Impact of Wnt/β-Catenin Inhibition on Cell Proliferation through CDC25A Downregulation in Soft Tissue Sarcomas

Simple Summary Growing evidence suggests that Wnt signaling may be crucial for tumorigenesis and progression of soft tissue sarcomas (STS). Inhibitors of this pathway are currently in clinical trials or pre-clinical studies in order to validate its utility in different neoplasia. One of this inhibitors, PRI-724, is showing promising results for advanced pancreatic adenocarcinoma or ovarian cancer. We found that PRI-724 is able to suppress cell viability/proliferation and to increase cell death rates of soft tissue sarcomas cells in vitro. CDC25A, a target gene of Wnt signaling pathway, is essential for STS proliferation because its downregulation via siRNA was able to mimic the effect of PRT-724 on cell cycle arrest and evaluation of NCBI/GenBank data confirmed its overexpression in STS patients’ samples. Moreover, in vitro administration of PRI-724 along with standard STS chemotherapeutic drugs improved the efficacy of chemotherapy, suggesting that Wnt inhibition could be a promising new therapeutic strategy in STS. Abstract The Wnt signaling pathway is an important cellular mechanism for regulating differentiation processes as well as cell cycle events, and different inhibitors of this pathway, for example, PRI-724, are showing promising results in clinical trials for treatment of advanced pancreatic adenocarcinoma or ovarian cancer. Growing evidence suggests that Wnt signaling may also be crucial for tumorigenesis and progression of soft tissue sarcomas (STS), a malignant neoplasm with few therapeutic options at an advanced state. Our study with several STS cell lines and primary cultures shows that inhibition of Wnt/β-catenin signaling with PRI-724 is able to suppress cell viability/proliferation and to increase cell death rates. TCF/β-catenin-mediated transcriptional activity is decreased in treated cells, leading to downregulation of its target genes CCND1 and CDC25A. The latter was critical because its downregulation via siRNA was able to mimic the effect of PRI-724 on cell cycle arrest and cell death induction. An evaluation of NCBI/GenBank data confirmed that CDC25A mRNA is elevated in STS patients. Importantly, PRI-724 in combination with standard STS chemotherapeutics doxorubicin or trabectedin enhanced their antitumoral effect in a synergistic manner according to isobolographic analysis, suggesting that Wnt inhibition through PRI-724 could be a beneficial combination regime in patients with advanced STS.


