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The majority of patients with prostate cancer treated with docetaxel develop resistance to it. To better understand the mechanism behind the acquisition of resistance, we conducted single-cell RNA-sequencing (scRNA-seq) of docetaxel-sensitive and -resistant variants of DU145 and PC3 prostate cancer cell lines. Overall, sensitive and resistant cells clustered separately. Differential gene expression analysis between resistant and sensitive cells revealed 182 differentially expressed genes common to both prostate cancer cell lines. A subset of these genes gave a gene expression profile in the resistant transcriptome-like–sensitive cells similar to the resistant cells. Exploration for functional gene pathways identified 218 common pathways between the two cell lines. Protein ubiquitination was the most differentially regulated pathway and was enriched in the resistant cells. Transcriptional regulator analysis identified 321 potential regulators across both cell lines. One of the top regulators identified was nuclear protein 1 (NUPR1). In contrast to the single-cell analysis, bulk analysis of the cells did not reveal NUPR1 as a promising candidate. Knockdown and overexpression of NUPR1 in the prostate cancer cells demonstrated that NUPR1 confers docetaxel resistance in both cell lines. Collectively, these data demonstrate the utility of scRNA-seq to identify regulators of drug resistance. Furthermore, NUPR1 was identified as a mediator of prostate cancer drug resistance, which provides the rationale to explore NUPR1 and its target genes for reversal of docetaxel resistance. Implications: Using single-cell sequencing of prostate cancer, we show that NUPR1 plays a role in docetaxel resistance.

This study identifies a novel pathway through which PCa selectively metastasizes to bone. PCa cells produce exosomes that transfer pyruvate kinase M2 to BMSCs, which in turn produce CXCL12 that promotes PCa invasion and growth., Prostate cancer (PCa) metastasizes selectively to bone through unknown mechanisms. In the current study, we identified exosome-mediated transfer of pyruvate kinase M2 (PKM2) from PCa cells into bone marrow stromal cells (BMSCs) as a novel mechanism through which primary tumor-derived exosomes promote premetastatic niche formation. We found that PKM2 up-regulates BMSC CXCL12 production in a HIF-1α-dependent fashion, which subsequently enhances PCa seeding and growth in the bone marrow. Furthermore, serum-derived exosomes from patients with either primary PCa or PCa metastasis, as opposed to healthy men, reveal that increased exosome PKM2 expression is associated with metastasis, suggesting clinical relevance of exosome PKM2 in PCa. Targeting the exosome-induced CXCL12 axis diminished exosome-mediated bone metastasis. In summary, primary PCa cells educate the bone marrow to create a premetastatic niche through primary PCa exosome-mediated transfer of PKM2 into BMSCs and subsequent up-regulation of CXCL12. This novel mechanism indicates the potential for exosome PKM2 as a biomarker and suggests therapeutic targets for PCa bone metastasis., Graphical Abstract

[This article][1] ([1][2]) is being retracted at the request of the authors. The images used in the second and third panels of CXCR4 siRNA in Fig. 1B were duplicated from the first and second panels of PC3-control cells in Fig. 1B of a previously published article that has since been retracted ([2][

Although docetaxel is the standard of care for advanced prostate cancer, most patients develop resistance to docetaxel. Therefore, elucidating the mechanism that underlies resistance to docetaxel is critical to enhance therapeutic intervention. Mining cDNA microarray from the PC-3 prostate cancer cell line and its docetaxel-resistant derivative (PC3-TxR) revealed decreased latexin (LXN) expression in the resistant cells. LXN expression was inversely correlated with taxane resistance in a panel of prostate cancer cell lines. LXN knockdown conferred docetaxel resistance to prostate cancer cells in vitro and in vivo, whereas LXN overexpression reduced docetaxel resistance in several prostate cancer cell lines. A mouse model of prostate cancer demonstrated that prostate cancer cells developed resistance to docetaxel in the bone microenvironment, but not the soft tissue microenvironment. This was associated with decreased LXN expression in prostate cancer cells in the bone microenvironment compared with the soft tissue microenvironment. It was identified that bone stromal cells decreased LXN expression through methylation and induced chemoresistance in prostate cancer in vitro. These findings reveal that a subset of prostate cancer develops docetaxel resistance through loss of LXN expression associated with methylation and that the bone microenvironment promotes this drug resistance phenotype. Implications: This study suggests that the LXN pathway should be further explored as a viable target for preventing or reversing taxane resistance in prostate cancer. Mol Cancer Res; 15(4); 457–66. ©2017 AACR.

