Bone Metastases In Mice Research Articles

Abstract PO2-27-02: Re-sensitizing the Refractory Breast Cancer Bone Metastasis to Endocrine Therapies

Abstract Approximately 90% of Breast Cancer (BrCa) related deaths are caused by metastasis and bone is the first and most frequent site of breast cancer metastatic expansion, which can lead to severe pain, immobility, fractures, and nerve damage, as well as the development of metastases in other organs, hence; a poor quality of life in patients. Currently, treatment options for these bone metastases (BoM) are limited because most patients with BoM shows reduced ER dependency which leads to resistance to standard endocrine or hormonal therapies for estrogen receptor-positive (ER+) BrCa. The long-term goal of the proposed research is to develop a strategy where metastatic cancer cells in bone can be dependent on ER signaling again thereby restoring their sensitivity to endocrine therapies. We observed a transient loss of ER expression in concert with enhanced activity of the transcriptional coactivators YAP/TAZ - the downstream effectors of the Hippo pathway (responsible for regulating cell proliferation and death) during bone colonization by BrCa cells. We hypothesize that the interaction between cancer cells and osteoblasts in the bone microenvironment leads to the activation of calcium signaling in cancer cells, resulting in the de-phosphorylation and nuclear localization of YAP/TAZ which subsequently suppresses the transcription of the estrogen receptor gene (ESR). Our research strategy is as presented below. First, we will employ a combination of 2D/3D co-culture models of BoM along with a mechanical loading system and determine the molecular mechanism driving the dynamical activation of YAP/TAZ in BrCa BoM. Using a unique ex vivo BM model called Bone In Culture Array (BICA) which replicates the in-vivo conditions, we will determine if verteporfin, a YAP/TAZ antagonist, can synergize and possibly enhance the effects of Fulvestrant, an ER antagonist in current use for certain types of BrCa in preventing BoM outgrowths and treating established BoM in mice. This research could ultimately lead to a favorable combination strategy to treat endocrine-resistant BoM in BrCa patients and prolong their survival. Citation Format: Anindita Das, Cheyenne Ernst. Re-sensitizing the Refractory Breast Cancer Bone Metastasis to Endocrine Therapies [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-27-02.

Interrogating Estrogen Signaling Pathways in Human ER-Positive Breast Cancer Cells Forming Bone Metastases in Mice.

Breast cancer bone metastases (BMET) are incurable, primarily osteolytic, and occur most commonly in estrogen receptor-α positive (ER+) breast cancer. ER+ human breast cancer BMET modeling in mice has demonstrated an estrogen (E2)-dependent increase in tumor-associated osteolysis and bone-resorbing osteoclasts, independent of estrogenic effects on tumor proliferation or bone turnover, suggesting a possible mechanistic link between tumoral ERα-driven osteolysis and ER+ bone progression. To explore this question, inducible secretion of the osteolytic factor, parathyroid hormone-related protein (PTHrP), was utilized as an in vitro screening bioassay to query the osteolytic potential of estrogen receptor- and signaling pathway-specific ligands in BMET-forming ER+ human breast cancer cells expressing ERα, ERß, and G protein-coupled ER. After identifying genomic ERα signaling, also responsibility for estrogen's proliferative effects, as necessary and sufficient for osteolytic PTHrP secretion, in vivo effects of a genomic-only ER agonist, estetrol (E4), on osteolytic ER+ BMET progression were examined. Surprisingly, while pharmacologic effects of E4 on estrogen-dependent tissues, including bone, were evident, E4 did not support osteolytic BMET progression (vs robust E2 effects), suggesting an important role for nongenomic ER signaling in ER+ metastatic progression at this site. Because bone effects of E4 did not completely recapitulate those of E2, the relative importance of nongenomic ER signaling in tumor vs bone cannot be ascertained here. Nonetheless, these intriguing findings suggest that targeted manipulation of estrogen signaling to mitigate ER+ metastatic progression in bone may require a nuanced approach, considering genomic and nongenomic effects of ER signaling on both sides of the tumor/bone interface.

In Silico Discovery of Stapled Peptide Inhibitor Targeting the Nur77-PPARγ Interaction and Its Anti-Breast-Cancer Efficacy.

