2-(4-Hydroxy-3-methoxyphenyl)-benzothiazole suppresses tumor progression and metastatic potential of breast cancer cells by inducing ubiquitin ligase CHIP

Breast cancer is the most common malignancy among women and has poor survival and high recurrence rates for aggressive metastatic disease. Notably, triple-negative breast cancer (TNBC) is a highly aggressive cancer and there is no preferred agent for TNBC therapy. In this study, we show that a novel agent, 2-(4-hydroxy-3-methoxyphenyl)-benzothiazole (YL-109), has ability to inhibit breast cancer cell growth and invasiveness in vitro and in vivo. In addition, YL-109 repressed the sphere-forming ability and the expression of stem cell markers in MDA-MB-231 mammosphere cultures. YL-109 increased the expression of carboxyl terminus of Hsp70-interacting protein (CHIP), which suppresses tumorigenic and metastatic potential of breast cancer cells by inhibiting the oncogenic pathway. YL-109 induced CHIP transcription because of the recruitment of the aryl hydrocarbon receptor (AhR) to upstream of CHIP gene in MDA-MB-231 cells. Consistently, the antitumor effects of YL-109 were depressed by CHIP or AhR knockdown in MDA-MB-231 cells. Taken together, our findings indicate that a novel agent YL-109 inhibits cell growth and metastatic potential by inducing CHIP expression through AhR signaling and reduces cancer stem cell properties in MDA-MB-231 cells. It suggests that YL-109 is a potential candidate for breast cancer therapy.

B reast cancer is the major cause of cancer death among women worldwide. Triple-negative breast cancer (TNBC), which has been reported to represent approximately 15% of all breast cancers 1 , is characterized by the absence of estrogen receptors (ERs), progesterone receptors (PRs), and human epidermal growth factor-2 (HER-2) expression 2 . TNBC is an aggressive cancer, characterized by rapid tumor growth, a high incidence of metastasis, an increased rate of distant recurrence, and a poor prognosis compared with other breast cancer subtypes 3 . Unlike ER/PR-positive or HER-2-overexpressing subtypes, the effective treatment options for TNBC are limited to cytotoxic therapies because of the lack of molecular targets. Moreover, TNBC cells show a profile that is similar to breast cancer stem cells, which have a strong resistance to chemotherapeutic drugs 4,5 . Therefore, new therapeutic options and strategies are required for TNBC therapy.
The carboxyl terminus of Hsp70-interacting protein (CHIP, also named STUB1) is a potential target for the treatment of TNBC. CHIP is a U-box-type ubiquitin E3 ligase that induces ubiquitylation and degradation of its substrates. These include several oncogenic proteins that suppress the tumorigenic and metastatic potential of breast cancer cells [6][7][8] . We previously reported that CHIP levels were much higher in MCF-7 cells, a non-aggressive cell line derived from human breast cancer cells, than in MDA-MB-231 cells, a highly aggressive cell line. Furthermore, CHIP levels are negatively correlated with the malignancy of human breast tumor tissues 9 . In addition, CHIP suppresses both tumor growth and metastasis in a nude mouse xenograft model. Thus, it has been suggested that the regulation of CHIP expression may represent a potential new clinical approach to TNBC therapy.
Aryl hydrocarbon receptor (AhR) has also recently emerged as a potential therapeutic target for breast cancer. The AhR is a basic helix-loop-helix transcription factor that was initially identified as a receptor for environmental toxins, such as dioxin 10 . Ligand binding to the receptor triggers formation of a heterodimeric nuclear AhR complex, which binds to dioxin response elements in target gene promoters to induce transcriptional activation 11 . Several studies have demonstrated that the AhR may be a potential drug target for several diseases, including endometrial, prostate, pancreatic, and ER-positive breast cancers [12][13][14][15][16][17] . In addition, the antitumor effects of compounds belonging to the 2-(4-amino-3-methylphenyl) benzothiazole group are mediated by AhR in ER-positive breast cancer cells [18][19][20] . Phortress, the lysine amide prodrug of 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole, has completed Phase I clinical evaluations 18,21 . In addition to 2-(4-aminophenyl) benzothiazoles, the relatively nontoxic selective AhR modulators (SAhRMs) are highly effective agents for inhibiting hormone-responsive breast cancer growth in animal models 17,22 . Although 2-(4-aminophenyl) benzothiazoles and SAhRMs are less effective against ER-negative breast cancer cells, AhR is also expressed in these cells 18,23,24 . Therefore, we hypothesized that ideal agents might exert the antitumor effects mediated by AhR signaling in both ER-positive and -negative breast cancer cells.
