Tumor Biology

, Volume 37, Issue 9, pp 12441–12453 | Cite as

Secretome of tumor-associated leukocytes augment epithelial-mesenchymal transition in positive lymph node breast cancer patients via activation of EGFR/Tyr845 and NF-κB/p65 signaling pathway

  • Eslam A. Elghonaimy
  • Sherif A. Ibrahim
  • Amal Youns
  • Zeinab Hussein
  • Mohamed Akram Nouh
  • Tahani El-mamlouk
  • Mohamed El-Shinawi
  • Mona Mostafa Mohamed
Original Article

Abstract

Epithelial-mesenchymal transition (EMT) is an essential process in breast cancer metastasis. The aim of the present study was to determine the role of secretions of tumor-associated leukocytes (TALs) isolated from negative and positive lymph nodes (nLNs and pLNs, respectively) breast cancer patients in regulating EMT mechanism and the associated signaling pathways. We found an increased infiltration of TALs, which was associated with downregulation of E-cadherin and over-expression of vimentin in the breast carcinoma tissues of pLNs as compared to nLNs patients and normal breast tissues obtained from healthy volunteers during mammoplasty. Furthermore, TALs isolated from pLNs breast cancer patients secreted an elevated panel of cytokines by up to 2–5-fold when compared with those isolated from nLNs patients. Secretome of TALs of pLNs possessed higher TARC, IGF-1, IL-3, TNF-β, IL-5, G-CSF, IL-4, and IL-1α with more than a fivefold compared to those of nLNs. Using the human breast cancer cell lines MCF-7 and MDA-MB-231, we found that cytokines secreted by TALs isolated from nLNs and pLNs breast cancer patients promoted EMT via upregulation of TGF-β and vimentin and downregulation of E-cadherin at messenger RNA (mRNA) levels in both cell lines and at protein level in MCF-7. While TGF-β is over-expressed by MDA-MB-231 seeded in media conditioned by secretome of TALs isolated from nLNs and pLNs breast cancer patients. The downstream TGF-β signaling transcription factors, Snail, Slug, and Twist, known to be associated with EMT mechanism were over-expressed by MCF-7 and MDA-MB-231 seeded in media conditioned by secretome of TALs isolated from nLNs and pLNs breast cancer patients. Acquisition of EMT in MCF-7 cells is mechanistically attributed to the activation of EGFR(Tyr845) and NF-κB/p65(Ser276) signaling which are significantly highly expressed by MCF-7 cells seeded in media conditioned by secretome of TALs isolated from pLNs compared to nLNs patients. Overall, this study provides implications of secretome of TALs and activated EGFR(Tyr845) and NF-κB/p65(Ser276) in EMT process that may be considered a therapeutic strategy to inhibit lymph node metastasis in breast cancer patients.

Keywords

Breast cancer Lymph node metastasis E-cadherin Vimentin EGFR(Tyr845) NF-κB/p65(Ser276) 

Supplementary material

13277_2016_5123_MOESM1_ESM.docx (12 kb)
Supplementary Table 1(DOCX 11 kb)
13277_2016_5123_Fig6_ESM.gif (680 kb)
Fig. S1

Expression level of total EGFR and NF-κB/p65 in breast cancer cell lines seeded in TALs condition media of nLNs and pLNs (TAL-CM-nLNs and TAL-CM-pLNs) patients. a Cell lysates (25 μg/lane) of MCF-7 and MDA-MB-231 cells seeded in control culture media, TAL-CM-nLNs and TAL-CM-pLNs were subjected to immunoblot analysis using antibodies against EGFR and NF-κB/p65 and β-actin as loading control. b Relative band intensities of EGFR and NF-κB/p65 were normalized to β-actin (50 kDa) expression. The data were analyzed using paired Student’s t test. Data represents mean ± SD. *p ≤ 0.05, **p ≤ 0.01, and ***represents p ≤ 0.001. Bands were visualized using ECL (Pierce, Paisley, UK) and BioSepctrum (UVP, Cambridge, UK) gel documentation system. (GIF 680 kb)

13277_2016_5123_MOESM2_ESM.tif (680 kb)
High-resolution image (TIFF 680 kb)

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Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2016

Authors and Affiliations

  • Eslam A. Elghonaimy
    • 1
  • Sherif A. Ibrahim
    • 1
  • Amal Youns
    • 1
  • Zeinab Hussein
    • 1
  • Mohamed Akram Nouh
    • 2
  • Tahani El-mamlouk
    • 1
  • Mohamed El-Shinawi
    • 3
  • Mona Mostafa Mohamed
    • 1
  1. 1.Department of Zoology, Faculty of ScienceCairo UniversityGizaEgypt
  2. 2.Department of Pathology, National Cancer InstituteCairo UniversityGizaEgypt
  3. 3.Department of General Surgery, Faculty of MedicineAin Shams UniversityCairoEgypt

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