E-Cadherin Expression in Melanoma Cells Restores Keratinocyte-Mediated Growth Control and Down-Regulates Expression of Invasion-Related Adhesion Receptors

Cell Culture

The isolation and culture of normal human melanocytes and melanoma cells was performed as previously described. 41,42 Briefly, melanocytes were cultured in MCDB153/L15 medium (v/v: 4/1) supplemented with CaCl₂ (2 mmol/L), insulin (5 μg/ml), EGF (5 ng/ml), 12-O-tetradecanoyl phorbol-13-acetate (10⁻⁷ mol/L), bovine pituitary extract (40 μg/ml), and 2% fetal bovine serum. Melanoma cells were cultured in melanocyte growth medium in the absence of EGF, phorbol ester, and bovine pituitary extract. WM115 is a vertical growth phase primary melanoma cell line, whereas, WM164, WM852, and 1205Lu are metastatic cells. All four cell lines are tumorigenic in severe combined immunodeficient mice and metastasis-competent. 42 Keratinocytes were grown in serum-free keratinocyte growth medium, containing modified MCDB153 supplemented with bovine pituitary extract (140 μg/ml), EGF (10 ng/ml), ethanolamine (0.1 mmol/L), hydrocortisone (5 × 10⁻⁷ mol/L), insulin (5 μg/ml), and O-phosphoryl ethanolamine (0.1 mmol/L). Primary human dermal fibroblasts were initiated as explant cultures from trypsin-treated and epidermis-stripped neonatal foreskin, and passaged in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum. Transcomplementing 293 cells, a cell line immortalized and transformed by adenovirus E1a and E1b, were obtained from the Vector Core of the Institute for Human Gene Therapy, University of Pennsylvania (Philadelphia, PA) and grown in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum. All tissue culture reagents were purchased from Sigma Chemical Co. (St. Louis, MO) except for EGF (Collaborative Biochemical Products, Bedford, MA) and Dulbecco’s modified Eagle’s medium (Life Technologies, Inc., Gaithersburg, MD).

Antibodies and Streptavidin Conjugates

Mouse monoclonal anti-human E-cadherin antibodies, HECD-1 and SHE78–7 were purchased from Zymed Laboratories (San Francisco, CA). SHE78–7 was used in neutralizing experiments for inhibition of E-cadherin-dependent adhesion. HECD-1 was used for flow cytometry. A third monoclonal antibody (mAb) against E-cadherin which recognizes the C-terminus of the molecule was obtained from Transduction Laboratories, Inc. (C20820, Lexington, KY) and used for immunoprecipitation. Anti-β-catenin mAb (C19220) was also purchased from Transduction Laboratories, Inc. Mel-5 is a mouse mAb of the immunoglobulin-2a (IgG_2a) subclass against tyrosinase-related protein-1 (a melanocytic marker) and was obtained from Signet (Dedham, MA). Fluorescein isothiocyanate-, Cy3-, and peroxidase-conjugated goat anti-mouse IgG as well as fluorescein isothiocyanate-conjugated streptavidin were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Mouse mAb SAP (IgG₁) directed to the β3 integrin subunit was generated by immunizing mice with platelet membranes. 43 Mouse mAb A32 (IgG₁) against MelCAM/MUC18 was previously characterized. 44 Antibody purification and biotinylation were performed following procedures described. 6,44

Construction of Replication-Deficient E-Cadherin Adenoviral Vector (E-cad/Ad5)

Full length human E-cadherin cDNA was a kind gift from Dr. David L. Rimm (Yale University, New Haven, CT). The adenoviral vector was constructed according to methods described by Graham and Prevec. 45 Briefly, full-length human E-cadherin cDNA was inserted into the multiple cloning site of pAd.cytomegalovirus (CMV)-Link.1 46 (obtained from the Vector Core, Institute for Human Gene Therapy, University of Pennsylvania, Philadelphia, PA) under the control of the CMV intermediate/early enhancer-promoter element and SV40 polyadenylation signal using a two-step subcloning strategy. Correct orientation of the insert was confirmed by restriction analyses. The resulting shuttle vector (E-cad/pAd.CMV-Link.1) was linearized and co-transfected with the ClaI-digested, E1–E3-deleted human adenoviral DNA dl7001 47 into 293 cells using calcium phosphate precipitation. When cytopathic effects were evident, individual plaques were picked and screened for incorporation of the E-cadherin sequence by Southern blotting. Positive plaques were repurified, propagated, and titrated in permissive 293 cells. Control adenoviral vector, lacZ/Ad5, encoding β-galactosidase was purchased from the Vector Core.

