While bone is a frequent target of breast cancer–associated metastasis, little is known about the effects of tumor–bone interactions on the efficacy of tumor-suppressing agents. Here we examined the effect of two FDA-approved dopamine modulators, fluphenazine and trifluoperazine, on mammary tumor cells, osteoclasts, osteoblasts, and osteocytes. These agents suppressed proliferation and migration of mammary tumor cells chiefly by antagonizing dopamine receptor D2 and reduced bone resorption by downregulating nuclear factor of activated T cells, cytoplasmic 1 (Nfatc1). Three-dimensional spheroid formation assays revealed that tumor cells have high affinity to osteocytes and type I collagen, and interactions with osteocytes as well as administration of fluphenazine and trifluoperazine downregulated Snail and suppressed migratory behaviors. Unlike the inhibitory action of fluphenazine and trifluoperazine on tumor growth, tumor–osteocyte interactions stimulated tumor proliferation by upregulating NFκB and Akt. In the bone microenvironment, osteocytes downregulated Snail and acted as an attractant as well as a stimulant to mammary tumor cells. These results demonstrate that tumor–osteocyte interactions strengthen dopamine receptor–mediated suppression of tumor migration but weaken its inhibition of tumor proliferation in the osteocyte-rich bone microenvironment.
Significance: These findings provide novel insight into the cellular cross-talk in the bone microevironment and the effects of dopamine modulators on mammary tumor cells and osteocytes. Cancer Res; 78(14); 3865–76. ©2018 AACR.
Bone is a frequent site of metastasis from cancers, as the bone microenvironment provides a rich bed of various hormones and cytokines for tumor growth (1). It is reported that TGFβ and its associated pathway drive a vicious cycle for tumor growth and bone resorption (2). Bone consists of many types of cells, including hematopoietic and mesenchymal stem cells in bone marrow, as well as bone-forming osteoblasts, bone-resorbing osteoclasts, and osteocytes, the most abundant cell type in the bone matrix (3, 4). Among bone marrow–derived cells of hematopoietic origin, T cells produce TGFβ and receptor activator of nuclear factor kappa B ligand (RANKL), which activate differentiation of macrophages to bone-resorbing osteoclasts (5). While the interactions of tumor cells with osteoclasts in the presence of TGFβ, RANKL, and interleukins have been extensively studied (6–8), interactions between tumor cells and osteocytes, which are thought to be a sensor for mechanotransduction (9), have not been fully explored.
In this study, we focused on two FDA-approved drugs, fluphenazine and trifluoperazine, and evaluated their roles in tumor–bone interactions. These two agents are modulators of dopaminergic signaling and currently used as antipsychotic medications (10, 11). The dopamine system is involved in many neurologic functions (12), but it is also important in other tissues, such as the cardiovascular and skeletal systems (13, 14). Dopaminergic signaling is mediated via G protein–coupled dopamine receptors (D1, D2, D3, D4, and D5), and regulates voluntary movements and reward responses (15). This pathway is integral for many neural functions, and its deficiency causes neurologic and psychiatric disorders such as Parkinson disease and schizophrenia (16). Dopaminergic signaling has recently been shown to be a potential target for preventing mammary tumor growth and bone metastasis using A77636, an agonist of DRD1 (17). Unlike fluphenazine and trifluoperazine, A77636 is not an FDA-approved drug. Furthermore, little is known about the effects of fluphenazine and trifluoperazine on tumor–bone interactions. In this study, we investigated their effects on mammary tumor cells, as well as bone-resorbing osteoclasts, bone-forming osteoblasts, and mechano-sensing osteocytes. Dopamine is reported to promote proliferation of bone-forming osteoblasts and mineralization of bone tissue (18).
To examine the efficacy of fluphenazine and trifluoperazine as well as interactions of mammary tumor cells with osteoclasts and osteocytes, we employed three dimensional (3D) spheroid cultures as well as monolayer cell cultures (19, 20). Interactions of tumor cells with bone cells were examined by premixing tumor and bone cells and forming a mosaic spheroid or fusing a pair of separately prepared spheroids consisting of tumor cells alone and bone cells alone. While we formed spheroids from 4T1.2 mammary tumor cells (21), RAW267.4 preosteoclast cells (22), MC3T3 osteoblast-like cells (23), and MLO-A5 osteocyte–like cells (24), our analysis of bone–tumor interactions was mostly focused on MLO-A5 cells in the presence and absence of fluphenazine and trifluoperazine. We used confocal microscopy to examine cellular morphology and viability of fluorescently labeled spheroids.
