The role of WNT10B in physiology and disease: A 10-year update.

WNT10B, a member of the WNT family of secreted glycoproteins, activates the WNT/ß-catenin signaling cascade to control proliferation, stemness, pluripotency, and cell fate decisions. WNT10B plays roles in many tissues, including bone, adipocytes, skin, hair, muscle, placenta, and the immune system. Aberrant WNT10B signaling leads to several diseases, such as osteoporosis, obesity, split-hand/foot malformation (SHFM), fibrosis, dental anomalies, and cancer. We reviewed WNT10B a decade ago, and here we provide a comprehensive update to the field. Novel research on WNT10B has expanded to many more tissues and diseases. WNT10B polymorphisms and mutations correlate with many phenotypes, including bone mineral density, obesity, pig litter size, dog elbow dysplasia, and cow body size. In addition, the field has focused on the regulation of WNT10B using upstream mediators, such as microRNAs (miRNAs) and long non-coding RNAs (lncRNAs). We also discussed the therapeutic implications of WNT10B regulation. In summary, research conducted during 2012-2022 revealed several new, diverse functions in the role of WNT10B in physiology and disease.

CYP1B1 Augments the Mesenchymal, Claudin-Low, and Chemoresistant Phenotypes of Triple-Negative Breast Cancer Cells.

Cytochrome P4501B1 (CYP1B1) is elevated in breast cancer. Studies indicate a relationship between CYP1B1 and aggressive cancer phenotypes. Here, we report on in vitro studies in triple-negative breast cancer cell lines, where knockdown (KD) of CYP1B1 was used to determine the influence of its expression on invasive cell phenotypes. CYP1B1 KD in MDA-MB-231 cells resulted in the loss of mesenchymal morphology, altered expression of epithelial-mesenchymal genes, and increased claudin (CLDN) RNA and protein. CYP1B1 KD cells had increased cell-to-cell contact and paracellular barrier function, a reduced rate of cell proliferation, abrogation of migratory and invasive activity, and diminished spheroid formation. Analysis of clinical breast cancer tumor samples revealed an association between tumors exhibiting higher CYP1B1 RNA levels and diminished overall and disease-free survival. Tumor expression of CYP1B1 was inversely associated with CLDN7 expression, and CYP1B1HI/CLDN7LOW identified patients with lower median survival. Cells with CYP1B1 KD had an enhanced chemosensitivity to paclitaxel, 5-fluorouracil, and cisplatin. Our findings that CYP1B1 KD can increase chemosensitivity points to therapeutic targeting of this enzyme. CYP1B1 inhibitors in combination with chemotherapeutic drugs may provide a novel targeted and effective approach to adjuvant or neoadjuvant therapy against certain forms of highly metastatic breast cancer.

MAP3K4 promotes fetal and placental growth by controlling the receptor tyrosine kinases IGF1R/IR and Akt signaling pathway.

Disruption of fetal growth results in severe consequences to human health, including increased fetal and neonatal morbidity and mortality, as well as potential lifelong health problems. Molecular mechanisms promoting fetal growth represent potential therapeutic strategies to treat and/or prevent fetal growth restriction (FGR). Here, we identify a previously unknown role for the mitogen-activated protein kinase kinase kinase 4 (MAP3K4) in promoting fetal and placental growth. We demonstrate that inactivation of MAP3K4 kinase activity causes FGR due in part to placental insufficiency. Significantly, MAP3K4 kinase-inactive mice display highly penetrant lethality prior to weaning and persistent growth reduction of surviving adults. Additionally, we elucidate molecular mechanisms by which MAP3K4 promotes growth through control of the insulin-like growth factor 1 receptor (IGF1R), insulin receptor (IR), and Akt signaling pathway. Specifically, MAP3K4 kinase inactivation in trophoblast stem (TS) cells results in reduced IGF1R and IR expression and decreased Akt activation. We observe these changes in TS cells also occur in differentiated trophoblasts created through in vitro differentiation of cultured TS cells and in vivo in placental tissues formed by TS cells. Furthermore, we show that MAP3K4 controls this pathway by promoting Igf1r transcript expression in TS cells through activation of CREB-binding protein (CBP). In the MAP3K4 kinase-inactive TS cells, Igf1r transcripts are repressed because of reduced CBP activity and increased histone deacetylase 6 expression and activity. Together, these data demonstrate a critical role for MAP3K4 in promoting fetal and placental growth by controlling the activity of the IGF1R/IR and Akt signaling pathway.

