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Ganglioside GD3 is highly expressed in human melanomas and enhances malignant properties of melanomas, such as cell proliferation and invasion activity. In this study, we analyzed the effects of GD3 expression on cell signals triggered by hepatocyte growth factor (HGF)/Met interaction and by adhesion to collagen type I (CL-I). Although stimulation of melanoma N1 cells (GD3+ and GD3−) with either HGF or adhesion to CL-I did not show marked differences in the phosphorylation levels of Akt at Ser473 and Thr308 between two types of cells, simultaneous treatment resulted in definite and markedly increased activation of Akt in GD3+ cells. Similar increases were also shown in Erk1/2 phosphorylation levels with the costimulation in GD3+ cells. When resistance to induced apoptosis by H2O2 was examined, only GD3+ cells treated with both HGF and adhesion to CL-I showed clearly low percentages of dead cells compared with GD3− cells or GD3+ cells treated with either one of the stimulants. Cell growth measured by 5-ethynyl-2‘ deoxyuridine uptake also showed synergistic effects in GD3+ cells. These results suggested that GD3 plays a crucial role in the convergence of multiple signals, leading to the synergistic effects of those signals on malignant properties of melanomas.
Although cancer cells generally obtain the ability to autonomously proliferate based on genetic changes in the key molecules involved in the regulation of cell growth, they are also supported by extrinsic signaling derived from growth factors and cell adhesion. It has been reported that multiple signals from environmental factors such as tumor niche is essential for the survival and proliferation for melanoma cells.[2, 3] Their importance increases particularly in cancer progression and cancer metastasis. Recently, a number of studies have been reported to indicate that multiple signaling pathways are present in melanoma cells, leading to the activation of common essential molecular systems involved in cell growth, such as Erks and Akt. Thus, blocking one of those pathways with molecular targeting drugs failed to suppress melanoma cell growth, indicating the prominent roles of particular signals, such as HGF/Met-mediated signaling.[7-9]
Gangliosides are expressed in neuroectoderm-derived human cancers such as malignant melanomas and neuroblastomas, and have been used as cancer-associated markers and/or targets of antibody therapies.[11, 12] Recently, there have been increasing reports implicating gangliosides in the malignant properties of various cancer cells.[13-15] Since we isolated ganglioside synthase cDNAs such as GM2/GD2 synthase, GD3 synthase, GM1 synthase and GM3 synthase, the roles of gangliosides in cancer cells have been analyzed based on the genetic manipulation of those genes.
In particular, disialyl gangliosides have been recognized as functional cell surface molecules in the enhancement of cell proliferation, migration, and metastasis. Ganglioside GD3 is expressed in the majority of human melanoma tissues and cell lines, enhanced cell proliferation and invasion, and cell adhesion to ECMs. Consequently, GD3 appears to converge two kinds of cell signals, that is, integrin-mediated adhesion signals and growth factor/receptor-mediated signals. Either one of them could not transduce sufficient signaling without the other.[22, 23] In these studies, we have used serum stimulation as a representative growth signal with no definite identification of particular growth factors.
In this study, we used the hepatocyte growth factor (HGF)/Met system as a representative growth factor/receptor system. However, HGF/Met-mediated signaling was not simply enhanced by GD3 expression. Only when HGF was used for stimulation together with adhesion to ECM, definitely increased signaling as well as correspondingly increased tumor phenotypes could be observed in only GD3-expressing cells, indicating that GD3 plays a key role in the synergism of the two major signals.
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Ganglioside GD3 is highly and specifically expressed in melanomas.[21, 25] To investigate the roles of GD3, we established a set of melanoma cell lines with or without GD3 expression using a GD3-deficient mutant, N1. Re-expression of GD3 with GD3 synthase cDNA resulted in the establishment of cell lines, which enabled us to identify phenotypic changes due to GD3 expression, and to investigate the molecular mechanisms of the enhanced malignant properties under GD3 expression.
From past data, induced gangliosides by GD3 synthase cDNA in N1 cells were almost exclusively GD3,[15, 27] and sometimes a very faint GD2 could be seen in thin-layer chromatography or flow cytometry. No other components could be seen. Therefore, the effects shown in the GD3 synthase transfectant cells should be due to GD3 itself. We have reported that GD3 is involved in malignant properties, such as cell growth and invasion and cell adhesion to the ECM.
Strong phosphorylation of FAK, p130Cas, paxillin, Akt, and Erks after treatment with FCS was observed[15, 27] in GD3+ cells. Increased phosphorylation of FAK and paxillin was also observed in GD3+ cells compared with those in GD3− cells during adhesion to CL-I. Although increased phosphorylation of Akt was also observed during adhesion to CL-I, the relative intensity in the phosphorylation of Akt was weaker than that with FCS stimulation, and the relative intensity of bands of phosphorylated Erk1/2 during adhesion to CL-I was not different between GD3+ cells and GD3− cells.
In this study, we showed the synergistic effects of adhesion- and growth factor/receptor-mediated stimulations on the activation of Akt and Erks in GD3+ cells. Furthermore, the suppressive effects of combined stimulation with HGF and adhesion to CL-I on induced apoptosis and the enhancing effects on cell growth were also observed. Although we have used FCS to examine growth factor signals in melanoma cells,[15, 24, 27] we focused on HGF as a growth factor in this study because HGF receptor Met was overexpressed in melanomas and overexpression of Met in human melanoma tissues was correlated with a poor clinical outcome of patients. Upregulation of Met expression enabled HGF to protect melanoma cells from apoptosis. Moreover, HGF upregulated the expression of CD44v6, and induced the expression of fibronectin in melanoma cells.
