KLF6 is not the major target of chromosome 10p losses in glioblastomas

Abstract

Dear Sir,Complete or partial deletions of chromosome 10 are critical events during the malignant progression of gliomas. 1 They are present in the large majority of patients with glioblastoma multiforme (GBM), the most malignant glioma. Recent evidence suggests that complete losses of one copy of chromosome 10 are more frequent in primary GBM and partial losses in secondary GBM. 2 More often deletions affect the long arm of chromosome 10 but in a large fraction of patients they are also present on the short arm. 3 A comparable frequency of deletions on 10p has been found in prostate cancer. 4 KLF6, a gene that is part of the Kruppel family of transcription factors, 5 is located on 10p and according to several reports is mutated in about 50% of these patients 6 (a recent report, however, does not confirm this finding 7 ).A recent report by Jeng and Hsu describes the presence of KLF6 mutations in astrocytic gliomas and concludes that "mutations of the KLF6 gene play a role in the pathogenesis of astrocytic gliomas." 8 We have investigated the loss of heterozygosity in the chromosomal region flanking KLF6 and looked for mutation of KLF6 in 34 patients with GBM and in 2 GBM cell lines. LOH studies were done using microsatellite markers KLF6-M1 and KLF6-M2 and methods described previously. 9 LOH with primer M1 (telomeric) was present in 16/34 cases (47%). LOH with primer M2 (centromeric) was found in 8/34 (24%). Overall at least one loss was present in 62% of the cases but only in 3 tumors allelic imbalance was present on both the markers flanking KLF6.We have analyzed the coding sequence of KLF6 in these 34 patients. The 4 exons were amplified using PCR primers and temperatures outlined in Table I.The sequence was found identical to normal in all patients, with one exception. In Patient 19, showing LOH on M1, exons 1, 3 and 4 could be amplified by PCR whereas exon 2 failed to be amplified. Using PCR primers on exons 1 and 4 we could amplify more than 5 kb of sequence of KLF6 in 2 independent DNAs but not in tumor DNA of Patient 19, suggesting the presence of a translocation interrupting the coding sequence of KLF6 in exon 2. In Patient 8, the sequence of lymphocyte DNA showed heterozygosity on codon 201 (CGG/CGA) and loss of heterozygosity on tumor DNA (CGA). Both codons however encode the same arginine residue (reverse sequences are shown in the Fig. 1).To test whether reduced expression of KLF6 was present in some patients, we analyzed by real-time PCR the expression of KLF6 in 8 patients, in 2 established cell lines deriving from malignant gliomas (U87 and U373) and in normal brain. RNA was extract with Eurozol (EuroClone, Wetherby, UK) and 1 g of RNA was reverse-transcribed with Expand Reverse Transcriptase (Roche Diagnostics, Milan, Italy). Real-time PCR was carried out using GeneAmp 5700 and TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA) and primers of KLF6 and GAPDH (Assay on Demand cat no. Hs00154550 and Hs99999905, respectively), all from Applied Biosystems. PCR conditions were 2 min ...

Absence of mutation in the putative tumor-suppressor gene KLF6 in colorectal cancers

“…Two of these polymorphims (G3023A and R201R) had been already reported (Kohler et al, 2004;Koivisto et al, 2004a, b;Montanini et al, 2004;Reeves et al, 2004;Boyault et al, 2005). We did not find mutation in KLF6 exon 2 in any of the 76 tumor samples analysed.…”

“…Indeed, no mutation was found in a larger series of hepatocellular carcinoma by another group (Boyault et al, 2005). Finally, astrocytic gliomas show the same difference with no mutation found in three different studies (Kohler et al, 2004;Koivisto et al, 2004b;Montanini et al, 2004) after a first report that described 9% of KLF6 mutations in the exon 2 (Jeng and Hsu, 2003).…”

KLF6 transcription factor protects hepatocellular carcinoma-derived cells from apoptosis

“…17 However, the extent of KLF6 loss in major human cancers has been recently subjected to passionate debate. Numerous studies now report the absence of somatic mutation nor KLF6 inactivation in colorectal carcinoma, 27 in glioblastomas, 28 in prostate cancer, 29,30 in meningiomas, 28,31 in astrocytic tumors, 32 and, finally, in HCC. 18 Here, we confirmed that KLF6 is not mutated, and expressed in HCC samples, and in HCC-derived cell lines.…”

Inactivation of the tumor suppressor Krüppel-like factor 6 (KLF6) by mutation or decreased expression in hepatocellular carcinomas

“…So the highly variable frequency of KLF6 mutations may be caused by differences in methodology and sample sets. For example, some reports showed that KLF6 mutations are absent in prostate cancers (Mühlbauer et al, 2003) and human astrocytic gliomas (Koivisto et al, 2004;Köhler et al, 2004;Montanini et al, 2004), while other authors reported high frequencies of KLF6 mutations in prostate cancer (55%) (Narla et al, 2001) and astrocytic gliomas (9%) (Jeng and Hsu, 2003). Therefore, extensive investigation in both HCC and other human cancers is needed to determine the frequency of KLF6 mutation and its role in hepatocarcinogenesis.…”