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Cancer Research 67, 5162, June 1, 2007. doi: 10.1158/0008-5472.CAN-06-2756
© 2007 American Association for Cancer Research
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Cell, Tumor, and Stem Cell Biology

Inhibition of Tumor Growth and Metastasis by Depletion of Vesicular Sorting Protein Hrs: Its Regulatory Role on E-Cadherin and ß-Catenin

Masafumi Toyoshima1,2, Nobuyuki Tanaka1,3, Jun Aoki1, Yoshinori Tanaka1, Kazuko Murata1, Masanao Kyuuma1, Hideyuki Kobayashi1, Naoto Ishii1, Nobuo Yaegashi2 and Kazuo Sugamura1

Departments of 1 Microbiology and Immunology and 2 Obstetrics and Gynecology, Tohoku University Graduate School of Medicine, Sendai, Japan and 3 Division of Immunology, Miyagi Cancer Center Research Institute, Miyagi, Japan

Requests for reprints: Nobuyuki Tanaka, Department of Microbiology and Immunology, Tohoku University Graduate School of Medicine, 2-1, Seiryo-machi, Aoba, Sendai 980-8575, Japan. Phone: 81-22-717-8096; Fax: 81-22-717-8097; E-mail: n-tanaka{at}mail.tains.tohoku.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Abnormally high signals from receptor tyrosine kinases (RTK) are associated with carcinogenesis, and impaired deactivation of RTKs may also be a mechanism in cancer. Hepatocyte growth factor–regulated tyrosine kinase substrate (Hrs) is one of the master regulators that sort activated receptors toward lysosomes and shut down their signals. Hrs contains a ubiquitin-interacting motif and is involved in the endosomal sorting of monoubiquitinated membrane proteins, such as growth factor receptor and E-cadherin. Here, we investigated the role of Hrs in determining the malignancy of cancer cells and discovered that the targeted disruption of Hrs by small interfering RNA effectively attenuated the proliferation, anchorage-independent growth, tumorigenesis, and metastatic potential of HeLa cells in vitro and in vivo. The restoration of Hrs expression increased cell proliferation and anchorage-independent growth in a mouse embryonic fibroblast line established from a Hrs knockout mouse. Further analysis revealed that Hrs depletion was associated with the up-regulation of E-cadherin and reduced ß-catenin signaling. The aberrant accumulation of E-cadherin most likely resulted from impaired E-cadherin degradation in lysosomes. These results suggest that Hrs may play a critical role in determining the malignancy of cancer cells by regulating the degradation of E-cadherin. [Cancer Res 2007;67(11):5162–71]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Cell growth is regulated by a variety of cell surface molecules, including cytokine receptors and adhesion molecules. Most growth factor receptors belong to the receptor tyrosine kinases (RTK) family. Because abnormally enhanced RTK signaling is associated with carcinogenesis, it is essential to understand how RTKs are activated and deactivated in cancer. One major deactivation pathway for RTKs is ligand-induced internalization by means of endocytosis followed by degradation in lysosomes (1). The phosphorylation and ubiquitination states of RTKs regulate their ligand-induced internalization and lysosomal degradation (2).

Hepatocyte growth factor (HGF)–regulated tyrosine kinase substrate (Hrs) is one of the master molecules in the vesicular transport and sorting of RTKs (2–6). Hrs contains a clathrin-binding domain and a ubiquitin-interacting motif (UIM) and is involved in the endosomal sorting of monoubiquitinated membrane proteins, including the RTKs, such as epidermal growth factor (EGF), transforming growth factor-ß, and HGF (2, 7–12). Two types of ubiquitination, monoubiquitination and polyubiquitination, are respectively involved in the lysosomal and proteosomal degradation processes (8, 13). Polyubiquitin chains formed via Lys48 have a well-characterized role in targeting proteins for degradation by the 26S proteasome, whereas those formed through the Lys29 or Lys63 of ubiquitin are involved in other cellular functions, including DNA repair and endocytosis (8, 14). By contrast, monoubiquitination is implicated in the endocytosis of plasma membrane proteins and their sorting to the multivesicular body (MVB; ref. 15). For example, monoubiquitinated EGF receptor is degraded in lysosomes (13), and its down-regulation is impaired in cells expressing Hrs mutants (16–19). E-cadherin is also phosphorylated, ubiquitinated, and degraded (20). In a recent study, the E3 ubiquitin ligase Hakai was shown to bind E-cadherin in a tyrosine phosphorylation-dependent manner (21). In addition, the ubiquitin tagging of E-cadherin is reported to be essential for its sorting to the lysosome (21), and Hrs is reported to mediate the lysosomal targeting of E-cadherin via its UIM domain (22).

