A CXCR4 inhibitor (balixafortide) enhances docetaxel-mediated antitumor activity in a murine model of prostate cancer bone metastasis.

BACKGROUND: Prostate cancer (PCa) bone metastases have been shown to be more resistant to docetaxel than soft tissue metastases. The proinflammatory chemokine receptor CXCR4 has been shown to confer resistance to docetaxel (DOC) in PCa cells. Balixafortide (BLX) is a protein epitope mimetic inhibitor of CXCR4. Accordingly, we hypothesized that BLX would enhance DOC-mediated antitumor activity in PCa bone metastases. METHODS: PC-3 luciferase-labeled cells were injected into the tibia of mice to model bone metastases. Four treatment groups were created: vehicle, DOC (5 mg/kg), BLX (20 mg/kg), and combo (receiving both DOC and BLX). Mice were injected twice daily subcutaneously with either vehicle or BLX starting on Day 1 and weekly intraperitoneally with DOC starting on Day 1. Tumor burden was measured weekly via bioluminescent imaging. At end of study (29 days), radiographs were taken of the tibiae and blood was collected. Serum levels of TRAcP, IL-2, and IFNγ levels were measured using ELISA. Harvested tibiae were decalcified and stained for Ki67, cleaved caspase-3, and CD34 positive cells or microvessels were quantified. RESULTS: Tumor burden was lower in the combo group compared to the DOC alone group. Treatment with the combination had no impact on the number of mice with osteolytic lesions, however the area of osteolytic lesions was lower in the combo group compared to the vehicle and BLX groups, but not the DOC group. Serum TRAcP levels were lower in the combo compared to vehicle group, but not the other groups. No significant difference in Ki67 staining was found among the groups; whereas, cleaved caspase-3 staining was lowest in the Combo group and highest in the BLX group. The DOC and combo groups had more CD34+ microvessels than the control and BLX groups. There was no difference between the treatment groups for IL-2, but the combo group had increased levels of IFNγ compared to the DOC group. CONCLUSIONS: Our data demonstrate that a combination of BAL and DOC has greater antitumor activity in a model of PCa bone metastases than either drug alone. These data support further evaluation of this combination in metastatic PCa.

Transcription factor network analysis based on single cell RNA-seq identifies that Trichostatin-a reverses docetaxel resistance in prostate Cancer.

BACKGROUND: Overcoming drug resistance is critical for increasing the survival rate of prostate cancer (PCa). Docetaxel is the first cytotoxic chemotherapeutical approved for treatment of PCa. However, 99% of PCa patients will develop resistance to docetaxel within 3 years. Understanding how resistance arises is important to increasing PCa survival. METHODS: In this study, we modeled docetaxel resistance using two PCa cell lines: DU145 and PC3. Using the Passing Attributes between Networks for Data Assimilation (PANDA) method to model transcription factor (TF) activity networks in both sensitive and resistant variants of the two cell lines. We identified edges and nodes shared by both PCa cell lines that composed a shared TF network that modeled changes which occur during acquisition of docetaxel resistance in PCa. We subjected the shared TF network to connectivity map analysis (CMAP) to identify potential drugs that could disrupt the resistant networks. We validated the candidate drug in combination with docetaxel to treat docetaxel-resistant PCa in both in vitro and in vivo models. RESULTS: In the final shared TF network, 10 TF nodes were identified as the main nodes for the development of docetaxel resistance. CMAP analysis of the shared TF network identified trichostatin A (TSA) as a candidate adjuvant to reverse docetaxel resistance. In cell lines, the addition of TSA to docetaxel enhanced cytotoxicity of docetaxel resistant PCa cells with an associated reduction of the IC50 of docetaxel on the resistant cells. In the PCa mouse model, combination of TSA and docetaxel reduced tumor growth and final weight greater than either drug alone or vehicle. CONCLUSIONS: We identified a shared TF activity network that drives docetaxel resistance in PCa. We also demonstrated a novel combination therapy to overcome this resistance. This study highlights the usage of novel application of single cell RNA-sequencing and subsequent network analyses that can reveal novel insights which have the potential to improve clinical outcomes.

