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, 10 (4), 511-526

Serum Amyloid A Predisposes Inflammatory Tumor Microenvironment in Triple Negative Breast Cancer

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Serum Amyloid A Predisposes Inflammatory Tumor Microenvironment in Triple Negative Breast Cancer

Rosa Mistica C Ignacio et al. Oncotarget.

Abstract

Acute-phase proteins (APPs) are associated with a variety of disorders such as infection, inflammatory diseases, and cancers. The signature profile of APPs in breast cancer (BC) is poorly understood. Here, we identified serum amyloid A (SAA) for proinflammatory predisposition in BC through the signature profiles of APPs, interleukin (IL) and tumor necrosis factor (TNF) superfamily using publicly available datasets of tumor samples and cell lines. Triple-negative breast cancer (TNBC) subtype highly expressed SAA1/2 compared to HER2, luminal A (LA) and luminal B (LB) subtypes. IL1A, IL1B, IL8/CXCL8, IL32 and IL27RA in IL superfamily and CD70, TNFSF9 and TNFRSF21 in TNF superfamily were highly expressed in TNBC compared to other subtypes. SAA is restrictedly regulated by nuclear factor (NF)-κB and IL-1β, an NF-κB activator highly expressed in TNBC, increased the promoter activity of SAA1 in human TNBC MDA-MB231 cells. Interestingly, two κB-sites contained in SAA1 promoter were involved, and the proximal region (-96/-87) was more critical than the distal site (-288/-279) in regulating IL-1β-induced SAA1. Among the SAA receptors, TLR1 and TLR2 were highly expressed in TNBC. Cu-CPT22, TLR1/2 antagonist, abrogated IL-1β-induced SAA1 promoter activity. In addition, SAA1 induced IL8/CXCL8 promoter activity, which was partially reduced by Cu-CPT22. Notably, SAA1/2, TLR2 and IL8/CXCL8 were associated with a poor overall survival in mesenchymal-like TNBC. Taken together, IL-1-induced SAA via NF-κB-mediated signaling could potentiate an inflammatory burden, leading to cancer progression and high mortality in TNBC patients.

Keywords: interleukin-1β; proinflammatory; serum amyloid A; triple negative breast cancer; tumor microenvironment.

Conflict of interest statement

CONFLICTS OF INTEREST The authors declare no conflicts of interest in this work.

