidney stromal (ZKS), we uncover that an inhibitor of canonical Wnt signaling, IWR-1-endo, is a potent regulator of HSCs. Coculture assays revealed that ZKS cells were in part supportive of maintenance, but not expansion, of gata2a:GFP+runx1:mCherry+ (gata2a+runx1+) HSCs. Transcriptome analysis revealed that, compared with candidate niche cells in the kidney, ZKS cells weakly expressed HSC maintenance factor genes, thpo and cxcl12, but highly expressed canonical Wnt ligand genes, wnt1, 7bb, and 9a. Thpo supplementation in ZKS culture slightly increased, but inhibition of canonical Wnt signaling by IWR-1-endo treatment largely increased the number of gata2a+runx1+ cells (>2-fold). Moreover, we found that gata2a+runx1+ cells can be maintained by supplementing both IWR-1-endo and Thpo without stromal cells. Collectively, our data provide evidence that IWR-1-endo can be used as a novel supporting factor for HSCs.

Uterine WNTS modulates fibronectin binding activity required for blastocyst attachment through the WNT/CA2+ signaling pathway in mice.

Adhesion of the implanting blastocyst involves the interaction between integrin proteins expressed by trophoblast cells and components present in the basement membrane of the endometrial luminal epithelium. Although several factors regulating integrins and their adhesion to fibronectin are already known, we showed that Wnt signaling is involved in the regulation of blastocyst adhesion through the trafficking of integrins expressed by trophoblast cells. Localization of Itgα5ß1 by immunofluorescence and FN-binding assays were conducted on peri-implantation blastocysts treated with either Wnt5a or Wnt7a proteins. Both Wnt5a and Wnt7a induced a translocation of Itgα5ß1 at the surface of the blastocyst and an increase in FN-binding activity. We further demonstrated that uterine fluid is capable of inducing integrin translocation and this activity can be specifically inhibited by the Wnt inhibitor sFRP2. To identify the Wnt signaling pathway involved in this activity, blastocysts were incubated with inhibitors of either p38MAPK, PI3K pathway or CamKII prior to the addition of Wnts. Whereas inhibition of p38MAPK and PI3K had not effect, inhibition of CamKII reduced FN-binding activity induced by Wnts. Finally, we demonstrated that inhibition of Wnts by sFRP2 reduced the binding efficiency of the blastocyst to uterine epithelial cells. Our findings provide new insight into the mechanism that regulates integrin trafficking and FN-binding activity and identifies Wnts as a key player in blastocyst attachment to the uterine epithelium.

BML-281 promotes neuronal differentiation by modulating Wnt/Ca2+ and Wnt/PCP signaling pathway.

Histone deacetylase (HDAC) inhibitors promote differentiation through post-translational modifications of histones. BML-281, an HDAC6 inhibitor, has been known to prevent tumors, acute dextran sodium sulfate-associated colitis, and lung injury. However, the neurogenic differentiation effect of BML-281 is poorly understood. In this study, we investigated the effect of BML-281 on neuroblastoma SH-SY5Y cell differentiation into mature neurons by immunocytochemistry (ICC), reverse transcriptase PCR (RT-PCR), quantitative PCR (qPCR), and western blotting analysis. We found that the cells treated with BML-281 showed neurite outgrowth and morphological changes into mature neurons under a microscope. It was confirmed that the gene expression of neuronal markers (NEFL, MAP2, Tuj1, NEFH, and NEFM) was increased with certain concentrations of BML-281. Similarly, the protein expression of neuronal markers (NeuN, Synaptophysin, Tuj1, and NFH) was upregulated with BML-281 compared to untreated cells. Following treatment with BML-281, the expression of Wnt5α increased, and downstream pathways were activated. Interestingly, both Wnt/Ca2+ and Wnt/PCP pathways activated and regulated PKC, Cdc42, RhoA, Rac1/2/3, and p-JNK. Therefore, BML-281 induces the differentiation of SH-SY5Y cells into mature neurons by activating the non-canonical Wnt signaling pathway. From these results, we concluded that BML-281 might be a novel drug to differentiation into neuronal cells through the regulation of Wnt signaling pathway to reduce the neuronal cell death.

Canonical Wnt Pathway Is Involved in Chemoresistance and Cell Cycle Arrest Induction in Colon Cancer Cell Line Spheroids.