Introduction
Soft tissue sarcomas (STS) are a heterogeneous group of malignant tumors of mesenchymal origin with 50 different histological subtypes. STS are part of the so-called rare cancers, since they have a low incidence on the population, representing only one per cent of all solid malignant neoplasia in adults [1,2]. Surgery with or without radiotherapy and chemotherapy is the first treatment option in patients with localized disease. Unfortunately, recurrence is frequent, reaching up to 60% or higher in high-risk localized populations. Therefore, systematic therapy for metastatic or unresectable patients is needed to offer some disease control [3]. The therapeutic options for inoperable or metastatic STS only offer a limited benefit on the prognosis and patients' survival making the search for new molecular targets and the development of novel therapeutic approaches a still unmatched goal.
In many cancers, including soft tissue sarcomas, the Wnt/β-catenin signaling pathway is found to have aberrant activation [4][5][6]. Its normal function is to regulate the development and homeostasis of many tissues, including those derived from mesenchymal cell lineages. The canonical the Wnt/βcatenin signaling pathway is activated with the binding of extracellular Wnt ligand to Frizzled (FZD) receptors and low-density lipoprotein receptor-related proteins-5/6 (LRP5/6) co-receptors. This triggers an intracellular signaling cascade that inactivates the Axin-APC-GSK3β destruction complex, allowing the accumulation of non-phosphorylated β-catenin in the cytoplasm. Free non-phosphorylated β-catenin then translocates to the nucleus and binds to the T-cell factor/lymphoid enhancer factor-1 (TCF/LEF) family of transcription factors and coactivator complexes, thereby inducing transcription of Wnt target genes [7].
Wnt pathway is frequently dysregulated through a variety of genetic and epigenetic mechanisms, such as loss of function mutations in APC gene, especially in colorectal carcinomas, or point mutations in β-catenin (CTNNB1), which prevent its GSK3-dependent phosphorylation in a variety of tumors [5,6,8].
In the context of sarcomas, Wnt canonical pathway has been shown to be upregulated by other mechanisms [8,9]. It is known that SS18-SSX, the oncogenic fusion protein in synovial sarcoma, drives constitutively active β-catenin signaling allowing tumor formation [8,10,11]. Moreover, an increase in Wnt receptor expression or a loss of expression of secreted inhibitors due to increased β-catenin activity is frequently observed in osteosarcoma. Therefore, the aberrant Wnt/β-catenin signaling disrupts normal bone development [8,12]. On the other hand, we and others have described that the Wnt/β-catenin signaling pathway is constitutively activated in a panel of STS cell lines and tumor-derived cells (liposarcomas, leiomyosarcomas, synovial sarcomas and fibrosarcomas), which confer them a highly proliferative and survival capacity, with CDC25A being a key target gene [11,13,14].
Many efforts have been made over the past decades in order to target the canonical Wnt signaling in cancer. There are multiple Wnt/β-catenin signaling inhibitors, including biological and small molecules, which have shown promising activities in cancer therapy by disrupting the pathway at different points [15][16][17][18][19]. The biological compounds include antibodies, RNA interference molecules and recombinant proteins that target Wnt proteins and extracellular modulators of the pathway [19,20]. Small molecules can be divided into four categories according to their mechanism of action [19]. At present, the Class I types (e.g., PKF118-310, CGP049090, CWP232228) are receiving increasing attention because they block the final step of the whole signaling pathway, i.e., the interaction of β-catenin with TCF. As a result, they more efficiently bypass all aberrant activations of the Wnt signaling pathway caused by any upstream dysregulation [16,18,19,[21][22][23]. Recently, our group showed that PKF118-310 effectively inhibits cell proliferation by inducing apoptosis in a panel of representative STS cell lines and primary cultures with upregulated Wnt/β-catenin signaling. Importantly, PKF118-310 simultaneously combined with doxorubicin, the standard first-line therapy in metastatic STS, increased its antitumoral effect in a synergistic manner [14].
Despite the fact that most of those Wnt inhibitors are in preclinical phases, a few of them, especially OMP-54F28 (Ipafricept) and PRI-724, are showing positive results in clinical trials for cancer treatment [15,16,[21][22][23][24][25][26]. A phase I trial of Ipafricept in combination with chemotherapy in ovarian cancer reported that 35% of patients showed complete response, 47% exhibited a partial response, and 18% had stable disease [27]. PRI-724 belongs to the Class II small molecule Wnt inhibitors, which are antagonists of β-catenin transcriptional coactivators. PRI-724 specifically inhibits CBP (CREB-binding protein)/β-catenin interaction, thereby reducing the recruiting of β-catenin with its coactivator CBP [26,28] (Figure 1a). In a phase Ib study, PRI-724 exhibited modest antitumoral activity when combined with gemcitabine as second-line therapy in patients with advanced pancreatic adenocarcinoma [29]. In addition, a phase II clinical trial is currently underway in which PRI-724 is evaluated in combination with chemotherapy and bevacizumab for the treatment of patients with newly diagnosed metastatic colorectal cancer [30].
Cancers 2020, 12, x 3 of 21 the Wnt signaling pathway caused by any upstream dysregulation [16,18,19,[21][22][23]. Recently, our group showed that PKF118-310 effectively inhibits cell proliferation by inducing apoptosis in a panel of representative STS cell lines and primary cultures with upregulated Wnt/β-catenin signaling. Importantly, PKF118-310 simultaneously combined with doxorubicin, the standard first-line therapy in metastatic STS, increased its antitumoral effect in a synergistic manner [14]. Despite the fact that most of those Wnt inhibitors are in preclinical phases, a few of them, especially OMP-54F28 (Ipafricept) and PRI-724, are showing positive results in clinical trials for cancer treatment [15,16,[21][22][23][24][25][26]. A phase I trial of Ipafricept in combination with chemotherapy in ovarian cancer reported that 35% of patients showed complete response, 47% exhibited a partial response, and 18% had stable disease [27]. PRI-724 belongs to the Class II small molecule Wnt inhibitors, which are antagonists of β-catenin transcriptional coactivators. PRI-724 specifically inhibits CBP (CREB-binding protein)/β-catenin interaction, thereby reducing the recruiting of β-catenin with its coactivator CBP [26,28] (Figure 1a). In a phase Ib study, PRI-724 exhibited modest antitumoral activity when combined with gemcitabine as second-line therapy in patients with advanced pancreatic adenocarcinoma [29]. In addition, a phase II clinical trial is currently underway in which PRI-724 is evaluated in combination with chemotherapy and bevacizumab for the treatment of patients with newly diagnosed metastatic colorectal cancer [30].  Normalized CI values at 48 h. (d) STS cells were treated with PRI-724 (2.5 µM) every 96 h. After 9 days, colonies reached saturation and quantification of colony-forming ability was determined by counting the colonies using the ImageJ software (ImageJ 1.51j8, Wayne Rasband, NIH, USA). The panel shows representative results of at least three independent experiments. (e) Cells were treated with PRI-724 (10 µM) for 48 and 72 h, fixed with ethanol, stained with propidium iodide, and DNA content was determined by flow cytometry. Columns show the extent of cell death induction with the inhibitor represented as fold increase at 48 and 72 h. Each column represents mean ± SEM of three independent experiments. Histograms of vehicle-treated (control) and PRI-724-treated cells (10 µM) for 72 h are shown. The fluorescence values used to calculate the peaks corresponding to the sub-G1 phase (cell death) are indicated on each histogram. * p < 0.05, ** p < 0.01 and *** p < 0.001 compared with vehicle-treated cells.
In the present work, we examined the antitumor efficacy of PRI-724 in a panel of prevalent STS cell lines and primary cultures. Our findings show that inhibition of CBP/β-catenin interaction suppresses the proliferation of a wide range of STS cell lines and patient-derived cells and promotes cell death, at least in part, via CDC25A downregulation. Furthermore, the combination of PRI-724 with conventional chemotherapeutic agents used in the treatment of metastatic STS, such as doxorubicin and trabectedin, increased their antitumoral effects in a synergistic manner, suggesting that CBP/β-catenin inhibitors, such as PRI-724, could also be a promising therapeutic option for STS patients, and should be further evaluated.

