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.

Construction of tellurium-doped mesoporous bioactive glass nanoparticles for bone cancer therapy by promoting ROS-mediated apoptosis and antibacterial activity

The commonly used treatment methods for bone cancer include chemotherapy, surgery and radiotherapy, but there are disadvantages such as nonspecific distribution, high toxicity of chemotherapy drugs, implantable infections and low monitoring. Nanoparticles are the new development direction of nanomedicine in cancer treatment. Structural characteristics of nanoparticles make it an excellent model for targeting and penetrating cancer-induced abnormal cell growth. In this study, a kind of novel and interesting tellurium ion doped mesoporous bioactive glasses (Te-MBG) nanoparticles were successfully synthesized by a simple sol–gel method, which had uniform spherical morphology (≈ 500 nm), high surface area (> 300 m2/g) and mesopore volume (> 0.30 cm3/g). Results found that Te doping does not affect the mineralization and degradation of the MBG nanoparticles. Meanwhile, compared to the undoped MBG, Te doped MBG not merely had the ability to promote MG63 cell apoptosis to inhibit bone cancer growth by ROS-mediated, but also had significant antibacterial activity. This all depends on the concentration of Te doping. It can be seen that Te-MBG nanoparticles can not only potentially fill bone defects caused by bone cancer removal, but also induce cancer cell apoptosis by tellurium release inducing reactive oxygen species (ROS) excessive production to inhibit bone cancer formation. This study provides a feasible strategy for the development of Te-MBG nanoparticles as well as their evaluation and basic research for bone cancer therapy.

Inflammatory conversion of quiescent osteoblasts by metastatic breast cancer cells through pERK1/2 aggravates cancer-induced bone destruction

Disruption of bone homeostasis caused by metastatic osteolytic breast cancer cells increases inflammatory osteolysis and decreases bone formation, thereby predisposing patients to pathological fracture and cancer growth. Alteration of osteoblast function induces skeletal diseases due to the disruption of bone homeostasis. We observed increased activation of pERK1/2 in osteolytic breast cancer cells and osteoblasts in human pathological specimens with aggressive osteolytic breast cancer metastases. We confirmed that osteolytic breast cancers with high expression of pERK1/2 disrupt bone homeostasis via osteoblastic ERK1/2 activation at the bone-breast cancer interface. The process of inflammatory osteolysis modulates ERK1/2 activation in osteoblasts and breast cancer cells through dominant-negative MEK1 expression and constitutively active MEK1 expression to promote cancer growth within bone. Trametinib, an FDA-approved MEK inhibitor, not only reduced breast cancer-induced bone destruction but also dramatically reduced cancer growth in bone by inhibiting the inflammatory skeletal microenvironment. Taken together, these findings suggest that ERK1/2 activation in both breast cancer cells and osteoblasts is required for osteolytic breast cancer-induced inflammatory osteolysis and that ERK1/2 pathway inhibitors may represent a promising adjuvant therapy for patients with aggressive osteolytic breast cancers by altering the shared cancer and bone microenvironment.

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.

Does vitamin D deficiency predict tumour malignancy in patients with bone tumours? Data from a multi-center cohort analysis

