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.

EGF induces the translocation of EGF receptor (EGFR) from the cell surface to the nucleus where EGFR activates gene transcription through its binding to an AT-rich sequence (ATRS) of the target gene promoter. However, how EGFR, without a DNA-binding domain, can bind to the gene promoter is unclear. In the present study, we show that RNA helicase A (RHA) is an important mediator for EGFRinduced gene transactivation. EGF stimulates the interaction of EGFR with RHA in the nucleus of cancer cells. The EGFR/RHA complex then associates with the target gene promoter through binding of RHA to the ATRS of the target gene promoter to activate its transcription. Knockdown of RHA expression in cancer cells abrogates the binding of EGFR to the target gene promoter, thereby reducing EGF/EGFR-induced gene expression. In addition, interruption of EGFR-RHA interaction decreases the EGFR-induced promoter activity. Consistently, we observed a positive correlation of the nuclear expression of EGFR, RHA, and cyclin D1 in human breast cancer samples. These results indicate that RHA is a DNA-binding partner for EGFR-mediated transcriptional activation in the nucleus.cyclin D1 | nuclear translocation | inducible nitric oxide synthase | transcription C ell surface EGF receptor (EGFR) has been shown to be localized in the nucleus (1-4). Nuclear EGFR has been demonstrated to contribute to cancer cell resistance to cetuximab and radiation treatment (5, 6) and to be negatively correlated with overall survival of patients with multiple cancer types (7-11). Moreover, nuclear EGFR interacts with signal transducer and activator of transcription 3 (STAT3), signal transducer and activator of transcription 5A (STAT5A), E2F1, DNA-dependent protein kinase (DNA-PK), and proliferating cell nuclear antigen (PCNA) and plays important roles in cell transformation, proliferation, and DNA repair and replication (12-16). Nuclear EGFR regulates gene expression by binding to an AT-rich sequence (ATRS) of the gene's promoter (13,16,17). Additionally, a recent unbiased protein-DNA interactome study indicates that EGFR is a DNAbinding protein (18). However, EGFR does not contain a DNAbinding domain, and evidence supporting direct binding of EGFR to the specific DNA sequence is lacking. Thus, identifying the DNA-binding partner for EGFR is crucial for understanding how EGFR regulates gene transcription in the nucleus.RNA helicase A (RHA), the human homolog of Drosophila maleless (MLE) that increases the transcription of male X-linked genes (19), is a multifunctional protein and is conserved in Drosophila and mammals (20)(21)(22). RHA belongs to the aspartateglutamate-alanine-aspartate (DEAD) box family of proteins and has the ability to bind to RNA and DNA (23,24). RHA regulates gene transcription by interacting with transcription factors (22) or by binding directly to the target gene promoter (25). Moreover, Drosophila MLE activates rox2 transcription by binding to an ATrich region of the gene promoter (26). Interestingly, this AT-rich region contains the previo...