- Email: [email protected]
Toll-like receptor 2 mediates invasion via activating NF-κB in MDA-MB-231 breast cancer cells
Biochemical and Biophysical Research Communications 379 (2009) 1027–1032
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Toll-like receptor 2 mediates invasion via activating NF-jB in MDA-MB-231 breast cancer cells Wenjie Xie *, Yongsheng Wang, Yafang Huang, Hongzhen Yang, Jiaping Wang, Zhuowei Hu Molecular Immunology and Pharmacology Laboratory, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, 1 Xian Nong Tan St., Beijing 100050, China
a r t i c l e
i n f o
Article history: Received 15 December 2008 Available online 12 January 2009
Keywords: Toll-like receptor 2 MDA-MB-231 cells MCF-7 cells Cell invasiveness NF-jB
a b s t r a c t MDA-MB-231 breast cancer cells have a high invasive potential, yet the mechanisms involved are not known. This study showed that Toll-like receptor 2 (TLR2) was highly expressed in MDA-MB-231 cells and played a critical role in cell invasion. Compared with the poorly invasive MCF-7 cells, MDA-MB231 cells expressed 10.5-fold more TLR2. Using TLR2 agonist pg-LPS and TLR2 neutralizing antibody, we found that TLR2 activation significantly promoted MDA-MB-231 invasion, whereas TLR2 blockade diminished this capacity. TLR2 activation enhanced the activity of NF-jB and induced phosphorylation of TAK1 and IjBa in the TLR2/NF-jB signaling pathway in MDA-MB-231, but not in MCF-7 cells. TLR2 activation increased IL-6, TGF-b, VEGF and MMP9 secretion, which are associated with TLR2-NF-jB signaling. We demonstrated that TLR2 is a critical receptor responsible for NF-jB signaling activity and highly invasive capacity of MDA-MB-231 cells. Ó 2009 Elsevier Inc. All rights reserved.
Cancer cell invasion is one of the fundamental steps in the metastatic process [1]. Highly invasive cancer cells are capable of passing through matrix membranes, yet the molecules involved in these mechanisms have not been identified. As we know that invasive capacity of highly metastatic MDA-MB-231 breast cancer cells is much higher than that of poorly metastatic MCF-7 breast cancer cells [2], the difference in metastatic potential between these two cell lines is important. It would be desirable to know which protein expressed in MDA-MB-231 cells is involved in their invasiveness. Toll-like receptors (TLRs) were first found in mammalian immune cells with a powerful capacity to initiate innate immune responses [3]. However, recent studies have also identified certain TLRs in non-immune cells: TLR2 in mesenchymal stem cells plays a role in the control of cell differentiation, and TLR4 in adipocytes is related to insulin resistance [4,5]. New evidence has shown that TLRs also exist in some mouse tumor cells [6,7]. For example, TLR4 on mouse colon tumor cells facilitates evasion of immune surveillance [6]. Moreover, TLR2 signaling promotes mouse hepatic tumor growth via IL-6 signaling [7]. We wonder if TLRs are expressed on human breast cancer cells and whether the cells have an effect on invasion. Transcriptional factor NF-jB plays a crucial role in human breast cancer cells, and its constitutive activity has been found in cancer cell invasion and metastasis [8,9]. Moreover, it has been re-
* Corresponding author. Address: Room12, Unit13, No. 7. BaiWanZhuang Street, XiCheng District, Beijing 100037, China. Fax: +86 10 68358685. E-mail address: [email protected] (W. Xie). 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.01.009
ported that NF-jB constitutive activity in MDA-MB-231 cells is much stronger than poorly invasive MCF-7 cells [9]. However, the factors that cause higher NF-jB constitutive activity in MDAMB-231 cells have not been fully elucidated. In the present study, we aimed to establish whether TLR2 was differentially expressed in MDA-MB-231 and MCF-7 cells, to activate NF-jB and promote cell invasion. Materials and methods Reagents and antibodies. Matrigel was purchased form Becton Dickinson Biosciences (San Jose, CA, USA). Pg-LPS (a TLR2 ligand, lipopolysaccharide from Porphyromonas gingivalis) and neutralizing anti-TLR2 antibodies were purchased from Invitrogen (San Diego, CA, USA). FITC-conjugated anti-TLR2 monoclonal antibody and its isotype FITC-IgG2a were all purchased from eBioscience Inc. (San Diego, CA, USA). Monoclonal anti-pNF-kBp65, pTAK1 and pIjBa antibodies were purchased form Cell Signaling Technology Inc. (Danvers, MA, USA). FITC-conjugated secondary antibody of goat anti-rabbit IgG (H+L) was purchased from BioLegend (San Diego, CA, USA). Cell lines and cell culture. MCF-7 and MDA-MB-231 cell lines were purchased from the American Type Culture Collection (Rockville, MD, USA). Both cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with high glucose supplement, with 10% FBS at 37 °C and 5% CO2. Qualitative RT-PCR for detection of TLR1–10 mRNA. Total RNA was extracted using TRIzol reagent (Invitrogen) and its quality and
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concentration were measured as described previously [13]. For RTPCR, first-strand cDNA was synthesized using 1lg total RNA with an oligo-dT primer and the avian myeloblastosis virus (AMV) reverse transcriptase (Promega, Madison, WI, USA). The PCR primers for TLR1 to TLR10 and b-actin were designed as described previously [13]. Evaluation of TLR2 expression. By using semi-quantitative RTPCR, six repeated samples were run for 32 cycles to amplify TLR2 cDNA. At the protein expression level, first we used Western blotting. Then, flow cytometry was used to quantify TLR2 expression. The two cell lines were separately stained with FITC-conjugated TLR2 antibody or isotype IgG according to the manufacturer’s instructions. Cells were analyzed with a MoFlo flow cytometer and the data were analyzed by Summit Software v4.3 (DAKO Inc. CO, USA). Transwell invasion assay. The bottoms of the inner transwells (8.0 lm pore size, 6.5 mm diameter; Corning Costar Inc. CA, USA) were coated with paraffin to avoid meniscus formation. Then, the bottoms of the transwells were coated with a thin layer of matrigel (2.5 mg/ml). The two cell lines in DMEM containing 0.2% BSA were seeded on top of this matrigel at a quantity of 50,000 cells per chamber. The cells were cultured in serum-free medium in the inner compartment inside the transwell. To the outer compartment outside the transwell, we added 600 ll DMEM with 10% FBS. After 24 h culture, the cells were fixed with 2% paraformaldehyde in 1 PBS and stained with 0.05% toluidine blue in 2% Na2CO3/water solution. After wiping off the inside of the chambers using cotton-tipped applicators, invasive cells remained on the bottom of the membranes and were counted under the microscope (magnification, 200) [14]. Intracellular phospho-NF-jBp65 staining for flow cytometry. Formaldehyde in 0.5–1 ml PBS was added to each cell sample, to a final concentration of 2%, and cells were fixed for 10 min at 37 °C. To make cells permeable, they were slowly added to ice-cold
100% methanol to a final concentration of 90%. Then, cells were incubated for 30 min on ice. Two milliliters of 2% BSA blocking buffer was added to each tube, and tubes was rinsed by centrifugation at 350g. The cells were resuspended in 100 ll blocking buffer and primary antibody was added to each tube, which was incubated on ice for 45 min. After two washings in blocking buffer to delete antibodies with non-specific attachment, the cells were resuspended in FITC-conjugated secondary antibody. After 30 min, cells were rinsed again and resuspended in 0.5 ml PBS for analysis by MoFlo flow cytometer (DAKO Inc. CO, USA) [15]. The strength of fluorescence was measured by the median fluorescence intensity (MFI). Fold change was calculated by dividing the MFI of the treated sample (MFI treated) by that of the control sample (MFI untreated), i.e., fold change percent % = (MFI treated/MFI untreated) 100 [15]. Western blotting. For pTAK1 or pIjBa detection, the cells were resuspended in lysis buffer (10 mM Tris–HCl, pH 8.0, 137 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 10 lg/ml leupeptin, 1 lg/ml aprotinin). For TLR2 detection, the cells were lysed in RIPA buffer (50 mM HEPES, 150 mM NaCl, 1% deoxycholate, 1% NP-40, 0.5% SDS and protease inhibitor). The cell lysates were centrifuged for 20 min at 16,000g, and then equal amounts of protein (30 lg) were separated by 10% SDS–PAGE, and transferred onto PVDF membranes (Millipore, Bedford MA, USA). After blocking in non-fat dried milk, membranes were probed with primary antibody in a solution of PBS containing 0.1% Tween-20. After three 10-min washes in PBS/ 0.1% Tween-20, blots were incubated with horseradish-peroxidase-conjugated secondary antibody. Immunoreactive bands were detected using the enhanced chemiluminescence system (Amersham, Piscataway, NJ, USA). ELISA. Cancer cells (1 105) were cultured in 24-well plates for 24 h and the supernatants were assayed for IL-6, TGF-b, VEGF and MMP9 using an ELISA kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.