The binding of peroxisome proliferator-activated receptor γ (PPARγ) to the orphan nuclear receptor Nur77 facilitates the ubiquitination and degradation of Nur77, and leads to aberrant fatty acid uptake for breast cancer progression. Because of its crucial role in clinical prognosis, the interaction between Nur77 and PPARγ is an attractive target for anti-breast-cancer therapy. However, developing an inhibitor of the Nur77-PPARγ interaction poses a technical challenge due to the absence of the crystal structure of PPARγ and its corresponding interactive model with Nur77. Here, ST-CY14, a stapled peptide,is identified as a potent modulator of Nur77 with a KD value of 3.247×10-8M by in silico analysis, rational design, and structural modification. ST-CY14 effectively increases Nur77 protein levels by blocking the Nur77-PPARγ interaction, thereby inhibiting lipid metabolism in breast tumor cells. Notably, ST-CY14 significantly suppresses breast cancer growth and bone metastasis in mice. The findings demonstrate the feasibility of exploiting directly Nur77-PPARγ interaction in breast cancer, and generate what to the best knowledge is the first direct inhibitor of the Nur77-PPARγ interaction available for impeding fatty acid uptake and therapeutic development.

ICAM1 promotes bone metastasis via integrin‐mediated TGF‐β/EMT signaling in triple‐negative breast cancer

Bone-related events caused by breast cancer bone metastasis substantially compromise the survival and quality of life of patients. Because triple-negative breast cancer (TNBC) lacks hormone receptors and Her2-targeted therapeutic options, progress in the treatment of TNBC bone metastasis has been very slow. Intercellular adhesion molecule 1 (ICAM1) is highly expressed in various cancers and plays an important role in tumorigenesis and metastasis. However, the effect and mechanism of ICAM1 in TNBC bone metastasis are still unknown. We found that ICAM1 was highly expressed in TNBC and correlated with prognosis in TNBC patients. Cell lines with high expression of ICAM1 exhibited enhanced bone metastasis in tumor-bearing mice, and silencing ICAM1 expression significantly inhibited bone metastasis in mice. ICAM1 interacted with integrins to activate the epithelial-to-mesenchymal transition program through TGF-β/SMAD signaling, ultimately enhancing cell invasiveness. Therefore, the findings of the present study provide a strong rationale for the application of ICAM1-targeted therapy in TNBC patients with bone metastasis.

Abstract P033: Bone microenvironment-suppressed T cells increase osteoclast formation and the development of osteolytic bone metastases in mice

Abstract Bone metastases are a highly debilitating complication in more than 70% of patients with advanced breast and prostate cancer, causing fractures, nerve compression, hypercalcemia. Currently approved treatments fail to cure bone metastases or increase patient survival. Immunotherapies activating T cells to fight cancer cells are changing cancer treatment, causing a durable response in some patients. However, it remains unclear whether immunotherapy could benefit patients with bone metastases. The bone microenvironment combines various immunosuppressive factors that could limit its efficacy. Also, T cells could increase bone resorption releases pro-metastatic growth factors from the bone matrix that can increase cancer cell growth. Using syngeneic mouse models, our study revealed that bone metastases from 4T1 breast cancer contain tumor-infiltrating lymphocyte (TILs) and that their development is increased in normal mice compared to immunodeficient and T-cell depleted mice (x3.6 and x1.6, respectively, p<0.05). This effect seemed caused by the TILs in bone, as T-cell depletion did not affect bone metastases from RM-1 prostate cancer cells that lack TILs and increased the volume of 4T1 orthotopic tumors. T cells from bone metastases expressed the pro-osteoclastic genes Rankl and Tnfa, and increased osteoclast formation ex vivo and in vivo, at the tumor-bone interface, contributing to bone metastasis development. This pro-osteoclastic effect is specific to inactivated T cells, since activated T cells, secreting IFNγ and IL-4, actually suppressed osteoclastogenesis, which could benefit patients. We confirmed that T cells in bone metastases were not activated, as >85% of T cells lacked the activation marker CD69. In addition, T cells from 4T1 bone metastases could not be activated in ex vivo cultures confirming the presence of immunosuppressive factors in this microenvironment. 4T1 bone metastases were associated with an increase of polymorphonuclear- and monocytic-MDSCs, metabolically active as they produced elevated levels of ROS and NO, respectively. While effective in other models, the PDE-5 inhibitor sildenafil and the bisphosphonate zoledronic acid did not affect the levels of MDSCs in bone metastases or their production of ROS and NO. Seeking other therapeutic targets, we found that 80% of monocytic-MDSCs are PD-L1+ in bone, which could trigger T-cell suppression since 70% express its receptor, PD-1. Collectively, our findings identified a new mechanism by which suppressed T cells increase osteoclastogenesis and bone metastases, and also provide a rationale for using immune checkpoint inhibitors since T-cell activation would increase their anti-cancer and their anti-osteoclastic properties. Citation Format: Danna L. Arellano, Patricia Juárez, Paloma S. Almeida-Luna, Felipe Olvera, Samanta Jiménez, Pierrick G. J. Fournier. Bone microenvironment-suppressed T cells increase osteoclast formation and the development of osteolytic bone metastases in mice [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P033.

