A potential tumor suppressor role for Hic1 in breast cancer through transcriptional repression of ephrin-A1

Abstract

The tumor suppressor gene hypermethylated in cancer 1 (HIC1), which encodes a transcriptional repressor, is epigenetically inactivated in various human cancers. In this study, we show that HIC1 is a direct transcriptional repressor of the gene encoding ephrin-A1, a cell surface ligand implicated in the pathogenesis of epithelial cancers. We also show that mouse embryos lacking both Hic1 alleles manifest developmental defects spatially associated with the misexpression of ephrin-A1, and that overexpression of ephrin-A1 is a feature of tumors arising in Hic1 heterozygous mice in which the remaining wild-type allele is epigenetically silenced. In breast cancer, we find that ephrin-A1 expression is common in vivo, but that in cell culture, expression of the EphA receptors is predominant. Restoration of HIC1 function in breast cancer cells leads to a reduction in tumor growth in vivo, an effect that can be partially rescued by co-overexpression of ephrin-A1. Interestingly, overexpression of ephrin-A1 in vitro triggers downregulation of EphA2 and EphA4 levels, resulting in an expression pattern similar to that seen in vivo. We conclude that Hic1 spatially restricts ephrin-A1 expression in development, and that upregulated expression of ephrin-A1 resulting from epigenetic silencing of HIC1 in cancer cells may be an important mechanism in epithelial malignancy.

Fig. 1

Effects of HIC1 overexpression on expression of ephrin-A1. (a) Western blot analysis of MCF-7 cell lysates treated with AdHIC1 for 0–14 days. The filter was probed with HIC1 antibody. GAPDH was used as loading control. (b) Quantitative real time RT-PCR assays for the ephrin-A1/β-actin mRNA ratio from cell lines treated with control (Adβ-Gal) or HIC1 expressing (AdHIC1) adenoviruses. Total RNA was prepared at 9 hrs post viral infection, followed by cDNA synthesis and expression analysis. The graph shows mean values and standard deviations of three independent experiments. (c) Western blot analysis of HIC1 and ephrin-A1 in lysates obtained from ZR-75-1 and MDA-MB453 cells infected with Adβ-gal or AdHIC1 adenoviruses. Equal loading was confirmed by immunoblotting for GAPDH. (d) Quantitative real time RT-PCR assays for the ephrin-A1/β-actin mRNA ratio from Hic1 wild-type and knockout MEF cells.

Fig. 2

Knockdown of endogenous HIC1 in the WI38 human fibroblast cells up-regulates ephrin-A1 expression. WI38 cells were either mock-transfected, transfected with HIC1 siRNA or transfected with non-target siRNA control (NT) as previously described. Total RNA was extracted and expression levels of HIC1 (a) and ephrin-A1 (b) mRNA were assessed by real-time quantitative RT-PCR. Values were normalized to human β-actin.

Fig. 3

HIC1 is a direct transcriptional repressor of the ephrin-A1 promoter. (a) Left, map of the ephrin-A1 promoter region, with the positions of selected consensus binding sites indicated above. The length of the different promoter constructs used in reporter assays is shown below. Right, reporter activity in MCF-7 cells, transiently transfected with the four pEFNA1P promoter constructs as listed in left panel. Activity of each promoter construct is shown as fold activation compared with a pGL3-basic empty vector control. (b) Reporter activity in MCF-7 cells transiently co-transfected with the pEFNA1P8.3 reporter and increasing amount of the pRcHIC1 expression vector. The results are normalized to those for empty control vectors and are expressed as a relative ratio of firefly luciferase to Renilla luciferase. (c) Reporter activity in MCF-7 cells transiently co-transfected with the pEFNA1P8.3 reporter and 8ng of each of the expression constructs: pCDNA3.1 (control), a HIC1 mutant lacking the POZ domain pRcΔPOZ (DPOZ), a mutant lacking the zinc finger domain pRcΔZFM (DZFD), and pRcHIC1 (HIC1). (d) Reporter activity in MCF-7 cells transiently co-transfected with 8ng of pRcHIC1, and each of the four pEFNA1P reporter constructs listed in Fig. 3a. Results are shown as mean, SEM, N=6.

Fig. 4

ChIP analysis of Hic1 at the mouse ephrin-A1 promoter in NIH3T3 cells. The mouse ephrin-A1 promoter is depicted with the predicted Hic1 binding sites shown above, and the distance in kb from the start of exon 1 is shown below. The area of the promoter covered by each primer pair (A-L) is shown below this scale. Amplification products for each primer pair, with (+) and without (−) anti-Hic1 antibody in the immunoprecipitation are shown in the lower panel.

Fig. 5

Abnormal expression of ephrin-A1 in Hic1 deficient mice. (a) Sagittal sections of E14.5 Hic1 wild-type (+/+) and knockout (−/−) embryos stained for ephrin-A1 by immunoperoxidase, detected by DAB and counterstained with hematoxylin. Scale bar = 500μm. (b) Dual label confocal immunofluorescence analysis of ephrin-A1 expression in axial sections of the developing telencephalon from E14.5 embryos. cp = cortical plate, v = ventricle. Upper panel, ephrin-A1 is abnormally expressed in the cortical plate of a Hic1^−/− embryo. Immunolocalization of Hic1 (Green) in cortical plate (cp) neurons in an E14.5 Hic1^+/+ embryo. Scale bar = 200μm. Lower panel, cortical plate neurons from a Hic1^−/− embryo stained with ephrin-A1 (green) and Tuj1 (red). Scale bar = 20μm. (c) Immunostaining of ephrin-A1 expression in E17.5 livers taken from Hic1 wild-type (+/+), heterozygous (+/−), and null (−/−) mouse embryos. Scale bar = 50μm.

Fig. 6

HIC1 inhibits tumorigenic behavior of human breast cancer cells in vivo. (a) Western blot analysis on ephrin-A1, EphA2 and EphA4 expression in lysates from MCF-7 and MDA-MB231 cells grown in vitro or in vivo. (b) Western blot analysis of ephrin-A1, EphA2 and EphA4 in lysates obtained from MDA-MB231 cells infected with Adβ-gal or AdEphrin-A1 adenoviruses. Equal loading was confirmed by immunoblotting for GAPDH. (c) Tumor volume of flank xenografts in athymic nude mice injected with MDA-MB231 cells infected with AdHIC1, Adβ-gal or AdEphrin-A1 adenoviruses. The black bars represent the mean values of tumor volume. Numbers below the x-axis indicate the number of mice that developed tumors. (d) Tumor volume of flank xenografts in athymic nude mice injected with MDA-MB231 cells infected with a combination of with AdHIC1 and Adβ-gal, or AdHIC1 and AdEphrin-A1 adenoviruses. The mean values of tumor volume and the number of mice that developed tumors are also indicated as in C.