MAP3K4 kinase activity dependent control of mouse gonadal sex determination†.

Sex determination requires the commitment of bipotential gonads to either a testis or an ovarian fate. Gene deletion of the kinase Map3k4 results in gonadal sex reversal in XY mice, and transgenic re-expression of Map3k4 rescues the sex reversal phenotype. Map3k4 encodes a large, multi-functional protein possessing a kinase domain and several, additional protein-protein interaction domains. Although MAP3K4 plays a critical role in male gonadal sex determination, it is unknown if the kinase activity of MAP3K4 is required. Here, we use mice expressing full-length, kinase-inactive MAP3K4 from the endogenous Map3k4 locus to examine the requirement of MAP3K4 kinase activity in sex determination. Although homozygous kinase-inactivation of MAP3K4 (Map3k4KI/KI) is lethal, a small fraction survive to adulthood. We show Map3k4KI/KI adults exhibit a 4:1 female-biased sex ratio. Many adult Map3k4KI/KI phenotypic females have a Y chromosome. XY Map3k4KI/KI adults with sex reversal display female mating behavior, but do not give rise to offspring. Reproductive organs are overtly female, but there is a broad spectrum of ovarian phenotypes, including ovarian absence, primitive ovaries, reduced ovarian size, and ovaries having follicles in all stages of development. Further, XY Map3k4KI/KI adults are smaller than either male or female Map3k4WT/WT mice. Examination of the critical stage of gonadal sex determination at E11.5 shows that loss of MAP3K4 kinase activity results in the loss of Sry expression in XY Map3k4KI/KI embryos, indicating embryonic male gonadal sex reversal. Together, these findings demonstrate the essential role for kinase activity of MAP3K4 in male gonadal sex determination.

GALNT3 Maintains the Epithelial State in Trophoblast Stem Cells.

O-GalNAc glycosylation is initiated in the Golgi by glycosyltransferases called GALNTs. Proteomic screens identified >600 O-GalNAc-modified proteins, but the biological relevance of these modifications has been difficult to determine. We have discovered a conserved function for GALNT3 in trophoblast stem (TS) cells, blastocyst trophectoderm, and human mammary epithelial cells (HMECs). The loss of GALNT3 expression in these systems reduces O-GalNAc glycosylation and induces epithelial-mesenchymal transition. Furthermore, Galnt3 expression is reduced in aggressive, mesenchymal claudin-low breast cancer cells. We show that GALNT3 expression controls the O-GalNAc glycosylation of multiple proteins, including E-cadherin in both TS cells and HMECs. The loss of GALNT3 results in the intracellular retention of E-cadherin in the Golgi. Significantly, re-expression of GALNT3 in TS cells increases O-GalNAc glycosylation and restores the epithelial state. Together, these data demonstrate the critical biological role of GALNT3 O-GalNAc glycosylation to promote the epithelial phenotype in TS cells, blastocyst trophectoderm, and HMECs.

Controlling Epithelial to Mesenchymal Transition through Acetylation of Histone H2BK5.

Large-scale epigenetic changes take place when epithelial cells with cell-cell adhesion and apical-basal polarity transition into invasive, individual, mesenchymal cells through a process known as epithelial to mesenchymal transition (EMT). Importantly, cancers with stem cell properties disseminate and form distant metastases by reactivating the developmental EMT program. Recent studies have demonstrated that the epigenetic histone modification, H2BK5 acetylation (H2BK5Ac), is important in the regulation of EMT. For example, in trophoblast stem (TS) cells, H2BK5Ac promotes the expression of genes important to the maintenance of an epithelial phenotype. This finding led to the discovery that TS cells and stem-like claudin-low breast cancer cells share similar H2BK5Ac-regulated gene expression, linking developmental and cancer cell EMT. An improved understanding of the role of H2BK5Ac in developmental EMT and stemness will further our understanding of epigenetics in EMT-related pathologies. Here, we examine the binders and regulators of H2BK5Ac and discuss the roles of H2BK5Ac in stemness and EMT.