Fig. 1. MDA-MB-231 cells expressed more TLR2 than poorly invasive MCF-7 cells. (A) Qualitative RT-PCR for 30–45 cycles using different primers was used to screen the profiles of TLR mRNAs, and TLR2 could be detected in both cell lines. b-Actin was used as a positive and molecular weight control. (B) Compared with MCF-7, MDA-MB-231 cells expressed more TLR2, as shown by semi-quantitative RT-PCR and protein analysis. (C) By using flow cytometry, TLR2 in MDA-MB-231 cells was nearly 10.5-fold higher than that in MCF-7 cells. Values were expressed as mean ± SD and one representative of six independent experiments is shown (n = 6 ##p < 0.01).
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Statistics. The values were expressed as mean ± SD. Statistical comparison was made using Student’s t test, and the level of significance was established at p < 0.05 and p < 0.01.
231 and MCF-7 cells positively correlated with cell invasive activity, which suggests that higher expression of TLR2 in MDA-MB-231 cells may be responsible for their invasive capacity.
Results
TLR2 mediated MDA-MB-231 cell invasion
TLR2 expression was higher in MDA-MB-231 than MCF-7 cells
To investigate cell invasive capacity, an invasion assay was developed in which only highly invasive cells could invade through the matrigel layer and migrate through the 8.0 lm holes. Compared with MCF-7, MDA-MB-231 cells showed a 14-fold higher invasive capacity (Fig. 2A). To further investigate the role that TLR2 played in MDA-MB-231 cells, we utilized TLR2 agonist pgLPS to activate TLR2 and monoclonal neutralizing antibody to block TLR2. The result showed that TLR2 exerted a significant positive effect on invasiveness of MDA-MB-231 cells (Fig. 2B). Pg-LPS stimulation significantly increased the number of invasive MDA-MB-231 cells, whereas blockade of TLR2 notably suppressed the number of invasive cells (Fig. 2B).
As TLRs have been identified in some mouse tumor cells [6,7], we sought to determine if they were expressed in MDA-MB-231 and MCF-7 human breast cancer cells. Qualitative PCR analysis revealed that MDA-MB-231 cells expressed mRNAs of many TLRs including TLR1, TLR2, TLR3, TLR5, TLR6, TLR8 and TLR9. In particular, mRNA for TLR2 and its coupled heterodimers TLR1 or TLR6 was seen in both cell lines (Fig. 1A). Furthermore, we used semi-quantitative PCR, Western blotting and flow cytometry to investigate the levels of TLR2 in the two cell lines. Notably, TLR2 mRNA in MDA-MB-231 cells was much higher than that in MCF-7 cells. TLR2 protein was highly expressed in MDA-MB-231 cells, but not in MCF-7 cells (Fig. 1B). Collectively, these results were confirmed by flow cytometry detection, and TLR2 protein expression was 10.5-fold higher in MDA-MB-231 than in MCF-7 cells (Fig. 1C). These results demonstrated that TLR2 existed in human breast cancer cell lines. It is the differential expression of TLR2 in MDA-MB-
TLR2 stimulated NF-jB activation Constitutive NF-jB activity has been found in human breast cancer cell lines and plays a pivotal role in breast cancer progression [9,16]. In immune cells, TLR2 stimulation leads to NF-jB
Fig. 2. TLR2 activation promoted MDA-MB-231 cell invasion. (A) 5 105 cells were cultured in each chamber for 24 h and only invading cells were stained. MDA-MB-231 cells showed a 12-fold higher capacity for invasion through the matrigel layer and 8.0 lm pores than MCF-7 cells did. (B) Utilizing TLR2 agonist pg-LPS (1 lg/ml) to activate TLR2, TLR2 activation increased the number of invasive MDA-MB-231 cells, whereas using monoclonal neutralizing antibody (10 lg/ml, TLR2 Ab) to block TLR2 significantly attenuated the number of invasive cells. IgG (10 lg/ml) treatment was an isotype control and cyclophosphamide (CTX) treatment was a positive control. pg-LPS + TLR2 neutralizing antibody showed no significant difference from the control. Values were expressed as mean ± SD (n = 6 ##p < 0.01; *p < 0.05; **p < 0.01).