Vitamin D deficiency is a global health concern that is estimated to afflict over one billion people globally. The major role of vitamin D is that of a regulator of calcium and phosphate metabolism, thus, being essential for proper bone mineralisation. Concomitantly, vitamin D is known to exert numerous extra-skeletal actions. For example, it has become evident that vitamin D has direct anti-proliferative, pro-differentiation and pro-apoptotic actions on cancer cells. Hence, vitamin D deficiency has been associated with increased cancer risk and worse prognosis in several malignancies. We have recently demonstrated that vitamin D deficiency promotes secondary cancer growth in bone. These findings were partly attributable to an increase in bone remodelling but also through direct effects of vitamin D on cancer cells. To date, very little is known about vitamin D status of patients with bone tumours in general. Thus, the objective of this study was to assess vitamin D status of patients with diverse bone tumours. Moreover, the aim was to elucidate whether or not there is an association between pre-diagnostic vitamin D status and tumour malignancy in patients with bone tumours.In a multi-center analysis, 25(OH)D, PTH and calcium levels of 225 patients that presented with various bone tumours between 2017 and 2018 were assessed. Collectively, 76% of all patients had insufficient vitamin D levels with a total mean 25(OH)D level of 21.43 ng/ml (53.58 nmol/L). In particular, 52% (117/225) of patients were identified as vitamin D deficient and further 24% of patients (55/225) were vitamin D insufficient. Notably, patients diagnosed with malignant bone tumours had significantly lower 25(OH)D levels than patients diagnosed with benign bone tumours [19.3 vs. 22.75 ng/ml (48.25 vs. 56.86 nmol/L); p = 0.04).In conclusion, we found a widespread and distressing rate of vitamin D deficiency and insufficiency in patients with bone tumours. However, especially for patients with bone tumours sufficient vitamin D levels seem to be of great importance. Thus, we believe that 25(OH)D status should routinely be monitored in these patients. Collectively, there should be an increased awareness for physicians to assess and if necessary correct vitamin D status of patients with bone tumours in general or of those at great risk of developing bone tumours.

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 693: Effects of a bone-anabolic agent on metastatic bone cancer growth

Abstract Introduction: Cancer cells colonized to bone pathologically alter the bone niche to support the cancers' growth and survival. Multiple myeloma (MM) and metastatic solid tumors, particularly breast cancer (BC), activate osteoclasts and suppress osteoblasts leading to many complications and an increased mortality risk. Current strategies primarily target osteoclasts. Known osteoclast activating factor PTHrP is expressed by MM cells and BC cells and may contribute to progression. We hypothesized that alteration of PTH receptor signaling should overcome cancers' inhibition of bone formation and decrease cancer growth in bone. BDDP, a novel PTH analog, is being developed for osteoporosis treatment. We determined the ability of BDDP to reverse osteoblast suppression and slow tumor growth in bone with MM and BC models. Methods: In vitro effects of BDDP on bone genes and bone mineralization were tested in a co-culture assay of osteoblastic MC3T3-E1 cells ± human MM.1s cells. After 3 days in proliferative media, they were cultured in differentiation media. At Day 7, the expression of genes with known roles in osteoblast differentiation were assayed by RT-PCR. At Day 25, nodular calcification was stained and measured by a colorimetric assay. In vivo effects of BDDP were evaluated in an intratibial transplant model. MDA-MB-231 BC cells expressing secreted luciferase were injected into the ipsilateral tibiae of Balb/cnu/nu mice. Mice received either BDDP (40μg/kg/d) or vehicle via osmotic pump for 30 days. After euthanasia, mice were subjected to Faxitron digital X-rays to detect osteolytic lesions and DEXA scans to assess bone mineral content (BMC) and bone mineral density (BMD). Tumor-injected and non-injected tibiae were subjected to micro-QCT for 3D tibiae imaging. Results: MM.1s-treated MC3T3-E1 cells showed approximately a 5-fold reduction in osteoblast differentiation gene RUNX2 and a 3-fold reduction in BGLAP. This effect was attenuated by 100 nM BDDP by approximately 4-fold in both Runx2 and BGLAP. BDDP increased nodular calcification. Nodular calcification was suppressed in MM.1s-treated MC3T3-E1 cells. This effect was reversed by BDDP. BDDP did not directly affect MM.1s and MDA-MB-231 growths in culture but showed dramatic tumor suppression in a MDA-MB-231 intratibial transplant model. Tumor burden assayed by serial serum luciferase assay showed BDDP-treated group to be 95% lower than control, at 3 and 4 weeks post-transplant. Tumor-injected tibiae had lytic lesions in all control mice, while there were no obvious lesions in BDDP-treated mice. There were no significant changes in the trabecular bone of non-injected tibiae, whole body composition, BMC or BMD. Conclusion: BDDP reverses osteoblastic suppression by MM cells in vitro, as well as inhibiting tumor growth and preventing local bone destruction in the BC intratibial transplant model, thereby making BDDP a promising potential treatment strategy for metastatic bone cancers. Citation Format: Teja Vallapuri, Colin Crean, John Chirgwin, G. David Roodman, Attaya Suvannasankha. Effects of a bone-anabolic agent on metastatic bone cancer growth [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 693.

