KLF6 Loss of Function in Human Prostate Cancer Progression Is Implicated in Resistance to Androgen Deprivation
2012; Elsevier BV; Volume: 181; Issue: 3 Linguagem: Inglês
10.1016/j.ajpath.2012.06.008
ISSN1525-2191
AutoresXiaomei Liu, Alejandro Gómez-Pinillos, Charisse Loder, Enrique Carrillo de Santa Pau, Ruifang Qiao, Pamela D. Unger, Ralf Kurek, Carole Oddoux, Jonathan Melamed, Robert E. Gallagher, John Mandeli, Anna C. Ferrari,
Tópico(s)Cancer-related gene regulation
ResumoInactivation of the transcription factor/tumor suppressor Krüppel-like factor 6 (KLF6) has been described in prostate cancer (PC). This study investigated the prevalence and significance of KLF6 exon 2 mutations and splice variants (SVs) in different stages of human PC progression. By using laser-capture microdissection and recombinant clone isolation of DNA sequences to enhance sensitivity, base changes were found in 20 (24.7%) of 81 PC tissues versus 1 (4%) of 25 normal prostate tissues (P = 0.02). Of 26 base changes, 54% produced nonsynonymous mutations. Only three mutations had driver characteristics (PCs, 4%; NPs, 0%). By using microdissection of fresh-frozen tissues and recombinant isolation of RNA sequences, SVs were found in 39 (75%) of 52 PCs and in 10 (45%) of 22 NPs (P = 0.01). Sixteen different SVs, including 13 unique SVs, were identified that used cryptic splicing sites and encoded nonfunctional KLF6 proteins. PCs that had survived hormone (androgen)-deprivation therapy (n = 21) had a significantly higher (P < 0.05) incidence, number, and expression level of nonfunctional SVs than either NPs (n = 22) or hormone-naïve PCs (n = 25). Forced expression of nonfunctional SVs conferred a survival advantage of androgen-dependent LNCaP cells under castration-simulated culture conditions. Together, these data suggest that decreased availability of functional KLF6 contributes to clinical PC progression. This decrease arises infrequently by somatic mutation and more commonly by the acquisition of SVs that provide a survival advantage under castrate conditions, enabling resistance to hormone therapy. Inactivation of the transcription factor/tumor suppressor Krüppel-like factor 6 (KLF6) has been described in prostate cancer (PC). This study investigated the prevalence and significance of KLF6 exon 2 mutations and splice variants (SVs) in different stages of human PC progression. By using laser-capture microdissection and recombinant clone isolation of DNA sequences to enhance sensitivity, base changes were found in 20 (24.7%) of 81 PC tissues versus 1 (4%) of 25 normal prostate tissues (P = 0.02). Of 26 base changes, 54% produced nonsynonymous mutations. Only three mutations had driver characteristics (PCs, 4%; NPs, 0%). By using microdissection of fresh-frozen tissues and recombinant isolation of RNA sequences, SVs were found in 39 (75%) of 52 PCs and in 10 (45%) of 22 NPs (P = 0.01). Sixteen different SVs, including 13 unique SVs, were identified that used cryptic splicing sites and encoded nonfunctional KLF6 proteins. PCs that had survived hormone (androgen)-deprivation therapy (n = 21) had a significantly higher (P < 0.05) incidence, number, and expression level of nonfunctional SVs than either NPs (n = 22) or hormone-naïve PCs (n = 25). Forced expression of nonfunctional SVs conferred a survival advantage of androgen-dependent LNCaP cells under castration-simulated culture conditions. Together, these data suggest that decreased availability of functional KLF6 contributes to clinical PC progression. This decrease arises infrequently by somatic mutation and more commonly by the acquisition of SVs that provide a survival advantage under castrate conditions, enabling resistance to hormone therapy. Krüppel-like factor 6 (KLF6) is a ubiquitously expressed member of a large