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Down regulation of PSA by C/EBPα is associated with loss of AR expression and inhibition of PSA promoter activity in the LNCaP cell Line
© Yin et al; licensee BioMed Central Ltd. 2006
Received: 06 February 2006
Accepted: 14 June 2006
Published: 14 June 2006
C/EBPα is a transcription factor essential for terminal differentiation of several cell types. It has not known if C/EBPα protein is expressed and functions in the prostate gland.
The presence of C/EBPα in normal and cancerous prostate epithelium was examined by immunochemistry. Over expression of C/EBPα in LNCaP cells was conducted with retrovirus-mediated transduction. PSA expression was examined by RT-PCR and western blot and PSA promoter activity by luciferase reporter assay.
In normal prostate C/EBPα was expressed in the basal layer of the epithelium. In prostate cancer C/EBPα was detected at low levels throughout the cancers and in advanced prostate cancer C/EBPα expression was associated with decreased expression of AR and PSA. Overexpression of C/EBPα inhibited epigenetically PSA expression and was accompanied by the loss of expression of AR. Transient increase of C/EBPα inhibited the PSA promoter/enhancer activity independently of expression of AR.
In LNCaP cells C/EBPα over expression inhibits expression of PSA by AR -dependent and independent mechanisms and by extinguishing AR expression provides a model for hormonal independent cell growth.
The CCAAT/enhancer binding proteins (C/EBPs) are a family of transcription factors that regulate cellular differentiation in a variety of tissues . The C/EBP transcription factors consist of an activation domain, a DNA-binding basic region, and a leucine-rich dimerization domain. The structure of C/EBPα and its isoforms has been well described. The predominant isoform is a 42 kDa protein with three transactivation domains in the N-terminal portion of the protein. A second isoform is a 30-kDa protein formed from a downstream initiation site that alters the transactivation domain. Both isoforms contain the same dimerization and DNA-binding domains and are influential in controlling terminal differentiation and growth of a variety of cells [2, 3]. C/EBPα is expressed in numerous tissues with the highest expression in the placenta, liver, lung, skeletal muscle, pancreas, small intestine, colon and peripheral blood leukocytes. The C/EBPs in general are important regulators of cellular differentiation and determinants of terminal cell function. C/EBPα in particular is required for myeloid differentiation; mice deficient in C/EBPα have an early block in granulocyte maturation with a lack of expression of the G-CSF receptor[4, 5]. The role of C/EBPα in hematopoietic differentiation is complex as at the same time that C/EBPα induces granulocyte differentiation while erythroid differentiation is inhibited . C/EBPα plays a role in hepatocytes and adipocytes mediating cell cycle arrest and transcription of hepatic-specific and adipose-specific genes respectively. C/EBPα interacts with regulators of cell cycle expression such as cdk2 and cdk4, directly interacts with E2F, and represses c-Myc expression with a resultant inhibition of adipocytes, hepatocyte, and granulocyte proliferation and induction of terminal differentiation [7–9]. In view of the expression of C/EBPα in terminally differentiated tissues and its role in regulating cell growth it is attractive to examine whether abnormalities of expression of C/EBPα have a role in carcinogenesis. Recently, C/EBPα has been shown to be important in a subset of patients with acute myelogenous leukemia. In patients with a t(8;21) translocation the resulting AML1-ETO fusion protein blocks C/EBPα expression and restoration of C/EBPα expression restores granulocyte differentiation . In other patients with morphologically similar leukemias, but without the t(8;21) translocation, heterozygous mutations in C/EBPα result in the production of an amino terminus truncated protein that acts in a dominant negative manner to block C/EBPα DNA binding . The resulting lack of transactivation of granulocyte target genes gives rise to the characteristic loss of differentiation seen in acute myelogenous leukemia.
