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Molecular Biology and Genetics |
The Wistar Institute, Philadelphia, Pennsylvania 19104 [R. A. S., K. S., B. V., M. H.]; Centre for Functional and Applied Genomics, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia [R. A. S., B. B. G., D. J. S.]; and Department of Dermatology, University of Tübingen, Tübingen, Germany 72076 [F. M.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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vß3 LM609 mAb, mitogen-activated protein kinase, or protein kinase C inhibitors, indicating that other signaling pathway(s) operate through vß3 integrin during conversion from RGP to VGP. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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vß3 appears to confer a tumorigenic phenotype (3)
. Five distinct steps have been proposed for the progression of melanoma, based on clinical and histopathological features: common acquired and congenital nevi with structurally normal melanocytes; dysplastic nevus with structural and architectural atypia; RGP3 melanoma without and VGP melanoma with competence for metastasis; and metastatic melanoma (4) . The genetic alterations responsible for the development and stepwise progression of melanoma are still unclear, but classification by gene expression profiling has been proposed to identify subsets of melanomas that correlate with phenotypic characteristics important for disease progression (5) . The transition from RGP to VGP melanoma is a biologically critical step in melanoma progression. A variety of changes can be observed in sections of melanoma lesions and in cultured cells that may help explain RGP to VGP progression (6) . Unlike RGP melanoma cells, VGP melanoma cells are tumorigenic and easily adapt to growth in culture. VGP melanoma cells are also less dependent on exogenous growth factors and have growth characteristics similar to those of metastatic cells, such as anchorage-independent growth in soft agar and tumorigenesis in immunodeficient mice. VGP primary melanomas display numerous cytogenetic abnormalities, suggesting considerable genomic instability. Only minor additional genetic changes may be required for further progression to metastatic dissemination because most VGP melanomas can be readily adapted to a metastatic phenotype through selection in growth factor-free media and induction of invasion through artificial basement membranes (7) . This suggests that microenvironmental factors such as cell-matrix and cell-cell signaling are critical for the metastatic phenotype.
Several adhesion molecules have been studied in melanoma progression. The most notable is the ß3 subunit of the vß3 vitronectin receptor (3)
. It is now recognized as a specific melanoma-associated marker that distinguishes RGP from VGP melanomas (8)
. ß3 is a prime candidate for prognostic studies (9)
because its expression correlates closely with clinical recurrence and mortality (10
, 11)
. The contribution of ß3 to metastatic behavior of melanoma was investigated by comparing cell variants with different levels of vß3 expression (12)
and by modulating vß3 function with antibodies and peptides (13
, 14)
. The strongest evidence for the role of ß3 in conversion of RGP to VGP melanoma comes from expression studies of the ß3 protein in RGP melanoma cell lines (15)
. Integrin vß3 was also demonstrated to promote melanoma cell survival (16)
.
The genetic determinants for ß3 to drive metastasis have not yet been determined. Therefore, we have searched for gene expression changes using a highly efficient cDNA-RDA subtractive hybridization method (17)
in ß3-positive and -negative melanoma cell populations. Our results indicate that ß3 overexpression up-regulates molecules associated with adhesion and that osteonectin/SPARC is critical for progression of melanoma cells from nontumorigenic RGP to tumorigenic VGP. Because osteonectin expression has previously been associated with progression in both human and mouse melanoma (18
, 19)
and is a marker correlated with increased incidence of distant metastases and decreased survival (20)
, we examined the modulation of vß3 integrin function on osteonectin gene induction in human melanoma cells.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Flow Cytometry.
