The first two authors contributed equally to this work.
Cancer Cell Biology
Epigenetic regulation and molecular characterization of C/EBPα in pancreatic cancer cells
Article first published online: 16 SEP 2008
Copyright © 2008 Wiley-Liss, Inc.
International Journal of Cancer
Volume 124, Issue 4, pages 827–833, 15 February 2009
How to Cite
Kumagai, T., Akagi, T., Desmond, J. C., Kawamata, N., Gery, S., Imai, Y., Song, J. H., Gui, D., Said, J. and Koeffler, H. P. (2009), Epigenetic regulation and molecular characterization of C/EBPα in pancreatic cancer cells. Int. J. Cancer, 124: 827–833. doi: 10.1002/ijc.23994
- Issue published online: 11 DEC 2008
- Article first published online: 16 SEP 2008
- Accepted manuscript online: 16 SEP 2008 12:00AM EST
- Manuscript Accepted: 21 AUG 2008
- Manuscript Received: 9 JUL 2008
- NIH grants. Grant Number: 5R01CA026038-30
- Parker Hughes Fund
- pancreatic cancer;
Molecular-targeted therapy is a hopeful approach for pancreatic cancer. Silencing of tumor suppressor genes can occur by histone deacetylation and/or DNA methylation in the promoter. Here, we identified epigenetically silenced genes in pancreatic cancer cells. Pancreatic cancer cell line, PANC-1 cells were treated either with or without 5Aza-dC (a DNA methyltransferase inhibitor) and suberoylanilide hydroxamic acid (SAHA, a histone deacetylase inhibitor), and mRNA was isolated from these cells. Oligonucleotide microarray analysis revealed that 30 genes including UCHL1, C/EBPα, TIMP2 and IRF7 were up-regulated after treatment with 5Aza-dC and SAHA in PANC-1. The induction of these 4 genes was validated by real-time PCR in several pancreatic cancer cell lines. Interestingly, expression of C/EBPα was significantly restored in 6 of 6 pancreatic cancer cell lines. Chromatin immunoprecipitation assay revealed that histone H3 of the promoter region of C/EBPα was acetylated in PANC-1 treated with SAHA; and bisulfate sequencing showed methylation of its promoter region in several pancreatic cancer cell lines. Forced expression of C/EBPα markedly suppressed clonal proliferation of PANC-1 cells. Co-immunoprecipitation assay showed the interaction of C/EBPα and E2F1; and the interaction caused the inhibition of E2F1 transcriptional activity. Immunohistochemical analysis revealed that C/EBPα localized in the cytoplasm in pancreatic adenocarcinoma cells, whereas it localized predominantly in the nucleus in normal pancreatic cells. Our data demonstrated that aberrant silencing, as well as, inappropriate cytoplasmic localization of C/EBPα causes dysregulation of its function, suggesting that C/EBPα is a novel candidate tumor suppressor gene in pancreatic cancer cells. © 2008 Wiley-Liss, Inc.
Pancreatic cancer is the fourth leading cause of cancer mortality in the United States.1 It is a devastating disease with a 5-yr survival of less than 5%.2 Chemotherapy is often either ineffective or effective for only a short duration. Further therapeutic approaches are needed in addition to conventional chemotherapy for this devastating disease.
Molecular-targeted therapy including activation of tumor suppressor genes and/or repression of oncogenes is an attractive therapeutic addition to regular chemotherapies. For example, the PML-RARα fusion protein resulting from a chromosomal translocation in acute promyelocytic leukemia cells is a transcriptional repressor, which acts to prevent cellular differentiation. Pharmacologic dose of all-trans retinoic acid (ATRA) inactivates this fusion protein, and treatment of patients with ATRA and chemotherapy results in durable complete remissions.3–5
Silencing of tumor suppressor genes (TSGs) can occur by aberrant histone deacetylation caused by histone deacetylase (HDAC) and/or DNA methylation in the promoter. Inhibitors of HDAC and/or DNA methyltransferase can promote expression of TSGs, leading to repression of growth of cancer cells. HDAC inhibitors are a therapeutically promising group of drugs.6 HADC inhibitors including suberoylanilide hydroxamic acid (SAHA) enhance acetylation of histones and lead to gene expression. Recently, the oral preparation of SAHA was approved for treatment of cutaneous T-cell leukemia. Inhibitor of DNA methyltransferase such as 5-Aza-2′-deoxycytidine (5Aza-dC) can restore the expression of aberrantly methylated genes.7 This compound also has been approved for the treatment of myelodysplastic syndrome.
