Cell surface molecules and tumor metastasis: Regulation of metastatic phenotypic diversity

Metastatic tumor cells are characterized by quantitative alterations in cell surface and other properties that confer to these cells their abilities to invade, disseminate, implant, survive and grow at secondary sites. Metastasis is also determined by a variety of host factors that prevent, allow or even stimulate metastatic processes. The emergence of diversified cell subpopulations in malignant tumors insures that some cells will ultimately become highly metastatic, resulting in tumor progression towards characteristics which are the most favorable for survival and growth. Unknown mechanisms appear to stimulate and then to control phenotypic diversification of tumor cell subpopulations. These mechanisms may be altered by genetic (mutational) and/or epigenetic (non-mutational) modifications that individually influence cells within a malignant neoplasm.     

Cyclooxygenase inhibitors modulate NK activities that control metastatic disease

Cyclooxygenase (COX) inhibitors have demonstrated efficacy in models of human cancer but the relevant mechanisms have not all been elucidated. Both Cox-dependent as well as Cox-independent mechanisms have been implicated. Using a syngeneic model of metastatic breast cancer, we have investigated the effect of Cox inhibitors on NK functions that are critical to the control of metastatic disease. NK recognition of target cells is governed by a balance of activating and inhibiting receptors that bind ligands including MHC class I. We now show that treatment of tumor cells with the nonselective COX-1/COX-2 inhibitor indomethacin or the selective COX-2 inhibitor celecoxib leads to decreased expression of the MHC class I molecules L^d and K^d . Downregulated class I expression is associated with concomitant increased sensitivity to NK cell-mediated lysis. Both COX inhibitors limit tumor metastasis and this therapeutic effect is dependent on NK but not T cell function. Antimetastatic activity is also lost in the absence of interferon- (IFN-). Both COX inhibitors also suppress local tumor growth of subcutaneously implanted mammary tumor cells in immune competent Balb/cByJ mice. This therapeutic activity is lost in the absence of either CD4+ or CD8+ T cells, but is not compromised by the loss of NK activity. Thus, the mechanism of tumor inhibition differs in the context of local versus metastatic disease. Taken together, these findings are consistent with a mechanism not previously described, whereby COX inhibitors may relieve MHC-mediated inhibition of NK cytotoxicity leading to recognition and lysis of metastatic tumor cells.     

Differential expression of MHC class II molecules in highly metastatic breast cancer cells is mediated by the regulation of the CIITA transcription Implication of CIITA in tumor and metastasis development

We analyzed the differential gene expression between variants of MDA-MB-435 human breast cancer cell line that share an identical genetic background but have different metastatic ability. The major histocompatibility complex class II was found down-regulated in highly metastatic cells and correlated with MHC transactivator (CIITA) expression. Constitutive CIITA expression observed in poorly metastatic is driven by promoters III and IV of CIITA gene. Conversely, both promoters were ineffective in highly metastatic cells. The MHC class II and CIITA expression was restored in these cells upon stimulation with IFNgamma or by the treatment with a hypomethylating agent. Both treatments induced USF-1 and IRF binding complexes to promoter IV but only IFNgamma induced the binding of 435-Lung2 nuclear proteins to an ARE-1 site at the promoter III. Neither Southern blot nor bisulfite sequencing of promoter IV demonstrated strong hypermethylation of this promoter at the IFNgamma-responsive elements such as GAS, E-box or IRF-1. We suggest that partial or hemimethylation of promoter IV is sufficient to silence the CIITA expression in highly metastatic cells and that this epigenetic mechanism is responsible for the lack of MHC-II expression. Forced CIITA expression restored the MHC-II antigen expression in 435-Lung2 cells and abrogates spontaneous lung metastasis in both SCID and nude mice but also affected the tumorigenicity in nude mice. The increase in NK cell infiltration in nude mice bearing CIITA-tumors correlated with sign of tumor cell apoptosis and the increase in the number of NK cells in the spleens, suggesting that NK cells might be responsible for the observed antitumor activity.     