In this study, we demonstrated that the novel agent 2-(4-hydroxy-3-methoxyphenyl)-benzothiazole (YL-109) has ability to inhibit breast cancer progression in TNBC, MDA-MB-231 cells, and ERpositive breast cancer MCF-7 cells. In addition, YL-109 suppresses the proliferation and invasiveness of MDA-MB-231 cells, both in vitro and in vivo. Moreover, YL-109 suppresses the properties of breast cancer stem cells. Furthermore, we demonstrated that YL-109 increases CHIP expression by the recruitment of AhR to an upstream region of the gene. Consistent with these observations, CHIP or AhR knockdowns inhibit the suppressive effects of YL-109 on anchorage-independent growth and invasiveness. Taken together, our findings indicate that YL-109 is a novel antitumor agent that can induce CHIP expression through AhR signaling, and that it represents a promising candidate for a new therapeutic strategy against TNBC.

Results
YL-109 inhibits cell proliferation, motility, and invasiveness in breast cancer cells. It has been reported that 2-(4-aminophenyl)benzothiazoles have anti-proliferative activity in MCF-7, ER-positive breast cancer cells [18][19][20] . Therefore, we investigated the effects of 2-(4hydroxy-3-methoxyphenyl)-benzothiazole, YL-109 ( Figure 1a) on cell proliferation in MCF-7 cells. YL-109 possesses characteristic hydroxyl group at C4, whereas 2-(4-aminophenyl)-benzothiazoles have the amino group at this position. YL-109 strongly inhibited cell proliferation of MCF-7 cells in a dose-dependent manner (IC 50 5 85.8 nM) (Figure 1b and c). Surprisingly, YL-109 had an antiproliferative effect in a dose-dependent manner (IC 50 5 4.02 mM) on MDA-MB-231 cells, known as TNBC cells, unlike 2-(4aminophenyl)-benzothiazoles (Figure 1b and c) 18 . We subsequently tested whether YL-109 inhibited anchorage-independent growth in poly-HEMA coated plates and colony formation in soft agar (Figure 1d  YL-109 inhibits both tumor growth and cancer metastasis of breast cancer cells in vivo. Using a nude mouse xenograft model, we investigated the effects of YL-109 in vivo. Mice treated with vehicle showed significantly enlarged tumors, whereas mice treated with YL-109 showed attenuated tumor growth using MCF-7 cells (Figure 2a). Interestingly, YL-109 also suppressed tumor growth in mice injected with MDA-MB-231 cells (Figure 2b). Next, to examine the effect of YL-109 against metastatic activity, we performed an in vivo lung metastasis assay using MDA-MB-231 cells. We discovered several metastatic tumors in the lungs of mice injected with MDA-MB-231 cells in the vehicle (Figure 2c). Compared with the vehicle control, YL-109 significantly reduced lung metastasis (Figure 2c). We also quantified lung metastasis using real-time RT-PCR, which confirmed our observations (Figure 2d). These results suggest that YL-109 suppresses tumor progression of breast cancer cells in vivo.
