Cancer Letters

Cancer Letters

Volume 434, 10 October 2018, Pages 184-195
Cancer Letters

Original Articles
Reciprocal feedback regulation of ST3GAL1 and GFRA1 signaling in breast cancer cells

https://doi.org/10.1016/j.canlet.2018.07.026Get rights and content

Highlights

  • ST3GAL1 silencing inhibits GDNF-induced activation of downstream kinases.

  • ST3GAL1-mediated O-linked sialylation of GFRA1 promotes its interaction with RET.

  • GDNF upregulates transcription of ST3GAL1 by Sp1 through GFRA1/RET/PI3K/AKT pathway.

  • ST3GAL1 knockdown augments anti-cancer efficacy of inhibitors of RET and/or ER.

Abstract

GFRA1 and RET are overexpressed in estrogen receptor (ER)-positive breast cancers. Binding of GDNF to GFRA1 triggers RET signaling leading to ER phosphorylation and estrogen-independent transcriptional activation of ER-dependent genes. Both GFRA1 and RET are membrane proteins which are N-glycosylated but no O-linked sialylation site on GFRA1 or RET has been reported. We found GFRA1 to be a substrate of ST3GAL1-mediated O-linked sialylation, which is crucial to GDNF-induced signaling in ER-positive breast cancer cells. Silencing ST3GAL1 in breast cancer cells reduced GDNF-induced phosphorylation of RET, AKT and ERα, as well as GDNF-mediated cell proliferation. Moreover, GDNF induced transcription of ST3GAL1, revealing a positive feedback loop regulating ST3GAL1 and GDNF/GFRA1/RET signaling in breast cancers. Finally, we demonstrated ST3GAL1 knockdown augments anti-cancer efficacy of inhibitors of RET and/or ER. Moreover, high expression of ST3GAL1 was associated with poor clinical outcome in patients with late stage breast cancer and high expression of both ST3GAL1 and GFRA1 adversely impacted outcome in those with high grade tumors.

Introduction

Membrane-associated sialoproteins such as mucin, E-cadherin, and integrins are extensively sialylated glycoproteins which have been shown to modulate cell-cell or cell-extracellular matrix interaction by steric hindrance [1,2]. Mucin-type proteins are highly expressed in many cancers including breast, ovary, and prostate. Mucin O-glycans begin with covalently α-linked N-acetylgalactosamine (GalNAc) to the -OH of serine or threonine, forming Tn antigen. Addition of galactose via β1, 3 linkage to GalNAc gives rise to a core 1 structure named T antigen, and attachment of a branching N-acetylglucosamine to core 1 forms core 2. Mucin O-glycans from normal breast are mostly composed of elongated core 2 structures. It has been shown that Mucin 1 O-glycans from breast cancer cell lines are often truncated with mainly core 1 structures based on mass spectrometry sequencing [3,4]. Moreover, the principal O-glycan species of Mucin 1 from the serum of breast cancer patients were sialylated core 1 type glycans, among which the most abundant glycan was NeuNAcα2-3Galβ1-3GalNAc (sialyl-3T) [5]. This was associated with overexpression of ST3 beta-galactoside alpha-2,3-sialyltransferase 1 (ST3GAL1) causing a change from core 2-based glycans to sialyl-3T on Mucin 1 [6,7].

ST3GAL1 catalyzes the transfer of sialic acid in an α2,3 linkage to Galβ1-3-GalNAc-Ser/Thr, and thus terminates further chain elongation, except extension with sialic acids [8]. Altered expression level or activity of this enzyme might lead to changes in the composition and length of O-glycans attached to mucin-type proteins. ST3GAL1 mRNA expression is elevated in primary breast carcinoma cells compared to normal or benign breast tissues [9]. In ductal carcinomas, the expression of ST3GAL1 appeared to correlate to histologic grade. Overexpression of ST3GAL1 in bladder cancer cells decreased expression of several genes involved in DNA repair, which might be associated with increased malignancy [10]. However, very little is known about the role of this enzyme in breast cancers.