We first examined the effect of HGF on enhancement of signaling in GD3+ cells because HGF activates the MAPK and Akt signaling pathways in human melanoma cells, as mentioned above. Although the phosphorylation levels of Met and Akt were increased depending on the concentration of added HGF, the phosphorylation levels of Met were not affected by GD3 expression in N1 cells. These results were quite different from those of breast cancer cell lines, in which GD2 expression induced cell growth due to constitutive activation of Met regardless of the presence of HGF. They showed increased phosphorylation of Akt and Erks. In contrast, constitutive Met activation without HGF was not detected in either GD3-expressing N1 cells or GD2-expressing N1 cells (data not shown). The phosphorylation levels of Akt and Erks with HGF were also not affected by GD3 expression in a melanoma cell line.
We then tried to elucidate whether HGF stimulation could enhance phosphorylation levels of Akt or Erks during cell adhesion to CL-I more strongly in GD3+ cells than in GD3− cells. Phosphorylation levels of Ser473 and Thr308 in Akt were higher in GD3+ cells than in GD3− cells during the adhesion to CL-I, while the phosphorylation levels were very low (Fig. 3B). Surprisingly, phosphorylation levels of Akt were increased remarkably and definitely in GD3+ cells compared with those in GD3− cells during costimulation with HGF and adhesion to CL-I (Fig. 3C). Phosphorylation levels of Erks showed similar results (Fig. 4). These results suggested that phosphorylation of Akt and Erks was synergistically enhanced by costimulation with HGF and adhesion to CL-I in GD3+ N1 cells.
As strong phosphorylation of Akt and Erks in GD3+ cells after costimulation with HGF and adhesion to CL-I was observed, we questioned whether this costimulation affects tumor phenotype, such as resistance to apoptosis and/or promotion of cell proliferation in GD3+ cells. To examine the effects of costimulation with HGF and adhesion to CL-I on apoptosis, we treated GD3+ and GD3− cells with H2O2 after stimulation with either adhesion to CL-I or HGF treatment, or both. In the case of simultaneous stimulation with HGF and CL-I, the percentage of apoptotic cells in total cells was significantly lower in GD3+ cells than in GD3− cells (Fig. 7B). We also examined the effects of simultaneous stimulation with HGF and CL-I on cell proliferation. The percentage of EdU positive cells was approximately two fold higher in GD3+ cells than in GD3− cells after 21 h of costimulation (Fig. 8B).
These results suggested that GD3 expression enhanced activation of Akt and Erks mainly when cells were costimulated with HGF and adhesion to CL-I, based on the convergence of two signaling pathways. Consequently, converged signals might bring about increased resistance to apoptosis and promotion of cell proliferation by synergistic effects of adhesion- and HGF/Met-mediated signaling.
Hepatocyte growth factor appeared to play a key role in the regulation of interaction between melanoma and its microenvironment. The growth- and metastasis-promoting effects of the microenvironment have been documented,[4, 5] and clustered fibroblasts in tumor cell-conditioned medium produced a large amount of HGF. A recent report has shown that stromal cell secretion of HGF in the tumor microenvironment resulted in activation of HGF receptor Met, and of MAPK and phosphatidylinositol-3-OH kinase–Akt signaling pathways in human melanoma cells. It led to immediate resistance to RAF inhibition treatment in BRAF-mutant melanomas,[6, 35] and the immunohistochemistry experiments revealed stromal cell expression of HGF in patients with BRAF-mutant melanoma.[6, 35] Of course, increased secretion of HGF from melanoma cells themselves might also be involved in the formation of the tumor microenvironment and progression of malignancy.
Our results indicated that GD3 expression in melanomas enhances the MAPK and Akt signaling pathways by costimulation with HGF and adhesion to CL-I, and eventually enhances the resistance to apoptosis and/or cell proliferation. Together with the reports described above,[4-6, 35] it is supposed that GD3 expression enhances cell adhesion to the environmental connective tissues and raises the sensitivity to HGF supplied from the tumor microenvironment, resulting in the convergence of multiple extrinsic signals in tumor tissues. These effects represent the driving source of GD3 expression for the development of micrometastases in the microenvironment of a secondary organ after the detachment of primary tumor cells, and confer high metastatic properties of melanomas.
Next question may be if GD3 itself is involved in the convergence of multiple extrinsic signals. As we previously reported,[15, 27] only GD3 is highly and constantly synthesized in GD3 synthase cDNA transfectant cells. Moreover, growth suppression of a melanoma cell line with anti-GD3-specific mAb R24 was reported by us. The fact that treatment of melanoma cells with mAb R24 could reduce the phosphorylation levels caused by costimulation of HGF and adhesion to CL-I suggests that GD3 itself can induce such synergistic effects, as shown in this study.
We reported previously that integrins were partially localized in membrane lipid rafts of GD3+ cells under cell suspension, resulting in the rapid activation of signal molecules on cell adhesion to the ECM. In this study, we showed that HGF/Met stimulation enhances phosphorylation levels of Akt and Erks in GD3+ cells during cell adhesion to CL-I. Downstream molecules of the CL-I/integrin-mediated signaling and HGF/Met-mediated signaling pathways are common, as shown in Figure 9. These common signal molecules may be shared and converged in the vicinity of cell membranes, leading to synergistic enhancement of the signaling. What kinds of molecules are physically associated with GD3 on the membrane, and how the convergence of multiple signals are brought about under GD3 remain to be investigated.