Abstract 1364: Osteoprotegerin production by breast cancer cells: Regulation by NF-kappaB and role in metastasis

Abstract

Abstract Human breast cancer cells and tissues express the protein osteoprotegerin (OPG). While many studies have investigated the role of OPG in cancer as a mediator of bone remodeling, few studies consider its impact outside the bone microenvironment. Further investigation is therefore required into endogenous OPG expression by breast cancer cells and its effects on tumor progression. OPG can be regulated by the transcription factor NF-kappaB in several cell types. We investigated whether OPG expression in MDA-MB-231 and MDA-MB-435 breast cancer cells was regulated by NF-kappaB. Cells were treated for 24 hours with 10ng/ml TNF-alpha or 10µM thymoquinone (TQ), to stimulate or inhibit NF-kappaB activity respectively. OPG expression was measured by real time RT-PCR (mRNA) and ELISA (protein). OPG levels were increased after treatment with TNF-alpha while treatment with TQ decreased expression. We used the chick embryo metastasis model to determine the effect of OPG on metastasis. Human breast cancer cells were inoculated onto the chick chorioallantoic membrane (CAM) where they form a tumor that metastasizes to the chick tissue. Metastasis is quantified by real time PCR for Human Alu repeat DNA sequences in the chick tissue. We transfected MDA-MB-231 cells with OPG-specific siRNA which reduced OPG mRNA expression up to 72 hours post-transfection. OPG knockdown or control (non-targeting siRNA) treated cells were inoculated onto the CAM at embryonic day 9; chick tissue was harvested at day 16 and analyzed for breast cancer cell metastasis. We found a significant reduction in metastasis of the OPG knockdown cells compared to the control. We performed genome-wide mRNA microarray data analysis to determine whether OPG expression correlates with breast cancer progression in human tumor samples. OPG expression in 1809 breast cancer tissues was not correlated with prognosis. However, analyses by breast cancer subtypes showed that high OPG mRNA expression correlated with poorer prognosis in basal (p=0.0028, n=383) and also HER2 positive tumor subtypes (p=0.021, n=408). In summary, our data suggest that OPG expression in breast cancer cells is regulated by NF-kappaB, a transcription factor associated with pro-tumor effects. Our in vivo studies suggest that OPG promotes metastasis. We are currently determining the mechanism whereby OPG exerts its effects. Since OPG levels correlate with poorer prognosis in basal and HER2 positive tumors, treatment strategies targeting OPG in the primary tumor may represent a potential therapeutic strategy for these disease subytpes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1364. doi:1538-7445.AM2012-1364