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. Author manuscript; available in PMC: 2016 Jan 1.
Published in final edited form as: Int J Cancer. 2014 May 20;136(1):11–20. doi: 10.1002/ijc.28948

Bone-induced c-kit expression in prostate cancer: a driver of intraosseous tumor growth

Leandro E Mainetti 1, Xiaoning Zhe 1, Jonathan Diedrich 1,2, Allen D Saliganan 1, Won Jin Cho 1, Michael L Cher 1,3, Elisabeth Heath 2, Rafael Fridman 3, Hyeong-Reh Choi Kim 3, R Daniel Bonfil 1,2,3
PMCID: PMC4199873  NIHMSID: NIHMS592292  PMID: 24798488

Abstract

Loss of BRCA2 function stimulates prostate cancer (PCa) cell invasion and is associated with more aggressive and metastatic tumors in PCa patients. Concurrently, the receptor tyrosine kinase c-kit is highly expressed in skeletal metastases of PCa patients and induced in PCa cells placed into the bone microenvironment in experimental models. However, the precise requirement of c-kit for intraosseous growth of PCa and its relation to BRCA2 expression remain unexplored. Here, we show that c-kit expression promotes migration and invasion of PCa cells. Alongside, we found that c-kit expression in PCa cells parallels BRCA2 downregulation. Gene rescue experiments with human BRCA2 transgene in c-kit-transfected PCa cells resulted in reduction of c-kit protein expression and migration and invasion, suggesting a functional significance of BRCA2 downregulation by c-kit. The inverse association between c-kit and BRCA2 gene expressions in PCa cells was confirmed using laser capture microdissection in experimental intraosseous tumors and bone metastases of PCa patients. Inhibition of bone-induced c-kit expression in PCa cells transduced with lentiviral short hairpin RNA reduced intraosseous tumor incidence and growth. Overall, our results provide evidence of a novel pathway that links bone-induced c-kit expression in PCa cells to BRCA2 downregulation and supports bone metastasis.

Keywords: c-kit, BRCA2, Prostate Cancer, Bone metastasis, Invasion

Introduction

Despite having a 5-year relative survival rate at nearly 100% when detected early, prostate cancer (PCa) patients eventually progress to castration-resistant prostate cancer (CRPC) and present bone metastasis in ≥ 84% of the cases1, 2. Currently, FDA-approved therapies available for metastatic bone disease in PCa patients are merely palliative3. The propensity of PCa cells to metastasize to bone suggests that bidirectional interactions between cancer cells and the bone microenvironment are pivotal in PCa progression.

The receptor tyrosine kinase (RTK) c-kit is deregulated in many human cancers through mutations (e.g., gastrointestinal-stromal tumors, acute myeloid leukemia, and testicular carcinoma) or overexpression (e.g., certain renal tumors, and some colorectal cancers), which result in constitutive activation or enhanced sensitivity to its ligand stem cell factor (SCF), respectively4, 5. We have previously reported higher expression of c-kit in metastatic bone lesions than primary tumors of PCa patients, and shown strong c-kit expression in intraosseous tumors formed by otherwise c-kit-negative human PCa cell lines6. We further confirmed the de novo expression of c-kit using two different intraosseous tumor models and three additional human PCa cell lines, which proved to be c-kit-negative when grown in vitro or as tumors in non-osseous tissues. However, despite such clear evidence suggesting that the induction of c-kit expression is triggered by the osseous microenvironment as a result of an adaptation of the PCa bone colonizing cells, the contribution of c-kit expression and activation in PCa cells to bone metastasis has not been confirmed up to date.

BRCA2 is typically recognized for its critical involvement in the maintenance of genomic integrity7. Interestingly, loss of BRCA2 function in PCa has been shown to promote invasion, and to be associated with progression to metastatic disease and worse prognosis in patients8, 9. A recent study has revealed a correlation between c-kit overexpression and loss of BRCA1, closely related to BRCA2, in a mouse breast cancer model10. However, whether this type of inverse association is also true for BRCA2 and in PCa is still not known.

