Effect of Dickkopf-1 (Dkk-1) and SP600125, a JNK Inhibitor, on Wnt Signaling in Canine Prostate Cancer Growth and Bone Metastases

Human Dickkopf-1 (Dkk-1) upregulates a noncanonical Wnt/JNK pathway, resulting in osteoclast stimulation, cell proliferation, and epithelial-to-mesenchymal transition (EMT) of cancer cells. Ace-1-Dkk-1, a canine prostate cancer (PCa) cell line overexpressing Dkk-1, was used to investigate Wnt signaling pathways in PCa tumor growth. SP600125, a JNK inhibitor, was used to examine whether it would decrease tumor growth and bone tumor phenotype in canine PCa cells in vitro and in vivo. Ace-1-VectorYFP-Luc and Ace-1-Dkk-1YFP-Luc cells were transplanted subcutaneously, while Ace-1-Dkk-1YFP-Luc was transplanted intratibially into nude mice. The effects of Dkk-1 and SP600125 on cell proliferation, in vivo tumor growth, and bone tumor phenotype were investigated. The mRNA expression levels of Wnt/JNK-related genes were measured using RT-qPCR. Dkk-1 significantly increased the mRNA expression of Wnt/JNK-signaling-related genes. SP600125 significantly upregulated the mRNA expression of osteoblast differentiation genes and downregulated osteoclastic-bone-lysis-related genes in vitro. SP600125 significantly decreased tumor volume and induced spindle-shaped tumor cells in vivo. Mice bearing intratibial tumors had increased radiographic density of the intramedullary new bone, large foci of osteolysis, and increased cortical lysis with abundant periosteal new bone formation. Finally, SP600125 has the potential to serve as an alternative adjuvant therapy in some early-stage PCa patients, especially those with high Dkk-1 expression.


Introduction
Prostate cancer (PCa) is the second most common cancer in men, with 248,530 newly diagnosed cases and 34,130 deaths in 2021 in the United States [1]. The incidence of this cancer increases with age. The majority of patients with advanced PCa die from metastatic disease, where bone is the most common metastatic site. PCa bone metastases are predominantly osteoblastic (new bone formation) [2]. Metastasis to bone is associated with nerve compression, severe pain, and decreased quality of life [3]. Even though studies have shown connections between bone metastasis and the bone microenvironment in prostate cancer, there are many details that need to be clarified.
previously developed in our laboratory, was used to elucidate the molecular role of Wnt signaling and Dkk-1 in canine PCa growth and bone tumor phenotype and growth.

Dkk-1 ELISA
Ace-1-Vector and Ace-1-Dkk-1 cells were seeded in 6-well culture plates in triplicate at a density of 500,000 cells per well and cultured in DMEM/F12 with 10% FBS and 1% penicillin/streptomycin. After 24 h, medium was collected, and Dkk-1 concentrations were measured using the DuoSet Human Dkk-1 ELISA Kit (R&D Systems, Minneapolis, MN, USA) [26]. The lowest standard of the assay was 62.5 pg/mL.

Immunohistochemistry
Ace-1-Vector and Ace-1-Dkk-1 were grown to 70% confluency, trypsinized, and centrifuged at 1000 RPM for 5 min. The cell pellet was then fixed in 10% neutral-buffered formalin for 24 h and embedded in paraffin. The specimens were sectioned (5 µm). Then they were deparaffinized, rehydrated, and incubated in antigen retrieval solution (Dako, Carpinteria, CA, USA) at 90 • C for 30 min before cooling down at room temperature for 15 min. The endogenous peroxidase was blocked using 3% hydrogen peroxide and serum-free protein block (Dako). Samples were incubated at room temperature for 1 h with monoclonal mouse anti-β-catenin (BD Pharmingen, San Jose, CA, USA, Cat. No. 610153, 1:250) in phosphate-buffered saline (PBS). Anti-mouse monoclonal (Vector Laboratories, Burlingame, CA, USA, Cat. No. BA-9200, 1:200) in PBS was applied as the secondary antibody. Slides were stained with a Vector ABC Elite complex (Dako) for 30 min, diaminobenzidine tetrahydrochloride (Dako) for 5 min, and Mayer's hematoxylin for 1 min. Image Pro Plus 9.0 (Media Cybernetics, Rockville, MD, USA) was used to perform batch analysis and quantify total cell staining (reported as optical density, OD). The values from 5 fields of approximately 700 cells were averaged.