MAP3K4 Controls the Chromatin Modifier HDAC6 during Trophoblast Stem Cell Epithelial-to-Mesenchymal Transition.

The first epithelial-to-mesenchymal transition (EMT) occurs in trophoblast stem (TS) cells during implantation. Inactivation of the serine/threonine kinase MAP3K4 in TS cells (TSKI4 cells) induces an intermediate state of EMT, where cells retain stemness, lose epithelial markers, and gain mesenchymal characteristics. Investigation of relationships among MAP3K4 activity, stemness, and EMT in TS cells may reveal key regulators of EMT. Here, we show that MAP3K4 activity controls EMT through the ubiquitination and degradation of HDAC6. Loss of MAP3K4 activity in TSKI4 cells results in elevated HDAC6 expression and the deacetylation of cytoplasmic and nuclear targets. In the nucleus, HDAC6 deacetylates the promoters of tight junction genes, promoting the dissolution of tight junctions. Importantly, HDAC6 knockdown in TSKI4 cells restores epithelial features, including cell-cell adhesion and barrier formation. These data define a role for HDAC6 in regulating gene expression during transitions between epithelial and mesenchymal phenotypes.

Implications of Mesenchymal Cells in Cancer Stem Cell Populations: Relevance to EMT.

The epithelial to mesenchymal transition (EMT) generates tumor cells having stem cell characteristics with phenotypes similar to cancer stem cells (CSCs). Evidence suggests CSCs are in an intermediate state of EMT expressing reduced levels of E-cadherin and exhibiting mesenchymal features including invasiveness associated with metastasis. These findings suggest mechanisms regulating EMT and stemness are closely integrated. Recent reports from multiple laboratories have identified novel mechanisms regulating EMT and stemness involving epigenetics, microenvironment, and dedifferentiation. Circulating tumor cells (CTCs) have also been shown to exhibit features of EMT, but it is unclear what fraction has CSCs properties. EMT characteristics of both CSCs and CTCs are associated with resistance to current clinical treatments, indicating therapies targeting the CSC in addition to the more differentiated tumor cells are required for durable responses. Thus, EMT characteristics of CTCs may prove useful biomarkers for effective therapies for many cancers.

SWI/SNF chromatin-remodeling factor Smarcd3/Baf60c controls epithelial-mesenchymal transition by inducing Wnt5a signaling.

We previously identified a gene signature predicted to regulate the epithelial-mesenchymal transition (EMT) in both epithelial tissue stem cells and breast cancer cells. A phenotypic RNA interference (RNAi) screen identified the genes within this 140-gene signature that promoted the conversion of mesenchymal epithelial cell adhesion molecule-negative (EpCAM-) breast cancer cells to an epithelial EpCAM+/high phenotype. The screen identified 10 of the 140 genes whose individual knockdown was sufficient to promote EpCAM and E-cadherin expression. Among these 10 genes, RNAi silencing of the SWI/SNF chromatin-remodeling factor Smarcd3/Baf60c in EpCAM- breast cancer cells gave the most robust transition from the mesenchymal to epithelial phenotype. Conversely, expression of Smarcd3/Baf60c in immortalized human mammary epithelial cells induced an EMT. The mesenchymal-like phenotype promoted by Smarcd3/Baf60c expression resulted in gene expression changes in human mammary epithelial cells similar to that of claudin-low triple-negative breast cancer cells. These mammary epithelial cells expressing Smarcd3/Baf60c had upregulated Wnt5a expression. Inhibition of Wnt5a by either RNAi knockdown or blocking antibody reversed Smarcd3/Baf60c-induced EMT. Thus, Smarcd3/Baf60c epigenetically regulates EMT by activating WNT signaling pathways.

Dynamic reprogramming of the kinome in response to targeted MEK inhibition in triple-negative breast cancer.

Kinase inhibitors have limited success in cancer treatment because tumors circumvent their action. Using a quantitative proteomics approach, we assessed kinome activity in response to MEK inhibition in triple-negative breast cancer (TNBC) cells and genetically engineered mice (GEMMs). MEK inhibition caused acute ERK activity loss, resulting in rapid c-Myc degradation that induced expression and activation of several receptor tyrosine kinases (RTKs). RNAi knockdown of ERK or c-Myc mimicked RTK induction by MEK inhibitors, and prevention of proteasomal c-Myc degradation blocked kinome reprogramming. MEK inhibitor-induced RTK stimulation overcame MEK2 inhibition, but not MEK1 inhibition, reactivating ERK and producing drug resistance. The C3Tag GEMM for TNBC similarly induced RTKs in response to MEK inhibition. The inhibitor-induced RTK profile suggested a kinase inhibitor combination therapy that produced GEMM tumor apoptosis and regression where single agents were ineffective. This approach defines mechanisms of drug resistance, allowing rational design of combination therapies for cancer.