YL-109 suppresses breast cancer progression by inducing CHIP expression. We previously demonstrated that CHIP suppresses tumorigenesis and the metastatic cellular phenotypes of breast cancer cells both in vitro and in vivo 9 . It has also been indicated that CHIP expression is significantly associated with prognostic parameters in breast cancer patients 25 . Therefore, we examined whether YL-109 induced CHIP expression. We observed that YL-109 increased both CHIP mRNA and protein levels (Figure 3a and b). Next, to investigate whether the antitumor effects of YL-109 were mediated by CHIP, we performed colony formation and Matrigel invasion assay using siRNA for CHIP (Figure 3c-e). YL-109-induced inhibition of cell growth and invasive potential were decreased by CHIP knockdown (Figure 3d and e). These results demonstrate that YL-109 inhibits breast cancer progression by inducing CHIP expression.
YL-109 activates AhR signaling to induce CHIP expression. Previous studies indicated that 2-(4-aminophenyl)-benzothiazoles activate AhR and increase transcription of AhR target genes, such as Cyp1a1 18-20 . We observed that YL-109 induced Cyp1a1 expression in MCF-7 and MDA-MB-231 cells ( Figure 4a). To explore AhR participation in the mechanisms underlying the induction of CHIP by YL-109, we examined whether CHIP expression levels were affected by knocking down of AhR with siRNA ( Figure 4b and c). We observed that YL-109-induced increases in CHIP mRNA were reduced by AhR knockdown (Figure 4c). We subsequently examined AhR recruitment to the promoter region in CHIP gene, which contains potential AhR response elements. Chromatin immunoprecipitation (ChIP) assay demonstrated that the interaction between AhR and the CHIP promoter is potentiated by YL-109 ( Figure 4d). Our data suggest that YL-109 induces CHIP expression through recruitment of AhR to upstream of CHIP gene.
YL-109 exerts antitumor effects through AhR signaling. To examine whether AhR signaling mediated the inhibition of cell proliferation by YL-109, we performed MTT assay using the AhR antagonist a-naphtoflavone (a-NF) and siRNA against AhR (Figure 5a (Figure 6c). Taken together, these results indicate that YL-109 inhibits the properties of breast cancer stem cells.

Discussion
In the present study, we revealed that the novel agent YL-109 has antitumor activity in TNBC cells. It was previously reported that 2-(4-aminophenyl)-benzothiazoles have anti-proliferative activity in ER-positive breast cancer cells, that is mediated by the AhR signaling pathway. AhR is expressed in breast cancer cells regardless of ER expression 23,24 . However, TNBC cells exhibit a poor response to benzothiazoles and SAhRMs because they do not activate AhRsignaling 18 . Consistent with previous reports on benzothiazole antitumor activity, YL-109 inhibited cell proliferation through AhR activation in ER-positive breast cancer cells. In addition, we observed that YL-109 could activate AhR signaling in MDA-MB-231 cells, which is necessary for YL-109 to exert antitumor effects in these cells. These results suggest that YL-109 inhibits breast cancer progression by AhR signaling activation in TNBC cells.
Our results also demonstrated that the YL-109-induced AhR signaling activation resulted in increased CHIP expression. We previously reported that CHIP suppresses tumor progression in human breast cancer by inhibiting oncogenic pathways 9 . In mice, tumor growth and metastasis were significantly inhibited by CHIP expression, whereas CHIP knockdowns in breast cancer cells resulted in rapid tumor growth and metastatic phenotypes. In this study, we observed that YL-109 treatment increased CHIP expression and CHIP knockdown inhibited the suppressive effects of YL-109 on both breast cancer cell growth and invasiveness. Moreover, YL-109 induced CHIP expression by recruiting AhR to an upstream region of the CHIP gene. We also found that the multiple AhR-responsive element sites exist in the promoter region of CHIP, which contain the core sequence 59-GCGTG-39. Taken together, these findings indicate www.nature.com/scientificreports that YL-109 inhibits breast cancer progression by inducing CHIP expression through AhR signaling. Our data demonstrated that YL-109 increased CHIP levels in MDA-MB-231 cells. We also observed that YL-109 inhibited anchorage-independent cell growth in soft agar as well as xenograft tumor growth, but not in poly-HEMA coated plates. For assays of in vitro cell growth, cells were detached and suspended, either in poly-HEMA coated plates for 24 h, or in soft agar for 3 weeks. In shortterm growth experiments such as the poly-HEMA assay, we did not observe cell growth inhibition by YL-109, because YL-109-induced inhibition of cell growth presumably requires the elevation of CHIP levels. Consistent with this, in long-term growth experiments such as soft agar colony formation and xenograft tumor growth, YL-109 inhibited cell growth of TNBC cells. Furthermore, this effect was repressed by the knockdown of CHIP or AhR in TNBC cells.
