ILEI requires oncogenic Ras for the epithelial to mesenchymal transition of hepatocytes and liver carcinoma progression

Abstract

In human hepatocellular carcinoma (HCC), epithelial to mesenchymal transition (EMT) correlates with aggressiveness of tumors and poor survival. We employed a model of EMT based on immortalized p19(ARF) null hepatocytes (MIM), which display tumor growth upon expression of oncogenic Ras and undergo EMT through the synergism of Ras and transforming growth factor (TGF)-beta. Here, we show that the interleukin-related protein interleukin-like EMT inducer (ILEI), a novel EMT-, tumor- and metastasis-inducing protein, cooperates with oncogenic Ras to cause TGF-beta-independent EMT. Ras-transformed MIM hepatocytes overexpressing ILEI showed cytoplasmic E-cadherin, loss of ZO-1 and induction of alpha-smooth muscle actin as well as platelet-derived growth factor (PDGF)/PDGF-R isoforms. As shown by dominant-negative PDGF-R expression in these cells, ILEI-induced PDGF signaling was required for enhanced cell migration, nuclear accumulation of beta-catenin, nuclear pY-Stat3 and accelerated growth of lung metastases. In MIM hepatocytes expressing the Ras mutant V12-C40, ILEI collaborated with PI3K signaling resulting in tumor formation without EMT. Clinically, human HCC samples showed granular or cytoplasmic localization of ILEI correlating with well and poorly differentiated tumors, respectively. In conclusion, these data indicate that ILEI requires cooperation with oncogenic Ras to govern hepatocellular EMT through mechanisms involving PDGF-R/beta-catenin and PDGF-R/Stat3 signaling.

Figure 1

ILEI expression in moderately or poorly differentiated experimental liver tumors and in ILEI-transduced hepatocytes. Epithelial MIM-R-GFP hepatocytes, or MIM-RT cells that have undergone EMT, were orthotopically transplanted into the spleen or injected by tail vein. Liver tumors were collected after 20 days and processed for histology and immunohistochemistry. (a) Sections from wild-type BALB/c livers (control), MIM-R-GFP- and MIM-RT-generated liver tumors and lung metastases induced after tail vein injection of MIM-RT cells were stained with hematoxylin and eosin (H&E) or immunohistochemically stained with anti-ILEI antibody. Insets show staining of tumor sections at 4-fold higher magnification to reveal endogenous ILEI localization. (b) ILEI-overexpressing MIM1-4-ILEI, MIM-C40-ILEI and MIM-R-ILEI cells or empty vector expressing MIM1-4-GFP, MIM-C40-GFP and MIM-R-GFP cells were analysed by immunoblotting using anti-ILEI and anti-β-actin antibodies. The expression of β-actin is shown as a loading control. ILEI, interleukin-like EMT inducer.

Figure 2

ILEI causes the disassembly of epithelial junctions, cytoskeletal reorganization and β-catenin activation in cooperation with oncogenic Ras. Phase contrast images of MIM-C40-GFP, MIM-C40-ILEI, MIM-R-GFP and MIM-R-ILEI cells are shown in the top panels (scale bar: 50 μm). The lower panels show confocal immunofluorescence images of these cell types after staining with specific antibodies against ZO-1, E-cadherin (E-cad), total β-catenin (total β-cat), activated, nondestructible β-catenin (nondestr. β-cat) and phalloidin to visualize β-actin. Blue-green staining: DNA. Scale bar in confocal images: 15 μm. Insets (green border): cells marked by white dots are shown without DNA staining to better visualize cytoplasmic versus nuclear staining of activated β-catenin (green asteriks in main panel mark DNA staining). ILEI, interleukin-like EMT inducer.

Figure 3

Expression of EMT markers and PDGF/PDGF-R isoforms in hepatocytes before and after ILEI overexpression. (a) Expression levels of transcripts for E-cadherin, the E-cadherin-repressing transcription factors Slug, Twist and ZEB-1, and the matrix metalloproteinase MMP-9 in MIM-R-GFP, MIM-R-ILEI and MIM-RT cells (positive EMT control), as determined by linear semi-quantitative RT–PCR. (b) Individual transcript levels of three PDGF isoforms (PDGF-A, -B and -C) and two PDGF-receptor isoforms (PDGF-Rα and PDGF-Rβ) were analysed by linear semi-quantitative RT–PCR in MIM1-4-GFP, MIM1-4-ILEI, MIM-C40-GFP, MIM-C40-ILEI, MIM-R-GFP and MIM-R-ILEI cells. Constitutive levels of RhoA mRNA are shown as a loading control. EMT, epithelial to mesenchymal transition; PDGF, platelet-derived growth factor; RT–PCR, Reverse transcriptase–PCR; ILEI, interleukin-like EMT inducer.

