Prostate Cancer Growth In Bone Research Articles

Total flavonoids of Litchi chinensis Sonn. seed inhibit prostate cancer growth in bone by regulating the bone microenvironment via inactivation of the HGFR/NF-κB signaling pathway

Ethnopharmacological relevanceLitchi chinensis Sonn. (Litchi) seed, a traditional Chinese medicine, is habitually used in the clinical treatment of prostate cancer (PCa)-induced bone pain. In our previous study, flavonoids have been identified as the active ingredient of litchi seed against PCa. However, its anti-tumor activities in bone and associated molecular mechanisms are still unclear. Aim of the studyTo investigate the effects and underlying mechanisms of total flavonoids of litchi seed (TFLS) on the growth of PCa in bone. Materials and methodsThe effect of TFLS on the growth of PCa in bone was observed using a mouse model constructed with tibial injection of luciferase-expressing RM1−luc cells. Conditioned medium (CM) from bone marrow stromal cells OP9 and CM treated with TFLS (T-CM) was used to investigate the effect on the proliferation, colony formation, and apoptosis of PCa cells (LNCaP, PC3, RM1). An antibody microarray was performed to detect cytokine expression in the supernatant fraction of OP9 cell cultures treated with TFLS or left untreated. Western blot assay was employed to determine the expression and activity of HGFR and its key downstream proteins, Akt, mTOR, NF-κB, and Erk, in PCa cells. The potential target was further verified using immunofluorescence and immunohistochemistry assays. ResultsTreatment with TFLS (80 mg/kg, 24 days) significantly suppressed the growth of RM1 cells in bone. CM from bone marrow stromal cells OP9 stimulated the proliferation and colony formation of the PCa cells as well as inhibited the apoptosis of PC3 cells, while T-CM reversed the effects mediated by OP9 cells in vitro. In an antibody array assay, TFLS regulated the majority of cytokines in OP9 cell culture supernatant, among which HGF, HGFR, IGF-1R, and PDGF-AA showed the greatest fold changes. Mechanistically, CM upregulated HGFR and promoted phosphorylation of NF-κB while T-CM induced reduction of HGFR and dephosphorylation of NF-κB in PC3 cells. Moreover, T-CM inhibited NF-κB entry into PC3 cell nuclei. Data from in vivo experiments further confirmed the inhibitory effects of TFLS on NF-κB. ConclusionTFLS suppresses the growth of PCa in bone through regulating bone microenvironment and the underlying mechanism potentially involves attenuation of the HGFR/NF-κB signaling axis.

GDF15 promotes prostate cancer bone metastasis and colonization through osteoblastic CCL2 and RANKL activation

Bone metastases occur in patients with advanced-stage prostate cancer (PCa). The cell-cell interaction between PCa and the bone microenvironment forms a vicious cycle that modulates the bone microenvironment, increases bone deformities, and drives tumor growth in the bone. However, the molecular mechanisms of PCa-mediated modulation of the bone microenvironment are complex and remain poorly defined. Here, we evaluated growth differentiation factor-15 (GDF15) function using in vivo preclinical PCa-bone metastasis mouse models and an in vitro bone cell coculture system. Our results suggest that PCa-secreted GDF15 promotes bone metastases and induces bone microarchitectural alterations in a preclinical xenograft model. Mechanistic studies revealed that GDF15 increases osteoblast function and facilitates the growth of PCa in bone by activating osteoclastogenesis through osteoblastic production of CCL2 and RANKL and recruitment of osteomacs. Altogether, our findings demonstrate the critical role of GDF15 in the modulation of the bone microenvironment and subsequent development of PCa bone metastasis.

Prostate cancer-secreted CCN3 uses the GSK3β and β-catenin pathways to enhance osteogenic factor levels in osteoblasts.

Prostate cancer osteoblastic bone metastases are incurable and associated with chronic bone pain and a high mortality rate. Osteoclast-targeting drugs intended to prevent skeletal-related events associated with prostate cancer bone metastases do not prolong overall survival. Improved understanding of the bone-derived factors that contribute to prostate cancer osteoblastic bone metastases is required to design treatments that will improve morbidities and overall survival. Activated osteoblasts stimulate prostate cancer growth in bone. In this study, we report that prostate cancer conditioned medium (CM) promoted bone morphogenetic protein (BMP)-2, -4 and -7 production and the expression of osteogenic transcription factors Runx2 and osterix in osteoblasts. Treating the prostate cancer CM with antibody against CCN3 (nephroblastoma-overexpressed), a cysteine-rich protein that belongs to the CCN family, reduced all of these increases. Incubation of osteoblasts with CCN3 facilitated phosphorylation of GSK3β and β-catenin. GSK3β and β-catenin inhibitors or siRNAs all abolished CCN3-induced promotion of BMPs, Runx2 and osterix expression in osteoblasts. Our results indicate that prostate cancer-secreted CCN3 enhances BMP, Runx2 and osterix expression in osteoblasts via the GSK3β and β-catenin signaling pathways. This understanding of the role played by CCN3 in osteoblastic prostate bone metastasis may lead to more efficient targeted therapies.

