Cancer Research AACR Membership Jordan
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
Cancer Research 68, 5040, July 1, 2008. doi: 10.1158/0008-5472.CAN-07-6575
© 2008 American Association for Cancer Research
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplementary Data
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Huang, G.
Right arrow Articles by Halmos, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Huang, G.
Right arrow Articles by Halmos, B.

Molecular Biology, Pathobiology, and Genetics

15-Hydroxyprostaglandin Dehydrogenase is a Target of Hepatocyte Nuclear Factor 3β and a Tumor Suppressor in Lung Cancer

Guosheng Huang1, Rosana Eisenberg2, Min Yan1, Stefano Monti4, Earl Lawrence1, Pingfu Fu3, Jaclyn Walbroehl5, Ester Löwenberg6, Todd Golub4, Jaime Merchan5, Daniel G. Tenen6, Sanford D. Markowitz1,7 and Balazs Halmos1

1 Division of Hematology/Oncology, Departments of 2 Pathology and 3 Epidemiology and Biostatistics, University Hospitals of Cleveland/Case Western Reserve University, Cleveland, Ohio; 4 The Broad Institute of Harvard University/MIT, Cambridge, Massachusetts; 5 Division of Hematology/Oncology, University of Miami Miller School of Medicine, Miami, Florida; 6 Beth Israel Deaconess Medical Center, Boston, Massachusetts; and 7 Howard Hughes Medical Institute, Chevy Chase, Maryland

Requests for reprints: Balazs Halmos, Division of Hematology/Oncology, Case Western Reserve University, Cleveland, OH 44106. Phone: 216-368-1175; Fax: 216-368-1166; E-mail: bxh60{at}case.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
The forkhead transcription factor hepatocyte nuclear factor 3β (HNF3β) is essential in foregut development and the regulation of lung-specific genes. HNF3β expression leads to growth arrest and apoptosis in lung cancer cells and HNF3β is a candidate tumor suppressor in lung cancer. In a transcriptional profiling study using a conditional cell line system, we now identify 15-PGDH as one of the major genes induced by HNF3β expression. 15-PGDH is a critical metabolic enzyme of proliferative prostaglandins, an antagonist to cyclooxygenase-2 and a tumor suppressor in colon cancer. We confirmed the regulation of 15-PGDH expression by HNF3β in a number of systems and showed direct binding of HNF3β to 15-PGDH promoter elements. Western blotting of lung cancer cell lines and immunohistochemical examination of human lung cancer tissues found loss of 15-PGDH expression in ~65% of lung cancers. Further studies using in vitro cell-based assays and in vivo xenograft tumorigenesis assays showed a lack of in vitro but significant in vivo tumor suppressor activity of 15-PGDH via an antiangiogenic mechanism analogous to its role in colon cancer. In summary, we identify 15-PGDH as a direct downstream effector of HNF3β and show that 15-PGDH acts as a tumor suppressor in lung cancer. [Cancer Res 2008;68(13):5040–8]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
Lung cancer remains the leading cause of cancer deaths within the United States, despite recent advances in its treatment (1). The minimal progress achieved in the treatment of this disease calls for a better understanding of the molecular mechanisms that lead to the development and maintenance of this malignancy (2, 3). We and others have shown the important role of the tissue-specific differentiation factor, CAAT/enhancer binding protein-{alpha}, as a tumor suppressor in lung cancer (4–7). We also identified hepatocyte nuclear factor 3β (HNF3β) as a downstream effector of CAAT/enhancer binding protein-{alpha} and a candidate tumor suppressor in lung cancers (8). HNF3β, also named Foxa2, belongs to the forkhead family of transcription factors and is essential in foregut development and in the regulation of several lung-specific genes (9). To gain additional insights into the downstream effects of HNF3β expression, we performed a microarray study on an HNF3β-inducible lung adenocarcinoma cell line and defined transcriptional changes secondary to conditional expression of HNF3β. This study identified the cyclooxygenase-2 (Cox-2) antagonist enzyme, 15-PGDH, as one of the most highly induced downstream targets of HNF3β in lung cancer cells and we show direct regulation of the 15-PGDH promoter by HNF3β. Furthermore, we show significant down-regulation of 15-PGDH expression in a tissue subtype–dependent manner in non–small cell lung cancer. We also show that 15-PGDH expression leads to strong in vivo tumor-suppressive effects via an antiangiogenic mechanism analogous to its function in colon cancer model systems. Our findings suggest that 15-PGDH is a downstream effector of HNF3β with in vivo tumor-suppressive effects in non–small cell lung cancer.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 References
 
Cell lines and cell culture. The following lung cancer cell lines, all from American Type Culture Collection, were used in this study: Calu-1, SK-MES-1, SW900, SK-LU-1, H23, H441, H358, A549, H322, H125, H292, H460, Calu-6, H69, and H211. Cell lines were grown in RPMI 1640 supplemented with 10% fetal bovine serum (FBS). The HNF3β-inducible stable cell line H358-HNF3β (tet-off) was previously generated from H358 lung cancer cells (8). H358-HNF3β (tet-off) cells were maintained in RPMI 1640 supplemented with 10% doxycycline-free FBS and 1 µg/mL of doxycycline. HNF3β expression can be induced by doxycycline withdrawal from the culture medium.

Oligonucleotide array analysis. Triplicate plates of H358-HNF3β (tet-off) cells were maintained in doxycycline-free RPMI 1640 for 0, 24, 48, 72, and 96 h. Total cellular RNA was isolated using the TRIzol method and was then processed and hybridized to Affymetrix Hu-133 microarrays and scanned. The expression value was calculated by application to the Affymetrix ".CEL" files of the robust multichip averaging procedure as implemented in Bioconductor (10, 11).

Quantitative reverse transcription-PCR assay. Total RNAs were collected using TRIzol from H358-HNF3β (tet-off) cells with or without the deprivation of doxycycline from culture medium. cDNAs were synthesized with Moloney murine leukemia virus reverse transcriptase (SuperScript III reverse transcriptase) with the use of oligo(dT) primers from Invitrogen (all primer sequences are listed in the Supplementary Methods). All samples were run in triplicate on Roche LightCycler using the Syber green probes (Roche) using the following variables: denaturation at 95°C for 10 min, followed by 45 cycles of amplification (95°C 10 s, 60°C 10 s, and 72°C 15 s), and cooled to 40°C at a transition rate of 20°C/s. Levels of glyceraldehyde-3-phosphate dehydrogenase expression were used as internal reference to normalize input cDNA. Ratios of level of each gene to glyceraldehyde-3-phosphate dehydrogenase were then calculated.

