Human Prostate Cancer Bone Metastases Research Articles

Targeting AXL inhibits the growth and metastasis of prostate cancer in bone.

After failing primary and secondary hormonal therapy, castration-resistant and neuroendocrine prostate cancer metastatic to the bone is invariably lethal, although treatment with docetaxel and carboplatin can modestly improve survival. Therefore, agents targeting biologically relevant pathways in PCa and potentially synergizing with docetaxel and carboplatin in inhibiting bone metastasis growth are urgently needed. Phosphorylated (activated) AXL expression in human prostate cancer bone metastases was assessed by immunohistochemical staining. We evaluated the effects of a novel soluble AXL signaling inhibitor, sAXL (batiraxcept or AVB-S6-500), on the tumor growth and lung metastases in PCa patient-derived xenograft models (PDX) that implanted intratibally. After injection of LuCaP cells into the tibiae, tumors were treated with batiraxcept and docetaxel or carboplatin alone or in combination, and tumor growth was monitored by serum PSA or bioluminescence. Tumor burden was quantified by human-specific Ku70 staining, and metastasis to the lung was determined using qPCR. Transcriptomic profiling, western blotting and immunohistochemistry were performed to identify treatment-regulated gene and protein profile changes. High AXL phosphorylation in human PCa bone metastases correlated with shortened survival. Batiraxcept alone or in combination with docetaxel or carboplatin significantly suppressed intratibial tumor growth and suppressed metastasis to the lung through multiple mechanisms, including repression of cancer stemness genes (CD44, ALDH1A1, TACSTD2, ATXN1) and the PI3K, JAK, MAPK, and E2F1/NUSAP1 signaling pathways. Our study provides a robust preclinical rationale and mechanisms of action for using batiraxcept as a single agent or in combination with docetaxel or carboplatin to treat lethal mPCa.

Animal models of bone metastatic prostate cancer.

Metastatic disease is a main cause of mortality in prostate cancer and remains to be incurable despite emerging new treatment agents. Development of novel treatment agents are confined within the boundaries of our knowledge of bone metastatic prostate cancer. Exploration into the underlying mechanism of metastatic tumorigenesis and treatment resistance will further expose novel targets for novel treatment agents. Up to date, many of these researches have been conducted with animal models which have served as classical tools that play a pivotal role in understanding the fundamental nature of cancer. The ability to reproduce the natural course of prostate cancer would be of profound value. However, currently available models do not reproduce the entire process of tumorigenesis to bone metastasis and are limited to reproducing small portions of the entire process. Therefore, knowledge of available models and understanding the strengths and weaknesses for each model is key to achieve research objectives. In this article, we take an overview of cell line injection animal models and patient derived xenograft models that have been applied to the research of human prostate cancer bone metastasis.

Animal Models for Bone Metastatic Prostate Cancer

Metastatic disease is a main cause of mortality in prostate cancer and remains to be uncurable despite emerging new treatment agents. Development of novel treatment agents are confined within the boundaries of our knowledge of bone metastatic prostate cancer. Exploration into the underlying mechanism of metastatic tumorigenesis and treatment resistance will further expose novel targets for novel treatment agents. Up to date, many of these researches have been conducted with animal models which have served as classical tools that play a pivotal role in understanding the fundamental nature of cancer. The ability to reproduce the natural course of prostate cancer would be of profound value. However, currently available models cannot reproduce the entire process of tumorigenesis to bone metastasis and are limited to reproducing small portions of the entire process. Therefore, knowledge of available models and understanding the strengths and weaknesses for each model is key to achieve research objectives. In this article, we take an overview of cell line injection animal models and patient-derived xenograft models that have been applied to the research of human prostate cancer bone metastasis.

Targeting the BMP Pathway in Prostate Cancer Induced Bone Disease.