CCL8 mediates crosstalk between endothelial colony forming cells and triple-negative breast cancer cells through IL-8, aggravating invasion and tumorigenicity.

Triple-negative breast cancer (TNBC) is an aggressive type of breast cancer with a poor prognosis for which no effective therapeutic measures are currently available. The present study aimed to investigate whether interactions with endothelial colony-forming cells (ECFCs) promote aggressive progression of TNBC cells. Herein, using an indirect co-culture system, we showed that co-culture increased the invasive and migratory phenotypes of both MDA-MB-231 TNBC cells and ECFCs. Through a cytokine antibody array and RT-PCR analysis, we revealed that co-culture markedly induced secretion of the chemokine C-C motif ligand (CCL)8 from ECFCs and that of interleukin (IL)-8 from MDA-MB-231 cells. CCL8 was crucial for ECFC-induced IL-8 secretion and invasion of MDA-MB-231 cells as well as for MDA-MB-231-enhanced MMP-2 secretion and angiogenesis of ECFCs. We suggest c-Jun as a transcription factor for CCL8-induced IL-8 expression in MDA-MB-231 cells. IL-8 was important for co-culture-induced CCL8 and MMP-2 upregulation and invasion of ECFCs. Notably, our findings reveal a positive feedback loop between CCL8 and IL-8, which contributes to the aggressive phenotypes of both ECFC and TNBC cells. Using an MDA-MB-231 cell-based xenograft model, we show that tumor growth and metastasis are increased by co-injected ECFCs in vivo. Increased expression of IL-8 was observed in tissues with bone metastases in mice injected with conditioned media from co-cultured cells. High IL-8 levels are correlated with poor recurrence-free survival in TNBC patients. Together, these results suggest that CCL8 and IL-8 mediate the crosstalk between ECFCs and TNBC, leading to aggravation of tumorigenicity in TNBC.

A Murine Bone Metastasis Model Using Caudal Artery Injection and Bioluminescence Imaging.

The current standard murine model of bone metastasis by using intracardiac injection (IC) has some limitations despite the great utility of this model. This fact emphasizes the need for a new murine model to accelerate basic research of bone metastasis. The present protocol provides instructions on caudal artery (CA) injection that is an easy-to-use method to reliably construct a murine bone metastasis model with a variety type of cancer cell lines. Bioluminescence imaging visualized that cancer cells injected via the caudal artery in the tail were efficiently delivered to a hind limb bone, where it is a common site affected with bone metastasis in mice. CA injection rarely causes stress-induced acute death in mice and enables us to inject a large number of cancer cells, thereby greatly increasing the frequency of bone metastasis in hind limb bones. Importantly, CA injection is technically as easy as tail vein injection and causes no lethal stress, indicating that it is a model that also contributes to animal welfare. CA injection model, therefore, could represent a powerful tool for many researchers to study molecular mechanisms of bone metastasis in mice.

