Vicious Cycle Of Bone Metastasis Research Articles

The role of IL-1B in breast cancer bone metastasis

Interleukin-1B (IL-1B) is a potent pro-inflammatory cytokine that plays multiple, pivotal roles, in the complex interplay between breast cancer cells and the bone microenvironment. IL-1B is involved in the growth of the primary tumours, regulation of inflammation within the tumour microenvironment, promotion of epithelial to mesenchymal transition (EMT), migration and invasion. Moreover, when breast cancer cells arrive in the bone microenvironment there is an upregulation of IL-1B which promotes the creation of a conducive niche for metastatic breast cancer cells as well as stimulating initiation of the vicious cycle of bone metastasis. Pre-clinical studies have demonstrated that inhibition of IL-1 signalling reduces bone metastasis from oestrogen receptor positive/triple-negative breast cancers in various mouse models. However, effects on primary tumours and soft tissue metastasis remain controversial with some studies showing increased tumour growth in these sites, whilst others show no effects. Notably, combining anti-IL-1 therapy with standard-of-care treatments, such as chemotherapy and immunotherapy, has been demonstrated to reduce the growth of primary tumours, bone metastasis, as well as metastatic outgrowth in other organs. This review focuses on the mechanisms by which IL-1B promotes breast cancer bone metastasis.

Insights into immuno-oncology drug development landscape with focus on bone metastasis.

Bone is among the main sites of metastasis in breast, prostate and other major cancers. Bone metastases remain incurable causing high mortality, severe skeletal-related effects and decreased quality of life. Despite the success of immunotherapies in oncology, no immunotherapies are approved for bone metastasis and no clear benefit has been observed with approved immunotherapies in treatment of bone metastatic disease. Therefore, it is crucial to consider unique features of tumor microenvironment in bone metastasis when developing novel therapies. The vicious cycle of bone metastasis, referring to crosstalk between tumor and bone cells that enables the tumor cells to grow in the bone microenvironment, is a well-established concept. Very recently, a novel osteoimmuno-oncology (OIO) concept was introduced to the scientific community. OIO emphasizes the significance of interactions between tumor, immune and bone cells in promoting tumor growth in bone metastasis, and it can be used to reveal the most promising targets for bone metastasis. In order to provide an insight into the current immuno-oncology drug development landscape, we used 1stOncology database, a cancer drug development resource to identify novel immunotherapies in preclinical or clinical development for breast and prostate cancer bone metastasis. Based on the database search, 24 immunotherapies were identified in preclinical or clinical development that included evaluation of effects on bone metastasis. This review provides an insight to novel immuno-oncology drug development in the context of bone metastasis. Bone metastases can be approached using different modalities, and tumor microenvironment in bone provides many potential targets for bone metastasis. Noting current increasing interest in the field of OIO, more therapeutic opportunities that primarily target bone metastasis are expected in the future.

Secreted microRNAs in bone metastasis

Bone metastasis is a common complication in several solid cancers, including breast, prostate, and lung. In the bone microenvironment, metastatic cancer cells disturb bone homeostasis leading to osteolytic or osteosclerotic lesions. Osteolytic lesions are characterized by an increased osteoclast-mediated bone resorption while osteosclerotic lesions are caused by enhanced activity of osteoblasts and formation of poor-quality bone. A common feature in bone metastasis is the complex interplay between the cancer cells and the cells of the bone microenvironment, which can occur already before the cancer cells enter the distant site. Cancer cells at the primary site can secrete soluble factors and extracellular vesicles to bone to create a "pre-metastatic niche" i.e., prime the microenvironment permissive for cancer cell homing, survival, and growth. Once in the bone, cancer cells secrete factors to activate the osteoclasts or osteoblasts and the so called "vicious cycle of bone metastases". These pathological cell-cell interactions are largely dependent on secreted proteins. However, increasing evidence demonstrates that secreted RNA molecules, in particular small non-coding microRNAs are critical mediators of the crosstalk between bone and cancer cells. This review article discusses the role of secreted miRNAs in bone metastasis development and progression, and their potential as non-invasive biomarkers.

