Abstract P3-01-11: Targeting RAGE inhibits breast cancer invasion and metastasis

Background: Breast cancer (BC) is treatable with early detection, but once metastasis occurs, there is no cure. Therefore, it is of upmost importance to find targetable biomarkers that are involved in modulating the multi-step metastatic process. The Receptor for Advanced Glycation Endproducts (RAGE) and its ligands are an inflammatory pathway that are involved in modulating breast cancer progression and metastasis. We recently demonstrated that genetic or pharmacological interruption of RAGE signaling affected tumor progression and metastasis. However, no studies have dissected the role of RAGE in tumor growth from the metastatic cascade. Here, we show for the first time in multiple metastatic breast cancer models that targeting RAGE impairs BC metastasis. Methods: We tested the anti-metastatic effect of RAGE in vitro using the RAGE inhibitor FPS-ZM1 or RAGE shRNA knockdown in cell invasion, proliferation, migration and sphere formation assays (with 4T1, Py8119 and E0771 mouse BC cells). For in vivo assays we used orthotopic BC models (4T1/BALB/c and E0771 & Py8119/C57BL6) and experimental metastasis assays (tail vein injection of 4T1/BALB/c and Py8119/C57BL6). To target RAGE, we used genetic (shRNA in 4T1 cells and RAGE knockout in C57BL6 mice) and pharmacological approaches (I.P. injection of FPS-ZM1). To investigate the synergistic effects of RAGE inhibition on progression and metastasis, we tested combination therapy of low-dose doxorubicin and FPS-ZM1. Results: Inhibition of RAGE with FPS-ZM1 (1-5uM) impaired tumor cell invasion of 4T1, E0771 and Py8119 cells. Similarly, in spheroid assays, treatment of cells with FPS-ZM1 resulted in fewer and smaller colonies. However, FPS-ZM1 treatment did not affect cell proliferation. In vivo studies revealed that FPS-ZM1 (1mg/kg) has a modest effect on tumor growth in 4T1/BALB/c injected mice but displayed a dramatic inhibitory effect on metastasis to the lungs. In experimental metastasis assays, tail-vein injection of 4T1 cells in BALB/c mice demonstrated that FPS-ZM1 treatment strongly impaired metastatic disease in mice compared to controls. To dissect the genetic role of RAGE in the tumor versus host, we test the effect of RAGE knockdown in 4T1 cells in experimental metastasis assays. RAGE shRNA impaired metastasis to the lungs, albeit to a lower degree seen with the RAGE inhibitor. To test the role of the host, we injected Py8119 cells into wild-type and RAGE knockout (RKO) mice. RKO mice displayed fewer metastatic burden compared to wild-type mice. Finally, in our combination treatment experiments, treatment of 4T1-injected BALB/c mice with Doxorubicin and FPS-ZM1 (alone and in combination), demonstrated that drug combination was more effective in inhibiting lung metastasis than either reagent alone. We are currently assessing how RAGE mechanistically drives these metastatic changes. Conclusion: Our data strongly suggests RAGE plays an important role in breast cancer metastasis, with less of an effect on tumor growth. Ongoing studies in our lab are testing which stage of the metastatic cascade RAGE is involved in, and the underlying mechanisms driving these processes. In conclusion, the use of RAGE inhibitors could represent a novel therapeutic approach for metastatic breast cancer. Citation Format: Gyong Ha Hwang, Melinda Magna, Barbara Mera, Toni Yeasky, Taekyoung Kwak, Lucas Outcault, Masaru Takabatake, Thuy-Mai Le, Marc E. Lippman, Barry I. Hudson. Targeting RAGE inhibits breast cancer invasion and metastasis [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-01-11.