![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Vol. 20, Issue 11, 2785-2795, June 1, 2009
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
SignalingFaculty of Medicine, Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia, Canada B3H1X5
Submitted November 21, 2008;
Revised March 12, 2009;
Accepted April 1, 2009
Monitoring Editor: William P. Tansey
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
(ER) signaling. We demonstrate for the first time that LKB1 binds to ER in the cell nucleus in which it is recruited to the promoter of ER-responsive genes. Furthermore, LKB1 catalytic activity enhances ER transactivation compared with LKB1 catalytically deficient mutants. The significance of our discovery is that we demonstrate for the first time a novel functional link between LKB1 and ER. Our discovery places LKB1 in a coactivator role for ER signaling, broadening the scientific scope of this tumor suppressor kinase and laying the groundwork for the use of LKB1 as a target for the development of new therapies against breast cancer. |
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
) in the development of breast carcinoma (Esteller et al., 2000; Shen et al., 2002; Fenton et al., 2006). Early evidence in the literature suggests that a loss of LKB1 expression leads to papillary breast carcinoma (Esteller et al., 2000). More recently, a study that evaluated the expression of LKB1 in 85 cases of breast cancers, the authors found that in a subset of high-grade in situ and invasive mammary carcinomas, the expression of LKB1 was completely lost as determined by immunohistochemistry (Fenton et al., 2006). In a separate study, 116 patients with confirmed breast carcinoma were evaluated for LKB1 protein expression, of which expression was low in approximately one third of the patients compared with control population of women. The authors of this study concluded that low expression of LKB1 correlated with higher histological grade, tumor size, and presence of lymph node status (Shen et al., 2002). Both studies conclude that LKB1 expression profile may serve as a prognostic marker for breast carcinoma; however, further investigation is warranted (Shen et al., 2002; Fenton et al., 2006). It should be noted that neither study determined whether LKB1 was mutated in the patients. These recent findings suggest a role for LKB1 in breast cancer however the molecular mechanisms by which this occurs is not fully understood.
Mutations in LKB1 are responsible for Peutz-Jeghers syndrome (PJS) (Hemminki et al., 1997; Ylikorkala et al., 1999; Marignani, 2005), an autosomal-dominant disease characterized by mucocutaneous pigmentation and benign gastrointestinal hamartomatous polyps (Hemminki, 1999). PJS patients are at a high risk of developing various cancers such as those of the gastrointestinal tract, breast, ovary, cervix, testis, lung, and pancreas (Avizienyte et al., 1999; Hruban et al., 1999; Wang et al., 1999; Esteller et al., 2000; Sanchez-Cespedes et al., 2002; Lim et al., 2003; Carretero et al., 2004; Nakanishi et al., 2004). PJS patients have a 54% greater risk of developing breast cancer than the general population (Giardiello et al., 2000), as well as a reported loss of heterozygosity of LKB1 in breast cancer (Bignell et al., 1998; Chen et al., 2000; Nakanishi et al., 2004; Yang et al., 2004). LKB1 contains a nuclear localization sequence that allows for LKB1 localization within the nuclear compartment of the cell (Nezu et al., 1999; Tiainen et al., 2002; Dorfman and Macara, 2008). Because a predominant number of mutations occur within the LKB1 kinase domain, the tumor suppressor function of LKB1 has been linked to its catalytic activity (Tiainen et al., 1999; Ylikorkala et al., 1999; Boudeau et al., 2003). More recently, we showed that LKB1 catalytic-deficient mutants exhibit oncogenic property, such that they are able to enhance the expression of the oncogene cyclin D1 in cells that lack expression cyclin D1-negative regulators p53 or p21WAF/CIP1 (Scott et al., 2007).
Aberrant ER signaling is a major contributor to the occurrence of ER-positive breast cancer (Sommer and Fuqua, 2001). ER is a member of the nuclear receptor (NR) family of transcription factors characterized by similar structural domains. There are two isoforms of ERs; ER (Walter et al., 1985) and ERβ (Kuiper et al., 1996). ER regulates genes such as cathepsin D, trefoil factor (pS2), c-myc, and cyclin D1 (reviewed in O'Lone et al., 2004). Whereas ER interacts with the estrogen response elements (EREs) in the promoters of the first two genes (classical pathway), it indirectly regulates the latter genes by interaction with DNA-bound transcription factors (nonclassical pathway). In general, upon stimulation with 17-β-estradiol (E2), ER dimerizes and translocate to the nucleus in which the dimer interacts with DNA and coregulator proteins to regulate gene transcription. Because alterations in LKB1 expression have been linked to breast cancer, we investigated the molecular mechanism by which LKB1 regulates ER-mediated signaling.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Glutathione Transferase (GST) Pull-Down Assay
ER expression plasmid was transcribed and translated (TnT) in vitro by using T7-coupled reticulocyte lysate and wheat germ extract (Promega, Madison, WI), in the presence of [35S]methionine. Labeled proteins were incubated with purified recombinant fusion proteins, GST-LKB1 and GST, on glutathione-Sepharose beads for 4 h at 4°C as described previously (Marignani et al., 2001). The samples were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and visualized by autoradiography.
Western Blotting and Antibodies
Cells were harvested in lysis buffer as described previously (Scott et al., 2007). Lysates were prepared for SDS-PAGE, and proteins were separated and transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA). ER (HC-20), LKB1 (Ley-37D), Lamin B1 (H-90), p300 (H-272), BRCA1 (D-9), and actin (I-19) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-ER (AER314) was from NeoMarkers (Fremont, CA). Brg1, MP-activated protein kinase (AMPK), and pAMPK(T172) antibodies were from Cell Signaling Technology (Danvers, MA).
Immunoprecipitation Assay
MCF7 cells were maintained in tissue culture dishes (10 cm) in phenol-free (PF)-DMEM (Sigma-Aldrich, St. Louis, MO) supplemented with 5% charcoal-stripped fetal bovine serum (csFBS) and transfected with FLAG-LKB1, ER expression plasmids. Cells were left untreated or treated with E2 (100 nM) for 45 min followed by lysis. LKB1 was immunoprecipitated (IP) from 500 µg of precleared total cell lysate (TCL) by using anti-LKB1.
