Formation Of Osteolytic Lesions Research Articles

From Tumor to Bone: Growth Factor Receptors as Key Players in Cancer Metastasis.

This review article explores the intricate correlation between growth factors and bone metastases, which play a crucial role in the development of several types of malignancies, namely breast, prostate, lung, and renal cancers. The focal point of our discussion is on crucial receptors for growth factors, including Epidermal Growth Factor Receptor (EGFR), Transforming Growth Factor-β (TGFβ), Vascular Endothelial Growth Factor Receptor (VEGFR), and Fibroblast Growth Factor Receptor (FGFR). These receptors, which are essential for cellular activities including growth, differentiation, and survival, have important involvement in the spread of cancer and the interactions between tumors and the bone environment. We discuss the underlying mechanisms of bone metastases, with a specific emphasis on the interaction between growth factor receptors and the bone microenvironment. EGFR signaling specifically enhances the process of osteoclast development and the formation of osteolytic lesions, especially in breast and lung malignancies. TGFβ receptors have a role in both osteolytic and osteoblastic metastases by releasing TGFβ, which attracts cancer cells and promotes bone remodeling. This is a crucial element in the spread of prostate cancer to the bones. The functions of FGFR and VEGFR in the processes of bone formation and tumor angiogenesis, respectively, highlight the complex and diverse nature of these interactions. The review emphasizes the possibility of targeted therapeutics targeting these receptors to interrupt the cycle of tumor development and bone degradation. Therapeutic approaches include focusing on the VEGF/VEGFR, EGF/EGFR, FGF/FGFR, and TGFβ/TGFβR pathways. These include a variety of compounds, such as small molecule inhibitors and monoclonal antibodies, which have shown potential to interfere with tumor-induced alterations in bone. The text discusses clinical trials and preclinical models, offering insights into the effectiveness and constraints of various treatments. Ultimately, this study provides a succinct but thorough summary of the present knowledge and treatment strategies focused on growth factor receptors in bone metastases. This highlights the significance of comprehending the signaling of growth factor receptors in the microenvironment where tumors spread to the bones, as well as the possibility of using targeted therapies to enhance the results for cancer patients with bone metastases. The advancement of treating bone metastases hinges on the development of treatments that specifically target the intricate relationships between malignancies and bone.

Impact of the host response and osteoblast lineage cells on periodontal disease

Periodontitis involves the loss of connective tissue attachment and alveolar bone. Single cell RNA-seq experiments have provided new insight into how resident cells and infiltrating immune cells function in response to bacterial challenge in periodontal tissues. Periodontal disease is induced by a combined innate and adaptive immune response to bacterial dysbiosis that is initiated by resident cells including epithelial cells and fibroblasts, which recruit immune cells. Chemokines and cytokines stimulate recruitment of osteoclast precursors and osteoclastogenesis in response to TNF, IL-1β, IL-6, IL-17, RANKL and other factors. Inflammation also suppresses coupled bone formation to limit repair of osteolytic lesions. Bone lining cells, osteocytes and periodontal ligament cells play a key role in both processes. The periodontal ligament contains cells that exhibit similarities to tendon cells, osteoblast-lineage cells and mesenchymal stem cells. Bone lining cells consisting of mesenchymal stem cells, osteoprogenitors and osteoblasts are influenced by osteocytes and stimulate formation of osteoclast precursors through MCSF and RANKL, which directly induce osteoclastogenesis. Following bone resorption, factors are released from resorbed bone matrix and by osteoclasts and osteal macrophages that recruit osteoblast precursors to the resorbed bone surface. Osteoblast differentiation and coupled bone formation are regulated by multiple signaling pathways including Wnt, Notch, FGF, IGF-1, BMP, and Hedgehog pathways. Diabetes, cigarette smoking and aging enhance the pathologic processes to increase bone resorption and inhibit coupled bone formation to accelerate bone loss. Other bone pathologies such as rheumatoid arthritis, post-menopausal osteoporosis and bone unloading/disuse also affect osteoblast lineage cells and participate in formation of osteolytic lesions by promoting bone resorption and inhibiting coupled bone formation. Thus, periodontitis involves the activation of an inflammatory response that involves a large number of cells to stimulate bone resorption and limit osseous repair processes.

'Educated' Osteoblasts Reduce Osteoclastogenesis in a Bone-Tumor Mimetic Microenvironment.