In the present study, we used a high-throughput microarray approach to screen genes silenced by histone deacetylation and DNA methylation in pancreatic cancer cells. Here, we identified the CEBPA gene, which is encoding the transcription factor CCAAT/enhancer binding protein α (C/EBPα), as an epigenetically silenced gene and analyzed its function as a TSG in pancreatic cancer cells.
Material and methods
Cell culture and drug treatment
Pancreatic cancer cell lines including PANC-1, AsPC-1, BxPC-3, Maraca, Capan-2, HPAC and HPAF-II were obtained from American Type Culture Collection (ATCC, Manassas, VA) and cultured under recommended conditions by ATCC.
A histone deacetylase inhibitor, SAHA was generously provided by Dr. V.M. Richen (Merck Pharmaceuticals, Darmstadt, Germany). An inhibitor of DNA methyltransferase, 5-Aza-2′-deoxycytidine (5Aza-dC) was purchased from Sigma-Aldrich (St. Louis, MO). Seven pancreatic cancer cell lines were treated with 5Aza-dC and SAHA, either alone or in combination as described in the text: 5Aza-dC (5μM) for 2 days or SAHA (2.5 μM) for 1 day. For combined treatment, cells were cultured with 5Aza-dC (5 μM) for 2 days and SAHA (2.5 μM) was added for the last 1 day. Control cells were cultured with the diluent alone.
Oligonucleotide microarray analysis
Complimentary RNA (cRNA) was synthesized by SuperScript Choice System (Invitrogen, Carlsbad, CA) according to the instructions except for using an oligo-dT primer containing a T7 RNA polymerase promoter site. The cRNA was labeled with the BioArray High Yield RNA Transcript Labeling kit (Enzo, New York, NY) and used as a probe. These samples were subjected to oligonucleotide microarray (Affymetrix, Santa Clara, CA). Hybridization, washing and signal detection were performed according to the manufacturer's protocols (Affymetrix). Fold change of signal intensities obtained by microarray analysis between drug treatment and control samples was calculated using Microarray Suite Software 5.0 (Affymetrix).
Real-time quantitative polymerase chain reaction
Total RNA was isolated using RNeasy kit (QIAGEN, Valencia, CA). Gene expression was quantified using real-time quantitative polymerase chain reaction (PCR) (TaqMan, iCycler, Bio-Rad, Hercules, CA) technique. Probes labeled with the reporter dye FAM in the 5′ end and the quencher dye TAMRA in the 3′ end were purchased from Applied Biosystems (Foster City, CA). Amplification reactions were performed with the Universal TaqMan PCR mastermix (Applied Biosystems) in triplicates in an iCycler iQ system (Bio-Rad). The thermal cycling conditions were as follows: 2 min at 50°C, 10 min at 95°C, followed by 45 cycles of 95°C for 15 sec and 60°C for 1 min.
For SybrGreen method, a melting curve analysis was performed following PCR to identify the correct product by its specific melting temperature. The PCR conditions were as follows: 94°C for 20 sec, 60°C for 10 sec, 65°C for 25 sec and 20 sec at melting temperature. Data were normalized to levels of 18S rRNA. The sequences of the primer and probe sets will be provided on request.
