Intravital imaging of Wnt/ß-catenin and ATF2-dependent signalling pathways during tumour cell invasion and metastasis.

Wnt signalling has been implicated as a driver of tumour cell metastasis, but less is known about which branches of Wnt signalling are involved and when they act in the metastatic cascade. Here, using a unique intravital imaging platform and fluorescent reporters, we visualised ß-catenin/TCF-dependent and ATF2-dependent signalling activities during human cancer cell invasion, intravasation and metastatic lesion formation in the chick embryo host. We found that cancer cells readily shifted between states of low and high canonical Wnt activity. Cancer cells that displayed low Wnt canonical activity showed higher invasion and intravasation potential in primary tumours and in metastatic lesions. In contrast, cancer cells showing low ATF2-dependent activity were significantly less invasive both at the front of primary tumours and in metastatic lesions. Simultaneous visualisation of both these reporters using a double-reporter cell line confirmed their complementary activities in primary tumours and metastatic lesions. These findings might inform the development of therapies that target different branches of Wnt signalling at specific stages of metastasis.

Small-molecule Wnt inhibitors are a potential novel therapy for intestinal fibrosis in Crohns disease.

Intestinal fibrosis and stricture formation is an aggressive complication of Crohns disease (CD), linked to increased morbidity and costs. The present study investigates the contribution of Wingless-Int-1 (Wnt) signalling to intestinal fibrogenesis, considers potential cross-talk between Wnt and transforming growth factor ß1 (TGFß) signalling pathways, and assesses the therapeutic potential of small-molecule Wnt inhibitors. ß-catenin expression was explored by immunohistochemistry (IHC) in formalin-fixed paraffin embedded (FFPE) tissue from patient-matched nonstrictured (NSCD) and strictured (SCD) intestine (n=6 pairs). Functional interactions between Wnt activation, TGFß signalling, and type I collagen (Collagen-I) expression were explored in CCD-18Co cells and primary CD myofibroblast cultures established from surgical resection specimens (n=16) using small-molecule Wnt inhibitors and molecular techniques, including siRNA-mediated gene knockdown, immunofluorescence (IF), Wnt gene expression arrays, and western blotting. Fibrotic SCD tissue was marked by an increase in ß-catenin-positive cells. In vitro, activation of Wnt-ß-catenin signalling increased Collagen-I expression in CCD-18Co cells. Conversely, ICG-001, an inhibitor of ß-catenin signalling, reduced Collagen-I expression in cell lines and primary CD myofibroblasts. TGFß increased ß-catenin protein levels but did not activate canonical Wnt signalling. Rather, TGFß up-regulated WNT5B, a noncanonical Wnt ligand, and the Wnt receptor FZD8, which contributed directly to the up-regulation of Collagen-I through a ß-catenin-independent mechanism. Treatment of CCD-18Co fibroblasts and patient-derived myofibroblasts with the FZD8 inhibitor 3235-0367 reduced extracellular matrix (ECM) expression. Our data highlight small-molecule Wnt inhibitors of both canonical and noncanonical Wnt signalling, as potential antifibrotic drugs to treat SCD intestinal fibrosis. They also highlight the importance of the cross-talk between Wnt and TGFß signalling pathways in CD intestinal fibrosis.

Proteomic Studies Reveal Disrupted in Schizophrenia 1 as a Player in Both Neurodevelopment and Synaptic Function.

A balanced chromosomal translocation disrupting DISC1 (Disrupted in Schizophrenia 1) gene has been linked to psychiatric diseases, such as major depression, bipolar disorder and schizophrenia. Since the discovery of this translocation, many studies have focused on understating the role of the truncated isoform of DISC1, hypothesizing that the gain of function of this protein could be behind the neurobiology of mental conditions, but not so many studies have focused in the mechanisms impaired due to its loss of function. For that reason, we performed an analysis on the cellular proteome of primary neurons in which DISC1 was knocked down with the goal of identifying relevant pathways directly affected by DISC1 loss of function. Using an unbiased proteomic approach, we found that the expression of 31 proteins related to neurodevelopment (e.g., CRMP-2, stathmin) and synaptic function (e.g., MUNC-18, NCS-1) is altered by DISC1 in primary mouse neurons. Hence, this study reinforces the idea that DISC1 is a unifying regulator of both neurodevelopment and synaptic function, thereby providing a link between these two key anatomical and cellular circuitries.