Simple SummaryPatients with late-stage bone metastatic breast cancer experience skeletal related events, including osteolytic lesion formation, due to overactive osteoclast bone resorption. It is well-known that osteoclast function is altered by breast cancer cells in bone. Breast cancer cells stimulate osteoblasts to secrete factors that initiate osteoclast differentiation and activation. Our lab has previously identified a novel subpopulation of osteoblasts in the bone-tumor microenvironment called “educated” osteoblasts (EOs) that alter breast cancer cell proliferation. The aim of this study was to identify how osteoclasts are affected by EOs during metastatic breast cancer progression in bone. Our results demonstrated that pre-osteoclast interaction with EOs reduces osteoclast formation and bone resorption in a bone-tumor mimetic microenvironment. Furthermore, we identified that altered osteoclast formation can be modulated, in part, by tumor necrosis factor alpha (TNFα). Overall, our data demonstrate osteoclastogenesis is reduced by EO cells, suggesting EO cells have a protective effect in bone and exert an inhibitory effect on tumor progression.Breast cancer (BC) metastases to bone disrupt the balance between osteoblasts and osteoclasts, leading to excessive bone resorption. We identified a novel subpopulation of osteoblasts with tumor-inhibitory properties, called educated osteoblasts (EOs). Here we sought to examine the effect of EOs on osteoclastogenesis during tumor progression. We hypothesized that EOs affect osteoclast development in the bone-tumor niche, leading to suppressed pre-osteoclast fusion and bone resorption. Conditioned media (CM) was analyzed for protein expression of osteoclast factors receptor activator of nuclear factor kappa-β ligand (RANKL), osteoprotegerin (OPG), and tumor necrosis factor alpha (TNFα) via ELISA. EOs were co-cultured with pre-osteoclasts on a bone mimetic matrix to assess osteoclast resorption. Pre-osteoclasts were tri-cultured with EOs plus metastatic BC cells and assessed for tartrate-resistance acid phosphatase (TRAP)-positive, multinucleated (≥3 nuclei), mature osteoclasts. Tumor-bearing murine tibias were stained for TRAP to determine osteoclast number in-vivo. EO CM expressed reduced amounts of soluble TNFα and OPG compared to naïve osteoblast CM. Osteoclasts formed in the presence of EOs were smaller and less in number. Upon co-culture on a mimetic bone matrix, a 50% reduction in the number of TRAP-positive osteoclasts formed in the presence of EOs was observed. The tibia of mice inoculated with BC cells had less osteoclasts per bone surface in bones with increased numbers of EO cells. These data suggest EOs reduce osteoclastogenesis and bone resorption. The data imply EOs provide a protective effect against bone resorption in bone metastatic BC.

Integrin alpha5 in human breast cancer is a mediator of bone metastasis and a therapeutic target for the treatment of osteolytic lesions

Bone metastasis remains a major cause of mortality and morbidity in breast cancer. Therefore, there is an urgent need to better select high-risk patients in order to adapt patient’s treatment and prevent bone recurrence. Here, we found that integrin alpha5 (ITGA5) was highly expressed in bone metastases, compared to lung, liver, or brain metastases. High ITGA5 expression in primary tumors correlated with the presence of disseminated tumor cells in bone marrow aspirates from early stage breast cancer patients (n = 268; p = 0.039). ITGA5 was also predictive of poor bone metastasis-free survival in two separate clinical data sets (n = 855, HR = 1.36, p = 0.018 and n = 427, HR = 1.62, p = 0.024). This prognostic value remained significant in multivariate analysis (p = 0.028). Experimentally, ITGA5 silencing impaired tumor cell adhesion to fibronectin, migration, and survival. ITGA5 silencing also reduced tumor cell colonization of the bone marrow and formation of osteolytic lesions in vivo. Conversely, ITGA5 overexpression promoted bone metastasis. Pharmacological inhibition of ITGA5 with humanized monoclonal antibody M200 (volociximab) recapitulated inhibitory effects of ITGA5 silencing on tumor cell functions in vitro and tumor cell colonization of the bone marrow in vivo. M200 also markedly reduced tumor outgrowth in experimental models of bone metastasis or tumorigenesis, and blunted cancer-associated bone destruction. ITGA5 was not only expressed by tumor cells but also osteoclasts. In this respect, M200 decreased human osteoclast-mediated bone resorption in vitro. Overall, this study identifies ITGA5 as a mediator of breast-to-bone metastasis and raises the possibility that volociximab/M200 could be repurposed for the treatment of ITGA5-positive breast cancer patients with bone metastases.

Synthesis of sophocarpine triflorohydrazone and its proliferation inhibition and apoptosis induction activity in myeloma cells through Notch3-p53 signaling activation.