Cells transduced with viral vectors were collected with versene, washed with DMEM, and resuspended in serum-free DMEM. Cells were seeded in duplicates at 2 x 105 cells/well in a 96-well plate and incubated with 10 µg/ml anti-ß3 mouse mAb SAP (11)
. After 45 min of incubation at 4°C with gentle shaking, cells were washed with PBS containing 0.1% BSA and 0.1% sodium azide to remove unbound antibodies before staining with 10 µg/ml FITC-conjugated rabbit antimouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA). After washing, cells were resuspended and analyzed by fluorescence-activated cell sorting using an Ortho Cytofluorograf 50 H connected to a 2150 Data Handling System (Ortho Diagnostics, Westwood, MA).
Isolation of RNA, cDNA Synthesis, and RDA.
Transduced cells were harvested and lysed using NP40 (21)
. DNA was removed by pelleting the nuclei, and total cytoplasmic RNA was prepared from the cleared supernatant by proteinase K/SDS treatment. Total RNA was recovered by phenol/chloroform extraction and ethanol precipitation. Polyadenylated RNA was selected using an Oligotex mRNA Midi Kit (Qiagen, Valencia, CA), and 1 µg was used as template for double-strand cDNA synthesis using a Life Technologies, Inc. SuperScript Choice System with 1 µg of oligodeoxythymidylic acid and 100 ng of random N6 primers. RDA is a subtractive hybridization procedure coupled with PCR amplification used to rapidly identify sequence differences between complex populations of DNA molecules (22
, 23)
. The complexity of the hybridizing material is reduced by restriction enzyme digestion, ligation of adaptors, and PCR of an amplimer product for sampling. Differential hybridization was coupled with amplification of DPs (DP1, DP2, and DP3) after each round due to removal of the adaptors from the "driver" but not the "tester" DNA representations. This approach results in the enrichment of sequences unique to the tester sample. The procedure was performed as described previously (22
, 23)
, with the following modifications: oligonucleotides were purified by high-pressure liquid chromatography (24)
; mung bean nuclease digestion of amplification products was omitted (25)
; and excess and cleaved adaptors from the RDA amplicons made from DpnII-digested cDNA representation and tester amplification products were purified using Millipore microcon 100 filters (26)
. Subtractive hybridizations were performed in 4-µl volumes using 40 µg of driver with the ratio of tester/cycle set at 1:100 for DP1, 1:500 for DP2, and 1:4000 for DP3. Twenty PCR amplification cycles were used to generate each of the DP products. Supplementation of the driver with ß3 coding sequences from the pcDNA1-ß3 plasmid (a gift from Dr. D. Cheresh, Scripps Research Institute, La Jolla, CA) was used to suppress amplification of the ß3 gene sequences derived from the adenovirus vector expression by adding 5 µg (22)
of ß3 plasmid cDNA generated as driver (27)
to each round of hybridization.
Clone Isolation and Sequencing.
Subtracted DP2 RDA products were digested with DpnII, purified using a microcon 100 filter, and cloned into the BamHI site of pBluescript SK+ (Stratagene, La Jolla, CA). DP3 products were digested with DpnII, separated by agarose gel electrophoresis, and purified using QiaexII resin (Qiagen) before cloning. DNA samples were sequenced from mini-extracted plasmids using BigDye terminator automated sequencing. Sequences obtained were compared with DNA databases using the BLASTN program (Australian National Genomic Information Service, University of Sydney, Sydney, Australia).
Radiolabeling of Probe and Northern Blotting.
Northern blotting was performed by fractionating 10 µg of each RNA sample on formaldehyde-agarose gels (21)
followed by soaking in 10x SSC, transfer to a nylon membrane, UV cross-linking, and hybridization. Efficiency of transfer and position of 18S and 28S rRNA bands were determined by UV shadowing of the nylon membrane. Loading and integrity of the RNA samples were tested using a GAPDH gene probe. Insert probes were prepared by radiolabeled PCR (28)
using 0.1 ng of plasmid clone DNA as template with SK (Stratagene) and KS-20mer (5'-CCTCGAGGTCGACGGTATCG-3') primers and fractionated on Sephadex G-50 Nick columns (Pharmacia, Piscataway, NJ). The PstI/XhoI fragment from the SPARC full-length cDNA (the pBS clone was a gift from R. Nischt, University of Cologne, Cologne, Germany) and GAPDH probes were generated by random labeling using a high prime DNA labeling kit and fractionated on Sephadex G-50 Quick Spin columns (Boehringer Mannheim; Roche, Indianapolis, IN).
