Acquisition of anchorage-independent growth is a hallmark of neoplastic transformation, but this property is not considered to be carcinogenic per se. In this work, we show that repetitive adhesion blockade events are sufficient to induce malignant transformation in nontumorigenic immortalized melanocytes (melan-a). Although we cannot exclude clonal selection as the principal mechanism responsible for tumorigenic conversion, several results indicate that adhesion impediment may trigger the carcinogenic process. All tumorigenic cell lines derived from melan-a have faster doubling times than their parental lineage (), but even after 80 in vitro
passages, melan-a cells were never tumorigenic in vivo
(data not shown). No negative influence of melan-a over its derived subline proliferation rate could be demonstrated either in vitro
(not shown) or in vivo
]. In addition, cell clones obtained after limiting the dilution of melan-a cells were not tumorigenic in vivo
, suggesting that transformed cells are not initially present in the parental cell line (not shown) and that repeated adhesion blockade is important to induce malignant phenotype. Epigenetic modifications are the most probable candidates to explain the myriad of morphologic and molecular alterations observed after repeated deadhesion cycles, and preliminary results from ongoing experiments in our laboratory indicate that such is the case, indeed.
Stressful conditions resulting from cell-cell and cell-substrate adhesion modifications have previously been associated with malignant transformation. Rubin [27
] has consistently shown that high-density cultures yield tumorigenic clones, which he attributes to clonal selection, without excluding a direct effect of adhesion alterations in the carcinogenic process. Another group has shown that the forced anchorage-independent growth of a nontumorigenic, immortalized epithelial cell line resulted in the acquisition of an anoikis-resistant phenotype and in tumorigenesis [28
]. As demonstrated by Zhu et al. [29
], selected anoikis-resistant melanoma cells showed increased metastatic potential and multiple alterations in their phenotypic properties. Diaz-Montero and McIntyre [30
] obtained anoikis resistance osteosarcoma sublines through modifications of culture conditions, attributing this phenotype to epigenetic events. Ongoing research in our laboratory has shown that adhesion alterations can also induce malignant transformation in the fibroblast cell line NIH 3T3, implying adhesion loss as a crucial factor for carcinogenesis in different cell types.
Interestingly, the proportion of spheroid formation for each cell line (melan-a and derived lineages; ) resembles the percentage of soft agar cell growth from different phases of melanocyte transformation [31
], where melan-a corresponds to dysplastic lesions and Tm melanoma cells correspond to invasive melanoma. These results reinforce the idea that the cell lines utilized in this work truly represent a continuum along the transformation process.
Integrins, which are the most important class of adhesion molecules related to ECM interactions, have been shown in most cellular types to impart survival signals on adhesion to substrate and surface clustering [32,33
]. However, Lewis et al. [34
] showed that loss of integrin-mediated cell adhesion may abrogate cell death in cells submitted to DNA damage, through p19Arf
and p53 signaling. In melan-a-derived cell lines, no modification of β1
, and αv
integrin expression was detected (not shown). Nevertheless, an electrophoretic migration shift was observed for β1
integrin chain (), which has been demonstrated by several groups, including ours, to be related to an aberrant N-glycosylation pattern [35,36
]. This alteration, caused by GnT-V overexpression, is associated with acquisition of migratory phenotype and tumor progression [37
]. The forced expression of GnT-V results in decreased fibronectin attachment of colon carcinoma cells [38
] possibly caused by aberrant β1
integrin chain glycosylation [35
], but this same, the glycosylation pattern was associated with increased fibronectin adhesion () in the melanoma cell lines presented here.
Although GnT-V expression was not investigated, a marked increase of its products (GlcNAcβ1,6Manα1,6-branched surface molecules) was demonstrated for all melan-a-derived cell lines in a progressive manner during the transformation process (, A and B). A qualitative alteration in the N-glycosylation protein profile was demonstrated for melanoma cells derived from melan-a (). The above studies and our findings suggest that GnT-V activity is causally associated with malignant transformation because melan-a cells submitted to sequential deadhesion cycles (but not yet tumorigenic) already show augmented levels of tri-antennary and tetra-antennary N-glycan products.
