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Cancer Genes and Networks

Therapeutic Targeting of CD146/MCAM Reduces Bone Metastasis in Prostate Cancer

Eugenio Zoni, Letizia Astrologo, Charlotte K.Y. Ng, Salvatore Piscuoglio, Janine Melsen, Joël Grosjean, Irena Klima, Lanpeng Chen, Ewa B. Snaar-Jagalska, Kenneth Flanagan, Gabri van der Pluijm, Peter Kloen, Marco G. Cecchini, Marianna Kruithof-de Julio and George N. Thalmann
DOI: 10.1158/1541-7786.MCR-18-1220 Published May 2019

Abstract

Prostate Cancer is the most common cancer and the second leading cause of cancer-related death in males. When prostate cancer acquires castration resistance, incurable metastases, primarily in the bone, occur. The aim of this study is to test the applicability of targeting melanoma cell adhesion molecule (MCAM; CD146) with a mAb for the treatment of lytic prostate cancer bone metastasis. We evaluated the effect of targeting MCAM using in vivo preclinical bone metastasis models and an in vitro bone niche coculture system. We utilized FACS, cell proliferation assays, and gene expression profiling to study the phenotype and function of MCAM knockdown in vitro and in vivo. To demonstrate the impact of MCAM targeting and therapeutic applicability, we employed an anti-MCAM mAb in vivo. MCAM is elevated in prostate cancer metastases resistant to androgen ablation. Treatment with DHT showed MCAM upregulation upon castration. We investigated the function of MCAM in a direct coculture model of human prostate cancer cells with human osteoblasts and found that there is a reduced influence of human osteoblasts on human prostate cancer cells in which MCAM has been knocked down. Furthermore, we observed a strongly reduced formation of osteolytic lesions upon bone inoculation of MCAM-depleted human prostate cancer cells in animal model of prostate cancer bone metastasis. This phenotype is supported by RNA sequencing (RNA-seq) analysis. Importantly, in vivo administration of an anti-MCAM human mAb reduced the tumor growth and lytic lesions. These results highlight the functional role for MCAM in the development of lytic bone metastasis and suggest that MCAM is a potential therapeutic target in prostate cancer bone metastasis.

Implications: This study highlights the functional application of an anti-MCAM mAb to target prostate cancer bone metastasis.

This article is featured in Highlights of This Issue, p. 1013

Introduction

Prostate cancer is the most common cancer and the second leading cause cancer-related deaths in men (1). Current treatments such as radiation therapy and androgen deprivation therapies (ADT) are effective when the cancer is still confined at the primary site (2). However, after the tumor progresses and acquires castration resistance, the development of incurable metastases is almost inevitable (3).

Prostate cancer metastases occur at specific sites with one of the most common locations being the bone, where the occurrence of both lytic and blastic lesions has been documented (4). Previous studies demonstrated that prostate cancer cells colonize the bone microenvironment within the so called “hematopoietic stem cell (HSC) niche” (3, 5). Prostate cancer metastasis–initiating cells (MIC) compete with the bone marrow cells for the occupancy of this microenvironment and induce an ectopic epithelial tissue-of-origin niche, referred as a “developmental prostate niche” (6).

Melanoma cell adhesion molecule (MCAM/CD146) is a cell-surface glycoprotein composed of five immunoglobulin-like domains, one transmembrane region, and a short cytoplasmic tail, which interacts with the cytoskeleton (7). Growing evidence supports the notion that high MCAM expression in a variety of carcinomas positively correlates with poor prognosis in prostate cancer (8), melanoma (9), pulmonary adenocarcinomas (10), epithelial ovarian cancer (11), and breast cancer (12). Overexpression of MCAM has been shown to increase tumorigenicity of human osteoblastic prostate cancer cells (LNCaP) in vivo (13). In adition, MCAM was shown to induce epithelial-to-mesenchymal transition (EMT) in breast cancer, a process thought to be involved in the initiation of metastasis (14).

Upon transplantation, MCAM-expressing subendothelial cells in human bone marrow stroma are capable of recapitulating hematopoiesis in heterotopic sites (15). This established the notion that MCAM-positive subendothelial cells are relevant to hematopoiesis and play key roles in the maintenance of the HSC niche. Previously, we identified a gene signature for the effect that prostate cancer cells exert on the bone stroma in a bone metastasis xenograft mouse model (6). This included MCAM as a potential mediator of the metastatic colonization and growth of prostate cancer cells in the bone in lytic and blastic bone metastases (6). The extracellular domain of MCAM can engage in heterophilic binding with various ligands (e.g., Laminin-411). It has also been proposed that homophilic binding with other MCAM molecules (16) might be occurring, suggesting that targeting MCAM on either tumor cells or stromal cells might have a functional effect on prostate cancer cell behavior.

In this study, we show that administration of an anti-MCAM mAb reduces intraosseous growth and lytic lesions in preclinical models of prostate cancer bone metastasis. We provide evidence that MCAM is strongly increased in androgen ablation–resistant metastases derived from prostate cancer and show that MCAM knockdown reduces prostate cancer cell proliferation and osteoblast-mediated induction of ALDH activity. Taken together, these findings suggest that MCAM supports the metastatic lytic phenotype in human prostate cancer cells and represents a possible therapeutic target in patients with prostate cancer bone metastasis.