Combinatorial effect of radium-223 and irreversible electroporation on prostate cancer bone metastasis in mice

Background Metastatic prostate cancer in bone is difficult to treat as the tumor cells are relatively resistant to hormonal or chemotherapies when compared to primary prostate cancer. Irreversible electroporation (IRE) is a minimally invasive ablation procedure that has potential applications in the management of prostate cancer in bone. However, a common limitation of IRE is tumor recurrence, which arises from incomplete ablation that allows remaining cancer cells to proliferate. In this study, we combined IRE with radium-223 (Ra-223), a bone-seeking radionuclide that emits short track length alpha particles and thus is associated with reduced damage to the bone marrow and evaluated the impact of the combination treatment on bone-forming prostate cancer tumors. Methods The antitumor activity of IRE and Ra-223 as single agents and in combination was tested in vitro against three bone morphogenetic protein 4 (BMP4)-expressing prostate cancer cell lines (C4-2B–BMP4, Myc-CaP–BMP4, and TRAMP-C2–BMP4). Similar evaluation was performed in vivo using a bone-forming C4-2B–BMP4 tumor model in nude mice. Results IRE and Ra-223 as monotherapy inhibited prostate cancer cell proliferation in vitro, and their combination resulted in significant reduction in cell viability compared to monotherapy. In vivo evaluation revealed that IRE with single-dose administration of Ra-233, compared to IRE alone, reduced the rate of tumor recurrence by 40% following initial apparent complete ablation and decreased the rate of proliferation of incompletely ablated tumor as quantified in Ki-67 staining (53.58 ± 16.0% for IRE vs. 20.12 ± 1.63%; for IRE plus Ra-223; p = 0.004). Histological analysis qualitatively showed the enhanced killing of tumor cells adjacent to bone by Ra-223 compared to those treated with IRE alone. Conclusion IRE in combination with Ra-223, which enhanced the destruction of cancer cells that are adjacent to bone, resulted in reduction of tumor recurrence through improved clearance of proliferative cells in the tumor region.

Bone Microenvironment-Suppressed T Cells Increase Osteoclast Formation and Osteolytic Bone Metastases in Mice.

ABSTRACTImmunotherapies use components of the immune system, such as T cells, to fight cancer cells, and are changing cancer treatment, causing durable responses in some patients. Bone metastases are a debilitating complication in advanced breast and prostate cancer patients. Approved treatments fail to cure bone metastases or increase patient survival and it remains unclear whether immunotherapy could benefit patients. The bone microenvironment combines various immunosuppressive factors, and combined with T cell products could increase bone resorption fueling the vicious cycle of bone metastases. Using syngeneic mouse models, our study revealed that bone metastases from 4T1 breast cancer contain tumor‐infiltrating lymphocyte (TILs) and their development is increased in normal mice compared to immunodeficient and T‐cell depleted mice. This effect seemed caused by the TILs specifically in bone, because T‐cell depletion increased 4T1 orthotopic tumors and did not affect bone metastases from RM‐1 prostate cancer cells, which lack TILs. T cells increased osteoclast formation ex vivo and in vivo contributing to bone metastasis vicious cycle. This pro‐osteoclastic effect is specific to unactivated T cells, because activated T cells, secreting interferon γ (IFNγ) and interleukin 4 (IL‐4), actually suppressed osteoclastogenesis, which could benefit patients. However, non‐activated T cells from bone metastases could not be activated in ex vivo cultures. 4T1 bone metastases were associated with an increase of functional polymorphonuclear and monocytic myeloid‐derived suppressor cells (MDSCs), potent T‐cell suppressors. Although effective in other models, sildenafil and zoledronic acid did not affect MDSCs in bone metastases. Seeking other therapeutic targets, we found that monocytic MDSCs are more potent suppressors than polymorphonuclear MDSCs, expressing programmed cell death receptor‐1 ligand (PD‐L1)+ in bone, which could trigger T‐cell suppression because 70% express its receptor, programmed cell death receptor‐1 (PD‐1). Collectively, our findings identified a new mechanism by which suppressed T cells increase osteoclastogenesis and bone metastases. Our results also provide a rationale for using immunotherapy because T‐cell activation would increase their anti‐cancer and their anti‐osteoclastic properties. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Optimization of a syngeneic murine model of bone metastasis