In the present study, we show that c-kit expression by PCa cells supports their migration and invasion and downregulates BRCA2. Conversely, genetic rescue with BRCA2 transgene reduces c-kit protein expression and migration and invasion of c-kit-transfected PCa cells, suggesting a novel cross-regulation between these two genes that supports PCa progression. Furthermore, we demonstrate the contribution of bone-induced c-kit expression by PCa cells to intraosseous tumor formation, and confirmed high and low expressions of c-kit and BRCA2 genes, respectively, in intraosseous PCa tumors in a mouse model and bone metastases from PCa patients. These findings may therefore result in new biomarkers and therapeutic targets for PCa patients presenting bone metastasis.

Material and Methods

Cell Culture and Plasmids/shRNA transfer

PC3 and C4-2B prostate carcinoma cells were obtained from American Type Culture Collection (ATCC) and provided by Dr. Leland Chung (Cedars-Sinai Medical Center, Los Angeles, CA), respectively. Human osteosarcoma-derived SAOS-2 cell line was purchased from ATCC. For studies involving ectopic expression of c-kit in vitro, PCa cells were stably transfected with an empty vector or the vector expressing the coding sequence of human c-kit (see Supporting Information Materials and Methods). Upregulation of BRCA2 in PCa cells was performed through transient transfection with the plasmid pcDNA3 236HSC WT expressing BRCA2 (Addgene)11 using Lipofectamine 2000. To discard non-specific effects on gene expression, cells were transiently transfected with the pcDNA3 empty vector (Invitrogen). To block bone-induced c-kit expression in PCa cells, we first screened short-hairpin RNA (shRNA) constructs to choose the most efficient in knocking down c-kit gene expression; we chose SAOS-2 cells, which express c-kit endogenously (see Supporting Information Materials and Methods). Stable knockdown of c-kit in parental PCa cell lines was achieved by transduction with lentiviral shRNA particles (Supporting Information Materials and Methods). Before using transduced PCa cells in vivo, blocking of c-kit gene induction was confirmed using cocultures of PC3 and bone marrow (BM) cells embedded in Matrigel (BD Biosciences), which mimic c-kit induction observed in intraosseous PCa tumors or metastases6.

Gene expression analysis

Total RNA was extracted using TRIzol (Invitrogen), and synthesized cDNA using standard techniques. Primers used for RT-PCR and real-time PCR studies are listed in Supporting Information Table S1. Semi-quantitative analysis of mRNA levels was done by real-time PCR using SYBR Green, and gene expression profile of c-kit-transfected and control PCa cells was analyzed with a gene-focused array (see Supporting Information Materials and Methods).

Western blotting

Immunoblotting analysis was conducted as previously described by us6 and in Supporting Information Materials and Methods.

Cell Proliferation, Migration, Invasion, and Anoikis

Cell viability and proliferation of PCa cells stably transfected with the vector expressing c-kit or the empty vector, were evaluated using the WST-1 assay in the presence or absence of 100 ng/ml SCF (PeproTech). Migratory and invasive abilities of c-kit-expressing and non-expressing PCa cells exposed or not exposed to SCF towards 5% FBS, used as chemoattractant, were assessed in 8 μm pore-sized Transwell inserts (BD Falcon), as previously described12, 13. Treatment with LY294002 (Cell Signaling) was included in some experiments to determine the contribution of PI3K/Akt to changes in migratory or invasive capacities. Anoikis assays were performed as described by Srikanth and colleagues14 (see Supporting Information Materials and Methods).

Establishment of intraosseous prostate tumors

We used the severe combined immunodeficiency (SCID) mouse-hu model as previously described15. Briefly, fragments of human male fetal femurs (Advanced Bioscience Resources) were implanted under the skin of 5-weeks-old male CB-17 scid/scid mice. Four weeks later, 1×105 PC3 or 1×106 C4-2B cells stably transduced with c-kit.shRNA were injected through the mice skin directly into the marrow side of the previously implanted bone (controls included PC3 and C4-2B cells transduced with scrambled shRNA [scr.shRNA]). Mice (six used per cell type) were euthanized based on biweekly whole body X-ray images using a piXarray100 digital specimen radiography system (Bioptix). Animal protocols were approved by the Animal Investigation Committee of Wayne State University.

Clinical Bone Metastasis Samples

CT-guided bone core biopsies were obtained from six bone-metastatic CRPC patients. Following fixation with 10% neutral buffer formalin, biopsies decalcified in 10% EDTA were processed for paraffin-embedding and H&E staining. The use of these human samples was approved by the Institutional Review Board and all human subjects provided written, informed consent.

