The Wnt signaling pathway, mediated through active beta-catenin, is responsible for initiating the majority of cases of human colorectal cancer (CRC), and we have previously shown that hyperactivation of this pathway by histone deacetylase inhibitors (HDACis), such as butyrate, can induce the death of CRC cells. An important cellular switch that mediates the effects of Wnt-signaling activation is variation in the association between beta-catenin and the transcriptional coactivators cAMP response element binding (CREB) binding protein (CBP) and p300. Association of CBP with beta-catenin is thought to activate a set of genes linked to cell proliferation, while the p300-mediated Wnt genetic program is believed to promote cell differentiation. Small molecule agents have been discovered that modulate CBP/p300 Wnt transcriptional programs by altering the association of CBP and p300 to beta-catenin. ICG-001 and ICG-427 inhibit CBP- and p300-mediated Wnt activity, respectively, while IQ-1 prevents the shift from CBP-mediated to a p300-mediated Wnt activity.Aim 1 of this proposal is designed to determine the role of CBP- and p300-mediated Wnt signaling in the response of CRC cells to HDACis. Aim 2 is to determine the role of CBP and p300 in the maintenance of high- and low-Wnt fractions in CRC cell line. Aim 3 will compare the effects of CBP- and p300-mediated Wnt activity on CRC initiation and progression.In Aim 1, cells will be cotreated with HDACis and ICG-001, ICG-427, or IQ-1 and the levels of Wnt activity, apoptosis, proliferation, differentiation, and CBP- or p300-beta-catenin binding measured. Aim 2 of this proposal may mirror similar heterogeneity observed in human tumors and which may be of clinical significance. Aim 3 will use CRC cell line model systems of initiation and progression: the normal colon cell lines CCD-841CoN, the adenoma line LT97, the primary colon carcinoma cell line SW480, and the lymph node metastasis cell line SW620. Cells will be treated with HDACis and the small molecule agents, and assayed as described above.We will also attempt to use changes in CBP- and p300-mediated Wnt signaling to shift colonic cells between cell type, modifying CBP- and p300-mediated gene expression in the LT97 adenoma line to shift the adenoma phenotype to more characteristic of the CCD-841CoN normal cells, or the SW480 carcinoma cells. We will use microarray analyses to determine the patterns of gene expression responsible for these CBP- or p300-mediated changes in colonic neoplastic phenotype.The findings generated from this study will lead to future, more in-depth projects to further dissect the action of CBP/p300 Wnt–mediated transcriptional programs in colonic neoplasia, with an emphasis on methods to modulate these genetic programs for chemopreventive effect. Colorectal cancers (CRCs) are amenable to a high degree of prevention -12 throuIn addition to butyrate, other HDACis have emerged as promising anticancer agents due to their ability to preferentially induce growth arrest, differentiation, and apoptosis in malignant cells ,20. ThusWnt signaling is induced by the binding of Wnt ligands to their cell surface receptors, resulting in inhibition of glycogen synthase kinase-3 beta (GSK-3beta) activity ,23. WhenAPC results in neuronal cell apoptosis in the Drosophila retina [Apc, the mouse version of APC, in murine neural crest cells results in massive apoptosis of cephalic and cardiac neural crest cells at 11.5 days post coitum [APC-/- CRC cells, induces apoptosis [While the majority of published studies associate the activation of Wnt signaling with proliferation and tumorigenesis, several in vivo studies -36 suppoa retina , (2) expa retina , (3) cont coitum , and (4)poptosis ,38, probpoptosis , and thapoptosis . These cpoptosis  proposedpoptosis .Based upon published studies by others -36, as wBased upon our findings in vitro ,2,21, weWe have established that the hyperactivation of Wnt signaling activity in NaB-treated CRC cells results from increased levels of Ser-37/Thr-41-dephosphorylated (active) beta-catenin, augmented formation of BCT complexes, as well as enhanced BCT complex-DNA binding . The HDAResults from our in vitro studies ,2 suggesAn important transcriptional control point that influences the levels and outcomes of Wnt-signaling activation is the association between beta-catenin and the transcriptional coactivators cAMP response element binding (CREB) binding protein (CBP) and p300 . Both CBAPC mutation initiated CRC, demonstrating preliminary in vivo efficacy of these agents [Interactions between CBP/p300 and Wnt signaling are optimally dissected using inhibitors known to be specific for CBP-Wnt and p300-Wnt transcriptional programs, the small molecule inhibitors ICG-001, ICG-427, and IQ-1. ICG-001 binds to CBP but not to p300, despite the significant homology between these two proteins [e agents . The tooOne factor that must be considered is the CBP/p300 status of colonic neoplastic cells, which has been associated with microsatellite instability (MSI) phenotypes . While mWith respect to mechanism(s) by which HDACis may modulate CBP/p300-mediated Wnt activity, we hypothesize that HDACis (1) result in the hyperacetylation of specific proteins that enhance CBP/p300-Wnt complex formation and activity, (2) alter gene expression and the target genes modulate CBP/p300-mediated Wnt activity, (3) result in a more open chromatin configuration, allowing enhanced access of CBP/p300-Wnt complexes to target DNA promoter/enhance regions, and/or (4) hyperacetylation of histone and nonhistone proteins resulting from HDACi inhibition complements the acetylation induced by the HAT proteins CBP and p300. Thus, treatment with HDACis can enhance either CBP- or p300-mediated Wnt activity. Whether HDACis activate the CBP or p300 pathways would be dependent upon which of those two pathways are most active in the cells at the time of HDACi treatment; thus, in this scenario, the small molecule inhibitors shift the cells toward either CBP-Wnt or p300-Wnt signaling, reinforcing whichever of the two pathways is activated in the cells. There are many possible mechanisms by which HDACis both generally upregulate Wnt activity and apoptosis as well as more specifically influencing CBP/p300-mediated Wnt activity; these mechanisms of action will be explored in a separate study building upon the findings of the current proposal. The specific aims of the current proposal are specifically focused upon using the small molecule inhibitors ICG-001, ICG-427, and IQ-1 to evaluate the requirement for CBP-Wnt and p300-Wnt interactions with respect to the induction of Wnt activity and of apoptosis by HDACis, and tumorigenic progression represented by a cell culture model of colonic neoplasia.In summary, CBP- and p300-mediated Wnt signaling likely influences the (1) differential response of CRC cell lines to HDACis, (2) maintenance of low- and high-Wnt fractions within single CRC cell lines, which may be representative of in vivo tumor heterogeneity, and (3) progression of colonic neoplasia from normal cells to metastatic carcinoma. In the current proposal, we will use in vitro models of CRC to explore the role of CBP/p300-mediated Wnt activity in these phenomena. The findings will lead to future projects to explore potential chemopreventive and therapeutic approaches based upon the modulation of CBP/p300-mediated Wnt activity. For example, it may be possible to modulate CBP- versus p300-mediated Wnt signaling to make colonic neoplasms more sensitive to the proapoptotic action of HDACis. Thus, pharmacological interventions based upon the small molecule inhibitors ICG-001, ICG-427, and/or IQ-1 can be used with butyrate derived from high fiber diets (chemoprevention) or therapeutic application of vorinostat (treatment and/or chemoprevention) to enhance the antineoplastic properties of these HDACis. If, for example, it is discovered that CBP-mediated Wnt activity promotes CRC cell proliferation and inhibits the apoptotic pathway induced by HDACis, then cotreatment with HDACis and a pharmacological agent similar in action to ICG-001 (CBP-Wnt inhibitor) will have greater antineoplastic efficacy than application of HDACis alone. Further, if, for example, p300-mediated Wnt signaling is shown to mediate the proapoptotic effects of HDAC-induced Wnt hyperactivation, then suppression of CBP-mediated Wnt activity with ICG-001 will enhance the p300-mediated pathway [We hypothesize that the effects of HDACis on Wnt signaling and cell fate are mediated by changes in the association of CBP and p300 with beta-catenin, and by modulation of BCT complex formation and Wnt activity. To investigate this hypothesis, we will first investigate the effects of CBP- and p300-mediated Wnt activity in the HWA HCT-116, and DLD-1 CRC cell lines, which respond to HDACis with a high-fold induction of Wnt activity and apoptosis , the LWA HT29 cell line, which exhibit a low-fold increase in Wnt activity and apoptosis after HDACi treatment , and SW620 cells, which exhibit a moderate-fold increase in Wnt activity and apoptosis after HDACi treatment. Although there are no clear divisions between HWA and LWA cell groups, based upon findings with 10 CRC cell lines, HWA is defined as a 10-fold or greater increase in Wnt activity and a 2-fold or greater increase in apoptosis; LWA is defined as a 1- to 4-fold increase in Wnt activity and a 1- to 1.5-fold increase in apoptosis, and a moderate fold increase in Wnt activity and apoptosis is between 5- and 10-fold for Wnt activity and 1.5- and 2-fold for apoptosis , this ceThe reported effects of CBP and p300 on Wnt activity are most likely mediated through changes in the association of these HAT proteins with Wnt signaling factors, particularly beta-catenin . CBP 5959 and p3To determine how CBP- or p300-mediated modulation of Wnt activity specifically influences the effects of HDACis on Wnt activity and cell physiology, CRC cells will be cotreated with HDACis and with ICG-001, ICG-427, or IQ-1 for 24 hours, or with each of the small molecule inhibitors alone. In addition, experiments will be conducted with the combination of ICG-001 and IQ-1. ICG-001 and ICG-427 inhibit CBP- and p300- mediated Wnt activity , respectThe appropriate levels of the small molecule effectors will be determined empirically. After treatment with these combinations of agents, coimmunoprecipitation assays, performed as described -3, will Further, expression of the receptor tyrosine kinases EphB2 and EphB4, which binds to EphrinB2, is controlled by CBP- and p300-mediated Wnt signaling, and these differences of expression influence cell physiology . EphB4 iAspects of CRC cell physiology that may be modulated by CBP/p300-mediated Wnt signaling will be determined. For example, it has been reported that inhibition of CBP-mediated Wnt activity stimulates apoptosis in CRC cells ,2, it isOur hypothesis is that the effects of HDACis on promoting apoptosis of CRC cells by the hyperactivation of Wnt signaling is dependent upon the specific induction of either CBP- or p300-mediated Wnt signaling, and that by specifically blocking CBP- or p300-mediated Wnt signaling with the appropriate small molecule inhibitor, we will also suppress the induction of apoptosis by the HDACis. To the extent that induction of Wnt activity by HDACis influences other aspects of colonic cell physiology , these phenomena would be specifically dependent upon CBP- or p300-mediated Wnt signaling as well. These findings, particularly, with respect to apoptosis, will have significant chemopreventive and/or therapeutic significance, because modulation of CBP/p300-mediated