Inhibition of CBP/β-Catenin Interaction Suppresses Cell Viability and Colony Formation of STS Cell Lines
To test the effect of CBP/β-catenin disruption on cell proliferation, STS cell lines and primary cultures representing different sarcoma histologies and mutational background were exposed to increasing concentrations of PRI-724 (0.1-50 µM) for 24, 48 and 72 h (Table S1). As shown in Figure 1b, after 48 h of treatment cell viability was reduced below 50% in 77.8% (7/9) of the cell lines tested. 93T449, AW, HT-1080 and CP0024 cell lines were more sensitive to treatment, with IC 50 values ranging from 5.18 to 9.77 µM, compared with SW982, SW872 and SK-UT-1, with IC 50 values ranging from 20.13 to 38.86 µM (Table S1 shows IC 50 values for 48 h treatment). Two cell lines, the commercial fibrosarcoma cell line SW684 and the primary liposarcoma culture ICP020, were more resistant to this Wnt inhibitor, since it was not able to achieve a 50% inhibition of cell viability at any of the concentrations studied. In addition, similar results were obtained when cell growth was continuously monitored in liposarcoma AW, 93T449 and leiomyosarcoma SK-UT-1 cells under PRI-724 treatment by using the xCELLigence system (Figure 1c). To assess whether disruption of CBP/β-catenin interaction altered more phenotypic properties of tumoral cells, colony formation assay was performed in two cell lines with different sensitivity to PRI-724. After 9 days of treatment, PRI-724 (2.5 µM) significantly decreased the colony-forming ability of leiomyosarcoma SK-UT-1 and liposarcoma 93T449 cells. As shown in Figure 1d, the reduction effect of PRI-724 was 52.04 ± 4.12% in SK-UT-1 cells and 99.59 ± 0.22% in 93T449 cells, the latter being the most sensitive cell line to this Wnt inhibitor according to the proliferation assay. Taken together, PRI-724 was able to reduce viability as well as proliferation of STS cells, and no correlation between antitumoral effects and histology of cell lines could be established.

Inhibition of CBP/β-Catenin Interaction Promotes Cell Death of STS Cell Lines
Cell cycle profiling was performed on STS cells treated with PRI-724 by flow cytometry (Figure 1e and Figure S1). Treatment with PRI-724 for 48 and 72 h increased the sub-G1 phase of STS cells in a time-dependent manner, being statistically significant in only two of the five cell lines tested. At 10 µM, CBP/β-catenin inhibitor provoked in fibrosarcoma HT-1080 and leiomyosarcoma SK-UT-1 the highest effect on the sub-G1 phase, thus raising cell death in vehicle-treated cells after 72 h 8-and 4-fold, respectively. This effect of CBP/β-catenin inhibition was more moderate in the rest of the cell lines tested, increasing cell death 2-fold after 72 h of treatment. Taken together, these results indicate that PRI-724 exhibits antiproliferative activity, at least in part, by triggering cell death in STS cell lines. Additional studies performed with Annexin V/PI and assessment of the cleavage of PARP by immunoblot, as well as measurement of caspase 3/7 activities ( Figure S2) ruled out that the involved mechanism of cell death provoked by PRI-724 treatment was apoptosis.

Inhibition of CBP/β-Catenin Interaction Reduces TCF/β-Catenin-Mediated Transcriptional Activity, but Not β-Catenin Subcellular Localization
To further evaluate the effects of PRI-724 treatment on STS, we chose representative cell lines covering a wide range of sensitivity to this inhibitor, i.e., 93T449, AW, HT-1080, CP0024 and SK-UT-1 (Table S1). As we previously reported, STS cell lines have basal TCF/β-catenin mediated transcriptional activity [14] being similar to that of human colon cancer cells, whose Wnt pathway is known to be strongly activated. To evaluate the effect of CBP/β-catenin disruption on β-catenin transcriptional activity, TCF reporter activity was evaluated in STS cells treated with PRI-724 by TOP/FOPflash luciferase reporter assay. As shown in Figure 2a, PRI-724 treatment for 24 h led to a reduction of TCF/β-catenin reporter activity in fibrosarcoma HT-1080 cells and 93T449 and AW liposarcoma cells. The inhibitory effect of PRI-724 on TCF/β-catenin reporter activity was rather small in HT-1080 and 93T449 cells (20% of decrease), while it inhibited by 50% the β-catenin transcriptional activity in AW cells. Several studies have shown that compounds that disrupt the interaction between β-catenin and TCF/LEF transcription factors prevent nuclear accumulation of β-catenin [14,31]. To investigate whether the disruption of β-catenin interaction with CBP had a similar effect on β-catenin localization, SK-UT-1, HT-1080 and 93T449 with strong nuclear β-catenin staining, CP0024 with moderate nuclear staining of β-catenin and AW with weak β-catenin nuclear staining were treated with the PRI-724 at 10 µM for 48 h. After treatment, intracellular location of β-catenin was determined by nuclear/cytoplasmic fractionation and immunofluorescence showing that disruption of CBP/β-catenin interaction was not able to reduce nuclear β-catenin levels in the STS cell lines studied, instead a mild increase in nuclear β-catenin was detected by immunofluorescence in 93T449 cells (   for 48 h were subjected to subcellular fractionation. Upper panel shows the immunoreactive bands of β-catenin in cytoplasmic and nuclear fractions in a representative immunoblot of at least three independent experiments. α-Tubulin and PARP were used as a cytoplasmic and nuclear marker, respectively. Intensity quantification of the bands are shown in the bottom graphs. (c) STS cells were treated with PRI-724 (10 µM) for 48 h and fixed with methanol: acetone (1:1). β-catenin was visualized with the primary antibody β-catenin (D10A8) [(XP Rabbit mAb #8480, Cell Signaling Technology, Beverly, MA, USA)] followed by the addition of the secondary antibody Alexa Fluor 488 (Goat a-R A11008, Invitrogen S.A, Barcelona, Spain). DAPI was added to visualize the nuclei. Panel shows representative images taken with confocal microscope, ZEISS LSM 710 (Carl Zeiss Microscope GmbH, Jena, Germany), with a 40× objective and power of 512 × 512 pixels of at least two independent experiments performed in duplicate.

