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01-09-2016 | Original Article

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

Authors: Eslam A. Elghonaimy, Sherif A. Ibrahim, Amal Youns, Zeinab Hussein, Mohamed Akram Nouh, Tahani El-mamlouk, Mohamed El-Shinawi, Mona Mostafa Mohamed

Published in: Tumor Biology | Issue 9/2016

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.

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Eccles SA, Welch DR. Metastasis: recent discoveries and novel treatment strategies. Lancet. 2007;369(9574):1742–57. doi:10.1016/S0140-6736(07)60781-8.CrossRefPubMedPubMedCentral

Shook D, Keller R. Mechanisms, mechanics and function of epithelial-mesenchymal transitions in early development. Mech Dev. 2003;120(11):1351–83.CrossRefPubMed

Visvader JE, Lindeman GJ. Cancer stem cells in solid tumours: accumulating evidence and unresolved questions. Nat Rev Cancer. 2008;8(10):755–68. doi:10.1038/nrc2499.CrossRefPubMed

Thiery JP, Acloque H, Huang RY, Nieto MA. Epithelial-mesenchymal transitions in development and disease. Cell. 2009;139(5):871–90. doi:10.1016/j.cell.2009.11.007.CrossRefPubMed

Lamouille S, Xu J, Derynck R. Molecular mechanisms of epithelial-mesenchymal transition. Nat Rev Mol Cell Biol. 2014;15(3):178–96. doi:10.1038/nrm3758.CrossRefPubMedPubMedCentral

Medici D, Hay ED, Olsen BR. Snail and slug promote epithelial-mesenchymal transition through beta-catenin-T-cell factor-4-dependent expression of transforming growth factor-beta3. Mol Biol Cell. 2008;19(11):4875–87. doi:10.1091/mbc.E08-05-0506.CrossRefPubMedPubMedCentral

Scheel C, Weinberg RA. Cancer stem cells and epithelial-mesenchymal transition: concepts and molecular links. Semin Cancer Biol. 2012;22(5–6):396–403. doi:10.1016/j.semcancer.2012.04.001.CrossRefPubMed

Wang X, Wang H, Li G, Song Y, Wang S, Zhu F, et al. Activated macrophages down-regulate expression of E-cadherin in hepatocellular carcinoma cells via NF-kappaB/slug pathway. Tumour Biol. 2014;35(9):8893–901. doi:10.1007/s13277-014-2159-7.CrossRefPubMed

Huber MA, Azoitei N, Baumann B, Grunert S, Sommer A, Pehamberger H, et al. NF-kappaB is essential for epithelial-mesenchymal transition and metastasis in a model of breast cancer progression. J Clin Invest. 2004;114(4):569–81.CrossRefPubMedPubMedCentral

10.

Huber MA, Beug H, Wirth T. Epithelial-mesenchymal transition: NF-kappaB takes center stage. Cell Cycle. 2004;3(12):1477–80.CrossRefPubMed

11.

Shostak K, Chariot A. NF-kappaB, stem cells and breast cancer: the links get stronger. Breast Cancer Res. 2011;13(4):214. doi:10.1186/bcr2886.CrossRefPubMedPubMedCentral

12.

Scartozzi M, Bearzi I, Pierantoni C, Mandolesi A, Loupakis F, Zaniboni A, et al. Nuclear factor-kB tumor expression predicts response and survival in irinotecan-refractory metastatic colorectal cancer treated with cetuximab-irinotecan therapy. J Clin Oncol. 2007;25(25):3930–5. doi:10.1200/JCO.2007.11.5022.CrossRefPubMed

13.

El-Ghonaimy EA, El-Shinawi M, Ibrahim SA, El-Ghazaly H, Abd-El-Tawab R, Nouh MA, et al. Positive lymph-node breast cancer patients—activation of NF-kappaB in tumor-associated leukocytes stimulates cytokine secretion that promotes metastasis via C-C chemokine receptor CCR7. FEBS J. 2015;282(2):271–82. doi:10.1111/febs.13124.

14.

Mohamed MM, El-Ghonaimy EA, Nouh MA, Schneider RJ, Sloane BF, El-Shinawi M. Cytokines secreted by macrophages isolated from tumor microenvironment of inflammatory breast cancer patients possess chemotactic properties. Int J Biochem Cell Biol. 2014;46:138–47. doi:10.1016/j.biocel.2013.11.015 S1357-2725(13)00353-1.CrossRefPubMed

15.