Dickkopf-3 regulates prostate epithelial cell acinar morphogenesis and prostate cancer cell invasion by limiting TGF-ß-dependent activation of matrix metalloproteases.

Dickkopf-3 (Dkk-3) is a secreted protein whose expression is downregulated in many types of cancer. Endogenous Dkk-3 is required for formation of acini in 3D cultures of prostate epithelial cells, where it inhibits transforming growth factor (TGF)-ß/Smad signaling. Here, we examined the effects of Dkk-3 on the expression and activity of matrix metalloproteases (MMPs), which mediate the effects of TGF-ß on extracellular matrix disassembly during tissue morphogenesis and promote invasion of tumor cells. Silencing of Dkk-3 in prostate epithelial cells resulted in increased expression and enzyme activity of MMP-2 and MMP-9. Inhibition of MMP-9 partially restored normal acinar morphogenesis in Dkk-3-silenced RWPE-1 prostate epithelial cells. In PC3 prostate cancer cells, Dkk-3 inhibited TGF-ß-dependent migration and invasion. Inhibition was mediated by the Dkk-3 C-terminal cysteine-rich domain (Cys2), which also inhibited TGF-ß-induced expression of MMP9 and MMP13. In contrast, Dkk-3, but not Cys2, increased formation of normal acini in Dkk-3-silenced prostate epithelial cells. These observations highlight a role for Dkk-3 in modulating TGF-ß/MMP signals in the prostate, and suggest that the Dkk-3 Cys2 domain can be used as a basis for therapies that target the tumor promoting effects of TGF-ß signaling in advanced prostate cancer.

Canonical and noncanonical Wnt signaling in neural stem/progenitor cells.

The first mammalian Wnt to be discovered, Wnt-1, was found to be essential for the development of a large part of the mouse brain over 25 years ago. We have since learned that Wnt family secreted glycolipoproteins, of which there are nineteen, which activate a diverse network of signals that are particularly important during embryonic development and tissue regeneration. Wnt signals in the developing and adult brain can drive neural stem cell self-renewal, expansion, asymmetric cell division, maturation and differentiation. The molecular events taking place after a Wnt binds to its cell-surface receptors are complex and, at times, controversial. A deeper understanding of these events is anticipated to lead to improvements in the treatment of neurodegenerative diseases and stem cell-based replacement therapies. Here, we review the roles played by Wnts in neural stem cells in the developing mouse brain, at neurogenic sites of the adult mouse and in neural stem cell culture models.

Downregulation of Dickkopf-3 disrupts prostate acinar morphogenesis through TGF-ß/Smad signalling.

Loss of tissue organization is a hallmark of the early stages of cancer, and there is considerable interest in proteins that maintain normal tissue architecture. Prostate epithelial cells cultured in Matrigel form three-dimensional acini that mimic aspects of prostate gland development. The organization of these structures requires the tumor suppressor Dickkopf-3 (Dkk-3), a divergent member of the Dkk family of secreted Wnt signalling antagonists that is frequently downregulated in prostate cancer. To gain further insight into the function of Dkk-3 in the prostate, we compared the prostates of Dkk3-null mice with those of control littermates. We found increased proliferation of prostate epithelial cells in the mutant mice and changes in prostate tissue organization. Consistent with these observations, cell proliferation was elevated in acini formed by human prostate epithelial cells stably silenced for Dkk-3. Silencing of Dkk-3 increased TGF-ß/Smad signalling, and inhibitors of TGF-ß/Smad signalling rescued the defective acinar phenotype caused by loss of Dkk-3. These findings suggest that Dkk-3 maintains the structural integrity of the prostate gland by limiting TGF-ß/Smad signalling.

A switch from canonical to noncanonical Wnt signaling mediates early differentiation of human neural stem cells.