Prostate cancer-derived MMP-3 controls intrinsic cell growth and extrinsic angiogenesis

Bone metastatic prostate cancer significantly impacts patient quality of life and overall survival, and despite available therapies, it is presently incurable with an unmet need for improved treatment options. As mediators of tumor progression, matrix metalloproteinases (MMPs) can degrade extracellular matrix components and regulate growth factor and cytokine bioactivity. Depending on tissue context, MMPs can either promote or inhibit tumorigenesis. Therefore, it is essential to study individual MMPs in specific cancer contexts and microenvironments to support the design and application of selective MMP inhibitors. Here we report that tumor-derived MMP-3 contributes to bone metastatic prostate cancer progression via intrinsic and extrinsic routes. MMP-3 ablation in prostate cancer cell lines significantly reduced in vitro growth combined with lowered AKT and ERK phosphorylation and total VEGFR1 and FGFR3 protein levels. In vivo, MMP-3 ablated tumors grew at a slower rate and were significantly less vascularized. Quantitative PCR analyses of wild type and MMP-3 silenced prostate cancer cells also demonstrate downregulation of a wide array of angiogenic factors. The extrinsic role for MMP-3 in angiogenesis was supported by in vitro endothelial tube formation assays where the lack of MMP-3 in prostate cancer conditioned media resulted in slower rates of tube formation. Taken together, our results suggest that tumor-derived MMP-3 contributes to prostate cancer growth in bone. These data indicate that selective inhibition of MMP-3 and/or targeting MMP generated products could be efficacious for the treatment of prostate to bone metastases.

Abstract 3481: Continuous load bearing exercise inhibits the development of prostate cancer bone metastasis

Abstract Background: Prostate cancer (PCa) preferentially metastasizes to bone, causing considerable mortality and morbidity. Exercise is a widely accepted intervention for improving bone health and can benefit overall health in patients with PCa. However, there are no direct studies defining the impact of various exercise regimens on the development of PCa bone metastasis. Hypothesis: Exercise regimens that induce osteogenic responses inhibit the growth of PCa cells in bone. Methods: Human PCa cells (PC3, 1x105 cells per injection) were intracardiacally injected into 8-week-old male BALB/c nude mice which were then subjected to right tibial dynamic mechanical loading (mimicking load bearing exercise, 9N peak force, 40 cycles with 10 second intervals, three times a week) or treadmill exercise (12 meters/minute, 30 minutes/day, 5 days/week) for 3 weeks. Another cohort of mice were firstly subjected to one week of mechanical loading before the inoculation of PC3 cells and then withdrawn from exercise regimen. The growth of skeletal tumours was tracked by bioluminescence for 3 weeks post-injection and bone structure was examined by micro-CT ex vivo. For mechanical loading experiments, comparison was made between loaded and contra-lateral non-loaded tibias, while treadmill exercise group was compared to sedentary controls in the treadmill study. Results: In the continuous mechanical loading group, tumours were detected in non-loaded tibias in all 12 mice (100%), whilst 8 out of 12 mice showed reduced skeletal tumours in the loaded tibias (67%), with 4 having no tumour in the loaded leg (33%). Tumour burden (Flux: photon/second) in the non-loaded tibias was 134% higher than that in the loaded tibias. Micro-CT analysis suggested that loaded tibias had significantly increased trabecular (BV/TV) and cortical bone volume and trabecular thickness (Tb.Th) (p<0.001). Surprisingly, treadmill exercised mice had higher tumour burden (~91%) in the hind limbs compared to the sedentary controls (P<0.05), with no statistically significant improvement in bone mass examined by micro-CT. In the loading withdrawal group, 6 out of 8 mice (75%) showed larger tumour size in the loading withdrawal tibias than in the non-loaded tibias, while only two mice (25%) had smaller or no tumour in the loading withdrawal tibias. Tumor burden were ~90% higher in the loading withdrawal tibias (p<0.05, t-test). This is associated with fast diminishing osteogenic response within a week after ceasing the loading stimulus, confirmed using micro-CT analysis by comparing percentage changes of BV/TV and Tb.Th among baseline, 1-week loading, and loading withdrawn groups. Conclusion: The continuity of high-intensity load bearing exercise is critical to prevent PCa growth in bone, possibly via maintaining the balance of bone remodelling towards osteoblastic bone formation. Mild aerobic exercise evaluated here was unable to significantly improve bone mass or achieve the benefits in inhibiting PCa bone metastases observed with high intensity loading. Citation Format: Ning Wang, Hector M. Arredondo, Alexandria R. Sprules, Colby L. Eaton. Continuous load bearing exercise inhibits the development of prostate cancer bone metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3481.