Infection of Melanoma Cells by Adenoviral Constructs

Optimal viral titer was defined as the minimum amount of virus required to yield the highest overall gene transfer efficiency without apparent alteration in cellular phenotype. In melanoma cells, 20 plaque forming units (pfu)/cell result in 100% gene transfer efficiency. Subconfluent melanoma cells were transduced with 20 pfu/cell of replication-deficient adenoviruses for 2 hours at 37°C in a minimum amount of serum-free Dulbecco’s modified Eagle’s medium sufficient to cover the culture vessels. Viral suspensions were then replaced by regular medium. Cells were allowed to recover for 24 hours before use.

Flow Cytometry

Cultured cells were detached with 10 mmol/L ethylenediaminetetraacetic acid in phosphate-buffered saline (PBS), washed once with 0.1% bovine serum albumin in PBS, and stained for 40 minutes with 10 μg/ml of primary mAb at 4°C. After final incubation with fluorescein isothiocyanate-conjugated goat anti-mouse IgG, cells were analyzed by fluorescence-activated cell sorting using an Ortho Cytofluorograf 50H connected to a 2150 Data Handling System (Ortho Diagnostics, Inc., Westwood, MA). As a negative control, unrelated mouse IgG was used.

Immunoprecipitation and Western Blot Analyses

Subconfluent monolayers of cells were scraped off, washed with PBS, and followed by extraction in lysis buffer containing 1% Triton X-100, 1% deoxycholic acid, 2 mmol/L CaCl₂, and protease inhibitors (10 μg/ml leupeptin, 10 μg/ml aprotinin, 1.8 mg/ml iodoacetamide, and 1 mmol/L phenylmethyl sulfonyl fluoride in PBS. After immunoprecipitation with anti-E-cadherin mAb- (2 μg/ml) or nonimmune mouse IgG-conjugated protein A Sepharose CL-4B (Pharmacia Biotech, Uppsala, Sweden), samples were washed three times with lysis buffer, boiled in Laemmli buffer containing β-mercaptoethanol, and then subjected to electrophoresis on a 6% sodium dodecyl sulfate-polyacrylamide gel. Immunoblotting was performed by sequential incubation with an anti-β-catenin mAb (0.5 μg/ml) and a peroxidase-labeled goat anti-mouse secondary antibody. For Western blot analyses, cell lysates were quantified by a BCA protein assay kit (Pierce, Rockford, IL). An equal amount (100 μg) of total protein from each sample was subjected to electrophoresis on a 6% sodium dodecyl sulfate-polyacrylamide gel, transblotted onto polyvinylidene difluoride membranes (Bio-Rad Laboratories, Richmond, CA), and probed with an anti-β3-integrin mAb (SAP) and a peroxidase-conjugated secondary antibody. Immunoreactive bands were detected using enhanced chemiluminescence (Amersham, Arlington Heights, IL).

Cell Adhesion Assay

Melanoma cells transduced with E-cad/Ad5 or lacZ/Ad5 were prelabeled with a fluorescent dye DiI (10 μg/ml; Molecular Probes, Eugene, OR) for 4 hours, and harvested by treatment with 0.01% trypsin in Hanks’ balanced salt solution containing 1 mmol/L calcium for 30 minutes at 37°C. Under these conditions, cadherins are specifically protected from proteolytic digestion. For blocking experiments, E-cad/Ad5-infected cells were incubated with E-cadherin blocking mAb SHE78–7 (5 μg/ml) at 4°C for 30 minutes, washed with Hanks’ balanced salt solution, and resuspended in assay medium containing 1% bovine serum albumin and 1 mmol/L calcium in Hanks’ balanced salt solution. A total of 2 × 10 5 cells in a volume of 400 μl was added to differentiated keratinocyte monolayers in 4-well chamber slides and allowed to adhere for 30 minutes. Keratinocyte differentiation was induced by preincubation in 2 mmol/L of calcium for 1 hour. After removal of nonadherent cells, slides were fixed. Numbers of adherent cells per high power field (×250) in triplicate wells were counted under a fluorescence microscope. Statistical analyses were done by Student’s t-test.