Chromatin immunoprecipitation (ChIP) assay
PANC-1 cells were plated at a density of 1 × 106 cells/10-cm dish. After 12 hr, cells were treated with either 2.5 μM SAHA or diluent as a control for 24 hr; cells were subsequently treated with formaldehyde (final concentration of 1%) for 10 min. After washing with PBS, the cells were resuspended in 0.5 ml of SDS lysis buffer (1% SDS, 10 mM ethylene diamine tetra acetic acid [EDTA], and 50 mM Tris-HCl [pH 8.1]), incubated on ice for 10 min, and sonicated at 3 times for 10 sec. After centrifugation (10 min at 15,000g), supernatants were 5-fold diluted by immunoprecipitation buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl [pH 8.1] and 16.7 mM NaCl); and 80 μl of 50% protein A sepharose slurry containing 20 μg sonicated salmon sperm DNA and 1 mg/ml BSA in TE buffer (10 mM Tris-HCl [pH 8.0] and 1 mM EDTA) was added and incubated, rocking for 30 min at 4°C. The sepharose was pelleted by centrifugation, and supernatants were recovered; and either 5 μg of anti-acetylated histone H3 antibody (Upstate, Lake Placid, NY) or normal rabbit serum were added, and incubated overnight at 4°C. Protein A Sepharose slurry (60 μl) was added, and samples were rocked for 1 hr at 4°C. After washing the sepharose, the immune complexes were eluted twice with 250 μl of elution buffer (1% SDS–0.1 M NaHCO3) for 15 min at room temperature. Twenty microliter of 5 M NaCl was added to the combined eluates, and the samples were incubated at 65°C for 4 hr. EDTA, Tris-HCl [pH 6.5], and proteinase K were added to the samples at a final concentration of 10 mM, 40 mM and 0.04 μg/μl, respectively; and the samples were incubated at 45°C for 1 hr. Immunoprecipitated DNA (both immunoprecipitation samples and input) was recovered by phenol/chloroform extraction and ethanol precipitation, and analyze by PCR using primers specific for C/EBPα. The optimal reaction conditions for PCR were determined for each primer pair. Primers were denatured at 95°C for 1 min and annealed at 62°C for 1 min, followed by elongation at 72°C for 1 min. PCR products were analyzed by 2.5% agarose/ethidium bromide gel electrophoresis. The primers used for C/EBPα ChIP analysis were: 5′-TGG ACA AGA ACA GCA ACG AG- 3′ (forward), 5′-TTG TCA CTG GTC AGC TCC AG- 3′ (reverse).
Methylation analysis of the CEBPA gene in pancreatic cancer cell lines
Genomic DNA was isolated from pancreatic cancer cell lines and modified by sodium bisulfate using EZ DNA Methylation Kit (Zymo Research, Orange, CA). The CpG island (−1423 to −1121) of the CEBPA gene was amplified from the bisulfate modified genomic DNA with specific primers (sense primer: 5′- TTG TTA GGT TTA AGG TTA TTG-3′, antisense primer: 5′- AAA CCC TAA AAC CCC TTA-3′) as described previously.8, 9 For PCR amplification, a total volume of 10 μL was used, containing modified genomic DNA, 0.5 μM of each primer, 5.0 μL of FailSafe PCR 2 × PreMix B (Epicentre Biotechnologies, Madison, WI) and 1.0 U platinum Taq (Invitrogen). PCR products were subcloned into pCR 2.1 vector (Invitrogen) and sequenced.
Transfection and colony formation assay
Colony formation assays were performed as described previously.10 Briefly, cells were plated at 2 × 104 per well using 6-well plates and transfected with 1 μg of either pcDNA3-C/EBPα11 or pcDNA3 (empty vector control) using Effectene Transfection Reagent (QIAGEN). After 48 hr, the cells were detached, replated on 10-cm tissue culture dish, and selected with G418 (800 μg/ml) for 2 weeks. The cells were stained with Crystal violet dye (0.1% crystal violet dissolved in 50% methanol). To quantify the number of surviving colonies, the colonies were counted and compared between empty vector- and C/EBPα expression vector-transfected samples.
Immunoprecipitation, Western blot analysis and immunohistochemistry
Cell lysates were extracted from PANC-1 cells transfected with either pcDNA3 or pcDNA3-C/EBPα using a cell lysis buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl and 0.5% NP-40). The cell lysate was incubated with anti-C/EBPα antibody (SC-61, Santa Cruz Biotechnology, Santa Cruz, CA) and Protein G Sepharose Fast Flow (Amersham Biosciences, Piscataway, NJ) at 4°C for 16 hr. The precipitated proteins were washed 3 times with PBS and eluted with the Laemmli sample buffer (Bio-Rad).
For Western blot analysis, these samples were subjected to sodium dodecyl sulfate polyacrylamide gels electrophoresis (SDS-PAGE) and then transferred to nitrocellulose membranes (Sigma-Aldrich). The filters were incubated with primary antibodies including either anti-C/EBPα (Research Diagnostics, Concord, MA) or anti-E2F1 (sc-251, Santa Cruz) antibodies followed by incubation with appropriate secondary antibody conjugated with horseradish peroxidase (Amersham Biosciences). Signals were developed by SuperSignal West Pico substrate (Pierce Biotechnology, Rockford, IL).
