A novel biphenyl urea derivate inhibits the invasion of breast cancer through the modulation of CXCR4

The increased migration and invasion of breast carcinoma cells are key events in the development of metastasis to the lymph nodes and distant organs. CXCR4, the receptor for stromal-derived factor-1, is reportedly involved in breast carcinogenesis and invasion. In this study, we investigated a novel biphenyl urea derivate, TPD7 for its ability to affect CXCR4 expression as well as function in breast cancer cells. We demonstrated that TPD7 inhibited the breast cancer proliferation and down-regulated the CXCR4 expression on breast cancer cells both over-expressing and low-expressing HER2, an oncogene known to induce the chemokine receptor. Treatments with pharmacological proteasome inhibitors partial suppressed TPD7-induced decrease in CXCR4 expression. Real-time PCR analysis revealed that down-regulation of CXCR4 by TPD7 also occurred at the translational level. Inhibition of CXCR4 expression by TPD7 further correlated with the suppression of SDF-1α-induced migration and invasion in breast tumour cells, knockdown of CXCR4 attenuated TPD7-inhibitory effects. In addition, TPD7 treatment significantly suppressed matrix metalloproteinase (MMP)-2 and MMP-9 expression, the downstream targets of CXCR4, perhaps via inactivation of the ERK signaling pathway. Overall, our results showed that TPD7 exerted its anti-invasive effect through the down-regulation of CXCR4 expression and thus had the potential for the treatment of breast cancer.


Introduction
Chemokines are expressed by many tumour types and can promote mitosis, modulate apoptosis, survival, and angiogenesis [1,2]. Stromal cell-derived factor-1(SDF-1 or CXCL12), which belongs to the CXC chemokine subfamily, is produced in two forms, SDF-1a (CXCL12a) and SDF-1b (CXCL12b), by alternative splicing of the SDF-1 gene. Interaction between the chemokine receptor CXCR4 and its ligand, SDF-1a, have also been shown to be involved in metastasis of several tumours [3][4][5][6][7]. CXCR4 over-expressing in human breast cancer tissues was linked to the nodal spread of breast cancer. In addition, the receptor has also been associated with metastatic disease and poor disease free survival [3]. The SDF-1a-CXCR4 interaction promotes tumour progression by several possible mechanisms. CXCR4/SDF-1 interactions trigger the activation of many downstream pathways, including Ca 2+ influx, activation of the MAPK/ERK-1/2 pathway, activation of phosphatidylinositol 3-kinase and Akt, as well as increased NF-jB (nuclear factor kappa-light-chain-enhancer of activated B cells) activity [8][9][10][11]. Because of its involvement in both metastasis and primary tumour growth, CXCR4 is an ideal target to investigate novel therapeutic interventions. Some studies have successfully shown that blockade of CXCR4 or CXCR4/SDF-1 interactions by siRNA and chemical inhibitors suppressed cancer cell proliferation, invasion and metastasis.
In the present studies, we investigated the effect of TPD7 (N-(4 0 -acetyl-3 0 ,5,6-trimethoxybiphenyl-3-yl)-N 0 -[4-(3-morpholin-4-ylpro poxy)phenyl]urea) as a novel regulator of CXCR4 expression and function in breast cancer. TPD7 was a novel biphenyl urea taspine derivatives designed and synthesized using dissection strategies in our laboratory [12] (Fig. 1A). Taspine was a natural alkaloid originally identified by screens of Radix et Rhizomaleonticis (Hong Mao Qi in Chinese) using cell membrane chromatography [13]. It has many pharmacologic actions such as bacteriostasis, antibiosis, antivirus, anti-inflammatory, antiulcer effects [14][15][16]. Previously, we found that taspine displayed anticancer and antiangiogenesis properties [17,18]. As one of taspine derivatives, TPD7 displayed significant inhibitory activity on proliferation of several different cancer cell lines. Because CXCR4 was known to mediate proliferation, invasion and metastasis of tumour cells, in this study, we investigated whether TPD7 could modulate the expression of CXCR4 and thus inhibited breast tumour cell proliferation and invasion.

Colony formation assay
MDA-MB-435s, MDA-MB-231 and SK-BR-3 cells were cultured in 6-well plates and fresh medium with or without TPD7 were added for 10-15 days. Colonies with cell numbers of >50 cells per colony were counted after staining with crystal violet solution. All the experiments were performed in triplicate wells in three independent experiments.

Western blot analysis
The MDA-MB-435s and MDA-MB-231 cells treated with or without TPD7 for 48 hrs were prepared by extracting proteins with RIPA lysis  buffer containing protease inhibitor cocktail and phosphatase inhibitor cocktail on ice. Cell lysates were analysed for Western blot analysis with primary antibodies, followed by enhanced chemiluminescence. Blots were reprobed with GAPDH to compare protein load in each lane.