In contrast, CHIP levels are considerably higher in MCF-7 cells than in MDA-MB-231 cells, and we observed that YL-109 did not affect CHIP expression in MCF-7 cells (data not shown). However, YL-109 was able to inhibit the growth of MCF-7 cells in both shortterm and long-term growth experiments. These observations suggest that CHIP does not contribute to the effects of YL-109 on cell growth in ER-positive breast cancer cells.
Previous studies have reported that the inhibition of ER signaling can result from cross talk with the ligand-activated AhR. AhR ligands strongly suppress estrogen-induced responses in the rodent uterus, mammary tumors, and human breast cancer cells 28 . Treatment of ER-positive breast cancer cells with 2,3,7,8-tetrachlorodeibenzo-pdioxin (TCDD) induces proteasome-dependent degradation of endogenous ERa 29 . In this study, we demonstrated that YL-109 inhibits cell proliferation through the AhR signaling pathway in MCF-7 The treatment options for patients with TNBC, including those with ER-negative breast cancer, have not yet been established. In this study, we have demonstrated that YL-109 has antitumor activity in TNBC cells, and thus represents a potential TNBC therapy. In addition, resistance to anti-estrogens like tamoxifen is a major clinical problem in the treatment of hormone-dependent breast cancer including ER-positive breast cancer. Our results indicated that YL-109 inhibited tumor growth using a different mechanism from anti-estrogens in ER-positive breast cancer cells. Therefore, we predict that YL-109 will also exert an antitumor effect in tamoxifenresistant breast cancer. Thus, YL-109 might have the ability to treat with several subtypes of breast cancer.
Drug resistance is a serious problem in breast cancer therapy, and cancer stem cells contributed to chemotherapeutic drug and radiation resistance 30 . Breast cancer stem cells form mammospheres and express stem cell markers. We observed that YL-109 markedly inhibited mammosphere formation and decreased the ALDH-positive cell population in MDA-MB-231 cells. Moreover, YL-109 significantly decreased Klf-4 and Hes1 expression in mammospheres derived from MDA-MB-231 cells. KLF-4 is essential for the maintenance of breast cancer stem cells and cell invasiveness 31 , and Hes1 is a target of Notch signaling, which may be important for the self-renewal of breast cancer stem cells 32,33 .  In summary, we demonstrated that YL-109 is a novel antitumor agent that suppresses tumor progression in TNBC cells and inhibits cancer stem cell properties, unlike other benzothiazoles. Furthermore, we show that YL-109 induces CHIP expression through AhR signaling. Taken together, we propose a new therapeutic strategy for breast cancer including TNBC, and prodrug of YL-109 improved water solubility and chemically stability may be helpful in development of new pharmacological treatments for TNBC.

Methods
Cell culture. MCF-7, MDA-MB-231 and BT-20 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) or RPMI1640 supplemented with 10% fetal bovine serum (FBS). For the experiments, cells were seeded in phenol red-free DMEM containing 4% charcoal-stripped FBS. After 24 h, the medium was exchanged for phenol red-free DMEM containing 1% charcoal-stripped FBS with or without ligands. Stealth TM RNAi reporter control was used as a negative control.
MTT assay. Cells were incubated in DMEM containing 4% charcoal-stripped FBS with DMSO or YL-109 (1 mM) for 96 h. The proliferation of cultured cells was measured by MTT assay using the MTT Cell Count Kit (Nakarai tesque).