Immunoblotting. Whole cell lysates were isolated using radioimmunoprecipitation assay buffer (Upstate Biotechnology) supplemented with protease inhibitor mixture (Roche Applied Sciences) and 20 µg of protein/lane were electrophoresed in 10% SDS-PAGE minigels. A dilution of 1:1,000 polyclonal goat anti-HNF3β antibody (Santa Cruz Biotechnology), 1:1,000 dilution of monoclonal mouse anti–15-PGDH antibody13, 1:1,000 polyclonal rabbit PARP antibody (Cell Signaling), and 1:500 monoclonal mouse β-actin antibody (Sigma) were used. Detection was performed using Western Lightning Chemiluminescence reagent (Perkin-Elmer Life Sciences).

Luciferase reporter assay. H358-HNF3β (tet-off) cells were grown in RPMI 1640/10% FBS containing 1 µg/mL of doxycycline. Duplicate samples of 1 x 105 cells in six-well plates were transfected using Fugene 6 (Roche) with 50 ng of phRL-CMV-Renilla luciferase plus one of the following plasmids: 0.5 µg of pcDNA3-pp5.9-Firefly luciferase, 0.5 µg of pcDNA3-pp2.2-firefly luciferase, or 0.5 µg of pcDNA3 empty vector. Cells were then deprived of doxycycline for 24 h posttransfection and cultured for an additional 0, 24, 48, 72, or 96 h. Cell extracts were prepared and luciferase assays were done on a luminometer (LMax II 384, Molecular Devices) by using the Dual Luciferase-Reporter Assay System (Promega) according to the manufacturer's instructions. Firefly luciferase activities were normalized to parallel Renilla luciferase activities to correct for differences in transfection efficiency.

Chromatin immunoprecipitation assay. H358-HNF3β (tet-off) cells were cultured in doxycycline-free medium for 0, 24, 48, 72, and 96 h. Cells (1 x 108) were cross-linked by 0.37% formaldehyde. Nuclear extracts were prepared (20% of each nuclear extract was saved before immunoprecipitation as input chromatin DNA) and immunoprecipitated by 5 µg of goat polyclonal HNF3β antibody (Santa Cruz Biotechnology) at 4°C for 6 h with rotation. Immune complexes were collected by incubating with protein A–agarose beads overnight at 4°C with rotation. Cross-links of the immunoprecipitated samples and input samples were reversed by heating at 65°C in the presence of NaCl and RNase A (Sigma) for 5 h followed by proteinase K (Roche Diagnostics) digestion. Binding of HNF3β to the 15-PGDH promoter was assessed by PCR and quantitative PCR with primer sets amplifying two HNF3β-binding sequence–containing regions spanning –3793 to –3778 bp and –446 to –430 bp of the 15-PGDH promoter, respectively (12, 13). These promoter regions were identified as putative HNF3β-binding regions by the use of MatInspector Software.

Electrophoretic mobility shift assay. Nuclear extracts were collected from H358-HNF3β (tet-off) cells cultured in doxycycline-free medium for 0 or 96 h. Double-stranded DNA (dsDNA) annealing was achieved by incubating complementary pairs of oligonucleotides at 95°C for 5 min followed by slow cooling to room temperature. Binding reactions were performed using Pierce LightShift Chemiluminescent EMSA Kit with 2 µg of nuclear extract and 20 fmol of biotin-labeled dsDNA. Unlabeled oligonucleotides (4 pmol) were used in the competition assays. The ENBA control system was used as experimental controls (Pierce). The mixture was analyzed by 6% DNA retardation gel (Invitrogen) in 0.5x Tris-borate EDTA buffer (Invitrogen), transferred to a positively charged nylon membrane, UV cross-linked, and detected by chemiluminescence. The gel-separated DNA-protein complex was also transferred to a nitrocellulose membrane for HNF3β immunoblotting.

MTS cell growth assay. H358 lung cancer cells were transfected with pcDNA6-empty vector (pcDNA6-EV), or expression vectors encoding wild-type (pcDNA6-WT-PGDH) or mutant (pcDNA6-Mu-PGDH) 15-PGDH by using Fugene 6 (Roche) according to the manufacturer's protocol. MTS cell growth assays were performed 48, 72, and 96 h after transfection according to the manufacturer's instructions.

Annexin/propidium iodide apoptosis assay. H358 cells were collected by trypsinization 72 h after transfection, and then washed with PBS, and stained with Annexin/propidium iodide according to the manufacturer's instructions (Roche Applied Science). Samples were analyzed on a fluorescence activated cell scan cytometer EPICS XL MCL Coulter.

Immunohistochemistry. Immunohistochemical staining was accomplished using the R.T.U. Vectastain Universal Quick Kit (Vector Lab) according to the manufacturer's instructions with the use of anti-HNF3β (1:500 dilution; Santa Cruz Biotechnology), anti–15-PGDH (1:500 dilution)13, or anti–vascular endothelial growth factor (VEGF) antibody (1:500 dilution; Santa Cruz Biotechnology).

Transient transfection. Calu-1 and SKLU-1 lung cancer cells were transfected with pcDNA3 (+)-ratHNF3β using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. Whole cell lysates collected 0, 24, 48, 72, or 96 h after transfection were used in immunoblotting for HNF3β and 15-PGDH. H358 cells were transfected using Fugene 6 (Roche). Whole cell lysates were collected 72 h posttransfection.

15-PGDH stable transfection and tumorigenesis assay. H358 lung cancer cells were transfected with pcDNA6-empty vector (pcDNA6-EV), or expression vectors encoding wild-type 15-PGDH (pcDNA6-WT-PGDH) by using Fugene 6 (Roche). Stable cell pools were selected by growing cells in regular culture medium containing 10 µg/mL of blasticidin. Cells at 70% to 80% confluency were trypsinized and washed thrice with serum-free RPMI 1640. Cells (5 x 106) resuspended in 200 µL of serum-free medium were injected s.c. behind the anterior forelimb of a 5-week-old BALB/c athymic mouse. Tumor diameters were measured weekly with a caliper and the tumor volume (in mm3) was calculated by the formula: volume = (width)2 x length / 2. The mean volumes of xenograft tumors were obtained from 10 replicate injections.

