Regulation of the inflammatory profile of stromal cells in human breast cancer: prominent roles for TNF-α and the NF-κB pathway.
BACKGROUND
Breast cancer progression is promoted by stromal cells that populate the tumors, including cancer-associated fibroblasts (CAFs) and mesenchymal stem/stromal cells (MSCs). The activities of CAFs and MSCs in breast cancer are integrated within an intimate inflammatory tumor microenvironment (TME) that includes high levels of tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β). Here, we identified the impact of TNF-α and IL-1β on the inflammatory phenotype of CAFs and MSCs by determining the expression of inflammatory chemokines that are well-characterized as pro-tumorigenic in breast cancer: CCL2 (MCP-1), CXCL8 (IL-8) and CCL5 (RANTES).


METHODS
Chemokine expression was determined in breast cancer patient-derived CAFs by ELISA and in patient biopsies by immunohistochemistry. Chemokine levels were determined by ELISA in (1) human bone marrow-derived MSCs stimulated by tumor conditioned media (Tumor CM) of breast tumor cells (MDA-MB-231 and MCF-7) at the end of MSC-to-CAF-conversion process; (2) Tumor CM-derived CAFs, patient CAFs and MSCs stimulated by TNF-α (and IL-1β). The roles of AP-1 and NF-κB in chemokine secretion were analyzed by Western blotting and by siRNAs to c-Jun and p65, respectively. Migration of monocytic cells was determined in modified Boyden chambers.


RESULTS
TNF-α (and IL-1β) induced the release of CCL2, CXCL8 and CCL5 by MSCs and CAFs generated by prolonged stimulation of MSCs with Tumor CM of MDA-MB-231 and MCF-7 cells. Patient-derived CAFs expressed CCL2 and CXCL8, and secreted CCL5 following TNF-α (and IL-1β) stimulation. CCL2 was expressed in CAFs residing in proximity to breast tumor cells in biopsies of patients diagnosed with invasive ductal carcinoma. CCL2 release by TNF-α-stimulated MSCs was mediated by TNF-RI and TNF-RII, through the NF-κB but not via the AP-1 pathway. Exposure of MSCs to TNF-α led to potent CCL2-induced migration of monocytic cells, a process that may yield pro-cancerous myeloid infiltrates in breast tumors.


CONCLUSIONS
Our novel results emphasize the important roles of inflammation-stroma interactions in breast cancer, and suggest that NF-κB may be a potential target for inhibition in tumor-adjacent stromal cells, enabling improved tumor control in inflammation-driven malignancies.
Department of Cell Research and Immunology, George S. Wise Faculty of Life Sciences, Tel Aviv University, P.O. Box 39040, Tel Aviv, 6997801, Israel. christina.katanov@weizmann.ac.il.
Christina
C
Katanov
Department of Cell Research and Immunology, George S. Wise Faculty of Life Sciences, Tel Aviv University, P.O. Box 39040, Tel Aviv, 6997801, Israel. slerrer@gmail.com.
Shalom
S
Lerrer
Department of Cell Research and Immunology, George S. Wise Faculty of Life Sciences, Tel Aviv University, P.O. Box 39040, Tel Aviv, 6997801, Israel. yulialiub@gmail.com.
Yulia
Y
Liubomirski
Department of Pathology, Tel Aviv Sourasky Medical Center and the Sackler School of Medicine, Tel Aviv University, 6 Weizmann Street, Tel Aviv, 64239, Israel. treleox2@yahoo.com.
Leonor
L
Leider-Trejo
Department of Cell Research and Immunology, George S. Wise Faculty of Life Sciences, Tel Aviv University, P.O. Box 39040, Tel Aviv, 6997801, Israel. tzipi@post.tau.ac.il.
Tsipi
T
Meshel
Institute of Oncology, Sheba Medical Center, Tel-Hashomer, Ramat Gan, 5262100, Israel. bar.jair@gmail.com.
Jair
J
Bar
Institute of Oncology, Sheba Medical Center, Tel-Hashomer, Ramat Gan, 5262100, Israel. rotemfe@gmail.com.
Rotem
R
Feniger-Barish
Institute of Oncology, Sheba Medical Center, Tel-Hashomer, Ramat Gan, 5262100, Israel. iris.kamer@gmail.com.
Iris
I
Kamer
Department of Cell Research and Immunology, George S. Wise Faculty of Life Sciences, Tel Aviv University, P.O. Box 39040, Tel Aviv, 6997801, Israel. galisoria@gmail.com.
Gali
G
Soria-Artzi
Department of Cell Research and Immunology, George S. Wise Faculty of Life Sciences, Tel Aviv University, P.O. Box 39040, Tel Aviv, 6997801, Israel. kahanihadar@gmail.com.
Hadar
H
Kahani
Department of Medicine and Pharmacology, Robert Wood Johnson Medical School and Graduate School of Biomedical Sciences, Rutgers, The State University of New Jersey, 195 Little Albany Street, New Brunswick, NJ, 08901, USA. banerjed@cinj.rutgers.edu.
Debabrata
D
Banerjee
Department of Cell Research and Immunology, George S. Wise Faculty of Life Sciences, Tel Aviv University, P.O. Box 39040, Tel Aviv, 6997801, Israel. aditbb@tauex.tau.ac.il.
Adit
A
Ben-Baruch
D015153:Blotting, Western; D001854:Bone Marrow Cells / Q000166:cytology; D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology*; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects; D018932:Chemokine CCL2 / Q000032:analysis; D018946:Chemokine CCL5 / Q000032:analysis; D017077:Culture Media, Conditioned / Q000494:pharmacology; D005260:Female; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D053583:Interleukin-1beta / Q000494:pharmacology; D016209:Interleukin-8 / Q000032:analysis; D048031:JNK Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D061986:MCF-7 Cells; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism*; D034622:RNA Interference; D015398:Signal Transduction; D051996:Transcription Factor RelA / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D015854:Up-Regulation / Q000187:drug effects
D018932:Chemokine CCL2; D018946:Chemokine CCL5; D017077:Culture Media, Conditioned; D053583:Interleukin-1beta; D016209:Interleukin-8; D016328:NF-kappa B; D051996:Transcription Factor RelA; D014409:Tumor Necrosis Factor-alpha; D048031:JNK Mitogen-Activated Protein Kinases