Nuclear Cytoplasmic Fractionation
MCF7 cells were trypsinized followed by centrifugation (700 x g), cells were resuspended in hypotonic buffer (10 mM HEPES, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol, 0.1% NP-40, 1 mM NaF, and 4 µg/ml protease inhibitors), incubated on ice, and centrifuged (700 x g) to separate the cytoplasmic fraction. To obtain the nuclear fraction, cells were homogenized in nuclear lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.2% NP-40, 0.04% SDS, 0.1% sodium deoxycholate, 50 mM NaF, 1 mM Na3VO4, and 4 µg/ml protease inhibitors), and ultracentrifuged (125,000 x g) to remove cellular debris.
Reporter Assay
Reporter assays were performed using Dual Luciferase kit (Promega), and promoter activity was analyzed using an Auto Lumat Plus luminometer (Berthold Technologies, Bad Wildbad, Germany) as described previously (Scott et al., 2007). Briefly, MCF7 or G361 cells were transfected with the following plasmids: LKB1 (50 ng), D194A/R304W (50 ng) LKB1 NR mutants (50 ng), pRL-tk (0.5 ng), ERE-luc (gift from Dr. Myles Brown, Harvard University, Boston, MA)/cyclin D1-luc (50 ng), ER/ERβ (Addgene plasmid 11356) (10 ng)/androgen receptor (AR; 10 ng), glucocorticoid receptor (GR; 50 ng), mouse mammary tumor virus luciferase (MMTV-luc; 50 ng) (gifts from Dr. Steven Balk, Harvard University), p300 (50 ng) (Addgene plasmid 10718; Addgene, Cambridge, MA), BRCA1, and p53 (indicated concentrations). Six hours after transfection, phenol-free DMEM 5% serum-stripped FBS (ssFBS) was used to replace the media. Twenty-four hours after transfection, cells were treated with E2 (10 nM), 4-hydroxy tamoxifen (4-OHT; 1 uM), dexamethasone (100 nM), or testosterone (100 nM) (Sigma-Aldrich). Control cells were left untreated. Cells were washed in phosphate-buffered saline (PBS) and harvested after 24 h in passive lysis buffer according to manufacturer's protocol (Promega).
Small Interfering RNA (siRNA) Transfection
MCF7 cells were transfected with LKB1 siRNA (100 nM; Target sequences 1, GCUCUUACGGCAAGGUGAA; 2, UGAAAGGGAUGCUUGAGUA; and 3 GAAGAAGGAAAUUCAACUA; Dharmacon, RNA Technologies, Lafayette, CO) by using DharmaFECT transfection reagent.
Flow Cytometry.
MCF7 maintained in PF-DMEM 5% csFBS for 72 h before transfection with LKB1 siRNA and myr-green fluorescent protein (GFP)-cytomegalovirus (CMV) as described previously (Scott et al., 2007). Twenty-four hours after transfection, cells were incubated in PF-DMEM, 3% ssFBS for 24 h before treatment with vehicle or E2 (100 nM) for 24 h and then harvested and prepared for flow cytometry. Propidium iodide was purchased from Sigma-Aldrich. Fluorescence-activated cell sorting (FACS) analysis was conducted on an FACSCalibur flow cytometer (Beckman Coulter, Fullerton, CA) with data acquired using CellQuest software. Cell cycle profiles were determined using ModFit LT.
Immunofluorescence
Cells were prepared for fluorescent microscopy as described previously (Marignani and Carpenter, 2001). Cells were incubated in primary antibody for 1 h, followed by incubation with appropriate secondary antibodies: Oregon green and rhodopsin (Invitrogen). Nuclear staining was accomplished using 4,6-diamidino-2-phenylindole (DAPI) for 2 min. Images were obtained using an Eclipse TE 2000-E inverted research microscope (Nikon, Tokyo, Japan), mounted with a Q-Imaging charge-coupled device camera and acquired using the Simple PCI software.
In Vitro Kinase Assay
LKBtide assays were conducted as described previously (Marignani et al., 2007). Briefly, HEK293 cells were transfected with the following expression plasmids: LKB1pEBG-2T, R304WpEBG-2T, and pEBG-2T vector alone together with MO25-MYC-CMV and STRAD-FLAG-CMV. Phosphotransferase activity of LKB1 toward the LKBtide peptide (150 µM) (Millipore, Billerica, MA) was determined using protein complex (0.5 µg) in the presence of 0.1 mM [
-32P]ATP (PerkinElmer Life and Analytical Sciences, Boston, MA) at 30°C for 15 min. Kinase assays in vitro were performed as described previously (Marignani et al., 2001). ER, AMPK, p300, BRCA1, and Brg1 were immunoprecipitated with appropriate antibodies from MCF7 cells for 3 h followed by incubation with purified LKB1/Strad/Mo25 complexes in the presence of [-32P]ATP. Purified recombinant human AMPK was from Cell Signaling Technology. Phosphorylation was visualized by autoradiography.
RNA Isolation and Polymerase Chain Reaction (PCR)
RNA was isolated using TRIzol reagent (Invitrogen) and reverse transcribed as per manufacturer's protocol (Ambion, Austin, TX). PCR was conducted at 95°C for 15 min, 24 cycles of 95°C for 30 s, 55°C for 30 s, 72°C for 1.5 min, and a final step of 72°C for 5 min (iCycler; Bio-Rad Laboratories, Hercules, CA) on cDNA by using primers listed on Supplemental Table 1.