Wnt signaling may evolve into therapeutic approaches to induce CRC cell apoptosis, either alone or in conjunction with stimulation of excessive Wnt activity by HDACis.We will also cotreat selected CRC cell lines with combinations of the small molecule agents  in the presence and absence of butyrate. For selected experiments, we will use vorinostat, to compare the findings with that obtained with butyrate. As described above, the ICG-001 and IQ-1 combination will inhibit CBP-mediated Wnt activity while simultaneously preventing the cells from switching to p300-mediated signaling. For all three aims, this will be an important control to distinguish effects of diminished CBP-mediated Wnt activity from that of lower CBP– and higher p300–mediated Wnt signaling. The combination of ICG-001 and ICG-427 will simultaneously block both CBP- and p300-mediated Wnt activity. These combinations will measure the extent that signaling from both CBP and p300 pathways is required for endogenous and butyrate-stimulated Wnt activity in CRC cells. These data will be confirmed by knockdown of CBP and p300 with siRNA. The objective of these latter experiments is to determine the extent that basal and HDACi-induced Wnt activity is mediated through CBP and p300, and evaluate if significant levels of CBP/p300-independent Wnt activity occur in these cell lines. If we demonstrate that significant Wnt signaling occurs even in the absence of CBP and p300 activity, we will ascertain whether CBP- or p300-mediated Wnt signaling is required for the proapoptotic action of the HDACi butyrate in CRC cells. The combinatorial treatments will be repeated, and apoptosis measured as described above.HDACis also influence the levels of, and activity of, p300 and CBP. For example, in Hela cells, butyrate induces the degradation of p300 through the 26S proteosome pathway ; in addiTherefore, using an anti-acetyl-CBP/p300 antibody, we will ascertain the effects of HDACi treatment on CBP/p300 autoacetylation in our cell lines. This will determine if there is a correlation between HAT factor autoacetylation and the ability of these factors to mediate the effects of HDACis on Wnt activity .t p300/CFor Aim 1, we expect to find that cells with higher levels of Wnt activity, and higher levels of induction of Wnt activity by HDACis, will exhibit a greater proportion of CBP-beta-catenin complexes compared with p300-beta-catenin complexes. If this expected result is observed, the major findings of Aim 1 would be correlated as follows. The greater increase in Wnt activity (measured by luciferase reporter assays) and apoptosis (measured by the Annexin V-PE Apoptosis Detection Kit I or the Caspase-Glo luciferase system) achieved in HWA cells treated with HDACis will be associated with a greater degree of CBP-beta-catenin complex formation (measured by co-immunoprecipitation) compared with LWA cells. Conversely, the weaker response of LWA cells to HDACis is expected to be correlated to a relatively higher association of p300 to beta-catenin compared to CBP-beta-catenin complexes. ICG-001, which interferes with the association of CBP and beta-catenin would therefore be expected to strongly suppress the marked upregulation of Wnt activity and apoptosis observed in HDACi-treated HWA cells; it is expected that ICG-001 and HDACi cotreatment would result in the following set of results: inhibition of CBP-beta-catenin complex formation, increased p300-beta-catenin complex formation, lower levels of Wnt signaling, and lower levels of apoptosis.The weak induction of Wnt signaling and apoptosis by HDACis in LWA cells would be relatively unaffected by ICG-001, because the LWA cells are expected to have minimal CBP Wnt activity even in the absence of ICG-001. However, treatment of HWA cells with HDACis and ICG-427/IQ-1, which inhibit p300 signaling and potentiate CBP- mediated Wnt activity, would be expected to enhance the induction of Wnt activity and apoptosis by HDACis; this would be correlated with an increase in CBP-beta-catenin complexes and a decrease in p300-beta-catenin complexes. It is possible that inhibition of p300-mediated Wnt activity by ICG-427/IQ-1 may enhance the sensitivity of LWA cells to HDACis by increasing CBP-beta-catenin complex formation and action. SW620 cells, which exhibit a phenotype midway between that of HWA and LWA cells, would also be expected to exhibit moderate upregulation of CBP-beta-catenin complex formation and activity after exposure to HDACis, and would be also expected to exhibit a moderate response to the small molecule inhibitors. These expected results would be generally consistent with our preliminary data, which showed that expression of a dominant negative form of p300 significantly enhanced Wnt activity in SW620 CRC cells, possibly by shifting Wnt complexes to CBP-mediated activity. Increased Wnt activity can be reasonably expected to be associated with enhanced levels of BCT complexes, and we have found that HDACis can enhance BCT complex formation in some CRC cell lines [However, the interactions between HDACis and the CBP and p300 signaling pathways are likely to be complex because (1) HDACis such as butyrate stimulate CRC cell differentiation and apoptosis, which is more consistent with the p300 Wnt pathway, (2) CBP-mediated Wnt activity is likely to be correlated with higher expression of survivin, cyclinD1, and EphB4 , which iThe existence of high- and low-Wnt fractions in CRC cells in culture is a stable and reproducible phenomenon , which mFlow cytometry will be used to separate HCT-116 cells into their constituent high- and low-Wnt fractions in the presence or absence of butyrate [6 cells per milliliter of alpha-minimal essential medium (αMEM), and samples analyzed by flow cytometry based upon fluorescence. Representative plots of relative cell numbers (counts) versus fluorescence in EGFP-TOP and EGFP-FOP transfected HCT-116 cells will be overlaid. The relative levels of CBP-beta-catenin and p300-beta-catenin complexes in each of the cell fractions will be ascertained through coimmunoprecipitation; the association of CBP and p300 with Tcf1 and Tcf4, as well as the formation of BCT complexes, will be similarly ascertained. Subsequently, the original mixed-cell populations will be cotreated with the HDACis and ICG-001, ICG-427, and/or IQ-1 , or with each of the small molecule inhibitors alone, followed by separation by flow sorting to determine if the proportion of cells in each fraction is altered when the CBP- or p300-mediated Wnt activity is repressed. The association of CBP and p300 with beta-catenin, Tcf1, and Tcf4, as well as the formation of BCT complexes, will be determined by coimmunoprecipitation assays in these cotreated cells. Levels of Wnt activity in the cell fractions will be ascertained using the OT/OF reporter system; we will calculate the Wnt fraction metrics described shown in Flow cytometric sorting of HCT-116 cells into high (“Wnt positive”) and low (“Wnt negative”) Wnt fractions based upon Wnt-specific GFP expression will be performed essentially as described . Cells wIn We expect that CBP-mediated Wnt activity will be associated with the high-Wnt fraction in untreated cells, and p300-mediated Wnt activity with the low-Wnt fraction. If so, the correlated findings will be similar to that described in Aim 1; the high-Wnt fraction of the cell population will be associated with higher levels of CBP-beta-catenin complexes, Wnt activity, and sensitivity to HDACis. However, as explained in Aim 1, it is possible that the induction of apoptosis through hyperactivated Wnt activity may occur through either CBP- or p300- mediated Wnt signaling, which needs to be determined empirically. Therefore, it is difficult to predict which of the two Wnt coactivators will be predominantly involved in changes in the proportion of high and low Wnt fractions induced by HDACis. This will be determined empirically, through the experiments outlined in Aim 2. Discovery of an association between CBP- or p300-mediated Wnt activity and the maintenance of high- and low-Wnt fractions in CRC cells in vitro would suggest the possibility of modulating CBP- and/or p300-mediated Wnt activity in vivo, to either repress Wnt signaling and proliferation at the invasive front of CRC tumors, which constitutes a high Wnt fraction component of the neoplasm, or in conjunction with HDACis, hyperactivate Wnt signaling in the high-Wnt activity fraction at the tumor’s invasive front, in order to target these potentially metastatic cells ,58 for aAim 1 of the current proposal focuses upon the role of CBP- and p300-mediated Wnt activity for the upregulation of Wnt signaling and apoptosis in CRC cells treated with HDACis; the major objective of Aim 2 is to determine the role of CBP/p300-mediated Wnt signaling in the maintenance, and response to HDACis, of high- and low-Wnt fractions within CRC cell populations. Thus, after determining the activity of CBP- and p300-mediated Wnt activity, and response to HDACis, between (Aim 1) and within (Aim 2) established carcinoma cell lines, Aim 3 extends these findings to evaluate CBP- and p300-mediated Wnt activity across four colonic cell lines ranging from normal to metastatic and thus representing an in vitro model of tumorigenic progression, and as a mechanism by which the tumorigenic phenotype of colonic cells can be modulated by altering the relative levels of CBP- versus p300-mediated Wnt signaling.Therefore, to contrast and compare the effects of CBP- and p300-mediated Wnt activity on colonic neoplastic progression , we will use, as an in vitro model system, the normal colon cell line CCD-841CoN, the adenoma line LT97 . Thus, tUnlike colon carcinoma cells, LT97 cells are incapable of growing in soft agar ; the LT9The first part of Aim 3 is to delineate the phenotypic characteristics of the four cell lines. SW480, SW620 cells and LT97 cells will be cultured as previously described ,2,7. CCDCoimmunoprecipitation will be used to determine the levels of CBP- and p300-beta-catenin complexes in these four cell lines in the presence and absence of the HDACis, treated as described above. The association of CBP and p300 with Tcf1 and Tcf4, as well as the formation of BCT complexes will also be measured by coimmunoprecipitation assays. Cells will be cotreated with HDACis  and with ICG-001, ICG-427, and/or IQ-1 , or with each of the small molecule agents alone, and the levels of Wnt activity, apoptosis, proliferation, differentiation, CBP- or p300-beta-catenin binding, and expression of survivin, c-myc, cyclin D1, EphB2, and EphB4 will be measured as described above. The same coimmunoprecipitation experiments will be performed to ascertain if the formation of complexes between CBP and p300 and Tcf proteins are altered in these cell lines. The levels of Wnt signaling present in each cell line will be measured using the luciferase reporter system. Repression of Wnt activity by one of the small molecule inhibitors would provide information as to whether the Wnt activity present in the cells is predominantly CBP- or p300-mediated, as explained previously. For example, repression by ICG-001 would specifically repress CBP-mediated Wnt activity; ICG-427 would specifically repress p300-mediated Wnt activity. In addition, determination of growth in soft agar will be performed as described [Consistent with our underlying hypothesis for Aim 3, we expect to observe differences between the four cell lines in the relative levels of CBP- versus p300-mediated Wnt activity, both in the presence and absence of HDACis. Thus, the relative levels of CBP–beta-catenin complexes versus p300–beta-catenin complexes