RNA extraction and PCR analysis
Total RNA of MDA-MB-435s and MDA-MB-231 cells treated with or without TPD7 were isolated using total RNA extracted kit according to the manufacturer's protocol. The RT-PCR was performed with PrimeScript RT Master Mix Perfect Real Time kit (TaKaRa DRR036A). Real-time PCR was performed with SYBR â Premix Ex TaqTM II and a Thermal Cycle Dice Real time system (TaKaRa). The result was analysed using the manufacturer's program (Thermal Cycler Dice TM Real Time System). The primer sequences were as following: GAPDH forward primer: 5 0 -GCACCGTCAAGGCTGAGAAC-3 0 ; GAPDH reverse primer: 5 0 -TGGT GAAGACGCCAGTGGA-3 0 ; CXCR4 forward primer: 5 0 -CCTGCCTGGTAT TGTCATCCTG-3 0 ; CXCR4 reverse primer: 5 0 -ACTGTGGTCTTGAGGGC CTTG-3 0 . Melt curve analysis was performed at the end of each PCR to confirm the specificity of the PCR product. Threshold cycle (Ct) values of CXCR4 in each sample were normalized with the GAPDH expression.

Wound healing assay
MDA-MB-435s and MDA-MB-231 cells were planted into 6-well plate and allowed to grow to 70% confluency in complete medium. Cells were then serum starved for 24 hrs, and cell monolayers were scratched with a pipette tip. Wounded monolayers were then washed several times with serum-free medium to remove floating cells and photographed in microscope. Cells were incubated in medium in the absence or presence of TPD7 for 48 hrs. After incubation, the growth medium was then changed to basal medium with or without 100 ng/ml SDF-1a. After 24 hrs, cell migrating into the wound surface and the average distance of migrating cells was determined under an inverted microscope.

Invasion assay
Cancer cells were suspended in medium and seeded into the Millicell chambers with polycarbonate membranes of 8-lm pore size coated with 100 ll 1 mg/ml Matrigel (Becton Dickinson, Franklin Lakes, NJ, USA). After preincubation with or without TPD7 for 48 hrs, Millicell chambers were then placed into 24-well plates in which were added the basal medium only or basal medium containing 100 ng/ml SDF-1a. After incubation, the upper surface of Millicell chambers was wiped off with a cotton swab and invading cells were fixed with 100% methanol and then stained with 0.2% crystal violet (Beijing Chemical Works, Beijing, China). The invading cell numbers were counted in five randomly selected microscope fields (9100).

siRNA analysis
A double-stranded siRNA against CXCR4 and nonspecific siRNA (control siRNA) were obtained from Shanghai GenePharma Co., Ltd.
(Shanghai, China) MDA-MB-435s and MDA-MB-231 cells were seeded in a 6-well plate and transfected with the siRNA against CXCR4 for 24 hrs at a final concentration of 50 nM with Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instructions. Transfection with a control siRNA was served as a negative control. Cells were subjected to RT-PCR to detect gene expression and western blotting to protein expression. The transfected cells were seeded for invasion assays.

Statistical analysis
All values are expressed as means AE SEM. Statistics was determined with ANOVA. Results were considered statistically significant if the P < 0.05.

TPD7 suppressed breast cancer cell proliferation and colony formation
To evaluate the effect of TPD7 on breast cancer cells, we observed its action on cell proliferation and colony formation. The results showed that TPD7 significantly inhibited cell proliferation of MDA-MB-435s, MDA-MB-231 and SK-BR-3 cells. The IC50 was 7.22 lM, 9.37 lM and 8.36 lM respectively (Fig. 1A). In colony formation assay, upon 10-15 days continuous culture, TPD7 also suppressed colony formation of MDA-MB-435s, MDA-MB-231 and SK-BR-3 cells (Fig. 1B). These findings indicated that TPD7 had potential anti-tumour properties in different type breast cancer cell lines.