Abstract 1087: Bone-resident neutrophils are mediators of prostate cancer growth in bone

Abstract Bone metastatic prostate cancer (BM-PCa) significantly reduces overall patient survival and is currently incurable. Current standard immune therapies have shown promising results for patients with less advanced disease (fewer than 20 bone lesions) suggesting that PCa growth in bone contributes to immunotherapeutic response. Gaining a better understanding of the immunogenicity of BM-PCa may present a new therapeutic approach for treating mCRPC. In bone, prostate cancer cells hijack the behavior of bone resident cells, inducing excessive bone degradation and release of bone-sequestered growth factors, such as transforming growth factor beta (TGFβ), that promote a “vicious cycle” of tumor progression. Findings from my lab revealed that: 1) BM-PCa stimulates bone stromal expression of Interleukin 8 (IL-8), a potent chemoattractant for polymorphonuclear neutrophils (PMNs), innate immune cells generated in bone marrow, and 2) PMNs heavily infiltrate the prostate tumor-bone microenvironment. Recent studies demonstrated the existence of two distinct PMN populations: anti-tumoral (N1) and immunosuppressive pro-tumoral (N2) PMNs, with emergence of the latter regulated by TGFβ. RealTime qPCR revealed that bone metastatic C42B prostate cancer significantly increases neutrophil expression of TGFβ receptor and MMP9, a trend previously identified as a marker of pro-tumoral neutrophils. Based upon these findings, we hypothesized that bone metastatic prostate cancer skews neutrophils to a pro-tumoral phenotype in a TGFβ-dependent manner. To test this, bone marrow-derived primary neutrophils were treated with conditioned media from metastatic metastatic prostate cancer cells (C42B) and poorly metastatic LNCaP and gene expression and oxidative burst were measured. Similar to previous findings, C42B stimulated reactive oxygen species (ROS) production by PMNs, a mechanism of immunosuppression utilized by N2 PMNs, and induced anti-inflammatory gene expression. In contrast, LNCaP cells failed to stimulate this response. C42B media promoted PMN expansion, a phenomenon that is reversed by TGFβ-blockade. Surprisingly, direct contact with neutrophils significantly increased luminescence of luciferase-expressing C42B cells independently of cell growth, when compared to LNCaP, demonstrating that neutrophils activate alternate protein signaling in metastatic cells compared to non-metastatic. In vivo intratibial models utilizing antibody-mediated neutrophil depletion validated our hypothesis that neutrophils are critical mediators of prostate cancer growth in bone. These results demonstrate the significant role of neutrophils in the progression of bone metastatic prostate cancer. Citation Format: Leah M. Cook, Diane Costanzo-Garvey, Tyler Keeley. Bone-resident neutrophils are mediators of prostate cancer growth in bone [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 1087.

Abstract B050: The role of activated beta-catenin/Wnt signaling in the progression of castration resistance of prostate cancer in bone

Abstract Bone metastases typically develop in patients with advanced prostate cancer (PCa). These metastases are osteoblastic (bone-forming) and constitute the main cause of morbidity and mortality. Androgen deprivation is commonly used to treat bone metastases of PCa, but responses to such therapy are short, and eventually the disease progresses to a castration-resistant form. Bone is the primary site of castration-resistant PCa (CRPC) progression. Further development of therapies for bone metastases of PCa requires an understanding of the mechanisms underlying the growth of CRPC in bone. Clinical and laboratory-based investigations have implicated a role for PCa cell-bone cell interaction in the growth of PCa and have demonstrated that osteoblasts produce factors that favor the growth and survival of PCa cells. Collectively, these observations strongly suggest that PCa cells interact with osteoblasts and that this interaction contributes to the growth of PCa in bone. Recently, a study showed 18% of patients with metastatic CRPC harbored alterations in the Wnt canonical pathway, including hotspot mutations of β-catenin (β-cat), the molecular node of the Wnt canonical pathway, that leads to pathway activation. This report is in agreement with previous publications of β-cat nuclear accumulation, an indirect measure of Wnt-canonical pathway activation, in 20 to 40% of advanced PCas. We previously reported that a patient-derived xenograft (PDX) MDA-PCa-118b (118b) developed in our lab harbors a β-cat mutation (D32G) and has an activated Wnt canonical pathway. 118b induces an osteoblastic reaction when it is implanted in the bone of mice in vivo. In vitro, 118b induces osteoblast proliferation in coculture. In contrast, the PCa cell line, PC3, induces osteolytic lesions in vivo and in vitro inhibits osteoblast proliferation in coculture. Strikingly, expression of β-cat mutated at D32G in PC3 cells reverses the osteoblast inhibitory effect of PC3 cells, resulting in increased proliferation of osteoblasts when grown in coculture. We therefore hypothesize that β-cat/Wnt signaling in PCa cells mediates the PCa-bone interaction that favors PCa growth in bone. Our future studies are focusing on studying the importance of β-cat mutations recently identified in patients with metastatic CRPC and determining whether these mutations also result in an osteoblastic phenotype, which in turn supports growth of prostate cancer in bone. Additionally, we are screening our collection of PCa PDX lines for increased β-cat expression through immunohistochemistry and will sequence DNA from PDX lines that demonstrate increased nuclear β-cat expression to test for activating mutations in β-cat. Our aim is to gain insight into the mechanism of the paracrine interaction between PCa cells and bone that supports the growth of PCa bone lesions, with the ultimate goal of developing therapies to block these interactions and reducing the morbidity and mortality associated with metastatic disease. Citation Format: Justin M. Roberts, Estefania Labanca, Peter Shepherd, Jun Yang, Michael W. Starbuck, Nora M. Navone. The role of activated beta-catenin/Wnt signaling in the progression of castration resistance of prostate cancer in bone [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 B050.