Activator Protein-1 (AP-1) Reporter Transfection
To investigate the activity of Wnt/JNK signaling, 10,000 Ace-1-Vector or Ace-1-Dkk-1 cells in 96-well plates in triplicate were transiently transfected with 0.1 µg of either an inducible AP-1 reporter construct, a negative control, or a positive control purchased from SABiosciences (Qiagen, Germantown, MD, USA, Cat. No. CCS-011L). Transient transfection was accomplished using a 0.5 µL lipofectamine 2000 transfection reagent (Invitrogen, Waltham, MA, USA) and Opti-MEM media (Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions. Cells were cultured for 24 h in DMEM/F12 and 0.1% bovine serum albumin (BSA) alone or with 20 µM of SP600125 (Santa Cruz Biotechnology, Dallas, TX, USA), a selective JNK inhibitor. A constitutively active Renilla luciferase was used as an internal control to normalize for transfection efficiency. The negative control was used to normalize for background luciferase reporter activity.

Cell Proliferation Assay
Ace-1-Vector and Ace-1-Dkk-1 cells were seeded in a 6-well plate in triplicate at a density of 200,000 cells per well. Cells were cultured in DMEM/F12 with 0.1% BSA and incubated at 37 • C and in 5% CO 2 . The cells were collected using 0.25% trypsin at 24, 48, and 72 h after the initial plating and counted with a Cellometer automated T4 cell counter (Nexcelom Bioscience, Lawrence, MA, USA) using trypan blue dye exclusion to differentiate between live and dead cells. The doubling time was calculated using the formula (t2-t1) − log(n 2 )/log(n 2 /n 1 ), where n is the cell number at given time points (t).

Wound Healing Assay
Cell migration was measured using a wound healing assay for Ace-1-Vector and Ace-1-Dkk-1 cells (control and SP600125 treated), which were grown to 100% confluence in 6-well plates in triplicate. Media were removed, cells were rinsed with sterile Dulbecco's phosphate-buffered saline (DPBS), and a sterile 200 µL pipet tip was used to scratch 3 separate wounds through the cells. The cells were rinsed with DPBS to remove cell debris and cultured in 1.5 mL of DMEM/F12 with 0.1% BSA alone or with 20 µM SP600125 (Santa Cruz Biotechnology) and incubated at 37 • C and in 5% CO 2 . Using an inverted-phase contrast microscope with a digital camera, images of the scratches were photographed at 0, 6, 12, and 24 h. The rate of wound closure was calculated using the slope of the line graph created from the time point data.

JNK Inhibitor (SP600125) Treatment In Vitro
Three different passages of Ace-1-Vector and Ace-1-Dkk-1 cells were grown to 90% confluence in a 6-well plate in triplicate. Cells were then cultured for 24 h in DMEM/F12 and 10% FBS with either 20 µM SP600125 (Santa Cruz Biotechnology) or an equal amount of the carrier, 0.1% dimethyl sulfoxide (DMSO). After a 24 h incubation period, the cells were collected to measure the mRNA levels of bone-related genes by quantitative reverse transcription PCR (RT-qPCR).

