Overexpression of E2F-5 correlates with a pathological basal phenotype and a worse clinical outcome

Abstract

The purpose of the present study is to identify genes that contribute to cell proliferation or differentiation of breast cancers independent of signalling through the oestrogen receptor (ER) or human epidermal growth factor receptor 2 (HER2). An oligonucleotide microarray assayed 40 tumour samples from ER(+)/HER2(-), ER(+)/HER2(+), ER(-)/HER2(+), and ER(-)/HER2(-) breast cancer tissues. Quantitative reverse transcriptase PCR detected overexpression of a cell cycle-related transcription factor, E2F-5, in ER-negative breast cancers, and fluorescence in situ hybridisation detected gene amplification of E2F-5 in 5 out of 57 (8.8%) breast cancer samples. No point mutations were found in the DNA-binding or DNA-dimerisation domain of E2F-5. Immunohistochemically, E2F-5-positive cancers correlated with a higher Ki-67 labelling index (59.5%, P=0.001) and higher histological grades (P=0.049). E2F-5-positive cancers were found more frequently in ER(-)/progesterone receptor (PgR)(-)/HER2(-) cancer samples (51.9%, P=0.0049) and in breast cancer samples exhibiting a basal phenotype (56.0%, P=0.0012). Disease-free survival in node-negative patients with E2F-5-positive cancers was shorter than for patients with E2F-5-negative cancers. In conclusion, we identify, for the first time, a population of breast cancer cells that overexpress the cell cycle-related transcription factor, E2F-5. This E2F-5-positive breast cancer subtype was associated with an ER(-)/PgR(-)/HER2(-) status, a basal phenotype, and a worse clinical outcome.

Figure 1

Quantification of E2F-5 mRNA levels by real-time RT–PCR. The expression level of E2F-5 is gradually higher in ER(+)/HER2(−), ER(+)/HER2(+), ER(−)/HER2(+), and ER(−)/HER2(−) breast cancer subtypes, respectively. ^*P=0.02 for ER(+)/HER2(−) vs ER(+)/HER2(+); ^**P=0.02 for ER(+) vs ER(−)/HER2(+); ^***P=0.04 for ER(+)/HER2(+) vs ER(−)/HER2(−).

Figure 2

Labelling of centromere 8 and E2F-5 by fluorescence in situ hybridisation (FISH). (A) The number of CEP8 and E2F-5 signals from the FISH experiments are plotted on the x- and y-axis, respectively. Five tumours, plotted on the upper left side of the solid line, have an E2F-5/CEP8 signal ratio of >2 and are therefore predicted to have undergone E2F-5 gene amplification. Data from the various breast cancer samples are labelled in the figure as follows: green circle dots – ER(+)/HER2(−); blue circle dots – ER(+)/HER2(+); orange circle dots – ER(−)/HER2(+); pink circle dots – ER(−)/HER2(−). Data from breast cancer cell lines (MCF-7, MDA-MB231) are indicated with purple square dots. (B) Centeromere 8 (CEP8) is labelled in green and E2F-5 is labelled in orange in ER(−)/HER2(−) cells.

Figure 3

Immunohistochemical study of E2F-5 expression in non-neoplastic and breast cancer tissues. Detection of E2F-5 in (A) squamous epithelium and (B) non-epithelial smooth muscle cells. In non-neoplastic mammary gland tissue, positive cells are detected in a resting state in the cytoplasm (C) as well as in the lactating phases (D) indicated by arrows. An arrow head (D) and inset indicate positive cells in nucleus. E2F-5 is also shown expressed in the cytoplasm of carcinoma cells (E). All of the metaplastic cancers tested were positive for E2F-5 (F). Scale bar indicates a length of 50 μm (A–F) and 10 μm (inset).

Figure 4

Kaplan–Meier survival analysis for patients with each of the four breast cancer subtypes analysed. (A) Disease-free survival was extended in the node-negative/E2F-5-negative cancer subtypes (blue, n=23) compared with the node-negative/E2F-5-positive subtype (pink, n=15), the node-positive/E2F-5-negative subtype (gray, n=13), and the node-positive/E2F-5-positive cancer subtype (green, n=6) (P=0.014). In the lymph node-negative subgroup (B–D), the E2F-5 positive group shows a shorter disease-free survival interval (pink, n=13, P=0.013) than the E2F-5-negative group (blue, n=23). There were no significant differences between the ER-positive (C; green, n=12) and -negative groups (C; blue, n=24, P=0.14), and the HER2-positive (D; orange, n=10) and -negative groups (D; blue, n=26, P=0.22).