Estrogen and growth factor receptor interactions in human breast and non-small cell lung cancer cells

Steroids 70 (2005) 372–381

Estrogen and growth factor receptor interactions in human breast and non-small cell lung cancer cells Richard J. Pietras a,∗ , Diana C. M´arquez a , Hsiao-Wang Chen a , Eugene Tsai a , Olga Weinberg a , Michael Fishbein b a b

Department of Medicine, Division of Hematology-Oncology, UCLA School of Medicine and Jonsson Comprehensive Cancer Center, Los Angeles, CA 90095-1678, USA Department of Pathology, UCLA School of Medicine and Jonsson Comprehensive Cancer Center, Los Angeles, CA 90095-1678, USA Available online 25 March 2005

Abstract Extranuclear estrogen receptors may mediate rapid effects of estradiol that communicate with nuclear receptors and contribute to proliferation of human cancers bearing these signaling proteins. To assess these growth-promoting pathways, we undertook controlled homogenization and fractionation of NIH-H23 non-small cell lung cancer cells. As many breast tumors, NIH-H23 cells express estrogen receptors (ER), with the bulk of specific estradiol binding in nuclear fractions. However, as in breast cells, a significant portion of specific, high-affinity estradiol-17␤ binding-sites are also enriched in plasma membranes of lung tumor cells. These estrogen binding-sites co-purify with plasma membranemarker enzymes and are not significantly contaminated by cytosol or nuclei. On further purification of membrane caveolae from lung tumor cells, proteins recognized by monoclonal antibodies to nuclear ER-␣ and to ER-␤ were identified in close association with EGF receptor in caveolae. In parallel studies, ER-␣ and ER-␤ are also detected in nuclear and extranuclear sites in archival human breast and lung tumor samples and are noted to occur in clusters at the cell membrane by using confocal microscopy to visualize fluorescent-labeled monoclonal antibodies to ER-␣. Data on site-directed mutagenesis of cysteine-447 in ER-␣ suggest that association of ER forms with membrane sites may depend on acylation of cysteine by palmitate. Estrogen-induced growth of MCF-7 breast cancer and NIH-H23 lung cancer cells in vitro correlated closely with acute hormonal activation of mitogen-activated protein kinase signaling and was significantly reduced by treatment with Faslodex, a pure anti-estrogen. Further, combination of Faslodex with selected growth factor receptor inhibitors elicited a more pronounced inhibiton of tumor cell growth. Thus, extranuclear forms of ER play a role in promoting downstream signaling for hormone-mediated proliferation and survival of breast, as well as lung, cancers and offer a new target for anti-tumor therapy. © 2005 Elsevier Inc. All rights reserved. Keywords: Estrogen receptor; HER-2 receptor; EGF receptor; Breast cancer; Lung cancer; Palmitoylation

1. Introduction Although lung cancer is now the leading cause of cancer death among women [1,2], breast cancer still constitutes the most commonly diagnosed malignancy in women after skin cancer. In the clinic, endocrine therapy has long been recognized as an important intervention in women with breast cancers that express estrogen receptor (ER), and treatment with tamoxifen has significantly enhanced patient survival [3]. More recently, several epidemiologic and clinical studies ∗

Corresponding author. Tel.: +1 310 825 9769; fax: +1 310 825 6192. E-mail address: [email protected] (R.J. Pietras).

0039-128X/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.steroids.2005.02.017

have provided evidence of a role for estrogens in the genesis and progression of lung cancer, especially non-small cell lung tumors [1]. In addition, among non-small lung tumor cells expressing either ER-␣ or ER-␤, treatment with estradiol stimulates DNA synthesis and cell proliferation, indicating that estrogen may also promote lung cancer growth [1,2]. The success of endocrine therapy for human cancers depends on close regulation of cell growth by steroids such as estrogen which bind estrogen receptors. These receptors are present in more than two-thirds of breast cancers at diagnosis [3]. According to prevailing theories of hormone action, estradiol-17␤ (E2 ␤) diffuses across plasma membranes and

R.J. Pietras et al. / Steroids 70 (2005) 372–381

binds with nuclear receptors [4,5]. The newly formed E2 ␤receptor complex may then regulate gene expression by binding estrogen-responsive elements in target genes or by modifying transcription via interaction with other nuclear proteins [6]. These nuclear events are believed to promote cell growth. However, in addition to this mechanism, many rapid effects of estrogen have been documented in breast [7,8] and other tissues, including lung [2,7–11]. The acute time course of these events suggests that they do not require precedent gene activation. Rather, many rapid effects of estrogens may be due to activation of extranuclear signaling pathways. There is increasing evidence that extranuclear forms of ER, including both ER-␣ and ER-␤, exist and participate in activation of downstream signaling pathways associated with gene regulation [8,9,11]. Estrogens trigger rapid stimulation of guanylate and adenylate cyclases [7,8], Ca2+ fluxes [8,12], nitric oxide synthase [10] and protein phosphorylation [13]. In addition, E2 ␤ activates within seconds mitogenactivated protein kinase (MAPK) signaling cascades in responsive tissues [13]. Although activation of these pathways is often considered to be restricted to transmembrane growth factor receptors, the presence of high-affinity receptors for E2 ␤ associated with plasma membranes of target cells was first reported more than two decades ago [14]. Moreover, proteins immunoreactive with antibodies to nuclear ER occur at the surface membrane of cells that exhibit rapid biologic responses to E2 ␤ [15–17]. ER-null cells transfected with expression constructs for ER have also been shown to express a portion of ER protein on their surface and to respond to estrogen with rapid membrane-initiated effects [18,19]. These data have led to a growing recognition that extranuclear ER are important in estrogen action in target cells [8,11,14,20,21]. The interaction of estrogen receptors with growth factor receptor signaling is another emerging area of investigation in elucidating factors that promote progression of human malignancy. Members of the EGFR/HER family of growth factor receptors, including epidermal growth factor receptor (EGFR), HER-2, HER-3 and HER-4, are often implicated in tumorigenesis [22]. EGFR is a 170 kD protein that, upon binding of ligand, undergoes receptor dimerization followed by autophosphorylation of its cytoplasmic domain at specific tyrosine residues. Functions of EGFR are closely linked to its family members by sharing of ligands and by crossactivation via receptor heterodimerization. Overexpression of HER-2 or related HER receptors occurs in two-thirds of breast tumors, while amplification and/or overexpression of HER-2 is found in 20–25% of breast cancers [23]. HER-2 overexpression is generally a marker of poor prognosis and associates with failure of anti-estrogen therapy in the clinic [24]. Of note, activation of EGF and HER-2 receptors often results in downstream protein kinase stimulation, including MAP and PI3 kinase/Akt kinases, with subsequent stimulation of specific transcription factors. Since several of these signaling molecules are shared with the ER signaling pathway, many sites for interaction between ER and HER signal transduction are possible [22,24].