The presence of cancer stem cells (CSCs) has been associated with the induction of drug resistance and disease recurrence after therapy. 5-Fluorouracil (5FU) is widely used as the first-line treatment of colorectal cancer (CRC). However, its effectiveness may be limited by the induction of drug resistance in tumor cells. The Wnt pathway plays a key role in the development and CRC progression, but it is not clearly established how it is involved in CSCs resistance to treatment. This work aimed to investigate the role played by the canonical Wnt/ß-catenin pathway in CSCs resistance to 5FU treatment. Using tumor spheroids as a model of CSCs enrichment of CRC cell lines with different Wnt/ß-catenin contexts, we found that 5FU induces in all CRC spheroids tested cell death, DNA damage, and quiescence, but in different proportions for each one: RKO spheroids were very sensitive to 5FU, while SW480 were less susceptible, and the SW620 spheroids, the metastatic derivative of SW480 cells, displayed the highest resistance to death, high clonogenic capacity, and the highest ability for regrowth after 5FU treatment. Activating the canonical Wnt pathway with Wnt3a in RKO spheroids decreased the 5FU-induced cell death. But the Wnt/ß-catenin pathway inhibition with Adavivint alone or in combination with 5FU in spheroids with aberrant activation of this pathway produced a severe cytostatic effect compromising their clonogenic capacity and diminishing the stem cell markers expression. Remarkably, this combined treatment also induced the survival of a small cell subpopulation that could exit the arrest, recover SOX2 levels, and re-grow after treatment.

The Roles of Secreted Wnt Ligands in Cancer.

Wnt ligands are secreted signaling proteins that display a wide range of biological effects. They play key roles in stimulating Wnt signaling pathways to facilitate processes such as tissue homeostasis and regeneration. Dysregulation of Wnt signaling is a hallmark of many cancers and genetic alterations in various Wnt signaling components, which result in ligand-independent or ligand-dependent hyperactivation of the pathway that have been identified. Recently, research is focusing on the impact of Wnt signaling on the interaction between tumor cells and their micro-environment. This Wnt-mediated crosstalk can act either in a tumor promoting or suppressing fashion. In this review, we comprehensively outline the function of Wnt ligands in different tumor entities and their impact on key phenotypes, including cancer stemness, drug resistance, metastasis, and immune evasion. Lastly, we elaborate approaches to target Wnt ligands in cancer therapy.

Identification of novel human Wnt target genes using adult endodermal tissue-derived organoids.

Canonical Wnt signaling plays a key role during organ development, homeostasis and regeneration and these processes are conserved between invertebrates and vertebrates. Mutations in Wnt pathway components are commonly found in various types of cancer. Upon activation of canonical Wnt signaling, ß-catenin binds in the nucleus to members of the TCF-LEF family and activates the transcription of target genes. Multiple Wnt target genes, including Lgr5/LGR5 and Axin2/AXIN2, have been identified in mouse models and human cancer cell lines. Here we set out to identify the transcriptional targets of Wnt signaling in five human tissues using organoid technology. Organoids are derived from adult stem cells and recapitulate the functionality as well as the structure of the original tissue. Since the Wnt pathway is critical to maintain the organoids from the human intestine, colon, liver, pancreas and stomach, organoid technology allows us to assess Wnt target gene expression in a human wildtype situation. We performed bulk mRNA sequencing of organoids immediately after inhibition of Wnt pathway and identified 41 genes as commonly regulated genes in these tissues. We also identified large numbers of target genes specific to each tissue. One of the shared target genes is TEAD4, a transcription factor driving expression of YAP/TAZ signaling target genes. In addition to TEAD4, we identified a variety of genes which encode for proteins that are involved in Wnt-independent pathways, implicating the possibility of direct crosstalk between Wnt signaling and other pathways. Collectively, this study identified tissue-specific and common Wnt target gene signatures and provides evidence for a conserved role for these Wnt targets in different tissues.

Wnt5a is a transcriptional target of Gli3 and Trps1 at the onset of chondrocyte hypertrophy.

During endochondral ossification, the differentiation of proliferating into hypertrophic chondrocytes is a key step determining the pace of bone formation and the future length of the skeletal elements. A variety of transcription factors are expressed at the onset of hypertrophy coordinating the expression of different signaling molecules like Bmps, Ihh and Wnt proteins. In this study, we characterized the murine Wnt5a promoter and provide evidence that two alternative Wnt5a transcripts, Ts1 and Ts2, are differentially expressed in the developing skeletal elements. Ts2 expression decreases while Ts1 expression increases during chondrocyte differentiation. The transcription factor Trps1 and the activator form of Gli3 (Gli3A), which is a mediator of Hedgehog signaling, activate Wnt5a expression. In Chromatin Immunoprecipitation and reporter gene assays, we identified two upstream regulatory sequences (URS) in the Wnt5a promoter mediating either activating or repressive functions. The activating URS1 is bound by Trps1 and Gli3A in vitro and in vivo to upregulate Wnt5a expression. Loss of both transcription factors decreases endogenous Wnt5a mRNA and protein levels during chondrocyte differentiation, thereby identifying Wnt5a as a target gene of Trps1 and Gli3A in chondrocytes.