Abstract B076: Osteoclasts influence androgen-related gene expression in prostate cancer cells

Abstract Background: The majority of progressive prostate cancer will develop metastases in the skeleton. In bone environment, the regulated destruction of bone matrix by osteoclasts is closely connected to the formation of new bone by osteoblasts. Although prostate cancer often forms sclerotic bone lesions, evidence from recent research show that prostate cancer cells also produce factors to promote osteoclastic action. However, little is known on how osteoclasts regulate the prostate cancer growth in bone. The aim of the present study was to investigate the role of osteoclasts in the crosstalk of bone microenvironment and prostate cancer cells. Methods: The interactions between prostate cancer cells and osteoclasts were studied in a coculture model using the transwell permeable supports system. Two castration-resistant prostate cancer cell lines (LNCaP-19 and PC3) were used together with osteoclasts that were transformed from the RAW 264.7 cells by stimulation with RANKL. After 48 hours coculturing, analysis on gene expression level was carried out with qRT-PCR. Results: The results show that osteoclasst directly influence gene expression patterns in prostate cancer cells, affecting genes generally involved in tumor progression, mainly in the osteolytic cell line PC3. In LNCaP-19 cells, which are sensitive to androgens, osteoclast stimulation upregulated expression of androgen receptor (AR), as well as the AR splice-variant ARV7. In contrast, in androgen-insensitive PC3 cells no such changes in AR and AR-V7 mRNA were detected. Instead, the expression of the steroidogenic enzymes CYP11A1 (converting cholesterol into pregnenolone) and CYP19A1 (converting testosterone into estrogen) were increased by osteoclast stimulation. In addition, osteoclast stimulation differentially affects expression of genes related to bone formation in the tumor cells. LNCaP-19, which gives osteoblastic lesions in mouse tibia, responds with increased expression of genes related to TGF-b signaling, osteogenesis, and bone matrix formation. In contrast, the osteolytic cell line PC3 downregulates expression of osteogenic genes in response to osteoclast stimulation. Conclusion: This study shows that there is a direct crosstalk between osteoclasts and prostate cancer cells, where the osteoclasts influence gene expression related to androgen signaling, steroidogenesis, and osteogenesis in different ways based on the androgen and bone-related characteristics of the prostate cancer cells. Since upregulated AR and induced ARV7 expression have been shown to assist the progression of castration-resistant prostate cancer cells, inhibiting this crosstalk by targeting osteoclasts may improve efficacy of androgen-targeting drugs. Citation Format: Junchi Huang, Jan-Erik Damber, Karin Welen. Osteoclasts influence androgen-related gene expression in prostate cancer cells [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 B076.

Dual targeting c-met and VEGFR2 in osteoblasts suppresses growth and osteolysis of prostate cancer bone metastasis

Prostate cancer characteristically induces osteoblastic bone metastasis, for which no therapies are available. A dual kinase inhibitor of c-Met and VEGFR-2 (cabozantinib) was shown to reduce prostate cancer growth in bone, with evidence for suppressing osteoblastic activity. However, c-Met and VEGFR2 signaling in osteoblasts in the context of bone metastasis remain unclear. Here we show using cultured osteoblasts that hepatocyte growth factor (HGF) and VEGF-A increased receptor activator of NFκB ligand (RANKL) and M-CSF, two essential factors for osteoclastogenesis. Insulin-like growth factor-1 (IGF1) also increased RANKL and M-CSF via c-Met transactivation. The conditioned media from IGF1-, HGF-, or VEGFA-treated osteoblasts promoted osteoclastogenesis that was reversed by inhibiting c-Met and/or VEGFR2 in osteoblasts. In vivo experiments used cabozantinib-resistant prostate cancer cells (PC-3 and C4-2B) to test the effects of c-Met/VEGFR2 inhibition specifically in osteoblasts. Cabozantinib (60 mg/kg, 3 weeks) suppressed tumor growth in bone and reduced expression of RANKL and M-CSF and subsequent tumor-induced osteolysis. Collectively, inhibition of c-Met and VEGFR2 in osteoblasts reduced RANKL and M-CSF expression, and associated with reduction of tumor-induced osteolysis, suggesting that c-Met and VEGFR2 are promising therapeutic targets in bone metastasis.

Radium-223 Inhibits Osseous Prostate Cancer Growth by Dual Targeting of Cancer Cells and Bone Microenvironment in Mouse Models.

