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The American Journal of Pathology logoLink to The American Journal of Pathology
. 2023 May;193(5):624–637. doi: 10.1016/j.ajpath.2023.01.004

Protein Kinase D2 and D3 Promote Prostate Cancer Cell Bone Metastasis by Positively Regulating Runx2 in a MEK/ERK1/2–Dependent Manner

Adhiraj Roy ∗,†, Sahdeo Prasad ∗, Yuzhou Chen ∗, Yapeng Chao ∗, Yu Liu ∗, Jinjun Zhao ∗,‡, Qiming Jane Wang ∗,∗
PMCID: PMC10155267  PMID: 36740185

Abstract

Advanced-stage prostate tumors metastasize to the bone, often causing death. The protein kinase D (PKD) family has been implicated in prostate cancer development; however, its role in prostate cancer metastasis remains elusive. This study examined the contribution of PKD, particularly PKD2 and PKD3 (PKD2/3), to the metastatic potential of prostate cancer cells and the effect of PKD inhibition on prostate cancer bone metastasis in vivo. Depletion of PKD2/3 by siRNAs or inhibition by the PKD inhibitor CRT0066101 in AR-positive and AR-negative castration-resistant prostate cancer cells potently inhibited colony formation and cell migration. Depletion or inhibition of PKD2/3 significantly blocked tumor cell invasion and suppressed the expression of genes related to bone metastasis in the highly invasive PC3-ML cells. The reduced invasive activity resulting from PKD2/3 depletion was in part mediated by the transcription factor Runx2, as its silencing decreased PKD2/3–mediated metastatic gene expression through the mitogen-activated protein kinase kinase/extracellular signal–regulated kinase 1/2 signaling axis. Furthermore, inhibition of PKD by CRT0066101 potently decreased the frequency of bone micrometastases in a mouse model of bone metastasis based on intracardiac injection of PC3-ML cells. These results indicate that PKD2/3 plays an important role in the bone metastasis of prostate cancer cells, and its inhibition may be beneficial for the treatment of advanced prostate cancer.

After skin cancer, prostate cancer is the most common cancer diagnosed in American men. It is also one of the leading causes of cancer-related deaths among men of all ethnicities. Despite effective initial management and improved therapeutic options in recent years, approximately one-half of patients with prostate cancer die of distal metastases.1 Bone is one of the most preferred sites of metastasis of prostate cancer cells, with up to 90% of patients with advanced disease and bone metastases.2 Bone metastases invariably lead to hypercalcemia, bone pain, fractures, and nerve compression, and an increased morbidity and mortality in patients with prostate cancer.3

Protein kinase D (PRKD, referred here as PKD) is a family of serine/threonine kinases belonging to a subfamily of the Ca2+/calmodulin–dependent protein kinase superfamily. Three isoforms of PKD (PKD1, PKD2, and PKD3) with high sequence homology have been identified. PKD can be activated in the canonical pathway through the phosphorylation of two conserved serine residues by classical or novel PKCs. PKD regulates many important cancer-associated protein targets, including β-catenin, androgen receptor, Wnt5, urokinase-type plasminogen activator, heat shock protein 27, class IIa histone deacetylases, vascular endothelial growth factor, matrix metalloproteinases (MMPs), and mitogen-activated protein kinase kinase/extracellular signal–regulated kinase (MEK/ERK).4 Its important role in cancer development provides a foundation for targeting PKDs using small molecules for cancer treatment. Several small-molecule inhibitors of PKD have been developed, including CID755673 and its analogues kb-NB142–70, SD-208, 1-naphthyl PP1, compound 139, CRT0066101, and others.5, 6, 7, 8, 9 Among them, CRT0066101, an aminopyridine arene, has emerged as a highly potent, cell-permeable, and in vivo active pan-PKD inhibitor. CRT0066101 blocks tumor cell proliferation, migration, and invasion, and inhibits the growth of pancreatic, breast, bladder, and colorectal tumor xenografts in mice.10, 11, 12, 13

Growing evidence supports an important role for PKD in prostate cancer.9,14 Aberrant PKD expression has been reported in human prostate carcinoma.15,16 In particular, PKD2 and PKD3 are frequently expressed in highly metastatic prostate cancer cells and promote prostate tumor cell proliferation and survival. PKD2 and PKD3 have also been implicated in prostate cancer cell invasion and migration by promoting NF-κB and urokinase-type plasminogen activator expression and increasing MMP-9 expression.17,18 PKD3 increases prostate cancer cell survival by prolonging the activation of Akt and ERK1/2.16 PKD3 has also been shown to encourage the production of tumor-promoting factors, as PKD3 knockdown inhibits the production of MMP-9, IL-6, IL-8, and GRO-α in prostate cancer cells.18 Despite these known functions, the role of PKDs in prostate cancer bone metastasis remains unknown.

The transcription factor Runx2 is involved in many cellular and physiological functions, including the regulation of bone formation, bone turnover, and epigenetic gene control during cell division. Abnormal or high expression of Runx2 transcription factor has been associated with tumor cells that metastasize to the bone.19 The current study investigated whether PKD plays a role in prostate cancer cell migration and invasion by modulating Runx2 expression. It further determined whether PKD inhibition prevents bone metastasis of prostate cancer cells in a mouse model of metastasis. Studies with PC3-ML cells (a subline derived from the PC3 cell line with 80% metastatic efficiency to the lumbar vertebrae and producing bone metastasis in mice)20 indicated that inhibition of PKD by CRT0066101 potently inhibited PC3-ML cell metastasis to the bone. Silencing of PKD2 and PKD3 (PKD2/3) or inhibition of PKD in PC3-ML cells abrogated tumor cell migration and invasion, in part by down-regulating bone metastatic genes via reduction of Runx2 protein expression. The findings identified PKD2/3–Runx2 as novel targets in bone metastasis of prostate cancer; targeted suppression of these enzymes may have significant therapeutic implications.

