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Brain tumor growth and progression is dependent upon vascularity, and is associated with altered ganglioside composition and distribution. In this study, we examined the influence of gangliosides on growth and vascularity in a malignant mouse astrocytoma, CT-2A. Ganglioside distribution was altered in CT-2A tumor cells using an antisense construct to β-1,4-N-acetylgalactosaminyltransferase (GalNAc-T), a key enzyme that uses the simple ganglioside GM3 as a substrate for the synthesis of the more complex gangliosides, GM2, GM1 and GD1a. GalNAc-T gene expression was significantly lower in CT-2A cells stably transfected with the antisense GalNAc-T plasmid, pcDNA3.1/TNG (CT-2A/TNG) than in either non-transfected CT-2A or mock-transfected (CT-2A/V) control tumor cells. GM3 was elevated from 16% to 58% of the total ganglioside distribution, whereas GM1 and GD1a were reduced from 17% and 49% to 10% and 17%, respectively, in CT-2A/TNG tumor cells. Growth, vascularity (blood vessel density and Matrigel assay) and vascular endothelial growth factor (VEGF) expression was significantly less in CT-2A/TNG tumors than in control CT-2A brain tumors. In addition, the expression of VEGF, hypoxia-inducible factor 1α (HIF-1α) and neuropilin-1 (NP-1) was significantly lower in CT-2A/TNG tumor cells than in control CT-2A tumor cells. These data suggest that gene-linked changes in ganglioside composition influence the growth and angiogenic properties of the CT-2A astrocytoma.
Ganglioside biosynthesis occurs in the Golgi by the step-wise addition of monosaccharides to the ceramide moiety (Keenan et al. 1974; Yusuf et al. 1984; Yu et al. 2004). β-1,4-N-Acetylgalactosaminyltransferase or GM2/GD2 synthase (GalNAc-T, EC 22.214.171.124) is a key enzyme required for the synthesis of complex gangliosides (Fig. 1a). Specifically, GalNAc-T catalyzes the conversion of lactosylceramide (LacCer), GM3 and GD3 to asialo-GM2 (GA2), GM2 and GD2, respectively (Walton and Schnaar 1986; Sango et al. 1995; Furukawa and Takamiya 2002). The structurally simple monosialoganglioside GM3, containing a single-terminal sialic acid, inhibits tumor progression through reduced angiogenesis and cell proliferation (Alessandri et al. 1997; Manfredi et al. 1999; Noll et al. 2001). The more complex gangliosides such as GD3, GM2, GM1 and GD1a, containing longer oligosaccharide chains, enhance angiogenesis and proliferation (Ziche et al. 1992; Manfredi et al. 1999; Zeng et al. 2000; Lang et al. 2001; Liu et al. 2004). Hence, the relative distribution of simple and complex gangliosides may be important for tumor progression and malignancy.
Figure 1. Complex ganglioside biosynthesis and the design of the antisense β-1,4-N-acetylgalactosaminyltransferase (GalNAc-T) plasmid, pcDNA3.1/TNG. (a) Synthesis of complex gangliosides through the ‘a’ metabolic pathway. Sialyltransferase I (ST1) adds a single sialic acid to the terminal galactose of lactosylceramide (LacCer) to form GM3, the precursor for the synthesis of complex gangliosides. GalNAc-T adds a β-linked N-acetylgalactosamine to the galactose of GM3 to form GM2. GM2 is converted to GM1 by the addition of a galactose, which is catalyzed by galactosyltransferase II (Gal T2). GM1 is converted to GD1a by the addition of a sialic acid to the terminal galactose of GM1, which is catalyzed by sialyltransferase IV (ST4). (b) A schematic representation of antisense GalNAc-T plasmid design. (c) An analysis of plasmid sequence by restriction enzyme digestion. Lane 1, DNA ladder; lane 2, pcDNA3.1/TNG digested with BamHI, which produced a single band at 5.9 kb; lane 3, pcDNA3.1/TNG double digested with BamHI and XbaI, which produced two bands at 5.0 and 0.9 kb; lane 4, pcDNA3.1 vector digested with XbaI, which produced a single band at 5.0 kb.
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The malignant mouse astrocytoma CT-2A, which is fast growing and highly vascularized, expresses low levels of the simple ganglioside GM3 and high levels of the complex gangliosides GM2, GM1 and GD1a (Seyfried et al. 1992). Although CT-2A tumor cells express minor quantities of LacCer, they do not synthesize gangliosides of the ‘asialo’ metabolic pathway (Seyfried et al. 1996; Bai and Seyfried 1997). In addition, CT-2A tumor cells do not express sialyltransferase II (ST2 or GD3-synthase) and thus do not synthesize GD3 and gangliosides of the ‘b’ metabolic pathway (Seyfried et al. 1996; Bai and Seyfried 1997). Hence, the CT-2A tumor is a good model for studying the role of GM3 and complex gangliosides of the ‘a’ metabolic pathway in tumor growth and vascularity.
