Transcriptional regulation of the protocadherin β cluster during Her-2 protein-induced mammary tumorigenesis results from altered N-glycan branching

Abstract

Changes in the levels of N-acetylglucosaminyltransferase V (GnT-V) can alter the function of several types of cell surface receptors and adhesion molecules by causing altered N-linked glycan branching. Using a her-2 mammary tumor mouse model, her-2 receptor signaling was down-regulated by GnT-V knock-out, resulting in a significant delay in the onset of her-2-induced mammary tumors. To identify the genes that contributed to this GnT-V regulation of early events in tumorigenesis, microarray analysis was performed using her-2 induced mammary tumors from wild-type and GnT-V-null mice. We found that 142 genes were aberrantly expressed (>2.0-fold) with 64 genes up-regulated and 78 genes down-regulated after deletion of GnT-V. Among differentially expressed genes, the expression of a subgroup of the cadherin superfamily, the protocadherin β (Pcdhβ) cluster, was up-regulated in GnT-V-null tumors. Altered expression of the Pcdhβ cluster in GnT-V-null tumors was not due to changes in promoter methylation; instead, impaired her-2-mediated signaling pathways were implicated at least in part resulting from reduced microRNA-21 expression. Overexpression of Pcdhβ genes inhibited tumor cell growth, decreased the proportion of tumor-initiating cells, and decreased tumor formation in vivo, demonstrating that expression of the Pcdhβ gene cluster can serve as an inhibitor of the transformed phenotype. Our results suggest the up-regulation of the Pcdhβ gene cluster as a mechanism for reduced her-2-mediated tumorigenesis resulting from GnT-V deletion.

FIGURE 1.

Microarray analysis of gene expression in her-2-induced tumors. A, total RNA was isolated from three wild-type and GnT-V knock-out tumor tissues and used for microarray analysis. A heat map of 57 transcripts differentially expressed (p < 0.05) between wild-type and GnT-V knock-out tumors was generated from the microarray data. Red indicates a high expression level, whereas green indicates a low expression level relative to wild-type. T1–3 represents three tumor tissues collected from three mice with GnT-V wild-type and knock-out backgrounds, respectively. B, total RNA was isolated from two wild-type and two GnT-V knock-out tumors and used for validation of transcript levels of the Pcdhβ cluster by qRT-PCR. For each transcript, the values are normalized to control (Gapdh or Rpl4) and expressed as means with error bars indicating ±1 S.D. C, comparison of -fold change (KO/WT) of transcripts from microarray and qRT-PCR analyses. D, mammary tumor sections from 10-week-old mice were immunostained with leukocytic phytohemagglutinin (L-PHA), anti-Pcdhβ4, and anti-Pcdhβ7, respectively.

FIGURE 2.

Increased transcript expression of the Pcdhβ gene cluster is related to absence of GnT-V activity. A, total RNA was isolated from wild-type and GnT-V knock-out tumor cells and used for detection of transcript levels of Pcdhβ genes. B, wild-type her-2 tumor cells were treated with swainsonine (SW; 1 μg/ml) for 24 h, and total RNA was isolated and used for detection of transcript levels of Pcdhβ by qRT-PCR. C, total RNA was isolated from GnT-V KO cells with GnT-V expression and used for detection of transcript levels of Pcdhβ. D, total RNA was isolated from control (scrambled siRNA)- and GnT-V siRNA-transfected MDA-MB231 cells and used for detection of transcript levels of GnT-V and Pcdhβ genes, respectively. For each transcript, the values are normalized to control (Gapdh or Rpl4) and expressed as means with error bars indicating ±1 S.D. of three independent experiments. *, Student's t test, p < 0.05; **, p < 0.01.

FIGURE 3.

Increased transcript expression of the Pcdhβ gene cluster is not primarily mediated by altered promoter methylation. A, her-2 tumor cells were treated with the demethylating reagent 5-aza-2′-deoxycytidine at two different concentrations for 3 days, and total RNA was isolated and used for detection of transcript levels of Pcdhβ. For each transcript, the values are normalized to control (Gapdh or Rpl4) and expressed as means with error bars indicating ±1 S.D. of three independent experiments. B, after the genomic DNA samples were purified and bisulfate-treated, methylation-specific PCR for the Pcdhβ genes was performed in two wild-type (samples 1 and 2) and two GnT-V knock-out (samples 3 and 4) tumors. The presence of a PCR product band in lane M or U indicates methylated or unmethylated genes, respectively. Data are representative of two independent experiments.

FIGURE 4.