Many cancers metastasize to the bones, particularly in cases of breast and prostate cancers. Due to the “vicious cycle” of cancer cells inducing bone resorption, which promotes further tumor growth, they are difficult to treat and may lead to extreme pain. These factors increase the urgency for emerging therapeutics that target bone metastases more specifically and effectively. Animal studies are essential to the development of any therapeutics, but also require robust animal models of human diseases. Robust animal models are often challenging to develop in the case of bone metastasis studies. Previous methods to induce bone metastasis include intracardiac, intravenous, subcutaneous via mammary fat pad, and intraosseous cancer cell injections, but these methods all have limitations. By contrast, the caudal artery route of injection offers more robust bone metastasis, while also resulting in a lower rate of vital organ metastases than that of other routes of tumor implantation. A syngeneic animal model of bone metastasis is necessary in many cancer studies, because it allows the use of immunocompetent animals, which more accurately mimic cancer development observed in immunocompetent humans. Here we present a detailed method to generate robust and easily monitored 4T1-CLL1 syngeneic bone metastases with over 95% occurrence in BALB/c mice, within two weeks. This method can potentially increase consistency between animals in bone cancer metastasis studies and reduce the number of animals needed for studying bone metastases in mice.

Bone-Induced Expression of Integrin β3 Enables Targeted Nanotherapy of Breast Cancer Metastases

Bone metastases occur in approximately 70% of metastatic breast cancer patients, often leading to skeletal injuries. Current treatments are mainly palliative and underscore the unmet clinical need for improved therapies. In this study, we provide preclinical evidence for an antimetastatic therapy based on targeting integrin β3 (β3), which is selectively induced on breast cancer cells in bone by the local bone microenvironment. In a preclinical model of breast cancer, β3 was strongly expressed on bone metastatic cancer cells, but not primary mammary tumors or visceral metastases. In tumor tissue from breast cancer patients, β3 was significantly elevated on bone metastases relative to primary tumors from the same patient (n = 42). Mechanistic investigations revealed that TGFβ signaling through SMAD2/SMAD3 was necessary for breast cancer induction of β3 within the bone. Using a micelle-based nanoparticle therapy that recognizes integrin αvβ3 (αvβ3-MPs of ∼12.5 nm), we demonstrated specific localization to breast cancer bone metastases in mice. Using this system for targeted delivery of the chemotherapeutic docetaxel, we showed that bone tumor burden could be reduced significantly with less bone destruction and less hepatotoxicity compared with equimolar doses of free docetaxel. Furthermore, mice treated with αvβ3-MP-docetaxel exhibited a significant decrease in bone-residing tumor cell proliferation compared with free docetaxel. Taken together, our results offer preclinical proof of concept for a method to enhance delivery of chemotherapeutics to breast cancer cells within the bone by exploiting their selective expression of integrin αvβ3 at that metastatic site. Cancer Res; 77(22); 6299-312. ©2017 AACR.

Halofuginone inhibits TGF-β/BMP signaling and in combination with zoledronic acid enhances inhibition of breast cancer bone metastasis.

More efficient therapies that target multiple molecular mechanisms are needed for the treatment of incurable bone metastases. Halofuginone is a plant alkaloid-derivative with antiangiogenic and antiproliferative effects. Here we demonstrate that halofuginone is an effective therapy for the treatment of bone metastases, through multiple actions that include inhibition of TGFβ and BMP-signaling.Halofuginone blocked TGF-β-signaling in MDA-MB-231 and PC3 cells showed by inhibition of TGF-β–induced Smad-reporter, phosphorylation of Smad-proteins, and expression of TGF-β-regulated metastatic genes. Halofuginone increased inhibitory Smad7-mRNA and reduced TGF-β-receptor II protein. Proline supplementation but not Smad7-knockdown reversed halofuginone-inhibition of TGF-β-signaling. Halofuginone also decreased BMP-signaling. Treatment of MDA-MB-231 and PC3 cells with halofuginone reduced the BMP-Smad-reporter (BRE)4, Smad1/5/8-phosphorylation and mRNA of the BMP-regulated gene Id-1. Halofuginone decreased immunostaining of phospho-Smad2/3 and phospho-Smad1/5/8 in cancer cells in vivo.Furthermore, halofuginone decreased tumor-take and growth of orthotopic-tumors. Mice with breast or prostate bone metastases treated with halofuginone had significantly less osteolysis than control mice. Combined treatment with halofuginone and zoledronic-acid significantly reduced osteolytic area more than either treatment alone. Thus, halofuginone reduces breast and prostate cancer bone metastases in mice and combined with treatment currently approved by the FDA is an effective treatment for this devastating complication of breast and prostate-cancer.