We hypothesized that fluphenazine and trifluoperazine suppress tumor growth and tumor-associated bone resorption via dopamine receptors. Because bone is one of the most frequent sites of metastasis from breast cancer, interactions of tumor cells with the most abundant cells in bone matrix is our research aim. We further hypothesized that tumor–osteocyte interactions may favor immobilization of tumor cells in the bone microenvironment and restrain efficacy of DRD2 inhibitors. Dopamine receptors are members of the DRD1-like family (DRD1 and DRD5) or DRD2-like family (DRD2, DRD3, and DRD4). To evaluate the role of DRD1-like and DRD2-like receptors in the responses to fluphenazine and trifluoperazine, we employed siRNAs specific to these receptors. In bone–tumor interactions, we focused on the role of Snail, a transcription factor involved in the epithelial-to-mesenchymal transition (EMT; ref. 25), ZO-1 (zonula occludens 1), as well as NFκB and AKT, two factors involved in tumor progression. ZO-1 is a tight junction–associated signaling protein, which mediates cell adhesion and is downregulated in EMT (26).
To evaluate direct and indirect tumor–bone interactions, we employed osteocyte-derived conditioned medium as well as a tumor–bone spheroid fusion assay. We examined the effects of secretory factors from osteocytes on tumor proliferation and tested the linkage of bone–tumor interactions to mesenchymal-to-epithelial transition (MET, a reversal of EMT) by determining and overexpressing Snail by plasmid transfection. Induction of MET at a potential site of metastasis for breast cancer cells has significant implications in regulating invasion and colonization of tumor cells in the bone microenvironment. These assays allowed us to evaluate the potential mechanism underlying bone–tumor interactions and their impact on the efficacy of fluphenazine and trifluoperazine. The results demonstrate that type I collagen and Snail play crucial roles in tumor–osteocyte interactions to enhance fluphenazine's and trifluoperazine's suppression of tumor migration and reduce their attenuation of tumor proliferation.
Materials and Methods
4T1.2 mouse mammary tumor cells (obtained from Dr. R. Anderson at Peter MacCallum Cancer Institute, Melbourne, Australia) were cultured in DMEM. RAW264.7 preosteoclast cells (ATCC TIB-71), MC3T3 osteoblast-like cells (Sigma-Aldrich), and MLO-A5 osteocyte–like cells (obtained from Dr. L. Bonewald at Indiana University, Indianapolis, IN) were grown in αMEM. Mycoplasma testing was conducted using a Lonza Mycoalert Plus Mycoplasma Kit (Lonza Inc.). For 4T1.2 cells, cell authentication testing was conducted using whole-exome DNA sequencing (Agilent SureSelect Mouse Exon). The culture media were supplemented with 10% FBS and antibiotics, and cells were maintained at 37°C and 5% CO2. Fluphenazine (Sigma-Aldrich) and trifluoperazine (Enzo Life Sciences) were employed as modulators of dopaminergic signaling. Fluphenazine (438 kDa) and trifluoperazine (407 kDa) are small synthetic agents used as antipsychotic medications in the treatment of schizophrenia. 4T1.2 cells were also treated with type I collagen (Corning). Cellular proliferation was examined using an MTT cell proliferation assay (Invitrogen) with the procedure described previously (17).
Transfection of Snail plasmid and siRNA for dopamine receptors D1 and D2 (DRD1 and DRD2)
For overexpressing Snail protein, 4T1.2 tumor cells were transfected with a plasmid consisting of Snail coding sequence (SnailHA_pcDNA3; Addgene), while a blank plasmid vector (FLAG-HA-pcDNA3.1; Addgene) was used as a control. 4T1.2 cells were also treated with siRNA specific to Drd1 (Life Technologies; siRNA: s65128) and Drd2 (161556). As a nonspecific control, a negative siRNA (Silencer Select #1, Life Technologies) was used. Cells were transiently transfected with siRNA in Opti-MEM I medium with Lipofectamine RNAiMAX (Life Technologies). Twenty-four hours later, the medium was replaced by regular culture medium with reagents. The efficiency of silencing was assessed with immunoblotting 24 hours after transfection.