family of mammalian Sp1/KLF transcription factors that play a critical role in the regulation of cellular development, differentiation, proliferation, and apoptosis.1Bieker J.J. Kruppel-like factors: three fingers in many pies.J Biol Chem. 2001; 276: 34355-34358Crossref PubMed Scopus (540) Google Scholar Some of the best known transcriptional targets of KLF6 include p21, E-cadherin,2DiFeo A. Narla G. Camacho-Vanegas O. Nishio H. Rose S.L. Buller R.E. Friedman S.L. Walsh M.J. Martignetti J.A. E-cadherin is a novel transcriptional target of the KLF6 tumor suppressor.Oncogene. 2006; 25: 6026-6031Crossref PubMed Scopus (68) Google Scholar and ATF3.3Huang X. Li X. Guo B. KLF6 induces apoptosis in prostate cancer cells through up-regulation of ATF3.J Biol Chem. 2008; 283: 29795-29801Crossref PubMed Scopus (102) Google Scholar The KLF6 gene is located on chromosome arm 10p and has four exons that encode a protein with 283 amino acids (AAs), including a transactivation domain (TAD) and three zinc fingers (ZFs) that constitute the DNA-binding domain (DBD).4Benzeno S. Narla G. Allina J. Cheng G.Z. Reeves H.L. Banck M.S. Odin J.A. Diehl J.A. Germain D. Friedman S.L. Cyclin-dependent kinase inhibition by the KLF6 tumor suppressor protein through interaction with cyclin D1.Cancer Res. 2004; 64: 3885-3891Crossref PubMed Scopus (131) Google Scholar, 5Rodríguez E. Aburjania N. Priedigkeit N.M. DiFeo A. Martignetti J.A. Nucleo-cytoplasmic localization domains regulate Krüppel-like factor 6 (KLF6) protein stability and tumor suppressor function.PLoS One. 2010; 5 (pii:e12639)Google Scholar A potential role for KLF6 in cancer emerged when a study of 22 localized prostate cancer (PC) specimens showed that 55% of the tumor cells had loss of heterozygosity.6Narla G. Heath K.E. Reeves H.L. Li D. Giono L.E. Kimmelman A.C. Glucksman M.J. Narla J. Eng F.J. Chan A.M. Ferrari A.C. Martignetti J.A. Friedman S.L. KLF6, a candidate tumor suppressor gene mutated in prostate cancer.Science. 2001; 294: 2563-2566Crossref PubMed Scopus (381) Google Scholar Furthermore, in specimens with loss of heterozygosity, 71% had a mutation in KLF6 exon 2 of the remaining allele. Of 30 total KLF6 mutations identified in these tumors, 28 (93%) were in exon 2 and 24 (86%) changed the AA sequence, whereas no mutations were found in a subset of matched normal epithelial counterparts (NECs). In addition, mutant KLF6 constructs were unable to transactivate p21 and augmented PC cell proliferation. These results supported the notion that KLF6 is a tumor suppressor gene frequently inactivated by somatic mutations in PC. However, these results were not confirmed in three subsequent studies, one of which found exon 2 base change (BC) in 10 (13%) of 75 PC specimens7Chen C. Hyytinen E.R. Sun X. Helin H.J. Koivisto P.A. Frierson Jr, H.F. Vessella R.L. Dong J.T. Deletion, mutation, and loss of expression of KLF6 in human prostate cancer.Am J Pathol. 2003; 162: 1349-1354Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar and two of which found no exon 2 BC in 135 PC specimens.8Muhlbauer K.R. Grone H.J. Ernst T. Grone E. Tschada R. Hergenhahn M. Hollstein M. Analysis of human prostate cancers and cell lines for mutations in the TP53 and KLF6 tumour suppressor genes.Br J Cancer. 2003; 89: 687-690Crossref PubMed Scopus (38) Google Scholar, 9Agell L. Hernandez S. de Muga S. Lorente J.A. Juanpere N. Esgueva R. Serrano S. Gelabert A. Lloreta J. KLF6 and TP53 mutations are a rare event in prostate cancer: distinguishing between Taq polymerase artifacts and true mutations.Mod Pathol. 