Inhibition of CBP/β-Catenin Interaction Reduces TCF/β-Catenin-Dependent Expression of Target Genes
To further investigate the effect of CBP/β-catenin disruption in STS cells, the expression of Wnt downstream signaling targets in STS treated with PRI-724 was assessed by RT-PCR. CCND1 is a well-known cell cycle regulator and CDC25A has been described as a major regulator of cell cycle progression in STS [13,14], with both of them being TCF/β-catenin-dependent target genes. Previous results showed that CDC25A gene levels in STS cells are higher than those of hMSCs, a reference cell line with low levels of endogenous Wnt signaling [14]. Interestingly, in APC-mutated-SK-UT-1 cells, endogenous CDC25A levels were higher than those found in two colorectal cancer cell lines with strong intrinsic Wnt signaling activity used as positive controls. Treatment with PRI-274 revealed a progressive time-dependent downregulation of CDC25A gene in all cell lines tested, whereas CCND1 gene expression decreased only during the first 48 h of treatment, recovering its expression afterwards ( Figure 3a and Figure S5). As expected, the downregulation effect of PRI-724 on CDC25A expression was less pronounced in SK-UT-1 cells (reduction of 20%, 44% and 51% after 24, 48 and 72 h, respectively) (Table S1). To evaluate CDC25A protein levels, immunofluorescence analysis was performed due to inconsistent data obtained by immunoblot with different CDC25A antibodies. In line with the results of mRNA expression, they confirmed a significant reduction of CDC25A protein levels in STS cells after 48 h of PRI-724 treatment (10 µM). CDC25A protein was detected in cytoplasmic as well as nuclear compartments in the analyzed STS cells (Figure 3b,c). Liposarcoma 93T449 and leiomyosarcoma CP0024 cells, which were more sensitive to disruption of CBP/β-catenin interaction (Table S1), had higher levels of CDC25A protein in the cytoplasm than in the nuclei. Conversely, in leiomyosarcoma SK-UT-1 cells, one of the most resistant cell lines to this Wnt inhibitor (Table S1), the CDC25A expression was greater in the nucleus than in the cytoplasm. In agreement with their sensibility to PRI-724, the greatest inhibitory effect of this compound on CDC25A protein levels was observed in liposarcoma 93T449 cells (Figure 3b,c) when compared with leiomyosarcoma CP0024 or SK-UT-1 cells. Thus, PRI-724 exerted an inhibitory effect on expression of CDC25A being in line with the decrease of β-catenin transcriptional activity shown with the reporter assay.  and incubated with the primary antibody CDC25A (M-191) (sc-7157, Santa Cruz) followed by the addition of the secondary antibody Alexa Fluor 488 (Goat a-R A11008, Invitrogen). DAPI was added to visualize the nuclei. Panel shows representative images taken with confocal microscope, ZEISS LSM 710 (Carl Zeiss Microscope GmbH, Jena, Germany), with a 40× objective and power of 512 × 512 pixels of at least two independent experiments performed in duplicate. (c) Nuclear and cytoplasmic CDC25A protein levels were quantified measuring the integrated density by ImageJ software. Each column represents mean ± SEM of two independent experiments performed in duplicate. * p < 0.05, ** p < 0.01 and *** p < 0.001 compared with vehicle-treated cells.