Materials and Methods

Cell lines and culture conditions

All the human cell lines employed in this study have been authenticated using highly polymorphic short tandem repeat (STR) loci. In addition, all the human cell lines have been previously validated in vitro and in vivo (17–19). PC-3M-Pro4 were cultured in DMEM (GibcoBRL) containing 4.5 g glucose/L supplemented with 10% FCII (Thermo Fisher Scientific), 1% penicillin–streptomycin (PS, Life Technologies). PC-3M-Pro4Luc2 and PC-3M-Pro4Luc2dTomato cells were cultured in the same medium supplemented with 0.8 mg/mL Neomycin (Santa Cruz Biotechnology) or Neomycin and 1 μg/mL Blasticidin (Sigma-Aldrich), respectively. C4-2B cells were cultured in T-medium DMEM (Sigma-Aldrich) supplemented with 20% F-12K nutrient mixture Kaighn's modification (GibcoBRL), 10% FCS, 0.125 mg/mL biotin, 1% insulin-transferrin-selenium (ITS), 6.825 ng/mL T3, 12.5 mg/mL adenine, 1% PS. The dTomato clones were supplemented with 1 μg/mL Blasticidin. Osteoblasts were derived and differentiated as we described previously (17). Culture was maintained in DMEM supplemented with 1% PS and 1% ITS. All cells were maintained at 37°C and 5% CO2.

Knockdown of MCAM with shRNA transfection

Short hairpin RNA (shRNA) constructs were obtained from Sigma's MISSION library [MCAM clone# TCRN0000151337 (shRNA#1), TCRN0000155692 (shRNA#2), TCRN0000154854 (shRNA#3)]. As a negative control, scrambled shRNA (SHC002, pLKO.1, shRNA-NT) with a lack of homology with any mammalian mRNA sequence was used.

RNA isolation and RT-qPCR

Total RNA was extracted using Trizol (Invitrogen) and cDNA synthesized by reverse transcription according to manufacturer's instructions (Promega). Real-time qPCR was performed with QuantStudio3 system (Thermo Fisher Scientific). ACTIN, HPRT, and GAPDH housekeeping genes were included for normalization. Primer sequences are reported in Supplementary Table S1. Data are displayed as 2−ΔCt when relative expression is indicated on the y-axis.

Western blot analysis

Anti-vimentin (Ab8979, Abcam) was diluted 1:1,000; anti–e-cadherin was diluted 1:1,000 (AF648, R&D Systems); anti-cripto was diluted 1:1,000 (clone no. PBL6900; ref. 17). Detection was performed with 1:10,000 secondary horseradish peroxidase (hrp) antibody (NA931VS, NA934VS, Sigma-Aldrich). Actin was detected with 1:20,000 hrp antibody (A3854, Sigma-Aldrich).

Flow cytometry, ALDEFLUOR, and viable cell sorting

Functional MCAM protein expression was determined by FACS with mouse IgG1 anti-human MCAM-Alexa647 clone P1H12 (BD Biosciences). Nonspecific binding was excluded by staining with an isotype control antibody (mouse IgG1, BD Biosciences). ALDH activity of the tumor cells was measured by the ALDEFLUOR Assay Kit (Stemcell Technologies) according to the manufacturer's instructions (17). Gating is obtained by acquisition of a control tube for each sample. Therefore, the percentage of ALDHhigh cells in the control gate correspond always to 0.01% of total. Subsequently the same gate is applied to the sample to assess the percentage of ALDHhigh cells in each experimental condition. After sorting, samples were controlled to assess purity of the sorted cell populations.

Proliferation assay

Cells were seeded at a density of 1,500 cells per well and growth monitored for 24, 48, 72, or 96 hours. For each time point, AU 490nm was measured 2 hours after incubation with 20 μL of 3-(4,5 dimethylthiazol- 2-yl)- 5 -(3 -carboxymethoxyphenyl)- 2 -(4 -sulfophenyl)- 2 Htetrazolium (MTS, Promega) at 37°C according to manufacturer's protocol. Data were normalized for the number of cells seeded. N = 5 per condition, performed at least in biological triplicates.

Animal experiments

CB17 SCID male mice, 5 to 6 weeks old were intraosseous injected with 50,000 PC-3M-Pro4Luc2dTomato cells bearing the stable shRNA#1 to knockdown MCAM expression or nontargeted (NT) shRNA control sequence. Sham-operated mice were included as additional control (data not shown). Body weight measure, bioluminescent imaging (BLI; NightOwl, Berthold) and X-ray assessment (Faxitron Bioptics), were conducted to monitor the healthy status of the animals, the growth of the tumor cells, and the progression of the lesions respectively. Bone morphometry was conducted as described by Bassett and colleagues (20). A dose of anti-MCAM mAb (10 mg/kg) was used (for both the rat anti-mouse and rat anti-human molecule) and animals injected intraperitoneally. Same dose was applied to the control IgG molecules. Monoclonal rat IgG1 anti-human MCAM (clone 2107) and rat anti-mouse MCAM mAb (clone 15) were kindly provided by Prothena Biosciences (21, 22). The anti-human MCAM antibody has previously been tested in two clinical trials (ClinicalTrials.gov Identifier: NCT02630901 and NCT02458677). Control IgG (rat Fc and human Fc) were purchased from BioXCell (BE0096 and BE0094). For zebrafish experiment, Tg(fli1:GFP)i114 zebrafish line (23) was handled according to local animal welfare regulations to standard protocols (http://www.ZFIN.org). Two days post fertilization, dechorionized zebrafish embryos were anesthetized and injected with PC-3M-Pro4Luc2dTomato cells as described previously (17). Data are representative of at least two independent and blind experiments with ≥30 embryos per group. Survival rate of control group lower than 80% was used as discard cut-off. Images were acquired with Leica SP8 confocal (Leica Microsystems).

