Background
The association between inflammation and cancer development has recently received wide recognition as a cancer hallmark [
1]. Persistent inflammation contributes to the progression of cancer by promoting tumor survival, proliferation, angiogenesis, metastasis, and immune evasion [
2]. The processes involved in cancer-associated inflammation entail complex interactions between the tumor cells and the tumor microenvironment. The key mediators include inflammatory cells and pro-inflammatory cytokines. Inflammatory cells, in particular tumor-associated macrophages (TAMs), are present in most malignant tumors, and high TAM density is correlated with poor prognosis [
2‐
4]. Amongst the cells in the tumor microenvironment and tumor cells themselves, TAMs serve as a major source of pro-inflammatory cytokines that influence tumor progression [
3]. Abundant at tumor sites, IL-1 is one of the most potent pro-inflammatory cytokines that can modulate the growth and invasive properties of tumor cells [
3]. IL-1 exists in two agonistic forms, IL-1alpha and -beta (IL1B). IL1B is active as a secreted form whereas IL-1alpha is active as an intracellular protein. Elevated IL1B levels in tumor and serum are associated with higher tumor grade and increased invasion in breast and pancreatic cancer and in myelogenous leukemia, and are correlated with poor patient outcome [
5‐
10]. Ultimately, the primary cause of mortality from cancer is due to metastasis, the spreading of primary tumor cells to distant sites to form secondary tumors. IL1B has been shown to play a significant role on tumor invasiveness and metastasis progression [
9,
11‐
13]. Studies in vivo showed reduced hepatic and lung metastasis of B16 melanoma cell xenografts in IL1B knockout mice [
11,
12]. In human breast carcinoma tissues, IL1B levels were found elevated in higher grade tumors [
14] and in invasive breast carcinoma versus ductal carcinoma in situ (DCIS) and benign lesions [
5].
IL1B has been shown to up-regulate Osteoprotegerin (OPG) expression in the breast cancer cell lines MCF-7 and MDA-MB-231 [
15]. OPG is a secreted member of the tumor necrosis factor (TNF) receptor super-family, prominently known for its role as a decoy receptor in bone resorption
in vivo, and for its inhibition of TNF-related apoptosis-inducing ligand (TRAIL) mediated apoptosis in vitro [
16,
17]. There is increasing evidence for a role of OPG in cancer, as OPG expression has been found elevated in more aggressive solid tumors [
18‐
21]. A number of studies support a tumor-promoting effect of OPG in breast cancer [
22]. OPG over-expression in MCF-7 (estrogen receptor, ER+) breast cancer cells resulted in increased tumor growth and osteolysis in mouse xenografts [
23]. Recently, we reported that siRNA-mediated OPG knockdown in triple-negative breast cancer cells reduced invasion and metastasis in a chick embryo in vivo model [
24]. Based on these findings we hypothesized that IL1B modulates breast cancer invasion and metastasis by OPG regulation.
Breast cancer metastasis poses significant treatment challenges. Furthering our understanding of the molecular processes involved is essential for novel therapeutic strategies for metastatic breast cancer. In this current study, we investigate the IL1B-mediated upstream signaling events involved in OPG expression, look into the involvement of macrophages in OPG expression, and examine the link between OPG and IL1B as a novel inflammatory pathway promoting breast cancer metastasis.
Methods
Reagents and cell culture
Recombinant human IL1B (200-01B) and IL-1R antagonist (IL1-RA, 200-01R) were purchased from Peprotech (Rocky Hill, NJ). p38 MAPK (8690), phospho-p38 MAPK (Thr180/Tyr182; 4511), p42/44 MAPK (9107S), phospho-p42/44 MAPK (Thr202/Tyr204; 9101) antibodies were purchased from Cell Signaling Technology (Beverly, MA). MAPK inhibitors SP600125, SB202190 and SB203580 were purchased from Sigma Aldrich (St Louis, MO), U0126 and BAY869766 were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA).
The human breast cancer lines: MDA-MB-231, MDA-MB-436, BT549, SKBR3, ZR75-1, HCC1954 were cultured in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals, Lawrenceville, GA), 2 mM L-glutamine, and 50 μg/mL gentamicin (Life Technologies, Carlsbad, CA). THP-1 monocyte cells were cultured in RPMI 1640 supplemented with 10% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 10 mM HEPES, and 1% antibiotic/antimycotic solution (15240062, Life Technologies). All cell lines were recently acquired from the ATCC (Manassas, VA). Cell lines were incubated in a humidified atmosphere of 5% CO2 at 37 °C.