CDC25A Is a Major Regulator of STS Cell Proliferation Mediated by β-Catenin Transcriptional Activity
To elucidate the role of CDC25A in STS proliferation we used those STS cells lines with a higher expression of CDC25A, SK-UT-1, HT-1080 and CP0024, as determined in a previous work [14]. A small interfering RNA (siRNA) to specifically downregulate CDC25A gene expression was used ( Figures S6 and S7). Fibrosarcoma HT-1080 and leiomyosarcoma CP0024 and SK-UT-1 cells were transiently transfected with CDC25A siRNA, and cell viability was continuously monitored using the xCELLigence system. As shown in Figure 4a, the inhibitory effect of PRI-724 at, 30 µM was evaluated, as this corresponded to the IC 50 concentration regarding proliferation inhibition of the most resistant cell line SK-UT-1. In fact, cell proliferation of CP0024 decreased by 66%, which was very similar to the 55% reduction induced by the downregulation (silencing) of CDC25A expression. However, CDC25A downregulation caused a moderate reduction of cell proliferation in SK-UT-1 and HT-1080 cell lines (22% and 49%, respectively) compared to that caused by CBP/β-catenin inhibition (70% and 89%, respectively). Interestingly, exogenous expression of CDC25A by co-transfecting cells with pCMV-CDC25A 24 h after siRNA transfection rescued cell index values after 48 h in all cells tested (Figure 4a white column). As CDC25A is an important regulator of cell cycle progression, we performed cell cycle analysis by flow cytometry to determinate the effect of CDC25A gene downregulation in the different phases of the cell cycle. As seen in Figure 4b, CDC25A downregulation inhibited cell proliferation in HT-1080 and SK-UT-1 cells by impairing cell cycle progression through the G1 checkpoint or by cell death induction. These results show that CDC25A is a relevant mediator of proliferation in STS and its downregulation leads to a decrease of cell growth as treatment with PRI-724.  To further address the clinical relevance of CDC25A in STS, its mRNA expression levels were examined in a broad series of patient samples (n = 149) gathered from a public TCGA data set [32] including 26 leiomyosarcomas, 89 liposarcomas, 34 myxofibrosarcomas and 9 control adipose tissue samples. As shown in Figure 5a CDC25A mRNA expression was similar between STS subtypes and higher when compared with control normal fat tissue samples. In conclusion, as shown by our results and public data, CDC25A is overexpressed in STS samples and seems to be involved in tumor To further address the clinical relevance of CDC25A in STS, its mRNA expression levels were examined in a broad series of patient samples (n = 149) gathered from a public TCGA data set [32] including 26 leiomyosarcomas, 89 liposarcomas, 34 myxofibrosarcomas and 9 control adipose tissue samples. As shown in Figure 5a CDC25A mRNA expression was similar between STS subtypes and higher when compared with control normal fat tissue samples. In conclusion, as shown by our results and public data, CDC25A is overexpressed in STS samples and seems to be involved in tumor proliferation. To further investigate this question, we analyzed the tumor same sample set by using Gene Set Enrichment Analysis (GSEA) in order to identify curated gene signatures whose expression changed significantly in CDC25A high STS patient samples. For that purpose, samples were separated in two groups, CDC25A high and CDC25A low, based on CDC25A first and last quartile of expression. As shown in Figure 5b,c and Table S3, gene signatures related with cell cycle regulation and DNA repair were significant enriched in the CDC25A high subgroup. Single-sample GSEA (ssGSEA) enrichment score represents the degree to which the genes in a particular gene set are coordinately up-or down-regulated within a sample. This score characterizes cell state in terms of the activity levels of biological processes and pathways rather than through the expression levels of individual genes. The ssGSEA performed on the entire sample revealed an up-down regulation of those pathways enriched in CDC25A high tumor subgroup. As shown in Figure 5d, samples nicely separate in two clusters (tumor and normal tissue) accompanied by a concordance with CDC25A expression levels. The samples with highest CDC25A levels showed higher scores in cell cycle and DNA repair related processes.

Inhibition of CBP/β-Catenin Interaction Enhances Antitumoral Effect of Standard Chemotherapeutic Agents in STS Cells
Combination of the CBP/β-catenin inhibitor PRI-724 with trabectedin or doxorubicin at different fixed dose ratio combinations, led to an enhanced reduction of STS cell viability, achieving synergistic effects (CI < 1) in most of cell lines tested after 48 h of treatment as seen in Table S2 and Figure 6. One of the combinations that exerted an antagonist effect was with PRI-724 and trabectedin in APC-mutated SK-UT-1 cell line.

Inhibition of CBP/β-Catenin Interaction Enhances Antitumoral Effect of Standard Chemotherapeutic Agents in STS Cells
Combination of the CBP/β-catenin inhibitor PRI-724 with trabectedin or doxorubicin at different fixed dose ratio combinations, led to an enhanced reduction of STS cell viability, achieving synergistic effects (CI < 1) in most of cell lines tested after 48 h of treatment as seen in Table S2 and Figure 6. One of the combinations that exerted an antagonist effect was with PRI-724 and trabectedin in APC-mutated SK-UT-1 cell line.