SHROOM3 is downstream of the planar cell polarity pathway and loss-of-function results in congenital heart defects.

Congenital heart disease (CHD) is the most common birth defect, and the leading cause of death due to birth defects, yet causative molecular mechanisms remain mostly unknown. We previously implicated a novel CHD candidate gene, SHROOM3, in a patient with CHD. Using a Shroom3 gene trap knockout mouse (Shroom3gt/gt) we demonstrate that SHROOM3 is downstream of the noncanonical Wnt planar cell polarity signaling pathway (PCP) and loss-of-function causes cardiac defects. We demonstrate Shroom3 expression within cardiomyocytes of the ventricles and interventricular septum from E10.5 onward, as well as within cardiac neural crest cells and second heart field cells that populate the cardiac outflow tract. We demonstrate that Shroom3gt/gt mice exhibit variable penetrance of a spectrum of CHDs that include ventricular septal defects, double outlet right ventricle, and thin left ventricular myocardium. This CHD spectrum phenocopies what is observed with disrupted PCP. We show that during cardiac development SHROOM3 interacts physically and genetically with, and is downstream of, key PCP signaling component Dishevelled 2. Within Shroom3gt/gt hearts we demonstrate disrupted terminal PCP components, actomyosin cytoskeleton, cardiomyocyte polarity, organization, proliferation and morphology. Together, these data demonstrate SHROOM3 functions during cardiac development as an actomyosin cytoskeleton effector downstream of PCP signaling, revealing SHROOM3's novel role in cardiac development and CHD.

Differential skeletal response in adult and aged rats to independent and combinatorial stimulation with pulsed electromagnetic fields and mechanical vibration.

Pulsed electromagnetic fields (PEMFs) and whole-body vibration (WBV) are proved to partially preserve bone mass/strength in hindlimb-unloaded and ovariectomized animals. However, the potential age-dependent skeletal response to either PEMF or WBV has not been fully investigated. Moreover, whether the coupled "mechano-electro-magnetic" signals can induce greater osteogenic potential than single stimulation remains unknown. Herein, 5-month-old or 20-month-old rats were assigned to the Control, PEMF, WBV, and PEMF + WBV groups. After 8-week treatment, single PEMF/WBV enhanced bone mass, strength, and anabolism in 5-month-old rats, but not in 20-month-old rats. PEMF + WBV induced greater increase of bone quantity, quality, and anabolism than single PEMF/WBV in young adult rats. PEMF + WBV also inhibited bone loss in elderly rats by primarily improving osteoblast and osteocyte activity, but had no effects on bone resorption. PEMF + WBV upregulated the expression of various canonical Wnt ligands and downstream molecules (p-GSK-3ß and ß-catenin), but had no impacts on noncanonical Wnt5a expression in aged skeleton, revealing the potential involvement of canonical Wnt signaling in bone anabolism of PEMF + WBV. This study not only reveals much weaker responsiveness of aged skeleton to single PEMF/WBV relative to young adult skeleton, but also presents a novel noninvasive approach based on combinatorial treatment with PEMF + WBV for improving bone health and preserving bone quantity/quality (especially for age-related osteoporosis) with stronger anabolic effects.

New roles for Wnt and BMP signaling in neural anteroposterior patterning.

During amphibian development, neural patterning occurs via a two-step process. Spemann's organizer secretes BMP antagonists that induce anterior neural tissue. A subsequent caudalizing step re-specifies anterior fated cells to posterior fates such as hindbrain and spinal cord. The neural patterning paradigm suggests that a canonical Wnt-signaling gradient acts along the anteroposterior axis to pattern the nervous system. Wnt activity is highest in the posterior, inducing spinal cord, at intermediate levels in the trunk, inducing hindbrain, and is lowest in anterior fated forebrain, while BMP-antagonist levels are constant along the axis. Our results in Xenopus laevis challenge this paradigm. We find that inhibition of canonical Wnt signaling or its downstream transcription factors eliminates hindbrain, but not spinal cord fates, an observation not compatible with a simple high-to-low Wnt gradient specifying all fates along the neural anteroposterior axis. Additionally, we find that BMP activity promotes posterior spinal cord cell fate formation in an FGF-dependent manner, while inhibiting hindbrain fates. These results suggest a need to re-evaluate the paradigms of neural anteroposterior pattern formation during vertebrate development.