Bone is the most frequent site of prostate cancer (PCa) metastasis; however, little is known about the role of the most common cell in bone, the osteocyte (OCy), in cancer biology. In this study we explored the crosstalk between PCa cells and OCys to determine if it contributes to PCa progression. PCa cells induced OCys to promote PCa proliferation, migration and invasion. A chemokine screen revealed that PCa cell induced OCys to produce growth-derived factor 15 (GDF15). Knockdown of GDF15 in OCys demonstrated that PCa cells conferred the ability on OCys to promote PCa proliferation, migration and invasion through GDF15. Consistent with this finding was the observation that the GDF15 receptor, GFRAL, was expressed on multiple PCa cell lines. Transcription factor array screening of PCa cells exposed to OCys with or without knockdown of GDF15 revealed that GDF15 in OCys promoted early growth response 1 (EGR1) expression in the PCa cells. Knockdown of EGR1 expression in PCa cells revealed it was required for the OCy-derived GDF15-mediated induction of in vitro PCa cell proliferation, migration and invasion. Subcutaneous co-injection of PCa cells and OCys into mice revealed that OCys promoted tumor growth in vivo, which was diminished by knockdown of GDF15 in the OCys. Knockdown of GDF15 in the tibiae diminished growth of PCa cancer cells injected into the tibiae, which was accompanied by decreased tumor cell proliferation and EGR1 expression. These results shed light on a novel mechanism through which PCa cells educate OCys to promote progression of PCa bone metastasis. They also suggest that targeting of GDF15-based and EGR1-based signaling pathways should be further explored for their potential to diminish progression of PCa bone metastasis.

Purpose: To investigate the efficacy and mechanisms of Notch signaling inhibition as an adjuvant to docetaxel in castration-resistant prostate cancer (CRPC) using a γ-secretase inhibitor (GSI), PF-03084014. Experimental Design: The effect of PF-03084014 on response to docetaxel was evaluated in docetaxel-sensitive and docetaxel-resistant CRPC cell lines in vitro and in murine models. Both soft tissue and bone sites were evaluated in vivo. Impacts on cell proliferation, apoptosis, cancer stem cells, and angiogenesis were evaluated. Results: The combination of PF-03084014 plus docetaxel reduced both docetaxel-sensitive and docetaxel-resistant CRPC tumor growth in soft tissue and bone greater than either agent alone. Antitumor activity was associated with PF-03084014–induced inhibition of Notch pathway signaling; decreased survival signals (cyclin E; MEK/ERK, PI3K/AKT, EGFR and NF-κB pathway; BCL-2, BCL-XL); increased apoptotic signals (BAK, BAX; cleaved caspase-3); reduced microvessel density; reduced epithelial–mesenchymal transition; and reduced cancer stem–like cells in the tumor. Conclusions: These results reveal that PF-03084014 enhances docetaxel-mediated tumor response and provides a rationale to explore GSIs as adjunct therapy in conjunction with docetaxel for men with CRPC. Clin Cancer Res; 21(20); 4619–29. ©2015 AACR. See related commentary by Zhang and Armstrong, p. 4505

Cross-talk between tumor cells and their microenvironment is critical for malignant progression. Cross-talk mediators, including soluble factors and direct cell contact, have been identified, but roles for the interaction of physical forces between tumor cells and the bone microenvironment have not been described. Here, we report preclinical evidence that tumor-generated pressure acts to modify the bone microenvironment to promote the growth of prostate cancer bone metastases. Tumors growing in mouse tibiae increased intraosseous pressure. Application of pressure to osteocytes, the main mechanotransducing cells in bone, induced prostate cancer growth and invasion. Mechanistic investigations revealed that this process was mediated in part by upregulation of CCL5 and matrix metalloproteinases in osteocytes. Our results defined the critical contribution of physical forces to tumor cell growth in the tumor microenvironment, and they identified osteocytes as a critical mediator in the bone metastatic niche. Cancer Res; 75(11); 2151–8. ©2015 AACR.

Integrins that contain an integrin αV subunit contribute to multiple functions that promote cancer progression. The goal of this study was to determine whether abituzumab (DI17E6, EMD 525797), a humanized monoclonal antibody (mAb) against integrin αV impacts, prostate cancer progression. To evaluate this, prostate cancer cells were treated with DI17E6 and its effects on proliferation, apoptosis, cell-cycle, adhesion, detachment, migration, invasion and phosphorylation of downstream targets, including FAK, Akt, and ERK, were determined. DI17E6 promoted detachment and inhibited adhesion of prostate cancer cells to several extracellular matrix (ECM) proteins and cells found in the bone microenvironment but had no impact on cell viability, cell-cycle, and caspase-3/7 activity. DI17E6 inhibited migration and invasion of prostate cancer cells. In addition, DI7E6 decreased phosphorylation of FAK, Akt, and ERK. These results indicate that inhibition of integrin αV with DI17E6 inhibits several prometastatic phenotypes of prostate cancer cells and therefore provide a rationale for further evaluation of DI17E6 for diminishing prostate cancer progression. Implications: This work identifies that therapeutic targeting of integrins containing an αV integrin unit inhibits cancer progression and thus may be of clinical benefit. Mol Cancer Res; 15(7); 875–83. ©2017 AACR.

