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

Abstract

Introduction: Interleukin-like epithelial-to-mesenchymal transition inducer (ILEI) is an essential cytokine in tumor progression that is upregulated in several cancers, and its altered subcellular localization is a predictor of poor survival in human breast cancer. However, the regulation of ILEI activity and the molecular meaning of its altered localization remain elusive.

Methods: The influence of serum withdrawal, broad-specificity protease inhibitors, different serine proteases and plasminogen depletion on the size and amount of the secreted ILEI protein was investigated by Western blot analysis of EpRas cells. Proteases with ILEI-processing capacity were identified by carrying out an in vitro cleavage assay. Murine mammary tumor and metastasis models of EpC40 and 4T1 cells overexpressing different mutant forms of ILEI were used-extended with in vivo aprotinin treatment for the inhibition of ILEI-processing proteases-to test the in vivo relevance of proteolytic cleavage. Stable knockdown of urokinase plasminogen activator receptor (uPAR) in EpRas cells was performed to investigate the involvement of uPAR in ILEI secretion. The subcellular localization of the ILEI protein in tumor cell lines was analyzed by immunofluorescence. Immunohistochemistry for ILEI localization and uPAR expression was performed on two human breast cancer arrays, and ILEI and uPAR scores were correlated with the metastasis-free survival of patients.

Results: We demonstrate that secreted ILEI requires site-specific proteolytic maturation into its short form for its tumor-promoting function, which is executed by serine proteases, most efficiently by plasmin. Noncleaved ILEI is tethered to fibronectin-containing fibers of the extracellular matrix through a propeptide-dependent interaction. In addition to ILEI processing, plasmin rapidly increases ILEI secretion by mobilizing its intracellular protein pool in a uPAR-dependent manner. Elevated ILEI secretion correlates with an altered subcellular localization of the protein, most likely representing a shift into secretory vesicles. Moreover, altered subcellular ILEI localization strongly correlates with high tumor cell-associated uPAR protein expression, as well as with poor survival, in human breast cancer.

Conclusions: Our findings point out extracellular serine proteases, in particular plasmin, and uPAR as valuable therapeutic targets against ILEI-driven tumor progression and emphasize the prognostic relevance of ILEI localization and a combined ILEI-uPAR marker analysis in human breast cancer.

Figure 1

Interleukin-like epithelial-to-mesenchymal transition inducer is processed extracellularly by serine proteases, most efficiently by plasmin. (A) Western blot analysis of interleukin-like epithelial-to-mesenchymal transition inducer (ILEI) in whole-cell lysate and conditioned medium (CM) of EpRas cells grown in 10% fetal calf serum (FCS) or under serum-free conditions. Loading was not normalized to cell numbers. Full-length ILEI runs at the size of 26 kDa, the processed form approximately 2 kDA lower. (B) Purified full-length ILEI-6xHis was incubated with serum-free Dulbecco’s modified Eagle’s medium (DMEM) or medium containing 10% FCS (DMEM 10%) for 5 hours. ILEI protein was repurified via its 6xHis affinity tag and subjected to Western blot analysis. (C) Western blot analysis of ILEI in CM of EpRas cells cultured in the presence of the broad-specificity inhibitors against cysteine proteases (E64), cathepsin B (CA-074), matrix metalloproteases (GM6001) and serine-type proteases (4-(2-aminoethyl)-benzolsulfonylfluorid-hydrochloride (AEBSF) and aprotinin) for 24 hours. (D) to (F) Purified intracellular, full-length ILEI-6xHis was subjected to Western blot analysis after incubation for 2 to 5 hours with the indicated amounts of purified (D) plasmin, uPA and tPA; (E) plasmin, thrombin and plasma kallikrein; and (F) plasmin, thrombin and neutrophil elastase (NE).

Figure 2

Removal of the propeptide is required for elevated tumor growth and lung colonization of EpC40 cells. (A) Schematic drawing of the interleukin-like epithelial-to-mesenchymal transition inducer (ILEI) protein and ILEI constructs used in this study. wt, Wild type. (B) ILEI and FLAG Western blot analysis of whole-cell lysates and conditioned medium (CM) of EpC40 cells overexpressing the ILEI constructs listed in (A). (C) and (D) Primary tumor growth capacity of EpC40 cells overexpressing empty vector or different ILEI forms was determined upon injection into the mammary gland fat pads of female nude mice (n = 5 per group). (C) Tumor growth rate by calculated tumor volume 41 days after injection (±SEM). (D) Tumor mass 41 days after injection (±SEM). (E) and (F) Lung colonization capacity of EpC40 cells overexpressing empty vector or different ILEI forms was determined upon intravenous injection of recultivated cells of mammary tumors into female nude mice (n = 5 per group). Lungs were dissected and analyzed for metastases 45 days after injection. (E) Hematoxylin and eosin–stained histological sections of representative lungs of each group. Scale bar, 2 mm. (F) The percentage of lung metastatic area of each group (±SEM).

Figure 3

Aprotinin suppresses increased tumor growth and lung colonization of wild-type interleukin-like epithelial-to-mesenchymal transition inducer–overexpressing EpC40 cells. Analysis of primary tumor growth and lung colonization capacity of EpC40 cells and derivatives in aprotinin (Ap)-treated mice was performed as described in the Figure 2 legend (n = 10 per group). Five mice in each group were administered with 4,000 kallikrein inactivator units of aprotinin daily. Error bars show mean ± SEM. (A) Tumor growth rate. (B) Tumor masses. (C) Lung colonization assay. Moribund mice were killed and analyzed for lung metastases. The experiment was terminated 80 days after tumor cell injection. The frequency of survival is represented in two separate Kaplan-Meier plots, with the upper showing untreated mice and the lower aprotinin-treated mice.