will differ between the four cell lines, as will effects of the small molecule inhibitors on both endogenous- and HDACi-stimulated Wnt activity. For example, one of the cell lines may exhibit relatively higher levels of CBP–beta-catenin complexes compared with the other lines; it would be expected that the Wnt activity present in this cell line would be sensitive to, and repressed by, ICG-001, because most of the Wnt signaling in this cell line would be CBP mediated. In contrast, another cell line showing relatively higher levels of p300–beta-catenin complexes would exhibit greater sensitivity to the Wnt activity-repressing effects of ICG-427, which targets p300-mediated signaling. Differential sensitivity of the cell lines to HDACis, with respect to CBP/p300-Wnt activity, would also be revealed by treatment with the small molecule inhibitors. For example, if ICG-001, but not ICG-427, represses induction of Wnt activity usually observed by treatment of a cell line with HDACis, that finding would support the hypothesis that HDACis predominantly influence CBP-mediated, but not p300-mediated, Wnt activity in those colonic cells.Therefore, one possibility is that as cells progress along the neoplastic continuum, CBP-mediated Wnt activity will predominate over p300-mediated activity, which is consistent with the finding that the CBP-Wnt inhibitor ICG-001 specifically inhibits the growth of neoplastic as opposed to normal colonic cells . Thus, iHowever, the greater sensitivity of LT97 adenoma cells to butyrate compared with carcinoma cell lines , suggestConcentrations of agents and treatment length will be determined empirically, until a defined phenotypic change is observed. We will measure Wnt signaling, apoptosis and apoptotic response to HDACis, proliferation and the growth suppressing effects of HDACis, differentiation, as well as growth in soft agar, as described above. We will also determine whether LT97 cells altered in the direction of a SW480-like phenotype are able to efficiently proliferate in the same cell culture medium as CRC cells. An endpoint for the process of phenotypic transition will be established; that is, a time point will be determined at which point any observed phenotypic modification of LT97 cells will be considered as “complete.” For example, acquisition of the ability to grow in soft agar or to proliferate independent of a specialized culture medium, would be suggestive of transition to a more tumorigenic, SW480 cell-like phenotype. Further, enhanced sensitivity to the growth suppressive and apoptosis-inducing effects of HDACis such as butyrate are also suggestive of a more advanced tumorigenic phenotype. Conversely, we will have established phenotypic differences distinguishing CCD-841Con cells from LT97 cells (see above), and evaluation of these metrics will be used to ascertain if CBP- or p300-signaling modifications transition LT97 cells toward a more normal, CCD-841CoN-like cellular phenotype. Our hypothesis, consistent to our expectations in the first part of Aim 3, is that each of the four cell lines is characterized by its own profile of CBP- versus p300-mediated Wnt activity, and that forcing LT97 cells to exhibit a CBP/p300 Wnt profile of CCD-841CoN or SW480 cells will promote LT97 cells to exhibit specific phenotypic characteristics of those cell lines, respectively.Dependent upon the differences that are observed between the four cell types, we expect that modulation of CBP/p300-mediated Wnt activity of LT97 cells, toward either that characteristic of CCD-841CoN cells or SW480 cells, will shift the LT97 cellular phenotype in those two directions, respectively. Once we have established a CBP/p300-mediated treatment regimen that is able to transition LT97 cells to lesser or greater tumorigenicity, we will conduct microarray analyses to determine the global changes in gene expression responsible for these CBP- or p300-mediated changes. LT97 cells will be treated with the particular combination of agents found to alter cell phenotype in the required directions (toward the CCD-841Con and SW480 phenotypes), or left untreated. Wnt signaling, apoptosis, and apoptotic response to HDACis, proliferation, differentiation, as well as growth in soft agar will be measured as described above. Measurements of the expression of survivin, cyclinD1, c-myc, EphB4, and EphB2 will also be performed; we would expect that transition of LT97 cells to a more tumorigenic phenotype would be characterized by increased expression of the first four genes and decreased expression of EphB2; this pattern would be expected to be reversed with transition of LT97 cells to a more normal colonic phenotype. However, this expectation is dependent upon whether a more tumorigenic phenotype is associated with increased CBP-mediated Wnt activity (see above).At the defined time point at which phenotypic transition is considered complete, mRNA will be isolated with Oligotex mRNA Direct Kit. Genus Biosystems will perform the microarray processing and data analyses for these experiments, using the Agilent human whole genome  oligo microarray. Targeted genes will be validated by Northern blot analyses or quantitative reverse transcription polymerase chain reaction (RT-PCR). A small set of selected discovered genes that are reasonably seen as major candidates for mediating the observed changes in cellular phenotype, particularly those genes that are known to be Wnt pathway targets, will be further analyzed. These three microarray measurements  will be repeated eight times to yield statistically significant quantitative data on changes in gene expression underlying the observed changes in cellular phenotype.We will determine, by Western blot analyses, whether expression of these genes are modulated by treatment of LT97 cells by the small molecule inhibitors; further, using DN-Tcf4 to block Wnt activity, we will determine whether expression of these selected genes are directly or indirectly dependent upon Wnt signaling. The expression of selected genes will then be directly up- or downregulated through overexpression (expression vectors) or knockdown (siRNA) in order to recapitulate the phenotype changes resulting from the treatments  above. If changes in the expression of specific CBP/p300 Wnt target genes can mimic phenotypic changes induced by modulation of CBP- or p300-mediated Wnt signaling, this will strongly suggest that the relevant genes are downstream effectors of CBP- or p300-mediated Wnt signaling, and are at least partially responsible for the changes in cellular phenotype induced by the CBP- or p300-targeted treatments.We expect that LT97 cells will exhibit Wnt activity and modulation of this activity by HDACis, consistent with the general literature; however, this would be inconsistent with the controversial possibility that the earliest stages of colonic initiation do not exhibit Wnt activity . We alsoThe initial evaluation of functional significance of identified genes will be performed as Aim 3 of the current proposal, and more in depth analyses of physiological relevance of these genes will be incorporated into future projects that will build upon the findings of the present study. Future studies will also determine whether observed differences in gene expression are observed in a broad spectrum of CRC cell lines, characterized by different Wnt pathway activating mutations. The development of treatment regimens that can either promote or inhibit tumorigenesis of early stage colonic neoplasms, and the determination of the patterns of gene expression that mediate these changes in tumorigenic potential, may lead to novel chemopreventive and therapeutic approaches against CRC. Thus, for example, a treatment regimen identified that induces LT97 cells to transition to a more CCD-841Con phenotype can serve as the basis for in vivo studies aimed at developing CBP/p300-targeted therapeutics effective at suppressing colonic tumorigenesis, reversing early stage tumorigenesis, or treating established CRC.We have extensive experience with the methodologies to be used in this proposal. The potential pitfalls for the specific aims, and alternative approaches that will be used, is outlined as follows. Aim 1: Some effects of HDACis on CRC cell physiology are independent of the modulation of Wnt activity. Wnt-mediated effects of HDACis, working through CBP/p300, may be obscured by opposing non-Wnt effects of HDACis. If necessary, CRC cells stably transfected with an inducible form of DN-Tcf4, which represses Wnt activity, can be used to separate Wnt-specific and non-Wnt-specific effects of HDACis on cell physiology . The rept-test will be used to determine statistical significance (P<.05). We have successfully used this biostatistical method to evaluate data from our previous studies on Wnt signaling that were generated by experimental methodologies identical to, or similar to, those proposed in for the current project [P values), the D’Agostino and Pearson test, or the Shapiro-Wilk test. Choice of normality test will depend upon sample, size; in most cases, Kolmogorov-Smirnov will be used. In the event that the data do not follow a normal distribution, the nonparametric Mann-Whitney U test will be used.Student’s  project ,2. When Research on this proposal is ongoing. To date, findings from this proposal, which cover all three specific aims, have been published in the Journal of Cancer ,62,63. TThis manuscript describes a R15 AREA grant proposal funded by the National Institutes of Health  upon initial submission. Our findings with respect to this research ,62,63 clThe ultimate objective of this line of research, and its clinical relevancy, is to determine whether modulators of CBP/p300-mediated Wnt signaling, in combination with HDACis such as butyrate and the FDA, vorinostat, exert more efficacious antineoplastic effects against CRC than the small molecules or HDACis alone. We therefore envision the possible use of combinatorial treatment with HDACis and small molecule inhibitors of CBP/p300-mediated Wnt signaling in the role of chemoprevention  of therapy to suppress tumor progression and/or as novel chemotherapeutic agents against advanced disease. This possibility would be advanced by in vivo studies demonstrating that specific combinations of HDACis and modulators of CBP/p300-Wnt activity result in marked suppression of intestinal tumorigenesis in mouse models of human CRC. Future in vitro experiments will be aimed at dissecting the molecular mechanisms by which CBP and p300 modulate Wnt activity, and promote cell differentiation and apoptosis, in a large range of CRC cell lines. Further, we will also dissect the mechanisms, whereby HDACis influence CBP/p300-mediated Wnt activity. More in depth microarray analyses will be used to determine the full genetic programs up- or downregulated by HDACis through the CBP- or p300-mediated Wnt signaling, and identify downstream targets of CBP-Wnt versus p300-Wnt signaling. Further, we propose that mouse models of CRC initiation and progression, such as the Cre-Lox model of APC inactivation  be used