Prostaglandin E2 ELISA. H358 stable cells expressing 15-PGDH and H358 empty vector control cells were seeded in a 12-well plate at a density of 1 x 105 cells/well. Culture medium was collected 72 h later and centrifuged briefly to remove cell debris. The prostaglandin E2 (PGE2) levels were examined by ELISA according to the manufacturer's instructions (Perkin-Elmer). The absorbance at 405 and 590 nm was read immediately on a microplate autoreader (EL311; BIO-TEK Instruments, Inc.). Each reading of absorbance at 405 nm was normalized by the reading of absorbance at 590 nm and the concentrations of PGE2 from each sample were calculated according the standard curve generated from the PGE2 standards provided.

Determination of microvessel density. Formalin-fixed paraffin-embedded sections were deparaffinized, rehydrated, and immunohistochemically stained with rat anti-mouse CD31 monoclonal antibody (clone MEC 13.3, 1:10 dilution; PharMingen). The microvessel density (MVD) of the xenograft tissue sections was evaluated at high-power field (x400). The mean microvessel count of the five most vascular areas was calculated as MVD for each section. The mean MVD from three animals in the wild-type 15-PGDH and empty vector control groups was obtained as the final MVD count.

Determination of VEGF. Concentration of the angiogenic growth factor VEGF was measured from H358 cell culture medium (in triplicate) at 72 h. Measurement was performed with the ELISA kits for VEGF 165 (R&D Systems) according to the manufacturer's instructions.

Endothelial cell proliferation assay. A total of 4 x 103 cells in 100 µL of basal medium with 1% FBS were seeded into each well of a 96-well plate, treated with 10% of the appropriate conditioned medium as the stimulant, and incubated at 37°C for 72 h; control cells were incubated in basal medium and 1% FBS, as above (14). No additional proliferation stimulus (VEGF or basic fibroblast growth factor) was added. After the 72-h incubation, WST-1 (10 µL) was added to each well, and after a 3-h incubation at 37°C, absorbance at 450 nm was determined for each well with a microplate reader (Dynex Technologies). The data presented are the average of triplicate experiments.

Matrigel tube formation assay. Each well of a prechilled 48-well cell culture plate was coated with 100 µL of unpolymerized Matrigel (7 µg/mL) and incubated at 37°C for 30 to 45 min (15). Human umbilical vascular endothelial cells were harvested with trypsin, and 4 x 104 cells were resuspended in 300 µL of endothelial cell basal medium supplemented with 1% FBS, and treated with 10% of the appropriate conditioned medium before plating onto the Matrigel-coated plates. After 12 h of incubation at 37°C, endothelial cell tube formation was assessed with an inverted photomicroscope (Nikon). Microphotographs of the center of each well at low power were taken at 40x magnification with the aid of imaging-capture software (NIS-Elements from Nikon). Tube formation in the microphotographs was quantitatively analyzed (total tube length); controls consisted of human umbilical vascular endothelial cells in basal medium supplemented with 1% FBS. The experiment was done in triplicate and the data presented represent the average of triplicate experiments.

Statistical analysis. Spearman correlation coefficient was used to estimate the association between two continuous measurements; and the association between two factors was examined by {chi}2 or Fisher's exact test. The differences of a continuous measurement between two groups were examined by Student's t test. All tests were two-sided and P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 References
 
Transcriptional profiling study identifies downstream changes upon HNF3β induction. We performed a transcriptional profiling study using our previously generated inducible H358-HNF3β (tet-off) stable cell lines (8). These clonally derived cell lines could strongly and reproducibly be induced to express HNF3β upon withdrawal of doxycycline from the medium. Two separate clones (clones 6108/4 and 6108/31) were induced by doxycycline withdrawal from the culture medium for 0, 24, 48, 72, and 96 hours. Triplicate specimens were collected for each time point and total cellular RNA was prepared with the use of Trizol and hybridized to Affymetrix Hu-133 arrays after cRNA generation from each triplicate specimen individually. Following appropriate processing and filtering, the microarray data on the triplicate samples were analyzed through the use of Affymetrix GeneChip software separately for each time point. A preliminary clustering analysis of the data suggested that the control and 24-hour specimens clustered separately from the samples from 48, 72, and 96 hours. This suggested that the HNF3β-induced signature becomes prominent after 24 hours. This finding was not surprising because in this system, HNF3β induction on the protein level is not observed until 24 to 48 hours of doxycycline withdrawal. Therefore, given the clustering analysis results, we carried out comparative marker selection by grouping the samples into a 0 + 24–hour class and a 48 + 72 + 96–hour class, and then comparing the relative expression between the two groups. Genes were ranked according to a two-group t statistic, permutation-based P values were computed, and the false discovery rate procedure was used to correct for multiple hypothesis testing (16, 17). Marker genes with a false discovery rate of <0.01 and at least 2-fold difference in expression levels between the two groups were selected as the group of HNF3β-regulated genes (Supplementary Tables S1 and S2).

HNF3β increases 15-PGDH expression in lung cancer cells. Among the genes induced by HNF3β, 15-PGDH was identified as overexpressed by four GeneChip probe sets with an approximate 3-fold increase. 15-PGDH is a metabolic enzyme of proliferative prostaglandins and through that an important antagonistic enzyme to Cox-2 (18). Given the important role of prostaglandin metabolism and Cox-2 in cancer, including lung cancer (19, 20), we focused our further studies on the delineation of 15-PGDH regulation by HNF3β. H358-HNF3β (tet-off) cells were maintained in the absence of doxycycline for up to 144 hours. Total RNAs were then collected for real-time reverse transcription-PCR. We observed an up-regulation of 15-PGDH mRNA as early as 12 hours after doxycycline withdrawal. The mRNA level peaked at 72 hours (10-fold) for clone 4 and at 96 hours (15-fold) for clone 31 (Fig. 1A ; Supplementary Fig. S1). Three other genes induced by HNF3β, including early growth response 4 (EGR4), complement component 5 (C5), and proteoglycan 1 (PRG1) were also confirmed by quantitative PCR (Fig. 1B). A significant increase of 15-PGDH protein was also found 24 hours after doxycycline withdrawal, reaching its peak level at 48 hours while starting to diminish at 72 hours (Fig. 1C). Transient transfection of HNF3β using squamous carcinoma Calu-1 cells and adenocarcinoma SKLU-1 cells (both with very weak expression of HNF3β and 15-PGDH) showed that HNF3β protein was induced at 24 hours after transfection and its level peaked at 48 hours in both cell lines; as expected with a transient transfection experiment, at 96 hours the expression decreased dramatically in Calu-1 cells whereas expression was totally absent in SKLU-1 (Fig. 1D). The expression of 15-PGDH became apparent at 48 hours after transfection and decreased in correlation with HNF3β expression in both cell lines (Fig. 1D). These data suggest that 15-PGDH expression is increased by HNF3β in a dose-dependent manner in human lung cancer cells and that 15-PGDH could be a potential downstream target of HNF3β's tumor suppressor activity.