Purpose: Cabozantinib, an orally available multityrosine kinase inhibitor with activity against mesenchymal epithelial transition factor (MET) and VEGF receptor 2 (VEGFR2), induces resolution of bone scan lesions in men with castration-resistant prostate cancer bone metastases. The purpose of this study was to determine whether cabozantinib elicited a direct antitumor effect, an indirect effect through modulating bone, or both. Experimental Design: Using human prostate cancer xenograft studies in mice, we determined the impact of cabozantinib on tumor growth in soft tissue and bone. In vitro studies with cabozantinib were performed using (i) prostate cancer cell lines to evaluate its impact on cell growth, invasive ability, and MET and (ii) osteoblast cell lines to evaluate its impact on viability and differentiation and VEGFR2. Results: Cabozantinib inhibited progression of multiple prostate cancer cell lines (Ace-1, C4-2B, and LuCaP 35) in bone metastatic and soft tissue murine models of prostate cancer, except for PC-3 prostate cancer cells in which it inhibited only subcutaneous growth. Cabozantinib directly inhibited prostate cancer cell viability and induced apoptosis in vitro and in vivo and inhibited cell invasion in vitro. Cabozantinib had a dose-dependent biphasic effect on osteoblast activity and inhibitory effect on osteoclast production in vitro that was reflected in vivo. It blocked MET and VEGFR2 phosphorylation in prostate cancer cells and osteoblast-like cells, respectively. Conclusion: These data indicate that cabozantinib has direct antitumor activity, and that its ability to modulate osteoblast activity may contribute to its antitumor efficacy. Clin Cancer Res; 20(3); 617–30. ©2013 AACR.

Once tumor cells metastasize to the bone, the prognosis for prostate cancer patients is generally very poor. The mechanisms involved in bone metastasis, however, remain elusive, because of lack of relevant animal models. In this manuscript, we describe step-by-step protocols for the xenograft mouse models that are currently used for studying prostate cancer bone metastasis. The different routes of tumor inoculation (intraosseous, intracardiac, intravenous and orthotopic) presented are useful for exploring the biology of bone metastasis.

Bone is the preferred metastasis site of advanced prostate cancer (PCa). Using an in vivo murine model of human PCa cell metastasis to bone, we noted that the majority of animals that develop skeletal metastasis have either spinal lesions or lesions in the bones of the hindlimb. Much less frequently, lesions develop in the bones of the forelimb. We therefore speculated whether the environment of the forelimb bones is not permissive for the growth of PCa. Consequently, data on tumor prevalence were normalized to account for the number of PCa cells arriving after intravascular injection, marrow cellularity, and number of hematopoietic stem cell niches. None of these factors were able to account for the observed differences in tumor prevalence. An analysis of differential gene and protein levels identified that growth arrest specific-6 (GAS6) levels were significantly greater in the forelimb versus hindlimb bone marrow. When murine RM1 cells were implanted into subcutaneous spaces in immune competent animals, tumor growth in the GAS6-/- animals was greater than in GAS6+/+ wild-type animals. In an osseous environment, the human PC3 cell line grew significantly better in vertebral body transplants (vossicles) derived from GAS6-/- animals than in vossicles derived from GAS6+/+ animals. Together, these data suggest that the differences in tumor prevalence after intravascular inoculation are a useful model to study the molecular basis of tumor dormancy. Importantly, these data suggest that therapeutic manipulation of GAS6 levels may prove useful as a therapy for metastatic disease.

BACKGROUND. Recently, we established paclitaxel-resistant prostate cancer cell lines (PC-3-TxR and DU145-TxR). To determine the mechanisms of paclitaxel resistance in PC-3TxR cells, we compared the gene expression profiles between PC-3 and PC-3-TxR cells. Our results indicated that expression of the C-terminal tensin like protein (CTEN, tensin 4) gene was down-regulated by 10-fold in PC-3-TxR cells. We investigated the possibility that CTEN overexpression restores paclitaxel sensitivity. METHODS. We investigated how knockdown and overexpression of CTEN in androgenindependent cell lines affect paclitaxel sensitivity by colony formation assay and growth inhibition assay. To determine the mechanisms by which CTEN affects paclitaxel sensitivity, we investigated the relationships between CTEN and F-actin or epidermal growth factor receptor (EGFR) in PC-3 cells. We also examined the association between expression of CTEN and grade of prostate cancer by immunohistochemistry using tissue microarray analysis. RESULTS. Down-regulation of CTEN, which is located in the cytoskeleton, played an important role in paclitaxel resistance in PC-3-TxR cells. Knockdown of CTEN expression in PC-3 cells induced paclitaxel resistance. Overexpression of CTEN in PC-3-TxR and DU145-TxR cells restored paclitaxel sensitivity. CTEN expression was inversely correlated with F-actin and EGFR expression. Then knockdown of actin and EGFR in PC-3-TxR cells recovered paclitaxel sensitivity, indicating that CTEN down-regulation mediates paclitaxel resistance through elevation of EGFR and actin expression. Moreover, CTEN expression was inversely correlated with Gleason score. CONCLUSIONS. These results strongly suggested that CTEN plays an important role in paclitaxel sensitivity and that CTEN expression level may be a prognostic predictive factor for PCa patients. Prostate 70: 48–60, 2010. # 2009 Wiley-Liss, Inc.