Materials and Methods

Materials

RPMI 1640 medium was purchased from Cellgro (Manassas, VA), and other culture materials were purchased from Invitrogen (Carlsbad, CA). Antibodies against PKD2, PKD3, Runx1, Runx2, and Runx3 were obtained from Cell Signaling Technology (Danvers, MA). Glyceraldehyde-3-phosphate dehydrogenase was purchased from Enzo Life Sciences (Farmingdale, NY). All other chemicals were purchased from MilliporeSigma (Burlington, MA), unless otherwise stated.

Cell Culture

Human prostatic tumor variant PC3-ML cells and PC3-ML cells infected with the ZsGreen lentivirus were kindly provided by Dr. Marcelo G. Kazanietz, Perelman School of Medicine at the University of Pennsylvania (Philadelphia, PA). C4-2B and 22Rv1 prostate cancer cells were obtained from ATCC (Manassas, VA). ATCC performed authentication for these cell lines using short tandem repeat profiling. All three cell lines were grown in RPMI 1640 media supplemented with 10% fetal bovine serum and 100 μg/mL penicillin/streptomycin in a 37°C incubator containing 5% carbon dioxide. All cell cultures were routinely tested for Mycoplasma by using the MycoAlert PLUS Mycoplasma Detection Kit (Lonza, Walkersville, MD).

Western Blot Analysis

Western blot analysis was performed as previously described.16 Briefly, the protein concentration was determined by using the Pierce BCA Protein Assay kit (Thermo Fisher Scientific, Rockford, IL) according to the manufacturer’s instructions. Equal amounts of total protein were resolved by using SDS-PAGE. After electrophoresis, proteins were transferred onto nitrocellulose membranes. Membranes were blocked with 5% non-fat dry milk in Tris-buffered saline containing 0.2% Tween 20 for 1 hour and then blotted with a primary antibody, followed by a secondary goat anti-rabbit or anti-mouse antibody conjugated to horseradish peroxidase. Blots were developed by using enhanced chemiluminescence reagent (GE Healthcare, Chicago, IL).

Matrigel Invasion Assays

The Matrigel invasion assay was conducted. Briefly, cells were seeded (1.5 × 104 cells per well) in RPMI medium containing 1% fetal bovine serum in the upper compartment of a Boyden chamber with a Matrigel-coated 12 μm pore polycarbonate membrane insert (Neuro Probe, Gaithersburg, MD). The RPMI medium containing 20% fetal bovine serum was used in the lower chambers. After an incubation period of 22 hours at 37°C, membranes were recovered, and cells on the upper side of the membrane were wiped off and stained with the Diff-Quik Stain Set (Dade Behring, Deerfield, IL). Similar procedures were performed by using a control chamber without Matrigel. Migratory cells in each well were counted by using phase-contrast microscopy in five random fields and analyzed. The percentage of invasion was determined by dividing the number of invaded cells by the total number of cells that migrated through the control insert.

Clonogenic Assay

PC3-ML cells (500 cells per well) were seeded in six-well plates and allowed to adhere overnight, transfected with siRNA against PKD2 and PKD3, and treated with CRT0066101. Cells were allowed to form colonies for 9 days and then stained with a 0.25% crystal violet solution. The number of colonies in each well was counted and analyzed.

Cell Viability Assay

Cell survival assay was measured by using the Cell Counting Kit-8 (MedChemExpress, Monmouth Junction, NJ) according to the manufacturer’s protocol. Briefly, cells were plated at a density of approximately 8000 cells per well in 96-well plates. Two days after siRNA transfection or compound treatment, CCK-8 reagent containing the highly water-soluble tetrazolium salt WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt] was added to cells in each well, followed by incubation for 2 to 3 hours. Cell proliferation/viability was determined by measuring the OD at 450 nm using a Synergy H1 hybrid reader (BioTek Instruments, Winooski, VT). Percentage of control was calculated as a measure of cell viability.

Wound Healing Assay

PC3-ML cells were grown to confluence in six-well plates. Wound healing assay was performed as described previously.18 Briefly, wounds were created by scraping the monolayer with a pipette tip and then washing with fresh medium to remove floating cells. The cells were transfected with PKD2 or PKD3 siRNA or exposed to CRT101 separately. To eliminate the interference of cell proliferation on migration, cells were pretreated with 0.5 μmol/L mitomycin C for 2 hours before wounding.18 Images of the wound were captured immediately and after 24 hours under an inverted phase contrast microscope. The wound gap was measured by using ImageJ software version 1.53c (NIH, Bethesda, MD; https://imagej.nih.gov/ij), and the percentage of wound healing was calculated.

Animals

The 4-week–old male athymic nu/nu mice were obtained from a breeding colony of Charles River Laboratories (Wilmington, MA). Animals were housed and maintained with food, light, and water in a standard manner. The animal protocol was approved by the University of Pittsburgh Institutional Animal Care and Use Committee.

Bone Metastasis in Nude Mice

Prostate cancer PC3-ML cell lines stably transfected with ZsGreen lentivirus were harvested from subconfluent cultures. The cells were washed, resuspended in phosphate-buffered saline, and kept on ice. A single-cell suspension (50,000 cells per mouse) with >90% viability was inoculated into the left cardiac ventricle of anesthetized athymic nude mice (n = 12). During cell inoculation, a needle was gently inserted into the ventricle. On the same day, animals were randomly assigned to two experimental groups: vehicle (5% dextrose, oral gavage daily) and CRT0066101 (80 mg/kg dissolved in 5% dextrose; oral gavage daily). Animals were sacrificed after 4 weeks by placing them in a carbon dioxide chamber, followed by cervical dislocation. The femur bones from each animal were dissected for further study. To confirm the delivery of cells into the systemic blood circulation, blue fluorescent 10 μm polystyrene beads (Molecular Probes, Eugene, OR) were injected separately into the left cardiac ventricle of mice in the same manner. After 24 hours, the animals were sacrificed, and the presence of fluorescent blue beads was observed in the kidneys and lungs under a fluorescent microscope.