Chromatin Immunoprecipitation
G361 and MCF7 cells were grown in DMEM supplemented with 8% FBS or PF-DMEM with 5% charcoal-stripped FBS, respectively. MCF7 cells were left untreated or treated with 100 nM E2 for 45 min. G361 cells were transfected with ER, LKB1, and R304W expression plasmids. Chromatin immunoprecipitation (ChIP) assays were conducted as described previously (Scott et al., 2007). Immunoprecipitation was performed at 4°C overnight using anti-LKB1 or anti-mouse surface immunoglobulin G (mIgG) (control) antibodies. PCR was performed using primers for the promoters of pS2, c-myc, and cathepsin D as listed in Supplemental Table 1.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). LKB1 interacts with the helicase domain of Brg1, facilitating the ATPase activity of this chromatin remodeler (Marignani et al., 2001). Given that Brg1 is necessary for ER-mediated gene transactivation (DiRenzo et al., 2000) and LKB1 enhances Brg1-ATPase activity, we investigated whether LKB1 interacts with the nuclear hormone receptor ER. Here, we TnT ER expression plasmid in vitro in the presence of [35S]methionine by using rabbit reticulocyte lysate and wheat germ agglutinin followed by GST pull-down assays (Figure 1A). We observed that recombinant GST-LKB1 protein bound to ER, whereas control recombinant GST protein did not (Figure 1A). The interaction was confirmed in MCF7 cells that express both endogenous LKB1 and ER (Figure 1B). Here, endogenous LKB1 and ER were IP using anti-LKB1 and anti-ER antibodies followed by Western blot analysis to confirm coimmunoprecipitation (Figure 1C).
|
Interaction interaction, MCF7 cells were transfected with LKB1 and ER expression plasmids. Cells were treated with E2 (100 nM) for 45 min before harvesting. Nuclear and cytoplasmic fractions were prepared, followed by immunoprecipitation of equal amounts of protein from each fraction with anti-LKB1 antibody. We observed that the interaction between LKB1 and ER was restricted to the nuclear fraction (Figure 1D). To further verify these observations, immunofluorescence microscopy was conducted in MCF7 cells left untreated or treated with E2. We observe the nuclear colocalization of endogenous LKB1 and ER (Figure 1E). These results confirm our Western blot analysis that shows coimmunoprecipitation of LKB1 and ER occurs in the nuclear fraction (Figure 1D).
LKB1 Functions as a Coactivator of ER
Because LKB1 interacts with the ER in the nuclear compartment of the cell, we investigated the possibility that LKB1 played a role in ER-mediated gene transactivation by performing reporter assays. Because we were interested in whether the catalytic activity of LKB1 was required for this function, we used LKB1 null cells to facilitate in the interpretations of results in the absence of endogenous LKB1. In addition, previous work by others have shown that ectopic expression of ER in cells that express endogenous ER, such as MCF7, may lead to aberrant ER signaling because the endogenous protein may function as a dominant-negative (Bocquel et al., 1989; Webb et al., 1992). Hence, we surveyed known ER-negative breast cancer cell lines for LKB1 expression. In contrast to previous reports (Shen et al., 2002), we observed LKB1 expression in MDA-MB-435S melanoma-derived cell line and MDA-MB-231 breast cancer cells (Figure 1B). Therefore, we conducted ERE-luciferase reporter gene assays in G361 melanoma cell line that do not express either LKB1 or ER. G361 cells were transfected with LKB1 and ER expression plasmids pERE-luc and pRL-tk, (Figure 2A). In the presence of LKB1, ER-mediated transactivation was enhanced in response to E2 treatment, compared with ER alone and LKB1 alone. To determine whether increasing concentrations of LKB1 altered ER-mediated transactivation of a reporter gene, G361 and MCF7 cells were transfected with increasing concentrations of LKB1 (50, 100, and 500 ng), followed by ERE-luc reporter assays. In G361 cells, optimal transactivation was observed at 50 ng LKB1, as squelching of the reporter gene, as described previously by Lee et al. (2005), occurred at higher concentrations (Figure 2B). In contrast, overexpression of LKB1 in MCF7 cells did not significantly alter ER-mediated gene transactivation in response to E2 treatment (Figure 2D). In the presence of 4-OHT, a selective estrogen receptor modulator of ER, as expected, transactivation of the reporter was abrogated both in the presence of ER alone and ER plus LKB1 (Figure 2, C and E). Knockdown of LKB1 expression in MCF7 cells using three separate siRNA duplexes suppressed ER-mediated transcriptional activity (Figure 2F).
|
-mediated transactivation was attributed to the introduction of LKB1 and not to changes in ER expression levels due to the introduction of LKB1, G361 and MCF7 cells were transfected with increasing concentrations of LKB1 expression plasmid (0, 0.5, 1.5, and 2 µg), while simultaneously maintaining the concentration of ER expression plasmid (2 µg) constant in G361 cells. Increased expression of LKB1 did not alter the expression of ectopic (G361 cells) or endogenous ER (MCF7) as determined by reverse transcription (RT)-PCR and Western blot analysis (Figure 3A). Furthermore, knockdown of LKB1 expression in MCF7 cells by siRNA did not affect ER expression (Figure 3A, bottom right). In addition, the introduction of increasing concentrations of ER expression plasmid (0, 0.5, 1.5, and 2 µg), while simultaneously maintaining the concentration of LKB1 expression plasmid (2 µg) constant in G361 cells did not alter LKB1 expression of ectopic LKB1 in G361 or endogenous LKB1 in MCF7 cells (Figure 3B).
|
activity in the presence of a known ER coactivator protein p300 (Hanstein et al., 1996), we conducted ERE-luc reporter assays as described above. We first tested whether expression of p300 in G361 cells maintained coactivator function of ER-ERE-luc (Figure 4A, left). When p300 and LKB1 expression plasmids were introduced simultaneously, we observe a synergistic effect, suggesting cooperation between LKB1 and p300 in enhancing ER activity (Figure 4A, right). However, coactivator function of LKB1 in the presence of known corepressor proteins p53 and BRCA1 (Yu et al., 1997; Fan et al., 1999) was suppressed in a concentration-dependent manner (Figure 4, B and C).
|
|
-mediated transactivation of the reporter, reporter assays were conducted using LKB1 catalytic-deficient mutants (LKB1 mutants) D194A and R304W described previously (Marignani et al., 2007; Scott et al., 2007). Both mutants did not alter ER-mediated transactivation of ERE-luc compared with ER alone, thereby implying that LKB1 catalytic activity contributes to ER-mediated transactivation of ERE-luc (Figure 6A). In the presence of 4-OHT transactivation of the reporter was abrogated both in the presence of ER alone and ER plus LKB1 or mutants (Figure 6B).