PTPRN2 and PLCβ1 promote metastatic breast cancer cell migration through PI(4,5)P2‐dependent actin remodeling

Altered abundance of phosphatidyl inositides (PIs) is a feature of cancer. Various PIs mark the identity of diverse membranes in normal and malignant cells. Phosphatidylinositol 4,5‐bisphosphate (PI(4,5)P₂) resides predominantly in the plasma membrane, where it regulates cellular processes by recruiting, activating, or inhibiting proteins at the plasma membrane. We find that PTPRN2 and PLCβ1 enzymatically reduce plasma membrane PI(4,5)P₂ levels in metastatic breast cancer cells through two independent mechanisms. These genes are upregulated in highly metastatic breast cancer cells, and their increased expression associates with human metastatic relapse. Reduction in plasma membrane PI(4,5)P₂ abundance by these enzymes releases the PI(4,5)P₂‐binding protein cofilin from its inactive membrane‐associated state into the cytoplasm where it mediates actin turnover dynamics, thereby enhancing cellular migration and metastatic capacity. Our findings reveal an enzymatic network that regulates metastatic cell migration through lipid‐dependent sequestration of an actin‐remodeling factor.     

Alteration of the metastatic potential of line 1 lung carcinoma cells: opposite effects of class I antigen induction by interferons versus DMSO or gene transfection.

Class I antigens are necessary for the recognition of tumor cells by cytotoxic T lymphocytes (CTL). The line 1 lung carcinoma is a spontaneous murine tumor deficient in class I antigen expression. Consistent with this, line 1 cells are highly metastatic in vivo. We investigated whether increasing class I antigen expression on line 1 cells could alter the metastatic potential of these tumor cells using an in vivo lung metastasis model. We used three methods to induce class I antigen expression on line 1 cells: gene transfection, treatment with dimethyl sulfoxide (DMSO), or treatment with interferon (IFN)-beta or -gamma. We found that line 1 cells expressing a transfected class I gene were significantly less metastatic than parental line 1 cells. DMSO-treated line 1 cells also formed significantly fewer metastases than parental line 1 cells. These results indicate that increased class I antigen expression decreases the metastatic potential of line 1 cells in vivo. However, we did not observe a significant decrease in the number of lung metastases in mice receiving line 1 cells treated with IFN-beta or -gamma, despite high levels of class I antigen expression. Thus, increasing class I antigen expression with IFN has an opposite effect on metastasis from class I antigen expression induced by transfection or DMSO. These results show that the method used to increase class I antigen expression is critical in terms of the in vivo effect observed. To investigate a possible mechanism for the differences observed in vivo between these class I expressing cells, we tested whether IFN alters or blocks susceptibility of line 1 cells to immune effector cells. We found IFN treatment increased the ability of line 1 cells to be recognized by CTL but concomitantly decreased the susceptibility of line 1 cells to NK cell lysis by a non-class I antigen-related mechanism. In contrast, transfected or DMSO-treated line 1 cells which were less metastatic in vivo were susceptible to both CTL and NK-mediated lysis. Taken together, these results suggest that immune intervention against metastasizing line 1 cells may involve NK cells and CTL.     

Characteristics of the NK cells in Syrian hamsters carrying tumors with varying metastatic and resistance-suppressing activity

Experiments in vivo and in vitro were made to study the effects of HETR-MLN-8 and HETR tumor cells differing in metastatic ability and inhibition of the natural host resistance to tumor on cytotoxic activity of NK from Syrian hamsters. Marked inhibition of cytotoxicity and ability for interferon activation was detected in NK isolated from tumors (as compared with blood), with that inhibition being far more pronounced in highly malignant HETR-MLN-8 tumors. This may indicate a direct inhibitory action of the tumor or its products on NK cytotoxicity. The in-vitro competition inhibition test yielded results showing that HETR-MLN-8 cells capable of in-vivo inhibition of the natural host resistance to the tumor also display much more demonstrable ability for in-vitro inhibition of NK cytotoxicity as compared to HETR cells.     

Phospholipase C treatment of certain human target cells reduces their susceptibility to NK lysis without affecting binding or sensitivity to lytic granules.