2008; 21: 1470-1478Crossref PubMed Scopus (43) Google Scholar Another mechanism for decreasing the availability of functional KLF6 is by altering the expression of splice variants (SVs). Alternative splicing has been detected in several solid tumors, including PCs.10Narla G. DiFeo A. Fernandez Y. Dhanasekaran S. Huang F. Sangodkar J. Hod E. Leake D. Friedman S.L. Hall S.J. Chinnaiyan A.M. Gerald W.L. Rubin M.A. Martignetti J.A. KLF6-SV1 overexpression accelerates human and mouse prostate cancer progression and metastasis.J Clin Invest. 2008; 118: 2711-2721Crossref PubMed Scopus (96) Google Scholar, 11Narla G. Difeo A. Reeves H.L. Schaid D.J. Hirshfeld J. Hod E. Katz A. Isaacs W.B. Hebbring S. Komiya A. McDonnell S.K. Wiley K.E. Jacobsen S.J. Isaacs S.D. Walsh P.C. Zheng S.L. Chang B.L. Friedrichsen D.M. Stanford J.L. Ostrander E.A. Chinnaiyan A.M. Rubin M.A. Xu J. Thibodeau S.N. Friedman S.L. Martignetti J.A. A germline DNA polymorphism enhances alternative splicing of the KLF6 tumor suppressor gene and is associated with increased prostate cancer risk.Cancer Res. 2005; 65: 1213-1222Crossref PubMed Scopus (192) Google Scholar, 12Hanoun N. Bureau C. Diab T. Gayet O. Dusetti N. Selves J. Vinel J.P. Buscail L. Cordelier P. Torrisani J. The SV2 variant of KLF6 is down-regulated in hepatocellular carcinoma and displays anti-proliferative and pro-apoptotic functions.J Hepatol. 2010; 53: 880-888Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 13Zaharieva E. Chipman J.K. Soller M. Alternative splicing interference by xenobiotics.Toxicology. 2012; 296: 1-12Crossref PubMed Scopus (31) Google Scholar, 14Ferrari A.C. Stone N.N. Kurek R. Mulligan E. McGregor R. Stock R. Unger P. Tunn U. Kaisary A. Droller M. Hall S. Renneberg H. Livak K.J. Gallagher R.E. Mandeli J. Molecular load of pathologically occult metastases in pelvic lymph nodes is an independent prognostic marker of biochemical failure after localized prostate cancer treatment.J Clin Oncol. 2006; 24: 3081-3088Crossref PubMed Scopus (45) Google Scholar In primary PC tissues, three KLF6 SVs involving different areas of the TAD and the ZF were identified.10Narla G. DiFeo A. Fernandez Y. Dhanasekaran S. Huang F. Sangodkar J. Hod E. Leake D. Friedman S.L. Hall S.J. Chinnaiyan A.M. Gerald W.L. Rubin M.A. Martignetti J.A. KLF6-SV1 overexpression accelerates human and mouse prostate cancer progression and metastasis.J Clin Invest. 2008; 118: 2711-2721Crossref PubMed Scopus (96) Google Scholar In addition, men with a germline single-nucleotide polymorphism located in intron 1 (−27 G>A) were at increased risk of PC.11Narla G. Difeo A. Reeves H.L. Schaid D.J. Hirshfeld J. Hod E. Katz A. Isaacs W.B. Hebbring S. Komiya A. McDonnell S.K. Wiley K.E. Jacobsen S.J. Isaacs S.D. Walsh P.C. Zheng S.L. Chang B.L. Friedrichsen D.M. Stanford J.L. Ostrander E.A. Chinnaiyan A.M. Rubin M.A. Xu J. Thibodeau S.N. Friedman S.L. Martignetti J.A. A germline DNA polymorphism enhances alternative splicing of the KLF6 tumor suppressor gene and is associated with increased prostate cancer risk.Cancer Res. 2005; 65: 1213-1222Crossref PubMed Scopus (192) Google Scholar This single-nucleotide polymorphism, found in 15.1% of sporadic PCs, generates a novel DNA-binding site for splicing factors that generate three alternatively spliced KLF6 isoforms in vitro, SV1 through SV3. In 15 PC specimens, the expression of KLF6 spliced isoforms was twofold higher than that of wild-type (wt) KLF6 mRNA compared with six normal prostate (NP) specimens. No differences in SV2 and SV3 expression were observed between primary PC tumors and metastases.10Narla G. DiFeo A. Fernandez Y. Dhanasekaran S. Huang F. Sangodkar J. Hod E. Leake D. Friedman S.L. Hall S.J. Chinnaiyan A.M. Gerald W.L. Rubin M.A. Martignetti J.A. KLF6-SV1 overexpression accelerates human and mouse prostate cancer progression and metastasis.J Clin Invest. 