From the 33,000 men in the U.S. who die from prostate cancer each year, the majority of these patients exhibit metastatic disease with bone being the most common site of metastasis. Prostate cancer bone metastases are commonly blastic, exhibiting new growth of unhealthy sclerotic bone, which can cause painful skeletal related events. Patient’s current care entails androgen deprivation therapy, anti-resorptive agents, radiation, and chemotherapy to help control the spread of the cancer but little intervention is available to treat blastic bone disease. The transforming growth factor beta (TGFβ) and bone morphogenetic protein (BMP) pathways are known to regulate bone growth and resorption of destructive lytic bone lesions, yet the role of TGFβ/BMP signaling in prostate cancer blastic vs lytic bone lesions are not fully understood. We hypothesized that to target the BMP/TGFβ pathway, a useful biomarker of bone lytic or blastic pathology would have superior response. We show distinct BMP vs. TGFβ signaling in clinical samples of human prostate cancer bone metastases with either lytic or blastic pathologies. BMPs exhibit distinct effects on bone homeostasis, so to examine the effect of BMP inhibition on healthy bone, we treated mice with the BMP receptor small molecule antagonist DMH1 and saw a modest temporary improvement in bone health, with increased trabecular bone. We next sought to use the BMP inhibitor DMH1 to treat bone metastasis engraftment seeded by a caudal artery injection of the lytic human prostate cell line PC3 in immunodeficient mice. The colonization by PC3 cells to the bone were restricted with DMH1 treatment and bone health was importantly preserved. We next proceeded to test BMP inhibition in an injury model of established bone metastasis via intratibial injection of the MYC-CaP mouse prostate cell line into FVBN syngeneic mice. DMH1 treated mice had a modest decrease in trabecular bone and reduced lymphocytes in circulation without affecting tumor growth. Taken together we show unique responses to BMP inhibition in metastatic prostate cancer in the bone. These studies suggest that profiling bone lesions in metastatic prostate cancer can help identify therapeutic targets that not only treat the metastatic tumor but also address the need to better treat the distinct tumor induced bone disease.

Fibroblast Growth Factor Receptor 1 Drives the Metastatic Progression of Prostate Cancer

BackgroundNo curative therapy is currently available for metastatic prostate cancer (PCa). The diverse mechanisms of progression include fibroblast growth factor (FGF) axis activation. ObjectiveTo investigate the molecular and clinical implications of fibroblast growth factor receptor 1 (FGFR1) and its isoforms (α/β) in the pathogenesis of PCa bone metastases. Design, setting, and participantsIn silico, in vitro, and in vivo preclinical approaches were used. RNA-sequencing and immunohistochemical (IHC) studies in human samples were conducted. Outcome measurements and statistical analysisIn mice, bone metastases (chi-square/Fisher’s test) and survival (Mantel-Cox) were assessed. In human samples, FGFR1 and ladinin 1 (LAD1) analysis associated with PCa progression were evaluated (IHC studies, Fisher’s test). Results and limitationsFGFR1 isoform expression varied among PCa subtypes. Intracardiac injection of mice with FGFR1-expressing PC3 cells reduced mouse survival (α, p < 0.0001; β, p = 0.032) and increased the incidence of bone metastases (α, p < 0.0001; β, p = 0.02). Accordingly, IHC studies of human castration-resistant PCa (CRPC) bone metastases revealed significant enrichment of FGFR1 expression compared with treatment-naïve, nonmetastatic primary tumors (p = 0.0007). Expression of anchoring filament protein LAD1 increased in FGFR1-expressing PC3 cells and was enriched in human CRPC bone metastases (p = 0.005). ConclusionsFGFR1 expression induces bone metastases experimentally and is significantly enriched in human CRPC bone metastases, supporting its prometastatic effect in PCa. LAD1 expression, found in the prometastatic PCa cells expressing FGFR1, was also enriched in CRPC bone metastases. Our studies support and provide a roadmap for the development of FGFR blockade for advanced PCa. Patient summaryWe studied the role of fibroblast growth factor receptor 1 (FGFR1) in prostate cancer (PCa) progression. We found that PCa cells with high FGFR1 expression increase metastases and that FGFR1 expression is increased in human PCa bone metastases, and identified genes that could participate in the metastases induced by FGFR1. These studies will help pinpoint PCa patients who use fibroblast growth factor to progress and will benefit by the inhibition of this pathway.

Human prostate cancer bone metastases have an actionable immunosuppressive microenvironment.

Bone metastases are devastating complications of cancer. They are particularly common in prostate cancer (PCa), represent incurable disease, and are refractory to immunotherapy. We seek to define distinct features of the bone marrow (BM) microenvironment by analyzing single cells from bone metastatic prostate tumors, involved BM, uninvolved BM, and BM from cancer-free, orthopedic patients, and healthy individuals. Metastatic PCa is associated with multifaceted immune distortion, specifically exhaustion of distinct Tcell subsets, appearance of macrophages with states specific to PCa bone metastases. The chemokine CCL20 is notably overexpressed by myeloid cells, as is its cognate CCR6 receptor on Tcells. Disruption of the CCL20-CCR6 axis in mice with syngeneic PCa bone metastases restores Tcell reactivity and significantly prolongs animal survival. Comparative high-resolution analysis of PCa bone metastases shows a targeted approach for relieving local immunosuppression for therapeutic effect.