TPD7 suppressed the expression of CXCR4 protein in breast cancer cells
We investigated the expression level of CXCR4 in the three breast cancer cell lines, namely SK-BR-3, MDA-MB-435s and MDA-MB-231, and found that CXCR4 protein expression was down-regulated by the TPD7 in a dose-dependent manner at the concentrations used in the experiments ( Fig. 2A-D). HER2 has been shown to induce the expression of CXCR4 in breast cancer cells [19], and it has been reported to regulate the expression of CXCR4 by stimulating CXCR4 translation and attenuating CXCR4 degradation [20]. We also examined whether TPD7 affected CXCR4 expression related to status of HER2 expression in breast cancer cells. We first investigated the expression level of HER2 in the above three different breast cancer cell lines. As shown in Figure 2, the results indicated that SK-BR-3 had high levels of HER2 expression as shown in western blotting, whereas MDA-MB-435s and MDA-MB-231cells had lack of HER2 expression (Fig. 2E), which were consistent with the previous reports [21][22][23][24]. In this experiment, down-regulation of CXCR4 expression were observed in all above cell lines, suggesting that the reduced expression of CXCR4 by TPD7 was irrespective of HER2 status in breast cancer.

Down-regulation of CXCR4 by TPD7 was partial mediated through its degradation
Because TPD7 could down-regulate CXCR4 expression, and CXCR4 has been shown to undergo ligand-dependent lysosomal degradation [25]. We next examined the ability of chloroquine, a lysosomal inhibitor, to block TPD7-induced degradation of CXCR4. The cells were pre-treated with chloroquine for 1 hr before exposure to TPD7 for 48 hrs. Our results showed that chloroquine had no effect on TPD7-induced degradation of CXCR4 (Fig. 3), suggesting that this was an unlikely basis for the suppression of TPD7 on CXCR4 expression.
Since CXCR4 has also been shown to undergo ubiquitination at its lysine residue followed by degradation [25,26], we next investigated whether TPD7 induced down-regulation of CXCR4 through proteasomal degradation. To determine this, we examined the ability of MG132, a proteasome inhibitor, to block TPD7-induced degradation of CXCR4. MDA-MB-435s and MDA-MB-231 cells were pre-treated with MG132 for 1 hr before being exposed to TPD7 for 48 hrs. As shown in Figure 3, MG132 could prevent TPD7-induced degradation of CXCR4, suggesting that this is one pathway for suppression of expression of CXCR4.

TPD7 down-regulated CXCR4 mRNA expression
We further investigated whether suppression occurred at the transcriptional level using RT-PCR and also by quantitative PCR (real-time PCR). Cells were treated with TPD7 for different concentrations and then examined for steady-state mRNA level of CXCR4. As shown in Figure 4, TPD7 induced down-regulation of CXCR4 mRNA expression in both MDA-MB-435s and MDA-MB-231 cells.

TPD7 inhibited SDF-1a-induced breast cancer cell migration and invasion
The expression of CXCR4/SDF-1 in breast tumours has been correlated with a poor prognosis, increased metastasis [4]. We found that both MDA-MB-435s and MDA-MB-231 cells migrated faster under the influence of SDF-1a and this effect was abolished on treatment with TPD7 ( Fig. 5A and B). To elucidate further the effect of TPD7 on SDF-1a induced cell invasion, we also found using an in vitro invasion assay, that treatment of TPD7 suppressed SDF-1a induced invasion of both MDA-MB-435s and MDA-MB-231 cells (Fig. 5C and D).

CXCR4 was a target of the inhibitory effect of TPD7 on cell invasion
To further elucidate the effect of CXCR4 on TPD7's cell invasion inhibition, we evaluated the effect on cell migration and invasion between wild tumour cells and CXCR4 knockdown cells, we observed the inhibition on cell mobility by wound healing assay and cell invasion by Millicell system. The results showed that treatment of TPD7 significantly decreased the migration and invasion ability of MDA-MB-435s and MDA-MB-231cells at the concentrations of 2.0-8.0 lM, respectively, and knockdown of CXCR4 by siRNA in the two cell lines significantly attenuated the inhibitory effects of TPD7 on migration and invasion, as compared with the control groups (Fig. 6). It indicated CXCR4 was a key factor in the cell migration by TPD7.

Effect of TPD7 on expression of MMP-2 and MMP-9 in breast tumour cells
Previous studies has shown that binding of SDF-1a to CXCR4 plays a role in tumour metastasis by increasing invasion associated with MMP-9 and MMP-2 activation [20,27,28]. MMPs are downstream targets of CXCR4-mediated signalling, CXCR4 promotes tumour migration and invasion through inducing expression of MMPs via the ERK signaling pathway [8,20,29]. Therefore, we detected the effect of TPD7 on expression of phosphorylated ERK1/2, MMP-2 and MMP-9. As shown in Figure 7, 48 hrs incubation with TPD7 reduced phosphorylated ERK1/2, MMP-2 and MMP-9 protein expression in a dose-dependent manner in MDA-MB-435s and MDA-MB-231 cells, suggesting that TPD7 perhaps suppressed the downstream targets of CXCR4, MMP-2 and MMP-9 via ERK pathway.