E-cadherin plays a critical role in many aspects of cell adhesion, epithelial development, and the establishment and maintenance of epithelial polarity. In an adhesive epithelial cell culture, E-cadherin is clustered at cell-cell contacts and is linked to the actin cytoskeleton by binding to the ß-catenin and {alpha}-catenin complex (23). Recent studies indicate that E-cadherin suppresses the invasion of various malignant tumors, and a reduction in E-cadherin expression is a critical molecular event that contributes to the dysregulation of cell adhesion, the triggering of cancer invasion, and metastasis (24, 25). Down-regulation of E-cadherin, either by transcriptional silencing or by protein degradation, also occurs during epithelial-mesenchymal transitions, organogenesis, and tumorigenesis (26). Recently, ß-catenin associated with E-cadherin was shown to have a critical role in the invasion-suppressing activity of E-cadherin.

On its nuclear translocation, ß-catenin is involved in the transduction of the Wnt signaling pathway, which is important for early embryo patterning, cell polarity, and tumor cell metastasis (27, 28). The subcellular localization of ß-catenin is determined by its interacting proteins, and E-cadherin is believed to trap ß-catenin in the cytoplasm (29, 30). Therefore, the loss of E-cadherin leads to the nuclear translocation of ß-catenin. The nuclear translocation of ß-catenin affects two critical cancer-promoting processes, invasion and metastasis, through activation of the Tcf/Lef transcription factor (31) and subsequent activation of growth-promoting genes, such as cyclin D1 and c-Myc (32). Taken together, these findings suggest that the loss of E-cadherin might be one of the frequent features in cancers of epithelial origin.

In this study, we investigated the role of Hrs in malignant cell phenotypes and documented that the targeted disruption of Hrs efficiently attenuates the proliferation, anchorage-independent growth, and metastatic potential of cells in vitro and in vivo. Hrs depletion was also associated with the up-regulation of E-cadherin and reduced ß-catenin signaling.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Clinical material and immunohistochemistry. Immunohistochemical analyses were done for surgical samples that contained cancer specimens from the stomach, colon, skin, liver, and cervix. The specimens were fixed in formalin and embedded in paraffin wax. Serial sections (4 µm thick) mounted on slides were dewaxed in xylene and rehydrated using graded ethanol washes. For antigen retrieval, slides were pretreated in an autoclave at 120°C for 5 min in citric acid buffer (pH 6.0). The sections analyzed contained both tumor and adjacent normal tissues, which acted as an internal control; in addition, no staining was confirmed in the omission of the primary antibody. Antigen-antibody complexes were detected by streptavidin-biotin signal amplification using the Histofine kit (Nichirei).

Antibodies and plasmids. The primary antibodies were as follows: anti-Hrs (33), anti-E-cadherin (HECD-1, Abcam; clone 36, BD Biosciences; and 67A4, Santa Cruz Biotechnology), anti-ß-catenin [mouse monoclonal antibody (mAb), Zymed Laboratories; rabbit polyclonal antibody, Sigma], anti-{alpha}-catenin (BD Biosciences), anti–glycogen synthase kinase-3ß (GSK-3ß) and anti–hemagglutinin A (HA; Cell Signaling Technology), anti-{alpha}-tubulin (Sigma), anti-Dvl-2, anti-lamin B, anti-Lamp-3/CD63, anti-EEA1 (Santa Cruz Biotechnology), and anti-ubiquitin (P4D1, Santa Cruz Biotechnology; FK-1, Biomol). Secondary antibodies were horseradish peroxidase (HRP)–labeled horse anti-mouse IgG and HRP-labeled goat anti-rabbit IgG (Cell Signaling Technology).

pCXN2-Hrs is an expression plasmid for wild-type (WT) Hrs (34). We generated two HA-tagged ubiquitin expression plasmids, one for WT ubiquitin (WT-Ub) and the other for a ubiquitin mutant (mono-Ub). In the mutant gene product, all the lysine residues were changed to arginine, thus preventing the protein from forming polyubiquitin chains. The ß-catenin–mediated signal reporter plasmids, TOP-tk and FOP-tk, were gifts from Dr. T. Noda (The Cancer Institute of Japanese Foundation for Cancer Research, Tokyo, Japan). The promoter activities of c-Myc were determined by using pHXLuc (35). pRL/TK and pME18S/Myc-NT/Hakai were kind gifts from Dr. K. Nakayama (Tohoku University, Sendai, Japan) and Dr. S. Higashiyama (Ehime University, Ehime, Japan), respectively.

Cell lines, culture, and transfection. The human cancer cell lines HeLa (cervix), MCF-7 (breast), PC-3 (prostate), HMV-1 (melanoma), and G361 (melanoma) were obtained from the Cell Resource Center for Biomedical Research, Tohoku University. A Hrs-deficient mouse embryonic fibroblastoid cell line (HRSd) and its subline, stably transfected with the WT Hrs gene (HRSw), were described previously (34, 36). Cells were grown in DMEM or RPMI 1640 containing 10% (v/v) FCS, glutamine, and antibiotics and maintained at 37°C in 7% CO2 in a humidified atmosphere. To transfect the cells with plasmids, Fugene 6 transfection reagent (Roche Diagnostics) or LipofectAMINE 2000 (Invitrogen) was used.

