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CXCL13–CXCR5 co-expression regulates epithelial to mesenchymal transition of breast cancer cells during lymph node metastasis

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Breast Cancer Research and Treatment Aims and scope Submit manuscript

An Erratum to this article was published on 17 February 2016

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.

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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

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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.

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Authors and Affiliations

  1. Immunology Laboratory, Department of Zoology, University of Calcutta, 35, Ballygunge Circular Road, Kolkata, 700019, West Bengal, India

    Subir Biswas, Suman Sengupta, Samir Jana, Gunjan Mandal & Arindam Bhattacharyya

  2. Materials Science Centre, Indian Institute of Technology Kharagpur, Kharagpur, India

    Sougata Roy Chowdhury

  3. Department of Pathology, North Bengal Medical College, Darjeeling, India

    Palash Kumar Mandal

  4. Department of Surgical Oncology, Saroj Gupta Cancer Centre and Research Institute, Kolkata, India

    Nipun Saha, Vivek Malhotra & Arnab Gupta

  5. Laboratory of Immunoregulation, Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia

    Dmitry V. Kuprash

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  1. Subir Biswas
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Corresponding author

Correspondence to Arindam Bhattacharyya.

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Suman Sengupta, Sougata Roy Chowdhury, and Samir Jana contributed equally to this work.

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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

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