Tumor metastasis is a primary source of morbidity and mortality in cancer. Adrenomedullin (AM) is a multifunctional peptide regulated by receptor activity-modifying proteins (RAMPs). We previously reported that the AM-RAMP2 system is involved in tumor angiogenesis, but the function of the AM-RAMP3 system remains largely unknown. Here, we investigated the actions of the AM-RAMP2 and 3 systems in the tumor microenvironment and their impact on metastasis. PAN02 pancreatic cancer cells were injected into the spleens of mice, leading to spontaneous liver metastasis. Tumor metastasis was enhanced in vascular endothelial cell-specific RAMP2 knockout mice (DI-E-RAMP2−/−). By contrast, metastasis was suppressed in RAMP3−/− mice, where the number of podoplanin (PDPN)-positive cancer-associated fibroblasts (CAFs) was reduced in the periphery of tumors at metastatic sites. Because PDPN-positive CAFs are a hallmark of tumor malignancy, we assessed the regulation of PDPN and found that Src/Cas/PDPN signaling is mediated by RAMP3. In fact, RAMP3 deficiency CAFs suppressed migration, proliferation, and metastasis in co-cultures with tumor cells in vitro and in vivo. Moreover, the activation of RAMP2 in RAMP3−/− mice suppressed both tumor growth and metastasis. Based on these results, we suggest that the upregulation of PDPN in DI-E-RAMP2−/− mice increases malignancy, while the downregulation of PDPN in RAMP3−/− mice reduces it. Selective activation of RAMP2 and inhibition of RAMP3 would therefore be expected to suppress tumor metastasis. This study provides the first evidence that understanding and targeting to AM-RAMP systems could contribute to the development of novel therapeutics against metastasis.
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This study was supported by Grants-in-Aid for Scientific Research (KAKENHI) from the Core Research for Evolutionary Science and Technology (CREST) of Japan Science and Technology Agency (JST) and the Japan Agency for Medical Research and Development (AMED), and research grants from Bristol-Myers Squibb Company, Japan Foundation for Applied Enzymology, the Naito Foundation, the Public Foundation of Chubu Science and Technology Center, Yamaguchi Endocrine Research Foundation, Hoyu Science Foundation, Takahashi Industrial and Economic Research Foundation, Akaeda Medical Research Foundation, Shinshu Public Utility Foundation for Promotion of Medial Sciences, and Japan Heart Foundation. KD received a scholarship from Otsuka Toshimi Scholarship Foundation. The authors are grateful to Prof. Sachie Hiratsuka for valuable comments and tumor cells.
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The authors declare that they have no conflict of interest.
All animal experiments were conducted in accordance with the ethical guidelines of Shinshu University. All animal handling procedures were performed in accordance with protocols approved by the Ethics Committee for Animal Care at Shinshu University.
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Dai, K., Tanaka, M., Kamiyoshi, A. et al. Deficiency of the adrenomedullin-RAMP3 system suppresses metastasis through the modification of cancer-associated fibroblasts. Oncogene 39, 1914–1930 (2020). https://doi.org/10.1038/s41388-019-1112-z