The presence of C/EBPα in normal prostate tissue  and the role of C/EBPα in the regulation of differentiation and growth in other tissues raises the issues as to whether C/EBPα is important in normal prostatic differentiation and if aberrations of expression of C/EBPα are seen in prostate cancer. Significant levels of C/EBPα mRNA can be detected in the prostate [11, 12]. While only one silent mutation of C/EBPα has been found in 33 prostate cancers  increased expression of C/EBPα RNA in cancerous prostate epithelium versus normal epithelium and stroma has been detected . To understand the function of C/EBPα in the prostate we examined first the distribution of C/EBPα in normal and cancerous prostatic epithelium. We then examined the effects of C/EBPα on the expression of the prostate specific antigen (PSA). PSA is a serine protease and a member of the kallkrein family. In normal prostate, PSA is produced in secretory epithelial cells to liquefy semen  and PSA is considered a marker of prostate epithelial differentiation [16, 17]. In prostate cancer, PSA is an important biomarker for the diagnosis, treatment evaluation, and prognosis [18, 19]. An elevated serum PSA often indicates the presence and/or recurrence of prostate cancer  and high levels of serum PSA is typical of patients with hormone refractory prostate cancer. In addition to semen liquefaction, PSA is thought to regulate prostate proliferation through the cleavage of insulin growth factor binding protein-3 (IGFBP-3) to activate IGF signaling [21–23]. Recent studies showed that PSA may contribute to bone metastasis by enhancing adhesion of prostate cancer cells to bone marrow endothelium and activating latent TGFβ 2 . The expression of PSA is mainly regulated by the androgen receptor (AR) [15, 25, 26]. Inhibition of AR signaling blocks the expression of PSA. The tumor suppressor genes PPAR gamma and p53 inhibited PSA expression through AR signaling [27, 28]. PSA is also regulated by androgen-independent mechanisms . In this study, we demonstrated the inhibition of PSA expression by C/EBPα via epigenetic loss of AR expression and direct interaction between C/EBPα and the PSA promoter/enhancer.
Cell culture and cell transduction by retrovirus expressing C/EBPα
The human prostate cancer cell line, LNCaP (ATCC, Rockville, MD), was maintained in RPMI 1640 medium (Mediatech Cellgro, Herndon, VA) supplemented with 10 % fetal bovine serum (FBS). LNCaP cells with stable expression of C/EBPα were established with a pantropic retroviral expression system (BD Biosciences Clontech, Palo Alto, CA). Briefly, the full length rat C/EBPα cDNA which shares 94 % homologuos to human C/EBPα was inserted into the retrovirus vector pLNCX and was co-transfected with Lipofectamine Plus (Invitrogen Life Technologies, Inc., Carlsbad, CA) into GP2-293 packaging cells with pVSV-G, expressing an envelope glycoprotein of the vesticular stomatitis virus. After 48 hours of transfection, medium was collected, filtered, and prostate cancer cell lines were transduced with a mixture of virus-containing medium and fresh medium at ratio of 1:2. Polybrene (Sigma-Aldrich, St. Louis, MO) was added to the medium at 8μg/ml for first 24 hours. Stable expressing clones were selected with geneticin at 400 μg/ml (Mediatech Cellgro, Herndon, VA) for 2–3 weeks.
Immunohistochemistry and immunofluorescence
Immunohistochemical staining of the tissue slides was performed by deparaffinization, treatment with 4% H2O2 to remove endogenous peroxidase activity, and antigen retrieval with 10 mM sodium citrate solution. After blocking the samples with 15% fetal bovine serum in phosphate buffer saline (PBS) for 30 min the sections were incubated at room temperature with antibodies against C/EBPα antibody (sc-61), androgen receptor (sc-7305), and PSA (sc-7316) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or against p63 (Lab Vision Co., Fremont, CA). The immunohistochemical staining was performed with multi-link reagent and AEC substrate (BioGenex, San Ramon, CA). For immunohistochemical staining of human prostate tissue, archived samples were obtained from the Department of Pathology of LSUHSC under a LSUHSC IRB approved protocol (Protocol No:E06-110).