373

In the present work, we present data demonstrating that human breast and non-small cell lung cancer cells contain extranuclear, as well as nuclear, receptors for estrogen. Activation of these extra-nuclear receptors appears to promote rapid stimulation of downstream kinase signaling and later cell proliferation. Biologic activity of the extranuclear ER pathway can be diminished by Faslodex, a pure anti-estrogen, and by disruption of associated membrane growth factor receptor signaling. The results suggest that estrogens may initiate extranuclear receptor signaling events leading to modulation of the growth and survival of breast and non-small cell lung cancers.

2. Experimental 2.1. Cell culture and proliferation in vitro Human MCF-7 breast cancer cells and NIH-H23 nonsmall cell lung cancer (NSCLC) cells (ATCC) were routinely maintained in RPMI 1640 medium with 10% fetal bovine serum (FBS). For estrogen-free conditions, medium was changed 48 h before experiments to phenol-red free RPMI 1640 with 1% dextran-coated, charcoal-treated (DCC) FBS [24]. To assess effects of estrogen on cell proliferation in selected experiments, cells were incubated with either 10 nM E2 ␤ or E2 ␣ (17␣-estradiol, Steraloids). After 72 h, cells were counted to estimate rates of cell proliferation, using data from 3 to 4 independent experiments. 2.2. Quantitative cell homogenization and sub-cellular fractionation Cell fractionation was done as before with methods designed to preserve the integrity of sub-cellular structures [25,26]. In brief, cells were harvested with ice-cold Versene in the presence of protease inhibitors, then homogenized using a Dounce homogenizer. Whole homogenate (H) was filtered through nylon mesh and centrifuged at 1000 × g for 10 min to yield crude nuclear (N) and post-nuclear supernate fractions. The N fraction was re-suspended in 31% sucrose in buffer, loaded on top of a discontinuous sucrose density-gradient and centrifuged at 67,000 × g for 2 h. Plasma membranes occurred predominantly at ρ = 1.13–1.16 (PM) [25,26]. The post-nuclear supernatant was centrifuged at 15,000 × g for 30 min, with the resulting pellet representing the mitochondria-lysosome fraction (ML). The supernate was centrifuged at 105,000 × g for 1 h to yield the microsomal pellet fraction (Ms) and the soluble cytosol fraction (S). Extracts from cell membranes were solubilized as before [25,26]. Protein was quantitated using the Pierce Protein Assay Kit (Rockford, IL). In sub-cellular fractions, activities of 5 -nucleotidase (EC 3.1.3.5) and lactate dehydrogenase (LDH) were determined as before [24–26]. Relative specific activity represents the specific activity of enzyme in a given fraction in relation

374

R.J. Pietras et al. / Steroids 70 (2005) 372–381

to that in homogenate. DNA was assessed by established methods [26]. Specific E2 ␤ binding was assessed in cell fractions using [[2,4,6,7,16,17]-3 H] estradiol-17␤ (NEN) as previously [24–26]. A 100-fold molar excess of unlabeled estradiol-17␤ was present with [3 H]estradiol-17␤ in paired samples to determine displaceable binding. To characterize ER forms associated with PM fractions, membrane proteins were separated by SDS-PAGE, then transferred to a nitrocellulose membrane for immunodetection by Western blot. Blots were probed as before [24] with ER-␣ antibodies, Ab1 against a ligand-binding domain (LBD) segment from amino acids 495-595 (Upstate Biotechnology, Lake Placid, NY) or Ab2 against a longer segment of LBD, from amino acids 302–595 (Neomarkers, Fremont, CA) or ER-␤ antibody (PA1-310; Affinity Bioreagents, Golden, CO). 2.3. ER activity in membrane caveolae fractions of ER-positive lung cancers Membrane caveolae fractions were isolated from ERpositive NIH-H23 lung cells, with detergent-free purification of caveolae fractions done as before [27]. Caveolae fractions were solubilized and separated by SDS-PAGE. Immunoblots were done with antibodies to ER-␣, ER-␤, flotillin-2, caveolins, EGFR, and HER-2 using established methods [21,24,28]. 2.4. To assess the occurrence of ER forms at plasma membranes of lung cancer cells To detect estrogen receptors at the plasma membrane of target cells, immunofluorescence methods were utilized as before in combination with confocal microscopy [15,18,19,21]. 2.5. Determination of p44/p42 MAP kinase (MAPK) activity Cells were maintained in estrogen-free conditions 48 h before experiments [24]. In confocal microscopy studies with lung cancer cells, the presence of phosphorylated MAPK was detected with or without estradiol at various times by use of primary phospho-MAPK polyclonal antibody (anti-phosphop44/p42 MAPK, Thr202/Tyr204, polyclonal antibody; New England Biolabs, Beverly, MA), and a fluorescent-labeled secondary anti-rabbit antibody as before [19,21]. 2.6. Assay of VEGF secretion by lung cancer cells Effects of estrogens, anti-estrogens (Faslodex; ICI 182,780) and the EGFR kinase inhibitor, Iressa, on secretion of VEGF was tested in vitro using lung tumor cells. VEGF secretion was quantitated in cell media by ELISA as described previously [29,30].