Phosphatidylinositol-specific phospholipase C (PI-PLC) is an enzyme that has the capacity to release glycosyl-phosphatidyl inositol (G-PI)-anchored proteins from the cells surface. Pretreatment of the human T-cell leukemia cell line Molt-4 with PI-PLC resulted in a decrease in the susceptibility to lysis by natural killer (NK) cells. Treatment of the erythroleukemia cell line K562 with PI-PLC had no effect on its NK susceptibility. PI-PLC-treated and untreated Molt-4 bound equally well to lymphocytes in target-binding studies with effector cell preparations enriched for NK cells. Susceptibility to cytolytic granules isolated from rat LGL tumor cells remained the same after treatment of Molt-4 or K562 with PI-PLC. Combined treatment of Molt-4 with PI-PLC and rlFN-alpha or rlFN-gamma resulted in additive reductions of the NK susceptibility, suggesting that PI-PLC and interferons act on different mechanisms to protect cells from NK lysis. When expression of a number of antigens on Molt-4 and K562 was analyzed in flow cytometry, only the expression of CD58 was reduced after PI-PLC treatment. The susceptibility of Con A blasts to MLR derived cytotoxic T-cells was not altered by treatment with phospholipase. These data suggest that PI-PLC treatment reduces the capacity of some target cells to activate NK cells upon contract. The mechanism behind this phenomenon is presently unclear.     

Transglutaminases: key regulators of cancer metastasis

The ability to metastasize represents the most important characteristic of malignant tumors. The biological details of the metastatic process remain somewhat unknown, due to difficulties in studying tumor cell behaviour with high spatial and temporal resolution in vivo. Several lines of evidence involve transglutaminases (TGs) in the key stages of tumor progression cascade, even though the molecular mechanisms remain controversial. TG expression and activity display a different role in the primary tumor or in metastatic cells. In fact, TG expression is low in the primary tumor mass, but augmented when cells acquire the metastatic phenotype. Nevertheless, in other cases, the use of inducers of TG transamidating activity seems to contrast tumor cell plasticity, migration and invasion. In the following review, the function of TGs in cancer cell migration into the extracellular matrix, adhesion to the capillary endothelium and its basement membrane, invasion and angiogenesis is discussed.     

A human NK cell activation/inhibition threshold allows small changes in the target cell surface phenotype to dramatically alter susceptibility to NK cells

NK cell activation is negatively regulated by the expression of target cell MHC class I molecules. We show that this relationship is nonlinear due to an NK cell activation/inhibition threshold. Ewing's sarcoma family tumor cell monolayers, which were highly susceptible to NK cells in vitro, developed a highly resistant phenotype when cultured as three-dimensional multicellular tumor spheroid structures. This suggested that tumor architecture is likely to influence the susceptibility to NK cells in vivo. Resistance of the multicellular tumor spheroid was associated with the increased expression of MHC class I molecules and greatly reduced NK cell activation, implying that a threshold of NK cell activation/inhibition had been crossed. Reducing MHC class I expression on Ewing's sarcoma family tumor monolayers did not alter their susceptibility to NK cells, whereas increased expression of MHC class I rendered them resistant and allowed the threshold point to be identified. This threshold, as defined by MHC class I expression, was predictive of the number of NK-resistant target cells within a population. A threshold permits modest changes in the target cell surface phenotype to profoundly alter the susceptibility to NK cells. Whereas this allows for the efficient detection of target cells, it also provides a route for pathogens and tumors to evade NK cell attack.     

Laminin synthesis by NK cells and modulation of its expression by TPA (12-O-tetradecanoylphorbol-13-acetate)

Natural killer (NK) cells have been suggested to play a major role in resistance against metastatic spread of tumors. This study was aimed at understanding whether laminin (LM), a component of the extracellular matrix involved in the mechanism of tumor invasion and cell interaction, is expressed by NK cells. The results indicate that NK cells can synthesize and display on the cell surface LM and that TPA can modulate its expression. Our findings suggest that the presence of LM on NK cells could be relevant in the control of tumor invasion by NK cells.     

Laminin synthesis by NK cells and modulation of its expression by TPA (12-O-tetradecanoylphorbol-13-acetate)

Natural killer (NK) cells have been suggested to play a major role in resistance against metastatic spread of tumors. This study was aimed at understanding whether laminin (LM), a component of the extracellular matrix involved in the mechanism of tumor invasion and cell interaction, is expressed by NK cells. The results indicate that NK cells can synthesize and display on the cell surface LM and that TPA can modulate its expression. Our findings suggest that the presence of LM on NK cells could be relevant in the control of tumor invasion by NK cells.     