Even though it is quite clear that several tumors accumulate glycoproteins bearing β1-6-branched N-linked oligosaccharides recognized by L-PHA, the precise function of this altered pattern of glycosylation in glycoprotein function remains elusive. An interesting hypothesis considering GnT-V as a transforming enzyme was proposed by Dennis et al. [39
], who had initially shown that its forced expression in a nontumorigenic cell line could convert those cells capable of forming tumors in nude mice [40
]. Based on more recent studies, Morgan et al. [41
] proposed that growth factor and cytokine receptors, which are also modified by GnT-V, exist as lattices on the cell surface. Maintenance of these lattices would depend on extracellular glycan-binding proteins, such as galectins. Complexes of glycoproteins and lectins would render cells more sensitive to growth factors, whose interaction with their cognate receptors usually lead to receptor dimerization/oligomerization. If correct, the prediction is that L-PHA binding correlates with autonomous growth. Integrins [35,42,43
] and cadherins [44,45
] are also substrates of GnT-V. Similarly to integrins, no differences in cadherin expression were detected by serial analysis of gene expression [46
], RT-PCR, or Western blot analysis (data not shown). The higher adhesiveness of tumorigenic cell lines (4C11 and Tm5), both to fibronectin and laminin (, B
), could be attributed to this aberrant glycosylation pattern present in β1
integrin chains. However, the true impact of this pattern of glycosylation in this model warrants further investigation.
PGs, another class of surface molecules, are also involved in neoplastic transformation in several cell types. HS and CS have been particularly implicated in tumor formation, including melanoma, because of their capacity to bind and modulate a large number of molecules that are important for tumor development, such as basic fibroblast growth factor and vascular endothelial growth factor [47
]. HS may either promote or inhibit tumor progression, depending on heparan fragments generated on digestion [48
]. In our model, the expression of HS was significantly decreased after transformation, although no structural modifications were discernible (; ). In parallel, an increased expression of heparanase was demonstrated not only in melan-a-derived melanoma cell lines, but also in melan-a maintained in suspension for 24 hours, indicating that heparanase may contribute to early changes involved in melan-a transformation. Using a different approach for the detection of heparanase in different melanoma cell lines, an increase in enzymatic activity was related to a higher metastatic phenotype [49
]. However, CS/DS levels were similar in both nontumorigenic and tumorigenic cell lines, but their disaccharide composition was clearly different from the parental cell lineage (; ). It is clear that transformation leads to an increase in the relative amounts of α-L
-iduronic acid, suggesting a possible upregulation of the β-D
-glucuronic acid C-5-epimerase. Furthermore, during transformation, the glucuronic acid-containing disaccharides show the presence of N
-sulfate, contrasting with the parental line that bears only N
-sulfate, indicating that malignant transformation leads to the expression of N
-sulfotransferase, as previously observed in brain tumors [50
In addition, the protein core expression levels of some PG subclasses were determined by semiquantitative RT-PCR. As shown in , perlecan levels increase during transformation, decorin and versican levels decrease, and syndecan-4 level does not change. Among extracellular PGs, decorin has emerged as an inhibitor of tumor progression, whereas perlecan seems to be a promoter of this process [51,52
]. Perlecan apparently supports the growth and invasion of tumor cells through its ability to store angiogenic factors [53
]. This last observation also corroborates our hypothesis that melan-a-derived melanoma cell lines were not selected by multiple cycles of adhesion blockade because perlecan increased during the transformation process even in the absence of a selective pressure related to angiogenesis.
Our model of melanocyte carcinogenesis allows the identification of morphologic and molecular alterations that precede full malignant transformation and reinforce micro-environmental role as a transforming factor, particularly the loss of cell-substrate adhesion.