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Effect of CD133 overexpression on bone metastasis in prostate cancer cell line LNCaP

Prostate cancer (PC) metastasizes to the bone, and a small number of cancer cells, described as cancer stem cells (CSCs), have the ability to differentiate into tumor cells. CSCs are responsible for tumor recurrence and metastases. In the present study, we examined whether ectopic overexpression of CD133, a key molecule maintaining the stability of CSCs in the human PC cell line, LnCaP, caused bone metastasis in a mouse model. Ectopic overexpression of CD133 was induced in LnCaP cells, and CSC-related protein expression was measured. Furthermore, a colony-forming assay was performed to compare results against the blank green fluorescent protein-expressing cells. Furthermore, epithelial to mesenchymal transition-related protein expression, cell migration and wound healing were investigated. To assess the role of CD133 in bone metastasis, CD133-overexpressing LnCaP cells were inoculated into mice via intracardiac injection, and bone metastasis was assessed via histological and immunohistochemical study. In addition, cytokine arrays were used to determine the cytokines involved in bone metastasis. Ectopic overexpression of CD133 in LnCaP cells increased CSC properties such as Oct-4 and Nanog expression and colony-forming ability. Furthermore, epithelial-to-mesenchymal transition (EMT) properties, including decreased E-cadherin and increased vimentin expression, wound gap distance, and cell migration increased. CD133 overexpression led to formation of bone metastatic tumors in mice, consistent with results of hematoxylin and eosin staining. In addition, an increase in expression of the macrophage-migration inhibitory factor was observed at the tumor margin in mice inoculated with CD133+ LNCaP cells. These findings suggest a regulatory role of CD133 in stem cell and EMT properties, and the sustained acquisition of osteolytic features in PC. Therefore, our results may facilitate development of a novel classification system and therapeutic strategies for bone metastasis of PC.


Introduction
Prostate cancer (PC) is the most commonly diagnosed form of cancer and the sixth leading cause of cancer-related deaths among men worldwide (1). The 5-year survival rate is approximately 100% in patients with localized PC, but it drops to 31% in those with distant metastasis (2). Similar to most other solid malignancies, PC may metastasize to distant organs such as the liver, lungs, and brain; however, it has an abnormally high propensity to metastasize to bones (3). Thus, it is desirable to gain a better understanding of mechanisms underlying PC metastasis to the bone for development and use of therapies to improve patient survival.
Studies have provided new insights into this advanced disease based on critical molecular and cellular events surrounding tumor progression, invasion, and metastasis to the bone and other sites (4). Epithelial-to-mesenchymal transition (EMT), in which cellular, morphological, and functional switches may transform adherent epithelial cells to migratory mesenchymal cells, is critical for tumorigenic progression and cancer metastasis. EMT may promote stem cell-related properties and generate cells with features related to tumor initiation (5). During EMT, tumor cells exhibit stem cell characteristics and confer invasive and migratory properties to primary tumor cells, ultimately resulting in metastasis (5).
It is hypothesized that tumors rely on a small portion of cells called cancer stem cells (CSCs) that have the ability to self-renew and generate multiple 'mature' tumor progenies (6). CSCs are typically dormant, and their regrowth is responsible for metastases (7). Primary tumors comprise a majority of differentiated epithelial cells and a small number of cells expressing stem cell markers (i.e., PC stem cells) (8).

Effect of CD133 overexpression on bone metastasis in prostate cancer cell line LNCaP
A previous study showed that CSCs derived from primary tumors exhibited increased invasion, self-renewal, and clonogenic properties (9). These cells may be identified using several CSC markers such as CD133, c-Met, and prostate stem cell antigens (10). A recent analysis involving a cohort of high-risk PC patients showed that the number of positive cancer cells for the a-6 and a-2 integrin subunits and the c-Met receptor in primary PC correlated with bone metastatic progression (11). CD133 (prominin-1), a 5-transmembrane glycoprotein, was originally recognized as a hematopoietic stem cell marker (12). CD133 is regarded as an important cell surface marker used to identify cancer-initiating cell subpopulations in brain tumors, colon carcinoma, head and neck cancer, hepatocellular carcinoma, thyroid carcinoma, and prostate carcinoma (13,14). Previous experiments have demonstrated a correlation between upregulated CD133 expression and progression of EMT in head and neck cancer cells and head and neck squamous cell carcinoma tissues (15). The CD133-mediated molecular mechanisms regulating cancer-initiating cells in bone metastasis of PC remain unclear.
