High EMT Signature Score of Invasive Non-Small Cell Lung Cancer (NSCLC) Cells Correlates with NFκB Driven Colony-Stimulating Factor 2 (CSF2/GM-CSF) Secretion by Neighboring Stromal Fibroblasts.
We established co-cultures of invasive or non-invasive NSCLC cell lines and various types of fibroblasts (FBs) to more precisely characterize the molecular mechanism of tumor-stroma crosstalk in lung cancer. The HGF-MET-ERK1/2-CREB-axis was shown to contribute to the onset of the invasive phenotype of Calu-1 with HGF being secreted by FBs. Differential expression analysis of the respective mono- and co-cultures revealed an upregulation of NFκB-related genes exclusively in co-cultures with Calu-1. Cytokine Array- and ELISA-based characterization of the "cytokine fingerprints" identified CSF2 (GM-CSF), CXCL1, CXCL6, VEGF, IL6, RANTES and IL8 as being specifically upregulated in various co-cultures. Whilst CXCL6 exhibited a strictly FB-type-specific induction profile regardless of the invasiveness of the tumor cell line, CSF2 was only induced in co-cultures of invasive cell lines regardless of the partnered FB type. These cultures revealed a clear link between the induction of CSF2 and the EMT signature of the cancer cell line. The canonical NFκB signaling in FBs, but not in tumor cells, was shown to be responsible for the induced and constitutive CSF2 expression. In addition to CSF2, cytokine IL6, IL8 and IL1B, and chemokine CXCL1 and CXCL6 transcripts were also shown to be increased in co-cultured FBs. In contrast, their induction was not strictly dependent on the invasiveness of the co-cultured tumor cell. In a multi-reporter assay, additional signaling pathways (AP-1, HIF1-α, KLF4, SP-1 and ELK-1) were found to be induced in FBs co-cultured with Calu-1. Most importantly, no difference was observed in the level of inducibility of these six signaling pathways with regard to the type of FBs used. Finally, upon tumor fibroblast interaction the massive induction of chemokines such as CXCL1 and CXCL6 in FBs might be responsible for increased recruitment of a monocytic cell line (THP-1) in a transwell assay.
Department of Lead Discovery, Boehringer Ingelheim RCV GmbH & Co KG, Vienna, Austria; Department of Microbiology, Immunobiology and Genetics, Center of Molecular Biology, Max F. Perutz Laboratories, University of Vienna, Vienna, Austria.
Albin
A
Rudisch
Department of Lead Discovery, Boehringer Ingelheim RCV GmbH & Co KG, Vienna, Austria.
Matthew Richard
MR
Dewhurst
Department of Lead Discovery, Boehringer Ingelheim RCV GmbH & Co KG, Vienna, Austria.
Luminita Gabriela
LG
Horga
Institute of Medical Genetics, Medical University of Vienna, Vienna, Austria.
Nina
N
Kramer
Department of Lead Discovery, Boehringer Ingelheim RCV GmbH & Co KG, Vienna, Austria.
Nathalie
N
Harrer
Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology and University of Tübingen, Stuttgart, Baden-Württemberg, Germany.
Meng
M
Dong
Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology and University of Tübingen, Stuttgart, Baden-Württemberg, Germany.
Heiko
H
van der Kuip
Department of Lead Discovery, Boehringer Ingelheim RCV GmbH & Co KG, Vienna, Austria.
Andreas
A
Wernitznig
Department of Lead Discovery, Boehringer Ingelheim RCV GmbH & Co KG, Vienna, Austria.
Andreas
A
Bernthaler
Institute of Medical Genetics, Medical University of Vienna, Vienna, Austria.
Helmut
H
Dolznig
Department of Lead Discovery, Boehringer Ingelheim RCV GmbH & Co KG, Vienna, Austria.
Wolfgang
W
Sommergruber
D000818:Animals; D015820:Cadherins / Q000378:metabolism; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics / Q000378:metabolism* / Q000473:pathology*; D002450:Cell Communication / Q000187:drug effects; D045744:Cell Line, Tumor; D016207:Cytokines / Q000378:metabolism; D020405:Dermis / Q000473:pathology; D058750:Epithelial-Mesenchymal Transition / Q000187:drug effects / Q000235:genetics*; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism*; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D053263:Gene Regulatory Networks / Q000187:drug effects; D017930:Genes, Reporter; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000378:metabolism*; D017228:Hepatocyte Growth Factor / Q000494:pharmacology; D006801:Humans; D007249:Inflammation / Q000473:pathology; D000090062:Kruppel-Like Factor 4; D008175:Lung Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D016328:NF-kappa B / Q000378:metabolism*; D009361:Neoplasm Invasiveness; D010641:Phenotype; D019859:Proto-Oncogene Proteins c-met / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D018874:Spheroids, Cellular / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D017154:Stromal Cells / Q000378:metabolism; D015854:Up-Regulation / Q000187:drug effects / Q000235:genetics
D015820:Cadherins; D016207:Cytokines; C000715467:KLF4 protein, human; C000715469:Klf4 protein, rat; D000090062:Kruppel-Like Factor 4; D016328:NF-kappa B; D012333:RNA, Messenger; D017228:Hepatocyte Growth Factor; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D019859:Proto-Oncogene Proteins c-met