Histological Analysis, Immunostaining, and Histomorphometry

Experimental intraosseous prostate tumors obtained using the SCID-hu model and bone marrow core biopsies from PCa patients were fixed with 4% paraformaldehyde for 24 h and decalcified in 10% EDTA pH 7.0 for 14 and 3 days, respectively. Paraffin embedding and H&E staining of 5-μm sections were performed using standard methods. Immunohistochemistry for cytokeratin6, 16 and histomorphometry to assess areas occupied by tumor and bone tissues17, were performed as previously described.

Immuno-laser Capture Microdissection, and Gene Expression Analysis

Five-μm sections of formalin-fixed paraffin-embedded intraosseous tumors obtained using the SCID-hu model or human bone core biopsies from PCa patients with confirmed diagnosis of bone metastasis were immunostained for cytokeratin. Tumor (cytokeratin-positive) and bone marrow stroma (cytokeratin-negative) tissue areas were selected from unmounted slides and micro-dissected using the Leica LMD6000 system (Leica Microsystems). The collected tissue material was then processed with TRIzol solution, and RNA was isolated and precipitated with chloroform/isopropanol. RNA was then re-suspended in nuclease-free water and amplified into antisense-RNA (aRNA) using MessageAmp II aRNA Amplification Kit (Invitrogen). cDNA was subsequently synthesized with iScript cDNA Synthesis Kit (Bio-Rad). PCR was then carried out using Choice Taq Blue Mastermix (Denville Scientific) followed by gel electrophoresis.

Statistical Analysis

Comparisons of means among more than two groups were analyzed using ANOVA with Bonferroni post-testing, and between two groups using two-tailed unpaired Student’s t-test. Chi-square test p-values were used to compare intraosseous tumor incidences in mice. Data were analyzed using Prism 5 (GraphPad Software Inc).

Results

C-kit promotes migration and invasion of prostate cancer cells

To address both the functional relevance and underlying mechanisms of c-kit expression and activation in PCa cells in intraosseous expansion, we stably transfected PC3 and C4-2B cells with full-length human c-kit. We first examined pooled populations of PC3-kit and C4-2B-kit cells for their migratory and invasive abilities with or without SCF stimulation. As shown in Fig. 1A, these analyses revealed that expression of c-kit and its activation with SCF drastically increased the migratory and invasive phenotype of both PC3 and C4-2B cells when compared with the cells transfected with the empty vector. C-kit expression did not affect proliferation or anoikis of the PCa cells. Similar results were obtained with three independent clones of the c-kit-expressing PCa cell lines (data not shown). To investigate the downstream effect of c-kit activation, PC3-kit and C4-2B-kit cells were serum-starved for 24 h and then stimulated with SCF for 20 min, and the cell lysates were examined for different intracellular signaling molecules using immunoblot analysis. In addition to c-kit, only Akt was differentially phosphorylated upon treatment with SCF (Fig. 1B). It should be noted that, although PC3 and C4-2B express endogenous SCF6, the levels of phosphorylated c-kit and Akt in the c-kit-transfected cells are below the detection limit of the immunoblot technique and only readily observed upon addition of exogenous ligand. To evaluate the significance of Akt phosphorylation in c-kit-mediated migration and invasion, we used LY294002, a chemical inhibitor of phosphatidylinositol 3-kinase (PI3K) activity18. Treatment with 10 μM LY294002 significantly reduced the migratory and invasive abilities of PC3- and C4-2B-kit cells treated with 100 ng/ml SCF (Fig. 1C). However, cell migration and invasion induced by c-kit transfection alone was not significantly reduced by LY294002 treatment (Fig. 1C). Of note, the concentration of LY294002 used did not affect the viability of the PCa cells (Supporting Information Fig. S1A). Consistently, immunoblot analysis of lysates derived from inhibitor-treated c-kit-expressing cells stimulated by SCF demonstrated reduced levels of phospho-Akt compared to untreated cells, in a dose-dependent manner (Supporting Fig. S1B). Together these data indicate that expression and activation of c-kit in PCa cells by SCF at higher levels stimulate their in vitro migration and invasion through a PI3K/Akt-dependent mechanism, which could partly facilitate expansion of PCa cells within the bone microenvironment, where soluble SCF is produced6. Conversely, lower levels of endogenous SCF can induce PCa cell migration and invasion even in the absence of PI3K/Akt signaling. These results are consistent with previous reports showing differences in the pathways RTKs activate, depending on the strength of the signal19, 20.