Figure 1
View larger version (33K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 1. Expression of 15-PGDH is induced by HNF3β in lung cancer cells. A, quantitative reverse transcription-PCR of 15-PGDH in H358-HNF3β (tet-off) cells (clones 6108-4 and 6108-31) maintained in doxycycline-free medium for 0, 12, 24, 48, 72, or 96 h. B, quantitative reverse transcription-PCR of EGR4, 15-PGDH, C5, and PRG-1 in H358-HNF3β (tet-off) cells (clone 6108-4) cultured in doxycycline-free medium for 0, 12, 24, 48, 72, or 96 h. Fold induction relative to 0 h control (A and B) was plotted against time courses after normalization by glyceraldehyde-3-phosphate dehydrogenase. C, immunoblotting of clone 6108-4 cells maintained in doxycycline-free medium for 0, 12, 24, 48, 72, or 96 h. D, Calu-1 and SKLU-1 cells were transfected with pcDNA3-ratHNF3β; whole cell lysate was collected 0, 24, 48, 72, or 96 h after transfection and Western blotted with antibodies against HNF3β and 15-PGDH.

 
HNF3β regulates 15-PGDH by direct binding to and modulation of the 15-PGDH promoter. To further determine the mechanism of how HNF3β regulates the expression of 15-PGDH, we compared the promoter activity of the 15-PGDH gene in the presence or absence of HNF3β. The promoter region of the 15-PGDH gene has been carefully mapped out previously (12, 21). Two firefly luciferase reporter constructs were used for transfection: pcDNA3(+)-pp5.9-Firefly luciferase (pp5.9), in which the expression of luciferase is driven by a fragment 5.9 kb upstream of the start codon of 15-PGDH (GenBank accession no. AF177983, NT_022792; ref. 22) and pcDNA3(+)-pp2.2-Firefly luciferase (pp2.2), in which the luciferase expression is driven by a fragment of the 15-PGDH promoter containing –1 to –2233 bp. phRL-CMV-Renilla luciferase reporter was used to normalize transfection efficiency. Promoter activity with either construct increased 24 hours after HNF3β induction with a 3-fold and a 7-fold change for pp2.2 and pp5.9, whereas the activity peaked at 96 hours with an 8-fold and 12-fold change for pp2.2 and pp5.9, respectively (Fig. 2A ). This suggested that the 15-PGDH promoter indeed is regulated by HNF3β and that there might be more than one regulation site for HNF3β that acts synergistically. The human 15-PGDH gene promoter contains two potential binding sites for the HNF3β transcription factor: –3793 to –3778 bp and –446 to –430 bp (MatInspector; Fig. 2B). Chromatin immunoprecipitation (ChIP) assays were performed to assess physical binding of HNF3β to these putative promoter elements. H358-HNF3β (tet-off) cells were used and HNF3β expression was induced. Protein-DNA complexes were cross-linked, DNA was fragmented, and nuclear extracts were prepared at different time points upon HNF3β induction and immunoprecipitation was then performed with an HNF3β-specific antibody. The expression level of HNF3β was increased 24 hours after induction as shown by the input control (Fig. 2C). The amount of immunoprecipitated HNF3β followed the same pattern as that of HNF3β expression (Fig. 2C). By using PCR, we detected the presence of both hypothesized 15-PGDH promoter elements in the precipitates (Fig. 2C), indicating the direct binding of HNF3β. Moreover, we observed that the level of these two promoter sequences increased along with the amount of HNF3β precipitated (Fig. 2D). Electrophoretic mobility shift assay (EMSA) assays further confirmed the interaction between the HNF3β and 15-PGDH promoters (Fig. 3A and B ). Although there was no binding seen using nuclear extracts collected from uninduced cells, strong binding was observed using nuclear extracts obtained after 96 hours of induction. This binding activity could be competed away by the use of complementary cold oligonucleotides but not by the use of oligonucleotides with mutations in the predicted HNF3β-binding site. Lastly, Western blotting showed specific HNF3β activity on transferred DNA-protein complexes, further confirming specificity.


Figure 2
View larger version (27K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 2. HNF3β increases 15-PGDH gene promoter activity by direct binding to two regions of the promoter. A, H358-HNF3β (tet-off) cells were transfected with 15-PGDH promoter conjugated luciferase reporters: pcDNA3-pp2.2-Firefly luciferase (pp2.2) or pcDNA3-pp5.9-Firefly luciferase (pp5.9; pp2.2 and pp5.9 contain 15-PGDH promoter sequences –1 to –2233 bp and –1 to –5950 bp, respectively). Twenty-four hours after transfection, cells were maintained in doxycycline-free RPMI 1640 for 0, 24, 48, 72, or 96 h. Dual luciferase reporter assays were conducted and Firefly luciferase activity was normalized by Renilla luciferase activity. Fold changes relative to 0 h control were plotted against time courses. B, a simplified structural map of the 15-PGDH gene with two predicted HNF3β binding sites. C and D, ChIP assays of H358-HNF3β (tet-off) cells. Cells were cultured in doxycycline-free medium for 0, 24, 48, 72, 96, and 120 h. ChIP assays were conducted with anti-HNF3β antibody. C, precipitated HNF3β was confirmed by Western blotting. 15-PGDH promoter elements were detected in precipitates by PCR (C) and further quantified by real-time PCR (D). ChIP, HNF3β precipitation; NE, nuclear extract (nuclear extract input was used as a control).

 

Figure 3
View larger version (35K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 3. EMSA assay shows HNF3β binding to 15-PGDH promoter sequences. A, EMSA using biotin-labeled oligonucleotides identical to HNF3β-binding sites (nos. 1 and 2) in 15-PGDH promoter and nuclear extracts from H358-HNF3β (tet-off) cells (clone 6108-4) maintained in doxycycline-free medium for 0 or 96 h. Unlabeled DNA competitor: c, cold complementary dsDNA; d, cold mutated dsDNA; e, cold complementary dsDNA containing sequences of the other binding site; f, cold mutated dsDNA containing sequences of the other binding site. Lanes 1-3, experimental controls using the EBNA control system containing biotin-labeled EBNA-binding sequence (c1) alone or in the presence of EBNA-1 protein extract (c2), and protein extract as well as 200-fold excess unlabeled ENBA binding sequence (c3). B, the mixture of binding reaction between biotin-labeled sequences and nuclear extracts from H358-HNF3β (tet-off) cells (clone 6108-4) maintained in doxycycline-free medium for 0 or 96 h was electrophoresed on DNA retardation gel and transferred to a nitrocellulose membrane, then immunoblotted with anti-HNF3β antibody.