Two-dimensional motility assay
A wound-healing scratch motility assay was utilized to evaluate two-dimensional cell motility (27). In brief, cells were grown on 12-well plates, and a plastic tip was used to scratch a gap onto the cell layer. After incubation, the areas newly occupied with cells in the scratched zone were imaged and measured with ImageJ (NIH, Bethesda, MD).
Osteoclast differentiation assay
Using RAW264.7 preosteoclast cells, an osteoclast differentiation assay was conducted in 96-well plates (28). During 6-day experiments of growing cells in 20 ng/mL of RANKL, the culture medium was exchanged once on day 4. Adherent cells were fixed and stained with a tartrate-resistant acid phosphate (TRAP)-staining kit (Sigma-Aldrich), according to the manufacturer's instructions. TRAP-positive multinucleated cells (>3 nuclei) were identified as mature osteoclasts and counted.
Western blot analysis
Cells were lysed with a radioimmunoprecipitation assay buffer supplemented with protease inhibitors (Santa Cruz Biotechnology) and phosphatase inhibitors (Calbiochem). Isolated proteins were fractionated using 10%–15% SDS gels and electrotransferred to polyvinylidene difluoride membranes (Millipore). We used antibodies against ATF4, p-Akt, AKT, caspase-3, cathepsin K, eIF2α, p-eIF2α, lamin B, Lc3A/B II, NFATC1, NFκB, p-NFκB, SNAIL, ZO-1 (Cell Signaling Technology), and β-actin (Sigma). Protein levels were assayed using a SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific).
Formation and viability of 3D spheroids
Cells were cultured in ultralow attachment 96-well plates (S-BIO) at 1 × 104 cells/well for 4T1.2 cells, 2 × 104 cells/well for A5 cells, and 2 × 104 cells/well for RAW cells. Cells were imaged every 24 hours, and area and roughness were calculated with ImageJ. To observe spheroid formation of mixtures of cancer cells with osteocyte and osteoblast cells, IncuCyte ZOOM real-time imaging microscope was used to image cells every hour with fluorescent staining (Incucyte CytoLight Green and Red, Essen Bioscience). In combination, 4T1.2, A5, and RAW cells were seeded at 1 × 104 cells/well, 3 × 103 cells/well, and 2 × 103 cells/well, respectively. A three-dimensional spheroid viability assay was conducted by plating 4T1.2, MLO-A5, and RAW 264.7 cells on ultralow attachment 96-well plates at concentrations of 1 × 104, 2 × 104, and 2 × 104 cells/well, then incubated for 24 hours. Treatment medium was exchanged, and spheroids were incubated for 72 hours. The cell viability was measured with CellTiter-Glo 3D Cell Viability Assay (Promega). To visualize 3D spheroid proliferation in response to medium exchange and drug treatment, a fluorescence-based cell proliferation kit was used (Click-iT EdU Alexa Fluor 488 Imaging Kit; Thermo Fisher). After 24 hours, cells were incubated with EdU for 3 hours, then fixed and labeled, as per manufacturer's instruction. Confocal microscope images were acquired with FV1000 (Olympus) and analyzed using ImageJ.
The experimental procedures using animals were approved by the Indiana University Animal Care and Use Committee and were in compliance with the Guiding Principles in the Care and Use of Animals endorsed by the American Physiological Society. Mice were housed five per cage and provided with mouse chow and water ad libitum. In the mouse model of mammary tumor (29), 23 BALB/c female mice (∼6 weeks, Harlan Laboratories) received subcutaneous injections of 4T1.2 cells (5.0 × 105 cells in 50 μL PBS) to the mammary fat pad on day 1. Fluphenazine or trifluoperazine (1 mg/kg body weight) was administered subcutaneously into the area of cell injection every day, while the placebo animals received a vehicle control. The animals were sacrificed on day 18, and the weight of each tumor was measured. In the mouse model of bone metastasis (30), 33 BALB/c female mice received injection of 4T1.2 cells (1.0 × 105 cells in 50 μL PBS) to the right iliac artery. Fluphenazine or trifluoperazine was administered daily via intraperitoneal injection at 2 mg/kg body weight, and the animals were sacrificed on day 17. A whole body X-ray of each mouse was taken, and the tibia and femur were harvested for mechanical testing, μCT imaging, and histology. To examine the role of type I collagen in the bone microenvironment, 10 BALB/c female mice were employed, and type I collagen was coinjected with 4T1.2 cells (5 × 105 cells in 20 μL PBS with 50 μg/mL type I collagen) into the intratibial cavity.