Normal and cancer pancreatic tissues were cut at 3 microns, deparaffinized and pretreated in Tris-HCl [pH 9.0]. These samples were incubated overnight with anti-C/EBPα antibody, washed, followed by the HRP-conjugated secondary antibody (Dako, Carpinteria, CA), and DAB chromogen. The tissues were counterstained with hematoxylin and then coverslipped.
PANC-1 cells were transiently co-transfected with 1 μg of pGL3B-3 × E2F (3 repeats of the E2F binding sequence), 0.2 μg of pRL-SV40, 0–0.6 μg of C/EBPα expression vector, and 0–30 ng of E2F1 expression vector.12 Lysates were harvested at 24 hr post-transfection and luciferase activity was measured with the Dual-Luciferase reporter 1000 assay system (Promega, Madison, WI). Transfection efficiency was normalized with renilla luciferase activity. Results represent the mean of triplicate transfections.
Statistical differences between means were analyzed by the t-test. p values <0.05 were considered to indicate a statistical difference.
Transcriptionally silenced genes in PANC-1 cells
To identify epigenetically silenced genes in pancreatic cancer cells, PANC-1 cells were treated either with or without 5Aza-dC and SAHA, and mRNA expression was determined by oligonucleotide microarray. As shown in Table I, 30 genes were identified as constitutively silenced in PANC-1 cells (fold change of level of transcripts ranged between 5.3 and 256). These genes were silent before treatment (raw level of the control cells <1,000) and were up-regulated with treatment (raw level of the treated cells >5,000). Nine of these genes including CTAG1B, CTAG2, CCL20, DHRS2, SSX2, MAGEA9, SSX1, SSX4 and GLRX do not have CpG island, whereas, the other genes have CpG island. Interestingly, 10 genes (33%) are known as cancer antigens, suggesting that many of these antigens are silenced by acetylation and/or methylation in PANC-1 cells. Silencing of these genes may allow pancreatic cancer cells to escape immune surveillance. Several of these genes including UCHL1, CEBPA, TIMP2 and IRF7 may be associated with aberrant behavior of these cancer cells.
|Fold change||Genbank||Gene name|
Validation induction of gene expression in several pancreatic cancer cell lines
Previously, we have demonstrated that expression of C/EBPα mRNA was induced after treatment of SAHA in PANC-1 cells.13 Here, we validated the induction of expression of the 4 genes in pancreatic cancer cell lines. Total RNA was harvested from 6 pancreatic cancer cell lines (PANC-1, AsPC-1, BxPC-3, HPAC, HPAF-II and MiaPaCa) after treatment either with or without 5Aza-dC and SAHA; and expression level of UCHL1, C/EBPα, TIMP2 and IRF7 mRNA was measured by quantitative RT-PCR. As shown in Figure 1, significant increased expression of the 4 genes was observed in several pancreatic cancer cell lines. Induction of UCHL1 mRNA occurred in PANC-1, AsPC-1 and HPAF-II cells (Fig. 1a); C/EBPα levels rose in all the cell lines (Fig. 1b); TIMP2 increased in PANC-1, AsPC-1, HPAC and HPAF-II (Fig. 1c), and expression of IRF7 increased in all cell lines except MiaPaCa cells (Fig. 1d). Taken together, the results suggested that these 4 genes were often epigenetically silenced in various pancreatic cancer cells.
Acetylation of histones and methylation of the promoter region of the CEBPA gene in pancreatic cancer cells
Our data, described above, showed that C/EBPα is most frequently up-regulated in pancreatic cancer cells treated with drugs; and therefore, we further investigated C/EBPα in pancreatic cancer cells. As shown in Figure 2a, induction of C/EBPα expression was observed after exposure to either 5Aza-dC or SAHA alone; and strong induction was found after their combined treatment in PANC-1 cells.
Next, we examined acetylation status of histones in the promoter region of the CEBPA gene. Chromatin immunoprecipitation (ChIP) assay showed that the promoter region of the CEBPA gene was precipitated with anti-acetylated histone H3 in SAHA-treated PANC-1 cells (Fig. 2b). The signal intensity increased 50-fold compared to untreated PANC-1 cells.
We also examined the methylation status of the CEBPA gene in pancreatic cancer cells. Bisulfate modified sequencing analysis revealed that the promoter region is often methylated in PANC-1, HPAC and MiaPaCa cell lines (Fig. 2c and data not shown). These results are consistent with the hypothesis that deacetylation and methylation of the promoter is associated with silencing of the gene.