 
15-PGDH is down-regulated in human lung cancer cells and human lung tumors and its expression correlates with HNF3β expression. We previously observed the loss of HNF3β in >50% of human lung cancer cell lines (8). Because the expression of 15-PGDH is regulated by HNF3β, we decided to investigate the expression of 15-PGDH in human lung cancers. We found a lack of 15-PGDH expression in 11 of 16 (69%) lung cancer cell lines (Fig. 4A ). A positive correlation between 15-PGDH and HNF3β expression was found to be significant (P < 0.05; Fig. 4A). Then, 78 paraffin-embedded, primary non–small cell lung tumors obtained from patients undergoing resection were stained by immunohistochemistry. 15-PGDH was strongly expressed in the nuclei of type I pneumocytes in normal lung tissues whereas no expression was found in either type II pneumocytes or bronchial epithelia (Fig. 4B). We set up a scoring system similar to our previously reported scoring system for CAAT/enhancer binding protein-{alpha} (4). The expression of 15-PGDH was undetectable or very weak (0–1+) in 49 out of 78 (63%) samples examined and there was a significant association between 15-PGDH expression and tumor histology (P = 0.006): 55.3% of patients with adenocarcinomas had 15-PGDH–positive tumors as compared with only 12.5% patients with squamous carcinoma. This might potentially reflect the cellular origin of these tumors with squamous cell cancers typically originating from more proximal airways (bronchial epithelium) in which 15-PGDH is not normally expressed versus adenocarcinomas originating from more peripheral (bronchiolar or alveolar) epithelium. We also completed an immunohistochemical study for HNF3β expression using 59 of the same tumor slides and found a significant association between HNF3β and 15-PGDH expression (P = 0.043, {chi}2 analysis). Twenty-four percent of the HNF3β-negative tumors and 50% of the HNF3β-positive tumors were 15-PGDH positive (Fig. 4C).


Figure 4
View larger version (39K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 4. 15-PGDH is down-regulated in human lung cancer cells and human non–small cell lung tumors. A, Western blotting of 15-PGDH and HNF3β in 16 lung cancer cell lines. B, immunohistochemical assay of 15-PGDH in normal lung epithelia (N) vs. lung cancers (T0, T1, T2, and T3). T0, T1, T3, and T3 were determined based on staining density, with T0 to T1 categorized as negative or weak staining (–) and T2 to T3 as strong staining (+). C, immunohistochemical assay of HNF3β in normal lung epithelium (N) vs. lung cancers (T0-3). Distribution of samples as per 15-PGDH versus HNF3β staining (right).

 
Tumor suppressor activity of 15-PGDH in lung cancer. Loss of 15-PGDH expression found in human lung cancer cells and lung tumor tissues, and the regulation of 15-PGDH by the tumor suppressor HNF3β, both suggest that 15-PGDH might have tumor suppressor activity in lung cancer similar to its role in other malignancies. To test this hypothesis, we first conducted in vitro assays using transient transfection of human 15-PGDH in H358 cells. H358 cells were transfected with pcDNA6 empty vector (EV), pcDNA6-Mu-PGDH (Mu) encoding an enzyme dead 15-PGDH mutant (Y151L and K155E; ref. 23) or pcDNA6-WT-PGDH (WT) encoding WT 15-PGDH. Whole cell lysates were collected 72 hours later and used in immunoblotting. Fragmentation of PARP (85 kDa) was undetectable in any of the three groups of transfected cells and the level of the intact caspase substrate in cells expressing WT 15-PGDH was similar to that of empty vector control cells or cells expressing the 15-PGDH mutant (Supplementary Fig. S2A). MTS cell growth assay performed 48, 72, and 96 h after transfection also showed no difference between the three groups (P > 0.05, Supplementary Fig. S2B). The percentage of apoptotic cells detected by Annexin/propidium iodide staining was also indistinguishable between WT 15-PGDH–expressing cells and control cells (Supplementary Fig. S2C). These in vitro data show no cell-autonomous growth effect of 15-PGDH on lung cancer cells. We then used stable transfection to reconstitute 15-PGDH expression in H358 lung cancer cells to test 15-PGDH function in vivo. H358 cells were transfected with an expression vector encoding WT 15-PGDH, or with a control empty expression vector. Expression of 15-PGDH was confirmed by immunoblotting of stable pools (Fig. 5A ). Similar MTS cell growth assays and Annexin/propidium iodide apoptosis flow cytometry assays were also performed on H358-PGDH stable cells and no significant difference was found between EV control cells and cells that express 15-PGDH (Fig. 5B). The PGE2 level in the culture medium of 15-PGDH–expressing H358-PGDH-WT cells was significantly lower than that of empty vector control cells (P < 0.001; Fig. 5C), confirming the negative regulation of PGE2 levels by 15-PGDH. Next, we performed a xenograft study by injecting groups of five athymic mice with H358-PGDH-WT cells or control H358-EV cells (two injections per mice). 15-PGDH markedly suppressed tumor formation in vivo: tumors arising from 15-PGDH–expressing cells were significantly and on average by 50% smaller than those from control cells (Fig. 5D).


Figure 5
View larger version (23K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 5. Tumor suppressor activity of 15-PGDH in vitro and in vivo. Stably transfected H358-PGDH cells (pools) were generated by transfection of H358 with pcDNA6-EV or pcDNA6-WT PGDH (WT) and selection with blasticidin. A, Western blotting of whole cell lysates with anti–15-PGDH shows strong expression of 15-PGDH in WT-PGDH–transfected cells. H358-PGDH stable cells were then used in MTS cell growth assays and Annexin/propidium iodide apoptosis assays (B). C, ELISA of extracellular PGE2 levels (*, P < 0.001). D, growth of xenograft tumors in athymic mice (*, P < 0.05; {dagger}, P = 0.054) when including an outlier in the EV group (no tumor growth in the first 2 wk and a tumor with 13.5 and 32 mm3 in volume at the 3rd and 4th weeks, respectively) and P < 0.05 when this outlier was excluded.

