Prostate Cancer Progression In Bone Research Articles

Abstract C062: Metabolic rewiring: A role for adenosine-inosine axis in African-American prostate cancer

Abstract African-American (AA) men have a 60% higher incidence of prostate cancer (PCa) compared to European-American (EA) men. An understanding of the altered biological responses and the factors that lead to health disparity in PCa is unclear. Metabolism defines the physiological state of the cell and metabolic reprogramming is a hallmark of cancer. Thus, studying the metabolic landscape will be crucial for understanding the biological changes that might contribute to this disparity. Analysis of 190 metabolites across PCa/benign adjacent tissue pairs from ancestry typed AA and EA men done in our laboratory, revealed altered levels of nucleosides adenosine and inosine. High inosine to adenosine ratio was observed in AA men compared to EA men. In line with this, the enzyme adenosine deaminase (ADA), which converts adenosine to inosine was found elevated in AA men, potentially explaining the high inosine to adenosine ratio in AA PCa. The consequences of the accumulation of these metabolites on AA PCa progression are unknown. This study will address the knowledge gap on the effects adenosine-inosine alterations in AA PCa and attempt to dissect its role in effecting an aggressive phenotype in PCa. To determine the role of elevated ADA in PCa, it was overexpressed in AA(MDA-PCa-2A) and EA PCa (LNCaP) cell lines (termed ADA OE). Phenotypic examination of these cells revealed high ADA enzyme activity decreases the cell-ECM adhesion. Molecular analyses revealed that upon ADA OE there is a reduction in Tenascin C(TNC), a matrix protein known for its anti-adhesive properties. TNC-rich microenvironment facilitates reactive stromal induction and high motility. This TNC-mediated reprogramming enhances the metastatic ability and PCa progression in bone. TNC induction was observed in stromal cells (DPT44) upon treatment with ADA OE conditioned media and inosine addition. PCa progression on bone was assessed using a cancer cell-bone in vitro co-culture system. High inosine and adenosine levels enabled better attachment of PCa cells to the bone. These findings suggest that an altered adenosine/inosine axis could facilitate adhesion decrease and modulate the stromal microenvironment to aid in the metastatic dissemination and migration towards the bone. Adenosine-inosine alterations could serve as a biomarker for metastatic PCa. We will further analyze the inosine and adenosine levels in patient samples and correlate them with various clinical outcomes in PCa. Our study so far shows high inosine to adenosine ratio in biopsy positive patient samples compared to biopsy negative samples. Our next steps of analysis include correlating the metabolite levels with (i)Biochemical recurrence (BCr) (ii)Time to attaining castration resistance (iii)Time to metastasis (iv)Response to Androgen deprivation in patients after BCr. Based on our current findings, we expect the inosine to adenosine ratio in plasma or urine to serve as a predictive marker for PCa incidence or progression in AA men. In addition, the enzyme ADA could serve as a potential therapeutic target for AA PCa. Citation Format: Christy Charles. Metabolic rewiring: A role for adenosine-inosine axis in African-American prostate cancer [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr C062.

Statins reduce castration-induced bone marrow adiposity and prostate cancer progression in bone.

A fraction of patients undergoing androgen deprivation therapy (ADT) for advanced prostate cancer (PCa) will develop recurrent castrate-resistant PCa (CRPC) in bone. Strategies to prevent CRPC relapse in bone are lacking. Here we show that the cholesterol-lowering drugs statins decrease castration-induced bone marrow adiposity in the tumor microenvironment and reduce PCa progression in bone. Using primary bone marrow stromal cells (BMSC) and M2-10B4 cells, we showed that ADT increases bone marrow adiposity by enhancing BMSC-to-adipocyte transition in vitro. Knockdown of androgen receptor abrogated BMSC-to-adipocyte transition, suggesting an androgen receptor-dependent event. RNAseq analysis showed that androgens reduce the secretion of adipocyte hormones/cytokines including leptin during BMSC-to-adipocyte transition. Treatment of PCa C4-2b, C4-2B4 and PC3 cells with leptin led to an increase in cell cycle progression and nuclear Stat3. RNAseq analysis also showed that androgens inhibit cholesterol biosynthesis pathway, raising the possibility that inhibiting cholesterol biosynthesis may decrease BMSC-to-adipocyte transition. Indeed, statins decreased BMSC-to-adipocyte transition in vitro and castration-induced bone marrow adiposity in vivo. Statin pre-treatment reduced 22RV1 PCa progression in bone after ADT. Our findings with statin may provide one of the mechanisms to the clinical correlations that statin use in patients undergoing ADT seems to delay progression to “lethal” PCa.

