A novel cell-penetrating peptide suppresses breast tumorigenesis by inhibiting β-catenin/LEF-1 signaling

The inhibition of β-catenin/LEF-1 signaling is an emerging strategy in cancer therapy. However, clinical targeted treatment of the β-catenin/LEF-1 complex remains relatively ineffective. Therefore, development of specific molecular targets is a key approach for identifying new cancer therapeutics. Thus, we attempted to synthesize a peptide (TAT-NLS-BLBD-6) that could interfere with the interaction of β-catenin and LEF-1 at nuclei in human breast cancer cells. TAT-NLS-BLBD-6 directly interacted with β-catenin and inhibited breast cancer cell growth, invasion, migration, and colony formation as well as increased arrest of sub-G1 phase and apoptosis; it also suppressed breast tumor growth in nude mouse and zebrafish xenotransplantation models, showed no signs of toxicity, and did not affect body weight. Furthermore, the human global gene expression profiles and Ingenuity Pathway Analysis software showed that the TAT-NLS-BLBD-6 downstream target genes were associated with the HER-2 and IL-9 signaling pathways. TAT-NLS-BLBD-6 commonly down-regulated 27 candidate genes in MCF-7 and MDA-MB-231 cells, which are concurrent with Wnt downstream target genes in human breast cancer. Our study suggests that TAT-NLS-BLBD-6 is a promising drug candidate for the development of effective therapeutics specific for Wnt/β-catenin signaling inhibition.

In the current study, we synthesized a small peptide to block the interaction between β -catenin and LEF-1. The effects on tumorigenesis and the downstream target gene profile in human breast cancer were investigated in vitro and in vivo. This novel small peptide may be a candidate in future approaches for human breast cancer therapy.

TAT-NLS-BLBD-6 inhibits the growth of breast cancer cells.
First, we synthesized successive short peptides of the β-catenin/LEF-1 binding domain 11 (BLBD), transactivator of transcription (TAT, YGRKKRRQRRR), and nuclear localization signal (NLS, RKRRK) protein to form a fusion peptide (Fig. 1a). BLBD peptides were derived from the first 76 amino acids of LEF-1, which are sufficient for the interaction with β -catenin. TAT is a cell-penetrating peptide from the human immunodeficiency virus, and it can deliver proteins, DNA, RNA, and nanoparticles into the cytoplasm in a short time with extremely high efficiency 19,20 . However, because stabilized β -catenin translocates into the nucleus to affect TCF-4/LEF-1 binding to Wnt target genes 21 , we synthesized TAT-NLS fusion peptides derived from LEF-1 to analyze their effects on β -catenin-mediated signaling in the nuclei of breast cancer cells. We chemically synthesized six peptides with a variable region of the β-catenin/LEF-1 binding domain and one mutated version of BLBD-6 to find the capacity of these peptides to suppress cancer cell growth (Fig. 1a). A growth assay was used to screen the small peptides for their ability to suppress cancer cell growth. The results indicated that two synthetic peptides (TAT-NLS-BLBD-3 and -6) inhibited the growth of MCF-7 and MDA-MB-231 cells ( Fig. 1b; also see Supplementary Fig. S1 online). BLBD-3 and BLBD-6 share a common sequence in the activation region of the β-catenin/LEF-1 binding domain (ATDEMIPF); thus, we mutated the activation region (GTDEAAAA, TAT-NLS-BLBD-6m) and found that TAT-NLS-BLBD-6m did not affect the growth of breast cancer cells compared with TAT-NLS-BLBD-6 ( Fig. 1b). Therefore, the activation region sequence (ATDEMIPF) is responsible for the growth inhibition of human breast cancer cells.
Next, we analyzed the time and dose dependence of TAT-NLS-BLBD-6 on cell growth. The results indicated that TAT-NLS-BLBD-6 inhibited breast cancer cell growth in a time-and dose-dependent manner (Fig. 1c,d).
We also examined the effect of combining BLBD-6 with drugs including E2 (1 μ M), BBP (1 μ M), and TAM (1 μ M) with BLBD-6 in breast cancer cells. TAT-NLS-BLBD-6 blocked the activity of E2 and BBP, but increased the response of TAM in breast cancer cells (Fig. 1e). Interestingly, TAT-NLS-BLBD-6 did not inhibit the growth of human normal mammary epithelial cell H184B5F5/M10 and embryonic kidney 293 (HEK293) cells (Fig. 1f). Together, these results suggested that the ATDEMIPF sequence of TAT-NLS-BLBD-6 inhibits growth of breast cancer cells, but not of normal cells such as H184B5F5/M10 and HEK293.
TAT-NLS-BLBD-6 specifically binds to β-catenin in the nucleus. Although TAT-NLS-BLBD-6 inhibited the growth of breast cancer cells, it was not clear whether TAT-NLS-BLBD-6 could enter into the nucleus and bind the β-catenin elements in vitro. We first determined the subcellular distribution of TAT-NLS-BLBD-6 peptide in the cell. Immunofluorescence staining indicated that TAT-NLS-BLBD-6 (100 μ mol/l) was located in the nuclei in both breast cancer cell lines (Fig. 2a). Next, we analyzed the peptide-protein interaction by immunoprecipitation and PLA assay. These assays indicated that TAT-NLS-BLBD-6 bound to the β -catenin elements in MCF-7 and MDA-MB-231 cell nuclei (Fig. 2b,c).