RNA sequencing

A biological triplicate for MCAM knockdown cells (shRNA#1) and control samples (shRNA-NT) of the cell line employed for the in vivo study was generated and total RNA extracted using RNeasy Mini Kit (Qiagen). Samples were measured with NextSeq500 (Illumina). Image analysis, base calling, and quality check was performed with the Illumina data analysis pipeline RTA v2.4.11 and Bcl2fastq v2.17. Sequence reads were aligned using STAR two-pass (24) to the human reference genome GRCh37 and gene counts quantified using the “GeneCounts” option. Per-gene counts-per-million (CPM) were computed and log2-transformed adding a pseudo-count of 1 to avoid transforming 0. Genes with log2-CPM <1 in more than three samples were removed. Unsupervised clustering was performed using the top 500 most variable genes, Euclidean distance as the distance metric and the Ward clustering algorithm, using the ConsensusClusterPlus (25) R package. Differential expression analysis between MCAM knockdown cells and control samples was performed using the edgeR (26) R package. Normalization was performed using the “TMM” (weighted trimmed mean) method and differential expression was assessed using the quasi-likelihood F-test. Genes with FDR <0.05 were considered differentially expressed. Genes differentially expressed by >2-fold were reported. Gene Set Enrichment Analysis (GSEA) was performed using the Preranked tool (27) for the, C2 (canonical pathways) and C5 (biological processes; ref. 28). Genes were ranked based on the F-statistic from the differential expression analysis, multiplying F-statistic of downregulated genes by −1. Pathways with FDR <0.25 were considered significant. As an alternative, pathway analysis was also performed for the set of differentially expressed genes using g:Profiler (29). Enrichment maps for GSEA and g:Profiler data were generated with Cytoscape (30). Sets of genes with P value cutoff 0.05 were included and similarity coefficient of 0.5 was applied. The RNA sequencing (RNA-seq) data have been deposited at the NCBI Sequence Read Archive under the accession SRP151808.

Analysis of publicly available dataset for transcriptomic and genomic evaluation and for gene expression–based survival calculation

mRNA data for MCAM expression were extracted with ShinyGEO (31) from the GSE6919 (32, 33), GSE6752 (32) and GSE101607 (34) dataset and analyzed with R. Gene expression–based survival analysis for MCAM was conducted with PROGgene (35) on GSE40272 (36).

Statistical analysis

Statistical analysis was performed using GraphPad Prism 6.0 using t test for comparison between two groups or ANOVA for comparison between more groups. Two-way ANOVA was used to examine the influence of knockdown and control cells on the dependent variable (time) in the in vivo study. Data are presented as mean ± SEM or mean ± SD. P values ≤0.05 are considered to be statistical significant (*, P ≤ 0.05; **, P < 0.01; ***, P < 0.001).

Study approval

Animal experiment was approved by the local ethical committee of Canton of Bern, Switzerland (permit number BE 55/16) and carried out in accordance with Swiss Guidelines for the Care and Use of Laboratory Animals.

Results

MCAM is highly expressed in prostate cancer and associated metastasis

MCAM expression correlates with poor prognosis in several types of cancer, including prostate cancer (37). We investigated the levels of MCAM in normal (N = 18), tumor (N = 65), and tumor adjacent tissues (N = 63) and prostate cancer metastasis (N = 20) in the GSE6919 and GSE6752 datasets (32, 33), which include transcriptional data for primary site and androgen ablation–resistant metastases. We found that MCAM was increased in tumor (P = 0.056) and tumor-adjacent tissue (P = 0.043) compared with normal samples (Fig. 1A and B). MCAM levels were similar in tumor and tumor adjacent tissues (Fig. 1C). Moreover, MCAM was strongly increased in prostate cancer metastases (N = 20) compared with primary tumors (N = 10; P < 0.001; Fig. 1D). Subsequent fractionation of MCAM expression in metastases from various soft tissues (lymph node, adrenal, liver, and lung) also revealed a significant increase in MCAM expression in these sites compared with the primary tumor site (P = 0.001; Fig. 1E). The relation between MCAM expression and prostate cancer recurrence in GSE40272 (36) suggested an involvement of high MCAM expression in disease relapse, although this did not reach significance (Supplementary Fig. S1A and S1B).

Figure 1.
Figure 1.

High expression of MCAM is associated with prostate cancer and prostate cancer metastasis. A–C, MCAM expression in primary normal, primary prostate cancer, and tumor adjacent tissue (GSE6919). Expression levels are presented as boxplots. D and E, MCAM expression in primary prostate cancer site compared with distant metastasis in samples from prostate cancer patients (GSE6752). F, Schematic representation of viable cell sorting; a fraction of MCAMHigh and MCAMLow cells (∼20% of parental gate) is isolated from the ALDHHigh and from the ALDHLow subpopulation, respectively. G, RT-qPCR on the sorted subpopulations for MCAM expression. H and I, Clonogenic assay and quantification of the colony forming capacity of the selected subpopulations after sorting. N = 3 technical replicates. Data are presented as mean ± SD (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Previously, it was demonstrated that a subpopulation of ALDHhigh cells isolated from the aggressive PC-3M-Pro4 prostate cancer cell line displays high clonogenicity in vitro and possesses the ability to generate bone metastases in preclinical mouse models, compared with nontumorigenic nonmetastatic ALDHlow (38). Therefore, we measured the expression of MCAM in selected subpopulations of human prostate cancer cells from the PC-3M-Pro4 cell line and tested whether it was possible to identify a subset of MCAMhigh cells with aggressive features. Using viable cell sorting, we identified four subsets of cells: ALDHhighMCAMhigh; ALDHhighMCAMlow; ALDHlowMCAMhigh; and ALDHlowMCAMlow (Fig. 1F; Supplementary Fig. S1C). Analysis of MCAM expression by RT-qPCR confirmed that we successfully isolated a subset of MCAMhigh cells by flow cytometry (Fig. 1G). However, when tested for colony forming capacity, the four subpopulations of cells displayed similar behavior (Fig. 1H and I).