2.7. Assay of aromatase, ER and HER receptors by immunohistochemistry in human tumors Specimens from the UCLA lung tumor bank were evaluated for expression of ER-␣, ER-␤, EGFR, and HER-2 and aromatase [23,24,31–33]. Human NSCLC specimens were formalin-fixed, paraffin-embedded tumor samples collected and processed by established methods. Antibodies to ER-␣, ER-␤, EGFR, HER-2, and aromatase were used to detect specific antigens as before [23,24,31–33]. Approval for use of human tumor specimens was obtained from the institutional review board, and all human subjects signed written informed consents. 2.8. Construction of expression vectors by in vitro mutagenesis Construction of full-length ER-␣ expression vector pCDNA3ER-WT was done by methods as before [34]. Mutation of the cysteine-447 residue in wild type ER to serine was achieved by use of the GeneTailor site-directed mutagenesis kit (Invitrogen) with pCDNA3ER-WT as the template [35]. The mutagenic primer pairs used to create the mutation were: C447S-F: 5 -CAGGGAGAGGAGTTTGTGAGCCTCAAATCTATTATTTTGC-3 and C447S-R: 5 -GCAAAATAATAGATTTGAGGCTCACAAACTCCTCTCCCTG-3 . Mutation was verified by standard sequencing approaches [34], and the new plasmid was termed pCDNA3ER-C447S. 2.9. Transfection of wild type and mutant ER and in vitro assays For studies on in vitro activity of wild type and mutant ER expression vectors, ER-null COS-7 cells were transfected with pCDNA3ER-wt and mutated pCDNA3ER-C447S with estrogen-free conditions as before [34]. In brief, cells were plated directly in microchamber slides and grown to 70% confluence. Transfection was done by using 2 ␮g of plasmid/slide and 12 ␮l of Polyfect transfection reagent (Qiagen). After 48 hours of transfection, cells were treated with or without 10 nM estradiol-17␤ for 10 min, then washed three times with cold PBS and fixed in 4% paraformaldehyde for confocal microscopy assays.

3. Results 3.1. Enrichment of high-affinity binding-sites specific for estradiol-17␤ in nuclear and extranuclear sub-fractions of lung cancer cells We have previously reported that specific [3 H]-estradiol17␤ (E2 ␤) binding occurs in sub-cellular fractions, including plasma membranes, of MCF-7 breast cancer cells after use of quantitative sub-cellular fractionation [21,24]. More-

R.J. Pietras et al. / Steroids 70 (2005) 372–381

375

Fig. 1. Distribution and relative specific activity of enzymes and specific [3 H]estradiol binding in plasma membrane (PM) and other sub-fractions of lung cancer cells, NIH-H23. Cells were grown in estrogen-free media, then disrupted [25,26]. (A) Yield of marker enzyme and E2 ␤ binding given as % homogenate, with mean ± S.E. (n = 3). Total recovery of protein, 5 -nucleotidase (5 -NUC), lactate dehydrogenase (LDH) and specific [3 H]E2 ␤ bound (E2 ␤) in crude nuclear (N), microsome-rich (Ms), mitochondria-lysosome (ML), and cytosol (S) fractions ranged from 96–101% of that in homogenates (WH). WH values were 30 ± 3 mg/108 cells for protein; 29 ± 3 nmol/min/mg protein for 5 -NUC; 20 ± 3 units/min/mg protein for LDH; and 85 ± 5 fmol/mg protein for specific [3 H]E2 ␤ binding. (B) Relative specific activity in PM represents specific activity of enzyme or E2 ␤ binding in a given fraction in relation to that in homogenates [25,26]. In further experiments to assess specific binding of [3 H]estradiol-17␤, NIH-H23 plasma membranes were incubated in Ca++ -free media with 0.25 M sucrose and proteinase inhibitors at 20 ␮g protein/ml for 2 h at 4 ◦ C with concentrations of [3 H]E2 ␤ ranging from 0.1 to 5.0 × 10−9 M; or in the presence of a 100-fold molar excess of unlabeled E2 ␤ plus [3 H]E2 ␤ [25,26]. In addition, ligand specificity of [3 H]estradiol-17␤ binding was tested by incubation in the presence of a 100-fold molar excess of competing steroids, E2 ␤, E2 ␣, progesterone (PRG) or testosterone (TST), with values determined in 3 experiments (refer to text).

over, the molecular size and binding affinity of specific estrogen-binding sites in extranuclear fractions of breast cells correspond with the properties of ER in breast cell nuclei [21,24]. To confirm these and other reports of extranuclear E2 ␤ binding-sites in target cells [8,9,11,15,20], we measured specific [3 H]E2 ␤ binding in sub-cellular fractions of NIH H23 NSCLC cells after controlled cell homogenization and sub-cellular fractionation [24–26]. In these experiments, more than 97% of total specific E2 ␤ binding was recovered in whole homogenates (WH), with distribution of specific [3 H]E2 ␤ binding in crude nuclear, microsomal, mitochondrialysosome and cytosol fractions (Fig. 1A). On purification of plasma membranes (PM) from crude nuclei, PM fractions had high activity of 5 -nucleotidase, a PM marker-enzyme, to 20-times homogenate levels (Fig. 1 A and B). Specific [3 H]E2 ␤ binding in membranes was also enriched to 25 times homogenate levels and represented up to 20% of WH binding. In confirmation of the purity of membranes, LDH activity, strongly enriched in cytosol, was not detected at significant levels in PM fractions. Further, cellular DNA recovery was 96 ± 2% of homogenate levels in nuclei, but no DNA was detected in PM fractions. To assess the affinity of specific estrogen binding in membranes from lung cancer cells, [3 H]-E2 ␤ binding by PM was analyzed by equilibrium binding methods [24–26] with membranes exposed to [3 H]-E2 ␤ at concentrations ranging from 0.1 to 5.0 × 10−9 M. Specific binding of the hormone by membranes was saturable, and Scatchard analyses of the data showed that the dissociation constant for binding estradiol was 2 × 10−10 M. Total binding sites in membranes at saturation corresponded to approximately 1.9 pmol E2 ␤ per mg membrane protein. In addition, [3 H]-E2 ␤ binding to mem-