It is believed that glycosylation is cell-type specific. Alteration of specific glycosyltransferase generates glycan microheterogeneity. Therefore, identifying specific targets of glycosyltransferases will help to elucidate how glycans on the cell surface affect the cell behavior. In this study, we identified GDNF receptor alpha 1 (GFRA1) to be a target protein of ST3GAL1 in breast cancer cell lines. The GFRA family consists of GFRA1-4, which is glycosylphosphatidylinositol (GPI)-anchored receptors with no transmembrane domain [11]. Glial cell line-derived neurotrophic factor (GDNF) family of ligands (GFL) comprises 4 members; GDNF, artemin, neurturin, and persephim [12]. The GFL family members preferentially interact with one or more of the GFRA family. Upon GFL binding to GFRA, the complex will bind and facilitate RET dimerization and subsequent phosphorylation, promoting neuron cell proliferation, migration, differentiation and neurite branching. GDNF preferentially interacts with GFRA1. RET is unable to bind GDNF on its own and GFRA1 is required for GDNF signaling [13,14]. Higher levels of GFRA1 mRNA are associated with later tumor stage and lymph node metastasis in breast cancers [15], and overexpression of GFRA1 and RET has been reported in ERα-positive breast cancers [[16], [17], [18], [19]]. Importantly, activation of GFRA1/RET signaling leads to ERα phosphorylation at Ser118/Ser167 and estrogen-independent transcriptional activation of ER-dependent genes [17]. This is consistent with the reports that GFRA1/RET signaling is a key determinant of response and resistance to endocrine treatment in ER-positive breast cancers [20] [19]. Based on NetOGlyc 3.1 prediction, there are several O-glycosylation sites on GFRA1 but none on RET [21]. We thus investigated whether O-linked sialylation on GFRA1 may affect GDNF-induced signaling in ER-positive breast cancer cells.

Section snippets

Clinical specimens

114 fresh primary breast cancer tumor and adjacent normal tissue specimens were collected during surgical resections performed at the Tri-Service General Hospital (Taipei, Taiwan). Informed consent was obtained from all subjects before their tissue were deposited. The sample were fully encoded and used under a protocol approved by the Institutional Review Board of Human Subjects Research Ethics Committee of the Tri-Service General Hospital and Chang Gung Memorial Hospital at Linkou, Taoyuan,

GFRA1 is a protein target of ST3GAL1

The nonsialylated Galβ1-3GalNAc-Ser/Thr core 1 O-glycan is a ligand for plant lectin peanut agglutinin (PNA) [23]. When this O-glycan is modified by the addition of sialic acid in α2-3 linkage, it will generate trisaccharide sialyl-3T, which is no longer recognized by PNA lectin. Using the lectin blot, several PNA labeling signals ranging from 55 kD to greater than 250 kD were increased in MCF7 cells (Fig. 1A), when ST3GAL1 was silenced to 33% of control at RNA level (Fig. 1B) and 23% of

Discussion

Although overexpression of ST3GAL1 has been reported to promote mammary tumorigenesis [37], little is known about its function and its protein targets. In this study, we identified GFRA1 as a substrate of ST3GAL1, which mediated its O-linked sialylation, facilitating its interaction with RET, thereby regulating the phosphorylation and downstream signaling of GDNF/GFRA1/RET pathway in breast cancer cells. On the other hand, we showed that GDNF upregulated the transcription of ST3GAL1, which was

Financial support

This work was supported by the grants from Ministry of Science and Technology, Taiwan, 104-2321-B-182A-003, 105-2321-B-182A-001, and Chang Gung Medical Foundation, OMRPG3C0014.

Conflicts of interest

The authors declare no conflict of interest related to the contents of this manuscript.

Acknowledgements

We thank Hsiao-Wei Wu for her excellent scientific illustration, and Dr. Jin-Yuh Shew for providing ZR-75-1 cells.

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