In the present study, we have demonstrated that CD133 plays a significant role by increasing stemness, enhancing EMT, and promoting bone metastasis in a PC cell line, LNCaP. Furthermore, we aimed to identify cytokines secreted by CD133+ LNCaP cells that play a critical role during bone metastasis of PC cells. Our results suggest that CD133+ PC cells play a central role in bone metastasis.
Cloning of human CD133 and establishment of stably transfected LNCaP Vec control and LNCap CD133+ cells. HT29 colon cancer cells were used as a source of CD133. Cloning of CD133 was performed as previously described (15). Plasmid pcDNA3.1/N-terminal green fluorescent protein (NT-GFP) lacking the CD133 insert was used as a transfection control. 293T cells were used as a positive control to evaluate the translated fusion product.
Colony-forming assays. Cells were seeded at a density of 1,000 cells/well in non-adherent 24-well culture plates coated with a 10% poly (2-hydroxyethyl methacrylate) (polyHEMA) solution (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) in absolute ethanol and then dried overnight. After seeding, cells were incubated in serum-free Dulbecco's modified Eagle's medium (DMEM) supplemented with 200 ng/ml epidermal growth factor (Sigma-Aldrich; Merck KGaA), 20 ng/ml of basic fibroblast growth factor (Sigma-Aldrich; Merck KGaA), and B-27 supplement (Invitrogen; Thermo Fisher Scientific, Inc.). After 5 days of incubation, the number of spheroids in each well was counted using a light microscope (Zeiss, Zena, Germany).
Cell migration assay. Cells were seeded at 1x10 5 cells/well in 6-well plates and cultured to approximately 90% confluency in 1 ml of DMEM supplemented with 10% FBS. The media were removed from the wells, and a straight transverse line was drawn through the adherent cells using a ruler and sterile 200-µl plastic micropipette tip to produce a uniform gap. Serum-free DMEM was added, and the distance between the gaps was measured immediately and 24 h later, following image capture of six random microscopic fields. The distance measured immediately was defined as 100%.
Cell invasion assay. Cell invasion was assessed using a chemotaxis chamber (Neuro Probe, Gaithersburg, MD, USA). The cells (5x10 4 cells in 0.35-ml serum-free DMEM per well) were seeded into the top chamber of a 10-well invasion chamber assay plate. DMEM supplemented with 10% FBS was placed in the lower chamber, and a matrigel-coated membrane was inserted between the two chambers. After incubation for 24 h at 37˚C, the membranes were fixed and stained with a Hemacolor rapid staining kit (Merck KGaA). Cells from five random microscopic fields (each 0.5 mm 2 ) were counted using a hematocytometer under a light microscope (Zeiss).
Immunofluorescence. To confirm morphological changes, cells were washed thrice with PBS, fixed in 4% paraformaldehyde (PFA) for 10 min at room temperature, and permeabilized with PBS containing 0.25% Triton X-100 (PBST) for 10-15 min at room temperature. After three washes with PBS, cells were blocked with 1% bovine serum albumin for 30 min. Samples were incubated overnight at 4˚C with the relevant antibodies anti-MIF (1:500; Cell Signaling Technology) and anti-F actin (1:500; Santa-Cruz Biotechnology Inc.) for 2 h at room temperature. After three washes with PBS, immune-labeled cells were counterstained with 50 µl of DAPI at 37˚C for 10 min. Cells were analyzed using a laser scanning confocal microscope (Leica Microsystems).
Immunoprecipitation. Immunoprecipitation analysis was carried out as previously described (16). Briefly, cells were lysed using a protein lysis buffer [1% (v/v) Triton X-100, 150 mM NaCl, 50 mM Tris-HCl, pH 8.0, 5 mM ethylenediaminetetraacetic acid (EDTA), and protease inhibitors], and cell lysates were incubated overnight with primary anti-β-catenin and protein A or G beads (GE Healthcare, Uppsala, Sweden) at 4˚C on a rotator. The beads were washed, boiled in Laemmli buffer, and processed for western blotting.
