Molecular biology is like criminology. Experts study the crime scene to identify a suspect, and then investigate all known associates. They try to determine where suspects and associates interact and understand the nature of their relationship. Csiszar and colleagues1 investigated the players and interactions involved in breast cancer metastasis, specifically the epithelial-to-mesenchymal transition (EMT). In this scenario, the prime suspect is Interleukin-like epithelial-to-mesenchymal transition inducer (ILEI), also known as FAM3C.

ILEI is a secreted cytokine known to be involved in tumor progression, though its exact mechanisms of action are unknown. The protein consists of three domains, an N-terminal signal peptide followed by a propeptide and a secreted domain. Upregulated by transforming growth factor b (TGFb), ILEI was first identified in an expression-profiling screen for TGFb-regulated EMT-specific genes. Elevated protein levels of ILEI are observed in many types of cancer and changes in subcellular localization (from granular to cytoplasmic) predict poor survival in human breast cancer.

Csiszar et al. used a range of classic molecular techniques to investigate the mechanisms of ILEI protein activation. They created several different ILEI expression constructs including a full-length wild-type version and mutants with altered protease cleavage sites and deleted propeptide domains. These were expressed in various cell lines including murine mammary epithelial cell line derivatives. Purified expression proteins and cell lines were used to conduct cleavage assays, secretion assays, binding experiments and expression studies. The following is a simplified summary of their key findings.

  1. Inactive (full-length) ILEI is tethered to fibronectin-containing fibers of the extracellular matrix through its propeptide. Researchers conducted ILEI binding assays using different ILEI transcripts and cell-free extracellular matrix.
  1. ILEI is processed into its shorter, secreted form by serine proteases. Full-length and mutated ILEI transcripts were used for in vitro protease cleavage assays. Plasmin, plasma kallikrein and neutrophil elastase cleaved ILEI at a consensus serine protease consensus cleavage site located between the propeptide and the secreted domain.
  1. Removal of the propeptide is necessary for ILEI-dependent tumor growth and metastasis activities. The inhibition of ILEI processing interferes with tumor growth and metastasis. Different ILEI transcripts were tested for activity using in vivo tumor formation and metastasis assays.
  1. Plasmin induces ILEI secretion both in a cleavage-dependent and a cleavage-independent manner. Upregulation of ILEI secretion by plasmin involves the activity of urokinase plasminogen activator-receptor (uPAR).
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In addition, Csiszar et al conducted a number of other exploratory investigations including subcellular localization experiments and gene expression studies. By correlating their results and outcome data from human breast cancer tissue arrays, they demonstrated that ILEI localization and combined ILEI-uPAR marker analysis have prognostic significance.

In conclusion, ILEI remains a prime suspect in breast tumor metastasis. Csiszar et al’s investigation has confirmed ILEI’s important role and provides some mechanistic insights into how the crime is committed. Their work identified some additional suspects; ILEI is regulated at multiple levels by proteases, specifically proteases involved in the plasminogen (Plg) and the uPAR pathways. These suspects, plasmin and uPAR, are likely potential therapeutic targets against ILEI-driven tumor progression. Metastatic crime scene investigation continues.

  1. Csiszar A, et al. (2014) “Interleukin-like epithelial-to-mesenchymal transition inducer activity is controlled by proteolytic processing and plasminogen-urokinase plasminogen activator receptor system-regulated secretion during breast cancer progression.” Breast Cancer Research 16(5) (pp. 433- 450).