RNA interference. The piGENE PUR hU6 vector expressing small interfering RNA (siRNA) against Hrs was custom constructed by Toyobo. The vector contains a DNA template for the synthesis of siRNA under the control of the human U6 promoter. The target sequence within human Hrs cDNA spans 302 to 320 bp (5'-AGGTAAACGTCCGTAACAA-3'). A target sequence within the Renilla luciferase gene (413–434 bp; 5'-GCAATAGTTCACGCTGAAAAG-3') was used as a control. To generate stable cell lines, piGENE PUR hU6/siHrs or a control plasmid was introduced into HeLa cells. After 48 h, 2 µg/mL puromycin (Invitrogen) was added to the medium to select puromycin-resistant clones. Eight clones with decreased Hrs expression [Hrs-RNA interference (RNAi) 1–8] and two control clones (controls 1 and 2) were maintained in 10% FCS-DMEM containing 2 mg/mL puromycin. Four Hrs-RNAi clones (2, 4, 6, and 7) were randomly selected from among the transfected lines for further experiments. To prepare the RNAis for retrovirus-mediated transfer, DNA templates expressing the human Hrs or a Renilla luciferase–specific short hairpin RNA (shRNA) were ligated into pSIREN-RetroQ (BD Biosciences). Newly generated pSIREN-RetroQ-Hrs or a control plasmid, pSIREN-RetroQ-RenillaLuc, was transduced into the Phoenix-Ampho packaging cell line (Orbigen). Replication-defective retrovirus was harvested from the cell supernatant 48 h after transfection and filtered (0.45 µm), and then polybrene (Sigma) was added at a final concentration of 8 µg/mL. The indicated cell lines were infected with retrovirus containing Hrs-RNAi or control plasmid and selected for an additional 5 days in the presence of 2 µg/mL puromycin.

For experiments involving the depletion of cellular E-cadherin, cells were treated with either the control siRNA duplex (AllStars Negative Control siRNA, Qiagen) or the E-cadherin–specific siRNA duplex (Hs_CDH1_12_HP Validated siRNA, Qiagen) using LipofectAMINE 2000.

Colony formation assays. WT, control, or Hrs-RNAi cells (1 x 103) were suspended in 2 mL of 0.47% SeaPlaque Agarose (Cambrex Bioscience) in DMEM supplemented with 10% FCS. The cell suspension was layered over 2 mL of medium containing 0.7% agarose in 35-mm dishes. Colonies were counted 3 weeks later under phase-contrast microscopy.

In vivo tumorigenesis and metastasis assays. All procedures were done according to protocols approved by the Institutional Committee for Use and Care of Laboratory Animals of Tohoku University. Eight-week-old female athymic nude mice (BALB/c Slc-nu/nu; Japan SLC) were used for the tumorigenesis assays. The indicated HeLa cells (1 x 106) were injected s.c. into the flank. The tumor size of the xenografts was periodically measured with a caliper, and tumor volume was estimated with the formula (width x length2) / 2.

For the metastasis assay, nonobese diabetic/severe combined immunodeficient/{gamma} null (NOG) mice maintained at the Central Institute for Experimental Animals (Kawasaki, Japan) were used. The indicated HeLa cells (1 x 105) in 0.2 mL PBS were injected into the lateral tail vein of NOG mice. Thirty days after the injection, the mice were euthanized, and the lungs were dissected out and preserved in buffered formalin. Serial sections of the lungs were stained with H&E. The number of pulmonary colonies was counted under a light microscope.

Immunofluorescence microscopy. For immunofluorescence staining, cells grown on 35-mm glass-bottomed dishes (Iwaki) were fixed in 4% formaldehyde in PBS at room temperature for 15 min. The fixed cells were washed and permeabilized with PBS containing 0.1% Triton X-100 for 10 min. The cells were washed once with PBS and then blocked in PBS containing 10% FCS and 0.1% Triton X-100 for 30 min at room temperature. The cells were incubated with primary antibodies in PBS containing 5% FCS overnight at 4°C, washed, and then incubated with fluorescence-conjugated secondary antibodies in PBS containing 5% FCS for 1 h at room temperature, washed thrice with PBS, and examined using a confocal microscope. To label lysosomes, cells were incubated with LysoTracker-Red DND-99 Reagent (diluted 1:5,000; Molecular Probes) for 30 min at 37°C. Confocal microscopy was done using an LSM 510 META (Carl Zeiss Microscope Systems).