BACKGROUND. Docetaxel induces an anti-tumor response in men with advanced prostate cancer (PCa); however, the side effects associated with docetaxel treatment can be severe, resulting in discontinuation of therapy. Thus, identification of an effective adjuvant therapy to allow lower doses of docetaxel is needed. Advanced PCa is typically accompanied by skeletal metastasis. Receptor activator of NFkB ligand (RANKL) is a key pro-osteoclastic factor. Targeting RANKL decreases establishment and progression of PCa growth in bone in murine models. METHODS. The efficacy of inhibiting RANKL, using a recombinant soluble RANK extracellular domain fused with the immunoglobulin Fc domain (RANK-Fc), was tested as an adjuvant therapy with docetaxel for PCa bone metastasis in a murine intra-tibial model. RESULT. The combination of RANK-Fc and docetaxel reduced tumor burden in bone greater than either treatment alone. CONCLUSION. The combination of docetaxel with a RANKL-inhibiting agent merits further investigation for treatment of advance PCa. Prostate 68: 820–829, 2008. # 2008 Wiley-Liss, Inc.

Prostate cancer frequently metastasizes to bone where it forms osteoblastic lesions through unknown mechanisms. Bone morphogenetic proteins (BMP) are mediators of skeletal formation. Prostate cancer produces a variety of BMPs, including BMP-6. We tested the hypothesis that BMP-6 contributes to prostate cancer–induced osteosclerosis at bone metastatic sites. Prostate cancer cells and clinical tissues produced BMP-6 that increased with aggressiveness of the tumor. Prostate cancer-conditioned medium induced SMAD phosphorylation in the preosteoblast MC3T3 cells, and phosphorylation was diminished by anti–BMP-6 antibody. Prostate cancer-conditioned medium induced mineralization of MC3T3 cells, which was blocked by both the BMP inhibitor noggin and anti–BMP-6. Human fetal bones were implanted in severe combined immunodeficient mice and after 4 weeks, LuCaP 23.1 prostate cancer cells were injected both s.c. and into the bone implants. Anti–BMP-6 or isotype antibody administration was then initiated. Anti–BMP-6 reduced LuCaP 23.1–induced osteoblastic activity, but had no effect on its osteolytic activity. This was associated with increased osteoblast numbers and osteoblast activity based on bone histomorphometric evaluation. As endothelin-1 has been implicated in bone metastases, we measured serum endothelin-1 levels but found they were not different among the treatment groups. In addition to decreased bone production, anti–BMP-6 reduced intraosseous, but not s.c., tumor size. We found that BMP-2, BMP-4, BMP-6, and BMP-7 had no direct effect on prostate cancer cell growth, but BMP-2 and BMP-6 increased the in vitro invasive ability of prostate cancer cell. These data show that prostate cancer promotes osteoblastic activity through BMP-6 and that, in addition to its bone effects, suggest that BMPs promote the ability of the prostate cancer cells to invade the bone microenvironment.

Prostate cancer produces painful osteoblastic bone metastases. Although prostate cancer cells produce numerous osteogenic factors, to date, none have been shown to mediate osteoblastic bone metastases in an in vivo model of prostate cancer. Wnts are a large family of proteins that promote bone growth. Wnt activity is antagonized by endogenous proteins including dickkopf-1 (DKK-1). We explored if prostate cancer cells mediate osteoblastic activity through Wnts using DKK-1 as a tool to modify Wnt activity. A variety of Wnt mRNAs were found to be expressed in prostate cancer cell lines and Wnt mRNA expression was increased in primary prostate cancer compared with nonneoplastic prostate tissue. In addition to expressing Wnts, PC-3 prostate cancer cells expressed the Wnt inhibitor DKK-1. To determine if DKK-1 masked Wnt-mediated osteoblastic activity in osteolytic PC-3 cells, the cells were stably transfected with DKK-1 short hairpin RNA. Decreasing DKK-1 enabled PC-3 cells to induce osteoblastic activity, including alkaline phosphatase production and mineralization, in murine bone marrow stromal cells indicating that DKK-1 blocked Wnt-mediated osteoblastic activity in PC-3 cells. Another prostate cancer cell line, C4-2B, induces mixed osteoblastic/osteolytic lesions. To determine if Wnts contribute to C4-2B's ability to induce mixed osteoblastic/osteolytic lesions, C4-2B cells were stably transfected with either empty vector or DKK-1 expression vector to block Wnt activity. The cells were then injected in the tibiae of mice and allowed to grow for 12 weeks. Blocking Wnt activity converted the C4-2B cells to a highly osteolytic tumor. Taken together, these data show that Wnts contribute to the mechanism through which prostate cancer induces osteoblastic activity.

