Abstract 4957: Post chemotherapy aggressiveness of breast cancer cells is related to TLR-3 / NOD-1 -2 activation.
Authors: Paulus Patrick , Ellinghaus Peter , Urbschat Anja , Zins Karin , Ockelmann Pia , Reissig Christin , Zacharowski Kai 
Published: August 2014

Abstract
Post chemotherapy relapsing breast cancer is often characterized by an increased aggressiveness and potential to metastasize. The mechanisms by which cancer cells escape chemotherapy are yet not completely understood. Toll-like receptor (TLR)-3, activated by RNAs, as well as NOD-1 and NOD-2, two newly described regulators of the innate immune system, play a role in tumor growth by regulating apoptosis or macrophage invasion into tumors. We hypothesize that chemotherapy leads to release of nucleotides from apoptotic cells which then in turn are capable of activating the pro-inflammatory TLR-3 pathway. The parallel activation of NOD-1/-2 via TRAF6 then leads to an amplification of the metastasizing potential of post chemotherapy breast cancer cells by increasing the recruitment of macrophages. Balb/c mice bearing 4T1 syngeneic breast cancer tumors were treated with a combination chemotherapy (CMF; cyclophosphamide 50 mg/kg, methotrexate 25 mg/kg, 5-fluoro-uracil 50 mg/kg; n=8) cycled twice i.p., solvent (NaCl; n=8) cycled twice i.p. or without therapy (n=8). After 21 days animals were sacrificed, tumor weight was measured, metastases were detected histologically, cell proliferation rate was analyzed by Ki-67 expression, macrophage count was evaluated immunohistologically and tissue mRNA expression was examined by RT-PCR. CMF treatment (376.0 ± 61.14 mg) significantly retarded tumor growth vs. untreated (620.6 ± 58.95 mg) or NaCl (665.8 ± 45.46 mg) treated controls (P&lt;0.05 respectively P&lt;0.01). Interestingly, pulmonary metastases occurred only in the CMF group. Proliferation as measured by Ki-67 expression however was not decreased in CMF treated animals and was even significantly higher as compared to untreated animals (34.76 ± 3.475 vs. 18.58 ± 2.666; P&lt;0.05). Moreover macrophage invasion into CMF treated tumors was significantly increased vs. untreated animals (35.25 ± 4.131 vs. 22.86 ± 2.511 cells/field; P&lt;0.05). TLR-3 (1471 ± 278.3 vs. 291.3 ± 33.4 or 512.7 ± 88.4; P&lt;0.001) mRNA expression as well as TLR-3 signaling members’ TRAF6 (1207 ± 145.0 vs. 418.1 ± 78.3 or 239.0 ± 32.8; P&lt;0.001) and Interferon beta1 (190.2 ± 25.5 vs. 54.50 ± 15.7 or 63.43 ± 33.4; P&lt;0.05) mRNA expression was significantly upregulated in CMF vs. untreated or NaCl treated mice. Increased cellular turnover upon chemotherapy leads to NOD-1 (74.54 ± 14.59 vs. 34.43 ± 6.46 or 26.11 ± 7.39; P&lt;0.05) activation vs. controls. Together with macrophage invasion, NOD-2 (82.61 ± 14.30 vs. 33.35 ± 5.33 or 42.17 ± 5.87; P&lt;0.01) expression is significantly upregulated vs. controls.
TLR-3 activation in 4T1 breast cancer tumors might lead to increased NOD-1/ -2 activation and simultaneously to increased macrophage invasion representing an independent indicator for the aggressiveness of breast tumors.
Supported by a grant from the Grants4Targets initiative from Bayer Pharma AG to P.P.
Citation Format: Patrick Paulus, Peter Ellinghaus, Anja Urbschat, Karin Zins, Pia Ockelmann, Christin Reissig, Kai Zacharowski. Post chemotherapy aggressiveness of breast cancer cells is related to TLR-3 / NOD-1 -2 activation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4957. doi:10.1158/1538-7445.AM2013-4957