Micro-CT was performed using Skyscan 1172 (Bruker-MicroCT; ref. 31). The harvested bone samples were wrapped in parafilm to maintain hydration and placed in a plastic tube and oriented vertically. Scans were performed at pixel size 8.99 μm. Using manufacturer-provided software, the images were reconstructed (nRecon v220.127.116.11) and analyzed (CTan v1.13).
Mechanical testing and histology
Mechanical testing was conducted using an ElectroForce loading device (TA Instruments). A sinusoidal force (1 N peak-to-peak force) was applied at 1 Hz, and the slope of force–displacement relationship was determined as tibial stiffness. For histology, bone samples were fixed in 10% neutral buffered formalin and decalcified in a 14% EDTA solution. They were then dehydrated through a series of graded alcohols, cleared in xylene, and embedded in paraffin. H&E staining was conducted on the sagittal sections, and the distribution of tumor cells in the tibial bone cavity was determined.
The data were expressed as mean ± SD. One-way ANOVA was employed to examine statistical significance among groups, and Fisher protected least significant difference was conducted post hoc to evaluate pairwise comparisons. Statistical significance for P < 0.05 was assumed.
Inhibition of proliferation and differentiation of RAW264.7 preosteoclast cells
To evaluate the effects of fluphenazine and trifluoperazine on bone-resorbing osteoclasts, we conducted in vitro analysis using RAW264.7 preosteoclast cells. First, the relative proliferation of RAW264.7 cells using an MTT cell proliferation assay was significantly decreased by fluphenazine in a dose-dependent manner (Fig. 1A). Second, in response to RANKL-driven osteoclastogenesis, the phosphorylation level of eIF2α was elevated, and genes related to osteoclast function, such as NFATc1 and Cathepsin K, were downregulated by treatment with 1, 5, and 10 μmol/L fluphenazine (Fig. 1B and C). The same trends were observed by trifluoperazine treatment (Fig. 1D–F). Third, the number of TRAP-positive, multinucleated cells induced by RANKL administration was decreased by fluphenazine and trifluoperazine (Fig. 1G and H). Finally, 3D viability of RAW264.7 spheroids was significantly reduced by 10 and 20 μmol/L trifluoperazine (Fig. 1I and J). Collectively, the result showed that fluphenazine and trifluoperazine inhibited proliferation and differentiation of RAW264.7 preosteoclast cells.
To evaluate the effects of fluphenazine and trifluoperazine on bone formation, we also examined bone-forming osteoblasts (MC3T3 osteoblast-like cells), as well as osteocytes (MLO-A5 osteocyte-like cells). Unlike their inhibitory effect to RAW264.7 cells, 5 μmol/L fluphenazine and trifluoperazine did not significantly reduce proliferation of MC3T3 cells (Supplementary Fig. S1A). These agents elevated the protein levels of p-eIF2α and ATF4, a transcription factor important for bone formation (Supplementary Fig. S1B). Furthermore, they elevated osteoprotegerin (Opg) mRNA and osteocalcin (Ocn) mRNA (Supplementary Fig. S1C). Opg is an inhibitory factor of osteoclastogenesis, while Ocn is a marker for bone formation. The same trends in cell proliferation and protein expression were found in MLO-A5 cells (Supplementary Fig. S1D and S1E). In a 3D spheroid formation assay, the agents did not reduce spheroid size or luminescence-based survival of MC3T3 and MLO-A5 cells (Supplementary Fig. S1F and S1G).
Inhibitory effects on proliferation and migration of 4T1.2 mammary tumor cells
Thus far, the results with RAW264.7 cells, MC3T3 cells, and MLO-A5 cells support the action of these agents in protecting bone from resorption. Next, we examined their effects on 4T1.2 mammary tumor cells. First, the result from an MTT cell proliferation assay revealed that these agents inhibited proliferation of 4T1.2 cells (Fig. 2A). The levels of p-eIF2α, a marker for integrated stress, cleaved caspase, an apoptosis marker, and LC3A/B II, an autophagy marker, were elevated (Fig. 2B–D). Of note, the elevation of the autophagy marker indicates induction of a prosurvival pathway along with induction of apoptosis. A 2D motility assay showed that both agents reduced the healing of the scratched wound in a dose-dependent manner at 1, 5, and 10 μmol/L (Fig. 2E; Supplementary Fig. S2A and S2B).