Western Blot of Cell Extracts, MAPK, PKC, and Antibody Inhibition.
SBcl2 and WM1552 cells were seeded in duplicate in 6-well plates and grown to subconfluence. After adenoviral gene transduction, the cells were overlaid with growth medium for 24 h and washed with serum-free medium, and medium was replaced with serum-free medium containing 5 µg/ml insulin for an additional 48 h. MAPK inhibitors PD98059 and wortmannin (Calbiochem, San Diego, CA) were added at 10 µM and 100 nM, respectively, for the 48-h time period. Cells were harvested by scraping and vigorous pipetting on ice in 150 µl of MAPK extraction buffer consisting of 10 mM Tris-acetate (pH 8.0), 0.5% NP40 (BDH Chemicals, Darmstadt, Germany), 1 mM EDTA, 2 mM phenylmethylsulfonyl fluoride, and 1 µg/ml each of aprotinin, leupeptin, and pepstatin, and insoluble material was removed by centrifugation at 13,000 x g for 10 min at 4°C. Total protein concentrations were estimated with a BCA kit (Bio-Rad, Hercules, CA) using BSA as standard. Equal amounts of protein were separated by 10% SDS-PAGE and electroblotted onto polyvinylidene difluoride membranes (NEN Polyscreen, Boston, MA). Membranes were blocked for 1 h at room temperature with 5% skim milk in 1x TBS/0.1% Tween 20 that was included in each antibody-binding step. Incubation with 1 µg/ml anti-osteonectin mouse mAb (Hematological Technologies Inc., Essex Junction, VT) overnight at 4°C was followed by rinsing in 1x TBS/0.1% Tween 20 and incubation with a 1:1000 dilution of peroxidase-labeled goat antimouse IgG (Kirkegaard & Perry Laboratories, Gaithersburg, MD). After extensive washing in 1x TBS/0.1% Tween 20, immunoreactive bands were identified by enhanced chemiluminescence (Amersham, Arlington Heights, IL).
A2058 cells were grown until approximately 80% confluence and washed with serum-free medium, and medium was replaced with serum-free medium containing 5 µg/ml insulin for 24 h. Cells were harvested in PBS without trypsin and transferred to the wells of a 6-well non-tissue culture plate (Falcon) in which the wells had been previously coated in duplicate for 4 h at 37°C with the following proteins: 5 µg/ml LM609 vß3 monoclonal blocking mAb (Chemicon Inc., Temecula, CA); 0.1 mg/ml poly-L-lysine; 2 µg/ml vitronectin; or 0.1mg/ml heat-denatured BSA. After incubation with serum-free medium containing 5 µg/ml insulin for an additional 24 h, cell extracts were prepared using MAPK extraction buffer. PKC inhibition of cells was performed in duplicate coated wells by treatment with 25 nM calphostin C (Sigma Chemical Co., St. Louis, MO). Protein concentration of the samples was normalized by incubating the membranes in a neat serum solution of intermediate filament antibody (29)
for 1 h at room temperature, followed by washes in 1x PBS/0.05% Tween 20. The membrane was then treated with peroxidase-labeled sheep antimouse IgG as described above.
Cell Adhesion Assay and in Vitro Reconstruction of Human Skin.