Animals. Five-week-old male athymic nude mice (BL-6/Nu, 20-25 g; Orient Bio Co., Ltd., Seoul, Korea) were housed under controlled light conditions and provided water ad libitum. All experimental procedures involving animals were performed in compliance with institutional and governmental requirements and approved by the Institutional Animal Care and Use Committee (approval no. CIACUC2015-A0032), Chosun University, Gwangju, Korea.
Intracardiac xenograft model. Twenty mice were subjected to intracardiac injections of LNCaP Vec or LNCaP CD133+ cells and 10 mice subjected PBS as mock mice to examine the ability of PC cells to metastasize to bone, respectively. Before injection, the cells were transfected with the pGL4.5 vector plasmid encoding luciferase (Promega Corporation, Madison, WI, USA). The stably transfected cells were grown in Hank's balanced salt solution (HBSS; Welgene) supplemented with hygromycin (2 mg/ml; Sigma-Aldrich; Merck KGaA) and, luciferase activity was measured using the Luciferase Assay System (Promega Corporation) and detected by luminometer GENios Plus (Tecan Group Ltd., Salzburg, Austria). The severe combined immunodeficient mice were anesthetized using isoflurane gas. Subsequently, LNCaP Vec+ or LNCaP CD133+ cells (1x10 5 cells per mouse) were injected into the left cardiac ventricle, as per the technique first described by Arguello et al (17) with some modifications. After 4 weeks, images of tumor-bearing tissues excised from mice during necropsy were obtained.
Bioluminescence imaging. After intracardiac injection, mice were weekly imaged via bioluminescence for 4-6 weeks using the IVIS ® imaging system (PerkinElmer, Waltham, MA, USA) at the Korea Basic Science Institute (Gwangju, Korea). Images were captured and processed using the Living Image ® v.4.2 software. Anesthesia was induced using inhaled isoflurane and maintained with 2% isoflurane mixed with oxygen/nitrogen via nose cone delivery. d-Luciferin (3 mg dissolved in water) was administered at 150 mg/kg in Dulbecco's phosphate-buffered saline (DPBS) via intraperitoneal injection. Optical imaging was acquired approximately 10-15 min later.
Histological analysis of mouse tissues. Tumor-bearing tissues were fixed in cold 4% PFA. Bone tissue was first decalcified using a sodium citrate solution before processing onto histological slides. Decalcified bones were cut at the midpoint and embedded in paraffin blocks. Fluorescence from serial paraffin sections was monitored via fluorescence microscopy (Leica Microsystems). Tissues were stained with hematoxylin and eosin (H&E) stain, and images were acquired using a microscope slide scanner (3D-HISTECH Ltd., Budapest, Hungary).
Cytokine profiling. Supernatants from LNCaP Vec and LNCaP CD133+ cells and sera from tumor-bearing mice were collected and assayed using a cytokine array kit (R&D Systems, Minneapolis, MN, USA), according to the manufacturer's instructions. The cytokines examined using this technique are listed in Table I. Cytokines were detected using the ECL detection kit (Amersham) and quantified via densitometric analysis using ImageJ software (Scion Corp, MD, USA).

Reverse transcription-quantitative PCR (qPCR) analysis.
Total RNA was extracted from cells using TRIzol ® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). cDNA was synthesized from 2 µg total RNA using the Super-Script II First-Strand Synthesis System (Invitrogen; Thermo Fisher Scientific, Inc.). mRNA levels were measured via qPCR, and the Gapdh gene was used as an endogenous control. Sequences of the primers used to target various genes are listed in Table II.
