The ubiquitin-like molecule interferon-stimulated gene 15 (ISG15) is a potential prognostic marker in human breast cancer

Abstract

Introduction: ISG15 is an ubiquitin-like molecule that is strongly upregulated by type I interferons as a primary response to diverse microbial and cellular stress stimuli. However, alterations in the ISG15 signalling pathway have also been found in several human tumour entities. To the best of our knowledge, in the current study we present for the first time a systematic characterisation of ISG15 expression in human breast cancer and normal breast tissue both at the mRNA and protein level.

Method: Using semiquantitative real-time PCR, cDNA dot-blot hybridisation and immunohistochemistry, we systematically analysed ISG15 expression in invasive breast carcinomas (n = 910) and normal breast tissues (n = 135). ISG15 protein expression was analysed in two independent cohorts on tissue microarrays; in an initial evaluation set of 179 breast carcinomas and 51 normal breast tissues; and in a second large validation set of 646 breast carcinomas and 10 normal breast tissues. In addition, a collection of benign and malignant mammary cell lines (n = 9) were investigated for ISG15 expression.

Results: ISG15 was overexpressed in breast carcinoma cells compared with normal breast tissue, both at the RNA and protein level. Recurrence-free (p = 0.030), event-free (p = 0.001) and overall (p = 0.001) survival analyses showed a significant correlation between ISG15 overexpression and unfavourable prognosis.

Conclusion: Therefore, ISG15 may represent a novel breast tumour marker with prognostic significance and may be helpful in selecting patients for and predicting response to the treatment of human breast cancer.

Figure 1

Abundance of the expression of ISG15 mRNA in breast cancer cell lines. Semiquantitative real-time PCR (LightCycler) of ISG15 expression was performed on reverse transcribed RNA from non-malignant (white bars) and malignant cell lines (black bars). ISG15 mRNA expression was low among the non-malignant mammary epithelial cell lines HMEC and MCF12A, whereas ISG15 mRNA transcript levels varied more and in summary were elevated in the investigated cancerous cell lines.

Figure 2

Upregulation of ISG15 mRNA in primary breast cancer. A collection of paraffin-embedded breast carcinomas (T; n = 25) and normal breast tissues (N; n = 14) was analysed for ISG15 expression by semiquantative real-time PCR. ISG15 was strongly upregulated at the transcript level in the tumourous tissues compared with the normal breast tissues.

Figure 3

Correlation between ISG15 mRNA and protein expression. An additional collection of paraffin-embedded breast carcinomas (T; n = 10) and corresponding normal breast tissues (N; n = 10) was analysed for ISG15 expression by semiquantitative real-time PCR and immunohistochemistry in the same samples. ISG15 mRNA upregulation correlated with ISG15 protein expression in the same tumour sample (T) compared with the corresponding normal breast tissue (N). Magnification: 400×.

Figure 4

Expression profiles were determined using cDNA dot-blot hybridisation analysis (BD Clontech) containing cDNA pairs derived from 50 matched normal breast tissues (N), tumourous breast tissues (T) and three lymph node metastastic tissues (marked with arrows). Upregulation of ISG15 was observed in primary breast tumours and in one of three metastatic lymph nodes, as compared with matched normal breast tissue.

Figure 5

Immunohistochemical expression of ISG15 in normal breast tissue, and in non-invasive and invasive breast tumours using a tissue microarray. (a, b) Normal breast tissue lacks ISG15 expression (IRS = 0). (c, d) In ductal carcinoma in situ ISG15 expression is more intense (IRS = 8) than in normal breast tissue. (e, f) In invasive breast carcinomas (here: ductal type) ISG15 expression was clearly more abundant (example shows staining with an IRS = 12) compared with normal breast tissue and ductal carcinoma in situ. (g, h) In tubular breast carcinomas (IRS = 4), a less frequent variant of invasive breast carcinomas with a more favourable prognosis than invasive ductal breast carcinomas, ISG15 expression was less abundant than in most invasive ductal breast carcinomas and stronger than in most normal breast tissues. Magnifications: a, c, e, g: 100×; b, d, f, h: 400×.

Figure 6

Correlation of ISG15 expression and patient prognosis according to univariate Kaplan-Meier analysis. Breast cancer patients overexpressing ISG15 show unfavourable prognosis in overall survival analysis (p = 0.001).

Figure 7

Correlation of ISG15 expression and patient prognosis according to univariate Kaplan-Meier analysis. Breast cancer patients overexpressing ISG15 exhibit unfavourable prognosis in event-free survival (p = 0.001).

Figure 8

Correlation of ISG15 expression and patient prognosis according to univariate Kaplan-Meier analysis. Breast cancer patients overexpressing ISG15 have an unfavourable prognosis in recurrence-free survival analysis (p = 0.030).