Human antigen R-regulated CCL20 contributes to osteolytic breast cancer bone metastasis

Breast cancer mainly spreads to bone, causing decreased survival of patient. Human antigen R (HuR) and chemokines are important molecules associated with mRNA stability and cell-cell interaction in cancer biology. Here, HuR knockdown inhibited bone metastasis and osteolysis of metastatic breast cancer cells in mice and HuR expression promoted the metastatic ability of cancer cells via CCL20 and GM-CSF. In contrast with the findings for GM-CSF, ELAVL1 and CCL20 expressions were markedly increased in breast tumor tissues and ELAVL1 expression showed a strong positive correlation with CCL20 expression in breast cancer subtypes, particularly the basal-like subtype. Metastasis-free survival and overall survival were decreased in the breast cancer patients with high CCL20 expression. We further confirmed the role of CCL20 in breast cancer bone metastasis. Intraperitoneal administration of anti-CCL20 antibodies inhibited osteolytic breast cancer bone metastasis in mice. Treatment with CCL20 noticeably promoted cell invasion and the secretion of MMP-2/9 in the basal-like triple-negative breast cancer cell lines, not the luminal. Moreover, CCL20 elevated the receptor activator of nuclear factors kappa-B ligand/osteoprotegerin ratio in breast cancer and osteoblastic cells and mediated the crosstalk between these cells. Collectively, HuR-regulated CCL20 may be an attractive therapeutic target for breast cancer bone metastasis.

A Novel Near-infrared Fluorescent Protein, iRFP720, Facilitates Transcriptional Profiling of Prostate Cancer Bone Metastasis in Mice.

Bone represents a frequent site of prostate cancer metastasis. As the molecular mechanism remains unclear, an accessible animal model is required. We established a novel murine metastasis model using near-infrared fluorescent protein iRFP720-labelled prostate cancer (PC3) cells. To clarify transcriptional alterations during metastasis, iRFP720-PC3 cells were intracardially injected into male mice. mRNA expression profiles of metastasis in bone using marrow cancer cells extracted by centrifugal separation and cell sorting were compared with those of parental cells by microarray. Differentially expressed genes were analyzed by pathway analysis. We identified 327 and 197 genes being up- and down-regulated, respectively. Pathway analysis revealed that the p53 signaling pathway, extracellular matrix receptor interaction, Mammalian target of rapamycin signaling pathway, cancer-related pathways, small cell lung cancer, and Escherichia coli infection response were altered. iRFP720 is useful for in vivo cell detection/isolation. The results of expression analysis may improve prostate cancer treatment strategies.

Semaphorin 4D Promotes Skeletal Metastasis in Breast Cancer.

Bone density is controlled by interactions between osteoclasts, which resorb bone, and osteoblasts, which deposit it. The semaphorins and their receptors, the plexins, originally shown to function in the immune system and to provide chemotactic cues for axon guidance, are now known to play a role in this process as well. Emerging data have identified Semaphorin 4D (Sema4D) as a product of osteoclasts acting through its receptor Plexin-B1 on osteoblasts to inhibit their function, tipping the balance of bone homeostasis in favor of resorption. Breast cancers and other epithelial malignancies overexpress Sema4D, so we theorized that tumor cells could be exploiting this pathway to establish lytic skeletal metastases. Here, we use measurements of osteoblast and osteoclast differentiation and function in vitro and a mouse model of skeletal metastasis to demonstrate that both soluble Sema4D and protein produced by the breast cancer cell line MDA-MB-231 inhibits differentiation of MC3T3 cells, an osteoblast cell line, and their ability to form mineralized tissues, while Sema4D-mediated induction of IL-8 and LIX/CXCL5, the murine homologue of IL-8, increases osteoclast numbers and activity. We also observe a decrease in the number of bone metastases in mice injected with MDA-MB-231 cells when Sema4D is silenced by RNA interference. These results are significant because treatments directed at suppression of skeletal metastases in bone-homing malignancies usually work by arresting bone remodeling, potentially leading to skeletal fragility, a significant problem in patient management. Targeting Sema4D in these cancers would not affect bone remodeling and therefore could elicit an improved therapeutic result without the debilitating side effects.