Enzyme-linked immunosorbent assay
5 × 105 breast cancer cells were seeded in 2 mL of medium in a 6 well plate and incubated for 48 h. Treatment with IL1B or IL-1RA was administered for the last 24 h. OPG protein from cell culture supernatant was measured using the OPG/TNFRSF11B DuoSet (R&D Systems, Minneapolis, MN). IL1B protein from cell culture supernatant was measured using the Human IL1B ABTS ELISA Development Kit (Peprotech).
Western blot
Protein extracts were obtained by cell lysis in M-PER (Pierce Biotechnology, Rockford, IL) and Halt protease inhibitor cocktail (Pierce Biotechnology). Proteins were separated by SDS-PAGE and blotted onto nitrocellulose membranes. Membranes were blocked with Blocking Buffer (LI-COR, Lincoln, NE) and incubated with specific antibodies. Protein signals were visualized using the Odyssey infra-red imaging scanner and software (LI-COR).
Real-time polymerase chain reaction
Total RNA was extracted from breast cancer cells using the RNeasy kit (Qiagen, Germantown, MD). cDNA was prepared from RNA (200 ng) in a 20 μL reaction using the iScript cDNA synthesis kit (Biorad, Hercules, CA). qRT-PCR reactions were performed in 25 μL mixtures containing 1 μL of cDNA, 2x iQ SYBR Green supermix (Biorad), and forward and reverse primers. cDNA’s diluted 1:3 were used for the qRT-PCR reactions of the 18S rRNA housekeeping gene. See Additional file
1: Table S1 for primer sequences. mRNA was normalized to 18S rRNA. Relative expression was determined by the ΔΔCT method [
25].
siRNA transfection
Breast cancer cells were transfected with OPG Stealth RNA siRNA (Stealth siRNAs for human OPG: HSS107349 [#1], HSS181651 [#2] and HSS181652 [#3]) or Negative Control Medium GC siRNA using Lipofectamine RNAiMax, according to the manufacturer’s instructions (all from Life Technologies).
Transwell co-culture assay
On Day 0, 1 ×106 THP-1 cells/well were seeded into a 6 well plate. On Day 1, the THP-1 cells were treated with 1 mM PMA to induce macrophage differentiation, and 5 × 105 breast cancer cells were seeded onto polycarbonate inserts for 6-well plates (pore size 0.4 μm; Corning Costar, Tewksbury, MA). On Day 2, 4 mL of fresh THP-1 medium was placed onto the THP-1 cells prior to the transfer of the transwell inserts containing the breast cancer cells. The co-cultures were incubated for 8 h in a humidified atmosphere of 5% CO2 at 37 °C.
Invasion assay
MDA-MB-436 cells were transfected with OPG or negative control siRNA as described above. Twenty-four hours post-transfection, cells were treated with IL1B (10 ng/mL) in medium containing 0.5% FBS for another 24 h. Following treatment, cells were seeded at 5 × 104 cells/well into a 96 well cell invasion chamber plate pre-coated with 1x Collagen IV using the Cultrex 96 Well Collagen IV Cell Invasion Assay (3458-096-K, Trevigen, Gaithersburg, MD),. The invasion assay was incubated for 24 h and analyzed according to the manufacturer’s instructions.
Public dataset analysis
Human breast cancer genome-wide mRNA expression datasets from patient sample series deposited for public access in a MIAME-compliant format were obtained through the Gene Expression Omnibus (GEO) database at the NCBI website (
http://www.ncbi.nlm.nih.gov/geo/), except for the Chin-124 set (E-TABM-158) from EMBL/EBI (
http://www.ebi.ac.uk/arrayexpress), and two TCGA sets from
https://gdc-portal.nci.nih.gov/. Datasets were analyzed using R2; a genomics analysis and visualization platform developed in the Department of Oncogenomics, Academic Medical Center, Amsterdam, The Netherlands (
http://r2.amc.nl/). Expression data were uploaded into R2 and analyzed as described previously [
26]. Briefly, gene transcript levels from Affymetrix array studies were determined from data image files using GeneChip operating software MAS5.0 and GCOS1.0, from Affymetrix (Santa Clara, CA). Samples were scaled by setting average intensity of the middle 96% of probe signals to a fixed value of 100 for every sample in the dataset, allowing comparisons between micro-arrays. The Illumina arrays for Servant-343 and Jonsdottir-94, and the Agilent arrays for TCGA-528 and −1097 underwent custom processing and normalization as described on their websites. All 29 public datasets were scrutinized for OPG (TNFRSF11B gene) mRNA expression and OPG-correlating genes. 13 datasets with a sample size < 100 and/or the absence of OPG mRNA expression or correlating genes were omitted from further analysis: Black-107 (GSE36771), Clynes-121 (GSE42568), Concha-66 (GSE29431), Desmedt-55 (GSE16391), Jonsdottir-94 (GSE46563), Loi-77 (GSE9195), Miller-116 (GSE5462), Minn-96 (GSE2603), Prat-156 (GSE50948), Quiles-61 (28844), Sotiriou-120 (GSE16446), Sotiriou-198 (GSE7390), and Wessels-60 (GSE41656). The TranscriptView tool (
http://bioinfo.amc.uva.nl/human-genetics/transcriptview/) was used to select probe-sets for the Affymetrix and Illumina datasets. No sequence data were available for the two TCGA datasets. Probes had to show unique mapping in an anti-sense position within (late) coding exons and/or the 3’ UTR of the gene. When multiple correct probe-sets were available for a gene, the probe-set with the highest average expression and the highest amount of present calls for that dataset were used. All probe-sets used meet these criteria. In no cases did additional probe-sets show a conflicting result for that dataset.
