The mirror neuron system (MNS), mainly including the premotor cortex (PMC), inferior frontal gyrus (IFG), superior parietal lobule (SPL), and rostral inferior parietal lobule (IPL), has attracted extensive attention as a possible neural mechanism of social interaction. Owing to high ecological validity, functional near-infrared spectroscopy (fNIRS) has become an ideal approach for exploring the MNS. Unfortunately, for the feasibility of fNIRS to detect the MNS, none of the four dominant regions were found in previous studies, implying a very limited capacity of fNIRS to investigate the MNS. Here, we adopted an experimental paradigm in a real-life situation to evaluate whether the MNS activity, including four dominant regions, can be detected by using fNIRS. Specifically, 30 right-handed subjects were asked to complete a table-setting task that included action execution and action observation. A double density probe configuration covered the four regions of the MNS in the left hemisphere. We used a traditional channel-based group analysis and also a ROI-based group analysis to find which regions are activated during both action execution and action observation. The results showed that the IFG, adjacent PMC, SPL, and IPL were involved in both conditions, indicating the feasibility of fNIRS to detect the MNS. Our findings provide a foundation for future research to explore the functional role of the MNS in social interaction and various disorders using fNIRS. Humans are highly social animals and social interaction is ubiquitous and essential for our survival . UnderstMirror neurons were originally discovered in area F5 of the monkey premotor cortex and subsequently in the rostral sector of the inferior parietal lobule (IPL) (PF/PFG) . PreviouFunctional near-infrared spectroscopy (fNIRS) is a promising, non-invasive neuroimaging approach. It is portable, cost-effective, highly flexible, and robust against head and body motions . This apConsidering the limited spatial coverage and spatial resolution, as well as the limited deep imaging capacity , it is fThe purpose of our study is to investigate whether the MNS activity, including four dominant regions , can be detected by using fNIRS. Based on the original definition of the MNS, the logic is to demonstrate that these regions are activated during both action execution and action observation. In the present study, we revised Koehler’s task and probe configuration to make it optimal for fNIRS to detect the MNS. Adopting the modified task and probe configuration, we proved the feasibility of fNIRS to investigate the MNS, using a traditional channel-based group analysis and also a ROI-based group analysis. In addition, a previous study found that neural synchronization was different between opposite gender dyads and same gender dyads in a social task , and in We recruited 30 healthy volunteers  to participate in this study. All participants were right-handed according to the Edinburgh Handedness Questionnaire  and had We adopted a table-setting task. The subject and the experimenter (actress) sat face to face at a table. They each had two placemats, one on their right-hand side and the other in front of them (center of the table). Each one also had a set of tableware consisting of five objects: a plate, a fork, a knife, a soup spoon, and a dessert spoon. The original positions of the tableware items were on their right-hand side. A monitor placed at a 45° angle in front of the subject presented cues.Based on the original definition of the MNS, each subject was asked to complete two experimental conditions: an action execution and an action observation. In the execution condition, the subjects had to move the five tableware items to their designated positions with their right hand in 15 s. In Block 1, each subject moved five tableware items from the placemat on the right of the subject to another placemat in front of the subject, and in Block 2, each subject returned the tableware items to their original positions. Block 3 was the same as Block 1 and Block 4 was the same as Block 2. The order of placement was fixed: plate, fork, knife, soup spoon, and dessert spoon. The subjects were told to imagine as preparing a meal in daily life and to execute every action at a normal, natural speed. The only difference between the observation condition and the execution condition was that all the actions were completed by the actress in the former condition. In the observation condition, the subjects were instructed to watch the actions of the actress carefully and remain still.Figure 