Luciferase reporter assay of PSA promoter/enhancer activity
Plasmids containing the PSA promoter and enhancer driving the luciferase reporter gene were the kind gift of Dr. Stephen P. Balk. One plasmid contained the proximal promoter, the distal enhancer from -6480 to +12 nucleotides, and had multiple androgen receptor elements (ARE). The other plasmid contained the proximal promoter from -632 to +12 nucleotides and had two ARE sites[25, 26]. Prostate cancer cell lines were grown in 24 well plates to about 70–80% confluence and were co-transfected either with 375 ng PSA promoter/enhance or proximal promoter-driven luciferase reporter plasmid and 0–100 ng pcDNA3-rat C/EBPα expression vector or mutated C/EBPα  using Lipofectamine Plus according to manufacturer's instruction. The cells were placed in medium with charcoal stripped FCS, 50 nM dihydrotestosterone added, and after 36 hours, the transfected cells were lysed with Passive Lysis Solution (Promega, Madison, WI). The luciferase activity was measured with the Dual-Luciferase® reporter assay system (Promega, Madison, WI) on a Monolight 2010 Luminometer (BD PharMingen, San Diego, CA). Renilla luciferase activity was used to normalize the transfection efficiency. The statistical significance was calculated with the unpaired Student T-test two-tailed analysis.
Cell sorting and RT-PCR
The C/EBPα-EGFP plasmid was constructed with a full length rat C/EBPα cDNA in the pcDNA3 vector. Briefly, pcDNA3-C/EBPα was digested with BamH1 and Sac1 and the insert containing C/EBPα was subcloned into pEGFP-N2 vector at Bgl2 and Sac1 sites. LNCaP cells were transiently transfected with C/EBPα-pEGFP by electroporation. After 48 hours, the cells were collected by trypsinization and sorted with the BD FACSVantage™ SE System at λex488 and λem522. The RNA was isolated from the sorted population of fluorescence-positive cells. RT-PCR was used to detect PSA mRNA expression. One μg of RNA was reversibly transcribed with Superscript™ II, RNase H- Reverse Transcriptase (Invitrogen, CA) and PCR was conducted with HotStarTaq DNA Polymerase (Qiagen, Valencia, CA) using RT product diluted at a ratio of 1:10. The primer pairs used for PSA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were, 5'-GGTGATGACTCCAGCCACGA-3'(PSA forward), 5'-GCGCACACACGTCATTGGAA-3' (PSA reverse), 5'-CTACTGGCGCT GCCAAGGCT-3' (GAPDH forward), and 5'-GCCATGAGGTCCACCACCCTGT-3' (GAPDH reverse). The conditions for the PCR were set at 30 s at 95°, 50 s at 57°C, and 1 min. at 72°C for 32 cycles following 15 min. at 95°C at the beginning of the PCR procedure to activate the hot-start taq DNA polymerase.
Western blot analysis
Whole cell extracts from prostate cancer cell lines were obtained with RIPA buffer (PBS, 1% Nonidet P-40 (NP-40), 0.25% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS)) containing 1X protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN). Protein concentration was determined by BCA Protein Assay Kit (Pierce Biotechnology, Rockford, IL). Cell proteins were separated by electrophoresis on 12% SDS-PAGE, transferred to Hybond ™ ECL nitrocellulose membrane (Amersham Biosciences, Piscataway, NJ), and blocked with 5% non-fat milk in 1X TBST (10 mM Tris-HCL, pH 8.0; 150 mM NaCl; 0.05% Tween-20). The blots were then incubated at room temperature with rabbit anti C/EBPα antibody for 2 hours, washed, and incubated with peroxidase-conjugated secondary antibody. The signal was detected with SuperSignal West Pico Substrate (Pierce Biotechnology, Rockford, IL). Antibodies against C/EBPα (sc-61), androgen receptor (sc-816 and sc-7305), PSA (sc-7638) were purchased from Santa Cruz Biotechnology, Inc.(Santa Cruz, CA). The antibody against the β-tubulin (MS-719) was from Lab Vision Co. (Fremont, CA).