Al-Raawi D, Abu-El-Zahab H, El-Shinawi M, Mohamed MM. Membrane type-1 matrix metalloproteinase (MT1-MMP) correlates with the expression and activation of matrix metalloproteinase-2 (MMP-2) in inflammatory breast cancer. Int J Clin Exp Med. 2011;4(4):265–75.PubMedPubMedCentral

16.

Nouh MA, Mohamed MM, El-Shinawi M, Shaalan MA, Cavallo-Medved D, Khaled HM, et al. Cathepsin B: a potential prognostic marker for inflammatory breast cancer. J Transl Med. 2011;9:1. doi:10.1186/1479-5876-9-1.CrossRefPubMedPubMedCentral

17.

El-Shinawi M, Abdelwahab SF, Sobhy M, Nouh MA, Sloane BF, Mohamed MM. Capturing and characterizing immune cells from breast tumor microenvironment: an innovative surgical approach. Ann Surg Oncol. 2010;17(10):2677–84. doi:10.1245/s10434-010-1029-9.CrossRefPubMedPubMedCentral

18.

Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248–54.CrossRefPubMed

19.

Mohamed MM, Cavallo-Medved D, Rudy D, Anbalagan A, Moin K, Sloane BF. Interleukin-6 increases expression and secretion of cathepsin B by breast tumor-associated monocytes. Cell Physiol Biochem. 2010;25(2–3):315–24. doi:10.1159/000276564.CrossRefPubMedPubMedCentral

20.

Ibrahim SA, Hassan H, Vilardo L, Kumar SK, Kumar AV, Kelsch R, et al. Syndecan-1 (CD138) modulates triple-negative breast cancer stem cell properties via regulation of LRP-6 and IL-6-mediated STAT3 signaling. PLoS One. 2013;8(12):e85737. doi:10.1371/journal.pone.0085737.CrossRefPubMedPubMedCentral

21.

Serrano-Gomez SJ, Maziveyi M, Alahari SK. Regulation of epithelial-mesenchymal transition through epigenetic and post-translational modifications. Mol Cancer. 2016;15:18. doi:10.1186/s12943-016-0502-x.CrossRefPubMedPubMedCentral

22.

Yu S, Yan C, Yang X, He S, Liu J, Qin C, et al. Pharmacoproteomic analysis reveals that metapristone (RU486 metabolite) intervenes E-cadherin and vimentin to realize cancer metastasis chemoprevention. Sci Rep. 2016;6:22388. doi:10.1038/srep22388.CrossRefPubMedPubMedCentral

23.

Shostak K, Zhang X, Hubert P, Goktuna SI, Jiang Z, Klevernic I, et al. NF-kappaB-induced KIAA1199 promotes survival through EGFR signalling. Nat Commun. 2014;5:5232. doi:10.1038/ncomms6232.CrossRefPubMedPubMedCentral

24.

Nathanson SD. Insights into the mechanisms of lymph node metastasis. Cancer. 2003;98(2):413–23.CrossRefPubMed

25.

Jatoi I, Hilsenbeck SG, Clark GM, Osborne CK. Significance of axillary lymph node metastasis in primary breast cancer. J Clin Oncol. 1999;17(8):2334–40.PubMed

26.

Markiewicz A, Welnicka-Jaskiewicz M, Seroczynska B, Skokowski J, Majewska H, Szade J, et al. Epithelial-mesenchymal transition markers in lymph node metastases and primary breast tumors—relation to dissemination and proliferation. Am J Transl Res. 2014;6(6):793–808.PubMedPubMedCentral

27.

Shriver CD, Hueman MT, Ellsworth RE. Molecular signatures of lymph node status by intrinsic subtype: gene expression analysis of primary breast tumors from patients with and without metastatic lymph nodes. J Exp Clin Cancer Res. 2014;33(1):782. doi:10.1186/s13046-014-0116-3.CrossRef

28.

DeNardo DG, Johansson M, Coussens LM. Immune cells as mediators of solid tumor metastasis. Cancer Metastasis Rev. 2008;27(1):11–8.CrossRefPubMed

29.

