TRAF2 in osteotropic breast cancer cells enhances skeletal tumour growth and promotes osteolysis.

NFκB plays an important role in inflammation and bone remodelling. Tumour necrosis factor receptor associated factor 2 (TRAF2), a key component of NFκB signalling, has been identified as an oncogene, but its role in the regulation of breast cancer osteolytic metastasis remains unknown. Here, we report that stable overexpression of TRAF2 in parental and osteotropic sub-clones of human MDA-MB-231 (MDA-231) breast cancer cells increased cell growth and motility in vitro, whereas TRAF2 knockdown was inhibitory. In vivo, TRAF2 overexpression in the parental MDA-231-P cells enhanced tumour growth after orthotopic injection into the mammary fat pad of mice but failed to promote the metastasis of these cells to bone. In contrast, overexpression of TRAF2 in osteotropic MDA-231-BT cells increased skeletal tumour growth, enhanced osteoclast formation and worsened osteolytic bone loss after intra-tibial injection in mice. Mechanistic and functional studies in osteotropic MDA-231-BT and osteoclasts revealed that upregulation of TRAF2 increased the ability of osteotropic MDA-231-BT cells to migrate and to enhance osteoclastogenesis by a mechanism dependent, at least in part, on NFκB activation. Thus, the TRAF2/NFκB axis is implicated in the regulation of skeletal tumour burden and osteolysis associated with advanced breast cancer.

MDA-231 human breast cancer cells increases tumour cell growth in bone, and enhances the ability of these cells to induce osteoclast formation and to cause osteolysis in mice. Thus, therapeutic targeting of TRAF2/NFκB signalling may be of value in protecting the skeleton from osteolytic bone damage associated with advanced breast cancer. TRAF2 enhances mammary tumour growth 7 , and we have observed that TRAF2 expression is upregulated in the human osteotropic MDA-231 breast cancer cell line when compared to their parental control (Fig. S1A). In view of this, we hypothesized that TRAF2 is implicated in bone metastasis, skeletal tumour growth and osteolysis associated with advanced breast cancer. We stably overexpressed (Fig. S1B,C) and knocked down (Fig. S1D,E) TRAF2 in the parental MDA-231-P and their osteotropic sub-clone MDA-231-BT (Fig. S1B-E), and assessed the growth and metastatic behaviour of these cells in vitro, ex vivo and in vivo. First, we confirmed that TRAF2 overexpression in parental MDA-231-P enhanced migration within 6 hours, whereas its knockdown in these cells was inhibitory (Fig. 1A,B). Overexpression of TRAF2 also increased the invasion of parental MDA-231-P after 48 hours (Fig. 1C,D). Of note, TRAF2 overexpression or knockdown had no effect on the viability of parental MDA-231-P (Fig. 1E). Next, we evaluated the mammary tumour growth and bone metastasis of parental MDA-231-P in mice after orthotopic injection (Fig. 1F,H). Overexpression of TRAF2 enhanced MDA-231-P growth in the mammary fat pads (Fig. 1G) but failed to cause osteolytic lesions in mice (Fig. 1H). We also failed to detect any MDA-231-P cells expressing green fluorescent protein in bone aspirates ex vivo (data not shown).

