Susan A. Chrysogelos

EGF receptor expression, regulation, and function in breast cancer.

SummaryEpidermal growth factor receptor (EGFR) overexpression correlates with both loss of estrogen receptor (ER) and poor prognosis in breast cancer. Interestingly, in normal breast EGFR appears to be expressed more frequently than in malignant tissue, and there may be a different relationship between ER and EGFR. A variety of cellular regulators, such as EGF, TGFα, phorbol esters, and steroid hormones, are capable of altering the level of EGFR expression in breast cells. However, much work remains to be done on the mechanistic details of EGFR regulation in this disease. The significance of EGFR as an oncogene in breast cancer is compounded by its potential interactions with other oncogenes such as c-erbB-2 and c-myc. Additionally, several recent studies have placed EGFR prominently in the signal transduction pathway, demonstrating that the EGFR-ligand system may play important roles throughout the course of malignant progression in breast cancer.

Estrogen suppression of EGFR expression in breast cancer cells: A possible mechanism to modulate growth

Epidermal growth factor receptor (EGFR) is a transmembrane receptor whose overexpression in breast cancer predicts for poor prognosis and is inversely correlated with expression of estrogen receptor (ER). This study was designed to investigate whether estrogen plays an active role in suppression of EGFR expression in estrogen‐responsive breast cancer cell lines expressing low levels of EGFR. Upon withdrawal of estrogen, EGFR mRNA and protein increased 3–6 fold in MCF‐7, T47D, and BT474 ER+ breast cancer cells. This was reversible upon addition of estradiol back to the culture media, but only after prolonged treatment. Nuclear run‐on assays and studies with the transcription inhibitor actinomycin D demonstrated that regulation is at the transcriptional level. These results indicate that in the presence of estrogen, ER+ breast cancer cells possess active mechanisms to suppress EGFR expression. Up‐regulation of EGFR in response to estrogen depletion and growth inhibition could represent an attempt to rescue cell growth by utilizing an alternative pathway. Indeed, we found that estrogen‐depleted breast cancer cells are more sensitive to the mitogenic effects of EGF and TGF‐α, and simultaneous blockade of both estrogen and EGFR signaling pathways induced cell death. J. Cell. Biochem. Suppl. 36: 232–246, 2001.

Estrogen induction of TGF-α is mediated by an estrogen response element composed of two imperfect palindromes

To investigate the molecular mechanisms underlying the two- to three-fold induction of human transforming growth factor-alpha (hTGF-alpha) mRNA and two- to five-fold induction of hTGF-alpha protein observed following estrogen treatment of hormone-responsive human breast cancer cell lines, the hTGF-alpha promoter was assayed for ERE-like sequences able to mediate estrogen induction of a heterologous gene. Transient co-transfection of a chloramphenicol acetyl transferase (CAT) construct consisting of either 1100 bp or 330 bp of hTGF-alpha promoter sequence and an estrogen receptor expression vector into either COS-7 cells or hormonally responsive MCF-7 human breast cancer cells resulted in a two- to five-fold induction of CAT activity by estrogen. Although no consensus estrogen response element (ERE) exists in the hTGF-alpha promoter, a sequence consisting of two imperfect ERE palindromes separated by 20 bp is located at -200 to -252. This sequence was inserted into a mouse mammary tumor virus (MMTV) based CAT construct and assayed for its ability to confer estrogen regulation of CAT expression to a heterologous promoter. Transient co-transfection of this construct with an estrogen receptor expression vector into either COS-7 cells or MCF-7 cells resulted in an average 30-fold estrogen induction of CAT activity. Gel shift assays with human recombinant estrogen receptor (ER) and 32P-labelled fragments revealed that the ER could specifically bind to this sequence. These results indicate that this 53 bp sequence can function as an ERE, and is likely to be responsible for the observed induction of TGF-alpha message and protein in response to estrogen. These data also indicate that the level of estrogen inducibility mediated by this element may be positively or negatively modulated by interaction or competition with other transcription factors.

Mechanisms of EGF receptor regulation in breast cancer cells.

Overexpression of the EGF receptor in breast cancer correlates with poor prognosis and failure on endocrine therapy for both ER−/EGFR+ and ER+/EGFR+ tumors, suggesting a role for EGFR in the progression to hormone independence. The identification of specific DNase I hypersensitive site patterns for the EGFR gene in ER+ vs. ER− cells implicates regions of the EGFR first intron in up-regulation of EGFR, while estrogen regulation studies indicate the involvement of a repressor(s) in the maintenance of low levels of EGFR. Based on these findings, a multi-step model is proposed for the progression of breast cancer from a hormone-dependent, ER+/EGFR- phenotype to an aggressive, hormone-independent, ER−/EGFR+ stage.