To delineate the role of SDF-1 and CXCR4 in metastatic prostate cancer (CaP), positive correlations were established between SDF-1 levels and tumor metastasis. Neutralization of CXCR4 limited the number and the growth of intraosseous metastasis in vivo. Together, these in vivo metastasis data provide critical support that SDF-1/CXCR4 plays a role in skeletal metastasis. Introduction: Previously we determined that the stromal-derived factor-1 (SDF-1)/CXCR4 chemokine axis is activated in prostate cancer (CaP) metastasis to bone. To delineate the role of SDF-1/CXCR4 in CaP, we evaluated SDF-1 levels in a variety of tissues and whether neutralization of SDF-1 prevented metastasis and/or intraosseous growth of CaPs. Materials and Methods: SDF-1 levels were established in various mouse tissues by ELISA, immunohistochemistry, and in situ hybridization. To assess the role of SDF-1/CXCR4 in metastasis, bone metastases were established by administering CaP cells into the left cardiac ventricle of nude animals in the presence or absence of neutralizing CXCR4 antibody. The effect of SDF-1 on intraosseous growth of CaP cells was determined using intratibial injections and anti-CXCR4 antibodies and peptides. Results: There was a positive correlation between the levels of SDF-1 and tissues in which metastatic CaP lesions were observed. SDF-1 levels were highest in the pelvis, tibia, femur, liver, and adrenal/kidneys compared with the lungs, tongue, and eye, suggesting a selective effect. SDF-1 staining was generally low or undetectable in the center of the marrow and in the diaphysis. SDF-1 mRNA was localized to the metaphysis of the long bones nearest to the growth plate where intense expression was observed near the endosteal surfaces covered by osteoblastic and lining cells. Antibody to CXCR4 significantly reduced the total metastatic load compared with IgG control-treated animals. Direct intratibial injection of tumor cells followed by neutralizing CXCR4 antibody or a specific peptide that blocks CXCR4 also decreased the size of the tumors compared with controls. Conclusions: These data provide critical support for a role of SDF-1/CXCR4 in skeletal metastasis. Importantly, these data show that SDF-1/CXCR4 participate in localizing tumors to the bone marrow for prostate cancer.

Metastasis is the major cause of treatment failure in various malignancies, including prostate cancer (PCa). Accumulating data has suggested that primary cancer cells prefer to spread to certain distant organs. To explore the mechanisms of different cancer cells preferentially metastasizing to certain organs, we established a prostate cancer lung metastasis model. Murine prostate cancer cells (RM1), which labeled with luciferase, were used in this study. RM1 cells were injected into left tibia of mouse (C57BL/6J), and monitored the tumor growth and metastasis every week by Bioluminescence Imaging. The mice would be sacrificed when metastatic tumor appeared in multi-organs. We isolated the lung metastatic tumor, made single cell suspension and amplified. The lung metastatic tumor cells were specifically metastasized to lung when the tumor cells were injected intratibially again. After several cycles we obtained highly lung metastatic tumor cells, named RM1-LM cells. Further, we investigated property and function changes in RM1-LM cells compared to parental cells (RM1) using in vitro assays. We firstly found the highly lung metastatic tumor cells showed more mesenchymal properties. Western blot results showed that hallmarks of EMT including E-cadherin, vimentin, snail, and ZO-1 were altered significantly in RM1-LM cells. Then, we observed that the highly lung metastatic tumor cells exhibited increased proliferative, colony formation, migratory and invasive abilities. Mechanistic analyses indicated that prostate cancer organ-specific metastasis mediated by the activation of GSK-3β/β-catenin pathway. The current study may provide potential therapeutic targets and diagnostic biomarkers of PCa metastasis. This work was supported by Natural Science Foundation of China (NSFC) Key Project (81130046); NSFC (81171993; 81272415); Guangxi Projects of China (2013GXNSFEA053004; 2012GXNSFCB053004). Note: This abstract was not presented at the meeting. Citation Format: Siyuan Qin, Qiong Song, Jinlu Dai, Haibo Tong, Evan Keller, Jian Zhang, Yi Lu. Activation of GSK3β/β-catenin pathway promotes organ-specific metastasis in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4939. doi:10.1158/1538-7445.AM2017-4939

Tumor cells secrete factors that modulate macrophage activation and polarization into M2 type tumor-associated macrophages, which promote tumor growth, progression, and metastasis. The mechanisms that mediate this polarization are not clear. Macrophages are phagocytic cells that participate in the clearance of apoptotic cells, a process known as efferocytosis. Milk fat globule- EGF factor 8 (MFG-E8) is a bridge protein that facilitates efferocytosis and is associated with suppression of proinflammatory responses. This study investigated the hypothesis that MFG-E8-mediated efferocytosis promotes M2 polarization. Tissue and serum exosomes from prostate cancer patients presented higher levels of MFG-E8 compared with controls, a novel finding in human prostate cancer. Coculture of macrophages with apoptotic cancer cells increased efferocytosis, elevated MFG-E8 protein expression levels, and induced macrophage polarization into an alternatively activated M2 phenotype. Administration of antibody against MFG-E8 significantly attenuated the increase in M2 polarization. Inhibition of STAT3 phosphorylation using the inhibitor Stattic decreased efferocytosis and M2 macrophage polarization in vitro, with a correlating increase in SOCS3 protein expression. Moreover, MFG-E8 knockdown tumor cells cultured with wild-type or MFG-E8-deficient macrophages resulted in increased SOCS3 expression with decreased STAT3 activation. This suggests that SOCS3 and phospho-STAT3 act in an inversely dependent manner when stimulated by MFG-E8 and efferocytosis. These results uncover a unique role of efferocytosis via MFG-E8 as a mechanism for macrophage polarization into tumor-promoting M2 cells.