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phosphorylation during activation [3]. We therefore sought to determine whether TLR2 activation in human breast cancer cells resulted in NF-jB activity. After TLR2 stimulation, the phosphorylation analysis by flow cytometry revealed that MDA-MB-231 cells with higher expression of TLR2 exhibited nearly a two-fold stronger phosphorylation of NF-jBp65 than the untreated group (Fig. 3A and B). However, blocking the TLR2 signal by neutralizing antibody decreased the level of NF-jBp65 (Fig. 3A and B). We further investigated TAK1 and IkBa, which are the key kinases in the TLR2/NF-jB pathway. Through examining the amounts of pTAK1 and pIkBa, which are activated states of TAK1 and IkBa, we found that after TLR2 activation, TAK1 and IkBa were all highly activated (Fig. 3C and D). Furthermore, compared with the MDA-MB-231 cell line with higher TLR2 expression, MCF-7 cells showed weak TLR2/ NF-jB signaling activity (Fig. 3C and D). Overall, TLR2 in invasive MDA-MB-231 cells had an obviously powerful regulatory capacity in NF-jB activation. TLR2 increased IL-6, TGF-b, VEGF and MMP9 secretion The results reported above indicated that TLR2 in MDA-MB-231 cells mediated invasion and played a major role in NF-jB activity. NF-jB-mediated cytokine production by breast cancer cells is
important for their biological behavior; e.g., IL-6 is indispensable for cancer cell invasion and proliferation [9,16]. We examined whether the cytokines in the supernatants of cultured MDA-MB231 cells were increased under the condition of TLR2 stimulation. After TLR2 activation by pg-LPS, amounts of IL-6 and TGF-b increased, respectively, up to five- and seven-fold (Fig. 4A and B). In contrast to TLR2 activation, TLR2 blockade significantly reduced IL-6 and TGF-b production (Fig. 4A and B). Cancer cell invasion also requires VEGF and MMP9 function [17]. We found that TLR2 stimulation also effectively enhanced VEGF and MMP9 excretion, whereas blockade of TLR2 decreased excretion (Fig. 4C and D). This result shows that TLR2 activation for cytokine secretion contributes to the invasion process. Discussion In this study, we identified that TLR2 was expressed in MCF-7 and MDA-MB-231 human breast cancer cells. Compared with nearly undetectable levels of TLR2 in poorly invasive MCF-7 cells, obviously greater amounts of TLR2 were expressed in MDA-MB231 cells, and this differential expression correlated with their invasive properties. We further observed that TLR2 activation could significantly enhance its invasive ability, whereas blockade
Fig. 3. TLR2 activation triggered NF-jB activity and increased amounts of pTAK1 and pIkBa in MDA-MB-231 cells. Each group was cultured for 6 h before flow cytometry. (A and B) MDA-MB-231 cells exhibited a nearly two-fold stronger NF-jB activity after treatment with pg-LPS (1 lg/ml), whereas pg-LPS stimulation had no such effect on MCF-7 cells. Blocking TLR2 by its neutralizing antibody (10 lg/ml, TLR2 Ab) decreased NF-jB activity in both MDA-MB-231 and MCF-7 cells. IgG (10 lg/ml) treatment was an isotype control. (C and D) After pg-LPS (1 lg/ml) stimulation, TAK1 and IjBa in MDA-MB-231 cells were highly activated. Especially compared with MDA-MB-231 cells, these signaling molecules under TLR2 regulation were not so significantly activated in MCF-7 cells. Values were expressed as mean ± SD (n = 6 *p < 0.05; **p < 0.01).
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Fig. 4. TLR2 activation increased IL-6, TGF-b, VEGF and MMP9 autocrine secretion in MDA-MB-231 cells. Cancer cells were cultured for 24 h and the supernatants were assayed by ELISA. (A and B) After TLR2 activation by pg-LPS (1 lg/ml), MDA-MB-231 cells secreted Il-6 or TGF-b up to five- or seven-fold, respectively, whereas TLR2 blockade by its neutralizing antibody (10 lg/ml, TLR2 Ab) reduced IL-6 and TGF-b level to half that in the untreated group. In addition, IL-6 and TGF-b autocrine secretion by MDA-MB231 cells was much higher than that in MCF-7 cells. (C and D) TLR2 stimulation effectively enhanced VEGF and MMP9 excretion in MDA-MB-231 and MCF-7 cells. Blockade of TLR2 decreased excretion of VEGF and MMP9 in MDA-MB-231 cells. Values were expressed as mean ± SD (n = 6 *p < 0.05; **p < 0.01; #p < 0.05; ##p < 0.01).
of TLR2 notably decreased this ability. Invasion through basal membranes is a critical metastatic step for cell detachment from primary loci and intrusion into distant organs [1]. Adhesive molecules such as b1 integrin and E-cadherin have been shown to be required for breast cancer cell invasion and metastasis, therefore, some adhesive molecules have become targets for breast cancer prevention [18,19]. Our findings indicated that TLR2 played an effective role in the invasive potential of MDA-MB-231 human breast cancer cells. Moreover, TLR2 can respond to pathogen-associated molecular patterns (PAMPs) such as LPS contained on pathogen cell-wall surfaces. TLR2 can also respond to endogenous ligands of damage-associated molecular patterns (DAMPs) such as heat shock protein 60 (Hsp60) and hyaluronic acid fragments, which are produced by tissue injury [20]. As solid malignancies are aggravated under conditions of chronic infection and tissue injury [21], our study suggests that besides inflammation, bacterial LPS may directly promote MDA-MB-231 cellular invasion via TLR2, and provide a direct link between bacterial infection and tumor progression. The present study showed that activation of TLR2 in MDA-MB231 cells increased constitutive NF-jB activity. NF-jB activity was first identified as an essential anti-apoptosis factor in cancer cells, and recently has been reported as an important factor during breast cancer cell invasion and metastasis [22]. Moreover, one study has reported that constitutive NF-jB activity in the highly invasive and metastatic MDA-MB-231 cells is much stronger than that in poorly invasive and metastatic breast cancer cells [9]. Our findings indicated that, in correlation with its differential expression in the two breast cancer cells, TLR2 activation effectively trig-