Extracellular Vesicles and Bone-Associated Cancer.

PURPOSE OF REVIEW: In this review, we describe the biology of extracellular vesicles (EV) and how they contribute to bone-associated cancers. RECENT FINDINGS: Crosstalk between tumor and bone has been demonstrated to promote tumor and metastatic progression. In addition to direct cell-to-cell contact and soluble factors, such as cytokines, EVs mediate crosstalk between tumor and bone. EVs are composed of a heterogenous group of membrane-delineated vesicles of varying size range, mechanisms of formation, and content. These include apoptotic bodies, microvesicles, large oncosomes, and exosomes. EVs derived from primary tumors have been shown to alter bone remodeling and create formation of a pre-metastatic niche that favors development of bone metastasis. Similarly, EVs from marrow stromal cells have been shown to promote tumor progression. Additionally, EVs can act as therapeutic delivery vehicles due to their low immunogenicity and targeting specificity. EVs play critical roles in intercellular communication. Multiple classes of EVs exist based on size on mechanism of formation. In addition to a role in pathophysiology, EVs can be exploited as therapeutic delivery vehicles.

Targeted Notch1 inhibition with a Notch1 antibody, OMP-A2G1, decreases tumor growth in two murine models of prostate cancer in association with differing patterns of DNA damage response gene expression.

Notch plays a protumorigenic role in many cancers including prostate cancer (PCa). Global notch inhibition of multiple Notch family members using γ-secretase inhibitors has shown efficacy in suppressing PCa growth in murine models. However, global Notch inhibition is associated with marked toxicity due to the widespread function of many different Notch family members in normal cell physiology. Accordingly, in the current study, we explored if specific inhibition of Notch1 would effectively inhibit PCa growth in a murine model. The androgen-dependent VCaP and androgen-independent DU145 cell lines were injected subcutaneously into mice. The mice were treated with either control antibody 1B7.11, anti-Notch1 antibody (OMP-A2G1), docetaxel or the combination of OMP-A2G1 and docetaxel. Tumor growth was measured using calipers. At the end of the study, tumors were assessed for proliferative response, apoptotic response, Notch target gene expression, and DNA damage response (DDR) expression. OMP-A2G1 alone inhibited tumor growth of both PCa cell lines to a greater extent than docetaxel alone. There was no additive or synergistic effect of OMP-A2G1 and docetaxel. The primary toxicity was weight loss that was controlled with dietary supplementation. Proliferation and apoptosis were affected differentially in the two cell lines. OMP-A2G1 increased expression of the DDR gene GADD45α in VCaP cells but downregulated GADD45α in Du145 cells. Taken together, these data show that Notch1 inhibition decreases PCa xenograft growth but does so through different mechanisms in the androgen-dependent VCaP cell line vs the androgen-independent DU145 cell line. These results provide a rationale for further exploration of targeted Notch inhibition for therapy of PCa.

Polarization of prostate cancer-associated macrophages is induced by milk fat globule-EGF factor 8 (MFG-E8)-mediated efferocytosis.

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.

Raf kinase inhibitor protein (RKIP) deficiency decreases latency of tumorigenesis and increases metastasis in a murine genetic model of prostate cancer.