Alterations in Fas expression are characteristic of,but not solely responsible for,enhanced metastatic competence

Dysregulation of the Fas pathway has been implicated in tumor progression; however, how alterations in Fas expression influence metastatic behavior remains unresolved. In this study, we investigated the link between Fas expression and metastatic capacity in two mouse tumor models: one was a sarcoma, which was used to analyze the consequences of loss of Fas function in experimental pulmonary metastases, and the other was a mammary carcinoma, where Fas expression was examined in matched pairs of primary and metastatic cell lines as well as by immunohistochemistry of tissues taken from primary and metastatic sites of spontaneous tumor development. In the sarcoma model, a Fas-resistant/refractory subline was produced in vitro from the parental line by biologic selection against Fas-responsive cells, and it was then compared with the poorly metastatic parental line and to an in vivo-derived subline that was highly metastatic for growth in the lungs. In both tumor models, an inverse correlation was demonstrated between Fas expression and metastatic phenotype. Subsequent studies in the sarcoma model revealed that although the Fas-resistant/refractory subline displayed significant metastatic ability, the parental line from which it was derived exhibited little to no additional metastatic activity if experimentally rendered Fas-resistant by molecular-based strategies or transplanted into a Fas ligand-deficient host. Therefore, these findings suggested that down-regulation of Fas was associated with the metastatic phenotype, but alterations in Fas expression alone were insufficient for acquisition of full metastatic potential. Rather, the ability of such Fas-resistant neoplastic subpopulations to achieve metastatic competence apparently required co-possession of additional malignant characteristics.     

GPI/AMF inhibition blocks the development of the metastatic phenotype of mature multi-cellular tumor spheroids

Epithelial–mesenchymal transition (EMT) and cellular invasiveness are two pivotal processes for the development of metastatic tumor phenotypes. The metastatic profile of non-metastatic MCF-7 cells growing as multi-cellular tumor microspheroids (MCTSs) was analyzed by determining the contents of the EMT, invasive and migratory proteins, as well as their migration and invasiveness potential and capacity to secrete active cytokines such as the glucose phosphate isomerase/AMF (GPI/AMF). As for the control, the same analysis was also performed in MCF-7 and MDA-MB-231 (highly metastatic, MDA) monolayer cells, and in stage IIIB and IV human metastatic breast biopsies. The proliferative cell layers (PRL) of mature MCF-7 MCTSs, MDA monolayer cells and metastatic biopsies exhibited increased cellular contents (2–15 times) of EMT (β-catenin, SNAIL), migratory (vimentin, cytokeratin, and fibronectin) and invasive (MMP-1, VEGF) proteins versus MCF-7 monolayer cells, quiescent cell layers of mature MCF-7 MCTS and non-metastatic breast biopsies. The increase in metastatic proteins correlated with substantially elevated cellular abilities for migration (18-times) and invasiveness (13-times) and with the higher level (6-times) of the cytokine GPI/AMF in the extracellular medium of PRL, as compared to MCF-7 monolayer cells. Interestingly, the addition of the GPI/AMF inhibitors erythrose-4-phosphate or 6-phosphogluconate at micromolar doses significantly decreased its extracellular activity (> 80%), with a concomitant diminution in the metastatic protein content and migratory tumor cell capacity, and with no inhibitory effect on tumor lactate production or toxicity on 3T3 mouse fibroblasts. The present findings provide new insights into the discovery of metabolic inhibitors to be used as complementary therapy against metastatic and aggressive tumors.     

Differential expression of tissue factor and tissue factor pathway inhibitor in metastatic melanoma lesions

Tissue factor (TF) is involved in tumor progression and metastatic potency in some malignant tumors and its function is regulated by tissue factor pathway inhibitor (TFPI) therefore the interaction of both molecules is crucial for their functional role. We evaluated the clinical relevance of TF and TFPI expression in benign and malignant melanocytic lesions. Expression of both was examined by immunoperoxidase staining using serial tissue sections in 16 nevi, 34 primary and 15 metastatic melanoma lesions. TF and TFPI were ubiquitously expressed in benign and malignant melanocytic lesions. This finding was confirmed by Western blot analysis using cultured human melanocytes, nevi cells (NCN) and melanoma cell lines. Although TF expression was not associated with malignant transformation and disease progression, TFPI expression in primary and metastatic melanoma lesions was significantly lower and weaker than that in nevi lesions in terms of intensity and percentage of stained cells. In addition, TFPI expression in metastatic lesions was significantly lower and weaker than that of TF. These results suggest that the relative expression of TF to TFPI may play a crucial role in the malignant transformation and metastatic potency in melanocytic cells.     