Anchorage-Dependent Growth Assay

Subconfluent cultures were trypsinized and seeded in triplicate 35-mm wells at 4 × 10 5 cells/well. Cells were refed twice weekly. At days 1, 4, and 7, cells were harvested and counted in a Coulter counter (Coulter Electronics; Luton, Beds, England). Statistical analyses were performed using the Student’s t-test.

Soft Agar Assay

Melanoma cells were suspended in MCDB153/L15 medium (v/v: 4/1) supplemented with 25 μg/ml bovine pituitary extract, 2 ng/ml EGF, 2 μg/ml insulin, 4% fetal bovine serum, and 0.25% agar and plated in triplicate at 6 × 10 4 cells/well in 6-well plates. After 3 weeks, colonies were counted using an inverted microscope. Student’s t-test was used for statistical analyses.

In Vivo Tumorigenicity

The tumorigenicity was examined in severe combined immunodeficient mice. Melanoma cells (5 × 10⁶/mouse) were suspended in 0.1 ml of growth medium and injected subcutaneously in the dorsal skin. Tumor size was monitored on days 3, 5, and 7. The size of tumors was determined as follows: (maximum dimensions × minimum dimensions)²/2.

Monolayer Co-Culture and Double Immunofluorescence

Melanocytic cells were detached by trypsinization, mixed with keratinocytes at a 1:10 ratio and seeded in 8-well chamber slides (Lab-Tek; Nunc, Inc., Naperville, IL). After 4 days in co-culture, double immunofluorescence was performed as described. 6 Briefly, cells were fixed, permeabilized, and incubated sequentially with antibodies Mel-5, Cy3-conjugated goat anti-mouse IgG, biotinylated SAP or A32, and fluorescein isothiocyanate-conjugated streptavidin. As a negative control, normal mouse serum was used instead of a primary antibody. All incubations were performed at room temperature for 1 hour. For cell growth experiments, slides were counterstained with Höechst reagent (Bisbenzimide; Sigma Chemical Co.). Cell growth was monitored by counting cells in five random high-power fields (×250). The ratio of keratinocytes and melanocytic cells was determined by the following equation: keratinocytes/melanocytic cells = (total number of Höechst positive nuclei − red cell number)/red cell number.

Cell Invasion in Three-Dimensional Skin Reconstructs

Skin reconstructs were prepared as previously described. 48,49 Invasion of melanoma cells was tested in artificial skin reconstructs, in which human foreskin dermal fibroblasts in rat tail collagen were placed on a precast collagen gel. After 6 days, the constricted collagen gels formed a concave surface, serving as a cradle for seeding of epidermal cells. Melanoma cells were then mixed with keratinocytes at a 1:5 ratio and seeded onto the dermal constructs. After 5 days, cultures were lifted to the air-liquid level for an additional 10 days to allow stratification of epidermal keratinocytes. The reconstructs were then harvested, fixed in paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Apoptosis was evaluated using the ApopTag in situ apoptosis detection kit (Oncor, Gaithersburg, MD).

Characterization of E-cad/Ad5-Transduced Melanoma Cells

Forty-eight hours after transduction with E-cad/Ad5 at 20 pfu/cell, more than 90% of the WM115 cells expressed E-cadherin, whereas control cells transduced with the lacZ reporter gene (lacZ/Ad5) or nontransduced cells remained negative by fluorescence analysis (Figure 1A) ▶ . Similar results were obtained with the additional three melanoma cell lines (WM164, WM852, and 1205Lu). The expression of exogenous E-cadherin in these melanoma cells was further confirmed by Western blotting (data not shown). Because cadherin function depends on the cytoplasmic anchorage, we tested the physical association of transduced E-cadherin with endogenous signal transduction proteins by immunoprecipitation. Forty-eight hours after transduction, extracts of melanoma cells (WM115 and 1205Lu) were immunoprecipitated with an E-cadherin mAb. After electrophoresis, samples were subjected to immunoblotting with an anti-β-catenin mAb. A band of 92 kd corresponding to β-catenin was detected in the E-cadherin-transduced cells, whereas no specific band was present in the cells transduced with lacZ/Ad5. Figure 1B ▶ shows representative data of WM115. In addition, the functional activity of the E-cadherin transgene products in melanoma cells was demonstrated by a fourfold increase in adhesion of E-cadherin-transduced WM115 melanoma cells to keratinocytes when compared to cells transduced with the control vector. Furthermore, the adhesion of E-cadherin-transduced cells to keratinocytes was significantly reduced in the presence of an E-cadherin blocking mAb (data not shown). Transduction of E-cadherin into four melanoma cell lines (WM115, WM164, WM852, and 1205Lu) reduced cell growth in monolayer by 25 to 50% (data not shown) and colony formation in soft agar by 15 to 30% (Figure 1C) ▶ . The E-cadherin-transduced cells grew as compact colonies of 40 to 60% smaller size when compared to lacZ-transduced or nontransduced cells (Figure 1C ▶ , inserts). The reduction in colony size was apparently due to the tight cell-cell adhesive interactions. In tumorigenicity assays, E-cadherin-overexpressing 1205Lu melanoma cells remained tumorigenic when injected subcutaneously into severe combined immunodeficient mice but the tumor size was significantly reduced by 60 to 70% in comparison to those arising from control vector-transduced counterparts (Figure 1D) ▶ .