RNA Extraction and Quantitative RT-PCR
Total RNA was extracted from the Ace-1-Vector and Ace-1-Dkk-1 cells treated with either 20 µM SP600125 or 0.1% DMSO using the RNA cultured cell HC kit (QuickGene by AutoGen, Holliston, MA, USA) according to the manufacturer's protocol for adherent tissue culture cells. RNA was also extracted from the mice xenografts (Section 2.9) to assess the JNK-related gene expression. Total RNA (0.5 µg) was reverse-transcribed using SuperScript II (Invitrogen), and RT-qPCR was performed in duplicate using the QuantiTect SYBR Green PCR Kit (Qiagen, Germantown, MD, USA, Cat. No. 204145) and a LightCycler ® 480 (Roche, Indianapolis, IN, USA). PCR cycling conditions were as follows: 95 • C 15 min (preincubation), followed by 45 cycles of 94 • C/15 s, 56 • C/20 s, and 72 • C/10 s and a melting curve beginning at 65 • C and going up to 93 • C at a ramp rate of 0.11 • C/s with continuous fluorescence monitoring. Primer concentrations were 0.5 µM each, and 2 µL cDNA was added to each PCR reaction (20 µL). Every experiment/plate included no RT controls of all of the samples. The housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and the following genes were quantified: bone morphogenetic protein-2, 4, and 7 (BMP2, BMP4, and BMP7); runt-related transcription factor 2 (RUNX2); receptor activator of nuclear factor kappa-B ligand (RANKL); osteoprotegerin (OPG); activating transcription factor 4 (ATF4); aldehyde dehydrogenase 1 family member 1 (ALDH1A1); Wnt family member 5A (WNT5A); Wnt family member 11 (Wnt11); WW domain containing transcription regulator 1 (WWTR1); mitogen-activated protein kinase kinase 7 (MAP2K7); Jun proto-oncogene (JUN); Fos proto-oncogene (FOS); tumor protein p53 (TP53); phosphatase and tensin homolog (PTEN); phosphatidylinositol 3-kinase (PIK3CA); TWIST; folate hydrolase 1 (FOLH1); transcription factor 4 (TCF4); mitogenactivated protein kinase 8 and 9 (MAPK8 and MAPK9); serine/threonine kinase 1 (AKT1); parathyroid hormone-related protein (PTHrP); and transforming growth factor beta (TGFβ) using canine-specific primers (Supplementary Table S1). GAPDH was not differentially expressed between comparison groups. The RT-qPCR results were analyzed using the LightCycler ® 480 Software (Roche Life Science, Pleasanton, CA, USA), and relative mRNA expression was calculated using the delta-delta Ct (∆∆Ct) method: all values were normalized to their corresponding GAPDH values (∆Ct). All primers were designed using the Primer-BLAST software with standard melting temperatures (57−63 • C; Opt. 60 • C) and PCR product sizes (70-200 nt, or up to 250 nt, when necessary). We designed primer pairs that gave the smallest amplicons possible, while at the same time crossing the largest introns possible (or spanning an exon-exon junction, if the introns were small). The primers used in this study were chosen from 3 to 4 different primer pairs designed for each gene, and the primer pair that had the best amplification (slope) and RT-qPCR product melting (single peak) characteristics for each gene was chosen. The primer pairs were designed using the Primer-BLAST software (http://www.ncbi.nlm.nih.gov/tools/primer-blast; last accessed date: 31 August 2017). To confirm primer specificity, all RT-qPCR products were verified by electrophoresis on a 2% agarose gel and stained with ethidium bromide to confirm a single amplification product of the expected size. The entire PCR reactions were then purified using the QIAquick PCR Purification Kit (Qiagen, Cat. No. 28106) and sequenced at the Plant-Microbe Genomics Facility at OSU using a 3730 DNA Analyzer (Applied Biosystems, Grand Island, NY, USA) and BigDye Terminator Cycle Sequencing chemistry (Applied Biosystems). Sequences were verified by a BLAST search using the NCBI website (https://blast.ncbi.nlm.nih.gov/Blast.cgi; last access date: 31 August 2017). When checked, efficiencies have always been >95%, with most of them being 99%-100% efficient, and the Ct values obtained were in the linear range of amplification.