Next, fluphenazine and trifluoperazine action through dopamine receptors was examined. RNA interference using Drd1 siRNA and Drd2 siRNA, two dopamine receptor genes, revealed that partial silencing of Drd2 significantly suppressed fluphenazine/trifluoperazine -induced inhibition of tumor proliferation and migration (Fig. 2F–H). Compared with the effect of Drd2 siRNA, that of Drd1 siRNA was less significant (Supplementary Fig. S2C–S2E). Using MDA-MB-231 breast cancer cells, we also confirmed that fluphenazine and trifluoperazine inhibited their proliferation (Supplementary Fig. S3A), elevated p-eIF2α, cleaved caspase-3, and LC3A/B II (Supplementary Fig. S3B), and decreased migration (Supplementary Fig. S3C). In MCF7 breast cancer cells, the same trends in proliferation (Supplementary Fig. S4A), protein expression (Supplementary Fig. S4B), and migration (Supplementary Fig. S4C) were observed. The result from the MTT cell proliferation assay was supported by an EdU cell proliferation assay, in which the relative proliferation rate of 4T1.2 cells, MDA-MB-231 cells, and MCF-7 cells was reduced by fluphenazine and trifluoperazine in a dose-dependent manner (Fig. 3A–C).
Expansion of the necrotic core in 4T1.2 spheroids by fluphenazine and trifluoperazine
We further examined the effects of these agents on tumor growth using 3D 4T1.2 tumor spheroids. We observed that the tumor spheroid developed a three-layered spherical structure composed of a necrotic core at the center, a quiescent shell around the necrotic core, and a proliferating zone on the surface (Fig. 3D). In response to 1, 5, and 10 μmol/L fluphenazine and trifluoperazine, the necrotic core expanded along incubation time in a dose-dependent manner (Fig. 3D–F). Consistent with the expansion of the necrotic core, the luminescence level in the 3D viability assay was decreased by drug treatment (Fig. 3G), associated with an increased level of cleaved caspase-3 (Fig. 3H).
Suppression of tumor growth and bone loss in a mouse model
After examining the actions of fluphenazine and trifluoperazine on bone cells and tumor cells in monolayer cell cultures and in 3D spheroids, we conducted in vivo analysis using mouse models of mammary tumor and bone metastasis. First, 4T1.2 cells were inoculated to the mammary fat pad, and development of the mammary tumor was monitored (Fig. 4A–D). The results revealed that the size and weights of the tumors were significantly reduced by daily administration of fluphenazine or trifluoperazine. Second, we induced the migration of tumor cells into the right hind limb by injecting 4T1.2 cells into the right iliac artery. Using μCT images, we reconstructed the 3D structure of trabecular bone in the distal femur (Fig. 4E). Compared with placebo, fluphenazine- and trifluoperazine-treated samples presented a denser distribution of trabecular bone segments in the distal femur, with an increase in trabecular thickness (Fig. 4F). Compared with placebo, the bone volume was significantly higher in the fluphenazine-treated group, but not in the trifluoperazine-treated group. In response to four-point bending mechanical test, the femur stiffness was greater in both fluphenazine- and trifluoperazine-treated groups (Fig. 4G).
Type I collagen is the most abundant protein in bone matrix. To evaluate the role of type I collagen in tumor growth and migration in the bone microenvironment, we coinjected type I collagen with 4T1.2 tumor cells into the left tibial cavity and conducted mechanical testing and histologic analysis. Mechanical testing revealed that compared with the contralateral tibia, tibial stiffness was significantly lowered after 2–3 weeks (Fig. 4H and I; Supplementary Fig. S5A). However, coinjection of collagen suppressed the reduction of stiffness. Histologic analysis showed that coinjection of collagen facilitated scattering tumor cells throughout the tibial bone cavity by reducing their aggregation (Fig. 4J; Supplementary Fig. S5B).