Tracking the intermediate stages of epithelial-mesenchymal transition in epithelial stem cells and cancer.

Epithelial-mesenchymal transition (EMT) is an essential developmental program that becomes reactivated in adult tissues to promote the progression of cancer. EMT has been largely studied by examining the beginning epithelial state or the ending mesenchymal state without studying the intermediate stages. Recent studies using trophoblast stem (TS) cells paused in EMT have defined the molecular and epigenetic mechanisms responsible for modulating the intermediate "metastable" stages of EMT. Targeted inactivation of MAP3K4, knockdown of CBP, or overexpression of SNAI1 in TS cells induced similar metastable phenotypes. These TS cells exhibited epigenetic changes in the histone acetylation landscape that cause loss of epithelial maintenance while preserving self-renewal and multipotency. A similar phenotype was found in claudin-low breast cancer cells with properties of EMT and stemness. This intersection between EMT and stemness in TS cells and claudin-low metastatic breast cancer demonstrates the usefulness of developmental EMT systems to understand EMT in cancer.

Efficiently identifying genome-wide changes with next-generation sequencing data.

We propose a new and effective statistical framework for identifying genome-wide differential changes in epigenetic marks with ChIP-seq data or gene expression with mRNA-seq data, and we develop a new software tool EpiCenter that can efficiently perform data analysis. The key features of our framework are: (i) providing multiple normalization methods to achieve appropriate normalization under different scenarios, (ii) using a sequence of three statistical tests to eliminate background regions and to account for different sources of variation and (iii) allowing adjustment for multiple testing to control false discovery rate (FDR) or family-wise type I error. Our software EpiCenter can perform multiple analytic tasks including: (i) identifying genome-wide epigenetic changes or differentially expressed genes, (ii) finding transcription factor binding sites and (iii) converting multiple-sample sequencing data into a single read-count data matrix. By simulation, we show that our framework achieves a low FDR consistently over a broad range of read coverage and biological variation. Through two real examples, we demonstrate the effectiveness of our framework and the usages of our tool. In particular, we show that our novel and robust 'parsimony' normalization method is superior to the widely-used 'tagRatio' method. Our software EpiCenter is freely available to the public.

MAP3K4/CBP-regulated H2B acetylation controls epithelial-mesenchymal transition in trophoblast stem cells.

Epithelial stem cells self-renew while maintaining multipotency, but the dependence of stem cell properties on maintenance of the epithelial phenotype is unclear. We previously showed that trophoblast stem (TS) cells lacking the protein kinase MAP3K4 maintain properties of both stemness and epithelial-mesenchymal transition (EMT). Here, we show that MAP3K4 controls the activity of the histone acetyltransferase CBP, and that acetylation of histones H2A and H2B by CBP is required to maintain the epithelial phenotype. Combined loss of MAP3K4/CBP activity represses expression of epithelial genes and causes TS cells to undergo EMT while maintaining their self-renewal and multipotency properties. The expression profile of MAP3K4-deficient TS cells defines an H2B acetylation-regulated gene signature that closely overlaps with that of human breast cancer cells. Taken together, our data define an epigenetic switch that maintains the epithelial phenotype in TS cells and reveals previously unrecognized genes potentially contributing to breast cancer.

Rho kinase inhibition rescues the endothelial cell cerebral cavernous malformation phenotype.