Characterization of MCAM knockdown cell lines and extravasation ability

We studied the functional role of MCAM in androgen receptor (AR)-negative and lytic prostate cancer PC-3M-Pro4 and PC-3M-Pro4 luc2dTomato cells, and in AR-positive and blastic C4-2B and C4-2BdTomato cells (17).

To investigate the function of MCAM, we used lentiviral delivery of three independent MCAM-targeting shRNAs (MCAM KD) and one control NT shRNA. RT-qPCR showed significant reduction of MCAM mRNA with each of the 3 MCAM shRNAs (shRNA#1; shRNA#2; shRNA#3) compared with the nontargeted control (shRNA-NT; Supplementary Fig. S2A). Measurement of functionally active protein by flow cytometry showed marked reduction of MCAM levels with shRNA#1 (approximately 30% reduction vs. NT control cells), whereas similar protein levels were measured for shRNA#2 and shRNA#3 (Fig. 2A). In C4-2B cells, RT-qPCR displayed strong reduction of MCAM mRNA by each of the 3 shRNAs compared with NT control (Supplementary Fig. S2B). Analysis of functionally active protein expression by flow cytometry showed higher reduction with shRNA#1 and shRNA#3 versus NT control, compared with shRNA#2 (Fig. 2B), in line with the transcriptional data.

Figure 2.
Figure 2.

MCAM knockdown in PC-3M-Pro4Luc2dTomato and C4-2BdTomato human prostate cancer cell lines. Functionally active protein expression is evaluated by flowcytometry and displayed in panel (A) for PC-3M-Pro4Luc2dTomato cells and (B) for C4-2BdTomato. Data are representative of at least three independent experiments. C, Evaluation of effect of MCAM knockdown on cell proliferation over 96 hours in PC-3M-Pro4Luc2dTomato cells and control (sh-NT) cells with three independent shRNAs. Data are representative of at least three independent experiments. **, P < 0.01 and ***, P < 0.001 with Bonferroni multiple comparison test. D, Proliferation assay on C4-2BdTomato cells with MCAM knockdown by three independent shRNAs compared with control nontargeted (sh-NT) cells. Data are represented as mean ± SD. ***, P < 0.001 with Bonferroni multiple comparison test. E, ALDEFLUOR assay on MCAM knockdown and nontargeted (NT) PC-3M-Pro4 cells to assess the percentage of ALDHhigh cells upon MCAM knockdown and data quantification (F). Data are representative of three independent experiments and represented as mean ± SD. G, ALDEFLUOR assay on MCAM knockdown and nontargeted (NT) C4-2BdTomato cells to assess the percentage of ALDHhigh cells upon MCAM knockdown and data quantification (H). Data are representative of three independent experiments and represented as mean ± SD.

We found that MCAM knockdown caused a reduction in cell proliferation in vitro in PC-3M-Pro4 cells (P < 0.01 for shRNA#1 and shRNA#3 at 72 hours and P < 0.001 for shRNA#1 at 96 hours with Bonferroni multiple comparison test, compared with NT control; Fig. 2C). Similarly, in C4-2B cells, MCAM knockdown reduced proliferation in vitro (P < 0.001 for shRNA#1, shRNA#2, and shRNA#3 at 72 hours and P < 0.001 for shRNA#3 at 96 hours with Bonferroni multiple comparison test; Fig. 2D). On the basis of these results and on the knockdown validation at the protein level, we selected the shRNA#1 to continue with subsequent in vitro and in vivo characterization.

To assess the role of MCAM in the modulation of ALDH activity in PC-3M-Pro4 cells, we employed the ALDEFLUOR assay (Fig. 2E). MCAM knockdown cells displayed a lower percentage of ALDHhigh cells compared with NT control, although this did not reach statistical significance (Fig. 2F). Similarly, no change was observed in C4-2B prostate cancer cells (Fig. 2G and H). This suggests that knockdown of MCAM does not cause depletion of the ALDHhigh population of human prostate cancer cells. Analysis of a panel of EMT markers (E-CAD, N-CAD, VIM, ZEB1, ZEB2, SNAIL1, SNAIL2 and TWIST) in the presence or absence of MCAM knockdown suggests that suppressing MCAM expression promotes an epithelial transcriptional phenotype as shown by the significant increase in the ratio E-cadherin/vimentin in both cell lines (P < 0.001 for PC-3M-Pro4 and P < 0.01 for C4-2B; Fig. 3A and B) and by the increase in the ratio E-Cad/N-Cad in PC-3M-Pro4 (P < 0.001 Supplementary Fig. S2C–S2E). However, evaluation of protein expression by Western blot analysis revealed a nonsignificant alteration in E-cadherin and vimentin expression (left, Fig. 3C–F) and in the ratio E-cadherin/vimentin (right, Fig. 3F). We used zebrafish to assess the impact of MCAM knockdown on the ability of prostate cancer cells to extravasate, which is an EMT feature, and to grow at distant sites (Supplementary Fig. S2F). Quantification of disseminated cells at 1 day postinjection and 4 days postinjection revealed similar behavior in knockdown and control (Supplementary Fig. S2G), supporting our analysis on E-cadherin and vimentin expression.

Figure 3.
Figure 3.