Neutrophils are mediators of metastatic prostate cancer progression in bone

Bone metastatic prostate cancer (BM-PCa) significantly reduces overall patient survival and is currently incurable. Current standard immunotherapy showed promising results for PCa patients with metastatic, but less advanced, disease (i.e., fewer than 20 bone lesions) suggesting that PCa growth in bone contributes to response to immunotherapy. We found that: (1) PCa stimulates recruitment of neutrophils, the most abundant immune cell in bone, and (2) that neutrophils heavily infiltrate regions of prostate tumor in bone of BM-PCa patients. Based on these findings, we examined the impact of direct neutrophil–prostate cancer interactions on prostate cancer growth. Bone marrow neutrophils directly induced apoptosis of PCa in vitro and in vivo, such that neutrophil depletion in bone metastasis models enhanced BM-PCa growth. Neutrophil-mediated PCa killing was found to be mediated by suppression of STAT5, a transcription factor shown to promote PCa progression. However, as the tumor progressed in bone over time, neutrophils from late-stage bone tumors failed to elicit cytotoxic effector responses to PCa. These findings are the first to demonstrate that bone-resident neutrophils inhibit PCa and that BM-PCa are able to progress via evasion of neutrophil-mediated killing. Enhancing neutrophil cytotoxicity in bone may present a novel therapeutic option for bone metastatic prostate cancer.

Prostate Tumor Cell-Derived IL1β Induces an Inflammatory Phenotype in Bone Marrow Adipocytes and Reduces Sensitivity to Docetaxel via Lipolysis-Dependent Mechanisms.

Adipocyte-tumor cell cross-talk is one of the critical mediators of tumor progression and an emerging facilitator of therapy evasion. Tumor cells that metastasize to adipocyte-rich bone marrow take advantage of the interplay between metabolic and inflammatory pathways to activate prosurvival mechanisms that allow them to thrive and escape therapy. Using in vitro and in vivo models of marrow adiposity, we demonstrate that metastatic prostate carcinoma cells engage bone marrow adipocytes in a functional cross-talk that promotes IL1β expression in tumor cells. Tumor-supplied IL1β contributes to adipocyte lipolysis and regulates a proinflammatory phenotype in adipocytes via upregulation of COX-2 and MCP-1. We further show that the enhanced activity of the IL1β/COX-2/MCP-1 axis and a resulting increase in PGE2 production by adipocytes coincide with augmented hypoxia signaling and activation of prosurvival pathways in tumor cells, revealing a potential mechanism of chemoresistance. The major consequence of this interplay is the reduced response of prostate cancer cells to docetaxel, a phenomenon sensitive to the inhibition of lipolysis. IMPLICATIONS: Studies presented herein highlight adipocyte lipolysis as a tumor-regulated metabolic event that engages proinflammatory cross-talk in the microenvironment to promote prostate cancer progression in bone. Understanding the impact of bone marrow adipose tissue on tumor adaptation, survival, and chemotherapy response is fundamentally important, as current treatment options for metastatic prostate cancer are palliative.

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

BackgroundThe 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.MethodsCatK 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.ResultsWe 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).ConclusionThe selective CatK inhibitor may prevent the establishment and progression of PCa in bone, thus making it a novel therapeutic approach for advanced PCa.

Osteoblastic Factors in Prostate Cancer Bone Metastasis

Prostate cancer bone metastasis is the lethal progression of the disease. The disease frequently presents with osteoblastic lesions in bone. The tumor-induced bone can cause complications that significantly hamper the quality of life of patients. A better understanding of how prostate cancer induces aberrant bone formation and how the aberrant bone affects the progression and treatment of the disease may improve the therapies for this disease. Prostate cancer-induced bone was shown to enhance tumor growth and confer therapeutic resistance in bone metastasis. Clinically, Radium-223, an alpha emitter that selectively targets bone, was shown to improve overall survival in patients, supporting a role of tumor-induced bone in prostate cancer progression in bone. Recently, it was discovered that PCa-induced aberrant bone formation is due, in part, from tumor-associated endothelial cells that were converted into osteoblasts through endothelial-to-osteoblast (EC-to-OSB) conversion by tumor-secreted BMP4. The unique bone-forming phenotype of prostate cancer bone metastasis plays a role in prostate cancer progression in bone and therapy resistance. Therapies that incorporate targeting the tumor-induced osteoblasts or EC-to-OSB conversion mechanism may reduce tumor-induced bone formation and improve therapy outcomes.