TAT-NLS-BLBD-6 induces apoptosis and inhibits invasion, migration, and colony formation.
To further investigate the biological effects of these peptides, we evaluated apoptosis, invasion, migration and colony formation. We used flow cytometry and the TUNEL assay to examine the effects of control, TAT-NLS-BLBD-6 and TAT-NLS-BLBD-6m apoptosis. Flow cytometry showed that, compared with control and TAT-NLS-BLBD-6m, TAT-NLS-BLBD-6 increased the sub-G1 phase region (pro-apoptotic effect) from 7.35% to 37.41% in MCF-7 and from 18.34% to 43.10% in MDA-MB-231 (Fig. 3a). The TUNEL assay showed that TAT-NLS-BLBD-6 also increased the effect of binding with BrdU and DNA fragmentation when compared with control or TAT-NLS-BLBD-6m in breast cancer cells (Fig. 3b). In addition, invasion, migration and colony-formation assays indicated that TAT-NLS-BLBD-6 inhibited the mobility and proliferation of breast cancer cells when compared with control or TAT-NLS-BLBD-6m ( Fig. 3c-e). We used normal cell lines to analyze the phenotypic features of TAT-NLS-BLBD-6. The results revealed that TAT-NLS-BLBD-6 had no effect on the phenotypic features including cell cycles, apoptosis, invasion, migration and colony formation assay in normal cell lines H184B5F5/M10 ( Supplementary Fig. S2 online). Therefore, TAT-NLS-BLBD-6 has the potential to mediate biological function in human breast cancer cells.