Parathyroid hormone 1 receptor signaling mediates breast cancer metastasis to bone in mice.

Bone metastases are a common complication of breast cancer. We have demonstrated that intermittent administration of parathyroid hormone (PTH[1-34]) reduces the incidence of bone metastases in murine models of breast cancer by acting on osteoblasts to alter the bone microenvironment. Here, we examined the role of signaling mediated by PTH 1 receptor (PTH1R) in both osteoblasts and breast cancer cells in influencing bone metastases. In mice with impaired PTH1R signaling in osteoblasts, intermittent PTH did not reduce bone metastasis. Intermittent PTH also did not reduce bone metastasis when expression of PTH1R was knocked down in 4T1 murine breast cancer cells by shRNA. In 4T1 breast cancer cells, PTH decreased expression of PTH-related protein (PTHrP), implicated in the vicious cycle of bone metastases. Knockdown of PTHrP in 4T1 cells significantly reduced migration toward MC3T3-E1 osteoblasts, and migration was further inhibited by treatment with intermittent PTH. Conversely, overexpression of PTHrP in 4T1 cells increased migration toward MC3T3-E1 osteoblasts, and this was not inhibited by PTH. In conclusion, PTH1R expression is crucial in both osteoblasts and breast cancer cells for PTH to reduce bone metastases, and in breast cancer cells, this may be mediated in part by suppression of PTHrP.

Progress in Targeted Alpha-Particle-Emitting Radiopharmaceuticals as Treatments for Prostate Cancer Patients with Bone Metastases.

Bone metastasis remains a major cause of death in cancer patients, and current therapies for bone metastatic disease are mainly palliative. Bone metastases arise after cancer cells have colonized the bone and co-opted the normal bone remodeling process. In addition to bone-targeted therapies (e.g., bisphosphonate and denosumab), hormone therapy, chemotherapy, external beam radiation therapy, and surgical intervention, attempts have been made to use systemic radiotherapy as a means of delivering cytocidal radiation to every bone metastatic lesion. Initially, several bone-seeking beta-minus-particle-emitting radiopharmaceuticals were incorporated into the treatment for bone metastases, but they failed to extend the overall survival in patients. However, recent clinical trials indicate that radium-223 dichloride (223RaCl2), an alpha-particle-emitting radiopharmaceutical, improves the overall survival of prostate cancer patients with bone metastases. This success has renewed interest in targeted alpha-particle therapy development for visceral and bone metastasis. This review will discuss (i) the biology of bone metastasis, especially focusing on the vicious cycle of bone metastasis, (ii) how bone remodeling has been exploited to administer systemic radiotherapies, and (iii) targeted radiotherapy development and progress in the development of targeted alpha-particle therapy for the treatment of prostate cancer bone metastasis.

Small extracellular vesicles deliver osteolytic effectors and mediate cancer-induced osteolysis in bone metastatic niche.

Extracellular vesicles (EVs) play critical roles in regulating bone metastatic microenvironment through mediating intercellular crosstalks. However, little is known about the contribution of EVs derived from cancer cells to the vicious cycle of bone metastasis. Here, we report a direct regulatory mode between tumour cells and osteoclasts in metastatic niche of prostate cancer via vesicular miRNAs transfer. Combined analysis of miRNAs profiles both in tumour‐derived small EVs (sEVs) and osteoclasts identified miR‐152‐3p as a potential osteolytic molecule. sEVs were enriched in miR‐152‐3p, which targets osteoclastogenic regulator MAFB. Blocking miR‐152‐3p in sEVs upregulated the expression of MAFB and impaired osteoclastogenesis in vitro. In vivo experiments of xenograft mouse model found that blocking of miR‐152‐3p in sEVs significantly slowed down the loss of trabecular architecture, while systemic inhibition of miR‐152‐3p using antagomir‐152‐3p reduced the osteolytic lesions of cortical bone while preserving basic trabecular architecture. Our findings suggest that miR‐152‐3p carried by prostate cancer‐derived sEVs deliver osteolytic signals from tumour cells to osteoclasts, facilitating osteolytic progression in bone metastasis.