Effects of MCAM knockdown on EMT markers and characterization of coculture with human osteoblasts. A and B, MCAM knockdown results in increased ratio of E-cadherin/vimentin and E-cadherin/N-cadherin. Data are representative of three independent samples and normalized with housekeeping gene. Data are represented as mean ± SD. C and D, Western blot analysis for E-cadherin expression and quantification. Calculation is normalized to expression of actin. Data are representative of three independent samples. Data are represented as mean ± SD. E and F, Western blot analysis for vimentin expression, quantification, and protein ratio E-cadherin/vimentin. Calculation is normalized to expression of housekeeping (actin). Data are representative of three independent samples. Data are represented as mean ± SD. G, Schematic representation of the coculture experiment. PC-3M-Pro4 cells expressing dTomato fluorescent protein are cultured in direct contact with differentiated human osteoblasts for 48 hours prior to viable cells sorting. H, Representative images of FACS analysis on ALDH activity on PC-3M-Pro4dTomato cells with nontargeted (sh-NT) without osteoblasts (−OB) or with osteoblasts (+OB). Assay was performed immediately after sorting. Same conditions are shown for the MCAM knockdown cells (two right panels, H). In each panel the small insert represents the control used for gating according from manufacturer's protocol. Quantification is displayed in I. Data are represented as mean ± SEM (**, P < 0.01 with Bonferroni Multiple Comparison Test).

MCAM is required for osteoblast-mediated induction of ALDH activity in prostate cancer cells and is increased upon castration

The osteoblastic microenvironment in bone functions as premetastatic niche by attracting bone-metastasizing prostate cancer cells (5). We have previously shown that coculture of human prostate cancer cells and mature human osteoblasts leads to increase in the percentage of highly metastatic ALDHhigh prostate cancer cells (17).

To investigate the role of MCAM in the context of the osteoblastic niche, we performed direct coculture of MCAM knockdown prostate cancer cells and human osteoblasts. After 48 hours of coculture, the dTomato-labelled PC-3M-Pro4 prostate cancer cells were separated from the osteoblasts by viable cell sorting (Fig. 3G). Immediately after sorting, we measured the ALDH activity of the sorted PC-3M-Pro4 MCAM knockdown (shRNA#1) and control prostate cancer cells (Sh-NT) that were either cocultured (Sh-NT+OB; shRNA#1+OB; Fig. 3H; each condition includes its own gating control, small insert in each panel) or not cocultured (Sh-NT-OB; shRNA#1-OB; Fig. 3H) with osteoblasts. In the absence of osteoblast coculture, NT and MCAM knockdown cells each displayed similar percentages of ALDHhigh cells (CTRL bars, Fig. 3I). However, when cocultured with osteoblasts (OB), the MCAM knockdown prostate cancer cells displayed a significant reduction in the percentage of ALDHhigh cells, when compared with NT prostate cancer cells cocultured with osteoblasts (P < 0.01; Fig. 3I, OB bars). This result indicates that MCAM plays a role in maintaining levels of ALDH activity in prostate cancer cells in the presence of osteoblast cells in coculture and suggests that it may play a similar role within the osteoblastic microenvironment. To test whether there is cross-talk between MCAM and the androgen signaling in the context of prostate cancer bone metastasis, we evaluated the expression level of MCAM and its extracellular matrix interaction partner laminin alpha 4 (LAMA4) in the GSE101607 dataset (34), which contains fresh-frozen bone metastasis samples from AR-driven (N = 32) and non-AR–driven (N = 8) tumors. We found a significant increase in MCAM (P = 0.044) and LAMA4 (P = 0.045) in non–AR-driven conditions compared with AR-driven (Fig. 4A and B). This reinforces the hypothesis that MCAM levels increase upon disease progression. Furthermore, we evaluated the effect of DHT administration in C4-2B cells under high-castration conditions in medium with Charcoal Stripped Serum (CSS) for 72 hours (ref. 39; Fig. 4C). The androgen response was compared in control and MCAM knockdown cells by evaluating the expression of 4 androgen responsive genes upon DHT stimulation (10 nmol/L; ref. 40). C4-2B cells displayed an intact AR machinery as indicated by the strong upregulation of FKBP5, KLK3, and TMPRSS2 and by the downregulation of OPRK1 (Fig. 4D; ref. 40). No difference was detected between control and knockdown cells, indicating that MCAM has no influence on the androgen responsiveness in C4-2B cells. Similarly, administration of the antiandrogen MDV3100 (enzalutamide) revealed no difference in the modulation of the AR responsive genes in control and knockdown cells (Fig. 4E). Interestingly, we observed a consistent downregulation of OPRK1 in MCAM knockdown cells compared with control. Finally, although no change was observed on MCAM expression upon MDV3100 treatment compared with DMSO control, DHT administration resulted in a strong reduction of MCAM expression compared with castration (EtOH control in CSS; Fig. 4F). This supports our dataset analysis and indicates that MCAM levels are higher upon castration.

Figure 4.
Figure 4.

Expression of MCAM in non-AR–driven prostate cancer and in vitro functional effect of DHT treatment. A and B, MCAM and LAMA4 expression in prostate cancer bone metastasis from AR-driven and non-AR–driven disease (in GSE101607). C, Schematic representation of in vitro castration and AR stimulation. N = 3 technical replicates. CSS; Charcoal Stripped Serum. D, Effect of DHT on AR-responsive genes. Results are expressed as the mean ± SD. E, Effect of MDV3100 on AR-responsive genes. F, Effect of AR stimulation or inhibition on MCAM expression. Results are expressed as the mean ± SD, *, P < 0.05; **, P < 0.01; and ***, P < 0.001 with t test; $$$, P < 0.001 between EtOH control and DHT in the respective cell clone.

