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Keywords:

  • breast cancer;
  • CXCR4;
  • migration;
  • miR-146a;
  • TRAIL

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is considered a promising agent for cancer therapy, as this molecule induces apoptosis specifically in various cancer cells. Apart from apoptosis, TRAIL also induces non-apoptotic signals, such as those for autophagy, proliferation and metastasis in cancer cells. In the present study, we report that TRAIL suppressed CXCR4-mediated human breast cancer MDA-MB-231 cell migration by up-regulating miR-146a expression through NF-κB. TRAIL receptor 1 (TRAIL-R1, DR4) was highly expressed in TRAIL-treated MDA-MB-231 cells. A neutralization antibody against DR4 specifically blocked TRAIL-induced NF-κB activation and miR-146a expression. These results were confirmed in a human breast cancer xenograft mouse model, suggesting that TRAIL significantly enhanced miR-146a expression and suppressed CXCR4 expression, indicating that TRAIL-induced miR-146a up-regulation is negatively associated with CXCR4 expression. These findings suggest that TRAIL-induced miR-146a expression suppresses CXCR4-mediated human breast cancer migration, and provide further insight into the non-apoptotic function of TRAIL in the prevention of metastasis as a therapy for breast cancer.


Abbreviations
CXCR4

CXC chemokine receptor 4

NF-κB

nuclear factor κB

rsTRAIL

recombinant soluble TRAIL

TRAIL

tumor necrosis factor-related apoptosis-inducing ligand

TRAIL-R

TRAIL receptor

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) is a typical member of the tumor necrosis factor family [1]. TRAIL induces apoptosis by binding to its receptors, TRAIL-R1 or TRAIL-R2 (also known as DR4 or DR5, respectively), in a wide variety of tumor cells but not in most normal cells, inducing either a caspase- or mitochondrial-dependent pathway [2-4]. Recent reports have primarily focused on the complicated mechanisms of TRAIL-induced apoptosis and the anti-tumor effects of this mechanism in vivo. Metastatic cancer is the major cause of death for patients diagnosed with some types of primary cancer such as colorectal cancer and breast cancer, but there are a few reports concerning the function of TRAIL in other tumor behaviors, such as metastasis, migration and invasion. The growth of breast cancer cells within the lungs and liver accounts for approximately 60–70% of the deaths associated with these cancers. Although breast cancer death rates are declining, primarily reflecting early diagnosis, this disease remains the second leading cause of cancer death in women. Furthermore, despite progress in the treatment of metastatic cancer in the past two decades, an efficient intervention strategy for breast cancer metastasis is still urgently needed to avoid over- or under-treatment of newly diagnosed patients.

It has been reported that TRAIL activates nuclear factor κB (NF-κB) in various cancer cell lines [5-7], but the mechanism underlying this activation remains unknown. Shetty et al. [8] demonstrated that TRAIL-induced NF-κB activation results in up-regulation of DR5 and a change in epithelial-derived cell sensitivity to TRAIL. In a previous study, we observed that over-expression of either the DR4 or DR5 death receptor induced apoptosis, concomitant with activation of NF-κB in 293T, MDA-MB-231 and HCT-116 cells [9], suggesting that death receptors mediate both apoptosis signaling pathways and NF-κB activation. NF-κB signaling is a key event in the multi-step process of carcinogenesis, progression, invasion and metastasis in many cancer types. However, the significance of the NF-κB pathway in complicated and tissue-specific cancer progression is less understood. Helbig et al. [10] reported that NF-κB promotes breast cancer cell migration and metastasis by inducing the expression of CXC chemokine receptor 4 (CXCR4), which plays an important role in development, organogenesis, hematopoiesis and immune responses. Breast carcinoma cells typically express high levels of CXCR4 and frequently colonize lung and bone tissue, which abundantly express stromal cell-derived factor-1, the preferred ligand for CXCR4 [11]. Thus, interfering with CXCR4/stromal cell-derived factor-1 interactions reduces the metastatic potential of the cells [12]. Recently, CXCR4 was reported to be a direct target of miR-146a in leukemic cell lines [13]. miR-146a and miR-146b inhibit both the migration and invasion of cancer cells [14], and Hurst et al. [15] reported that breast cancer metastasis suppressor 1 (BRMS1) suppressed metastasis through up-regulation of miR-146a in the breast cancer cell line MDA-MB-231. Although regulation of CXCR4 via a BRMS1–miR-146 axis was not directly tested, a microarray analysis revealed a reduction in CXCR4 expression in MDA-MB-231 and MDA-MB-435 cells following BRMS1 re-expression [15]. Furthermore, Taganov et al. [16] demonstrated that miR-146a is an NF-κB-sensitive endotoxin-responsive gene.