PTK7 proteolytic fragment proteins function during early Xenopus development.

Protein Tyrosine Kinase 7 (PTK7) is as a critical regulator of canonical and non-canonical Wnt-signaling during embryonic development and cancer cell formation. Disrupting PTK7 activity perturbs vertebrate nervous system development, and also promotes human cancer formation. Observations in different model systems suggest a complex cross-talk between PTK7 protein and Wnt signaling. During Xenopus laevis nervous system development, we previously showed that PTK7 protein positively regulates canonical Wnt signaling by maintaining optimal LRP6 protein levels, but PTK7 also acts in concert with LRP6 protein to repress non-canonical Wnt activity. PTK7 is a transmembrane protein, but studies in cancer cells showed that PTK7 undergoes "shedding" by metalloproteases to different proteolytic fragments. Some PTK7 proteolytic fragments are oncogenic, being localized to alternative cytoplasmic and nuclear cell compartments. In this study we examined the biological activity of two proteolytic carboxyl-terminal PTK7 proteolytic fragments, cPTK7 622-1070 and cPTK7 726-1070 during early Xenopus nervous system development. We found that these smaller PTK7 proteolytic fragments have similar activity to full-length PTK7 protein to promote canonical Wnt-signaling via regulation of LRP6 protein levels. In addition to cancer systems, this study shows in vivo proof that these smaller PTK7 proteolytic fragments can recapitulate full-length PTK7 protein activity in diverse systems, such as vertebrate nervous system development.

Beta-catenin non-canonical pathway: A potential target for inflammatory and hyperproliferative state via expression of transglutaminase 2 in psoriatic skin keratinocyte.

Psoriasis is a chronic, local as well as a systemic, inflammatory skin condition. Psoriasis influences the quality of life up to 3.8% of the population and occurs often between 15 and 30 years of age. Specific causes are linked to psoriasis, including the interleukin IL-23/IL-17 Axis, human antigen leucocyte (HLA), and tumor necrosis factor-α (TNF-α). Secukinumab is a monoclonal antibody that specifically binds and neutralizes IL-17A required in the treatment of Psoriasis. The signaling pathways of Wnt govern multiple functions of cell-like fate specification, proliferation, polarity, migration, differentiation with their signaling controlled rigorously, given that dysregulation caused by various stimuli, can lead to alterations in cell proliferation, apoptosis, and human inflammatory disease. Current data has supported non-canonical Wnt signaling pathways in psoriasis development, particularly Wnt5a activated signaling cascades. These interconnected factors are significant in interactions between immune cells, keratinocytes, and inflammatory factors due to a higher degree of transglutaminase 2, mediated by activation of the keratinocyte hyperproliferation of the psoriatic patient's epidermis. This study discusses the pathology of Wnt5a signaling and its involvement in the epidermal inflammatory effects of psoriasis with other related pathways.

Repression of MAP3K1 expression and JNK activity by canonical Wnt signaling.

Morphogenesis is a complex and highly coordinated process orchestrated by temporal spatial activity of developmental pathways. How the different pathways interact to guide the developmental program remains an intriguing and open question. MAP3K1-JNK and Wnt are signaling pathways crucial for embryonic eyelid closure, an epithelial morphogenetic event conserved in mammals. Here we used a mouse model of eyelid development and genetic and biochemistry tools to investigate the relationships between the two pathways. We found that Wnt activation repressed MAP3K1 expression. Using Axin-LacZ reporter mice, spatial Wnt activity was detected in the leading edge of the developing eyelid. Conditional knockout of Wntless (Wls) in ocular surface ectoderm blocked eyelid formation, and significantly increased MAP3K1 expression in eyelid cells at the nasal canthus region. Conversely, knockout of Dkk2, encoding a canonical Wnt antagonist, resulted in an increase of Wnt activity in cells at the upper eyelid margin near the nasal canthus. Up-regulation of Wnt signaling in the Dkk2-knockout embryos corresponded to down-regulation of MAP3K1 expression. In vitro data showed that Wnt3a treatment decreased MAP3K1 promoter activity, whereas activation of Wnt by lithium chloride inhibited MAP3K1 expression, and attenuated MAP3K1-mediated JNK activity. Our data identify a unique signal crosstalk between Wnt signaling and the MAP3K1-JNK pathway in epithelial morphogenesis.