Multiple myeloma is indicated by the presence of excessive monoclonal plasma cells in bone marrow, which result in the formation of osteolytic lesions. The present study investigated SCA as anti-proliferative agent for myeloma cells and explored the mechanism associated. Effect of SCA on viabilities of KRASA12 and AMO-1 cells was evaluated by MTT assay and apoptotic ratio using flow cytometry. Protein expression was investigated by western blotting and expression of genes related to Notch3-p53 signaling axis using RT-PCR assay. Increase in SCA concentration caused a significant (P < .01) reduction in KRASA12 and AMO-1 cell viability. The KRASA12 and AMO-1 cell viabilities were reduced to 29% and 21%, respectively on treatment with 21 μM doses of SCA. SCA treatment of KRASA12 and AMO-1 cells significantly (P < .05) increased apoptosis compared with untreated cells. The Bcl-2 (26 kDa) protein expression was reduced whereas the Bax (21 kDa) and cleaved caspase-3 levels elevated in SCA treated KRASA12 and AMO-1 cells. Treatment with SCA significantly promoted Hes1, p53 (53 kDa) and Hey1 mRNA expression in KRASA12 and AMO-1 cells. Treatment of KRASA12 and AMO-1 cells with SCA led to a marked reduction in Notch3 protein expression. SCA inhibits KRASA12 and AMO-1 myeloma cell proliferation by promoting pro-apoptotic proteins. Moreover, SCA treatment suppressed Hes1 and Hey1 mRNA expression and targeted Notch3 expression. Therefore, SCA may be studied further for development of treatment for myeloma.

Therapeutic Targeting of CD146/MCAM Reduces Bone Metastasis in Prostate Cancer.

Prostate Cancer is the most common cancer and the second leading cause of cancer-related death in males. When prostate cancer acquires castration resistance, incurable metastases, primarily in the bone, occur. The aim of this study is to test the applicability of targeting melanoma cell adhesion molecule (MCAM; CD146) with a mAb for the treatment of lytic prostate cancer bone metastasis. We evaluated the effect of targeting MCAM using in vivo preclinical bone metastasis models and an in vitro bone niche coculture system. We utilized FACS, cell proliferation assays, and gene expression profiling to study the phenotype and function of MCAM knockdown in vitro and in vivo. To demonstrate the impact of MCAM targeting and therapeutic applicability, we employed an anti-MCAM mAb in vivo. MCAM is elevated in prostate cancer metastases resistant to androgen ablation. Treatment with DHT showed MCAM upregulation upon castration. We investigated the function of MCAM in a direct coculture model of human prostate cancer cells with human osteoblasts and found that there is a reduced influence of human osteoblasts on human prostate cancer cells in which MCAM has been knocked down. Furthermore, we observed a strongly reduced formation of osteolytic lesions upon bone inoculation of MCAM-depleted human prostate cancer cells in animal model of prostate cancer bone metastasis. This phenotype is supported by RNA sequencing (RNA-seq) analysis. Importantly, in vivo administration of an anti-MCAM human mAb reduced the tumor growth and lytic lesions. These results highlight the functional role for MCAM in the development of lytic bone metastasis and suggest that MCAM is a potential therapeutic target in prostate cancer bone metastasis. IMPLICATIONS: This study highlights the functional application of an anti-MCAM mAb to target prostate cancer bone metastasis.

Highlights of This Issue

TP53 is the most commonly mutated gene in solid tumors, and loss of p53 function is strongly correlated with resistance to chemotherapy in head and neck cancer. In this study, Diab and colleagues demonstrate that inhibition of WEE1, a kinase involved in regulating cell cycle transition during S phase and mitosis, has increased efficacy in p53-deficient cells due to incomplete resolution of DNA damage and stalled replication forks. These lesions are carried over across more than one round of cell division, thereby magnifying the effects of genotoxic therapy.Prostate cancer represents an increasing clinical burden, and metastatic prostate cancer has thus far proven incurable. Metastatic prostate cancer primarily localizes to the bones, inducing osteopathy and bone-related adverse events in patients. Here, Zoni and colleagues demonstrate greatly reduced incidences of bone metastatic lesions by blocking MCAM. Ablation of MCAM or blockade with an MCAM-specific monoclonal antibody reduced interactions between prostate cancer cells and osteoblasts in vitro, and significantly reduced the formation of osteolytic lesions as well as overall tumor growth in vivo. Taken together, these data support the potential utility of targeting MCAM to slow or prevent prostate cancer bone metastasis.Cancer cell motility is a key factor in disease progression, and expression of plasminogen activator inhibitor 1 (PAI1) is associated with increased cellular migration and poor prognosis. Here, Humphries and colleagues demonstrate that increased PAI1 expression reprograms cellular energetics, increasing reliance on glycolysis and inducing mitochondrial fission, cytoskeletal rearrangement, and invasiveness both in vitro and in vivo. Targeting this pathway may present a novel means of blocking cell migration and tumor progression.For decades, prostate cancer diagnosis and prognosis have been largely based on Gleason scoring, a histologic grading scale of the disruption of prostate architecture as the disease increases in severity. While effective, the scale is somewhat subjective and can be difficult to apply to the limited tissue samples obtained from biopsy. Here, Randall and colleagues detail a mass spectrometry imaging workflow based on lipid and metabolite abundance that tightly correlate with increasing Gleason score. The data suggest that lipidomics and metabolomics could represent a higher-fidelity, faster means of scoring prostate cancer aggressiveness to support diagnostic efforts in the clinic.