2008; 118: 2711-2721Crossref PubMed Scopus (96) Google Scholar, 11Narla G. Difeo A. Reeves H.L. Schaid D.J. Hirshfeld J. Hod E. Katz A. Isaacs W.B. Hebbring S. Komiya A. McDonnell S.K. Wiley K.E. Jacobsen S.J. Isaacs S.D. Walsh P.C. Zheng S.L. Chang B.L. Friedrichsen D.M. Stanford J.L. Ostrander E.A. Chinnaiyan A.M. Rubin M.A. Xu J. Thibodeau S.N. Friedman S.L. Martignetti J.A. A germline DNA polymorphism enhances alternative splicing of the KLF6 tumor suppressor gene and is associated with increased prostate cancer risk.Cancer Res. 2005; 65: 1213-1222Crossref PubMed Scopus (192) Google Scholar SV1 through SV3 have been characterized in some detail. SV1 lacks part of the TAD and all three ZFs, SV2 has a partial loss of the TAD and ZF1 but retains ZF2 and ZF3, and SV3 lacks ZF2 and ZF3 but preserves an intact TAD and ZF1.5Rodríguez E. Aburjania N. Priedigkeit N.M. DiFeo A. Martignetti J.A. Nucleo-cytoplasmic localization domains regulate Krüppel-like factor 6 (KLF6) protein stability and tumor suppressor function.PLoS One. 2010; 5 (pii:e12639)Google Scholar Functional analysis indicated that SVs lacking ZF1 lose the nuclear localizing signal and remain cytoplasmic, whereas SVs that retain ZF1 localize to the nucleus, such as wt-KLF6, irrespective of the status of ZF2 and ZF3.5Rodríguez E. Aburjania N. Priedigkeit N.M. DiFeo A. Martignetti J.A. Nucleo-cytoplasmic localization domains regulate Krüppel-like factor 6 (KLF6) protein stability and tumor suppressor function.PLoS One. 2010; 5 (pii:e12639)Google Scholar In vitro assays showed that forced expression of SV1 in PC cells increased proliferation by antagonizing wt-KLF6 activity and decreasing endogenous p21 levels.11Narla G. Difeo A. Reeves H.L. Schaid D.J. Hirshfeld J. Hod E. Katz A. Isaacs W.B. Hebbring S. Komiya A. McDonnell S.K. Wiley K.E. Jacobsen S.J. Isaacs S.D. Walsh P.C. Zheng S.L. Chang B.L. Friedrichsen D.M. Stanford J.L. Ostrander E.A. Chinnaiyan A.M. Rubin M.A. Xu J. Thibodeau S.N. Friedman S.L. Martignetti J.A. A germline DNA polymorphism enhances alternative splicing of the KLF6 tumor suppressor gene and is associated with increased prostate cancer risk.Cancer Res. 2005; 65: 1213-1222Crossref PubMed Scopus (192) Google Scholar In contrast, in vivo overexpression of SV1 in PC3M xenografts did not affect local tumor growth, but it enhanced angiogenesis and metastases.10Narla G. DiFeo A. Fernandez Y. Dhanasekaran S. Huang F. Sangodkar J. Hod E. Leake D. Friedman S.L. Hall S.J. Chinnaiyan A.M. Gerald W.L. Rubin M.A. Martignetti J.A. KLF6-SV1 overexpression accelerates human and mouse prostate cancer progression and metastasis.J Clin Invest. 2008; 118: 2711-2721Crossref PubMed Scopus (96) Google Scholar The functional activity of SV2 is unclear. Although some investigators found that overexpressed SV2 in PC3 cells remained cytoplasmic and was unable to induce p21, other groups found that SV2 induced endogenous p21 and ATF3 transcription and produced apoptosis.3Huang X. Li X. Guo B. KLF6 induces apoptosis in prostate cancer cells through up-regulation of ATF3.J Biol Chem. 2008; 283: 29795-29801Crossref PubMed Scopus (102) Google Scholar, 12Hanoun N. Bureau C. Diab T. Gayet O. Dusetti N. Selves J. Vinel J.P. Buscail L. Cordelier P. Torrisani J. The SV2 variant of KLF6 is down-regulated in hepatocellular carcinoma and displays anti-proliferative and pro-apoptotic functions.J Hepatol. 