Decalcification of Bone and Analysis of Micrometastases

The cleaned bones were then transferred to a 4% paraformaldehyde solution. After 24 hours, bones were transferred to fresh paraformaldehyde for an additional 24 hours. Bone was decalcified by transferring the bone to a 0.5 mol/L EDTA (pH 7.2) solution for 10 days. The EDTA solution was changed every day to a fresh solution and maintained at 4°C. The bone was placed in a rocker during the decalcification process. After 10 days, bones were incubated in 30% sucrose for 24 hours. To obtain sections using a cryostat, bones were frozen in optimum cutting temperature medium by placing over dry ice. Femoral bones were cut entirely and made available for analysis. Images were acquired from each bone section by using a confocal microscope connected to a multispectral imaging system. Digital images were analyzed, and micrometastases were determined.

Bone Marrow Cultures

After the mice were sacrificed, their femur bones were dissected. Muscles on the bone surface were gently cleaned, and the bone marrow was flushed out with RPMI media using 25-gauge needles in a sterilized Eppendorf tube. Cells were dissociated using a syringe, kept on ice, washed once with RPMI medium, and then incubated with RPMI media supplemented with 10% fetal bovine serum, 2 mmol/L glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, and 50 μg/mL gentamycin. The medium was replaced with fresh RPMI medium twice per week. After 10 days of culture, the presence of ZsGreen fluorescent colonies was determined by using an inverted microscope.

Quantitative Real-Time RT-PCR

Total RNA from PC3-ML cells was extracted by using TRIzol LS reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. RNA quality was assessed by the 260/280 ratio using a NanoDrop spectrophotometer (Thermo Fisher Scientific). One microgram of total RNAs was used to generate cDNA by using an iScript cDNA synthesis kit (Bio-Rad, Richmond, CA). Real-time PCR was subsequently performed by using iTaq Universal SYBR Green Supermix (Bio-Rad) with the primers listed in Table 1 on a CFX96 Real-Time PCR Detection System (Bio-Rad). The data were normalized to glyceraldehyde-3-phosphate dehydrogenase as an internal control.

Table 1.

List of Real-Time PCR Primers Used in the Study

Target Primer sequence
Runx1 F: 5′-AACCTCGAAGACATCGGCAG-3′
R: 5′-GGCTGAGGGTTAAAGGCAGT-3′
Runx2 F: 5′-GAGTGGACGAGGCAAGAGTT-3′
R: 5′-GGATGAGGAATGCGCCCTAA-3′
Runx3 F: 5′-CGGGGACCCTAACAACCTTC-3′
R: 5′-GTGGGGGTGGTAACCTATGC-3′
OPN F: 5′-GCCCATCCCGTAAATGAAAAAG-3′
R: 5′-GCTGACAACCAAGCCCTCCCAG-3′
OCN F: 5′-GCTTTGTTTACTTGTCAGGTTGGG-3′
R: 5′-CCCTGTGTCCTTAGCAGGCAGGGA-3′
MMP-13 F: 5′-TTTTGAGACCCTGCTGAAACAA-3′
R: 5′-GTCTTTCCGCAGAGATTACC-3′
FAK F: 5′-GGTGCCTGGAGAGTGCAC-3′
R: 5′-GCCGGGGTCGCCCCGCCGA-3′
MMP-9 F: 5′-CCATCACTTTCCCTTGGCT-3′
R: 5′-ACCAGCATGAGAAAGGGCTT-3′
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F, forward primer; FAK, focal adhesion kinase; MMP, matrix metalloproteinase; OCN, osteocalcin; OPN, osteopontin; R, reverse primer.

Immunofluorescence Assay

The cells were seeded on glass coverslips coated with poly-d-lysine (Fisher Scientific, Pittsburgh, PA). After treatment, cells were fixed in 4% paraformaldehyde, blocked with 5% normal goat serum containing 0.1% Triton X-100, and incubated with primary antibody followed by Alexa Fluor 488–conjugated secondary antibody. Images were captured by using a FluoView 1000 confocal microscope (Olympus, Bethlehem, PA) equipped with a 60× oil-immersion objective lens (numerical aperture, 1.43).

Statistical Analysis

Data analysis was performed by using the t-test for comparison between two groups (two-tailed). All statistical analyses were performed by using GraphPad Prism 8 software (GraphPad Software, La Jolla, CA). All values are presented as the means ± SEM of at least three independent experiments. Statistical significance was set at P < 0.05 being considered statistically significant (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001).

