Abstract
We investigated the expression of –CXC chemokine ligand 13 (CXCL13) and its receptor –CXC chemokine receptor 5 (CXCR5) in 98 breast cancer (BC) patients with infiltrating duct carcinoma, out of which 56 were found lymph node metastasis (LNM) positive. Interestingly, co-expression of CXCL13 and CXCR5 showed a significant correlation with LNM. Since, epithelial to mesenchymal transition (EMT) is highly associated with metastasis we investigated EMT-inducing potential of CXCL13 in BC cell lines. In CXCL13-stimulated BC cells, expression of various mesenchymal markers (Vimentin, N-cadherin), EMT regulators (Snail, Slug), and matrix metalloproteinase-9 (MMP9) was increased, whereas the expression of epithelial marker E-cadherin was found to be decreased. In addition, expression of receptor activator of nuclear factor kappa-B ligand (RANKL), which is known to regulate MMP9 expression via Src activation, was also significantly increased after CXCL13 stimulation. Using specific protein kinase inhibitors, we confirmed that CXCL13 stimulated EMT and MMP9 expression via RANKL–Src axis in BC cell lines. To further validate this observation, we examined gene expression patterns in primary breast tumors and detected significantly higher expression of various mesenchymal markers and regulators in CXCL13–CXCR5 co-expressing patients. Therefore, this study showed the EMT-inducing potential of CXCL13 as well as demonstrated the prognostic value of CXCL13–CXCR5 co-expression in primary BC. Moreover, CXCL13–CXCR5–RANKL–Src axis may present a therapeutic target in LNM positive BC patients.
Similar content being viewed by others
Abbreviations
- ANOVA:
-
Analysis of variance
- AP:
-
Alkaline phosphatase
- BC:
-
Breast cancer
- BCIP:
-
5-Bromo-4-chloro-3′-indolyphosphate
- BSA:
-
Bovine serum albumin
- CXCL13:
-
–CXC chemokine ligand 13
- CXCR5:
-
–CXC chemokine receptor 5
- DAB:
-
3-3′-Diaminobenzidine
- DAPI:
-
4′,6-Diamidino-2-phenylindole
- DMEM:
-
Dulbecco’s modified Eagle’s medium
- DPX:
-
Distyrene plasticizer and xylene
- E-cadh:
-
E-cadherin
- ECM:
-
Extracellular matrix
- EMT:
-
Epithelial to mesenchymal transition
- ER:
-
Estrogen receptor
- FAK:
-
Focal adhesion kinase
- FBS:
-
Foetal bovine serum
- FITC:
-
Fluorescein isothiocyanate
- HRP:
-
Horse-radish-peroxidase
- IDC:
-
Infiltrating duct carcinoma
- IHC:
-
Immunohistochemistry
- LNM:
-
Lymph node metastasis
- MMLV:
-
Moloney murine leukemia virus
- MMP2:
-
Matrix metalloproteinase-2
- MMP9:
-
Matrix metalloproteinase-9
- MRM:
-
Modified radical mastectomy
- NBT:
-
Nitro-blue tetrazolium
- N-cadh:
-
N-cadherin
- p :
-
Probability
- PBS:
-
Phosphate buffered saline
- PR:
-
Progesterone receptor
- p-Src:
-
Phosphorylated-Src
- PVDF:
-
Polyvinylidine difluoride
- RANKL:
-
Receptor activator of nuclear factor kappa-B ligand
- RIPA:
-
Radio-immunoprecipitation assay
- SDS-PAGE:
-
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
- SGCC & RI:
-
Saroj Gupta Cancer Centre and Research Institute
- SD:
-
Standard deviation
- t-Src:
-
Total-Src
- TNF:
-
Tumor necrosis factor
- WB:
-
Western blot
References
Balkwill F (2004) Cancer and the chemokine network. Nat Rev Cancer 4:540–550
Gupta SK, Lysko PG, Pillarisetti K, Ohlstein E, Stadel JM (1998) Chemokine receptors in human endothelial cells. Functional expression of CXCR4 and its transcriptional regulation by inflammatory cytokines. J Biol Chem 273:4282–4287
Zlotnik A, Yoshie O (2000) Chemokines: a new classification system and their role in immunity. Immunity 12:121–127
Patel J, Channon KM, McNeill E (2013) The downstream regulation of chemokine receptor signaling: implications for atherosclerosis. Mediators Inflamm. doi:10.1155/2013/459520
Latek D, Modzelewska A, Trzaskowski B, Palczewski K, Filipek S (2012) G protein-coupled receptors-recent advances. Acta Biochim Pol 59:515–529
Zlontik A (2004) Chemokines in neoplastic progression. Semin Cancer Biol 14:181–185
Longo-Imedio MI, Longo N, Trevino I, Lazaro P, Sanchez-Mateos P (2005) Clinical significance of CXCR3 and CXCR4 expression in primary melanoma. Int J Cancer 117:861–865
Murphy C, McGurk M, Pettigrew J et al (2005) Nonapical and cytoplasmic expression of interleukin-8, CXCR1, and CXCR2 correlates with cell proliferation and microvessel density in prostate cancer. Clin Cancer Res 11:4117–4127