Abstract A026: Expression of fibroblast growth factor receptor 1 isoforms and activation of different pathways in prostate cancer progression

Abstract Bone metastases typically develop in patients with advanced prostate cancer (PCa). Androgen deprivation is commonly used as treatment, but responses are short, and eventually the disease progresses to a castration-resistant form (CRPC). The fibroblast growth factor (FGF) axis is commonly altered during PCa progression. Our group and others have implicated the FGF axis in the pathogenesis of PCa progression in bone, identified it as a candidate target for therapy, and suggested that FGF receptor (FGFR)-1 mediates a positive feedback loop between PCa cells and bone cells. RNA sequencing studies of 183 human PCa samples indicated that the mean expression of FGFR-1 is the highest of all the FGFR family genes studied. Analyses of these FGFR-1 transcripts identified eight different protein-coding transcripts to be the most abundantly expressed with diverse human PCa tissue samples expressing different FGFR-1 isoforms. The overall goal of this project is to investigate implications of FGFR1/FGFR1 isoforms expression in the pathogenesis of PCa bone metastases. When we assessed the expression of the two best-characterized FGFR-1 isoforms (alpha (NM_023110.2) and beta (NM_023105.2), which represent the most abundant protein coding transcripts found in PCa, we observed that all patient-derived xenografts (PDXs) studied express significantly higher levels of FGFR-1 alpha than beta while PCa cell lines mostly express the beta isoform. Mining The Cancer Genome Atlas (TCGA) PCa data, we identified distinct patterns of gene expression associated with each FGFR-1 isoform (alpha and beta). Briefly, FGFR-1 beta (but not alpha) is significantly associated with pathways including the MAP-kinase signaling cascade, signaling by FGFR in disease and pathways in cancer. In vitro studies of FGF signaling activation in PCa cells expressing FGFR-1 isoforms alpha, beta or empty vector, confirmed these results. Male SCID mice injected intracardially with PC3-FGFR-1 alpha exhibited higher death rates compared to animals injected with either PC3-FGFR-1 beta or PC3-empty vector. Furthermore, higher number of metastases per mice as well as higher number of mice with bone metastases were observed in animals injected with PC3-FGFR-1 alpha. The fact that different prostate tumors express different FGFR-1 isoforms suggests that FGFR1 alpha and beta isoforms activate different genes or pathways in PCa cells and this may underlie, at least in part, PCa heterogeneity, pattern of progression, and differences in response to FGFR1 inhibitors. Further studies will warrant the understanding of the implications of FGFR-1 isoforms expression in the pathogenesis of PCa. Citation Format: Estefania Labanca, Jun Yang, Peter Shepherd, Justin Roberts, Michael Starbuck, Bradley Broom, Matthew Iyer, Christopher Logothetis, Arul Chinnaiyan, Nora Navone. Expression of fibroblast growth factor receptor 1 isoforms and activation of different pathways in prostate cancer progression [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A026.

A 3D in vitro model of patient-derived prostate cancer xenograft for controlled interrogation of in vivo tumor-stromal interactions

Patient-derived xenograft (PDX) models better represent human cancer than traditional cell lines. However, the complex in vivo environment makes it challenging to employ PDX models to investigate tumor-stromal interactions, such as those that mediate prostate cancer (PCa) bone metastasis. Thus, we engineered a defined three-dimensional (3D) hydrogel system capable of supporting the co-culture of PCa PDX cells and osteoblastic cells to recapitulate the PCa-osteoblast unit within the bone metastatic microenvironment in vitro. Our 3D model not only maintained cell viability but also preserved the typical osteogenic phenotype of PCa PDX cells. Additionally, co-culture cellularity was maintained over that of either cell type cultured alone, suggesting that the PCa-osteoblast cross-talk supports PCa progression in bone, as is hypothesized to occur in patients with prostatic bone metastasis. Strikingly, osteoblastic cells co-cultured with PCa PDX tumoroids organized around the tumoroids, closely mimicking the architecture of PCa metastases in bone. Finally, tumor-stromal signaling mediated by the fibroblast growth factor axis tightly paralleled that in the in vivo counterpart. Together, these findings indicate that this 3D PCa PDX model recapitulates important pathological properties of PCa bone metastasis, and validate the use of this model for controlled and systematic interrogation of complex in vivo tumor-stromal interactions.

RSK promotes prostate cancer progression in bone through ING3, CKAP2, and PTK6-mediated cell survival.