branes was ligand-specific, with suppression by excess unlabeled E2 ␤, but not by estradiol-17␣, progesterone or testosterone (data not shown) [see [24–26,36]. 3.2. Identification of estrogen receptor forms in sub-cellular fractions and in intact cells Membrane rafts and caveolae appear to offer a matrix for integration and organization of many signaling complexes important for cell growth regulation [16,37]. Localization of ER and EGFR/HER receptors in caveolae domains from lung tumor cells may allow for interactions between these signaling pathways. To assess the potential localization of ER forms in caveolae domains, we isolated caveolae from NSCLC cells using an established detergent-free purification method [27]. The results show that ER-␤ and ER-␣ co-purify with flotillin2 and caveolin, known markers for caveolae from cancer cells (Fig. 2A). Using Western blot methods [23,24], caveolae showed predominant enrichment of a 59 kD protein that binds ER-␤ antibody, as well as notable enrichment of a 67 kD protein that binds nuclear ER-␣ antibody (Fig. 2A). Using intact cells, fluorescent-labeled ER antibodies show a ring-like distribution in distinct clusters at the surface membrane of tumor cells, suggesting a membrane-associated receptor protein (Fig. 2B). 3.3. Assay of ER and aromatase by immunohistochemistry in lung and breast tumors To evaluate by independent means the occurrence and subcellular distribution of ER forms in human breast and lung cancers, archival formalin-fixed, paraffin-embedded tumor

376

R.J. Pietras et al. / Steroids 70 (2005) 372–381

Fig. 2. Identification of ER in intact lung cancer cells and in caveolae membranes from lung cancer cells, NIH-H23 [27]. (A) Proteins from sucrose gradient fractions 4–7 for isolation of caveolae [27] analyzed by PAGE, transferred to nitrocellulose membranes and immunoblotted with antibodies to nuclear ER␣ (67 kD), ER␤ (59 kD), EGF receptor (EGFR), caveolin and flotillin. Caveolin and flotillin, markers of caveolae-related fractions, were enriched in samples 4–7, the sucrose gradient region enriched with EGFR and ER. Relative positions of the proteins on gels corresponded to known molecular sizes. (B) Fluorescentlabeled antibody to ER␣ cross-reacts with membranes of intact, non-permeabilized NCI-H23 cells, possibly clustered in caveolae/lipid raft domains. Controls with excess unlabelled antibody show no binding [21].

specimens from the clinic were assayed for ER-␣ and ER␤ expression by immunohistochemistry. The findings show that both ER-␤ and ER-␣ occur in tumor cell nuclei, but specific staining of ER forms was also detected at extranuclear sites. Representative results are shown in Fig. 3. Further studies on the prevalence of ER in nuclear and extranuclear sites in these cancers are underway. In parallel studies, we also assessed the presence of aromatase, a critical enzyme for estrogen synthesis that regulates the conversion of androgen to estrogen [32,33]. Both breast and lung tumor specimens exhibited significant expression of aromatase (Fig. 3).

3.4. Rapid effects of estradiol on activation of MAP kinase in breast and lung cancer cells Post-receptor signal transduction events, such as stimulation of MAPK, extracellular signal-regulated kinase ERK-1 (p44) and ERK-2 (p42) [8,13], may contribute to proliferative effects of E2 ␤ in target cells including those from breast and lung. We previously reported that estradiol elicited a rapid phosphorylation of MAPK in MCF-7 cells [21]. Thus, we assessed similar estrogen-induced effects in NIH-H23 cells in vitro. E2 ␤, but not 17␣-estradiol (E2 ␣), promotes phosphorylation of MAPK isoforms, with effects evident within 2 min

Fig. 3. Immunohistochemical (IHC) detection of ER-␣, ER-␤ and aromatase in tumor sections from post-menopausal women. Excised tumors were fixed in formalin and embedded in paraffin prior to preparation for IHC by standard methods. Appropriate tissue and reagent controls were done in parallel to confirm specificity of the immunoassays. Both ER-␣ (a) and ER-␤ (b) occur in non-small cell lung tumor cell nuclei, but specific staining of ER forms was also detected at extranuclear sites, including membrane-associated staining in non-small cell lung (b) and in human breast adenocarcinoma cells (c). Representative results are shown, based on a review of 10 independent specimens. In parallel studies, aromatase, a critical enzyme for estrogen synthesis well known to occur in breast tissues, was also detected in non-small cell lung tumor specimens (d).

R.J. Pietras et al. / Steroids 70 (2005) 372–381

377

Fig. 4. Estrogen promotes phosphorylation and nuclear translocation of p42/p44 MAPK. NCI-H23 NSCLC cells were grown with estrogen-free condition for 48 h before treatment with estradiol (2 nM) for 2,10 or 30 min or with estradiol plus Faslodex (10 nM) at indicated times. Treatments are compared with 0 time or appropriate control. Fluorescent-labeled secondary antibody that reacts with primary antibody to phospho-p42/p44 MAPK is seen by confocal microscopy. Estrogen-induced increase in MAPK phosphorylation and in nuclear localization of MAPK by 2–10 min is blocked by Faslodex.

(Fig. 4A). The estrogen-induced increase in MAPK phosphorylation and in nuclear localization of MAPK by 2–10 min is significantly blocked by the pure anti-estrogen, Faslodex (Fig. 4B) [21]. 3.5. Site-directed mutagenesis of Cys447 in ER impairs membrane association of the receptor and blocks estrogen-induced rapid activation of MAP kinase Previous work demonstrated that ER-␣ is a palmitoylated protein [35,38] and that point mutation of Cys447 in ER disrupts palmitoylation of the receptor [35]. To assess whether this alteration affects membrane localization of ER and receptor-mediated signaling, COS-7 cells, normally de-

void of ER expression, were transiently transfected with a control vector, a wild-type ER-␣ vector or the ER-␣ mutant in which Cysteine-447 is changed to serine by use of sitedirected mutagenesis (ER-␣-Cys447Ser). To visualize the cellular distribution of wild type and mutant ER-␣, transfected COS-7 cells were evaluated by immunofluorescence staining with confocal microscopy (see Fig. 5). Using antiER-␣ antibody, significant membrane-associated ER-␣ occurred in COS-7 cells expressing wild type ER-␣, while no membrane-associated ER-␣ was detected in cells expressing the mutant ER-␣. Further, ER-␣ was not detected in COS-7 cells transfected with control vector (not shown). The findings suggest that Cys447 is important for membrane localization of a portion of cellular ER.