Prostate cancer (CaP) forms osteoblastic skeletal metastases with an underlying osteoclastic component. However, the importance of osteoclastogenesis in the development of CaP skeletal lesions is unknown. In the present study, we demonstrate that CaP cells directly induce osteoclastogenesis from osteoclast precursors in the absence of underlying stroma in vitro. CaP cells produced a soluble form of receptor activator of NF-kappaB ligand (RANKL), which accounted for the CaP-mediated osteoclastogenesis. To evaluate for the importance of osteoclastogenesis on CaP tumor development in vivo, CaP cells were injected both intratibially and subcutaneously in the same mice, followed by administration of the decoy receptor for RANKL, osteoprotegerin (OPG). OPG completely prevented the establishment of mixed osteolytic/osteoblastic tibial tumors, as were observed in vehicle-treated animals, but it had no effect on subcutaneous tumor growth. Consistent with the role of osteoclasts in tumor development, osteoclast numbers were elevated at the bone/tumor interface in the vehicle-treated mice compared with the normal values in the OPG-treated mice. Furthermore, OPG had no effect on CaP cell viability, proliferation, or basal apoptotic rate in vitro. These results emphasize the important role that osteoclast activity plays in the establishment of CaP skeletal metastases, including those with an osteoblastic component.

BACKGROUND Prostate cancer frequently metastasizes to bone. However, unlike many other tumors that produce osteolytic lesions, prostate cancer produces osteoblastic lesions through unknown mechanisms. In the current study, we explored the ability and mechanism of an osteotropic prostate cancer cell line (C4-2B) to induce mineralization. METHODS C4-2B cells were grown in promineralization media. Mineral deposition was characterized using von Kossa staining, calcium retention, alizarin red staining, Raman spectroscopy, and electron microscopy. Expression of osteoblast-related proteins was determined by RT-PCR. The nuclear level of the bone-specific transcription factor Cbfa1 was determined using western analysis and the effect of inhibiting Cbfa1 function, using a “decoy” Cbfa1 response element oligo, on mineralization was determined. RESULTS The studies demonstrated that C4-2B cells, but not its nonosteotropic parent cell line LNCaP, has an osteoblastlike phenotype including production of alkaline phosphatase, osteocalcin, osteonectin, bone sialoprotein, osteoprotegerin (OPG), and OPG ligand. Most importantly, the C4-2B cells produced hydroxyapatite mineral in vitro. Furthermore, C4-2B cells expressed high nuclear levels of the bone-specific transcription factor Cbfa1, compared to LNCaP cells, which accounts for their ability to produce bone-specific proteins. Inhibition of Cbfa1, using decoy DNA Cbfa1 response elements, abrogated the ability of C4-2B to produce mineral. Finally, we determined that C4-2B cells express bone morphogenic protein-7, a known inducer of Cbfa1 expression. CONCLUSIONS These data demonstrate a novel mechanism through which prostate cancer cells may directly contribute to the osteoblastic component that characterize their skeletal metastatic lesions. Prostate 47:212–221, 2001. © 2001 Wiley-Liss, Inc.

Due to its bone anabolic activity, methods to increase Wnt activity, such as inhibitors of dickkopf-1 and sclerostin, are being clinically explored. Glycogen synthase kinase (GSK3β) inhibits Wnt signaling by inducing β-catenin degradation, and a GSK3β inhibitor, AR79, is being evaluated as an osteoanabolic agent. However, Wnt activation has the potential to promote tumor growth; therefore, the goal of this study was to determine if AR79 has an impact on the progression of prostate cancer. Prostate cancer tumors were established in subcutaneous and bone sites of mice followed by AR79 administration, and tumor growth, β-catenin activation, proliferation, and apoptosis were assessed. Additionally, prostate cancer and osteoblast cell lines were treated with AR79, and β-catenin status, proliferation (with β-catenin knockdown in some cases), and proportion of ALDH+CD133+ stem-like cells were determined. AR79 promoted prostate cancer tumor growth, decreased phospho-β-catenin, increased total and nuclear β-catenin, and increased tumor-induced bone remodeling. Additionally, AR79 treatment decreased caspase-3 and increased Ki67 expression in tumors and increased bone formation in normal mouse tibiae. Similarly, AR79 inhibited β-catenin phosphorylation, increased nuclear β-catenin accumulation in prostate cancer and osteoblast cell lines, and increased proliferation of prostate cancer cells in vitro through β-catenin. Furthermore, AR79 increased the ALDH+CD133+ cancer stem cell–like proportion of the prostate cancer cell lines. In conclusion, AR79, while being bone anabolic, promotes prostate cancer cell growth through Wnt pathway activation. Implications: These data suggest that clinical application of pharmaceuticals that promote Wnt pathway activation should be used with caution as they may enhance tumor growth. Mol Cancer Res; 11(12); 1597–610. ©2013 AACR.