Reduced inhibitory effects of fluphenazine and trifluoperazine by interactions with RAW264.7 cells
The results so far indicate that the two agents in this study significantly alter the fate of tumor growth as well as bone remodeling. To understand the action of these agents in the bone microenvironment, we next examined interactions of 4T1.2 spheroids with RAW264.7 cells in the presence and absence of these agents. First, we evaluated indirect interactions using the conditioned medium isolated from RAW264.7 preosteoclast cells. RAW264.7 conditioned medium expanded the quiescent zone with an increase in live cells (Supplementary Fig. S6A). The 3D viability assay revealed that the conditioned medium elevated the number of live cells in the presence and absence of fluphenazine and trifluoperazine at 1 and 5 μmol/L (Fig. 5A and B). In response to RAW264.7 preosteoclast-derived conditioned medium, expression of SNAIL, a transcription factor for inducing EMT, was upregulated (Fig. 5C). In a 3D coculture, 4T1.2 cells did not mix with RAW264.7 cells, and they formed two separate spheroids (Supplementary Fig. S6B and S6C). In a spheroid fusion assay, 20 μmol/L fluphenazine and trifluoperazine reduced the size of RAW264.7 spheroids.
Regulation of Snail and p-Akt by interactions with MLO-A5 cells
To further understand the interactions in the bone microenvironment, we examined 4T1.2 spheroids with osteocytes. Incubation of 4T1.2 spheroids with conditioned medium from MLO-A5 osteocyte cells showed a decrease in surface roughness and an increase in relative proliferation (Fig. 5D and E; Supplementary Fig. S6D). Confocal microscopy of 4T1.2 spheroids illustrated that, compared with control, MLO-A5–conditioned medium increased the number of Click-iT EdU–labeled dividing cells (Fig. 5E). Western blot analysis revealed that SNAIL was downregulated in 4T1.2 cells by MLO-A5–conditioned medium as well as fluphenazine and trifluoperazine (Fig. 5F–H). Of note, RAW264.7-conditioned medium elevated the protein level of SNAIL (Fig. 5C). However, the level of p-AKT was elevated by MLO-A5–conditioned medium and reduced by fluphenazine and trifluoperazine (Fig. 5F–H). The level of SNAIL was further reduced by simultaneous application of osteocyte-derived conditioned medium and fluphenazine/trifluoperazine (Fig. 5H). Unlike RAW264.7 cells, MLO-A5 cells freely mixed with 4T1.2 cells, and these two types of cells formed a single integrated spheroid (Fig. 5I). In the presence of MLO-A5 spheroid, the diameter of the 4T1.2 spheroid was slightly decreased, and administration of 20 μmol/L fluphenazine and trifluoperazine further reduced the diameter (Fig. 5J).
Reduction in Snail by incubation with type I collagen
Osteocytes are surrounded by bone matrix, in which type I collagen is the most abundant protein. In response to MLO-A5–derived conditioned medium, downregulation of SNAIL and upregulation of p-AKT were sustained for 24 hours (2, 3, 6, 12, and 24 hours) (Fig. 6A). To evaluate the potential role of type I collagen in interactions of tumor spheroids with MLO-A5 osteocytes or their conditioned medium, we cultured 4T1.2 tumor spheroids in the presence of type I collagen at 0.1 to 10 μg/mL (Fig. 6B) and measured their protein expression in 2D culture (Fig. 6C). Compared with tumor spheroids in the control medium, the medium with type I collagen made spheroids smaller and more circular with smoother surfaces (Fig. 6B, D, and E). Associated with those phenotypic alterations, SNAIL protein was downregulated, and 4T1.2 cells became less migratory in a dose-dependent manner (Fig. 6C, F, and G). Furthermore, the protein levels of ZO-1, p-NFκB, and p-AKT were elevated (Fig. 6C). In the wound -healing assay, motility of 4T1.2 cells was reduced by collagen, fluphenazine, and trifluoperazine, and the simultaneous application of collagen with fluphenazine/trifluoperazine further suppressed the migratory behavior of tumor cells (Fig. 6F and G).
Partial suppression of A5 medium effects by Snail overexpression
The results so far show that direct and indirect interactions of tumor cells with osteocytes shrink tumor spheroid size and alter expression of SNAIL and AKT. SNAIL is downregulated by fluphenazine and trifluoperazine as well as tumor–osteocyte interactions. We further evaluated the role of Snail using a wound-healing assay, in which migratory behaviors of 4T1.2 cells were suppressed by MLO-A5 conditioned medium (Fig. 7A and B). When Snail plasmid was transfected into 4T1.2 cells to elevate its expression, ZO-1 is downregulated (Fig. 7C), and the surface of the tumor spheroids became rougher, regardless of treatment with MLO-A5 conditioned medium or fluphenazine/trifluoperazine (Fig. 7D and E). Furthermore, compared with the vector control, Snail overexpression partially suppressed A5 medium–driven inhibition of cellular motility (Fig. 7F and G).