Downregulation of STRAP inhibits invasion, tumorigenicity, and metastasis of CRC cells. Mechanistically, STRAP binds with GSK-3β and reduces the phosphorylation, ubiquitylation, and degradation of β-catenin through preventing its binding to the destruction complex. This leads to an inhibition of Wnt/β-catenin signaling and reduction in the expression of downstream targets, such as Cyclin D1, matrix metalloproteinases 2 and 9, and ß-TrCP. In human CRC specimens, higher STRAP expression correlates significantly with β-catenin expression with increased nuclear levels . Together, these results suggest that STRAP increases invasion and metastasis of CRC partly through inhibiting ubiquitin-dependent degradation of β-catenin and promoting Wnt/β-catenin signaling.Serine-Threonine Kinase Receptor-Associated Protein (STRAP) interacts with a variety of proteins and influences a wide range of cellular processes. Aberrant activation of Wnt/β-catenin signaling has been implicated in the development of colorectal cancer (CRC). Here, we show the molecular mechanism by which STRAP induces CRC metastasis by promoting β-catenin signaling through its stabilization. We have genetically engineered a series of murine and human CRC and lung cancer cell lines to investigate the effects of STRAP on cell migration and invasion  Wnt/β-catenin signaling plays a pivotal role in many human malignancies, especially in colorectal cancers (CRC) . Even thSerine-Threonine Kinase Receptor-Associated Protein (STRAP) is a WD40 domain-containing protein  that facin vitro and in vivo. In an attempt to understand the mechanism, we have observed that STRAP stabilizes β-catenin by inhibiting its ubiquitin-dependent degradation, thus resulting in the inhibition of the expression of its downstream target genes. Most interestingly, in support of these results we have observed that both STRAP and β-catenin is co-upregulated in high percent of human CRC . Thus, our results provide evidence of how STRAP is involved in the contribution to CRC development and progression by a unique mechanism.Our present study shows that knockdown of STRAP reduces CRC cell invasion and metastasis in vitro, we performed cell counting and soft agar assays. Downregulation of STRAP significantly inhibited CRC cell growth in liquid culture as well as in soft agar in both MC38 and CT26 cells . Furthermore, we found that the expression of both STRAP and β-catenin were much higher in AJCC stage I than that in other stages  cell lines A549 and H460; and HEK-293T cells were maintained in 7% serum-containing medium supplemented with penicillin and streptomycin.6-Ubiquitin expression construct was a gift from Dr. Christoph Eglert . TOP/FOP Flash reporter constructs were gifts from Dr. Eric Fearon .HA-tagged or Flag-tagged STRAP (in pCDNA3 vector) has been described previously . Myc-tagSelleckchem.com and TOCRIS bioscience , respectively. Lithium chloride was obtained from Calbiochem . Cycloheximide was purchased from Sigma . Human and mouse Wnt3a was purchased from R & D Systems . Antibodies were purchased as follows: anti-CyclinD1, anti-HA, anti-c-Myc, anti-RhoA, and anti-poly (ADP) ribose polymerase (PARP) from Santa Cruz Biotechnology ; anti-STRAP and anti-β-catenin from BD Transduction Labs ; anti-phospho-β-catenin (Ser33/37/Thr41), anti-GSK-3β, anti-APC and anti-phospho-GSK-3β (S9) from Cell Signaling Technology ; and anti-β-actin and anti-Flag antibodies from Sigma .Proteasomal inhibitor MG132 and GSK3β inhibitor SB415286 were purchased from Cell lines (as indicated) were infected with STRAP shRNA lentivirus and selected with puromycin. STRAP knockdown polyclonal populations of SW480, HCT116, and RKO were generated using similar protocol. The expression of STRAP was verified by western blotting.Western blot and immunoprecipitation analyses were performed as previously described . For subMC38 and CT26 cells were plated in 12-well plates. After 48 h, cells were counted every day for 5 days and the average cell numbers from triplicate wells were plotted.5 cells from STRAP knockdown stable clones and vector control cells derived from MC38 and CT26 cell lines were subcutaneously injected into C57BL/6 and Balb/c mice, respectively. All animal experiments were performed in accordance with IACUC and state and federal guidelines for the humane treatment and care of laboratory animals. The animals were monitored for tumor formation every 3 days for a total of 3–5 weeks and the tumors were measured as discussed previously [MC38 and CT26 cells were plated for soft agarose assays as described previously . For xeneviously .Migration and invasion assays were performed as previously described . MC38 an™ Reverse Transcription Supermix . Real-time PCR was carried out using 2.5 ul cDNA with FastStart SYBR Green Master  following the manufacturer's instruction. The primer sequences of STRAP, β-catenin, β-TrCP, MMP2, MMP7, MMP9 and GAPDH were shown in Total RNA was extracted from MC38, CT26, H460 and A549 cells using Trizol reagent and RT-PCR amplification was performed using iScript2000 following the manufacturer's protocol. β-galactosidase (25 ng/well) was used as a control for the transfection efficiency. After approximately 44 hours, cells were harvested and luciferase assays were performed using Monolight™ 3010  according to the manufacturer's protocol. Transfection of each group was performed in triplicate. Luciferase reading values were normalized with β-gal values and triplicates were averaged.Cells were seeded in 12-well plates. After overnight incubation, TOP/FOP Flash reporter plasmid (0.5 ug/well) were transfected into cells with LipofectamineFor gelatin zymography, cells were seeded in 6-well plates. After overnight incubation, cells were treated with 900 ul serum-free media for another 16 hours and the media were collected. After normalized with total protein of the cell lysate, the media were subjected to zymography analyses with non-reduced SDS-PAGE using 10% gels containing 0.1% gelatin according to the protocol as discussed elsewhere .6-ubiquitin, β-catenin-HA and STRAP-Flag with lipofectamine2000 . Forty hours after transfection, the cells were treated with 25 μM of MG132 for 6 hours. Then the cells were lysed in highly denaturing conditions with 6 M guanidine hydrochloride buffer and ubiquitylated proteins were pulled down from the lysates with Ni–NTA agarose resin following the manufacturer's protocol. The bound proteins were eluted with 2X Laemmli buffer containing 250 mM imidazole. The eluants were analyzed by western blotting.HEK-293T cells were transiently transfected with combinations of expression constructs his5 cells in 100ul PBS were injected into the spleens. 5 min after injection, spleens were removed. C57BL/6 mice injected with MC38 cells were sacrificed after 3 weeks, whereas Balb/c mice injected with CT26 cells were sacrificed after 4 weeks for analysis of tumor formation in the liver. For cecum injection, 1 × 105 LM10 cells, derived from MC38 cells, were suspended in 50 ul PBS and were injected subserosally into ceca using a 30-G needle under stereomicroscope. Mice were monitored and sacrificed 48 days after injection when some mice became moribund. The tumors in ceca, livers, and lymph nodes were examined for primary and metastatic tumor growth.Splenic injection and cecum injection models were described in our previous reports , 43. Brihttp://www.proteinatlas.org/ENSG00000168036-CTNNB1): cytoplasmic/membranous/nuclear, cytoplasmic/membranous, nuclear, and none.Paraffin-embeded mouse xenograft tissue and human colon cancer tissue microarray slides were subjected to immunostaining. The slides were stained as we described previously . Anti-STt-test or analysis of variance (ANOVA) with Bonferroni post hoc test. Spearman rank correlation coefficient analysis was performed to analyze the correlation between STRAP and β-catenin expression in human CRC samples. Chi-square was used to analyze β-catenin nuclear accumulation in different STRAP expression groups in human CRC samples. Kruskal-Wallis test was used to analyze the associations between the expression of STRAP or β-catenin and clinical information, including AJCC stage, pathology grade and age. Statistical analysis was performed with SPSS software for windows . A 2-sided p value of less than .05 was considered statistically significant.The data are presented as mean ± S.D. Statistical analyses were performed by Student's


In vitro, Lv-shβ-catenin significantly decreased the expression of β-catenin, MMP2 and MMP9, and secretion of TGF-β1. In vivo, intratracheal treatment with Lv-shβ-catenin significantly reduced expression of β-catenin in the lung and levels of TGF-β1 in bronchoalveolar lavage fluid, and notably attenuated pulmonary fibrosis as evidenced by hydroxyproline content and collagen I\III synthesis in silica-administered mice. These results indicate that blockade of the Wnt/β-catenin pathway can prevent the development of silica-induced lung fibrosis. Thus Wnt/β-catenin pathway may be a target in prevention and treatment of silicosis.Silicosis is a form of occupational lung disease caused by inhalation of crystalline silica dust. While the pathogenesis of silicosis is not clearly understood, the Wnt/β-catenin signaling pathway is thought to play a major role in lung fibrosis. To explore the role of Wnt/β-catenin pathway in silicosis, we blocked Wnt/β-catenin pathway both in silica-treated MLE-12 cells  and in a mouse silicosis model by using a lentiviral vector expressing a short hairpin RNA silencing β-catenin (Lv-shβ-catenin).  Silicosis is a form of occupational lung disease caused by inhalation of crystalline silica dust ,2. HowevThe Wnt signaling pathway is highly conserved in evolution. It can be divided into three distinct pathways, and the “canonical” β-catenin dependent Wnt signaling pathway is best characterized ,8. This in vitro and in vivo. In addition, intratracheal of Lv-shβ-catenin suppressed β-catenin expression and attenuated lung fibrosis in the mouse silicosis model.In the present study, we generated a lentiviral vector expressing a short hairpin RNA (shRNA) that targeted β-catenin gene of mouse (Lv-shβ-catenin). A mouse pulmonary epithelial cell line MLE-12 and a mouse silicosis model were used to estimate the effect of β-catenin inhibition on silicosis. We observed that Lv-shβ-catenin decreased the expression of β-catenin, meanwhile the expression of MMP2, MMP9 and secretion of TGF-β1 both http://ihome.ust.hk/~bokcmho/siRNA/siRNA.html). A negative control sequence cited by many other papers was chosen [Five siRNA sequences were designed against the mouse β-catenin sequence in GenBank NM_007614.3) using on-line siRNA software (.3 using s chosen . The len2. MLE-12 cells were plated in 6-well plates (2 × 106 cells/well), and the Lv-sh-β-catenin -1, -2, -3, -4, -5 (KD) and NC were incubated with cells at the multiplicity of infection (MOI = 50) for 8 h. After this, the medium was replaced with fresh medium without FBS and the incubation was continued for another 72 h. Cells of each group were given serum-free medium containing 200 μg/mL of silica, and incubated for 18 h [MLE-12 cell line was purchased from ATCC and grown in equal parts DMEM and F12 medium  with 10% FBS (Gibco) at 37 °C, 5% COfor 18 h . Total m6 cells/mL in serum-free DMEM and F12 medium in 6-well plates. After 2 h at 37 °C, cells were given serum-free medium containing 200 μg/mL of silica, and incubated for 18 h [After MLE-12 cells infected with Lv-shβ-catenin or Lv-shβ-catenin-NC, cell viability was measured using a MTT Cell Viability Assay Kit  according to the manufacturer’s protocol. To detect anti-fibrogenic role by Lv-shβ-catenin in MLE-12 cells that had been treated by silica, normal MLE-12 cells, MLE-12 infected with Lv-shβ-catenin, or MLE-12 cells infected with Lv-shβ-catenin-NC were plated at a density of 1 × 10for 18 h . Then su2 was purchased from U.S. Silica Company . The silica content of the SiO2 was > 99%, the dust particle size distribution: 97% < 5 μm diameter, 80% < 3 μm diameter. All experiments and surgical procedures were approved by the Animal Care and Use Committee at the China Medical University, which complies