Human prostate cancer frequently metastasizes to bone marrow. What defines the cellular and molecular predilection for prostate cancer to metastasize to bone marrow is not well understood. CD4(+)CD25(+) regulatory T (Treg) cells contribute to self-tolerance and tumor immune pathology. We now show that functional Treg cells are increased in the bone marrow microenvironment in prostate cancer patients with bone metastasis, and that CXCR4/CXCL12 signaling pathway contributes to Treg cell bone marrow trafficking. Treg cells exhibit active cell cycling in the bone marrow, and bone marrow dendritic cells express high levels of receptor activator of NFκB (RANK), and promote Treg cell expansion through RANK and its ligand (RANKL) signals. Furthermore, Treg cells suppress osteoclast differentiation induced by activated T cells and M-CSF, adoptive transferred Treg cells migrate to bone marrow, and increase bone mineral intensity in the xenograft mouse models with human prostate cancer bone marrow inoculation. In vivo Treg cell depletion results in reduced bone density in tumor bearing mice. The data indicates that bone marrow Treg cells may form an immunosuppressive niche to facilitate cancer bone metastasis and contribute to bone deposition, the major bone pathology in prostate cancer patients with bone metastasis. These findings mechanistically explain why Treg cells accumulate in the bone marrow, and demonstrate a previously unappreciated role for Treg cells in patients with prostate cancer. Thus, targeting Treg cells may not only improve anti-tumor immunity, but also ameliorate bone pathology in prostate cancer patients with bone metastasis.

Bone metastasis is often the penultimate harbinger of death for many cancer patients. Bone metastases are often associated with fractures and severe pain resulting in decreased quality of life. Accordingly, effective therapies to inhibit the development or progression of bone metastases will have important clinical benefits. To achieve this goal understanding the mechanisms through which bone metastases develop and progress may provide targets to inhibit the metastases. In the past few years, there have been advances in both understanding the mechanisms through which bone metastases develop and how they impact bone remodeling. Additionally, gains in promising clinical strategies to target bone metastases have been developed. In this prospectus, we will discuss some of these advances.

The most frequent site of prostate cancer metastasis is the bone. Adhesion to bone-specific factors may facilitate the selective metastasis of prostate cancer to the skeleton. Therefore, we tested whether prostate cancer bone metastasis is mediated by binding to type I collagen, the most abundant bone protein. We observed that only bone metastatic prostate cancer cells bound collagen I, whereas cells that form only visceral metastases failed to bind collagen. To confirm the relationship between collagen adhesion and bone metastatic potential, a collagen-binding variant of human LNCaP prostate cancer cells was derived through serial passage on type I collagen (LNCaPcol). Fluorescence-activated cell sorting analysis showed that LNCaPcol cells express increased levels of the integrin collagen I receptor α2β1 compared with LNCaP cells. Antibodies to the α2β1 complex inhibited LNCaPcol binding to collagen, confirming that integrins mediated the attachment. Correspondingly, LNCaPcol cells displayed enhanced chemotactic migration toward collagen I compared with LNCaP cells, an activity that could be blocked with α2β1 antibodies. To directly test the role of α2β1-dependent collagen binding in bone metastasis, LNCaP and LNCaPcol cells were injected into the tibia of nude mice. After 9 weeks, 7 of 13 (53%) mice injected with LNCaPcol developed bone tumors, whereas 0 of 8 mice injected with LNCaP cells had evidence of boney lesions. LNCaPcol cells were found to express increased levels of the metastasis-promoting RhoC GTPase compared with parental LNCaP. We conclude that collagen I attachment mediated by α2β1 initiates motility programs through RhoC and suggest a mechanism for prostate cancer metastasis to the bone. (Cancer Res 2006; 66(17): 8648-54)

Prostate cancer frequently metastasizes to bone resulting in the formation of osteoblastic metastases through unknown mechanisms. Vascular endothelial growth factor (VEGF) has been shown recently to promote osteoblast activity. Accordingly, we tested if VEGF contributes to the ability of prostate cancer to induce osteoblast activity. PC-3, LNCaP, and C4-2B prostate cancer cell lines expressed both VEGF-165 and VEGF-189 mRNA isoforms and VEGF protein. Prostate cancer cells expressed the mRNA for VEGF receptor (VEGFR) neuropilin-1 but not the VEGFRs Flt-1 or KDR. In contrast, mouse pre-osteoblastic cells (MC3T3-E1) expressed Flt-1 and neuropilin-1 mRNA but not KDR. PTK787, a VEGFR tyrosine kinase inhibitor, inhibited the proliferation of human microvascular endothelial cells but not prostate cancer proliferation in vitro. C4-2B conditioned medium induced osteoblast differentiation as measured by production of alkaline phosphatase and osteocalcin and mineralization of MC3T3-E1. PTK787 blocked the C4-2B conditioned medium–induced osteoblastic activity. VEGF directly induced alkaline phosphatase and osteocalcin but not mineralization of MC3T3-E1. These results suggest that VEGF induces initial differentiation of osteoblasts but requires other factors, present in C4-2B, to induce mineralization. To determine if VEGF influences the ability of prostate cancer to develop osteoblastic lesions, we injected C4-2B cells into the tibia of mice and, after the tumors grew for 6 weeks, administered PTK787 for 4 weeks. PTK787 decreased both intratibial tumor burden and C4-2B–induced osteoblastic activity as measured by bone mineral density and serum osteocalcin. These results show that VEGF contributes to prostate cancer–induced osteoblastic activity in vivo.