Prostate cancer has a proclivity to metastasize to bone. The mechanism by which prostate cancer cells are able to survive and progress in the bone microenvironment is not clear. Identification of molecules that play critical roles in the progression of prostate cancer in bone will provide essential targets for therapy. Ribosomal S6 protein kinases (RSK) have been shown to mediate many cellular functions critical for cancer progression. Whether RSK plays a role in the progression of prostate cancer in bone is unknown. IHC analysis of human prostate cancer specimens showed increased phosphorylation of RSK in the nucleus of prostate cancer cells in a significant fraction of human prostate cancer bone metastasis specimens, compared with the primary site or lymph node metastasis. Expression of constitutively active myristylated RSK in C4-2B4 cells (C4-2B4/RSK) increased their survival and anchorage-independent growth compared with C4-2B4/vector cells. Using an orthotopic bone injection model, it was determined that injecting C4-2B4/RSK cells into mouse femurs enhanced their progression in bone compared with control cells. In PC3-mm2 cells, knockdown of RSK1 (RPS6KA1), the predominant RSK isoform, but not RSK2 (RPS6KA2) alone, decreased anchorage-independent growth in vitro and reduced tumor progression in bone and tumor-induced bone remodeling in vivo. Mechanistic studies showed that RSK regulates anchorage-independent growth through transcriptional regulation of factors that modulate cell survival, including ING3, CKAP2, and PTK6. Together, these data provide strong evidence that RSK is an important driver in prostate cancer progression in bone. RSK, an important driver in prostate cancer progression in bone, has promising potential as a therapeutic target for prostate cancer bone metastasis.

Abstract 2030: Culturing patient-derived prostate cancer xenografts using a three-dimensional hydrogel system for drug testing and mechanistic studies

Abstract Advanced prostate cancer (PCa) typically metastasizes to bone and current treatments are mainly palliative. While a mechanistic understanding of PCa progression in bone is needed to develop new therapies, the complex phenotypes in PCa are poorly reflected by cell lines and cell line-based xenograft models, impeding progress in this endeavor. To overcome this limitation, a panel of PCa patient-derived xenograft (PDX) models was established at the University of Texas MD Anderson Cancer Center. While these PDX models better recapitulate the phenotypic signature of the original tumors as compared to cell lines, limitations exist, including the inability to manipulate these cells ex vivo before implantation in vivo and issues associated with the foreign animal physiology. Based on previous studies demonstrating the potential of using hyaluronan (HA)-based hydrogels for the 3D engineering of PCa cell line-derived ectotumors suitable for mechanistic studies, we evaluated the ability of these hydrogels to maintain viability of the PCa PDX tumors in vitro and optimize culture conditions. Using the MDA PCa 183 and 118b PDX tumors as proof-of-concept, tumors freshly harvested were digested and encapsulated within a HA-based hydrogel, comprised of thiol-modified HA crosslinked with poly(ethylene glycol)-diacrylate. Tumor cells remained as multicellular aggregates or ectotumors in 3D, and were viable even up to 2 weeks in culture. Furthermore, cells were positive for Ki-67, indicating that the hydrogel system is able to retain the proliferative capacity of the tumor cells. In probing for the androgen receptor (AR) expression, we found that while the hydrogel-encapsulated MDA PCa 183 cells stained positive for AR, it was not the case for the MDA PCa 118b, as would be expected in vivo, demonstrating that the hydrogel system is also able to preserve critical phenotypic characteristics of the original tumors. Lastly, to evaluate this novel system as a potential platform for rapid drug testing, we treated the 3D PDX ectotumors to docetaxel, and compared their response against a traditional bone metastatic PCa cell line, C4-2B. Interestingly, the 3D PDX ectotumors exhibited a significantly greater resistance to the drug than the C4-2B cells, reminiscent of chemoresistant tumors in vivo. We demonstrate for the first time that PCa PDX tumors are amenable to in vitro culture with the use of a 3D HA-based hydrogel system. The hydrogel supported the maintenance of multicellular ectotumors of PCa cells, which remained viable and phenotypically similar to the tumors of origin. Furthermore, a differential drug response to docetaxel was observed for both the 3D PCa PDX ectotumors tested as compared to the C4-2B cell line, the clinical relevance of which is currently being investigated. This model may have broad applicability for the culture of other bone metastatic cancers and enable mechanistic studies of disease biology. Citation Format: Eliza L.S. Fong, Mariane Martinez, Jun Yang, Leland M.K. Chung, Antonios G. Mikos, Nora M. Navone, Daniel A. Harrington, Mary C. Farach-Carson. Culturing patient-derived prostate cancer xenografts using a three-dimensional hydrogel system for drug testing and mechanistic studies. [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 2030. doi:10.1158/1538-7445.AM2014-2030

Abstract 4877: MMP-3 generates a novel PTHrP peptide that impacts osteoblast behavior and contributes to metastatic tumor growth in bone

