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Short Communications |
From the Wistar Institute*
and the Department of
Pathology,
University of Pennsylvania,
Philadelphia, Pennsylvania
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cadherins comprise a growing family of surface membrane glycoproteins that mediate cell-cell adhesion in a calcium-dependent fashion.7 The extracellular domains of cadherins serve as molecular zippers connecting neighboring cells,8 whereas the cytoplasmic tails are linked noncovalently to the actin cytoskeleton via catenins.9 Cadherins are not just biological glues. Signal mediators such as phosphokinases,10 phosphatases,11 and their substrates12 are localized to the cadherin-catenin complexes. The organization of a hierarchy of multiple components allows cadherins to act as signaling receptors for intercellular communication with subsequent modulation of growth13-16 and differentiation17-19 in both normal and malignant cells. The physical association of the adenomatous polyposis coli tumor suppressor protein with catenins20,21 and the ability of proto-oncogenes including c-erbB2,22 Wnt-1,23 and epidermal growth factor (EGF) receptor24 to modulate E-cadherin expression also indicate an important link between tumor initiation and cell adhesion.
A tumor/invasion suppressor role of E-cadherin has been established in various human carcinomas.25 Studies have demonstrated an association between E-cadherin down-regulation and dedifferentiation, invasiveness, and lymph node or distant metastasis in breast,26 colorectal,27 prostate,28 bladder,29 renal,30 hepatocellular,31 ovarian,32 pancreatic,33 endometrial,34 and squamous cell carcinoma.35 Furthermore, the functional modification of E-cadherin using blocking antibodies27,31,36 demonstrated that disruption of E-cadherin-mediated homotypic cell adhesion facilitates tumor invasion, presumably by releasing the malignant cells from the primary site. Conversely, activation of E-cadherin resulted in growth retardation13,14,16,37 and inhibition of the invasive and metastatic phenotype in carcinoma cells.36,38-40
To obtain direct information on the role of E-cadherin-mediated adhesion and/or signal transduction in melanoma, we transduced E-cadherin-negative melanoma cell lines with full-length E-cadherin cDNA using an adenoviral vector. Our results show that functional restoration of E-cadherin in melanoma cells retards cell growth in anchorage-dependent and -independent cultures, reduces tumorigenicity in vivo, and confers keratinocyte-dependent normal melanocytic phenotype. The transduced cells grew in a controlled manner in the presence of keratinocytes and showed down-regulation of invasion-related antigens, such as MelCAM/MUC18 and ß3 integrin subunit. In a three-dimensional reconstruction model, ectopic E-cadherin expression in melanoma cells inhibited invasion and induced apoptosis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 x 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-ß-catenin mAb (C19220) was also purchased from Transduction Laboratories, Inc. Mel-5 is a mouse mAb of the immunoglobulin-2a (IgG2a) 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 ß3 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.146 (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 dl700147 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 ß-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 ß-mercaptoethanol, and then subjected to electrophoresis on a 6% sodium dodecyl sulfate-polyacrylamide gel. Immunoblotting was performed by sequential incubation with an anti-ß-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-ß3-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 x 105 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 (x250) 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 x 105 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 x 104 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 x 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 x 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 (x250). 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).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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-ß-catenin mAb. A band of 92 kd corresponding to ß-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)
.
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Valyi-Nagy et al5
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.
|
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 ß3
subunit of the 
vß3 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)
.
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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).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although our data demonstrated that E-cadherin induced inhibition of
growth in human melanoma cells in vitro (Figure 1C)
and
in vivo (Figure 1D)
, there is no evidence that
down-regulation of E-cadherin initiates melanoma. Earlier cytogenetic
analyses of melanoma cell lines failed to identify frequent
abnormalities at 16q where the E-cadherin gene is
mapped.52
Clearly, with new markers currently available,
additional genetic evidence is required to establish a true tumor
suppressor role of E-cadherin in human melanoma. Our data demonstrated
that functional expression of E-cadherin in melanoma cells resulted in
growth retardation as well as inhibition of cell motility and local
invasion. However, malignant properties such as soft agar growth and
tumor formation remained relatively intact. Recently, Rubinfeld et
al53
demonstrated accumulation of ß-catenin as a result
of gene mutations in human melanoma cells, which in turn activated
aberrant Lef/Tcf-dependent gene transcription, including transcription
factors c-jun and fra-1, and
urokinase-type plasminogen activator receptor.54
E-cadherin may exert its growth-suppressive effect directly through
tight cell-cell adhesion or indirectly by sequestrating ß-catenin and
thus preventing aberrant Lef/Tcf-related transcriptional activation.