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).

Longitudinal evaluation of Wnt inhibitors and comparison with others serum osteoimmunological biomarkers in osteolytic bone metastasis

Bone and the immune system are closely linked: bone regulates the hematopoietic stem cells, which are precursors of immune cells, and several immunoregulatory cytokines influence the differentiation of bone cells, thus defining the osteoimmunological system. Cytokines and growth factors produced by immune and bone cells promote tumors in bone, supporting the vicious cycle of bone metastasis. Therefore osteoimmunological molecules linking the immune and bone systems could have diagnostic and prognostic potential for bone metastases. The osteoimmunologic Wnt pathway has been recently described as an important pathway with a vital role in bone carcinogenesis and metastatic progression. We examined the Wnt inhibitor DKK-1, sclerostin and several other osteoimmunological biomarkers involved in bone metastatic progression: RANKL, OPG, OPN, matrix metalloproteinase MMP-3 and the Receptor of Advanced Glycosylated End-products sRAGE. OPN and sclerostin proved good biomarkers of metastatic bone progression; the RANKL/OPG ratio was a good indicator of bone erosion in the metastatic process, while sRAGE had a protective role against metastatic progression in bone. These results serve to define a panel of new osteoimmunological biomarkers that could be useful in assessing the progress of osteolytic bone metastases.

MTOR inhibitor and bone-targeted drugs break the vicious cycle between clear-cell renal carcinoma and osteoclasts in an in vitro co-culture model

The skeleton is one of the most common sites of metastatic spread from advanced clear-cell renal carcinoma (ccRCC). Most of the bone lesions observed in RCC patients are classified as osteolytic, causing severe pain and morbidity due to pathological bone destruction. Nowadays, it is well known that cancer induced bone loss in lytic metastasis is caused by the triggering of a vicious cycle between cancer and bone resident cells that leads to an imbalance between bone formation and degradation. Targeting the mammalian target of rapamycin (mTOR) is an efficient treatment option for metastatic renal carcinoma patients. Moreover, bone targeted therapy could benefit bone metastatic cancer patients caused by advanced RCC. However, more data is needed to support the hypothesis of the beneficial effect of a combined therapy. The aim of this work is to investigate the effect of targeting mTOR and the sequential combination with bone targeted therapy as a strategy to break the vicious cycle between ccRCC cells and osteoclasts. A previously optimized fully human co-culture model is used to mimic the crosstalk between Caki-2 cells (ccRCC) and osteoclasts. Cells are treated at fixed timing with everolimus, zoledronic acid and denosumab as single or sequential combined treatment. We show that Caki-2 cells can induce osteoclast cells differentiation from isolated human monocytes, as demonstrated by specific tartrate-resistant acid phosphatase (TRAP) staining and f-actin ring formation, in a statistically significant manner. Moreover, differentiated osteoclasts proved to be functionally active by pit formation assay. Caki-2 cells co-cultured with osteoclasts acquire a more aggressive phenotype based on gene expression analysis. Interestingly, the sequential combined treatment of everolimus and zoledronic acid is the most effective in the inhibition of both Caki-2 cells survival and osteoclastogenic potential, making it an effective strategy to inhibit the vicious cycle of bone metastasis. At preclinical level, this observation confirms the value of our co-culture model as a useful tool to mimic the bone microenvironment and to assess drug sensitivity in vitro. A better understanding of the molecular mechanisms involved in tumor-bone cells crosstalk will be investigated next.

Current concepts and trends in the treatment of bone metastases in patients with advanced prostate cancer.