Abstract 2116: Tumor-specific effects of estrogen drive a murine model of human estrogen receptor-positive breast cancer bone metastasis independent of bone microenvironment changes

Abstract Estrogen receptor α-positive (ER+) breast cancers (BrCAs) have the greatest predilection for forming bone metastases (BMETs). To begin to query ERα's role in osteolytic BrCA BMET progression, tumoral vs bone microenvironment effects of 17β-estradiol (E2) were determined in a human xenograft murine model of ER+ BrCA BMET. For these studies, naïve or 17β-estradiol (E2)-treated female athymic mice aged 4 weeks (young) or 15 weeks (mature) were inoculated with 1x105 human ER+ MCF-7 BrCA cells via the left cardiac ventricle 2 days post-placement of 60-day release E2 pellets (0.05, 0.10, 0.18, 0.36, &amp; 0.72 mg E2). For studies examining E2 effects on bone, MCF-7 cells were not inoculated. Osteolytic lesion formation was assessed by hind limb radiographs, and E2 effects on bone were assessed by DXA (Faxitron), μCT (Scanco), and biochemical markers of bone formation or resorption (P1NP and TRAcP, respectively; Immunodiagnostic Systems). BMET incidence and lesion size in young mice were E2 dose-dependent, achieving 100% incidence at the highest dose tested, with smaller lesions and lower incidence occurring in response to lower doses, and no lesions forming in the absence of E2 even after 8 months. In contrast, E2 effects on the bone microenvironment were not dose dependent, and resulted in identical increases in bone mineral density (BMD) and bone volume (BV/TV), primarily via an increase in bone formation. To further query the importance of tumoral vs bone effects of E2 in driving ER+ BrCA progression, the tumoral effects of an identical E2 dose (0.72 mg) were compared in the skeletally immature young mice, which accrued increased bone (BV/TV) due to increases in bone formation in response to E2, vs skeletally mature mice, where E2-induced increases in bone were less marked and primarily driven by decreases in bone resorption. In these two very different bone milieus, progression of E2-dependent BMET was identical. While the relative roles of E2-induced tumor cell proliferation vs osteolytic factor secretion cannot be distinguished here, the development of osteolytic lesions similar in size to those occurring in ER- models driven by tumoral secretion of osteolytic factors, despite a marked increase in bone volume, suggests that tumoral effects of E2 may drive osteolytic ER+ BrCA BMET progression in a bone-specific fashion. Citation Format: Julia N. Cheng, Jennifer B. Frye, Susan A. Whitman, Andrew G. Kunihiro, Madison M. Egan, Julia A. Brickey, Janet L. Funk. Tumor-specific effects of estrogen drive a murine model of human estrogen receptor-positive breast cancer bone metastasis independent of bone microenvironment changes [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 2116.

MDA-MB-231 breast cancer cells fuel osteoclast metabolism and activity: A new rationale for the pathogenesis of osteolytic bone metastases

Recent progress in dissecting the molecular paracrine circuits of cancer and stromal cells in bone metastases (BM) are offering new options to improve current merely palliative approach. The study of tumor-stroma metabolic interplay may further ameliorate this scenario. In this context, we demonstrated that highly glycolytic MDA-MB-231 cancer cells, that form osteolytic BM in vivo, release a large amount of lactate at a significantly higher level than MCF7 cells. Thus, we speculated that lactate released from carcinoma cells is uptaken and metabolically used by osteoclasts, the key players of osteolysis associated with BM. First, we demonstrated that the release of lactate at the bone site is mediated by monocarboxylate transporter 4 (MCT4), as revealed by immunostaining and MCT4 localization at the plasma membrane of tumor cells in mouse model of BM and in human tissue sections of BM. Then, we showed that in vitro lactate is uptaken by osteoclasts to be used as a fuel for the oxidative metabolism of osteoclasts, ultimately enhancing Type I collagen resorption. The passive transport of lactate into osteoclasts was mediated by MCT1: MCT1 expression is significantly upregulated during osteoclast differentiation and Type I collagen resorption is significantly impaired when osteoclasts are treated with 7-(N-benzyl-N-methylamino)-2-oxo-2H-chromene-3-carboxylic acid, an MCT-1 inhibitor. Together, these data demonstrate that lactate released by glycolytic breast carcinoma cells in the bone microenvironment promotes the formation of osteolytic lesions, and provide the rationale for further studies on the use of MCT1 targeting as a novel therapeutic approach in advanced cancer patients with BM.

Bisphosphonate guidelines for treatment and prevention of myeloma bone disease.

