Abstract 3162: NOD1 augments cancer cell metastatic potential through p38 MAP kinase activation
Authors: Jiang Henry , Najmeh Sara , Berube Julie , Leone Arielle , Savage Paul , Giannias Betty , Bourdeau France , Rousseau Simon , Park Morag , Ferri Lorenzo Edwin 
Published: August 2015

Abstract
INTRODUCTION: Strong clinical evidence demonstrates a link between acute bacterial infection and metastasis. We and others have shown that activation of certain membrane pattern recognition receptors (PRRs), such as Toll-like receptors, on cancer cells can be implicated in this process. There is emerging data that a cytoplasmic PRR, the nucleotide oligomerization domain receptor 1 (NOD1), may also play an important and complementary role in the immune response to bacterial infection, however its role in cancer progression is entirely unknown. We sought to determine the influence of NOD1 activation on metastasis.
METHODS: NOD1 expression in human and murine colon (HT29, MC38) and lung (A549, H59) cancer cells were confirmed using flow cytometry (FC) and immunoblotting (WB). A series of in vitro and in vivo functional assays, including adhesion, migration and hepatic intravital microscopy (IVM), was conducted to assess the effect of NOD1 activation and inhibition. C12-iE-DAP, a highly selective NOD1 ligand derived from gram-negative bacterial wall, was used to simulate NOD1-activation under infectious condition. ML130, a specific NOD1 inhibitor, was used to block C12-iE-DAP activation. Stable knockdown (KD) of NOD1 in HT29 and A549 cells were constructed with shRNA lentiviral transduction and the functional assays were thus repeated. The predominant signaling pathway, downstream cytokines and cell adhesion molecules of NOD1-activation were identified using WB in the presence of kinase inhibitors.
RESULTS: WB and FC showed abundant NOD1 expression in all tested cell lines. Cancer cells stimulated with C12-iE-DAP doubled the in vitro adhesion to collagen I, IV and fibronectin, as well as in vitro migration, an effect completely attenuated with ML130 inhibition and NOD1 knockdown. Using a murine hepatic adhesion assay under IVM, we observed a doubling in the amount of in vivo capture of cancer cells within hepatic sinusoids of C57BL6 mice in the C12-iE-DAP group compared to control, an effect again abrogated by ML130 inhibition and NOD1 knockdown. Immunoblotting using HT29 cells under a panel of kinase inhibitors revealed that NOD1 activation is p38 dependent. Subsequently, the use of p38 inhibitor, BIRB0796, abolished C12-iE-DAP mediated adhesion to collagen I and fibronectin. Using human cytokine quantification array, we determined that NOD1 activation led to increase in IL-8, 15, MCP1 and RANTES, all of which have been shown to link to cancer progression or to chemoattract neutrophils, which we have recently shown to mediate metastasis.
CONCLUSION: Our data demonstrate that NOD1 activation by the gram-negative bacterial wall component, C12-iE-DAP is important in enhancing the metastatic potential of cancer cells, and its mechanism is dependent on p38 activation and the upregulation of downstream cytokines. This is the first study to implicate NOD1 in metastasis, and thus identify this receptor as a putative therapeutic target.
Citation Format: Henry Jiang, Sara Najmeh, Julie Berube, Arielle Leone, Paul Savage, Betty Giannias, France Bourdeau, Simon Rousseau, Morag Park, Lorenzo Edwin Ferri. NOD1 augments cancer cell metastatic potential through p38 MAP kinase activation. [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 3162. doi:10.1158/1538-7445.AM2015-3162