c-Myb interferes with inflammatory IL1α-NF-κB pathway in breast cancer cells

Abstract

The transcription factor c-Myb can be involved in the activation of many genes with protumorigenic function; however, its role in breast cancer (BC) development is still under discussion. c-Myb is considered as a tumor-promoting factor in the early phases of BC, on the other hand, its expression in BC patients relates to a good prognosis. Previously, we have shown that c-Myb controls the capacity of BC cells to form spontaneous lung metastasis. Reduced seeding of BC cells to the lungs is linked to high expression of c-Myb and a decline in expression of a specific set of inflammatory genes. Here, we unraveled a c-Myb-IL1α-NF-κB signaling axis that takes place in tumor cells. We report that an overexpression of c-Myb interfered with the activity of NF-κB in several BC cell lines. We identified IL1α to be essential for this interference since it was abrogated in the IL1α-deficient cells. Overexpression of IL1α, as well as addition of recombinant IL1α protein, activated NF-κB signaling and restored expression of the inflammatory signature genes suppressed by c-Myb. The endogenous levels of c-Myb negatively correlated with IL1α on both transcriptional and protein levels across BC cell lines. We concluded that inhibition of IL1α expression by c-Myb reduces NF-κB activity and disconnects the inflammatory circuit, a potentially targetable mechanism to mimic the antimetastatic effect of c-Myb with therapeutic perspective.

Keywords: Breast cancer; IL1α; Inflammation; NF-κB; Transactivation; c-Myb.

Fig. 1

c-Myb overexpression interferes with NF-κB signaling in breast cancer cells lines. (A) Heatmap of MYB mRNA expression in human breast cancer cell lines aligned according to their constitutive NF-κB activity as measured in . (B) Transactivation of NF-κB-luc reporter in human MDA-MB-436 and MDA-MB-231 cells transiently overexpressing MYB, luciferase activity was normalized to β-galactosidase activity and expressed as relative light units. Average normalized relative light units (RLU) from 3 independent experiments are shown, comparison to mock transfected cells. (C) Transactivation of NF-κB-luc reporter in murine 4T1 cells transiently overexpressing wt Myb and Myb harboring M303V amino acid substitution. Luciferase activity was normalized to the protein concentration. Average normalized relative light units (RLU) from 3 independent experiments are shown in comparison to mock-transfected cells. (D) NF-κB target genes that are differentially expressed in 4T1 Myb^high cells compared to mock control cells, heatmap of RNA sequencing data. Significant differences (*P < 0.05, **P < 0.01) are indicated.

Fig. 2

IL1α is downregulated by c-Myb and its overexpression inversely impacts the inflammatory signature and NF-κB pathway. (A) mRNA levels of indicated genes in JSH-23-treated (20 µM, 24 h) E0771.LMB cells were normalized to Gapdh, relative fold change to DMSO-treated cells is shown for each gene. An average of 3 independent experiments is shown. Secreted IL1α protein levels in 4T1 cells overexpressing wt c-Myb (Myb^high 5 and 8) (B) and MDA-MB-231c-Myb knock-out cells (MYB KO) (C) as measured by Cytometric Bead Array (CBA). An average of 3 independent experiments is shown, comparison to mock-transfected and scrambled-transfected cells, respectively (D) Myb (left) and Il1a (right) mRNA expression in E0771.LMB cells overexpressing wt c-Myb (Myb^high pool, Myb^high C2) and M303V mutant c-Myb (M303V pool). Data were normalized to Gapdh, fold change values relative to wt cells from 3 independent experiments are shown, comparison to mock-transfected cells. Clones overexpressing mouse Il1a (IL1α up) were derived from 4T1 (E) and E0771.LMB (F) cells. Secreted IL1α protein levels were measured in 3 biological replicates by CBA, higher IL1α concentrations are compared to mock-transfected cells. (G) Expression of selected NF-κB family members/targets in E0771.LMB IL1α up cells as determined by qPCR, normalized to Gapdh and shown in triplicates as a heatmap. (H) IκBα levels in E0771.LMB cells cotransfected with pcDNA3.1 (mock), resp. pcDNA3.IL1a (IL1α), and pIκBα-miRFP703 vectors. Graph shows average frequency of miRFP703+ cells from 3 independent experiments. (I) mRNA levels of indicated genes in IL1α up E0771.LMB cells as determined by qPCR were normalized to Gapdh and expressed as fold change to wt cells. Significance calculated from 3 independent experiment is shown, comparison to mock-transfected cells. Proteins levels of Cxcl1 and Icam1 were determined in E0771.LMB IL1α up cells by CBA (J) and flow cytometry, respectively (K). Significant differences (*P < 0.05, **P < 0.01, ***P < 0.001) are indicated.