BACKGROUND: Raf kinase inhibitor protein (RKIP) has been shown to act as a metastasis suppressor gene in multiple models of cancer. Loss of RKIP expression promotes invasion and metastasis in cell transplantation animal models. However, it is unknown if RKIP expression can impact the progression of cancer in an autochthonous model of cancer. The goal of this study was to determine if loss of RKIP expression in a genetic mouse model of prostate cancer (PCa) impacts metastasis. METHODS: Endogenous RKIP expression was measured in the primary tumors and metastases of transgenic adenocarcinoma of the mouse prostate (TRAMP(+) ) mice. RKIP knockout mice (RKIP(-/-) ) were crossbred with (TRAMP(+) ) mice to create RKIP(-/-) TRAMP(+) mice. Mice were euthanized at 10, 20, and 30 weeks for evaluation of primary and metastatic tumor development. To determine if loss of RKIP alone promotes metastasis, RKIP was knocked down in the low metastatic LNCaP prostate cancer cell line. RESULTS: Endogenous RKIP expression decreased in TRAMP(+) mice as tumors progressed. Primary tumors developed earlier in RKIP(-/-) TRAMP(+) compared to TRAMP(+) mice. At 30 weeks of age, distant metastases were identified only the RKIP(-/-) TRAMP(+) mice. While prostate epithelial cell proliferation rates were higher at 10 and 20 weeks in RKIP(-/-) TRAMP(+) compared to TRAMP(+) mice, by 30 weeks there was no difference. Apoptosis rates in both groups were similar at all timepoints. Decreased RKIP expression did not impact the metastatic rate of LNCaP in an orthotopic PCa model. CONCLUSIONS: These results demonstrate that loss of RKIP decreases latency of tumor development and promotes distant metastasis in the TRAMP mouse model in the context of a pro-metastatic background; but loss of RKIP alone is insufficient to promote metastasis. These findings suggest that in addition to its known metastasis suppressor activity, RKIP may promote tumor progression through enhancing tumor initiation. Prostate 75:292-302, 2015. © 2014 Wiley Periodicals, Inc.

NF-kappaB in breast cancer cells promotes osteolytic bone metastasis by inducing osteoclastogenesis via GM-CSF.

Advanced breast cancers frequently metastasize to bone, resulting in osteolytic lesions, yet the underlying mechanisms are poorly understood. Here we report that nuclear factor-kappaB (NF-kappaB) plays a crucial role in the osteolytic bone metastasis of breast cancer by stimulating osteoclastogenesis. Using an in vivo bone metastasis model, we found that constitutive NF-kappaB activity in breast cancer cells is crucial for the bone resorption characteristic of osteolytic bone metastasis. We identified the gene encoding granulocyte macrophage-colony stimulating factor (GM-CSF) as a key target of NF-kappaB and found that it mediates osteolytic bone metastasis of breast cancer by stimulating osteoclast development. Moreover, we observed that the expression of GM-CSF correlated with NF-kappaB activation in bone-metastatic tumor tissues from individuals with breast cancer. These results uncover a new and specific role of NF-kappaB in osteolytic bone metastasis through GM-CSF induction, suggesting that NF-kappaB is a potential target for the treatment of breast cancer and the prevention of skeletal metastasis.

'Educated' Osteoblasts Reduce Osteoclastogenesis in a Bone-Tumor Mimetic Microenvironment.

Breast cancer (BC) metastases to bone disrupt the balance between osteoblasts and osteoclasts, leading to excessive bone resorption. We identified a novel subpopulation of osteoblasts with tumor-inhibitory properties, called educated osteoblasts (EOs). Here we sought to examine the effect of EOs on osteoclastogenesis during tumor progression. We hypothesized that EOs affect osteoclast development in the bone-tumor niche, leading to suppressed pre-osteoclast fusion and bone resorption. Conditioned media (CM) was analyzed for protein expression of osteoclast factors receptor activator of nuclear factor kappa-ß ligand (RANKL), osteoprotegerin (OPG), and tumor necrosis factor alpha (TNFα) via ELISA. EOs were co-cultured with pre-osteoclasts on a bone mimetic matrix to assess osteoclast resorption. Pre-osteoclasts were tri-cultured with EOs plus metastatic BC cells and assessed for tartrate-resistance acid phosphatase (TRAP)-positive, multinucleated (≥3 nuclei), mature osteoclasts. Tumor-bearing murine tibias were stained for TRAP to determine osteoclast number in-vivo. EO CM expressed reduced amounts of soluble TNFα and OPG compared to naïve osteoblast CM. Osteoclasts formed in the presence of EOs were smaller and less in number. Upon co-culture on a mimetic bone matrix, a 50% reduction in the number of TRAP-positive osteoclasts formed in the presence of EOs was observed. The tibia of mice inoculated with BC cells had less osteoclasts per bone surface in bones with increased numbers of EO cells. These data suggest EOs reduce osteoclastogenesis and bone resorption. The data imply EOs provide a protective effect against bone resorption in bone metastatic BC.