Immunoprecipitation, immunoblotting, and biotinylation assay. Immunoprecipitation and immunoblotting were carried out as described (34). The biotinylation assay was done as described, with minor modifications (21, 37). Briefly, HeLa cells grown at 95% confluence on 6-cm plastic dishes were incubated with 0.5 mg/mL EZ-Link sulfo-NHS-SS-biotin (Pierce) at 4°C for 30 min, washed with quenching solution (50 mmol/L NH4Cl in PBS containing 1 mmol/L MgCl2 and 0.1 mmol/L CaCl2) to quench free sulfo-NHS-SS-biotin, and then washed several more times with PBS. The cells were then starved by incubation in serum-free DMEM at 37°C for 1 h, which also allowed trafficking to resume. Next, 10 ng/mL HGF was added to the medium and the cells were incubated for 4 h. They were further incubated in glutathione solution (60 mmol/L glutathione, 83 mmol/L sodium chloride, 83 mmol/L sodium hydroxide, and 10% FCS) for 20 min at 0°C to remove cell surface biotin groups (37). Biotinylated proteins that were sequestered inside the cells by endocytosis were protected from glutathione stripping. The cells were then incubated in serum-free DMEM at 37°C for the indicated times, scraped from the dish, and lysed in radioimmunoprecipitation assay buffer with protease inhibitors. The cell extracts were spun to obtain a detergent-soluble supernatant, which was incubated with streptavidin beads (Invitrogen) to collect the biotinylated proteins. These samples were fractionated by SDS-PAGE and immunoblotted with the anti-E-cadherin mAb (clone 36).

Transient transfection and reporter gene assay. HeLa cells were transfected in 24-well plastic dishes using LipofectAMINE 2000. Reporter gene assays were determined using the Dual-Luciferase Reporter Assay System (Promega). To determine the endogenous ß-catenin/Tcf transcriptional activities, the cells were transfected with the reporter construct TOP-tk or FOP-tk. To determine the endogenous c-Myc transcriptional activities, the cells were cotransfected with pHXLuc (35) and pRL/TK. At 24 h after the transfection, the cells were lysed in 100 µL passive lysis buffer and the soluble fractions were used for luciferase assays following the manufacturer's instructions.

Statistical analysis. Statistical significance was determined using the paired Student's t test. Differences were considered significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Strong expression of Hrs in cancer tissues. Human and mouse Hrs are reported to be ubiquitously expressed in a variety of tissues and cell lines (3, 33, 38) and to be involved in the down-regulation of RTKs (4), suggesting possible involvement of Hrs in tumorigenesis. Here, we first verified the expression levels of Hrs in normal and malignant human tissues by immunohistochemistry, with five specimens of each tissue. As assessed by immunohistochemistry, Hrs was expressed more strongly in tumors than in the normal tissue in all five of the stomach, colon, and liver specimens, three of the five cervix, and four of the five skin melanoma specimens (Fig. 1A–E ). More than 10 human cancer cell lines that have been maintained in our laboratory, including those of epithelial and mesenchymal origins, also showed obvious expression of Hrs protein and mRNA (data not shown).


Figure 1
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  		Figure 1. Expression of Hrs in human tumor specimens. Immunohistochemistry of five representative human cancer tissues was done by using a rabbit anti-Hrs polyclonal antibody. Representative H&E and Hrs staining photographs of gastric cancer (A), colon cancer (B), and skin melanoma (C). Magnification, x5. White bar, 200 µm. Hrs was mainly expressed in cytoplasm, not in nucleus. Representative photographs of liver cancer (D) and cervical cancer (E). Magnification, x20. Green bar, 50 µm.

 
Suppressed expression of Hrs induces inhibition of cell colony formation. Because Hrs expression was stronger in cancerous than healthy tissues, we addressed its effect on tumor cell growth. We first generated genetically stable HeLa clones transfected with Hrs siRNA (Hrs-RNAi 4, 2, 6, and 7) or a control siRNA (controls 1 and 2) as described in Materials and Methods. In soft agar colony formation assays, the control clones and WT HeLa cells grew at 310 to 406 colonies per well, whereas all four Hrs-RNAi clones showed significantly lower numbers of colonies (14–18 per well; Fig. 2A, top ). The Hrs-RNAi colonies were detectable only microscopically, whereas the control and WT HeLa grew up into large colonies by 3 weeks (Fig. 2A, bottom). We also confirmed that little Hrs was expressed in the Hrs-RNAi clones (Fig. 2A, middle). To confirm the enhancing effect of Hrs on colony formation, we used mouse embryonic fibroblast (MEF) sublines that were generated from a Hrs knockout mouse (HRSd) or its Hrs revertant (HRSw). The average number of colonies and colony sizes were significantly greater for HRSw than HRSd (Fig. 2B).