Intratibial Injection and JNK Inhibitor (SP600125) Treatment
Fifteen 6-week-old athymic (NCR-nu/nu) male nude mice were purchased from OSUCCC TVSR. All mice were injected with 5 × 10 5 of Ace-1-Dkk-1 YFP-Luc cells, into the left tibial bone, as described [28]. The mice were treated (IP) with 5 mg/kg of an equal volume of either SP600125 (n = 8) or vehicle (n = 7) every other day for 2 weeks. The mice were euthanized on day 15 after transplantation.

Bioluminescent Imaging
Mice were intraperitoneally injected weekly with 0.1 mL of 40 mg/mL D-luciferin (Caliper Life Sciences, Hopkinton, MA, USA) dissolved in PBS prior to imaging. The mice were anesthetized in an induction chamber with a 3% isoflurane/oxygen mixture and maintained at 2% isoflurane using a nose-cone delivery system during imaging. Bioluminescent in vivo imaging was performed using IVIS 100 (Caliper Life Sciences), and photon signal intensity was quantified using the Living Image software, version 2.50 (Caliper Life Sciences). Imaging was performed every 2 min until peak photon signal was achieved (approximately 10 to 15 min postinjection). Bioluminescence was expressed as total photons/sec/region of interest.

Faxitron Imaging
On day 15 (euthanasia), radiographic images of the left tibia from each mouse were obtained using an LX-60 Faxitron laboratory radiography system (Faxitron X-ray Corp., Wheeling, IL, USA). The tibias were imaged at 28 KV for 5 s to qualitatively evaluate the effect of SP600125 on bone tumor growth in mice bearing Ace-1-Dkk-1 YFP-Luc intratibial tumors.

Histopathological Studies
The mice bearing subcutaneous tumors were euthanized after 4 weeks of treatment with either SP600125 or vehicle. Organs and subcutaneous xenografts were collected and examined to confirm metastases and tumor growth. All xenografts were weighed and divided into 2 halves: 1 half was immediately snap-frozen in liquid nitrogen and kept at −80 • C, and the other half was fixed in 10% natural-buffered formalin for 48 h, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE).
The mice bearing intratibial tumors were euthanized at day 15 after they were transplanted with the tumor cells. Tibial bones were decalcified in a mild decalcifier (formaldehyde, methanol, and formic acid) (Leica Biosystems Inc., Buffalo Grove, IL, USA, 60089) at 37 • C for 4 h before histopathological processing. Images were acquired using an Olympus BX51 microscope (Olympus America, Inc., Melville, NY, USA, 11747) equipped with a Nikon digital camera (Nikon Inc., Melville, NY, USA, 11747).

Statistical Analysis
Data were analyzed using GraphPad PRISM 6.0 (GraphPad, San Diego, CA, USA). Results are displayed as means ± standard deviation. Normalized gene expression, AP-1 reporter activity, proliferation, and migration data were analyzed using a 2-way ANOVA, followed by Sidak's multiple comparisons test to perform pairwise comparisons. The data from the Dkk-1 ELISA and β-catenin OD were analyzed by comparing the Ace-1-Dkk-1 group with the Ace-1-Vector group using Student's t-test. The in vitro experiments were repeated in triplicate, and the values were expressed as the ratio between SP600125 treated and control groups (y-axis) ± SD. Data were analyzed using unpaired t-test to compare results between the 2 groups (SP600125 and control) in most in vitro and in vivo experiments. One-way ANOVA was used to analyze the results of RT-qPCR. p-Value <0.05 was considered statistically significant.

AP-1 Reporter Activity
Ace-1-Dkk-1 cells had approximately a threefold increase in AP-1 reporter activity compared with Ace-1-Vector cells. Treatment with the selective JNK inhibitor, SP600125, reduced the AP-1 reporter activity of Ace-1-Dkk-1 cells almost to the Ace-1-Vector level (p < 0.0001). For the Ace-1-Vector cells, treatment did not result in significant changes in reporter activity ( Figure 2).

Cell Proliferation Assay
Proliferation of Ace-1-Vector and Ace-1-Dkk-1 cells was similar in vitro ( Figure 3A). No significant change in proliferation was seen in either Ace-1-Vector or Ace-1-Dkk-1 cells after treatment with SP600125.

