Integration of Single-Cell RNA-Sequencing and Network Analysis to Investigate Mechanisms of Drug Resistance.

Innate resistance and therapeutic-driven development of resistance to anticancer drugs is a common complication of cancer therapy. Understanding mechanisms of drug resistance can lead to development of alternative therapies. One strategy is to subject drug-sensitive and drug-resistant variants to single-cell RNA-seq (scRNA-seq) and to subject the scRNA-seq data to network analysis to identify pathways associated with drug resistance. This protocol describes a computational analysis pipeline to study drug resistance by subjecting scRNA-seq expression data to Passing Attributes between Networks for Data Assimilation (PANDA), an integrative network analysis tool that incorporates protein-protein interactions (PPI) and transcription factor (TF)-binding motifs.

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.

Effects of Analgesics on Tumor Growth in Mouse Models of Prostate Cancer Bone Metastasis.

Murine models of tumor development often require invasive procedures for tumor implantation, potentially causing pain or distress. However, analgesics are often withheld during implantation because of concerns that they may adversely affect tumor development. Previous studies examining the effects of analgesics on the development and metastasis of various tumor lines show that the effect of analgesics depends on the tumor line and analgesic used. A blanket statement that analgesics affect the general growth of tumors is not adequate scientific justification for withholding pain relief, and pilot studies or references are recommended for each specific tumor cell line and treatment combination. In this study, we evaluated the effects of 2 commonly used analgesics on tumor growth in 2 models of prostate cancer (PCa) bone metastasis. We hypothesized that a one-time injection of analgesics at the time of intratibial injection of tumor cells would not significantly impact tumor growth. Either C57BL/6 or SCID mice were injected subcutaneously with an analgesic (carprofen [5 mg/kg], or buprenorphine [0.1 mg/kg]) or vehicle (0.1 mL of saline) at the time of intratibial injection with a PCa cell line (RM1 or PC3, n = 10 to 11 per group). Tumor growth (measured by determination of tumor burden and the extent of bone involvement) and welfare (measured by nociception, locomotion, and weight) were monitored for 2 to 4 wk. Neither carprofen or buprenorphine administration consistently affected tumor growth or indices of animal welfare as compared with the saline control for either cell line. This study adds to the growing body of literature demonstrating that analgesia can be compatible with scientific objectives, and that a decision to withhold analgesics must be scientifically justified and evaluated on a model-specific basis.

Enrichment Preferences of Singly Housed Zebrafish (Danio rerio).

Despite the increasing popularity of zebrafish (Danio rerio) as an animal model, the environmental enrichment preferences of this species have been largely unexplored. We sought to determine the preferences of mature female zebrafish that were singly housed with or without access to one of 10 inanimate forms of enrichment. As a marker of preference, in-tank fish location was observed by video recording. All subjects showed a preference for the front of the tank when caretakers entered the room, demonstrating an effect of human presence on tank location. Among the 10 enrichment items tested, subjects showed the strongest preference for mirrored paper on the side of the tank when compared with the barren half of the tank. Fish also were observed interacting with PVC pipe, marbles, and tulle. Given the preference for enrichment imitating social interaction, we conducted a second study to assess the value of visual exposure of conspecifics in adjacent tanks. The experimental zebrafish were then provided one of 3 conditions-a singly housed neighbor fish, group-housed neighbor fish, or no neighbor fish. All zebrafish housed next to neighboring fish showed a preference to be on the side of the tank nearer to the other fish. Overall, our data indicate that singly housed zebrafish prefer enrichment items that resemble or promote social behaviors. Therefore items such as mirrored paper or housing next to conspecifics should be strongly considered as enrichment strategies for singly housed zebrafish.

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.

Immune mediators in the tumor microenvironment of prostate cancer.

Prostate cancer tissue is composed of both cancer cells and host cells. The milieu of host components that compose the tumor is termed the tumor microenvironment (TME). Host cells can be those derived from the tissue in which the tumor originates (e.g., fibroblasts and endothelial cells) or those recruited, through chemotactic or other factors, to the tumor (e.g., circulating immune cells). Some immune cells are key players in the TME and represent a large proportion of non-tumor cells found within the tumor. Immune cells can have both anti-tumor and pro-tumor activity. In addition, crosstalk between prostate cancer cells and immune cells affects immune cell functions. In this review, we focus on immune cells and cytokines that contribute to tumor progression. We discuss T-regulatory and T helper 17 cells and macrophages as key modulators in prostate cancer progression. In addition, we discuss the roles of interleukin-6 and receptor activator of nuclear factor kappa-B ligand in modulating prostate cancer progression. This review highlights the concept that immune cells and cytokines offer a potentially promising target for prostate cancer therapy.

