Abstract 5109: The CARMA3-Bcl10-MALT1 signalosome mediates NF-κB activation and cellular invasion in AGTR1-positive breast cancer
Authors: Ekambaram Prasanna , Hubel Nathaniel , Lee Jia-Ying , Klei Linda , Concel Vincent , Delekta Philip , Tomlins Scott , McAllister-Lucas Linda , Lucas Peter 
Published: August 2015

Angiotensin II (Ang II) is a potent vasoconstrictor and vascular pro-inflammatory mediator, known classically for its role in promoting arterial dysfunction. Many of these pathogenic effects result from activation of NF-κB in response to stimulation of the type-1 Ang II receptor (AGTR1), a G protein-coupled receptor. We previously discovered that a complex composed of three proteins, CARMA3, Bcl10 and MALT1 (CBM signalosome) mediates AGTR1-dependent activation of NF-κB in vascular cells. Despite its principal role in vascular pathobiology, we recently found that AGTR1 is aberrantly overexpressed in 10-20% of breast cancers (characterized as Luminal, ER+, PR+, Her2-). In this context, AGTR1 promotes cellular invasion and in vivo tumorigenesis, but the mechanisms driving this effect of AGTR1 have remained unclear. We hypothesized that stimulation of AGTR1 in this subset of breast tumors induces CBM-dependent NF-κB activation, thus contributing to tumorigenesis.

To develop experimental models for studying the impact of AGTR1 in breast cancer (BC), we screened a panel of BC cell lines and found overexpression of endogenous AGTR1 in BT549 cells, among a few others, but no expression in most lines. To complement the BT549 line, we stably over-expressed exogenous AGTR1 in ZR75 cells (an AGTR1-, luminal line) to produce the ZR75-AGTR1 line. All BC lines tested, irrespective of AGTR1 status, expressed CARMA3, Bcl10 and MALT1.

We found that Ang II treatment induces time-dependent phosphorylation of IκB, indicating NF-κB activation, in both BT549 and ZR75-AGTR1 cells, but not in control ZR75 cells. We then employed an NF-κB reporter assay to generate further evidence of NF-κB activation in ZR75-AGTR1 cells. We also demonstrated induction of several endogenous NF-κB target genes using real-time Q-PCR. Importantly, Ang II-dependent NF-κB activation is effectively blocked by pretreatment with losartan (AGTR1 antagonist) or with IKK-VI (IKKβ inhibitor), as well as by siRNA-mediated knockdown of Bcl10 or MALT1. These data indicate that Ang II dependent NF-κB activation in these cells is dependent on activation of AGTR1, proceeds through the canonical NF-κB machinery, and requires the CBM signalosome. Moreover, we showed that AGTR1 activation triggers a dramatic increase in migration and invasion of both BT549 and ZR75-AGTR1 cells, and that this response is completely blocked by siRNA-mediated Bcl10 knockdown.

These results demonstrate for the first time that AGTR1 stimulation leads to NF-κB activation in a subset of breast cancer cells, and link this response to aggressive phenotypic behavior. These studies have significant clinical implications since AGTR1 antagonists, currently used for treatment of hypertension, might be effectively re-deployed for treatment of breast cancers that harbor AGTR1 overexpression. Inhibition of CBM-dependent signaling may also represent a novel treatment approach.

Citation Format: Prasanna Ekambaram, Nathaniel Hubel, Jia-Ying Lee, Linda Klei, Vincent Concel, Philip Delekta, Scott Tomlins, Linda McAllister-Lucas, Peter Lucas. The CARMA3-Bcl10-MALT1 signalosome mediates NF-κB activation and cellular invasion in AGTR1-positive breast cancer. [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 5109. doi:10.1158/1538-7445.AM2015-5109