Upregulation of TRAF2 enhances breast cancer-induced osteolysis in vivo.
To test if TRAF2 is implicated in the regulation of osteolytic activity of breast cancer cells, we assessed skeletal tumour growth and osteolysis in mice after intra-tibial injection of the osteotropic sub-clone MDA-231-BT. Overexpression of TRAF2 in these cells markedly increased tumour growth in bone ( Fig. 2A,B) and enhanced their ability to cause osteolysis (Fig. 2C,E). As shown in Fig. 2, panel D, detailed microCT analysis of bone samples from this experiment revealed significant reduction in trabecular bone volume (BV/TV) that is accompanied by reduced trabecular number and thickness, increased trabecular separation and decreased trabecular connectivity (increased trabecular pattern factor and SMI). We also detected significant increase in cortical porosity (Fig. 2E) whereas cortical area and thickness remained unchanged (data not shown).
Cancer-specific TRAF2 regulates osteotropic breast cancer -bone cell crosstalk. Breast cancer cells contribute to osteolysis through secretion of pro-inflammatory factors 4 . To explore the role of TRAF2 in this process, we took advantage of an in vivo supracalvarial injection and ex-vivo calvarial osteoblast organ models to assess osteolysis in response to MDA-231-BT conditioned medium in adult immuno-competent mice (unlike the MDA-231-BT nude mouse model described above) (Fig. 3A,D). Conditioned medium from MDA-231-BT cells overexpressing TRAF2 (MDA-231-BT-T2 OE ) induced osteolytic bone damage in calvarial bone in vivo (Fig. 3B,C) and ex vivo (Fig. 3E,F) that is characterized by significant loss in bone volume (p < 0.01). Histomorphometric analysis of the calvarial bone from the organ culture showed that conditioned medium from TRAF2 overexpressing cells increased osteoclast number (Fig. 3G, left panel) without affecting the number of osteoblasts (Fig. 3G, right panel). Next, we employed a quantitative proteomic approach to identify the tumour-derived factor(s) responsible. Analysis of protein level of human cytokines and chemokines in conditioned medium revealed that TRAF2 overexpression in the osteotropic MDA-231-BT-T2 OE is associated with upregulation of a total of 48 secreted proteins in the conditioned medium (Fig. 3G). The identified proteins are common tumour-derived factors that have previously been found to be involved in the regulation of inflammation, angiogenesis, innate immunity and tumorigenesis ( Fig. 3G and Table S1). Further evaluation of the role of these proteins in breast cancer, osteoclastogenesis and/or osteoblast differentiation revealed a subset of 21 proteins that are likely to be implicated in the regulation of the TRAF2-driven breast cancer-induced osteoclast and osteoblast changes that we have observed in our models ( Fig. 3H and Table S2).
Cancer-specific TRAF2 regulates osteoblast and osteoclast changes associated with breast cancer. Previous studies have shown that TRAF2 expression in bone cells plays an important role in osteoclast differentiation and osteoblast apoptosis. Here, we carried out in vitro functional studies to examine the effects of knockdown and overexpression of cancer-specific TRAF2 on osteoclast and osteoblast changes associated with breast cancer. These experiments showed that TRAF2 overexpression significantly increased the ability of osteotropic MDA-231-BT cells (Fig. 4A) and their conditioned medium (20% v/v, Fig. 4B) to enhance RANKL-induced osteoclast formation in bone marrow cultures, whereas TRAF2 knockdown in these cells was inhibitory ( Fig. 4A-C). Exposure of mouse primary osteoblasts and the human osteoblast-like cells Saos-2 cells to conditioned medium from the osteotropic MDA-231-BT cells overexpressing TRAF2 for 2 (mouse osteoblasts) or 12 (human Saos-2) days had no effect on osteoblast differentiation (Fig. 4D) or growth (Fig. 4E) in vitro. However, a modest yet significant reduction in bone nodule formation was detected in human Saos-2 cultures after 12 days of continuous exposure to conditioned medium from the osteotropic MDA-231-BT cells overexpressing TRAF2 when compared to control (Fig. 4F,G).

TRAF2 regulates the behaviour of osteotropic breast cancer cells by engaging IKKβ and IKKε.
Previous studies have shown that TRAF2-driven NFκB activation in breast cancer cells is primarily mediated by the expression and kinase activity of IKKβ and IKKε 3,6,7,29 . In view of this, we knocked down IKKβ and IKKε in the osteotropic MDA-231-BT cells overexpressing TRAF2 (Fig. S2) and we investigated the effects of these changes on these cells metastatic and osteolytic behaviour in vitro. As shown in Fig and C) and reduced the ability of these cells to enhance RANKL-induced osteoclast formation in bone marrow cultures ( Fig. 5D-F). Knockdown of IKKβ or IKKε expression had no effect on the growth of MDA-231-BT cells overexpressing TRAF2 (Fig. 5B).

