Research ArticleMetastatic breast cancer induces an osteoblast inflammatory response
Introduction
Breast cancer is the second deadliest form of cancer for women in the United States, largely due to its tendency to metastasize. Once metastasis occurs, the relative 5-year survival rate drops precipitously from over 90% to less than 10% depending on the site of the metastasis. For breast cancer, the skeleton is the preferred site of metastasis. Nearly 50% of primary and about 70% of secondary metastases target bone [1], [2], [3]. Within the skeletal system, breast cancer cells most frequently colonize the ends of long bones, ribs, and vertebrae; these areas contain rich microvasculature closely juxtaposed to metabolically active trabecular bone surfaces [2].
The metaphyseal area at the ends of long bones contains a complex network of bone cells, hematopoietic cells, and stromal cells. The entry of breast cancer cells into the marrow cavity disturbs the status quo, in particular, the interaction between osteoblasts and osteoclasts. In the adult skeleton, these two cell types are responsible for the slow and continuous turnover of bone [4]. When metastatic breast cancer cells invade the bone microenvironment, the balance is upset in favor of net bone loss. Indeed, breast cancer metastasis usually results in osteolytic lesions due to activated osteoclasts that degrade bone matrix. The bone loss can cause severe pain, pathologic fractures, spinal cord and nerve compression, hypercalcemia, and bone marrow suppression [2]). In addition, growth factors released from the matrix promote cancer cell proliferation and contribute to what has been described as “the vicious cycle”[5]. In particular, transforming growth factor-beta (TGF-β) and insulin growth factor-1 (IGF-1), which are released from the matrix during bone degradation, stimulate the production of parathyroid hormone-related protein (PTHrP) that fosters cancer cell growth [5], [6], [7], [8].
Furthermore, growth factors released from the matrix, substances secreted by the cancer cells and osteoblasts contribute to the metastatic microenvironment. Some osteoblast factors, such as receptor activator of nuclear factor kappa B ligand (RANKL) and osteoprotogerin (OPG), are part of the normal osteoblast-osteoclast signaling cross talk. Others, such as interleukin-6 (IL-6), IL-8, and monocyte chemoattractant protein-1 (MCP-1), may indicate an osteoblast inflammatory response. It has been known for a long time that chronic inflammation, which occurs as part of the cancer cell's interaction with the stromal environment, supports cancer progression and metastasis [9]. Fibroblasts, endothelial cells, cells of the blood and lymph vasculature, as well as transient cells of the innate and adaptive immune systems all affect cancer cell growth and metastasis. Under ordinary conditions, communication within the stromal network is carried out by cytokines, chemokines, and other peptides. A disruption of homeostasis by trauma, microorganisms, foreign materials, or cancer cells results in drastic changes in the levels and types of cytokines expressed [10]. The stromal environment in metaphyseal bone is no exception. Any circumstance that changes the balance between osteoblasts and osteoclasts may lead to bone loss. For example, osteomyelitis brought about by M. tuberculosis or S. aureus is associated with uncontrolled inflammation and especially high levels of IL-8, RANTES, and MCP-1 [11]. Titanium transplant-induced bone loss has been traced to an osteoblast stress response with high levels of IL-8, MCP-1, and IL-6 [12]. These cytokines have been shown to attract and activate osteoclasts as well as cells of the immune system, thus perpetuating bone loss [13].
While it is likely that tumor-infiltrating lymphocytes, neutrophils, and macrophages are a potent source of inflammatory molecules, we present evidence in this paper that metastatic breast cancer cells can directly induce osteoblasts to express increased levels of inflammatory stress response molecules, specifically IL-6, IL-8 (macrophage inflammatory protein-2 [MIP-2], KC), and MCP-1. Moreover, the osteoblast response was mediated by soluble factors and occurred independently of direct cancer cell–osteoblast contact. Their ultimate target is the osteoclast.
Current therapies are directed at blocking osteoclast activity. Bisphosphonates such as clodronate, ibandronate, pamidronate, and zoledronic acid are the current standard of care for most metastases to bone. These synthetic analogues of inorganic pyrophosphates inhibit osteoclast activity and slow lesion formation. Although they reduce skeletal-related events, they are not curative. They do not lead to restoration of the bone and do not eliminate the cancer cells [14]. The osteoblasts appear to be functionally paralyzed [15].
We have previously reported that osteoblasts exposed to metastatic breast cancer cells or their conditioned media show an increase in apoptosis, suppression of production of bone matrix proteins, and a change in morphology [16], [17], [18]. The results of this study indicate that the osteoblasts also switch into an inflammatory mode in the presence of the breast cancer cells. These osteoblast-produced inflammatory cytokines, different from those made by the cancer cells, can target osteoclast precursors that are effectors of osteolysis. Therefore, osteoblasts contribute to the osteolytic phenotype due to loss of bone deposition functions as well as to increased production of osteoclast activating cytokines.
Section snippets
Osteoblasts
hFOB 1.19 cells are human fetal osteoblasts that have been immortalized with a temperature-sensitive SV40 large T antigen. At a permissive temperature of 34 °C, they proliferate; incubation at 39 °C show their growth and fosters osteoblast differentiation [19]. These cells, a gift from Dr. Thomas Spelsberg, were cultured at 34 °C, 5% CO2 in hFOB growth medium, which consists of Dulbecco's Modified Eagle's Medium (DMEM):Ham's F-12 (1:1) (Sigma, St. Louis, MO), 10% fetal bovine serum (FBS)
The production of IL-6, IL-8, and MCP-1 by hFOB 1.19 cells was increased in the presence of breast cancer conditioned medium and was dose-dependent
When hFOB 1.19 were approximately 90% confluent, they were rinsed with PBS and treated with either VM or CM. Twenty-four hours later, the culture media were collected and screened with a RayBio® Human Cytokine Array. Among the 62 cytokines in the screen, IL-6, IL-8, and MCP-1 levels were readily detected (Fig. 1A). In this array, no cytokines were detected in the BCCM alone (Fig. 1B). Medium from VM-treated osteoblasts also had no detectable levels of cytokines in this array (data not shown).
In
Discussion
We found that osteoblasts responded to conditioned medium from MDA-MB-231 human metastatic breast cancer cells with enhanced cytokine expression. Specifically, the osteoblasts increased production of IL-6, MCP-1, and IL-8 (human), three cytokines characterized as osteoblast inflammatory stress proteins. These molecules may help to provide a favorable tumor cell environment as well as initiate osteoclastogenesis. This finding supports the idea that breast cancer metastases create a unique niche
Acknowledgments
This work was supported by the U.S. Army Medical and Material Research Command Breast Cancer Program (DAMD 17-02-1-0358 and W81XWH-06-1-0432 to AMM, W81XWH-06-1-0363 to KMB); National Foundation for Cancer Research, Center for Metastasis Research; The Susan G. Komen Breast Cancer Foundation, BCTR104406; The American Institute of Cancer Research, #06A027-REV2; Sigma Xi, Undergraduate Research Grants; President's Fund, and Schreyer Honors College research awards to M.K. and E.C. The authors would
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Submitted in partial fulfillment of the honors thesis requirement of the Schreyer Honors College.