Fig. 5. Site-directed mutagenesis of cysteine-447 in ER-␣ impairs membrane association of ER and blocks estrogen-induced MAPK activation. COS-7 cells, normally devoid of ER expression, were transiently transfected with wild-type ER-␣ vector (WT) or ER-␣ mutant vector (C447S). After 48 h, cells were plated, then treated without (CN; vehicle, left) or with (10 nM E2, right) estradiol for 10 min, followed by cold washes and fixation. Using the IHC, ER-␣ was detected with primary antibody (A, anti-ER-␣) and phospho-MAPK was detected similarly in parallel experiments (B, anti-MAPK). Secondary antibody for both antigens was green fluorescent Alexa-IgG; and DAPI stained nuclei blue. (A) Cellular distribution of WT and mutant ER-␣ shown. In CN-treated cells, WT ER was weakly detected at surface membranes [(A)(a)], but none occurred at membranes of cells transfected with C447S ER-mutant [(A)(b)]. After 10 nM E2, more pronounced membrane association of WT-ER occurred in ring-like arrays [(A)(e)], but mutant ER was limited to cytoplasm [(A)(f)]. (B) Activation of MAPK is shown. In CN-treated cells, neither WT [(B)(c)] nor C447S mutant [(B)(d)] ER-transfected cells showed significant MAPK activation. However, E2 for 10 min stimulated MAPK in cells with WT ER [(B)(g)] but not in cells bearing C447S ER mutant [(B)(h)]. Similarly, MAPK activation was not detected in normal COS-7 cells treated with E2 (not shown).

378

R.J. Pietras et al. / Steroids 70 (2005) 372–381

Fig. 6. Enhanced inhibition of non-small cell lung tumor cell growth (NIHH23) in vitro by dual therapy with an anti-estrogen (Faslodex) and a tyrosine kinase inhibitor with specificity for EGFR (TKI; Iressa). NIH-H23 human NSCLC cells were treated in vitro with Iressa at the doses indicated over 72 h in the absence or in the presence of Faslodex (10 nM), a pure anti-estrogen. As noted, NIH-H23 cells express both estrogen receptors and EGFR.

To determine whether mutation of Cys447 in ER affected estrogen-induced rapid changes in MAP kinase activity, COS-7 cells were transiently transfected as above with wild type or mutant ER-␣. Estradiol elicited activation of MAP kinase in COS-7 cells expressing wild type ER-␣, but it was ineffective in inducing MAP kinase activation in cells expressing ER-␣-Cys447Ser (see Fig. 5). In parallel studies, MCF-7 breast tumor cells and NIH-H23 lung cancer cells were treated with the palmitoyl-acyltransferase inhibitor 2-bromohexadecanoic acid [35] at 10 ␮M for 10 min before treatment with estradiol (10 nM) in vitro. In both cell lines, the palmitoyl-acyltransferase inhibitor blocked estrogen-induced growth of the cells to 33–35% of controls treated in the absence of the inhibitor (P < 0.001; not shown). Together, these data further implicate membrane-associated ER in contributing to the regulation of proliferation in malignant breast and lung cells. 3.6. Efficacy of pure anti-estrogen, Faslodex, alone and combined with growth factor receptor inhibitory agents in blocking growth and progression of human NSCLC To assess potential interactions between ER and EGFR/HER receptors in regulating cell proliferation, we conducted experiments with Iressa, a tyrosine kinase inhibitor targeted to EGFR [39], administered alone and in combination with Faslodex, a pure anti-estrogen [40]. Data from in vitro experiments with NIH-H23 NSCLC cells show, as expected, that Iressa has modest inhibitory effects when given as a single agent (Fig. 6). However, when combined with Faslodex, Iressa appears to elicit more pronounced growth suppression (P < 0.001) (Fig. 6). It is notable that similar

Fig. 7. VEGF secretion by NSCLC cells. Estradiol (E2) (10 nM) enhanced secretion of VEGF by more than 2-fold in human NSCLC cells, NIH-H23, incubated in vitro for 48 h. Effects of estrogen were significantly blocked by 10 nM Faslodex (Fx). Moreover, Iressa (TKI) at 10 ␮M reduced basal levels of VEGF secretion as compared with control, and Iressa in combination with Faslodex (Fx/TKI) may be more effective than either inhibitor given alone. Ligands that stimulate EGFR/HER receptors may also be important in regulating VEGF and tumor proliferation [29,30,41].

receptor interactions have been found for combination of Faslodex with Herceptin in human breast tumors that overexpress HER-2 receptors [40,41]. 3.7. Efficacy of pure anti-estrogen, Faslodex, alone and combined with growth factor receptor inhibitory agents in blocking secretion of VEGF by human NSCLC NIH-H23 cells produce and secrete VEGF into the extracellular media (Fig. 7). Treatment with estradiol-17␤ (10 nM) enhanced the secretion of VEGF by more than 2-fold in human NIH-H23 cells incubated in vitro for 48 h (Fig. 7). In confirming specificity of this effect, VEGF secretion induced by estrogen was significantly blocked by Faslodex. Further, the tyrosine kinase inhibitor, Iressa, reduced basal levels of VEGF secretion as compared to control, and Iressa with Faslodex was more effective than either inhibitor alone in blocking VEGF release from NSCLC cells (Fig. 7). This provides further evidence for co-operativity between ER and growth factor receptor pathways in regulating cell functions vital to tumor progression.

