Epigenetic repression of PDZ-LIM domain-containing protein 2: implications for the biology and treatment of breast cancer

Abstract

The NF-kappaB transcription factor plays a pivotal role in breast cancer progression and therapy resistance. However, the mechanisms by which the tightly regulated NF-kappaB becomes constitutively activated during breast cancer pathogenesis remain obscure. Here, we report that PDZ-LIM domain-containing protein 2 (PDLIM2), an essential terminator of NF-kappaB activation, is repressed in both estrogen receptor-positive and estrogen receptor-negative breast cancer cells, suggesting one important mechanism for the constitutive activation of NF-kappaB. Indeed, PDLIM2 reexpression inhibited constitutive NF-kappaB activation and expression of NF-kappaB-targeted genes in those breast cancer cells. Importantly, PDLIM2, but not its mutants defective in NF-kappaB termination, could suppress in vitro anchorage-independent growth and in vivo tumor formation of those malignant breast cells. In addition, we have shown that PDLIM2 repression involves promoter methylation. Accordingly, treatment of the breast cancer cells with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine reverses the methylation of the PDLIM2 promoter, restored PDLIM2 expression, and suppressed tumorigenicities of human breast cancer cells both in vitro and in vivo. These studies thus provide important mechanistic insights into breast cancer pathogenesis. These studies also suggest a tumor suppression function of PDLIM2 and a therapeutic strategy for breast cancer.

FIGURE 1.

Repression of PDLIM2 expression in ER-positive or ER-negative breast cancer cells. A, Protein repression of PDLIM2 in breast cancer cells. Protein expressions of PDLIM2 in the indicated nontumorigenic breast epithelial cell lines, ER-positive breast cancer cell lines and ER-negative breast cancer cell lines were analyzed by immunoblotting using PDLIM2 specific antibody. Hsp90 was used as a loading control. B, RNA repression of PDLIM2 in breast cancer cells. The relative levels of PDLIM2 mRNAs in the indicated breast cancer cells were analyzed by real-time PCR using β-actin mRNA as a control and represented as percentile of that in HMT3522 S-1 cells (set as 100). The data presented are the mean ± S.D. (error bars) (n ≥3).

FIGURE 2.

PDLIM2 inhibition of NF-κB constitutive activation in breast cancer cells. A and B, dose-dependent inhibition of NF-κB constitutive activation in breast cancer cells by transiently expressed PDLIM2. MCF-7 and MDA-MB-231 cells were transfected with κb-TATA driven firefly luciferase reporter and thymidine kinase-driven Renilla luciferase reporter in the presence of increasing amounts of Myc-PDLIM2 followed by measure of luciferase activity. The luciferase activities were presented as the percentile of that in cells without PDLIM2 transfection (denoted as 100). The data presented are the mean ± S.D. (error bars) (n = 3). The protein expression levels of transfected myc-tagged PDLIM2 and endogenous Hsp90 were detected by direct immunoblotting using Myc antibody. C and D, inhibition of NF-κB constitutive activation in breast cancer cells by stably infected PDLIM2. MCF-7 and MDA-MB-231 cells stably expressing Myc-PDLIM2 or an empty vector were transfected with κb-TATA-driven luciferase reporter followed by measure of luciferase activity. The luciferase activities were presented as the percentile of that in the vector-expressing cells (denoted as 100). The data presented are the mean ± S.D. (error bars) (n = 3). The protein levels of stably expressed PDLIM2 were also examined by immunoblotting. E, inhibition of NF-κB-targeted genes by PDLIM2. The MCF-7 and MDA-MB-231 stably expressing cells described in D were also used for real-time PCR analysis to measure the expression levels of Bcl-2 and cyclin D1, which were represented as percentile of that in vector control cells (set as 100). The data presented are the mean ± S.D. (error bars) (n = 3). F, PDLIM2 inhibition of p65 in breast cancer cells. The transfected MDA-MB-231 cells were treated with 10 μm MG132 for the indicated time periods followed by subcellular fractionation and immunoblotting assays to determine p65 expression in the soluble nuclear fraction and insoluble nuclear fraction. Expression of Myc-PDLIM2, Sp1, and lamin B was also detected.