In the present study, we demonstrate that TRAIL up-regulates miR-146a expression in an NF-κB-dependent manner, which, in turn, inhibits CXCR4 expression and ultimately limits the migration of MDA-MB-231 cells. These data reveal a potential mechanism by which TRAIL suppresses tumor cell migration via alterations in the expression of miRNAs and downstream expression of CXCR4 in breast cancer cells.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

TRAIL inhibits MDA-MB-231 cell migration

We first examined the sensitivity of various cancer cell lines to recombinant soluble TRAIL (rsTRAIL) using a cell proliferation assay (MTS). As shown in Fig. S1A, treatment with 1.0 μg·mL−1 rsTRAIL for 24 h induced differential viability in the six cell lines tested. Three of these cell lines, including MDA-MB-231, U251 MG and A549, were sensitive to rsTRAIL. To examine the role of TRAIL in the migration of sensitive cells, a metastasis assay was performed using an in vitro scratch wound assay. As shown in Fig. 1A, rsTRAIL treatment suppressed migration of MDA-MB-231 cells in a dose-dependent manner, but no change was observed in the other two cell lines, U251 MG and A549, in response to TRAIL (Fig. S2). This result was confirmed using a Transwell migration assay. As shown in Fig. 1B, rsTRAIL significantly inhibited the migration of MDA-MB-231 cells (one tenth of the control, < 0.01), suggesting a negative regulatory role for TRAIL in MDA-MB-231 chemotaxis.

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Figure 1. TRAIL inhibits the migration of MDA-MB-231 cells. (A). Wound healing assay for the inhibitory effect of TRAIL on cell migration. The cells were pre-treated with various concentrations of rsTRAIL for 1 h. A scrape wound was created, and the cells were cultured for 18 h. (B) Transwell assay for migration of MDA-MB-231 cells with rsTRAIL or NaCl/Pi treatment. The cells were pre-treated with rsTRAIL for 1 h, followed by culturing in a Transwell system for 18 h. The cells that migrated were photographed, and four fields from three repeat experiments were counted using a light microscope. The data were expressed as a percentage of the control (basal migration without TRAIL pre-treatment). All the experiments were performed in triplicate. The error bars represent the standard deviation of mean values obtained from triplicate experiments. Double asterisks indicate a statistically significant difference compared with control (P < 0.01).

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TRAIL suppresses CXCR4 expression in MDA-MB-231 cells

Several studies have shown that CXCR4 induces tumor cell migration and invasion [10, 17, 18]. To determine the role of CXCR4 in TRAIL-mediated inhibition of MDA-MB-231 cell migration, we examined the expression of CXCR4 in MDA-MB-231 cells treated with TRAIL. As shown in Fig. 2A, TRAIL significantly reduced CXCR4 expression in MDA-MB-231 cells in a time-dependent manner up to 24 h post-treatment, demonstrating a more than 75% reduction in CXCR4 expression. In contrast, no change in CXCR4 expression was detected in the other cell lines tested, including MCF-7, U251 MG, A549, THP-1 and U937 cells. These data show that the TRAIL-mediated down-regulation of CXCR4 expression is cell type-specific. To investigate whether CXCR4 plays a role in TRAIL-mediated inhibition of metastasis, we assessed the influence of CXCR4 on MDA-MB-231 cell migration. CXCR4 expression was detected using flow cytometry assay with antibody that specifically recognizes CXCR4 on the cell surface. Both cell fractions, those that remained in the upper chamber and those that migrated to the lower chamber in a Transwell system, were collected for analysis of CXCR4 expression. As shown in Fig. 2B, cell-surface expression of CXCR4 in the MDA-MB-231 cells in the lower chamber was higher than that in the cells remaining in the upper chamber (76.3% versus 26.5%). To further assess the function of CXCR4, MDA-MB-231 cells were transfected with CXCR4 dsRNA (si-CXCR4) or control dsRNA (si-ctrl), and cell migration was analyzed using a Transwell system. The results showed that, although CXCR4 expression was reduced to 50% (Fig. 2C) at 48 h after transfection, the number of si-CXCR4-transfected MDA-MB-231 cells passing through the membrane was significantly lower than the number of si-ctrl-transfected cells passing through the membrane in the Transwell system. Similarly, the number of si-CXCR4-transfected MDA-MB-231 cells migrating into the wound space was markedly reduced compared to the controls (Fig. 2D,E). These data suggest that CXCR4 is a key molecule involved in the regulation of MDA-MB-231 cell migration.

image

Figure 2. TRAIL-mediated down-regulation of CXCR4 inhibits the migration of MDA-MB-231 cells. (A) Western blot assay for expression of CXCR4 in the indicated cell lines treated with rsTRAIL for 24 h. The cells were divided into two groups: TRAIL-sensitive and TRAIL-insensitive cells. The cell lysates were subjected to Western blot assay using an anti-CXCR4 antibody. All the experiments were performed in triplicate. (B) Cell-surface expression of CXCR4 in MDA-MB-231 cells from the top and bottom chambers of the Transwell apparatus, as detected using flow cytometry. (C) Effect of CXCR4 siRNA on the expression of CXCR4 protein. (D, E) Migration of MDA-MB-231 cells in wound-healing (D) and Transwell (E) assays. Both migration assays were performed using cells transfected with siRNA for 48 h. The wound surface was photographed after 18 h cultivation. The data were generated through a comparison of the healed wounds in the control group with those in the CXCR4 siRNA-transfected group. In the Transwell assay, the data were expressed as a percentage of the control (basal migration with control siRNA transfection). The error bars represent the standard deviation of mean values obtained from triplicate experiments.

