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. 2010 Apr 15;70(8):3361-71.
doi: 10.1158/0008-5472.CAN-09-2719.

The bisecting GlcNAc on N-glycans inhibits growth factor signaling and retards mammary tumor progression

Yinghui Song  1 , Jason A Aglipay, Joshua D Bernstein, Sumanta Goswami, Pamela Stanley
Affiliations

The bisecting GlcNAc on N-glycans inhibits growth factor signaling and retards mammary tumor progression

Yinghui Song et al. Cancer Res. .

Abstract

The branching of complex N-glycans attached to growth factor receptors promotes tumor progression by prolonging growth factor signaling. The addition of the bisecting GlcNAc to complex N-glycans by Mgat3 has varying effects on cell adhesion, cell migration, and hepatoma formation. Here, we show that Chinese hamster ovary cells expressing Mgat3 and the polyoma middle T (PyMT) antigen have reduced cell proliferation and growth factor signaling dependent on a galectin lattice. The Mgat3 gene is not expressed in virgin mammary gland but is upregulated during lactation and is expressed in mouse mammary tumor virus (MMTV)/PyMT tumors. Mice lacking Mgat3 that cannot transfer the bisecting GlcNAc to N-glycans acquire PyMT-induced mammary tumors more rapidly and have an increased tumor burden, increased migration of tumor cells, and increased early metastasis to lung. Tumors and tumor-derived cells lacking Mgat3 exhibit enhanced signaling through the Ras pathway and reduced amounts of functionally glycosylated alpha-dystroglycan. Constitutive overexpression of an MMTV/Mgat3 transgene inhibits early mammary tumor development and tumor cell migration. Thus, the addition of the bisecting GlcNAc to complex N-glycans of mammary tumor cell glycoprotein receptors is a cell autonomous mechanism serving to retard tumor progression by reducing growth factor signaling.

Keywords: LEC10; MMTV/PyMT mammary tumors; Mgat3; bisecting GlcNAc; invasion; metastasis.

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Conflict of interest statement

Conflicts of interest were disclosed.

Figures

Figure 1
Figure 1
Mgat3 retards cell proliferation. A, complex N-glycans of CHO and LEC10 showing the reactions catalysed by Mgat3 and Mgat5 (top). LEC10 cells are resistant to ricin, and hypersensitive to E-PHA (bottom). B, glycoproteins expressing the bisecting GlcNAc bind well to biotinylated-E-PHA (top) and biotinylated-L-PHA (bottom). C, proliferation of CHO, Lec4, Lec8 and LEC10 in medium with 7.5% serum. D, proliferation of CHO/PyMT, Lec4/PyMT, Lec8/PyMT and LEC10B/PyMT in medium with 7.5% serum. Bars STDEV; ***p<0.0001, **p<0.01 two-tailed Student t test comparing CHO with LEC10B.
Figure 2
Figure 2
Galectin-regulated PDGF signaling is reduced by Mgat3. A, western blot of pErk-1/2 and Erk-1/2 in PyMT CHO cells. The N-glycans are typical of the cell line. Symbols in Fig. 1A. B, ratios of pErk-1/Erk-1 and pErk-2/Erk-2 after 50 ng/ml PDGF-AB (n=5). Bars SEM, **p<0.001, two-tailed Student’s t test; *p<0.05 one-tailed Student’s t test comparing CHO to Lec4 or LEC10B. C, western blot of pErk-1/2 and Erk-1/2 in CHO/PyMT cells after treatment with 0.5M lactose or sucrose. D, effects of lactose on PDGF signaling. Fold-activation of pErk1/Erk1 at 5 and 10 min compared to 0 min after 50 ng/ml PDGF (n=3). Bars SEM.
Figure 3
Figure 3
Mgat3 is expressed in lactating mammary gland and MMTV/PyMT tumors. A, RT-PCR of total RNA from the fourth mammary gland of 4 month virgin or lactating females. B, glycoproteins (~80 μg) from lactating mammary gland of the same females bound E-PHA. Lec4 glycoproteins lack and kidney glycoproteins (~5 μg) carry the bisecting GlcNAc. C, representative RT-PCR of total RNA from tumors of Mgat3+/−/PyMT(n=13) and Mgat3−/−/PyMT(n=7) females at 17 weeks. D, glycoproteins (~80 μg) with the bisecting GlcNAc bound E-PHA.
Figure 4
Figure 4
Tumor burden is increased in the absence of Mgat3. A, Mgat3+/−/PyMT(Control) and Mgat3−/−/PyMT mammary glands were palpated beginning at week 6. Times to first and first 5 palpable tumors are shown. *p<0.05, two-tailed Student’s t test. Bars SEM. B, percent Mgat3+/−/PyMT(Control) and Mgat3−/−/PyMT mammary glands tumor-free to 17 weeks (Kaplan-Meier plot, ***p<0.0001, Mantel Cox log rank test). C, weight of the largest 3 tumors from Mgat3+/−/PyMT(Control) and Mgat3−/−/PyMT females at 17 weeks. *p<0.05, two-tailed Student’s t test. Bars SEM. D, weight of each tumor from Mgat3−/−/PyMT(n=60) and Control(n=69) females ***p<0.0001, Chi-squared test.
Figure 5
Figure 5
Increased expression of pErk-1/2 and early pulmonary metastases in the absence of Mgat3. A, western blot of pErk-1/2 and Erk1/2 in tumors from Mgat3+/−/PyMT and Mgat3−/−/PyMT females. Ratios of pErk-1/Erk-1 and pErk-2/Erk-2 in histograms. *p<0.05, two-tailed Student’s t test. Bars SEM. B, EGF and PDGF-AB signaling in Mgat3+/−/PyMT and Mgat3−/−/PyMT TECs in the presence and absence of 10 μM UO126, Bars SEM. C, functionally-glycosylated α-DG (IIH6) in 15 week tumors and TEC lines. D, ratio absolute number PyMT/actin(X103) transcripts. *p<0.05, two-tailed Student’s t test. Bars SEM (left panel). Ratio absolute number PyMT/actin(X103) transcripts versus tumor area from the fourth mammary gland of the same mice (right panel).
Figure 6
Figure 6
Constitutive overexpression of Mgat3 inhibits early mammary tumor development. A, RT-PCR of total RNA from the fourth mammary gland of 5-week virgin transgenic (Tg) or non-Tg females; kidney cDNA, positive control. B, glycoproteins with bisected N-glycans from the other mammary gland of the same females bound E-PHA. C, tumor lesion areas in control and MMTV-Mgat3-PyMT transgenic mice *p<0.05; one-tailed Student’s t test. Bars SEM. D, cells collected in control (n=2) or EGF-containing needles (n=4) inserted for 4h into mammary tumors (n=4–6) of ~1.5 cm.
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