Brain tumor invasiveness and malignancy is positively associated with the degree of tumor vascularity and angiogenic potential (Leon et al. 1996; Chaudhry et al. 2001). Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen (Claffey et al. 1996; Ferrara and Davis Smyth 1997) and is a biomarker of angiogenesis in brain tumors (Chaudhry et al. 2001). VEGF is a secreted 40–45-kDa homodimer that exists in four major isoforms (VEGF121, VEGF165, VEGF189 and VEGF206) produced from alternative mRNA splicing. Although VEGF primarily enhances the survival and proliferation of endothelial cells, VEGF is also known to influence tumor cell growth (Podar et al. 2001). The transcription factor, hypoxia-inducible factor 1 (HIF-1), regulates gene expression in various biological processes to include angiogenesis, growth, cell survival and glycolysis (Lee et al. 2004). Under hypoxic conditions, HIF-1 binds to the hypoxia response element (HRE) upstream of the VEGF gene to initiate gene transcription. Although HIF-1 is a heterodimer of HIF-1α and HIF-1β subunits, HIF-1α expression is altered by hypoxia whereas HIF-1β is constitutively expressed. Both VEGF and HIF-1α are up-regulated in brain tumors and their expression is associated with tumor progression and angiogenesis (Zagzag et al. 2000; Chaudhry et al. 2001).
The biological functions of VEGF are mediated through two different tyrosine kinase receptors, VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1) (Ferrara and Davis Smyth 1997). Both VEGFR-1 and VEGFR-2 bind VEGF with high affinity and are expressed predominately on endothelial cells, yet both receptors can be expressed on tumor cells (Herold-Mende et al. 1999). Recently neuropilin-1 (NP-1), a 130–135-kDa glycoprotein, was found to bind VEGF, suggesting a possible role in vascularity. In contrast to VEGFR-1 and VEGFR-2, NP-1 lacks an intracellular tyrosine kinase domain and acts as a co-receptor enhancing the binding of VEGF165 to VEGFR-2 (Soker et al. 1998). NP-1 is associated with VEGF expression and tumor vascularity in human astrocytomas (Ding et al. 2000). Modulation of VEGF and/or VEGF receptor function may have therapeutic potential for brain cancer management.
Although previous studies have shown that gangliosides influence growth and angiogenesis, the mechanisms involved remain unclear (Manfredi et al. 1999; Zeng et al. 2000). In this study, we examined the effects of shifting the distribution of GM3 and complex gangliosides (GM1 and GD1a) on the growth and angiogenic properties of the CT-2A astrocytoma. An antisense GalNAc-T plasmid, pcDNA3.1/TNG, was produced and transfected into CT-2A tumor cells (CT-2A/TNG). This elevated GM3 while reducing GM1 and GD1a. Brain tumor growth and vascularity was significantly lower in CT-2A/TNG tumors than in control CT-2A and CT-2A/V (mock-transfected or vector alone) tumors. In addition, we observed a reduction in HIF-1α and NP-1 mRNA expression in the CT-2A/TNG tumor cells, indicating a possible mechanism by which gangliosides may modulate tumor angiogenesis.
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We showed that a gene-linked shift in ganglioside distribution significantly influenced growth and vascularity in the rapidly growing, highly vascularized malignant mouse astrocytoma, CT-2A. Specifically, the down-regulation of GalNAc-T expression in the CT-2A tumor cells caused a shift in the distribution of gangliosides synthesized through the ‘a’ metabolic pathway. This resulted in elevation of the ganglioside GM3 with concomitant reductions of the complex gangliosides GM1 and GD1a. Also, down-regulation of GalNAc-T expression affected the expression of other glycosyltransferases in the ‘a’ metabolic pathway. As the end product of a ganglioside pathway regulates the expression of biosynthetic enzymes within the same pathway, it is possible that the changes in ST1, Gal T2 and ST4 expression resulted from lower levels of the ‘a’ pathway end product, GD1a (Yusuf et al. 1987). The shift in ganglioside distribution significantly reduced growth, VEGF expression, and blood vessel number in the CT-2A brain tumor grown in vivo. The reduced growth and vascularity seen in vivo was also associated with reductions in expression of angiogenic biomarkers (VEGF, HIF-1α and NP-1) and in cell proliferation in vitro. As asialogangliosides are largely undetectable in the CT-2A tumor cells (Seyfried et al. 1996; Bai and Seyfried 1997), the observed effects of GalNAc-T down-regulation are likely to arise from the shift in distribution of ‘a’ metabolic pathway gangliosides. We do not, however, exclude the possibility that alterations in glycosyltransferase expression may influence other glycolipids and glycoproteins. Our data also support previous findings in other tumor models indicating that genetic manipulation of ganglioside biosynthesis, which shifts the relative distribution of GM3 and complex gangliosides, influences tumor growth and angiogenesis (Manfredi et al. 1999; Zeng et al. 2000).
Although the relationship between gangliosides, brain tumor growth and angiogenesis is complicated, our findings in the CT-2A brain tumor can provide new insight on this phenomenon. The gene-linked shift in ganglioside distribution may reduce CT-2A growth and vascularity through direct effects on angiogenesis, on tumor cell proliferation or through indirect effects on cell surface receptors that modulate both angiogenesis and proliferation.