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TRAIL-induced miR-146a expression suppresses CXCR4 expression

Recently, CXCR4 was implicated as a direct target of miR-146a in leukemic cell lines [13]. To verify whether CXCR4 was regulated by miR-146a in the cancer cells used in our study, miR-146a mimics were transfected into MDA-MB-231 cells, and CXCR4 expression was analyzed using a Western blot assay. The results indicated that CXCR4 expression was markedly reduced when miR-146a was over-expressed in MDA-MB-231 cells (Fig. 3A). To further establish the link between miR-146a and CXCR4 expression in TRAIL-treated cancer cells, miR-146a expression was determined in MDA-MB-231 cells treated with rsTRAIL. As shown in Fig. 3B, TRAIL treatment resulted in a threefold increase of miR-146a expression in MDA-MB-231 cells in a time-dependent manner at 24 h post-treatment.

image

Figure 3. TRAIL-mediated up-regulation of miR-146a suppresses the migration of MDA-MB-231 cells through CXCR4 targeting. (A) Western blot analysis of miR-146a mimic-transfected MDA-MB-231 cells. The cells were transfected with synthetic miR-146a mimics and mimic controls. The cell lysates were subjected to SDS/PAGE and Western blot analysis. (B) miR-146a expression in MDA-MB-231 cells treated with TRAIL. The cells were treated with or without rsTRAIL for the indicated times, and the miR-146a levels were detected using real-time PCR and reported as the fold change after normalization to U6 RNA. For the migration assay, the cells were transiently transfected with synthetic miR-146a mimics or mimic controls and grown for 48 h. (C, D) Cell migration was then analyzed using wound-healing (C) or Transwell (D) assays. The transfected cells were seeded in the top chamber of a Transwell apparatus at 2.5 × 105 cells per chamber. (E) Quantification of the migration of miR-146a-expressing cells through an 8 μm pore. Double asterisks indicate a statistically significant difference compared with control (P < 0.01). (F) Real-time PCR analysis of miR-146a expression in the cells remaining in the top chamber and those migrating to the bottom chamber. The error bars represent the standard deviation of mean values obtained from triplicate experiments.

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In addition, a scratch assay showed that over-expression of miR-146a significantly reduced MDA-MB-231 cell migration compared with control cells (Fig. 3C), and the Transwell assay demonstrated that over-expression of miR-146a suppressed the migration of MDA-MB-231 cells from the upper to the lower chamber (Fig. 3D,E). To further characterize the effects of miR-146a on breast cancer cell migration, MDA-MB-231 cells were added to the upper chamber and allowed to migrate into the lower chamber for 12 h. The cell fractions were then harvested from the upper and lower chambers, followed by analysis of miR-146a expression. As shown in Fig. 3F, miR-146a expression in the cells in the upper chambers was significantly increased (approximately twofold, < 0.01) compared with that in cells in the lower chambers. These data indicate that TRAIL inhibits the migration of breast cancer cells by up-regulating the expression of miR-146a, which directly targets CXCR4.

To determine the influence of the effects of miR-146a on inhibition of migration and TRAIL-induced apoptosis, MDA-MB-231 cells were pre-treated with the zVAD-fmk caspase inhibitor prior to TRAIL treatment. The results indicated that, although zVAD-fmk pre-treatment inhibited TRAIL-mediated apoptosis, TRAIL treatment still increased the expression of miRNA-146a, reduced the expression of CXCR4, and inhibited tumor metastasis in MDA-MB-231 cells (Fig. S3). Therefore, TRAIL-mediated apoptosis does not affect cancer cell metastasis.

TRAIL induces miR-146a expression in an NF-κB-dependent manner

TRAIL is known to activate NF-κB in MCF-7 and MDA-MB-231 epithelial cell lines [8]. Because miR-146a is an NF-κB-sensitive endotoxin-responsive gene [16], we further investigated whether TRAIL-mediated NF-κB activation is responsible for the increased miR-146a expression in MDA-MB-231 cells. The cells were transfected with a luciferase reporter plasmid harboring an NF-κB DNA-binding site, and subsequently treated with rsTRAIL for 6 h, followed by a dual luciferase reporter assay. As shown in Fig. 4A, treatment with TRAIL increased the NF-κB transcriptional activity by approximately twofold in MDA-MB-231 cells compared to the control, whereas transfection with an IκBα dominant-negative (IκBα-DN) plasmid completely inhibited NF-κB activity in TRAIL-treated 293T cells.

