Nuclear expression of epidermal growth factor receptor is a novel prognostic value in patients with ovarian cancer

Abstract

The epidermal growth factor receptor (EGFR) has previously been detected in the nucleus of cancer cells and primary tumors. We have reported that EGFR translocates from the plasma membrane to the nucleus. Accumulation of nuclear EGFR is linked to increased DNA synthesis and proliferation; however, the pathological significance of nuclear EGFR is not completely understood. Here, we sought to determine the predictive value of EGFR for the survival of ovarian cancer patients, through the examination of 221 cases of ovarian cancer tissues by immunohistochemical analysis to determine nuclear EGFR expression. In addition, we also examined cyclin D1 and Ki-67 through immunohistochemisty. Furthermore, we examined nuclear EGFR levels in ovarian cancer cell lines treated with EGF, and primary ovarian tumor tissue using immunofluorescence analysis. Nuclear fractions extracted from serum-starved cells treated with or without EGF were subjected to SDS-PAGE and Western blot analyses. We found that 28.3% of the cohort had high levels of nuclear EGFR, while 22.5% had low levels of nuclear EGFR, and 49.2% were negative for nuclear EGFR. Importantly, there was an inverse correlation between high nuclear EGFR, cyclin D1, and Ki-67 with overall survival (P < 0.01, P < 0.09, P < 0.041). Additionally, nuclear EGFR correlated positively with increased levels of cyclin D1 and Ki-67, both indicators for cell proliferation. Our findings indicate a pathological significance of nuclear EGFR that might be important for predicting clinical prognosis of ovarian cancer patients.

Figure 1

Western blot analysis of EGFR nuclear translocation in ovarian cancer cell lines following EGF stimulation. OVCA420, OVCA433, and OVCAR3 cells were treated without and with EGF (50 ng/mL) for 30 min and subjected to cell fractionation, SDS–PAGE, and Western blot. Lamin B, tubulin, and calregulin were used for cell fractionation controls and loading controls.

Figure 2

Confocal analysis of nuclear accumulation of EGFR in the OVCA420 cell line following EGF stimulation. OVCA420 cells were starved for overnight, treated without and with EGF (50 ng/mL) for 30 min. Nuclear accumulation of EGFR is shown in the bottom line merged image. Yellow spots indicate the nuclear EGFR. Scale bar: 20 µm. White box represents area of insert.

Figure 3

Nuclear EGFR correlated with expression of Ki-67 and cyclin D1 in ovarian carcinomas. 400×. (A) Case I is a representative sample of a tumor positive for nuclear EGFR. (B) Case II is a representative sample of a tumor negative for nuclear EGFR.

Figure 4

(A) Immunofluorescence of nuclear EGFR and cyclin D1 staining in ovarian cancer patient tumor tissues. Red, EGFR; green, cyclin D1; blue, nuclei, stained by DAPI; merge shows EGFR, cyclin D1, and DAPI. (B) Immunofluorescence of nuclear EGFR and cyclin D1 staining in ovarian cancer patient tumor tissues. Red, EGFR; green, Ki-67; blue, nuclei, stained by DAPI; merge shows EGFR, Ki-67, and DAPI. White bar is the scale bar, 20 µm. Box represents area of insert (yellow box).

Figure 5

(A) Correlation between high nuclear EGFR expression with poor patient survival in primary ovarian cancers, P < 0.01. (B) Correlation between Ki-67 expression with poor patient survival in primary ovarian cancers, P < 0.04. (C) No correlation between cyclin D1 expression with poor patient survival in primary ovarian cancers, P = 0.09. Survival curves were calculated by the method of Kaplan and Meier. P-values were analyzed by the SPSS test. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]