Gene expression analysis of human breast cancer and normal tissues
qPCR was performed to characterize the OPG, IL1B and CCL2 mRNA expression profiles using the TissueScan Breast Tissue qPCR Array (OriGene, Technologies, Rockville MD) containing cDNA from normal and breast cancer tissue from different disease stages. See Additional file
1: Table S1 for primer sequences. Data were presented as delta Ct values normalized against β-actin.
Immunohistochemistry
Human breast cancer tissue microarray slides (Abcam, Cambridge, MA) were separately immunostained for OPG (Abcam) and CD68 (Dako North America, Inc. Carpinteria, CA). Following deparaffinization in xylene and rehydration, slides were subjected to antigen retrieval (10 mmol/L citrate buffer; pH 6.0), followed by 3% hydrogen peroxide incubation, and blocking in 1.5% goat serum. Slides were then incubated with diluted primary antibody followed by incubation with HiDef amplifier secondary antibody, and detection with HiDef polymer (Cell Marque Laboratories, Rocklin, CA) and DAB substrate (3,3-diaminobenzidine; Cell Marque Laboratories). Counterstaining was performed with hematoxylin. The tissue sections were scored semi-quantitatively by a pathologist based on staining intensity. Staining and analyses were performed by the University of Hawaii Cancer Center-Pathology Shared Resource.
Statistical analysis
Results are presented as mean ± SD. Statistical analyses were determined using a Student’s
t-test, as indicated in the text. Analyses were performed and graphs were plotted in Prism V6 (GraphPad Software). Correlations between OPG and CCL2/IL1B mRNA expression (Fig.
4) were calculated using R2. Briefly, a Pearson test was performed on 2log-transformed expression values (with the significance of a correlation determined by t = r/sqrt((1-r^2)/(n-2)), where r is the correlation value and n is the number of samples, and distribution measure is approximately as t with n-2° of freedom). Correlations were only calculated for datasets when ≥ 10% of samples had a present call for both genes. For all statistical analyses,
p < 0.05 was considered significant.
Discussion
Persistent inflammation is linked with cancer development and progression [
43]. It has been shown that treatment with the inflammatory cytokine IL1B promotes the invasiveness of breast cancer cells in vitro [
38,
39]. In our previous work we reported that the invasive and metastatic capacity of TNBC cells was reduced upon OPG knockdown [
24]. In this study, we investigated OPG in the context of its role in breast cancer and inflammation, with a particular focus on IL1B. Here we provide mechanistic insight into the IL1B-OPG signaling axis and reveal a potential role for OPG in the invasion-promoting effects of IL1B. We show that OPG secretion is induced by IL1B in a p38- and p42/44-dependent manner, independent of breast cancer subtype or basal OPG levels. Macrophages, but possibly also breast cancer cells themselves, may serve as local IL1B sources to influence OPG secretion. Also, we show that IL1B-mediated breast cancer cell invasion, and the induction of MMP3 and IL1B itself, occurs in an OPG-dependent manner.
Elevated OPG secretion has been detected in aggressive tumors with poor patient outcome, including breast, lung, prostate, gastric and bladder cancers [
19,
20,
44‐
46]. In breast cancer cells, OPG over-expression resulted in enhanced tumor growth [
23] and increased pulmonary metastasis in mice [
47]. In agreement with these reports, we have shown that TNBC cells, representing the most aggressive breast cancer subtype, secreted higher basal OPG levels than non-TNBC cells. Similarly, we showed that TNBC cells secreted higher IL1B levels than non-TNBC cells, which exhibited little to no IL1B secretion. These results are consistent with a study indicating that the non-TNBC cell line HCC1954 produces low IL1B amounts [
48]. We found that regardless of the basal OPG and IL1B levels, all breast cancer cell lines remained responsive to IL1B-mediated OPG induction, with the highest induction of OPG secretion in the non-TNBC cells (Fig.