Figure 2
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  		Figure 2. Suppression of Hrs expression by siRNA inhibits cell proliferation and anchorage-independent growth. HeLa cell clones stably expressing anti-luciferase shRNA (control clones: 1 and 2) or anti-Hrs shRNA (Hrs-RNAi clones: 2, 4, 6, and 7) were assayed for colony formation in soft agar. A, top, the numbers of colonies were counted. Bottom, representative colonies. Middle, Hrs expression by each clone was shown by immunoblotting. B, top, HRSd, which lacks Hrs, and HRSw, a Hrs revertant subline of HRSd, were also used in the colony formation assay. Bottom, representative colonies. C, four human cancer cell lines, which were infected with retrovirus bearing Hrs-RNAi plasmids (bullet) or anti-luciferase (control; {square}), were used for cell growth analysis. D, cell growth curves for HRSd and HRSw. Hrs expression in each cell line was quantified by immunoblotting with the anti-Hrs antibody (C and D). Points, mean; bars, SE. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

 
Next, we examined the effect of Hrs expression on the proliferation of different types of cell lines (HeLa, PC-3, G361, and HMV-1). The cells were infected with retrovirus containing Hrs-RNAi or control plasmid and selected with puromycin for 5 days as described in Materials and Methods. Their growth curves were measured by cell counting. Although there was no difference in their growth curves in the early stages after cell seeding, the Hrs-depleted cells showed significantly decreased proliferation compared with the Hrs-expressing control cells when they reached near-confluent states (Fig. 2C). The enhanced growth effect of Hrs was also seen in the Hrs revertant HRSw cells (Fig. 2D).

Suppression of Hrs expression inhibits tumorigenesis and metastasis of HeLa cells. To address the in vivo effects of Hrs on tumorigenicity, the indicated HeLa cell lines (1 x 106) were s.c. injected into nude mice. The tumor volumes of the control and WT HeLa clones rapidly increased over the 3 weeks following the injections, whereas those of the Hrs-RNAi clones showed a slower increase (Fig. 3A ).


Figure 3
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  		Figure 3. Suppression of Hrs expression by stable siRNA inhibits tumorigenesis and tumor metastasis in vivo. A, tumor volumes from s.c. xenografts of WT, control, and Hrs-RNAi clones (five animals per cell line; total 35) were calculated from bidimensional measurements on the indicated days after injection. Metastatic colonies quantified in serial sections of lung from NOG mice (five animals per cell line; total 30). B, cells were injected into the tail vein, and the lung tissue was examined 30 d later. C, representative H&E staining of pulmonary colonies showing control and Hrs-RNAi clones. Bar, 100 µm. Points and columns, mean; bars, SE. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

 
The metastatic potential of the control and Hrs-RNAi HeLa clones were compared by injecting cultured cells into the tail vein of NOG mice. All the mice were healthy at the time of sacrifice, 30 days after the injection. In accordance with the fact that cervical cancers show high incidence of lung metastasis, HeLa cells primarily establish lung metastasis foci, but not other organs, when inoculated in vivo (39). We therefore examined the lungs of the mice histologically for metastatic lesions. Mice that received injections of the two control subclones showed an average of 267 and 215 visible metastatic nodules per mouse, respectively, whereas injection of any of the four Hrs-RNAi clones induced significantly fewer visible metastatic nodules (1.6–5 nodules per mouse; Fig. 3B). The nodules were small in the mice that received injections of the Hrs-RNAi clones, but the mice that received injections of the control clones had numerous large metastases in their lungs (Fig. 3C).

Effects of Hrs on expression of E-cadherin and subcellular localization of ß-catenin. Based on the above observations, we expected that Hrs might affect certain cell surface molecules, such as cytokine receptors and adhesion molecules, which contribute to colony formation, tumorigenesis, and metastasis. In differential expression assays of various cell surface molecules in the Hrs-RNAi HeLa and control clones, we found that E-cadherin was markedly up-regulated in the Hrs-RNAi HeLa clones (Fig. 4A, left ). As E-cadherin is a main binding partner of ß-catenin and plays an important role in ß-catenin nuclear localization and the subsequent up-regulation of the transcriptional activities of the Tcf/Lef DNA-binding factors (30, 40, 41), we investigated the subcellular localization of ß-catenin by two methods. First, we measured the protein levels of ß-catenin in the nuclear and cytosolic fractions. Hrs depletion markedly increased the protein level of cytosolic ß-catenin, whereas it decreased that of nuclear ß-catenin, and these changes were accompanied by an augmentation of E-cadherin in whole-cell lysates. The purity of the nuclear and cytoplasmic fractions was confirmed by immunoblotting with antibodies against the nuclear lamin B and cytoplasmic {alpha}-tubulin, respectively (Fig. 4A, right).


Figure 4
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  		Figure 4. Suppression of Hrs expression increases E-cadherin and changes the subcellular distribution and signaling of ß-catenin. A, whole-cell lysates (left) and nuclear or cytoplasmic fractions (right) of control or Hrs-RNAi HeLa clones were subjected to immunoblotting with the indicated antibodies. The purity of the nuclear and cytoplasmic fractions was confirmed by immunoblotting with antibodies against the nuclear lamin B and cytoplasmic {alpha}-tubulin proteins, respectively (right). B, control and Hrs-RNAi HeLa clones were stained with an anti-ß-catenin antibody and analyzed by confocal microscopy. White bar, 10 µm. C, transcriptional activities of ß-catenin–mediated signaling (TOP/FOP) and c-Myc in control or Hrs-RNAi HeLa clones represented as fold activation compared with control, in triplicate wells. D, Hrs-RNAi HeLa cells were transiently transfected with anti-E-cadherin or control siRNA and then cotransfected with TOP-tk or FOP-tk to determine the fold activation of TOP/FOP. Columns, mean; bars, SE.