AP-1 Reporter Activity
Ace-1-Dkk-1 cells had approximately a threefold increase in AP-1 reporter activity compared with Ace-1-Vector cells. Treatment with the selective JNK inhibitor, SP600125, reduced the AP-1 reporter activity of Ace-1-Dkk-1 cells almost to the Ace-1-Vector level (p < 0.0001). For the Ace-1-Vector cells, treatment did not result in significant changes in reporter activity (Figure 2).

AP-1 Reporter Activity
Ace-1-Dkk-1 cells had approximately a threefold increase in AP-1 reporter activity compared with Ace-1-Vector cells. Treatment with the selective JNK inhibitor, SP600125, reduced the AP-1 reporter activity of Ace-1-Dkk-1 cells almost to the Ace-1-Vector level (p < 0.0001). For the Ace-1-Vector cells, treatment did not result in significant changes in reporter activity ( Figure 2).

Cell Proliferation Assay
Proliferation of Ace-1-Vector and Ace-1-Dkk-1 cells was similar in vitro ( Figure 3A). No significant change in proliferation was seen in either Ace-1-Vector or Ace-1-Dkk-1 cells after treatment with SP600125.

Wound Healing Assay
Untreated Ace-1-Vector and Ace-1-Dkk-1 cells had similar rates of wound healing in vitro ( Figure 3B). However, treatment with SP600125 reduced Ace-1-Dkk-1 migration by approximately 67% (p < 0.05), but had no effect on the rate of migration for Ace-1-Vector cells.

Wound Healing Assay
Untreated Ace-1-Vector and Ace-1-Dkk-1 cells had similar rates of wound healing in vitro ( Figure 3B). However, treatment with SP600125 reduced Ace-1-Dkk-1 migration by approximately 67% (p < 0.05), but had no effect on the rate of migration for Ace-1-Vector cells.

Intratibial Injection and JNK Inhibitor (SP600125) Treatment
Intratibial injections were performed with Ace-1-Dkk-1 YFP-Luc cells to evaluate the effect of SP600125 on tumor growth in the bone. The growth of bone tumors was measured by bioluminescent imaging on days 7 and 14 after tumor cell transplantation. There was no difference in bioluminescence between SP600125-treated mice and control mice (data not shown), demonstrating similar growth of viable tumor cells. However, the radiographs between the two groups had marked differences. The control tumors had smooth external bone surfaces with minimal periosteal new bone and intramedullary new bone formation with mild to moderate bone lysis. Bone tumors from mice treated with SP600125 had irregular shapes with dense intramedullary bone formation (increased radio-opacity), abundant periosteal new bone formation in the tibias and fibulas, and large foci of bone lysis ( Figure 10).
Histopathology confirmed the radiographic findings. Both groups of mice had growth of well-differentiated PCa cells with both osteoclastic bone lysis and new medullary woven bone. The SP600125-treated tumors were interpreted to be more invasive with cortical bone lysis and secondary periosteal new bone formation. SP600125 also may have induced the formation of a denser medullary bone. The tumor cells in both groups grew in the diaphyseal regions, replacing most of the bone marrow ( Figure 11A,B). not shown), demonstrating similar growth of viable tumor cells. However, the radiographs between the two groups had marked differences. The control tumors had smooth external bone surfaces with minimal periosteal new bone and intramedullary new bone formation with mild to moderate bone lysis. Bone tumors from mice treated with SP600125 had irregular shapes with dense intramedullary bone formation (increased radio-opacity), abundant periosteal new bone formation in the tibias and fibulas, and large foci of bone lysis ( Figure 10). Histopathology confirmed the radiographic findings. Both groups of mice had growth of well-differentiated PCa cells with both osteoclastic bone lysis and new medullary woven bone. The SP600125-treated tumors were interpreted to be more invasive with cortical bone lysis and secondary periosteal new bone formation. SP600125 also may have induced the formation of a denser medullary bone. The tumor cells in both groups grew in the diaphyseal regions, replacing most of the bone marrow ( Figure 11A,B).