Bone metastases have a major impact on quality of life and survival of patients with advanced prostate cancer. In the last decade, the development and approval of substances inhibiting the vicious cycle of bone metastases have enabled the reduction of complications caused by bone metastases in patients with castration-resistant prostate cancer. These drugs have raised awareness of the importance of skeletal-related events which in the meantime represent an important end point also in trials using agents not specifically designed for bone lesions. Second-generation antihormonal drugs such as enzalutamide or abiraterone have been shown to have a positive impact on the incidence of skeletal complications and therefore provide an important tool in the armamentarium used for treating bone metastases. Radiopharmaceuticals such as radium-223 dichloride ([223Ra]) have been demonstrated not only to reduce skeletal-related events and bone-related pain, but also to prolong overall survival, thereby being the first bone-targeting agent showing a survival benefit. As previous studies have not provided an obvious benefit of bone-targeted lesions in castration-sensitive disease, the use of these agents is not recommended. In oligometastatic prostate cancer, the role of local treatment of metastases using stereotactic radiation or radiosurgery is a matter of intense debates and may play an increasing role in the future.

Abstract 1959: MTA1 promotes tumor progression and bone metastasis in prostate cancer via positive regulation of MUC1 and CTSB

Abstract About 80% of the time that prostate cancer cells metastasize, they spread to the bones. Metastasis involves activation of invasive programs through genetic and epigenetic alterations, including changes in expression of chromatin remodeler proteins. We have previously identified metastasis-associated protein 1 (MTA1), a chromatin remodeler, as a component of vicious cycle of bone metastasis and confirmed significantly higher expression of MTA1 in bone metastatic samples from patients. In an attempt to dissect molecular mechanisms of MTA1 action, we performed integrative analysis of our bone metastasis signature data along with MTA1 ChIP-Seq data, which revealed mucin 1 (MUC1) and cathepsin B (CTSB) as strong potential candidates responsible for MTA1-driven invasiveness.Here, we show that loss of function studies with MTA1 in PC3M cells exhibit reduced expression for MUC1 and CTSB. We also demonstrate that depletion of MTA1 in PC3M aggressive prostate cancer cellsdoes not affect cell proliferation but leads to decrease in colony forming ability, invasive and migratory property of these cells. When PC3M cells silenced for MTA1 and tagged with luciferase are used to generate subcutaneous or intracardiac xenografts, it leads to reduced tumor progression or decreased metastasis to bone, respectively as evident by bioluminescent measurements and histology of tumors. Ongoing experiments aim to validate the role of MUC1 and CTSB in MTA1-driven invasiveness and metastasis in prostate cancer. Citation Format: Avinash Kumar, Swati Dhar, Gisella Campanelli, Nasir A. Butt, Christian R. Gomez, Jason M. Schallheim, Anait S. Levenson. MTA1 promotes tumor progression and bone metastasis in prostate cancer via positive regulation of MUC1 and CTSB [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1959. doi:10.1158/1538-7445.AM2017-1959

Bone Metastasis from Solid Tumors: Biologic and Clinical State of the Art

Bone metastases are a frequent and debilitating consequence for many tumors, of which breast, lung, prostate, and kidney cancer are the most common. The dialog among cancer cells, bone microenvironment, and immune system regulates bone metastasis formation. Indeed, bone and immune system are strictly linked to each other because bone regulates the hematopoietic stem cells from which all cells of the immune system derive. Many immunoregulatory cytokines influence the fate of bone cells and promote the growth of tumor cells in bone, contributing to sustain the vicious cycle of bone metastasis. Bone is an attractive soil for cancer cells, which can remain dormant for years or directly form bone lesions. The fate of cancer cells after their arrival to bone marrow depends on a complex cross-talk among cancer, bone, and immune cells in the microenvironment. This review provides an overview of the different steps occurring during bone metastatic process, with particular attention to the osteoimmunology field. Furthermore, we will revise the current available clinical approaches for the therapy of bone metastatic patients.