1 depicts the experimental design.Each condition consisted of eight blocks, and every block contained a 15-s task and a 30-s rest period. In the execution condition, the subjects were instructed to take a glimpse at the monitor when the cue words appeared. Upon seeing “execute,” the subjects began to execute the tasks according to the instructions and upon seeing “rest,” the subjects looked at the cross in the center of the table and avoided any actions. In the observation condition, the monitor was turned toward the actress, and the cues were the same as those in the execution condition. The subjects appropriately responded according to the actress’s actions. When the actress executed the actions, the subjects watched carefully, and when the actress stopped, the subjects rested. The order of the two conditions was counterbalanced across the subjects. Additionally, all the subjects spent some time practicing before the formal experiment to ensure that they were familiar with the rules and could adequately complete the entire task. In this study, the changes in oxyhemoglobin (HbO) and deoxyhemoglobin (HbR) concentration in each channel were measured by an optical system . This system utilizes a three-wavelength technology at 780, 805, and 830 nm.Figure 2). The probe sets were positioned on the left hemisphere only because the left hemisphere is dominant when the subjects perform a right-handed action (We employed a double density probe configuration that was mixed by two standard probe sets (a 4 × 3 probe set and a 4 × 2 probe set). Two double density probe sets  were attached to a regular swimming cap that was placed on the head of each subject in an identical manner  for the corresponding Brodmann area and the probability of finding the scalp position in these areas. To facilitate the subsequent analysis, we used the Brodmann area possessing maximum probability as this channel’s location . To allow comparison of our results with previous findings, the Brodmann atlas was utilized in our study. If one channel is located in areas A, B, and C, the Brodmann atlas in the NIRS_SPM toolbox produces a list  was removed, leaving 390 s for each condition, which was down sampled to 10 Hz . In ordet-tests . The reason why we chose one-sided t-test was because we had a hypothesis about the direction of the effect, that was, the task period yielded an increased hemodynamic response compared with the rest period in both conditions.First, at the individual level, the general linear model (GLM) was used to detect the hemodynamic response of HbO in all 54 channels under the two conditions, and for each subject, channel, and condition, a beta-value was generated. Second, at the group level, one-sided, one-sample t-tests  were pert-tests were used. All analysis steps were performed for both conditions. The entire data analysis framework is presented in Figure 3.Oxyhemoglobin changes were analyzed in three steps. The first step was the same as channel-based group analysis. According to the GLM, two beta maps (for two conditions) that included all the channels for each subject were generated. Second, for a given ROI, we extracted the beta-values of the channels which were located in the ROI based on the location map of each subject and weighted the contribution of each channel to the ROI according to its probability . Thus, ft-test was performed to explore the differences between genders. This effect was analyzed only for the HbO signal.The actress was fixed and half of the subjects were male and the other half were female, thus constituting both same gender pairs and opposite gender pairs. For each condition and for each mirror-related channel or ROI, a two-sided, two-sample p < 0.01, FDR-corrected).We first analyzed channel by channel to explore whether the activated channels in execution condition are also activated in observation condition. The results showed that 18 channels were significantly activated in both conditions , and in observation condition, the activation reached a significant level with a liberal threshold . The t-values of all channels in both conditions are shown in Figure 4.As described in the spatial localization section, each subject was assigned a location map in which each channel had only one identity based on the maximum probability from the Brodmann atlas. According to the location maps of 30 subjects, we found that the anatomical location of these activated channels were mainly PMC (BA6), IFG (BA44/45), SPL (BA7), frontal eye fields (BA8), dorsolateral prefrontal cortex (BA9), and associative visual cortex (BA19). The rostral IPL (BA40) was significantly activated in execution condition . These results were consistent with the results of traditional channel-based group analysis.Using ROI-based group analysis, we found that three of the four ROIs, the PMC (BA6), IFG (BA44/45), and SPL (BA7), were significantly activated during both action execution and action observation. The rostral IPL was also significantly activated in execution condition, but in observation condition, the activation reached a marginal significant level .To evaluate the specificity of significant activation detected by fNIRS, we selected a control region, angular gyrus (BA39). Using the ROI-based group analysis, we found that angular gyrus was activated in execution condition but not activated in observation condition . In this figure, we can obviously see the task period (gray background) yielded an increased hemodynamic response compared with the rest period in both conditions. We also found that the magnitude of the hemodynamic response in execution condition was greater than that in observation condition.In order to observe the neural response to task intuitively, we presented time series of both experimental conditions across all subjects from an example ROI , adjacent PMC (BA6), SPL (BA7), and rostral IPL (BA40). The data indicated that the activity of the four regions could indeed be detected by fNIRS, using the traditional channel-based group analysis and also using the ROI-based group analysis. These findings prove that it is feasible for fNIRS to comprehensively investigate MNS activity.Here, we detected the activity of the MNS using fNIRS where Koehler’s work did not. This reason may due to two differences between the two studies. First, in our study, the actions in observation condition were presented by a real person, while in Koehler’s study, they were presented by video. Some studies have shown that the neural response is higher when observing live actions compared to video actions . The secIn addition to Koehler’s work, another related study explored the brain activity during both action execution and action observation with fNIRS . SubjectIn our study, we used two methods to demonstrate the feasibility of fNIRS to detect the MNS, and the results of the two methods were consistent. When using traditional channel-based group analysis, due to anatomical inconsistency across 30 subjects, the anatomical label at group level, for a given channel, was defined by distribution of all the subjects in different areas. For example, 80% (24/30) of the subjects showed that channel 1 was located in BA6 and 20% (6/30) of the subjects showed it was located in BA8, so we defined the channel as BA6. The significantly activated channels in the present study showed a tolerable consistency with an average of 80%. ROI-based group analysis provides an opportunity to achieve a better spatial consistency. Just as every coin has two sides, ROI-based group analysis solves the problem of inconsistency at the cost of spatial scale. Each of the two methods has its own scope of application. Maybe there will be a better method to guide the probe placement in the future to ensure that the same channel can fall on the same Brodmann area, even the same MNI coordinates of all subjects.Figure 6), we found that the execution condition had a greater hemodynamic response compared to the observation condition. Thus far, some studies have shown that there is a significantly greater response in mirror-related areas for action execution than for action observation , using the traditional channel-based group analysis and also using the ROI-based group analysis. Although the difference between the two conditions in the rostral IPL did not reach a significant level, the tendency to show a greater response in execution condition was consistent with other regions.When presenting the time series of both conditions from an example ROI  which we chose meets both of the two criteria.The experimental paradigm we adopted closely resembles a real-life situation. The majority of previous neuroimaging studies investigating the MNS focused on functional magnetic resonance imaging (fMRI). However, the ecological validity of these studies is low, which requires participants to lie in a narrow, noisy, motionless scanner and restricts head movements to a range of 2 mm . These tHere, probabilistic registration needs to be emphasized. fNIRS is a transcranial brain mapping technique, which has no anatomical brain information . In an fIn our study, the PMC is defined as BA6. In the Brodmann atlas, BA6 