Discussion
TRAF2 has diverse functions in inflammation 3,29,30 , and its upregulation in mammary epithelial cells enhances their oncogenic transformation and promotes tumour growth 7 . In addition, TRAF2, through its interaction with NFκB, is implicated in the regulation of bone cell differentiation and bone remodelling 2,4,25-27,31-33 . Nonetheless, the contribution of TRAF2 to bone metastasis, skeletal tumour growth and osteolysis associated with advanced breast cancer has not been investigated. Our present in vivo, ex vivo and in vitro investigation showed that TRAF2 overexpression in parental and osteotropic sub-clones of MDA-231 human breast cancer cells increased tumour cell growth and motility, whereas its knockdown is inhibitory. TRAF2 overexpression also enhanced the ability of osteotropic breast cancer cells and their derived factors to induce osteoclast formation, inhibit osteoblast differentiation and to cause osteolysis by a mechanism that depends at least in part on NFκB activation. Malignant breast cancer cells accumulate oncogenic alterations that significantly affect their growth in the primary site and metastatic activity at distant sites such as bone 34,35 . The majority of studies thus far implicate the TRAF2/NFκB pathway in these processes were limited to the role of TRAF2 in mammary tumour gro wth 6,10,12,17,19,[21][22][23] . Here, and in broad agreement with these reports, we have found that TRAF2 overexpression promoted both mammary and skeletal tumour growth in mice after orthotopic and intra-tibial injections. NFκB signalling in breast cancer cells facilitates tumour growth and spread through induction of inflammatory mediators 36 . In vitro examination of the metastatic behavior of parental and osteotropic cells confirmed that TRAF2 overexpression is linked to increased tumour cell motility and invasion, whereas knockdown of TRAF2 was inhibitory. Examination of the levels of secreted proteins in conditioned medium from these cultures revealed that TRAF2 overexpression in osteotropic cells is associated with elevated levels of various pro-tumour and pro-migratory factors including uPAR, VEGF, IL-1, TSP-1 and Serpine 1, each with functions that enable primary and metastatic breast cancer cells to spread (Table S1).
Bone metastasis in advanced breast cancer is predominantly osteolytic 37 . Malignant breast cancer cells in bone acquire new characteristics influenced by matrix-derived factors 24,[37][38][39][40] . In the present study, we provide evidence to support the notion that cancer-specific TRAF2 contributes to breast cancer cell behavior in bone. The evidence for this comes from the experiments that showed that (A) TRAF2 is highly expressed in the osteotropic MDA-231-BT cells when compared to parental control, (B) knockdown of TRAF2 in osteotropic MDA-231-BT inhibited cell migration and reduced the ability of these cells to enhance osteoclastogenesis, (C) conditioned medium from osteotropic MDA-231-BT overexpressing TRAF2 reduced osteoblast ability to form bone nodules in vitro, and (D) upregulation of TRAF2 worsened breast cancer-induced osteolysis. Microarray analysis of secreted proteins in conditioned medium from osteotropic MDA-231-BT overexpressing TRAF2 indicates that these effects were likely due to increase in the level of tumour-derived NFκB-mediated osteolytic factors, such as TNFα, IL-1, granulate macrophage colony stimulating factor (GM-CSF), M-CSF, CD40L, IL-17, IL-11, vascular endothelial growth factor (VEGF) ( Table S1). TRAF2 overexpression also enhanced the level of tumour-derived TNFα, a known an inhibitory factor of osteoblast survival and differentiation 4,25,33 . Thus, it is reasonable to speculate that TRAF2 in osteotropic breast cancer cells orchestrates the expression of a set of tumour-derived factors that function cooperatively to alter the balance between osteoblasts and osteoclasts. Nevertheless, it is important to note that we cannot exclude the role of well-established systemic and bone-derived osteolytic mediators such as RANKL, insulin growth factor 1 (IGF-1) and parathyroid hormone-related protein (PTHrP) in TRAF2-induced osteolysis in our models.
A number of studies have shown that TRAF2 oncogenic activity in parental breast cancer cells is driven by IKKβ and IKKε 3,6,7,29,41 . In the skeleton, selective inhibition of IKKε or IKKβ have been found to inhibit osteoclastic bone resorption 28,42-44 and promote bone formation 45,46 . Here, we provided further evidence that IKKβ and IKKε are also implicated in TRAF2-driven metastatic and osteolytic behavior of the osteotropic MDA-231-BT cells. To this end, we showed that selective silencing of either IKKβ or IKKε reduced the ability of TRAF2 overexpressing MDA-231-BT cells to migrate and to enhance RANKL-induced osteoclastogenesis. Altogether, these results demonstrate that TRAF2 contributes to breast cancer related osteolysis through its regulation of both the canonical and non-canonical NFκB signalling pathway.
In conclusion, our studies showed that the TRAF2/IKK/NFκB axis in osteotropic breast cancer cells contributes to breast cancer associated osteolytic bone damage. We also confirmed that cancer-specific expression of TRAF2 regulates mammary tumour growth and showed for the first time that TRAF2 is implicated in skeletal tumour burden. When combined with previous studies that demonstrated that TRAF2 plays an important role in breast cancer initiation 3,6,7,29 , our present findings indicate that targeting of TRAF2 may have a potential therapeutic value in halting breast cancer metastasis and protecting the skeleton from the osteolysis associated with advanced breast cancer.   Animal experiments. All experimental protocols were approved by the Ethics Committee at the University of Edinburgh (Scotland, United Kingdom) and L' Aquila (Italy) and were conducted in accordance with the United Kingdom Home Office regulations.