Prostatic Acid Phosphatase Is a Progenitor Cell Marker That Persists After Androgen Ablation

Introduction: Prostatic Acid Phosphatase (PAP), a protein phosphatase and 5’ecto-nucleotidase, is expressed in prostate cancer (PCa) bone metastases and correlates with poor survival. Growing evidence suggests that PAP is not regulated by androgens, but rather by factors in the tumor microenvironment.Hypothesis: We hypothesized that PAP is a marker for a more progenitor type PCa cell and its expression is androgen-independent, persisting in castration-resistant disease.Methods: Protein expression of PAP and three androgen-regulated proteins, the Androgen Receptor (AR), Prostate-Specific Antigen (PSA), and ETS-related gene (ERG) protein, was assessed with immunohistochemistry in human fetal prostate (9.5 - 20 weeks of gestational age), archival human PCa bone metastases, and human PCa cell lines. VCaP cells were treated in vitro with dihydrotestosterone (DHT) and the effects on AR and PAP protein expression determined with Western Blotting. PAP-expressing PCa cell lines (LNCaP, C42B, and VCaP) were inoculated subcutaneously (s.c.) into SCID mice. To model tumor-bone interaction, LNCaP and MC3T3 osteoblast cells were co-inoculated s.c. into SCID mice. A VCaP castration study with surgical or sham castration was performed after tumors were palpable and effects of castration on tumor growth and protein expression determined.Results: PAP expression was observed in the fetal prostate as early as 11.5 weeks of gestational age prior to PSA and AR expression. Strong PAP expression was noted in all human PCa bone metastases examined, both treatment-naive and castrate-resistant (n=10). In vitro, VCaP cells expressed high levels of AR and PAP protein and DHT treatment increased AR and decreased PAP protein expression. In vivo, PAP expression was observed in all tumor models; LNCaP (low PAP expression), C42B (moderate PAP expression) and VCaP (high PAP expression). Castrated VCaP tumors underwent tumor stasis, were significantly smaller compared to intact mice, had decreased AR, PSA and ERG expression but persistent expression of PAP. Double staining of tumors for PAP and AR demonstrated a population of cells that were positive for PAP but negative for AR expression in hypoxic areas near necrosis. Inoculation of LNCaP cells with MC3T3 osteoblastic cells increased PAP expression in vivo.Conclusions: PAP is expressed early in human fetal prostate development prior to the secretion of significant androgens or expression of AR. In mouse xenograft tumors and human PCa bone metastases, androgens did not significantly regulate PAP expression. Both hypoxia and stroma increased PAP expression. These data demonstrate that PAP is a marker of early progenitor cells, is persistently expressed after castration and is upregulated by tumor microenvironmental factors. PAP may be a suitable target for the treatment of castration-resistant metastatic disease.

Multiple pathways coordinating reprogramming of endothelial cells into osteoblasts by BMP4

SummaryCell type transition occurs during normal development and under pathological conditions. In prostate cancer bone metastasis, prostate cancer-secreted BMP4 induces endothelial cell-to-osteoblast (EC-to-OSB) transition. Such tumor-induced stromal reprogramming supports prostate cancer progression. We delineate signaling pathways mediating EC-to-OSB transition using EC lines 2H11 and SVR. We found that BMP4-activated pSmad1-Notch-Hey1 pathway inhibits EC migration and tube formation. BMP4-activated GSK3β-βcatenin-Slug pathway stimulates Osx expression. In addition, pSmad1-regulated Dlx2 converges with the Smad1 and β-catenin pathways to stimulate osteocalcin expression. By co-expressing Osx, Dlx2, Slug and Hey1, we were able to achieve EC-to-OSB transition, leading to bone matrix mineralization in the absence of BMP4. In human prostate cancer bone metastasis specimens and MDA-PCa-118b and C4-2b-BMP4 osteogenic xenografts, immunohistochemical analysis showed that β-catenin and pSmad1 are detected in activated osteoblasts rimming the tumor-induced bone. Our results elucidated the pathways and key molecules coordinating prostate cancer-induced stromal programming and provide potential targets for therapeutic intervention.