Cell adhesion assays were performed with melanoma cells infected with either LacZ/Ad5 or ß3/Ad5. Cells were harvested after 48 h, counted, and seeded in triplicate at 104 cells/well in a 96-well non-tissue culture plate that had previously been coated overnight at 4°C with the following matrix proteins: 5 µg/ml vitronectin; 10 µg/ml fibronectin; 5 µg/ml osteonectin (Calbiochem); or heat-denatured BSA. Cells were allowed to adhere for 1 h at 37°C, washed gently in PBS to remove unbound cells, fixed in 3% paraformaldehyde, and stained with crystal violet as described previously (30)
. After extensive washing to remove nonspecifically bound dye, cells were lysed in 1% SDS, and absorbance was read at 595 nm.
Skin reconstructs were generated as described previously (31) . They consist of a "dermis" of collagen type I embedded with fibroblasts and an "epidermis" with keratinocytes and melanocytes or melanoma cells. The melanoma cells had been transduced with adenoviral vector 48 h before mixing with the keratinocytes at a 1:5 ratio. After 10–14 days of air exposure, during which the keratinocytes formed multiple layers, skin reconstructs were harvested by fixing overnight with 4% paraformaldehyde, dehydration, and embedding in paraffin. Sections were stained with H&E for histological analysis.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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demonstrated that transduced cells expressed the ß3 molecule. The highest level of expression was found at 20 pfu/cell, which was consistent with the results of previous gene transduction studies (15)
. For additional experiments, the cells transduced with 20 pfu/cell were chosen to look for changes associated with ß3 expression.
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To avoid amplification of the ß3 gene DPs detected in our initial analysis, we doped the driver sample with ß3 coding sequences (22
, 23
, 27)
. The PCR fragment profiles resulting from the ß3 coding sequence-doped RDA products were separated by agarose gel electrophoresis and are shown in Fig. 2
. The complex pattern of the initial driver (Lane 2) and tester (Lane 3) cDNA amplification products was sequentially reduced by subtractive hybridization coupled with PCR amplification as seen in the first (Lane 4, DP1)-, second (Lane 5, DP2)-, and third (Lane 6, DP3)-round DPs. A similar experiment was performed using the WM1552C cell line, in which only two rounds of subtractive hybridization were completed. The DP2 fragments generated from the SBcl2 and WM1552C cDNA were cloned as separate pools, whereas the SBcl2 DP3 fragments were isolated from the gel before cloning and sequencing.
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, Lane 6) were identified by comparison of their sequence with published nucleotide databases. The most intense of these fragments was a 450-bp ß3/Ad5 vector SV40 sequence that was not competed out when using the ß3 coding region. This served as an internal control for the fidelity of the RDA assay. Sequencing the DP3 cloned fragments then identified 14 known genes and 1 insert of unknown identity (Table 1)
. Seventy-two SBcl2 and 24 WM1552C DP2 isolates were sequenced to determine the range of genes whose expression levels may have been affected by ß3 (data not shown). Fifty-five genes were identified, some of which overlapped with those found in the DP3 cycle. When confirming the induction of several genes by Northern blotting including KIAA0108, integrins 5 and ß5, and Smad7, expression of osteonectin mRNA (32)
was also observed in the ß3-transduced SBcl2 cells (Fig. 3A)
.
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shows the results of both SBcl2 and WM1552C cells transduced with either LacZ (Lanes 1 and 7) or ß3 (Lanes 2 and 8) and sampled 72 h postinfection with anti-osteonectin antibodies. A 3–4-fold increase in protein levels was seen in both cell lines. To test for the involvement of the MAPK pathway in the ß3-mediated induction of osteonectin, SBcl2 cells were treated with the MAPK inhibitors PD98059 (Lanes 3 and 4) and wortmannin (Lanes 5 and 6). Transduction with ß3 continued to show increased levels of osteonectin synthesis relative to LacZ, indicating that ß3-induced changes do not affect the MAPK pathway.