Results

Expression and Activity of PKD2 and PKD3 Are Required for the Viability, Migration, and Invasion of Prostate Cancer Cells

PKD has emerged as a promising therapeutic target for various cancers, including prostate cancer. However, its precise role in prostate cancer metastasis, particularly bone metastasis, and the mechanisms by which it regulates this process remain largely unknown. The study sought to define the role of PKD tumor metastasis to the bone by using a PC3-ML bone metastasis model. PC3-ML, a subline of PC3, displays 80% metastatic efficiency in the lumbar vertebrae and produces bone metastasis when injected into mice.20 First, the expression of PKD isoforms in PC3-ML cells, compared with the normal prostate epithelial cell line RWPE-1 and the androgen-dependent prostate cancer cell line LNCaP, was examined by Western blot analysis (Figure 1A). RWPE-1 and LNCaP cells expressed all three PKD isoforms, whereas PC3-ML cells expressed only PKD2 and PKD3. Next, the functional input of PKD2/3 in PC3-ML proliferation and migration/invasion was assessed. PKD2 and PKD3 were silenced by two separate siRNAs targeting different regions of the PKD2 or PKD3 genes, and their activities were inhibited by the pan-PKD inhibitor CRT0066101 (CRT101). The cells were examined for their long-term colony-forming ability. As shown in Figure 1B, silencing of PKD2 or PKD3 dramatically decreased the number of colonies formed compared with the control, and inhibition of PKD by CRT101 had similar effects. Quantitative analysis confirmed these findings (Figure 1B). Accordingly, overexpression of a catalytically active PKD2 mutant (ie, PKD2-CA) promoted colony formation by twofold compared with the empty vector control (Figure 1C). Taken together, these data indicate that PKD2/3 plays an essential role in the clonogenic potential of PC3-ML cells.

Figure 1.

Figure 1

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Depletion of protein kinase D2 and D3 (PKD2/3) or inhibition of PKD suppresses the proliferation, migration, and invasion of PC3-ML prostate cancer cells. A: Expression of PKD1, PKD2, and PKD3 was analyzed in RWPE-1, LNCaP, and PC3-Ml cells by Western blot analysis. B and C: Depletion or inhibition of PKD blocked clonogenic ability of PC3-ML cells. PC3-ML (1000 cells per well) transfected with PKD2 or PKD3 siRNAs or exposed to CRT101 (5 μmol/L) (B) or transfected with an empty vector (EV) or a catalytically active PKD2 (PKD2-CA) plasmid (C). Left panel: Crystal violet stain. Right panel: Colony numbers from five random fields. D: Depletion or inhibition of PKD inhibited PC3-ML prostate tumor cell migration. Cells transfected with PKD2 or PKD3 siRNAs for 48 hours or treated with CRT0066101 (5 μmol/L) for 24 hours were stained with crystal violet (left panel), and wound closure was measured by using ImageJ software (right panel). E: Depletion or inhibition of PKD blocked PC3-ML prostate tumor cell invasion. PC3-ML cells were transfected with PKD2 or PKD3 siRNAs, and treated with the PKD inhibitor CRT0066101 (5 μmol/L). Right panel: Percent invasion was calculated as the percentage of the cells invaded through Matrigel inserts versus the total cells migrated through the control inserts. F: Matrigel invasion assay was performed on cells transfected with a control (EV) or a PKD2-CA plasmid. Cells invaded through Matrigel were imaged (left panel) and quantified (right panel). Data shown in the bar graphs are the average of three independent experiments with error bars representing SEM (B–F). ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001. Original magnification, ×10 (D–F).

Next, the effects of silencing of PKDs in PC3-ML cells on tumor cell migratory and/or invasive were determined. PC3-ML cells were transfected with PKD2/3 siRNAs or treated with CRT101, followed by wound healing assay. As shown in Figure 1D, silencing of PKDs resulted in 22% to 35% inhibition of cell migration compared with the control. Incubation of cells with CRT101 for 24 hours resulted in 87% inhibition of wound closure compared with the control. These data indicate that PKD2 and PKD3, along with their inhibitors, potently decreased the migration of prostate cancer cells. The effects of PKD2/3 depletion or inhibition on PC3-ML cell invasion were further determined. As shown in Figure 1E, silencing of PKD2 and PKD3 in PC3-ML cells for 22 hours resulted in approximately 40% inhibition of cell invasion compared with the control, whereas inhibition of PKD by CRT0066101 resulted in >60% decrease in invasion of PC3-ML cells. Accordingly, overexpression of catalytically active PKD2-CA doubled the number of cells that invaded through the Matrigel (Figure 1F). Thus, these data clearly show that PKD2 and PKD3 are required for PC3-ML cell invasion in vitro.

In addition to PC3-ML cells, the effects of silencing or inhibiting PKD2/3 on the viability of two additional AR-positive castration-resistant prostate cancer cell lines, C4-2B and 22Rv1, were examined. C4-2B expressed high levels of PKD1 and less PKD2/3, whereas 22Rv1 predominantly expressed PKD2 but less PKD3 and minimal PKD1 (Figure 2A). Knockdown of PKD2/3 by siRNAs or inhibition of PKD by CRT101 significantly reduced cell viability of C4-2B and 22Rv1 cells compared with the control cells transfected with nontargeting siRNA (Figure 2B). In contrast, knockdown of PKD1 only marginally reduced the viability in C4-2B cells when examined at 48 and 72 hours after transfection (Figure 2C). Thus, depletion of PKD2/3 had greater impact on cell viability in cells expressing higher levels of PKD2/3 (22Rv1), whereas knockdown PKD1 did not have such an effect in cells expressing higher levels of PKD1 (C4-2B); this finding indicates differential contributions of PKD2/3 and PKD1 to prostate cancer cell proliferation/survival and a minor role of PKD1 in this context. Moreover, knockdown of PKD2/3 or treatment with CRT101 also significantly reduced C4-2B cell migration at 24 hours after wounding compared with the control cells (Figure 2D). Similarly, depleting PKD2 or PKD3 dramatically decreased the number of colonies formed compared with the untransfected control (Figure 2E). Inhibition of PKD by CRT101 similarly blocked C4-2B cell clonogenicity (Figure 2E). These data suggest that PKD2 and PKD3 are required for the migration and clonogenicity of AR-positive castration-resistant prostate cancer cells.

Figure 2.

