Overexpression of the ERBB2 gene in human breast cancer is associated with a poor prognosis and resistance to hormonal treatment and chemotherapy. Oestrogen receptor (ER) positive tumour-derived cell lines are known to express relatively low levels of ERBB2 protein under oestrogenic conditions, but markedly higher levels following withdrawal of oestrogens or administration of tamoxifen. Expression of the closely related ERBB3 gene, which co-operates with ERBB2 in cellular transformation, is now shown to respond to oestrogenic manipulation in a similar way, both responses being mediated largely by transcriptional changes. Six previously undescribed DNase I hypersensitive sites occur within the ®rst intron of ERBB2 in cells that overexpress the gene. A 409 base pair DNA fragment containing one of these sites conferred ER dependent oestrogen inhibition on the ERBB2 promoter in two types of transient transfection assay. DNase I footprinting revealed four separate transcription factor binding sites within this fragment consistent with a role as a transcriptional enhancer. These ®ndings implicate intron 1 sequences in the control of ERBB2 expression for the ®rst time and demonstrate that one site within this region is involved in mediating the transcriptional response to oestrogens. Additionally, there is likely to be synergism between ERBB2 and ERBB3 signalling when both are overexpressed in response to oestrogen inhibition, thereby driving transformed cell behaviour.

Overexpression of the ERBB2 proto-oncogene in breast tumours, which occurs in 25 ± 30% of patients, correlates with poor prognosis. In oestrogen receptor (ER) positive breast epithelial cells oestrogens reduce ERBB2 mRNA and protein levels, an e ect that is reversed in the presence of anti-oestrogens such as tamoxifen and ICI 182780. Our previous studies have shown that the major e ect of oestrogen on ERBB2 expression is at the level of transcription and that this is mediated through a region within the ERBB2 ®rst intron which can act as an oestrogen-suppressible enhancer in ER positive breast cells. In vitro footprinting of the smallest DNA fragment that retained full activity revealed four transcription factor binding sites. We report here that two of these sites are recognized by AP-2 proteins and the other two are bound by a variety of bZIP factors, including CREB and ATF1, with a major complex containing ATFa/ JunD. However, by using ER mutants it is clear that repression occurs essentially o the DNA. Indeed, the essential domain of the ER responsible for repression of the ERBB2 enhancer is a region termed AF2 which is required for the ligand-dependent association of non-DNA binding cofactors. We further demonstrate that one of these ER cofactors, SRC-1, can relieve oestrogen repression of the ERBB2 enhancer and conclude that these data ®t with a model whereby the ER and the ERBB2 enhancer compete for this limiting, non-DNA binding cofactor. Thus, in oestrogenic conditions SRC-1 preferentially binds to the ER which e ectively sequesters it thereby reducing enhancer activity, but in antioestrogenic media the cofactor is released from the ER and is therefore available to activate the ERBB2 enhancer. Oncogene (2000) 19, 490 ± 497.

Purpose: HER-2/neu oncogene is overexpressed in 10 -30% of epithelial ovarian cancers and is associated with a poor prognosis. The E1A gene product of adenovirus type 5 down-regulates HER-2/neu and causes tumor regression in animal models. In the current study, we sought to determine the toxicity and biological activity of E1A-lipid complex in ovarian cancer patients.Experimental Design: A Phase I trial involving intraperitoneal (i.p.) administration of E1A-lipid complex was initiated in ovarian cancer patients to assess biological activity (E1A gene transfer/transcription/translation and HER-2/neu expression) and to determine the maximum tolerated dose. Successive cohorts received E1A-lipid complex at doses of 1.8, 3.6, and 7.2 mg DNA/m 2 , given as weekly i.p. infusions for 3 of 4 weeks (each cycle) up to a maximum of six cycles. Peritoneal fluid was sampled at baseline and twice monthly for cellularity, cytology, CA-125, and biological activity Results: Fifteen patients, with a median age of 57 years (range, 43-81) were recruited. Three (1.8 mg DNA/m 2 ), 4 (3.6 mg DNA/m 2 ), and 8 patients (7.2 mg DNA/m 2 ) received i.p. E1A. A total of 91 infusions (range, 1-18) was administered. Abdominal pain was the dose-limiting toxicity, and the maximum-tolerated dose was 3.6 mg DNA/m 2 . E1A gene transfer and expression was observed in all of the patients and at all of the dose levels. HER-2/neu down-regulation could be demonstrated in the tumor cells of 2 patients (18%). There was no correlation between dose and biological activity.Conclusions: I.P. EIA-lipid complex gene therapy is feasible and safe. Future studies, either alone or in combination with chemotherapy, particularly in patients with minimal residual disease, should be evaluated.

The transcriptional regulation of the human EGFR3 and ERBB2 genes has been extensively studied, particularly in the context of their overexpression in breast cancer. Here we summarize published work detailing the transcription factors which interact with the promoters of these and the rat ERBB2 homologue, neu, genes and discuss their possible relevance to gene activation in cancer. In addition we review the biologically significant molecules which modulate expression of these genes and discuss the nuclear factors involved in mediating these responses. We also describe novel therapies which may result from these studies and highlight directions for future research into the control of expression of the EGFR and ERBB2 genes in the normal mammary gland and in breast cancer.