|
Is Not a Substrate of LKB1-mediated transactivation (Figure 2), and LKB1 mutants attenuated ER-mediated transactivation (Figure 6), we tested whether ER was a substrate for LKB1. LKB1/Strad/MO25, R304W/Strad/MO25 complexes, and GST were prepared from HEK293 cells as described previously (Marignani et al., 2007; Scott et al., 2007) (Figure 7A) and tested for catalytic activity toward the LKBtide peptide (Figure 7A) and purified recombinant human AMPK protein followed by Western blot analysis using anti-phospho-AMPK antibody (pT172) (Figure 7B). Endogenous ER was immunoprecipitated from MCF7 cells (Figure 7C, top) followed by incubation with purified complexes in the presence of [-32P]ATP (Figure 7C, middle). To test whether known coregulator proteins of ER may be substrates for LKB1, BRCA1, p300, Brg1 (negative control) (Marignani et al., 2001), and AMPK (positive control) (Hawley et al., 2003) were immunoprecipitated from MCF-7 cells by using the appropriate antibodies (Figure 7D, top) followed by incubation with purified LKB1 complex (Figure 7A) followed by kinase assays in vitro (Figure 7D, bottom).
|
Signaling-mediated transcription of genes through the nonclassical pathway, whereby ER tethers to transcription factors that bind to the promoters of genes, reporter assays were conducted using cyclin D1 luciferase (CD1-luc) (Scott et al., 2007). We observed that ER enhances CD1-luc in the presence of E2 consistent with a previous report (Sabbah et al., 1999), whereas coexpression of LKB1 and ER followed by E2 treatment enhanced transactivation of cyclin D1-luc compared with LKB1 alone or ER alone (Figure 8A). Transactivation of cyclin D1-luc by LKB1 mutants did not differ from ER alone. Given that G361 cells are wild type for expression of cyclin D1 regulators p53 and p21WAF/CIP, we did not expect LKB1 mutants to function as described previously in DLD1p21–/–p53–/– colorectal cancer (Scott et al., 2007).
|
-mediated transcription in G361 cells (Figure 2A), we tested whether LKB1 could alter ER-ligand dependent cell cycle progression (Ikeda and Inoue, 2004). For these studies, LKB1 expression in MCF7 cells was ablated using siLKB1#2 (Figure 2F), followed by treatment with E2 (100 nM) for 24 h, and then flow cytometry to evaluate the cell cycle profile of asynchronous cells. As expected, abrogation of LKB1 expression resulted in an overall increase in the percentage of cells found in S phase for untreated cells with a corresponding reduction in the percentage of cells in G0/G1 phase (Figure 8B), agreeing with previous work that shows the introduction of LKB1 into LKB1 null G361 cells arrests cells in G1 phase of cell cycle (Tiainen et al., 1999). Compared with control E2-treated cells, we did not observe a difference in the percentage of cells in S phase from siLKB1 cells. Similar cell cycle profiles were observed for siLKB1#1 and #3 (data not shown).
LKB1 Is Recruited to the Promoters of ER-regulated Genes
Because LKB1 enhances ER transcriptional activity, we examined whether endogenous LKB1 interacts with the promoters of ER activated genes pS2, cathepsin D (Augereau et al., 1994), and c-myc (Dubik and Shiu, 1988) (Figure 9A). We conducted ChIP in MCF7 cells that were either left untreated or were treated with E2. When endogenous LKB1 was immunoprecipitated using anti-LKB1 antibody, we observed recruitment of LKB1 to the pS2, cathepsin D, and c-myc promoters in both untreated and E2-treated cells, compared with corresponding control regions (cr) (Figure 9B). To determine whether catalytic-deficient mutants were recruited to these promoters, G361 cells were transfected with ER, LKB1, and R304W expression plasmids or vector control followed by ChIP. Both LKB1 and R304W were recruited to all three gene promoters, compared with the vector control (Figure 9C).
|
-responsive genes, we asked whether LKB1 altered the expression of pS2, cathepsin D, and c-myc. To test this, we depleted the expression of LKB1 in MCF7 cells by siRNALKB1#2 (Figure 2F), followed by RT-PCR (Supplemental Table 1). In cells depleted of 
75% of LKB1 (Figure 9D, left), we observed a >50% reduction in pS2, cathepsin D, and c-myc expression in response to E2 treatment, compared with cells not depleted of LKB1 expression (Figure 9D, right; RT-PCR expressed relative to glyceraldehyde-3-phosphate dehydrogenase [GAPDH]). |
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
signaling. We demonstrate that LKB1 binds to ER in a ligand-independent manner (Figure 1A) in the nuclear compartment of the cell (Figure 1, B and C). A function of this interaction is that LKB1 enhances ER-mediated transactivation in a ligand-dependent manner (Figure 2, A–C), because abrogation of LKB1 expression by siRNA resulted in a suppression of ER activity (Figure 2F). We confirmed by mRNA and protein analysis that the introduction of LKB1 did not alter the overall expression of ER (Figure 3); thus, the observed enhancement in ER activity was not due to increased ER levels but due to the interaction of LKB1 with ER. Equally important, the introduction of ER into cells did not alter the overall expression of LKB1 (Figure 3). Our discovery is consistent with a previous report (Dutertre and Smith, 2003) that demonstrates coactivator proteins such as p160 interacting with ER independently of ligand binding; however, p160-enhanced ER function was dependent of ligand (Dutertre and Smith, 2003). Furthermore, we show that LKB1 did not alter the transcriptional activity of ERβ, AR, or GR (Figure 5), suggesting that LKB1 is selective for ER.
Because coregulator proteins function to alter ER activity, we investigated the effect of LKB1 in the presence of known ER coregulatory proteins. In the presence of ER coactivator p300 (Hanstein et al., 1996), LKB1 synergizes ER-mediated transactivation of ERE-luc (Figure 4A). Synergy between ER coactivator proteins has been described, such as GRIP1, CARM1, and p300 (Chen et al., 2000), and between SRC1 and CBP (Smith et al., 1996). In the presence of ER corepressor proteins p53 and BRCA1 (Yu et al., 1997; Fan et al., 1999), the enhanced transactivation we observe with LKB1 and ER alone is antagonized when either of the ER corepressor proteins is introduced, suggesting that these corepressor proteins compete with LKB1 for ER (Figure 4, B and C). Previously, our laboratory discovered a functional interaction between LKB1 and Brg1, whereby the interaction enhanced Brg1 ATPase activity, necessary for chromatin remodeling and therefore gene transactivation (Marignani et al., 2001). Furthermore, LKB1 induces p21WAF/CIP1 (Tiainen et al., 2002), which in turn enhances ER activity (Fritah et al., 2005). Coregulator proteins function by participating in chromatin remodeling or assist in the assembly of other coregulatory proteins. Because LKB1 exhibits both these functions, it is likely that LKB1 may be part of coactivator complexes with functionally diverse roles that are dependent on the availability of other coregulatory proteins (Szapary et al., 1999).