Bone Microenvironment Changes in Latexin Expression Promote Chemoresistance.

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.

Phase II studies of two different schedules of dasatinib in bone metastasis predominant metastatic breast cancer: SWOG S0622.

Bone metastases from breast cancer are common, causing significant morbidity. Preclinical data of dasatinib, an oral small molecule inhibitor of multiple oncogenic tyrosine kinases, suggested efficacy in tumor control and palliation of bone metastases in metastatic breast cancer (MBC). This clinical trial aimed to determine whether treatment with either of 2 dose schedules of dasatinib results in a progression-free survival (PFS) >50 % at 24 weeks in bone metastasis predominant MBC, to evaluate the toxicity of the 2 dosing regimens, and explore whether treatment results in decreased serum bone turnover markers and patient-reported "worst pain." Subjects with bone metastasis predominant MBC were randomly assigned to either 100 mg of dasatinib once daily, or 70 mg twice daily, with treatment continued until time of disease progression or intolerable toxicity. Planned accrual was 40 patients in each arm. The primary trial endpoint was PFS, defined as time from registration to progression or death due to any cause. Median PFS for all eligible patients (79) was 12.6 weeks (95 % CI 9.1-16.7). Neither cohort met the threshold for further clinical interest. There were no significant differences in PFS by randomized treatment arm (p = 0.85). Toxicity was similar in both cohorts, with no clear trend in serum biomarkers of bone turnover or patient-reported pain. Dasatinib was ineffective in controlling bone-predominant MBC in a patient population, unselected by molecular markers. Further study of dasatinib in breast cancer should not be pursued unless performed in molecularly determined patient subsets, or rational combinations.

Diffuse Infiltrative Gastrointestinal Lipomatosis in a Guinea Pig (Cavia porcellus).

An intact adult male guinea pig (Cavia porcellus) went into cardiopulmonary arrest during a surgical procedure, and efforts at resuscitation were unsuccessful. Gross examination revealed a gastric rupture along the greater curvature of the stomach, which was associated with free blood and ingesta in the abdominal cavity, and a 2-cm nodular, partially circumferential, soft-to-firm mass within the pyloric region. Histologically, the pyloric mass was composed of sheets of infiltrative adipocytes expanding the muscular wall. Similar infiltrative sheets of adipocytes were present adjacent to the rupture site and within the small intestine, cecum, and colon. These findings are consistent with diffuse infiltrative lipomatosis, an exceedingly rare condition in human and veterinary species. This report is the first description of this rare disease in guinea pigs, and the concurrent involvement of both the stomach and intestines has not been reported in any veterinary species.

Notch Pathway Inhibition Using PF-03084014, a γ-Secretase Inhibitor (GSI), Enhances the Antitumor Effect of Docetaxel in Prostate Cancer.

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.

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.

Recent advances in bone-targeted therapies of metastatic prostate cancer.

Prostate cancer is one of the most common malignancies affecting men worldwide, with bone being the most common site of metastasis in patients that progress beyond organ confinement. Bone metastases are virtually incurable and result in significant disease morbidity and mortality. Bone provides a unique microenvironment whose local interactions with tumor cells offer novel targets for therapeutic interventions. Several attractive molecules or pathways have been identified as new potential therapeutic targets for bone metastases caused by metastatic castration-resistant prostate cancer. In this review, we present the recent advances in molecular targeted therapies for prostate cancer bone metastasis focusing on therapies that target the bone cells and the bone microenvironment. The therapies covered in this review include agents that inhibit bone resorption, agents that stimulate bone formation, and agents that target the bone matrix. Suggestions to devise more effective molecular targeted therapies are proposed. Hopefully, with better understanding of the biology of the disease and the development of more robust targeted therapies, the survival and quality of life of the affected individuals could be significantly improved.

Survey of Raf kinase inhibitor protein (RKIP) in multiple cancer types.