Discussion
Metastases, rather than primary tumours, are responsible for breast cancer deaths, many experimental and clinical studies have demonstrated that SDF-1a/CXCR4 axis play a key role in regulating the directional migration of breast cancer cells to sites of metastasis. CXCR4 may be a useful prognostic indicator and a potential therapeutic target in cancer therapies in patients with breast cancer. In this study, we sought to elucidate whether the small molecule compound TPD7 could inhibit breast cancer growth and invasion, and demonstrated the biologically relevant modulation of oncogenic signaling by TPD7.
In this study, we firstly showed that TPD7 had significant inhibitory effect on breast cancer cell proliferation and invasion in vitro. We have been suggested that TPD7 treatment reduced CXCR4 signaling, and therefore cell migration and invasion. The expression of CXCR4 was investigated in breast cancer cells treated with TPD7. The epidermal growth factor receptor, c-erbB2, and its encoding gene, HER2/ neu, have also been implicated in the positive regulation of CXCR4 expression at the post-transcriptional level [19,30]. Our results indicated that TPD7 down-regulated the expression of CXCR4 not only in high HER2 expressing SK-BR-3 but also low HER2 expressing MDA-MB-435s and MDA-MB-231 cells. Because the reduced expression of CXCR4 by TPD7 was irrespective of HER2 status in breast cancer, we used two of these cell lines, MDA-MB-435s and MDA-MB-231, to gain insights into the mechanisms of TPD7 on down-regulation of CXCR4 in further studies.
Various reports suggest that expression of CXCR4 may be increased by inflammatory cytokines such as TNF [31] and VEGF [32]. VEGF, a major angiogenic factor, is also a requisite autocrine factor for breast carcinoma invasion in vitro. Previous findings indicate that a VEGF autocrine pathway induces CXCR4 expression in breast carcinoma cells, thus promoting their directed migration towards specific chemokines [32]. Here, we found that VEGF expression was minimally affected after TPD7 treatment (data not shown), thus suggesting that down-regulation of CXCR4 expression by TPD7 was not because of modulation of autocrine VEGF. Recently, more evidence has been presented to show the ligand-dependent downregulation of the CXCR4 expression by lysosomal degradation [26], which involves atrophin-interacting protein 4-mediated ubiquitination and degradation [25]. Our results, however, indicated that downregulation of CXCR4 by TPD7 was induced not through lysosomal Data were expressed as mean AE SEM. *P < 0.05, **P < 0.01 compared with cells transfected with control siRNA. degradation but partial through proteasomal degradation, suggesting that proteasomal degradation was one pathway for suppression of CXCR4. In addition, we found that down-regulation of CXCR4 by TPD7 also occured at both the transcriptional and translational levels.
We also observed that TPD7 suppressed the ligand induced invasion of breast cancers, and this correlated with the down-regulation of CXCR4. To further validate this, knockdown of CXCR4 expression in MDA-MB-435s and MDA-MB-231 cells by siRNA significantly attenuated TPD7-inhibitory effects, thus suggesting that TPD7 had a potential to suppress tumour migration and invasion through its action on CXCR4.
The expression and activity of MMPs against matrix macromolecules have been linked to the development of malignant phenotypes [33] and the promotion of cell metastasis [34]. MMP-2 and MMP-9, two major MMPs, play important roles in cancer cell invasion and metastasis [35,36]. Recently, a growing body of evidence has been presented to show that SDF-1a/CXCR4 stimulation also leads to the secretion of various proteases, such as MMP-2 and MMP-9 by the breast cancer cells, which are then used to degrade the extracellular matrix, thereby facilitating cellular motility. These secreted proteases are also thought to facilitate the intravasation of cancer cells into the bloodstream. Furthermore, further studies show that MMPs are downstream targets of CXCR4-mediated signaling, CXCR4 regulation of MMPs expression is likely mediated by ERK signal pathways [28]. In this study, we showed that MMP-2, MMP-9 and p-ERK1/2 activity was strongly decreased, which indicating that TPD7 inhibited MMP-2 and MMP-9 expression, the downstream targets of CXCR4, perhaps via inactivation of the ERK signaling pathway.
In summary, TPD7 inhibited breast cancer cell proliferation and invasiveness in vitro. The underlying mechanism was through downregulating the expression of CXCR4, a key receptor involved in the cross-talk between tumour cells and its microenvironment, which contributed to its anti-invasive effects. Based upon these results, TPD7 might be promising candidate as CXCR4 inhibitor, the data supported further development of TPD7 as an adjuvant therapy agent for breast cancer. Further in vivo studies on the orthotopic breast cancer and metastasis to other organs are being planned to demonstrate the relevance of these observations to cancer treatment.