Figure 2

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Depletion of protein kinase D2 and D3 (PKD2/3) or inhibition of PKD reduces the viability, migration, and clonogenicity of AR-positive castration-resistant prostate cancer cells. A: Expression of PKD1, PKD2, and PKD3 in the castration-resistant prostate cancer cell lines C4-2B and 22Rv1 was analyzed by Western blot analysis. B: Depletion or inhibition of PKD2/3 reduced the viability of C4-2B and 22Rv1 cells transfected with PKD2 or PKD3 siRNAs or treated with CRT101 (5 μmol/L). C: Effects of PKD1 knockdown on the viability of C4-2B cells. Cell viability at 48 and 72 hours after PKD1 siRNA transfection was measured by using the CCK-8 assay. D: Depletion or inhibition of PKD2/3 suppressed C4-2B cell migration. Cells were transfected with PKD2 or PKD3 siRNAs or treated with CRT0066101 (5 μmol/L). Representative data from one of three independent experiments with seven to nine determinations at each time point are shown. E: Knockdown or inhibition of PKD2/3 blocked clonogenicity of C4-2B and 22Rv1 cells. PKD2 and PKD3 siRNA-transfected or CRT101-treated C4-2B and 22Rv1 cells were stained by crystal violet and quantified. Representative data from one of two independent experiments with triplicate determinations are shown. Data are expressed as means ± SEM from three independent experiments (B and C). ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001.

PKD2 and 3 Modulate the Expression of Bone Metastatic Genes in Prostate Cancer Cells

A number of metastatic genes, including osteopontin (OPN), osteocalcin (OCN), alkaline phosphatase, integrin 1β, talin1, focal adhesion kinase (FAK), MMP-9, IL-1α, and IL-1β, are known to contribute to the bone metastasis of cancer cells. These molecules regulate the process of bone metastasis and the phenomenon of tumor dormancy in the bone marrow, as well as create metastatic niches.21,22 The study evaluated the impact of silencing PKD2/3 on the expression of a panel of metastasis-related genes in prostate cancer cells. Several bone metastatic genes, including FAK, OCN, and OPN, decreased as a result of PKD2/3 silencing (Figure 3A). Collagenase I, the negative control, did not display any significant change in expression, either in response to silencing PKD2 and PKD3 or by CRT0066101 treatment (data not shown).

Figure 3.

Figure 3

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Depletion or inhibition of protein kinase D (PKD) blocks the expression of Runx2-regulated bone metastatic genes in prostate cancer cells. A: PC3-ML cells were transfected with PKD2 or PKD3 siRNA or treated with CRT101 (5 μmol/L). Transcript levels relative to the control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were determined. B: PC3-ML cells were transfected with empty vector (EV), Flag–catalytically active PKD2 mutant (PKD2-CA), and Flag–dominant negative PKD2 mutant (ie, PKD2-DN). Transcript levels of alkaline phosphatase (ALP), focal adhesion kinase (FAK), osteocalcin (OCN), and osteopontin (OPN) were determined by using real-time RT-qPCR. Top right: Confirmation of the overexpressed PKD2-CA and PKD2-DN in PC3-ML by Western blot analysis. C: Runx expression in prostate cancer cells. Whole cell extracts of RWPE-1, LNCaP, and PC3-ML cells were subjected to Western blot analysis for Runx1-3 expression. D: PC3-ML were transfected with a control siRNA (si-NT) or a Runx2 siRNA (si-Runx2), followed by real time RT-qPCR for the indicated genes. Left: Western blot analysis confirmed the knockdown of Runx2. E: Matrigel invasion assay was performed on PC3-ML cells transfected with si-NT and si-Runx2. Representative images are shown (top). Percent invasion is shown in the bar graph (bottom). Each real-time quantitative PCR experiment was repeated with duplicates at least two to three times, and data represent the means ± SEM from all independent experiments. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001. Original magnification, ×10 (E).

The impact of PKD2 on the expression of bone metastatic genes was further examined by overexpressing catalytically active PKD2-CA and dominant negative PKD2 mutant (ie, PKD2-DN) in PC3-ML cells. As shown in Figure 3B, PKD2-CA overexpression increased the expression of alkaline phosphatase, FAK, OCN, and OPN, whereas PKD2-DN blocked or did not affect the expression of these genes. Overexpression of PKD2-CA and PKD2-DN proteins was confirmed by Western blot analysis (Figure 3B). The regulation of bone metastasis genes by PKD2 and PKD3 implies a potential role of PKD in prostate cancer bone metastasis.

PKDs Regulates Bone Metastatic Gene Expression through Runx2

Runx2 knockdown decreases the migratory and invasive abilities of cancer cells.23 Therefore, whether silencing Runx2 inhibits the invasion of prostate cancer cells was investigated next. First, the expression levels of Runx1, Runx2, and Runx3 were determined in PC3-ML cells and then compared with those in other cell lines, including prostate epithelial RWPE-1 and androgen-dependent LNCaP prostate cancer cells. Runx1 and Runx2 were found to be highly expressed in RWPE-1 and PC3-ML cells, whereas Runx3 was undetectable in all three cell lines (Figure 3C). Runx1 and Runx2 mRNA levels were higher in PC3-ML cells than in LNCaP cells (data not shown).

Runx2 is a key regulator of bone metastasis in multiple cancers, including prostate cancer.19 Therefore, the role of Runx2 in PKD-regulated tumor metastasis in prostate cancer cells was investigated. Whether Runx2 was directly involved in metastatic gene expression in prostate cancer cells was examined first. As shown in Figure 3D, silencing of Runx2 resulted in the down-regulation of the same bone metastasis–associated genes, MMP-9, OCN, and OPN, which were regulated by PKD2/3. Functionally, knockdown of Runx2 resulted in approximately 20% inhibition of cell invasion compared with the control (Figure 3E). Thus, Runx2 contributes to the invasive properties of prostate cancer cells.