Dynamic modeling of bone metastasis, microenvironment and therapy: Integrating parathyroid hormone (PTH) effect, anti-resorptive and anti-cancer therapy

Bone is a common site for the development of metastasis, as its microenvironment provides the necessary conditions for the growth and proliferation of cancer cells. Several mathematical models to describe the bone remodeling process and how osteoclasts and osteoblasts coupled action ensures bone homeostasis have been proposed and further extended to include the effect of cancer cells. The model proposed here includes the influence of the parathyroid hormone (PTH) as capable of triggering and regulating the bone remodeling cycle. It also considers the secretion of PTH-related protein (PTHrP) by cancer cells, which stimulates the production of receptor activator of nuclear factor kappa-B ligand (RANKL) by osteoblasts that activates osteoclasts, increasing bone resorption and the subsequent release of growth factors entrapped in the bone matrix, which induce tumor growth, giving rise to a self-perpetuating cycle known as the vicious cycle of bone metastases. The model additionally describes how the presence of metastases contributes to the decoupling between bone resorption and formation. Moreover, the effects of anti-cancer and anti-resorptive treatments, through chemotherapy and the administration of bisphosphonates or denosumab, are also included, along with their corresponding pharmacokinetics (PK) and pharmacodynamics (PD). The simulated models, available at http://sels.tecnico.ulisboa.pt/software/, are able to describe bone remodeling cycles, the growth of bone metastases and how treatment can effectively reduce tumor burden on bone and prevent loss of bone strength.

The flavonoid p-hydroxycinnamic acid mediates anticancer effects on MDA-MB-231 human breast cancer cells in vitro: Implications for suppression of bone metastases.

Tumor invasion into bone tissues is associated with osteoclast and osteoblast recruitment, resulting in the liberation of growth factors from the bone matrix, which can feed back to enhance tumor growth resulting in the vicious cycle of bone metastasis. Activated nuclear factor-κB (NF-κB) in breast cancer cells has been shown to play a crucial role in the osteolytic bone metastasis of breast cancer in stimulating osteoclastogenesis. The flavonoid p-hydroxycinnamic acid (HCA) mediates bone anabolic and anti-catabolic effects by stimulating osteoblastic bone formation and suppressing osteoclastic bone resorption. However, the capacity of HCA to ameliorate the negative effects of breast cancer on bone cells has not been investigated. The present study was undertaken to determine the anticancer effects of HCA on MDA-MB-231 human breast cancer bone metastatic cells in vitro models. Proliferation of MDA-MB‑231 cells was suppressed by culture with HCA (10-1000 nM) due to G1 and G2/M phase cell cycle arrest. The suppressive effects of HCA were mediated through signaling pathways that are related to NF-κB, extracellular signal-regulated kinase (ERK), protein kinase C, calcium signaling, phosphatidylinositol 3-kinase (PI3K) and nuclear transcription activity. HCA was also found to induce death of confluent cancer cells. Furthermore, co-culture with MDA-MB-231 cells suppressed mineralization and stimulated osteoclastogenesis in bone marrow cells. These alterations were prevented by HCA (10-250 nM). The present study demonstrates that HCA possesses anticancer properties in MDA-MB-231 human breast cancer cells and alleviates the negative effects on osteoblastogenesis and osteoclastogenesis in vitro. HCA may have important applications in the treatment of breast cancer bone metastasis.

The Impact of Immune System in Regulating Bone Metastasis Formation by Osteotropic Tumors.

