Immune Netw.  2017 Apr;17(2):121-127. 10.4110/in.2017.17.2.121.

Augmented Serum Amyloid A1/2 Mediated by TNF-induced NF-κB in Human Serous Ovarian Epithelial Tumors

• Choi H
• Ignacio RM
• Lee ES
• Wilson AJ
• Khabele D
• Son DS

• KMID: 2376870
• DOI: http://doi.org/10.4110/in.2017.17.2.121

Abstract

Tumor necrosis factor-α (TNF) is well known to be involved in the immune system and ovarian inflammation. Ovarian cancer is an inflammation-related malignancy that lacks early screening strategies, resulting in late diagnosis followed by high mortality. Based on our previous data, TNF induced abundant serum amyloid A (SAA), an acute phase protein linked to inflammation, in ovarian granulosal cells. To date, the regulation and expression of SAA in ovarian cancer is not fully elucidated. Here, we investigated the relationship between TNF and SAA by comparing human normal ovarian tissues and serous ovarian tumors. We found that SAA1/2 was significantly expressed in tumor tissues, but no or trace expression levels in normal tissues. TNF was also significantly upregulated in ovarian tumor tissues compared to normal tissues. Moreover, TNF significantly increased SAA1/2 levels in human ovarian cancer cell lines, OVCAR-3 and SKOV-3, in a time-dependent manner. Since the SAA1 promoter contains two nuclear factor (NF)-κB sites, we examined whether TNF regulates SAA1 promoter activity. Deletion analysis revealed that the proximal NF-κB site (−95/−85) played a critical role in regulating TNF-induced SAA1 promoter activity. Within 2 h after intraperitoneal injection of lipopolysaccharide, a product known to stimulate release of TNF, SAA preferably localized to ovarian epithelial cells and the thecal-interstitial layers compared to granulosal cell layers. Based on Gene Expression Omnibus (GEO) database, SAA1/2 and TNF were dominantly expressed in advanced grade ovarian cancer. Taken together, the accumulation of SAA1/2 in ovarian cancer could be mediated by TNF-induced NF-κB activation.

Keyword

Serum amyloid A; Ovarian cancer; Tumor necrosis factor; NF-κB

MeSH Terms

Acute-Phase Proteins
Amyloid*
Cell Line
Delayed Diagnosis
Epithelial Cells
Gene Expression
Humans*
Immune System
Inflammation
Injections, Intraperitoneal
Mass Screening
Mortality
Necrosis
Ovarian Neoplasms
Serum Amyloid A Protein
Tumor Necrosis Factor-alpha
Acute-Phase Proteins
Amyloid
Serum Amyloid A Protein
Tumor Necrosis Factor-alpha

Figure

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  Figure 1 SAA1/2 is a dominant isoform in human ovarian tumor tissues. (A) Expression levels of SAA1/2 mRNA determined by real-time PCR in five different ovarian normal (N1-N5) and tumor tissues (T1-T5). (B) Expression levels of SAA4 mRNA in normal ovarian tissues and tumor specimens determined by real-time PCR. (C) Visual confirmation of SAA1/2 and SAA4 expression by RT-PCR. After isolating total RNA, RT-PCR and real-time PCR were carried out using primers for SAA1/2 and SAA4. GAPDH was used as a loading control.

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  Figure 2 TNF enhances SAA1/2 expression in ovarian cancer. (A) Expression levels of TNF mRNA determined by real-time PCR in five different ovarian normal (N1-N5) and tumor tissues (T1-T5). (B) Correlation between mRNA levels of TNF and SAA isoforms. (C) Time-dependent effects of TNF on SAA1/2 mRNA by real-time PCR in ovarian cancer cell lines (OVCAR-3 and SKOV-3). Cells were incubated with TNF (10 ng/ml) for 0, 1, 3, 6, 12 and 24 hours. *Indicates significant increase (p≤0.05) compared to its own control as calculated by the paired Student's t test.

• 

  Figure 3 TNF increases SAA1 promoter activity via NF-κB signaling. (A) DNA sequences of the human SAA1 promoter. (B) Effect of TNF and IκB on luciferase activity of SAA1 promoter in SKOV-3 cells. Results were normalized to the protein level and represented as a fold increase compared to empty vector (pA) controls. (C) Effects of TNF on luciferase activity of the SAA1 promoter with NF-κB site deletions. After SKOV-3 cells were transfected with SAA1 luciferase vectors overnight, cells were treated with TNF (10 ng/ml) for 6 h, followed by luciferase assay. Results were normalized to the protein level and represented as a fold increase compared to non-treated control. Grey and white colors indicate NF-κB like (lower case for unmatched DNA sequence) and consensus sites, respectively. *Indicates significant increase (p≤0.05) compared to its own control as calculated by the paired Student's t test.

• 

  Figure 4 Ovarian localization of SAA after LPS treatment in mice, and GEO profiles of SAAs and proinflammatory cytokines in human ovarian tumor tissues. (A) LPS-induced SAA3 accumulation in ovarian epithelial and thecal-interstitial layers of mouse ovary. Six week-old mice were given vehicle (saline) or LPS (100 µg/mouse, ip). Ovaries were collected at 2 hrs after LPS treatment and ovarian localization of SAA3 mRNA was investigated by using in situ hybridization with a DIG-labeled RNA probe. Sense probe of SAA did not express any signal (data not shown). (B) GEO profiles of SAAs and proinflammatory cytokines in normal ovarian tissues (NOT), benign ovarian tumor (BOT) and serous ovarian cancer (SOC) based on the NCBI GEO database (GSE51088). Data values were expressed as the mean±SEM. *indicate significantly higher (p≤0.05) within groups as analyzed by Tukey's pairwise comparisons.

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