Figure 4

ILEI modulates migratory behavior of hepatocytes but has little effect on proliferation. (a–c) Proliferation kinetics were determined by cumulative cell numbers. (a) Proliferation kinetics of untreated MIM-R-GFP, MIM-R-ILEI and MIM-R-ILEI-dnP cells. (b) Proliferation kinetics of MIM1-4-GFP and MIM1-4-ILEI cells as well as (c) MIM-R-GFP and MIM-R-ILEI cells during treatment with TGF-β1 (1 ng/ml). (d) Control cells (MIM-C40-GFP and MIM-R-GFP), ILEI-overexpressing cells (MIM-C40-ILEI and MIM-R-ILEI) and ILEI-overexpressing MIM-R-GFP cells harboring a dominant-negative PDGF-Rα (MIM-R-ILEI-dnP) after transmigration through Transwell filters. Migrated cells were visualized by Hoechst staining under UV-light and counted (numbers represent transmigrated cell numbers per standard microscopic field). Error bars denote s.e.m. ILEI, interleukin-like EMT inducer.

Figure 5

ILEI enhances tumor formation in synergy with oncogenic Ras and induces tumorigenicity in nontumorigenic hepatocytes expressing the PI3K-hyperactivating Ras mutant V12-C40. Tumors were generated in SCID mice by subcutaneous injection of MIM-R-GFP (a, white bars), MIM-R-ILEI-dnP (a, dark gray bars), MIM-R-ILEI cells (a, black bars) and MIM-C40-ILEI (b, light gray bars). Control MIM-C40-GFP hepatocytes (b, hatched bars) failed to form tumors. (a and b) Kinetics of tumor formation as determined by calculation of tumor volumes. (c) Weights of tumor tissues collected after 20 days (MIM-R-GFP, white bar; MIM-R-ILEI, black bar; MIM-R-ILEI-dnP, dark gray bar) or 49 days (MIM-C40-ILEI, light gray bar). Statistically significant weight differences (P<0.0005) between MIM-R-GFP and MIM-R-ILEI tumors are indicated with ***. (a–c) One representative experiment out of three is shown. Error bars denote s.e.m. SCID, severe combined immunodeficiency; ILEI, interleukin-like EMT inducer.

Figure 6

Ras plus ILEI expression increases nuclear β-catenin levels in vivo, which requires PDGF-R signaling. (a) Experimental tumors obtained after subcutaneous injection of MIM-R-GFP, MIM-R-GFP-dnP, MIM-R-ILEI or MIM-R-ILEI-dnP cells into SCID mice were collected after 20 days and processed for histology and immunohistochemistry using anti-E-cadherin (E-cad), anti-β-catenin (total β-cat) or anti-activated, nondestructible β-catenin (nondestr. β-cat) antibodies. Bar, 50 μm. Insets show staining of tumor sections at fivefold higher magnification to reveal details of E-cadherin localization at cell membranes and nuclear β-catenin. (b) Quantitative evaluation of cells for nuclei expressing activated, nondestructible β-catenin. Error bars depict s.e.m. from three independent experiments. SCID, severe combined immunodeficiency; ILEI, interleukin-like EMT inducer.

Figure 7

The cooperation of ILEI and Ras activates Stat3 in a PDGF-dependent fashion. (a) Subcutaneous tumors were generated in SCID mice, collected after 20 days and processed for immunohistochemistry, employing anti-ILEI, anti-phospho-Stat3 (pY-Stat3) and anti-total Stat3 (total Stat3) antibodies for staining. Bar: 50 μm. Insets: fivefold enlarged images to reveal details in granular versus cytoplasmic ILEI staining and nuclear pY-Stat3 staining. (b) Quantitative evaluation of cell nuclei positive for tyrosine-phosphorylated Stat3 (pY-Stat3; gray bars) and total Stat3 (black bars). Statistically significant differences (P<0.05) in expression of pY-Stat3 are indicated with asterisk. SCID, severe combined immunodeficiency; ILEI, interleukin-like EMT inducer; PDGF, platelet-derived growth factor.

Figure 8

Intensity and pattern of ILEI expression predicts tumor dedifferentiation and prognosis of human HCCs. A tissue array containing 69 human HCC samples and respective control liver tissues were immunohistochemically stained with anti-ILEI antibody. (a) Representative examples shown are for no (negative), weak or strong granular and cytoplasmic ILEI staining of the HCC tissue array. Scale bar: 200 μm. (b) The intensity of granular (gran; left panel) and cytoplasmic (cyto; right panel) ILEI staining (weak including negative or strong) was correlated with postoperative histological grading (pG) of the HCC in the array ranging from well-differentiated (pG1) to moderately (pG2) and poorly dedifferentiated HCCs (pG3). pG2 and pG3 were grouped together because of low sample numbers for pG3. Whereas strong granular staining slightly correlated with more differentiated HCC, strong cytoplasmic staining correlated with poorly differentiated HCC and thus bad prognosis. The comparison of the tumor grading distribution between the groups cytoplasmic weak and cytoplasmic strong was significant (P<0.05) showing a higher percentage of pG1 and pG2–3 tumors in the group cytoplasmic strong than in group cytoplasmic weak. ILEI, interleukin-like EMT inducer; HCC, hepatocellular carcinoma.