1c and d). Taking into account the association of higher OPG and IL1B basal levels in TNBC cells, we treated the TNBC type MDA-MB-436 cell line with the IL1B receptor antagonist IL-1RA. This resulted in the partial repression of basal OPG secretion suggesting an autocrine loop, by which IL1B produced by cells is linked to the higher basal expression of OPG.
The mitogen-activated protein kinase (MAPK) signaling pathway is often activated in cancer [
49,
50]. Indeed, p38 and p42/44 MAPK signaling have been associated with breast cancer invasion and progression. In breast cancer cells it has been reported that elevated p38 MAPK signaling can drive invasiveness and chemoresistance of HER2-overexpressing cells [
35]. Another study has reported that patients with lymph node-positive breast carcinoma showed shorter progression-free survival when their primary tumors expressed high levels of phosphorylated p38 [
32]. In a study examining primary human breast tumors, 11 out of 23 samples showed active p42/44, significantly elevated relative to adjacent matched normal breast tissue [
51]. Interestingly, it has been reported that TNBC, basal-like type breast cancer cell lines, relative to breast cancer cell lines representative of the other subtypes, exhibit a greater sensitivity to p42/44 MEK1/2 inhibitors [
52,
53]. In our inhibitor studies we confirmed that both p38 and p42/44 MAPK activities mediated IL1B-induced OPG secretion. This effect was observed independent of breast cancer subtype. Therefore our results show that IL1B-induced OPG secretion is regulated by the p38 and p42/44 MAPK pathways in breast cancer cells.
In an analysis of publicly available human breast cancer genome-wide mRNA expression datasets from patient samples we showed that OPG mRNA expression was significantly correlated with IL1B mRNA expression. This correlation is consistent with in vitro experiments showing that IL1B and OPG levels were either both elevated or both relatively lower in TNBC cells and non-TNBC cells, respectively. CCL2 mRNA expression was also found to be significantly correlated with OPG mRNA expression. CCL2 is a potent chemo-attractant involved in macrophage tissue infiltration. TAMs are present in many solid tumors, including breast cancer. Clinical studies have indicated TAM levels are correlated with breast cancer prognosis [
54], and experimental evidence showed that CCL2 levels are significantly associated with TAM numbers [
36,
55] and TAM retention [
56], implicating CCL2 in breast cancer progression. Furthermore, we showed relative to normal breast tissue that elevated levels of CCL2, IL1B and OPG mRNA levels were detected in stage I breast cancer human tissue samples. We asked whether macrophages could serve as an IL1B source to influence OPG expression in breast cancer cells. Upon co-culture with THP-1 macrophages, OPG was significantly induced in breast cancer cells. This induction was partially repressed in the presence of IL-1RA, indicating that the effects on OPG were specific to IL1B. The potential causative link between macrophages and elevated OPG levels were further supported by the immunohistochemical analyses of pan-macrophage marker CD68 and OPG which indicated a co-occurrence of these two markers in human primary breast tumors. Studies have shown that IL1B and the p38 and p42/44 MAPK pathways play important roles in tumor cell progression [
9,
31,
34,
35,
38,
39,
51‐
53,
57‐
59]. Given our data demonstrating that OPG is subject to regulation by IL1B-p38 and -p42/44 signaling, we sought to investigate OPG function in IL1B-mediated breast cancer invasion. Our results showed that IL1B treatment significantly elevated the invasiveness of MDA-MB-436 cells. Interestingly, these effects were inhibited upon OPG knockdown. MMPs play key roles in promoting the invasive properties of tumor cells [
40]. Particularly, MMP3 has been shown to drive the formation of mammary tumors in mice when over-expressed in mammary epithelial cells [
41]. Additionally, MMP3 mRNA levels had been reported to be elevated in TNBC cells versus non-TNBC cells, where treatment with exogenous IL1B could lead to MMP3 up-regulation [
39]. In line with this published study, treatment of MDA-MB-436 with IL1B cells induced MMP3 expression. We show IL1B treatment can lead to IL1B up-regulation (Fig.
4d). Induction of both MMP3 and IL1B were repressed in cells treated with OPG siRNA (Figs.
4c-e), this is in agreement with our previous study showing reduced invasion upon OPG knockdown in TNBC cell lines [
24]. The effects of OPG knockdown on IL1B induction suggest that IL1B expression may also be subject to regulation by OPG. Further studies are needed to define this cross-regulation between OPG and IL1B. In any case, the interdependent effects of OPG and IL1B expressions further support the significance of OPG in inflammatory-driven tumor progression.