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Departments of 1 Clinical Preventive Medicine, 2 Respiratory Medicine, and 3 Thoracic Surgery, Nagoya University School of Medicine, Nagoya, Japan
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Krüppel-like factor (KLF) family members are transcriptional factors, which bind GC box and up-regulate and/or down-regulate the expression of target genes. Functionally, the KLF family members are shown to be involved in cell differentiation, development, growth related signal transduction, cell proliferation, and apoptosis (8) . KLF6 contains a proline- and serine-rich NH2-terminal activation domain, and like other KLFs, three COOH-terminal C2H2 zinc fingers. Whereas several members of the KLF family have been shown to be involved in carcinogenesis, such as down-regulation of KLF4 found in colon cancer (9) , and KLF5 and KLF10 in breast cancer (10 , 11) , KLF6 has also been suggested to be a candidate TSG at 10p15, with frequent mutations observed in prostate adenocarcinoma (12) . Furthermore, KLF6 was also shown to transactivate WAF1, which encodes a cyclin-dependent kinase inhibitor of the cell cycle via a p53-independent pathway (12) .
Because the KLF6 gene is located on chromosome 10p15, which has been indicated to be a site showing frequent allelic loss in NSCLC (7) , we hypothesized that the KLF6 gene might be the target TSG for NSCLC as well. Moreover, a cDNA microarray analysis that showed frequent KLF6 gene down-regulation in lung adenocarcinoma also seemed to support our hypothesis (13) . To determine this, we analyzed 105 NSCLCs for genetic mutation and 65 NSCLCs for mRNA expression. Although somatic mutations were not detected in the coding sequence of KLF6, expression of KLF6 mRNA was down-regulated in 9 of 9 (100%) cell lines and 55 of 65 (85%) surgical specimens compared with normal lung tissues. We detected LOH at the KLF6 locus in 21 of 62 (34%) informative cases. Comparing the LOH status with KLF6 expression, we found that 14 of 14 (100%) samples with LOH showed KLF6 down-regulation, and that 23 of 31 (74%) samples that retained both alleles also showed KLF6 down-regulation. Furthermore, we demonstrated that the exogenously transfected KLF6 suppressed tumor growth of lung cancer cells and revealed that this was due to apoptosis. Our studies suggested that the frequent down-regulation of KLF6 in NSCLC might be involved in lung cancer development.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Ethical approval was obtained from each of the five hospitals, and fully informed consent was obtained from all of the patients before tissue collection. The material comprised 62 adenocarcinomas, 27 squamous cell carcinomas, 4 large cell carcinomas, 2 adenosquamous cell carcinomas, and 1 undifferentiated carcinoma.
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Mutational Analysis.
We developed 7 primer sets from the KLF6 genomic sequence (GenBank accession no. AF001461) to cover the entire coding region of exon 1–4. Primer sets used were as follows (numbers after "ex" indicate exon number, and "S" and "AS" indicate sense and antisense, respectively. Exon 1 and 2 were analyzed with two and three primer sets, respectively): KLF6-ex1–1S, 5'-TGGCAGCGGAGCTTTGAATA-3', and KLF6-ex1–1AS, 5'-TCCTGGAAGATGCTGCACAT-3'; KLF6-ex1–2S, 5'-GAGCCTGGAGTTTGCATGAA-3', and KLF6-ex1–2AS, 5'-TGCGTTTACCTGTTGCCAGT-3'; KLF6-ex2–1S, 5'-TTCATTTTGCACAGACCTGC-3', and KLF6-ex2–1AS, 5'-TAAACTTGGCCGTGGGAGAAAGTT-3'; KLF6-ex2–2S, 5'-TTACAACTTAGAGACCAACAGCCT-3', and KLF6-ex2–2AS, 5'-CATTTCCCTTGTCACCTGGCTT-3'; KLF6-ex2–3S, 5'-GTTTTGGTCAGCTCGGGAAAATTG-3', and KLF6-ex2–3AS, 5'-AACTGAGCAGGGAACCTTCT-3'; KLF6-ex3–1S, 5'-TGAAGTCATGGGCTGCTTGT-3', and KLF6-ex3–1AS, 5'-CCTGGTCATCACATTCCCAA-3'; and KLF6-ex4–1S, 5'-GGAACCTAACTTCTTCTGTG-3', and KLF6-ex4–1AS, 5'-GGTGCTATGCCGCTTCTTACAGGA-3'. Twenty ng of genomic DNA was used as a template for PCR. In PCR for KLF6, we used a modified "Touchdown PCR" (15)
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For cell lines, PCR products were directly sequenced using an Applied Biosystems model 377 DNA sequencer (Perkin-Elmer, Norwalk, CT) with a PCR primer and a BigDye terminator 3.0 Cycle sequencing FS Ready Reaction kit (PE Applied Biosystems, Foster City, CA). For surgical specimens, PCR and single-strand conformation polymorphism analysis was performed as described previously (16) .
