Correlations between transmembrane 4 L6 family member 5 (TM4SF5), CD151, and CD63 in liver fibrotic phenotypes and hepatic migration and invasive capacities.

Transmembrane 4 L6 family member 5 (TM4SF5) is overexpressed during CCl4-mediated murine liver fibrosis and in human hepatocellular carcinomas. The tetraspanins form tetraspanin-enriched microdomains (TEMs) consisting of large membrane protein complexes on the cell surface. Thus, TM4SF5 may be involved in the signal coordination that controls liver malignancy. We investigated the relationship between TM4SF5-positive TEMs with liver fibrosis and tumorigenesis, using normal Chang hepatocytes that lack TM4SF5 expression and chronically TGFβ1-treated Chang cells that express TM4SF5. TM4SF5 expression is positively correlated with tumorigenic CD151 expression, but is negatively correlated with tumor-suppressive CD63 expression in mouse fibrotic and human hepatic carcinoma tissues, indicating cooperative roles of the tetraspanins in liver malignancies. Although CD151 did not control the expression of TM4SF5, TM4SF5 appeared to control the expression levels of CD151 and CD63. TM4SF5 interacted with CD151, and caused the internalization of CD63 from the cell surface into late lysosomal membranes, presumably leading to terminating the tumor-suppressive functions of CD63. TM4SF5 could overcome the tumorigenic effects of CD151, especially cell migration and extracellular matrix (ECM)-degradation. Taken together, TM4SF5 appears to play a role in liver malignancy by controlling the levels of tetraspanins on the cell surface, and could provide a promising therapeutic target for the treatment of liver malignancies.

Introduction

The plasma membrane is structurally important for signal transduction between the intracellular and extracellular environments. A diverse set of membrane proteins with specific membrane domains facilitates this signal transduction [1]. In addition to lipid rafts, which are small, dynamic, and heterogeneous membrane compartments enriched with sterol- and sphingolipids [2], tetraspanin-enriched microdomains (TEMs) are independent organizations of large protein complexes that include tetraspanins, integrins, and growth factor receptors contribute to adhesion, proliferation, and migration [3].

Tetraspanins are linked to the progression of a variety of cancers [4]. Currently, 33 mammalian tetraspanins (TM4SFs) have been identified. These proteins weigh between 20 and 30 kDa and have variable sequence homology. However, all these proteins contain four common transmembrane domains, two cytosolic tails, a short extracellular loop (SEL), and a long extracellular loop (LEL) [1]. CD151 (Tspan24) was first identified as a promoter of metastasis [5]; its expression is increased in liver cancer, compared to normal cells [6]. CD151 functions in cellular migration, invasion, angiogenesis, and drug resistance by forming protein complexes with integrins [4], [7], [8].

CD63 (Tspan30) is a tumor suppressor expressed in endosomes and lysosomes and on the cell surface [9]. The trafficking of CD63 between the cell surface and the internal membranes occurs via AP2, clathrin-coated pit-mediated endocytosis, or caveolae-mediated endocytosis, and it requires specific amino acid motifs present in the CD63 protein [9]. The cell surface expression of CD63 is mediated by tumor-associated antigen L6, L6-Ag [10]. CD63 is abundantly expressed as a surface antigen in the early stage of melanoma, but its expression decreases with malignant progression [11], suggesting a negative correlation between CD63 surface levels and invasiveness.

TM4SF5 is related to the tetraspanins by having four transmembrane domains, but belong to transmembrane 4 L6 family member 5 due to no CCG motifs in the second extracellular loop [12], [13]. Being similar to tetraspanins, TM4SF5 has an intracellular loop, two extracellular loops, and cytosolic NH2- and COOH-terminal tails [12], [13]. TM4SF5 is induced by TGFβ1/Smads signaling pathway in fibrotic livers of CCl4-administarated mice [14]. More than 80% of HCC is known to be associated with advanced fibrosis or cirrhosis [15], [16]. TM4SF5 is highly expressed in hepatocellular cancer tissues, and enhances their aberrant proliferation, migration, and invasion of hepatocytes [13]. TM4SF5 mediates adhesion-dependent focal adhesion kinase (FAK)/c-Src activation to direct motility and invasive capacity [17], [18]. Although TM4SF5 does not belong to the genuine tetraspanin family [12], TM4SF5 can form TEMs with other tetraspanins and can play a role in the regulation of metastasis. Furthermore, any hierarchy among these tetraspan(in)s has not been reported.

Here in this study, we examined the correlations between TM4SF5, CD151, and CD63 expression using normal Chang hepatocytes that do not express TM4SF5, chronically TGFβ1-treated Chang cells that do express TM4SF5 [14], and other hepatocyte cells. We found that TM4SF5 expression could override CD151 functions, and TM4SF5 acted antagonistically to CD63 during liver fibrosis development and during hepatic migration/invasive extracellular matrix (ECM) -degradation.

Materials and Methods

Cell Culture

Normal human hepatocyte Chang cells and chronically TGFβ1-treated Chang cells (Chang-TGFβ1), hepatocellular carcinoma Huh7, Hep3B, SNU449, non-small cell lung cancer (NSCLC) HCC827 cells were described previously [19]. Chang, SNU449, and HCC827 cells do not express TM4SF5, whereas Chang-TGFβ1, Huh7, and Hep3B cells express TM4SF5 [19]. Cells including stable Huh7-shScramble (TM4SF5-expressing) or Huh7-shTM4SF5 (TM4SF5-suppressed) cells were maintained in RPMI-1640 (WelGene, Daegu, Korea) containing 10% FBS and antibiotics (Invitrogen, Grand Island, NY, USA).