TAT-NLS-BLBD-6 inhibits tumor growth in the xenograft and xenotransplantation models.
To evaluate the effects of the TAT-NLS-BLBD-6 peptides in vivo, we established a xenograft model in nude mice and a zebrafish xenotransplantation model. For the xenograft model, 1 × 10 7 MCF-7-YFP and MDA-MB-231-GFP cells were subcutaneously injected into the right flanks of nude mice. After 1 week of implantation, the mice were treated with the control, TAT-NLS-BLBD-6m (1 mg/kg) and TAT-NLS-BLBD-6 (1 and 10 mg/kg) peptide through intratumoral injection, and the fluorescence density was analyzed by In Vivo Imaging System (IVIS) 35 days after inoculation. TAT-NLS-BLBD-6 inhibited tumor growth without having any effect on body weight when compared with the control peptide ( Fig. 4a,b, also see Supplementary Fig. S3 online). In addition, we obtained tumor sections and confirmed that they originated from the injected breast cancer cells, which were positive for YFP or GFP. Immunohistochemistry staining revealed that TAT expression was high and located in the nuclei in the tumors injected with TAT-NLS-BLBD-6 compared with those injected with control peptide (Fig. 4c).
Various preclinical approaches have been used to inhibit Wnt/β-catenin signaling pathways in cancer progression. These modalities, including protein depletion by a neutralizing anti-Dickkopf-1 (DKK1) antibody 31 , the soluble antagonist secreted Frizzled-related protein 2 (sFRP-2) 15,32 , and disheveled PDZ peptides 33 , inhibit tumor growth. Our results found that TAT-NLS-BLBD-6 inhibited cancer cell growth, invasion, and migration in vivo and in vitro, but not in normal cells. Therefore, we believe that using TAT-NLS-BLBD-6 (ATDEMIPF) peptide may be an effective therapeutic approach for human breast cancer without harm to normal cells. Consistent with our finding regarding the role of the interaction binding sequence, A 17 TDEMIPF 24 , structure analysis found that the LEF-1 residues responsible for binding to β -catenin are Asp19, Met21, Ile22, and Phe24 34 . The ATDEMIPF had the greatest effect on progression of human breast cancer compared with other sequences.
Previous investigations have reported that environmental hormone factors, such as phthalates (e.g., BBP), stimulate breast cancer through activation of the Wnt/β-catenin signaling pathway that contributes to tumorigenesis 35 and epithelial-mesenchymal transition 36 . Phthalates are important in breast cancer progression; they can induce translocation of β -catenin into the nucleus and activate the response transcription factors LEF-1/ TCF to induce downstream target gene expression. Therefore, developing new drugs to block Wnt/β-catenin signaling will inhibit cancer progression induced by phthalates. In the present study, we demonstrated that TAT-NLS-BLBD-6 combined with BBP blocked cell growth induced by BBP. This result suggests that TAT-NLS-BLBD-6 is an inhibitor for breast cancer progression induced by phthalates.
HER-2 is a tyrosine-protein kinase 2 of the ErbB family 37 . Early study found that the β-catenin/LEF-1 was activated by HER-2 to induce proliferation, invasion and migration and survival of cancer cells 38,39 . In addition, IL-9 is a cytokine produced by T-cells 40 , which was the down-regulated gene of Wnt 41 . Interesting, we results showed that the TAT-NLS-BLBD-6 downstream target genes were associated with IL-9 and HER-2 signaling pathways. This global gene profiles and IPA software results were confirmed with previous study and demonstrate that TAT-NLS-BLBD-6 is an inhibitor of β-catenin/LEF-1 signaling pathway.
In summary, our study found that TAT-NLS-BLBD-6, a cell-penetrating pentapeptide, blocks the interaction between β -catenin and LEF-1 and also efficiently attenuates tumorigenesis in vitro and in vivo. These results suggest that TAT-NLS-BLBD-6 is an effective Wnt signaling inhibitor and may be a potential therapeutic agent of human breast cancer.
Immunoprecipitation and western blotting. Immunoprecipitation and western blotting were performed as described previously 42,43 . MCF-7 and MDA-MB-231 cells were harvested in 4 °C phosphate-buffered saline and cell pellets were lysed with RIPA lysis buffer (Millipore, Bedford, MA, USA) for 30 min on ice. Cell lysis supernatant liquid was obtained by centrifugation at 10,000 × g for 10 min, incubated with protein-G beads (Roche, Indianapolis, IN) and anti-β -catenin, and subjected to western blotting. For the western blotting assay, cellular extract proteins were separated by SDS-polyacrylamide gel (SDS-PAGE) and transferred to nitrocellulose membrane (Millipore) using a dry transfer apparatus (Bio-Rad). After blocking nonspecific binding with 5% Scientific RepoRts | 6:19156 | DOI: 10.1038/srep19156 milk buffer, the membrane was incubated with primary antibodies: anti-TAT (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-LEF-1(Epitomics, Burlingame, CA, USA) and anti-β -catenin (Epitomics, Burlingame, CA, USA). The proteins were visualized using ECL (Amersham Pharmacia Biotech) and coupled using the Bio-Rad Chemiluminescent Detection System.

Immunofluorescence, TUNEL staining, and proximity ligation assay (PLA). MCF-7 and
MDA-MB-231 cells were cultured in 35-mm plates with cover slides on the plate bottom and treated with NLS-BLBD-6, TAT-BLBD-6 and TAT-NLS-BLBD-6 peptide for 24 hr. Subsequently, cells were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100 for 20 min at room temperature. For the immunofluorescence assay, the slides were incubated with primary antibodies, anti-β -catenin (Epitomics) and anti-TAT (Santa Cruz), for 6 hr, and Alexa-488 fluorescence secondary antibodies for 1 hr. DAPI (Sigma, St Louis, MO) was used to stain the nucleus for 1 min at room temperature. For the TUNEL assay, apoptosis was analyzed by the Apo-BrdU-red DNA Fragmentation Assay kit, according to the manufacturer's protocol (BioVision, Mountain View, CA, USA). For the PLA, the protein-protein interaction assay was analyzed by Duolink ® using PLA ® Technology, according to the manufacturer's protocol (Olink Bioscience, Uppsala, Sweden). Briefly, the slides were incubated with primary antibodies, anti-β -catenin (Epitomics) and anti-TAT (Santa Cruz), and secondary antibodies, Duolink PLA Rabbit MINUS and PLA Mouse PLUS proximity probes. Finally, the proximity ligation was performed by the Duolink detection reagent kit (Olink Bioscience). The immunofluorescence image and TUNEL staining was photographed by a microscope (IX-71, Olympus, Tokyo, Japan).
Cell cycle analysis. MCF-7 and MDA-MB-231 cells were plated in 6-well plates for 24 hr, and the medium was replaced with fresh culture medium containing 100 μ mol/l peptide. After incubation for 24 hr, the cells were harvested by trypsinization and then fixed with 70% 4 °C ethanol. Intracellular DNA was stained with 50 ng/ml propidium iodide in the dark for 30 min at room temperature, and the percentages of sub-G1 cells were determined by flow cytometry (BD LSRII analyzer; BD Biosciences). Tumor growth analysis in vivo. All animal experiments were approved by the Kaohsiung Medical University Institutional Animal Care and Use Committee (IACUC Approval No: 101156) and we accordance with the approved guidelines. Female nude mice (4-5 weeks old) were obtained from the National Laboratory Animal Center (Taipei, Taiwan). Xenograft tumor models were established by subcutaneous injection of 1 × 10 7 MCF-7-YFP or MDA-MB-231-GFP cells into the right flanks of mice. Mice with palpable tumors were randomly divided into three groups with five animals in each group. The mice were treated with control peptide, 0, 1 and 10 mg/kg TAT-NLS-BLBD-6 by intratumoral injection once every 2 days for 35 days. The fluorescence density was analyzed by an In Vivo Imaging System (IVIS) (Berthold Technologies, Bad Wildbad, Germany), and the tumor volumes (V) of nude mice were calculated by: V = length × diameter 2 × 0.5. For zebrafish xenotransplantation, zebrafishes (Danio rerio) were maintained at 28 °C in an air incubator, and 1 × 10 4 MCF-7-GFP or MDA-MB-231-GFP cells combined with TAT-NLS-BLBD-6 or control peptide were injected into the zebrafish embryos according to the previously described protocol 44 . The fluorescence density was measured 24 and 28 hr after injection using an epifluorescence microscope.

