Proteomic Analysis of Exosomes from Mutant KRAS Colon Cancer Cells Identifies Intercellular Transfer of Mutant KRAS

Activating mutations in KRAS occur in 30% to 40% of colorectal cancers. How mutant KRAS alters cancer cell behavior has been studied intensively, but non-cell autonomous effects of mutant KRAS are less understood. We recently reported that exosomes isolated from mutant KRAS-expressing colon cancer cells enhanced the invasiveness of recipient cells relative to exosomes purified from wild-type KRAS-expressing cells, leading us to hypothesize mutant KRAS might affect neighboring and distant cells by regulating exosome composition and behavior. Herein, we show the results of a comprehensive proteomic analysis of exosomes from parental DLD-1 cells that contain both wild-type and G13D mutant KRAS alleles and isogenically matched derivative cell lines, DKO-1 (mutant KRAS allele only) and DKs-8 (wild-type KRAS allele only). Mutant KRAS status dramatically affects the composition of the exosome proteome. Exosomes from mutant KRAS cells contain many tumor-promoting proteins, including KRAS, EGFR, SRC family kinases, and integrins. DKs-8 cells internalize DKO-1 exosomes, and, notably, DKO-1 exosomes transfer mutant KRAS to DKs-8 cells, leading to enhanced three-dimensional growth of these wild-type KRAS-expressing non-transformed cells. These results have important implications for non-cell autonomous effects of mutant KRAS, such as field effect and tumor progression.K-RAS (KRAS) is a small, monomeric GTPase whose biological activity is specified by its nucleotide binding state. Multiple lines of evidence highlight the importance of KRAS in colorectal cancer (CRC).¹ For example, activating missense mutations in KRAS, which lock the protein into the GTP-bound state, occur in 30% to 40% of CRCs and are strongly associated with poor prognosis (1, 2). Also, mutant KRAS negatively predicts responsiveness to anti-EGF receptor (EGFR) therapy (3).Early attempts to decipher the neoplastic consequences of mutant KRAS relied on overexpression studies. A drawback of these studies is their failure to simulate the genetic conditions present in human tumors, where there is often one wild-type (WT) and one mutant KRAS allele (1). More recently, KRAS mutant CRC cell lines have been engineered to selectively contain either the wild-type or the mutant KRAS allele (4), and a single mutant Kras allele has been activated in the intestine using genetically engineered mice (5). Detailed studies using these complementary approaches demonstrate a wide range of tumor-promoting effects of mutant KRAS (reviewed in Ref. 6). Much of what is known about mutant KRAS pertains to its ability to alter the behavior of a transformed cell in a cell autonomous manner. With the exception of increased tumor vascularity via increased tumor-derived VEGF expression (7, 8), non-cell autonomous effects of mutant KRAS have been much less studied.Exosomes are 30- to 100-nm secreted vesicles that have emerged as a novel mode of intercellular communication (9). We recently reported that exosomes purified from conditioned medium of mutant KRAS CRC cells contained higher levels of the EGFR ligand amphiregulin (AREG) and enhanced invasiveness of recipient cancer cells relative to exosomes from isogenically matched wild-type KRAS cells (10). These results prompted us to perform a comprehensive analysis of exosomes purified from these cells. Herein, we show that mutant KRAS induces many changes in exosomal protein composition. Notably, we show that (i) KRAS is contained within exosomes, (ii) exosomes can transfer mutant KRAS to cells expressing only wild-type KRAS, and (iii) mutant KRAS-containing exosomes enhance wild-type KRAS cell growth in collagen matrix and soft agar. These results have important implications for the progression of CRC tumors by providing a mechanism by which the tumor microenvironment may be influenced by non-cell autonomous signals released by mutant KRAS-expressing tumor cells.