MCAM knockdown reduces prostate cancer lytic bone metastasis in a preclinical mouse model

To test the impact of MCAM knockdown on bone metastasis of prostate cancer cells, we performed intraosseous inoculation of PC-3M-Pro4 MCAM knockdown cells and NT control cells expressing luciferase 2 (Luc2) in male mice (sham-operated mice were included as an additional control). Animals were monitored by BLI during the course of the experiment (4 weeks; Fig. 5A; all animals, at day 7 and day 28 displayed in Supplementary Fig. S3A). Luciferase activity of MCAM knockdown and NT control cells was assessed by serial cell dilution to confirm that there was no external influence of the shRNAs employed on BLI signal detection (Supplementary Fig. S3B). Body weight of the animals was monitored along the course of the experiment (Supplementary Fig. S3C) and the development of bone lesions was assessed by X-ray measurements (Fig. 5B; data for all mice, including sham, at day 7 and day 28 are displayed in Supplementary Fig. S3D). We found that MCAM knockdown strongly reduced the lytic phenotype of PC-3M-Pro4 cells compared with NT control. This was confirmed by bone morphometric analysis (Fig. 5C; Supplementary Fig. S3E; analysis at day 28) and histologic evaluation (Fig. 5D; Supplementary Fig. S3F). Bone morphometry revealed that bone area in knockdown was similar to sham and significantly higher in mice injected with MCAM knockdown cells compared with those injected with NT control cells (P < 0.05; Fig. 5E) despite the fact that BLI measurement revealed similar tumor burden between MCAM knockdown and NT control cells (Fig. 5F). Finally, we used RT-qPCR to measure the expression of a panel of genes previously identified as regulators of multiple steps of the bone metastatic cascade (TDGF1/CRIPTO, PMEPA1, COL1a, VEGFa, DKK1, PTHLH, MSF; refs. 17, 41). We found a strong inhibition of the oncogene CRIPTO/TDGF1 (42) in MCAM knockdown cells compared with NT control (P < 0.05) and a general modulation of molecules involved in bone remodeling (e.g., PTHLH and DKK1; Fig. 5G). The downmodulation of CRIPTO/TDGF1 in MCAM knockdown cells was confirmed also at protein level (Fig. 5H). Taken together, our data suggest that MCAM influences the expression of molecules that are important modulators of bone remodeling and bone metastasis.

Figure 5.
Figure 5.

CB17 SCID male mice inoculated with PC-3M-Pro4Luc2 human prostate cancer cells with nontargeting shRNA (sh-NT) or MCAM-targeting shRNA (shRNA#1) or sham animals. 50,000 cells/animal. See Supplementary information for representation of all the single animals. A–C, Representative BLI images and bone measurements with X-ray (B) and bone morphometric analysis (C). D, Representative histologic analysis (hematoxylin and eosin staining) of sections of tibia (T, tumor; B, bone; BM, bone marrow). E, Quantification of the bone area at day 28. Data are represented as mean ± SD. F, Quantification of tumor burden by BLI imaging. G, Expression of genes related to bone metastasis in MCAM KD and nontargeted control (NT) prostate cancer cells. N = 3 independent experiments, represented as ± SD. H, Western blotting for Cripto expression. Data are representative of three independent protein isolations.

MCAM knockdown impacts expression of genes that regulate hematopoiesis and bone remodeling

To identify the putative mechanisms of action of MCAM, we performed RNA-seq on MCAM knockdown cells. Unsupervised hierarchical clustering demonstrated a separation between MCAM knockdown and control NT samples (Fig. 6A; heatmap and volcano plot in top and bottom, respectively). Differential expression analysis between MCAM knockdown and NT control revealed that 55 different genes were significantly upregulated (>2-fold increase, FDR < 0.05) and 99 significantly downregulated (>2-fold decrease, FDR < 0.05; Supplementary Table S2 and Supplementary Table S3). We performed GSEA (27) to identify biological processes and pathways modulated by MCAM knockdown (Fig. 6B). We found that the upregulated genes in MCAM knockdown cells were enriched for the process related to negative regulation of hematopoiesis [normalized enrichment score (NES) = 1.68, P = 0.02; Fig. 6C; Supplementary Table S6, for the list of 14 genes involved in the pathway). This supports our previous findings (6) and the notion that increasing the number of HSC niches increases metastatic growth in the bone marrow (5). In addition, we found an enrichment of Gene Ontology processes of biomineral tissue development (NES = 1.8, P = 0.002) and bone mineralization (NES = 1.68, P = 0.008) among the genes upregulated upon MCAM knockdown (Fig. 6D–F). This supports the hypothesis that MCAM knockdown disrupts signaling pathways associated to bone remodeling and are in accordance with our in vivo experiment.

Figure 6.
Figure 6.

RNA-seq and GSEA. A, Heatmap illustrating hierarchical clustering of the samples, based on the 500 most variable genes and Volcano plot of −log10(P) against log2(fold change). Red dots are those with at least 2-fold difference and FDR ≤ 0.05 (i.e., differentially expressed genes). B, GSEA performed on C5_biological processes. Red bars, upregulated pathways; blue bars, downregulated pathways. Numbers on the left (of the blue bars) or on the right (of the red bars) indicate the amount of genes enriched for each gene sets. Data are sorted for NES (x-axis). C, GSEA plot for negative regulators of hematopoiesis. NES and P value are indicated in the plot. D, GSEA enrichment plot for biomineral tissue development. E, GSEA enrichment plot for bone mineralization. The plots C–E illustrate profiles of the NES and positions of the genes on the rank-ordered list in GSEA. Statistical P value is also indicated. F, Enrichment map, for C5 (biological processes) displays sets of genes upregulated (red) or downregulated (blue) in MCAM knockdown cells.

