Estrogen receptor signaling pathways in human non-small cell lung cancer
Introduction
Lung cancer is the most common cause of cancer mortality in both male and female patients in the US. It is estimated that more than 180,000 new cases of non-small cell lung cancer (NSCLC) will be diagnosed this year in the US, and about 165,000 patients will succumb to the disease. Survival rates from NSCLC are unacceptably low [1], [2], and new therapeutic options are urgently needed. The etiology of most NSCLC is not fully defined, but several studies suggest a role of estrogens in progression [2], [3], [4], [5]. Clearly, cigarette smoking remains the primary risk factor for lung cancer with 85–90% of all lung cancer patients having smoked cigarettes at some time. Remaining lung cancer cases occur in nonsmokers, mainly women. In the past, men had higher lung cancer incidence due to higher smoking rates. However, rates of smoking in women have increased, with a 600% increase in the death rate from lung cancer leading to a “full blown epidemic” as noted by the US Surgeon General [2]. Mortality from lung cancer in women now exceeds that from breast cancer.
Estrogen status appears to be a significant factor in lung cancer in women, with evidence that exogenous and endogenous estrogen may play a role in development of lung cancer, especially adenocarcinoma [2], [5], [6]. Women have naturally higher circulating estrogen levels than men that may increase their susceptibility to lung cancer. In addition, estrogen biosynthesis due to activity of aromatase is reported in lung, suggesting that estrogens are produced locally in women and men and could affect tumor development [4], [7]. Despite earlier conflicting reports on the presence of ER in lung [8], new work clearly shows that both ERα and ERβ mRNA and protein are expressed in malignant lung epithelial cells [4], [9], [10], [11]. Moreover, these receptors may play important biologic roles in lung and respond to antiestrogens. In lung tumor cells in the laboratory, estrogens stimulate cell proliferation and enhance tumor progression in vivo. Blockade of this pathway by competition for estrogen binding to ER is the basis of the therapeutic tamoxifen, a partial agonist that limits proliferative effects of estrogen in breast cancer. However, in the uterine endometrium, tamoxifen has more prominent agonist effects and promotes growth, and it appears to have similar effects in lung [9], [11]. In contrast, we and others expect that new agents, such as Faslodex, an antiestrogen that downregulates ER [12], and aromatase inhibitors [13], downregulators of local estrogen production in tissues, may have previously unsuspected use to suppress lung cancer [4], [13].
Extranuclear and nuclear ER were postulated in early concepts of steroid interaction with target cells, such as breast and ovary [14], with the transcriptional activity of estrogen mediated by high-affinity ER in cell nuclei [15]. On estrogen binding in target cells, ER is phosphorylated and undergoes a conformational change that allows receptor dimerization and association of estrogen–ER complexes with specific estrogen response elements (ERE) in DNA, leading to transcription. Nuclear actions of estrogen are dependent, in part, on the subtype (ERα, ERβ) of ER, the gene promoter, and the steroid receptor coactivator and co-repressor proteins that modulate transcription [15], [16]. In addition, ER also regulates gene expression without direct binding to DNA. This occurs by protein–protein interaction with other transcription factors, such as AP-1, and with extranuclear signaling complexes that, in turn, modulate downstream gene transcription. Extranuclear signaling, such as MAPK activation, has a rapid onset and is mediated by ER in or tethered to membrane. In lung, as in breast, extranuclear ER appear to derive from the same transcript as nuclear ER [4], [17], [18], [19]. Nuclear and extranuclear ER act in concert with growth factor signaling pathways, such as EGFR [20], [21], to promote growth and survival [4], [11], [22]. The EGFR family of receptors, including EGFR (HER-1) and HER-2, are also implicated in lung cancer pathogenesis [23], [24]. This receptor axis is associated with progression of malignancy, inhibition of apoptosis and angiogenesis. EGF receptor antibodies or EGFR tyrosine kinase inhibitors elicit growth inhibition of lung tumors expressing these receptors. Moreover, EGFR/HER receptors regulate ligand-independent ER activation. Molecular details of interaction between ER and EGFR/HER are emerging, and ER is an important locus for signal convergence [4], [21], [22], [25]. In gene knockout mice lacking ERα, both estrogen- and EGF-stimulated growth in target tissue is blocked, while, in knockout mice lacking ERβ, disruption of normal lung development occurs [26]. Thus, ER may mediate transcription by integrating signals from growth factor pathways as well as from estrogen binding [27], [28], [29], [30]. Although some reports suggest that extranuclear ER is an alternative protein [31], [32], most studies indicate that extranuclear and nuclear ER derive from the same transcript [17], [33], [34], [35]. Post-translational ER modification can elicit membrane targeting [36], [37], [38], and association of ER with other adaptor or signaling proteins (e.g. shc, c-src or MNAR) may occur [39], [40], [41].
Section snippets
Cell culture
Human non-small cell lung cancer cells (NCI-H23 [H23], A549) and breast cancer cells (MCF-7 and SKBR3) were from ATCC. MCF-7 tumor cells with HER-2 overexpression (MCF-7/HER-2) were prepared as before [28]. Cell lines were routinely maintained in RPMI 1640 medium with 10% fetal bovine serum (FBS, Invitrogen/Life Technologies, Carlsbad, CA), 2 mM l-glutamine and 1% Antibiotic-Antimycotic solution 100× (Mediatech, Herndon, VA). For estrogen-free conditions, media were changed 48 h before
ERα and ERβ are both expressed in archival human NSCLC specimens from the clinic
The prevalence of ERα and ERβ in human NSCLC specimens was assessed by standard IHC methods using archival formalin-fixed, paraffin-embedded human tumor specimens. Fig. 1 presents representative examples of IHC staining patterns for ERα and ERβ. Appropriate tissue and reagent controls were done to confirm specificity [43]. For example, in the absence of antibodies to ERα or ERβ, no specific staining was observed (see Fig. 1A and D). Staining in tumor cell nuclei was observed for both ERα (Fig. 1
Discussion
Data from the present study confirm earlier work showing that estrogen as well as growth factors promote the progression of human NSCLC [4], [9], [10], [11]. Although previously not recognized, it is now known that estradiol promotes the growth of NSCLC [4], [11]. NSCLC cells harbor a complete estrogen signaling system, including estrogen receptors, estrogen-responsive elements in DNA and steroid hormone receptor coactivators. Using controlled homogenization and quantitative subcellular
Acknowledgments
We thank Dr. Hermes J. Garbán for his support in the preparation of this manuscript. Research supported by funding from NCI Lung Cancer SPORE Program at UCLA (P50 CA90388), Hamburger Fund of the Jonsson Cancer Center, Stiles Program in Integrative Oncology (in vitro work) and National Lung Cancer Partnership.
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