Wnt/ß-catenin signaling is essential for neurogenesis but less is known about ß-catenin-independent Wnt signals. We show here that Wnt/activator protein-1 (AP-1) signaling drives differentiation of human embryonic stem cell and induced pluripotent stem cell-derived neural progenitor cells. Neuronal differentiation was accompanied by a reduction in ß-catenin/Tcf-dependent transcription and target gene expression, increased levels and/or phosphorylation of activating transcription factor 2 (ATF2), cyclic AMP response element-binding protein, and c-Jun, and increased AP-1-dependent transcription. Inhibition of Wnt secretion using the porcupine inhibitors IWP-2 and Wnt-C59 blocked neuronal differentiation, while activation or inhibition of Wnt/ß-catenin signaling had no effect. Neuronal differentiation increased expression of several Wnt genes, including WNT3A, silencing of which reduced differentiation. Addition of recombinant Wnt-3a to cells treated with IWP-2 or Wnt-C59 increased AP-1 levels and restored neuronal differentiation. The effects of Wnt-3a could not be blocked by addition of Dkk-1 or IWR-1, suggesting the involvement of noncanonical signaling. Consistent with this, restoration of neuronal differentiation by Wnt-3a was reduced by inhibition of Jun N-terminal kinase (JNK) and by gene silencing of ATF2. Together, these observations suggest that ß-catenin-independent Wnt signals promote neural stem/progenitor cell differentiation in a signaling pathway involving Wnt-3a, JNK, and ATF2.

Distinct expression and activity of GSK-3α and GSK-3ß in prostate cancer.

Glycogen synthase kinase (GSK-3) is upregulated in many types of tumor, including prostate cancer. GSK-3 inhibitors reduce prostate tumor cell growth; however, it is not clear if both isoforms, GSK-3α and GSK-3ß, are involved. Here, we compared their expression in prostate tumors and used gene silencing to study their functions in 22Rv1 prostate cancer cells. Compared to normal prostate, GSK-3α and GSK-3ß were upregulated in 25/79 and 24/79 cases of prostate cancer, respectively, with GSK-3α elevated in low Gleason sum score tumors and GSK-3ß expressed in high Gleason tumors, and both isoforms correlating with high expression of the androgen receptor (AR). Gene silencing of GSK-3α and, to a lesser extent, GSK-3ß reduced AR transcriptional activity. In addition, silencing of GSK-3ß, but not GSK-3α, reduced Akt phosphorylation. Acute and chronic silencing of either isoform reduced 22Rv1 growth in colony formation assays; however, this did not correlate with effects on AR activity. The GSK-3 inhibitor CHIR99021 reduced 22Rv1 colony formation by 50% in normal growth medium and by 15% in hormone-depleted medium, suggesting that GSK-3 is required both for hormone-dependent and hormone-independent proliferation. In addition, CHIR99021 enhanced growth inhibition by the AR antagonists bicalutamide and MDV3100. Finally, expression of GSK3A and GSK3B mRNAs correlated with a gene expression signature for androgen-regulated genes. Our observations highlight the importance of the GSK-3/AR signaling axis in prostate cancer and support the case for development of isoform-specific GSK-3 inhibitors and their use, in combination with AR antagonists, to treat patients with prostate cancer.

Distinct roles for Wnt-4 and Wnt-11 during retinoic acid-induced neuronal differentiation.

Retinoic acid and Wnt/ß-catenin signals play important roles during neuronal differentiation but less is known about noncanonical Wnt signals in this context. We examined retinoic acid and Wnt signaling in two human embryonal carcinoma cell lines, NTERA-2 (clone D1), which undergoes neuronal differentiation in response to retinoic acid, and 2102Ep, which does not. Retinoic acid treatment inhibited ß-catenin/Tcf activity in NTERA-2 cells but not in 2102Ep cells. Inhibition occurred downstream of ß-catenin but did not involve competition between retinoic acid receptors and ß-catenin for binding to p300 or Tcf-4. Ectopic expression of FZD1 partially restored inhibition in 2102Ep cells, suggesting the involvement of Wnt ligands. Retinoic acid treatment of NTERA-2 cells induced the expression of Wnt-4 and Wnt-11, both of which were able to inhibit ß-catenin/Tcf activity. Wnt-4 and Wnt-11 were found at cell borders in islands of cells that expressed OCT4 and GFAP and were predominantly negative for Nestin, PAX6, and GATA6. Gene silencing of Wnt-4, but not Wnt-11, reduced retinoic acid downregulation of OCT4 and Nanog and upregulation of PAX6, ASCL1, HOXC5, and NEUROD1, suggesting that Wnt-4 promotes early neuronal differentiation. Gene expression analysis of NTERA-2 cells stably overexpressing Wnt-11 suggested that Wnt-11 potentiates retinoic acid induction of early neurogenesis. Consistent with this, overexpression of Wnt-11 maintained a population of proliferating progenitor cells in cultures treated with retinoic acid for several weeks. These observations highlight the distinct roles of two noncanonical Wnts during the early stages of retinoic acid-induced neuronal differentiation.