gered NF-jB activation more in MDA-MB-231 than in MCF-7 cells. We also showed that pTAK1 and pIkBa, which are two key kinases in the TLR2/NF-jB signaling pathway, were significantly higher after TLR2 stimulation, and these results indicate a validating TLR2/NF-jB signal in invasive MDA-MB-231 cells [3]. Our study indicated that, after TLR2 stimulation, breast cancer cells produced higher amounts of Il-6, TGF-b, VEGF and MMP9, whereas TLR2 blockade significantly decreased excretion of these cytokines. Autocrine secretion of cytokines by cancer cells exerts an important effect in the control of cancer cell behavior such as adhesion and invasion [11,12,23]. For example, IL-6 contributes to lung and breast cancer cell malignancy and effusion [10,11]; TGF-b induces cancer cell invasion in breast cancer [23]; and VEGF and MMP9 are regarded as indispensable cytokines for breast cancer cell invasion and adhesion [17]. Consistent with previous findings that TLR2 mediates high invasive capacity and augments NF-jB activity in MDA-MB-231 cells, we showed that TLR2 also promotes excretion of these cytokines in MDA-MB-231 cells. Therefore, we found that TLR2-triggered NF-jB promoted production of these cytokines, thus leading to MDA-MB-231 cell invasion. Since blocking NF-jB strongly suppresses MDA-MB-231 cell invasion [9,24], we conclude that TLR2/NF-jB signaling in invasive MDA-MB-231 cells contributes to their invasive activity via autocrine secretion of cytokines. In conclusion, we report herein that TLR2 mediates MDA-MB231 invasive properties by controlling activation of NF-jB, which is a very important factor for breast cancer cell transcriptional cytokines. TLR2/NF-jB signaling in MDA-MB-231 cells contributes to the underlying mechanism of invasive potential, which suggests
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that TLR2 is a potential hallmark and a therapeutic target in highly invasive breast cancer cells. Our results may also be relevant for breast cancer therapy. Post-mastectomy bacterial infection and inflammatory breast cancer are associated with PAMPs and DAMPs, such as Hsp60, which can directly activate TLR2 in cancer cells [20,25]. Thus pathogen eradication and anti-inflammatory therapy are important for preventing poor prognosis in breast cancer. Acknowledgment The first author expresses his gratitude to Professor Wei Wu and Professor Xiaoning Wang for their helpful discussion. References [1] K. Pantel, R.H. Brakenhoff, Dissecting the metastatic cascade, Nat. Rev. Cancer 4 (2004) 448–456. [2] Y. Zuo, S.K. Shields, C. Chakraborty, Enhanced intrinsic migration of aggressive breast cancer cells by inhibition of Rac1 GTPase, Biochem. Biophys. Res. Commun. 15 (2006) 361–367. [3] K. Takeda, T. Kaisho, S. Akira, Toll-like receptors, Annu. Rev. Immunol. 21 (2003) 335–376. [4] M. Pevsner-Fischer, V. Morad, M. Cohen-Sfady, L. Rousso-Noori, A. ZaninZhorov, S. Cohen, I.R. Cohen, D. Zipori, Toll-like receptors and their ligands control mesenchymal stem cell functions, Blood 109 (2007) 1422–1432. [5] M.J. Song, K.H. Kim, J.M. Yoon, J.B. Kim, Activation of Toll-like receptor 4 is associated with insulin resistance in adipocytes, Biochem. Biophys. Res. Commun. 4 (2006) 739–745. [6] B. Huang, J. Zhao, H. Li, K.L. He, Y. Chen, L. Mayer, J.C. Unkeless, H. Xiong, Tolllike receptors on tumor cells facilitate evasion of immune surveillance, Cancer Res. 65 (2005) 5009–5014. [7] B. Huang, J. Zhao, S. Shen, H. Li, K.L. He, G.X. Shen, L. Mayer, J. Unkeless, D. Li, Y. Yuan, G.M. Zhang, H. Xiong, Z.H. Feng, Listeria monocytogenes promotes tumor growth via tumor cell Toll-like receptor 2 signaling, Cancer Res. 67 (2007) 4346–4352. [8] M.A. Huber, N. Azoitei, B. Baumann, S. Grunert, A. Sommer, H. Pehamberger, N. Kraut, H. Beug, T. Wirth, NF-jB is essential for epithelial-mesenchymal transition and metastasis in a model of breast cancer progression, J. Clin. Invest. 114 (2004) 569–581. [9] B.K. Park, H. Zhang, Q. Zeng, J. Dai, E.T. Keller, T. Giordano, K. Gu, V. Shah, L. Pei, R.J. Zarbo, L. McCauley, S. Shi, S. Chen, C.Y. Wang, NF-jB in breast cancer cells promotes osteolytic bone metastasis by inducing osteoclastogenesis via GMCSF, Nat. Med. 13 (2007) 62–69.
[10] Z.T. Schafer, J.S. Brugge, IL-6 involvement in epithelial cancers, J. Clin. Invest. 117 (2007) 3660–3663. [11] P. Sansone, G. Storci, S. Tavolari, T. Guarnieri, C. Giovannini, M. Taffurelli, C. Ceccarelli, D. Santini, P. Paterini, K.B. Marcu, P. Chieco, M. Bonafe, IL-6 triggers malignant features in mammospheres from human ductal breast carcinoma and normal mammary gland, J. Clin. Invest. 117 (2007) 3988–4002. [12] A.T. Hooper, G. Akiri, D. Jin, S.V. Shmelkov, E. Chuang, S.J. Shin, Y. Wu, D.J. Hicklin, S. Rafii, L.T. Vahdat, VEGF receptor expression on reactive breast cancer stroma: paving the way for tumor invasion, J. Clin. Oncol. 23 (2005) 9642–9650. [13] I. Kokkinopoulos, W.J. Jordan, M.A. Ritter, Toll-like receptor mRNA expression patterns in human dendritic cells and monocytes, Mol. Immunol. 42 (2005) 957–968. [14] L. Connolly, P. Maxwell, Image analysis of transwell assays in the assessment of invasion by malignant cell lines, Br. J. Biomed. Sci. 59 (2002) 11–14. [15] P.O. Krutzik, G.P. Nolan, Intracellular phospho-protein staining techniques for flow cytometry: monitoring single cell signaling events, Cytometry 55 (2003) 61–70. [16] M. Karin, Y. Cao, F.R. Greten, Z.W. Li, NF-jB in cancer: from innocent bystander to major culprit, Nat. Rev. Cancer 2 (2002) 301–310. [17] S.H. Kim, N.H. Cho, K. Kim, J.S. Lee, B.S. Koo, J.H. Kim, J.H. Chang, E.C. Choi, Correlations of oral tongue cancer invasion with matrix metalloproteinases (MMPs) and vascular endothelial growth factor (VEGF) expression, J. Surg. Oncol. 93 (2006) 330–337. [18] E.S. Yao, H. Zhang, Y.-Y. Chen, B. Lee, K. Chew, D. Moore, C. Park, Increased b1 integrin is associated with decreased survival in invasive breast cancer, Cancer Res. 67 (2007) 659–664. [19] L. Mauro, S. Catalano, G. Bossi, M. Pellegrino, I. Barone, S. Morales, C. Giordano, V. Bartella, I. Casaburi, S. Ando, Evidences that leptin up-regulates E-cadherin expression in breast cancer: effects on tumor growth and progression, Cancer Res. 67 (2007) 3412–3421. [20] M.E. Bianchi, DAMPs, PAMPs and alarmins: all we need to know about danger, J. Leukoc. Biol. 81 (2007) 1–5. [21] L.M. Coussens, Z. Werb, Inflammation and cancer, Nature 420 (2002) 860–867. [22] G. Sethi, K.S. Ahn, B. Sung, B.B. Aggarwal, Pinitol targets nuclear factor-jB activation pathway leading to inhibition of gene products associated with proliferation, apoptosis, invasion, and angiogenesis, Mol. Cancer Ther. 7 (2008) 1604–1614. [23] M.B. Buck, C. Knabbe, TGF-b signaling in breast cancer, Ann. NY Acad. Sci. 1089 (2006) 119–126. [24] B. Sung, M.K. Pandey, Y. Nakajima, H. Nishida, T. Konishi, M.M. Chaturvedi, B.B. Aggarwal, Identification of a novel blocker of IkBa kinase activation that enhances apoptosis and inhibits proliferation and invasion by suppressing nuclear factor-jB, Mol. Cancer Ther. 7 (2008) 191–201. [25] A.R. Ferrara, Inflammatory breast cancer: challenges and developments, Radiol. Technol. 80 (2008) 49–60.