Bone metastases are frequent and debilitating consequence for many tumors, such as breast, lung, prostate, and kidney cancer. Many studies report the importance of the immune system in the pathogenesis of bone metastasis. Indeed, bone and immune system are strictly linked to each other because bone regulates the hematopoietic stem cells from which all cells of the immune system derive, and many immunoregulatory cytokines influence the fate of bone cells. Furthermore, both cytokines and factors produced by immune and bone cells promote the growth of tumor cells in bone, contributing to supporting the vicious cycle of bone metastasis. This review summarizes the current knowledge on the interactions among bone, immune, and tumor cells aiming to provide an overview of the osteoimmunology field in bone metastasis from solid tumors.

Abstract LB-105: Radium-223 dichloride monotherapy and combination therapy with zoledronic acid or doxorubicin improve survival in a mouse model of breast cancer bone metastasis.

Abstract Breast cancer metastasis to bone results in significant morbidity and poor prognosis. Radium-223 dichloride is an alpha-emitting calcium mimetic that localizes to bone and provides targeted radiation therapy. A phase III clinical study on prostate cancer patients with bone metastases showed that radium-223 dichloride improved overall survival (ALSYMPCA, Parker et al. ECCO/ESMO 2011). We have previously reported that radium-223 decreases osteolysis and tumor burden in bone in a mouse model of breast cancer bone metastasis in preventive and micro-metastatic settings (Suominen et al. AACR Annual Meeting 2012), as well as, in mice with established bone metastases (Suominen et al. AACR Annual Meeting 2011). Here, we investigated the effects of radium-223 dichloride monotherapy compared to and in combination with either doxorubicin or zoledronic acid on survival in a mouse model of established breast cancer bone metastasis. Human MDA-MB-231(SA)/GFP cells were inoculated intracardially into nude mice, and 15 days later, a single dose of vehicle, radium-223 dichloride (300 kBq/kg, iv injection) and/or zoledronic acid (0.1 mg/kg, sc injection) was administered. Doxorubicin (5 mg/kg, ip injection) was administered once weekly. Radium-223 dichloride monotherapy extended time to sacrifice (P = 0.039), unlike doxorubicin or zoledronic acid monotherapy which did not improve survival as compared to the vehicle group. Radium-223 dichloride in combination with zoledronic acid (P = 0.004) or doxorubicin (P < 0.001) also extended time to sacrifice as compared to the vehicle but did not provide additional survival benefit as compared to the radium-223 dichloride monotherapy. Histological examination revealed that radium-223 dichloride treatment induced tumor cell necrosis in bone metastases. Therefore, the effect of radium-223 dichloride in inducing double-strand breaks in cancer cells was evaluated by immunohistochemical staining of γ-H2AX molecules. A 3-fold increase in the number of tumor cells with double-strand breaks in the radium-223 dichloride-treated as compared to the vehicle control mice was observed (P < 0.001). This finding supports our previous observations that radium-223 dichloride has an effect on both tumor cells and osteoclasts. In conclusion, radium-223 dichloride therapy alone or in combination with doxorubicin or zoledronic acid increases survival in breast cancer bone metastasis mouse model via dual action by targeting tumor growth and osteolysis, both important players in the destructive vicious cycle of bone metastasis. Our findings strongly support the development of radium-223 dichloride for the treatment of patients with bone metastatic breast cancer. Citation Format: Mari I. Suominen, Jukka P. Rissanen, Rami Kakonen, Katja M. Fagerlund, Esa Alhoniemi, Dominik Mumberg, Karl Ziegelbauer, Jussi M. Halleen, Sanna-Maria Kakonen, Arne Scholz. Radium-223 dichloride monotherapy and combination therapy with zoledronic acid or doxorubicin improve survival in a mouse model of breast cancer bone metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-105. doi:10.1158/1538-7445.AM2013-LB-105