Cerebral cavernous malformations (CCM) are vascular lesions causing seizures and stroke. Mutations causing inactivation of one of three genes, ccm1, -2, or -3, are sufficient to induce vascular endothelial cell defects resulting in CCM. Herein, we show that loss of expression of the CCM1, -2, or -3 proteins causes a marked increase in expression of the GTPase RhoA. Live cell imaging with a RhoA-specific biosensor demonstrates increased RhoA activity with loss of CCM1, -2, or -3, with an especially pronounced RhoA activation in both the cytosol and the nucleus with loss of CCM1 expression. Increased RhoA activation was associated with Rho kinase-dependent phosphorylation of myosin light chain 2. Functionally, loss of CCM1, -2, or -3 inhibited endothelial cell vessel-like tube formation and extracellular matrix invasion, each of which is rescued by chemical inhibition or short hairpin RNA knockdown of Rho kinase. The findings, for the first time, define a signaling network for CCM1, -2, and -3 in CCM pathology, whereby loss of CCM1, -2, or -3 protein expression results in increased RhoA activity, with the activation of Rho kinase responsible for endothelial cell dysregulation. The results define Rho kinase as a therapeutic target to rescue endothelial cells from loss of CCM protein function.

Trophoblast stem cell maintenance by fibroblast growth factor 4 requires MEKK4 activation of Jun N-terminal kinase.

Trophoblast differentiation during placentation involves an epithelial-mesenchymal transition (EMT) with loss of E-cadherin and gain of trophoblast invasiveness. Mice harboring a point mutation that renders inactive the mitogen-activated protein kinase kinase kinase MEKK4 exhibit dysregulated placental development with increased trophoblast invasion. Isolated MEKK4 kinase-inactive trophoblast stem (TS) cells cultured under undifferentiating, self-renewing conditions in the presence of fibroblast growth factor 4 (FGF4) display increased expression of Slug, Twist, and matrix metalloproteinase 2 (MMP2), loss of E-cadherin, and hyperinvasion of extracellular matrix, each a hallmark of EMT. MEKK4 kinase-inactive TS cells show a preferential differentiation to Tpbp alpha- and Gcm1-positive trophoblasts, which are indicative of spongiotrophoblast and syncytiotrophoblast differentiation, respectively. FGF4-stimulated Jun N-terminal kinase (JNK) and p38 activity is markedly reduced in MEKK4 kinase-inactive TS cells. Chemical inhibition of JNK in wild-type TS cells induced a similar EMT response as loss of MEKK4 kinase activity, including inhibition of E-cadherin expression and increased expression of Slug, MMP2, Tpbp alpha, and Gcm1. Chromatin immunoprecipitation analyses revealed changes in AP-1 composition with increased Fra-2 and decreased Fra-1 and JunB binding to the regulatory regions of Gcm1 and MMP2 genes in MEKK4 kinase-inactive TS cells. Our results define MEKK4 as a signaling hub for FGF4 activation of JNK that is required for maintenance of TS cells in an undifferentiated state.

MEKK4 stimulation of p38 and JNK activity is negatively regulated by GSK3beta.

The MAPK kinase kinase MEKK4 is required for neurulation and skeletal patterning during mouse development. MEKK4 phosphorylates and activates MKK4/MKK7 and MKK3/MKK6 leading to the activation of JNK and p38, respectively. MEKK4 is believed to be auto-inhibited, and its interaction with other proteins controls its dimerization and activation. TRAF4, GADD45, and Axin each bind and activate MEKK4, with TRAF4 and Axin binding to the kinase domain and GADD45 binding within the N-terminal regulatory domain. Here we show that similar to the interaction with TRAF4 and Axin, the kinase domain of MEKK4 interacts with the multifunctional serine/threonine kinase GSK3beta. GSK3beta binding to MEKK4 blocks MEKK4 dimerization that is required for MEKK4 activation, effectively inhibiting MEKK4 stimulation of the JNK and p38 MAPK pathways. Inhibition of GSK3beta kinase activity with SB216763 results in enhanced MEKK4 kinase activity and increased JNK and p38 activation, indicating that an active state of GSK3beta is required for binding and inhibition of MEKK4 dimerization. Furthermore, GSK3beta phosphorylates specific serines and threonines in the N terminus of MEKK4. Together, these findings demonstrate that GSK3beta binds to the kinase domain of MEKK4 and regulates MEKK4 dimerization. However, unlike TRAF4, Axin, and GADD45, GSK3beta inhibits MEKK4 activity and prevents its activation of JNK and p38. Thus, control of MEKK4 dimerization is regulated both positively and negatively by its interaction with specific proteins.

Ablation of MEKK4 kinase activity causes neurulation and skeletal patterning defects in the mouse embryo.