 
These findings of an inhibitory role of 15-PGDH in in vivo tumorigenic growth, but not in in vitro cultured cells, are consistent with what has been found in colon cancers, suggesting a possible cell heterologous mechanism of 15-PGDH function, in which 15-PGDH prevents cell growth by lowering PGE2 level and thus inhibiting angiogenesis as opposed to directly affecting cellular growth (13). To test this, we compared the microvessel densities between xenografts derived from 15-PGDH expressing H358 cells and those from control H358 cells (H358-EV), and found significantly reduced MVD in tumor tissues with 15-PGDH overexpression (Fig. 6A ). At 400x microscopic magnification, the mean ± SD of MVD was 2.87 ± 0.70 for xenograft tumors with 15-PGDH overexpression compared with 4.80 ± 0.35 for xenografts without 15-PGDH overexpression (P < 0.05; Fig. 6B). Because PGE2 levels are believed to mediate their angiogenic effect through modulation of VEGF expression via EP receptor activation (24), we decided to test VEGF levels from conditioned medium collected from H358-WT-PGDH cells and indeed found that VEGF levels were 25% lower than that from H358-EV cells (P < 0.01; Fig. 6C). Next, we performed immunohistochemistry on xenografted tumors to assess in vivo changes in VEGF expression. These studies showed a significant decrease of VEGF expression in 15-PGDH expressing WT H358 cells (Fig. 6A). Last, we performed endothelial proliferation and functional assays (Matrigel tube formation assay) using conditioned medium obtained from H358-WT and H358-EV cells (Fig. 6D). Although no significant difference was seen on endothelial cell proliferation, H358-WT conditioned medium significantly reduced endothelial cell function consistent with a paracrine effect of 15-PGDH expressing lung cancer cells on endothelial cell function, likely by PGE2-mediated reduction in VEGF levels.


Figure 6
View larger version (31K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 6. Effects of 15-PGDH on tumor angiogenesis. A, 5-µmol/L-thick formalin-fixed paraffin-embedded xenograft tissue sections were deparaffinized, rehydrated, and stained with rat anti-mouse CD31, anti-VEGF or anti–15-PGDH antibody. B, MVD was evaluated at x400 microscopic magnification (*, P < 0.05). C, effects of 15-PGDH expression on VEGF production (*, P < 0.01). D, no significant change in proliferation of human umbilical vein endothelial cells was observed in the presence of conditioned medium of H358-EV vs. H358-WT cells whereas Matrigel tube formation assays showed reduced capillary tube formation of human umbilical endothelial cells grown in the presence of conditioned medium of H358-WT vs. H358-EV cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 References
 
Our studies show that 15-PGDH is a direct target and a downstream effector enzyme of the candidate tumor suppressor, HNF3β, in lung cancer cells. The loss of 15-PGDH expression correlates with histologic subtype in non–small cell lung cancer specimens. Our studies also reveal potent in vivo tumor-suppressive properties of 15-PGDH in lung cancer cells mediated by an antiangiogenic mechanism. The 15-PGDH gene is located on chromosome 4q34-35 and encodes a 29 kDa protein that is active as a homodimer. Via NAD+-dependent oxidation of the 15(S)-hydroxyl group of prostaglandins and lipoxins, 15-PGDH is a key enzyme responsible for their biological inactivation (18). The enzyme is widely distributed in various mammalian tissues among which the lung is one of the most active tissues and genetic deletion of 15-PGDH leads to increased tissue levels of PGE2. We previously showed that the forkhead transcription factor, HNF3β acts as a candidate tumor suppressor in non–small cell lung cancer, and here, we show that 15-PGDH is one of the most highly induced genes by HNF3β. Although it remains to be determined as to what extent the tumor-suppressing activity of HNF3β is mediated by 15-PGDH, our data clearly show that 15-PGDH expression is directly regulated by HNF3β in human lung cancer cells and also show in vivo growth-suppressive properties of 15-PGDH per se via an antiangiogenic mechanism, likely mediated by the reduction of PGE2 levels and as a consequence, regulation of VEGF expression. Our studies therefore identify 15-PGDH as a candidate tumor suppressor in human lung cancers. Despite its ubiquitous expression in normal alveolar epithelium, 15-PGDH is lost in 69% of human lung cancer cell lines and in 63% of human lung tumor tissues, which strongly suggests a connection between the lack of 15-PGDH expression and tumor development/progression. The fact that the absence of 15-PGDH is more commonly observed in squamous carcinoma (87.5%) than in adenocarcinoma (44.7%) may indicate potentially cell type–specific tumor-suppressive mechanisms and could be of significant utility in both diagnosis and choosing therapeutic strategies. Whether the lack of 15-PGDH expression observed is indeed of functional relevance will need to be ultimately determined by in vivo genetic studies using 15-PGDH knockout mice as previously done in colon cancer (25). Similar to our data, others have noted down-regulation of 15-PGDH in lung tumors (26, 27). One major limitation of both sets of data is the use of bulk RNA and protein derived from resected specimens not allowing a valid evaluation of its distribution, in particular, considering the mix of tissue types a lung specimen is made up of. The immunohistochemical method we have developed allows more detailed mapping of 15-PGDH expression.