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Toll-like receptor 2 mediates invasion via activating NF-jB in MDA-MB-231 breast cancer cells Wenjie Xie *, Yongsheng Wang, Yafang Huang, Hongzhen Yang, Jiaping Wang, Zhuowei Hu Molecular Immunology and Pharmacology Laboratory, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, 1 Xian Nong Tan St., Beijing 100050, China
a r t i c l e
i n f o
Article history: Received 15 December 2008 Available online 12 January 2009
Keywords: Toll-like receptor 2 MDA-MB-231 cells MCF-7 cells Cell invasiveness NF-jB
a b s t r a c t MDA-MB-231 breast cancer cells have a high invasive potential, yet the mechanisms involved are not known. This study showed that Toll-like receptor 2 (TLR2) was highly expressed in MDA-MB-231 cells and played a critical role in cell invasion. Compared with the poorly invasive MCF-7 cells, MDA-MB231 cells expressed 10.5-fold more TLR2. Using TLR2 agonist pg-LPS and TLR2 neutralizing antibody, we found that TLR2 activation significantly promoted MDA-MB-231 invasion, whereas TLR2 blockade diminished this capacity. TLR2 activation enhanced the activity of NF-jB and induced phosphorylation of TAK1 and IjBa in the TLR2/NF-jB signaling pathway in MDA-MB-231, but not in MCF-7 cells. TLR2 activation increased IL-6, TGF-b, VEGF and MMP9 secretion, which are associated with TLR2-NF-jB signaling. We demonstrated that TLR2 is a critical receptor responsible for NF-jB signaling activity and highly invasive capacity of MDA-MB-231 cells. Ó 2009 Elsevier Inc. All rights reserved.
Cancer cell invasion is one of the fundamental steps in the metastatic process [1]. Highly invasive cancer cells are capable of passing through matrix membranes, yet the molecules involved in these mechanisms have not been identified. As we know that invasive capacity of highly metastatic MDA-MB-231 breast cancer cells is much higher than that of poorly metastatic MCF-7 breast cancer cells [2], the difference in metastatic potential between these two cell lines is important. It would be desirable to know which protein expressed in MDA-MB-231 cells is involved in their invasiveness. Toll-like receptors (TLRs) were first found in mammalian immune cells with a powerful capacity to initiate innate immune responses [3]. However, recent studies have also identified certain TLRs in non-immune cells: TLR2 in mesenchymal stem cells plays a role in the control of cell differentiation, and TLR4 in adipocytes is related to insulin resistance [4,5]. New evidence has shown that TLRs also exist in some mouse tumor cells [6,7]. For example, TLR4 on mouse colon tumor cells facilitates evasion of immune surveillance [6]. Moreover, TLR2 signaling promotes mouse hepatic tumor growth via IL-6 signaling [7]. We wonder if TLRs are expressed on human breast cancer cells and whether the cells have an effect on invasion. Transcriptional factor NF-jB plays a crucial role in human breast cancer cells, and its constitutive activity has been found in cancer cell invasion and metastasis [8,9]. Moreover, it has been re-
* Corresponding author. Address: Room12, Unit13, No. 7. BaiWanZhuang Street, XiCheng District, Beijing 100037, China. Fax: +86 10 68358685. E-mail address: [email protected] (W. Xie). 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.01.009
ported that NF-jB constitutive activity in MDA-MB-231 cells is much stronger than poorly invasive MCF-7 cells [9]. However, the factors that cause higher NF-jB constitutive activity in MDAMB-231 cells have not been fully elucidated. In the present study, we aimed to establish whether TLR2 was differentially expressed in MDA-MB-231 and MCF-7 cells, to activate NF-jB and promote cell invasion. Materials and methods Reagents and antibodies. Matrigel was purchased form Becton Dickinson Biosciences (San Jose, CA, USA). Pg-LPS (a TLR2 ligand, lipopolysaccharide from Porphyromonas gingivalis) and neutralizing anti-TLR2 antibodies were purchased from Invitrogen (San Diego, CA, USA). FITC-conjugated anti-TLR2 monoclonal antibody and its isotype FITC-IgG2a were all purchased from eBioscience Inc. (San Diego, CA, USA). Monoclonal anti-pNF-kBp65, pTAK1 and pIjBa antibodies were purchased form Cell Signaling Technology Inc. (Danvers, MA, USA). FITC-conjugated secondary antibody of goat anti-rabbit IgG (H+L) was purchased from BioLegend (San Diego, CA, USA). Cell lines and cell culture. MCF-7 and MDA-MB-231 cell lines were purchased from the American Type Culture Collection (Rockville, MD, USA). Both cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with high glucose supplement, with 10% FBS at 37 °C and 5% CO2. Qualitative RT-PCR for detection of TLR1–10 mRNA. Total RNA was extracted using TRIzol reagent (Invitrogen) and its quality and
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concentration were measured as described previously [13]. For RTPCR, first-strand cDNA was synthesized using 1lg total RNA with an oligo-dT primer and the avian myeloblastosis virus (AMV) reverse transcriptase (Promega, Madison, WI, USA). The PCR primers for TLR1 to TLR10 and b-actin were designed as described previously [13]. Evaluation of TLR2 expression. By using semi-quantitative RTPCR, six repeated samples were run for 32 cycles to amplify TLR2 cDNA. At the protein expression level, first we used Western blotting. Then, flow cytometry was used to quantify TLR2 expression. The two cell lines were separately stained with FITC-conjugated TLR2 antibody or isotype IgG according to the manufacturer’s instructions. Cells were analyzed with a MoFlo flow cytometer and the data were analyzed by Summit Software v4.3 (DAKO Inc. CO, USA). Transwell invasion assay. The bottoms of the inner transwells (8.0 lm pore size, 6.5 mm diameter; Corning Costar Inc. CA, USA) were coated with paraffin to avoid meniscus formation. Then, the bottoms of the transwells were coated with a thin layer of matrigel (2.5 mg/ml). The two cell lines in DMEM containing 0.2% BSA were seeded on top of this matrigel at a quantity of 50,000 cells per chamber. The cells were cultured in serum-free medium in the inner compartment inside the transwell. To the outer compartment outside the transwell, we added 600 ll DMEM with 10% FBS. After 24 h culture, the cells were fixed with 2% paraformaldehyde in 1 PBS and stained with 0.05% toluidine blue in 2% Na2CO3/water solution. After wiping off the inside of the chambers using cotton-tipped applicators, invasive cells remained on the bottom of the membranes and were counted under the microscope (magnification, 200) [14]. Intracellular phospho-NF-jBp65 staining for flow cytometry. Formaldehyde in 0.5–1 ml PBS was added to each cell sample, to a final concentration of 2%, and cells were fixed for 10 min at 37 °C. To make cells permeable, they were slowly added to ice-cold
100% methanol to a final concentration of 90%. Then, cells were incubated for 30 min on ice. Two milliliters of 2% BSA blocking buffer was added to each tube, and tubes was rinsed by centrifugation at 350g. The cells were resuspended in 100 ll blocking buffer and primary antibody was added to each tube, which was incubated on ice for 45 min. After two washings in blocking buffer to delete antibodies with non-specific attachment, the cells were resuspended in FITC-conjugated secondary antibody. After 30 min, cells were rinsed again and resuspended in 0.5 ml PBS for analysis by MoFlo flow cytometer (DAKO Inc. CO, USA) [15]. The strength of fluorescence was measured by the median fluorescence intensity (MFI). Fold change was calculated by dividing the MFI of the treated sample (MFI treated) by that of the control sample (MFI untreated), i.e., fold change percent % = (MFI treated/MFI untreated) 100 [15]. Western blotting. For pTAK1 or pIjBa detection, the cells were resuspended in lysis buffer (10 mM Tris–HCl, pH 8.0, 137 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 10 lg/ml leupeptin, 1 lg/ml aprotinin). For TLR2 detection, the cells were lysed in RIPA buffer (50 mM HEPES, 150 mM NaCl, 1% deoxycholate, 1% NP-40, 0.5% SDS and protease inhibitor). The cell lysates were centrifuged for 20 min at 16,000g, and then equal amounts of protein (30 lg) were separated by 10% SDS–PAGE, and transferred onto PVDF membranes (Millipore, Bedford MA, USA). After blocking in non-fat dried milk, membranes were probed with primary antibody in a solution of PBS containing 0.1% Tween-20. After three 10-min washes in PBS/ 0.1% Tween-20, blots were incubated with horseradish-peroxidase-conjugated secondary antibody. Immunoreactive bands were detected using the enhanced chemiluminescence system (Amersham, Piscataway, NJ, USA). ELISA. Cancer cells (1 105) were cultured in 24-well plates for 24 h and the supernatants were assayed for IL-6, TGF-b, VEGF and MMP9 using an ELISA kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.