Depletion or Inactivation of PKD Accelerates Runx2 Degradation

To gain insights into the regulation of Runx2 by PKD in prostate cancer cells, the effects of altering PKD expression or activity on Runx2 mRNA and protein expression were examined. As shown in Figure 4A, knockdown of PKD2 by siRNA resulted in a small decrease in Runx2 mRNA levels, whereas knockdown of PKD3 did not significantly affect Runx2 transcription (Figure 4A). Interestingly, inactivation of PKD by CRT101 provided a >50% decrease in runx2 mRNA, possibly due to inhibition of both PKD2 and PKD3. Note that the effect of PKD3 on Runx2 transcription should not be completely excluded because the PKD3 siRNAs provided only partial knockdown of PKD3 (Figure 4B). Depletion or inactivation of PKD had no effect on Runx1 expression (Supplemental Figure S1A). The siRNAs were effective in knocking down PKD2 and PKD3 (Supplemental Figure S1, B and C). These data indicate that depletion of either PKD2 or PKD3 has a marginal effect on Runx2 transcription in prostate cancer cells, although a greater effect was observed when inhibiting both.

Figure 4.

Figure 4

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Inactivation or knockdown of protein kinase D2 (PKD2) causes down-regulation of Runx2 protein in prostate cancer cells. A and B: Transcript and protein levels of Runx1/2 and PKD2/3 were analyzed by using quantitative real-time RT-PCR (A) or Western blot analysis (B) in PC3-ML cells transfected with PKD2 or PKD3 siRNAs or treated with CRT101 (5 μmol/L). C: Immunofluorescence staining in tumor cells transfected with PKD2 and PKD3 siRNAs or treated with CRT101. The nuclei are counterstained with DAPI. Representative images from three independent experiments are shown. D:Top: PC3-ML cells were treated with cycloheximide with or without CRT101 for indicated times, and cell lysates were immunoblotted for Runx2. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as loading control. Bottom: Half-life of Runx2 protein was measured from the blots above. Data are expressed as means ± SEM of three independent experiments (A and D). ∗P < 0.05; ∗∗P < 0.01; ∗∗∗∗P < 0.0001. Original magnification, ×20 (C). DMSO, dimethyl sulfoxide.

The expression and localization of Runx2 was examined by immunoblotting and immunofluorescence staining before or after knockdown of PKD2 or PKD3 and inhibition of PKD activity by CRT101. As shown in Figure 4B, the knockdown or inactivation of PKD significantly down-regulated Runx2 at the protein level in PC3-ML cells. Accordingly, the overall intensity of Runx2 staining was significantly diminished upon depletion of PKD2/3 or inactivation of PKD, whereas its nuclear localization was not altered (Figure 4C). These data suggest that PKD regulates Runx2 protein expression, mainly at the posttranscriptional level, in prostate cancer cells. To further this analysis, whether PKD regulates the stability of Runx2 protein was examined. The cells were treated with either dimethyl sulfoxide or CRT101 at different time points in the presence of cycloheximide, an inhibitor of protein synthesis, and the levels of Runx2 protein were examined by Western blot analysis (Figure 4D). The data showed that the inhibition of PKD by CRT101 accelerated the degradation of Runx2. The protein half-life of Runx2 in the presence of CRT101 was reduced to 3.2 hours from 17.6 hours (dimethyl sulfoxide) (Figure 4D). Taken together, PKD positively regulates Runx2 at the posttranslational level by stabilizing Runx2 protein.

PKD Modulates Runx2 Activity through Ras/ERK1/2 Mitogen–Activated Protein Kinase Signaling Pathway

Emerging evidence suggests that PKDs modulate a plethora of cellular functions involved in tumorigenesis via the Ras/Raf/MEK/ERK1/2 mitogen–activated protein kinase (MAPK) signaling cascade.24 To gain mechanistic insights into PKD-regulated Runx2 activity in bone metastasis of prostate cancer, whether PKDs modulate Runx2 and the downstream target genes responsible for prostate cancer bone metastasis via the MEK/ERK1/2 axis was investigated. PC3-ML cells were transfected with an empty vector or a plasmid carrying constitutively active PKD2 (Flag-PKD2CA), and the activation of ERK1/2 and expression of Runx2 were determined by Western blot analysis (Figure 5A). Indeed, overexpression of active PKD2 increased both phosphor-ERK1/2 and Runx2 levels, and this effect was attenuated in PC3-ML cells treated with U0126, a highly selective inhibitor of MAPK/ERK kinases (Figure 5B). In line with these observations, treatment of PC3-ML cells with U0126 significantly down-regulated the mRNA levels of Runx2 and several genes responsible for bone metastasis, including FAK, OCN, and OPN (Figure 5C). Moreover, overexpression of PKD2 in U0126-treated PC3-ML cells rescued the reduced expression of Runx2, alkaline phosphatase, FAK, OCN, and OPN caused by U0126, as analyzed by quantitative real-time RT-PCR and Western blot analysis (Figure 5D). Taken together, these results suggest that PKD modulates Runx2 activity through the MEK/ERK1/2 MAPK signaling pathway.

Figure 5.

