Abstract P6-07-02: The CARMA3-Bcl10-MALT1 signalosome mediates pro-angiogenic IL-6 and IL-8 paracrine signaling in GPCR+ breast cancer
Authors: Kang H , Ekambaram P , McAllister-Lucas LM , Lucas PC 
Published: May 2019

Abstract
Background: The overexpression of selected G-protein coupled receptors (GPCRs) has been linked to the pathogenesis of multiple cancer subtypes. We recently demonstrated that overexpression of two such GPCRs, the angiotensin II receptor type I (AGTR1) and protease-activated receptor type 1 (PAR1), occurs in a substantial fraction of luminal breast cancers and is associated with treatment resistance and poor prognosis. Further, experimental models demonstrate that overexpression of these receptors in breast cancer cell lines promotes aggressive features that include EMT, invasion, migration, and loss of ER expression. In addition to these cell intrinsic effects, we find that GPCR overexpression in breast cancer cells can impact the tumor microenvironment. Specifically, we recently reported that conditioned media from angiotensin II-stimulated AGTR1+ breast cancer cells induces endothelial chemotaxis in vitro and tumor angiogenesis in vivo. We also demonstrated that this pro-angiogenic phenotype requires the CARMA3-Bcl10-MALT1 (CBM) signalosome, a signaling complex that links upstream GPCR stimulation with downstream NF-κB activation. We hypothesized that stimulation of either AGTR1 or PAR1 induces CBM-dependent secretion of NF-κB responsive, pro-angiogenic factors from these GPCR+ breast cancer cells which then exert proangiogenic effects on neighboring endothelial cells through paracrine signaling.
Methods: To identify CBM-dependent secreted factors, we evaluated the AGTR1+ BT549 cell line, +/- Bcl10 or MALT1 siRNA knockdown, for expression of 770 genes of significance to solid tumor pathogenesis using the NanoString PanCancer Progression Panel followed by Ingenuity Pathway Analysis (IPA). RT-PCR and ELISA were used to validate hits and determine if the CBM signalosome controls expression of the same genes in the PAR1+ cell lines, MCF7-N55 and MDA-MB-231.
Results: We identified IL-6 and IL-8 signaling pathways as the two most significantly downregulated angiogenesis pathways following either Bcl10 or MALT1 knockdown. Using quantitative RT-PCR and ELISA, we confirmed that IL-6 and IL-8 gene expression and protein secretion are significantly induced in response to stimulation of BT549 cells by angiotensin II and MCF7-N55 and MDA-MB-231 cells by TRAP6, a PAR1 agonist. siRNA-mediated MALT1 knockdown in BT549 cells led to a significant reduction of IL-6 and IL-8 gene expression upon angiotensin II stimulation; similarly, CRISPR/Cas9-mediated MALT1-deletion in MCF7-N55 cells resulted in failure of these cells to secrete IL-8 upon TRAP6 stimulation.
Conclusions: The GPCR-CBM-cytokine signaling pathway provides a common druggable target to curb pro-angiogenenic paracrine signaling in GPCR+ breast cancers. Importantly, the CBM signalosome has also been shown to be required for IL-8 dependent upregulation of VEGF in endothelial cells, indicating that inhibition of the signalosome could exert complementary effects on both cancer cells and endothelial cells to effectively limit the pro-angiogenic phenotype driven by GPCR overexpression. Several small-molecule MALT1 inhibitors are now available and can be tested for their efficacy as angiogenesis inhibitors in the setting of GPCR+ breast cancer.
Citation Format: Kang H, Ekambaram P, McAllister-Lucas LM, Lucas PC. The CARMA3-Bcl10-MALT1 signalosome mediates pro-angiogenic IL-6 and IL-8 paracrine signaling in GPCR+ breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-07-02.