 
Next, we investigated the differences in the ß-catenin nuclear and cytosolic translocation in the two lines by immunofluorescence microscopy. Consistent with the cell fractionation findings, the control HeLa clones displayed the nuclear accumulation of ß-catenin. In contrast, in the Hrs-RNAi clones, ß-catenin was dispersed throughout the cell (Fig. 4B). The reduced nuclear localization of ß-catenin in the Hrs-RNAi clones was associated with decreased ß-catenin transcriptional activity, which was detected with the ß-catenin-Tcf/Lef–responsive reporter plasmid introduced into the transfected cells (Fig. 4C, left). In addition, we checked the activation of c-Myc, which is downstream of ß-catenin signaling, by luciferase assay and confirmed that its activity was decreased in the Hrs-RNAi HeLa cells (Fig. 4C, right). On the other hand, the amounts of other components involved in Wnt signaling, such as {alpha}-catenin, Dsh, and GSK-3ß proteins, were not significantly different irrespective of Hrs expression level (data not shown). These results suggest that Hrs depletion induces the up-regulation of E-cadherin, resulting in reduced ß-catenin signaling in the Hrs-RNAi clones. To confirm the inhibitory effect of E-cadherin on ß-catenin signaling, we asked whether suppression of E-cadherin could recover the reduced ß-catenin signaling in a Hrs-RNAi clone. Transfection of this clone with E-cadherin siRNA resulted in a 2-fold increase in ß-catenin signaling, measured as TOP/FOP (Fig. 4D).

Involvement of Hrs in the down-regulation of E-cadherin via lysosomal trafficking. A Hrs mutant lacking its UIM blocks the sorting of E-cadherin into lysosomes in Madin-Darby canine kidney cells (22). To clarify the role of Hrs on the traffic of E-cadherin, we used MCF-7 cells, in which E-cadherin can be clearly chased by the confocal study (42). We used MCF-7 cells infected with the Hrs-RNAi retrovirus and showed that the depletion of Hrs leads to a defect in the sorting of E-cadherin from the early endosome to the MVB outer membrane. The infected cells were stimulated with HGF for 8 h and then analyzed using immunofluorescence. In the control MCF-7 cells, several E-cadherin–positive puncta colocalized with Lamp-3/CD63, a late endosomal marker, but this colocalization was not seen in the Hrs-RNAi MCF-7 cells (Fig. 5A, top ). Similarly, the colocalization of E-cadherin with lysosomes was observed in the control MCF-7 cells but not in the Hrs-RNAi MCF-7 cells (Fig. 5A, bottom).


Figure 5
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  		Figure 5. Hrs contributes to the down-regulation of E-cadherin via the trafficking of E-cadherin to the lysosome. A, MCF-7 cells expressing shRNA for Hrs (Hrs-RNAi) or for luciferase (control) were seeded onto glass-bottom dishes. The cells were starved in serum-free medium with cycloheximide for 1 h and incubated with HGF for 8 h. The cells were fixed and immunolabeled for CD63 and E-cadherin (67A4), or they were stained with LysoTracker to label lysosomes and then fixed and immunolabeled for E-cadherin. Coincident staining appears yellow (arrow) in the overlaid images. Bar, 10 µm. B, HeLa cells infected with retrovirus bearing the Hrs-RNAi or the anti-luciferase (control) plasmid were surface biotinylated and incubated with HGF for 4 h, allowing E-cadherin from the cell surface to be internalized into the cytoplasm as described in Materials and Methods. The biotinylated proteins were recovered on streptavidin beads and analyzed by SDS-PAGE. Intact E-cadherin (120 kDa) was detected by immunoblotting. The relative amounts of internalized E-cadherin were quantified by densitometry and expressed as the percentage of the initial amount of E-cadherin. Points, mean of three separate experiments; bars, SE.

 
We next asked whether the impairment of the lysosomal translocation of E-cadherin affects the degradation of E-cadherin in Hrs-depleted cells. For this study, HeLa cells were infected with Hrs-RNAi or the control retrovirus, and their cell surface E-cadherin was biotinylated. The cells were stimulated with HGF to induce internalization of the surface E-cadherin, incubated for the indicated periods, and the internalized E-cadherin was detected by immunoprecipitation. Degradation of the internalized E-cadherin was significantly delayed and suppressed in the Hrs-RNAi HeLa cells (Fig. 5B), suggesting that Hrs is involved in the regulation of E-cadherin degradation through the lysosomal pathway.