Figure 1.

Figure 1

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C-kit expression and activation in prostate cancer cells stimulates their migration and invasion. A, In vitro proliferation, anoikis, Transwell migration, and Matrigel invasion of PC3 and C4-2B cells stably transfected with c-kit or empty vector (EV), treated or untreated with 100 ng/ml SCF. Values are mean ± S.E. *, P < 0.01; **, P < 0.001; ***, P < 0.0001 Bonferroni post hoc applied to significant effect of group ANOVA (P ≤0.0001). B, Immunoblot analysis for different intracellular signaling molecules and phosphorylated forms in whole cell lysates from kit- and EV-transfected PC3 or C4-2B cells. In addition to c-kit, only Akt became phosphorylated upon treatment with SCF. GAPDH served as loading control. C, PC3 or C4-2B cells stably transfected with c-kit exposed to SCF and treated with 10 μM LY294002 significantly reduced their migratory and invasive abilities. Bars represent mean ± SE number of cells traversing the filters. ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05 by Bonferroni post hoc applied to significant effect of group ANOVA (P <0.0001).

Activation of c-kit leads to downregulation of BRCA2 in prostate cancer

To identify genes regulated by c-kit, we used an oncogenes and tumor suppressor genes-focused real-time PCR array to compare c-kit-transfected PCa cells treated with SCF and empty vector-transfected control cells (Supporting Information Table S2). The expression of BRCA2, a gene commonly involved in DNA double-strand break repair7, was downregulated more than 180-fold in c-kit-expressing PC3 and C4-2B cells stimulated with SCF (Fig. 2A). This was validated using RT-PCR (Fig. 2B) and immunoblot analyses (Fig. 3A–B).

Figure 2.

Figure 2

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C-kit expression and activation in prostate cancer cells downregulate BRCA2 expression. A, Heat map showing genes up- and down-regulated in PC3-kit and C4-2B-kit cells treated with 100 ng/ml SCF compared to PC3-EV and C4-2B-EV, respectively, using an oncogene and tumor suppressor gene-focused qPCR array. Two-fold or greater changes in gene expression with a P < 0.05 are shown as log2-transformed fold change. Red and green indicate increased and decreased mRNA expression, respectively. B, RT-PCR analysis confirms downregulation of BRCA2 gene expression by c-kit expression and activation by SCF in PC3 and C4-2B cells. GAPDH was used as loading control. C, Gene expression of c-kit and BRCA2 of parental PC3 or C4-2B cells, and bone marrow (BM) cells in 3D cultures in Matrigel alone or together, as analyzed by real-time PCR. Values are mean ± S.E. *P < 0.0001 calculated by two-tailed Student’s t test.

Figure 3.

Figure 3

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Restoration of BRCA2 expression in c-kit-expressing prostate cancer cells inhibits c-kit expression, PI3K/Akt activation, and migratory and invasive abilities. A, In vitro migration and invasion assays of PC3-EV and –kit treated with 100 ng/ml SCF and C4-2B-EV and –kit treated with 100 ng/ml SCF after transient transfection with BRCA2 (shown as “/BRCA2”). Values are mean ± S.E. ** P<0.001, * P<0.05 by Bonferroni post hoc applied to significant effect of group ANOVA (P <0.005). Immunoblotting confirms BRCA2 overexpression at the protein level in c-kit-expressing PC3 and C4-2B cells treated with SCF used in the migration and invasion assays after transfection with BRCA2 (bottom). B, Immunoblot analysis for BRCA2, c-kit, Akt and its activated form phosphorylated at Ser473. After transient transfection of SCF-treated PC3-kit or C4-2B-kit cells with BRCA2, protein expression levels of c-kit and phospho-Akt diminished. GAPDH served as loading control.

Because c-kit is upregulated by interaction of PCa cells with the bone microenvironment, we examined for BRCA2 levels in cocultures of PC3 or C4-2B and BM cells embedded in a Matrigel droplet. Consistent with our previous studies, c-kit was induced in PCa cells only when cocultured with BM cells6. Interestingly, in those same conditions BRCA2 was downregulated (Fig. 2C). Overall, these results indicate a negative regulation of BRCA2 gene by activated c-kit, which not only occurs in PCa cells with ectopic c-kit expression, but also in a model mimicking bone-induced c-kit expression in PCa cells.