is defined as the premotor and supplementary motor cortex. In other words, the PMC occupies part of BA6, which lies in the lateral cerebral cortex. The medial part of BA6 is the site of the supplementary motor cortex. The probe location in our study, which covered the frontal cortex, was placed mainly close to the lateral frontal cortex, and the channels located in BA6 can be attributed to the PMC. Taking a step back, some studies suggest that the supplementary motor cortex also belongs to the MNS .When acquiring fNIRS data, changes in HbO and HbR concentration were measured simultaneously. However, there is controversy regarding which signal to select to analyze brain activation. In our study, we mainly focus on the HbO signal because it is often observed to have a higher amplitude than the HbR signal . In otheSome people might question whether activation in the observation condition comes from movements of the subject. Before the experiment, all the subjects were asked to remain still during the observation condition; during the experiment, an experimenter monitored the action of subjects all the time; and after the experiment, we ensured that the subjects did not produce any explicit movements by watching their experimental videos. In our study, the subjects and the actress sat face to face, meaning the subjects observed actions with a third person view. When the experiment was completed, the subject’s self-report about perspective was submitted. In total, 28 out of 30 subjects reported that it was very easy to understand the actress’s actions by mental rotation, and only two subjects felt slightly uncomfortable, which did not affect their ability to complete the tasks. In addition, all the subjects thought that sitting face to face was closer to a real-life situation.t-value of the rostral IPL was the smallest among all four regions, which may support the reason that we propose. Thus, this is the limitation of our study, and perhaps additional studies can address this issue by optimizing the probe placement or increasing the effective sample size. The second limitation is the lack of a control condition. Without a control condition, it is relatively weak to interpret the activation as mirror-related responses. It is possible that the common activation may be caused by other factors presented in the two conditions, e.g., attention, visual information, and so on. In the future, a more rigorous study is needed.In the current study, among the four key regions, the response of rostral IPL (BA40) was the weakest. In the observation condition, activation of the rostral IPL reached a significant level with a liberal threshold. We tried to explore a probable reason. After analyzing the location data, we realized that because of the limited number of optodes and because we used the double density configuration, the probe sets did not cover the optimal position of the IPL. The classic mirror area is in the rostral IPL (BA40), but the probe sets mainly covered the posterior part of the IPL (BA39). There is no channel that covers the BA40 in one subject due to individual differences in brain shape and size. Thus, the activity of the mirror neurons in the rostral IPL (BA40) was most likely not well detected. Regardless of whether in the execution condition or in the observation condition, the By employing the modified task and probe configuration described in this study, we demonstrated the feasibility of using fNIRS to investigate the activity of the MNS. This study provides a foundation for future research to explore the functional role of the MNS, which correlates with social interaction, in real-life situations using fNIRS. Furthermore, an increasing number of studies have shown that some neurological and psychiatric disorders are closely related to MNS dysfunction, including autism spectrum disorder (ASD)  and schiThis study was carried out in accordance with the recommendations of ethics committee of the State Key Laboratory of Cognitive Neuroscience and Learning at Beijing Normal University with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the ethics committee of the State Key Laboratory of Cognitive Neuroscience and Learning at Beijing Normal University.P-PS, C-ZZ, and Y-HJ designed the research. P-PS, F-LT, and Y-HJ performed the experiments. P-PS, F-LT, ZZ, and YZ analyzed the data. P-PS, F-LT, and C-ZZ drafted the work. The other authors revised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