Catalytically deficient LKB1 mutants D194A and R304W failed to enhance ER-mediated transactivation, indicating that LKB1 catalytic activity is necessary for this function (Figure 6). Thus, we tested whether LKB1 phosphorylated ER because previous reports suggest phosphorylation of ER enhances transcription activity by several mechanisms (Weigel and Moore, 2007). From kinase assays, we determined that LKB1 does not phosphorylate ER (Figure 7B). Others have reported that alteration in the phosphorylation state of coregulator proteins modulate nuclear receptor activity (Frigo et al., 2006). It is conceivable that LKB1 phosphorylates an ER coregulator protein, thereby enhancing ER activity. A previous study suggested that a novel nuclear protein kinase that interacts with AR enhances AR activity by phosphorylating coregulatory proteins and not AR (Moilanen et al., 1998). We demonstrate that LKB1 does not phosphorylate ER coregulatory proteins p300 and BRCA1, compared with Brg1 (negative control) (Marignani et al., 2001) and AMPK (positive control) (Lizcano et al., 2004; Sakamoto et al., 2004) (Figure 7C), indicating that a plausible substrate of LKB1 may be a coregulatory protein of hormone receptor signaling other than the coregulatory proteins we tested in our system.
In addition to the classical pathway of gene regulation, ER regulates genes such as cyclin D1 by an alternative mechanism (Sabbah et al., 1999; Castro-Rivera et al., 2001), that is, the nonclassical pathway (Nilsson et al., 2001; Sommer and Fuqua, 2001), whereby ER associates with transcription factors that bind to DNA. Increases in cyclin D1 expression leads to enhanced cellular proliferation, promoting atypical lobular-alveolar development and contributing to the formation of mammary adenocarcinomas (Wang et al., 1994). Clinical studies show >50% of breast tumors express elevated levels of cyclin D1 (Barnes and Gillett, 1998) and display elevated levels of ER activity (Shoker et al., 2001). Previously, we showed recruitment of LKB1 to the promoter of cyclin D1 in cells lacking the expression of cyclin D1 negative regulators p53 and p21WAF1/CIP1 (Scott et al., 2007). Our current results demonstrate that LKB1 enhances ER-mediated transactivation of the cyclin D1 promoter in G361 cells (Figure 8A). Hence, it is possible that recruitment of LKB1 to the promoter of cyclin D1 facilitates the protein–DNA or protein–protein interactions necessary for ER activity. Previous work by others show that ER associates with the p160-CBP coactivators, which are in turn recruited by jun/fos to the AP-1 site (Kushner et al., 2000). Perhaps LKB1 may function in a similar manner by recruiting ER to the cyclin D1 promoter, along with additional coactivator proteins. Interestingly, ablation of LKB1 expression in MCF7 cells by siRNA resulted in an increase in the percentage of cells transitions to S phase of cell cycle compared with control untreated cells, as measure by flow cytometry (Figure 8B). In response to E2 treatment, however, the percentage of cells in S phase of cell cycle did not differ between control and siLKB1 cells (Figure 8B). These data are supported by recent studies conducted in three-dimensional acini cultures of 4-OHT–inducible MCF10A-MycERtm cells. In this study, the reduction of LKB1 expression by short hairpin RNA led to an increase in c-myc–mediated cell proliferation as measured by Ki-67 labeling of acini (Partanen et al., 2007). The authors of this study correlate reduced LKB1 expression with disruption of quiescent 4-OHT–induced MCF10A-MycERtm cell architecture for up to 10 d, confirming cell polarity plays a protective role against apoptosis (Partanen et al., 2007). Together, these data suggest that the tumor suppressor activity of LKB1 is likely distinct from its hormone receptor coactivator function and further highlight the multitasking and therefore multifunctional activities of LKB1 (Marignani, 2005).
We show that LKB1 is recruited to the promoters of ER-regulated genes independently of E2 treatment, specifically cathepsin D, pS2/TFF1, and c-Myc (Shang and Brown, 2002; Métivier et al., 2003) (Figure 9, B and C). Furthermore, we confirmed by siRNA that LKB1 contributes to the expression of cathepsin D, pS2/TFF1, and c-Myc because abrogation of LKB1 expression attenuated expression of pS2 in the absence of ligand and expression of all three ER targets—pS2, cathepsin D, and c-myc—in the presence of E2 ligand (Figure 9D). In addition to wild-type LKB1, ChIP assays confirm the recruitment of catalytic deficient mutant R304W to the promoters of cathepsin D, pS2/TFF1, and c-Myc, supporting our position that a plausible substrate of LKB1 may be a coregulatory protein of hormone receptor signaling. Overall, these results suggest that LKB1 through its association with ER is involved in the expression of downstream targets of ER and that LKB1 tumor suppressor function, that is, preventing cells from transitioning from G1 phase to S phase of cell cycle (Tiainen et al., 1999) is distinct from its ER coactivator function.
Our discovery defines a new function for the tumor suppressor serine threonine kinase LKB1, as a coactivator of ER. It is paradoxical that LKB1, a tumor suppressor, enhances ER activity, which would likely contribute to breast cancer risk. This said, there are reports in the literature for tumor suppressor proteins functioning as nuclear receptor coactivators. Briefly, despite being the archetypical tumor suppressor, retinoblastoma (Rb) also has the distinction of being a coactivator of nuclear hormone receptors ER, AR, progesterone receptor (PR), and GR (Singh et al., 1995; Lu and Danielsen, 1998; Yeh et al., 1998; Batsche et al., 2005). Interestingly, Rb is the first example of a tumor suppressor to function in a role as an activator of nuclear hormone receptor signaling. The regulation of the GR by Rb is mediated through Rb association with SWI/SNF chromatin remodeling complex, specifically Brg1(Singh et al., 1995), whereas up-regulation of AR-mediated transcription by Rb is independent of Rb–Brg1 interaction (Lu and Danielsen, 1998). A second tumor suppressor, p21WAF/CIP1, was found to bind to ER, enhancing ER-dependent transcription of target genes by forming a ternary complex composed of p21WAF/CIP1, CREB-binding protein, and ER (Fritah et al., 2005). The tumor suppressor tuberous sclerosis 2 (TSC2) has also been shown to function as a coactivator of nuclear hormone receptors, specifically the retinoid X receptors, peroxisome proliferator receptor (PPAR) , and vitamin D receptor (VDR) (Henry et al., 1998). In these studies, TSC2 weakly associated with the receptors and up-regulated both PPAR- and VDR-mediated transactivation. However, TSC2 functioned as a corepressor of GR-mediated transcription (Henry et al., 1998). Finally, in addition to binding to LKB1 (Marignani et al., 2001), Brg1 is reported to bind to nuclear hormone receptors including the ER, AR, GR, and PR (Trotter and Archer, 2008). Interestingly, when bound to ER, AR, and PR, Brg1 functions as a coactivator protein and when bound to GR, Brg1 functions as a corepressor protein for this receptor (Trotter and Archer, 2008).