A deficiency in SUMOylation activity disrupts multiple pathways leading to neural tube and heart defects in Xenopus embryos.

BACKGROUND: Adenovirus protein, Gam1, triggers the proteolytic destruction of the E1 SUMO-activating enzyme. Microinjection of an empirically determined amount of Gam1 mRNA into one-cell Xenopus embryos can reduce SUMOylation activity to undetectable, but nonlethal, levels, enabling an examination of the role of this post-translational modification during early vertebrate development. RESULTS: We find that SUMOylation-deficient embryos consistently exhibit defects in neural tube and heart development. We have measured differences in gene expression between control and embryos injected with Gam1 mRNA at three developmental stages: early gastrula (immediately following the initiation of zygotic transcription), late gastrula (completion of the formation of the three primary germ layers), and early neurula (appearance of the neural plate). Although changes in gene expression are widespread and can be linked to many biological processes, three pathways, non-canonical Wnt/PCP, snail/twist, and Ets-1, are especially sensitive to the loss of SUMOylation activity and can largely account for the predominant phenotypes of Gam1 embryos. SUMOylation appears to generate different pools of a given transcription factor having different specificities with this post-translational modification involved in the regulation of more complex, as opposed to housekeeping, processes. CONCLUSIONS: We have identified changes in gene expression that underlie the neural tube and heart phenotypes resulting from depressed SUMOylation activity. Notably, these developmental defects correspond to the two most frequently occurring congenital birth defects in humans, strongly suggesting that perturbation of SUMOylation, either globally or of a specific protein, may frequently be the origin of these pathologies.

Functions of the APC tumor suppressor protein dependent and independent of canonical WNT signaling: implications for therapeutic targeting.

The acquisition of biallelic mutations in the APC gene is a rate-limiting step in the development of most colorectal cancers and occurs in the earliest lesions. APC encodes a 312-kDa protein that localizes to multiple subcellular compartments and performs diverse functions. APC participates in a cytoplasmic complex that promotes the destruction of the transcriptional licensing factor ß-catenin; APC mutations that abolish this function trigger constitutive activation of the canonical WNT signaling pathway, a characteristic found in almost all colorectal cancers. By negatively regulating canonical WNT signaling, APC counteracts proliferation, promotes differentiation, facilitates apoptosis, and suppresses invasion and tumor progression. APC further antagonizes canonical WNT signaling by interacting with and counteracting ß-catenin in the nucleus. APC also suppresses tumor initiation and progression in the colorectal epithelium through functions that are independent of canonical WNT signaling. APC regulates the mitotic spindle to facilitate proper chromosome segregation, localizes to the cell periphery and cell protrusions to establish cell polarity and appropriate directional migration, and inhibits DNA replication by interacting directly with DNA. Mutations in APC are often frameshifts, insertions, or deletions that introduce premature stop codons and lead to the production of truncated APC proteins that lack its normal functions and possess tumorigenic properties. Therapeutic approaches in development for the treatment of APC-deficient tumors are focused on the inhibition of canonical WNT signaling, especially through targets downstream of APC in the pathway, or on the restoration of wild-type APC expression.

Progesterone and Wnt4 control mammary stem cells via myoepithelial crosstalk.

Ovarian hormones increase breast cancer risk by poorly understood mechanisms. We assess the role of progesterone on global stem cell function by serially transplanting mouse mammary epithelia. Progesterone receptor (PR) deletion severely reduces the regeneration capacity of the mammary epithelium. The PR target, receptor activator of Nf-κB ligand (RANKL), is not required for this function, and the deletion of Wnt4 reduces the mammary regeneration capacity even more than PR ablation. A fluorescent reporter reveals so far undetected perinatal Wnt4 expression that is independent of hormone signaling. Pubertal and adult Wnt4 expression is specific to PR+ luminal cells and requires intact PR signaling. Conditional deletion of Wnt4 reveals that this early, previously unappreciated, Wnt4 expression is functionally important. We provide genetic evidence that canonical Wnt signaling in the myoepithelium required PR and Wnt4, whereas the canonical Wnt signaling activities observed in the embryonic mammary bud and in the stroma around terminal end buds are independent of Wnt4. Thus, progesterone and Wnt4 control stem cell function through a luminal-myoepithelial crosstalk with Wnt4 acting independent of PR perinatally.