Abstract Prostate cancer commonly metastasizes to bone, generating incurable mixed lesions that are both osteolytic and osteogenic. To identify new therapeutic targets we performed gene expression analysis and found that matrix metalloproteinase-3 (MMP-3), also known as stromelysin-1 was highly expressed in the prostate tumor-bone microenvironment. In keeping with the literature, immunohistochemical analysis of human bone metastases revealed MMP-3 was largely localized to the stromal compartment. To test whether stromal/host derived MMP-3 contributed to prostate cancer progression in bone, we generated immunocompromized MMP-3 null mice. Using an in vivo intratibial model of prostate to bone metastases (PaIII), we found that tumor growth, as measured by luminescence, and tumor-induced bone remodeling (μCT, histomorphometry) were significantly mitigated (p<0.05) in MMP-3 null mice compared to wild type controls. Using a candidate approach to examine potential mechanisms, we focused on parathyroid hormone-related protein (PTHrP), a powerful regulator of osteoblast behavior in the vicious cycle. We identified that MMP-3 processes mature PTHrP1-36 to yield unique PTHrP1-17, PTHrP18-26, and PTHrP27-36 fragments in vitro. To test the biological significance of the fragments we focused on testing their impact on stromal cell behavior. Using Boyden chamber migration assays, we observed a significant increase in migration of murine MSCs and MC3T3-E1 pre-osteoblasts in response to 10nM PTHrP1-17 compared to that of PTHrP1-36. Further analysis via confocal microscopy revealed a reduction in actin stress fibers as well as variations in lamellipodia and vinculin organization of pre-osteoblasts and primary osteoblasts treated with PTHrP1-17, and live cell microscopy indicated that these effects were transient with the cells reverting to the usual phenotype in a 24-hour period. Reduced phosphorylation of Rho-associated protein kinase (ROCK) substrates such as the myosin light chain suggest that PTHrP1-17 may mediate pre-osteoblast migration by modulating ROCK activity. Our data demonstrate that host MMP-3 contributes to prostate tumor growth and tumor induced changes in bone remodeling in vivo. Further, we have identified that mature PTHrP1-36 is subject to cleavage by MMP-3 resulting in PTHrP1-17, PTHrP18-26, and PTHrP27-36 fragments of which PTHrP1-17 significantly stimulates migration of murine MSCs and MC3T3-E1 pre-osteoblasts. Collectively, these data indicate that specific inhibition of MMP-3 and/or targeting MMP generated neo-epitopes would be efficacious for the treatment of prostate to bone metastases. Citation Format: Jeremy Steven Frieling, Lizzie Atomi Pamen, Shengyu Yang, Conor C. Lynch. MMP-3 generates a novel PTHrP peptide that impacts osteoblast behavior and contributes to metastatic tumor 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 4877. doi:10.1158/1538-7445.AM2014-4877

Targeting the RANKL Pathway: Putting the Brakes on Prostate Cancer Progression in Bone

Article Tools UNDERSTANDING THE PATHWAY Article Tools OPTIONS & TOOLS Export Citation Track Citation Add To Favorites Rights & Permissions COMPANION ARTICLES No companion articles ARTICLE CITATION DOI: 10.1200/JCO.2013.50.1544 Journal of Clinical Oncology - published online before print September 16, 2013 PMID: 24043735 Targeting the RANKL Pathway: Putting the Brakes on Prostate Cancer Progression in Bone Isla P. GarrawayxIsla P. GarrawaySearch for articles by this author Show More Jonsson Comprehensive Cancer Center; David Geffen School of Medicine; Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles; and Greater Los Angeles Veterans Affairs Medical Center, Los Angeles, CA https://doi.org/10.1200/JCO.2013.50.1544 First Page Full Text PDF Figures and Tables © 2013 by American Society of Clinical Oncology

Abstract 4092: Identifying the role of TGFβ in osteogenic prostate cancer to bone metastases; an integrated biological/mathematical modeling study.