In summary, we demonstrate for the first time a functional link between LKB1 and the hormone receptor ER. Our discovery now places LKB1 in a coactivator role for ER signaling, broadening the scientific scope of this tumor suppressor kinase and laying the groundwork for further investigations into the role of LKB1 in breast disease.
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
Address correspondence to: Paola A. Marignani (pmarigna{at}dal.ca).
Abbreviations used: 4-OHT, 4-hydroxy tamoxifen; E2, 17β-estradiol; ER, estrogen receptor ; ERE, estrogen response element; PJS, Peutz-Jeghers syndrome; TnT, transcribed and translated.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Avizienyte, E. et al. (1999). LKB1 somatic mutations in sporadic tumors. Am. J. Pathol 154, 677–681.
Barnes, D., and Gillett, C. (1998). Cyclin D1 in breast cancer. Breast Cancer Res. Treat 52, 1–15.[CrossRef][Medline]
Batsche, E., Desroches, J., Bilodeau, S., Gauthier, Y., and Drouin, J. (2005). Rb enhances p160/SRC coactivator-dependent activity of nuclear receptors and hormone responsiveness. J. Biol. Chem 280, 19746–19756.
Bignell, G. R., Barfoot, R., Seal, S., Collins, N., Warren, W., and Stratton, M. R. (1998). Low frequency of somatic mutations in the LKB1/Peutz-Jeghers syndrome gene in sporadic breast cancer. Cancer Res 58, 1384–1386.
Bocquel, M. T., Kumar, V., Stricker, C., Chambon, P., and Gronemeyer, H. (1989). The contribution of the N- and C-terminal regions of steroid receptors to activation of transcription is both receptor and cell-specific. Nucleic Acids Res 17, 2581–2595.
Boudeau, J., Kieloch, A., Alessi, D. R., Stella, A., Guanti, G., and Resta, N. (2003). Functional analysis of LKB1/STK11 mutants and two aberrant isoforms found in Peutz-Jeghers syndrome patients. Hum. Mutat 21, 172.[CrossRef][Medline]
Carretero, J., Medina, P. P., Pio, R., Montuenga, L. M., and Sanchez-Cespedes, M. (2004). Novel and natural knockout lung cancer cell lines for the LKB1/STK11 tumor suppressor gene. Oncogene 23, 4037–4040.[CrossRef][Medline]
Castro-Rivera, E, Samudio, I., and Safe, S. (2001). Estrogen regulation of cyclin D1 gene expression in ZR-75 breast cancer cells involves multiple enhancer elements. J. Biol. Chem 276, 30853–30861.
Chen, D., Huang, S.-M., and Stallcup, M. R. (2000). Synergistic, p160 coactivator-dependent enhancement of estrogen receptor function by CARM1 and p300. J. Biol. Chem 275, 40810–40816.
DiRenzo, J., Shang, Y., Phelan, M., Sif, S., Myers, M., Kingston, R., and Brown, M. (2000). BRG-1 is recruited to estrogen-responsive promoters and cooperates with factors involved in histone acetylation. Mol. Cell Biol 20, 7541–7549.
Dorfman, J., and Macara, I. G. (2008). STRAD{alpha} Regulates LKB1 Localization by Blocking Access to Importin-{alpha}, and by Association with Crm1 and Exportin-7. Mol. Biol. Cell 19, 1614–1626.
Dubik, D., and Shiu, R. P. (1988). Transcriptional regulation of c-myc oncogene expression by estrogen in hormone-responsive human breast cancer cells. J. Biol. Chem 263, 12705–12708.
Dutertre, M., and Smith, C. L. (2003). Ligand-independent interactions of p160/steroid receptor coactivators and CREB-binding protein (CBP) with estrogen receptor-{alpha}: regulation by phosphorylation sites in the A/B region depends on other receptor domains. Mol. Endocrinol 17, 1296–1314.
Esteller, M., Avizienyte, E., Corn, P. G., Lothe, R. A., Baylin, S. B., Aaltonen, L. A., and Herman, J. G. (2000). Epigenetic inactivation of LKB1 in primary tumors associated with the Peutz-Jeghers syndrome. Oncogene 19, 164–168.[CrossRef][Medline]
Fan, S., Wang, J. A., Yuan, R., Ma, Y., Meng, Q., Erdos, M. R., Pestell, R. G., Yuan, F., Auborn, K. J., Goldberg, I. D., and Rosen, E. M. (1999). BRCA1 inhibition of estrogen receptor signaling in transfected cells. Science 284, 1354–1356.
Fenton, H., Carlile, B., Montgomery, E., Carraway, H., Herman, J., Sahin, F., Su, G., and Argani, P. (2006). LKB1 protein expression in human breast cancer. Appl. Immunohistochem. Mol. Morphol 14, 146–153.[CrossRef][Medline]
Frigo, D. E. et al. (2006). p38 Mitogen-activated protein kinase stimulates estrogen-mediated transcription and proliferation through the phosphorylation and potentiation of the p160 coactivator glucocorticoid receptor-interacting protein 1. Mol. Endocrinol 20, 971–983.
Fritah, A., Saucier, C., Mester, J., Redeuilh, G., and Sabbah, M. (2005). p21WAF1/CIP1 selectively controls the transcriptional activity of estrogen receptor {alpha}. Mol. Cell Biol 25, 2419–2430.