Malignant Hypercalcemia

Malignancy-associated hypercalcemia (MAH) is one of the clinical emergencies in medical oncology, arising early or, more often, during the late phases of disease. Prevalence cannot be estimated accurately because previous figures of 5-30% of all cancer patients have progressively reduced thanks to the widespread use of bisphosphonates for the prevention of skeletal events. The classic distinction of humoral vs. osteolytic hypercalcemia is still relevant from an etiological point of view, but should not be considered as a rigid alternative since both mechanisms may be active in the same patients and the activation of the RANKL pathway is a common pathogenetic mechanism. Parathyroid hormone-related protein mimics the effects of PTH on the bone and kidney (tubular calcium resorption) and may represent an attractive druggable target, but additional agents (cytokines or other mediators) as well as ectopic production of 1,25(OH)₂D₃ may give an important contribution to humoral hypercalcemia. Conversely, bone invasion by cancer cells determines massive bone reabsorption due to the release of proteolytic enzymes and pro-osteolytic agents with paracrine activity on adjacent bone and stromal cells. When cancer patients develop headache, confusion, de-hydration and tremors hypercalcemia should be suspected although slow rise of calcium levels may produce more indolent symptoms. Bisphosphonates (with or without hydration and diuretics) may efficiently control MAH but only if an active treatment for the underlying cancer is promptly started. The anti-RANKL monoclonal antibody denosumab represents a novel agent able to revert the vicious cycle of bone metastases and data from phase III studies are currently showing promising activity in reverting bone resorption with manageable toxicity.

Hydroxyapatite nanoparticle-containing scaffolds for the study of breast cancer bone metastasis

Breast cancer frequently metastasizes to bone, where it leads to secondary tumor growth, osteolytic bone degradation, and poor clinical prognosis. Hydroxyapatite Ca 10(PO 4) 6(OH) 2 (HA), a mineral closely related to the inorganic component of bone, may be implicated in these processes. However, it is currently unclear how the nanoscale materials properties of bone mineral, such as particle size and crystallinity, which change as a result of osteolytic bone remodeling, affect metastatic breast cancer. We have developed a two-step hydrothermal synthesis method to obtain HA nanoparticles with narrow size distributions and varying crystallinity. These nanoparticles were incorporated into gas-foamed/particulate leached poly(lactide-co-glycolide) scaffolds, which were seeded with metastatic breast cancer cells to create mineral-containing scaffolds for the study of breast cancer bone metastasis. Our results suggest that smaller, poorly-crystalline HA nanoparticles promote greater adsorption of adhesive serum proteins and enhance breast tumor cell adhesion and growth relative to larger, more crystalline nanoparticles. Conversely, the larger, more crystalline HA nanoparticles stimulate enhanced expression of the osteolytic factor interleukin-8 (IL-8). Our data suggest an important role for nanoscale HA properties in the vicious cycle of bone metastasis and indicate that mineral-containing tumor models may be excellent tools to study cancer biology and to define design parameters for non-tumorigenic mineral-containing or mineralized matrices for bone regeneration.

Abstract 4279: 3D in vitro tri-culture system mimicking the vicious cycle of breast cancer colonization of bone