Abstract Approximately 90% of patients that succumb to prostate cancer will have evidence of bone metastasis. Prostate to bone metastases are incurable with patients often experiencing intense pain. In bone, prostate cancer cells derive factors necessary for growth and survival by manipulating bone forming osteoblasts and bone resorbing osteoclasts, resulting in areas of excessive bone formation/osteogenesis and osteolysis, respectively. Our group and others have defined transforming growth factor beta (TGFβ) as being a key factor in the progression of bone metastases. Therapeutic inhibition of TGFβ however, presents a dilemma since TGFβ can have differential effects on various cell types in the tumor-bone microenvironment. Furthermore, the straightforward nature of current biological assays does not accurately reflect the complexity of a heterogeneous tumor and the potential impact that TGFβ inhibition may have on other cellular components, either individually or in tandem. Our in vitro experimental results show that prostate cancer-derived conditioned media promotes osteoblast migration in a TGFβ-dependent manner and also stimulates osteoblast proliferation, suggesting that TGFβ may play a role in prostate tumor-induced osteogenesis. Using an osteogenic intratibial PAIII model, we have observed that inhibition of TGFβ prevented prostate cancer progression in bone. To determine the mechanism, an integrated approach where experimental results inform a predictive mathematical model, allowing for exploration of complex interactions mediated by TGFβ. Our model is based on the evolutionary game theory (EGT) paradigm where the interactions between key cell types and their role on the evolutionary dynamics of the tumor can be studied. We consider a heterogeneous tumor made of three subpopulations: TGFβ ligand- and receptor-producing cells, TGFβ receptor producing, and TGFβ independent, and also incorporates resident stromal cells – osteoblasts and osteoclasts. Crucially, the fitness of each of cell type as mediated by TGFβ can readily be explored. This model suggests that TGFβ inhibition can hinder tumor growth but, in some instances, can lead to tumors that become less reliant on TGFβ. However, TGFβ inhibition may prevent prostate cancer-induced osteogenesis/osteolysis by regulation of osteoblast/osteoclast precursor recruitment. Histological analyses will determine the robustness of the mathematical approach and the impact of TGFβ inhibition on the tumor-bone microenvironment. The goal of these studies is to develop a method to predict the efficacy of therapeutically targeting TGFβ in prostate to cancer bone metastases. Citation Format: Leah M. Cook, Arturo Araujo, David Basanta, Conor C. Lynch. Identifying the role of TGFβ in osteogenic prostate cancer to bone metastases; an integrated biological/mathematical modeling study. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4092. doi:10.1158/1538-7445.AM2013-4092

Phase 3, randomized, placebo‐controlled study of zibotentan (ZD4054) in patients with castration‐resistant prostate cancer metastatic to bone

Endothelin-1 and the endothelin A (ET(A) ) receptor have been implicated in prostate cancer progression in bone. This study aimed to determine whether the specific ET(A) receptor antagonist, zibotentan, prolonged overall survival (OS) in patients with castration-resistant prostate cancer and bone metastases who were pain-free or mildly symptomatic for pain. Patients were randomized 1:1 to zibotentan 10 mg/day or placebo, plus standard prostate cancer treatment. The primary endpoint was OS. Secondary endpoints included times to pain progression, chemotherapy use, new bone metastases, and safety. Efficacy endpoints were analyzed using a log-rank test. A total of 594 patients were randomized (zibotentan, n = 299; placebo, n = 295). Median OS was 24.5 months in zibotentan-treated patients versus 22.5 months for placebo, but the difference did not reach statistical significance (hazard ratio, 0.87; 95.2% confidence interval, 0.69-1.10; P = .240). No statistically significant differences were observed for any secondary efficacy endpoints. Peripheral edema (44%) and headache (31%) were the most commonly reported adverse events in the zibotentan group. Cardiac failure events were higher in the zibotentan group than placebo (any grade, 5.7% and 1.7%; Common Terminology Criteria for Adverse Events grade ≥3, 3.0% and 1.0%, respectively); these were manageable and reversible. In this large, randomized, placebo-controlled phase 3 trial, treatment with zibotentan 10 mg/day did not lead to a statistically significant improvement in OS in this patient population. Zibotentan had an acceptable safety profile.

Abstract 1767: Targeting prostate cancer osteoblastic progression with VEGF121/rGel, a single agent targeting osteoblasts, osteoclasts, and tumor neovasculature

Abstract Purpose: A hallmark of prostate cancer (PCa) progression is the development of osteoblastic bone metastases, which respond poorly to available therapies. We previously reported that VEGF121/rGel targets osteoclast precursors and tumor neovasculature. Here we tested the hypothesis that targeting non-tumor cells expressing the receptors for VEGF121, namely VEGFR-1 and VEGFR-2, can inhibit tumor progression in a clinically relevant model of osteoblastic PCa. Experimental Design: We examined the effect of VEGF121/rGel on osteoblast precursors and several PCa cell lines in vitro; on osteoid formation in a mouse calvaria culture assay ex vivo; and on PCa osteoblastic progression in the femurs of mice injected with MDA PCa 118b, a PCa xenograft obtained from a bone metastasis in a patient with castrate-resistant PCa. Results: VEGF121/rGel was cytotoxic in vitro to osteoblast precursor cells. This cytotoxicity was specific as VEGF121/rGel internalization into osteoblasts was VEGF121 receptor driven. Furthermore, VEGF121/rGel significantly inhibited PCa-induced bone formation in a mouse calvaria culture assay. In vivo, systemic administration of VEGF121/rGel significantly inhibited the osteoblastic progression of PCa cells in the femurs of nude mice. Microcomputed tomography analysis revealed that VEGF121/rGel restored the bone volume fraction of tumor-bearing femurs to values similar to those of the contralateral (non-tumor bearing) femurs. VEGF121/rGel significantly reduced the number of tumor-associated osteoclasts but did not change the numbers of peritumoral osteoblasts. Importantly, VEGF121/rGel-treated mice had significantly less tumor burden than control mice did. Our results thus indicate that VEGF121/rGel inhibits osteoblastic tumor progression by targeting angiogenesis, osteoclastogenesis, and bone formation. Conclusions: Targeting VEGFR-1- or VEGFR-2-expressing cells is effective in controlling the osteoblastic progression of PCa in bone. These findings provide the basis for an effective multitargeted approach for metastatic PCa. We believe that VEGF121/rGel in combination with tumor cell-targeting therapies (e.g., chemotherapy) constitutes a novel strategy for advanced PCa. Research conducted, in part, by the Clayton Foundation for Research. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1767. doi:10.1158/1538-7445.AM2011-1767