Purpose: Radium-223 dichloride (radium-223, Xofigo), a targeted alpha therapy, is currently used for the treatment of patients with castration-resistant prostate cancer (CRPC) with bone metastases. This study examines the mode-of-action and antitumor efficacy of radium-223 in two prostate cancer xenograft models.Experimental Design: Mice bearing intratibial LNCaP or LuCaP 58 tumors were randomized into groups (n = 12-17) based on lesion grade and/or serum PSA level and administered radium-223 (300 kBq/kg) or vehicle, twice at 4-week intervals. X-rays and serum samples were obtained biweekly. Soft tissue tumors were observed macroscopically at sacrifice. Tibiae were analyzed by gamma counter, micro-CT, autoradiography and histology.Results: Radium-223 inhibited tumor-induced osteoblastic bone growth and protected normal bone architecture, leading to reduced bone volume in LNCaP and abiraterone-resistant LuCaP 58 models. Furthermore, radium-223 resulted in lower PSA values and reduced total tissue and tumor areas, indicating that treatment constrains prostate cancer growth in bone. In addition, radium-223 suppressed abnormal bone metabolic activity as evidenced by decreased number of osteoblasts and osteoclasts and reduced level of the bone formation marker PINP. Mode-of-action studies revealed that radium-223 was deposited in the intratumoral bone matrix. DNA double-strand breaks were induced in cancer cells within 24 hours after radium-223 treatment, and PSA levels were significantly lower 72 hours after treatment, providing further evidence of the antitumor effects.Conclusions: Taken together, radium-223 therapy exhibits a dual targeting mode-of-action that induces tumor cell death and suppresses tumor-induced pathologic bone formation in tumor microenvironment of osseous CRPC growth in mice. Clin Cancer Res; 23(15); 4335-46. ©2017 AACR.

Inhibition of the insulin-like growth factor-1 receptor potentiates acute effects of castration in a rat model for prostate cancer growth in bone

Prostate cancer (PCa) patients with bone metastases are primarily treated with androgen deprivation therapy (ADT). Less pronounced ADT effects are seen in metastases than in primary tumors. To test if acute effects of ADT was enhanced by concurrent inhibition of pro-survival insulin-like growth factor 1 (IGF-1), rats were inoculated with Dunning R3327-G tumor cells into the tibial bone marrow cavity and established tumors were treated with castration in combination with IGF-1 receptor (IGF-1R) inhibitor NVP-AEW541, or by each treatment alone. Dunning R3327-G cells were stimulated by androgens and IGF-1 in vitro. In rat tibia, Dunning R3327-G cells induced bone remodeling, identified through increased immunoreactivity of osteoblast and osteoclast markers. Tumor cells occasionally grew outside the tibia, and proliferation and apoptotic rates a few days after treatment were evaluated by scoring BrdU- and caspase-3-positive tumor cells inside and outside the bone marrow cavity, separately. Apoptosis was significantly induced outside, but unaffected inside, the tibial bone by either castration or NVP-AEW541, and the maximum increase (2.7-fold) was obtained by the combined treatment. Proliferation was significantly reduced by NVP-AEW541, independently of growth site, although the maximum decrease (24%) was observed when NVP-AEW541 was combined with castration. Tumor cell IGF-1R immunoreactivity was evaluated in clinical PCa bone metastases (n = 61), and positive staining was observed in most cases (74%). In conclusion, IGF-1R inhibition may be evaluated in combination with ADT in patients with metastatic PCa, or in combination with therapies for the subsequent development of castration-resistant disease, although diverse responses could be anticipated depending on metastasis site.

Tasquinimod inhibits prostate cancer growth in bone through alterations in the bone microenvironment.

Tasquinimod (ABR-215050) is an orally active quinoline-3-carboxamide analog that inhibits occurrence of experimental metastasis and delays disease progression of castration resistant prostate cancer in humans. Its mechanism of action is not fully elucidated, but previous studies show immunomodulatory and anti-angiogenic effects. The aim of the present study was to investigate the tumor inhibiting effect of tasquinimod in bone of castrated mice as well as to elucidate its working mechanism related to bone microenvironment. Effects of tasquinimod on prostate cancer metastasis to bone was studied in an intratibial xenograft model. Animals were treated with tasquinimod and tumor establishment and growth, immunological status, as well as markers for bone remodeling were analyzed. Direct effects of tasquinimod on osteoblasts were studied in vitro. Establishment and growth of tumors in the bone after intratibial implantation in castrated mice was suppressed by tasquinimod treatment. The treatment effect was linked to decreased potential for immunosuppression in the pre-metastatic niche in bone (lower levels of CD206 and Arg1 expression in combination with increased iNOS expression) as well as in the tumor microenvironment (less Gr1 and CD206 staining). The shift to a pro-inflammatory, anti-tumorigenic milieu was also reflected in serum by increased levels of IFN-γ, CCL4, IL-5, LIX, IP-10, and MCP-1 as well as decreased TGF-β. Tasquinimod treatment also affected expression of factors involved in the pre-metastatic niche in the bone microenvironment (Lox, Cdh2, Cdh11, and Cxcl12). In addition, tasquinimod treatment caused a decreased osteogenic response indicated by decreased expression of Ocn, Runx2, and Col1a2 and increased expression of osteoclast stimulating CSF2. In vitro studies on mouse osteoblasts showed impaired osteoblast mineralization upon tasquinimod treatment. The present study shows that tasquinimod reduces establishment and progression of tumor growth in bone likely through a combination of effects on the pre-metastatic niche, homing, immunological status, and osteogenesis. It was concluded that tasquinimod interferes with the metastatic process, presumably by inhibition of tumor establishment. Hence, our data suggest that tasquinimod might be most effective in inhibiting the occurrence of new metastatic lesions.