We also explored the mechanism of how 15-PGDH could be a potential tumor suppressor by both in vitro cell growth assays and in vivo tumorigenic experiments. We observe no in vitro effect of 15-PGDH overexpression on growth or apoptosis in H358 lung cancer cells, although we did find that 15-PGDH expression markedly reduces tumor formation in a xenograft model suggestive of a cell-heterologous manner in 15-PGDH tumor suppressor activity in which 15-PGDH prevents tumor growth by inhibiting tumor angiogenesis, analogous to its functional role in colon cancer (13). Overexpression of 15-PGDH in H358 lung cancer cells with modest endogenous 15-PGDH expression further decreases the level of secreted PGE2. Although the observed reduction in PGE2 levels was modest, similar changes in PGE2 levels in other studies were shown to be of functional significance. For example, in a study of Cox-2 knockout mice, PGE2 levels in the mammary gland were reduced by ~20% in heterozygous versus wild-type animals, and this change was associated with a significant reduction in tumor multiplicity (28). This finding was further corroborated by our in vitro and in vivo findings of reduced VEGF expression. Furthermore, our MVD analysis of mouse xenograft tissues and in vitro endothelial cell function studies supports the role of 15-PGDH expression in reducing tumor angiogenesis via modulation of PGE2, and secondarily, VEGF levels, although it cannot be excluded that some of its effects are at least in part mediated by some alternative mechanisms. Because 15-PGDH is the rate-limiting enzyme catalyzing the degradation of PGE2 synthesized by Cox-2 and acts as a physiologically negative regulator of prostaglandin levels, its important functional role in cancers is not surprising (18, 29). The suppression by 15-PGDH of in vivo tumorigenic growth but not of growth in cell culture, is consistent with suggestions from several models that the tumor-promoting effect of increased prostaglandin synthesis is principally mediated via increased tumor angiogenesis (30, 31). Although there was a strong correlation noted between HNF3β and 15-PGDH expression, a fair number of PGDH-negative tumors still express HNF3β, suggestive of alternative mechanisms for 15-PGDH silencing. The 15-PGDH promoter contains a CpG island in the region –163 to +140 relative to the start ATG codon, and promoter methylation of the 15-PGDH promoter has been previously noted in breast and prostate cancer (32, 33). These suggest that promoter methylation is a potential mechanism for the deregulation of 15-PGDH in non–small cell lung cancer and should lead to further investigation of the methylation of the 15-PGDH promoter. The chromosomal locus of 15-PGDH, 4q34-35, was found to be one of the most commonly lost regions in the genome-wide allelotyping study of Girard and colleagues, suggestive of an important unidentified tumor suppressor at this locus (34). Our results suggest that the 15-PGDH gene might be a prime candidate for such. Thus, 15-PGDH is a tumor suppressor whose activity is increased by HNF3β-regulated expression. We postulate that the loss of 15-PGDH activity could provide a mechanism for tumor progression and drug resistance. It has been shown that 15-PGDH expression can be restored in certain colon cancer cell lines either by restoring transforming growth factor-β signaling or by inhibiting EGFR signaling (13, 28), and a recent study also showed the regulation of 15-PGDH in lung cancer cells by EGF signaling (26). It is also intriguing that Wolfrum and colleagues recently showed that the activation of phosphoinositide-3-kinase AKT by insulin induced Foxa2 phosphorylation, nuclear exclusion, and thereby, inhibition of Foxa2-dependent transcriptional activity in liver cells, potentially suggestive of the existence of an EGFR-AKT-Foxa2 pathway modulating 15-PGDH activity in lung cancer cells (35). These findings suggest several potential ways of modulating 15-PGDH expression and activity, and raise the possibility of 15-PGDH as a potential target in developing lung cancer therapy. Modulation of prostaglandin metabolism, in particular, through the use of Cox-2 inhibitors, has been an area of active investigation in cancer research over the last few years (19, 36). Although the tumor-suppressive and preventative effects of Cox-2 inhibition seem valid, their use is associated with increased thromboembolic phenomena limiting their clinical utility. The prothrombotic effects of Cox-2 inhibition are felt to be at least in part mediated by prostaglandin I2. Because 15-PGDH does not alter the levels of the rapidly hydrolyzable, antithrombotic prostaglandin, prostaglandin I2, its modulation might avoid some of the cardiovascular side effects associated with Cox-2 inhibition and lead to treatment options with a better risk-benefit profile.


   Disclosure of Potential Conflicts of Interest
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
References
 
No potential conflicts of interest were disclosed.


   Acknowledgments
 
Grant support: Young Clinical Scientist Award from the Flight Attendant Medical Research Institute (B. Halmos), NIH R21 CA119545-02 (J. Merchan), NIH/NCI "Specialized Programs of Research Excellence in Human Lung Cancer" P50 CA90578-04 (B. Halmos and D.G. Tenen), and NIH U54 CA116867 and NIH RO1 CA127306 (S.D. Markowitz).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Received 12/10/07. Revised 4/22/08. Accepted 4/24/08.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 

