Novel nuclear interaction of EGFR and STAT3 in the activation of iNOS/NO pathway
Authors: Hui-Wen Lo, Weiya Xia, Mehmet Gunduz, Mohamed Ali Seyed, Yongkun Wei, Norishisa Hanada, Sheng-Chieh Hsu, Mien-Chie Hung
Journal: Cancer Research (2004)

Proc Amer Assoc Cancer Res, Volume 45, 2004

1440 

EGF receptor (EGFR) has been detected as a full-length protein in the nucleus of highly proliferative tumor cells and primary tumor specimens for the past decade. The nuclear function of EGFR, however, was not revealed until recently by our laboratory (Nat. Cell Biol. 3:802, 2001) to involve direct activation of cyclin D1 gene promoter. Despite with significant transactivational activity, EGFR lacks a putative DNA binding domain and, therefore, requires transcription co-factor with DNA-binding ability for its transcriptional function. Additionally, in some tumor types, EGFR over-expression has been shown to correlate with high levels of nitric oxide (NO), both properties commonly associate with tumorigenesis and tumor growth. However, the molecular mechanism underlying this correlation remains unknown. We report here, for the first time, that nuclear EGFR directly activates inducible NO synthase (iNOS) gene via its physical and functional cooperation with signal transducers and activators of transcription 3 (STAT3), following EGF stimulation. Cell-surface EGFR rapidly translocated into the nucleus of tumor cells following activation by EGF and interacted significantly with STAT3 as shown by immunofluorescent microscopy/deconvolution and co-immunoprecipitaiton analyses. Nuclear EGFR/STAT3 complex bound to iNOS gene promoter in vitro and in vivo, as indicated by gel shift/supershift assays and chromatin immunoprecipitation (ChIP) methods, respectively. Using RT-PCR and western blot analyses, we observed EGF significantly enhanced iNOS gene expression by 5-fold in MDA-MB-468 breast cancer cells. Consistently, EGF increased the activity of a 7.2 kb iNOS promoter by approximately 30-fold compared to that of the promoter-less construct. Promoter deletion/mutation studies further identified a region in the iNOS promoter to be required for its responsiveness to EGF and to contain both EGFR and STAT3 binding sites. Immunohistochemical analyses of a cohort of 130 breast carcinomas showed that patients with high nuclear EGFR in their tumors had worse overall survival than those stained negative (33.4 + 14.6 vs. 47.8 + 15.3 months; p=0.009). Consistently, we found a direct correlation (p<0.001) between high levels of nuclear EGFR and Ki-67, a measure of active proliferation. In contrast, no significant correlation was found between the levels of non-nuclear EGFR and nuclear EGFR suggesting a complex mechanism underlying nuclear transport of cell-surface EGFR. Together, these findings describe a novel mode of transcriptional control that involves cooperated efforts of STAT3 and its novel transcription partner, nuclear EGFR. Our work also indicates that nuclear EGFR is an important prognostic indicator for breast cancer patients as well as a positive regulator of the NO pathway, suggesting de-regulated iNOS expression may, in part, contribute to the malignant behavior in tumors with increased nuclear EGFR.