FIGURE 3.

Suppression of tumorigenicities of breast cancer cells by PDLIM2, but not its mutants defective in NF-κB termination. A and B, suppression of the in vitro anchorage-independent growth of human breast cancer cells by PDLIM2 but not its mutants defective in NF-κB termination. MCF-7 and MDA-MB-231 cells stably expressing empty vector, PDLIM2, or its LIM or PDZ deletion mutant were plated in soft agar, and colony numbers were counted at day 21 after plating. The data presented are the mean ± S.D. (error bars) (n ≥ 6; *, p < 0.05; **, p < 0.01). WT, wild type. C and D, suppression of the in vivo tumor formation of human breast cancer cells by PDLIM2 but not its mutants defective in NF-κB termination. The MCF-7 and MDA-MB-231 stable cell lines were subcutaneously inoculated into the mammary fat pads of the SCID mice for the tumor formation. For the MCF-7 cell inoculation, the mice were subcutaneously implanted with a slow release pellet of 25 mg of estrogen at least 3–4 days before the cell injection. The data presented are the mean ± S.D. (error bars) (n ≥ 4; **, p < 0.01). E and F, expression levels of stably infected PDLIM2 and its mutants. The indicated stable cell lines were examined by immunoblotting using anti-Myc antibody. Expression of endogenous Hsp90 protein was used as a loading control.

FIGURE 4.

DNA methylation-mediated repression of PDLIM2 in breast cancer cells. A, 5-aza-dC-mediated recovery of PDLIM2 expression in human breast cancer cells by RT-PCR analysis. MCF10A, MCF-7, and MDA-MB-231 cells were treated with 5 μm 5-aza-dC or vehicle for 48 h, followed by RT-PCR to determine mRNA levels of PDLIM2 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). B, 5-aza-dC-mediated recovery of PDLIM2 expression in human breast cancer cells by real time PCR analysis. PDLIM2 mRNA levels in the 5-aza-dC- or mock-treated cells were also analyzed by real-time PCR. The PDLIM2 inductions by 5-aza-dC are represented as percentile of that in mock-treated MCF10A cells (set as 100). The data presented are the mean ± S.D. (error bars) (n = 3). C, methylation of the PDLIM2 promoter in breast cancer cells. The indicated cell lines were treated with 5 μm 5-aza-dC or vehicle for 5 days, followed by the bisulfite genomic DNA sequencing. Each circle represents a CpG site; open circles represent unmethylated CpG dinucleotides, and filled circles represent methylated CpG sites. The ratios of the filled area in circles represent percentiles of the methylation in the CpG sites. The position of each CpG nucleotide relative to the PDLIM2 transcription initiation site (+1) is indicated at the top.

FIGURE 5.

In vitro and in vivo suppression of breast cancer cell tumorigenicities by 5-aza-dC. A and B, growth suppression of breast cancer cells by 5-aza-dC. MCF-7 and MDA-MB-231 cells were treated with 5 μm 5-aza-dC or vehicle for the indicated time points, followed by cell growth assay. The data presented are the mean ± S.D. (error bars) (n = 3). C and D, in vitro tumor suppression of breast cancer cells by 5-aza-dC. MCF-7 and MDA-MB-231 cells were treated with 5-aza-dC or vehicle for 48 h before being plated in soft agar. After plating, drug or vehicle diluted in culture medium was added on the top of agarose every 3 days. Colony growth was scored after 21 days. The data presented are the mean ± S.D. (error bars) (n = 3; **, p < 0.01). E, in vivo tumor suppression of breast cancer cells by 5-aza-dC. Female SCID mice were inoculated with MCF-7 or MDA-MB-231 at the mammary fat pads. The receiving mice were injected intraperitoneally with 5-aza-dC (5 mg/kg body weight) or vehicle upon cell inoculation. The 5-aza-dC administrations were repeated 48 h later for four more times. Of note, the mice inoculated with MCF-7 cell were subcutaneously implanted with a slow release pellet of 25 mg of estrogen prior to the cell injection. The data presented are the mean ± S.D. (error bars) (n ≥ 3; **, p < 0.01).