Multiple myeloma (MM) is a haematological malignancy characterised by the clonal proliferation of plasma cells in the bone marrow. More than 80% of patients with MM display evidence of myeloma bone disease (MBD), characterised by the formation of osteolytic lesions throughout the axial and appendicular skeleton. MBD significantly increases the risk of skeletal-related events such as pathologic fracture, spinal cord compression and hypercalcaemia. MBD is the result of MM plasma cells-mediated activation of osteoclast activity and suppression of osteoblast activity. Bisphosphonates (BP), pyrophosphate analogues with high bone affinity, are the only pharmacological agents currently recommended for the treatment and prevention of MBD and remain the standard of care. Pamidronate and zoledronic acid are the most commonly used BP to treat MBD. Although generally safe, frequent high doses of BP are associated with adverse events such as renal toxicity and osteonecrosis of the jaw. As such, optimal duration and dosing of BP therapy is required in order to minimise BP-associated adverse events. The following guidelines provide currently available evidence for the adoption of a tailored approach when using BP for the management of MBD.

Abstract P6-18-01: Site-specific activation of curcuminoids in the breast cancer bone metastases microenvironment

Abstract The majority of women with advanced breast cancer (BCa) develop incurable osteolytic bone metastases. Our laboratory has previously demonstrated that curcuminoids, bioactive compounds isolated from turmeric rhizomes, prevent the development of lytic bone lesions in a murine xenograft model of human BCa bone metastasis and inhibit tumor cell secretion of TGFβ-stimulated parathyroid-related protein (PTHrP), a signaling pathway known to drive bone metastasis progression. In both humans and mice, glucuronidated phase II metabolites of curcumin, which are thought to be biologically inactive, are the primary form detected in circulation after consuming curcuminoid-containing foods or supplements. This has led to an untested postulate that curcuminoids may be deglucuronidated at sites of action to form bioactive, aglycone (free) curcumin. Studies were therefore undertaken to test this hypothesis in the context of the murine BCa bone metastasis model, assessing site-specific deconjugation of curcuminoids in the tumor-bone microenvironment. Effects of curcumin-glucuronide (G-CURC) vs. curcumin (CURC) on TGFβ-stimulated PTHrP secretion by bone-tropic MDA-MB-231 (MDA) cells was determined by immunoradiometric assay. G-CURC and CURC levels were quantified by LC-MS in plasma and bone marrow specimens isolated from curcuminoid-treated female nude mice. Endogenous β-glucuronidase enzyme expression was localized by immunohistochemical (IHC) staining in paraffin-embedded sections of decalcified, MDA metastases-containing hind limbs of nude mice inoculated, via intracardiac injection 21 days prior, with MDA cells. Glucuronide deconjugation activity of bone marrow lysates was determined by colorimetric assay. In contrast to the inhibitory effects of a naturally occurring curcuminoid mixture or pure CURC, G-CURC did not alter TGFβ-stimulated PTHrP secretion, confirming its postulated lack of biologic activity. In mice treated with curcuminoids, the majority of circulating curcumin was conjugated (91%). In contrast, the majority of curcumin in the bone marrow (56%) was unconjugated free (aglycone) curcumin (p &amp;lt;0.01). IHC staining of MDA tumor-bearing hind limbs of nude mice demonstrated expression of β-glucuronidase (GUSB), an enzyme that deconjugates compounds in mice and humans, by bone marrow cells, but not by tumor cells. Consistent with IHC, bone marrow from female nude mice displayed significantly higher (30-fold) deconjugation enzyme activity compared to MDA cells (p &amp;lt; 0.01). Female C3H/HeJ mice, which possess a partial loss-of-function mutation in the GUSB gene, had significantly decreased (66% lower) deconjugation enzyme activity compared to normal female C57BL/6 (p &amp;lt; 0.001) or nude (p &amp;lt; 0.01) mice. In addition, treatment with the β-glucuronidase inhibitor, saccharolactone (SL), also decreased deconjugation enzyme activity in marrow lysates from female nude mice. These results suggest that curcuminoids, in the setting of breast cancer bone metastases, may act as a pro-drug, becoming activated within the tumor-bone microenvironment by glucuronidase-expressing hematopoietic bone marrow cells, to limit the progression of osteolytic lesion formation in a murine model of BCa bone metastasis. Supported by: R01CA174926-01, R01AT006896, and R03CA159382. Citation Format: Kunihiro AG, Frye JB, Brickey JA, Luis PB, Schneider C, Funk JL. Site-specific activation of curcuminoids in the breast cancer bone metastases microenvironment [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 P6-18-01.

Abstract P4-06-08: Characterization of an estrogen-dependent murine model of human estrogen receptor-positive breast cancer bone metastasis

