Cyclin D1 is required for transformation by activated Neu and is induced through an E2F-dependent signaling pathway

Abstract

The neu (c-erbB-2) proto-oncogene encodes a tyrosine kinase receptor that is overexpressed in 20 to 30% of human breast tumors. Herein, cyclin D1 protein levels were increased in mammary tumors induced by overexpression of wild-type Neu or activating mutants of Neu in transgenic mice and in MCF7 cells overexpressing transforming Neu. Analyses of 12 Neu mutants in MCF7 cells indicated important roles for specific C-terminal autophosphorylation sites and the extracellular domain in cyclin D1 promoter activation. Induction of cyclin D1 by NeuT involved Ras, Rac, Rho, extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38, but not phosphatidylinositol 3-kinase. NeuT induction of the cyclin D1 promoter required the E2F and Sp1 DNA binding sites and was inhibited by dominant negative E2F-1 or DP-1. Neu-induced transformation was inhibited by a cyclin D1 antisense or dominant negative E2F-1 construct in Rat-1 cells. Growth of NeuT-transformed mammary adenocarcinoma cells in nude mice was blocked by the cyclin D1 antisense construct. These results demonstrate that E2F-1 mediates a Neu-signaling cascade to cyclin D1 and identify cyclin D1 as a critical downstream target of neu-induced transformation.

FIG. 1

Cyclin D1 protein levels are induced by Neu. (A) Western blot analysis of NIH 3T3 cell lines DHFR/G8 (expresses wild-type Neu [NeuWt]), B104-1-1 (expresses NeuT), and neuΔC-1 (contains a carboxy-terminal deletion of NeuT). α-Tubulin is shown as a protein loading control. Right, schematic representation of the Neu mutants showing the signal peptide (SP), cysteine-rich domains (CRD), transmembrane domain (TM), tyrosine kinase (TK), and carboxy terminus (CT). Within the CT are shown the five autophosphorylation sites (P1 to P5). The mutation within the transmembrane domain (Glu664) is shown. (B) Cyclin D1 protein levels were assessed in human breast cancer cell lines that have amplification of neu (MDA-MB-453 and BT-483) compared with cells with wild-type neu (MDA-MB-231 and HBL-100). Cells were deprived of serum (0.5% serum) for 24 h and then refed serum (10% serum) for 0, 4, or 8 h. Neu protein levels are indicated, with GDI blotting for protein loading control. (C) Western blot for endogenous cyclin D1 in MCF7 cells transfected with the NeuT expression vector, with comparison made to transfection of the empty expression vector cassette. (D) Mammary tumors of MMTV-neu and MMTV-NDL transgenic animals were analyzed for cyclin D1 protein levels by Western blotting (lower panel). Cyclin D1 immune-complex assays were conducted with the cyclin D1-specific antibody DCS-11. Phosphorylation of the GST-pRB substrate is indicated by the arrow (upper panel). NBE, normal breast epithelium. (E) The relative cyclin D1 protein levels and kinase activity for each tumor (MMTV-neu in blue and MMTV-NDL in red). Fold induction is shown in comparison with the mean derived from assays of three normal mammary glands, indicated by dashed lines. (F) Representative cyclin D1 immunohistochemical staining of MMTV-neu mammary gland tumors, with positive tumor cells appearing brown (top, yellow arrow) and negative cells appearing blue (red arrow). Normal mammary gland from the same animal demonstrated little nuclear cyclin D1 positivity (bottom).

FIG. 2

Neu stimulates the cyclin D1 promoter in MCF7 cells. (A) The −1745CD1LUC reporter was transfected with increasing amounts of the Neu expression vector (pSV₂NeuT) into MCF7 cells. Luciferase activity (relative light units) is shown with the activity induced by equal amounts of control vector cassette. (B) Neu induces the cyclin D1 promoter but not the cyclin A promoter. Cotransfection experiments were conducted with plasmids for the cyclin D1, c-fos, and cyclin A promoters linked to the luciferase reporter gene. The c-fos serum response element (SRE) linked to the minimal TATA box was also assessed. Induction by pSV₂NeuT is shown. (C and D) Expression vectors encoding different neu mutants, previously described for their transforming ability, were assessed for their effects on cyclin D1 promoter activity in MCF7 cells. Data are shown as the mean ± standard error of the mean of the number of experiments shown in parentheses. wt, wild type.

FIG. 3

Effects of inhibitors on NeuT induction of cyclin D1. To examine the intracellular signaling pathways involved in Neu induction of the cyclin D1 promoter, the −1745CD1LUC reporter was introduced into MCF7 cells with the NeuT expression vector. (A) Cotransfection experiments were conducted using increasing amounts of dominant negative expression vector, and the inhibition of Neu-induced promoter activation is shown as percent activity. Comparison was made between the effect of the dominant negative mutants for N17Ras, N17Rac, or N19Rho and equal amounts of empty expression vector cassette. (B) The chemical inhibitors of the MEK/ERK pathway (PD098059) (n = 8) and the p38 pathway (SB203580) (n = 8) were added to the culture medium and compared with equal volumes of DMSO vehicle. The expression vector encoding the inhibitor of JNK signaling (JIP-1) (n = 4) for each concentration of plasmid is shown compared with equal amounts of empty expression vector cassette. Inhibition is significant at P < 0.05.