Raf kinase inhibitor protein (RKIP), an inhibitor of several signaling pathways, has been shown to have metastasis suppressor gene activity and promote apoptosis. While first identified in prostate cancer, RKIP's anti-metastasis properties have now been demonstrated in multiple tumor types. Furthermore, loss of RKIP expression is observed in many cancers as they progress. In this review, we provide a survey of the many tumor types in which RKIP function or expression has been evaluated. Particular attention is focused on the expression of RKIP in clinical tissues and its prognostic significance. A PubMed search through May 2014 identified 56 publications detailing RKIP expression in clinical cancer tissues. The majority of studies revealed that loss of RKIP expression has prognostic value for overall survival, disease free survival, and presence of metastasis for most solid tumor cancers; whereas, RKIP expression correlated with tumor grade or stage in approximately only 50% of the publications. In summary, RKIP loss is a frequent occurrence in many solid tumor cancers and may serve as a viable prognostic biomarker.

A review on the diagnosis and treatment of hepatocellular carcinoma with a focus on the role of Wnts and the dickkopf family of Wnt inhibitors.

Hepatocellular carcinoma (HCC) is one of the most prevalent cancers worldwide. There are multiple etiologic factors including viral and environmental influences that can lead to HCC. Successful screening for early HCC is challenging due to the lack of well characterized and specific biomarkers. However, achieving successful screening is critically important as early diagnosis can potentially provide curative opportunities. Once HCC is advanced, there are multiple therapeutic venues, but most eventually fail, therefore developing new targeted therapies may provide greater chance for effective therapies. Along these lines, the Wnt pathway has been identified as contributing to the development and progression of HCC. Wnts can modify HCC growth and invasive ability. A key factor in the Wnt pathway is the dickkopf (DKK) family of Wnt inhibitors. DKKs have also been shown to modulate HCC progression. Additionally, several studies have suggested that DKK expression in tissue and serum has diagnostic and prognostic value.

Cabozantinib inhibits prostate cancer growth and prevents tumor-induced bone lesions.

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.

Activation of the Wnt pathway through AR79, a GSK3ß inhibitor, promotes prostate cancer growth in soft tissue and bone.

UNLABELLED: 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 (GSK3ß) inhibits Wnt signaling by inducing ß-catenin degradation, and a GSK3ß 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, ß-catenin activation, proliferation, and apoptosis were assessed. Additionally, prostate cancer and osteoblast cell lines were treated with AR79, and ß-catenin status, proliferation (with ß-catenin knockdown in some cases), and proportion of ALDH(+)CD133(+) stem-like cells were determined. AR79 promoted prostate cancer tumor growth, decreased phospho-ß-catenin, increased total and nuclear ß-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 ß-catenin phosphorylation, increased nuclear ß-catenin accumulation in prostate cancer and osteoblast cell lines, and increased proliferation of prostate cancer cells in vitro through ß-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.

A novel canine model for prostate cancer.

BACKGROUND: No existing animal model fully recapitulates all features of human prostate cancer. The dog is the only large mammal, besides humans, that commonly develops spontaneous prostate cancer. Canine prostate cancer features many similarities with its human counterpart. We sought to develop a canine model of prostate cancer that would more fully represent the features of human prostate cancer than existing models. METHODS: The Ace-1 canine prostate cancer cell line was injected transabdominally under transrectal ultrasound (TRUS) guidance into the prostates of immunosuppressed, intact, adult male dogs. Tumor progression was monitored by TRUS imaging. Some dogs were subjected to positron emission tomography (PET) for tumor detection. Time of euthanasia was determined based on tumor size, impingement on urethra, and general well-being. Euthanasia was followed by necropsy and histopathology. RESULTS: Ace-1 tumor cells grew robustly in every dog injected. Tumors grew in subcapsular and parenchymal regions of the prostate. Tumor tissue could be identified using PET. Histological findings were similar to those observed in human prostate cancer. Metastases to lungs and lymph nodes were detected, predominantly in dogs with intraprostatic tumors. CONCLUSIONS: We have established a minimally invasive dog model of prostate cancer. This model may be valuable for studying prostate cancer progression and distant metastasis.

Transcriptional regulation of RKIP expression by androgen in prostate cells.