Inhibition of Prostate Cancer Osteoblastic Progression with VEGF121/rGel, a Single Agent Targeting Osteoblasts, Osteoclasts, and Tumor Neovasculature

A hallmark of prostate cancer (PCa) progression is the development of osteoblastic bone metastases, which respond poorly to available therapies. We previously reported that VEGF(121)/rGel targets osteoclast precursors and tumor neovasculature. Here we tested the hypothesis that targeting nontumor cells expressing these receptors can inhibit tumor progression in a clinically relevant model of osteoblastic PCa. Cells from MDA PCa 118b, a PCa xenograft obtained from a bone metastasis in a patient with castrate-resistant PCa, were injected into the femurs of mice. Osteoblastic progression was monitored following systemic administration of VEGF(121)/rGel. VEGF(121)/rGel was cytotoxic in vitro to osteoblast precursor cells. This cytotoxicity was specific as VEGF(121)/rGel internalization into osteoblasts was VEGF(121) receptor driven. Furthermore, VEGF(121)/rGel significantly inhibited PCa-induced bone formation in a mouse calvaria culture assay. In vivo, VEGF(121)/rGel significantly inhibited the osteoblastic progression of PCa cells in the femurs of nude mice. Microcomputed tomographic analysis revealed that VEGF(121)/rGel restored the bone volume fraction of tumor-bearing femurs to values similar to those of the contralateral (non-tumor-bearing) femurs. VEGF(121)/rGel significantly reduced the number of tumor-associated osteoclasts but did not change the numbers of peritumoral osteoblasts. Importantly, VEGF(121)/rGel-treated mice had significantly less tumor burden than control mice. Our results thus indicate that VEGF(121)/rGel inhibits osteoblastic tumor progression by targeting angiogenesis, osteoclastogenesis, and bone formation. Targeting VEGF receptor (VEGFR)-1- or VEGFR-2-expressing cells is effective in controlling the osteoblastic progression of PCa in bone. These findings provide the basis for an effective multitargeted approach for metastatic PCa.

Phase III study of the efficacy and safety of zibotentan (ZD4054) in patients with bone metastatic castration-resistant prostate cancer (CRPC).

117 Background: Endothelin-1 and the endothelin A (ETA) receptor have been implicated in prostate cancer progression in bone. Zibotentan, a specific ETA receptor antagonist, had a promising signal for prolonged overall survival (OS) in a phase II study of patients with CRPC and bone metastases who were pain free or mildly symptomatic for pain. The aim of this phase III study was to confirm the efficacy and safety of zibotentan in a similar but larger population. Methods: Patients with CRPC and bone metastases were randomized 1:1 to zibotentan 10 mg/day po or placebo, plus standard of care including chemotherapy. The primary endpoint was OS. Secondary endpoints included times to pain progression, chemotherapy use, new bone metastases, and safety. Efficacy endpoints were analyzed using a log-rank test. At least 263 deaths were required for formal analysis. If the true hazard ratio (HR) for zibotentan versus placebo was 0.67, the analysis would have 90% power to demonstrate a statistically significant effect in OS at the 5% level. Results: A total of 594 patients were randomized (299 to zibotentan; 295 to placebo). Baseline group demographics were similar. Mean age was ∼71 yrs, and 64% were Caucasian. Although median OS was longer in zibotentan-treated patients than those receiving placebo (median 24.5 vs 22.5 months), the difference did not reach significance (HR [95.2% confidence interval]; 0.87 [0.69–1.10]: P=0.240). No significant differences were observed for any secondary endpoints. The most commonly reported AEs in the zibotentan group, peripheral edema and headache, were consistent with the pharmacologic action of zibotentan as a vasodilator. Cardiac failure events, actively solicited following a phase II signal, were higher in the zibotentan group (any grade, 5.7%; Common Terminology Criteria for Adverse Events [CTCAE] grade ≥3, 3.0%) than placebo (any grade, 1.7%; CTCAE grade ≥3, 1.0%), but were manageable and reversible. Conclusions: In this placebo-controlled phase III trial treatment with zibotentan 10 mg/day did not lead to a significant improvement in OS in patients with CRPC and bone metastases. Zibotentan had an acceptable safety profile. [Table: see text]

