Expression of Globo H and SSEA3 in breast cancer stem cells and the involvement of fucosyl transferases 1 and 2 in Globo H synthesis

Abstract

We examined the expression in breast cancer stem cells (BCSCs) of Globo H, a potential tumor-associated antigen for immunotherapy of epithelial cancers including breast cancer. Flow cytometry revealed Globo H expression in 25/41 breast cancer specimens (61.0%). Non-BCSCs from 25/25 and BCSCs from 8/40 (20%) expressed Globo H. We showed the expression of stage-specific embryonic antigen 3 (SSEA3), the pentasaccharide precursor of Globo H, in 31/40 (77.5%) tumors. Non-BCSCs from 29/40 [corrected] and BCSCs from 25/40 (62.5%) expressed SSEA3. Like Globo H, SSEA3 expression in normal tissues was predominately at the secretory borders of epithelium, where access to the immune system is restricted. Immunization of mice with Globo H-KLH and alpha-GalCer induced antibodies reactive with Globo H and SSEA3, suggesting that a Globo H-based vaccine will target tumor cells expressing Globo H or SSEA3. We next sought to reduce Globo H expression by siRNA targeting fucosyltransferase (FUT) 1 and 2, which mediate alpha-1,2 linkage of fucose to SSEA3 to generate Globo H. We showed both genes to be involved in the biosynthesis of Globo H. Moreover, FUT2 expression in BCSCs was significantly lower than in non-BCSCs harvested from a primary human breast cancer in NOD/SCID mouse, whereas FUT1 was slightly lower in BCSCs. Thus, the lower expression of Globo H in BCSCs may be attributed to less FUT2/FUT1, and to reduced SSEA3 in BCSCs compared with non-BCSCs. Our findings provide insight into further development of a Globo H-based vaccine and FUT1/FUT2-targeted therapy for breast cancer.

Fig. 1.

Production of anti-Globo H and anti-SSEA3 by Globo H-KLH vaccine. Mice were immunized s.c. with 0.6 μg Globo H-KLH with or without 2 μg α-GalCer, weekly for 3 weeks. The mouse sera were obtained at Day 10 after the third immunization, and anti-Globo H (GH) or anti-SSEA3 antibody was detected with glycochip assay as described in SI Materials and Methods.

Fig. 2.

siRNA-induced knockdown of the FUT1 gene resulted in diminished expression of Globo H antigen in MB157 cells. Knockdown of FUT1 by siRNA was used to examine the involvement of FUT1 in Globo H biosynthesis. (A) The efficiency of siFUT1 in MB157 cells was evaluated by qRT-PCR analysis. Total RNA was extracted 72 h after infection with control lentivirus (control) or siFUT1-encoding vector (siFUT1). The level of FUT1 mRNA expressions was determined and plotted as fold-change relative to the control. (B) (Left) Globo H expression was determined by flow cytometry with the AlexaFluor488-VK-9 antibodies. (Right) The fold-change in MFI of Globo H relative to the control. AlexaFluor488-conjugated-mIgG3 was used as the isotype control in all experiments.

Fig. 3.

Silencing of FUT2 but not FUT1 mRNA in T-47D cells reduced the level of Globo H expression. Knockdown of FUT1 or FUT2 by siRNA was used to examine the involvement of these two FUTs in Globo H biosynthesis. (A) The efficiency of siFUT1 and siFUT2 in T-47D cells was evaluated by qRT-PCR analysis. Total RNA was extracted 72 h after infection with control lentivirus (control), siFUT1-encoding vector (siFUT1), or siFUT2-encoding vector (siFUT2). The levels of FUT1 and FUT2 mRNA expressions were determined and plotted as fold-change relative to the control. Infection of cells with siFUT1 lentivirus did not affect FUT2 expression, nor did siFUT2 alter FUT1 expression. (B) Left: Alteration in the expression of Globo H antigen was examined by flow cytometry with the AlexaFluor488-VK-9 antibodies. Right: The fold-change in MFI of Globo H relative to the control. AlexaFluor488-conjugated-mIgG3 was used as the isotype control in all experiments. P value was calculated by using the student t test.

Fig. 4.

BCSCs express lower level of FUT2 relative to non-BCSCs. Total RNA was extracted from BCSCs and non-BCSCs isolated from the engrafted tumors and reverse-transcribed to cDNA. Expressions of FUT1 and FUT2 were determined by q-PCR. Levels of FUT1 and FUT2 mRNA in BCSCs were then normalized to that of the corresponding mRNA in non-BCSCs.