Multiple Pathways Coordinating Reprogramming of Endothelial Cells into Osteoblasts by BMP4

Cell type transition occurs during normal development and under pathological conditions. In prostate cancer bone metastasis, prostate cancer-secreted BMP4 induces endothelial-to-osteoblast (EC-to-OSB) transition, leading to aberrant bone formation. Such tumor-induced stromal reprogramming supports prostate cancer progression. Here, we delineate signaling pathways mediating EC-to-OSB transition using endothelial cell lines 2H11 and SVR. We found that for EC-to-OSB transition to occur, inhibition of angiogenesis, through the Smad1-Notch-Hey1 pathway that inhibits EC migration and tube formation, together with activation of osteogenesis, through the p38MAPK(p44/42ERK,AKT)-GSK3β-βcatenin-Slug pathway that stimulates osterix and osteocalcin expression, are required. In addition, Smad1-regulated Dlx2 plays a role in converging the Smad1 and β-catenin pathways during EC-to-OSB transition. Importantly, by just co-expressing the four transcription factors, osterix, Dlx2, Slug and Hey1, we were able to achieve EC-to-OSB transition, leading to bone matrix mineralization even in the absence of BMP4. Thus, the interplay of cell fate determinants and transcription factors mediates BMP4-induced EC-to-OSB transition during stromal reprogramming. In human prostate cancer bone metastasis specimens, immunohistochemical analysis showed that β-catenin and pSmad1 are detected in activated osteoblasts rimming the tumor-induced bone. Similarly, in MDA-PCa-118b and C4-2b-BMP4 osteogenic xenografts, β-catenin and pSmad1 are expressed in EC-OSB hybrid cells that rim the tumor-induced bone. Our results elucidated the pathways and key molecules coordinating prostate cancer-induced stromal programming and provide potential targets for therapeutic intervention.

SUN-143 Prostatic Acid Phosphatase Is Not Regulated by Androgens During Prostate Development and Tumorigenesis

INTRODUCTION: Prostatic acid phosphatase (PAP) is a soluble factor secreted by prostate luminal epithelial cells. PAP expression correlates with prostate cancer (PCa) bone metastases and poor survival. The androgenic regulation of PAP in prostate development and tumorigenesis is not fully understood. We investigated the relationship between PAP and androgens in human prostate specimens and in vivo. HYPOTHESIS AND OBJECTIVES: We hypothesized that PAP expression was independent of androgens. Our objectives were to determine the immunohistochemical expression of PAP in human fetal prostate tissue, human PCa bone metastases, and xenograft and surgical castration mouse models. METHODS: Immunohistochemical staining for PAP and three androgen-regulated proteins, the Androgen Receptor (AR), Prostate-Specific Antigen (PSA), and ETS-related gene (ERG) protein, was carried out on human fetal prostate (9.5, 11.5, 13, 16.5, 18 and 20 weeks of gestational age), archival human PCa bone metastases, and PCa mouse models. For xenograft studies, PAP-expressing PCa cell lines, LNCaP, C42B, and VCaP cells, were inoculated subcutaneously into SCID mice. A castration study with surgical or sham castration was performed after VCaP tumors were palpable. Mouse tumor growth and weight were measured biweekly, and tumor tissue isolated after mouse sacrifice. RESULTS: PAP expression was observed in the fetal prostate as early as 11.5 weeks of gestational age. Strong PAP expression was noted in all human PCa bone metastases examined, both treatment-naive and castrate-resistant (n=10). However, AR and ERG expression was absent in two of four castrate-resistant specimens. PSA was weakly expressed in human castration-resistant bone metastatic prostate specimens. In vivo, PAP expression was observed in all tumor models; however, the expression of PAP differed among androgen-sensitive models; LNCaP (low PAP), C42B (moderate PAP) and VCaP (high PAP). Castrated VCaP tumors underwent tumor stasis and were significantly smaller compared to intact mice. Strong expression of PAP was observed after castration. In contrast, AR, PSA, and ERG expression were reduced in castrated VCaP tumors compared to tumors from intact mice. Double staining of tumors for PAP and AR demonstrated a population of cells that were positive for PAP but negative for AR expression located in hypoxic areas near necrosis. CONCLUSIONS: Our findings demonstrated that PAP is expressed early in normal human fetal prostate development prior to the secretion of significant androgens or expression of AR. In mouse xenografts and human PCa bone metastases, androgens did not significantly regulate PAP expression. These data demonstrate that PAP is a marker of early progenitor cells in the normal prostate and is persistently expressed after castration. PAP may be a suitable target for the treatment of castration-resistant metastatic disease.

Notch3 promotes prostate cancer-induced bone lesion development via MMP-3.