Abstract Introduction: Breast cancer patients with estrogen receptor-positive (ER+) tumors have the highest rate of developing clinically evident osteolytic bone metastasis. However, much of our knowledge of breast cancer bone metastasis has been derived from ER- murine xenograft models, as robust pre-clinical models of ER+ bone metastasis are currently lacking. Thus we sought to develop and characterize a murine xenograft model of breast cancer bone metastases using an estrogen-dependent ER+ MCF-7 cell line. Methods: 28-day-old female nude mice were implanted with the following 60-day extended-release 17β-estradiol (E2) pellets: 0.72, 0.36, 0.18, 0.10, or 0.05 mg; with or without 1x105 MCF-7 cells (ATCC) inoculated via the left cardiac ventricle. Non-estradiol-supplemented female nude mice were also inoculated with either MCF-7 cells or a well-characterized, bone-tropic human ER- MDA-MB-231 cell line (referred to as MDA-SA), used as a comparison model. Radiographs and dual-energy X-ray absorptiometry's (DXAs) of hind femurs and tibias were obtained weekly to assess osteolytic lesion formation and BMD, respectively. Statistical significance was analyzed using one-way ANOVA with Tukey's post-hoc testing. Results: Using E2 doses previously reported in the literature to promote MCF-7 orthotopic tumors and bone metastases (0.72 mg 60-day 17β- E2 pellets), bone metastases developed with 100% incidence (n = 13) by day 28 in the MCF-7 model, with osteolytic lesion area increasing to 10.3±0.8 mm2 by day 42 (as compared with 100% incidence on day 21 in non-estrogen-supplemented mice inoculated with MDA-SA (n=8) with osteolytic lesion area of 10.7±2.0 mm2). However, histologic assessment of hind limbs of MCF-7 tumor bearing mice suggested that this estrogen dose stimulated a significant increase in bone mass. Therefore, the estrogen-dependency of bone metastasis formation and changes in BMD were explored. Bone metastasis incidence was estrogen-dependent; falling to less than 100% in mice with E2 doses less than 0.72 mg (50-75%, n = 4-12). BMD of distal femurs of mice supplemented with estradiol was significantly greater than naïve controls on day 28 (average increase of 65.3%, p&amp;lt;0.05), and was not dose dependent over the range of doses tested (0.05-0.72 mg pellets). Conclusion: Despite a marked increase in bone density, ER+ MCF-7 cells formed radiographically detectable osteolytic lesions with 100% incidence by day 28 in 0.72 mg 60-day 17β- E2 supplemented nude mice. It is possible that estrogen-stimulated increases in BMD contributed to the longer time required to achieve ER+ osteolytic lesion sizes comparable to those in standard ER- models. However, lower doses did not mitigate effects of estrogen on BMD in these 1-month-old mice, while lowering the incidence of bone metastasis formation. While further optimization is planned, intracardiac inoculation of MCF-7 cells in female nude mice provides a robust model of estrogen-dependent ER+ breast cancer bone metastases. Source of Research Support: R01 CA174926-01 R03 CA181893-01. Citation Format: Cheng JN, Frye JB, Whitman SA, Kunihiro AG, Funk JL. Characterization of an estrogen-dependent murine model of human estrogen receptor-positive breast cancer bone metastasis [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 P4-06-08.

Lysyl Oxidase Is a Strong Determinant of Tumor Cell Colonization in Bone.

Lysyl oxidase (LOX) is a secreted copper-dependent amine oxidase whose primary function is to drive collagen crosslinking and extracellular matrix stiffness. LOX in colorectal cancer synergizes with hypoxia-inducible factor-1 (HIF-1) to promote tumor progression. Here we investigated whether LOX/HIF1 endows colorectal cancer cells with full competence for aggressive colonization in bone. We show that a high LOX expression in primary tumors from patients with colorectal cancer was associated with poor clinical outcome, irrespective of HIF-1 In addition, LOX was expressed by tumor cells in the bone marrow from colorectal cancer patients with bone metastases. In vivo experimental studies show that LOX overexpression in colorectal cancer cells or systemic delivery of the conditioned medium from LOX-overexpressing colorectal cancer cells promoted tumor cell dissemination in the bone marrow and enhanced osteolytic lesion formation, irrespective of HIF-1 Conversely, silencing or pharmacologic inhibition of LOX activity blocked dissemination of colorectal cancer cells in the bone marrow and tumor-driven osteolytic lesion formation. In vitro, tumor-secreted LOX supported the attachment and survival of colorectal cancer cells to and in the bone matrix, and inhibited osteoblast differentiation. LOX overexpression in colorectal cancer cells also induced a robust production of IL6. In turn, both LOX and IL6 were acting in concert to promote RANKL-dependent osteoclast differentiation, thereby creating an imbalance between bone resorption and bone formation. Collectively, our findings show that LOX supports colorectal cancer cell dissemination in the bone marrow and they reveal a novel mechanism through which LOX-driven IL6 production by colorectal cancer cells impairs bone homeostasis. Cancer Res; 77(2); 268-78. ©2016 AACR.

The hypoxic cancer secretome induces pre-metastatic bone lesions through lysyl oxidase.