Northern Blot Analysis.
Northern blot was performed as described previously (17)
. The cDNA probes synthesized with reverse transcription-PCR were a 197-bp fragment covering from nucleotide 140 to nucleotide 337 of the KLF6 gene and a 494-bp fragment covering the entire coding sequence of the WAF1 gene. The mRNA levels of KLF6, WAF1, and ß-actin were quantified with an imaging analyzer (BAStation; Fujifilm, Tokyo, Japan), and the relative amounts of KLF6 and WAF1 were normalized with the amounts of ß-actin reprobed on the same membrane. Equal or less than 0.6 (KLF6) or 0.4 (WAF1) expression of a normal lung was defined as down-regulation.
Laser Capture Microdissection.
Laser capture microdissection was performed as described previously using a PixCell laser capture microscope (Arcturus Engineering, Mountain View, CA; Ref. 15
). In general, 2000 hits with a laser pulse were used to obtain >2000 cells. The cells were immersed in 40 µg of digestion buffer, containing 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.4 mg/ml proteinase K, and 1% Tween 20, and digested at 37°C overnight. After digestion, the enzyme was heat inactivated (95°C for 10 min), and the extract was directly used for allelotyping PCR. Genomic DNA extracted from the noncancerous lung tissues served as the normal control for LOH analysis.
LOH Analysis.
Genomic DNAs extracted from the microdissected tumor specimens were amplified by PCR, which was carried out in a 20-µl volume containing extracted DNA from at least 100 microdissected nuclei, 4 pmol of each primer for KLF6M1 and KLF6M2 (12)
, 200 µM of each deoxynucleoside triphosphate, and TaqDNA polymerase (Takara Bio, Otsu, Japan) containing 10 mM Tris (pH 8.3), 50 mM KCl, and 1.5 mM MgCl2. The analysis was performed using the ABI Genescan and Genotyper software packages (Perkin-Elmer). Relative allele ratio <0.65 was determined as LOH.
Transient Transfection and Colony Formation Assay.
Full-length KLF6-cDNA was amplified with reverse transcription-PCR using cDNA synthesized from normal lung RNA and a primer set of KLF6-forward, 5'-AGCGAATTCGACATGGACGTGCTCCCCATG-3' (italicized nucleotides indicate artificial EcoRI site), and KLF6-reverse, 5'-AGCTCTAGATCAGAGGTGCCTCTTCATGTG-3' (italicized nucleotides indicate artificial XbaI site). The PCR product was double-digested with EcoRI and XbaI and subcloned into the EcoRI and XbaI sites of pcDNA3.1 (+) (Invitrogen, Carlsbad, CA). Preparation of p53-construct is described elsewhere (18)
. The entire insert was confirmed by sequencing from both directions. For colony formation assay, 4 x 105 NCI-H1299 cells or 3 x 105 NCI-H2009 cells were seeded into six-well plates a day before transfection. Transient transfection of the KLF6 and p53 genes was performed with Lipofectamine PLUS reagent according to the manufacturer’s protocol (Invitrogen). Lung cancer cells in six-well dishes were transfected with 1 µg per well of the expression vector. After transfection, cells were trypsinized, replated, and cultured in 600 µg/ml G418 (G7034; Sigma, St. Louis, MO) supplemented medium (RPMI 1640 and 10% fetal bovine serum) for 2 weeks. The numbers of the G418-resistant colonies were counted after staining with methylene blue in ethanol/H2O (50/50%). The surviving colonies of the vector control were set at 100%. The data represent the mean ± SD of three independent experiments, each carried out in triplicate plates.