The A2058 melanoma cell line is known to express high levels of vß3 integrin and can be inhibited in attachment to substratum-bound vitronectin using the LM609 vß3 blocking mAb (33)
. The expression levels of osteonectin protein produced in these cells were assessed after plating on vitronectin, LM609 mAb, poly-L-lysine, or heat-denatured BSA-coated plastic dishes. The levels of osteonectin were reduced 10-fold on culturing these cells with LM609 blocking antibody as compared with treatment with vitronectin alone; also, the antibody was able to reduce osteonectin production when coated together with vitronectin (Fig. 3, C and D)
. Inhibition of vß3 function supports the induction of osteonectin through active ß3 integrin expression. The calphostin C nonspecific PKC inhibitor was used to determine whether this pathway was involved in the expression of osteonectin through vß3 integrin-vitronectin binding, as has been seen for vß3 integrin-LM609 ligation induction of other molecules (34)
; however, expression levels were not affected (Fig. 3D)
.
In Vitro Effect of Osteonectin on Melanoma Cell Adhesion to ECM Proteins.
To test the effects of osteonectin protein on cell function, we examined the adhesion of SBcl2 cells after ß3 overexpression. Vitronectin, fibronectin, osteonectin, or heat-denatured BSA was used to coat plastic dishes. Attachment of LacZ- or ß3-transduced SBcl2 cells was tested. Cells expressing ß3 integrin showed a 2-fold increase in adhesion to both vitronectin- and fibronectin-coated wells as compared with LacZ-expressing cells; however, osteonectin alone did not significantly affect adhesion (Fig. 4)
. When vitronectin and osteonectin were mixed before coating, cell binding was reduced by >30%. Inhibition of adhesion of the vitronectin/osteonectin mixture was not seen in LacZ-transduced SBcl2 cells nor when osteonectin was mixed with fibronectin.
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. In contrast, SBc12 cells transduced with ß3 were deeply invasive in the dermis (Fig. 5, B and D)
. The cells expressed ß3 and osteonectin, confirming the up-regulation of osteonectin by ß3 seen in monolayer cultures.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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vß3 dimer formation promotes melanoma tumorigenicity through escape from apoptosis (15
, 16) , other pathways remain to be investigated. This study has found a range of genes whose expression was up-regulated on ectopic expression of ß3 integrin. SBcl2 and WM1552C RGP melanoma cells were used as a test system to profile gene expression responsible for aggressive behavior. Both DP3 and DP2 cycle gene fragment products amplified from the cDNA-RDA procedure were analyzed to increase the range of genes whose expression may be affected by induction of the ß3 integrin subunit (25
, 35)
. The DP3 cycle favors the identification of more abundant gene transcripts with most dramatic changes in gene expression. Fourteen genes and one unknown transcript were selectively amplified as products of the DP3 cDNA-RDA procedure in the SBcl2 cells transduced with ß3. Of these genes, osteonectin is expressed by melanoma cells (32) but not by normal melanocytes. This pattern has been associated with progression in both human and mouse melanoma (18 , 19) . Expression of osteonectin has been correlated with increased incidence of distant metastases and decreased survival (20) ; however a relationship with primary tumor growth has not yet been determined. Studies of invasive meningiomas and prostate carcinomas have also demonstrated a correlation between osteonectin production and tumor progression (36 , 37) . Addition of osteonectin protein to culture medium of carcinoma cells stimulated cell migration and invasion (38 , 39) . Antisense RNA down-regulation of osteonectin transcripts in melanoma cells reduced the invasive and adhesive capacities in vitro and inhibited tumor formation in vivo (40) . The loss of aggressive growth properties was associated with loss of matrix metalloproteinase-2 expression. Osteonectin can activate matrix metalloproteinase-2 in invasive breast cancer cells (41) . Thus, osteonectin may be involved in cell-matrix interactions during tissue remodeling, morphogenesis, migration, and proliferation by acting as an antiadhesive molecule stimulating invasion; it may also modulate angiogenesis (42) .