Dickkopf-3: An Update on a Potential Regulator of the Tumor Microenvironment.

Dickkopf-3 (Dkk-3) is a member of the Dickkopf family protein of secreted Wingless-related integration site (Wnt) antagonists that appears to modulate regulators of the host microenvironment. In contrast to the clear anti-tumorigenic effects of Dkk-3-based gene therapies, the role of endogenous Dkk-3 in cancer is context-dependent, with elevated expression associated with tumor promotion and suppression in different settings. The receptors and effectors that mediate the diverse effects of Dkk-3 have not been characterized in detail, contributing to an ongoing mystery of its mechanism of action. This review compares the various functions of Dkk-3 in the tumor microenvironment, where Dkk-3 has been found to be expressed by subpopulations of fibroblasts, endothelial, and immune cells, in addition to epithelial cells. We also discuss how the activation or inhibition of Dkk-3, depending on tumor type and context, might be used to treat different types of cancers.

Mechano-transduction in osteoblastic cells involves strain-regulated estrogen receptor alpha-mediated control of insulin-like growth factor (IGF) I receptor sensitivity to Ambient IGF, leading to phosphatidylinositol 3-kinase/AKT-dependent Wnt/LRP5 receptor-independent activation of beta-catenin signaling.

The capacity of bones to adjust their mass and architecture to withstand the loads of everyday activity derives from the ability of their resident cells to respond appropriately to the strains engendered. To elucidate the mechanisms of strain responsiveness in bone cells, we investigated in vitro the responses of primary mouse osteoblasts and UMR-106 osteoblast-like cells to a single period of dynamic strain. This stimulates a cascade of events, including activation of insulin-like growth factor I receptor (IGF-IR), phosphatidylinositol 3-kinase-mediated phosphorylation of AKT, inhibition of GSK-3beta, increased activation of beta-catenin, and associated lymphoid-enhancing factor/T cell factor-mediated transcription. Initiation of this pathway does not involve the Wnt/LRP5/Frizzled receptor and does not culminate in increased IGF transcription. The effect of strain on IGF-IR is mimicked by exogenous des-(1-3)IGF-I and is blocked by the IGF-IR inhibitor H1356. Inhibition of strain-related prostanoid and nitric oxide production inhibits strain-related (and basal) AKT activity, but their separate ectopic administration does not mimic it. Strain-related IGF-IR activation of AKT requires estrogen receptor alpha (ERalpha) with which IGF-1R physically associates. The ER blocker ICI 182,780 increases the concentration of des-(1-3)IGF-I necessary to activate this cascade, whereas estrogen inhibits both basal AKT activity and its activation by des-(1-3)IGF-I. These data suggest an initial cascade of strain-related events in osteoblasts in which strain activates IGF-IR, in association with ERalpha, so initiating phosphatidylinositol 3-kinase/AKT-dependent activation of beta-catenin and altered lymphoid-enhancing factor/T cell factor transcription. This cascade requires prostanoid/nitric oxide production and is independent of Wnt/LRP5.

Opposing prognostic relevance of junction plakoglobin in distinct prostate cancer patient subsets.