Skeletal disorders and neural tube closure defects represent clinically significant human malformations. The signaling networks regulating normal skeletal patterning and neurulation are largely unknown. Targeted mutation of the active site lysine of MEK kinase 4 (MEKK4) produces a kinase-inactive MEKK4 protein (MEKK4(K1361R)). Embryos homozygous for this mutation die at birth as a result of skeletal malformations and neural tube defects. Hindbrains of exencephalic MEKK4(K1361R) embryos show a striking increase in neuroepithelial cell apoptosis and a dramatic loss of phosphorylation of MKK3 and -6, mitogen-activated protein kinase kinases (MKKs) regulated by MEKK4 in the p38 pathway. Phosphorylation of MAPK-activated protein kinase 2, a p38 substrate, is also inhibited, demonstrating a loss of p38 activity in MEKK4(K1361R) embryos. In contrast, the MEK1/2-extracellular signal-regulated kinase 1 (ERK1)/ERK2 and MKK4-Jun N-terminal protein kinase pathways were unaffected. The p38 pathway has been shown to regulate the phosphorylation and expression of the small heat shock protein HSP27. Compared to the wild type, MEKK4(K1361R) fibroblasts showed significantly reduced phosphorylation of p38 and HSP27, with a corresponding heat shock-induced instability of the actin cytoskeleton. Together, these data demonstrate MEKK4 regulation of p38 and that substrates downstream of p38 control cellular homeostasis. The findings are the first demonstration that MEKK4-regulated p38 activity is critical for neurulation.

MEKK4 is an effector of the embryonic TRAF4 for JNK activation.

TRAF4 has previously been shown to activate JNK through an unknown mechanism. Here, we show that endogenous TRAF4 and MEKK4 associate in both human K562 cells and mouse E10.5 embryos. TRAF4 interacts with the kinase domain of MEKK4. However, this association does not require MEKK4 kinase activity. The interaction of MEKK4 and TRAF4 are further demonstrated by the colocalization of TRAF4 and MEKK4 in cells. Importantly, although TRAF4 has little or no ability to activate JNK independently, coexpression of TRAF4 and MEKK4 results in synergistic activation of JNK that is inhibited by a kinase-inactive mutant of MEKK4, MEKK4K1361R. MEKK4 binds the TRAF domain of TRAF4 and MEKK4/TRAF4 activation of JNK is inhibited by expression of the TRAF domain. Furthermore, TRAF4 stimulates MEKK4 kinase activity by promoting MEKK4 oligomerization and JNK activation can be stimulated by chemical induction of MEKK4 dimerization. The findings identify MEKK4 as the MAPK kinase kinase for TRAF4 regulation of the JNK pathway.

CCM1 and CCM2 protein interactions in cell signaling: implications for cerebral cavernous malformations pathogenesis.

Cerebral cavernous malformations (CCMs) are sporadically acquired or inherited vascular lesions of the central nervous system consisting of clusters of dilated thin-walled blood vessels that predispose individuals to seizures and stroke. Familial CCM is caused by mutations in KRIT1 (CCM1) or in malcavernin (CCM2), the murine ortholog of which was concurrently characterized as osmosensing scaffold for MEKK3 (OSM). The roles of the CCM proteins in the pathogenesis of the disorder remain largely unknown. Here, we use co-immunoprecipitation, fluorescence resonance energy transfer and subcellular localization strategies to show that the CCM1 gene product, KRIT1, interacts with the CCM2 gene product, malcavernin/OSM. Analogous to the established interactions of CCM1 and beta1 integrin with ICAP1, the CCM1/CCM2 association is dependent upon the phosphotyrosine binding (PTB) domain of CCM2. A familial CCM2 missense mutation abrogates the CCM1/CCM2 interaction, suggesting that loss of this interaction may be critical in CCM pathogenesis. CCM2 and ICAP1 bound to CCM1 via their respective PTB domains differentially influence the subcellular localization of CCM1. Furthermore, we expand upon the established involvement of CCM2 in the p38 mitogen-activated protein kinase signaling module by demonstrating that CCM1 associates with CCM2 and MEKK3 in a ternary complex. These data indicate that the genetic heterogeneity observed in familial CCM may reflect mutation of different molecular members of a coordinated signaling complex.