Figures

Figure 1
Figure 1. Acute-phase protein signatures in BC tissues and cell lines
(A) Heatmap for APP expression profiles in human BC tissues from TCGA-based dataset using Gitools 2.3.1. (B) Statistical analysis of APP expression intensity in human BC tissues. (C) Heatmap for RNA expression levels of APPs based on analysis of GEO dataset (Accession: GSE12777) with 51 human BC cell lines using Gitools 2.3.1. (D) Intersection of APP signature between human BC tissues and cell lines. Red, yellow, blue and green dots specify high expression levels in BL-, HER2 (H2)-, LA- and LB-BC subtypes, respectively. Pink letters specify high expression levels in both BL- and HER2-BC subtypes. ML; mesenchymal-like, LAR; luminal androgen receptor and TNBC; triple-negative breast cancer.
Figure 2
Figure 2. Interleukin superfamily signatures in BC tissues and cell lines
(A) Heatmap for IL superfamily expression profiles in human BC tissues from TCGA-based dataset using Gitools 2.3.1. (B) Statistical analysis of IL superfamily expression intensity in human BC tissues. (C) Heatmap for RNA expression levels of IL superfamily based on analysis of GEO dataset (Accession: GSE12777) with 51 human BC cell lines using Gitools 2.3.1. (D) Intersection of IL superfamily signature between human BC tissues and cell lines. Red, yellow, blue and green dots specify high expression levels in BL-, HER2 (H2)-, LA- and LB-BC subtypes, respectively. Pink letters specify high expression levels in both BL- and HER2-BC subtypes. ML; mesenchymal-like, LAR; luminal androgen receptor and TNBC; triple-negative breast cancer.
Figure 3
Figure 3. Interleukin receptor superfamily signatures in BC tissues and cell lines
(A) Heatmap for IL receptor superfamily expression profiles in human BC tissues from TCGA-based dataset using Gitools 2.3.1. (B) Statistical analysis of IL receptor superfamily expression intensity in human BC tissues. (C) Heatmap for RNA expression levels of IL receptor superfamily based on analysis of GEO dataset (Accession: GSE12777) with 51 human BC cell lines using Gitools 2.3.1. (D) Intersection of IL receptor superfamily signature between human BC tissues and cell lines. Red, yellow, blue and green dots specify high expression levels in BL-, HER2 (H2)-, LA- and LB-BC subtypes, respectively. Pink letters specify high expression levels in both BL- and HER2-BC subtypes. ML; mesenchymal-like, LAR; luminal androgen receptor and TNBC; triple-negative breast cancer.
Figure 4
Figure 4. TNF superfamily signatures in BC tissues and cell lines
(A) Heatmap for TNF superfamily expression profiles in human BC tissues from TCGA-based dataset using Gitools 2.3.1. (B) Statistical analysis of TNF superfamily expression intensity in human BC tissues. (C) Heatmap for RNA expression levels of TNF superfamily based on analysis of GEO dataset (Accession: GSE12777) with 51 human BC cell lines using Gitools 2.3.1. (D) Intersection of TNF superfamily signature between human BC tissues and cell lines. Red, yellow, blue and green dots specify high expression levels in BL-, HER2 (H2)-, LA- and LB-BC subtypes, respectively. Pink letters specify high expression levels in both BL- and HER2-BC subtypes. ML; mesenchymal-like, LAR; luminal androgen receptor and TNBC; triple-negative breast cancer.
Figure 5
Figure 5. TNF receptor superfamily signatures in BC tissues and cell lines
(A) Heatmap for TNF receptor superfamily expression profiles in human BC tissues from TCGA-based dataset using Gitools 2.3.1. (B) Statistical analysis of TNF receptor superfamily expression intensity in human BC tissues. (C) Heatmap for RNA expression levels of TNF receptor superfamily based on analysis of GEO dataset (Accession: GSE12777) with 51 human BC cell lines using Gitools 2.3.1. (D) Intersection of TNF receptor superfamily signature between human BC tissues and cell lines. Red, yellow, blue and green dots specify high expression levels in BL-, HER2 (H2)-, LA- and LB-BC subtypes, respectively. Pink letters specify high expression levels in both BL- and HER2-BC subtypes. ML; mesenchymal-like, LAR; luminal androgen receptor and TNBC; triple-negative breast cancer.
Figure 6
Figure 6. SAA receptor and TLR family signatures in BC tissues and cell lines
(A) Heatmap for SAA receptor and TLR family expression profiles in human BC tissues from TCGA-based dataset using Gitools 2.3.1. (B) Statistical analysis of SAA receptor and TLR family expression intensity in human BC tissues. (C) Heatmap for RNA expression levels of SAA receptor and TLR family based on analysis of GEO dataset (Accession: GSE12777) with 51 human BC cell lines using Gitools 2.3.1. (D) Intersection of SAA receptor and TLR family signature between human BC tissues and cell lines. Red, yellow, blue and green dots specify high expression levels in BL-, HER2 (H2)-, LA- and LB-BC subtypes, respectively. Pink letters specify high expression levels in both BL- and HER2-BC subtypes. ML; mesenchymal-like, LAR; luminal androgen receptor and TNBC; triple-negative breast cancer.
Figure 7
Figure 7. IL-1β increases human SAA1 promoter activity via NF-κB signaling
(A) DNA sequence and homology of the human SAA1 and SAA2 promoters. (B) Effects of IL-1β on luciferase activity in deletion constructs of the SAA1 promoter. After transfection with deletion constructs of SAA1 (SAA1-P401, SAA1-P319, SAA1-P139 and SAA1-P85) luciferase vectors in MDA-MB231 TNBC cells overnight, a luciferase promoter activity assay was performed at post-treatment of IL-1β (10 ng/ml) for 6 h. (C) Effects of IL-1β on luciferase activity in NF-κB mutated constructs of the SAA1 promoter. Site-directed mutants were generated from the SAA1-P319LUC using primers with mutant κB-like sites (-287/-278) and κB-consensus site (-95/-86). After transfection with SAA1-P319LUC and its mutant κB-site luciferase vectors in MDA-MB231 TNBC cells overnight, a luciferase promoter activity assay was performed at post-treatment of IL-1β (10 ng/ml) for 6 h. Results were normalized to the protein level and expressed as a fold increase compared to non-treated control. Gray circles indicate κB site mutants. *, ** indicate significant (p < 0.05) increase compared to each control, when a Student's-t test was analyzed. Also, significant (p < 0.05) change exists between * and ** groups. Representative results are shown from triplicated experiments.
Figure 8
Figure 8. Abrogated effects of Cu-CPT22, a TLR1/2 inhibitor, on IL-1β-induced SAA1 and SAA1-induced CXCL8 promoter activities and overall survival (OS) of SAA1/2, TLR2 and CXCL8 expression levels
(A) Effect of Cu-CPT22, a TLR1/2 inhibitor, on IL-1β-induced SAA1 promoter activity. After transfection with SAA1-P319 luciferase vectors in MDA-MB231 TNBC cells overnight, a luciferase promoter activity assay was performed at post-treatment of IL-1β (10 ng/ml) for 6 h with a pre-treatment of Cu-CPT22 (1 μM) for 0.5 h. (B) Effect of Cu-CPT22, a TLR1/2 inhibitor, on SAA1-induced CXCL8/IL8 promoter activity. After transfection with human CXCL8 promoter (-322/+10) luciferase vectors in MDA-MB231 TNBC cells overnight, a luciferase promoter activity assay was performed at post-treatment of recombinant human SAA1 (500 ng/mL) for 6 h with a pre-treatment of Cu-CPT22 (1 μM) for 0.5 h. Results were normalized to the protein level and expressed as a fold increase compared to non-treated control. *, # indicate significant (p ≤ 0.05) increase and decrease, respectively, when ANOVA test was analyzed. Representative results are shown from triplicated experiments. (C) Kaplan-Meier OS for SAA1/2, TLR2 and CXCL8 in ML-TNBC patients (n = 73). The black and red lines indicate low and high expression levels, respectively.

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