Immunohistochemical analysis of bone specimens. Paraffin sections were deparaffinized in three solutions of xylene and rehydrated in a graded series of ethanol solutions. For antigen retrieval, slides were placed in 0.01 M citrate buffer at pH 6.0 and heated in a steamer for 30 min. Endogenous peroxidases were quenched by incubating the samples with 3% hydrogen peroxide for 20 min at room temperature. Sections were incubated overnight at 4˚C using a 1:50 dilution of the primary antibody against MIF (Santa-Cruz Biotechnology Inc.). Sections were then incubated for 30 min with a biotinylated secondary antibody (LSAB system HRP kit; DakoCytomation, Glostrup, Denmark), rinsed in PBS, and incubated for 30 min with a streptavidin-peroxidase conjugate (LSAB; DakoCytomation). The reaction was developed for 5 min using 3, 30-diaminobenzidine tetrahydrochloride (Sigma-Aldrich; Merck KGaA). Slides were counterstained in hematoxylin, dehydrated, and covered with a cover slip. Negative and positive controls were simultaneously analyzed. Positive controls comprised mammary tissues. The slides were captured using a microscope slide scanner (3D-HISTECH Ltd., Budapest, Hungary).
Statistical analysis. One-way analysis of variance (ANOVA) followed by Sidak's multiple comparison test (unless specifically mentioned otherwise) were used for statistical analyses. P<0.05 was considered to indicate a statistically significant difference. Data are expressed as the mean ± standard deviation (SD) unless specified otherwise. Data were analyzed using the SPSS v.20.0 software program for Windows (SPSS, Inc., Chicago, IL, USA). GraphPad Prism v.6.00 software program for Windows (GraphPad, La Jolla, CA, USA) was used to analyze data from in vitro and in vivo experiments.

Stable overexpression of CD133 in LNCaP cells.
To evaluate the effect of CD133 overexpression in vitro, we established a stable cell line overexpressing CD133 under the control of a constitutive promoter. We transfected LNCaP cells with either CD133 or an empty vector tagged with GFP. A representative CD133 clone was selected from LNCaP CD133+ cells and compared against control cells transfected with the empty vector (LNCaP Vec ). Transiently transfected 293T Vec or 293T CD133+ cells were used as a positive control. Basal expression of CD133 protein in LNCaP Vec cells was very low; however, the basal expression of CD133 was high in stable CD133-transfected cells (LNCaP CD133+ ) and transiently transfected 293T cells (Fig. 1A). Green fluorescence was observed in the cytosol and membrane of LNCaP CD133+ cells (Fig. 1B).
To explore the function of CD133 in cancer stemness, we evaluated the acquisition of CSC properties such as increased ability to form tumor spheres and expression of stem cell markers such as Oct-4, SOX2, and NANOG. The expression levels of Oct-4 and NANOG were significantly elevated in LNCaP CD133+ cells, consistent with the increase in stemness properties (Fig. 1C). Stemness was also confirmed by a colony-formation assay. Colony-forming ability was significantly elevated in LNCaP CD133+ cells, in line with the elevated expression of stemness factors (Fig. 1D). These results suggest that the increase in colony-forming ability and Oct-4 and NANOG expression conferred stem cell-like properties during ectopic overexpression of CD133.
EMT-related properties in CD133-overexpressing LNCaP cells. CD133 overexpression in LNCaP cells, as determined by  wound healing and cell invasion assays, induced a significant increase in cell migration and invasion. After 24 h, the artificial wound gaps became significantly narrower in LNCaP CD133+ cells than in LNCaP Vec cells ( Fig. 2A). Furthermore, the invasive abilities of LNCaP CD133+ cells significantly increased (Fig. 2B). To monitor morphological changes in LNCaP cells, immunofluorescence analysis for F-actin was performed using confocal microscopy. The number of spindle and discontiguous cells observed in the LNCaP CD133+ group was higher than that observed in the LNCaP Vec group (Fig. 2C).
To identify factors associated with altered invasiveness and migration caused by ectopic overexpression of CD133, we monitored levels of epithelial and mesenchymal markers in PC cells. Protein expression analyses revealed a slightly lower expression of epithelial marker E-cadherin in LNCaP CD133+ cells than in LNCaP Vec cells (Fig. 2D). In contrast, expression of the mesenchymal marker vimentin was significantly upregulated in LNCaP CD133+ cells. Further, analysis of β-catenin-associated signal transduction was carried out via immunoprecipitation in LnCaP CD133+ cells (Fig. 2E). CD133 overexpression led to a significant increase in binding between β-catenin and TCF-4, an EMT-inducing transcriptional factor in cancer cells in compared with LnCaP vec cells as a negative control. These data strongly support the idea that CD133 is essential for increased and decreased expression of vimentin and E-cadherin, respectively, leading to subsequent EMT and metastasis in PC cells.