Cao YW, Wan GX, Sun JP, Cui XB, Hu JM, Liang WH, et al. Implications of the Notch1-snail/slug-epithelial to mesenchymal transition axis for lymph node metastasis in infiltrating ductal carcinoma. Kaohsiung J Med Sci. 2015;31(2):70–6. doi:10.1016/j.kjms.2014.11.008.CrossRefPubMed

30.

Grzegrzolka J, Biala M, Wojtyra P, Kobierzycki C, Olbromski M, Gomulkiewicz A, et al. Expression of EMT markers Slug and Twist in breast cancer. Anticancer Res. 2015;35(7):3961–8.PubMed

31.

Cho HJ, Park SM, Kim IK, Nam IK, Baek KE, Im MJ, et al. RhoGDI2 promotes epithelial-mesenchymal transition via induction of snail in gastric cancer cells. Oncotarget. 2014;5(6):1554–64. doi:10.18632/oncotarget.1733.CrossRefPubMedPubMedCentral

32.

Kang R, Zhao S, Liu L, Li F, Li E, Luo L, et al. Knockdown of PSCA induces EMT and decreases metastatic potentials of the human prostate cancer DU145 cells. Cancer Cell Int. 2016;16:20. doi:10.1186/s12935-016-0295-4.CrossRefPubMedPubMedCentral

33.

Ricciardi M, Zanotto M, Malpeli G, Bassi G, Perbellini O, Chilosi M, et al. Epithelial-to-mesenchymal transition (EMT) induced by inflammatory priming elicits mesenchymal stromal cell-like immune-modulatory properties in cancer cells. Br J Cancer. 2015;112(6):1067–75. doi:10.1038/bjc.2015.29.CrossRefPubMedPubMedCentral

34.

Amin A, Mokhdomi TA, Bukhari S, Wani SH, Wafai AH, Lone GN, et al. Tectorigenin ablates the inflammation-induced epithelial-mesenchymal transition in a co-culture model of human lung carcinoma. Pharmacol Rep. 2015;67(2):382–7. doi:10.1016/j.pharep.2014.10.020.CrossRefPubMed

35.

Al-haidari AA, Syk I, Jirstrom K, Thorlacius H. CCR4 mediates CCL17 (TARC)-induced migration of human colon cancer cells via RhoA/rho-kinase signaling. Int J Color Dis. 2013;28(11):1479–87. doi:10.1007/s00384-013-1712-y.CrossRef

36.

Olkhanud PB, Baatar D, Bodogai M, Hakim F, Gress R, Anderson RL, et al. Breast cancer lung metastasis requires expression of chemokine receptor CCR4 and regulatory T cells. Cancer Res. 2009;69(14):5996–6004. doi:10.1158/0008-5472.CAN-08-4619.CrossRefPubMedPubMedCentral

37.

Li CW, Xia W, Huo L, Lim SO, Wu Y, Hsu JL, et al. Epithelial-mesenchymal transition induced by TNF-alpha requires NF-kappaB-mediated transcriptional upregulation of Twist1. Cancer Res. 2012;72(5):1290–300. doi:10.1158/0008-5472.CAN-11-3123.CrossRefPubMedPubMedCentral

38.

Dinarello CA. Why not treat human cancer with interleukin-1 blockade? Cancer Metastasis Rev. 2010;29(2):317–29. doi:10.1007/s10555-010-9229-0.CrossRefPubMedPubMedCentral

39.

Ji X, Li J, Xu L, Wang W, Luo M, Luo S, et al. IL4 and IL-17A provide a Th2/Th17-polarized inflammatory milieu in favor of TGF-beta1 to induce bronchial epithelial-mesenchymal transition (EMT). Int J Clin Exp Pathol. 2013;6(8):1481–92.PubMedPubMedCentral

40.

Cohen EN, Gao H, Anfossi S, Mego M, Reddy NG, Debeb B, et al. Inflammation mediated metastasis: immune induced epithelial-to-mesenchymal transition in inflammatory breast cancer cells. PLoS One. 2015;10(7):e0132710. doi:10.1371/journal.pone.0132710.CrossRefPubMedPubMedCentral

41.