Prostate cancer cell‐intrinsic interferon signaling regulates dormancy and metastatic outgrowth in bone

The latency associated with bone metastasis emergence in castrate‐resistant prostate cancer is attributed to dormancy, a state in which cancer cells persist prior to overt lesion formation. Using single‐cell transcriptomics and ex vivo profiling, we have uncovered the critical role of tumor‐intrinsic immune signaling in the retention of cancer cell dormancy. We demonstrate that loss of tumor‐intrinsic type I IFN occurs in proliferating prostate cancer cells in bone. This loss suppresses tumor immunogenicity and therapeutic response and promotes bone cell activation to drive cancer progression. Restoration of tumor‐intrinsic IFN signaling by HDAC inhibition increased tumor cell visibility, promoted long‐term antitumor immunity, and blocked cancer growth in bone. Key findings were validated in patients, including loss of tumor‐intrinsic IFN signaling and immunogenicity in bone metastases compared to primary tumors. Data herein provide a rationale as to why current immunotherapeutics fail in bone‐metastatic prostate cancer, and provide a new therapeutic strategy to overcome the inefficacy of immune‐based therapies in solid cancers.

Abstract P5-05-15: Activation of osteocytes connexin43 hemichannels suppresses breast cancer bone metastasis, a potential therapeutic target

Abstract Bone is a fertile place for metastasized breast cancer cell (BCC), however, mechanical loading is reported to inhibit BCC growth. Osteocytes, representing over 95% of bone cells, are mechanosensitive; they coordinate bone remodeling and modulate the extracellular environment. Mechanical loading activates osteocityc connexin 43 (Cx43) hemichannels, which mediates communication between the intracellular and extracellular space with message of molecules below 1 kDa. We have shown that ATP released by osteocytic hemichannels through the activation of BCC P2X7 receptor, inhibits BCC growth and migration. Here, we evaluate if activation of Cx43 hemichannels in osteocytes plays a critical role in inhibiting breast cancer bone metastasis. Our lab develops a monoclonal antibody, MHC2, which binds to extracellular domain of Cx43 and increases the activity of osteocytic hemichannels. This antibody was immune-reactive in HeLa cells transfected with Cx43 and osteocytes cell line MLO-Y4. It also labeled mouse osteocytes in situ. MHC2 antibody enhanced the activity of Cx43 hemichannels in cells and in vivo osteocytes and promoted the ATP release from osteocytic MLO-Y4 cells. Moreover, conditioned media collected from MHC2-treated osteocytes reduced BCC migration in vitro. The weekly injection of MHC2 reduced the breast cancer growth in bone in WT mice, but not in specific osteocyte Cx43 knockout mice. MHC2 antibody increase levels of lymphocytes T cytotoxic (CD3+/CD8+) and helper (CD3+/CD4+) in tumor and draining lymph nodes. Moreover, the weekly injection of MHC2, in intracardiac injected tumor cells model, in both immunocompetent and immune-compromised mice increased survival rate and reduced tumor spreading. As is known, osteocytic Cx43 hemichannels are permeable to ATP. We showed that P2X7 specific agonist benzoyl ATP reduced in vitro breast cancer cell migration, increased cytoplasmic free Ca2+ and induced formation of tubulin bundles, an indicator of cytoskeleton reorganization. The effect of benzoyl ATP required activation of CamKII in breast cancer cells. Our results suggest that Cx43 hemichannels are likely a potential therapeutic target in treating breast cancer growth and metastasizing to the bone, probably through the enhancement of the extracellular ATP level and the activation of P2X7/CamKII axis. Citation Format: Manuel A. Riquelme, Sumin Gu, Jeffery B. Chavez, Zhiqiang An, Jean X. Jiang. Activation of osteocytes connexin43 hemichannels suppresses breast cancer bone metastasis, a potential therapeutic target [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-05-15.