Keeley EC, Mehrad B, Strieter RM (2010) CXC chemokines in cancer angiogenesis and metastases. Adv Cancer Res 106:91–111
Belperio JA, Keane MP, Arenberg DA et al (2000) CXC chemokines in angiogenesis. J Leukoc Biol 68:1–8
Vicari AP, Caux C (2002) Chemokines in cancer. Cytokine Growth Factor Rev 13:143–154
Murakami T, Cardones AR, Hwang ST (2004) Chemokine receptors and melanoma metastasis. J Dermatol Sci 36:71–78
Tanaka T, Bai Z, Srinoulprasert Y, Yang BG, Hayasaka H, Miyasaka M (2005) Chemokines in tumor progression and metastasis. Cancer Sci 96:317–322
Shayan R, Achen MG, Stacker SA (2006) Lymphatic vessels in cancer metastasis: bridging the gaps. Carcinogenesis 27:1729–1738
Li YM, Pan Y, Wei Y et al (2004) Up-regulation of CXCR4 is essential for HER2-mediated tumor metastasis. Cancer Cell 6:459–469
Bachelder RE, Wendt MA, Mercurio AM (2002) Vascular endothelial growth factor promotes breast carcinoma invasion in an autocrine manner by regulating the chemokine receptor CXCR4. Cancer Res 62:7203–7206
Muller A, Homey B, Soto H et al (2001) Involvement of chemokine receptors in breast cancer. Nature 410:50–56
Wiseman BS, Werb Z (2002) Stromal effects on mammary gland development and breast cancer. Science 296:1046–1049
Kim M, Koh YJ, Kim KE et al (2010) CXCR4 signaling regulates metastasis of chemoresistant melanoma cells by a lymphatic metastatic niche. Cancer Res 70:10411–10421
Rhodes LV, Short SP, Neel NF et al (2011) Chemokine receptor CXCR4 mediates estrogen-independent tumorigenesis, metastasis, and resistance to endocrine therapy in human breast cancer. Cancer Res 71:603–613
Dhawan P, Richmond A (2002) Role of CXCL1 in tumorigenesis of melanoma. J Leukoc Biol 72:9–18
Zhou Y, Zhang J, Liu Q et al (2005) The chemokine GRO-alpha (CXCL1) confers increased tumorigenicity to glioma cells. Carcinogenesis 26:2058–2068
Singh S, Singh AP, Sharma B, Owen LB, Singh RK (2010) CXCL8 and its cognate receptors in melanoma progression and metastasis. Future Oncol 6:111–116
Lin Y, Huang R, Chen L, Li S, Shi Q, Jordan C, Huang RP (2004) Identification of interleukin-8 as estrogen receptor-regulated factor involved in breast cancer invasion and angiogenesis by protein arrays. Int J Cancer 109:507–515
Meijer J, Zeelenberg IS, Sipos B, Roos E (2006) The CXCR5 chemokine receptor is expressed by carcinoma cells and promotes growth of colon carcinoma in the liver. Cancer Res 66:9576–9582
Bürkle A, Niedermeier M, Schmitt-Gräff A, Wierda WG, Keating MJ, Burger JA (2007) Overexpression of the CXCR5 chemokine receptor, and its ligand, CXCL13 in B-cell chronic lymphocytic leukemia. Blood 110:3316–3325
Panse J, Friedrichs K, Marx A et al (2008) Chemokine CXCL13 is overexpressed in the tumor tissue and in the peripheral blood of breast cancer patients. Br J Cancer 99:930–938
Airoldi I, Cocco C, Morandi F, Prigione I, Pistoia V (2008) CXCR5 may be involved in the attraction of human metastatic neuroblastoma cells to the bone marrow. Cancer Immunol Immunother 57:541–548
Razmkhah M, Jaberipour M, Safaei A, Talei AR, Erfani N, Ghaderi A (2012) Chemokine and chemokine receptors: a comparative study between metastatic and non-metastatic lymph nodes in breast cancer patients. Eur Cytokine Netw 23:72–77
El Haibi CP, Sharma PK, Singh R, Johnson PR, Suttles J, Singh S, Lillard JW Jr (2010) PI3Kp110-, Src-, FAK-dependent and DOCK2-independent migration and invasion of CXCL13-stimulated prostate cancer cells. Mol Cancer 9:85
Guarino M (2007) Epithelial-mesenchymal transition and tumor invasion. Int J Biochem Cell Biol 39:2153–2160
Markiewicz A, Ahrends T, Wełnicka-Jaskiewicz M et al (2012) Expression of epithelial to mesenchymal transition-related markers in lymph node metastases as a surrogate for primary tumor metastatic potential in breast cancer. J Transl Med 10:226
Thiery JP, Sleeman JP (2006) Complex networks orchestrate epithelial-mesenchymal transitions. Nat Rev Mol Cell Biol 7:131–142
Cavallaro U, Christofori G (2004) Cell adhesion and signalling by cadherins and Ig-CAMs in cancer. Nat Rev Cancer 4:118–132
Thiery JP, Acloque H, Huang RY, Nieto MA (2009) Epithelial-mesenchymal transitions in development and disease. Cell 139:871–890
Fata JE, Kong YY, Li J et al (2000) The osteoclast differentiation factor osteoprotegerin-ligand is essential for mammary gland development. Cell 103:41–50