Cell growth analysis
Growth curves were generated by plating the retrovirus infected cells suspended in RPMI 1640 containing 10% FBS and 400 μg/ml of geneticin in 12-well plates at initial cell densities of about 40,000 cells/well. Every two days cells were collected from triplicate wells by trypsinization. The cells were then resuspended in Isoton® II (Beckman Coulter Company, Hialeah, FL) and counted on a Coulter Z1 (Beckman Coulter Company, Hialeah, FL).
Immunohistochemical identification of C/EBPα in normal and cancerous prostate cancer
C/EBPα expression inhibits expression of AR and PSA
Further examination of the effects of C/EBPα expression on the transcriptional activity of PSA
C/EBPα is expressed in many tissues including white and brown adipose tissue, myeloid cells, lung, prostate, ovary, and colon [11, 30]. The role of C/EBPα in adipose, myeloid, and hepatocyte terminal differentiation has been well established. In myeloid cells, for example, the expression of C/EBPα is regulated and C/EBPα expression leads, in turn, to expression of genes necessary for cell differentiation and to limited myeloid proliferation [4, 7, 31, 32]. However, all hematopoiesis is not affected similarly. While C/EBPα stimulates the differentiation of myeloid cells, such as granulocytes, increased expression of C/EBPα significantly blocks the differentiation of erythroid precursors . The regulation by C/EBPα in proliferation and differentiation might contribute to the development of the malignant phenotype. The aberrant expression of C/EBPα has been described in myeloid leukemias [10, 13]. In a subset of myeloid leukemias mutations of C/EBPα result in the synthesis of abnormal proteins that act as dominant negative proteins to block the action of C/EBPα with a resulting block of differentiation and loss of the limits on proliferation . In other leukemias the fusion protein formed from the AML1-ETO translocation down-regulates transcription of the C/EBPα gene .
However, only to varying extents has the role of C/EBPα in the differentiation of some of the other tissues been defined. To even a lesser extent has the presence of aberrant expression of C/EBPα in malignancies of these various organs been described. For example, C/EBPα has a gradient of expression in the small intestine of mice with the greatest expression in the most proximal portions of the intestine and without any expression detected in mouse colonic epithelium . In the mouse intestine C/EBPα was found in the epithelium of the villi and not in the crypt cells suggesting that here too C/EBPα expression is associated with terminal differentiation. In human colon and colonic cancers, however, C/EBPα can be detected suggesting that there is some species specificity and a slight decrease of expression has been detected in more advanced colon cancer . In the rat ovary expression of C/EBPα increases with differentiation of the follicular cells ; to date there are no reports of altered C/EBPα expression in ovarian cancer. In addition to the detection of mutations in C/EBPα in acute myelogenous leukemia of subtype FAB classification M2, mutations have been detected in myelodysplastic disorders but only silent mutations have been detected in non-hematologic tumors including one lung cancer and one prostate cancer . In the lung C/EBPα is expressed in the basal layer of normal lung tissue and expression of C/EBPα is recognized to contribute to type II cell differentiation . However the role of expression of C/EBPα on the differentiation of lung cells is still unknown.
