BackgroundIt is widely recognized that inflammation promotes breast cancer invasion and metastasis. Given the complex nature of the breast tumor inflammatory microenvironment, much remains to be understood of the molecular mechanisms that govern these effects. We have previously shown that osteoprotegerin knockdown in breast cancer cells resulted in reduced invasion and metastasis. Here we present novel insight into the role of osteoprotegerin in inflammation-driven tumor progression in breast cancer by investigating the link between osteoprotegerin, macrophages and the potent pro-inflammatory cytokine Interleukin-1beta.MethodsWe used human breast cancer cell lines to investigate the effects of Interleukin-1beta treatment on osteoprotegerin secretion as measured by ELISA. We analyzed public datasets containing human breast cancer genome-wide mRNA expression data to reveal a significant and positive correlation between osteoprotegerin mRNA expression and the mRNA expression of Interleukin-1beta and of monocyte chemoattractant protein CC-chemokine ligand 2. Osteoprotegerin, Interleukin-1beta and CC-chemokine ligand 2 mRNA levels were also examined by qPCR on cDNA from normal and cancerous human breast tissue. We determined the effect of Interleukin-1beta–producing macrophages on osteoprotegerin expression by co-culturing breast cancer cells and differentiated THP-1 macrophages. Immunohistochemistry was performed on human breast tumor tissue microarrays to assess macrophage infiltration and osteoprotegerin expression. To demonstrate that osteoprotegerin mediated functional effects of Interleukin-1beta we performed cell invasion studies with control and OPG siRNA knockdown on Interleukin-1beta-treated breast cancer cells.ResultsWe report that Interleukin-1beta induces osteoprotegerin secretion, independent of breast cancer subtype and basal osteoprotegerin levels. Co-culture of breast cancer cells with Interleukin-1beta-secreting macrophages resulted in a similar increase in osteoprotegerin secretion in breast cancer cells as Interleukin-1beta treatment. Macrophage infiltration correlates with osteoprotegerin secretion in human breast tumor tissue samples. We show that osteoprotegerin secretion is regulated by Interleukin-1beta in a p38- and p42/44-dependent manner. We also demonstrate that osteoprotegerin knockdown represses Interleukin-1beta expression, Interleukin-1beta-mediated breast cancer cell invasion and MMP3 expression.ConclusionsThese data indicate a novel role for osteoprotegerin as a mediator of inflammation- promoted breast cancer progression.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-017-0606-y) contains supplementary material, which is available to authorized users.

It is widely recognized that inflammation promotes breast cancer invasion and metastasis. Given the complex nature of the breast tumor inflammatory microenvironment, much remains to be understood of the molecular mechanisms that govern these effects. We have previously shown that osteoprotegerin (OPG) knockdown in breast cancer cells resulted in reduced invasion and metastasis. Here we present novel insight into the role of OPG in inflammation-driven tumor progression in breast cancer cells by investigating the link between OPG and the potent pro-inflammatory cytokine IL1B (IL1B). We used human breast cancer cell lines to investigate the effects of IL1B treatment on OPG expression. We analyzed public datasets containing human breast cancer genome-wide mRNA expression data to reveal a significant and positive correlation between OPG mRNA expression and the mRNA expression of IL1B as well as CCL2 (a monocyte chemoattractant protein). We determined the effect of macrophages (which produce IL1B) on OPG expression by co-culturing breast cancer cells and differentiated THP-1 macrophages. In order to demonstrate that OPG mediated functional effects of IL1B we performed cell invasion studies with control and OPG siRNA knockdown breast cancer cells treated with IL1B. We report that OPG expression is induced by IL1B, independent of breast cancer subtype and basal OPG levels. Co-culture of breast cancer cells with IL1B-secreting macrophages resulted in a similar increase in OPG expression in breast cancer cells as IL1B treatment. We show that OPG expression is regulated by IL1B in a p38-dependent manner. We also demonstrate that OPG knockdown represses IL1B expression, IL1B-mediated breast cancer cell invasion and MMP3 expression.It is widely recognized that inflammation promotes breast cancer invasion and metastasis. Given the complex nature of the breast tumor inflammatory microenvironment, much remains to be understood of the molecular mechanisms that govern these effects. We have previously shown that osteoprotegerin (OPG) knockdown in breast cancer cells resulted in reduced invasion and metastasis. Here we present novel insight into the role of OPG in inflammation-driven tumor progression in breast cancer cells by investigating the link between OPG and the potent pro-inflammatory cytokine IL1B (IL1B). We used human breast cancer cell lines to investigate the effects of IL1B treatment on OPG expression. We analyzed public datasets containing human breast cancer genome-wide mRNA expression data to reveal a significant and positive correlation between OPG mRNA expression and the mRNA expression of IL1B as well as CCL2 (a monocyte chemoattractant protein). We determined the effect of macrophages (which produce IL1B) on OPG expression by co-culturing breast cancer cells and differentiated THP-1 macrophages. In order to demonstrate that OPG mediated functional effects of IL1B we performed cell invasion studies with control and OPG siRNA knockdown breast cancer cells treated with IL1B. We report that OPG expression is induced by IL1B, independent of breast cancer subtype and basal OPG levels. Co-culture of breast cancer cells with IL1B-secreting macrophages resulted in a similar increase in OPG expression in breast cancer cells as IL1B treatment. We show that OPG expression is regulated by IL1B in a p38-dependent manner. We also demonstrate that OPG knockdown represses IL1B expression, IL1B-mediated breast cancer cell invasion and MMP3 expression. Citation Format: Tsang Mui Chung S, Geerts D, Roseman K, Renaud A, Connelly L. Osteoprotegerin mediates tumor-promoting effects of Interleukin-1beta in breast cancer cells [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-08-10.