Tumour metastasis is a complex process involving reciprocal interplay between cancer cells and host stroma at both primary and secondary sites, and is strongly influenced by microenvironmental factors such as hypoxia. Tumour-secreted proteins play a crucial role in these interactions and present strategic therapeutic potential. Metastasis of breast cancer to the bone affects approximately 85% of patients with advanced disease and renders them largely untreatable. Specifically, osteolytic bone lesions, where bone is destroyed, lead to debilitating skeletal complications and increased patient morbidity and mortality. The molecular interactions governing the early events of osteolytic lesion formation are currently unclear. Here we show hypoxia to be specifically associated with bone relapse in patients with oestrogen-receptor negative breast cancer. Global quantitative analysis of the hypoxic secretome identified lysyl oxidase (LOX) as significantly associated with bone-tropism and relapse. High expression of LOX in primary breast tumours or systemic delivery of LOX leads to osteolytic lesion formation whereas silencing or inhibition of LOX activity abrogates tumour-driven osteolytic lesion formation. We identify LOX as a novel regulator of NFATc1-driven osteoclastogenesis, independent of RANK ligand, which disrupts normal bone homeostasis leading to the formation of focal pre-metastatic lesions. We show that these lesions subsequently provide a platform for circulating tumour cells to colonize and form bone metastases. Our study identifies a novel mechanism of regulation of bone homeostasis and metastasis, opening up opportunities for novel therapeutic intervention with important clinical implications.

NOD/SCID-GAMMA mice are an ideal strain to assess the efficacy of therapeutic agents used in the treatment of myeloma bone disease.

Animal models of multiple myeloma vary in terms of consistency of onset, degree of tumour burden and degree of myeloma bone disease. Here we describe five pre-clinical models of myeloma in NOD/SCID-GAMMA mice to specifically study the effects of therapeutic agents on myeloma bone disease. Groups of 7–8 week old female irradiated NOD/SCID-GAMMA mice were injected intravenously via the tail vein with either 1x106 JJN3, U266, XG-1 or OPM-2 human myeloma cell lines or patient-derived myeloma cells. At the first signs of morbidity in each tumour group all animals were sacrificed. Tumour load was measured by histological analysis, and bone disease was assessed by micro-CT and standard histomorphometric methods. Mice injected with JJN3, U266 or OPM-2 cells showed high tumour bone marrow infiltration of the long bones with low variability, resulting in osteolytic lesions. In contrast, mice injected with XG-1 or patient-derived myeloma cells showed lower tumour bone marrow infiltration and less bone disease with high variability. Injection of JJN3 cells into NOD/SCID-GAMMA mice resulted in an aggressive, short-term model of myeloma with mice exhibiting signs of morbidity 3 weeks later. Treating these mice with zoledronic acid at the time of tumour cell injection or once tumour was established prevented JJN3-induced bone disease but did not reduce tumour burden, whereas, carfilzomib treatment given once tumour was established significantly reduced tumour burden. Injection of U266, XG-1, OPM-2 and patient-derived myeloma cells resulted in less aggressive longer-term models of myeloma with mice exhibiting signs of morbidity 8 weeks later. Treating U266-induced disease with zoledronic acid prevented the formation of osteolytic lesions and trabecular bone loss as well as reducing tumour burden whereas, carfilzomib treatment only reduced tumour burden. In summary, JJN3, U266 or OPM-2 cells injected into NOD/SCID-GAMMA mice provide robust models to study anti-myeloma therapies, particularly those targeting myeloma bone disease.

Interaction between bone marrow stromal cells and neuroblastoma cells leads to a VEGFA-mediated osteoblastogenesis.

The potential role of osteoblasts in bone and bone marrow (BM) metastases in neuroblastoma (NBL) remains unclear. In this study, we examined the effect of NBL cells on the osteoblastic differentiation of BM-derived mesenchymal stromal cells (BMMSC). We show that the presence of NBL cells enhanced the osteoblastic differentiation of BMMSC driven by bone morphogenetic protein (BMP)-4, in the absence of any effect on NBL cell proliferation. Expression profiles of BMMSC driven toward osteoblastic differentiation revealed an increase in vascular endothelial growth factor A (Vegfa) expression in the presence of NBL cells. We demonstrated that NBL cells increased BMMSC-derived VEGFA mRNA and protein and that this was enhanced by BMP-4. However, in similar conditions, neither the addition of an mVEGFA blocking antibody nor exogenous recombinant (r) mVEGFA affected osteoblastic differentiation. In contrast, siRNA- mediated knock-down of VEGFA in BMMSC prevented osteoblastic differentiation in BMP-4-treated cocultures, an effect that was not reversed in the presence of rmVEGFA. An analysis of murine bones injected with hNBL cells revealed an increase of mVEGFA producing cells near tumor cells concomitantly with an increase in Vegfa and Runx2 mRNA. This coincided with an increase in osteoclasts, in Rankl/Opg mRNA ratio and with the formation of osteolytic lesions. Thus NBL cells promote osteoblastogenesis in the BM by increasing VEGFA expression in BMMSC. Our study provides a new insight into the role of VEGFA in NBL metastases by pointing to the role of stroma-derived intracrine VEGFA in osteoblastogenesis.