Both oncogenic and tumor suppressor functions have been described for junction plakoglobin (JUP), also known as γ-catenin. To clarify the role of JUP in prostate cancer, JUP protein expression was immunohistochemically detected in a tissue microarray containing 11 267 individual prostatectomy specimens. Considering all patients, high JUP expression was associated with adverse tumor stage (P = 0.0002), high Gleason grade (P < 0.0001), and lymph node metastases (P = 0.011). These associations were driven mainly by the subset without TMPRSS2:ERG fusion, in which high JUP expression was an independent predictor of poor prognosis (multivariate analyses, P = 0.0054) and early biochemical recurrence (P = 0.0003). High JUP expression was further linked to strong androgen receptor expression (P < 0.0001), high cell proliferation, and PTEN and FOXP1 deletion (P < 0.0001). In the ERG-negative subset, high JUP expression was additionally linked to MAP3K7 (P = 0.0007) and CHD1 deletion (P = 0.0021). Contrasting the overall prognostic effect of JUP, low JUP expression indicated poor prognosis in the fraction of CHD1-deleted patients (P = 0.039). In this subset, the association of high JUP and high cell proliferation was specifically absent. In conclusion, the controversial biological roles of JUP are reflected by antagonistic prognostic effects in distinct prostate cancer patient subsets.

The neuron-specific isoform of glycogen synthase kinase-3beta is required for axon growth.

Glycogen synthase kinase-3 (GSK-3) has become an important target for the treatment of mood disorders and neurodegenerative disease. It comprises three enzymes, GSK-3alpha, beta and the neuron-specific isoform, beta2. GSK-3 regulates axon growth by phosphorylating microtubule-associated proteins including Tau. A genetic polymorphism that leads to an increase in the ratio of GSK-3beta1 to GSK-3beta2 interacts with Tau haplotypes to modify disease risk in Parkinson's and Alzheimer's disease. We have examined the roles of each isoform of GSK-3 in neurons. Silencing of GSK-3beta2 inhibited retinoic acid-induced neurite outgrowth in SH-SY5Y neuroblastoma cells and axon growth in rat cortical neurons. Inhibition of neurite outgrowth was prevented by co-expression of GSK-3beta2 but not by co-expression of GSK-3alpha or GSK-3beta1. Ectopic expression GSK-3beta2 enhanced the effects of retinoic acid on neurite length and induced neurite formation in the absence of retinoic acid. GSK-3beta2 phosphorylated Tau at a subset of those sites phosphorylated by GSK-3beta1. In addition, Axin, which regulates responses to Wnt signals, associated more readily with GSK-3beta1 than with GSK-3beta2. Our results suggest that GSK-3 inhibitors that target the Axin-binding site in GSK-3 will preserve the beneficial effects of GSK-3beta2 on axon growth.

Wnt-11 promotes neuroendocrine-like differentiation, survival and migration of prostate cancer cells.

BACKGROUND: Wnt-11 is a secreted protein that modulates cell growth, differentiation and morphogenesis during development. We previously reported that Wnt-11 expression is elevated in hormone-independent prostate cancer and that the progression of prostate cancer from androgen-dependent to androgen-independent proliferation correlates with a loss of mutual inhibition between Wnt-11- and androgen receptor-dependent signals. However, the prevalence of increased expression of Wnt-11 in patient tumours and the functions of Wnt-11 in prostate cancer cells were not known. RESULTS: Wnt-11 protein levels in prostate tumours were determined by immunohistochemical analysis of prostate tumour tissue arrays. Wnt-11 protein was elevated in 77/117 of tumours when compared with 27 benign prostatic hypertrophy specimens and was present in 4/4 bone metastases. In addition, there was a positive correlation between Wnt-11 expression and PSA levels above 10 ng/ml. Androgen-depleted LNCaP prostate cancer cells form neurites and express genes associated with neuroendocrine-like differentiation (NED), a feature of prostate tumours that have a poor prognosis. Since androgen-depletion increases expression of Wnt-11, we examined the role of Wnt-11 in NED. Ectopic expression of Wnt-11 induced expression of NSE and ASCL1, which are markers of NED, and this was prevented by inhibitors of cyclic AMP-dependent protein kinase, consistent with the known role of this kinase in NED. In contrast, Wnt-11 did not induce NSE expression in RWPE-1 cells, which are derived from benign prostate, suggesting that the role of Wnt-11 in NED is specific to prostate cancer. In addition, silencing of Wnt-11 expression in androgen-depleted LNCaP cells prevented NED and resulted in apoptosis. Silencing of Wnt-11 gene expression in androgen-independent PC3 cells also reduced expression of NSE and increased apoptosis. Finally, silencing of Wnt-11 reduced PC3 cell migration and ectopic expression of Wnt-11 promoted LNCaP cell invasion. CONCLUSIONS: These observations suggest that the increased level of Wnt-11 found in prostate cancer contributes to tumour progression by promoting NED, tumour cell survival and cell migration/invasion, and may provide an opportunity for novel therapy in prostate cancer.