Invasion, migration, and colony-formation assays.
Histologic study of the tumor. Tumor tissues were sectioned to a thickness of 5 μ m and mounted on microscope slides. Tissue slides were stained with a Dako LSAB kit (Dako, Carpinteria, CA) according to the manufacturer's protocol. The TAT antibody (Santa Cruz) was used for immunohistochemistry, and the nuclei were stained with hematoxylin and eosin. The fluorescence images were captured by a fluorescence microscope (IX-71, Olympus, Tokyo, Japan).
Human oligonucleotide DNA microarray. Total RNA was extracted from cells using the Trizol reagent (Invitrogen, Carlsbad, CA, USA). The RNA concentration and purity were checked by OD 260 /OD 280 (> 1.8) and OD 260 /OD 230 (> 1.6), and the yield and quality were accessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The Human Whole Genome OneArray ® v6 (Phalanx Biotech Group, Taiwan) contains 32,679 DNA oligonucleotide probes, and each probe is a 60-mer designed in the sense direction. Among the probes, 31,741 probes correspond to the annotated genes in the RefSeq v51 and Ensembl v65 databases. In addition, 938 control probes are also included. The detailed descriptions of the gene array list are available from http://www.phalanx.com.tw/Products/HOA_Probe.php Scientific RepoRts | 6:19156 | DOI: 10.1038/srep19156 Microarray analysis. Fluorescent aRNA targets were prepared from 1 μ g total RNA samples using the OneArray ® Amino Allyl aRNA Amplification kit (Phalanx Biotech Group, Taiwan) and Cy5 dyes (Amersham Pharmacia, Piscataway, NJ, USA). Fluorescent targets were hybridized to the Human Whole Genome OneArray ® with Phalanx hybridization buffer using the Phalanx Hybridization System. After 16 hr of hybridization at 50 °C, non-specific binding targets were washed away by three different washing steps (wash I 42 °C 5 min; wash II 42 °C, 5 min, 25 °C 5 min; wash III rinse 20 times), and the slides were dried by centrifugation and scanned by an Agilent G2505C scanner (Agilent Technologies, Santa Clara, CA, USA). The Cy5 fluorescence intensities of each spot were analyzed by GenePix 4.1 software (Molecular Devices). The signal intensity of each spot was loaded into the Rosetta Resolver System ® (Rosetta Biosoftware) to process data analysis. The error model of Rosetta Resolver System ® could remove both systematic and random errors from the data. We filtered out spots for which the flag was less than 0. Spots that passed the criteria were normalized by the 50% media scaling normalization method.
The technically repeated data were tested by the Pearson correlation coefficient calculation to check the reproducibility (R value > 0.975). Normalized spot intensities were transformed to gene expression log 2 ratios between the control and treatment groups. The spots with log 2 ratio ≥ 1 or log 2 ratio ≤ −1 and P-value < 0.05 were tested for further analysis.
Quantitative RT-PCR. Total RNA was isolated by Trizol reagent (Invitrogen, Carlsbad, CA, USA) and reverse transcribed to produce cDNA using the Deoxy + HiSpec reverse transcriptase kit (Yeastern, Taipei, Taiwan) according to the manufacturer instructions. Q-PCR was analyzed with SYBR Green Master Mix (Applied Biosystems, Stockholm, Sweden) and subjected to quantitation in an Applied Biosystems LightCycler instrument. The primers used for PCR are given in Supplementary Table S5 online. The Q-PCR data were normalized to 18S cDNA levels.