Identification and characterization of a negative regulatory element within the epidermal growth factor receptor gene first intron in hormone‐dependent breast cancer cells

The epidermal growth factor receptor (EGFR) exhibits an inverse correlation with estrogen receptor (ER) expression in the majority of breast cancers, predicting a poor response to endocrine therapy and poor survival rate. Inappropriate overexpression of EGFR in breast cancer is associated with a more aggressive phenotype. Transcriptional regulation is the major regulatory mechanism controlling EGFR overexpression in breast cancer cells. We have identified a region within the first intron of the EGFR gene that mediates transcriptional repression of EGFR gene expression in ER +/low EGFR expressing but not in ER−/high EGFR expressing breast cancer cells. Utilizing transient transfections of homologous and heterologous promoter‐reporter constructs, we localized optimal repressive activity to a 96 bp intron domain. The 96 bp fragment displayed differential DNA‐protein complex formation with nuclear extracts from ER + vs. ER− breast cancer cells. Moreover, factors interacting with this intron negative regulatory element appear to be estrogen‐regulated. Consequently, our results suggest that we have identified a potential mechanism by which maintenance of low levels of EGFR expression and subsequent EGFR upregulation may be attributed to the loss of transcriptional repression of EGFR gene expression in hormone‐dependent breast cancer cells. J. Cell. Biochem. 85: 601–614, 2002.

Growth and EGFR Regulation in Breast Cancer Cells by Vitamin D and Retinoid Compounds.

The effect of 1,25-dihydroxyvitamin D3, analog C (1,25-(OH)2-16-en-23-yn-26,27-F6-vitamin D3), 9-cis retinoic acid, and all-trans retinoic acid on the growth and expression of EGFR in MCF7, T47D, BT474, and BT549 breast cancer cells was examined. Significant growth inhibition was noted in MCF7, T47D, and BT474 cells by 8 days of treatment, while BT549 cells showed none. MCF7, T47D, and BT549 cells treated with 1,25-dihydroxyvitamin D3 demonstrated a 50% decrease in EGFR mRNA within 2 h which was sustained to 72 h, while BT474 cells demonstrated a 200–500% increase. EGFR protein levels correlated with these mRNA changes in BT474 and BT549 cells. Measurement of mRNA stability in vitamin D treated BT474 cells indicated that there was no change in EGFR mRNA half-life. Transfection of an EGFR promoter containing reporter plasmid demonstrated vitamin D induced changes in reporter gene activity that paralleled the changes observed in EGFR mRNA and protein. Electrophoretic mobility shift assays using a putative vitamin D response element within this region of the EGFR promoter demonstrated specific VDR binding. These results indicate that the vitamin D effect on EGFR expression in breast cancer cells has a transcriptional component likely mediated through a vitamin D responsive promoter sequence. They also suggest that growth inhibition and EGFR down-regulation by vitamin D and retinoids may be related events in some breast cancer cells, but not in all.

Breast Cancer: Influence of Endocrine Hormones, Growth Factors and Genetic Alterations

Breast cancer is a disease whose frequency as well as pathologic characteristics vary markedly with age and sex. Women develop breast cancer with an incidence of approximately 1 in 10 in the United States, about 100 times the frequency in men. In women, the incidence of breast cancer increases with increasing age, but the rate of increase drops off sharply at the age of menopause (Pike et al., 1981). Breast cancer is more likely in postmenopausal than in premenopausal women to be positive for the receptor for estrogen (Ottman et al., 1981). Estrogen receptor positive breast cancer, whether postmenopausal or premenopausal, is associated with better prognosis than receptor negative breast cancer (Sunderland and McGuire, 1990). These statistics have contributed to the view that exposure of the mammary gland to ovarian estrogens (and progestins) is critical to onset and malignant progression of breast cancer. Indeed, perimenarchal loss of ovarian function can result in a decrease in breast cancer risk by a factor of 100 to about that found in men (Brown, 1981). Furthermore, ovariectomy and/or antiestrogenic and antiprogestational drugs have been successfully used in treatment of breast cancer (lino et al., 1990).