NFκB is implicated in breast cancer bone metastasis and skeletal remodelling. However, the role of IKKβ, a key component of the canonical NFκB pathway, in the regulation of breast cancer osteolytic metastasis has not been investigated. Here, we describe the cancer-specific contribution of IKKβ to bone metastasis, skeletal tumour growth and osteolysis associated with breast cancer. IKKβ is highly expressed in invasive breast tumours and its level of expression was higher in patients with bone metastasis. IKKβ overexpression in parental MDA-MD-231 breast cancer cells, promoted mammary tumour growth but failed to convey osteolytic potential to these cells in mice. In contrast, IKKβ overexpression in osteotropic sub-clones of MDA-MB-231 cells with differing osteolytic phenotypes increased incidence of bone metastasis, exacerbated osteolysis and enhanced skeletal tumour growth, whereas its knockdown was inhibitory. Functional and mechanistic studies revealed that IKKβ enhanced the ability of osteotropic MDA-MB-231 cells to migrate, increase osteoclastogenesis, and to inhibit osteoblast differentiation via a mechanism mediated, at least in part, by cytoplasmic sequestering of FoxO3a and VEGFA production. Thus, tumour-selective manipulation of IKKβ and its interaction with FoxO3a may represent a novel strategy to reduce the development of secondary breast cancer in the skeleton. Bone metastases and skeletal related events in patients with metastatic breast cancer are an important cause of morbidity, which place significant demands on health care resources , 2. ClinThe nuclear factor-κB (NFκB) signaling pathway plays an important role in breast cancer progression, inflammation and bone cell activity . The IκBIKKβ is also implicated in breast cancer tumorigenesis and metastasis. High levels of expression and activity of IKKβ correlate with drug and radiotherapy resistance and poor clinical outcome in breast cancer patients –16. A plThere has been an increasing interest in the therapeutic targeting of the IKK/NFκB signalling pathway for the treatment of cancer associated bone disease. Both the canonical and non-canonical NFκB signalling pathways play a role in breast cancer bone metastasis , and we p = 0.00032, n = 185) and it is strongly associated with disease recurrence  and bone metastasis relapse . To further evaluate the clinical importance and potential role of IKKβ in breast cancer bone metastasis, we performed detailed immunohistochemical assessment of IKKβ expression in breast tumours and lytic lesions in bone in biopsies from breast cancer patients who developed bone metastasis. As shown in Figure in situ . Stable over-expression of IKKβ   and became morbid and were sacrificed before 55 days compared to only 50% from the mock control group (p < 0.05)  Figure  compared) Figure . Of note) Figure , middle ) Figure , right c) Figure , left an) Figure , right. ) Figure . Collectin vivo, ex vivo and in vitro. All mice that received intra-tibial injection of human MDA-231-BT2 breast cancer cells suffered from osteolysis and bone damage as assessed by micro-CT analysis . Conversely, calvarial bone exposed to conditioned medium from IKKβ deficient MDA-231-BT1 cells suffered less bone damage in vivo . Further detailed histological analysis of the calvarial bone from the ex vivo experiment described in Figure p < 0.05, figure not shown) and higher bone volume  when compared to mice injected with conditioned medium from mock control cells. Representative photomicrographs of histological sections showing osteoclasts and osteolysis are shown in Figure ex vivo experiments using calvarial organ culture exposed to conditioned medium from osteotropic human MDA-231-BT1 cells . In contrast, both knockdown and pharmacological inhibition of IKKβ in these cells significantly reduced FoxO3a cytoplasmic sequestering and enhanced FoxO3a nuclear localisation, whereas IKKβ overexpression was inhibitory   Figure . We alsoin vitro, ex vivo and in vivo. Furthermore, IKKβ overexpression significantly enhanced skeletal tumour burden and increased the incidence of bone metastasis, exacerbated osteolysis, worsened cachexia and increased mortality in mice injected with two independent osteotropic sub-clones of MDA-231 cells with different metastatic abilities. Conversely, knockdown of IKKβ in the aggressive osteotropic sub-clone of MDA-231 cells reduced their ability to cause osteolysis in both immuno-deficient and wild type mice. Although the effect of IKKβ knockdown/overexpression on breast cancer bone metastasis that we observed in this study were limited to the osteotropic MDA-231 breast cancer cells used, it is tempting to speculate that