This study presented that tumor–osteocyte interactions downregulated expression of Snail in 4T1.2 tumor cells and reduced the migratory behavior of 3D tumor spheroids. The interactions also elevated DNA replication in the proliferating surface of tumor spheroids by activating NFκB and Akt pathways. Compared with MLO-A5 osteocyte cells, RAW264.7 preosteoclast cells showed weaker affinity to tumor cells and Snail overexpression. Tumor–osteocyte interactions as well as type I collagen suppressed the inhibitory effect of fluphenazine and trifluoperazine on the proliferation of tumor cells, while they enhanced fluphenazine/trifluoperazine's suppression of tumor cell migration. Collectively, osteocytes appear to act as an attractant of tumor cells to the collagen-rich bone matrix, while osteoclasts act as a desensitizer of the action of fluphenazine and trifluoperazine on tumor cells in the 3D bone-like microenvironment. In the proposed mechanism (Fig. 7H), Drd2 and osteocytes both suppress Snail, but they have opposing effects on tumor proliferation markers Akt and NFκB. Collagen-rich osteocytes promote tumor cell aggregation, while Drd2 attenuates migratory tumor behavior.
Fluphenazine is an antagonist of dopamine receptors D1 and D2, while trifluoperazine is an antagonist of D2 dopamine receptor (32). Trifluoperazine is also known to antagonize calmodulin (33). We have previously reported that A77636, a selective D1 dopamine receptor agonist, prevents tumor growth and bone resorption (17). It is reported that the role of D1 is opposite to that of D2, and thus an agonist of D1, like A77636, may exert the same effect of an antagonist of D2, like fluphenazine and trifluoperazine. We have previously reported that A77636′s action is in part mediated by the elevated phosphorylation of eIF2α (17). A77636 stimulated dopamine- and cAMP-regulated phosphoprotein (DARPP32) that inhibits protein phosphate 1 and elevates the level of p-eIF2α. Furthermore, elevated p-eIF2α was shown to suppress tumor cell migration via inhibition of Rac1 and prevent bone resorption via suppression of NFATc1. Consistent with our previous study, we have also observed dose-dependent upregulation of p-eIF2α in 4T1.2 tumor cells and RAW264.7 preosteoclast cells by fluphenazine and trifluoperazine. In response to partial RNA silencing of Drd1 and Drd2, the result indicated that fluphenazine/trifluoperazine -driven suppression of proliferation and migration of tumor cells was mainly mediated by Drd2.
In evaluating interactions of tumor cells to MLO-A5 cells and RAW264.7 cells, we employed 3D spheroid assays as well as monolayer cell cultures with conditioned media. Experiments with conditioned media allowed us to evaluate the effects of secretory factors, while spheroid coculturing and fusion led to evaluation of direct interactions more relevant to the physiologic microenvironment than monolayer cultures. In response to fluphenazine and trifluoperazine, the necrotic core of tumor spheroids was enlarged, and ATP availability on the proliferating surface was reduced in a 3D viability assay. Unlike RAW264.7 cells, which did not fuse well with 4T1.2 cells, MLO-A5 cells fused with 4T1.2 cells to merge completely. Conditioned medium from RAW264.7 cells spread tumor spheroids and expanded the proliferating zone on the spheroid surface. The conditioned medium from MLO-A5 cells, however, reduced surface roughness and slightly shrank the tumor spheroids. Although tumor spheroid size was reduced by fusion with MLO-A5, MLO-A5–conditioned medium elevated DNA replication of the proliferating zone of tumor spheroids. Taken together, MLO-A5 cells appeared to capture and nurture tumor cells.
To understand the mechanism of action in tumor–osteocyte interactions, we evaluated the role of type I collagen, the most abundant protein in bone matrix and rich in osteocyte-derived conditioned medium. We observed that type I collagen acted as an attractant and dwarfing agent of migratory tumor cells and converted them to less migratory, more proliferative cells. When collagen was heat-denatured, it lost its ability to attract migratory tumor cells. It is conceivable that interactions of type I collagen with tumor cells are mediated by integrins such as α1β1, α2β1, and α11β1, and other bone matrix proteins may assist interactions via binding to collagens (34). Consistent with in vitro results, histologic analysis showed that coinjection of type I collagen with tumor cells promoted the mixing and scattering of tumor cells, diffusing through the bone marrow cavity to form small but widely spread clusters. Collagen coinjection also reduced the weakening of bone stiffness measured by in vivo mechanical testing.