VGP melanoma cells have acquired the ability to invade the dermis. This process requires separation of the melanoma cells from neighboring keratinocytes, attachment to the basement membrane, proteolytic degradation of the basal lamina, and proliferation in the dermis. The induction of osteonectin during this phase is expected to provide the cells with the appropriate matrix to migrate on.
Our finding that osteonectin inhibits vitronectin-mediated binding of melanoma cells suggests that the melanoma cells can readily detach from substrate despite expression of the vitronectin receptor vß3. Because melanoma cells also secrete vitronectin (43)
, it appears that the secretion of these two molecules establishes a balance of adhesion and counter-adhesion. Similarly, binding of osteonectin to vitronectin can modulate attachment of endothelial cells (44)
. In human glioma cells, expression of osteonectin correlates with the angiogenic potential of the cells (45)
.
It is evident that integrins are involved in adhesion and signaling. Stimulation of integrins triggers intracellular signaling events that can be integrated with those originating from growth factor receptors to organize the cytoskeleton, stimulate MAPK cascades, and regulate gene expression (2 , 33 , 34 , 46) . Ligand binding to integrins can activate protein kinases, in particular, activation and autophosphorylation of the cytoplasmic focal adhesion kinase. ß3 integrin appears to participate in specific signaling events that are poorly delineated. Potentially, there are cell type-specific functions of signaling pathways (47) . An increase in osteonectin protein production that accompanies ß3 integrin expression in RGP melanoma cells is independent of MAPK and PKC, suggesting that other, as yet undefined pathways are important. It is notable that the small GTPase RhoC and osteonectin are up-regulated in highly metastatic melanoma cells, suggesting that enhanced expression of genes involved in ECM assembly (48) are critical. Additional studies will need to establish a link between RhoC signaling and osteonectin expression in melanoma.
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FOOTNOTES |
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1 During this work, R. A. S. was supported by an SSP Award from the University of Queensland and a Queensland Cancer Fund travel grant; B. B. G. is a Queensland Cancer Fund Ph.D. scholar. The Center for Functional and Applied Genomics is a Special Research Center of the Australian Research Council. This work was also Supported by NIH Grants CA-47159 and CA-10815 and the Australian NHMRC-102565. 
2 To whom requests for reprints should be addressed, at Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Australia. Phone: 61-7-3365-1831; Fax: 61-7-3365-4388; E-mail: R.Sturm{at}imb.uq.edu.au
3 The abbreviations used are: RGP, radial growth phase; DP, difference product; mAb, monoclonal antibody; MAPK, mitogen-activated protein kinase; pfu, plaque-forming unit; RDA, representational difference analysis; TBS, Tris-buffered saline; VGP, vertical growth phase; PKC, protein kinase C; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ECM, extracellular matrix.
Received 12/18/00. Accepted 11/ 1/01.
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REFERENCES |
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vß3 integrin and intercellular adhesion molecule-1 expression in cutaneous malignant melanoma lesions. Cancer Res., 57: 1554-1560, 1997.Vß3 integrin in human melanoma cell invasion. Proc. Natl. Acad. Sci. USA, 89: 1557-1561, 1992.v-integrin antibody that blocks integrin function inhibits the development of a human melanoma in nude mice. J. Cell Sci., 108: 2825-2838, 1995.[Abstract]
vß3 promotes M21 melanoma growth in human skin by regulating tumor cell survival. Cancer Res., 59: 2724-2730, 1999.6Aß1 induces CD81-dependent cell motility without engaging the extracellular matrix migration substrate. Mol. Biol. Cell, 8: 2253-2265, 1997.vß3 mediates chemotactic and haptotactic motility in human melanoma cells through different signaling pathways. J. Biol. Chem., 271: 3247-3254, 1996.Vß3. Int. J. Cancer, 91: 300-308, 2001.[Medline]
V integrins and vitronectin in human melanoma cell growth. Cold Spring Harbor Symp. Quant. Biol., 57: 233-240, 1992.[Medline]
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