Abstract P3-06-01: Therapeutic targeting of RAGE in the tumor and tumor microenvironment inhibits breast progression and metastasis
Authors: Kwak T , Drews-Elger K , Ergonul A , Braley A , Hwang GH , El-Ashry D , Slingerland JM , Lippman ME , Hudson BI 
Published: March 2017

Background: The Receptor for Advanced-Glycation End-products (RAGE) is highly expressed in various cancers and its expression is correlated with poorer outcomes in breast cancer. We have previously implicated RAGE in breast cancer, but whether RAGE drives breast cancer progression and metastasis either through tumor cell intrinsic effects, non-tumor cells of the tumor microenvironment, or both, is not fully understood. More importantly, studies are lacking that target RAGE therapeutically in cancer, and may therefore represent a novel treatment for breast cancer metastasis.

Methods: Using multiple human and murine breast cancer models we dissected the tumor intrinsic versus tumor microenvironment role of RAGE in metastasis. RAGE was targeted in tumor cells using multiple shRNAs, in non-tumor cells by global gene knockout in mice, and both by therapeutically targeting with the novel RAGE inhibitor FPS-ZM1. In vivo orthotopic models included the NSG (NOD-SCID-gamma) xenograft mouse model (with MDA-MB-231 cells; herein 231), BALBc (4T-1 and 67NR), and C57BL6 wild-type and RAGE knockout (RAGE -/-) mice (with MMTV-PyMT spontaneous breast cancer derived AT-3 cells).

Results: We first tested how RAGE impacts tumor cell intrinsic mechanisms using either RAGE shRNAs or FPS-ZM1 in 231, 4175 (231 isogenic highly metastatic cells) and 4T-1 cells. RAGE shRNA and FPS-ZM1 both decreased RAGE MAP-kinase signaling, transwell invasion and soft agar colony formation, without affecting proliferation. In vivo, RAGE shRNA knockdown in 231 cells did not affect tumor growth, but inhibited metastasis to lung and liver. RAGE shRNA knockdown in 4175 cells, decreased orthotopic tumor growth, and reduced tumor angiogenesis and tumor recruitment of leukocyte / macrophages. Furthermore, RAGE shRNA knockdown dramatically decreased metastasis of 4175 cells to lung and liver in a time and sized matched manner compared to shRNA controls. Similarly, RAGE knockdown in 4T-1 cells reduced cell invasion and colony formation, and inhibited lung metastasis from the orthotopic site in BALBc immunocompetent mice.

To test the non-tumor cell microenvironment role of RAGE, we performed syngeneic studies with orthotopically injected AT-3 cells in RAGE +/+ and RAGE -/- C57BL6 mice. RAGE -/- mice displayed striking impairment of tumor cell growth compared to RAGE +/+ mice, along with decreased MAP-kinase signaling, tumor angiogenesis and inflammatory cell recruitment.

Finally, to test the combined inhibition of RAGE in both tumor cell intrinsic and non-tumor cells of the microenvironment, we performed in vivo treatment of 4175 tumors with FPS-ZM1 (1mg/kg, twice per week). Compared to vehicle, FPS-ZM1 inhibited primary tumor growth, inhibited tumor angiogenesis and inflammatory cell recruitment, and most importantly prevented metastasis to lung and liver.

Conclusion: These data clearly demonstrate a role for RAGE in breast cancer progression and metastasis through distinct effects in the tumor cell and non-tumor cells of the tumor microenvironment. Furthermore, our data from drug inhibitor studies highlight the combined targeting of RAGE in the tumor and tumor microenvironment, and as a viable therapeutic means for breast and other metastatic cancers.

Citation Format: Kwak T, Drews-Elger K, Ergonul A, Braley A, Hwang GH, El-Ashry D, Slingerland JM, Lippman ME, Hudson BI. Therapeutic targeting of RAGE in the tumor and tumor microenvironment inhibits breast progression and metastasis [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-06-01.