Discussion
In a previous work, we demonstrated that the Wnt signaling pathway is constitutively activated in a broad range of STS cell lines and primary cultures independently of the sarcoma subtype [14]. In this study, we explore the antitumoral effects of Wnt pathway inhibition on STS cell lines and patient-derived primary cultures using the small molecule PRI-724, which blocks CBP/β-catenin interaction and is currently in clinical trials for different cancer types but not for STS.
PRI-724 was able to strongly reduce cell proliferation in vitro by inducing nonapoptotic cell death or by impairing cell cycle progression through the G1 checkpoint ( Figure 1). This effect was less pronounced in cell lines with mutations in the APC gene (SK-UT-1 and SW684), and this is probably related to the elevated activation of the pathway as a consequence of this mutation, which is not completely reverted by PRI-724. Our results are in agreement with Ma and colleagues, who found that ICG-001, a first generation of CBP/β-catenin inhibitor, is able to reduce viability in colorectal carcinoma cells SW480 [33]. Moreover, as pointed by Emami and colleagues, who found the same effects of ICG-001 on cell viability using SW480 and HCT116 cells [34], most probably PRI-724 would be cytostatic at lower concentrations and cytotoxic, increasing caspase activity, at higher concentrations. Accordingly, in our study, caspase activity decreased upon treatment with PRI-724 (10 µM), which would be consistent with an inability to induce apoptotic processes due to cell cycle arrest.
At the molecular level, PRI-724 treatment caused attenuation of TCF/β-catenin mediated transcriptional activity in STS cells, and it was not accompanied by a change of the subcellular localization of β-catenin (Figure 2), whereas disruption of TCF/β-catenin interaction by PKF118-310 provokes destabilization of β-catenin and reduction of its nuclear localization as our previous study indicated [14,31]. However, it has to be taken into account that both compounds interact with the Wnt pathway through different molecular mechanisms. Once in the nucleus, β-catenin must recruit one of the two transcriptional coactivators CBP (cAMP-response-element-binding protein (CREB)-binding protein) or p300 (E1A-binding protein) to induce transcription of Wnt target genes. For several years, the functions of these two coactivators have been described as redundant due to their high degree of homology. Despite this, emerging studies [35][36][37][38] indicate that CBP and p300 are not interchangeable, and it has been reported that each of these two coactivators is responsible for a distinct transcriptional program guiding the cell to either proliferate or retain potency in the case of CBP or to initiate differentiation in case of p300 [33,37,39,40]. In this context, PKF118-310 blocks the binding of β-catenin to the TCF/LEF complex, thereby decreasing the amount of bound β-catenin in the nucleus, whereas PRI-724 selectively blocks the CBP/β-catenin interaction without interfering with the interaction of β-catenin with p300 [34]. This fact would explain that, although the inhibitory effect of PRI-724 on total TCF/β-catenin-mediated transcriptional activity was mild and not statistically significant in HT-1080 and 93T449 cells (Figure 2a), PRI-724 strongly reduced their proliferation (Figure 1; Table S1) most probably by inhibiting the expression of TCF/CBP/β-catenin dependent target genes which are related to cell proliferation, as is CDC25A (Figure 3). As PRI-724 leaves the TCF/LEF binding site unaltered, β-catenin can still bind to TCF/LEF and recruit transcriptional cofactors other than CBP, thereby avoiding a decrease of nuclear β-catenin localization. Such a differential regulation of TCF/LEF activity has also been observed in the case of the formerly mentioned CBP/β-catenin antagonist ICG-001. This compound induces apoptosis through downregulation of the protein survivin, whose expression is as well only increased by the transcriptional coactivator CBP but not p300 [33]. Altogether, our results point to a cellular model with a nonredundant function of CBP and p300, which seem to be able to trigger a different set of transcriptional routes.
As a result of the attenuation of CBP/β-catenin mediated transcriptional activity, PRI-724 significantly reduced the expression of TCF/β-catenin dependent target genes related with cell proliferation such as CCND1, a cell cycle regulator well-known by its role in tumorigenesis [41,42], and CDC25A. The reduction of CDC25A expression at mRNA and protein level is of special clinical relevance, since CDC25A has been described as a major cell proliferation mediator in STS, both in vitro and in vivo [13]. We observed that, in general terms, CDC25A was more strongly downregulated by PRI-724 than CCND1 (Figure 3a). However, in SK-UT-1 cells, which were rather resistant to the antiproliferative effect of PRI-724, downregulation of CDC25A levels were also rather weak. In contrast, in 93T449 cells a drastic downregulation of CDC25A was paralleled by an only weak downregulation of CCND1, and these cells were highly sensitive to PRI-724. Accordingly, depletion of CDC25A by a specific siRNA significantly decreased STS proliferation through cell death induction or G1 arrest (Figure 4). Additionally, in this work CDC25A protein was found in both nuclear and cytoplasmic compartments in all analyzed STS cells and, interestingly, those STS cell lines in which CBP/β-catenin inhibition by PRI-724 most effectively inhibited cell proliferation were the ones with higher levels of CDC25A in cytoplasm (Figure 3b). In fact, the extent of CDC25A protein reduction in cytoplasm after treatment with PRI-724 determined the extent of sensitivity to this Wnt inhibitor (e.g., 93T449 cells) (Figure 3b). These results are in line with previously published data indicating that cytoplasmic localization of CDC25A is related to anti-apoptotic and pro-survival effects on ovarian and skin tumor cells [43,44], and therefore leads to a dependency of tumor progression on CDC25A. Taken together, the fact that inhibition of CBP/β-catenin interaction by PRI-724 suppressed cell proliferation to a similar extent to that provoked by CDC25A downregulation further strengthens the role of CDC25A as a major regulator of STS cell proliferation and its importance as pivotal molecular target of this compound.
High CDC25A mRNA levels have been reported previously by us and others in a wide range of representative Wnt-activated STS cell lines [13,14]. In parallel, Bertucci and colleagues reported a positive correlation between Wnt activation and expression of CDC25A mRNA in a large series of sarcoma patient samples [45]. The results of available public data analysis, comparing STS patient samples with normal tissues and evaluating gene signatures and pathways enriched in CDC25A high STS patient samples confirmed these results ( Figure 5) and highlight that this gene target is actually of clinical relevance in STS independently of STS subtype.
Although the Wnt signaling pathway was discovered over 30 years ago, it was not before the last decade that drug development focused on this pathway as a new therapeutic approach in oncology. However, clinical Wnt modulator candidates must take into account the different roles of the pathway, which maintain the fine balance between cell proliferation versus differentiation. Therefore, only few specific therapeutic agents targeting this pathway have entered clinical trials until today. On the other hand, a growing body of evidence indicates that a combination therapy with such compounds may be more effective than monotherapy in most malignancies [26,[28][29][30]. For this reason, several clinical trials are currently testing PRI-724 in combination with conventional chemotherapeutics. Our work demonstrates that combination of PRI-724 with trabectedin or doxorubicin, both standard chemotherapeutic agents in advanced STS, may also have a beneficial clinical effect. In fact, the combination of PRI-724 with either of these drugs increased their antitumor activity in a synergistic manner in most of the STS cells studied. Interestingly, it has been reported that CBP/β-catenin inhibitors are able to improve the chemotherapy-associated toxicity in preclinical models [46].
Wnt inhibitor, PRI-724, was purchased from Selleck Chemicals LLC (Houston, TX, USA). Doxorubicin was kindly provided by the Pharmacology Department of Son Espases University Hospital. Trabectedin was provided by Pharmamar (Madrid, Spain).