Wnt-11 as a Potential Prognostic Biomarker and Therapeutic Target in Colorectal Cancer.

The expression of the secreted factor Wnt-11 is elevated in several types of cancer, including colorectal cancer, where it promotes cancer cell migration and invasion. Analysis of colorectal cancer gene expression databases associated WNT11 mRNA expression with increased likelihood of metastasis in a subset of patients. WNT11 expression was correlated with the expression of the Wnt receptors FZD6, RYK, and PTK7, and the combined expression of WNT11, FZD6 and RYK or PTK7 was associated with an increased risk of 5-year mortality rates. Immunohistochemical analysis of Wnt-11 in a cohort of 357 colorectal cancer patients found significantly higher Wnt-11 levels in tumors, compared with benign tissue. Elevated Wnt-11 levels occurred more frequently in rectal tumors than in colonic tumors and in tumors from women than men. In univariate analysis, increased Wnt-11 expression was also associated with tumor invasion and increased 5-year mortality. High Wnt-11 levels were not associated with high levels of nuclear ß-catenin, suggesting Wnt-11 is not simply an indicator for activation of ß-catenin-dependent signaling. Expression of Wnt-11 in colorectal cancer cell lines expressing low endogenous Wnt-11 inhibited ß-catenin/Tcf activity and increased ATF2-dependent transcriptional activity. WNT11 gene silencing and antibody-mediated inhibition of Wnt-11 in colorectal cancer cell lines expressing high Wnt-11 reduced their capacity for invasion. Together, these observations suggest that Wnt-11 could be a potential target for the treatment of patients with invasive colorectal cancer.

A Sox2-Sox9 signalling axis maintains human breast luminal progenitor and breast cancer stem cells.

Increased cancer stem cell content during development of resistance to tamoxifen in breast cancer is driven by multiple signals, including Sox2-dependent activation of Wnt signalling. Here, we show that Sox2 increases and estrogen reduces the expression of the transcription factor Sox9. Gain and loss of function assays indicate that Sox9 is implicated in the maintenance of human breast luminal progenitor cells. CRISPR/Cas knockout of Sox9 reduces growth of tamoxifen-resistant breast tumours in vivo. Mechanistically, Sox9 acts downstream of Sox2 to control luminal progenitor cell content and is required for expression of the cancer stem cell marker ALDH1A3 and Wnt signalling activity. Sox9 is elevated in breast cancer patients after endocrine therapy failure. This new regulatory axis highlights the relevance of SOX family transcription factors as potential therapeutic targets in breast cancer.

The Tumor Suppressor ING5 Is a Dimeric, Bivalent Recognition Molecule of the Histone H3K4me3 Mark.

The INhibitor of Growth (ING) family of tumor suppressors regulates the transcriptional state of chromatin by recruiting remodeling complexes to sites with histone H3 trimethylated at lysine 4 (H3K4me3). This modification is recognized by the plant homeodomain (PHD) present at the C-terminus of the five ING proteins. ING5 facilitates histone H3 acetylation by the HBO1 complex, and also H4 acetylation by the MOZ/MORF complex. We show that ING5 forms homodimers through its N-terminal domain, which folds independently into an elongated coiled-coil structure. The central region of ING5, which contains the nuclear localization sequence, is flexible and disordered, but it binds dsDNA with micromolar affinity. NMR analysis of the full-length protein reveals that the two PHD fingers of the dimer are chemically equivalent and independent of the rest of the molecule, and they bind H3K4me3 in the same way as the isolated PHD. We have observed that ING5 can form heterodimers with the highly homologous ING4, and that two of three primary tumor-associated mutants in the N-terminal domain strongly destabilize the coiled-coil structure. They also affect cell proliferation and cell cycle phase distribution, suggesting a driver role in cancer progression.