The studies presented here provide additional information to the study that examined C/EBPα expression by gene array  and in which expression of C/EBPα RNA was increased by three fold in cancerous prostate epithelium versus normal epithelium. C/EBPα expression at the protein level in malignant and non-malignant prostate glands has not been previously investigated. In the normal prostate, C/EBPα is predominately expressed in the basal layers of the epithelium with little C/EBPα detected by immunohistochemistry in the more differentiated secretory epithelium expressing AR and PSA. This is a distinctly different distribution than seen in other tissues where C/EBPα is expressed in differentiated cells. It is interesting that in our immunohistochemistry studies, p63 was found to co-localize with the expression of C/EBPα in normal prostate gland. p63 is a marker of the prostate gland basal layer cell and a potential marker for prostate stem cells, which are characterized by active cellular proliferation [37, 38]. Further, in prostate cancer the detection of C/EBPα in the basal layer was lost, but C/EBPα staining could be detected in cancerous cells throughout pseudoglandular structures. In advanced prostate cancer with the loss of glandular like structures, C/EBPα was still expressed in some cancer cells with a similar intensity of staining as in the non-malignant epithelium. Interestingly, in the immunohistochemical staining of prostate tissue arrays for C/EBPα and AR, the ratio of C/EBPα to androgen receptor expression increased with increasing Gleason scores (data not shown) supporting our observation that forced overexpression of C/EBPα in LNCaP cells induced loss of AR expression.
To address the questions derived from our immunohistochemical staining, we first selected the PSA promoter/enhancer as a molecular marker to investigate the role of C/EBPα in the prostate. PSA seemed a reasonable choice: PSA is as a marker of prostate differentiation and is expressed in the terminally differentiated epithelium lining the lumen of tubules in the normal prostate gland as is AR; expression of PSA is androgen-dependent; and increased levels of PSA in the serum is an important biomarker for prostate cancer. The observation that C/EBPα was expressed in the basal epithelium suggested that C/EBPα may suppress PSA expression. In view of very low level of constitutional expression of C/EBPα protein in the LNCaP cells, a widely used prostate cancer cell line that expresses AR and PSA, exogenous C/EBPα was expressed by use of a retrovirus system and two pooled clones of LNCaP cells with stable expression of C/EBPα were established. Clone1 was found to express neither AR nor PSA immediately upon establishment of the clone. Clone 2 developed epigenetic loss of AR and PSA expression with serial passage. Given the rapidity in which AR expression is lost it is unlikely that the retrovirus itself caused the loss of AR expression. In addition, we have examined AR and PSA expression in LNCaP cells stably transfected with pcDNA3-C/EBPα and also noted decreased expression of PSA and AR (data not shown). However, the stable expression of C/EBPα introduced with a non-virus vector could not be maintained in LNCaP cells despite continued selection pressure with geneticin.
In the LNCaP clones with stable expression of C/EBPα, concomitant with the expression of C/EBPα we observed the loss of AR expression. Both C/EBPα and AR are important for the control of growth of prostate cells. The continued growth of the clones overexpressing C/EBPα itself was a surprise. In lung cancer cells and other cell types restoration or increased expression of C/EBPα caused growth arrest . In contrast, in the LNCaP clones expressing C/EBPα we did not observe any signs of growth arrest. The resistance to C/EBPα-induced cell growth arrest may have taken place during clonal selection. However, the establishment of stable expression of C/EBPα itself in several prostate cell lines meant that cells could escape from the negative regulation of cell proliferation by C/EBPα. This may explain the difference of cell fates between transient and stably expression of C/EBPα. A recent similar example has been observed in hepatocytes where the suppression of cell proliferation by C/EBPα is lost during oncogenesis by dephosphorylation of C/EBPα which leads to failure of C/EBPα to interact with cdk2 and E2F as well as sequestering retinoblastoma protein [39, 40]. Similar loss of the interaction between C/EBPα and cell cycle related proteins was found in established prostate cell lines (date not shown). In addition, the expression of C/EBPα in the actively proliferating basal cell layer of the prostate suggests that C/EBPα does not cause growth arrest in normal prostate epithelium.