H-2Kb antigen expression has no effect on natural killer susceptibility and tumorigenicity of a murine hepatoma

Recent reports suggested a correlation between decreased expression of tumor cell MHC class I Ag and increased susceptibility to NK cells. These studies led to the hypothesis that tumor cells displaying reduced levels of MHC class I Ag have reduced tumorigenicity in vivo because they are eliminated from the host by endogenous NK cells. The present studies use the murine hepatoma BW7756 and a spontaneous H-2Kb loss variant, Hepa-1, to test this hypothesis. The parental BW7756 tumor is highly malignant in syngeneic C57L/J hosts while Hepa-1 cells do not give rise to tumors, suggesting that the loss of H-2Kb Ag expression correlates with decreased tumorigenicity and NK susceptibility. Hepa-1 cells were therefore transfected with an H-2Kb gene to generate H-2Kb Ag expressing clones. The resulting clones were tested for tumorigenicity. Syngeneic or NK-deficient C57BL/6-beige/beige mice challenged with Hepa-1 or the H-2Kb transfectants rejected the cells, suggesting that reexpression of H-2Kb Ag does not restore tumorigenicity and that NK cells are not involved in Hepa-1 rejection. In vitro H-2Kb Ag-negative and -positive Hepa-1 cells are equally susceptible to tilorone-boosted NK cells, indicating that MHC class I Ag expression also does not affect in vitro NK susceptibility. Tumor challenged athymic nude and sublethally irradiated syngeneic mice develop tumors demonstrating that T cells are probably responsible for rejection of the Hepa-1 tumor, and that H-2Kb Ag expression has no effect on rejection. Inasmuch as the expression of H-2Kb Ag on Hepa-1 cells does not effect tumorigenicity or in vitro NK susceptibility, the previously reported association between reduced MHC class I Ag levels and increased NK susceptibility is not universally applicable.     

Expression of the alpha7beta1 laminin receptor suppresses melanoma growth and metastatic potential.

The alpha7beta1 integrin is a laminin-binding receptor that was originally identified in melanoma. Here, we show that, in clonally derived mouse K1735 melanoma variant cell lines with high (M-2) and low (C-23) metastatic potential, elevated expression of alpha7 correlates with reduced cell motility, metastasis, and tumor growth. Both cell lines showed similar beta1 integrin-dependent adhesion to laminin-1 and the E8 laminin fragment. However, the highly metastatic M-2 cells rapidly migrated on laminin, whereas the nonmetastatic C-23 cells were minimally motile. Laminin-binding integrin profiles showed that the M-2 cells expressed moderate amounts of alpha1 and abundant alpha6 but low or undetectable levels of alpha2 and alpha7. By contrast, C-23 cells expressed low or undetectable levels of alpha1, alpha2, and alpha6 but had up-regulated levels of alpha7. Consistent with the protein data, Northern blot analysis showed that levels of alpha7 mRNA were highest in the poorly metastatic variant cells, whereas alpha6 message was not detected; in contrast, alpha6 mRNA was elevated in the highly metastatic cells, whereas alpha7 message was not detected. Forced expression of alpha7 in the M-2 cells suppressed cell motility, tumor growth, and metastasis. Collectively, these results indicate that, during melanoma progression, acquisition of a highly tumorigenic and metastatic melanoma phenotype is associated with loss of the alpha7beta1 laminin receptor.     

Analysis of the mechanisms involved in NK resistance induced by a new tumor factor NK-RIF

The mechanisms involved in susceptibility or resistance of neoplasic cells to lysis by NK cells are not well known. We have recently described a 12-kDa factor (NK-RIF), produced and released by different tumor cell lines, making K562 resistant to NK lysis without affecting the cytotoxic function of NK effector cells. In this paper we further study the mechanism involved in NK resistance of K562 mediated by NK-RIF and its biological implications. The results show that NK-RIF does not affect the binding capacity of target and effector cells nor the levels of HLA class I antigen expression on the target cells, as a proof that resistance to NK-mediated lysis is not always associated with a defect in target effector binding or with an increased MHC class I antigen expression. However NK-RIF-treated K562 loses its capacity to induce NK cell activation and the subsequent capacity to release NKCF and makes K562 resistant to lysis by NKCF. Therefore our results show that induction of resistance to NK cytotoxicity can be the result of the modulation of target structures responsible for inducing effector cell activation without affecting target/effector binding molecules. This indicates that the structures involved in adherence and activation of NK cells have a different nature and that molecules other than HLA participate in NK resistance.     

