Deletion, mutation, and loss of expression of KLF6 in human prostate cancer

Abstract

Kruppel-like factors (KLFs) are a group of transcription factors that appear to be involved in different biological processes including carcinogenesis. In a recent study, KLF6 was reported as a tumor suppressor gene in prostate cancer because of its frequent loss of heterozygosity (LOH) and mutation as well as functional suppression of cell proliferation. Loss of chromosomal locus spanning KLF6 is relatively infrequent in other published studies of prostate cancer, however. To clarify the role of KLF6 in prostate cancers, particularly those that are high grade, we examined KLF6 for deletion, mutation, and loss of expression in 96 prostate cancer samples including 21 xenografts/cell lines. Loss of heterozygosity occurred in 4 (19%) of 21 xenografts/cell lines and 8 (28%) of 29 informative tumors. Fourteen of the 96 (15%) samples showed 15 somatic sequence changes in the KLF6 gene, including 7 that changed KLF6 peptide sequences, 4 that did not, and 4 that were located in untranslated regions. Expression levels of KLF6 were significantly lost in 4 of 20 (20%) xenografts/cell lines of prostate cancer, as detected by RT-PCR and Northern blot analysis. These findings indicate that significant genetic alterations of KLF6 occur in a minority of high-grade prostate cancers.

Figure 1.

Detection of allelic status at the KLF6 locus in prostate cancer cell lines and xenografts (A) and LOH in prostate cancer specimens (B) using microsatellite markers. Markers D10S189 and D10S591 were used for panel A and B respectively. Sample names are shown at the top of each panel. In panel A, ++ denotes two different alleles whereas + denotes one allele. N, normal; T, primary tumor, P, primary tumor from patients whose metastases were also used; M, metastasis.

Figure 2.

Detection of LOH at the KLF6 locus in cell lines and xenografts of prostate cancer by the method of HOMOD analysis. Marker names are listed at the top with the order from telomere to centromere. Cases with LOH are boxed. Black boxes indicate one allele and white boxes indicate two alleles.

Figure 3.

LOH of the KLF6 locus in prostate cancer specimens. Only cases informative for at least one marker are shown. Markers are shown at the top, and samples at the left. N, normal; T and P, primary tumor; M, metastasis.

Figure 4.

Detection of KLF6 mutations in prostate cancer samples. A: Examples of SSCP analysis. Asterisks denote samples with shifted bands. B: DNA sequence determination in representative samples with mutations. Case 53 has a mutation of 565C>T(G189S) and xenograft LAPC-3 has a mutation of –4G>T.

Figure 5.

Loss of expression for KLF6 in some prostate cancer cell lines or xenografts, as detected by duplex RT-PCR (A) and Northern blot analysis (B). In each panel, samples are listed at the top, gene names at the left, and DNA/RNA sizes at the right. Prostate 1 and prostate 2 were normal prostates from different individuals; PZ-HPV-7, PWR1E, and RWPE1 are immortalized cell lines from normal prostate epithelium; and BRF-55T is an immortalized cell line from a benign prostatic hyperplasia.