Prostate cancer metastases primarily localize in the bone where they induce a unique osteoblastic response. Elevated Notch activity is associated with high-grade disease and metastasis. To address how Notch affects prostate cancer bone lesions, we manipulated Notch expression in mouse tibia xenografts and monitored tumor growth, lesion phenotype, and the bone microenvironment. Prostate cancer cell lines that induce mixed osteoblastic lesions in bone expressed 5–6 times more Notch3, than tumor cells that produce osteolytic lesions. Expression of active Notch3 (NICD3) in osteolytic tumors reduced osteolytic lesion area and enhanced osteoblastogenesis, while loss of Notch3 in osteoblastic tumors enhanced osteolytic lesion area and decreased osteoblastogensis. This was accompanied by a respective decrease and increase in the number of active osteoclasts and osteoblasts at the tumor-bone interface, without any effect on tumor proliferation. Conditioned medium from NICD3-expressing cells enhanced osteoblast differentiation and proliferation in vitro, while simultaneously inhibiting osteoclastogenesis. MMP-3 was specifically elevated and secreted by NICD3-expressing tumors, and inhibition of MMP-3 rescued the NICD3-induced osteoblastic phenotypes. Clinical osteoblastic bone metastasis samples had higher levels of Notch3 and MMP-3 compared to patient matched visceral metastases or osteolytic metastasis samples. We identified a Notch3-MMP-3 axis in human prostate cancer bone metastases that contributes to osteoblastic lesion formation by blocking osteoclast differentiation, while also contributing to osteoblastogenesis. These studies define a new role for Notch3 in manipulating the tumor microenvironment in bone metastases.

Abstract 4523: A novel syngeneic mouse model of prostate cancer bone metastasis: Mechanisms of chemotaxis and bone colonization

Abstract Background: Bone metastasis in human prostate cancer remains a major clinical problem since no effective therapy exists. The RANKL/RANK pathway plays a predominant role in the interaction between metastasized prostate cancer cells and osteoclasts that increases the bone turnover. The current therapies, including targeting RANKL with denosumab, address the growth of prostate tumor cells that have already colonized the bone, but are largely ineffective in prolonging the survival of human prostate cancer patients with bone metastasis. Further, a major impediment to prostate cancer bone metastasis research is the lack of an animal model that spontaneously recapitulates human prostate cancer bone metastasis in the context of an intact immune system. Results: To overcome this major limitation, we have developed a novel syngeneic mouse model to study prostate cancer bone metastasis. Both the CXCL12/CXCR4 and RANKL/RANK pathways have been reported to be overexpressed / dysregulated in human prostate cancer bone metastatic samples. Data generated utilizing our immune-intact mouse model shows that the CXCL12/CXCR4 and RANKL/RANK pathways co-operate with each other to drive prostate cancer bone metastasis. Studies have shown that targeting the CXCL12/CXCR4 and RANKL/RANK pathways individually affects the immune system, thereby making our syngeneic mouse model an indispensable tool for studying the critical co-operation between these 2 pathways in the manifestation of human prostate cancer bone metastasis. Extending our earlier findings that RANKL drives PCa metastases in immune-deficient mice (Chu et al., 2014) to immune-intact C57/Bl6 mice, we found that MPC3 mouse PCa cells with RANKL overexpression (MPC3-Luc-GFP-RANKL) develop 70-80% limb and jaw within 4 weeks of intra-cardiac injection in these syngeneic mice. Control MPC3 cells had no bone metastasis. Bone lesions visualized by luciferase imaging and X-ray were confirmed by micro CT and immunohistochemistry. RANKL signaling drove bone and visceral metastases via the downstream CXCL12/CXCR4 signaling axis. MPC3-Luc-GFP-RANKL cells showed increased CXCR4 protein levels by immunohistochemistry. Metastatic bone marrow flush showed dramatically increased levels of CXCL12 mRNA compared with control mice (MPC3-Luc-GFP-EV). Conclusion: In sum, 1) circulating PCa cells induce a marked CXCL12 elevation after colonizing bone, triggering chemotaxis and recruiting CXCR4-positive PCa cells to migrate to bone; and 2) osteomimetic PCa cells with increased RANKL expression interact with osteoclasts to enhance bone resorption and turnover, releasing additional growth factors and chemokines for PCa cell growth and survival in bone. Citation Format: Srinivas Nandana, Murali Gururajan, Manisha Tripathi, Chia-Yi Chu, Haiyen Zhau, Stephen Shiao, Leland Chung. A novel syngeneic mouse model of prostate cancer bone metastasis: Mechanisms of chemotaxis and bone colonization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4523.

