Volume 126, Issue 4, Pages 1090-1103 (April 2004) Kruppel-like factor 6 (KLF6) is a tumor-suppressor gene frequently inactivated in colorectal cancer  Helen L. Reeves, Goutham Narla, Olagunju Ogunbiyi, Asif I. Haq, Amanda Katz, Sharon Benzeno, Eldad Hod, Noam Harpaz, Shlomit Goldberg, Sigal Tal-Kremer, Francis J. Eng, Michael J.P. Arthur, John A. Martignetti, Scott L. Friedman  Gastroenterology  Volume 126, Issue 4, Pages 1090-1103 (April 2004) DOI: 10.1053/j.gastro.2004.01.005

Figure 1 (A) LOH and mutation status of KLF6 and TP53 in sporadic cancers. LOH of the KLF6 and TP53 loci was analyzed using microsatellite markers from 10p15 and 17p13, respectively. The relative positions of these markers, as per the Ensemble database (March 2003), are indicated in megabases. KLF6M1, M2, and M4 flank the KLF6 locus by 40 kb centromerically, 10 kb, and 20 kb telomerically, respectively. The cases are ranked according to degree of loss in the KLF6 region. The Dukes’ stage of each cancer is shown, as is the presence of a coding mutation (+), no mutation (−), or a variation in normal DNA (p) in KLF6, P53, or K-Ras. Large black circle, LOH; large grey circle, noninformative (NI) markers; open circle, no evidence of loss; ∅, additional alleles in tumor compatible with microsatellite instability (MSI); small grey circle, a PCR reaction that failed 3 or more times. (B) LOH and mutational status of KLF6 and TP53 in IBD-related cancers. LOH, Dukes’ stage, and mutation status are summarized as in A. In addition, APC mutational data are included (fs, frameshift mutation; m, missense mutation). Data have also been included for cases in which DNA was available and isolated from colitic epithelium. c, colitis; d, colitis with evidence of dysplasia. (C) Representative microsatellite marker electropherograms. The electropherograms for each of the 5 markers used in the study are shown for the first 6 cases. A relative allele ratio, XLOH, of <0.7 was defined as LOH. Case 1 shows LOH of only KLF6M1, whereas case 2 has LOH of only KLF6M2. Gastroenterology 2004 126, 1090-1103DOI: (10.1053/j.gastro.2004.01.005)

Figure 1 (A) LOH and mutation status of KLF6 and TP53 in sporadic cancers. LOH of the KLF6 and TP53 loci was analyzed using microsatellite markers from 10p15 and 17p13, respectively. The relative positions of these markers, as per the Ensemble database (March 2003), are indicated in megabases. KLF6M1, M2, and M4 flank the KLF6 locus by 40 kb centromerically, 10 kb, and 20 kb telomerically, respectively. The cases are ranked according to degree of loss in the KLF6 region. The Dukes’ stage of each cancer is shown, as is the presence of a coding mutation (+), no mutation (−), or a variation in normal DNA (p) in KLF6, P53, or K-Ras. Large black circle, LOH; large grey circle, noninformative (NI) markers; open circle, no evidence of loss; ∅, additional alleles in tumor compatible with microsatellite instability (MSI); small grey circle, a PCR reaction that failed 3 or more times. (B) LOH and mutational status of KLF6 and TP53 in IBD-related cancers. LOH, Dukes’ stage, and mutation status are summarized as in A. In addition, APC mutational data are included (fs, frameshift mutation; m, missense mutation). Data have also been included for cases in which DNA was available and isolated from colitic epithelium. c, colitis; d, colitis with evidence of dysplasia. (C) Representative microsatellite marker electropherograms. The electropherograms for each of the 5 markers used in the study are shown for the first 6 cases. A relative allele ratio, XLOH, of <0.7 was defined as LOH. Case 1 shows LOH of only KLF6M1, whereas case 2 has LOH of only KLF6M2. Gastroenterology 2004 126, 1090-1103DOI: (10.1053/j.gastro.2004.01.005)