Restoration of BRCA2 reduces migratory and invasive capacity, c-kit expression, and PI3K/Akt activation in c-kit-expressing prostate cancer cells

Having shown that c-kit activation in PCa cells leads to enhanced migratory and invasive abilities and downregulation of BRCA2, we wondered whether BRCA2 could inhibit migration and invasion of c-kit-expressing PCa cells. We transiently transfected PC3-kit and C4-2B-kit cells with wild-type BRCA2 and assessed their capacity to traverse no-coated and Matrigel-coated 8 μm pore-sized Transwell inserts. As shown in Fig. 3A, BRCA2 overexpression in c-kit-transfected PC3 and C4-2B cells stimulated with SCF reduced their migratory and invasive abilities nearly to the basal level shown by control PC3-EV and C4-2B-EV cells not expressing c-kit.

Because the migratory and invasive abilities of c-kit-expressing PCa cells stimulated with SCF are enhanced, and BRCA2 showed an inhibitory effect on those processes, we hypothesized that BRCA2 may obstruct PI3K/Akt signaling triggered by high concentrations of SCF. We found that transient transfection of c-kit-expressing PCa cells with wild-type BRCA2 not only resulted in diminished levels of p-Akt in PC3-kit and C4-2B-kit cells stimulated with SCF, but also of c-kit at the protein level (Fig. 3B). Of note, c-kit mRNA level was unaffected by restoration of BRCA2 (data not shown). Non-specific effects that might be ascribed to the transfection method used itself were ruled out, as the use of a control vector comprising an identical backbone, but without the BRCA2 insert, did not affect c-kit expression in c-kit-expressing PCa cells (Supporting Information Fig. S2).

Taken together, these results imply an inhibitory effect of BRCA2 on migration and invasion, and PI3K/Akt signaling stimulated by expression and activation of c-kit in PCa cells. Moreover, c-kit reduction by BRCA2 re-expression suggests the existence of a cross-regulation mechanism.

Obstruction of bone-induced expression of c-kit in prostate cancer cells subdues their intraosseous growth

To determine the relevance of c-kit expression in PCa cells growing within bone, we stably knocked down the gene’s expression in parental PC3 and C4-2B cells through lentiviral transduction of a c-kit short-hairpin RNA (kit.shRNA; clone ID: V2LHS_76974). The kit.shRNA contained in the lentiviral particle was selected from a panel of five shRNA sequences targeting unique regions of the c-kit gene (Supporting Information Table S3), which were transiently transfected using electroporation in the osteosarcoma cell line SAOS-2, which has endogenous c-kit expression. A reduction in c-kit expression was observed with all the kit.shRNA sequences with highest silencing effect shown with clone ID: V2LHS_76974, whereas scrambled shRNA (scr.shRNA) did not affect basal c-kit expression in a significant manner (Fig. 4A–B). The efficient silencing effect of clone ID: V2LHS_76974 was further confirmed in parental PC3 and C4-2B cells transduced with lentiviral particles using the PCa-BM cell coculture in Matrigel (Fig. 4C), which mimics c-kit induction observed in intraosseous PCa tumors or metastases6. Having verified the obstruction of c-kit induction in PCa cells in vitro, we transduced parental PC3 and C4-2B cells with lentiviral shRNA particles targeting human c-kit (V2LHS_76974) or scr.shRNA (control) and assessed their growth within bone xenografts transplanted in SCID mice (SCID-hu model). As shown in Fig. 5A–C, a significant reduction in incidence and extent of intraosseous tumors was found with kit.shRNA-transduced PCa cells, as compared to control groups injected with PCa cells transduced with scr.shRNA-lentiviral particles. Concomitantly, bone/tissue areas were significantly larger in bone xenografts injected with parental PC3 or C4-2B cells transduced with kit.shRNA (Fig. 5B). We also found that the percentage of cancer proliferating (Ki-67-positive) cells was significantly lower in existing intraosseous tumors formed by both PC3- and C4-2B-kit.shRNA cells than in their respective controls (Supporting Information Fig. S3). Tumor apoptosis was low and similar in all groups (data not shown). In addition, histological analyses of intraosseous tumors clearly revealed discohesive and infiltrating tumor cells with high grade nuclear features in scr.shRNA-transduced parental PC3 and C4-2B cells, whereas kit.shRNA-transduced PC3 and C4-2B cells formed fewer tumors with limited and circumscribed growth and lower grade nuclear features (Fig. 5C). Moreover, we confirmed significant reductions in c-kit expression and increase in BRCA2 at the mRNA and protein levels in the few tumors that grew in bone xenografts injected with kit.shRNA-transduced parental PC3 or C4-2B cells, compared to controls (Figs. 5D and E). These data suggest that bone-induced c-kit expression/activation in PCa cells plays a crucial role in their intraosseous expansion.