Figure 4

Interleukin-like epithelial-to-mesenchymal transition inducer binds to soluble and deposited fibronectin-containing extracellular matrix complexes via its propeptide. (A) Western blot analysis of wild-type (wt) or cleavage mutant (FD) interleukin-like epithelial-to-mesenchymal transition inducer (ILEI) immunoprecipitated (IP) from conditioned medium of overexpressing EpRas cells via their FLAG epitope tags. Coprecipitation of extracellular soluble fibronectin was probed using a fibronectin-specific antibody. (B) Immunofluorescence analysis of purified WT and cleavage mutant (FD) ILEI-6xHis proteins after incubation with prepared cell-free extracellular matrix (ECM) deposited by NIH 3T3 fibroblasts. Bound ILEI proteins were visualized via the 6xHis affinity tag using penta-His antibody (green). ECM was counterstained with a fibronectin-specific antibody (red). The purification fraction of empty vector expressing EpRas cells was used as control. Scale bar, 5 μm.

Figure 5

Plasminogen-urokinase plasminogen activator receptor signaling rapidly upregulates interleukin-like epithelial-to-mesenchymal transition inducer secretion by mobilizing its intracellular protein pool. (A) to (D) Western blot analysis of interleukin-like epithelial-to-mesenchymal transition inducer (ILEI) in whole-cell lysates and conditioned medium (CM) of EpRas cells not treated or treated with plasmin for 16 hours after serum withdrawal (A), harvested 24 hours after serum withdrawal and incubation with plasmin for the indicated periods of time (B), treated with indicated concentrations of plasmin, thrombin or plasma kallikrein for 24 hours after serum withdrawal (C) and treated with indicated concentrations of neutrophil elastase (NE) for 24 hours after serum withdrawal (D). (E) Fold change ± SEM in ILEI secretion of EpRas cells after plasmin, thrombin, plasma kallikrein and NE treatments calculated by semiquantification of Western blots of three independent experiments. (F) and (G) Western blot analysis of ILEI in whole-cell lysates and CM of EpRas cells treated with indicated concentrations of plasmin, tissue plasminogen activator (tPA) or urokinase plasminogen activator (uPA) for 24 hours after serum withdrawal (F) and in control and urokinase plasminogen activator receptor (uPAR) short-hairpin RNA EpRas cells not treated or treated with plasmin or transforming growth factor β-1 (TGFβ-1) for 24 hours after serum withdrawal (G).

Figure 6

Interleukin-like epithelial-to-mesenchymal transition inducer secretion levels correlate with altered subcellular localization of the protein. (A) Western blot analysis of interleukin-like epithelial-to-mesenchymal transition inducer (ILEI) in whole-cell lysates and conditioned medium (CM) of wild-type ILEI-overexpressing EpC40 cells (EpC40-wt) harvested 32 hours after serum withdrawal with or without treatment with plasmin (10 mU/ml) for 8 hours before harvest or aprotinin (10 μM) for 32 hours before harvest. CM of the last 24 hours were collected. (B) to (D) Immunofluorescence analysis of EpC40-wt cells as described in (A) without treatment (B) or after plasmin treatment (C) or aprotinin treatment (D). ILEI is visualized via its FLAG tag (green), Golgi is marked by a GM130 antibody (red) and genomic DNA is counterstained with DAPI (4′,6-diamidino-2-phenylindole, blue). Scale bar, 10 μm. (E) to (L) Immunohistochemistry for ILEI localization on representative 3-μm-thick sections of paraffin-embedded EpC40 mammary tumors (n = 5 per group) overexpressing the empty vector control (cont.; (E) and (F)) or the following ILEI constructs: EpC40-wt (G) and (H), EpC40-ΔN-RS (I) and (J) and EpC40-FD (K) and (L). (E, G, I and K) Tumors from nontreated animals. (F, H, J and L) tumors from aprotinin-treated animals. Overexpressed ILEI is shown via its FLAG epitope tag (brown), and genomic DNA is counterstained with hematoxylin (blue). Scale bar, 10 μm.

Figure 7

Cytoplasmic interleukin-like epithelial-to-mesenchymal transition inducer localization correlates with high tumor-cell associated urokinase plasminogen activator receptor expression, and a combined ILEI-uPAR marker analysis shows enhanced prognostic power in human breast cancer. (A) Immunohistochemistry for interleukin-like epithelial-to-mesenchymal transition inducer (ILEI) localization (granular or cytoplasmic, upper images) and urokinase plasminogen activator receptor (uPAR) expression (low or high, lower images) on representative samples from two human breast cancer tissue arrays. ILEI/uPAR signal is in purple, and genomic DNA is in blue (counterstained with hematoxylin and eosin). Scale bar, 50 μm. (B) Distribution of tumor-cell associated uPAR protein expression levels in 88 patients with different subcellular ILEI localizations. (C) Evaluation of ILEI localization (left panel), tumor-cell associated uPAR expression (middle panel) and a combined analysis of the two markers (right panel) for metastasis-free survival depicted in Kaplan-Meier plots.