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Figure 4. NF-κB-dependent regulation of miR-146a expression. (A) TRAIL-induced NF-κB activation was measured in 293T cells using a dual-luciferase reporter assay. (B) miR-146a promoter activity in 293T cells. miR-146a promoter-luc, pRL-TK and either IκB-DN expression vector or empty expression vector were co-transfected into 293T cells for 42 h. The cells were treated with rsTRAIL for 6 h, and the cell lysates were then subjected to a luciferase activity assay. miR-146a promoter-luc, NF-κB p65 expression vector ,respectively were co-transfected with PRL-TK into 293T cells without TRAIL treatment. All the relative luciferase activities were normalized to Renilla luciferase activity by co-transfection with the Renilla luciferase vector pRL-TK. (C, D) Expression of IκBα (C) and miR-146a (D) in TRAIL-treated MDA-MB-231 cells infected with lentivirus expressing IκBα-DN. The MDA-MB-231 cells were infected with lentivirus expressing IκBα-DN or empty virus; the results of real-time PCR are shown as the fold change in miR-146a expression between the TRAIL-treated and untreated cells. The error bars represent the standard deviation of mean values obtained from triplicate experiments.

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To examine whether miR-146a expression is dependent on NF-κB activation, we analyzed the miR-146a promoter activity in these cancer cell lines. There are two functional NF-κB binding sites upstream of the predicted human pri-miR-146a start site [16, 19]. A DNA fragment containing these NF-κB binding sites was inserted into the pGL-Basic luciferase reporter plasmid, and relative promoter activity was detected in 293T cells transfected with the recombinant reporter, followed by a luciferase assay. As shown in Fig. 4B, there was a fivefold increase in miR-146a promoter activity upon rsTRAIL stimulation compared to the non-treated cells. As expected, over-expression of the p65 subunit of NF-κB induced an approximately six- to sevenfold increase in promoter activity. However, expression of IκBα-DN significantly suppressed miR-146a promoter activity in the TRAIL-treated cells (< 0.05), suggesting that TRAIL up-regulates miR-146a promoter activity in an NF-κB-dependent manner. We further assessed whether TRAIL-induced NF-κB activation increases miR-146a expression. MDA-MB-231 cells were infected with a recombinant lentivirus expressing IκBα-DN for 48 h, and miR-146a expression was detected by Western blot analysis. As shown in Fig. 4C,D, IκBα degradation was blocked and possibly stabilized through over-expression of IκBα-DN in MDA-MB-231 cells infected with the lentivirus vector for 24 h, whereas TRAIL-induced miR-146a expression was completely abrogated in MDA-MB-231 cells treated with rsTRAIL for 12 or 24 h. These data suggest that TRAIL induces miR-146a expression in an NF-κB-dependent manner in MDA-MB-231 breast cancer cells.

TRAIL-induced miR-146a expression is mediated through DR4

To further explore whether the TRAIL-induced up-regulation of miR-146a is associated with TRAIL receptor expression, the surface expression of DR4 (TRAIL-R1) and DR5 (TRAIL-R2) was assessed by Western blot analysis of MDA-MB-231 cells treated with rsTRAIL. As shown in Fig. 5A, both DR4 and DR5 were highly expressed in MDA-MB-231 cells; however, expression of DR4 but not DR5 was increased in a time-dependent manner with rsTRAIL treatment. This result was consistent with the levels of receptor-interacting protein 1 (RIP1) and phosphorylated IκBα expression, which peaked at 6 h (over a sixfold increase) and reduced to basal levels at 24 h post-treatment. To confirm that DR4 mediates TRAIL-induced NF-κB activation, a DR4-specific neutralizing antibody was added to cultured MDA-MB-231 cells prior to rsTRAIL treatment. As shown in Fig. 5B, the neutralizing antibody against DR4 decreased RIP1 expression and NF-κB activation. Moreover, miR-146a expression was not increased in the DR4-inhibited MDA-MB-231 cells (Fig. 5C). Taken together, these data indicate that TRAIL-induced miR-146a expression is mediated through DR4 (TRAIL-R1) but not DR5 (TRAIL-R2).

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Figure 5. TRAIL activates the NF-κB signaling pathway. (A) Western blot analysis for the proteins involved in the TRAIL-mediated NF-κB pathway. MDA-MB-231 cells were treated with rsTRAIL at the indicated times. The cell lysates were analyzed by immunoblotting using antibodies against DR4, DR5, RIP1, IκBα, phosphorylated IκBα and GAPDH. The densitometric analysis showed changes in the absorbance of the DR4, RIP1 and IκBα phosphorylation bands comparing with GAPDH. GAPDH expression was used for normalization. (B) RIP1 expression and IκBα and IKK phosphorylation in MDA-MB-231 cells pre-treated with a DR4-specific neutralizing antibody for 2 h, followed by rsTRAIL treatment. (C) Real-time PCR results showing the inhibitory effect of the DR4-neutralizing antibody on miR-146a expression induced through TRAIL. The error bars represent the standard deviation of mean values obtained from triplicate experiments.