Figure 4.

Figure 4

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Screening of shRNA constructs targeting c-kit. A, SAOS-2 cells were transiently transfected using electroporation with pGIPZ vectors containing 5 shRNA sequences targeting human c-kit (kit.shRNA) or scrambled shRNA (scr.shRNA) with not known sequence homology to any cellular RNA. After 3-day culture, c-kit gene expression reduction was observed in cells transfected with kit.shRNA, but not with scr.shRNA, as determined by RT-PCR. GAPDH served as the loading control. B, Intensity levels of bands obtained for c-kit mRNA in agarose gels, as analyzed using Image Lab™ software included in the ChemiDoc XRS+ system used for gel imaging. Values are mean ± S.E. **** P<0.0001, *** P<0.001, **P<0.01, *P<0.05 by Bonferroni post hoc applied to significant effect of group ANOVA (P <0.0001). C, Parental PC3 and C4-2B cells were stably transduced with lentiviral particles containing kit.shRNA or scr.shRNA, and cocultured with human bone marrow cells embedded in Matrigel for 72 h. While cocultures of prostate cancer cells transduced with scr.shRNA resulted in induction of c-kit expression, as revealed by RT-PCR analysis, cocultures involving prostate cancer cells transduced with kit.shRNA and cells cultured alone did not reveal c-kit expression at the RNA level. GAPDH served as the loading control.

Figure 5.

Figure 5

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Blocking of bone-induced c-kit expression in prostate cancer cells inhibits their intraosseous growth. A, Intraosseous tumor incidence of parental PC3 and C4-2B stably transduced with short-hairpin RNA targeting c-kit (kit.shRNA) or scrambled shRNA (scr.shRNA) in the SCID-hu model, using a cutoff of area occupied by tumor of 15%, as assessed by histomorphometry. ** P<0.002, * P<0.02 calculated by Chi-square test. B, Histomorphometrical analysis of tumor and bone areas in bone xenografts injected with parental PC3 or C4-2B cells stably transduced with kit.shRNA or scr.shRNA. Values are mean ± S.E. *P <0.05, **P <0.02, ***P<0.003, ***P<0.0003, Student’s t test. C, Representative images showing macroscopic, X-ray, and histological (H&E) appearance of bone xenografts injected with kit.shRNA- and scr.shRNA-transduced parental PC3 cells. Arrow indicates a small tumor formed by kit.shRNA-transduced PC3 cells within bone marrow, as compared to a large invasive tumor formed by scr.shRNA-transduced PC3 cells. Scale bars, 50 μm. D, Gene expression of c-kit and BRCA2 in tumors formed by injection of kit.shRNA-transduced and control scr.shRNA-transduced parental PC3 cells into bone xenografts, as analyzed by real-time PCR. Values are mean ± S.E. * P < 0.0001 calculated by two-tailed Student’s t test. E, Immunoblot analysis for c-kit, phospho-c-kit, and BRCA2 in homogenates obtained from tumors formed by injection of kit.shRNA- and scr.shRNA-transduced parental PC3 cells into bone xenografts. GAPDH served as loading control.

C-kit and BRCA2 expression in experimental and human intraosseous prostate tumors

We next sought to determine whether the inverse association between c-kit and BRCA2 observed in PCa cells in in vitro systems, also occurred in intraosseous PCa tumors/metastases. Using immuno-Laser capture microdissection (LCMD), we found that c-kit gene expression was evident in parental PC3 and C4-2B (cytokeratin-positive) cells growing in bone xenografts in SCID mice, as shown by c-kit-transfected PC3 and C4-2B cells grown in vitro used as control (Fig. 6A). No c-kit expression was detected in peritumoral BM stroma (Fig. 6A). We confirmed a similar pattern of c-kit gene expression in tumor foci in clinical specimens of bone metastasis (Figs. 6A and B). These results are in agreement with our previous immunostaining results for c-kit6. Notably, in each of the experimental intraosseous tumors and human PCa bone metastases analyzed, BRCA2 gene expression was not evident in PCa cells, while it showed only weak expression in BM stroma from some clinical metastases (Fig. 6A). Overall, these results support the in vitro data, revealing a clear association between c-kit expression and expansion of PCa cells within the bone microenvironment in in vivo situations, and supporting the clinical relevance of our findings.