Figure 2 (A) KLF6 wild-type and mutant alleles in normal and tumor DNA. The case number, nucleotide substitution, and amino acid translation and sequence chromatogram are shown for paired normal and tumor DNA for each mutation described. The forward sequence chromatograms are shown. An asterisk indicates the mutation. (B) KLF6 normal DNA sequence variations. A number of heterozygous KLF6 sequence variations present in normal DNA were not seen in their paired tumor samples. The chromatograms for those used to support allelic imbalance in the tumor DNA relative to normal are shown in both directions. (C) Restriction digest to confirm the mutations coding for P172L and T179I. The BfaI recognition site C▾TAG is created by the C3462T point mutation identified in patient 2. Thus, the single cut dividing the ∼300-base pair PCR product in the second lane confirms the presence of this mutation in the tumor DNA and not the normal DNA. The BsmFI recognition site GGGAC(N)10▾ is destroyed by the C3483T point mutation. The restriction digest performed after generating a PCR product from patient 39 confirms its presence in the tumor (lane 4) but not the normal DNA (lane 3). Gastroenterology 2004 126, 1090-1103DOI: (10.1053/j.gastro.2004.01.005)

Figure 2 (A) KLF6 wild-type and mutant alleles in normal and tumor DNA. The case number, nucleotide substitution, and amino acid translation and sequence chromatogram are shown for paired normal and tumor DNA for each mutation described. The forward sequence chromatograms are shown. An asterisk indicates the mutation. (B) KLF6 normal DNA sequence variations. A number of heterozygous KLF6 sequence variations present in normal DNA were not seen in their paired tumor samples. The chromatograms for those used to support allelic imbalance in the tumor DNA relative to normal are shown in both directions. (C) Restriction digest to confirm the mutations coding for P172L and T179I. The BfaI recognition site C▾TAG is created by the C3462T point mutation identified in patient 2. Thus, the single cut dividing the ∼300-base pair PCR product in the second lane confirms the presence of this mutation in the tumor DNA and not the normal DNA. The BsmFI recognition site GGGAC(N)10▾ is destroyed by the C3483T point mutation. The restriction digest performed after generating a PCR product from patient 39 confirms its presence in the tumor (lane 4) but not the normal DNA (lane 3). Gastroenterology 2004 126, 1090-1103DOI: (10.1053/j.gastro.2004.01.005)

Figure 3 (A) Wild-type KLF6 is antiproliferative in both fibroblast and CRC cell lines. The percentage inhibition of cell growth relative to empty vector transfected cells is shown for CCD-18 fibroblasts and HCT116, HT29, HCT15, and SW480 colon cancer cell lines. The antiproliferative effects in CCD-18 fibroblasts and HT29 cells were present after 24 hours maintained in 10% serum. For HCT116, HCT15, and SW480, the significant antiproliferative effect was detected at 18 hours posttransfection in cells maintained in 1% serum. (1, CCD-18 fibroblasts; 2, HT29; 3, HCT116; 4, SW480; 5, HCT15 colon cancer cell lines.) (B and C) KLF6 has growth suppressive properties that the tumor-derived mutations lack. The nature and relative expression levels of wild-type KLF6 and its mutant isoforms are shown in B, and relative proliferation as assessed by 3H-thymidine incorporation into DNA in CCD-18 colonic fibroblasts 24 hours after transient transfection is shown in C. KLF6 has a significant antiproliferative effect, while some of the mutants are significantly pro-proliferative relative to expression vector alone (∗∗∗P < 0.001, analysis of variance, Bonferroni correction applied, n = 4). The endogenous levels of p21 are differentially regulated by wild-type and mutant isoforms. A representative experiment is shown in C. (1, pCI-neo; 2, KLF6 wild type; 3, K74R; 4, G131S; 5, P148L; 6, P149S; 7, W162X; 8, G163D; 9, P166S; 10, P172L; 11, T179I.) Gastroenterology 2004 126, 1090-1103DOI: (10.1053/j.gastro.2004.01.005)