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TRAIL-induced miR-146a expression down-regulates CXCR4 in a human breast cancer xenograft mouse model

To evaluate the effect of TRAIL on expression of miR-146a and CXCR4 in vivo, we established a tumor model in nude mice bearing human breast cancer cell line MDA-MB-231 xenografts. When a mean tumor volume of approximately 50 mm3 was reached, the mice were intra-tumorally injected three times with rsTRAIL or NaCl/Pi as a control. Subsequently, the miR-146a expression in the tumor specimens dissected from the tumor-bearing animals was detected using a quantitative RT-PCR analysis. As shown in Fig. 6A, the miR-146a level, as expected, was significantly increased by approximately sixfold in the TRAIL-treated tumor tissue compared to the control (< 0.01), indicating that TRAIL induces miR-146a expression in breast cancer cells in vivo. The Western blot analysis revealed that CXCR4 protein expression in the TRAIL-treated tumor tissue was reduced to approximately 70% compared to the control (Fig. 6B). These data indicate that the TRAIL-induced up-regulation of miR-146a down-regulates CXCR4 expression in human breast cancer cells, indicating a potential mechanism by which TRAIL suppresses tumor cell migration via alterations in the expression profile of miRNAs and CXCR4 in breast cancer cells.

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Figure 6. TRAIL increases miR-146a expression and reduces CXCR4 expression in human breast cancer xenograft mouse models. (A) Quantitative PCR of miR-146a expression. The middle line represents the median value (50%). (B) Western blot analysis of expression of CXCR4 in breast cancer xenografts in TRAIL-injected and control groups. Six athymic nude mice per group were intra-tumorally injected with or without 15 mg·kg−1 rsTRAIL every other day. The mice were killed at 9 h after the third injection, and the tumor tissues were sampled from the two groups for real-time PCR or Western blot analyses. The error bars represent the standard deviation of mean values obtained from triplicate experiments.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Given that expression of miR-146a, which is involved in the inhibition of migration and invasion, is regulated through the NF-κB signaling pathway [14], a pathway in which TRAIL is implicated, we hypothesized that TRAIL may exert an anti-metastasis function through regulation of miR-146a expression in cancer cells. In the present study, we observed that miR-146a was up-regulated after TRAIL treatment, and MDA-MB-231 cancer cell migration was suppressed through down-regulation of CXCR4 in an NF-κB-dependent manner. These results indicate a potential mechanism for TRAIL-mediated suppression of metastasis through alterations in the expression of metastasis-associated miRNAs. Treatment with a lower concentration (25 ng·mL−1) of rsTRAIL induced more than 50% cell death in MDA-MB-231 cells, and the remaining living cells appeared insensitive to TRAIL, even at higher concentrations. Therefore, we determined whether TRAIL exerts other biological effects on these cells. Because TRAIL-resistant cancer cells possess a high metastatic potential, we determined whether TRAIL blocked migration of these cells. Interestingly, in vitro migration of MDA-MB-231 cells treated with TRAIL was dramatically suppressed. However, TRAIL treatment had no effect on migration of U251 MG cells, which showed the same susceptibility to TRAIL-mediated apoptosis (Fig. S2), suggesting that the TRAIL-induced suppression of cancer cell migration and invasion is cell-specific.

Tumor cells may spread from the primary tumor tissue and develop resistance to anti-cancer drugs through loss of relevant drug targeting during transformation. For example, some cancer cells resist TRAIL-induced apoptosis [20]. Thus, a critical question in clinical trials is whether the TRAIL/TRAIL-R system serves as an effector mechanism in any physiological and/or pathophysiological process.

The current view suggests that CXCR4 is a crucial molecule in cancer cell metastasis [17, 18], and the motility of cancer cells, including breast tumor metastasis, is promoted by both a high level of CXCR4 expression and enhancement of chemokine (C-C motif) ligand 12 expression [21, 22]. In the present study, we observed a reduction in CXCR4 expression in TRAIL-treated MDA-MB-231 cells, but this reduced expression did not reflect apoptosis in these cells, which was regulated by pre-incubation with rsTRAIL for 1 h. These results also indicate that the TRAIL-induced down-regulation of CXCR4 expression is cell type-specific. There was no change in expression of CXCR4 in other TRAIL-resistant or -sensitive cells, including human breast cancer MCF-7 cells, in response to TRAIL treatment, potentially reflecting cell lines with different metastatic potencies. Indeed, MDA-MB-231 cell lines exhibit a dramatically higher invasive phenotype than MCF-7 cells [23, 24]. Therefore, these results may provide an alternative mechanism by which TRAIL contributes to the modulation of tumor malignancy.