Metastatic breast tumors break down bone

Cancer Most breast cancer cells activate the breakdown of bone to promote metastases. Wang et al. found that the ABL kinases enhanced the ability of breast cancer cells to invade and break down bone in mice. In breast cancer cells, the ABL kinases activated pathways that triggered the transcription of genes encoding factors that activate osteoclasts (cells that break down bone) and those that enhanced the survival of breast cancer cells in the bone micro-environment. An ABL-specific inhibitor decreased bone metastasis in mice. Sci. Signal. 9 , ra12 (2016).

Excess TGF-β mediates muscle weakness associated with bone metastases in mice.

Cancer-associated muscle weakness is poorly understood and there is no effective treatment. Here, we find that seven different mouse models of human osteolytic bone metastases, representing breast, lung and prostate cancers, as well as multiple myeloma exhibited impaired muscle function, implicating a role for the tumor-bone microenvironment in cancer-associated muscle weakness. We found that TGF-β, released from the bone surface as a result of metastasis-induced bone destruction upregulated NADPH oxidase 4 (Nox4), resulting in elevated oxidization of skeletal muscle proteins, including the ryanodine receptor/calcium (Ca2+) release channel (RyR1). The oxidized RyR1 channels leaked Ca2+, resulting in lower intracellular signaling required for proper muscle contraction. We found that inhibiting RyR1 leak, TGF-β signaling, TGF-β release from bone or Nox4 all improved muscle function in mice with MDA-MB-231 bone metastases. Humans with breast cancer- or lung cancer-associated bone metastases also had oxidized skeletal muscle RyR1 that is not seen in normal muscle. Similarly, skeletal muscle weakness, higher levels of Nox4 protein and Nox4 binding to RyR1, and oxidation of RyR1 were present in a mouse model of Camurati-Engelmann disease, a non-malignant metabolic bone disorder associated with increased TGF-β activity. Thus, metastasis-induced TGF-β release from bone contributes to muscle weakness by decreasing Ca2+-induced muscle force production.

Abstract B53: RANK- and AR-mediated signaling axes in prostate cancer metastasis

Abstract Introduction and Objective: The development and progression of prostate cancer (PCa) is strongly associated with androgen receptor (AR)-mediated signaling. In our lab we found in addition to AR, Receptor Activator of NF-κB (RANK) plays a coordinated role in driving PCa progression. PCa cells are initially androgen-sensitive (AS) and respond to androgen-deprivation therapy (ADT). With time, while some PCa cells remain AR positive others are AR-negative, but they exhibit a common androgen-insensitive (AI) phenotype with increased invasiveness and metastatic potential. Our lab showed activation of RANK ligand (RANKL)-RANK signal network in human PCa specimens correlated with poor prognosis of PCa patients. The objectives of this study are: 1) to compare RANK- and AR-mediated signaling axes in a PCa cell line and to study their coordinated regulation with growth under 2-D versus 3-D cultures; 2) to investigate the behaviors of PCa cells with both activated RANK- and AR- signaling axes; 3) to determine if RANKL-RANK signal network activates integrin expression and promotes the attachment of PCa cells to type 1 collagen (Col1), rich in bone matrix. Methods: Stable RANKL-expressing LNCaP cells, LN-RANKL, were obtained and their AR expression were compared under 2-D versus 3-D cultures. Expression of AR and its functionality, at day 7, was further confirmed using AR promoter- and PSA promoter-luciferase activities. RANKL-RANK-mediated expression of cell adhesion, mediated by integrins, is determined. AR and RANK-mediated adhesion will be assessed in the presence and absence of isotype-specific integrins and extracellular matrices. Results: Using the suspension 3-D growth model, we found that a functional AR expression is restored in LNCaP-RANKL cells when compared to their growth under 2-D condition. We observed a downregulation of AP4 transcription factor, as possible mechanism negatively regulating AR gene transcription. We showed that down-regulation of AP4 using sh- or si-RNA in 2-D culture also restored functional AR expression and increased vimentin expression. It did not, however, affect the expression of stem cell markers such as CD133, CD44, and CD49f. We further investigated metastatic behavior of these cell lines in suspension model. LNCaP-RANKL cells grown on osteoblastic matrices travelled much faster and longer distance compared to LNCaP-RANKL cells grown on 2-D or the control LNCaP-Neo cells. LNCaP-RANKL cells grown in suspension showed higher and faster adhesion to Col1 coated plate in comparison with their parental cells. Cell adhesion was similar between the two cell lines on plastic- and fibronectin-coated plates. This observation is in agreement with higher expression of α2β1, which was confirmed by FACS and qRT-PCR, and a higher propensity for bone metastasis in mice. Conclusions: 3-D culture systems are established to study changes in RANK- and AR-mediated signal network and results are correlated with tumor growth and metastasis in mice. Our data suggest, in 3-D suspension model, AR expression is restored through AP-4 downregulation and increased aggressiveness of PCa. RANK-mediated signal also increased PCa cells to adhere to Col1 and exhibit increased cell motility on collagen matrices. Given that RANKL and AR are co-expressed in clinical human PCa specimens, our model allows us better define RANK- and AR-signal axes at a molecular level in human PCa. Citation Format: Shabnam Ziaee, Leland W.K Chung. RANK- and AR-mediated signaling axes in prostate cancer metastasis. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B53. doi:10.1158/1538-7445.CHTME14-B53