Western Blot Analysis.
Preparations of total cell lysates and Western blotting were performed as described previously (19)
. First antibodies used were anti-zf9 (KLF6) antibody (R-173; Santa-Cruz Biotechnology, Santa Cruz, CA), anti-p53 antibody (NCL-p53-DO7; Novocastra Laboratories, Newcastle, United Kingdom), anti-p21 (WAF1) antibody (H-164; Santa-Cruz Biotechnology), anti-caspase-3 antibody (Cell Signaling Technology, Beverly, MA), antipoly(ADP-ribose) polymerase antibody (Cell Signaling Technology, Inc.), and anti-ß-actin antibody (AC-15; Sigma, St. Louis, MO).
Apoptosis Assay.
For the Annexin V staining, the Annexin V-Biotin Apoptosis Detection kit (BV-K 109–3; MBL, Nagoya, Japan) was used as described previously (18)
. In brief, cells (0.5 x 105) were plated on cover glasses on 24-well plates the day before transfection. Twenty-four h after cotransfection with a construct and pEGFP-F vector (Clontech Laboratories Inc., Palo Alto, CA) as a cotransfection marker, cells were incubated with Annexin V-biotin for 5 min at room temperature. They were then incubated with streptavidin-Alexa 568 (Molecular Probes, Eugene, OR). The cover glasses were inverted on a drop of Mounting Medium (Shandon, Pittsburgh, PA) on slide glasses and observed with a confocal laser-scanning microscope (MRC1024, Bio-Rad Laboratories, Hercules, CA). For the terminal deoxynucleotidyl transferase-mediated nick end labeling assay, an in situ cell death detection kit (Roche, Mannheim, Germany) was used as described previously (18)
. Briefly, 36 h after cotransfection with pEGFP-F vector, the cells were fixed with 4% paraformaldehyde in PBS and terminal deoxynucleotidyl transferase-mediated nick end labeling reaction was performed according to the manufacturer’s protocol. For quantification, stained cells were counted, and the results were presented as percentage of green fluorescent protein-positive cell number. Three independent x200 fields containing a minimum of 300 green fluorescent protein-positive cells on three replicate slides were evaluated for each condition.
Statistical Analysis.
Fisher’s exact tests were used for the correlation analysis, and paired t tests were used for significance. Statistical calculations were performed using a computer statistical package (StatView version 5.0; SAS Institute Inc., Cary, NC).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Expression of the KLF6 Gene.
To determine whether the KLF6 gene was down-regulated in NSCLCs, we performed Northern blot analysis for the 9 cell lines and 65 surgical specimens that were available for RNA. Whereas all of the 9 cell lines showed down-regulation of KLF6 mRNA compared with normal lung tissue, 55 of 65 (84%) showed down-regulation of KLF6 mRNA (Fig. 1)
. We compared the KLF6 expression with the patient characteristics but did not find any association between the KLF6 expression and the histology or surgical-pathological stages of lung tumors (data not shown). Next, to determine whether the KLF6 gene is silenced by promoter hypermethylation, we tested 2 NSCLC cell lines, NCI-H1299 and NCI-H2009, which were shown to be down-regulated for KLF6 with a demethylation reagent, 5-azacytidine. However, we did not detect an induction of KLF6 expression in these cell lines after treatment of 5-azacytidine (data not shown).