4. Discussion The biologic activity of estrogen in target tissues is generally considered to be mediated through receptors that act in the cell nucleus [4]. However, many studies provide evidence for extranuclear forms of ER that elicit rapid alterations in signaling that appear to be communicated to downstream sites, including the nucleus [8,9,11,20]. This work

R.J. Pietras et al. / Steroids 70 (2005) 372–381

extends these observations in studies with breast and lung cancer cells and suggests that targeting of extranuclear, as well as nuclear, forms of ER may elicit potent anti-tumor effects [8,11,21,34,40,41]. As with affinity-binding strategies used to identify peptide hormone receptors at the plasma membrane, estrogenbinding sites were first identified at the external membranes of target cells by exposure to estradiol covalently bound to an inert support [14]. Early work on purification of estrogenbinding components from uterus and liver membranes suggested that the binding moiety was a protein with high-affinity binding for E2 ␤ and with a molecular size in the range of nuclear ER [9,25,26]. In addition, these extranuclear receptors had a notable capacity to form dimers after ligand binding [26]. To determine whether extranuclear receptors had antigenic homology with nuclear ER, Pappas et al. [15] used antibodies prepared to different functional epitopes of nuclear ER and demonstrated significant surface labeling in intact pituitary cells by confocal scanning laser-microscopy. Using endothelial cells, Russell et al. [17] independently confirmed that surface binding-sites for estradiol react specifically with antibodies directed to ER-␣. To evaluate the source and distribution of extranuclear ER, Razandi et al. [18] transfected cDNA for ER-␣ and ER-␤ in CHO cells that do not express these genes. Expression of a single cDNA encoding either receptor gave rise both to nuclear and membrane-associated ER, suggesting that extranuclear and nuclear ER derive from a single transcript. The affinity of receptors for E2 ␤ in both sites was nearly identical, but a greater number of receptors were detected in cell nuclei. In addition, both extranuclear ER-␣ and ER-␤ receptors could signal to MAPK which was necessary for activating DNA synthesis [18]. These findings have been confirmed by use of ER knockdown techniques [cf. [11,19]]. The present work elucidates further molecular properties of extranuclear ER in tumor cells. After use of controlled, quantitative cell fractionation to preserve the integrity of sub-cellular structures [24–26], the bulk of specific E2 ␤ binding in MCF-7 and NIH-H23 cells is found in nuclear fractions. However, a significant portion of specific E2 ␤-binding sites also occurs in extranuclear sites, including plasma membranes. These E2 ␤ binding-sites co-purify with plasma membrane-marker enzyme and are free from contamination by cytosol or nuclei. In addition, monoclonal antibodies against nuclear ER identify membrane-associated ER in MCF-7 and NIH-H23 cells, a finding consistent with studies with other cell types [15–18]. In both breast and lung cancer cells, membrane-associated protein reactive with antibodies to nuclear ER-␣ occurred at 67 kDa, a molecular size comparable to that of nuclear ER-␣. However, levels of ER-␣ expression in lung tumor cells are substantially less than those found in MCF-7 cells. Additional studies with MCF-7 and endothelial cells indicate that other protein species, notably at 46 kDa, also occur [17,38,42,43], with truncated receptor forms due, in part, to limited protein degradation or alternative translation [43]. In addition, antibody to ER-␤ reacts

379

with proteins at the expected size of 58–62 kD in extranuclear fractions of NIH-H23 cells. This result confirms independent studies that identified enrichment of ER-␤ forms in human NSCLC cells [1,2]. These data suggest that extranuclear ER contain common structural elements, at least in key molecular domains, with ER-␣, ER-␤ and various splice variants. The classical receptors contain several hydrophobic regions, but it is not known if these are sufficient to allow disposition as an integral membrane protein [42]. Extranuclear ER may also be complexed with other trans-membrane molecules coupled to signaling cascades [10,44]. This notion is supported by reports of ER forms in vesicular invaginations of plasma membrane [45] and in association with caveolin and other components of caveolae and caveolae-related lipid rafts, such as EGFR and HER-2 [16,19], plasma membrane microdomains involved in assembly of signaling complexes. Further, phosphorylation of transmembrane growth factor receptors, such as members of the EGFR/HER family, results in phosphorylation of Shc which, in turn, associates with Grb2-SOS complexes leading to activation of the Ras/Raf/MAPK pathway. In MCF-7 cells, estradiol is reported to induce formation of a protein complex that includes ER-␣ and Shc [46], and such complexes may be due to clustering of these molecules in caveolae-related lipid rafts [40]. Li et al. [38] and Acconcia et al. [47] report that posttranslational modification of ER may play a critical role in membrane association of ER. Protein acylation commonly occurs with linkage of palmitate to the S-atom of cysteine. Although ER has 13 cysteines, only two, Cys447 and Cys530 in the ligand-binding domain, appear readily accessible for S-acylation. The amino acid sequence surrounding Cys447 in ER is noted to have considerable homology with that of S-palmitoylated Cys133 of caveolin-1 [47]. In early experiments aimed at assessing functional roles of cysteine residues in the receptor, ER mutated at Cys447 was found to have an affinity for binding estradiol similar to that of wild type ER. However, ER mutated at Cys447 was deficient in its ability to activate estradiol-induced transcription [48]. Transfection of ER mutated at Cys447 (C447S) or Cys530 (C530S) in COS-7 cells also resulted in altered sub-cellular localization of the mutant receptor as compared with that of wild-type ER [49]. As reported in the present work and in earlier studies [35,47], palmitoylation at Cys447 may be vitally important for ER membrane anchorage in tumor cells and in the mediation of estrogen-induced cell proliferation. ER plays a central role in regulating cell proliferation in several responsive cells, including human breast epithelium [3]. Estrogen action leading to growth involves activation of early response genes, cell cycle-regulatory gene products and growth factors [3–5]. However, the initial targets of estrogens leading to regulation of the expression of these molecules remain to be identified. Estradiol stimulation of breast and endothelial cells is associated with rapid activation of MAPK [17,18,50], as well as other important signaling cascades [8,11,13] that are postulated to promote the later activation of