Pheno-SELEX: Engineering Anti-Metastatic Aptamers through Targeting the Invasive Phenotype Using Systemic Evolution of Ligands by Exponential Enrichment.

Multiple methods (e.g., small molecules and antibodies) have been engineered to target specific proteins and signaling pathways in cancer. However, many mediators of the cancer phenotype are unknown and the ability to target these phenotypes would help mitigate cancer. Aptamers are small DNA or RNA molecules that are designed for therapeutic use. The design of aptamers to target cancers can be challenging. Accordingly, to engineer functionally anti-metastatic aptamers we used a modification of systemic evolution of ligands by exponential enrichment (SELEX) we call Pheno-SELEX to target a known phenotype of cancer metastasis, i.e., invasion. A highly invasive prostate cancer (PCa) cell line was established and used to identify aptamers that bound to it with high affinity as opposed to a less invasive variant to the cell line. The anti-invasive aptamer (AIA1) was found to inhibit in vitro invasion of the original highly invasive PCa cell line, as well as an additional PCa cell line and an osteosarcoma cell line. AIA1 also inhibited in vivo development of metastasis in both a PCa and osteosarcoma model of metastasis. These results indicate that Pheno-SELEX can be successfully used to identify aptamers without knowledge of underlying molecular targets. This study establishes a new paradigm for the identification of functional aptamers.

CTEN/tensin 4 expression induces sensitivity to paclitaxel in prostate cancer.

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-3-TxR 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 androgen-independent 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.

Single-Cell Transcriptomics Analysis Identifies Nuclear Protein 1 as a Regulator of Docetaxel Resistance in Prostate Cancer Cells.

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.

A glycolytic mechanism regulating an angiogenic switch in prostate cancer.

The generation of an "angiogenic switch" is essential for tumor growth, yet its regulation is poorly understood. In this investigation, we explored the linkage between metastasis and angiogenesis through CXCL12/CXCR4 signaling. We found that CXCR4 regulates the expression and secretion of the glycolytic enzyme phosphoglycerate kinase 1 (PGK1). Overexpression of PGK1 reduced the secretion of vascular endothelial growth factor and interleukin-8 and increased the generation of angiostatin. At metastatic sites, however, high levels of CXCL12 signaling through CXCR4 reduced PGK1 expression, releasing the angiogenic response for metastastic growth. These data suggest that PGK1 is a critical downstream target of the chemokine axis and an important regulator of an "angiogenic switch" that is essential for tumor and metastatic growth.

Macrofluidic recirculating model of skeletal metastasis.

While microfluidic systems model aspects of metastasis, they are limited to artificially created tissues of limited complexity. We set out to develop an in vitro model of tumor cell migration from a primary tumor to a distant site that allows use of tissue. Accordingly, we created a macrofluidic model using culture plate wells connected with type I collagen-coated large bore tubing and has recirculating media. Green fluorescent protein-positive prostate carcinoma cells in a hydrogel or excised tumor xenografts from mice were placed into primary tumor sites and either human bone stromal cells (HS-5) in a hydrogel or human-derived bone chips were seeded into metastatic sites. Cells from the primary sites migrated to and grew in metastatic sites. Bone enhanced growth at metastatic sites and established a CXCL12 gradient that was higher in metastatic versus primary sites. AMD3100-mediated inhibition of CXCL12 function reduced the number of cells targeting the bone at the metastatic sites. In summary, we have developed a macrofluidic metastasis model that allows incorporation of tumor and metastatic microenvironment tissues and models chemotaxis. This system allows for incorporation of tumor heterogeneity and inclusion of an intact microenvironment. These features will facilitate identification of mechanisms and therapeutics for bone metastasis.

Prostate cancer promotes a vicious cycle of bone metastasis progression through inducing osteocytes to secrete GDF15 that stimulates prostate cancer growth and invasion.

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.

Primary prostate cancer educates bone stroma through exosomal pyruvate kinase M2 to promote bone metastasis.

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.

Targeting cathepsin K diminishes prostate cancer establishment and growth in murine bone.