over-expressing IKKβ would likely have increased the ability of parental MDA-231 to metastasise to bone after intra-cardiac injection in mice. When combined with previous studies on the role of IKKβ in primary breast tumour development  was applied. Mice were euthanized by carbon dioxide inhalation after 55 days from tumour-cell injection. The primary tumours and the lungs of the animals were dissected and processed as described in [Four week-old female BALB/c-ribed in .5 /100 μL PBS) were injected into the left ventricle (16 mice per group) [in vivo micro-computed tomography analysis  [Four week-old female BALB/c-nu/nu athymic mice were anesthetized and cancer cells (1 × 10r group) . The devBelgium) . Radiogr4 cells/ 20μL PBS) in the left leg [Seven four week-old female BALB/c-nu/nu athymic mice received intra-tibial injection cancer cells . Tissue culture medium was changed every 48h hours and fresh control medium or medium containing test agents was added and the cultures were terminated after 7 days. For studies involving conditioned medium, conditioned medium form breast cancer cells (20% v/v) was prepared and added to the calvarial organ cultures. Calvarial bone density was assessed using microCT at a resolution of 5 mm.Neonatal mouse calvarias were isolated from 7 day-old mice, incubated in standard alpha-MEM for 24 hour and divided into equal halves along the median sagittal suture. Excised calvarial half was placed into culture on stainless steel rafts in 48-well plates in the presence and absence of cancer cells  using a Reichert-Jung 1150/Autocut microtome, incubated with antibodies and visualised using the Ultra-Vision Detection System anti-Polyvalent HRP/DAB kit according to the manufacturer’s instructions. Metastatic bones were embedded in methyl-methacrylate without decalcification. Von gieson-Von Kossa staining was performed to evaluate the tumour burden, Hematoxilin and Eosin staining for evaluating osteoclast parameters and toluidine blue staining for osteoblast. Histomorphometric measurements were carried out using an interactive image analysis system .2O2 and incubated for 40 min at room temperature with the anti-IKKβ rabbit polyclonal antibody . The staining signals were revealed using the Dako LSAB+ System-HRP. Slides were counterstained with Mayer’s hematoxylin. Human placenta was used as positive control whereas negative controls were performed in parallel by omitting primary antibody for each case. IKKβ expression was evaluated in invasive primary carcinoma, in situ carcinoma (where present) and bone metastases and recorded as percentage of positively stained cells and staining intensity .Paraffin embedded material from human breast cancer and matched bone metastases were retrieved from the archive of Pathology Department of San Salvatore Hospital . Histological sections were deparaffinized, incubated with 0.07 M citrate buffer (pH 6), 15 min at 98° C, for antigen retrieval, treated with 3% HCancer cells were transfected with siRNA  according to the manufacturer’s instructions. Small interfering RNAs  as a pool of four annealed double-stranded RNA oligonucleotides for IKKβ (M-003503-03) and their non-targeting control no. 3 (D001201-03)  were used. The efficiency of knockdown was determined by Western blot analysis.5 cell/cm2) were transfected with the 5 μg pMX retroviral vector (empty or containing IKKβ), 5 μg pCMV-Gag-pol vector, 5 μg pCMV-VSV-G vector and 40 μl polyethylenimine. pLKO.1 puro lentiviral plasmid empty  or containing IKKβ-shRNA  were purchased from Thermo-Dharmacon . To generate IKKβ-shRNA expressing lentivirus 293T-packaging cells (1 × 105 cell/cm2) were transfected with the 5 μg pLKO.1 lentiviral vector (empty or containing IKKβ), 5 μg psPAX2 vector, 5 μg pCMV-VSV-G vector and 40 μl polyethylenimine. Medium containing viral particles was collected, filtered applied immediately to MDA-231 cells, which had been plated 18 hours before infection at a density of 1 × 104 cells/cm2. Polybrene  was added to a final concentration of 5 mg/ml, and the retroviruses supernatants were incubated with the MDA-231 cells. Twenty-four hours after infections, cells were selected with 1mg/ml puromycin for 72 hours.To generate IKKβ expressing retroviruses 293T packaging cells (1 × 105 cells/well) for up to 21 days in αMEM supplemented with 10% FCS, penicillin and streptomycin, β-glycerol phosphate (10 μM) and L-ascorbic acid (50 μg/ml). Osteoblast number, differentiation and bone nodule formation were determined by AlamarBlue assay, alkaline phosphatase (Alk Phos) assay and alizarin red (ALZ) staining, respectively. Activity of Alk Phos was assessed by a colorimetric assay, which measures the conversion of p-nitrophenyl phosphate to p-nitrophenyl in cell lysate and activity was normalized to cell number as determined by the AlamarBlue assay as previously described [Primary osteoblasts were isolated from the calvarial bones of 2-day-old mice by sequential collagenase digestion. For bone nodule assay, osteoblasts were seeded into 12-well plates  cells were flushed from the long bones of 3–5 month old mice and were incubated for 48 hours in αMEM supplemented with mouse M-CSF (100 ng/ml). For osteoclast generation, M-CSF-generated bone marrow macrophages (osteoclast precursor cells) were cultured in αMEM supplemented with M-CSF (25 ng/ml) and RANKL (100 ng/ml). For studies involving conditioned medium, conditioned medium form cancer cultures was prepared in αMEM and added to osteoclast cultures (10% v/v). Osteoclasts were identified using TRAcP staining and manually counted on a Zeiss Axiovert light microscope.The migration of cancer cells was assessed by wound healing assay . BrieflyCell monolayer was gently scraped in standard lysis buffer (0.1% (w/v) SDS, 0.5% (w/v) sodium deoxycholate, 1% Triton X-100, 1 μM EDTA, 2% (v/v) protease inhibitor cocktail, 10 mM of sodium fluoride and 2% (v/v) phosphatase inhibitor cocktail. For studies involving Cytoplasmic/Nuclear fractions, cell monolayer was scraped in Cytoplasmic lysis buffer  supplemented with protease and phosphatase inhibitor cocktail. Cell pellets were then re-suspended in complete RIPA lysis buffer and protein concentration was measured using BCA assay . Total protein (70 mg) was resolved by SDS-PAGE on 12% polyacrylamide SDS gels, transferred onto PVDF membranes  and immunoblotted with appropriate antibodies. The immuno-complexes were visualised by an enhanced chemiluminescence detection kit  using horseradish peroxidase-conjugated secondary antibody , and then visualised using chemiluminescence  on a Syngene GeneGnome imaging system.Levels of vascular endothelial growth factor (VEGF) in MDA-231 and 4T1 conditioned medium was determined by enzyme linked immunosorbent assay (ELISA)  and Proteome Profiler Human XL Cytokine Array Kit , according to the manufacturer’s instructions.Nuclear extracts from bone and cancer cells were prepared using a nuclear extract kit  and DNA binding was measured using TRANSAM ELISA kit for p65 NFκB , according to the manufacturer’s instructions.® TotalPrep RNA Amplification Kit , according the manufacturer’s instructions, and then quantified using a Bioanalyser 2100. 750 ng of high-quality labeled cRNA per sample was hybridized to Illumina HumanHT-12v3 Expression Beadarrays in triplicate  using Whole-Genome Expression Direct Hybridisation kit (Illumina) and scanned with a 500GX (Illumina). The raw and normalized microarray data have been deposited in the Gene Expression Omnibus (GEO) database under accession number GSE71444.Ribonucleic acid (RNA) was extracted using RNeasy Mini Kit with RNAse-free DNAse treatment , according the manufacturer’s instructions. 500 ng of total RNA from each sample was amplified and biotinylated using IlluminaP values were derived from Wilcoxon test and were two-tailed. Immunohistochemical analysis of IKKβ was also performed in cohort of 13 primary breast carcinomas and 5 matched bone metastasis samples obtained from the Pathology Unit, San Salvatore hospital , with informed consent from all subjects.Retrospective analysis of IKKβ expression with respect to primary tumour growth and bone relapse was conducted using published cohorts of data. The MSK82, EMC192 and EMC286 datasets were directly integrated with batch-correction using ComBat. Exhaustive survival analysis was performed by considering all possible cut-points using the survivALL package. p-value value of 0.05 or below was considered statistically significant. The half maximal inhibitory concentration (IC50) values were calculated using GraphPad Prism 4.0 for windows. Other comparisons were performed using unpaired two-sided Student’s t test or analysis of variance (ANOVA) followed by Dunnet’s post test using SPSS for Windows version 11. Statistical analysis of the microarray data was performed using R and BioConductor packages. Quantile normalisation was performed using the lumi package. Comparisons between Kaplan-Meier curves were performed using the a log-rank (Mantel-Cox) test. Meta-analysis was performed using Review Manager (RevMan), version 5.3 .Results were reported as mean ± standard deviation (SD) as indicated in figure legends. A