Prostate cancer (CaP) metastases selectively develop in bone as opposed to other sites through unknown mechanisms. Interleukin-6 (IL-6) is considered to contribute to CaP progression and is produced at high levels in osteoblasts. We hypothesized that osteoblast-derived IL-6 in the bone microenvironment contributes to the fertile soil for CaP growth. Accordingly, human CaP cells, LNCaP, C4-2B and VCaP, were treated with conditioned medium (CM) collected from human osteoblast-like HOBIT cells grown in androgen-depleted medium. We found that CM induced proliferation, prostate-specific antigen (PSA) protein and mRNA expression in a dose-dependent manner in these cell lines as determined by ELISA and real-time PCR, respectively. CM also activated the PSA promoter in these cells. Both HOBIT and primary osteoblast (POB) cells produced high levels of IL-6 measured by bioassay. LNCaP, C4-2B and VCaP cells expressed IL-6, but at much lower levels then the HOBIT and POB and they also expressed the IL-6 receptor mRNA, indicating they can respond to IL-6. Anti-IL-6 antibody added to HOBIT or POB CM dose-dependently inhibited the CM-induced cell proliferation and PSA expression in these CaP cell lines. HOBIT CM induced nuclear translocation of the AR and this was inhibited by anti-IL-6 antibody. Additionally, the antiandrogen bicalutamide inhibited HOBIT CM-induced cell proliferation. These results demonstrate that osteoblasts promote CaP growth through IL-6-mediated activation of the AR. Furthermore, these data underscore the importance of cross-talk between tumor and the bone microenvironment in the development of CaP bone metastases.

Human prostate cancer has a high predisposition to metastasize to bone, resulting in the formation of osteoblastic metastases. The mechanism through which prostate cancer cells promote osteoblastic lesions is undefined. Vascular endothelial growth factor (VEGF) has been implicated as a mediator of osteoblast activity. In the present study, we examined if prostate cancer cells promote osteoblastic activity through VEGF. We found that LNCaP and C4-2B prostate cancer cell lines and primary tumor and metastatic prostate cancer tissues from patients expressed VEGF. Bone morphogenetic proteins (BMPs), which are normally present in the bone environment, induced VEGF protein and mRNA expression in C4-2B cells. Furthermore, BMP-7 activated the VEGF promoter. Noggin, a BMP inhibitor, diminished VEGF protein expression and promoter activity in C4-2B cells. Conditioned media (CM) from C4-2B cells induced pro-osteoblastic activity (increased alkaline phosphatase, osteocalcin, and mineralization) in osteoblast cells. Both noggin alone and anti-VEGF antibody alone diminished C4-2B CM-induced pro-osteoblastic activity. Transfection of C4-2B cells with VEGF partially rescued the C4-2B CM-induced pro-osteoblastic activity from noggin inhibition. These observations indicate that BMPs promote osteosclerosis through VEGF in prostate cancer metastases. These results suggest a novel function for VEGF in skeletal metastases. Specifically, VEGF promotes osteoblastic lesion formation at prostate cancer bone metastatic sites.

Prostate cancer (PCa) frequently metastasizes to bone forming predominantly osteoblastic lesions with underlined osteolytic components. Because current therapies are generally ineffective in PCa skeletal metastases, patients seek alternative medicines such as herbal medicines, which have been used for many centuries in Asian countries. The purpose of this study was to identify Chinese herbal medicines that may be effective in PCa skeletal metastasis. We first screened 130 Chinese herbs (ethanol extracts) for anti-proliferation effects by non-radioactive cell proliferation assay in LNCaP and C4-2B PCa cells. We found five extracts with anti-proliferation activity including extract from Scutellaria baicalensis Georgi (Huang Qin, HQ), which had the strongest effect. We next evaluated HQ's ability to induce apoptosis of LNCaP and C4-2B cells. HQ, in a dose-dependent manner, induced apoptosis of both cell lines based on TUNEL assay. In addition, HQ induced apotosis-related gene caspase-3 and suppressed bcl-2 expressions in both cell lines. Therefore, we tested if HQ (1mg per mouse three times a week for 8 weeks intraperitoneally) could diminish establishment of skeletal metastases in mice. We injected C4-2B cells into tibia of SCID mice. The animals were randomized into two groups to receive either HQ or vehicle only (n=8/group). At 8 weeks post-tumor injection, mice were sacrificed and tumor establishment in the skeleton was determined using radiography, dual energy x-ray absorptiometry and histology. Radiographic and histological lesions were present in 7/8 vehicle-treated mice and in 6/8 HQ-treated mice. However, the radiographically- and histologically-determined areas of the tumors in the HQ-treated mice were approximately 47% that of the vehicle-treated animals. We conclude that HQ has efficacy for inducing PCa cells apoptosis in vitro and has the ability to diminish growth of PCa in murine bone. Supported by NSF key project 81130046, NSF projects 81171993 and 81272415. Citation Format: Chaoqian Li, Hongwei Guo, Yi Lu, Jinlu Dai, Evan Keller, Jian Zhang. Extract from Scutellaria baicalensis Georgi inhibits prostate cancer growth in bone. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3198. doi:10.1158/1538-7445.AM2014-3198