Increased transcript expression of the Pcdhβ gene cluster is caused by impaired her-2-mediated downstream signaling. A, wild-type tumor cells were grown for 2 days with or without MEK inhibitor PD98059 (PD; 10 mol/liter), PI3K inhibitor wortmannin (WM; 1 mol/liter), or both inhibitors. Cells were then collected, and total RNA was isolated and used for detection of transcript levels of Pcdhβ. B, GnT-V knock-out tumor cells were grown in serum-free medium for 2 days and stimulated with EGF (100 ng/ml), neuregulin (NRG) (50 ng/ml), or serum-containing medium for 2 days. Cells were collected for detection of phospho-PKB (p-PKB) and phospho-ERK (p-ERK) using immunoblot (left panel) and transcripts of the Pcdhβ cluster (right panel). After wild-type her-2 cells were treated with control (scrambled) and her-2/neu siRNA oligonucleotides (50 nm) for 48 h, respectively, cells were collected and subjected to detection of transcripts of her-2/neu using qRT-PCR (C); her-2/neu, phospho-PKB (p-PKB), and phospho-ERK (p-ERK) using immunoblot (D); and transcripts of the Pcdhβ gene cluster (E). For each transcript, the values are normalized to control (Gapdh or Rpl4) and expressed as means with error bars indicating ±1 S.D. of three independent experiments. *, Student's t test, p < 0.05; **, p < 0.01.

FIGURE 5.

MiR-21 is implicated in the regulation of her-2 signaling-mediated gene expression of Pcdhβ cluster. A, total RNA was pooled from four tumors of GnT-V wild-type and knock-out mice, and expression of mature miR-21 was detected by qRT-PCR. B, total RNA was isolated from MDA-MB231 and SK-BR3 tumor cells with and without GnT-V siRNA expression, and expression of GnT-V and mature miR-21 was detected by qRT-PCR. After wild-type her-2 tumor cells were treated with control and anti-miR-21 siRNA oligonucleotides (50 nm) for 48 h, respectively, cells were collected and subjected to detection for transcripts of miR-21 (C), the Pcdhβ cluster (D), and miR-21 target genes as indicated (E). For each transcript, the values are normalized to control (Gapdh or Rpl4) and expressed as means with error bars indicating ±1 S.D. of three independent experiments. *, Student's t test, p < 0.05; **, p < 0.01.

FIGURE 6.

Tumor-suppressive effects of the Pcdhβ cluster. A, wild-type her-2 tumor cells and MDA-MB231 cells were transfected with either Pcdhβ4 or Pcdhβ19 cDNA and grown in selection medium for 2–3 weeks for colony formation. The number of colonies in six random fields was counted and expressed as the mean with error bars indicating ±1 S.D. * represents Student's t test, p < 0.001. B, transfected cells were grown in soft agar for 2–3 weeks, and the number of colonies in six random fields was counted and expressed as the mean with error bars indicating ±1 S.D. *, p < 0.05. C, Pcdhβ4-transfected wild-type her-2 tumor cells (2 × 10⁶) were injected into mammary fat pads of SCID mice (n = 5), and secondary tumor growth was observed for up to 8 weeks. *, p < 0.05. D, secondary tumors in SCID mice formed by injection of Pcdhβ4-expressing cells were dissected at week 8 and photographed (left panel), and the weight of tumors was quantified and expressed as the mean with error bars indicating ±1 S.D. (n = 5; right panel). *, p < 0.05. E, ALDEFLUOR assays of Pcdhβ4-expressing her-2 tumor cells (left panel) and xenografts formed by injection of Pcdhβ4-expressing her-2 cells (right panel) were performed, and the percentage of ALDEFLUOR-positive cells (tumor initiating cells) was determined using similar gating criteria. Data are representative of two independent experiments. DEAB, diethylaminobenzaldehyde.

FIGURE 7.

Schematic indicating how the expression of the Pcdhβ gene cluster is regulated by GnT-V. Pathways implicated in the regulation of the Pcdhβ gene cluster are depicted based on the observations presented in this study. GnT-V expression levels regulate N-glycosylation of her-2 and her-2-induced signaling pathways. Knock-out of GnT-V results in inhibited expression of N-linked β(1,6) branching on her-2 and impaired her-2-induced signaling pathways, which leads to up-regulation of the Pcdhβ gene cluster. The increased Pcdhβ expression is mediated indirectly at least in part by miR-21, one of the downstream effectors regulated by her-2 signaling, and contributes to the inhibition of her-2-induced tumor onset.