S-20 : Exploiting the type-1 interferon pathway as a biomarker and therapeutic target for metastatic cancer

It has been well described that the initiation and progression of some cancers is tightly controlled by tumour immunosurveillance. Our recent findings suggest that in breast cancer, the balance of pro-tumour and anti-tumour immune responses is critical in dictating metastatic cell survival and outgrowth. We revealed that tumour cells secrete type I IFN and that loss of this signalling is a tumour cell intrinsic mechanism of evading metastasis-specific immune responses and promoting bone metastasis. We have demonstrated that restored type I IFN signalling in mice bearing 4T1.2 breast tumour cells significantly reduces bone metastasis and that metastasis suppression is linked to NK cell activation and decreased accumulation of immune suppressor cells [1] . The key role of the host immune system in responding to tumour cell-derived IFN has also been verified using mice deficient in the type I IFN receptor (Ifnar1). In these studies, the lack of host Ifnar1 increased the incidence of bone metastasis in mice bearing breast tumours of varying metastatic potential. Our current investigations also extend to orthotopic models of prostate cancer where similar observations of increased metastasis are seen in Ifnar1 null animals. Subsequent ex-vivo studies have confirmed a crucial role of IFN signalling to NK cells in tumour cell recognition. These findings have clinical relevance as decreased primary tumour expression of the type I IFN signature or key interferon regulatory factors predict an increased risk of metastatic relapse in breast cancer patients. Additionally, we have now demonstrated potent anti-metastatic effects of IFN-based therapeutics alone or in combination with chemotherapeutics. Our studies are now focused on refining the prognostic markers that best predict metastatic relapse in patients and investigating the therapeutic window where restoring such immune responses is critical in metastasis suppression.

Zoledronic acid at the time of castration prevented castration-induced bone metastasis in mice.

Androgen deprivation therapy (ADT) is known to cause bone loss in a majority of patients with castration-resistant prostate cancer (CRPC). A study published in this issue of Endocrine-Related Cancer by Ottewell and colleagues shows that ADT increased bone resorption and triggered growth of disseminated prostate cancer (CaP) cells to form bone metastasis using an in vivo model. However, prevention of bone decay by weekly administration of zoledronic acid (ZOL) at the time of castration prevented ADT-induced tumor growth in bone. Recently, two publications from Japan have demonstrated that ZOL combined with ADT improved outcomes for patients with treatment-naïve CaP with bone metastasis. The mechanistic cause for these patients having an improved overall survival compared with those who were treated with ZOL after ADT initiation or before metastasis development was never explained. Ottewell and colleague's study now suggests that it is the bone loss caused by ADT that promoted bone metastasis, and if ZOL is administered at the time of ADT initiation, it would prevent this bone loss and prolong skeletal-related event-free survival.