Fig. 3

Suppression of NF-κB and the inflammatory circuit by c-Myb depends on IL1α. IL1α and NF-κB inhibition in IL1α up cells reversed the up-regulation of inflammatory genes: Cxcl1 (A), Icam1 (B), Tnfrsf9 (C) upon treatment with IRAK1/4, IL1-Ra and JSH-23. An average from 3 independent experiments is shown. Indicated concentration of inhibitors were added to E0771.LMB IL1α up 28 cells (IL1α up 28), after 24 h Cxcl1 concentrations were determined by CBA and Icam1 and Tnfrsf9 surface expression was measured by flow cytometry and expressed as frequency of positive cells, comparison to vehicle (DMSO)-treated cells. (D) qPCR detection of signature genes in JSH-23-treated (10 µM, 24 h) E0771.LMB IL1α up cells (IL1α up 28). Values were normalized to Gapdh and expressed as a fold change of DMSO-treated cells. An average of 3 independent experiments is shown. E0771.LMB (E) and 4T1 (F) cells overexpressing wt c-Myb (E0071.LMB Myb^high C2, 4T1 Myb^high 7) supplemented with recombinant mIL1α (1 ng/mL, 24 h) induces the expression of the signature genes. mRNA levels of indicated genes are determined by qPCR relative to vehicle-treated cells (ctrl) and normalized to Gapdh. An average of 3 independent experiments is shown. (G) Dose-dependent increase in surface Icam1 upon IL1α stimulation (24 h) of E0771.LMB cells overexpressing wt c-Myb (Myb^high C2) as determined by flow cytometry. (H) Surface Icam1 expression is restored by recombinant IL1α in 4T1 cells overexpressing wt c-Myb (Myb^high 7) concomitant with loss of IκBα-miRFP703. Myb^high 7 cells cotransfected with pEGFP-C1 (GFP) and pIκBα−miRFP703 were treated with 1 ng/mL IL1α for 24h, Icam1 was stained and determined in GFP+ cells by flow-cytometry in parallel with IκBα levels. (I) Two 4T1 IL1α KO clones (IL1α KO CD34 and AB20), and scrambled control cells, were cotransfected with pcDNA3.c-Myb (c-Myb)/pcDNA3 (mock) and the NF-κB reporter. Luciferase activity (RLU) was measured 18 h later and normalized to β-galactosidase activity. An average of 3 independent experiments is shown. (J) Putative MYB-binding sites (MBSs, red) in the murine Il1a promoter sequence (980 bp upstream TSS, green). Primer pair used for cloning Il1a-luc reporter construct is indicated in pink. (K) Transactivation assay using 4T1 cells cotransfected with c-Myb or empty vector and Il1a-luc reporter. Il1a promoter activity is expressed as luciferase relative light units normalized to total protein levels. An average of 3 independent experiments is shown. Significant differences (*P < 0.05, **P < 0.01, ***P < 0.001) are indicated.

Fig. 4

IL1A facilitates transendothelial migration and inversely correlates with MYB in BC cells. (A) Monocyte-assisted TEM of 4T1 cells across primary lung ECs in vitro is enhanced by ectopic IL1α (IL1α up clones 20 and 21). –/+ bone marrow derived CD115+ monocytes. (B) Human IL1A overexpressed by MDA-MB-231 potentiates TEM across HUVECs in vitro. (C) Concentrations of IL1α protein in CM from a panel of human BC cell lines as measured by CBA. Average values of 3 independent experiments are shown. (D) Endogenous c-Myb levels in indicated BC cell lines as determined by immunoblotting, α-tubulin was used as a loading control. (E) Inverse correlation between IL1A and MYB mRNA levels as determined by RNAseq in BC cell lines in a Cancer Cell Line Encyclopedia (CCLE). r = Pearson correlation coefficient, CI = confidence interval. (F) Myb and Il1a transcript levels in tumors developed in genetically engineered mouse models of BC overexpressing Myb (WapCre; Brca1^F/F; Trp53^F/F; Col1a1^{invCAG-Myb2-IRES-Luc/+} mice, “WB1P_Myb”) and controls (WapCre; Brca1^F/F; Trp53^F/F mice, “WB1P_ctrl”) as determined by RNAseq involved in GSE112665 . (G) Proposed role of c-Myb protein in the regulation of NF-κB activity in BC. High level of c-Myb repress IL1α expression which in turn leads to a decline in NF-κB activity and TEM ability of the BC cells. Significant differences (*P < 0.05, ***P < 0.001) are indicated.