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. We found that 55 of the 65 (85%) primary tumors had down-regulation of WAF1. Among the 55 tumors with WAF1 down-regulation, 48 (87%) showed simultaneous KLF6 down-regulation, although it was not statistically significant (P = 0.18; Table 2
). Furthermore, we analyzed the 65 tumors for p53 alteration and found that 27 cases had missense mutation, 12 had nonsense or frameshift mutation, and 26 had wild-type p53 (20)
. However, we did not find any significant correlation between p53 mutation and WAF1 expression status (P > 0.99; data not shown). Meanwhile, among the 9 cell lines, 6 cell lines (NCI-H157, -H460, -H920, -H1155, -H1299, and -H2009) showed down-regulation of WAF1 mRNA, whereas 3 did not (NCI-H226, -H358, and -H1666; Fig. 1A
).
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Next, we compared the LOH status with the expression status of KLF6 mRNA in the 63 samples for which we were able to perform both analyses, with 45 cases being informative and 18 cases not informative (Table 3)
. Of the 45 informative cases, 31 (69%) showed heterozygosity, and 14 (31%) cases showed allelic loss. Whereas all 14 cases with LOH showed down-regulation, 23 of the 31 (75%) cases with heterozygosity indicated down-regulation of the KLF6 gene. We detected a significant correlation between LOH status and KLF6 down-regulation in the informative cases (P = 0.04).
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). KLF6 also showed strong suppression for colony formation in both the NCI-H1299 (18 ± 7%) and NCI-H2009 (18 ± 9%) cells (P < 0.05).
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. First, we tested whether p53 induces WAF1 expression in NSCLCs. Of the 3 cell lines, WAF1 up-regulation was found in NCI-H1299 and NCI-H2009 but not in NCI-H920 (Fig. 3)
. Next, we transfected KLF6 into these 3 cell lines but did not find the induction of WAF1 in any of these cells (Fig. 3)
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, KLF6 transfection into the NCI-H1299 cells showed significantly higher percentages of Annexin V-positive cells than the empty pcDNA3.1 (+) vector. The percentage of Annexin V-positive cells in KLF6-transfected cells was equal to that in the p53-transfected cells (Fig. 4, A and B)
. Next, we performed terminal deoxynucleotidyl transferase-mediated nick end labeling assay, which detects the end stage of apoptosis. Transfection of the NCI-H1299 cells with KLF6 resulted in significantly higher percentages of terminal deoxynucleotidyl transferase-mediated nick end labeling-positive cells than the empty pcDNA3.1 (+) vector (Fig. 4, C and D)
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All 9 of the NSCLC cell lines examined and 55 of 65 (85%) primary NSCLCs showed down-regulation of the KLF6 with Northern blot analysis, including 37 of 44 (84%) adenocarcinomas. The frequency of NSCLCs with KLF6 down-regulation seems to be consistent with a previous study using a cDNA microarray and reverse transcription-PCR assays, which demonstrated that 8 of 14 (57%) primary adenocarcinomas showed down-regulation of KLF6 (13) . Meanwhile, with respect to allelic loss at 10p15, Girard et al. (7) reported that 40% of NSCLC cell lines showed LOH at 10p15 using D10S591 and D10S189 microsatellite markers by comparing them with the corresponding EBV-transformed B lymphoblastoid cell lines. We found that 21 of 62 (34%) informative cases showed LOH at the KLF6 gene locus. Thus, the present study also showed that NSCLCs have LOH at 10p15 with a frequency similar to other studies.