Understanding multiple myeloma

Understanding multiple myeloma

Abstract BS1-2: Reactive Stroma as Therapeutic Targets in Breast Cancer Bone Metastasis

Abstract Metastasis represents the most devastating stage of cancer progression. Bone metastasis is a frequent occurrence in breast cancer, affecting more than 70% of late stage cancer patients. Discovering bone metastasis genes that are clinically relevant and functionally important are critical for the development of novel therapeutics for high-risk breast cancer patients. We apply a multidisciplinary approach to analyze the molecular basis of breast cancer bone metastasis, combining functional genomics tools with animal models and clinical analysis of cancer metastasis. Candidate bone metastasis genes were identified from gene expression profiling of highly bone metastatic cells derived after in vivo selection in animal models. Functional characterization of these genes revealed their novel role in mediating tumor-stromal interactions essential for the formation of osteolytic bone metastasis. We discovered a novel metalloprotease-initiated, EGFR-dependent paracrine signaling cascade among tumor cells, osteoblasts and osteoclasts that promotes the maturation of bone-degrading osteoclasts and formation of osteolytic lesions. Using in vivo imaging technology, we showed that TGFbis released from bone matrix upon bone destruction, and signals to breast cancer to further enhance their malignancy in developing bone metastasis. We furthered identified Jagged1 as a TGFb target genes in tumor cells that engaged bone stromal cells through the activation of Notch signaling to provide a positive feedback to promote tumor growth and to activate osteoclast differentiation. More recently, we identified osteoclast-derived miRNAs as biomarkers and therapeutic targets of osteolytic bone metastasis. Importantly, pharmaceutical inhibitors against EGFR, TGFb and Notch signaling pathways and miRNAs blocking osteoclast differentiation significantly reduce the development of bone metastasis, suggesting novel avenues for clinical management of skeletal complications of breast cancer. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr BS1-2.

CCR1 blockade reduces tumor burden and osteolysis in vivo in a mouse model of myeloma bone disease

AbstractThe chemokine CCL3/MIP-1α is a risk factor in the outcome of multiple myeloma (MM), particularly in the development of osteolytic bone disease. This chemokine, highly overexpressed by MM cells, can signal mainly through 2 receptors, CCR1 and CCR5, only 1 of which (CCR1) is responsive to CCL3 in human and mouse osteoclast precursors. CCR1 activation leads to the formation of osteolytic lesions and facilitates tumor growth. Here we show that formation of mature osteoclasts is blocked by the highly potent and selective CCR1 antagonist CCX721, an analog of the clinical compound CCX354. We also show that doses of CCX721 selected to completely inhibit CCR1 produce a profound decrease in tumor burden and osteolytic damage in the murine 5TGM1 model of MM bone disease. Similar effects were observed when the antagonist was used prophylactically or therapeutically, with comparable efficacy to that of zoledronic acid. 5TGM1 cells were shown to express minimal levels of CCR1 while secreting high levels of CCL3, suggesting that the therapeutic effects of CCX721 result from CCR1 inhibition on non-MM cells, most likely osteoclasts and osteoclast precursors. These results provide a strong rationale for further development of CCR1 antagonists for the treatment of MM and associated osteolytic bone disease.

Angiotensin‐(1‐7) attenuates metastatic prostate cancer and reduces osteoclastogenesis

Angiotensin-(1-7) [Ang-(1-7)] is an endogenous, heptapeptide hormone with anti-proliferative and anti-angiogenic properties. The primary objective of this study was to determine whether Ang-(1-7) effectively reduces prostate cancer metastasis in mice. Human PC3 prostate cancer cells were injected into the aortic arch via the carotid artery of SCID mice pre-treated with Ang-(1-7) or injected into the tibia of athymic mice, administered Ang-(1-7) for 5 weeks beginning 2 weeks post-injection. Tumor growth and volume were determined by bioluminescent and magnetic resonance imaging. The presence of tumors was confirmed by hematoxylin and eosin staining; TRAP histochemistry was used to identify osteolytic lesions. The effect of Ang-(1-7) on osteoclastogenesis was assessed in differentiated bone marrow cells. Pre-treatment with Ang-(1-7) prevented metastatic tumor formation following intra-aortic injection of PC3 cells, while 83% of untreated mice developed tumors in metastatic sites. Circulating VEGF was significantly higher in control mice compared to mice administered Ang-(1-7). A 5-week regimen of the heptapeptide hormone attenuated intra-tibial tumor growth; Ang-(1-7) was significantly higher in the tibia of treated mice than in control animals. Osteoclastogenesis was reduced by 50% in bone marrow cells differentiated in the presence of Ang-(1-7), suggesting that the heptapeptide hormone prevents the formation of osteolytic lesions to reduce tumor survival in the bone microenvironment. These findings suggest that Ang-(1-7) may serve as an anti-angiogenic and anti-metastatic agent for advanced prostate cancer. By extension, the heptapeptide hormone may provide effective therapy for bone metastasis produced from primary tumors of the lung and breast.