Cell Viability Assay (MTT)
The methylthiazoletetrazolium (MTT) method was used to measure cell viability using the CellTiter 96 ® AQueous One Solution (Promega, Madison, WI, USA) following the manufacturer's instructions. Briefly, 5000 cells were seeded in 96-well plates for 24 h and exposed to increasing concentrations of the Wnt inhibitor for 24, 48 and 72 h followed by addition of CellTiter reagent. Absorbance at 490 nm was detected using a multi-well scanning spectrophotometer (Synergy H1 microplate reader, Bio-tek, Winooski, VT, USA). The mean percentage of cell viability relative to vehicle-treated cells was estimated, the compound concentration resulting in 50% inhibition of cell viability (IC50) was determined using GraphPad software (GraphPad Software Inc., San Diego, CA, USA).

Real-Time Cell Analysis (RTCA)
Cells were seeded in 96-well E-plates with integral sensor electrode arrays (ACEA Biosciences, San Diego, CA, USA). After 24 h, the cells were treated with different concentrations of the Wnt inhibitor and cell proliferation was monitored noninvasively, in real time and label-free using the xCELLigence Real-Time Cell Analyzer (ACEA Biosciences) for up to 96 h. The 96-well E-plates used with the xCELLigence system have gold electrodes on their bottom surface, serving as sensors of alternating current modified by the number of adherent cells and cell status (including cell viability, morphology, and adhesion). A dimensionless parameter termed cell index (CI) was derived as a relative change in measured electrical impedance to represent cell status. CI normalized to the time of medium switch/treatment was presented in xCELLigence recordings.

Colony-Forming Assay
Cellular ability to grow and form colonies was evaluated by clonogenic assay. Briefly, 500 cells were seeded in 6-well plates and treated with PRI-724 (2.5 µM) for 9 days (medium was changed every 96 h). Once colonies reached saturation cells were fixed with cold methanol, washed with PBS and stained with crystal violet 1% (Sigma-Aldrich, Madrid, Spain) for 20 min. Cellular colony-forming ability was quantified using the ImageJ software.

Cell Cycle Analysis
Flow cytometry was performed to analyze the effect of Wnt inhibitor on cell cycle. Briefly, 30,000 cells were seeded in 24-well plates. After 24,48

Western Blotting and Antibodies
Cells were seeded in 6-well plates, and after 48 h of treatment with vehicle or Wnt inhibitor, nuclear and cytoplasmic fractions were obtained using Nuclear Extract kit (Active Motif) and following the manufacturer's instructions. The antibodies used were the following: β-catenin (#8480, Cell Signaling Technology, 1:1000 dilution), PARP (#9542S, Cell Signaling Technology, 1:1000 dilution) and α-tubulin (#T9026, Sigma-Aldrich, 1:1000 dilution). The secondary antibodies were: Donkey anti-Rabbit (E 365D5) and Donkey anti-Mouse (E 510KC) (LI-COR, 1:10,000 dilution). Western blotting was performed, subjecting 10 µg of each lysed fraction to SDS-PAGE, transferring onto a PVDF membrane (Immobilon-FL, Merck Millipore, Cork, Ireland) and incubating with the indicated antibodies. Immunoreactivity intensity of the bands was analyzed with the Odyssey imaging system from LI-COR and quantified by Quantity One 1-D Analysis Software (BIO-RAD, Hercules, CA, USA).

Immunofluorescence
Cells were grown in a Lab-Tek II Chamber Slide (Lab-Tek) chamber, and 48 h after treatment with vehicle or Wnt inhibitor cells were washed with PBS plus 0.2% BSA and fixed with methanol: acetone (1:1), blocked with a 5% BSA solution, incubated with the primary antibody β-Catenin (D10A8) (#8480, Cell Signaling Technology, 1:250 dilution), and CDC25A (M-191) (sc-7157, Santa Cruz, 1:250 dilution) and with the secondary antibody (Alexa Fluor 488 Goat a-R A11008, Invitrogen, 1:150 dilution). DAPI (Fisher Scientific) was used to visualize the nuclei. Five images per condition of each individual experiment were taken using the confocal microscope, ZEISS LSM 710, with a 40X objective and power of 512 × 512 pixels. The ImageJ software was used to quantify β-catenin and CDC25A staining intensity. Briefly, using the DAPI channel, the nuclei of the cells in each field of view were selected manually and the software automatically measured the integrated density of selected areas.