Comparing the mRNA expression level with LOH status of KLF6, all 14 cases with LOH showed down-regulation of KLF6. Moreover, 23 of 31 (75%) surgical specimens with heterozygosity also showed down-regulation of KLF6. Despite the frequent down-regulation of KLF6, no somatic mutation was detected in the coding region in NSCLCs compared with sporadic pituitary tumors (21) , astrocytic gliomas (22) , and prostate adenocarcinomas (12 , 23) , with the frequencies of mutation reported to be 5%, 5–11%, and 15–55%, respectively. Only a rare polymorphism was seen in 1 surgical specimen (KD623), which has not been identified before. Thus, a point mutation does not seem to be a common mechanism to inactivate KLF6 in NSCLCs.
NCI-H920 had a mutation (C to A) at 4 bp upstream from the starting codon and showed down-regulation of KLF6, which was identical to a mutation that was found in a prostate adenocarcinoma (23) . Whether this mutation could alter KLF6 expression or translation remains unclear; this mutation might be associated with down-regulation of KLF6. Conversely, KLF6 was also suggested to be a methylation-silenced gene in esophageal squamous cell carcinoma, because 5-aza-deoxycytidine treatment for the KYSE30 and KYSE410 cells induced up-regulation of KLF6 (24) . However, in our study, KLF6 expression was not induced by another demethylation reagent, 5-azacytidine, in the NCI-H1299 or -H2009 cells. Thus, although our results suggest that LOH may partly contribute to its down-regulation, additional studies seem to be necessary to identify the mechanism for the strong down-regulation of KLF6 expression in NSCLCs.
Transfection of the KLF6 gene expression construct markedly inhibited colony formation of NCI-H1299 and -H2009. Because NCI-H1299 is a p53-null cell line (25) , our data also indicate that KLF6 inhibits tumor cell growth through a p53-independent pathway. In prostate adenocarcinoma cells, KLF6 was also shown to suppress tumor growth by demonstrating reduced [3H]thymidine incorporation into DNA, possibly due to up-regulation of WAF1 (12) . However, we did not detect WAF1 up-regulation by KLF6 transient transfection in NCI-H920, -H1299, or -H2009, although the latter 2 cell lines were strongly suppressed in colony formation assay. These data suggest that KLF6 does not induce WAF1 expression in NSCLC cells, indicating that growth inhibition may not be due to G1-S arrest of the cell cycle. Indeed, we found that the transfection of KLF6 induced a marked apoptotic cell death in p53-null NSCLC cell line, NCI-H1299. To our knowledge, the present study is the first report demonstrating that KLF6 induces apoptosis in a human cancer cell line.
Regarding other KLF family members, apoptosis has been implicated in bladder cancer cells by KLF4 (26)
, and in pancreatic epithelial cells by KLF10, which mediates apoptotic effects of transforming growth factor ß1 (27)
. Because KLF6 has been shown to up-regulate transforming growth factor ß1 and its type I and II receptors (28)
, the induction of apoptosis by KLF6 in NSCLC cells may also be via the transactivation of transforming growth factor ß1. Other putative transcriptional targets of KLF6 may include the genes encoding a placental glycoprotein (29)
, collagen 
1 (30)
, urokinase type plasminogen activator (31)
, inducible nitric oxide synthase (32)
, and corneal keratin-12 (33)
. Additional studies need to determine the target gene of KLF6 for induction of apoptosis in NSCLC cells.
In conclusion, KLF6 is frequently down-regulated in NSCLCs, and exogenously induced KLF6 induces apoptosis and inhibits tumor cell growth, indicating that KLF6 might be a tumor suppressor gene like other genes in the KLF family. Our present study provides the basis for additional analysis of KLF6 and other KLFs in carcinogenesis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
Requests for reprints: Yoshitaka Sekido, Department of Clinical Preventive Medicine, Nagoya University School of Medicine, Tsurumai 65, Showa-Ku, Nagoya 466-8560, Japan. Phone: 81-52-744-1974; Fax: 81-52-744-1975; E-mail: ysekido{at}med.nagoya-u.ac.jp
Received 1/19/04. Revised 3/ 9/04. Accepted 3/22/04.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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