Abstract Breast cancer frequently metastasizes to bone where it causes osteolytic lesions. Once colonization of bone occurs, an interconnected series of interactions between breast cancer cells and those of the bone microenvironment result in uncontrolled tumor growth. These interactions have been collectively interpreted in terms of the “vicious cycle of bone metastasis”. According to this paradigm, cancer cells secrete pro-inflammatory factors that lead to osteoclast (OC, bone resorbing cells) activation, through intermediary stimulation of osteoblasts (OB, bone building cells). OBs increase production of inflammatory cytokines, which further stimulates OC to differentiate into mature OC that actively resorb bone leading to severe osteopathologies. A complete understanding of cancer in bone thus requires a full appreciation of a network of biochemical signaling occurring between OB, OC and the breast cancer cells at tissue and cellular levels. We hypothesize that breast cancer interaction with the bone microenvironment, simulating a vicious cycle model can be developed in a 3D in vitro bioreactor system. This vicious cycle model would enable further characterization and manipulation of the interactions among the three cell types. A novel bioreactor, based on the principle of simultaneous growth and dialysis, designed and developed in our laboratory, was used to study this three-way interaction between OB, OC and breast cancer cells. We created an in vitro ‘bone-remodeling’ mimic to which metastatic cancer cells can be added and monitored over time. We have been able to differentiate OCs from hematopoietic progenitor cells isolated from murine bone marrow. The pre-osteoclasts cells when introduced onto the OB (MC3T3-E1) tissue grown in the bioreactor, differentiated to form multinucleated bone resorbing OCs. The OCs, positive for TRAP and actin ring staining; digested the OB matrix. From an initial inoculum of 75×104 pre-OCs, the bioreactor cultures yielded a recovery of approximately 2% mature OCs. Re-infusing this co-culture system with pre-OBs resulted in re-filling of OC degraded matrix with proliferating OBs. When metastatic breast cancer cells (MDA-MB-231GFP) were introduced into the OB-OC co-culture, the breast cancer cells migrated towards sites of active remodeling and clustered as an aggregation of cells that further degraded the OB matrix, an observation similar to that described for an in vivo scenario. This 3D in-vitro cell culture system mimicking the vicious cycle of bone metastases will help to further understand the cellular and molecular events that lead to successful breast cancer colonization of bone. Positive outcomes of the work will help clarify the etiology of metastasis but also create a much-needed in vitro tool for study of cancer therapeutics. This work was supported by US Army Medical and Materiel Command Breast Cancer Idea Program (W81XWH-06-1-0432). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4279. doi:10.1158/1538-7445.AM2011-4279

Knockdown of c-Jun N-terminal kinase 1 expression in murine mammary cancer cells leads to an increase in tumor growth and bone metastasis.

Abstract Abstract #1156 The c-Jun N-Terminal Kinase (JNK) family of proteins are involved in cell proliferation and apoptosis depending on the specific isoform, cell type, and activating stimuli. The JNK proteins are known as the predominant c-Jun kinases, resulting in AP-1 activation and increased expression of AP-1 transcriptional targets. More specifically, it has been reported that osteoclast differentiation is impaired in jnk1-/- mice. The purpose of this study was to determine whether expression of JNK1 in cancer cells will influence the bone microenvironment during metastatic growth. The 4T1.2 murine mammary cancer cell line was studied as an established model for mammary cancer mediated bone metastasis. Using a JNK1α1 shRNA approach, we observed 75% reduction in expression Orthotopic injection of the shJNK1α1 cells into the mammary fat pad of Balb/c mice significantly increased tumor growth rates and shortened survival compared to the respective empty vector cell line (p<0.0001). Similarly, intra-cardiac injection of the shJNK1α1 cells led to a significant decrease in survival (p=0.003) and an increase in incidence of metastatic lesions in the ribcage (p=0.04). Serum levels of Cathepsin K were assayed as an indicator of bone resorption and was significantly higher in the mice injected with the knockdown cells compared to empty vector controls (p=0.03). Finally, immunohistochemical analysis of bone lesions showed higher expression of ADAM (A Disintegrin and Metalloproteinase) 10 suggesting that the more aggressive phenotype observed with the shJNK1a1 cells may be attributed to the increase in ADAM10 protein expression. Previous studies have shown that ADAM10 that cleaves RANKL into its active soluble form. Further, RANKL expression has been reported to be elevated in breast carcinomas, resulting in osteolysis during metastasis. Indeed our qPCR analysis detected an approximate 2 fold increase in RANKL levels in the shJNK1α1 cells, consistent with this assertion. In summary, these data support that reduction in JNK1a1 expression enhances bone metastasis and ADAM10 expression. We hypothesize that increased RANKL cleavage by ADAM 10 allows RANKL to bind its receptors expressed on nearby osteoclasts further perpetuating the vicious cycle of bone metastasis. These data suggest that bone metastasis may be controlled by inhibiting ADAM10 activity to reduce RANKL induced osteolysis and tumor progression. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1156.