Giardiello, F. M., Brensinger, J. D., Tersmette, A. C., Goodman, S. N., Petersen, G. M., Booker, S. V., Cruz-Correa, M., and Offerhaus, J. A. (2000). Very high risk of cancer in familial Peutz-Jeghers syndrome. Gastroenterology 119, 1447–1453.[CrossRef][Medline]
Hanstein, B., Eckner, R., DiRenzo, J., Halachmi, S., Liu, H., Searcy, B., Kurokawa, R., and Brown, M. (1996). p300 is a component of an estrogen receptor coactivator*complex. Proc. Natl. Acad. Sci. USA 93, 11540–11545.
Hawley, S. A., Boudeau, J., Reid, J. L., Mustard, K. J., Udd, L., Makela, T. P., Alessi, D. R., and Hardie, D. G. (2003). Complexes between the LKB1 tumor suppressor, STRADalpha/beta and MO25alpha/beta are upstream kinases in the AMP-activated protein kinase cascade. J. Biol 2, 28.[CrossRef][Medline]
Hemminki, A. (1999). The molecular basis and clinical aspects of Peutz-Jeghers syndrome. Cell Mol. Life Sci 55, 735–750.[CrossRef][Medline]
Hemminki, A. et al. (1997). Localization of a susceptibility locus for Peutz-Jeghers syndrome to 19p using comparative genomic hybridization and targeted linkage analysis. Nat. Genet 15, 87–90.[CrossRef][Medline]
Henry, K. W., Yuan, X., Koszewski, N. J., Onda, H., Kwiatkowski, D. J., and Noonan, D. J. (1998). Tuberous sclerosis gene 2 product modulates transcription mediated by steroid hormone receptor family members. J. Biol. Chem 273, 20535–20539.
Hruban, R. H., Petersen, G. M., Goggins, M., Tersmette, A. C., Offerhaus, G. J., Falatko, F., Yeo, C. J., and Kern, S. E. (1999). Familial pancreatic cancer. Ann. Oncol 10, (suppl 4), 69–73.
Ikeda, K., and Inoue, S. (2004). Estrogen receptors and their downstream targets in cancer. Arch. Histol. Cytol 67, 435–442.[CrossRef][Medline]
Kuiper, G. G., Enmark, E., Pelto-Huikkom, M, Nilssonm, S, and Gustafsson, J. A. (1996). Cloning of a novel receptor expressed in rat prostate and ovary. Proc. Natl. Acad. Sci. USA 93, 5925–5930.
Kushner, P. J., Agard, D. A., Greene, G. L., Scanlan, T. S., Shiau, A. K., Uht, R. M., and Webb, P. (2000). Estrogen receptor pathways to AP-1. J. Steroid Biochem. Mol. Biol 74, 311–317.[CrossRef][Medline]
Lee, J. E., Kim, K., Sacchettini, J. C., Smith, C. V., and Safe, S. (2005). DRIP150 Coactivation of estrogen receptor {alpha} in ZR-75 breast cancer cells is independent of LXXLL motifs. J. Biol. Chem 280, 8819–8830.
Lim, W. et al. (2003). Further observations on LKB1/STK11 status and cancer risk in Peutz-Jeghers syndrome. Br. J. Cancer 89, 308–313.[CrossRef][Medline]
Lizcano, J. M. et al. (2004). LKB1 is a master kinase that activates 13 kinases of the AMPK subfamily, including MARK/PAR-1. EMBO J 23, 833–843.[CrossRef][Medline]
Lu, J., and Danielsen, M. (1998). Differential regulation of androgen and glucocorticoid receptors by retinoblastoma protein. J. Biol. Chem 273, 31528–31533.
Marignani, P. A. (2005). LKB1, the multitasking tumour suppressor kinase. J. Clin. Pathol 58, 15–19.
Marignani, P. A., and Carpenter, C. L. (2001). Vav2 is required for cell spreading. J. Cell Biol 154, 177–186.
Marignani, P. A., Kanai, F., and Carpenter, C. L. (2001). LKB1 associates with Brg1 and is necessary for Brg1-induced growth arrest. J. Biol. Chem 276, 32415–32418.
Marignani, P. A., Scott, K. D., Bagnulo, R., Cannone, D., Ferrari, E., Stella, A., Guanti, G., Simone, C., and Resta, N. (2007). Novel splice isoforms of STRADalpha differentially affect LKB1 activity, complex assembly and subcellular localization. Cancer Biol. Ther 6, 1627–1631.[Medline]
Métivier, R., Penot, G., Hübner, M., Reid, G., Brand, H., Kos, M., and Gannon, F. (2003). Estrogen receptor- directs ordered, cyclical, and combinatorial recruitment of cofactors on a natural target promoter. Cell 115, 751–763.[CrossRef][Medline]
Moilanen, A.-M., Karvonen, U., Poukka, H., Janne, O. A., and Palvimo, J. J. (1998). Activation of androgen receptor function by a novel nuclear protein kinase. Mol. Biol. Cell 9, 2527–2543.
Nakanishi, C., Yamaguchi, T., Iijima, T., Saji, S., Toi, M., Mori, T., and Miyaki, M. (2004). Germline mutation of the LKB1/STK11 gene with loss of the normal allele in an aggressive breast cancer of Peutz-Jeghers syndrome. Oncology 67, 476–479.[CrossRef][Medline]
Nezu, J., Oku, A., and Shimane, M. (1999). Loss of cytoplasmic retention ability of mutant LKB1 found in Peutz-Jeghers syndrome patients. Biochem. Biophys. Res. Commun 261, 750–755.[CrossRef][Medline]
Nilsson, S., Makela, S., Treuter, E., Tujague, M., Thomsen, J., Andersson, G., Enmark, E., Pettersson, K., Warner, M., and Gustafsson, J. A. (2001). Mechanisms of estrogen action. Physiol. Rev 81, 1535–1565.
O'Lone, R., Frith, M. C., Karlsson, E. K., and Hansen, U. (2004). Genomic targets of nuclear estrogen receptors. Mol. Endocrinol 18, 1859–1875.