BACKGROUND: The processes of prostate cancer (PCa) invasion and metastasis are facilitated by proteolytic cascade involving multiple proteases, such as matrix metalloproteinases, serine proteases and cysteine proteases including cathepsin K (CatK). CatK is predominantly secreted by osteoclasts and specifically degrades collagen I leading to bone destruction. PCa and breast cancer preferentially metastasize to the bone. Importantly, CatK expression level is greater in PCa bone metastatic sites compared to primary tumor and normal prostate tissues. However, the underlying mechanism of CatK during PCa metastases into the bone remains to be elucidated. We investigated the functional role of CatK during the PCa establishment and growth process in the murine bone. METHODS: CatK mRNA expression was validated by RT-PCR, protein expression by immunoblotting in PCa LNCaP, C4-2B, and PC3 cells as well as in PCa tissues. Its protein production was measured using ELISA assay. The effect of both knockdowns via siRNA and CatK inhibitor was compared in regard to PCa cell invasion. We further studied the dose-dependent CatK inhibitor effect on conditioned media-induced bone resorption. In setting up an animal model, C4-2B cells were injected into the tibiae of SCID mice. The animals treated with either vehicle or CatK inhibitor for 8 weeks at the time of tumor cell injection (tumor establishment model; protocol I) or 4 weeks after tumor cell injection (tumor progression model; protocol II) were applied to histological and histomorphometric analyses. RESULTS: We confirmed CatK expression in PCa LNCaP, C4-2B, and PC3 cells as well as in PCa tissues. Furthermore, we observed the inhibitory effects of a selective CatK inhibitor on PCa cell invasion. The CatK inhibitor dose-dependently inhibited PCa-conditioned media-induced bone resorption. Upon injection of C4-2B cells into the tibiae of SCID mice, the selective CatK inhibitor significantly prevented the tumor establishment in protocol I, and reduced the tumor growth in bone in protocol II. It also decreased serum PSA levels in both animal models. The inhibitory effects of the CatK inhibitor were enhanced in combination with zoledronic acid (ZA). CONCLUSION: The selective CatK inhibitor may prevent the establishment and progression of PCa in bone, thus making it a novel therapeutic approach for advanced PCa.

Inhibition of interleukin-6 with CNTO328, an anti-interleukin-6 monoclonal antibody, inhibits conversion of androgen-dependent prostate cancer to an androgen-independent phenotype in orchiectomized mice.

Initially, prostate cancer is androgen dependent. However, most cases progress to an androgen-independent state through unknown mechanisms. Interleukin-6 (IL-6) has been associated with prostate cancer progression including activation of the androgen receptor (AR). To determine if IL-6 plays a role in the conversion of prostate cancer from androgen dependent to androgen independent, we established androgen-dependent LuCaP 35 human prostate cancer xenografts in nude mice, castrated the mice, and blocked IL-6 activity using a neutralizing antibody (CNT0328) for a period of 18 weeks. IL-6 inhibition increased survival of mice and inhibited tumor growth, as reflected by decreased tumor volume and prostate-specific antigen levels, compared with that in mice receiving isotype control antibody. To test the effect of IL-6 inhibition on the conversion from androgen dependent to androgen independent, tumor cells from the treated mice were assessed for their androgen dependence both in vitro and by implanting them into sham-operated or orchiectomized mice. Tumor cells derived from the isotype-treated animals converted to androgen-independent state, whereas tumor cells from the anti-IL-6 antibody-treated mice were still androgen dependent in vitro and in vivo. Although there was no difference in AR levels between the androgen-independent and androgen-dependent tumors, IL-6 inhibition promoted both apoptosis and inhibited cell proliferation in tumors and blocked the orchiectomy-induced expression of histone acetylases, p300 and CBP, which are AR cofactors. These data show that IL-6 contributes to the development of androgen independence in prostate cancer and suggest that it mediates this effect, in part, through modulation of p300 and CBP.

Bone morphogenetic protein-6 promotes osteoblastic prostate cancer bone metastases through a dual mechanism.

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 cells promote osteoblastic bone metastases through Wnts.

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.

Vascular endothelial growth factor contributes to prostate cancer-mediated osteoblastic activity.

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.