miRNAs are evolutionarily conserved non-protein-coding RNAs that widely regulate gene expression. An increasing number of studies have revealed that miRNAs are closely associated with tumorigenesis and progression [25, 26], indicating that miRNAs play a key role in modulating the TRAIL-mediated signaling pathway. Previous studies have shown that miRNAs also serve as regulators of death receptor signaling, in addition to affecting the sensitivity of cancer cells to TRAIL-induced apoptosis [27-31]. We hypothesized that, by altering the expression of miRNAs, TRAIL modulates many tumor behaviors, including metastasis. To our knowledge, there are no reports on the role of miRNAs in the metastasis of cancer cells treated with TRAIL. In the present study, we focused on miR-146a because this miRNA profoundly inhibits the invasion and metastasis of MDA-MB-231 cells [14, 15]. Moreover, a recent study reported that CXCR4 is a predicted target of miR-146a in normal megakaryopoiesis [13]. Because miR-146a is involved in regulation of the NF-κB signaling pathway, which is also activated through TRAIL, we investigated whether miR-146a is a downstream regulator of cancer cell metastasis. We observed that TRAIL up-regulated miR-146a expression in a time-dependent manner. Furthermore, transduction of miR-146a into MDA-MB-231 cells inhibited the migration of MDA-MB-231 cells via targeting CXCR4. Although the mechanism underlying the TRAIL-induced suppression of metastasis is complicated, our data revealed a previously unknown mechanism by which TRAIL regulates breast cancer cell migration through alterations in the expression profile of miRNAs.

Although miRNAs add another layer of complexity to our understanding of TRAIL-induced suppression of metastasis, it remains unclear how TRAIL regulates the expression of miR-146a. Recent studies have shown that TRAIL activates the NF-κB signaling pathway in several cancer cell lines, but whether NF-κB activation blocks TRAIL-induced apoptosis is controversial. For example, a previous study suggested that NF-κB activation does not affect TRAIL-R1-induced apoptosis [32]. However, in another study, NF-κB activation interfered with TRAIL-induced cell death in breast cancer MCF-7 cells [33]. Although the effects on TRAIL resistance are not fully understood, TRAIL-induced NF-κB activation may exert an unknown biological function in cancer cells. The emerging role of NF-κB activation in promoting transcription of the miR-146a gene has also expanded our perspectives. Thus, we anticipated that a potential TRAIL/NF-κB/miR-146a/CXCR4 axis may exist in MDA-MB-231 cells. This idea is supported by the fact that over-expression of IκBα-DN suppressed TRAIL-induced up-regulation of miR-146a by 50%, an event that was mediated through DR4. Therefore, this study provides new insight into TRAIL-induced NF-κB activation, which promoted transcription of miR-146a, and, in turn, limited the migration of MDA-MB-231 cells via targeting CXCR4. In addition, we observed that TRAIL induced a time-dependent increase in RIP1 expression, despite reductions in IκBα phosphorylation. This result is consistent with recent reports that recruitment of RIP1 to the TRAIL—death-inducing signaling complex (DISC) induced IκB degradation and the downstream phosphorylation of p38 and IκB [34, 35].

An siRNA directed against CXCR4 was shown to inhibit breast cancer metastasis in vivo [21]. We observed that TRAIL suppressed the expression of CXCR4 through up-regulation of miR-146a expression in a human breast cancer xenograft mouse model, suggesting that TRAIL-up-regulated miR-146a expression may suppress breast cancer metastasis. As the tumor micro-environment is far more complicated than the culture of cells in vitro, other mechanisms that modulate the TRAIL-induced up-regulation of miR-146a via targeting CXCR4 may exist in vivo. Unfortunately, the metastatic phenomenon of MDA-MB-231 cells is very difficult to observe in the xenograft model. After observation for 6 months, the xenografts of mice grew largely without showing pulmonary metastasis, the survival states of mice were worse, and the mice died easily. In addition, the subcutaneous xenografts of mice shrunk significantly after TRAIL treatment, and it is possible that mouse survival may be improved. However, we were unable to determine whether the extended survival was related to down-regulation of CXCR4 by miR-146a. Thus, we were unable to compare the metastasis and survival in the xenograft model of MDA-MB-231 cells between TRAIL treatment and controls.

In summary, the present study demonstrated that, as a potential apoptosis-based cancer therapy, TRAIL suppresses metastasis through miR-146a up-regulation in MDA-MB-231 cells. As TRAIL does not induce cytotoxic effects in MDA-MB-231 cells under physiological conditions, TRAIL may be able to inhibit the migration of living cells, thus providing a better understanding of the non-apoptotic function of TRAIL and its application in breast cancer therapy and prognosis. In addition, the results of the present study provide insight into the role of miRNAs in TRAIL-mediated downstream signaling pathways. To our knowledge, this report is the first to suggest that TRAIL suppresses human breast cancer metastasis via up-regulation of miR-146a via targeting CXCR4. These data provide a novel mechanistic insight into the use of TRAIL for the treatment of breast cancer. Because miR-146a suppresses the invasion of some cancer cells and also plays a role in the regulation of immune and inflammatory responses, these results suggest that TRAIL-induced miR-146a up-regulation may have a broad effect on tumorigenesis and cancer progress. Therefore, strategies to up-regulate miR-146a levels in vivo may be used as a therapy for preventing tumor metastasis and progression.