Re-Expression of E-Cadherin Restores Keratinocyte-Dependent Growth Control and Melanoma-Associated Antigen Down-Regulation in Melanoma Cells

Valyi-Nagy et al 5 had shown that normal melanocytes, co-cultured with human keratinocytes at a physiological 1:5 or 1:10 ratio, maintained the initial seeding ratio throughout a 14-day observation period, despite continuing proliferation of both cell types. E-cadherin-transduced WM115 melanoma cells when seeded with keratinocytes exhibited similar controlled growth as normal melanocytes (Figure 2) ▶ . In contrast, lacZ/Ad5-transduced melanoma cells rapidly propagated resulting in a significant increase of the ratio beginning at approximately 4 days after seeding (Figure 2) ▶ . Similar results were obtained using 1205Lu melanoma cells. In control experiments, when E-cadherin-transduced WM115 melanoma cells were cultured separately from keratinocytes, the ratios between the two cell types increased (although at a slower rate in comparison to those of lacZ-transduced cells; data not shown) indicating that keratinocytes regulate growth of E-cadherin-transduced melanoma cells through direct cell-cell contact.

To investigate whether E-cadherin contributes to the phenotypic plasticity of melanocytic cells in response to keratinocyte contact-mediated control, we tested cell surface antigen expression in E-cadherin-transduced WM115 and 1205Lu melanoma cells co-cultured with keratinocytes. Figure 3 ▶ shows representative data from WM115. Nontransduced or lacZ/Ad5-transduced melanoma cells retained the expression of melanoma-associated antigens such as the cell-cell adhesion molecules, MelCAM/MUC18 and the β3 subunit of the αvβ3 vitronectin receptor (Figure 3) ▶ . By contrast, E-cadherin-transduced melanoma cells expressed neither antigen at detectable levels after 7 days in co-culture. In the absence of keratinocytes, E-cadherin transduction of melanoma cells had no effect on the expression of these invasion-related adhesion molecules (Figure 4) ▶ .

E-Cadherin Expression in Melanoma Cells Inhibits Invasion and Triggers Apoptosis in Three-Dimensional Skin Reconstructs

To determine whether the E-cadherin-induced down-regulation of tumor-associated antigens on the melanoma cell surface has biological consequences, a three-dimensional reconstruct was used to test the invasive capacity of the melanoma cells. This model consists of a dermal compartment containing fibroblasts in a collagen gel, which is separated from an epidermal compartment composed of melanocytic cells and keratinocytes by a naturally formed basement membrane. 49 Control vector-transduced 1205Lu metastatic melanoma cells grew deep into the dermis, forming strands of cell nests (Figure 5 ▶ , B and D), whereas, E-cadherin-transduced melanoma cells remained in the epidermis and upper dermis (Figure 5 ▶ , A and C). Those melanoma cells located in the upper dermis showed typical signs of apoptotic death (Figure 5C) ▶ , including nuclear condensation and apoptotic bodies. Free 3′-OH ends resulting from DNA fragmentation were detected in the apoptotic cells using the ApopTag in situ apoptosis detection kit (Oncor) (Figure 5E) ▶ , whereas, invading cells in control reconstructs showed no evidence of apoptosis (Figure 5F) ▶ . Similar results were obtained using WM115 vertical growth phase melanoma cells (data not shown).