Osteoprotegerin (OPG) is a secreted member of the Tumor Necrosis Factor family. In addition to the functional roles of OPG in bone metabolism and apoptosis, there is growing evidence of the tumor promoting role of OPG in breast cancer. We have previously shown OPG can promote the invasion and metastasis of triple negative breast cancer cells. To understand the regulation of OPG, we are investigating the molecular mechanisms that control OPG expression in breast cancer cells. Breast cancer cell lines MDA-MB-231 and MDA-MB-436 (triple negative); SKBR-3 and HCC1954 (Human Epidermal Growth Factor Receptor 2 positive; HER2+); ZR75-1 and MCF-7 (Estrogen Receptor positive; ER+) were treated with Interleukin1beta (IL1-beta) for 24 hours. Levels of OPG mRNA and secreted OPG protein were analyzed by real time RT-PCR and ELISA, respectively. IL1-beta induced OPG expression regardless of basal OPG levels, with the exception of MDA-MB-231 which exhibited relatively moderate OPG induction. To explore the signaling mechanisms downstream of IL1-beta, we treated breast cancer cells with MAP kinase inhibitors in the presence of IL1-beta and examined the effects on OPG protein induction. Both p38 MAPK inhibitors, SB203580 and SB202190, were observed to partially blocked OPG induction by IL1-beta. Western blot analysis confirmed IL1-beta induced p38 phosphorylation. Additionally, we examined the effects of NFkappaB p65 binding to potential NFKappaB binding sites within the OPG promoter upon IL1-beta treatment by ChIP assay in SKBR3 and MDA-MB-436 cells. The highest fold increased binding was observed to occur within the -188 and -4675bp regions upon cytokine treatment in SKBR3 cells. No detectable binding was observed in MDA-MB-436 cells. We also looked at whether OPG expression levels could be altered by incubating breast cancer cells with macrophages, a cell type found in the breast tumor microenvironment. We co-cultured cells from each subtype with THP-1 human macrophages for 8 hours and extracted mRNA from the breast cancer cells or incubated the breast cancer cells for an additional 16 hours alone to measure secreted OPG protein. We found that co-culture with macrophages significantly increased OPG mRNA and protein expression in MCF-7 (ER+) and SKBR-3 (HER2+) cells but there was no increase in the MDA-MB-231 cells (triple negative). Overall, we show that IL1-beta can induce OPG expression in breast cancer cells and this signaling may be mediated by activating p38 MAP kinases. The ChIP analysis indicates signaling may involve activation of classical NFkappaB signaling. The similar effects of cytokine induced OPG induction observed in the co-culture of breast cancer cells with THP-1 suggests a mechanism by which inflammatory signaling could increase OPG expression in vivo. Citation Format: Stephanie Tsang Mui Chung, Ashleigh Renaud, Kim Roseman, Nalani Yadav, Linda Connelly. Inflammatory signaling regulates osteoprotegerin expression in breast cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B58.

Osteoprotegerin (OPG) is a regulator of bone metabolism that is also produced by breast tumor cells. There is accumulating evidence that OPG has tumor-promoting effects in breast cancer. We have recently demonstrated that OPG can promote invasion and metastasis of triple negative breast cancer cells. We are now investigating the expression of OPG across major breast cancer subtypes. We cultured 3 breast cancer cells lines from each of the major subtypes: MCF-7, ZR-75-1 and T47-D (Estrogen Receptor positive; ER+); SKBR-3, HCC1954 and HCC202 (Human Epidermal Growth Factor Receptor 2 positive; HER2+) and MDA-MB-231, MDA-MB-436 and BT-549 (triple negative). We incubated cells for 24 hours and then measured OPG mRNA expression by real time RT-PCR and secreted OPG protein in cell culture supernatant by ELISA. The triple negative cells that we tested expressed the highest levels of OPG mRNA and protein. A much lower level of OPG expression was observed in the HER2+ cells. Contrasting results were observed with the ER+ cells. While there was no expression from the ZR-75-1 or T47-D cells after 24 hours incubation, the MCF-7 cells produced levels of OPG similar to those observed with the triple negative breast cancer cells. We also looked at whether OPG expression levels could be altered by incubating breast cancer cells with macrophages, a cell type found in the breast tumor microenvironment. We co-cultured cells from each subtype with THP-1 human macrophages for 8 hours and extracted mRNA from the breast cancer cells or incubated the breast cancer cells for an additional 16 hours alone to measure secreted OPG protein. We found that co-culture with macrophages significantly increased OPG mRNA and protein expression in MCF-7 (ER+) and SKBR-3 (HER2+) cells but there was no increase in the MDA-MB-231 cells (triple negative). In conclusion, while OPG was expressed at comparatively high levels in triple negative breast cancer cells, there were generally low levels or no expression in the ER+ and HER2+ cells tested. This suggests that the tumor-promoting effects of OPG may contribute to the more aggressive phenotype in triple negative cells. However OPG expression in ER+ and HER2+ cells can be stimulated through co-culture with macrophages. Thus conditions within the breast tumor microenvironment could amplify the effects of OPG in these tumor subtypes. Citation Format: Stephanie Tsang, Ashleigh Renaud, Kim Roseman, Yuko Imaizumi, Nalini Yadav, Michael Weichhaus, Linda Connelly. Level of Osteoprotegerin expression is breast cancer subtype specific. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3210. doi:10.1158/1538-7445.AM2015-3210