with the National Institute of Health Guide for the Care and Use of Laboratory Animals.Equal proportions of male and female C57BL/6 mice at 6–8 weeks of age, weighing 16–18 g, were obtain from SLAC Laboratory Animal Co. Ltd. . The permit number of animal Care and Use Committee of China Medical University is CMU62043013. The animals were housed at an environmental temperature of 24 ± 1 °C and a 12/12 h light/dark cycle, with free access to food and water. SiO7 transducing units (TU) of Lv-shβ-catenin in a total volume of 0.1 mL of sterile physiological saline; (4) silica + Lv-shβ-catenin-NC group: instillation of a mixed suspension of 1 mg of silica dust and 5 × 107 TU Lv-shβ-catenin-NC in a total volume of 0.1 mL of sterile physiological saline. Mice were anesthetized with an intraperitoneal injection of 0.1 mL/mouse with 10% chloral hydrate. The skin of the neck was opened and blunt dissection exposed the trachea. Either physiological saline, silica in physiological saline, or silica with Lv-shβ-catenin or Lv-shβ-catenin-NC in physiological saline, was instilled into the lungs using a 7-gauge needle inserted into the trachea through the epiglottis of the larynx. The site of surgery was sutured and the mice were allowed to recover until they were sacrificed. At 7, 28 and 56 days post-instillation, six mice of each group were anesthetized with anesthetic ether, sacrificed by decapitation, and the lungs were removed. Bronchoalveolar lavage fluid (BALF) was obtained by cannulating the trachea, injecting and retrieving 1 mL aliquots of sterile physiological saline. Then BALF was centrifuged at 1000 rpm for 1 min at 4 °C, and stored at –80 °C.Animals were divided randomly into the following four experimental groups (n = 18 per group): (1) saline control group: instillation of 0.1 mL of sterile physiological saline; (2) silica group: instillation of a suspension of 1 mg silica dust in a total volume of 0.1 mL sterile physiological saline; (3) silica + Lv-shβ-catenin group: instillation of a mixed suspension of 1 mg of silica dust and 5 × 10Quantitative real-time PCR (qRT-PCR) analysis was performed as previously described . BrieflyFor western blot analysis, total protein was extracted from MLE-12 cells and lung homogenates with M-PER and T-PER Protein Extraction Reagents , respectively which containing 0.1 mM of phenylmethyl sulfonyl fluoride  according to manufacturer’s protocol. 20–40 µg proteins were separated by 10% SDS-PAGE under a constant voltage of 120 V for 2 h and then transferred to a nitrocellulose membrane at a constant voltage of 300 V for 1 h using a Mini-PROTEAN tetra system . After blocking with phosphate buffer saline containing 0.05% Tween-20 (PBST) and 5% non-fat dry milk, the membranes were incubated at 4 °C overnight with TBST and 5% milk containing primary antibodies at following dilution ratios: anti-β-catenin antibody ; anti-MMP9 antibody ; anti-MMP2 antibody  and anti-GAPDH antibody . The membranes were then washed three times with PBST and incubated with horseradish peroxidase-conjugated anti-rabbit IgG, HRP-linked antibody  for 1 h at room temperature (RT), followed by washing with PBST. Proteins were visualized by chemiluminescence .The ELISA plate was coated with 100 µL of capture antibody in coating buffer per well of ELISA kit  and incubated overnight at 4 °C. The plate was washed with 250 µL of wash buffer. Then the well was blocked with 200 µL of assay diluent, incubated 1 h at RT. A volume of 100 µL cell supernatants or BALF or the different dilutions of standard (for standard curve) was added to each well, incubated 2 h at RT. The well was incubated at RT with 100 µL detection antibody 1 h, followed by 100 µL of avidin-HRP for 30 min and 100 µL of substrate solution for 15 min. Before plate reading at 450 nm, 50 µL of Stop Solution was added. The ELISA assay was performed in triplicate.The lung samples were measured for hydroxyproline content using a hydroxyproline kit from Nanjing Jian Cheng Institute  following instruction from the manufacturer. The results were calculated as milligrams of hydroxyproline per gram of wet lung weight.Following gross inspection of each mouse, small pieces of lung tissue from the middle of the lobes, along with the hilar lymph nodes, were fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned at 5 µm. The tissue sections were stained with hematoxylin and eosin (HE). Silicotic nodules were graded as following: cellular nodules as Stage I; fibrotic cellular nodules as Stage II; cellular fibrotic nodules as Stage III; fibrotic nodules as Stage IV .2O2 solution for 30 min to block endogenous peroxidase activity. After overnight incubation at 4 °C with rabbit polyclonal anti-collagen I and III antibodies , anti-MMP9 antibody , anti-MMP2 antibody  diluted in phosphate-buffered saline (PBS), antigen-antibody complexes were detected using Streptavidin/Peroxidase (SP) HistostainTM-Plus Kits . Peroxidase activity was revealed using a 3,3’-diaminobenzidine tetrahydrochloride Substrate Kit (Beijing Zhongshan Golden Bridge Biotechnology Ltd.). The sections were counterstained with hematoxylin for 3 min, rinsed and mounted with glycerin gelatin for histological examination. Brown particles in the cytoplasm or the cellular membrane were considered a positive reaction. The collagen I, collagen III, MMP2 and MMP9 proteins were analyzed quantitatively using MetaMorph/DP10/BX41-type image analysis software . In 10 × 40 fields, three to five fields were randomly selected for each section. The integrated optical density (IOD) average represented the quantitative expression of collagen I, collagen III, MMP2 and MMP9.For immunohistochemical examination, all sections were deparaffinized in xylene followed by 100% ethanol and then placed in a freshly prepared methanol plus 3% Hp < 0.05 was considered statistically significant.SPSS 17.0 software was used to conduct statistical analyses. The differences between values were evaluated through one-way analysis of variance (ANOVA) followed by pair-wise comparison with the Student-Newman-Keuls test. MLE-12 cells were infected with Lv-shβ-catenin -1, -2, -3, -4,-5, or -NC, respectively. Cell viability was above 80% of control at 24 to 96 h of virus infection (data not shown). After 72 h cell image was taken by fluorescence microscopy. We observed the expression of green fluorescent protein in a large number of MLE-12 cells, which indicates a decent infection effect by Lv-shβ-catenin (KD) and Lv-shβ-catenin-NC (NC) A. The exp < 0.05) (p < 0.05) (p < 0.05)  B,D and M < 0.05) C,D insil < 0.05) E.Lv-shβ-catenin in the lung of silica—administrated mice. The results demonstrate that expression of β-catenin mRNA in the silica+Lv-shβ-catenin group was significantly lower than in the saline control, silica, and silica+Lv-shβ-catenin-NC groups (p < 0.05) at the three time points after instillation (We used western blot assays to examine the silencing effects of illation .p < 0.05) at 7 days after instillation. TGF-β1 content of the silica + Lv-shβ-catenin group was significantly lower than in the silica group and the silica + Lv-shβ-catenin-NC group (p < 0.05), and significantly higher than that of the saline control group (p < 0.05) at 7 days after instillation  at 28 and 56 days after instillation. Hydroxyproline content of the silica + Lv-shβ-catenin group was significantly lower than that of the silica group and the silica + Lv-shβ-catenin-NC group (p < 0.05), and significantly higher than that of the saline control group (p < 0.05) at 28 and 56 days after instillation  were observed at 7 days after instillation. There were less cellular nodules (Stage I) in the lung of mice in the silica + Lv-shβ-catenin group. At 28 days after instillation, primarily cellular nodules and fibrotic cellular nodules (Stage I+ and II) were observed in the silica and the silica + Lv-shβ-catenin-NC groups. There were mainly cellular nodules (Stage I) in the lung of mice in the silica + Lv-shβ-catenin group. Compared to the silica and the silica + Lv-shβ-catenin-NC groups, nodules in the lung of mice in the silica + Lv-shβ-catenin group was less in count and smaller in size. In the silica and the silica + Lv-shβ-catenin-NC groups, fibrotic cellular nodules (Stage II+ and III) were observed at 56 days after the instillation. There were still mostly cellular nodules (Stage I+) and fibrotic cellular nodules (Stage II) in the lung of mice from the silica + Lv-shβ-catenin group, and the number of nodules was increased .p < 0.05).To further observe the degree of fibrosis in the lung, immunohistochemical examination of collagen I, collagen III, MMP2 and MMP9 was performed. Representative images are shown in p < 0.05), but was significantly lower than that of the silica and silica + Lv-shβ-catenin-NC groups (p < 0.05) (p < 0.05), but was significantly lower than that of the silica and silica + Lv-shβ-catenin-NC groups (p < 0.05)  A. Altera < 0.05) B. At 7 d < 0.05) C. Altera < 0.05) D.Pulmonary fibrosis is the most important pathological change in silicosis. Although the pathogenesis of silicosis has not been clearly explained, recent studies have suggested that the Wnt/β-catenin signaling pathway is involved in the development of pulmonary fibrosis ,15. Follin vitro and in vivo.Accordingly, elevated expression of β-catenin plays a key role in the Wnt/β-catenin signaling pathway. To investigate whether inhibition of β-catenin expression attenuates lung fibrosis, we used RNAi technology to construct a recombinant lentiviral vector, Lv-shβ-catenin, which expressed shRNA specific against mouse β-catenin. We confirmed the β-catenin-silencing efficacy of shβ-catenin in two different cell lines, MLE-12 and HEK-293T cells (data not shown), as well as in the mouse model of silicosis. The results showed that a recombinant shβ-catenin lentivirus was successfully constructed, which could interfere with the expression of β-catenin both in vitro and in vivo experiments showed that silencing β-catenin suppressed the expression of MMP2 and MMP9 at both mRNA and protein levels, which provide a possible mechanism underlying the role of Wnt/β-catenin signaling pathway in lung fibrosis. The cytokine TGF-β1 also contributes to the progression of lung fibrosis [MMP2 and MMP9 are expressed in many types of cells in the lung tissue, such as bronchiolar epithelial cells, fibroblasts, macrophages, and so on . These Mfibrosis ,24. We fin vitro, attenuation of MMPs expression in the lung and TGF-β1 secretion in BALF by Lv-shβ-catenin was observed in mice of silicosis, however, presented earlier than onset of alteration in lung fibrosis (7 days versus 28 days and 56 days after silica instillation), which suggests the role of MMPs and TGF-β1 in the development of lung fibrosis. Therefore, we may conclude that Lv-shβ-catenin could prevent the development of silica-induced lung fibrosis. This study provides a new molecular basis for understanding the mechanism of silicosis. The Wnt/β-catenin signaling pathway may represent a novel target for prevention and treatment of silicosis.An experimental mouse model of silicosis was induced by an intratracheal administration of silica dust in the present study. This method was proved to result in varying degrees of fibrotic silicosis . Lv-shβ-Our findings suggest that blockade of the Wnt/β-catenin signaling pathway by silencing β-catenin prevents the development of silica-induced lung fibrosis, which is related to the expression of MMP2 and MMP9, and secretion of TGF-β1 in the lung. Our data support the concept that the Wnt/β-catenin signaling pathway may represent a novel target for prevention and treatment of silicosis.