Bone marrow macrophages support prostate cancer growth in bone

Resident macrophages in bone play important roles in bone remodeling, repair, and hematopoietic stem cell maintenance, yet their role in skeletal metastasis remains under investigated. The purpose of this study was to determine the role of macrophages in prostate cancer skeletal metastasis, using two in vivo mouse models of conditional macrophage depletion. RM-1 syngeneic tumor growth was analyzed in an inducible macrophage (CSF-1 receptor positive cells) ablation model (MAFIA mice). There was a significant reduction in tumor growth in the tibiae of macrophage-ablated mice, compared with control non-ablated mice. Similar results were observed when macrophage ablation was performed using liposome-encapsulated clodronate and human PC-3 prostate cancer cells where tumor-bearing long bones had increased numbers of tumor associated-macrophages. Although tumors were consistently smaller in macrophage-depleted mice, paradoxical results of macrophage depletion on bone were observed. Histomorphometric and micro-CT analyses demonstrated that clodronate-treated mice had increased bone volume, while MAFIA mice had reduced bone volume. These results suggest that the effect of macrophage depletion on tumor growth was independent of its effect on bone responses and that macrophages in bone may be more important to tumor growth than the bone itself. In conclusion, resident macrophages play a pivotal role in prostate cancer growth in bone.

Abstract 1172: Targeting c-Met and VEGFR2 in the stromal compartment of prostate cancer bone metastasis

Abstract Advanced-stage prostate cancer patients commonly develop bone metastasis, accounting for significant morbidity and mortality. Metastatic prostate cancer cells characteristically induce osteoblastic reactions, for which no targeted therapies are currently available. Recent clinical and experimental data showed that dual inhibition of c-Met and VEGF receptor (VEGFR)-2 kinase activity reduced prostate cancer growth in bone with indications for suppressing osteoblastic activities, while the specific functions of c-Met and VEGFR2 in osteoblasts remain unclear. In our in vitro studies using three types of osteoblasts (MC3T3-E1 subclone 4, hFOB1.19 and murine calvarial osteoblasts), hepatocyte growth factor (HGF, a ligand for c-Met) and VEGFA increased expression of receptor activator of nuclear factor κB ligand (RANKL) and macrophage colony stimulating factor (M-CSF), two essential factors for osteoclastogenesis. In addition, siRNA-mediated knockdown of c-Met or VEGFR2 in osteoblasts suppressed HGF- or VEGFA-dependent gene expression, respectively. We demonstrate that insulin growth factor (IGF) increased RANKL and M-CSF expression in osteoblasts via c-Met transactivation. Furthermore, the conditioned media from IGF-, HGF-, or VEGFA-treated osteoblasts promoted in vitro osteoclastogenesis that was suppressed by inhibition of c-Met and VEGFR2 in osteoblasts. Subsequently, we examined whether cabozantinib, a dual kinase inhibitor of c-Met and VEGFR2 currently in clinical trials for metastatic prostate cancer, could suppress prostate cancer bone metastasis via inhibition of osteoblasts. To examine the effects of cabozantinib specifically in osteoblasts, cabozantinib-resistant PC-3 prostate cancer cells were generated and implanted in the tibiae of male nude mice. Cabozantinib (60mg/kg) or control diluent was administered via daily oral gavage for 3 weeks. In agreement with our in vitro data, cabozantinib suppressed tumor growth in bone (determined by in vivo bioluminescence) and tumor-induced osteolysis (determined by X-ray). Tumor cells were isolated and in vitro cultured to demonstrate that cabozantinib resistance was maintained during 3-week in vivo growth. Osteoblasts and osteoclasts numbers were reduced in the bones of cabozantinib-treated mice, and these histological changes were accompanied by significantly lower levels of RANKL and M-CSF levels in bone. Collectively, inhibition of c-Met and VEGFR2 in osteoblasts reduces RANKL and M-CSF expression, associated with decreased osteoclastogenesis and tumor-induced osteolysis. Therefore, we conclude that c-Met and VEGFR2 are promising therapeutic targets in the stromal compartment of prostate cancer bone metastasis, suggesting hat the effects of cabozantinib on skeletal-related events of prostate cancer are, at least in part, mediated by suppression of osteoblast function. Citation Format: Changki Lee, Preston Campbell, Young Mi Whang, Jamey D. Young, Florent Elefteriou, Serk In Park. Targeting c-Met and VEGFR2 in the stromal compartment of prostate cancer bone metastasis. [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 1172. doi:10.1158/1538-7445.AM2014-1172

Abstract 3198: Extract from Scutellaria baicalensis Georgi inhibits prostate cancer growth in bone