CD133 promotes LNCaP-induced bone metastasis in vivo.
Both of LNCaP Vec and LNCaP CD133+ cells were labeled with luciferase, and the cells showed a similar luciferase activities (Fig. 3A). An then we injected LNCaP Vec and LNCaP CD133+ cells into the left cardiac ventricle of male nude mice at 0.5x10 6 cells/mouse. The mice were imaged weekly, starting from week 4 after intracardiac injection. The results of PBS (mock) injection and injection of LNCaP Vec and LNCaP CD133+ cells are shown in the whole-mouse image in Fig. 3B. A large hot-spot and a small spot of bioluminescence were observed in the whole body following LNCaP CD133+ cell injection. Only 20% of animals inoculated with LNCaP Vec cells developed skeletal tumors (n = 10) as compared with 80% of those inoculated with LNCaP CD133+ cells (Table III).
We performed H&E staining to examine the histological features of skeletal tumors formed by LNCaP Vec /LNCaP CD133+ cells inoculated into mice via intracardiac injection. As shown in Fig. 3C, gross examination of excised spines in mice injected with LNCaP Vec or LNCaP CD133+ cells showed that the tumor mass occupied the primary spongiosum (trabecular epiphysis) and displaced the bone marrow cells. In particular, an apparent margin between the spinal cord and metastatic tumors was observed in LNCaP Vec -inoculated mice. However, metastatic tumors invading the spinal cord and discursive osteolytic features of vertebrae were observed in LNCaP CD133+ cell-inoculated mice. We aimed to identify the secreted molecules that played a critical role in LNCaP CD133+ cell-induced bone metastasis through a cytokine screening using a proteome array cytokine kit (Fig. 3D). CD133 overexpression in LNCaP cells led to a marked increase in the release of interleukin 8 and MIF from mouse serum. MIF expression increased in the supernatants of LNCaP CD133+ cells. These data suggest that ectopic overexpression of CD133 might lead to a significantly increased risk of bone metastatic cancer. Further, increased MIF expression may contribute to PC metastasis in the bone.

Expression of MIF in primary PC and metastatic lesions.
To investigate the role of MIF in bone metastasis of LNCaP cells, its mRNA and protein expression levels were analyzed. MIF protein (Fig. 4A) and mRNA (Fig. 4B) levels were higher in LNCaP CD133+ cells than in LNCaP Vec cells. To monitor the cellular distribution of MIF in LNCaP cells, MIF immunofluorescence was performed. MIF expression in the membrane and cytosol was higher in LNCaP CD133+ cells than in LNCaP Vec cells (Fig. 4C).
In the immunohistochemical study, MIF expression increased around the tumor mass and trabecular epiphysis region of bones inoculated with LNCaP CD133+ cells. These observations suggest that ectopic overexpression of CD133 leads to increased MIF expression in CD133+ cells and activation of bones adjacent to the tumor, particularly in LNCaP cell-injected bones, and that CD133 expression is associated with PC metastasis to the bone.

Discussion
Bone is a common site of distant metastasis in PC. Association of bone and metastatic cancer cells is important in site-specific manifestation of PC (18). There are no curative treatments for the disease at this stage, and bone metastasis remains a devastating complication of advanced PC, despite advances in understanding of mechanisms underlying the basic molecular biology of this process. The existence and identification of CSCs reveal the pivotal roles that these cells play in metastasis (19). In the present study, we identified CSC-like cells following ectopic overexpression of CD133 in the PC cell line LNCaP. This phenomenon gave rise to genetic variation in PC cells and altered expression of several factors related to stemness, including NANOG and Oct-4, resulting in increased colony-forming abilities. Furthermore, genetic modulation of CD133 altered expression of EMT-related factors increased vimentin expression and decreased E-cadherin expression, resulting in the generation of a complex with TCF-4 and β-catenin. Although the role of EMT in PC remains unclear, several factors involved in EMT or related pathways have been identified as key molecules in cancer cell invasion and metastasis. There is the strong correlation between CD133 and EMT factors that may explain the invasiveness and migration of LNCaP CD133+ cells.
