Exosome-delivered EGFR regulates liver microenvironment to promote gastric cancer liver metastasis

ARTICLE Received 9 May 2016 | Accepted 21 Feb 2017 | Published 10 Apr 2017 DOI: 10.1038/ncomms15016 OPEN Exosome-delivered EGFR regulates liver microenvironment to promote gastric cancer liver metastasis Haiyang Zhang1, Ting Deng1, Rui Liu1, Ming Bai1, Likun Zhou1, Xia Wang1, Shuang Li1, Xinyi Wang1, Haiou Yang1, Jialu Li2, Tao Ning1, Dingzhi Huang1, Hongli Li1, Le Zhang1, Guoguang Ying1 & Yi Ba1 The metastatic organotropism has been one of the cancer’s greatest mysteries since the ‘seed and soil’ hypothesis. Although the role of EGFR in cancer cells is well studied, the effects of secreted EGFR transported by exosomes are less understood. Here we show that EGFR in exosomes secreted from gastric cancer cells can be delivered into the liver and is integrated on the plasma membrane of liver stromal cells. The translocated EGFR is proved to effectively activate hepatocyte growth factor (HGF) by suppressing miR-26a/b expression. Moreover, the upregulated paracrine HGF, which binds the c-MET receptor on the migrated cancer cells, provides fertile ‘soil’ for the ‘seed’, facilitating the landing and proliferation of metastatic cancer cells. Thus, we propose that EGFR-containing exosomes derived from cancer cells could favour the development of a liver-like microenvironment promoting liver-specific metastasis. 1 Tianjin Medical University Cancer Institute and Hospital, Tianjin Key Laboratory of Cancer Prevention and Therapy, National Clinical Research Center for Cancer, Tianjin 300060, China. 2 Department of Gastroenterology, Tianjin First Center Hospital, Tianjin 300192, China. Correspondence and requests for materials should be addressed to G.Y. (email: ) or to Y.B. (email: ). NATURE COMMUNICATIONS | 8:15016 | DOI: 10.1038/ncomms15016 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms15016 T he fact that certain tumours are inclined to metastasize to specific organs has been recognized for over a century1. The Paget’s ‘seed and soil’ hypothesis suggests that the successful growth of metastatic cancer cells largely depends on the properties of target organs (soil) and cancer cells (seeds)1,2. Liver is the organ where various types of metastatic tumours take place3,4; however, the knowledge on the mechanism that liver promotes cancer cell colonization and growth is still absent. Exosomes are small vesicles that are secreted from cells and have been found to mediate signalling transduction between neighbouring or distant cells5,6. Exosomes (30–200 nm) and shedding vesicles (200–1,000 nm) are two main forms of extracellular vesicles. The previous study has shown that exosomes bear surface receptors or ligands of the original cells; therefore, they have the tendency to specifically interact with target cells7. Although exosomes are well known to deliver microRNAs (miRNAs) and messenger RNAs8–10, the role of proteins in exosomes, especially membrane proteins, has not been fully understood yet. Epidermal growth factor receptor (EGFR) is located in the cytomembrane, which is well known to play a dominant role in tumorigenesis and development. Recent studies showed that EGFR can be secreted from cells via the transport of vesicles and these EGFR-containing exosomes are proved to regulate signalling pathways of endothelial cells and T cells11–13. Moreover, microvesicles containing EGFRvIII are found to merge with the plasma membranes of cancer cells lacking this type of receptor and the share of EGFR mutants between cancer cells promote tumour development14. Hepatocyte growth factor (HGF) was first discovered in mouse liver and has been found to be linked with tumour development. Serum HGF is upregulated in various types of cancer, which is a potential biomarker for prognosis15–17. C-MET is the receptor of HGF and is widely expressed in various types of cancer. The HGF-cMET pathway is involved in cell invasion, proliferation and angiogenesis, and is believed to be a novel target for cancer therapy18,19. Gastric cancer (GC) with liver metastasis is one of the main forms in advanced GC20,21; however, the molecular mechanism in this process remains unclear. Liver has adequate supply of blood and may provide nutrition for cancer cells; however, the role of paracrine growth factors has not been the cause for concern. Liver-derived HGF may contribute to the landing and fast growth of metastatic GC cells. In the present study, we first find that c-MET, but not HGF, is highly expressed in the liver metastases of GC, suggesting that GC metastases mainly bind with liver paracrine HGF. Exosomes, derived from GC cells, are proved to activate liver HGF by suppressing miR-26a and miR-26b; the two miRNAs directly target the 30 -untranslated region (UTR) of HGF mRNA. Subsequently, we show that secreted EGFR, which is found in the exosomes of GC serum and GC cells, is finally located in membrane of mixed liver cells, including stromal cells. In addition, EGFR-absent exosomes lost the ability to regulate miR-26/HGF pathway in the liver. Moreover, in vivo studies provide direct evidence that liver HGF plays a key role in determining the ratio of hepatotropic metastasis as well as the growth of liver metastases. Hence, exosomes secreted from primary gastric tumour regulate liver micro-environment to promote liver metastasis and the upregulated liver paracrine HGF provides fertile ‘soil’ for the metastatic cancer cells. Results EGFR is located in the serum exosomes of GC. Although EGFR is well known to be upregulated in tumour tissues, few studies have been focused on circulatory EGFR delivered by exosomes. We first isolated serum exosomes (sr-exosomes) by high-speed centrifugation and determined EGFR levels. As is shown in Fig. 1a, the sizes of these exosomes were mostly around 100 nm. 2 EGFR is enriched in sr-exosomes of GC patients but not in exosomes of normal human serum (n ¼ 20); full-length EGFR was detected at 185 kDa (Fig. 1b). In addition, the content of exosome EGFR was increased in serum of stage IV GC patients (n ¼ 20, Fig. 1b). These results illustrated that GC sr-exosomes contains EGFR oncoprotein, which may play an important role in the development of GC. The expression of HGF and c-MET in GC liver metastases. Although HGF has been reported to be upregulated in various types of cancer, the expression pattern of HGF in tumour metastases is little known. To explore whether HGF is expressed in the liver metastases of GC, we determined HGF expression by using immunohistochemistry and western blotting. The results showed that HGF is highly expressed in para-carcinoma tissues and liver but not in the GC metastases (Fig. 1c,d). However, the HGF receptor, c-MET and phosphorylated c-MET (p-c-MET) is obviously expressed in GC liver metastases (Fig. 1c). Liver metastases from 30 patients were detected and the positive detection rate of HGF is only 15%, whereas the positive rate of c-MET is 90% (Fig. 1e). These data suggested that the cancer cells in the (...truncated)