Abstract P3-01-27: Ectonucleotidase ATP hydrolysis facilitates breast cancer bone metastasis

Abstract Bone is the most common site for breast cancer metastasis. Osteocytes are the most abundant bone cell and can signal nearby cells by releasing ATP through connexin 43 hemichannels. We previously found that this signaling pathway hindered breast cancer growth in bone. In this study, we investigated the role of purinergic signaling on breast cancer bone metastasis in patient samples. Surprisingly, no ATP receptors were differentially expressed in bone metastases compared to other sites. However, enzymes that degrade ATP into adenosine (CD39/ENTPD1 and CD73/NT5E) were upregulated, as was the ADORA3 adenosine receptor (Limma, p=0.037, 0.016, 0.0019, respectively). We next investigated whether this expression signature in primary tumors predicts bone metastasis. In primary ER+ breast cancer, which predominately metastasizes to bone, high expression of these genes is correlated with lower distant metastasis-free survival (HR = 1.97, 1.49, 2.03, logrank p=0.0017, 0.0004, 0.0012). Moreover, this expression pattern is correlated with a bone metastasis signature (Spearman R=0.28, p=1.2 × 10−20). In a pharmacologic screen of 750 cancer cell lines, breast cancer lines and especially ER+ ones, are uniquely sensitive to a pan-adenosine receptor inhibitor (Limma, p=2.9 × 10−5) and ADORA3/2B inhibitor (p=5.4 × 10−5). Our findings suggest that an expression program to convert ATP to adenosine facilitates bone metastasis and begins in the primary tumor. ADORA3 inhibition is proposed as a pharmacologic strategy for preventing or treating bone metastasis. Furthermore, bone metastases should be analyzed in anti-CD39 and CD73 clinical trials. Citation Format: Daniel B Shropshire, Jaime Benavides, Jean X Jiang. Ectonucleotidase ATP hydrolysis facilitates breast cancer bone metastasis [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-01-27.

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.

The Role of Vitamin D and the Vitamin D Receptor in Bone Oncology

SummaryVitamin D deficiency is a global health problem of enormous and increasing dimensions. In the past decades, numerous studies have centered on the role of vitamin D in the pathogenesis and course of many diseases including several types of cancer. Indeed, vitamin D has been widely acknowledged to be involved in the regulation of cell proliferation, differentiation and apoptosis in numerous cancer cells. While the full range of molecular mechanisms involveld in cancer cell growth and progression remains to be elucidated, recent research has deepened our understanding of the processes that may be affected by vitamin D or vitamin D deficiency.In this review, we consider the properties of bone that enable cancer cells to grow and thrive within the skeleton, and the role of vitamin D and the vitamin D receptor in the process of primary and secondary cancer growth in bone.

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.

Abstract P6-12-10: Bone seeking matrix metalloproteinase-2 inhibitors prevent bone metastatic breast cancer growth