Experimental procedures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Cell lines and cell culture

The human breast cancer cell lines MDA-MB-231 and MCF-7, glioma cell line U251 MG, the non-small-cell lung carcinoma cell line A549, acute monocytic leukemia cell line THP-1, leukemic monocyte lymphoma cell line U937, and embryonic kidney fibroblast cell lines 293T were obtained from the American Type Culture Collection (Manassas, VA, USA). The MDA-MB-231 cells were cultured in Leibovitz's L-15 medium containing 10% fetal bovine serum. The MCF-7 and U937 cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum. The U251 MG cells were grown in minimum essential medium/Earle's buffered salt solution containing 10% fetal bovine serum. The A549 cells were cultured in F-12K medium with 10% fetal bovine serum, and the THP-1 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum and 0.05 mm β-mercaptoethanol. The 293T cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Neither antibiotics nor antimycotics were used. All the cell cultures were maintained at 37 °C in a humidified atmosphere of 5% CO2. The rsTRAIL was purified by Ni+-agarose affinity chromatography from stable proteins expressed in Escherichia coli after transformation with pET28b vector (Novagen, Germany) containing the extracellular domain of TRAIL. rsTRAIL protein quantification was performed using the bicinchoninic acid assay.

Plasmids and siRNAs

A reporter plasmid containing NF-κB binding sites (NF-κB-Luc) and the recombinant lentivirus expressing IκBα-DN (a dominant-negative mutant of IκBα, with mutations S32A and S36A) have been described previously [9]. The NF-κB p65 expression vector [36] was a kind gift from Jae Won Rhee (Department of Biochemistry and Institute of Life Science and Biotechnology, College of Science, Yonsei University, Seoul , Republic of Korea). The PGL3-miR-146a promoter reporter plasmid was generated by insertion of a PCR fragment encompassing 560 bp upstream of the predicted transcriptional start site of the pri-miR-146a mRNA into the pGL-Basic vector [16]. The protocol for generating virus particles was described previously [37]. CXCR4 siRNA was purchased from Dharmacon (Lafayette, CO, USA), and the DR4-specific neutralizing antibody was purchased from R&D Systems (Minneapolis, MN, USA). The miR-146a mimics (dsRNA oligonucleotides) were obtained from GenePharma (Shanghai, China) and used for over-expression of miR-146a in the MDA-MB-231 cells. Negative control mimics (Shanghai GenePharma Co., Shanghai, China) were transfected to serve as matched controls. The cells were transfected with mir-146a mimics, miRNA control, siRNA at a final concentration of 10 nm using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.

Cell viability assay

The cell viability was measured using an MTS assay. Briefly, 1 × 104 cells per well were plated in a 96-well microplate and exposed to various concentrations of recombinant soluble TRAIL (rsTRAIL) (Xingpeng Biotech Co., Shenzhen, China) for 0, 3, 6, 9, 12 and 24 h. The MTS assay was performed using a CellTiter 96® AQueous non-radioactive kit (Promega, Madison, WI, USA) according to the manufacturer's instructions. Cells without any treatment were used as a negative control. The cell viability was converted and expressed as a percentage of the negative control, and the results are expressed as the mean attenuance for samples performed in duplicate (= 3).

In vitro migration scratch assay

Cell migration was analyzed using an in vitro migration scratch assay. Briefly, MDA-MB-231 cells were plated in six-well plates, and the confluent cells were scratched using a standard 10 μL standard pipette tip. The cells were then washed twice (5 min each time) with sterile NaCl/Pi at room temperature to remove the cellular debris. To determine the effect of TRAIL on MDA-MB-231 cell migration, cells were cultured in complete medium (L-15 medium containing 10% fetal bovine serum) in the absence or presence of 5, 12.5 and 25 ng·mL−1 rsTRAIL. One hour later, the medium was replaced with fresh complete medium (L-15 medium containing 10% fetal bovine serum), and the cells were incubated for another 18 h. To analyze the effects of CXCR4 and miR-146a on the migration of the MDA-MB-231 cells, the confluent cells were transfected with si-CXCR4, miR-146a mimics or control oligonucleotides and incubated for 48 h. Images of the scratches were captured using a digital camera system coupled to a microscope.

Transwell migration assay

Cells were pre-treated with rsTRAIL for 1 h or transfected with si-CXCR4, miR-146a mimics or a negative control; after 48 h, the cells were harvested using trypsinization and washed once with Hank's balanced salt solution (Invitrogen). To measure cell migration, 8 nm pore-size culture inserts (Transwell; Costar, High Wycombe, UK) were placed into the wells of a 24-well culture plate, separating the upper and the lower chambers. A 0.6 mL aliquot of complete medium containing 20% fetal bovine serum was added to the lower chamber, and 2.5 × 105 cells were added to the upper chamber in 0.4 mL of serum-free L-15 medium (Gibco, USA). After 18 h of incubation at 37 °C with 5% CO2, the number of cells that migrated through the pores was counted in 10 independent visual fields using a microscope (Zeiss, Jena, Germany) with a 10× objective. The cell morphology was observed by hematoxylin staining.