Extract Preparation and Western Blots

Subconfluent cells in media containing 10% FBS, or cells transiently transfected with short hairpin RNA (shRNA, control shRNA or shRNA against TM4SF5, CD151, or CD63) separately or in combination with each shRNA or cDNA plasmid encoding for FLAG-TM4SF5, Strep-TM4SF5, CD151, or CD63, for 48 h were harvested for whole cell lysates using radio-immunoprecipitation assay (RIPA) lysis buffer containing 0.1% SDS, 0.5% deoxycholate, 1% NP-40, and proteinase inhibitors [19]. Tissue extracts from human or mouse livers were also prepared as previously reported [19]. The primary antibodies included anti-α-tubulin (Sigma, St Louis, MO, USA), anti-CD151, anti-CD63, anti-pY416c-Src, anti-FLAG (Cell Signaling Technol. Danvers, MA, USA), anti-FAK (BD Transduct. Lab., Bedford, MA, USA), anti-pY397FAK (Abcam, Cambridge, UK), anti-c-Src, anti-pY577FAK (Santa Cruz Biotech., Santa Cruz, CA, USA), and anti-TM4SF5 [19].

Coimmunoprecipitations

Whole cell extracts for coimmunoprecipitation were prepared by using a lysis buffer (10 mM Tris, pH 7.5, 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2) including 1% Brij97 and protease inhibitors. The whole cell lysates were then immunoprecipitated with anti-FLAG antibody-precoated agarose beads (Sigma) overnight. Alternatively, whole cell extracts from Chang-TGFβ1 cells, or extracts from Chang or HCC827 cells transiently transfected with Strep-tagged mock or TM4SF5 were incubated with either normal immunoglobulin (IgG), anti-CD151 antibody (Cell Signaling Technol.), or biotin-precoated beads (IBA, Hanover, Germany) for 2 h. Immunoprecipitated proteins were boiled in 2× SDS-PAGE sample buffer before Western blot analysis.

Flow Cytometry

Cells were analyzed by flow cytometry for tetraspanin expression profiles, as previously reported [20]. Primary antibodies used included anti-CD151, anti-CD63, anti-CD9, and anti-TM4SF5 (Clone # 27, anti-TM4SF5 mAb, described below).

TM4SF5 Antibody

Generation of anti-TM4SF5 monoclonal antibodies were described previously [21]. Briefly, a recombinant TM4SF5 LEL domain (amino acid residues 113 to 157) fused with Fc (i.e., an antibody crystallizable) and myc at the C-terminus was expressed in HEK293E cells, purified by affinity chromatography using protein A/G-agarose (Millipore, Billerica, MA, USA), and used as an antigen to select antibodies. An anti-TM4SF5 monoclonal antibody (human Fc-fusion form) was purified by affinity chromatography using protein A/G-agarose and used in flow cytometry or immunohistochemistry analysis.

Indirect Immunofluorescence

Cells grown on glass coverslips without or with transient transfection with FLAG-TM4SF5 or CD151 for 48 h or cells on coverslips were immunostained using antibody against CD151, CD63, TM4SF5, and/or FLAG, in addition to DAPI staining for nucleus. In cases, LAMP2, a lysosomal marker, was immunostained using anti-LAMP2 (Abcam, Cambridge, UK). Immunofluorescent images were acquired on a fluorescent microscope (BX51TR, Tokyo, Olympus) or a confocal laser scanning microscope (Nikon C2, Nikon, Tokyo, Japan).

Transwell Migration Assay

Cells transfected with diverse shRNA or plasmids were processed for the Transwell migration assay using 8-µm pore transwells (Corning, Corning, NY, USA), as described previously [22]. The migration assay was performed for 18 h with normal 10% FBS-containing media in the lower chambers. Migrating cells were stained and visualized using microscopy, and representative images were obtained. Mean values ± standard deviation were evaluated from randomly saved images and were graphed.

ECM-Degradation Analysis

ECM-degradation by cells on coverslips precoated with Oregon Green 488-conjugated-gelatin (Invitrogen) was analyzed for 18 h, as described in a previous report [22].

TM4SF5 promoter assay

The transcriptional activity of the TM4SF5 promoter (−3.2∼+0.5 kb fragment in pGL3) was analyzed, as previously described [14]. The total DNA amount for each transfection was normalized using control DNA.

Immunohistochemistry of Murine Fibrotic or Human Tumor Liver Tissues

All animal procedures were performed in accordance with the procedures of the Seoul National University Laboratory Animal Maintenance Manual and Institutional Review Board (IRB) agreement approved by Institute of Laboratory Animal Resources Seoul National University (ILARSNU). Four-week-old mice (BALB/c, Orient. Co. Ltd, Seungnam, Korea) were housed in a specific pathogen-free room with controlled temperature and humidity. Mice aged 5 weeks (n = 5) were injected intraperitoneally with or without CCl4 (Sigma, 1 mg/kg) in 40% olive oil, three times per week for 2 weeks. Double-immunostaining of human liver tissues were obtained from each patient as reported previously after informed content [19], or murine liver tissues were incubated with primary antibodies specific for TM4SF5 [21] (Clone # 27, anti-TM4SF5 mAb, see above), CD63, and CD151 (Cell Signaling Technol.) with or without DAPI staining. Alternatively, the tissues were processed for Masson’s trichrome or hematoxylin and eosin staining, as previously described [23].

Statistical Methods

Student’s t-tests were performed for statistical comparisons of mean values to determine significance. A P value less than 0.05 was considered statistically significant.