The loss AR expression occurs in several prostate cancer cell lines, such DU145, PPC1 and PC3, and occurs frequently during the clinical evolution of prostate cancer [41, 42]. The data from gene expression profiles shows that expression of AR RNA is absent in 30 to 40 % of prostate cancer samples (GEO accession: GSE1431) and the proportion of prostate cancers not expressing AR is increased in metastatic prostate cancer (GEO accession:GSE3325). LNCaP cell lines with loss of AR expression exhibit stronger tumorgenicity and a greater metastatic potential in xenografts than AR positive LNCaP cells . In another prostate cancer cell line that lacked AR expression restoration of that expression restored AR-dependent cell growth . We would predict that the loss of AR in our C/EBPα overexpressing LNCaP clones would cause a transformation of cells from androgen-dependent to androgen-independent growth and that the cells would exhibit more aggressive growth, invasion, and metatstatic potential. Indeed, our initial analysis of cell growth showed a more rapid growth of the C/EBPα expressing Clone 2 LNCaP cells compared with the control cells at passage 8 when AR was barely detectable. In addition, our immunohistochemical findings of reduced expression of AR in advanced prostate cancer specimens especially those that express C/EBPα is in agreement with recent demonstrations of altered AR expression especially in androgen-independent prostate cancer .
The mechanism(s) for the loss of AR expression both in prostate cell lines and prostate cancer tissue are not completely understood and expression may vary in the same patient at different sites of recurrence . Methylation of the AR gene promoter has been reported in advanced hormone-independent prostate cancer tissue  and the addition of 5-Aza-2'-deoxycytidine(5-AZA) into DU145 cells with a heavily methylated AR promoter restored AR mRNA expression . In our C/EBPα overexpressing LNCaP cells, treatment with 5-AZA at 10 μM increased expression of AR and PSA in early passages of clone 2 but not clone 1 cells (data not shown). However, increased expression of AR by 5-AZA was seen in the corresponding control cells suggesting that methylation affecting AR expression was occurring in both C/EBPα expressing and non-expressing cells.
The immunohistochemical result demonstrating C/EBPα expression in the basal layer of the prostate epithelium with no expression of PSA in these cells led us to examine the effect of C/EBPα on the expression of the PSA promoter/enhancer. The inhibition of expression was not cell type specific as similar studies in CV1 and COS7 cells of co-transfection of C/EBPα and PSA promoter reporter gene demonstrated similar results (data not shown). A recent report shows that inhibition of PSA expression in LNCaP cells was dependent on an interaction between AR and C/EBPα  while our results indicated that the inhibition was independent of any direct interaction with AR. With transient expression of C/EBPα in prostate cells, ALVA 101 and the non-prostate cell line CV-1 we could not show inhibition of the MMTV promoter which contains a cryptic AR (data not shown). Nor did we observe that an increased expression of AR affected the suppression of the PSA promoter by C/EBPα. In addition, a standard search for transcription factor binding sites by TESS or TFSEARCHING showed that more than 10 potential C/EBPα binding sites in the PSA promoter. Preliminary studies with a gel shift assay indicated that C/EBPα could bind to at least 3 of these sites. Therefore, direct binding of C/EBPα to PAS promoter may explain the suppression of the PSA promoter by C/EBPα. We can not exclude that C/EBPα might also stimulate expression of coactivators. On the other hand, the results also suggest that the C-terminal leucine zipper region was more important than the transactivating domains.
We are beginning to explore, amongst the various genes whose expression in prostate cancer cells is uniformly altered by forced expression of C/EBPα, those genes that may contribute to the loss of AR expression and which may provide therapeutic targets for the difficult clinical problem of hormonal independence.
C/EBPα was differentially displayed between normal and cancerous prostate. Long term of stably expression of C/EBPα in prostate cancer cells stimulates cell proliferation. Down-regualtion of PSA expression by C/EBPα depends on the loss of AR expression in AR posive LNCaP cells and on the universal inhibiton of PSA promoter.
This study is supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Research Grant DK-41279, CA88046 and CA90578 from the National Cancer Institute to D.G.T., and by the Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center, Shreveport, LA.
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