Zaynagetdinov R, Sherrill TP, Gleaves LA, McLoed AG, Saxon JA, Habermann AC, et al. Interleukin-5 facilitates lung metastasis by modulating the immune microenvironment. Cancer Res. 2015. doi:10.1158/0008-5472.CAN-14-2379.PubMedPubMedCentral

42.

Kowanetz M, Wu X, Lee J, Tan M, Hagenbeek T, Qu X, et al. Granulocyte-colony stimulating factor promotes lung metastasis through mobilization of Ly6G+Ly6C+ granulocytes. Proc Natl Acad Sci U S A. 2010;107(50):21248–55. doi:10.1073/pnas.1015855107.CrossRefPubMedPubMedCentral

43.

Cui YH, Suh Y, Lee HJ, Yoo KC, Uddin N, Jeong YJ, et al. Radiation promotes invasiveness of non-small-cell lung cancer cells through granulocyte-colony-stimulating factor. Oncogene. 2015. doi:10.1038/onc.2014.466.

44.

Julien S, Puig I, Caretti E, Bonaventure J, Nelles L, van Roy F, et al. Activation of NF-kappaB by Akt upregulates snail expression and induces epithelium mesenchyme transition. Oncogene. 2007;26(53):7445–56. doi:10.1038/sj.onc.1210546.CrossRefPubMed

45.

Wu Y, Deng J, Rychahou PG, Qiu S, Evers BM, Zhou BP. Stabilization of snail by NF-kappaB is required for inflammation-induced cell migration and invasion. Cancer Cell. 2009;15(5):416–28. doi:10.1016/j.ccr.2009.03.016.CrossRefPubMedPubMedCentral

46.

Stanisavljevic J, Porta-de-la-Riva M, Batlle R, de Herreros AG, Baulida J. The p65 subunit of NF-kappaB and PARP1 assist Snail1 in activating fibronectin transcription. J Cell Sci. 2011;124(Pt 24):4161–71. doi:10.1242/jcs.078824.CrossRefPubMed

47.

Tsubaki M, Komai M, Fujimoto S, Itoh T, Imano M, Sakamoto K, et al. Activation of NF-kappaB by the RANKL/RANK system up-regulates snail and twist expressions and induces epithelial-to-mesenchymal transition in mammary tumor cell lines. J Exp Clin Cancer Res. 2013;32:62. doi:10.1186/1756-9966-32-62.CrossRefPubMedPubMedCentral

48.

Jiang C, Lin X. Analysis of epidermal growth factor-induced NF-kappaB signaling. Methods Mol Biol. 2015;1280:75–102. doi:10.1007/978-1-4939-2422-6_6.CrossRefPubMed

49.

Lo HW, Hsu SC, Xia W, Cao X, Shih JY, Wei Y, et al. Epidermal growth factor receptor cooperates with signal transducer and activator of transcription 3 to induce epithelial-mesenchymal transition in cancer cells via up-regulation of TWIST gene expression. Cancer Res. 2007;67(19):9066–76. doi:10.1158/0008-5472.CAN-07-0575.CrossRefPubMedPubMedCentral

50.

Corkery B, Crown J, Clynes M, O’Donovan N. Epidermal growth factor receptor as a potential therapeutic target in triple-negative breast cancer. Ann Oncol. 2009;20(5):862–7. doi:10.1093/annonc/mdn710.CrossRefPubMed

51.

Cai J, Tian AX, Wang QS, Kong PZ, Du X, Li XQ, et al. FOXF2 suppresses the FOXC2-mediated epithelial-mesenchymal transition and multidrug resistance of basal-like breast cancer. Cancer Lett. 2015;367(2):129–37. doi:10.1016/j.canlet.2015.07.001.CrossRefPubMed

Title: 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
Authors: Eslam A. Elghonaimy
Sherif A. Ibrahim
Amal Youns
Zeinab Hussein
Mohamed Akram Nouh
Tahani El-mamlouk
Mohamed El-Shinawi
Mona Mostafa Mohamed
Publication date: 01-09-2016
Publisher: Springer Netherlands
Published in: Tumor Biology / Issue 9/2016
Print ISSN: 1010-4283
Electronic ISSN: 1423-0380
DOI: https://doi.org/10.1007/s13277-016-5123-x

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