Among the genes downregulated in MCAM knockdown cells, we found an enrichment of genes involved in G0 and early G1 (NES = −1.5, P = 0.02) and in E2F-mediated regulation of DNA replication (NES = −1.5, P = 0.01; Supplementary Fig. S4A and S4B; Supplementary Table S5 and S6). These data reinforce our finding that MCAM knockdown cells displayed reduced proliferation in vitro. Similar results were obtained using an alternative gene set analysis method g:Profiler (Supplementary Fig. S4C and S4D).

Targeting MCAM with a mAb reduces intraosseous growth and diminishes the extension of lytic lesions in an intraosseous model

To test the effect of targeting MCAM on both the tumor and the stroma in a preclinical intraosseous model of prostate cancer bone metastasis, we employed an anti-human and anti-mouse MCAM mAb. Five animals per experimental group (anti-human, anti-mouse, anti-human + mouse, control IgG rat, control IgG human) were pretreated with 10 mg/kg of mAbs and controls IgG intraperitoneally the day prior to intrabone injection of prostate cancer cells. After intrabone injection, all animals received administration of the mAbs or controls IgG every second day at a dose of 10 mg/kg intraperitoneally. Animals were monitored by BLI during the course of the experiment (5 weeks, Fig. 7A, all animals at end of experiment displayed in Supplementary Fig. S5A). Administration of anti-human MCAM mAb resulted in significantly smaller intraosseous growth (P < 0.05; Fig. 7B). Body weight was evaluated, and development of bone lesions was monitored during the experiment with X-ray (Fig. 7C and D, all animals displayed in Supplementary Fig. S5B and statistic in Supplementary Fig. S5C). In addition, luciferase activity of prostate cancer cells was evaluated prior to in vivo experimentation (Supplementary Fig. S5D). Bone morphometric analysis and histologic evaluation revealed a higher bone area in animals treated with anti-human mAbs compared with other experimental groups (P < 0.05; Fig. 7E and F, all animals displayed in Supplementary Fig. S6A–S6D; statistical evaluation displayed in Supplementary Fig. S6B and S6C). Evaluation of the kidney histology revealed normal and comparable histologic characteristics in all the experimental groups (Supplementary Fig. S7A). Contralateral bones as control of normal histologic features were collected and displayed in Supplementary Fig. S7B.

Figure 7.
Figure 7.

CB17 SCID male mice inoculated with PC-3M-Pro4Luc2 human prostate cancer cells intrabone and treated with anti-MCAM mAb. A, Representative BLI images of separate experimental groups (5 animal/group, see Supplementary Materials and Methods for representation of all the single animals. 50,000 cells/animal; in the label descriptions of the group names; H, human; M, mouse). B, Quantification of tumor burden by BLI imaging. Data are represented as mean ± SEM. C, Representative X-ray images of separate experimental groups. See Supplementary Information for representation of all the single animals. D, Body weight of animals measured over the course of the experiment. Data are represented as mean ± SD. E, Representative images of bone morphometric analysis. See Supplementary information for representation of all the single animals and image processing. F, Quantification of the bone area at the end of experiment. Data are represented as mean ± SD (*, P < 0.05 with one way ANOVA and Bonferroni multiple comparison test).

Discussion

In this study, we provide evidence that MCAM drives lytic metastatic human prostate cancer and has potential as a therapeutic target in metastatic prostate cancer.

Our in vivo experiments revealed that MCAM knockdown in prostate cancer cells reduces their ability to generate lytic lesions upon inoculation into the bone. We found that MCAM knockdown cells and NT control cells displayed similar tumor burden in vivo despite showing different cell proliferation rates in vitro. This supports the notion that, in the presence of the bone microenvironment, MCAM has a prominent role in modulating the process of bone remodeling and suggests that, in this context, other mechanisms sustain the proliferation of tumor cells. Treatment with an anti-MCAM mAb revealed significant impact on lytic lesions in the mice bearing prostate cancer cells in the bone. This finding is in line with the results of our in vivo experiment with MCAM knockdown. However, the administration of anti-MCAM mAb also resulted in lower tumor burden in the intraosseous model. This might be related to the residual level of MCAM in our knockdown line (approximately 30% knockdown on the protein level) compared with the administered mAb. We showed here that the reduction in MCAM expression is sufficient to influence the expression of the oncogenic driver CRIPTO/TDGF1 (43). This suggests that in MCAM knockdown in prostate cancer cells, the proliferation in vitro might be supported by TDGF1. Consistent with this possibility, we have previously shown that TDGF1 knockdown decreases cell proliferation in vitro and bone metastases in vivo and that TDGF1 mRNA increases upon direct coculture of human prostate cancer cells with human osteoblasts (17). Although MCAM knockdown does not prevent prostate cancer growth in bone, it does impact on the lytic phenotype. This role of MCAM is supported by our RNA-seq data and transcriptional analysis on a set of genes that was previously shown to regulate bone metastasis in lytic prostate cancer cells (41). MCAM knockdown reduces the mRNA of the osteolytic factor PTHLH (44) and the Wnt inhibitor dickkopf-1 (DKK-1; ref. 45). DKK-1 expression was proposed as an early event in skeletal metastasis, thus favoring osteolysis at the metastatic site (45). In addition, the concurrent downregulation of PTHLH upon MCAM knockdown might explain the effect on cell proliferation measured in vitro, given that PTHLH was shown to play a role in prostate cancer cells' proliferation (46).