Figure 6.

Figure 6

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Expression of c-kit along with loss of BRCA2 expression in prostate cancer intraosseous tumors and clinical bone metastasis. A, RT-PCR analysis for c-kit and BRCA2 in RNA obtained from tumor (pan-cytokeratin [pCK]-positive cells) and stroma areas isolated using immuno-laser capture microdissection from experimental intraosseous tumors formed by parental PC3 or C4-2B cells (SCID-hu model), and bone metastases from prostate cancer (PCa) patients. Cultured PC3 and C4-2B cells transfected with c-kit or empty vector (EV) were run in parallel. GAPDH was used as loading control. B, Histological section of a patient’s PCa bone metastasis stained with H&E (top) and immunostained for pan-cytokeratin before (bottom left) and after (bottom right) immuno-laser capture microdissection. Scale bars, 50 μm. C, Model of c-kit/BRCA2 regulation in the bone microenvironment: Colonizing prostate cancer (PCa) cells respond to the bone milieu by increasing c-kit expression (the factor[s] involved in this process need to be defined). Tumor-associated c-kit is activated by secreted and membrane-tethered SCF (sSCF and mSCF, respectively) produced by PCa, fibroblasts, and osteoblast cells, which in turn results in downregulation of BRCA2 expression. C-kit activation leads to enhanced PCa cell migration and invasion, favoring intraosseous tumor expansion.

Discussion

Management of metastatic CRPC patients continues to be challenging, with some improvements in palliative treatments but little advances in therapies that, as of yet, only extend overall survival for a few months. In this scenario, it has been of interest the use of different drugs targeting RTKs, which showed encouraging results in many preclinical models of bone metastasis21–24. Clinical studies in CRPC patients with bone metastases with the groundbreaking RTK inhibitor imatinib mesylate have led to inconclusive results25, 26. However, recent studies with the small molecule inhibitor of multiple RTKs Cabozantinib (a.k.a. XL184) have shown significantly prolonged progression free survival and improved bone scans in metastatic CRPC patients, bringing new excitement to the field27. C-kit, which we have previously found to be highly expressed in PCa bone metastases and intraosseous PCa xenografts6, is also targeted by this RTK inhibitor. In this manuscript we describe how PCa cells are functionally affected by c-kit expression and activation, and we show the actual contribution of bone-induced c-kit expression to intraosseous expansion of PCa. We also report a novel a cross-regulation mechanism between c-kit and BRCA2, suggesting new targets for therapeutic intervention against PCa bone metastasis.

Our findings using c-kit-negative PCa cell lines indicate that c-kit expression is induced specifically in cancer cells and not in interacting bone cells. Our analysis of the abundance of c-kit transcripts of RNA in PCa cells suggests a regulation of the gene expression by bone microenvironment at the transcriptional level. Among multiple factors or mechanisms that have been related to regulation of c-kit in other cell types28–32, microRNA-221 is the only one that has been related to modulation of c-kit expression in aggressive forms of PCa33. However, we believe that inherent conditions of the bone microenvironment, including low oxygen content33 and TGF-β released from the osseous matrix by tumor-related osteolysis34, should be investigated as potential players in bone-induced c-kit expression in PCa cells. In fact, Notch signaling, which has been reported to upregulate c-kit expression34 and to be overexpressed in metastatic PCa35, 36, is triggered by hypoxic stimulus37, 38 and TGF-β39. Moreover, four hypoxia responsive elements (HREs) have been recently identified within the promoter region of the c-kit gene40. Additional studies are now under way to confirm the actual contribution of a Notch-dependent pathway in the induction of c-kit in PCa bone metastasis.