Intra-tibial injection in mice.
The effect of TRAF2 overexpression on skeletal tumour growth and local osteolysis was studied by intra-tibial injection of osteotropic MDA-MB-231 (MDA-231-BT) breast cancer cell-line in 4-week-old female Balb/c nu/nu mice. Briefly, mice were divided into two groups and received intra-tibial injection of human breast cancer cells mock (MDA-231-BT mock ) or cells overexpressing TRAF2 (MDA-231-BT-T2 OE ) (5 × 10 4 cells) in the left leg or a sham injection of Phosphate buffered saline (PBS) into the right leg. Animals were euthanized 14 days post injection and bones were analyzed by micro-computed tomography (microCT, Skyscan 1172 scanner) 48 . Skeletal tumor growth was measured on 2D microCT images using Brucker ctAN and Image J (1.34 s; NIH, Bethesda, MD, USA) and results were expressed as a percentage of total metaphyseal area.
Supracalvarial injection in mice. The effects of TRAF2 overexpression on breast cancer-induced osteolysis was studied by supracalvarial injection of conditioned medium in immuno-competent 3-week-old C57Bl/6 female mice. Mice were injected subcutaneously over the calvarial bones with conditioned medium (50μL) from human mock (MDA-231-BT mock ) or cells overexpressing TRAF2 (MDA-231-BT-T2 OE ) breast cancer cells on 5 consecutive days. The calvarial bone was assessed using microCT at a resolution of 8μm 48 . Bone organ culture system. Ex vivo osteolysis was studied in mouse calvarial bone using an adaptation of the mouse calvarial organ culture 49 . Mouse calvarias were isolated from 7-day-old mice, divided into equal halves and cultured on stainless steel rafts in MDA-BT-mock or MDA-BT-T2OE conditioned medium (20% v/v). Tissue culture medium was refreshed every 48 hours and the cultures were terminated after 7 days. Bone volume was assessed by microCT (resolution, 5μm) as previously described 48 . Bone histomorphometry was performed on the calvarial bone and Hematoxilin and Eosin staining was used to evaluate osteoclasts and toluidine blue staining for osteoblasts. Histological sections were analysed using software based on the Aphelion Image Analysis tool kit (Adcis, He0rouville-Saint-Clair, France).
Osteoblast cultures. Primary osteoblasts were isolated from the calvarial bones of 2-day-old mice 50 .
Osteoclast cultures. Osteoclast formation was studied in RANKL and M-CSF stimulated bone marrow cultures. Bone marrow (BM) cells were obtained from the marrow of 3-month-old mice and M-CSF-dependent osteoclast precursor cells generated as previously described 51 . M-CSF-dependent osteoclast precursors were plated into 96 well plates (15 × 10 3 cells/well) in alpha-MEM supplemented with M-CSF (25 ng/ml) and RANKL (100 ng/ml) for 6 hours prior addition of MDA-231 breast cancer cells (300 cells/well) or their conditioned medium (20% v/v). Cultures were stained for Tartrate-Resistant Acid Phosphatase (TRAcP) 50 .
Western Blotting. Western blot analysis was used to detect protein expression in breast cancer cells. Cells were lysed in a standard buffer (0.1% (w/v) Sodium dodecyl sulfate, 0.5% (w/v) sodium deoxycholate, 1% Triton X-100, 1 μM Ethylenediaminetetraacetic acid, 2% (v/v) protease inhibitor cocktail, 10 µM of sodium fluoride and 2% (v/v) phosphatase inhibitor cocktail). Protein concentration was determined using BCA assay (Pierce, USA). Total protein lysate (60μg) was resolved by SDS-PAGE (BioRAD, United Kingdom), immunoblotted with antibodies according to manufacturer's instructions, detected using rabbit monoclonal antibodies (all at 1:1000 dilution, cell Signalling Technology, USA) and immuno-complexes were visualised by enhanced chemiluminescence (Amersham, UK) on a Syngene GeneGnome imaging system. The intensity of the bands was quantified using GeneSnap software (Syngene, UK) and level of actin was used for normalization.

Measurement of levels of tumour-derived factors.
Level of tumour-derived factors in conditioned medium from human MDA-231 breast cancer cells was determined by Proteome Profiler Human XL Cytokine Array Kit (ARY022, R&D Systems, Abingdon, UK), according to the manufacturer's instructions.