380

R.J. Pietras et al. / Steroids 70 (2005) 372–381

transcription, DNA synthesis and growth [18,21]. It is likely that primary E2 ␤-induced activation of extranuclear ER will also affect subsequent hormonal interactions with nuclear ER to promote activation of transcription and cell proliferation. In similar fashion, estrogens appear to regulate, in part, differentiation of normal lung epithelium [51] and to stimulate growth of non-small cell lung tumors, especially adenocarcinomas [1,2]. Our studies suggest that activation of extranuclear forms of ER contribute to promotion of tumorigenesis of breast and non-small cell lung cancers. Moreover, membrane-associated ER appears to co-localize with HER receptors in these tumors. Treatment of breast and lung cancer cells with Faslodex is an effective anti-tumor therapy in vitro, but combination of Faslodex with HER receptor inhibitors elicits more profound growth-suppressive effects. Thus, dual targeting of ER and growth factor receptor signaling pathways may provide a more effective and prolonged anti-tumor effect [40,41]. The molecular details of cross-communication between estrogen and growth factor receptors are emerging [9,24,41], and membrane ER may be in a pivotal cellular location to enhance convergence among diverse signaling pathways. In promoting a hypothesis of estrogen action via both nuclear and extranuclear receptors, this work may lead to development of previously unsuspected antitumor therapies targeted to breast and non-small cell lung cancers.

Acknowledgments We thank AstraZeneca Pharmaceuticals for generously providing Iressa and Faslodex for use in these studies, and we acknowledge Dr. H. Garban and Dr. C.M. Szego for helpful discussions and comments. Support for this work was provided by US Army Breast Cancer Research Program (DAMD17-03-1-0381), UCLA Specialized Program of Research Excellence in Lung Cancer (NIH grant P50 CA90388), the Hamburger Fund of the Jonsson Cancer Center Foundation, a Howard Hughes Fellowship (O.W.) and the Stiles Program in Integrative Oncology.

References [1] Stabile LP, Davis AL, Gubish CT, Hopkins TM, Luketich JD, Christie N, et al. Human non-small cell lung tumors and cells derived from normal lung express both estrogen receptor alpha and beta and show biological responses to estrogen. Cancer Res 2002;62:2141–50. [2] Stabile L, Siegfried J. Estrogen receptor pathways in lung cancer. Curr Oncol Rep 2004;6:259–67. [3] Henderson BE, Ross R, Bernstein L. Estrogens as a cause of human cancer. Cancer Res 1988;48:246–53. [4] Evans RM. The steroid and thyroid hormone receptor superfamily. Science 1988;240:889–95. [5] Murdoch FE, Gorski J. The role of ligand in estrogen receptor regulation of gene expression. Mol Cell Endocrinol 1991;78:C103–8.

[6] McKenna NJ, O’Malley BW. An issue of tissues: divining the split personalities of selective estrogen receptor modulators. Nat Med 2000;6:960–2. [7] Aronica S, Kraus W, Katzenellenbogen BS. Estrogen action via the cAMP signaling pathway: stimulation of adenylate cyclase and cAMP-regulated gene transcription. Proc Natl Acad Sci U S A 1994;91:8517–21. [8] Levin ER. Cellular functions of the plasma membrane estrogen receptor. Trends Endocrinol Metab 1999;10:374–7. [9] Szego CM, Pietras RJ. Lysosomal functions in cellular activation: propagation of the actions of hormones and other effectors. Int Rev Cytol 1984;88:1–302. [10] Mendelsohn M, Karas R. The protective effects of estrogen on the cardiovascular system. N Engl J Med 1999;340:1801–11. [11] Watson CS, Gametchu B. Membrane-initiated steroid actions and the proteins that mediate them. Proc Soc Exp Biol Med 1999;220: 9–19. [12] Pietras RJ, Szego CM. Endometrial cell calcium and oestrogen action. Nature 1975;253:357–9. [13] Migliaccio A, Di Domenico M, Castoria G, de Falco A, Bontempo P, Nola E, et al. Tyrosine kinase/p21ras/MAP-kinase pathway activation by estradiol-receptor complex in MCF-7 cells. EMBO J 1996;15:1292–300. [14] Pietras R, Szego C. Specific binding sites for oestrogen at the outer surfaces of isolated endometrial cells. Nature 1977;265:69–72. [15] Pappas TC, Gametchu B, Watson CS. Membrane estrogen receptors identified by multiple antibody labeling and impeded-ligand binding. FASEB J 1995;9:404–10. [16] Chambliss K, Yuhanna I, Mineo C, Liu P, German Z, Sherman T, et al. Estrogen receptor alpha and endothelial nitric oxide synthase are organized into a functional signaling module in caveolae. Circ Res 2000;87:E44–52. [17] Russell KS, Haynes MP, Sinha D, Clerisme E, Bender JR. Human vascular endothelial cells contain membrane binding sites for estradiol, which mediate rapid intracellular signaling. Proc Natl Acad Sci U S A 2000;97:5930–5. [18] Razandi M, Pedram A, Greene G, Levin E. Cell membrane and nuclear estrogen receptors (ERs) originate from a single transcript: studies of ER␣ and ER␤ expressed in Chinese Hamster Ovary cells. Mol Endocrinol 1999;13:307–19. [19] Marquez D, Pietras RJ. Membrane-associated estrogen receptors and breast cancer. In: Watson CS, editor. The Identities of Membrane Steroid Receptors. Kluwer Academic Publishers; 2002. p. 1–10. [20] Nemere I, Farach-Carson MC. Membrane receptors for steroid hormones: a case for specific cell surface binding sites for vitamin D metabolites and estrogens. Biochem Biophys Res Commun 1998;248:443–9. [21] Marquez D, Pietras RJ. Membrane-associated binding sites for estrogen contribute to growth regulation in human breast cancer cells. Oncogene 2001;20:5420–30. [22] Bange J, Zwick E, Ullrich A. Molecular targets for breast cancer therapy and prevention. Nat Med 2001;7:548–52. [23] Slamon DJ, Godolphin W, Jones L, Holt J, Wong SG, Keith DE, et al. Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science 1989;244:707–11. [24] Pietras RJ, Arboleda J, Wongvipat N, Ramos L, Parker MG, Sliwkowski MX, et al. HER-2 tyrosine kinase pathway targets estrogen receptor and promotes hormone-independent growth in human breast cancer cells. Oncogene 1995;10:2435–46. [25] Pietras R, Szego C. Metabolic and proliferative responses to estrogen by hepatocytes selected for plasma membrane binding-sites specific for estradiol-17␤. J Cell Physiol 1979;98:145–59. [26] Pietras R, Szego C. Partial purification and characterization of oestrogen receptors in subfractions of hepatocyte plasma membranes. Biochem J 1980;191:743–60. [27] Song KS, Li Shengwen, Okamoto T, Quilliam LA, Sargiacomo M, Lisanti MP. Co-purification and direct interaction of ras with cave-