Fig. 1. MDA-MB-231 cells expressed more TLR2 than poorly invasive MCF-7 cells. (A) Qualitative RT-PCR for 30–45 cycles using different primers was used to screen the profiles of TLR mRNAs, and TLR2 could be detected in both cell lines. b-Actin was used as a positive and molecular weight control. (B) Compared with MCF-7, MDA-MB-231 cells expressed more TLR2, as shown by semi-quantitative RT-PCR and protein analysis. (C) By using flow cytometry, TLR2 in MDA-MB-231 cells was nearly 10.5-fold higher than that in MCF-7 cells. Values were expressed as mean ± SD and one representative of six independent experiments is shown (n = 6 ##p < 0.01).
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Statistics. The values were expressed as mean ± SD. Statistical comparison was made using Student’s t test, and the level of significance was established at p < 0.05 and p < 0.01.
231 and MCF-7 cells positively correlated with cell invasive activity, which suggests that higher expression of TLR2 in MDA-MB-231 cells may be responsible for their invasive capacity.
Results
TLR2 mediated MDA-MB-231 cell invasion
TLR2 expression was higher in MDA-MB-231 than MCF-7 cells
To investigate cell invasive capacity, an invasion assay was developed in which only highly invasive cells could invade through the matrigel layer and migrate through the 8.0 lm holes. Compared with MCF-7, MDA-MB-231 cells showed a 14-fold higher invasive capacity (Fig. 2A). To further investigate the role that TLR2 played in MDA-MB-231 cells, we utilized TLR2 agonist pgLPS to activate TLR2 and monoclonal neutralizing antibody to block TLR2. The result showed that TLR2 exerted a significant positive effect on invasiveness of MDA-MB-231 cells (Fig. 2B). Pg-LPS stimulation significantly increased the number of invasive MDA-MB-231 cells, whereas blockade of TLR2 notably suppressed the number of invasive cells (Fig. 2B).
As TLRs have been identified in some mouse tumor cells [6,7], we sought to determine if they were expressed in MDA-MB-231 and MCF-7 human breast cancer cells. Qualitative PCR analysis revealed that MDA-MB-231 cells expressed mRNAs of many TLRs including TLR1, TLR2, TLR3, TLR5, TLR6, TLR8 and TLR9. In particular, mRNA for TLR2 and its coupled heterodimers TLR1 or TLR6 was seen in both cell lines (Fig. 1A). Furthermore, we used semi-quantitative PCR, Western blotting and flow cytometry to investigate the levels of TLR2 in the two cell lines. Notably, TLR2 mRNA in MDA-MB-231 cells was much higher than that in MCF-7 cells. TLR2 protein was highly expressed in MDA-MB-231 cells, but not in MCF-7 cells (Fig. 1B). Collectively, these results were confirmed by flow cytometry detection, and TLR2 protein expression was 10.5-fold higher in MDA-MB-231 than in MCF-7 cells (Fig. 1C). These results demonstrated that TLR2 existed in human breast cancer cell lines. It is the differential expression of TLR2 in MDA-MB-
TLR2 stimulated NF-jB activation Constitutive NF-jB activity has been found in human breast cancer cell lines and plays a pivotal role in breast cancer progression [9,16]. In immune cells, TLR2 stimulation leads to NF-jB
Fig. 2. TLR2 activation promoted MDA-MB-231 cell invasion. (A) 5 105 cells were cultured in each chamber for 24 h and only invading cells were stained. MDA-MB-231 cells showed a 12-fold higher capacity for invasion through the matrigel layer and 8.0 lm pores than MCF-7 cells did. (B) Utilizing TLR2 agonist pg-LPS (1 lg/ml) to activate TLR2, TLR2 activation increased the number of invasive MDA-MB-231 cells, whereas using monoclonal neutralizing antibody (10 lg/ml, TLR2 Ab) to block TLR2 significantly attenuated the number of invasive cells. IgG (10 lg/ml) treatment was an isotype control and cyclophosphamide (CTX) treatment was a positive control. pg-LPS + TLR2 neutralizing antibody showed no significant difference from the control. Values were expressed as mean ± SD (n = 6 ##p < 0.01; *p < 0.05; **p < 0.01).