The colocalization of Hrs and E-cadherin has been shown in the late endosome (22), but it is still unknown in the early endosome. To investigate the mechanism of Hrs-mediated regulation of E-cadherin, we attempted to detect any colocalization of Hrs and E-cadherin in the early endosome. Although a small amount of E-cadherin was seen intracellularly as puncta, E-cadherin did not colocalize with endogenous Hrs, which appeared as small vesicles in the cytoplasm of unstimulated MCF-7 (Fig. 6A, 0 min ). After stimulation with HGF to promote the endocytosis of E-cadherin, the colocalization of E-cadherin with Hrs on endosomes labeled with the early endosome marker EEA1 gradually increased (Fig. 6A, 30–120 min).


Figure 6
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  		Figure 6. Endocytosis and monoubiquitination of E-cadherin in MCF-7 cells. A, MCF-7 cells were starved in serum-free medium with cycloheximide for 1 h and then stimulated with HGF for the indicated time periods (0–120 min). Cells were fixed and labeled for E-cadherin, Hrs, and EEA1. Bar, 10 µm. B and C, MCF-7 cells were cotransfected with pME18S/Myc-NT/Hakai and HA-tagged WT-Ub or HA-tagged mono-Ub. The cells were starved in serum-free medium for 90 min and then incubated with HGF for 4 h. Lysates were immunoprecipitated with the anti-E-cadherin antibody (HECD-1) and immunoblotted with anti-HA, P4D1, an anti–monoubiquitin + polyubiquitin antibody, or FK-1, an anti-polyubiquitin antibody.

 
Along with growth factor receptors, E-cadherin is tyrosine phosphorylated and ubiquitinated by the E3 ligase Hakai and internalized into the cytoplasm (21). We next analyzed the ubiquitination of the endogenous E-cadherin complex. We generated two kinds of expression vectors for ubiquitin, one WT (WT-Ub) and one bearing mutations that prevented it from forming polyubiquitin chains (mono-Ub). MCF-7 cells were cotransfected with Hakai and HA-tagged WT-Ub or mono-Ub and then stimulated with HGF for 4 h. Their lysates were subjected to immunoprecipitation and immunoblotting with anti-E-cadherin and anti-HA antibodies, respectively. The strongest band was detected at 130 kDa, which is consistent with monoubiquitinated E-cadherin (Fig. 6B). To confirm the monoubiquitination of E-cadherin, two kinds of anti-ubiquitin antibodies were used for immunoblotting: P4D1, specific for both polyubiquitinated and monoubiquitinated proteins, and FK-1, specific only for polyubiquitinated proteins. Ubiquitinated E-cadherin was readily detected by P4D1 but not by FK-1 (Fig. 6C), supporting the idea that E-cadherin is monoubiquitinated.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Deregulation of growth factor RTKs is linked to a large number of malignancies (1). Hrs is required for the degradation of RTKs and other signaling receptors (2, 9, 10, 19), and the depletion of Hrs elevates RTK signals by impairing the down-regulation of RTKs (10, 16–19). Furthermore, Hrs interacts with Merlin, the product of the NF2 tumor suppressor gene (43), and the growth-suppressing effect of Merlin is dependent on Hrs (44). For these reasons, we anticipated that Hrs could have oncosuppressive effects.

Our present study, however, indicates that Hrs contributes to promoting the malignant characteristics of cancer cells in vivo as well as in vitro. Hrs was highly expressed in cancer lesions of human stomach, colon, skin, liver, and cervix. The silencing of Hrs by RNAi in HeLa cells reduced their colony formation in soft agar cultures. Similarly, the in vitro proliferation of Hrs-positive cancer cell lines, such as HeLa, G361, PC-3, and HMV-1, was suppressed when they were infected with Hrs-RNAi retrovirus, particularly as they approached confluence. Consistent with this, HRSw, a revertant clone of HRSd, a MEF cell line derived from a Hrs-targeted mouse, induced a significant enhancement of colony formation and cell proliferation in vitro. These observations suggest that Hrs plays important roles in the induction of anchorage-independent growth and the reduction of the contact inhibition of cell growth, which are in vitro malignant phenotypes.

To investigate further the contribution of Hrs to the generation of in vivo malignant cell phenotypes, we used HeLa cell clones that stably express Hrs-RNAi or a control plasmid. The tumorigenic and metastatic abilities of the Hrs-RNAi HeLa cell clones were significantly lower than those of the control HeLa cell clones, indicating that Hrs is critically involved in the malignancy of HeLa cells. Therefore, we hypothesized that Hrs invests cancer cells with oncogenic activities by repressing oncosuppressive signals or inhibiting the degradation of certain tumor suppressors and thereby preventing their recycling to the membrane.