TCF Reporter Assay, Small Interfering RNA (siRNAs), Plasmids and Transfections
Transient transfection was performed using the Lipofectamine Plus (Invitrogen) method. TCF reporter system plasmids (pTOPFLASH and pFOPFLASH) and the empty vector pcDNA3.1 were kindly donated by Dr. Gabriel Capellà, expression plasmid pCMV-CDC25A (HG11291-M-N) was purchased from Abyntek Biopharma, S.L (Bizkaia, Spain). Luciferase reporter assay was performed using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer's instructions. Luciferase activity was quantified by multi-well scanning spectrophotometer (Synergy H1 microplate reader, Bio-tek) 24 h after transfection and treatment with Wnt inhibitor. Firefly luciferase activity was normalized to the corresponding Renilla luciferase activity.
Cells seeded with added transfection complex were incubated for 24 h or 72 h at 37 • C in a humidified incubator containing 5% CO 2 .

Data Collection and Analysis
Raw data from Barretina et al. 2010 public data set (GSE21122) was collected from the National Center for Biotechnology Information (NCBI)/GenBank GEO web site. After quality control, raw data was normalized by RMA [51] in R using Bioconductor and related packages.
Gene Set Enrichment Analysis (GSEA) was performed based on CDC25A first and last quartile of expression in STS tumor samples using the GSEA 4.0.3 software (www.broadinstitute.org/gsea). Gene sets were obtainged from H (Hallmark gene sets) and C2 (Curated gene sets) collections from MSigDB (Molecular Signature Database) [52,53]. Significant enriched signatures were identified using FDR q-value < 0.25 and nominal p-value < 0.05.

Statistical Analysis
The antiproliferative effect of the combination of Wnt inhibitor with doxorubicin and trabectedin was assessed by Loewe's isobolographic analysis (1927,1928,1953), which considered three types of interactions: additivity (CI = 1), synergy (CI < 1) and antagonism (CI > 1). Each combination was evaluated by cell viability assay to determine the 50% inhibition of cell viability (IC 50 ) value. By plotting the IC 50 value of doxorubicin or trabectedin on the Y-axis and the IC 50 value of PRI-724 on the X-axis combination isobolograms were obtained. The line connecting these two points it is known as line of additivity.
Results are expressed as mean ± SEM from n independent experiments performed in duplicate or triplicate. Statistical evaluations were assessed by GraphPadPrism, GraphPad Software, Inc. To detect the difference of quantitative values between the different groups, an ANOVA (parametric), along with a Bonferroni test for multiple comparisons, or Kruskal-Wallis (non-parametric) were used. Differences were considered statistically significant at p < 0.05 and are indicated by: *** p < 0.001, ** p < 0.01, and * p < 0.05.

Conclusions
In summary, our study provides evidence that the Wnt inhibitor PRI-724 reduces STS cell proliferation in vitro by inducing cell death or G1 arrest, at least in part, through CDC25A downregulation. Moreover, these results are highly translational because they demonstrate that inhibition of CDC25A, which is highly expressed in a broad series of patient samples, could represent a potential new therapeutic drug target (Figure 7). Moreover, concomitant treatment with the Wnt inhibitor PRI-724 and trabectedin or doxorubicin is able to enhance the chemotherapeutic effect of standard STS drugs. This new strategy could benefit sarcoma patients with molecular alterations in the Wnt signaling pathway, especially if CDC25A deregulation is observed.  Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1, Figure S1: Inhibition of CBP/β-catenin interaction promotes cell death in STS cells, Figure S2: Determination of cellular apoptosis upon treatment with PRI-724, Figure S3: Inhibition of CBP/β-catenin interaction does not reduce β-catenin subcellular localization, Figure S4: Inhibition of CBP/β-catenin interaction does not alter β-catenin nuclear levels in STS cells, Figure S5: Inhibition of CBP/β-catenin interaction decreases the expression of TCF/β-catenin dependent target genes, Figure S6: CDC25A mRNA expression levels in STS cells after transfection of CDC25A siRNA for 48, 72 and 96 h, Figure S7: CDC25A mRNA expression levels in STS cells,    Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6694/12/9/2556/s1, Figure S1: Inhibition of CBP/β-catenin interaction promotes cell death in STS cells, Figure S2: Determination of cellular apoptosis upon treatment with PRI-724, Figure S3: Inhibition of CBP/β-catenin interaction does not reduce β-catenin subcellular localization, Figure S4: Inhibition of CBP/β-catenin interaction does not alter β-catenin nuclear levels in STS cells, Figure S5: Inhibition of CBP/β-catenin interaction decreases the expression of TCF/β-catenin dependent target genes, Figure S6: CDC25A mRNA expression levels in STS cells after transfection of CDC25A siRNA for 48, 72 and 96 h, Figure S7: CDC25A mRNA expression levels in STS cells,