Discussion
Nineteen Wnts were identified in humans and mice that bind to 10 cognate recepto from the FZD family that regulate several signaling cascades. The two best characteriz are the canonical and the noncanonical Wnt signaling cascades. Canonical Wnt signali depends on the cytoplasmic stabilization and subsequent nuclear translocation of catenin for the transcription of Wnt-specific genes associated with stemness, cell prolif ation, differentiation, migration, and apoptosis [29,30]. The noncanonical Wnt signali pathways are classified into either the planar cell polarity (PCP) or the Wnt-calcium pa way [31]. Previously, we showed that Dkk-1 overexpression in Ace-1 cells activated t noncanonical Wnt/JNK signaling pathway, resulting in increased tumor growth and t incidence of bone metastasis [5]. Ace-1 cells are a relevant model of canine prostate canc as they form localized and metastatic prostate cancer in immunosuppressed beagle [2 and left ventricular injection of Ace-1 cells in rodent results in almost exclusive metasta

Discussion
Nineteen Wnts were identified in humans and mice that bind to 10 cognate receptors from the FZD family that regulate several signaling cascades. The two best characterized are the canonical and the noncanonical Wnt signaling cascades. Canonical Wnt signaling depends on the cytoplasmic stabilization and subsequent nuclear translocation of β-catenin for the transcription of Wnt-specific genes associated with stemness, cell proliferation, differentiation, migration, and apoptosis [29,30]. The noncanonical Wnt signaling pathways are classified into either the planar cell polarity (PCP) or the Wnt-calcium pathway [31].
Previously, we showed that Dkk-1 overexpression in Ace-1 cells activated the noncanonical Wnt/JNK signaling pathway, resulting in increased tumor growth and the incidence of bone metastasis [5]. Ace-1 cells are a relevant model of canine prostate cancer, as they form localized and metastatic prostate cancer in immunosuppressed beagle [29], and left ventricular injection of Ace-1 cells in rodent results in almost exclusive metastasis to the bone [32]. This cell line has also been used in dogs as a model for developing therapeutic strategies for human prostate cancer patients [33]. In the present study, we reported increased Wnt/JNK signaling in Ace-1-Dkk-1 cells and showed that Wnt/JNK signaling resulted in increased tumor growth.
The canonical pathway is initiated by the binding of Wnt ligands to the FZD receptor and LRP5/6 to activate signaling through β-catenin [34]. While the noncanonical Wnt/JNK pathway is less understood, it has been reported that Wnt ligands also bind to the FZD receptor in the absence of LRP5/6, resulting in phosphorylation of disheveled (Dsh) and JNK activation [35]. In the present study, Dkk-1 was secreted from the Ace-1-Dkk-1 cells and significantly decreased β-catenin compared with Ace-1-Vector cells. Moreover, Dkk-1 significantly increased AP-1 activity, a downstream product of the noncanonical Wnt/JNK pathway, in Ace-1-Dkk-1 compared with Ace-1-Vector cells. Thus, the effect of Dkk-1 in inhibiting canonical Wnt and activating noncanonical Wnt/JNK signaling pathways was demonstrated. Blocking noncanonical Wnt/JNK using a JNK inhibitor (SP600125) markedly decreased the AP-1 activity in Ace-1-Dkk-1, but not in Ace-1-Vector cells, suggesting that this pathway is not active in the Ace-1-Vector cells.
The expression of BMPs by PCa was reported to induce osteoblast differentiation through both the canonical (Wnt/β-catenin) and noncanonical (Wnt/JNK) signaling pathways in C4-2B cells (PCa cell line), and Dkk-1 blocked the osteoblastic differentiation in vitro [36]. Dkk-1 was highly expressed in breast cancer patients who predominantly developed osteolytic bone metastases [34,37]. Dkk-1 has been shown to induce osteolysis and inhibit osteoblast differentiation in PCa bone metastases by inhibiting the Wnt signaling pathway. Moreover, Dkk-1 decreased the osteoblastic differentiation and mineralization in PCa cell lines in vitro and has the ability to switch the bone metastasis phenotype from osteoblastic to osteolytic [38]. In this study, Dkk-1 significantly downregulated BMP2 expression in vitro; therefore, we speculated that the osteolytic appearance of Ace-1-Dkk-1 arises from the BMP2 inhibition effect of Dkk-1.