Forced expression of C/EBPα in PANC-1 cells decreased their clonogenic growth
Since pancreatic cancer cells had low levels of C/EBPα, the C/EBPα expression vector was transfected into PANC-1 cells. Western blot analysis showed strong expression of the protein in the C/EBPα-transfected PANC-1 cells (Fig. 3a). Clonogenic growth of PANC-1 cells transfected with C/EBPα expression vector was markedly suppressed compared to the control cells (Figs. 3b and 3c). These results indicated that C/EBPα inhibits growth and can act as a TSG in PANC-1 cells.
C/EBPα interacts with E2F1 and inhibits E2F1 transcriptional activity
Previously, we found an interaction of C/EBPϵ and E2F1 and showed that C/EBPϵ repressed the transcriptional activity of E2F1 in hematopoietic cells.12 To explore the possibility of a similar function of C/EBPα in pancreatic cancer cells, we performed co-immunoprecipitation and luciferase assays. Co-immunoprecipitation assay showed that strong signal of E2F1 was detected after immunoprecipitation of C/EBPα, suggesting that C/EBPα is interacting with E2F1 in the PANC-1 cells (Fig. 4a).
To determine if C/EBPα could blunted the transcriptional activity of E2F1, PANC-1 cells were transiently co-transfected with the luciferase reporter plasmid pGL3B/3xE2F (contains 3 repeats of E2F binding sequence), E2F1 and increasing amount of C/EBPα expression plasmid (0–0.6 μg), as well as, pRL-SV40. The luciferase activity was decreased by C/EBPα in a dose-dependent manner (Fig. 4b). These findings indicate that the anti-proliferative activity of C/EBPα might, in part, be mediated by inhibiting E2F1 activity.
Expression of C/EBPα in pancreatic cancer tissue
To examine the expression of C/EBPα in human pancreatic tissues, normal and carcinoma tissues were stained with anti-C/EBPα antibody. Immunohistochemistry revealed that C/EBPα signal was strongly detected in the nucleus in normal pancreatic cells (Fig. 5a). Interestingly, C/EBPα was detected in the cytoplasm and to a lesser extent in the nucleus of pancreatic carcinoma cells (Fig. 5b). Because C/EBPα has its anti-proliferative effects when behaving as a nuclear transcription factor, the inappropriate localization of the protein in the cytoplasm may provide a mechanism by which some pancreatic cancer cells escape the control of this transcription factor.
Inactivation of TSGs plays an important role in carcinogenesis.14, 15 Silencing can occur by epigenetic alterations including aberrant hypermethylation of the promoter region and chromatin modification accompanied by histone deacetylation14, 16, 17; these 2 epigenetic changes are closely linked to each other.16, 17 Treatment with a DNA demethylating agent and a HDAC inhibitor has revealed genes that have been epigenetically silenced in various types of cancer.18–21 In this study, we identified epigenetically silenced genes in pancreatic cancer cells in order to identify candidate TGSs.
Several human tumor antigens have been identified in cancer cells, and they are categorized into various classes: differentiation antigens (e.g., tyrosinase and MelanA/MART-1), antigens derived from overexpression and/or amplification (e.g., HER-2/neu), gene mutation (e.g., p53), viral origin antigens (e.g., E6 and E7),22 and cancer/testis (CT) antigens (e.g., MAGE, GAGE and NY-ESO-1). Among them, the CT antigens have received particular attention as potential vaccine targets because of their unique tissue expression.23, 24 The CT antigens are intriguing targets due to the ability of reactivating their expression by epigenetic therapy. Our microarray analysis showed that several CT antigens were up-regulated by treatment of 5Aza-dC and SAHA in PANC-1 cells. Silencing of these genes might allow pancreatic cancer cells to escape from immune system. Thus, inhibitors of DNA methyltransferase (5Aza-dC) and histone deacetylase (SAHA) may induce a T-cell-mediated anti-tumor effect. Furthermore, pretreatment with these agents may enhance vaccine therapy for cancers.
A prior study identified silenced genes in pancreatic cancer cells using microarray analysis.20 Several genes including UCHL1, NPTX2 and GAGEs were also obtained in our analysis. Here, we focused on 4 genes including UCHL1, TIMP2, IRF7 and CEBPA.