We found that in human patients with prostate cancer, MCAM is significantly elevated in androgen ablation–resistant metastases relative to primary tumors. This is in line with our findings that MCAM is elevated upon castration. Moreover, this matches previous clinical literature reporting that MCAM expression is strongly related to poor prognosis in a variety of carcinomas including prostate cancer (37).

We have shown that although it is possible to isolate a fraction of MCAMhigh and MCAMlow cells, their molecular features do not necessarily overlap with those of “bulk” ALDHhigh and ALDHlow cells as shown by our clonogenic assay. This suggests that further subpopulations of cells might display independent phenotypic characteristics as we showed here for the MCAMhigh fraction.

Our findings also suggest that the modulation of MCAM expression might impact on the transcriptional program of EMT-related genes. This is in line with previous studies showing that MCAM promotes EMT and EMT-related drug resistance (47). However, our data on protein expression highlight a lack of significant effect on the detection of E-cadherin and vimentin at the protein level, possibly due to residual MCAM protein. Extravasation is one of the features of EMT and MCAM knockdown prostate cancer cells behaved similarly to control cells when injected into zebrafish. Treatment with DHT and MDV3100 revealed no difference in the response of control and knockdown cells to androgen stimulation or blockade. However, administration of DHT resulted in a strong decrease in MCAM expression compared with castration. This finding and our analysis of two prostate cancer bone metastasis subsets of AR-driven and non-AR–driven disease supports the involvement of MCAM during disease progression and reinforce the hypothesis of targeting MCAM in advanced disease.

In conclusion, we have analyzed the role of MCAM in prostate cancer cells by investigating its biological function with models that recapitulate the presence and absence of androgens and the ostoblastic niche in vitro and the context of the bone microenvironment in vivo. Although additional studies are required to dissect the molecular function of MCAM, our data indicate that MCAM is required for producing the lytic phenotype in prostate cancer bone metastasis. Moreover, we showed here that treatment with anti-MCAM mAb has a significant impact on prostate cancer cells' growth intrabone and diminishes the extension of lytic lesions. Our findings affirm the potential utility of MCAM-targeting agents that are able to interfere with its biological function for use in treating metastatic disease to the bone. The combination of these agents with currently available drugs that target prostate cancer growth might lead to better treatment for patients with prostate cancer, especially those with life-threatening metastatic disease.

Disclosure of Potential Conflicts of Interest

K. Flanagan is the head of cell biology at Prothena Biosciences. No potential conflicts of interest were disclosed by the other authors.

Authors' Contributions

Conception and design: E. Zoni, L. Astrologo, K. Flanagan, G. van der Pluijm, M.G. Cecchini, M. Kruithof-de Julio, G.N. Thalmann

Development of methodology: E. Zoni, S. Piscuoglio, L. Chen, E.B. Snaar-Jagalska, M. Kruithof-de Julio, G.N. Thalmann

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): E. Zoni, L. Astrologo, J. Melsen, J. Grosjean, I. Klima, L. Chen, E.B. Snaar-Jagalska, G. van der Pluijm, P. Kloen, M. Kruithof-de Julio, G.N. Thalmann

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): E. Zoni, L. Astrologo, C.K.Y. Ng, S. Piscuoglio, J. Melsen, M. Kruithof-de Julio, G.N. Thalmann

Writing, review, and/or revision of the manuscript: E. Zoni, L. Astrologo, C.K.Y. Ng, S. Piscuoglio, E.B. Snaar-Jagalska, K. Flanagan, G. van der Pluijm, M. Kruithof-de Julio, G.N. Thalmann

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): E. Zoni, M. Kruithof-de Julio, G.N. Thalmann

Study supervision: E. Zoni, E.B. Snaar-Jagalska, M. Kruithof-de Julio, G.N. Thalmann

Acknowledgments

The authors thank Guido de Roo and Sabrina Veld from the Flow Cytometry Facility (Department of Hematology, Leiden University Medical Center, Leiden, the Netherlands). We also would like to thank Stefan Müller, Bernadette Nyfeler, and Thomas Schaffer from the FACS laboratory (Department of BioMedical Research, University of Bern, Bern, Switzerland). Monoclonal anti-human MCAM and rat anti-mouse MCAM antibodies were kindly provided by Prothena Biosciences. This project received support by the Swiss National Science Foundation (310030_156933 to G.N. Thalmann and 31003A_169352 to M. Kruithof-de Julio) and by the Dutch Cancer Society, grant no. UL2015-7599 KWF to M. Kruithof-de Julio. Additional financial support was provided by the Swiss Cancer League (KFS-3995-08-2016 to S. Piscuoglio) and the Swiss National Science Foundation (Ambizione PZ00P3_168165 to S. Piscuoglio). In memory of Marco G. Cecchini.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Footnotes

  • Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/).

  • ↵†Deceased.

  • Received November 14, 2018.
  • Revision received December 6, 2018.
  • Accepted February 6, 2019.
  • Published first February 11, 2019.

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Therapeutic Targeting of CD146/MCAM Reduces Bone Metastasis in Prostate Cancer
Eugenio Zoni, Letizia Astrologo, Charlotte K.Y. Ng, Salvatore Piscuoglio, Janine Melsen, Joël Grosjean, Irena Klima, Lanpeng Chen, Ewa B. Snaar-Jagalska, Kenneth Flanagan, Gabri van der Pluijm, Peter Kloen, Marco G. Cecchini, Marianna Kruithof-de Julio and George N. Thalmann
Mol Cancer Res May 1 2019 (17) (5) 1049-1062; DOI: 10.1158/1541-7786.MCR-18-1220
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