The expressions of genes associated with osteoblast differentiation, including BMP4, BMP7, RUNX2, and OPG, in both Ace-1-Dkk-1 and Ace-1-Vector were investigated in vitro. BMP4 was reported to induce osteoblast differentiation in PCa-118b (PCa cell line) [39]. BMP7 was highly expressed in metastatic bone lesions of prostate cancer, and expression was related to osteoblastic metastasis [40]. Moreover, RUNX2 upregulation was associated with osteoblast differentiation through the canonical Wnt pathway [41]. OPG is a decoy receptor expressed by osteoblast that inhibits the binding of RANKL on osteoblast to its receptor RANK on osteoclast and negatively regulates osteoclast differentiation and reduces bone resorption [7]. We found that SP600125 upregulated all of these genes in vitro. These results indicated that SP600125 restored the canonical Wnt signaling in Ace-1-Dkk-1 cells.
The upregulation of cell-survival-related genes, including ATF4 and WWTR1, was demonstrated in vitro in Ace-1-Vector cells with SP600125 treatment. ATF4 expression is upregulated by tumor microenvironment stress, including hypoxia and nutrient deprivation. This gene promotes cancer cell adaptation to such conditions by transcriptionally upregulating genes essential for redox balance, angiogenesis, and autophagy. Moreover, ATF4 has been implicated in cancer progression and drug resistance [42]. WWTR1 is a downstream transcriptional coactivator of the Hippo pathway and has been reported to be overexpressed in various human cancers, which correlated to cancer aggressiveness by promoting malignant cell proliferation and inhibiting apoptosis [43]. Our data demonstrated that SP600125 upregulated the in vitro expression of these genes in Ace-1-Vector cells, which could enhance tumor cell survival.
The upregulation of PTEN expression in Ace-Dkk-1 cells was also observed in this study. PTEN is a tumor suppressor gene that is inactivated by mutation or deletion in advanced PCa. PTEN exerts its tumor suppressor function by inducing cell cycle arrest through inhibition of the PI3K signaling pathway [44]. JNK overexpression was found to downregulate PTEN expression. Decreased PTEN expression promotes cell proliferation, decreases apoptosis, and enhances tumor angiogenesis [45]. In the present study, SP600125 upregulated PTEN expression in Ace-1-Dkk-1 cells in vitro, which may be due to JNK inhibition.
Interestingly, Dkk-1 downregulated the in vitro expression of FOLH1 in Ace-1-Dkk-1 cells. Low-to-moderate expression of FOLH1, a prostate-specific membrane antigen (PSMA), was observed in osteoblastic activity [46]. FOLH1 was reported to be upregulated in PCa, and its expression has been correlated with cancer aggressiveness. Moreover, FOLH1 may be a potential marker for PCa therapeutics [47]. In men with PCa, high FOLH1 expression has been correlated with low androgen expression [48]. Most canine prostate cancers are discovered at a late stage in progression and are typically androgen independent [49,50]. Consequently, the decrease in the expression of FOLH1 in Ace-1-Dkk-1 cells could alter the phenotype of bone metastases from osteoblastic to osteolytic.
Dkk-1 significantly upregulated PIK3CA in Ace-1-Dkk-1 YFP-LUC xenografts compared with Ace-1-Vector YFP-LUC xenografts. Oncogenic activation of the phosphatidylinositol-3kinase (PI3K) pathway is a common event in PCa that promotes tumorigenesis, disease progression, and therapeutic resistance [51]. In addition, genetic alterations of the PI3K pathway are common in PCa patients with up to 42% of primary versus 100% of metastatic prostatic tumor samples [51,52]. Dkk-1 contributed to tumor growth through the regulation of Wnt signaling and PI3K/AKT signaling in cancer cells [53]. Therefore, it is possible that the increased tumor growth in Ace-1-Dkk-1 YFP-LUC xenografts was related to the upregulated expression of PIK3CA.