Poly-HEMA. One gram of poly-(2-hydroxyethyl methacrylate) (poly-HEMA) (Sigma-Aldrich) was dissolved in 25 mL 99.5% ethanol and mixed overnight at 37uC. The poly-HEMA stock solution were added to 12-well plates and plates were left to dry for a few hours. After drying, the plates were washed with PBS. Cells were plated in the poly-HEMA-coated 12-well plates at a density of 1 3 10 5 cells (MCF-7) or 1.  In vivo lung metastasis analysis. BALB/cAjcl-nu/nu female mice at 4-5 weeks of age were purchased from CLEA Japan. The tail vein of each mouse was injected with MDA-MB-231 cells (5 3 10 5 cells). Forty-two days after injection, the mice were sacrificed and lung metastases were examined by hematoxylin-eosin (H&E) staining and immunohistochemistry for human cytokeratin and quantified by real-time RT-PCRs using primers specific for human HPRT that did not cross-react with the mouse homologue. Mouse b-actin was used for normalization.
Immunohistochemistry. Immunohistochemistry was performed under contract with Genostaff Co., Ltd. Tissues were fixed, dehydrated and embedded in paraffin  were cut at 5 mm thickness. For antigen retrieval, the sections were incubated with EDTA buffer [10 mM Tris-HCl and 1 mM ethylenediaminetetraacetic acid (EDTA)] (pH 9.0) at 95uC for 20 min, and then treated with 0.3% hydrogen peroxide. After blocking, the sections were incubated with anti-human-cytokeratin clone MNF-116 (Abcam) (10 mg/ml, overnight at 4uC). Immunostaining was performed with EnVision TM system (Dako). Staining of H&E was performed by standard protocol.
Real-time RT-PCR. Tissues and cells were homogenized in 1 mL of Sepazol and total RNA was extracted according to the manufacturer's instructions (Nacalai tesque). cDNA was synthesized from total RNA using RevaTraAce reverse transcriptase (Toyobo) and oligo dT primer. Real-time PCRs were performed to amplify fragments representing for the indicated mRNA expression using the Thermal Cycle DiceTM TP800 (Takara) and SYBR Premix Ex Taq (Takara). The primer sequences can be found in Supplementary Table S1.
Chromatin immunoprecipitation (ChIP) assay. This was done essentially as described previously 34 . The DNA was amplified by real-time PCR as described above.
The primers for real-time PCR are as follows: forward primer: 59-TCACATGCTTCTCTGCTCTG-39; reverse primer: 59-GACTGTTGGTAGAGTGGAAG-39 for CHIP gene upstream region. Samples were normalized based on the amount of input DNA.
Mammosphere formation assay. MDA-MB-231 cells were plated onto 6-well ultralow-attachment plates (Corning Costar) at 5 3 10 3 cells per well. Cells were maintained in serum-free with a CnT-27 medium and growth additives (CellnTEC Advanced cell systems) 27 . After 7 days, over 100 mm spheres were counted.
ALDH assays. The ALDEFLUOR kit (Stem Cell Technologies, Durham, NC, USA) was used to detect cancer stem cell population with high aldehyde dehydrogenase (ALDH) enzyme activity, as previously reported 35 . Briefly, MDA-MB-231 cells were treated with YL-109 (1 mM) for 4 days. The cells were then incubated in ALDH assay buffer containing the ALDH substrate (BAAA, 1 mM) for 30 min at 37uC. As a negative control, cells were stained under identical conditions in the presence of diethylaminobenzaldehyde (DEAB, 15 mM), a specific ALDH inhibitor. FACS Aria II cell sorter (BD Biosciences) was used to measure the ALDH-positive cell population.
Statistical analysis. Data are representative of at least three different experiments. Significance of differences was determined by Student t-test analyses or by two-way ANOVA with Bonferroni's post hoc test (tumor xenograft experiments).