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phosphorylation during activation [3]. We therefore sought to determine whether TLR2 activation in human breast cancer cells resulted in NF-jB activity. After TLR2 stimulation, the phosphorylation analysis by flow cytometry revealed that MDA-MB-231 cells with higher expression of TLR2 exhibited nearly a two-fold stronger phosphorylation of NF-jBp65 than the untreated group (Fig. 3A and B). However, blocking the TLR2 signal by neutralizing antibody decreased the level of NF-jBp65 (Fig. 3A and B). We further investigated TAK1 and IkBa, which are the key kinases in the TLR2/NF-jB pathway. Through examining the amounts of pTAK1 and pIkBa, which are activated states of TAK1 and IkBa, we found that after TLR2 activation, TAK1 and IkBa were all highly activated (Fig. 3C and D). Furthermore, compared with the MDA-MB-231 cell line with higher TLR2 expression, MCF-7 cells showed weak TLR2/ NF-jB signaling activity (Fig. 3C and D). Overall, TLR2 in invasive MDA-MB-231 cells had an obviously powerful regulatory capacity in NF-jB activation. TLR2 increased IL-6, TGF-b, VEGF and MMP9 secretion The results reported above indicated that TLR2 in MDA-MB-231 cells mediated invasion and played a major role in NF-jB activity. NF-jB-mediated cytokine production by breast cancer cells is
important for their biological behavior; e.g., IL-6 is indispensable for cancer cell invasion and proliferation [9,16]. We examined whether the cytokines in the supernatants of cultured MDA-MB231 cells were increased under the condition of TLR2 stimulation. After TLR2 activation by pg-LPS, amounts of IL-6 and TGF-b increased, respectively, up to five- and seven-fold (Fig. 4A and B). In contrast to TLR2 activation, TLR2 blockade significantly reduced IL-6 and TGF-b production (Fig. 4A and B). Cancer cell invasion also requires VEGF and MMP9 function [17]. We found that TLR2 stimulation also effectively enhanced VEGF and MMP9 excretion, whereas blockade of TLR2 decreased excretion (Fig. 4C and D). This result shows that TLR2 activation for cytokine secretion contributes to the invasion process. Discussion In this study, we identified that TLR2 was expressed in MCF-7 and MDA-MB-231 human breast cancer cells. Compared with nearly undetectable levels of TLR2 in poorly invasive MCF-7 cells, obviously greater amounts of TLR2 were expressed in MDA-MB231 cells, and this differential expression correlated with their invasive properties. We further observed that TLR2 activation could significantly enhance its invasive ability, whereas blockade
Fig. 3. TLR2 activation triggered NF-jB activity and increased amounts of pTAK1 and pIkBa in MDA-MB-231 cells. Each group was cultured for 6 h before flow cytometry. (A and B) MDA-MB-231 cells exhibited a nearly two-fold stronger NF-jB activity after treatment with pg-LPS (1 lg/ml), whereas pg-LPS stimulation had no such effect on MCF-7 cells. Blocking TLR2 by its neutralizing antibody (10 lg/ml, TLR2 Ab) decreased NF-jB activity in both MDA-MB-231 and MCF-7 cells. IgG (10 lg/ml) treatment was an isotype control. (C and D) After pg-LPS (1 lg/ml) stimulation, TAK1 and IjBa in MDA-MB-231 cells were highly activated. Especially compared with MDA-MB-231 cells, these signaling molecules under TLR2 regulation were not so significantly activated in MCF-7 cells. Values were expressed as mean ± SD (n = 6 *p < 0.05; **p < 0.01).
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Fig. 4. TLR2 activation increased IL-6, TGF-b, VEGF and MMP9 autocrine secretion in MDA-MB-231 cells. Cancer cells were cultured for 24 h and the supernatants were assayed by ELISA. (A and B) After TLR2 activation by pg-LPS (1 lg/ml), MDA-MB-231 cells secreted Il-6 or TGF-b up to five- or seven-fold, respectively, whereas TLR2 blockade by its neutralizing antibody (10 lg/ml, TLR2 Ab) reduced IL-6 and TGF-b level to half that in the untreated group. In addition, IL-6 and TGF-b autocrine secretion by MDA-MB231 cells was much higher than that in MCF-7 cells. (C and D) TLR2 stimulation effectively enhanced VEGF and MMP9 excretion in MDA-MB-231 and MCF-7 cells. Blockade of TLR2 decreased excretion of VEGF and MMP9 in MDA-MB-231 cells. Values were expressed as mean ± SD (n = 6 *p < 0.05; **p < 0.01; #p < 0.05; ##p < 0.01).
of TLR2 notably decreased this ability. Invasion through basal membranes is a critical metastatic step for cell detachment from primary loci and intrusion into distant organs [1]. Adhesive molecules such as b1 integrin and E-cadherin have been shown to be required for breast cancer cell invasion and metastasis, therefore, some adhesive molecules have become targets for breast cancer prevention [18,19]. Our findings indicated that TLR2 played an effective role in the invasive potential of MDA-MB-231 human breast cancer cells. Moreover, TLR2 can respond to pathogen-associated molecular patterns (PAMPs) such as LPS contained on pathogen cell-wall surfaces. TLR2 can also respond to endogenous ligands of damage-associated molecular patterns (DAMPs) such as heat shock protein 60 (Hsp60) and hyaluronic acid fragments, which are produced by tissue injury [20]. As solid malignancies are aggravated under conditions of chronic infection and tissue injury [21], our study suggests that besides inflammation, bacterial LPS may directly promote MDA-MB-231 cellular invasion via TLR2, and provide a direct link between bacterial infection and tumor progression. The present study showed that activation of TLR2 in MDA-MB231 cells increased constitutive NF-jB activity. NF-jB activity was first identified as an essential anti-apoptosis factor in cancer cells, and recently has been reported as an important factor during breast cancer cell invasion and metastasis [22]. Moreover, one study has reported that constitutive NF-jB activity in the highly invasive and metastatic MDA-MB-231 cells is much stronger than that in poorly invasive and metastatic breast cancer cells [9]. Our findings indicated that, in correlation with its differential expression in the two breast cancer cells, TLR2 activation effectively trig-
