Identification of Krüppel-like factor 4 as a potential tumor suppressor gene in colorectal cancer

Abstract

Krüppel-like factor 4 (KLF4 or GKLF) is an inhibitor of the cell cycle. The gene encoding KLF4 is localized on chromosome 9q, previously shown to exhibit allelic loss in colorectal cancer (CRC). In this study, we show that the mean level of KLF4 mRNA in a panel of 30 CRC was 52% that of paired normal colonic tissues. Similarly, the levels of KLF4 mRNA and protein in a panel of six established CRC cell lines were significantly lower than those of an untransformed colonic epithelial cell line. Using highly polymorphic DNA markers that flank the KLF4 locus, we found evidence for loss of heterozygosity (LOH) in two of eight surgically resected CRC specimens. In addition, LOH was observed in five of six CRC cell lines with one additional cell line exhibiting hemizygous deletion in the KLF4 gene. We also found that the 5'-untranslated region of KLF4 was hypermethylated in a subset of resected CRC specimens and cell lines. Lastly, the open-reading frame of KLF4 in two of three CRC cell lines examined contained several point mutations that resulted in a diminished ability to activate the p21(WAF1/Cip1) promoter. These findings indicate that KLF4 is a potential tumor suppressor gene in CRC.

Figure 1

Synthesis of 3′-UTR probes for KLF4 and KLF5 and demonstration of probe specificity used for colon cancer profiling array. (a) Represents the design of KLF4 and KLF5 3′-UTR probes. RT–PCR reactions were performed to amplify the two probes using specific primers in the 3′-UTR of the two cDNAs and total RNA from human colon. (b) Northern blot analysis of total RNA from HCT116 cells using the KLF4 and KLF5 3′-UTR probes and full-length human p21^WAF1/Cip1 and ubiquitin cDNA probes. Total RNA (10 μg) was used in each lane. The positions of the 28S and 18S ribosomal RNAs are identified. ORF = openreading frame; UTR = untranslated region

Figure 2

Expression analysis of KLF4, KLF5 and p21^WAF/Cip1 in paired human CRC and normal colonic tissues. (a) ³²P-labeled cDNA probes for KLF4, KLF5, p21^WAF1/Cip1 and ubiquitin were hybridized to human colon cancer arrays that contained cDNAs from adenocarcinoma of the colorectum (T) and matched normal colonic tissues (N) of 30 patients who underwent resection for CRC. (b) Densitometric quantification of mRNA levels for KLF4, KLF5 and p21^WAF1/Cip1 shown in panel a by phosphor imaging analysis. Results represent mean ratios of the 30 samples after normalizing the signals of the respective mRNA to those from the internal control ubiquitin mRNA. * = Statistically significant when compared to normal control; N = normal; T = tumor

Figure 3

Northern and Western blot analyses of KLF4 mRNA and protein in normal human colonic epithelial cells and CRC cell lines. (a) Total RNA (15 μg) from the indicated cell lines was analysed. FHC was an untransformed human colonic epithelial cell line, whereas the rest of the cell lines were derived from human CRCs. The membrane was stripped and hybridized to the control glyceraldehydes-3-phosphate dehydrogenase (GAPDH) probe. (b) Relative levels of KLF4 mRNA in each cell line after normalization to the GAPDH control. (c) Western blot analysis of equal quantities of proteins extracted from each cell line was performed for KLF4. β-actin was used as a loading control

Figure 4

LOH of the KLF4 locus in surgically resected CRC specimens and colon cancer cell lines. (a) LOH analyses on surgically resected CRC and matched normal colonic tissues from eight patients and six colon cancer cell lines were conducted as described in Materials and methods. Filled circles indicate LOH. (b) Shows the chromosomal locations in Mb for the three polymorphic markers used for the LOH studies in relationship to the KLF4 locus. The thick horizontal arrow indicates the direction of transcription for the KLF4 gene

Figure 5

Southern blot analysis of KLF4 in genomic DNA from colon cancer cell lines. (a) Genomic DNA extracted from the indicated colon cancer cell lines was digested with EcoRI or HindIII and probed with full-length KLF4 cDNA. (b) Is a diagram of the surrounding genomic structure of KLF4. The locations of the EcoRI (E) and HindIII (H) restriction sites are illustrated. The thick horizontal bar indicates a 7 kb deletion of the KLF4 locus in RKO cells based on results of the Southern blot analyses

Figure 6

MSP analysis of the KLF4 5′-UTR in surgically resected CRC specimens and colon cancer cell lines. MSP was conducted as described in Materials and methods using primers specific for the unmethylated (U) or methylated (M) KLF4 5′-UTR in tissues derived from patients (a) or colon cancer cell lines (b). The expected product for the unmethylated DNA is 275 bp and that for the methylated product is 271 bp. H₂O was used as a negative control. IVD is in vitro methylated human genomic DNA control. T indicates tumor and N the matched normal colonic tissues

Figure 7

DNA sequence of the KLF4 coding region in colon cancer cell lines. Point mutations in the coding region of KLF4 in reverse-transcribed cDNAs from HCT116 (a) and HT29 (b) cell lines are shown. The wild-type (WT) sequence is shown for comparison. The changes in nucleotide and amino-acid sequences are highlighted and underlined

Figure 8

Functional analysis of mutant KLF4 from HT20 cells. (a) Wild-type (WT) and mutant KLF4 cDNAs from HT29 cells (HT29) were cloned into expression vectors, transfected into NIH3T3 cells and analysed by immunofluorescence for KLF4 (panels a and d). The nuclei were stained with ToPRO3 (panels b and e). Panels c and f are merged images. (b) NIH3T3 cells were cotransfected with the 2.4 kb p2^WAF1/Cip1 promoter-luciferase reporter and vector construct alone, or expression construct containing wild-type (WT) or mutant KLF4 cDNA from HT29 cells (HT29). Shown are the mean relative luciferase activities after normalizing for the internal control renilla luciferase activities. Vertical bars are standard deviations. N = 6; *P <0.05 when compared to WT-transfected cells