KLF4 is an important regulator of cell-fate decision, including DNA damage response and apoptosis. We identify a novel interplay between protein modifications in regulating KLF4 function. Here we show that arginine methylation of KLF4 by PRMT5 inhibits KLF4 ubiquitylation by VHL and thereby reduces KLF4 turnover, resulting in the elevation of KLF4 protein levels concomitant with increased transcription of KLF4-dependent p21 and reduced expression of KLF4-repressed Bax. Structure-based modelling and simulations provide insight into the molecular mechanisms of KLF4 recognition and catalysis by PRMT5. Following genotoxic stress, disruption of PRMT5-mediated KLF4 methylation leads to abrogation of KLF4 accumulation, which, in turn, attenuates cell cycle arrest. Mutating KLF4 methylation sites suppresses breast tumour initiation and progression, and immunohistochemical stain shows increased levels of both KLF4 and PRMT5 in breast cancer tissues. Taken together, our results point to a critical role for aberrant KLF4 regulation by PRMT5 in genome stability and breast carcinogenesis. Krüppel-like factor 4 plays an important role in regulating responses to DNA damage, cell-fate decision and apoptosis. Here the authors show that aberrant regulation by methyltransferase PRMT5 results in failure to arrest the cell cycle and genome instability, pointing to a role in carcinogenesis. Krüppel-like factor 4  is an important regulator of cell-fate decisions, including DNA damage response, inflammation, apoptosis and stem cell renewal13168910111211214PRMT5 resulted in early embryonic lethality and suppression of pluripotency in ES cells by reprogramming a set of genes that orchestrate stem cell self-renewal and differentiationPRMT5 in worms leads to genome instability in response to ionizing irradiation2024257PRMT5 is a mammalian protein arginine methyltransferase that catalyses the addition of methyl groups to the guanidine nitrogen atoms of arginine161617We recently reported that KLF4 is a rapidly turned over protein with its half-life governed by VHL-VBC ubiquitin protein ligasein vitro . KLF4, but not a control precipitate showed a signal at the expected protein size . We confThe above interaction and enzymatic assays suggest a direct interaction between KLF4 and PRMT5, which is facilitated by residues lying in the regions K382-T388 of KLF4 and P465-H510 of PRMT5 . Our strKLFs contain three highly conserved zinc-finger motifs Zf1–Zf3;  that bin3234active site on insertion through a narrow tunnel created by residues L312, F327 and W579, where it forms salt bridges with highly conserved E435 and E444 ; (i) first KLF4 was steered into the large cavity formed by the PRMT5 dimer so that the experimentally detected binding regions were in close proximity; (ii) then R377 was steered into the active site through the narrow tunnel formed by L312, F327 and W579, to tightly pin down KLF4 on PRMT5; (iii) by keeping R377 at the active site, an initial bound conformation was generated on the basis of the surface chemical properties of the binding regions . R377 waAs explained above, R374 has been originally positioned in the vicinity of both E435 and E444 at the active site of PRMT5. However, as the simulations evolved, the interaction with E444 became prevalent. A strong interaction network comprising the positively charged residues R381, K382, R383 on KLF4 and negatively charged residues E483, E489 on PRMT5 became prominent. This interaction network is shown in Taken together, the above MD results highlight the significance of the charged residue stretch RKR (381–383) adjacent to the methylation sites of KLF4. Both PRMT5 subunits of the dimer are involved in the KLF4 binding, while one of them (the first) is juxtaposed to catalyse the methylation of KLF4 arginines. This particular subunit is engaged in tight interactions with KLF4, whereas the second seems to stabilize Zf1 in a proper orientation. In our simulations, Zf2 and Zf3 of KLF4 bound to the outer surface of the PRMT5 dimer, potentially giving them the freedom to bind DNA.Methylation by PRMT5 has been reported to regulate proteins that orchestrate various cellular processes, including cell-cycle control, signal transduction, DNA damage response and DNA repair, immune response activation and tumorigenesis1617242529The above results in Both KLF4 and PRMT5 are thought to be critical in the maintenance of genome stability29372029373940739To test the role of PRMT5 in KLF4-dependent p21 transcription, we measured p21 RNA levels after overexpression of KLF4 in the absence or presence of siRNA against PRMT5. Knockdown of PRMT5 leads to impaired KLF4 mediated p21 activation . PRMT5 oin vivo mouse xenograft study. As shown in Protein levels for both PRMT5 and KLF4 need to be tightly regulated, since aberrant expressions of PRMT5 or KLF4 have been correlated with poor prognosis in various types of tumors123134244453To dissect the possible molecular mechanisms how an aberrant interplay of PRMT5 and KLF4 contributes to breast tumor initiation and progression, we performed gene array assays and identified the downstream gene network responding to the aberrant KLF4 methylation by PRMT5 by using a customized breast cancer gene profile with quantitative real-time PCR (qPCR) array from SABiosciences 4849. The48484951The above characterizations based on the cultured-cell model have demonstrated the biological consequence and physiological relevance of crosstalk regulation of KLF4 by ubiquitylation and protein methylation in the maintenance of genome stability as well as tumorigenesis. While our results suggest the importance of the interplay between KLF4 and PRMT5 in determining the cellular fate after the exposure to DNA damage signal, we observed that the aberrant KLF4 abundance due to the deregulated KLF4 regulation by PRMT5 could directly enhance the breast tumor initiation and progression. In order to further explore the possible clinical relevance of KLF4-PRMT5 axis in tumorigenesis, we decided to examine if disrupted regulation of KLF4 by PRMT5 is a driving factor for breast tumor initiation and progression. To this end, we measured the protein expression levels of PRMT5 and KLF4 in human breast tumor and adjacent normal tissue by immunohistochemistry (IHC). As shown in p21 and Bax. Since the levels of p21 and Bax are crucial in determining whether cells undergo cell-cycle arrest or apoptosis, the interplay between KLF4 and PRMT5 has important repercussions for tumorigenesis and the DNA damage response . The p53+/+ and p53−/− HCT116 cells were provided by Lin Zhang. The U2OS cells were provided by Robert W. Sobol. The MCF10A and MCF7 cells were provided by Dr Yi Huang. The viral packaging line Pheonix-A was provided by Dr E.V. Prochownik. The HEK293T, HeLaS3, U2OS, MDA-MB-231 and Pheonix-A cells were maintained in DMEM (Invitrogen) with 10% FBS. The BTR cells were maintained as described previouslyHeLa S cells stably expression FLAG-tagged KLF4 were washed twice with PBS and then incubated at room temperature for 30 min with freshly resuspended 2.5 mM DSP (Lomant's reagent). After the crosslinking reaction was stopped by adding Tris pH7.5 to a final concentration of 50 mM for 10 min, nuclear extract from HeLa cells was prepared. KLF4-interacting proteins were purified from the nuclear extract by immunopurification on M2 agarose (Sigma) and washing twice with BC buffer  containing 300 mM KCl and once with BC buffer containing 100 mM KCl. The complex was eluted with FLAG peptide in BC buffer containing 100 mM KCl and the resulting elute was treated with 50 mM DTT at 37 °C for 30 min to reverse the crosslinking. The decrosslinked elute was then separated on SDS-PAGE followed by silver staining. The interest bands were cut out for mass spectrum analysis.The lentivirus-based tet inducible system containing pLenti TetR and pLenti CMV/TO Puro DEST #2 was ordered form Addgene. The p21-Luc reporter plasmid was the gift from C. Seiser . The Lefty1-Luc reporter plasmid was the gift from H. Niwa (Kobe University). The KLF4 full-length and deletion mutant constructs were generated by PCR amplification of the full-length or partial coding sequence of human KLF4 and subsequent subcloning into mammalian expression vectors with FLAG or HA tag. The KLF4 methylation deficient mutant construct was generated using the site-directed mutagenesis kit (Stratagene). The primer sequences are as following, 5′-GGAGCCCAAGCCAAAGAAGGGAAAAAAATCGTGGCCCCGGAAAAG-3′ (forward primer), and 5′-CTTTTCCGGGGCCACGATTTTTTTCCCTTCTTTGGCTTGGGCTCC-3′ (reverse primer). The Myc-tagged PRMT5 full-length and deletion mutant constructs were generated as described previously−1) or blasticidin (10 μg ml−1). The KLF4 inducible cell lines (KLF4-WT and KLF4-3K in both MCF10A and MCF7) were generated by sequential infection with pLenti TetR and pLenti CMV/TO-KLF4 and subsequent selection by both blastidicin and puromycin.pLKO.1-PRMT5-shRNA (RHS3979-201825392 from Open Biosystem) or pLenti 6-V5-KLF4 was co-transfected with pVSV-G, pRRE and pRSV-REV into HEK293T cells using Lipofectamine 2000 ref. . The pacsiRNAs (Sigma) specifically targeted to PRMT5 or luciferase were synthesized and transfected into cells using Lipofectamine 2000. Cells were collected at 48 h post-transfection for immunoblotting assay. The synthesized siRNA sequences are as following: Luciferase, 5′- CGT ACGCGGAATACTTCGA-3′; PRMT5, 5′-GCCCAGTTTGAGATGCCTTAT-3′.Cells were harvested and lyzed in RIPA lysis buffer (Upstate Biotechnology) containing protease inhibitor cocktail (Sigma). The protein concentration was determined using Bio-Rad Protein Assay Reagent (Bio-Rad). Immunoblotting was performed using the antibody to KLF4 , p53 , HA tag  (Santa Cruz), Myc tag , PARP , cleaved caspase-3  , FLAG tag , β-actin  (Sigma), PRMT5 , p21 , V5 tag , and HRP-conjugated goat anti-mouse or anti-rabbit secondary antibody . Signals were detected with ECL reagents (Amersham Pharmacia). Semi-quantification of data was performed using software NIH Image J. For immunoprecipitation assay, cells were lyzed in TNE buffer  supplemented with protease inhibitor cocktail. Cell lysates were incubated with the antibody overnight at 4 °C on a