Abstract Bone metastasis is a common event during breast cancer progression. The resultant lesions are painful and currently, despite medical advances, are incurable. The progression of bone metastatic breast cancer is critically dependent on interactions with the surrounding microenvironment. Therefore, identifying the underpinning molecular mechanisms is vital for the development of new therapies. Rationale: Gene expression analysis and validation in human and murine specimens of bone metastases revealed that matrix metalloproteinases, such as MMP-2, are highly expressed in the bone metastatic microenvironment and significantly associated with aggressive breast cancer and poorer overall survival. In bone, tumor or host derived MMP-2 contributes to breast cancer growth and does so by processing substrates including type I collagen and transforming growth factor beta (TGFβ) latency proteins. These data provide strong rationale for the application of MMP-2 inhibitors to treat the disease. However, in vivo, MMP-2 is systemically expressed. Therefore, to overcome potential toxicities noted with previous broad-spectrum MMP inhibitors (MMPIs), we used highly selective bisphosphonic based MMP-2 inhibitors (BMMPIs) that allowed for specific bone targeting. Methods: We utilized a novel chemical approach to synthesize bone seeking MMP inhibitors (BMMPIs) on a bisphosphonic backbone, with specificity for MMP-2 in the nanomolar range (IC50=140 nM). Results: In vitro, we tested the effect of BMMPIs at varying doses (1nM-100μM) on the viability of the major cellular components of the cancer-bone microenvironment, namely breast cancer cells, (PyMT, 4T1, MDA-MB-231, MCF-7), osteoblasts (MC3T3) and osteoclasts (primary monocytes and RAW 264.7). In vivo, we demonstrated using two bone metastatic models (PyMT-R221A-Luc and 4T1-Luc) that BMMPI treatment significantly reduced tumor growth and tumor associated bone destruction. Additionally, BMMPIs are superior in promoting tumor apoptosis compared to the standard of care bisphosphonate, zoledronate. MMP activity was also lower in the BMMPI treated groups (using tumor burden to normalize values). μCT/Xray/Histomorphometry analysis also illustrated the significant beneficial effects of the BMMPIs in reducing the size of osteolytic lesions (up to 80% by μCT; p<0.05). We demonstrated MMP-2 selective inhibition in the bone microenvironment using specific and broad spectrum MMP probes. Further, compared to zoledronate, BMMPI treated mice had significantly lower levels of TGFβ signaling and MMP generated type I collagen carboxy-terminal (ICTP) fragments. Taken together, our data show the feasibility of selective inhibition of MMPs in the bone metastatic breast cancer microenvironment. Conclusions. MMP-2 specific inhibition was achieved in the bone microenvironment. BMMPIs significantly inhibit breast cancer growth in bone, they are able to induce breast cancer cell apoptosis and prevent cancer induced bone destruction. Given that bisphosphonates are well tolerated in the clinical setting, we predict that BMMPIs could be translated to the clinical setting for the treatment and eradication of bone metastatic breast cancer. Citation Format: Tauro M, Laghezza A, Tortorella P, Lynch CC. Bone seeking matrix metalloproteinase-2 inhibitors prevent bone metastatic breast cancer growth [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-12-10.

Sunitinib treatment reduces tumor growth and limits changes in microvascular properties after minor surgical intervention in an in vivo model of secondary breast cancer growth in bone.

Surgical interventions can alter the balance between pro- and anti-angiogenic growth factors and thereby modulate tumor growth. Since the microcirculatory properties of tumors underlie organ-specific differences, the microhemodynamic characteristics of bone metastasis have not yet been fully described. Angiogenesis inhibitors are increasingly being used to treat advanced stages of cancer. We hypothesized that the anti-angiogenic drug sunitinib abrogates alterations in microvascular properties following a minor surgical intervention in an in vivo model of secondary breast cancer growth in the bone. Intravital microscopy was performed over 25 days using a xenograft model of breast cancer tumor growth in the bone to determine changes in microvascular properties during sunitinib treatment. Mastectomy was performed on day 5 to evaluate the effect of a minor surgical trauma on tumor growth and microvascular properties. Anti-angiogenic therapy resulted in reduced tumor growth, decreased vascular density, and increased vascular diameters. Blood flow velocity remained constant while microvascular permeability temporarily increased after the surgical intervention. Administration of sunitinib reduced tumor growth and altered microcirculatory properties in a time-dependent manner. The observed dramatic increase in microvascular permeability after the surgical intervention may have implications for local tumor growth, and metastatic dissemination. J. Surg. Oncol. 2016;113:515-521. © 2016 Wiley Periodicals, Inc.

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.