R.J. Pietras et al. / Steroids 70 (2005) 372–381

[28]

[29]

[30]

[31]

[32]

[33]

[34]

[35]

[36] [37] [38]

[39]

[40]

olin, an integral membrane protein of caveolae microdomains. J Biol Chem 1996;271:9690–7. Koleske AJ, Baltimore D, Lisanti MP. Reduction of caveolin and caveolae in oncogenically transformed cells. Proc Natl Acad Sci U S A 1995;92:1381–5. Petit AM, Rak J, Hung MC, Rockwell P, Goldstein N, Fendly B, et al. Neutralizing antibodies against epidermal growth factor and ErbB2/neu receptor tyrosine kinases down-regulate vascular endothelial growth factor production by tumor cells in vitro and in vivo: angiogenic implications for signal transduction therapy of solid tumors. Am J Pathol 1997;151:1523–30. Li D, Williams JI, Pietras RJ. Squalamine and cisplatin block angiogenesis and growth of human ovarian cancer cells with or without HER-2 gene overexpression. Oncogene 2002;21:2805–14. Dabbs D, Landreneau R, Liu Y, Raab S, Maley R, Tung M, et al. Detection of estrogen receptor by immunohistochemistry in pulmonary adenocarcinoma. Ann Thorac Surg 2002;73:403–6. Pezzi V, Mathis J, Rainey W, Carr B. Profiling transcript levels for steroidogenic enzymes in fetal tissues. J Steroid Biochem Mol Biol 2003;87:181–9. Kinoshita Y, Chen S. Induction of aromatase (CYP19) expression in breast cancer cells through a nongenomic action of estrogen receptor␣. Cancer Res 2003;63:3546–55. Marquez D, Lee J, Lin T, Pietras RJ. Epidermal growth factor receptor and tyrosine phosphorylation of estrogen receptor. Endocrine 2001;16:73–81. Acconcia F, Ascenzi P, Fabozzi G, Visca P, Marino M. Spalmitoylation modulates human estrogen receptor-alpha functions. Biochem Biophys Res Commun 2004;316:878–83. Pietras RJ, Szego CM. Estrogen receptors in uterine plasma membrane. J Steroid Biochem 1979;11:1471–83. Anderson RG. The caveolae membrane system. Annu Rev Biochem 1998;67:199–225. Li L, Haynes M, Bender JR. Plasma membrane localization and function of the estrogen receptor alpha variant (ER46) in human endothelial cells. Proc Natl Acad Sci U S A 2003;100:4807–12. Ciardiello F, De Vita F, Orditura M, Tortora G. The role of EGFR inhibitors in non-small cell lung cancer. Curr Opin Oncol 2004;16:130–5. Pietras RJ, Marquez D, Chen H-W, Ayala R, Ramos L, Slamon D. Improved anti-tumor therapy with Herceptin and Faslodex for

[41]

[42]

[43]

[44]

[45]

[46]

[47]

[48]

[49]

[50]

[51]

381

dual targeting of HER-2 and estrogen receptor signaling pathways in human breast cancers with overexpression of HER-2/neu gene. Breast Cancer Res Treat 2003;82(Suppl 1):12–3. Pietras RJ. Interactions between estrogen and growth factor receptors in human breast cancers and the tumor-associated vasculature. The Breast J 2003;9:361–73. Green S, Walter P, Greene G, Krust A, Goffin C, Jensen E, et al. Cloning of the human oestrogen receptor cDNA. J Steroid Biochem 1986;24:77–83. Flouriot G, Brand H, Denger S, Metivier R, Kos M, Reid G, et al. Identification of a new isoform of the human estrogen receptor-alpha (hER-alpha) that is encoded by distinct transcripts and that is able to repress hER-alpha activation function 1. EMBO J 2000;19:4688–700. Simoncini T, Hafezi-Moghadam A, Brazil DP, Ley K, Chin WW, Liao JK. Interaction of oestrogen receptor with the regulatory subunit of phosphatidylinositol-3-OH kinase. Nature 2000;407:538–41. Pietras RJ, Szego CM. Specific internalization of estrogen and binding to nuclear matrix in isolated uterine cells. Biochem Biophys Res Commun 1984;123:84–90. Song RX, Barnes C, Zhang Z, Bao Y, Kumar R, Santen R. The role of Shc and insulin-like growth factor 1 receptor in mediating the translocation of estrogen receptor alpha to the plasma membrane. Proc Natl Acad Sci U S A 2004;101:2076–81. Acconcia F, Bocedi A, Ascenzi P, Marino M. Does palmitoylation target estrogen receptors to plasma membrane caveolae? IUBMB Life 2003;55:33–5. Reese JC, Katzenellenbogen BS. Mutagenesis of cysteines in the hormone binding domain of the human estrogen receptor alterations in binding and transcriptional activation by covalently and reversibly attaching ligands. J Biol Chem 1991;266:10880–7. Neff S, Sadowski C, Miksicek RJ. Mutational analysis of cysteine residues within the hormone-binding domain of the human estrogen receptor identifies mutants that are defective in both DNA-binding and subcellular distribution. Mol Endocrinol 1994;8:1215–23. Improta-Brears T, Whorton A, Codazzi F, York J, Meyer T, McDonnell D. Estrogen-induced activation of mitogen-activated protein kinase requires mobilization of intracellular calcium. Proc Natl Acad Sci U S A 1999;96:4686–91. Patrone C, Cassel TN, Pettersson K, Piao YS, Cheng G, Ciana P, et al. Regulation of postnatal lung development and homeostasis by estrogen receptor beta. Mol Cell Biol 2003;23:8542–52.