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

Targeting IL-6 and RANKL signaling inhibits prostate cancer growth in bone

In prostate cancer metastases to bone, cancer cell-derived cytokines stimulate RANKL expression by cells of the osteoblast lineage, which in turn activates osteoclastic bone resorption. However, it is unclear whether cells of the osteoblast lineage signal back to prostate cancer cells, and if so, whether such direct cross-talk can be targeted therapeutically. Using the human prostate cancer cell line, PC3, we identified two novel signalling pathways acting between cells of the osteoblast lineage and cancer cells. First, exposure to RANKL stimulated the expression and release of IL-6 by PC3 cells in vitro (which is known to promote RANKL expression by osteoblasts). Second, treatment of PC3 cells with IL-6 increased the expression of RANK, the cognate receptor of RANKL, and enhanced the RANKL-induced release of IL-6 by PC3 cells. Third, targeted disruption of IL-6 signaling with tocilizumab, a clinically available antibody against the human IL-6 receptor, inhibited skeletal tumor growth in vivo and reduced serum RANKL levels as well as RANK expression by PC3-derived bone tumors. Similar effects were achieved when RANK expression was knocked down in PC3 cells. In contrast, disruption of IL-6 or RANK/RANKL signalling had no effect on PC3 tumor growth in soft tissues, indicating that these signalling pathways act specifically within the bone microenvironment. In conclusion, prostate cancer cells and cells of the osteoblast lineage communicate via two inter-dependent signaling pathways, which through auto-amplification strongly enhance metastatic prostate cancer growth in bone. Both pathways may be targeted for effective therapeutic intervention.

Cabozantinib Inhibits Growth of Androgen-Sensitive and Castration-Resistant Prostate Cancer and Affects Bone Remodeling

Cabozantinib is an inhibitor of multiple receptor tyrosine kinases, including MET and VEGFR2. In a phase II clinical trial in advanced prostate cancer (PCa), cabozantinib treatment improved bone scans in 68% of evaluable patients. Our studies aimed to determine the expression of cabozantinib targets during PCa progression and to evaluate its efficacy in hormone-sensitive and castration-resistant PCa in preclinical models while delineating its effects on tumor and bone. Using immunohistochemistry and tissue microarrays containing normal prostate, primary PCa, and soft tissue and bone metastases, our data show that levels of MET, P-MET, and VEGFR2 are increasing during PCa progression. Our data also show that the expression of cabozantinib targets are particularly pronounced in bone metastases. To evaluate cabozantinib efficacy on PCa growth in the bone environment and in soft tissues we used androgen-sensitive LuCaP 23.1 and castration-resistant C4-2B PCa tumors. In vivo, cabozantinib inhibited the growth of PCa in bone as well as growth of subcutaneous tumors. Furthermore, cabozantinib treatment attenuated the bone response to the tumor and resulted in increased normal bone volume. In summary, the expression pattern of cabozantinib targets in primary and castration-resistant metastatic PCa, and its efficacy in two different models of PCa suggest that this agent has a strong potential for the effective treatment of PCa at different stages of the disease.

Inhibition of CCL2 Signaling in Combination with Docetaxel Treatment Has Profound Inhibitory Effects on Prostate Cancer Growth in Bone

The C-C chemokine ligand 2 (CCL2) stimulates migration, proliferation, and invasion of prostate cancer (PCa) cells, and its signaling also plays a role in the activation of osteoclasts. Therefore targeting CCL2 signaling in regulation of tumor progression in bone metastases is an area of intense research. The objective of our study was to investigate the efficacy of CCL2 blockade by neutralizing antibodies to inhibit the growth of PCa in bone. We used a preclinical model of cancer growth in the bone in which PCa C4-2B cells were injected directly into murine tibiae. Animals were treated for ten weeks with neutralizing anti-CCL2 antibodies, docetaxel, or a combination of both, and then followed an additional nine weeks. CCL2 blockade inhibited the growth of PCa in bone, with even more pronounced inhibition in combination with docetaxel. CCL2 blockade also resulted in increases in bone mineral density. Furthermore, our results showed that the tumor inhibition lasted even after discontinuation of the treatment. Our data provide compelling evidence that CCL2 blockade slows PCa growth in bone, both alone and in combination with docetaxel. These results support the continued investigations of CCL2 blockade as a treatment for advanced metastatic PCa.

Effect of transforming growth factor beta (TGF-β) receptor I kinase inhibitor on prostate cancer bone growth

Transforming growth factor beta 1 (TGF-β1) has been implicated in the pathogenesis of prostate cancer (PCa) bone metastasis. In this study, we tested the antitumor efficacy of a selective TGF-β receptor I kinase inhibitor, LY2109761, in preclinical models. The effect of LY2109761 on the growth of MDA PCa 2b and PC-3 human PCa cells and primary mouse osteoblasts (PMOs) was assessed in vitro by measuring radiolabeled thymidine incorporation into DNA. In vivo, the right femurs of male SCID mice were injected with PCa cells. We monitored the tumor burden in control- and LY2109761-treated mice with MRI analysis and the PCa-induced bone response with X-ray and micro-CT analyses. Histologic changes in bone were studied by performing bone histomorphometric evaluations. PCa cells and PMOs expressed TGF-β receptor I. TGF-β1 induced pathway activation (as assessed by induced expression of p-Smad2) and inhibited cell growth in PC-3 cells and PMOs but not in MDA PCa 2b cells. LY2109761 had no effect on PCa cells but induced PMO proliferation in vitro. As expected, LY2109761 reversed the TGF-β1-induced pathway activation and growth inhibition in PC-3 cells and PMOs. In vivo, LY2109761 treatment for 6weeks resulted in increased volume in normal bone and increased osteoblast and osteoclast parameters. In addition, LY2109761 treatment significantly inhibited the growth of MDA PCa 2b and PC-3 in the bone of SCID mice (p<0.05); moreover, it resulted in significantly less bone loss and change in osteoclast-associated parameters in the PC-3 tumor-bearing bones than in the untreated mice. In summary, we report for the first time that targeting TGF-β receptors with LY2109761 can control PCa bone growth while increasing the mass of normal bone. This increased bone mass in nontumorous bone may be a desirable side effect of LY2109761 treatment for men with osteopenia or osteoporosis secondary to androgen-ablation therapy, reinforcing the benefit of effectively controlling PCa growth in bone. Thus, targeting TGF-β receptor I is a valuable intervention in men with advanced PCa.