FIG. 4

Promoter sequences involved in Neu induction of the cyclin D1 promoter. (A) Schematic representation of the cyclin D1 promoter, with the sequences homologous to E2F and Sp1 binding sites indicated. LUC, luciferase. (B) pSV₂NeuT was transfected with cyclin D1 5′ promoter constructs into MCF7 cells. ∗ represents significant difference from the −163CD1LUC reporter for P < 0.05. (C) The heterologous constructions, encoding the E2F or Sp1 site of the cyclin D1 promoter linked to the minimal TK promoter, were transfected with pSV₂NeuT into MCF7 cells. Comparison is made with the effect on the minimal TK reporter. In each case, fold stimulation reflects induction with NeuT compared with the empty expression vector cassette, with the number of experiments shown in parentheses.

FIG. 5

Sp1/Sp3 and E2F-1 proteins bind the neuT-responsive elements of the cyclin D1 promoter. (A) The ³²P-labeled cyclin D1 Sp1-like sequence was incubated with MCF7 cell nuclear extracts, and the effects of 100-fold excess of cognate competitor (lane 2), wild-type canonical Sp1 binding site competitor (lane 3), and an unrelated oligonucleotide competitor (lane 4) were determined. Specific antibodies to the Sp proteins or equal amounts of control IgG (lane 5) were added as indicated above the lanes (lanes 5 to 10). Arrows indicate the predicted proteins constituting the bands (A to C) identified through supershift or inhibition of DNA binding. (B) EMSA with extracts prepared from baculovirus-infected Sf9 cells. The ³²P-labeled adenovirus E2F site (lanes 1 to 4) and wild-type (lanes 5 to 8) or mutant (lanes 9 to 10) cyclin D1 E2F sites were incubated with E2F and DP proteins as indicated above the lanes. Relative binding compared to the adenovirus E2F site for each E2F-DP complex is indicated below each lane. (C) CHIP assays were performed with the −1745 CD1LUC MCF7 cell line or wild-type MCF7 cells (lanes 5 to 8). PCR was performed with cyclin D1-specific primers on water (lane 2), control plasmid (lane 3), or immunoprecipitation buffer (lane 4) or after immunoprecipitation of formaldehyde cross-linked cell extracts with either IgG control (lanes 5 and 7) or E2F-1-specific antibody (lanes 6 and 8). The specific cyclin D1 promoter band is shown (arrow).

FIG. 6

Induction of E2F-1, Sp1, and Sp3 transactivation by NeuT. The effects of wild-type E2F-1, the E132 mutant E2F-1, the Y411C mutant E2F-1, and the DP-1 dominant negative mutant on NeuT-induced (A) or basal (B) cyclin D1 promoter activity were assessed in MCF7 cells and compared with effects of the respective empty vectors. (C) The E2F-1 expression vector was transfected with cyclin D1 5′ promoter constructs into MCF7 cells. ∗ represents significant difference from the −163CD1LUC reporter for P < 0.05. (D) Schematic representation of the GAL4 constructs and the heterologous luciferase reporter containing five upstream activator binding sites for the GAL4 DNA binding domain. The reporter (UAS)₅E1BTATALUC (2.4 μg) was transfected with expression vectors for GAL4–E2F-1, GAL4–E2F-1(Δ413-417) (the pRB-binding-defective E2F-1 mutant), GAL4-Sp1, GAL4-Sp3, PAG236, and either pSV₂NeuT (600 ng) or empty expression vector cassette in MCF7 cells. Comparison was made between the effect of the NeuT expression vector and equal amounts of the parental vector. Data are mean fold induction ± standard error of the mean for the number of separate experiments indicated in parentheses.

FIG. 7

Cyclin D1 antisense inhibits neu-induced focus formation. Transformation assays were conducted with NeuT in Rat-1 cells. The activating NeuT mutant (2.5 μg) was introduced alone or in conjunction with one of the antisense or dominant negative expression plasmids listed. The cyclin D1 antisense (pBPSTR-1CD1AS) (A) and the dominant negative mutants for N17Ras, N17Rac, N19Rho, MEKC, and E2F-1 (E2F-1 E132) (2.5 or 5 μg) (B) were assessed in comparison to the empty expression vector cassette. The effect on transformation is shown for 1:1 and 1:2 molar ratios of NeuT vector to dominant negative or antisense plasmid. Panel A shows a representative assay with the effect of cyclin D1 antisense. The transformation induced by NeuT is shown as 100% in black bars throughout. The results are shown as percentage of transformation by NeuT for independent transformation assays compared with the effect of empty vector cassette (mean ± standard error of the mean).

FIG. 8

Cyclin D1 antisense inhibits growth of NeuT-transformed mammary epithelial cells in nude mice. Immunodeficient mice received subcutaneous injections into each flank with transfected NAFA cells in PBS. Sites injected with NAFA cells transfected with control vector (right flank, yellow arrow) showed development of tumors, whereas in the same animal, the left flank (red arrow), injected with NAFA cells transfected with cyclin D1 antisense, did not show the development of tumors. (A and B) Two examples of mice injected in both flanks; (C) hematoxylin and eosin staining of the right flank tumor of the mouse in panel A, demonstrating adenocarcinoma; (D) Western blot of implanted cells of the mouse in panel A, demonstrating reduced cyclin D1 protein levels in cells transfected with the cyclin D1 antisense (CD1 AS) compared with the control vector (control). GDI blotting confirmed equivalent protein loading, and keratin-8 blotting confirmed that the tissues were of epithelial origin.