BACKGROUND/AIMS: Raf kinase inhibitory protein (RKIP) is a scaffolding molecule in the PEBP family that sequesters certain signaling molecules away from their pathways, thereby abrogating intracellular growth signals. RKIP has been assigned multiple functions and is associated with an increasing number of diseases through its involvement with signal transduction pathways. We previously demonstrated that RKIP is highly expressed in human normal prostate epithelial cells and plays a pivotal role during prostate cancer (PCa) progression. Whether RKIP is subject to endocrine regulation has not been reported. METHODS: The effect of dihydrotestosterone (DHT) on RKIP expression in normal prostate epithelial cells was determined by real-time RT-PCR and Western blot. Report assay was performed to determine whether the regulation of RKIP by androgens is at the transcriptional level. The binding of androgen receptor (AR) to the RKIP promoter was determined by EMSA and Chromatin Immunoprecipitation (ChIP) assays. To determine whether RKIP was regulated by androgen in vivo, we examined RKIP expression level in response to castration in 6-8 week old C57BL/6 male mice. RESULTS: Here we report that DHT positively regulates the transcription of RKIP in the normal prostate epithelial cells. The anti-androgen bicalutamide blocked androgen-mediated regulation of RKIP, which indicates that this regulation is mediated through AR. Transfection of the cells with a RKIP promoter-driven luciferase reporter vector showed that DHT increased RKIP promoter activity in parallel with changes in expression. EMSA demonstrates that AR binds to a putative ARE in the RKIP promoter, which was further validated by ChIP assay. Importantly, these data are further supported by our in vivo experiment where castrated mice had less RKIP expression in their prostate glands than sham-operated mice. CONCLUSIONS: Collectively, the results establish RKIP as a novel androgen target gene. Androgens induce RKIP expression through AR-mediated transcriptional modulation of the RKIP promoter in the prostate. This is the first demonstration of endocrine regulation of the metastasis suppressor gene RKIP.

Reversal of chemotherapy-induced leukopenia using granulocyte macrophage colony-stimulating factor promotes bone metastasis that can be blocked with osteoclast inhibitors.

Hematopoietic growth factors are used to reverse chemotherapy-induced leukopenia. However, some factors such as granulocyte macrophage colony-stimulating factor (GM-CSF) induce osteoclast-mediated bone resorption that can promote cancer growth in the bone. Accordingly, we evaluated the ability of GM-CSF to promote bone metastases of breast cancer or prostate cancer in a mouse model of chemotherapy-induced leukopenia. In this model, GM-CSF reversed cyclophosphamide-induced leukopenia but also promoted breast cancer and prostate cancer growth in the bone but not in soft tissue sites. Bone growth was associated with the induction of osteoclastogenesis, yet in the absence of tumor GM-CSF, it did not affect osteoclastogenesis. Two osteoclast inhibitors, the bisphosphonate zoledronic acid and the RANKL inhibitor osteoprotegerin, each blocked GM-CSF-induced tumor growth in the bone but did not reverse the ability of GM-CSF to reverse chemotherapy-induced leukopenia. Our findings indicate that it is possible to dissociate the bone-resorptive effects of GM-CSF, to reduce metastatic risk, from the benefits of this growth factor in reversing leukopenia caused by treatment with chemotherapy.

Prostate cancer induces bone metastasis through Wnt-induced bone morphogenetic protein-dependent and independent mechanisms.

Prostate cancer (PCa) is frequently accompanied by osteosclerotic (i.e., excessive bone production) bone metastases. Although bone morphogenetic proteins (BMP) and Wnts are mediators of PCa-induced osteoblastic activity, the relation between them in PCa bone metastases is unknown. The goal of this study was to define this relationship. Wnt3a and Wnt5a administration or knockdown of DKK-1, a Wnt inhibitor, induced BMP-4 and 6 expression and promoter activation in PCa cells. DKK-1 blocked Wnt activation of the BMP promoters. Transfection of C4-2B cells with axin, an inhibitor of canonical Wnt signaling, blocked Wnt3a but not Wnt5a induction of the BMP promoters. In contrast, Jnk inhibitor I blocked Wnt5a but not Wnt3a induction of the BMP promoters. Wnt3a, Wnt5a, and conditioned medium (CM) from C4-2B or LuCaP23.1 cells induced osteoblast differentiation in vitro. The addition of DKK-1 and Noggin, a BMP inhibitor, to CM diminished PCa CM-induced osteoblast differentiation in a synergistic fashion. However, pretreatment of PCa cells with DKK-1 before collecting CM blocked osteoblast differentiation, whereas pretreatment with Noggin only partially reduced osteoblast differentiation, and pretreatment with both DKK-1 and Noggin had no greater effect than pretreatment with DKK-1 alone. Additionally, knockdown of BMP expression in C4-2B cells inhibited Wnt-induced osteoblastic activity. These results show that PCa promotes osteoblast differentiation through canonical and noncanonical Wnt signaling pathways that stimulate both BMP-dependent and BMP-independent osteoblast differentiation. These results show a clear link between Wnts and BMPs in PCa-induced osteoblast differentiation and provide novel targets, including the noncanonical Wnt pathway, for therapy of PCa.