Downregulation of Transcription Factor Sp1 Suppresses Malignant Properties of A549 Human Lung Cancer Cell Line with Decreased β4-Galactosylation of Highly Branched <i>N</i>-Glycans

Abstract

Dramatic changes in the glycan structures of cell surface proteins have been observed upon malignant transformation of cells as induced by the altered expression levels of glycosyltransferases. Such changes are closely associated with the malignant properties of cancer cells. Transcription factor Sp1 regulates the gene expression of various molecules including glycosyltransferases. Herein, we investigated whether or not Sp1-downregulation affects to N-glycosylation of glycoproteins and malignant properties of A549 human lung cancer cell line. We established a stable clone whose Sp1-expression level was reduced to 50% of a control clone by RNA interference. Lectin blotting revealed that the β4-galactosylation of highly branched N-glycans decreases mainly in cell adhesion molecule, E-cadherin. The analysis of underlying mechanism for decreased β4-galactosylation of N-glycans showed that the gene expression level of β4-galactosyltransferase (β4GalT) 1 decreases dramatically by downregulation of Sp1 without changes in those of β4GalT2 and N-acetylglucosaminyltransferase V. Mutations in the Sp1-binding sites of the β4GalT1 gene promoter showed that the promoter activity decreases significantly, indicating that the gene expression is regulated by Sp1. These results indicate that the β4-galactosylation of highly branched N-glycans decreases by downregulation of Sp1 through the reduced expression of the β4GalT1 gene. Furthermore, the Sp1-downregulated cells showed the suppression of the anchorage-independent growth in soft agar and migratory activity when compared to the control cells. The present study demonstrates that downregulation of Sp1 suppresses the malignant properties of A549 cells through the decreased β4-galactosylation of highly branched N-glycans. Key words Sp1; N-glycan; β4-galactosylation; β4-galactosyltransferase 1; lung cancer; malignant propertyIn general, the carbohydrate structures of glycans attached to cell surface proteins change dramatically by malignant transformation.1,2) The most prominent change is the increment of highly branched N-glycans containing the Galβ1 4GlcNAcβ1 6Man group, which is synthesized by N-acetylglucosaminyltransferase (GlcNAcT) V and β4-galactosyltransferase (β4GalT). The increment of highly branched N-glycans is associated with malignant properties of cancer cells.3,4) On the other hand, the GlcNAcT V-deficient mice, in which the amounts of highly branched N-glycans are reduced, showed that the growth and metastasis of mammary tumor induced by polyoma virus middle T oncogene are suppressed.5) Therefore, the biosynthetic pathway of highly branched N-glycans is considered to be important for expressing the malignant properties of cancer cells.The gene expression levels of several glycosyltransferases including GlcNAcT IVa, GlcNAcT V, β4GalT1, β4GalT5, fucosyltransferase (FUT) I, FUT II, and α-2,6-sialyltransferase I have been shown to increase in cancer cells, [6][7][8][9][10][11][12] and the changes in glycosylation induced by the altered expression levels of the glycosyl...

Novel Screening Method for Anti-Colon Cancer Drugs Using Two Sensor Cell Lines with Human &beta;4-Galactosyltransferase 4 Gene Promoters

“…The results of luciferase assay were analyzed with the analysis of variance followed by the Bonferroni correction for multiple comparisons. The results of quantitative real-time RT-PCR analysis were analyzed by Student's t-test [10,17].…”

“…The promoter activities of the sensor cells were determined by luciferase assay as described previously [10,14,17].…”

“…The cell lysates were prepared from the hGT4-0.3-sensor cells treated with 20 μM U0126 or DMSO for 24 h. Immunoblot analysis using the antibodies against p44/42 MAPK and phosphorylated p44/42 MAPK was conducted, and the band intensity was quantified as the method described previously [8,17].…”

“…h. The expression levels of the β4GalT4 gene were examined by quantitative real-time RT-PCR analysis as described previously [8,10,17]. The gene-specific primers used were as follows: β4GalT4, F: 5'-GCGAAGACGATGACCTCAGACTC-3', R: 5'-CTCCAGACTCGTGACACTTGGTGTA-3'; glyceraldehyde 3-phosphate dehydrogenase (G3PDH), F: 5'-GCACCGTCAAGGCTGAGAAC-3', R:…”

Suppression of Malignant Potentials of A549 Human Lung Cancer Cell Line by Downregulation of the β4-Galactosyltransferase 1 Gene Expression

“…β4GalT family consists of seven members with different substrate specificity and tissue distribution. 6) Among β4GalT family, the transcription of the human β4GalT1, β4GalT3, β4GalT4, and β4GalT5 genes was reported to be regulated by Sp1. [7][8][9][10] Recently, we demonstrated that downregulation of Sp1 suppresses the malignant potentials of A549 human lung cancer cell line with the reduced β4-galactosylation of highly branched N-glycans on cell surface glycoproteins, which is resulted in the decreased expression of the β4GalT1 gene.…”