  1. Jemal A, Siegel R, Ward E, Murray T, Xu J, Thun MJ. Cancer statistics, 2007. CA Cancer J Clin 2007;57:43–66.[Abstract/Free Full Text]
  2. Breathnach OS, Freidlin B, Conley B, et al. Twenty-two years of phase III trials for patients with advanced non-small-cell lung cancer: sobering results. J Clin Oncol 2001;19:1734–42.[Abstract/Free Full Text]
  3. Sato M, Shames DS, Gazdar AF, Minna JD. A translational view of the molecular pathogenesis of lung cancer. J Thorac Oncol 2007;2:327–43.[Medline]
  4. Halmos B, Huettner CS, Kocher O, Ferenczi K, Karp DD, Tenen DG. Down-regulation and antiproliferative role of C/EBP{alpha} in lung cancer. Cancer Res 2002;62:528–34.[Abstract/Free Full Text]
  5. Basseres DS, Levantini E, Ji H, et al. Respiratory failure due to differentiation arrest and expansion of alveolar cells following lung-specific loss of the transcription factor C/EBP{alpha} in mice. Mol Cell Biol 2006;26:1109–23.[Abstract/Free Full Text]
  6. Costa DB, Dayaram T, D'Alo F, et al. C/EBP{alpha} mutations in lung cancer. Lung Cancer 2006;53:253–4.[CrossRef][Medline]
  7. Tada Y, Brena RM, Hackanson B, Morrison C, Otterson GA, Plass C. Epigenetic modulation of tumor suppressor CCAAT/enhancer binding protein {alpha} activity in lung cancer. J Natl Cancer Inst 2006;98:396–406.[Abstract/Free Full Text]
  8. Halmos B, Basseres DS, Monti S, et al. A transcriptional profiling study of CCAAT/enhancer binding protein targets identifies hepatocyte nuclear factor 3β as a novel tumor suppressor in lung cancer. Cancer Res 2004;64:4137–47.[Abstract/Free Full Text]
  9. Costa RH, Kalinichenko VV, Lim L. Transcription factors in mouse lung development and function. Am J Physiol 2001;280:L823–38.
  10. Gentleman RC, Carey VJ, Bates DM, et al. Bioconductor: open software development for computational biology and bioinformatics. Genome Biol 2004;5:R80.[CrossRef][Medline]
  11. Irizarry RA, Hobbs B, Collin F, et al. Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics 2003;4:249–64.[Abstract]
  12. Greenland KJ, Jantke I, Jenatschke S, Bracken KE, Vinson C, Gellersen B. The human NAD+-dependent 15-hydroxyprostaglandin dehydrogenase gene promoter is controlled by Ets and activating protein-1 transcription factors and progesterone. Endocrinology 2000;141:581–97.[Abstract/Free Full Text]
  13. Yan M, Rerko RM, Platzer P, et al. 15-Hydroxyprostaglandin dehydrogenase, a COX-2 oncogene antagonist, is a TGF-β-induced suppressor of human gastrointestinal cancers. Proc Natl Acad Sci U S A 2004;101:17468–73.[Abstract/Free Full Text]
  14. Merchan JR, Chan B, Kale S, Schnipper LE, Sukhatme VP. In vitro and in vivo induction of antiangiogenic activity by plasminogen activators and captopril. J Natl Cancer Inst 2003;95:388–99.[Abstract/Free Full Text]
  15. Merchan JR, Jayaram DR, Supko JG, He X, Bubley GJ, Sukhatme VP. Increased endothelial uptake of paclitaxel as a potential mechanism for its antiangiogenic effects: potentiation by Cox-2 inhibition. Int J Cancer 2005;113:490–8.[CrossRef][Medline]
  16. Benjamini Y, Hochberg Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc 1995;57:289–300.
  17. Gould J, Getz G, Monti S, Reich M, Mesirov JP. Comparative Gene Marker Selection suite. Bioinformatics 2006;22:1924–5.[Abstract/Free Full Text]
  18. Tai HH, Cho H, Tong M, Ding Y. NAD+-linked 15-hydroxyprostaglandin dehydrogenase: structure and biological functions. Curr Pharm Des 2006;12:955–62.[CrossRef][Medline]
  19. Sandler AB, Dubinett SM. COX-2 inhibition and lung cancer. Semin Oncol 2004;31:45–52.[Medline]
  20. Brown JR, DuBois RN. Cyclooxygenase as a target in lung cancer. Clin Cancer Res 2004;10:4266–9s.[CrossRef]
  21. Nandy A, Jenatschke S, Hartung B, Milde-Langosch K, Bamberger AM, Gellersen B. Genomic structure and transcriptional regulation of the human NAD+-dependent 15-hydroxyprostaglandin dehydrogenase gene. J Mol Endocrinol 2003;31:105–21.[Abstract]
  22. International Human Genome Sequencing Consortium. Finishing the euchromatic sequence of the human genome. Nature 2004;431:931–45.[CrossRef][Medline]
  23. Ensor CM, Tai HH. Bacterial expression and site-directed mutagenesis of two critical residues (tyrosine-151 and lysine-155) of human placental NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase. Biochim Biophys Acta 1994;1208:151–6.[CrossRef][Medline]
  24. Fukuda R, Kelly B, Semenza GL. Vascular endothelial growth factor gene expression in colon cancer cells exposed to prostaglandin E2 is mediated by hypoxia-inducible factor 1. Cancer Res 2003;63:2330–4.[Abstract/Free Full Text]
  25. Myung SJ, Rerko RM, Yan M, et al. 15-Hydroxyprostaglandin dehydrogenase is an in vivo suppressor of colon tumorigenesis. Proc Natl Acad Sci U S A 2006;103:12098–102.[Abstract/Free Full Text]
  26. Yang L, Amann JM, Kikuchi T, et al. Inhibition of epidermal growth factor receptor signaling elevates 15-hydroxyprostaglandin dehydrogenase in non-small-cell lung cancer. Cancer Res 2007;67:5587–93.[Abstract/Free Full Text]
  27. Ding Y, Tong M, Liu S, Moscow JA, Tai HH. NAD+-linked 15-hydroxyprostaglandin dehydrogenase (15-PGDH) behaves as a tumor suppressor in lung cancer. Carcinogenesis 2005;26:65–72.[Abstract/Free Full Text]
  28. Howe LR, Chang SH, Tolle KC, et al. HER2/neu-induced mammary tumorigenesis and angiogenesis are reduced in cyclooxygenase-2 knockout mice. Cancer Res 2005;65:10113–9.[Abstract/Free Full Text]
  29. Tai HH, Ensor CM, Zhou H, Yan F. Structure and function of human NAD(+)-linked 15-hydroxyprostaglandin dehydrogenase. Adv Exp Med Biol 2002;507:245–50.[Medline]
  30. Tsujii M, Kawano S, Tsuji S, Sawaoka H, Hori M, DuBois RN. Cyclooxygenase regulates angiogenesis induced by colon cancer cells. Cell 1998;93:705–16.[CrossRef][Medline]
  31. Wang D, DuBois RN. Cyclooxygenase 2-derived prostaglandin E2 regulates the angiogenic switch. Proc Natl Acad Sci U S A 2004;101:415–6.[Free Full Text]
  32. Wolf I, O'Kelly J, Rubinek T, et al. 15-hydroxyprostaglandin dehydrogenase is a tumor suppressor of human breast cancer. Cancer Res 2006;66:7818–23.[Abstract/Free Full Text]
  33. Lodygin D, Epanchintsev A, Menssen A, Diebold J, Hermeking H. Functional epigenomics identifies genes frequently silenced in prostate cancer. Cancer Res 2005;65:4218–27.[Abstract/Free Full Text]
  34. Girard L, Zochbauer-Muller S, Virmani AK, Gazdar AF, Minna JD. Genome-wide allelotyping of lung cancer identifies new regions of allelic loss, differences between small cell lung cancer and non-small cell lung cancer, and loci clustering. Cancer Res 2000;60:4894–906.[Abstract/Free Full Text]
  35. Wolfrum C, Besser D, Luca E, Stoffel M. Insulin regulates the activity of forkhead transcription factor Hnf-3β/Foxa-2 by Akt-mediated phosphorylation and nuclear/cytosolic localization. Proc Natl Acad Sci U S A 2003;100:11624–9.[Abstract/Free Full Text]
  36. Reckamp KL, Krysan K, Morrow JD, et al. A phase I trial to determine the optimal biological dose of celecoxib when combined with erlotinib in advanced non-small cell lung cancer. Clin Cancer Res 2006;12:3381–8.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 CarcinogenesisHome page
A. E. Moore, A. Greenhough, H. R. Roberts, D. J. Hicks, H. A. Patsos, A. C. Williams, and C. Paraskeva
HGF/Met signalling promotes PGE2 biogenesis via regulation of COX-2 and 15-PGDH expression in colorectal cancer cells
Carcinogenesis, October 1, 2009; 30(10): 1796 - 1804.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
A. Thiel, A. Ganesan, J. Mrena, S. Junnila, A. Nykanen, A. Hemmes, H.-H. Tai, O. Monni, A. Kokkola, C. Haglund, et al.
15-Hydroxyprostaglandin Dehydrogenase Is Down-regulated in Gastric Cancer
Clin. Cancer Res., July 15, 2009; 15(14): 4572 - 4580.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Prevention ResearchHome page
S. M. Dubinett,, J. T. Mao, and S. Hazra
Focusing Downstream in Lung Cancer Prevention: 15-Hydroxyprostaglandin Dehydrogenase
Cancer Prevention Research, September 1, 2008; 1(4): 223 - 225.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplementary Data
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Huang, G.
Right arrow Articles by Halmos, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Huang, G.
Right arrow Articles by Halmos, B.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online