Abstract PL06-04: Targeting the tumor ecosystem: Translating the molecular biology of chemokine interactions to the clinic.

Abstract There has been an increasing recognition that the tumor microenvironment contains host non-cancer cells in addition to cancer cells, interacting in a dynamic fashion over time. The cancer cells compete and/or cooperate with non-tumor cells, and the cancer cells may compete and/or cooperate with each other. It has been demonstrated that these interactions can alter the genotype and phenotype of the host cells as well as the cancer cells. The interaction of these cancer and host cells to remodel the normal host organ microenvironment may best be conceptualized as an evolving ecosystem. Describing tumors as ecological systems defines new opportunities for novel cancer therapies (“ecological therapy”). The ecology literature is filled with stories of how changing the ecosystem resulted in the death of a species. A key method, therefore, to kill cancer cells in the tumor ecosystem may be to inhbit other cell species within the environment that are supporting the growth and survival of cancer cells. Chemokines are a family of small and secreted proteins that play pleiotropic roles in inflammation-related diseases, including cancer. Among the identified 50 human chemokines, chemokine (C-C motif) ligand 2 (CCL2) is of particular importance in cancer development since it serves as one of the key mediators of interactions between tumor and host cells. CCL2 is produced by cancer cells and multiple different host cells within the tumor microenvironment. In the bone marrow microenvironment, for example, it is produced by osteoblasts, osteoclasts, and endothelial cells. CCL2 mediates tumorigenesis in many different cancer types and has been reported to promote cancer cell proliferation, migration, invasion, and survival, via binding to its functional receptor CCR2. Most importantly, however, CCL2 induces the recruitment of monocytes to the tumor and their subsequent differentiation into tumor associated macrophages (TAMS). Upon CCL2 stimulation, CD11b(+) cells demonstrated a significant increase in the mannose receptor (CD206) and the CD14(+)/CD206(+) double-positive cells, suggesting a polarization of macrophages toward the CD206(+) M2-type phenotype. TAMs subsequently are major mediators of angiogenesis and matrix remodeling. CCL2 protects macrophages from autophagic death by activating survivin through a PI3K/AKT (phosphatidylinositol 3-kinase/protein kinase B)-dependent mechanism, increasing their ability to survive in the hypoxic tumor microenvironment. CCL2 induces up-regulation of the antiapoptotic proteins cFLIP(L) (cellular caspase-8 (FLICE)-like inhibitory protein), Bcl-2, and Bcl-X(L) and inhibits the cleavage of caspase-8 and subsequent activation of the caspase-cascade, thus protecting cells from apoptosis under serum deprivation stress. Multiple studies have revealed that inhibition of CCL2 substantially decreases macrophage infiltration, decreases osteoclast function, and inhibits prostate cancer growth in bone in preclinical animal models. In one study, for example, utilizing neutralizing antibodies to human CCL2 (CNTO888) and the mouse homologue CCL2/JE (C1142), treatment with anti-CCL2/JE antibody (2 mg/kg, twice weekly i.p.) attenuated prostate cancer cell PC-3Luc-mediated overall tumor burden in an in vivo model of prostate cancer metastasis by 96% at 5 weeks post intracardiac injection. These preclinical data have been successfully translated to the clinic. SWOG study S0916, for example, was a phase II, window trial of an anti-CCR2 antibody in patients with bone metastases. This biomarker driven study was designed to test the hypothesis that inhibiting the CCL-2-CCR2 axis in patients with cancer metastatic to bone osteoclast function would be inhibited as measured by a decrease in urinary NTX by 25% or more from baseline. Patients were given three doses of antibody two weeks apart and urinary NTX was measured at baseline, two weeks, 4 weeks, and 6 weeks. Preliminary results suggest that administration of anti-CCR2 antibody resulted in decreases in urinary NTX in a significant number of patients. These data suggest that altering the tumor microenvironment through inhibition of CCL2 may be a viable strategy for interfering with cancer cell activity and growth. This example of translation from bench to bedside demonstrating how an understanding chemokine effects on the tumor ecosystem can serve as a paradigm for other cytokines, chemokines, and cell interactions within the tumor microenvironment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr PL06-04.