rotator. Protein A/G plus agarose (Pierce) was then added and the reactions were further incubated at 4 °C for 2 h. After five washes with TNE buffer without Triton X-100 but supplemented with protease inhibitor cocktail, complexes were released from agarose by boiling for 5 min in 2 × SDS-PAGE loading buffer. Immunoblotting was performed to detect protein expression or ubiquitin conjugates.Cells grown on glass bottom culture dishes (MatTek) were transfected with pEGFP-KLF4. Twenty-four hours after transfection, cells were fixed with 4% paraformaldehyde in PBS for 10 min and rendered permeable by further treatment with 0.2% Triton X-100 for 5 min. The PRMT5 antibody  was diluted in PBSB (1% BSA in PBS) and incubated with cells for 1 h. After three washes with PBSB, cells were incubated with Alexa 594-labelled anti-rabbit IgG  diluted in PBSB for 1 h. Cells were washed, mounted with UltraCruz DAPI containing mounting medium (Santa Cruz), viewed, and photographed under a FluoView 1200 confocal microscope (Olympus).Renilla luciferase control vector using Lipofectamine 2000 according to the manufacturer's instruction. Firefly and Renilla luciferase activities were measured using a dual luciferase kit (Promega). The firefly luciferase activity for each sample was normalized based on transfection efficiency as determined by Renilla luciferase activity. Each experiment was performed in triplicate and repeated at least three times.Cells were plated in 24-well plates. After 24 h, cells were co-transfected with various plasmids together with reporter plasmid and E. coli strain BL21 and purified using glutathione resin following standard protocol. 2 μg of purified recombinant protein was incubated at 4 °C for 1 h with 500 μg of cell lysates from HEK293T cells transfected with HA-PRMT5. Glutathione resin was added and KLF4 interacting protein was pulled down.GST-fused KLF4 wide-type or mutant was expressed in the 3H-SAM (Perkin Elmer). Reactions were incubated at 30 °C for 1 h and terminated by addition of SDS sample buffer. Tritiated protein was separated by SDS-PAGE and transferred to polyvinylidene difluoride. For autoradiography, blots were treated with En3Hance Spray (Perkin Elmer) and exposed to film at minus 80 °C for 7 days.FLAG-tagged PRMT5 was immunoprecipitated from transfected HEK293T cells, incubated with 2 μg of GST-fused KLF4 wide-type or mutant and 2 μCi Apoptotic cell death was measured by flow cytometric analysis after staining using FITC-conjugated Annexin V/PI Kit (BD PharMingen) following the manufacturer's instructions. In the population of Annexin V-positive cells, PI-negative or PI-positive cells were considered to be early apoptotic or late apoptotic.−1 and changed every three days. After two weeks of culture at 39.5 °C, colonies were fixed with glutaraldehyde (6.0% v/v), stained with crystal violet (0.5% w/v) and counted using a digital colony counter pen.BTR cells were seeded in a six-well plates and infected with pLenti 6-V5-KLF4 or pLenti 6-V5-KLF4-mut. Forty-eight hours after infection, the culture medium was supplemented with blasticidin at 10 μg mlTotal RNA was isolated from proliferating cells of MCF7 or MCF10A using the Trizol (Invitrogen) according to the manufacturer's instructions. RNA was reversed transcribed using High Capacity RNA-to-cDNA Master Mix (Invitrogen). Real-time PCR was performed on a StepOnePlus Real-Time PCR System (ABI) in duplicates with Fast SYBR Green Master Mix (Applied Biosystems). The cycle threshold (CT) defines the number of PCR cycles required for the fluorescent signal to cross the threshold.ΔΔCT) method and generated heatmap by using the online analysis tools provided by Qiagen SABiosciences . The resulting values were reported as fold change; only genes showing twofold or greater change were considered.For PCR arrays, the breast cancer genes profile quantitative real-time PCR array PAHS-131Z (Qiagen SABiosciences) plus custom 27 PCR primer were used to simultaneously examine the mRNA levels of 111 genes plus 6 housekeeping control genes. Arrays were performed on StepOne Plus real-time polymerase chain reaction system (Applied Biosystems). Data were analyzed via the comparative CT (6 of MCF-7 cells mixed with matrigel (1:1) were injected into the mammary fat pads of 8-week-old female Crl:Nu-Foxn1(nu) nude mice. Two days before the injection, each mouse was implanted with 17β-estradiol (E2) pellets (0.72 mg/pellet) . Tumor size was measured weekly. The animal study was approved by the Institutional Animal Care and Use Committee (IACUC) from University of Pittsburgh.In an orthotopic model, 5 × 10How to cite this article: Hu, D. et al. Interplay between arginine methylation and ubiquitylation regulates KLF4-mediated genome stability and carcinogenesis. Nat. Commun. 6:8419 doi: 10.1038/ncomms9419 (2015).Supplementary Figures 1 -18, Supplementary Tables 1-4, Supplementary Discussion, Supplementary Methods and Supplementary References.Protocol for the series of MD simulations performed to generate the PRMT5-KLF4 complex


These findings suggest that ilimaquinone and ethylsmenoquinone exert their anti-cancer activity by blocking the Wnt/β-catenin pathway and have significant potential as therapies for multiple myeloma.Deregulation of Wnt/β-catenin signaling promotes the development of a broad range of human cancers, including multiple myeloma, and is thus a potential target for the development of therapeutics for this disease. Here, we used a cell-based reporter system to demonstrate that ilimaquinone and ethylsmenoquinone (formerly smenorthoquinone), sesquiterpene-quinones from a marine sponge, inhibited β-catenin response transcription induced with Wnt3a-conditioned medium, by down-regulating the level of intracellular β-catenin. Pharmacological inhibition of glycogen synthase kinase-3β did not abolish the ilimaquinone and ethylsmenoquinone-mediated β-catenin down-regulation. Degradation of β-catenin was consistently found in RPMI-8226 multiple myeloma cells after ilimaquinone and ethylsmenoquinone treatment. Ilimaquinone and ethylsmenoquinone repressed the expression of cyclin D1, c-myc, and axin-2, which are β-catenin/T-cell factor-dependent genes, and inhibited the proliferation of multiple myeloma cells. In addition, ilimaquinone and ethylsmenoquinone significantly induced G Hippospongia metachromia +. Anal. calcd. for C23H32O4Na.To a stirred solution of ilimaquinone (400 mg) in ethanol (21 mL) was added 1M-KOH solution (10.5 mL) at room temperature. The reaction mixture was heated to 70 °C and stirred for 1 h. The reaction mixture was quenched with 1M-HCl solution and extracted with ethylacetate. The combined organic layer was washed with brine and dried over anhydrous MgSOHEK293, RPMI-8226, and Wnt3a-secreting L cells were obtained from the American Type Culture Collection and maintained in DMEM supplemented with 10% fetal bovine serum (FBS), 120 μg/mL penicillin, and 200 μg/mL streptomycin. HEK293-FL reporter (TOPFlash), and control cells were established as previously described . Wnt3a-cCytosolic fractions were prepared as previously described . ProteinTotal RNA was isolated with Trizol reagent  in accordance with the manufacturer’s instructions. Synthesis of cDNA, reverse transcription, and PCR were performed as previously described . The ampCells were inoculated into 96-well plates and treated with ilimaquinone and ethylsmenoquinone for 24 h. Cell viability from each treated sample was measured in triplicate by using the Celltiter-Glo assay kit  according to the manufacturer’s instructions.Cells were treated with ilimaquinone and ethylsmenoquinone for 24 h. Cells were then collected, washed with cold phosphate buffered saline (PBS), and fixed in 70% ethanol at 4 °C overnight. They were then centrifuged at 2000 rpm for 5 min, resuspended in PBS, and incubated with propidium iodide (100 μg/mL) and RNase A (50 μg/mL) at room temperature for 30 min in the dark. Cells were then analyzed using a Cellometer cytometer .After cells were treated with ilimaquinone and ethylsmenoquinone for 24 h, they were washed with cold PBS and resuspended in staining buffer containing Annexin V-FITC and propidium iodide, provided in the apoptosis detection kit (BD Transduction Laboratories), according to the manufacturer’s instructions. Flow cytometric data were obtained using a FACScalibur Flow Cytometer equipped with BD Cell Quest Pro software.0/G1 phase and apoptosis. Ilimaquinone and ethylsmenoquinone therefore have the potential to be developed into effective cancer preventive agents and antineoplastic therapeutics for multiple myeloma.We investigated the anticancer effect of ilimaquinone and ethylsmenoquinone on multiple myeloma cells and revealed their mechanism of action by using a cell-based reporter system. Ilimaquinone and ethylsmenoquinone suppressed the Wnt/β-catenin pathway by promoting the degradation of β-catenin via a mechanism independent of destruction complex, thereby inhibiting the proliferation of multiple myeloma cells through inducing cell cycle arrest in G