Flow cytometry

The cells were plated in the upper chamber of the Transwell system. After 18 h, the non-migrated cells in the top chamber and the cells that had migrated to beneath the membrane were collected. The-cell surface CXCR4 expression was evaluated using flow cytometry (FACS Caliber; Becton Dickinson, San Jose, CA, USA), followed by incubation with an anti-CXCR4 antibody and fluorescein isothiocyanate-conjugated anti-mouse secondary antibody (R&D Systems). Phycoerythrin-labeled mouse IgG2B (R&D Systems) was used as a control.

Real-time PCR

miRNAs were extracted from MDA-MB-231 cells or tumor tissue using a mirVana miRNA isolation kit (Ambion, Austin, TX, USA) according to the manufacturer's instructions. Total RNA was reverse-transcribed using a TaqMan microRNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA) in a reaction mixture containing an miR-specific stem-loop reverse transcription primer. Quantification of mature miRNAs was performed using the TaqMan miRNA assay kit (Applied Biosystems) containing TaqMan primers in a universal PCR master mix. Quantitative PCR was performed at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The threshold crossing value was determined for each transcript and normalized to that for the internal control. Quantification of miRNAs was performed using the comparative Ct method. The experiments were performed using an ABI Prism 7900 system (Applied Biosystems). The cycle threshold values were converted to relative gene expression levels using the inline image method [38].

Western blot assay

The proteins extracted from cells were separated by 10% SDS/PAGE and transferred to poly(vinylidene difluoride) membranes (Amersham Biosciences, Piscataway, NJ, USA). Using standard techniques, the membranes were blocked and incubated overnight with antibodies against CXCR4 (Abcam, Cambridge, MA, USA), DR4, DR5, RIP1, GAPDH, and phosphorylated and total IκBα (Cell Signaling Technology, Beverly, MA, USA). After three washes with TBST, the membranes were incubated with a biotinylated goat anti-rabbit IgG complex or goat anti-mouse IgG complex (Zhongshan Goldenbridge Biotechnology Co. Ltd, Beijing, China) for 2 h at room temperature. After thorough washing with TBST buffer, the specific proteins were visualized using enhanced chemiluminescence reagents (Hyperfilm ECL; Amersham Biosciences), followed by exposure to X-ray film for the appropriate durations.

Luciferase reporter assay

For the NF-κB activation luciferase assay, 293T cells were co-transfected with NF-κB-Luc, pRL-TK and either the IκBα-DN expression vector or empty vector. For the miR-146a promoter luciferase reporter assay, 293T cells were co-transfected with miR-146a promoter-luc, pRL-TK (Promega) and either the IκBα-DN expression vector or empty vector. The cells were stimulated with or without rsTRAIL (25 ng·mL−1) for 6 h before harvesting. At 48 h after transfection, the cells were lysed with lysis buffer, and a luciferase activity assay was performed using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer's instructions. The luciferase activity was measured using a microplate luminometer (Berthold Centro LB 960, Berthold Technologies, Germany). The firefly luciferase activity was normalized against Renilla luciferase activity.

Animal experiment

All animal experiments were approved by the Committee of Use and Care of Animals, Chinese Academy of Medical Sciences. Male BALB/c nude mice (6 weeks old), with a mean weight of 18 g, were purchased from the Wei Tong Li Hua Experimental Animal Center (Beijing, China). A total of 2 × 106 MDA-MB-231 cells were suspended in 0.1 mL of NaCl/Pi and injected subcutaneously in the right back of the mice. When the tumor volumes reached approximately 50 mm, on the 10th day post-injection, the animals were divided into two groups (= 6). The animals in the experimental animal were injected with 0.1 mL rsTRAIL (15 mg·kg−1) intra-tumorally every other day, and the animals in the control group were injected with 0.1 mL NaCl/Pi. At 9 h after the 3rd injection, the animals were killed, and the solid tumors were removed and frozen in liquid nitrogen for real-time PCR and Western blot analyses.

Statistical analysis

All the data were obtained from at least three independent experiments. The data are expressed as mean values ± SD and were compared using Student's t test. Statistical significance was defined as a P value < 0.05.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

This work was partially funded through grants from the State Key Basic Research Program of China (grant number 2007CB507404), the Natural Science Foundation of China (grant numbers 30972684, 30972699 and 81001315) and the Doctoral Degree Program of the Ministry of Education (grant number 20101196110027).

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  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
FilenameFormatSizeDescription
febs12323-sup-0001-FigS1-S3.zipapplication/ZIP737K

Fig. S1. Effect of rsTRAIL on the viability of tested cells measured by MTS assay.

Fig. S2. TRAIL has no effect on the migration of U251 cells as analyzed by an in vitro scratch assay.

Fig. S3. Effects of caspase inhibitor zVAD-fmk on the expression of miR-146a and CXCR4, and migration in the MDA-MB-231 cell line.

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.