In differential gene expression analyses, we found a significantly increased expression of E-cadherin in Hrs-RNAi HeLa clones compared with control clones. In addition, we showed that the up-regulation of E-cadherin resulted in the relocalization of ß-catenin from the nucleus to the cytoplasm and reduced ß-catenin signaling. E-cadherin is a tumor suppressor protein with a well-established role in cell-cell adhesion. Loss of E-cadherin expression is associated with the invasion and metastasis of several epithelial cancers (24, 25). One of the mechanisms that E-cadherin uses to suppress tumorigenesis is to bind and antagonize the nuclear signaling activity of ß-catenin (41, 45). Previous studies show that transient or stable expression of E-cadherin strongly reduces ß-catenin–mediated Tcf/Lef signaling via the recruitment of ß-catenin from nuclear and cytoplasmic pools to the plasma membrane (29, 41, 45). Hence, we think that the accumulation of E-cadherin in Hrs-RNAi HeLa clones traps ß-catenin in the cytoplasm, thereby suppressing the ß-catenin signaling for Tcf/Lef activation. We confirmed that E-cadherin has this suppressive effect by knocking E-cadherin down in Hrs-RNAi HeLa cells, which resulted in the near doubling of ß-catenin signaling for Tcf/Lef activation. In addition, because c-Myc, one of the target genes in the Wnt pathway, is activated by ß-catenin (32), we tested c-Myc activation by luciferase assays and showed decreased c-Myc activation in Hrs-RNAi cells. The attenuation of the ß-catenin signaling pathway might explain, at least in part, why the Hrs-RNAi HeLa cells expressed less malignant phenotypes than the control HeLa cells.

Taking the physiologic role of Hrs into account, the accumulation of E-cadherin in Hrs-depleted cells seemed likely to be mediated by impaired lysosomal degradation of E-cadherin. Because E-cadherin degradation is reported in lysosomes but not in proteasomes (22), we confirmed the colocalization of E-cadherin with late endosomes and lysosomes in control MCF-7 cells but not in Hrs-RNAi MCF-7 cells. Furthermore, the depletion of Hrs led to the suppression and delay of the signal-induced degradation of E-cadherin, resulting in the intracellular accumulation of E-cadherin. In fact, E-cadherin molecules are not static but undergo constitutive endocytosis and are recycled back to the same plasma membrane domain, even at the lateral membrane of confluent cells (23, 37, 42, 46). Hence, the impaired degradation of E-cadherin could result in the gradual accumulation of intracellular E-cadherin under physiologic conditions.

Hrs binds directly to ubiquitinated cargo proteins by way of its UIM, leading to their endosomal sorting (2, 6). The ubiquitination of a cargo protein requires its tyrosine phosphorylation (7). Similarly, E-cadherin is phosphorylated on tyrosine residues, and the tyrosine-phosphorylated E-cadherin is ubiquitinated by an E3 ligase, Hakai, resulting in its endocytosis (21), and a relationship between the monoubiquitination of E-cadherin and its degradation in lysosomes has been suggested (21, 47, 48). Here, we obtained evidence using both anti-ubiquitin antibodies and a ubiquitin mutant that E-cadherin is monoubiquitinated but rarely polyubiquitinated. Thus far, monoubiquitination is implicated in endocytosis (13) and membrane trafficking, whereas polyubiquitination targets proteins for destruction by proteasomes (8, 13). Therefore, the present study together with previous reports indicates that E-cadherin is mainly monoubiquitinated and that Hrs binds to the ubiquitin of E-cadherin to recruit it to lysosomes to be degraded.

Another point this report highlights is that several human cancers are associated with highly elevated expression of Hrs. This finding may be consistent with a former report indicating that Hrs is highly expressed in growth hormone–secreting adenoma (49). The spectra of Hrs expression seem to at least partially correlate with that of Wnt signal-related cancers, such as colorectal, gastric, and hepatocellular carcinomas (50). Nevertheless, our present study clearly indicates that Hrs expression does not significantly alter the Wnt pathways. We therefore speculate that Hrs may confer its tumorigenic effect via the E-cadherin–dependent manner. It is intriguing to determine whether Hrs may be a novel tumor marker. Further accumulation of human cancer scrutiny is required. On the other hand, it is tempting to consider whether Hrs may be a potential therapeutic target of malignancies. We need to pave the way toward a novel approach for future clinical interventions.

In conclusion, our findings identified Hrs as an oncogenic factor that is required for the maintenance of such malignant cell phenotypes as anchorage-independent growth, tumorigenesis in nude mice, and metastasis. We think that all the malignant cell phenotypes associated with Hrs expression are mediated by the Hrs-dependent lysosomal degradation of E-cadherin, which results in the promotion of ß-catenin signaling. Hrs was originally identified as a signal-transducing factor (33, 36). Further studies analyzing the signal transduction associated with Hrs in cancer cells will uncover a clearer understanding of how Hrs contributes to oncogenic cell phenotypes.


   Acknowledgments
 
Grant support: Grant-in-aid for Scientific Research from the Japan Society for the Promotion of Science, grant-in-aid from the 21st Century Center of Excellence Program Special Research Grant, grant-in-aid for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports, and Culture of the Japanese Government, and a grant-in-aid from Takeda Medical Research Foundation.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Megumi Chiba for generous contributions of time and technical assistance, Keiko Abe for assistance with the pathologic analysis, and Lamichhane Aayam for critically reading the manuscript.

Received 7/25/06. Revised 2/28/07. Accepted 3/20/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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