Secretion of cytokines and growth factors as a general cause of constitutive NFκB activation in cancer

Abstract

The constitutive activation of nuclear factor jB (NFjB) helps a variety of tumors to resist apoptosis and desensitizes them to chemotherapy, but the causes are still largely unknown. We have analysed this phenomenon in eight mutant cell lines derived from human 293 cells, selected for NFjB-dependent expression of a marker gene, and also in seven tumor-derived cell lines. Conditioned media from all of these cells stimulated the activation of NFjB (up to 30-fold) in indicator cells carrying an NFjB-responsive reporter. Therefore, secretion of extracellular factors as the cause of constitutive activation seems to be general. The mRNAs encoding several different cytokines and growth factors were greatly overexpressed in the tumor and mutant cells. The pattern of overexpression was distinct in each cell line, indicating that the phenomenon is complex. Two secreted factors whose roles in the constitutive activation of NFjB are not well defined were investigated further as pure proteins: transforming growth factor b2 (TGFb2) and fibroblast growth factor 5 (FGF5) were both highly expressed in some mutant clones and tumor cell lines, each activated NFjB alone, and the combination was synergistic. Our data indicate that a group of different factors, expressed at abnormally high levels, can contribute singly and synergistically to the constitutive activation of NFjB in all of the mutant and tumor cell lines we studied. Since several NFjB target genes encode secreted proteins that induce NFjB, autocrine loops are likely to be ubiquitously important in the constitutive activation of NFjB in cancer. We provide the first evidence of the general, complex, and synergistic activation of NFjB in tumor and mutant cell lines through the action of secreted factors and suggest that the same explanation is likely for the constitutive activation of NFjB in cancers.

Mutant human cells with constitutive activation of NF-κB

“…Several studies in recent years have shown that autocrine production of cytokines confers constitutive NF-B activity in colon, prostate, pancreatic, and other types of cancer (53). Our recent results reveal that the constitutive activation of NF-B in most of the mutants is caused by deregulated expression of secretion of cytokines and other ligands, and some of these have been identified (54). Although the loss of cytoplasmic negative regulators such as SODD, CARD-inhibitor of NF-Bactivating ligands, apoptosis signal-regulating kinase 1, rafkinase inhibitor protein, PKC-interacting cousin of thioredoxin (PICOT), and phosphatase and tensin homolog (PTEN) might also result in the constitutive activation of NF-B, we find that SODD, PICOT, and PTEN are not missing in our constitutive mutants.…”

Tissue factor as an effector of angiogenesis and tumor progression in hematological malignancies

“…Furthermore, recent data suggest that in a variety of tumors, several NFkB target genes encode secreted growth factors that induce NFkB activation. 64 Thus, autocrine loops might be also important in the constitutive activation of NFkB in cancer. In this work, clinical studies demonstrated a higher incidence of recurrence and fatal outcome among patients in which a coordinated increase in the expression and activation of the above-mentioned proteins was observed.…”

Mutagenesis by reversible promoter insertion to study the activation of NF-κB

“…In fact, the relA insertional mutant has already provided important evidence for a positive-feedback loop involving secreted factors and NF-B. We have previously reported that elevated secretion of certain growth factors can cause constitutive NF-B activation in many cancer cell lines, and probably, also in tumors (13,14). Others have reported that Ap1 causes elevated growth factor secretion and the ensuing activation of NF-B which, in turn, cooperates with Ap1 (25).…”

“…Because several different cytokines are under NF-B transcriptional control, a positive-feedback loop can be established. We used an NF-B reporter cell line (13) to test whether medium conditioned by the mutant clones contains NF-Bactivating factors. Treatment of the reporter cells with medium conditioned by 3B22͞35 cells caused a robust elevation of luciferase activity (Fig.…”

“…Conditioned media from confluent cultures, collected after 24 h, was applied overnight to 293IL1R NF-B indicator cells, and a luciferase assay was carried out (13). Transient transfection of subconfluent cultures with a mixture of luciferase reporter plasmid (pE-selectin-luciferase) and pSV2-␤gal was performed by using the Lipofectamine Plus method (Invitrogen), and reporter assays were conducted (14).…”