Incubation with type I collagen downregulated Snail, a transcription factor involved in EMT, and upregulated ZO-1, p-AKT, and p-NFκB. Snail is regulated by various signals at the level of transcription as well as posttranslation (35). Zo-1 is associated with adhesion and tight junction linked cell–cell communication, while Akt and NFκB contribute to various mechanisms, including cellular proliferation. Bone matrix contains a variety of matrix proteins, and, in addition to type I collagen, the involvement of other proteins need to be evaluated. These results raise the possibility that matrix proteins, including type I collagen, may act as inducers of immobilization and proliferation of tumor cells in the bone microenvironment, which is commonly observed in MET (36), the reversal of EMT. Further investigation of molecular and cellular behaviors of tumor cells is needed to understand the role of tumor–osteocyte interactions in MET.
Clinical reports on the usage of dopamine modulators on the risk of breast cancer vary. In approximately 140,000 cases of dopamine-deficient Parkinson disease patients, administration of dopamine antagonist is shown to lower rates of breast cancer, even factoring in depression and suicide risk (37). In a separate study of approximately 53,000 cases, usage of dopamine antagonists increased the risk of breast cancer by 16% (38). In schizophrenia patients (∼6,000 cases), results vary widely, ranging from 52% risk increase to 40% risk decrease (39). No clinical data are available for potential effects of fluphenazine and trifluoperazine on bone metastasis.
The results herein establish a link between bone cell secretions and tumor activity, although there are several limitations that should be noted. Dopamine receptor modulators fluphenazine and trifluoperazine and osteocyte-conditioned medium both reduced spheroid size and tumor migration, but they had opposing effects on cell proliferation. This study used mouse-derived cell lines of osteoblasts, osteoclasts, and osteocytes, as well as a triple-negative mammary tumor mouse cell line. Human cell lines as well as other breast cancer types should be used to translate these findings toward clinical relevance. Although these existing drugs are well-studied for use in psychiatric patients, their side effects must be carefully considered, or they might be selectively delivered to tumor cells. While fluphenazine and trifluoperazine were analyzed as antagonists to DRD2, these agents may have limited selectivity and interact with other DRDs.
Much previous work has shown the importance in osteoclast feedback with tumor cells in bone metastasis, but our work explores the role of osteocytes. This study demonstrates that tumor–osteocyte interactions decrease Snail expression and increase Akt and NFκB signaling in tumor cells, and those interactions alter the efficacy of dopamine-modulating fluphenazine and trifluoperazine. In the bone microenvironment, tumor–osteocyte interactions strengthen inhibition by fluphenazine and trifluoperazine on tumor migration but weaken their suppression of tumor proliferation. Furthermore, the result presents a mechanistic rationale for possible future research directions in using collagen-based or other osteocyte-derived secretory factors to capture or target metastatic tumor cells. Those factors might also be useful in disrupting the communication between osteocytes and metastatic tumor cells.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Conception and design: S. Liu, A. Chen, A. Jalali, K. Minami, H. Nakshatri, B.-Y. Li, H. Yokota
Development of methodology: S. Liu, Y. Fan, A. Chen, H. Yokota
Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): S. Liu, Y. Fan, A. Chen, A. Jalali, K. Ogawa
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): S. Liu, Y. Fan, A. Chen, A. Jalali, K. Minami, K. Ogawa, H. Yokota
Writing, review, and/or revision of the manuscript: S. Liu, A. Chen, A. Jalali, K. Minami, B.-Y. Li, H. Yokota
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): S. Liu, Y. Fan, A. Chen, A. Jalali, K. Minami, H. Nakshatri, H. Yokota
Study supervision: S. Liu, A. Jalali, H. Yokota
The authors appreciate Chie Deguchi and Rika Kondo for technical support. This study was in part supported by funds from 100 Voices of Hope (to H. Nakshatri and H. Yokota) and NIH/NIAMS (NIH R01 AR052144; to H. Yokota).