Partanen, J. I., Nieminen, A. I., Makela, T. P., and Klefstrom, J. (2007). Suppression of oncogenic properties of c-Myc by LKB1-controlled epithelial organization. Proc. Natl. Acad. Sci. USA 104, 14694–14699.
Sabbah, M., Courilleau, D., Mester, J., and Redeuilh, G. (1999). Estrogen induction of the cyclin D1 promoter: involvement of a cAMP response-like element. Proc. Natl. Acad. Sci. USA 96, 11217–11222.
Sakamoto, K., Goransson, O., Hardie, D. G., and Alessi, D. R. (2004). Activity of LKB1 and AMPK-related kinases in skeletal muscle: effects of contraction, phenformin, and AICAR. Am. J. Physiol. Endocrinol. Metab 287, E310–E317.
Sanchez-Cespedes, M., Parrella, P., Esteller, M., Nomoto, S., Trink, B., Engles, J. M., Westra, W. H., Herman, J. G., and Sidransky, D. (2002). Inactivation of LKB1/STK11 is a common event in adenocarcinomas of the lung. Cancer Res 62, 3659–3662.
Scott, K. D., Nath-Sain, S., Agnew, M. D., and Marignani, P. A. (2007). LKB1 Catalytically Deficient Mutants Enhance Cyclin D1 Expression. Cancer Res 67, 5622–5627.
Shang, Y., and Brown, M. (2002). Molecular determinants for the tissue specificity of SERMs. Science 295, 2465–2468.
Shen, Z., Wen, X. F., Lan, F., Shen, Z. Z., and Shao, Z. M. (2002). The tumor suppressor gene LKB1 is associated with prognosis in human breast carcinoma. Clin. Cancer Res 8, 2085–2090.
Shoker, B. S., Jarvis, C., Davies, M.P.A., Iqbal, M., Sibson, D. R., and Sloane, J. P. (2001). Immunodetectable cyclin D1is associated with oestrogen receptor but not Ki67 in normal, cancerous and precancerous breast lesions. Br. J. Cancer 84, 1064–1069.[CrossRef][Medline]
Singh, P., Coe, J., and Hong, W. (1995). A role for retinoblastoma protein in potentiating transcriptional activation by the glucocorticoid receptor. Nature 374, 562–565.[CrossRef][Medline]
Smith, C., Oñate, S., Tsai, M., and O'Malley, B. (1996). CREB binding protein acts synergistically with steroid receptor coactivator-1 to enhance steroid receptor-dependent transcription. Proc. Natl. Acad. Sci. USA 93, 8884–8888.
Sommer, S., and Fuqua, S. A. (2001). Estrogen receptor and breast cancer. Semin. Cancer Biol 11, 339–352.[CrossRef][Medline]
Szapary, D., Huang, Y., and Simons, S. S., Jr. (1999). Opposing effects of corepressor and coactivators in determining the dose-response curve of agonists, and residual agonist activity of antagonists, for glucocorticoid receptor-regulated gene expression. Mol. Endocrinol 13, 2108–2121.
Tiainen, M., Vaahtomeri, K., Ylikorkala, A., and Makela, T. P. (2002). Growth arrest by the LKB1 tumor suppressor: induction of p21(WAF1/CIP1). Hum. Mol. Genet 11, 1497–1504.
Tiainen, M., Ylikorkala, A., and Makela, T. P. (1999). Growth suppression by Lkb1 is mediated by a G(1) cell cycle arrest. Proc. Natl. Acad. Sci. USA 96, 9248–9251.
Trotter, K., and Archer, T. K. (2008). The BRG1 transcriptional coregulator. Nucl. Recept. Signal 6, 1–12.
Walter, P., Green, S., Greene, G., Krust, A., Bornert, J. M., Jeltsch, J. M., Staub, A., Jensen, E., Scrace, G., and Waterfield, M. (1985). Cloning of the human estrogen receptor cDNA. Proc. Natl. Acad. Sci. USA 82, 7889–7893.
Wang, T. C., Cardiff, R. D., Zukerberg, L., Lees, E., Arnold, A., and Schmidt, E. V. (1994). Mammary hyperplasia and carcinoma in MMTV-cyclin D1 transgenic mice. Nature 369, 669–671.[CrossRef][Medline]
Wang, Z. J., Ellis, I., Zauber, P., Iwama, T., Marchese, C., Talbot, I., Xue, W. H., Yan, Z. Y., and Tomlinson, I. (1999). Allelic imbalance at the LKB1 (STK11) locus in tumours from patients with Peutz-Jeghers' syndrome provides evidence for a hamartoma-(adenoma)-carcinoma sequence. J. Pathol 188, 9–13.[CrossRef][Medline]
Webb, P., Lopez, G. N., Greene, G. L., Baxter, J. D., and Kushner, P. J. (1992). The limits of the cellular capacity to mediate an estrogen response. Mol. Endocrinol 6, 157–167.
Weigel, N., and Moore, N. L. (2007). Kinases and protein phosphorylation as regulators of steroid hormone action. Nucl. Recept. Signal 5, e005.[Medline]
Yang, T. L., Su, Y. R., Huang, C. S., Yu, J. C., Lo, Y. L., Wu, P. E., and Shen, C. Y. (2004). High-resolution 19p13.2–13.3 allelotyping of breast carcinomas demonstrates frequent loss of heterozygosity. Genes Chromosomes Cancer 41, 250–256.[CrossRef][Medline]
Yeh, S., Miyamoto, H., Nishimura, K., Kang, H., Ludlow, J., Hsiao, P., Wang, C., Su, C., and Chang, C. (1998). Retinoblastoma, a tumor suppressor, is a coactivator for the androgen receptor in human prostate cancer DU145 cells. Biochem. Biophys. Res. Commun 248, 361–367.[CrossRef][Medline]
Ylikorkala, A. et al. (1999). Mutations and impaired function of LKB1 in familial and non-familial Peutz-Jeghers syndrome and a sporadic testicular cancer. Hum. Mol. Genet 8, 45–51.
Yu, C.-L., Driggers, P., Barrera-Hernandez, G., Nunez, S. B., Segars, J. H., and Cheng, S.-Y. (1997). The tumor suppressor p53 is a negative regulator of estrogen receptor signaling pathways. Biochem. Biophys. Res. Commun 239, 617–620.[CrossRef][Medline]
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