Abstract 1086: Phosphoproteomic analysis of signaling pathways driving bone metastasis in prostate cancer

Abstract Osteoblasts play a fundamental role in prostate cancer bone metastasis, whereby dysregulated bone remodelling results in bone pain, fractures and increased mortality. Although it remains unclear how early on in the disease pathway prostate cancer cells populate the endosteal niche, osteoblasts almost certainly play a role in the homing of tumor cells to this microenvironment. In support of this, prostate cancer cells have been shown to home to osteoblast-rich areas in animal models of bone metastasis and human prostate cancer bone metastases are typically osteoblastic on radiography. We have shown that conditioned medium from mature, mineralising human hFOB1.19 osteoblasts (hFOB-CM) is rich in pro-inflammatory cytokines including IL-6, IL-8 and various chemokine ligands. hFOB-CM promotes a strong migratory and invasive phenotype in a panel of prostate cancer cell lines (including androgen-sensitive and -insensitive lines), with invading cells displaying increased actomyosin contractility and amoeboid-like traits, namely a rounded cell morphology, cytoskeletal reorganization and phosphorylation of myosin light chain. We now aim to comprehensively interrogate the intracellular signaling pathways driving endosteal homing in prostate cancer cells by systems-wide ITRAQ mass spectrometry-based phosphoproteomic analysis of prostate cancer cells treated with hFOB-CM. This approach will identify individual signaling pathways or combinations of pathways to target with existing kinase inhibitors to inhibit osteoblast-driven invasion and endosteal homing during prostate cancer metastasis. Ultimately, targeting tumor cell responses to stromal signals may prove critical in the search for better therapies to treat this frequently lethal disease. Citation Format: Benjamin Abbott, Andrew Pierce, Anthony D. Whetton, Paul A. Townsend. Phosphoproteomic analysis of signaling pathways driving bone metastasis in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1086.

Prostatic Acid Phosphatase Alters the RANKL/OPG System and Induces Osteoblastic Prostate Cancer Bone Metastases.

Prostate cancer (PCa) is unique in its tendency to produce osteoblastic (OB) bone metastases. There are no existing therapies that specifically target the OB phase that affects 90% of men with bone metastatic disease. Prostatic acid phosphatase (PAP) is secreted by PCa cells in OB metastases and increases OB growth, differentiation, and bone mineralization. The purpose of this study was to investigate whether PAP effects on OB bone metastases are mediated by autocrine and/or paracrine alterations in the receptor activator of nuclear factor κ-B (RANK)/RANK ligand (RANKL)/osteoprotegerin (OPG) system. To investigate whether PAP modulated these factors and altered the bone reaction, we knocked down PAP expression in VCaP cells and stably overexpressed PAP in PC3M cells, both derived from human PCa bone metastases. We show that knockdown of PAP in VCaP cells decreased OPG while increasing RANK/RANKL expression. Forced overexpression of PAP in PC3M cells had the inverse effect, increasing OPG while decreasing RANK/RANKL expression. Coculture of PCa cells with MC3T3 preosteoblasts also revealed a role for secretory PAP in OB-PCa cross talk. Reduced PAP expression in VCaP cells decreased MC3T3 proliferation and differentiation and reduced their OPG expression. PAP overexpression in PC3M cells altered the bone phenotype creating OB rather than osteolytic lesions in vivo using an intratibial model. These findings demonstrate that PAP secreted by PCa cells in OB bone metastases increases OPG and plays a critical role in the vicious cross talk between cancer and bone cells. These data suggest that inhibition of secretory PAP may be an effective strategy for PCa OB bone lesions.

Abstract 1564: Reactive endosteum in prostate cancer bone metastases: role of tenascin-C in regulating cancer cell adhesion and proliferation

Abstract The objective of this study is to assess the regulatory roles of reactive endosteum associated with foci of bone metastatic prostate cancer cells. Previous studies evaluated tissue arrays of human prostate cancer bone metastases and we identified a “reactive endosteum” spatially associated with foci of prostate cancer cells on trabecular bone. Reactive endosteum was characterized by elevated expression of tenascin-C, a glycoprotein deposited in the extracellular matrix of reactive stroma at sites of wound repair and in the primary tumor microenvironment in adult tissues. In general, tenascin-C is stromal derived in adult tissues. Of interest, tenascin-C is also deposited at sites of fracture repair and at sites of Brodie abscess osteomyelitis-associated inflammation in human bone. Tenascin-C has many diverse functions: it regulates cell adhesion, migration and proliferation in different pathological states while playing an important role in neuropatterning and osteogenesis during development. Moreover, tenascin-C has been shown to regulate several signal transduction pathways. To evaluate prostate cancer-bone interactions, we developed an in vitro, 3D, osteogenic organoid model composed of human mesenchymal stem cells induced to osteogenesis that were combined with human prostate VCaP cells in organ culture. In this model, foci of VCaP cells associated preferentially to regions of high tenascin-C deposition. VCaP cells also preferentially bound to purified human tenascin-C deposited on culture plates in a dose-dependent manner. VCaP cells also exhibited an elevated growth rate on osteo-mimetic plates coated with human tenascin-C as compared to control. Moreover, VCaP cells preferentially bound to human tenascin-C coated bovine trabecular bone cubes in vitro and initiated colony formation. Evaluation of potential mediators identified integrin alpha 9 beta 1 as the key mediator of attachment to human tenascin-C. Neutralization of this integrin inhibited adhesion. Evaluation of potential signaling pathways have implicated activation of EGF receptor (EGFR), Wnk1, and STAT6 pathways. Human tenascin-C exhibits EGF-like repeats and fibronectin type III domains that may mediate activation of these pathways. Of interest, adhesion and growth of VCaP cells on tenascin-C also induced elevated tenascin-C expression in these cells, a novel finding. Additional 3D organoid and in vivo xenograft studies with other cell types support the finding of induced expression of tenascin-C in epithelial cells associated with reactive stroma or matrix. In summary, our studies characterize elevated tenascin-C at sites of a reactive endosteum associated with metastatic prostate cancer foci. Data suggests that tenascin-C mediates adhesion and proliferation of cancer cells via activation of several pathways. These data may aid in developing novel therapeutic approaches to treat metastatic disease. Citation Format: Rebeca San Martin, Kenneth Pienta, David R. Rowley. Reactive endosteum in prostate cancer bone metastases: role of tenascin-C in regulating cancer cell adhesion and proliferation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1564.