Recent studies have shown that EMT may induce differentiation of cancer cells into a CSC-like state. EMT plays a critical role in the process of development and wound healing; however, recent studies have linked EMT with human pathology, including cancer metastasis (20). However, no research has yet revealed the mechanisms underlying this phenomenon or identified the source of cells with both EMT and stem cell properties. In the present study, we show for the first time that PC cells via ectopic overexpression of CD133 may present mesenchymal characteristics and stem cell properties. In addition, LNCaP cells were originated from not bone metastatic PC but lymph node, it confirmed that ectopic overexpression of CD133 in LNCaP led to promote the ability of bone metastasis in the present study. These results imply that CSCs may induce local and distant metastases to the bone by acquiring mesenchymal features, which may greatly facilitate systemic dissemination from the primary mass. Furthermore, the redundant regulation of β-catenin/TCF-4 signaling during tumor metastasis through several EMT-inducing signals suggests that this expression is critical in the initiation and maintenance of EMT. According to recent studies, the microenvironment at the invasive front of cancer cells, especially secretion of factors such as hepatocyte growth factor by stromal myofibroblasts near dedifferentiated cancer cells, plays a key role in the nuclear translocation of β-catenin and activation of β-catenin/TCF-4 signaling (16).
We explored factors that play a critical role in CD133+ PC cell-induced bone metastasis. LNCaP Vec and LNCaP CD133+ cells were inoculated into athymic nude mice via intracardiac injection, and showed an osteolytic bone metastasis with a similar result of previous study (21). And then, secreted cytokines with paracrine and endocrine actions were evaluated using mouse serum in array kits. Histological analysis revealed that CD133+ cells generated larger and more aggressive tumors than their wild-type counterparts, while LNCaP CD133+ cells showed higher expression of MIF than LNCaP Vec cells. Moreover, MIF is a pleiotropic inflammatory cytokine with chemokine-like functions and may bind and signal via several receptors, including CD74, C-X-C motif chemokine receptor 2 (CXCR2), and CXCR4. MIF may play a central role in bone metastasis of PC and perform critical biological functions (22). MIF is known to induce osteoclast differentiation and contribute to the progression of various bone and periodontal diseases (23). A previous study has shown that binding of both MIF and stromal cell-derived factor 1 chemokines to CXCR4 may trigger chemotaxis of osteoclast precursors and enhance osteoclastogenesis (24). The interaction of MIF with chemokine receptors may explain its ability to induce macrophage migration and osteoclastogenesis. Our current findings support the conclusion that while a physiological target is yet unidentified, tautomerase activity is important for MIF functionality and may contribute to tumor growth and metastasis. Tropism of LNCaP CD133+ cells to bone suggests that CSCs may preferentially interact with specific cells in the bone microenvironment, and the most likely candidates are osteoclasts that cause an increase in bone lysis at sites of bone metastases, as demonstrated by histomorphometric evidence.
With respect to clinical applications, CD133 may serve as a candidate marker to detect bone metastasis risk in patients with PC. The inability to predict development of metastatic disease in patients is a major challenge in PC management and has resulted in excessive therapy in some patients and delayed or insufficient therapy in others (25). The nature of the association between circulating tumor cells and bone metastasis remains controversial. The presence of circulating PC cells in the blood is an indication of dissemination of tumor cells from the primary site. Only a subset of circulating tumor cells may possess the necessary properties to target bone.
In light of the significant progress in the field of metastasis and stem cell research, we suggest a CSC-based model for bone metastasis in PC. CD133 regulates CSC and EMT properties in PC and sustains acquisition of osteolytic features through MIF secretion. These data demonstrate the role of EMT and CSCs in cancer metastasis to the bone. Thus, our findings may facilitate development of a novel classification system and therapeutic strategies for bone metastasis.