In Silico Approach for Immunohistochemical Evaluation of a Cytoplasmic Marker in Breast Cancer.

Breast cancer is the most frequently diagnosed cancer in women and the second most common cancer overall, with nearly 1.7 million new cases worldwide every year. Breast cancer patients need accurate tools for early diagnosis and to improve treatment. Biomarkers are increasingly used to describe and evaluate tumours for prognosis, to facilitate and predict response to therapy and to evaluate residual tumor, post-treatment. Here, we evaluate different methods to separate Diaminobenzidine (DAB) from Hematoxylin and Eosin (H&E) staining for Wnt-1, a potential cytoplasmic breast cancer biomarker. A method comprising clustering and Color deconvolution allowed us to recognize and quantify Wnt-1 levels accurately at pixel levels. Experimental validation was conducted using a set of 12,288 blocks of m × n pixels without overlap, extracted from a Tissue Microarray (TMA) composed of 192 tissue cores. Intraclass Correlations (ICC) among evaluators of the data of 0.634 , 0.791 , 0.551 and 0.63 for each Allred class and an average ICC of 0.752 among evaluators and automatic classification were obtained. Furthermore, this method received an average rating of 4.26 out of 5 in the Wnt-1 segmentation process from the evaluators.

CRISPR-Mediated Reactivation of DKK3 Expression Attenuates TGF-ß Signaling in Prostate Cancer.

The DKK3 gene encodes a secreted protein, Dkk-3, that inhibits prostate tumor growth and metastasis. DKK3 is downregulated by promoter methylation in many types of cancer, including prostate cancer. Gene silencing studies have shown that Dkk-3 maintains normal prostate epithelial cell homeostasis by limiting TGF-ß/Smad signaling. While ectopic expression of Dkk-3 leads to prostate cancer cell apoptosis, it is unclear if Dkk-3 has a physiological role in cancer cells. Here, we show that treatment of PC3 prostate cancer cells with the DNA methyltransferase (DNMT) inhibitor decitabine demethylates the DKK3 promoter, induces DKK3 expression, and inhibits TGF-ß/Smad-dependent transcriptional activity. Direct induction of DKK3 expression using CRISPR-dCas9-VPR also inhibited TGF-ß/Smad-dependent transcription and attenuated PC3 cell migration and proliferation. These effects were not observed in C4-2B cells, which do not respond to TGF-ß. TGF-ß signals can regulate gene expression directly via SMAD proteins and indirectly by increasing DNMT expression, leading to promoter methylation. Analysis of genes downregulated by promoter methylation and predicted to be regulated by TGF-ß found that DKK3 induction increased expression of PTGS2, which encodes cyclooxygenase-2. Together, these observations provide support for using CRISPR-mediated induction of DKK3 as a potential therapeutic approach for prostate cancer and highlight complexities in Dkk-3 regulation of TGF-ß signaling.

Frizzled-8 integrates Wnt-11 and transforming growth factor-ß signaling in prostate cancer.

Wnt-11 promotes cancer cell migration and invasion independently of ß-catenin but the receptors involved remain unknown. Here, we provide evidence that FZD8 is a major Wnt-11 receptor in prostate cancer that integrates Wnt-11 and TGF-ß signals to promote EMT. FZD8 mRNA is upregulated in multiple prostate cancer datasets and in metastatic cancer cell lines in vitro and in vivo. Analysis of patient samples reveals increased levels of FZD8 in cancer, correlating with Wnt-11. FZD8 co-localizes and co-immunoprecipitates with Wnt-11 and potentiates Wnt-11 activation of ATF2-dependent transcription. FZD8 silencing reduces prostate cancer cell migration, invasion, three-dimensional (3D) organotypic cell growth, expression of EMT-related genes, and TGF-ß/Smad-dependent signaling. Mechanistically, FZD8 forms a TGF-ß-regulated complex with TGF-ß receptors that is mediated by the extracellular domains of FZD8 and TGFBR1. Targeting FZD8 may therefore inhibit aberrant activation of both Wnt and TGF-ß signals in prostate cancer.