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.

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.

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.

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.

Protective effect of stromal Dickkopf-3 in prostate cancer: opposing roles for TGFBI and ECM-1.

Aberrant transforming growth factor-ß (TGF-ß) signaling is a hallmark of the stromal microenvironment in cancer. Dickkopf-3 (Dkk-3), shown to inhibit TGF-ß signaling, is downregulated in prostate cancer and upregulated in the stroma in benign prostatic hyperplasia, but the function of stromal Dkk-3 is unclear. Here we show that DKK3 silencing in WPMY-1 prostate stromal cells increases TGF-ß signaling activity and that stromal cell-conditioned media inhibit prostate cancer cell invasion in a Dkk-3-dependent manner. DKK3 silencing increased the level of the cell-adhesion regulator TGF-ß-induced protein (TGFBI) in stromal and epithelial cell-conditioned media, and recombinant TGFBI increased prostate cancer cell invasion. Reduced expression of Dkk-3 in patient tumors was associated with increased expression of TGFBI. DKK3 silencing reduced the level of extracellular matrix protein-1 (ECM-1) in prostate stromal cell-conditioned media but increased it in epithelial cell-conditioned media, and recombinant ECM-1 inhibited TGFBI-induced prostate cancer cell invasion. Increased ECM1 and DKK3 mRNA expression in prostate tumors was associated with increased relapse-free survival. These observations are consistent with a model in which the loss of Dkk-3 in prostate cancer leads to increased secretion of TGFBI and ECM-1, which have tumor-promoting and tumor-protective roles, respectively. Determining how the balance between the opposing roles of extracellular factors influences prostate carcinogenesis will be key to developing therapies that target the tumor microenvironment.

Identification of Noncanonical Wnt Receptors Required for Wnt-3a-Induced Early Differentiation of Human Neural Stem Cells.

Wnt proteins preferentially activate either ß-catenin-dependent or ß-catenin-independent signals, but the activity of a particular Wnt also depends on cellular context and receptor availability. We previously reported that Wnt-3a induces neural differentiation of human embryonic stem cell-derived neural stem cells (NSCs) in a ß-catenin-independent manner by activating a signal involving JNK and the AP-1 family member ATF-2. Here, we report the results of a gene silencing approach to identify the Wnt receptors that mediate this response to Wnt-3a. Silencing of ROR2 increased neuronal differentiation, as measured by expression of the genes DCX, NEUROD1, and NGN1, suggesting ROR2 signals normally prevent differentiation. Silencing of the other Wnt receptors singly did not affect Wnt-3a-induced neuronal differentiation. However, pairwise silencing of ROR1 and FZD4 or FZD5 and of LRP6 and FZD4 or FZD5 inhibited neuronal differentiation, as detected by reductions in the expression of neuronal genes and immunocytochemical detection of DCX, NEUROD1 and DCX. Ectopic expression of these receptors in HEK 293 cells increased ATF2-dependent transcription. In addition, ROR1 coimmunoprecipitated with FZD4 and LRP6 in transfected HEK 293 cells and colocalized with FZD4 and with LRP6 at the cell surface of transfected L cells. Wnt-3a did not appear to affect these interactions but did alter the interactions between LRP6 and FZD4/5. Together, these observations highlight roles for ROR1, LRP6, FZD4, and FZD5 in neural stem cell differentiation and provide support for a model in which dynamic interactions among these receptors mediate Wnt-3a activation of ATF2 signaling.

Dickkopf-3 alters the morphological response to retinoic acid during neuronal differentiation of human embryonal carcinoma cells.

Dickkopf-3 (Dkk-3) and Dkkl-1 (Soggy) are secreted proteins of poorly understood function that are highly expressed in subsets of neurons in the brain. To explore their potential roles during neuronal development, we examined their expression in Ntera-2 (NT2) human embryonal carcinoma cells, which differentiate into neurons upon treatment with retinoic acid (RA). RA treatment increased the mRNA and protein levels of Dkk-3 but not of Dkkl-1. Ectopic expression of both Dkk-3 and Dkkl-1 induced apoptosis in NT2 cells. Gene silencing of Dkk-3 did not affect NT2 cell growth or differentiation but altered their response to RA in suspension cultures. RA treatment of NT2 cells cultured in suspension resulted in morphological changes that led to cell attachment and flattening out of cell aggregates. Although there were no significant differences in the expression levels of cell adhesion molecules in control and Dkk-3-silenced cells, this morphological response was not observed in Dkk-3-silenced cells. These findings suggest that Dkk-3 plays a role in the regulation of cell interactions during RA-induced neuronal differentiation.

A screen for transcription factor targets of glycogen synthase kinase-3 highlights an inverse correlation of NFκB and androgen receptor signaling in prostate cancer.

Expression of Glycogen Synthase Kinase-3 (GSK-3) is elevated in prostate cancer and its inhibition reduces prostate cancer cell proliferation, in part by reducing androgen receptor (AR) signaling. However, GSK-3 inhibition can also activate signals that promote cell proliferation and survival, which may preclude the use of GSK-3 inhibitors in the clinic. To identify such signals in prostate cancer, we screened for changes in transcription factor target DNA binding activity in GSK-3-silenced cells. Among the alterations was a reduction in AR DNA target binding, as predicted from previous studies, and an increase in NFκB DNA target binding. Consistent with the latter, gene silencing of GSK-3 or inhibition using the GSK-3 inhibitor CHIR99021 increased basal NFκB transcriptional activity. Activation of NFκB was accompanied by an increase in the level of the NFκB family member RelB. Conversely, silencing RelB reduced activation of NFκB by CHIR99021. Furthermore, the reduction of prostate cancer cell proliferation by CHIR99021 was potentiated by inhibition of NFκB signaling using the IKK inhibitor PS1145. Finally, stratification of human prostate tumor gene expression data for GSK3 revealed an inverse correlation between NFκB-dependent and androgen-dependent gene expression, consistent with the results from the transcription factor target DNA binding screen. In addition, there was a correlation between expression of androgen-repressed NFκB target genes and reduced survival of patients with metastatic prostate cancer. These findings highlight an association between GSK-3/AR and NFκB signaling and its potential clinical importance in metastatic prostate cancer.