Beta-actin in human colon adenocarcinoma cell lines with different metastatic potential

Human colon adenocarcinoma LS180 parental cell line and selected variants, characterized by different metastatic capacity were used to examine, whether a correlation exists between beta-actin expression, its subcellular distribution and metastatic potential of these cells. Cytosolic fraction (supernatant 105000 x g), isolated from the tumor cells was used as a source for actin quantification. The higher level of beta-actin was observed in the cytosol of three selected sublines to compare with LS180 parental line. Statistically significant increase of beta-actin level in highly motile EB3 cells variant should be underlined to compare with the other sublines. Distinct differences in the phenotype of adenocarcinoma cell variants were found, such as the changes in cells shape, cells spreading and ability to attach to the surface of culture dish. Actin cytoskeleton was visualized with fluorescence microscopy application and microfilaments rhodamine-conjugated phalloidin staining. beta-actin subcellular localization was done by immunofluorescence staining with monoclonal anti-beta actin antibodies. In the elongated cells (LS180, 3LNLN), this isoactin is dispersed in the whole cell body and concentrates in pseudopods and at the leading edges, when in the rounded variant (EB3) beta-actin dominates mainly in cortical ring under cellular membrane and it is also seen in the subtle protrusions. Summary of our former (Nowak et al., 2002, Acta Biochim. Polon., 49: 823) and current data lead to the conclusion that there is a distinct correlation between metastatic capacity of examined human colon adenocarcinoma cells, the state of actin polymerization, actin cytoskeleton organization and beta-actin expression.     

Diversification and progression of malignant tumors

Tumor-cell diversification mechanisms insure that malignant neoplasms contain diversified tumor-cell subpopulations. Because of the instability of tumor cell phenotypes, some malignant cells will evolve with the most favorable properties for their progression to highly metastatic cells. The rates of cellular phenotypic diversification vary greatly among different tumors, and they are probably modulated, in part, by genetic and chromosome defects and by epigenetic events that may vary widely depending upon the nature of the tumor cells and their microenvironments. As tumor diversification and selection proceed, the most malignant cell subpopulations may eventually become dominant and gradually lose their microenvironmental responsiveness. Tumor-cell diversification mechanisms may be similar or identical to normal, developmentally regulated diversification mechanisms that are used during embryonic cell diversification and differentiation.     

Contribution of alpha-D-galactopyranosyl end groups to attachment of highly and low metastatic murine fibrosarcoma cells to various substrates

There are much greater numbers of cell surface terminal, non-reducing alpha-D-galactorpyranosyl groups in highly malignant (metastatic) cells than are found in low malignant cells derived from the same murine fibrosarcoma. We have examined the contribution of these residues to attachment of the cells to various collagens and to plastic. Removal of these carbohydrate groups with alpha-galactosidase or blocking them with lectins from Griffonia simplicifolia seeds or with anti-blood group B antiserum all dramatically inhibited the attachment of both the highly malignant and the low malignant cells. Following removal with the enzyme, the alpha-D-galactopyranosyl end groups were rapidly resynthesized. This resynthesis was inhibited by tunicamycin, an inhibitor of de novo glycoprotein synthesis. This antibiotic also impaired cell attachment and, when used in addition to treatment with alpha-galactosidase, it inhibited cell attachment more than did treatment with the enzyme alone. The effects of all treatments on cell attachment were greater for the highly malignant than for the low malignant cells. With the latter cells, inhibition by lectin was seen only in the absence of serum, whereas the adhesion of highly malignant cells was affected in both the presence and the absence of serum. On their surface membrane the highly malignant cells express much more than do the low malignant cells of a glycoprotein that cross-reacts immunologically with laminin. The basement membrane glycoprotein laminin promotes cell attachment to collagen, and both glycoproteins contain terminal, non-reducing alpha-D-galactopyranosyl groups. Attachment of cells is a requirement for the formation of a metastasis, and thus the laminin-like molecule and the alpha-D-galactopyranosyl end groups (whether on the laminin-related moiety or on other cell surface molecules) may both be important for expression of the most malignant phenotype.