Differential expression of S100A2 and S100A4 in lung adenocarcinomas: clinicopathological significance, relationship to p53 and identification of their target genes

Abstract

Previous studies suggest that some S100 proteins are involved in the progression of certain types of cancer. However, no comprehensive data is currently available on the expression of S100 family genes in lung adenocarcinomas. Oligonucleotide array, quantitative reverse transcription-polymerase chain reaction and western blot analyses of lung adenocarcinoma cell lines and bronchiolar epithelial cells (SAEC and NHBE) revealed that S100A2 and S100A4 were the most strikingly downregulated and upregulated members of the S100 family, respectively. Immunohistochemical analyses of 94 primary lung adenocarcinomas showed that positive S100A2 expression (33/94, 35.1%) was significantly associated with lymphatic invasion (P=0.0233) and positive S100A4 expression (19/94, 20.2%) with vascular invasion (P=0.0454). Interestingly, a strong inverse relationship was found between S100A4 and p53 expression (P=0.0008). Survival analyses showed that S100A4 positivity was associated with poor patient prognosis (P=0.042). S100A2 positivity was not associated with patient survival when the whole patient group was analyzed; however, S100A2 positivity was a favorable prognostic indicator in patients with p53-negative tumors (P=0.0448). Finally, we used oligonucleotide array analyses and identified potential S100A2 and S100A4 target genes involved in cancer progression: S100A2 induced RUNX3 and REPRIMO; S100A4 induced EZRIN, RUNX1 and WISP1; S100A2 repressed EGFR, NFKB2 and RELA2; and S100A4 repressed ANXA10 and IL1RN. Thus, the present study demonstrates involvement of S100A2 and S100A4 in the progression of lung adenocarcinomas and an inverse association between S100A4 and p53 expression, and provides a list of targets regulated by S100A2 and S100A4.

Figure 1

Expression analyses of S100A2 and S100A4 in bronchial epithelial cells (NHBE and SAEC) and nine lung adenocarcinoma cell lines. Levels of S100A2 (a) and S100A4 (b) mRNA were determined by real‐time reverse transcription–polymerase chain reaction analyses. Protein levels of S100A2 (c) and S100A4 (d) were examined by western blot analysis. The lower panels in (c) and (d) represent β‐actin expression (arrows) serving as an internal control. Lanes: 1, NHBE; 2, SAEC; 3, LC‐2/ad; 4, ABC‐1; 5, A549; 6, H460; 7, HLC1; 8, H1299; 9, PC3; 10, VMRC‐LCD; 11, RERF‐LC‐KJ.

Figure 2

Immunohistochemical analyses of S100A2 and S100A4 expression in primary lung adenocarcinoma specimens. (a) S100A2 immunoreactivity was absent in normal lung tissue, but in some cases it was observed in basal cells of the bronchial epithelium, typically in inflamed areas near invading tumor cells. (b) S100A2‐positive tumor. S100A2 immunoreactivity was found in both the cytoplasm and nuclei of cancer cells. (c) S100A4 staining was found in both lymphocytes and fibroblasts. (d) S100A4‐positive tumor. S100A4 immunoreactivity was found in both the cytoplasm and nuclear membranes of cancer cells.

Figure 3

Patient survival according to the expression of S100A2 and S100A4. (a) Patients with S100A2‐positive tumors tended to have better prognoses than those with S100A2‐negative tumors, but the difference was not statistically significant (P = 0.1142). (b) S100A4 expression was significantly associated with poor prognosis (P = 0.0269).

Figure 4

Patient survival according to the expression of S100A2 in patients with p53‐positive or p53‐negative tumors. (a) S100A2 positivity was not associated with prognosis of patients with p53‐positive tumors (P = 0.6517). (b) S100A2 positivity was significantly associated with favorable outcome in patients with p53‐negative tumors (P = 0.0448).