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.

Cellular Plasticity in Prostate Cancer Bone Metastasis.

Purpose. Experimental data suggest that tumour cells can reversibly transition between epithelial and mesenchymal states (EMT and MET), a phenomenon known as cellular plasticity. The aim of this review was to appraise the clinical evidence for the role of cellular plasticity in prostate cancer (PC) bone metastasis. Methods. An electronic search was performed using PubMed for studies that have examined the differential expression of epithelial, mesenchymal, and stem cell markers in human PC bone metastasis tissues. Results. The review included nineteen studies. More than 60% of the studies used ≤20 bone metastasis samples, and there were several sources of heterogeneity between studies. Overall, most stem cell markers analysed, except for CXCR4, were positively expressed in bone metastasis tissues, while the expression of EMT and MET markers was heterogeneous between and within samples. Several EMT and stemness markers that are involved in osteomimicry, such as Notch, Met receptor, and Wnt/β pathway, were highly expressed in bone metastases. Conclusions. Clinical findings support the role of cellular plasticity in PC bone metastasis and suggest that epithelial and mesenchymal states cannot be taken in isolation when targeting PC bone metastasis. The paper also highlights several challenges in the clinical detection of cellular plasticity.

Efficient delivery of micro RNA to bone-metastatic prostate tumors by using aptamer-conjugated atelocollagen in vitro and in vivo

Bone is the primary site of skeletal metastasis in prostate cancer (PCa). Atelocollagen (ATE)-mediated siRNA delivery system can be used to silence endogenous genes involved in PCa metastatic tumor cell growth. However, we hope that the delivery system can target PCa cells to reduce damage to the bone tissue and improve the therapeutic effect. RNA aptamer (APT) A10-3.2 has been used as a ligand to target PCa cells that express prostate-specific membrane antigen (PSMA). APT was investigated as a PSMA-targeting ligand in the design of an ATE-based microRNA (miRNA; miR-15a and miR-16-1) vector to PCa bone metastasis. To observe the targeted delivery and transfection efficiency of ATE–APT in PSMA-overexpressing cells, luciferase activity and biodistribution of nanoparticles in Balb/c mice was analyzed. The anticancer effect of nanoparticles in vivo was investigated using the survival times of human PCa bone metastasis mice model. Luciferase assays of pGL-3 expression against PC3 (PSMA−) and LNCaP (PSMA+) cells showed that the transfection efficiency of the synthesized DNA/ATE–APT complex was higher than that of the DNA/ATE complex. The anticancer efficacy of miRNA/ATE–APT was superior to those of other treatments in vivo. This PSMA-targeted system may prove useful in widening the therapeutic window and allow for selective killing of PCa cells in bone metastatic foci.