UCHL1 is a member of the carboxyl-terminal ubiquitin hydrolase family. This gene has been previously identified as a putative tumor-suppressor gene whose promoter has been reported to be methylated as a cancer-specific event in head and neck cancer.25 TIMP2 is one of the tissue inhibitors of matrix metalloproteinases (TIMPs), a family of natural inhibitors which control the activity of matrix metalloproteinases in the extracellular matrix. The role of TIMPs in cancer has been the subject of conflicting reports; some investigators find the protein to have antitumor activity, others show it can increase tumor growth.26 Enhanced expression of the gene in cancer cells can slow proliferation and invasiveness of tumor cells.26, 27 In addition, hypermethylation of the gene has been found in several types of cancer cells.28, 29 IRF7 is activated through the c-Jun N-terminal kinase/stress-activated protein kinase stress response pathway in response to various DNA-damaging chemotherapeutic agents.30 The promoter region of the gene has been shown to be methylated, and silenced in a fibrosarcoma cell line.31 In addition, IRF7 is the target gene of BRCA1, a known TSG for breast and ovarian tissues, and presence of IRF7 may be an important effecter of interferon gamma or BRCA1-mediated growth suppression in response to either viral infection or DNA damage.32
Transcription factor C/EBPα plays a critical role in the regulation of mitotic growth arrest and differentiation in numerous cell types, including preadipocytes, myeloid cells, hepatocytes, keratinocytes and pneumocytes.33–39 About 10–15% of acute myeloblastic leukemia (AML) samples have inactivating mutations of C/EBPα, and forced expression of C/EBPα in AML cells can induce terminal differentiation11, 40; and the gene is often methylated in AML cells.41 Breast cancer cells often have low expression of C/EBPα, and transduction of these cells with a C/EBPα expression vector markedly slowed their growth.42 Expression of C/EBPα is silenced in lung cancer cells, as well as, head and neck squamous cell carcinoma.8, 9, 43 Here, we showed for the first time that C/EBPα is methylated in several pancreatic cancer cell lines and inhibits PANC-1 cell growth. Although cancer cell lines often possess a much higher frequency of aberrantly methylated genes, these findings strongly suggested that C/EBPα is a TSG in pancreatic cancer cells. Further studies in a larger cohort of patient samples will clarify the frequency of methylation of the gene.
We explored mechanisms by which C/EBPα suppressed growth by using PANC-1 pancreatic cancer cells in reporter assay using a concatamerized E2F binding site. E2F is a transcriptional factor whose target genes are necessary for the G1/S transition.44, 45 C/EBPα inhibited the ability of the E2F transcription factor to stimulate the reporter gene. This inhibition may be caused by the ability of C/EBPα to bind E2F1. The retinoblastoma (Rb) and E2F proteins are key regulators of the cell cycle and differentiation.44, 45 Rb is a well-established TSG involved in the regulation of cell cycle progression. Hypophosphorylated Rb sequesters the E2F transcriptional factors whose target genes are necessary for the G1/S transition.44, 45 Recently, we reported that the forced expression of C/EBPα in pancreatic cancer cells decreased the expression of phosphorylated Rb.13 Taken together, forced expression of C/EBPα may retard growth of pancreatic cancer cells by effecting both E2F1 and Rb.
Our immunohistochemical staining showed that C/EBPα signal was strongly detected in the nucleus in normal pancreatic islet cells but not in normal pancreatic duct (data not shown). Interestingly, the signal was detected in the cytoplasm and to a lesser extent the nucleus of pancreatic carcinoma cells. Aberrant cytoplasmic localization of C/EBPα is also found in breast cancer tissue.42 In general, transcription factors including C/EBPα function in the nucleus, suggesting that deregulation of nuclear localization of C/EBPα leads to functional deficiency and results in cell abnormalities. Although the C/EBPα gene is methylated in pancreatic cancer cell lines, primary tumor samples showed expression of the protein; but displayed cytoplasmic localization. These findings suggest that several mechanisms for inactivating C/EBPα function might be involved in the carcinogenesis of pancreatic cells.
In summary, our data demonstrated that aberrant silencing, as well as, inappropriate cytoplasmic localization of C/EBPα causes dysregulation of its functions, suggesting that C/EBPα is a novel candidate TSG dysregulated in pancreatic cancer cells. Its re-activation or re-localization may be a promising therapeutic approach to this cancer.
We thank members of our laboratory for their helpful discussions. H.P.K. is the holder of the Mark Goodson endowed Chair in Oncology Research and is a member of the Jonsson Cancer Center and the Molecular Biology Institute, UCLA.