Zhang L, Teng Y, Zhang Y et al (2012) C-Src-mediated RANKL-induced breast cancer cell migration by activation of the ERK and Akt pathway. Oncol Lett 3:395–400
Armstrong AP, Miller RE, Jones JC, Zhang J, Keller ET, Dougall WC (2008) RANKL acts directly on RANK-expressing prostate tumor cells and mediates migration and expression of tumor metastatic genes. Prostate 68:92–104
Neve RM, Chin K, Fridlyand J et al (2006) A collection of breast cancer cell lines for the study of functionally distinct cancer subtypes. Cancer Cell 10:515–527
Jemal A, Siegel R, Xu J, Ward E (2010) Cancer statistics, 2010. CA Cancer J Clin 60:277–300
Weigelt B, Peterse JL, van’t Veer LJ (2005) Breast cancer metastasis: markers and models. Nat Rev Cancer 5:591–602
Sen U, Sankaranarayanan R, Mandal S, Ramanakumar AV, Parkin DM, Siddiqi M (2002) Cancer patterns in eastern India: the first report of the Kolkata cancer registry. Int J Cancer 100:86–91
http://www.breastcancerindia.net/bc/statistics/stati.htm. Accessed 15 Jun 2013
Singh S, Singh R, Singh UP et al (2009) Clinical and biological significance of CXCR5 expressed by prostate cancer specimens and cell lines. Int J Cancer 125:2288–2295
Singh S, Singh R, Sharma PK et al (2009) Serum CXCL13 positively correlates with prostatic disease, prostate-specific antigen and mediates prostate cancer cell invasion, integrin clustering and cell adhesion. Cancer Lett 283:29–35
Del Grosso F, Coco S, Scaruffi P et al (2011) Role of CXCL13–CXCR5 crosstalk between malignant neuroblastoma cells and Schwannian stromal cells in neuroblastic tumors. Mol Cancer Res 9:815–823
Ebisuno Y, Tanaka T, Kanemitsu N et al (2003) Cutting edge: the B cell chemokine CXC chemokine ligand 13/B lymphocyte chemoattractant is expressed in the high endothelial venules of lymph nodes and Peyer’s patches and affects B cell trafficking across high endothelial venules. J Immunol 171:1642–1646
Peinado H, Portillo F, Cano A (2004) Transcriptional regulation of cadherins during development and carcinogenesis. Int J Dev Biol 48:365–375
Dong C, Wu Y, Yao J et al (2012) G9a interacts with Snail and is critical for Snail-mediated E-cadherin repression in human breast cancer. J Clin Invest 122:1469–1486
Fujita N, Jaye DL, Kajita M, Geigerman C, Moreno CS, Wade PA (2003) MTA3, a Mi-2/NuRD complex subunit, regulates an invasive growth pathway in breast cancer. Cell 113:207–219
El-Haibi CP, Singh R, Gupta P, Sharma PK, Greenleaf KN, Singh S, Lillard JW Jr (2012) Antibody microarray analysis of signaling networks regulated by Cxcl13 and Cxcr5 in prostate cancer. J Proteomics Bioinform 5:177–184
Zhao Y, Li X, Sun X, Zhang Y, Ren H (2012) EMT phenotype is induced by increased Src kinase activity via Src-mediated caspase-8 phosphorylation. Cell Physiol Biochem 29:341–352
Acknowledgments
Financial supports were made by Department of Science and Technology, Govt. of India (Sanction No.-INT/RFBR/P-82), Council of Scientific and Industrial Research (37/1455/10/EMR-II), and Russian Foundation for Basic Research, Russian Federation (Sanction No.-10-04-92657) for the work and Council of Scientific and Industrial Research—JRF/NET Fellowship Grant [No. 9/028(842)/2011-EMR-I] to Subir Biswas. We thank the patients and their families who participated in this study and also thank the nursing staffs of SGCC & RI. We are thankful to Shravasti Roy, for her help in acquiring histopathological information of the patients, Prakriti Roy and Mayuri Nath for their help in sample collection, Kayum Alam for his help in acquisition of real-time PCR data, Subhadip Kundu and Soumya Chatterjee for initial project design, Soham Mitra, Tarun Keswani, Shauryabrota Dalui for their help in running the experiments.
Conflict of interest
The authors declared that they do not have any conflict of interest.
Author information
Authors and Affiliations
Corresponding author
Additional information
Suman Sengupta, Sougata Roy Chowdhury, and Samir Jana contributed equally to this work.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Biswas, S., Sengupta, S., Roy Chowdhury, S. et al. CXCL13–CXCR5 co-expression regulates epithelial to mesenchymal transition of breast cancer cells during lymph node metastasis. Breast Cancer Res Treat 143, 265–276 (2014). https://doi.org/10.1007/s10549-013-2811-8
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10549-013-2811-8