We also found that c-kit expression by PCa cells contributes to migration and invasion, similar to what was previously reported for other cancer cell types41, 42. Studies have related c-kit expression and activation to increased cell proliferation and/or stimulation of anti-apoptotic signaling pathways41, 43–45. In our case, we purposely selected transfected PCa cells that not only express c-kit, but show proliferation rates similar to control PCa cells transfected with the empty vector. However, our in vivo studies with parental PC3 and C4-2B cells in the SCID-hu model revealed that suppression of bone-induced c-kit expression robustly reduced invasion and proliferation of PCa cells. Together, these findings indicate that these mechanisms are truly triggered in c-kit-expressing PCa cells and are critical for intraosseous tumor expansion.

Another major discovery of this study relates to the inverse association between c-kit and BRCA2 expressions in PCa cells. Similar findings have been reported in normal mammary epithelium and in a mouse model of breast cancer, where an association between c-kit expression and loss of BRCA1 was demonstrated10, 46. Here, we confirmed downregulation of BRCA2 in every scenario where c-kit upregulation occurred, namely PC3 and C4-2B cells genetically modified to express c-kit, parental PCa cells cultured with BM cells, and cancer cells isolated from intraosseous prostate tumors and clinical PCa bone metastases. Moreover, we found that BRCA2 re-expression in c-kit-transfected PCa cells downregulated c-kit expression at the protein level, without affecting c-kit mRNA expression levels. These results suggest that BRCA2 can modulate c-kit protein expression either at the translational (e.g., through binding of specific miRNAs to sites in the 3′UTR of c-kit mRNA) or post-translational (e.g., proteasomal or lysosomal degradation of c-kit protein) levels. Moreover, BRCA2 re-expression in c-kit-transfected PCa cells inhibited c-kit-enhanced invasiveness to levels similar to those shown by c-kit-non-expressing cells. Accordingly, loss of BRCA2 has been shown by others to promote migration and invasion of PCa cells8, 47. However, the studies performed by others were not related to c-kit and only addressed primary prostate tumors, with no prior evidence of cross-regulation between c-kit and BRCA2 or association with PCa bone metastasis, which are key findings of our research. Furthermore, we found that gene restoration of BRCA2 in c-kit-transfected PCa cells resulted in reduced phosphorylation of Akt, which could be a direct consequence of diminished expression of c-kit that can bind SCF or, as shown by others in breast cancer with BRCA1, a result of negative regulation of Akt activation mediated by direct binding leading to ubiquitination of p-Akt and degradation48. The relevance of our findings is supported by clinical studies in PCa patients with germline inactivating mutations in the BRCA2 gene associated with more aggressive clinical course and decreased survival9, 49, suggesting that inactivation or loss of BRCA2 contributes to a more malignant phenotype. Future studies will be needed to understand the bidirectional regulation between c-kit and BRCA2, and find the mechanisms emanating from the bone microenvironment that trigger c-kit transcription in PCa cells and allow autocrine and paracrine activation by SCF (Fig. 6C).

In summary, our findings indicate that c-kit expression and activation induced in PCa cells by the bone microenvironment are key contributors to bone metastasis, and that a novel cross-regulation between c-kit and BRCA2 takes place in that process. These results are timely considering that they could contribute to the development of new biomarkers and potential therapeutic targets for PCa patients with skeletal metastasis, for whom no curative treatments are available as of yet.

Supplementary Material

Supp Material

Novelty and Impact.

This study reveals for the first time a functional role of bone-induced c-kit in intraosseous growth of prostate cancer (PCa) cells. Moreover, we show that activation of c-kit in PCa cells results in downregulation of BRCA2, while re-expression of the latter reduces c-kit protein expression. Our findings suggest a novel cross-regulation between c-kit and BRCA2, which may contribute to the development of novel biomarkers and potential therapeutic targets for PCa patients with bone metastatic disease.

Acknowledgments

This work was supported by the National Cancer Institute (R21CA162232 to R.D.B) and Wayne State School of Medicine (start-up funds to R.D.B.).

Abbreviations

aRNA

antisense-RNA

ATCC

American Type Culture Collection

BM

bone marrow

CRPC

castration-resistant prostate cancer

GAPDH

glyceraldehyde-3-phosphate dehydrogenase

LCMD

Laser capture microdissection

PCa

prostate cancer

PI3K

phosphatidylinositol 3-kinase

RTK

receptor tyrosine kinase

SCF

stem cell factor

SCID

severe combined immunodeficiency

shRNA

short-hairpin RNA

Footnotes

Additional Supporting information may be found in the online version of this article.

The authors disclose no potential conflicts of interest.

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