Figure 5

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Protein kinase D (PKD) regulates bone metastatic gene expression through mitogen-activated protein kinase kinase/extracellular signal–regulated kinase (ERK). A: Catalytically active PKD2 mutant (PKD2-CA) overexpression increased ERK activity. PC3-ML cells were transfected with an empty vector (EV) or Flag–PKD2-CA, followed by Western blot analysis for the indicated genes. B: U0126 blocked ERK1/2 phosphorylation in PC3-ML cells. C and D: PC3-ML cells were treated with the mitogen-activated protein kinase kinase inhibitor U0126. The transcript levels of Runx2, alkaline phosphatase (ALP), focal adhesion kinase (FAK), osteocalcin (OCN), and osteopontin (OPN) were determined by using quantitative real-time RT-PCR. Data are expressed as means ± SEM of three independent experiments (C and D). ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001. DMSO, dimethyl sulfoxide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Inhibition of PKD Blocks Accumulation of PC3-ML Cells in the Mouse Bone Marrow and Reduces Tumor Metastasis of Prostate Cancer Cells to Bone in Mice

PKD has been implicated in a variety of cancer-associated biological processes, including cancer cell growth, apoptosis, motility, and angiogenesis.4 However, its role in bone metastasis in vivo in the context of prostate cancer has not been elucidated. A mouse model of bone metastasis generated by cardiac injection of tumor cells was used to determine the potential role of PKD in bone metastasis by targeted inhibition of PKD using CRT101. This is a valuable model for bone metastasis research, as well as a standard and time-honored technique to investigate human cancer bone metastasis in vivo. Because fluorescent cells are required to detect the circulation and homing of cells in the bone, fluorescent PC3-ML ZsGreen cells were generated by stably expressing ZsGreen in PC3-ML cells and inoculating into the left cardiac ventricle of athymic nude mice as described previously.25 After the injection of cells, the animals were randomly divided into two treatment groups: vehicle and CRT101. Treatment with vehicle and the PKD inhibitor CRT101 (80 mg/kg) was administered orally daily for 4 weeks (Figure 6A). At the end of the experiment, the animals were sacrificed, and femur bones from each mouse were dissected. One femur bone from each mouse was used for decalcification followed by cryosectioning; the bone marrow of the other femur bones was flushed out and cultured in RPMI media. The body weights of animals recorded twice a week showed no significant changes during the experiment (at all time points, P > 0.05) (Figure 6B).

Figure 6.

Figure 6

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Protein kinase D (PKD) inhibitor CRT0066101 impairs metastatic dissemination of PC3-ML cells to the athymic mouse bone. A: Schematic representation for the treatment of mice. Male athymic nude mice (n = 12) were inoculated by intracardiac injection with the ZsGreen PC3-ML cell lines. Animals were treated with CRT101 (80 mg/kg, gavage daily) or vehicle (5% dextrose). B: Body weight was recorded twice a week (all P > 0.05, nonparametric U-test). C: Representative micrographs of cultured cells from each treatment group are shown. D: After 28 days, skeletal micrometastasis was identified by confocal microscopy. Representative images showing ZsGreen PC3-ML cell micrometastases (top). Percent decrease in skeletal micrometastasis on CRT101-treated animals after 28 days of injection was quantified (bottom). ∗∗∗P < 0.001. Original magnification, ×10 (C and D). BF, bright field.

First, whether CRT101 affects the migration and accumulation of PC3-ML cells in the bone marrow was determined. The presence of fluorescent ZsGreen-labeled cells in the bone marrow was analyzed in mice treated with or without CRT101. At the end of the experiment, the bone marrow cells were flushed out from the femurs and cultured in vitro. After 10 days of culture, a significant number of ZsGreen-labeled cells were found in the bone marrow of mice treated with vehicle compared with ZsGreen fluorescent cells detected in the bone marrow of CRT101-treated mice (Figure 6C).

Direct inoculation of prostate cancer cells in the blood circulation of immunodeficient mice has been shown to generate metastatic foci, primarily in the femur, tibia, jaws, and ribs.20 Therefore, PC3-ML cells were innoculated into the left cardiac ventricle of athymic nude mice. After completion of the treatment period of 28 days, the mice were sacrificed, and their femurs were harvested, fixed, and analyzed for the presence of micrometastatic foci. As shown in Figure 5D, microscopic tumors were found in the femur upon intracardiac inoculation with PC3-ML ZsGreen cells. Quantification analysis revealed that treatment of CRT101 in PC3-ML ZsGreen inoculated mice severely impaired the formation of micrometastases in the bone (Figure 6D). This result indicates that the inhibition of PKD impairs the accumulation of prostate cancer cells.

Discussion

Metastasis is the spread of cancer cells from the primary tumor, which initiates new tumors in the surrounding tissues and distant organs. To date, several signaling molecules that contribute to the metastasis of different cancer cells have been discovered. However, the mechanisms that lead to bone metastasis in prostate cancer are not completely understood. Although PKD is known to play important roles in cell proliferation, survival, migration, gene regulation, protein trafficking, and immune responses, its role in prostate bone metastasis has not been investigated. In this study, PKD was identified as a potential mediator of bone metastasis in prostate cancer. Depletion of PKD2/3 or inhibition of PKD blocked tumor proliferation, migration, and invasion at the cellular level, and inhibition of PKD reduced the formation of tumor micrometastases in a mouse bone metastasis model.

Several lines of evidence support the idea that PKDs contribute to the proliferation of multiple cancer cell types, including prostate cancer.16,26,27 To investigate the molecular effect of PKD2/3 on these properties, endogenous PKD2/3 were silenced by using siRNAs or inhibited by treatment of PKD inhibitor CRT101 in PC3-ML cells that predominantly express PKD2 and PKD3. Depletion of PKD2/3 resulted in the suppression of cell growth and proliferation, as seen in previous studies.18 This result was further substantiated by pharmacologic inhibition of PKD activity with a pan-PKD inhibitor, CRT101, which reduced colony formation. Similar effects on cell viability were observed for two AR-positive castration-resistant prostate cancer cell lines, C4-2B and 22Rv1. Because opposing effects of PKD1 on cell proliferation/viability have been reported in prostate cancer cells,15 the effects of PKD1 knockdown on cell viability were examined in C4-2B cells that predominantly express PKD1. Interestingly, knockdown PKD1 did not increase cell viability but rather inhibited it, although the overall effects were much smaller compared with those of PKD2/3, indicating a less significant role of PKD1 in prostate cancer cell viability. Taken together, these results indicate that PKD2/3, especially PKD2, are the main isoforms that contribute to the survival and proliferation of prostate cancer cells.