One caveat is that E-cadherin-induced growth retardation in melanoma
cell lines renders our study on keratinocyte-mediated growth control
more difficult to interpret. Nevertheless, a specific growth regulation
by keratinocytes was evident because E-cadherin-transduced melanoma
cells when physically separated from keratinocytes exhibited increased
relative cell ratios over time.
In our co-culture model, keratinocytes down-regulated
melanoma-associated cell adhesion molecules, MelCAM/MUC18 and ß3
integrin subunit, in E-cadherin-transduced melanoma cells (Figure 3)
.
This suggests that E-cadherin-mediated signal transduction may
contribute to the regulation of cell adhesion molecules expression.
Indeed, cross-talk between adhesion receptors has been described
recently. Hodivala and Watt55
provided evidence that
cadherins play a role in integrin down-regulation during keratinocyte
terminal differentiation. Monier-Gavelle and Duband56
reported that ß1 and ß3 integrins control cell surface distribution
and activity of N-cadherin in migrating neural crest cells. Forced
expression of 5 integrin subunit in primary quail myoblasts also
up-regulates N-cadherin expression.57
Therefore,
cross-talk between adhesion molecules may represent one of the
mechanisms through which specific cellular functions are coordinated.
Tumor invasion is the hallmark of malignancy. However, the invasive
phenotype does not originate solely from the cancer cells themselves
but also depends on microenvironmental factors including local growth
factors, cytokines, and interactions between the cancer cells and
surrounding host cells.58
Our skin reconstruct model,
which allows evaluation of the invasive potential of melanoma cells in
a physiological milieu, demonstrated an anti-invasive effect of
E-cadherin (Figure 5)
. This effect may rely in a decreased mobility of
melanoma cells due to E-cadherin-enhanced adhesion to adjacent
keratinocytes, or alternatively, to E-cadherin-induced modulation of
invasion-related cell adhesion molecules (such as MelCAM/MUC18 and ß3
integrin subunit) and proteolytic enzymes important for matrix
degradation.59
The importance of adhesion receptors for
the tumorigenic phenotype was demonstrated in our previous study where
overexpression of the ß3 integrin subunit in nontumorigenic and
noninvasive radial growth phase primary melanoma cells induced
conversion and progression to the tumorigenic and invasion-competent
vertical growth phase primary melanoma.48
The observed
down-regulation of MelCAM/MUC18 and ß3 integrin subunit in
co-cultures further pointed to the plasticity of the antigenic
phenotype of malignant cells suggesting plausible means to modulate
expression of tumor-associated molecules and reverse natural
progression.
It remains, however, unclear whether signals from keratinocytes for the control of growth and expression of melanoma-associated adhesion receptors of melanocytes/melanoma cells are transduced directly through E-cadherin/catenin pathway or indirectly through other molecules whose function requires E-cadherin-mediated cell-cell adhesion. It is hoped that once the molecular details of these mechanisms are discovered, it may be possible to activate them in the absence of keratinocytes. Therapeutic applications of this strategy could lead to a static tumor without further proliferation, invasion, and metastasis.
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Acknowledgements |
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Footnotes |
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Supported by National Institutes of Health grants CA-76674, CA-25874, and CA-15479, by the Wistar Cancer Center Core Grant (CA-10815), and NASA grant NAG-9–832.
M.-Y. Hsu’s current address: Department of Pathology, Thomas Jefferson University, Philadelphia, PA 19107.
F. E. Meier’s current address: Department of Dermatology, University of Tübingen, Tübingen, Germany.
J.-Y. Hsu’s current address: Sidney Kimmel Cancer Center, San Diego, CA 92121.
Accepted for publication January 6, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 A. Slominski, D. J. Tobin, S. Shibahara, and J. Wortsma |
), and normal human
melanocytes as positive control
(
) were co-cultured
with keratinocytes at an initial seeding ratio of 1:10. On days 1, 4,
and 7, co-cultures were stained with mAb Mel5, an anti-TRP-1 antibody,
to identify melanocytic cells and then counterstained with
Höechst dye to visualize all cells. Cell ratios were determined
by counting under a fluorescence microscope. E-cadherin- and
lacZ-transduced melanoma cells showed significant differences in cell
ratios (P < 0.05 on days 4
and 7 by Satterthwaite’s method for unequal
variances).