As brain tumor growth is correlated with the degree of tumor vascularity (Leon et al. 1996; Chaudhry et al. 2001; Mukherjee et al. 2004; Kieran 2005), the shift in CT-2A ganglioside distribution may reduce growth and vascularity through effects on the angiogenic properties of the microenvironment. For example, shed gangliosides from tumors in the ECM can either enhance or suppress angiogenic responses through autocrine and paracrine effects on tumor cells and tumor-associated host cells (endothelial and macrophages) (Yohe et al. 1985; Ziche et al. 1989; Ziche et al. 1992; Koochekpour et al. 1996; Olshefski and Ladisch 1996; Alessandri et al. 1997; Lang et al. 2001). Our results with the CT-2A/TNG tumor cells support these findings as we found that these cells shed gangliosides and that the number and size of blood vessels was less in the in vivo Matrigel plugs containing CT-2A/TNG tumor cells than in those containing control CT-2A tumor cells. Previous studies in the rabbit cornea model of angiogenesis showed that GM3 inhibits endothelial cell migration and proliferation, whereas complex gangliosides (GT1b, GM1, GD3, etc.) counteracted the inhibitory effects of GM3 (Ziche et al. 1989, 1992; Alessandri et al. 1997). Although GD3 is considered the most pro-angiogenic ganglioside, CT-2A tumors do not express GD3. GD1a also stimulates endothelial cell proliferation and migration in vitro (Lang et al. 2001). We suggest that the gene-linked elevation of GM3 and the reduction of complex gangliosides, reduces the level of CT-2A angiogenesis in part through direct effects on the tumor microenvironment. We do not, however, exclude the possibility that the shift in ganglioside distribution may influence angiogenesis in the CT-2A brain tumor through other mechanisms to include integrin function and cellular immune responses (Yohe et al. 1985; McKallip et al. 1999; Wang et al. 2002).
In addition to the modulation of tumor–host interactions in the microenvironment, the shift in CT-2A ganglioside distribution could also influence angiogenesis through effects on the angiogenic properties of the CT-2A tumor cell itself. Support for this possibility comes from our findings that the expression of angiogenic biomarkers (VEGF, HIF-1α and NP-1) was lower in the CT-2A/TNG tumor cells than in the control CT-2A cells. VEGF, HIF-1α and NP-1 expression is associated with the function of cell surface receptors (Semenza 2000; Parikh et al. 2003; Lee et al. 2004; Bos et al. 2005; Kaur et al. 2005). Gangliosides are known modulators of cell surface receptor function and signaling (Zhou et al. 1994; Yates and Rampersaud 1998; Meuillet et al. 2000; Miljan and Bremer 2002; Liu et al. 2004). It is therefore possible that the shift in CT-2A ganglioside distribution indirectly influences VEGF, HIF-1α and NP-1 expression through the modulation of cell surface receptors. As VEGF expression is also regulated by HIF-1α, the overall reduction in VEGF expression in the CT-2A/TNG cells may result from both lower HIF-1α expression and modulation of receptor function. We suggest that the gene-linked elevation of GM3 and the reduction of complex gangliosides, reduces CT-2A growth and vascularity in part by decreasing the angiogenic potential of the CT-2A tumor cell.
Besides influencing angiogenesis, the shift in CT-2A ganglioside distribution may also influence growth and vascularity through effects on tumor cell proliferation. Support for this possibility comes from our findings that proliferation was less in the CT-2A/TNG tumor cells than in the control CT-2A cells. GM3 inhibits epidermal growth factor receptor (EGFR) signaling, whereas GD1a enhances EGFR signaling through effects on receptor phosphorylation and dimerization (Zhou et al. 1994; Liu et al. 2004). Moreover, treatment of human glioma cells with GM3 inhibited cell proliferation and induced apoptosis, suggesting a role of GM3 as a growth regulator (Noll et al. 2001). It is therefore possible that the shift in CT-2A ganglioside distribution influences tumor cell proliferation in part through the modulation of growth factor receptor function.
Finally, the shift in CT-2A ganglioside distribution may influence growth and vascularity through indirect effects on cell surface receptors that modulate both angiogenesis and proliferation. For example, gangliosides influence the EGFR signaling that regulates both cell proliferation and VEGF expression (Goldman et al. 1993; Maity et al. 2000; Meuillet et al. 2000; Miljan and Bremer 2002; Liu et al. 2004). It is therefore possible that the ganglioside shift in the CT-2A brain tumor simultaneously modulates cell proliferation and angiogenesis through effects on EGFR function. Further studies will be needed to confirm these interesting possibilities.
Taken together, our findings show that a shift in the distribution of ‘a’ metabolic pathway gangliosides in the CT-2A brain tumor significantly influences growth and vascularity, and also provide new insight into the possible mechanisms underlying these effects. Moreover, we suggest that the relative distribution of simple and complex gangliosides is important for tumor progression and malignancy. As gangliosides influence numerous processes involved in tumor progression (proliferation, invasion and angiogenesis), gangliosides are potential targets for the management of brain tumors and other types of cancer.