Integrin αvβ6 Promotes an Osteolytic Program in Cancer Cells by Upregulating MMP2

The molecular circuitries controlling osseous prostate metastasis are known to depend on the activity of multiple pathways, including integrin signaling. Here, we demonstrate that the αvβ6 integrin is upregulated in human prostate cancer bone metastasis. In prostate cancer cells, this integrin is a functionally active receptor for fibronectin and latency-associated peptide-TGF-β1; it mediates attachment and migration upon ligand binding and is localized in focal contacts. Given the propensity of prostate cancer cells to form bone metastatic lesions, we investigated whether the αvβ6 integrin promotes this type of metastasis. We show for the first time that αvβ6 selectively induces matrix metalloproteinase 2 (MMP2) in vitro in multiple prostate cancer cells and promotes osteolysis in vivo in an immunodeficient mouse model of bone metastasis through upregulation of MMP2, but not MMP9. The effect of αvβ6 on MMP2 expression and activity is independent of androgen receptor in the analyzed prostate cancer cells. Increased levels of parathyroid hormone-related protein (PTHrP), known to induce osteoclastogenesis, were also observed in αvβ6-expressing cells. However, by using MMP2 short hairpin RNA, we demonstrate that the αvβ6 effect on bone loss is due to upregulation of soluble MMP2 by the cancer cells, not due to changes in tumor growth rate. Another related αv-containing integrin, αvβ5, fails to show similar responses, underscoring the significance of αvβ6 activity. Overall, these mechanistic studies establish that expression of a single integrin, αvβ6, contributes to the cancer cell-mediated program of osteolysis by inducing matrix degradation through MMP2. Our results open new prospects for molecular therapy for metastatic bone disease.

Abstract 2436: Fibroblast growth factor receptor blockade in prostate cancer bone metastases.

Abstract Castrate-resistant progression and bone metastases (BM) are hallmarks of advanced prostate cancer (PCa) for which there is no definitive cure. We previously reported that human PCa BM highly express fibroblast growth factor (FGF)-9 (J Clin Invest. 2008;118:2697). We now discovered that human PCa xenografts growing in the bone of mice induce bone cells to express components of the FGF/FGF receptor (FGFR) complex, Fgfr1, Fgfr2 and Fgf-2, as assessed by mouse-specific RT-PCR in bone bearing MDA PCa 118b tumors and in the contralateral bone (P&amp;lt;0.001). Similar results were found in 2 other human PCa xenografts. We then tested antitumor activity of dovitinib (Novartis), a receptor tyrosine kinase inhibitor (TKI) active against FGFR1-3 (IC50 &amp;lt;40 nM) in MDA PCa 118b bone tumors. We found that treated mice had significantly smaller tumors (P=0.016) than the controls had as assessed by magnetic resonance imaging. By human- and mouse-specific RT-PCR analyses we found that 7 days’ treatment reduces FGFR1 expression in tumor-bearing bones (P&amp;lt;0.01). Based on this evidence, a clinical study of dovitinib in men with castrate-resistant PCa and BM was initiated (Identifier: NCT00831792). In this study, serial bone marrow biopsy specimens are collected before and 8 weeks after treatment. To date, 29 of 40 patients have been enrolled. Preliminary results demonstrate tumor regression in BM and soft tissue metastases and progression-free survival of &amp;gt;20 weeks in a subset of patients (∼25%). Dovitinib inhibits vascular endothelial growth factor receptors (VEGFRs) 1-3, with IC50s (∼10 nM) similar to those of FGFR1-3. To assess the relative antitumor activity of FGFR blockade (with respect to VEGFR), we tested a TKI, AZD4547 (AstraZeneca), which inhibits FGFR1-3 (IC50s &amp;lt;3 nM) but not VEGFRs. We found that AZD4547 had antitumor activity against MDA PCa 118b bone tumors and reduces the volume of normal bones and of FGFR1 expression in tumor-associated bone cells (P&amp;lt;0.001). Accordingly, we observed a decrease in the blood levels of bone-specific alkaline phosphatase of men enrolled in the dovitinib trial, and this reduction was associated with the time men remained on treatment (P=0.035) (eg, those who benefited from treatment). No association was found between PSA blood levels or urinary N-telopeptides and response to therapy. Finally, we found that PCa cells and/or osteoblasts express FGFR1 in a fraction of human PCa BM (assessed by immunohistochemistry). We conclude that dovitinib therapy benefits a subset (∼25%) of men with PCa BM and that FGFR blockade contributes to this antitumor effect, at least partially, by blockade of FGFR-mediated PCa-bone cross talk. Ongoing studies focused on further understanding the mechanism of this antitumor activity will help identify likely responders and elucidate FGF signaling interaction with other candidate targets (eg, AR, c-Met, IGF, Src, VEGF). Citation Format: Xinhai Wan, Paul Corn, Jun Yang, Eleni Efstathiou, Elsa M. Li Ning Tapia, Fen Wang, Wallace McKeehan, Christopher Logothetis, Patricia Troncoso, Nora M. Navone. Fibroblast growth factor receptor blockade in prostate cancer bone metastases. [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 2436. doi:10.1158/1538-7445.AM2013-2436