Because PKD2 and PKD3 coordinate to promote prostate cancer cell invasion,17 they may be directly involved in the invasion and migration of cancer cells. To explore this, PKDs were silenced and the migration of PC3-ML prostate cancer cells was analyzed using wound healing and invasion assays. Depletion of PKD2 and PKD3 in prostate cancer PC3-ML cells reduced cell migration and invasion. Although PC3-ML does not express PKD1, overexpression of PKD1 in prostate cancer LNCaP cells increases aggregation but reduces motility and invasiveness through interaction with and phosphorylation of E-cadherin.28 PKD2 silencing inhibits the migration of doxorubicin-resistant MCF7 cells without affecting chemoresistance.29 Zou et al17 also reported that PKD2/3 coordinates to promote prostate cancer cell invasion through p65 NF-κB and histone deacetylase 1–mediated urokinase-type plasminogen activator expression and activation.

The current study found that depletion of PKD resulted in a decrease in the transcription factor Runx2, which is involved in bone metastasis in prostate cancer.19 Runx2 is a member of the mammalian Runt homology domain transcription factor family. Runx proteins are involved in cell lineage determination during development and in various cancers.30 Runx2 is critical for the regulation of genes that support bone formation and is abnormally expressed in tumors that metastasize to bone.31 It activates several metastatic genes, including MMPs, vascular endothelial growth factor, OPN, and RANKL, in metastatic cancer cells.32 Depletion of Runx2 blocked the migration and invasion of prostate cancer cells and suppressed the expression of various bone metastasis–associated genes in PC3-ML cells. These data suggest that in Runx2-expressing prostate cancer cells, such as PC3-ML, Runx2 can act either independently or downstream of PKD2/3 to promote prostate cancer cell metastasis. In cells that do not express Runx2, such as LNCaP cells, PKD may act on different targets and have a different impact on cell migration and invasion, as shown for PKD1 in LNCaP cells.28 Mechanistically, the current data indicate that PKD regulated Runx2 mainly at the posttranscriptional level by stabilizing Runx2 protein in part through the MEK/ERK1/2 signaling pathway. However, transcriptional regulation of Runx2 by PKD may also be involved.

The current study also found that the PKD inhibitor CRT101 suppressed invasion and metastasis markers in prostate cancer cells. This finding is supported by several previous reports. Borges et al11 showed that treatment with CRT101 inhibits the growth of primary breast tumors and metastasis both in vivo and in vitro. CRT101 induces apoptosis in pancreatic cancer cells by suppressing the NF-κB pathway. It also inhibited the growth of pancreatic tumors in a xenograft model, correlating with changes in cyclin D1, survivin, and cellular inhibitor of apoptosis protein 1.13 It has also been shown to inhibit the migration and invasion of U87MG cells by inhibiting p44/42 MAPK and, to a smaller extent, p54/46 JNK and p38 MAPK activation.33 In the current study, CRT101 suppressed the invasion and migration of prostate cancer cells by blocking Runx2 expression at both transcriptional (minor effect) and posttranscriptional (major effect) levels. Importantly, we also found that the inhibitor CRT101 suppressed the bone metastasis of prostate cancer cells in a mouse metastasis model using intracardiac injection. These results indicate that the inhibition of PKD, either by genetic depletion or pharmacologic inhibition, suppressed bone metastasis in prostate cancer cells.

In conclusion, this study showed that PKD2/3 plays an important role in prostate cancer metastasis through the transcription factor Runx2. It also showed, for the first time, that a PKD inhibitor, CRT101, inhibited the migration and accumulation of prostate cancer cells to the bone in a mouse metastasis model. These findings may have significant therapeutic implications in bone metastatic diseases, as CRT101 has shown promising anticancer activities against various cancers.11,13,34

Acknowledgment

We thank Dr. Alvaro Gutierrez-Uzquiza in the Department of Systems Pharmacology and Translational Therapeutics, University of Pennsylvania, for invaluable discussions and guidance on intracardiac injection of prostate cancer cells in animals and Dr. Xuejing Zhang for help with the real-time PCR analysis.

Footnotes

Supported by NIH grant R01CA229431 (Q.J.W.).

Disclosures: None declared.

Supplemental material for this article can be found at http://doi.org/10.1016/j.ajpath.2023.01.004.

Author Contributions

Q.J.W. conceptualized the study; A.R., S.P., Y. Chao, J.Z., Y. Chen, Y.L. performed experiments; A.R., S.P., Y. Chao, J.Z., Y. Chen, Y.L., Q.J.W. analyzed data; Q.J.W. provided resources; A.R., S.P., Q.J.W. wrote the manuscript; A.R. and Q.J.W. reviewed and edited the manuscript; Q.J.W. supervised the study; and Q.J.W. acquired funding. All authors have read and agreed to the published version of the manuscript.

Supplemental Data

Supplemental Figure S1.

Supplemental Figure S1

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The effect of protein kinase D2 and D3 (PKD2/3) knockdown or inactivation on Runx1 mRNA expression in prostate cancer cells. PC3-ML cells were transfected with PKD2 or PKD3 siRNA for 48 hours or treated with CRT101 (5 μmol/L) for 24 hours. The transcript levels of Runx1 (A), PKD2 (B), and PKD3 (C) were determined by quantitative real-time RT-PCR with glyceraldehyde-3-phosphate dehydrogenase as the internal control. Data are expressed as means ± SEM from two independent experiments with triplicate determinations. ∗∗∗∗P < 0.0001.

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