This is in line with the in vitro results of the alternative WNT receptors ROR1 and ROR2, which foster invasion. In breast cancer, the value of prognostic markers established in primary tumors cannot directly be translated to metastases. Our results revealed β-catenin-independent WNT signaling to be associated with poor prognosis in patients with breast cancer liver metastasis.Liver metastasis development in breast cancer patients is common and confers a poor prognosis. So far, the prognostic significance of surgical resection and clinical relevance of biomarker analysis in metastatic tissue have barely been investigated. We previously demonstrated an impact of WNT signaling in breast cancer brain metastasis. This study aimed to investigate the value of established prognostic markers and WNT signaling components in liver metastases. Overall N = 34 breast cancer liver metastases (with matched primaries in 19/34 cases) were included in this retrospective study. Primaries and metastatic samples were analyzed for their expression of the estrogen (ER) and progesterone receptor, HER-2, Ki67, and various WNT signaling-components by immunohistochemistry. Furthermore, β-catenin-dependent and -independent WNT scores were generated and analyzed for their prognostic value. Additionally, the influence of the alternative WNT receptor ROR on signaling and invasiveness was analyzed in vitro. ER positivity  and high Ki67  in the primaries had prognostic impact. However, only Ki67 remained prognostic in the metastatic tissue . Additionally, the β-catenin-independent WNT score correlated with reduced overall survival only in the metastasized situation  contains supplementary material, which is available to authorized users. The 5-year overall survival (OS) for breast cancer patients in Europe increased over time from 78.4 % 1999–2001) to 82.4 % (2005–2007) owing to the availability of more efficient treatment modalities nowadays 999–2001 . Neverthparallel progression or branched evolution) . Individual treatment strategies are summarized in Supplemental Table The OS had a time to 50 % at risk of 95.5 months 95 % CI [73.6–127.9]. Survival after diagnosis of liver metastasis (OS liver metastasis) revealed a time to 50 % at risk of 15.91 months 95 % CI [9.83–27.06].p = 0.0481). Patients who underwent surgery for resectable liver metastases  had a significantly improved survival compared to patients without liver surgery  . Survival was significantly shorter in older patients at the time of diagnosis of breast cancer .The presence of extrahepatic distant metastases on diagnosis lead to a significantly shorter survival . In the matched samples, 13/19 (68.42 %) of the breast cancer primaries revealed PGR expression, whereas only 6/19 (31/0.59 %) liver metastases were positive. Thus, PGR expression also decreased during the development of liver metastasis, however no correlation was detected . Within the matched samples, HER-2 expression was 21.05 % in primaries: 4/19 and 26.32 % in metastases: (5/19) and significantly correlated .ER and PGR expression were detected in 15/19 (78.95 %) and 13/19 (68.42 %) of the breast cancer primaries and in 19/34 (55.88 %) and 11/34 (32.35 %) of the liver metastases. 4/19 (21.05 %) of the primaries demonstrated HER-2 positivity, whereas in the liver metastases 9/34 (26.47 %) were positive. Focusing on the 19 available pairs of matched primaries and liver metastases, the expression of ER was significantly lower in the metastases (primaries 15/19 (78.95 %); metastases: 11/19 (57.89 %) and significantly correlated  . The same is true for the liver metastases (median 30 %), for which survival was significantly shorter when Ki67 staining was above the median   demonstrated a proliferation index of Ki67 ≥ 10 %. In 31/34 (91.18 %) of the liver metastases, a proliferation index of Ki67 [10 %] was detected with a range from 0 to 80. Owing to the fact that almost all primary and metastases samples were positive for the proliferation index Ki67, the median expression was taken as cutoff to perform survival analyses. Taking the primaries’ median proliferation index Ki67 of 20 %, a higher proliferation index Ki67 was associated with shorter survival  of the breast cancer primaries. Three samples were graded as 1+, one sample as 2+ and fifteen samples as 4+  expressed nuclear Phospho-c-Jun. Twenty-one samples were graded as 1+, eight as 2+, and two samples as 3+ and 4+, respectively  of the primary samples  nor the β-catenin-independent WNT score alone  were of prognostic value. In contrast, in the liver metastases, a high β-catenin-independent WNT score  proved to be unfavorable analyzed from the time point after liver metastasis, whereas the β-catenin-dependent WNT score was not prognostic   was two (95 % CI [0.45–6.1]) in the primary tumors and one (95 % CI [0.0–7.0]) in the liver metastases. The median β-catenin-independent WNT score was four (95 % CI [1.45–8.1]) in the primaries and five (95 % CI [2.7–9.2]) in the liver metastases . In the matched liver metastases samples, the β-catenin-dependent WNT score decreased to one (95 % CI [0.0–7.0]) and the β-catenin-independent WNT score increased to 5 (95 % CI [1.9–9.1]). In the breast cancer primaries, neither the β-catenin-dependent WNT score  or metachronous (n = 29) liver metastases of breast cancer patients as well as in available matched primary tumors (n = 19). While biomarker analysis identified the proliferation index Ki67 to be prognostic in both types of tissue (primary and metastatic), ER status was shown to lose its prognostic value: Surprisingly, ER positivity in the metastatic samples was not prognostic anymore analyzed from the time point of liver metastasis, although it was prognostic in the matched primary tumors analyzed from the time point of the first diagnosis of the primary as expected. This discrepancy may be attributed to the fact that metastatic breast cancer cells are more resistant to anti-hormonal treatment than non-metastatic cells . FurtherAdditionally, current investigations detected a genomic evolution with gain of, tissue-specific and de novo mutations in the metastatic samples in comparison to the primary tumor –56. ThisNonetheless, both (genetic) adaptation of the tumor cells to the metastatic host organ during the process of metastasis and the influence of the new microenvironment are very obvious explanations for our finding that ER expression as a biomarker changes its prognostic capacity in the metastatic tissue.In this line, the β-catenin-independent WNT score gains prognostic impact in the metastatic tissue of the liver, which further implies the above described mechanisms, in particular the adaptation during the process of metastasis and to the new host microenvironment. Both processes presuppose an enormous plasticity of the metastatic cells with subsequent differential gene/protein expression at the different metastatic steps and sites of metastases. While during progression in the primary tumor, pre-metastatic cells have to gain mesenchymal characteristics (EMT), they have to regain epithelial features (MET) and polarization of the pre-metastatic cells in the newly nascent metastatic tissue at the distant sites. The latter process is well described during embryonic development for the establishment of the mesoderm. In that case, mesenchymal cells also regain epithelial characteristics (MET) in a mesenchymal neighborhood that is WNT/PCP-signaling-dependent. Furthermore, WNT/PCP-signaling regulates not only the establishment but also the maintenance of the epithelial polarity and orientation of a single cell in the overall context of the tissue. Recent findings of β-catenin-independent WNT components in cancer also strengthen their role during metastasis. For example, in breast cancer, the WNT/PCP pathway was essential in the tumor-stroma communication via exosomes to gain metastatic features . We demoAll these findings underline the role of β-catenin-independent signaling in the later stages of metastasis. However, so far this has received little attention compared to WNT/β-catenin signaling.However, further studies are required for a more specific characterization of the role of the microenvironment in the development of liver metastases and liver-specific mutations or pathway activations. Here we clearly demonstrate a role of the WNT/β-catenin-independent signaling pathway during this process.Taken together, the presented data clearly demonstrate that biomarkers derived from studies in primary tumors cannot simply be translated to the metastatic tissue because of the parallel evolution of the metastatic cells and the organ-specific growth conditions accountable by their specific microenvironments. Furthermore, we revealed the significance of β-catenin-independent WNT signaling at least for liver metastasis. However, so far the WNT/β-catenin independent signaling has received little attention compared to WNT/β-catenin signaling. In conclusion, we suggest that prognostic biomarkers should be implemented in the clinical decision making or stratification of patients with liver metastasis of breast cancer in multimodal treatment strategies. This may include the resection of breast cancer liver metastases in selected patients.(A) Kaplan–Meier curves depicting cancer-specific survival in month for all patients. Patients with limited metastatic spread being resected had a better long-term survival (p = 0.0207) compared to non-resected patients. (B) Workflow of this study showing the included patient cohort and tissues available of breast cancer primaries and liver metastasesSupplementary material 1 (DOCX 852 kb) Supplemental Fig. (A) Immunoblot of active β-catenin expression in cytosolic (C) and nuclear (N) fractions of MCF-7, SK-BR-3 and MDA-MB-231 cells. HDAC is included as control for successful fractionation. (B) Immunofluorescence staining of ROR2 (green) in MCF-7 empty vector (ctl) and ROR2-overexpressing (pROR2) cells. F-Actin  staining (red) was used to visualize the cytoskeleton of the cells. (C) Measurement of the cell index as a marker for cell proliferation via xCELLigence for MCF-7 and SK-BR-3 empty vector (ctl) and pROR2 cells. One representative image out of three biologically and technically independent experiments is shown. (D) Expression of active β-catenin, total β-catenin and HDAC in cytosolic (C) and nuclear (N) fractions of MCF-7 empty vector and ROR2-overexpressing cells was analyzed by immunoblotting. (E) Flow cytometry of MDA-MB-231 cells  confirming the knockdown of ROR1. (F) Immunoblot analysis of ROR2 expression in MDA-MB-231 nonsense control (ctl) and ROR1 knockdown (shROR1) cells. Pos. Ctl = positive control. (G) MCF-7 cells were transiently transfected with ROR2-Flag and/or myc-tagged Dvl1, Dvl2 or Dvl3 and stimulated with either control supernatant (sn) or Wnt5a-conditioned medium, which was collected from 3T3murine fibroblasts transduced with pMSCV-Xenopus WNT-5a or an empty control. Lysates were co-immunoprecipitated using anti-Flag magnetic beads and precipitated proteins were detected with anti-myc and anti-Flag antibodies. The bottom lanes show the input levels in the lysatesSupplementary material 2 (DOCX 823 kb) Supplemental Fig. Supplementary material 3 (DOCX 22 kb) Supplemental Fig. Supplementary material 4 (DOCX 16 kb)Supplementary material 5 (DOCX 24 kb)