There was no difference in in vitro cell proliferation between Ace-1-Vector and Ace-1-Dkk-1 cells. Therefore, we speculate that the increased in vivo subcutaneous growth in Ace-1-Dkk-1 YFP-LUC compared with Ace-1-Vector YFP-LUC xenografts was due to a paracrine interaction with the tumor microenvironment, which is consistent with a previous study [4]. However, SP600125 reduced the tumor growth of Ace-1-Dkk-1 YFP-LUC but not Ace-1-Vector YFP-LUC xenografts. SP600125 treatment led to the acquisition of a spindle-shaped tumor cell appearance in both Ace-1-Dkk-1 YFP-LUC and Ace-1-Vector YFP-LUC xenografts. This was suggestive of an EMT phenotype that should be confirmed. It may be therapeutically advantageous to use SP600125 in combination with other drugs in the early stages of PCa.
In vivo, SP600125 downregulated JUN, TP53, and TWIST expressions in Ace-1-Dkk-1 YFP-LUC xenografts. JUN was identified as a master regulator gene in cell proliferation, differentiation, and apoptosis [54]. JUN overexpression induced oncogenic transformation, increased tumor formation, and invasion in human breast cancer cells [55]. The TP53 tumor suppressor gene is mutated (30%) in PCa, and TP53 mutations were associated with tumor progression in PCa [56,57]. A previous study revealed PTEN loss concurrently with TP53 structural rearrangements in PCa cases [58]. The levels of TP53 expression and types of TP53 mutation were found to be closely related to the prognosis in triple-negative breast cancer. Low TP53 expression in missense mutation patients and high TP53 expression in TP53 deletion mutation patients were associated with poor prognosis [59]. In this study, we found that SP600125 downregulated TP53 expression in Ace-1-Dkk-1 YPF-LUC xenografts and reduced tumor sizes in vivo. Low TP53 expression is not always a negative prognostic indicator, and its prognostic values can be varied on TP53 mutation types. Thus, TP53 mutation in PCa should be further investigated. TWIST is an important gene in cancer metastasis due to its contribution in EMT, angiogenesis, and chromosomal instability [60,61], and its downregulation is commonly associated with decreased metastasis [62].
Our previous work showed that Dkk-1 inhibited the canonical Wnt-induced osteoblastic bone metastases [4]. In the current study, SP600125 did not alter tumor growth in the bone but increased the Ace-1-Dkk-1 YFP-Luc osteoblastic and osteolytic phenotype of the bone tumors with increased cortical invasion and secondary induction of periosteal new bone formation. The intratibial woven bone formation induced by SP600125 through the inhibition of Wnt/JNK signaling and the activation of the canonical Wnt signaling indicates that the bone microenvironment plays a pivotal role in new bone formation through a paracrine mechanism.

Conclusions
Overexpression of human Dkk-1 increased tumor growth in vivo and upregulated the noncanonical Wnt/JNK signaling pathway in Ace-1-Dkk-1 cells, resulting in downstream alterations in gene expression involved in the osteoblast differentiation, cell proliferation, and microscopic appearance of the cells. Thus, Ace-1-Vector and Ace-1-Dkk-1 cells are useful models for studying the biological and molecular characteristics of canonical Wnt and noncanonical Wnt/JNK signaling pathways in PCa, respectively. In addition, SP600125 could be an alternative adjuvant therapy for decreasing tumor size in dogs and humans with spontaneous PCa that expresses high levels of Dkk-1. However, SP600125 may have the potential to increase the local invasiveness of bone metastases, and the phenotype and drug responsiveness of the tumor cells may depend on the local microenvironment.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.