Activation of MCP-1/CCR2 axis promotes prostate cancer growth in bone

Prostate cancer (PCa) frequently metastasizes to bone resulting in a mixture of osteolytic and osteoblastic lesions. We have previously reported that monocyte chemotactic protein-1 (MCP-1) is chemotactic for PCa cells, and its receptor, CCR2 expression, correlates with pathological stages. However, the role of MCP-1/CCR2 axis on PCa progression in bone remains unclear. We first evaluated the serum levels of MCP-1 in patients with bone metastases or localized PCa by enzyme-linked immunosorbent assay. We found that MCP-1 levels were elevated in patients with bone metastases compared to localized PCa. We further determined the effects of knockdown CCR2 or MCP-1 on PCa cell invasion and the tumor cell-induced osteoclast activity in vitro, respectively. PCa C4-2B and PC3 cells were transfected stably with either CCR2 short hairpin RNA (shRNA) or a scrambled RNA. CCR2 knockdown significantly diminished the MCP-1-induced PCa cell invasion. In addition, the MCP-1 production was knocked down by MCP-1 shRNA in C4-2B and PC3 cells. Conditioned media (CM) was collected and determined for the CM-induced osteoclast formation in vitro. MCP-1 knockdown significantly decreased the PCa CM-induced osteoclast formation. Finally, MCP-1 knockdown PC3 cells were implanted into the tibia of SCID mice for 4 weeks. Tumor volume was determined by histopathology and bone histomorphometry. MCP-1 knockdown diminished PC3 tumor growth in bone. We concluded that activation of MCP-1/CCR2 axis promotes PCa growth in bone. This study suggests that MCP-1 may be a target for PCa progression.

Targeting Bone Metastasis in Prostate Cancer with Endothelin Receptor Antagonists

Recent advances in the understanding of prostate cancer biology and its progression to bone metastasis have led to the development of drugs directed against precise molecular alterations in the prostate tumor cell and host cells in the normal bone environment such as osteoclasts and osteoblasts. Endothelins (ETs) and their receptors have emerged as a potential target in prostate cancer bone metastasis. By activating the ETA receptor, ET-1 is pathogenically involved in facilitating several aspects of prostate cancer progression, including proliferation, escape from apoptosis, invasion, and new bone formation, processes that are general to many malignancies. Notwithstanding, there are a number of features specifically driven by the ET axis in prostate cancer, such as creating and perpetuating a unique interaction between the metastatic prostate cancer cell and the bone microenvironment (osteoblast, osteoclast, and stroma) or altering the equilibrium in pain modulation. These features have led to the preferential clinical evaluation of atrasentan (ABT-627) as a biological therapy in prostate carcinoma, first in hormone-refractory prostate cancer. Biological activity of atrasentan in patients with prostate cancer has been shown by the suppression of biochemical markers of prostate cancer progression in bone, and clinical activity is evidenced by a consistent trend demonstrating a delay in time to disease progression when compared with placebo, especially in patients with bone metastases. Further studies of atrasentan and other selective ET-1 antagonists (ZD4054) are ongoing.

Direct evidence that PTHrP expression promotes prostate cancer progression in bone

Parathyroid hormone-related protein (PTHrP) is an oncoprotein that is expressed in many malignancies as well as normal tissues. At essentially every site of expression, PTHrP regulates cell growth and proliferation. We and other investigators have previously reported that PTHrP is widely expressed by prostate cancer. For this tumor, there are substantial in vitro and correlative data that PTHrP expression regulates the progression of the tumor, especially in bone, but little direct data. We studied the effects of PTHrP expression on prostate cancer behavior directly in a mouse model of human prostate cancer cells that were transfected to express different forms of the polypeptide and then injected intraskeletally. Skeletal progression of the prostate cancer cells was evaluated radiologically and by measurement of serum tumor markers. PTHrP transfection converted a non-invasive cell line into one that progressed in the skeleton: Injection of the PTHrP transfected cells resulted in greater tumor progression in bone when compared to non-transfected cells, and this effect was also influenced by non-amino terminal peptides of PTHrP. Serum measurements of PTHrP, IL-6, IL-8, and calcium reflected tumor burden. Our experiments provide direct in vivo evidence that PTHrP expression results in the skeletal progression of prostate cancer cells.