gered NF-jB activation more in MDA-MB-231 than in MCF-7 cells. We also showed that pTAK1 and pIkBa, which are two key kinases in the TLR2/NF-jB signaling pathway, were significantly higher after TLR2 stimulation, and these results indicate a validating TLR2/NF-jB signal in invasive MDA-MB-231 cells [3]. Our study indicated that, after TLR2 stimulation, breast cancer cells produced higher amounts of Il-6, TGF-b, VEGF and MMP9, whereas TLR2 blockade significantly decreased excretion of these cytokines. Autocrine secretion of cytokines by cancer cells exerts an important effect in the control of cancer cell behavior such as adhesion and invasion [11,12,23]. For example, IL-6 contributes to lung and breast cancer cell malignancy and effusion [10,11]; TGF-b induces cancer cell invasion in breast cancer [23]; and VEGF and MMP9 are regarded as indispensable cytokines for breast cancer cell invasion and adhesion [17]. Consistent with previous findings that TLR2 mediates high invasive capacity and augments NF-jB activity in MDA-MB-231 cells, we showed that TLR2 also promotes excretion of these cytokines in MDA-MB-231 cells. Therefore, we found that TLR2-triggered NF-jB promoted production of these cytokines, thus leading to MDA-MB-231 cell invasion. Since blocking NF-jB strongly suppresses MDA-MB-231 cell invasion [9,24], we conclude that TLR2/NF-jB signaling in invasive MDA-MB-231 cells contributes to their invasive activity via autocrine secretion of cytokines. In conclusion, we report herein that TLR2 mediates MDA-MB231 invasive properties by controlling activation of NF-jB, which is a very important factor for breast cancer cell transcriptional cytokines. TLR2/NF-jB signaling in MDA-MB-231 cells contributes to the underlying mechanism of invasive potential, which suggests
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that TLR2 is a potential hallmark and a therapeutic target in highly invasive breast cancer cells. Our results may also be relevant for breast cancer therapy. Post-mastectomy bacterial infection and inflammatory breast cancer are associated with PAMPs and DAMPs, such as Hsp60, which can directly activate TLR2 in cancer cells [20,25]. Thus pathogen eradication and anti-inflammatory therapy are important for preventing poor prognosis in breast cancer. Acknowledgment The first author expresses his gratitude to Professor Wei Wu and Professor Xiaoning Wang for their helpful discussion. References [1] K. Pantel, R.H. Brakenhoff, Dissecting the metastatic cascade, Nat. Rev. Cancer 4 (2004) 448–456. [2] Y. Zuo, S.K. Shields, C. Chakraborty, Enhanced intrinsic migration of aggressive breast cancer cells by inhibition of Rac1 GTPase, Biochem. Biophys. Res. Commun. 15 (2006) 361–367. [3] K. Takeda, T. Kaisho, S. Akira, Toll-like receptors, Annu. Rev. Immunol. 21 (2003) 335–376. [4] M. Pevsner-Fischer, V. Morad, M. Cohen-Sfady, L. Rousso-Noori, A. ZaninZhorov, S. Cohen, I.R. Cohen, D. Zipori, Toll-like receptors and their ligands control mesenchymal stem cell functions, Blood 109 (2007) 1422–1432. [5] M.J. Song, K.H. Kim, J.M. Yoon, J.B. Kim, Activation of Toll-like receptor 4 is associated with insulin resistance in adipocytes, Biochem. Biophys. Res. Commun. 4 (2006) 739–745. [6] B. Huang, J. Zhao, H. Li, K.L. He, Y. Chen, L. Mayer, J.C. Unkeless, H. Xiong, Tolllike receptors on tumor cells facilitate evasion of immune surveillance, Cancer Res. 65 (2005) 5009–5014. [7] B. Huang, J. Zhao, S. Shen, H. Li, K.L. He, G.X. Shen, L. Mayer, J. Unkeless, D. Li, Y. Yuan, G.M. Zhang, H. Xiong, Z.H. Feng, Listeria monocytogenes promotes tumor growth via tumor cell Toll-like receptor 2 signaling, Cancer Res. 67 (2007) 4346–4352. [8] M.A. Huber, N. Azoitei, B. Baumann, S. Grunert, A. Sommer, H. Pehamberger, N. Kraut, H. Beug, T. Wirth, NF-jB is essential for epithelial-mesenchymal transition and metastasis in a model of breast cancer progression, J. Clin. Invest. 114 (2004) 569–581. [9] B.K. Park, H. Zhang, Q. Zeng, J. Dai, E.T. Keller, T. Giordano, K. Gu, V. Shah, L. Pei, R.J. Zarbo, L. McCauley, S. Shi, S. Chen, C.Y. Wang, NF-jB in breast cancer cells promotes osteolytic bone metastasis by inducing osteoclastogenesis via GMCSF, Nat. Med. 13 (2007) 62–69.
[10] Z.T. Schafer, J.S. Brugge, IL-6 involvement in epithelial cancers, J. Clin. Invest. 117 (2007) 3660–3663. [11] P. Sansone, G. Storci, S. Tavolari, T. Guarnieri, C. Giovannini, M. Taffurelli, C. Ceccarelli, D. Santini, P. Paterini, K.B. Marcu, P. Chieco, M. Bonafe, IL-6 triggers malignant features in mammospheres from human ductal breast carcinoma and normal mammary gland, J. Clin. Invest. 117 (2007) 3988–4002. [12] A.T. Hooper, G. Akiri, D. Jin, S.V. Shmelkov, E. Chuang, S.J. Shin, Y. Wu, D.J. Hicklin, S. Rafii, L.T. Vahdat, VEGF receptor expression on reactive breast cancer stroma: paving the way for tumor invasion, J. Clin. Oncol. 23 (2005) 9642–9650. [13] I. Kokkinopoulos, W.J. Jordan, M.A. Ritter, Toll-like receptor mRNA expression patterns in human dendritic cells and monocytes, Mol. Immunol. 42 (2005) 957–968. [14] L. Connolly, P. Maxwell, Image analysis of transwell assays in the assessment of invasion by malignant cell lines, Br. J. Biomed. Sci. 59 (2002) 11–14. [15] P.O. Krutzik, G.P. Nolan, Intracellular phospho-protein staining techniques for flow cytometry: monitoring single cell signaling events, Cytometry 55 (2003) 61–70. [16] M. Karin, Y. Cao, F.R. Greten, Z.W. Li, NF-jB in cancer: from innocent bystander to major culprit, Nat. Rev. Cancer 2 (2002) 301–310. [17] S.H. Kim, N.H. Cho, K. Kim, J.S. Lee, B.S. Koo, J.H. Kim, J.H. Chang, E.C. Choi, Correlations of oral tongue cancer invasion with matrix metalloproteinases (MMPs) and vascular endothelial growth factor (VEGF) expression, J. Surg. Oncol. 93 (2006) 330–337. [18] E.S. Yao, H. Zhang, Y.-Y. Chen, B. Lee, K. Chew, D. Moore, C. Park, Increased b1 integrin is associated with decreased survival in invasive breast cancer, Cancer Res. 67 (2007) 659–664. [19] L. Mauro, S. Catalano, G. Bossi, M. Pellegrino, I. Barone, S. Morales, C. Giordano, V. Bartella, I. Casaburi, S. Ando, Evidences that leptin up-regulates E-cadherin expression in breast cancer: effects on tumor growth and progression, Cancer Res. 67 (2007) 3412–3421. [20] M.E. Bianchi, DAMPs, PAMPs and alarmins: all we need to know about danger, J. Leukoc. Biol. 81 (2007) 1–5. [21] L.M. Coussens, Z. Werb, Inflammation and cancer, Nature 420 (2002) 860–867. [22] G. Sethi, K.S. Ahn, B. Sung, B.B. Aggarwal, Pinitol targets nuclear factor-jB activation pathway leading to inhibition of gene products associated with proliferation, apoptosis, invasion, and angiogenesis, Mol. Cancer Ther. 7 (2008) 1604–1614. [23] M.B. Buck, C. Knabbe, TGF-b signaling in breast cancer, Ann. NY Acad. Sci. 1089 (2006) 119–126. [24] B. Sung, M.K. Pandey, Y. Nakajima, H. Nishida, T. Konishi, M.M. Chaturvedi, B.B. Aggarwal, Identification of a novel blocker of IkBa kinase activation that enhances apoptosis and inhibits proliferation and invasion by suppressing nuclear factor-jB, Mol. Cancer Ther. 7 (2008) 191–201. [25] A.R. Ferrara, Inflammatory breast cancer: challenges and developments, Radiol. Technol. 80 (2008) 49–60.