Stage-associated overexpression of the ubiquitin-like protein, ISG15, in bladder cancer

Abstract

Bladder cancer is among the most prevalent malignancies, and is characterised by frequent tumour recurrences and localised inflammation, which may promote tissue invasion and metastasis. Microarray analysis was used to compare gene expression in normal bladder urothelium with that in tumours at different stages of progression. The innate immune response gene, interferon-stimulated gene 15 kDa (ISG15, GIP2), was highly expressed at all stages of bladder cancer as compared to normal urothelium. Western blotting revealed a tumour-associated expression of ISG15 protein. ISG15 exhibited a stage-associated expression, with significantly (P<0.05) higher levels of ISG15 protein in muscle-invasive T2-T4 tumours, compared with normal urothelium. Although ISG15 is involved in the primary immune response, ISG15 expression did not correlate with bladder inflammation. However, immunohistochemical staining revealed expression of ISG15 protein in both cancer cells and stromal immune cells. Interestingly, a significant fraction of ISG15 protein was localised to the nuclei of tumour cells, whereas no nuclear ISG15 staining was observed in ISG15-positive stromal cells. Taken together, our findings identify ISG15 as a novel component of bladder cancer-associated gene expression.

Figure 1

Microarray analysis of ISG15 gene expression in normal urothelium and bladder tumour. (A) ISG15 gene expression in normal urothelium (n=9) and in tumours from patients with Ta grade bladder tumours (n=45) (HG-U133A). (B) ISG15 gene expression in normal urothelium (n=18), Ta tumours (n=28), T1 tumours (n=20), and T2–T4 tumours (n=45) (EOS Hu03 microarray).

Figure 2

(A) Densitometric analysis of ISG15 protein expression by Western blotting analysis. The relative expression of each tumour group compared to the normal level is shown in the diagram (All T=all tumours). All ISG15 levels are normalised to actin expression. (B) Western blotting analysis of ISG15 protein expression in 10 paired samples; T2–T4 tumour (T) and normal bladder (N) from the same cancer patient (upper panel). The actin protein expression was measured as a control for equivalent protein loading (lower panel).

Figure 3

Immunohistochemical staining of ISG15 expression in bladder tumours. Tissue material obtained from two patients; 1518-1 (Ta tumour) and 1014-1 (T2–T4 tumour). Original magnification: × 20.

Figure 4

A tumour-infiltrating blood vessel with ISG15-positive immune cells. Tissue material obtained from patient 795-13 with a Ta tumour. Original magnification: × 40.

Figure 5

(A) Immunohistochemical analysis of ISG15 expression using tissue microarray normal urothelium: N=10; Ta tumours: N=20; T2–T4 tumours: N=20. (B) Distribution of samples with ISG15-positive nuclei (>50%) among normal urothelium samples, Ta tumours, and T2–T4 tumours. (C) Nuclear ISG15 expression of a Ta tumour. Original magnification: × 40.