2010; 53: 880-888Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar In one study, SV3 localized to the nucleus, but functional analysis was not performed.5Rodríguez E. Aburjania N. Priedigkeit N.M. DiFeo A. Martignetti J.A. Nucleo-cytoplasmic localization domains regulate Krüppel-like factor 6 (KLF6) protein stability and tumor suppressor function.PLoS One. 2010; 5 (pii:e12639)Google Scholar Collectively, the data previously summarized suggest that local (cis) changes in exon or intron gene sequences alter the availability of functional KLF6 and may play a role in PCs. However, the apparent prevalence of mutations significantly differs among human PC studies, and the prevalence of splicing has not been established, making the significance of either unclear. The main goals of this study were to more incisively assess the prevalence of KLF6 exon 2 mutations and alternative KLF6 mRNA splicing in human PC progression. In addition, because mRNA splicing is highly regulated and responsive to environmental conditions,13Zaharieva E. Chipman J.K. Soller M. Alternative splicing interference by xenobiotics.Toxicology. 2012; 296: 1-12Crossref PubMed Scopus (31) Google Scholar we evaluated whether it might play a role in tumor cell adaptation to castrate conditions. Human tissue specimens from patients who consented were obtained from the tissue banks of the Department of Pathology, Mount Sinai Hospital, New York, NY; Stadtische Klinikum Frankfurt am Main, Offenbach, Germany; New York University Cooperative Prostate Cancer Tissue Resource & Prostate Cancer Serum and Genetics Repository; and the National Disease Research Interchange.14Ferrari A.C. Stone N.N. Kurek R. Mulligan E. McGregor R. Stock R. Unger P. Tunn U. Kaisary A. Droller M. Hall S. Renneberg H. Livak K.J. Gallagher R.E. Mandeli J. Molecular load of pathologically occult metastases in pelvic lymph nodes is an independent prognostic marker of biochemical failure after localized prostate cancer treatment.J Clin Oncol. 2006; 24: 3081-3088Crossref PubMed Scopus (45) Google Scholar Except for the latter, all samples were consecutively collected. This study (CA098135-010) was approved by the Institutional Review Boards at Mount Sinai School of Medicine (020137) and New York University (numbers 1245 and 8723). In total, 159 samples were processed, including 116 primary PCs (Gleason score of 6, 72%; score of 7, 9%; score of 8, 14%; score of 9, 1%; and score of 10, 4%), 18 NECs, 14 metastases (mets), and 29 NPs from cadavers. Of the 116 PCs, 70 (18 with associated NECs) were formalin fixed, paraffin embedded (FFPE), and 46 were frozen in liquid nitrogen. Of the 14 mets, eight were FFPE hormone-naïve lymph nodes (HN-LNs) and six were frozen castration-resistant PC (CRPC) mets from bone marrow,2DiFeo A. Narla G. Camacho-Vanegas O. Nishio H. Rose S.L. Buller R.E. Friedman S.L. Walsh M.J. Martignetti J.A. E-cadherin is a novel transcriptional target of the KLF6 tumor suppressor.Oncogene. 2006; 25: 6026-6031Crossref PubMed Scopus (68) Google Scholar skull, epidural mass, and peritoneal/pleural effusion cells. All 29 NPs were frozen samples. Sections of FFPE specimens (8 to 10 μm thick) were mounted on 10to 20 superfrost/snow-treated slides (Fisher Scientific, Pittsburgh, PA) and stained with Mayer's H&E to map ≥5000 cells of each PC and NEC. Sections were processed by a standard deparaffinization/dehydration-rehydration protocol and stained with hematoxylin for laser-capture microdissection (LCM). Sections of frozen specimens (7 to 15 μm thick) in Tissue-Tek OCT (Fisher Scientific) were processed on RNase-free superfrost/snow-treated slides (Fisher Scientific). LCM was performed using the Pixcell II System (Arcturus Engineering, Sunnyvale, CA). Recovered cells were suspended in 50 μL of DNA extraction buffer (1 mmol/L EDTA; 10 mmol/L Tris, pH 8; 1% Tween-20; and 1 mg/mL proteinase-K) and incubated at 50°C overnight, followed by 10 minutes at 95°C. Total RNA from LCM cells or homogenized microdissected samples was extracted using the Stratagene Absolutely RNA Microprep kit (Agilent Technologies, Palo Alto, CA). DNA solution (5 μL) was mixed with 45 μL of High Fidelity PCR Supermix (Invitrogen, San Diego, CA) and 100 μmol/L forward and reverse primers. First-round PCR product, 1 μL, was similarly amplified using two sets of primers: 2AF (5′-CGGGCGCAATGTTATCTGTCCTTC-3′) and 2AR (5′-AATGGGGTCGGAGGTAAA-3′) and 2BF (5′-AGCAGCGAATCCTCTGACAG-3′) and 2BR (5′-CCCTCCAGGGCTGGTGCA-3′) to spare exon 2 in two fragments of 400 and 422 bp, respectively; and 2ANF (5′-TCTGTCCTTCATTCTTGCAT-3′), 2ANR (5′-TTGGCCGTGGGAGAAAGTTC-3′), 2BNF (5′-CTCCCACGGCCAAGTTTAC-3′), and 2BNR (5′-GTCCGCTGGTGTGCTTTCAA-3′) for nested PCR. Nested-PCR amplicons were purified with the QIAquick PCR Purification Kit (QIAGEN, Germantown, MD) after insertion into the pCR2.1-TOPO DNA plasmid (Invitrogen); constructs were incubated overnight with Mach-1 competent cells and plated on LB Amp plates (Roche, Indianapolis, IN) with 40 μL of 40 mg/mL X-Gal at 37°C for 24 hours. Plasmids from positive clones12Hanoun N. Bureau C. Diab T. Gayet O. Dusetti N. Selves J. Vinel J.P. Buscail L. Cordelier P. Torrisani J. The SV2 variant of KLF6 is down-regulated in hepatocellular carcinoma and displays anti-proliferative and pro-apoptotic functions.J Hepatol. 2010; 53: 880-888Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 13Zaharieva E. Chipman J.K. Soller M. Alternative splicing interference by xenobiotics.Toxicology. 2012; 296: 1-12Crossref PubMed Scopus (31) Google Scholar, 14Ferrari A.C. Stone N.N. Kurek R. Mulligan E. McGregor R. Stock R. Unger P. Tunn U. Kaisary A. Droller M. Hall S. Renneberg H. Livak K.J. Gallagher R.E. Mandeli J. Molecular load of pathologically occult metastases in pelvic lymph nodes is an independent prognostic marker of biochemical failure after localized prostate cancer treatment.J Clin Oncol. 2006; 24: 3081-3088Crossref PubMed Scopus (45) Google Scholar, 15Wong W.C. Kim D. Carter H. Diekhans M. Ryan M.C. Karchin R. CHASM and SNVBox: toolkit for detecting biologically important single nucleotide mutations in cancer.Bioinformatics. 2011; 27: 2147-2148Crossref PubMed Scopus (90) Google Scholar, 16Parsons D.W. Jones S. Zhang X. Lin J.C. Leary R.J. Angenendt P. Mankoo P. Carter H. Siu I.M. Gallia G.L. Olivi A. McLendon R. Rasheed B.A. Keir S. Nikolskaya T. Nikolsky Y. Busam D.A. Tekleab H. Diaz Jr, L.A. Hartigan J. Smith D.R. Strausberg R.L. Marie S.K. Shinjo S.M. Yan H. Riggins G.J. Bigner D.D. Karchin R. Papadopoulos N. Parmigiani G. Vogelstein B. Velculescu V.E. Kinzler K.W. An integrated genomic analysis of human glioblastoma multiforme.Science. 2008; 321: 1807-1812Crossref PubMed Scopus (4616) Google Scholar, 17Adzhubei I.A. Schmidt S. Peshkin L. Ramensky V.E. Gerasimova A. Bork P. Kondrashov A.S. Sunyaev S.R. A method and server for predicting damaging missense mutations.Nat Methods. 2010; 7: 248-249Crossref PubMed Scopus (9508) Google Scholar, 18Liu X.M. Gomez-Pinillos A. Liu X.J. Johnson E.M. Ferrari A.C. Induction of bicalutamide sensitivity in prostate cancer cells by an epigenetic Pura-mediated decrease in androgen receptor levels.Prostate. 2010; 70: 179-189PubMed Google Scholar were purified with the QIAprep Spin Miniprep Kit (QIAGEN). The KLF6 insert was confirmed by PCR (30 cycles) and sequenced in both directions with the nested-PCR primers using a 3730xl DNA Analyzer (Applied Biosystems, Carlsbad, CA). DNA sequence results were assembled using Sequencher software version 4.2 (GeneCodes Corporation, Ann Arbor, MI) and compared with the KLF6 GenBank DNA genomic sequence (AF284036). We defined a mutation as any BC identified in both forward- and reverse-sequence products in two or more clones per specimen and not present in its NEC or any NP. To distinguish driver from passenger somatic missense mutations, Cancer-specific High-throughput Annotation of Somatic Mutations software (CHASM) was applied.15Wong W.C. Kim D. Carter H. Diekhans M. Ryan M.C. Karchin R. CHASM and SNVBox: toolkit for detecting biologically important single nucleotide mutations in cancer.Bioinformatics. 2011; 27: 2147-2148Crossref PubMed Scopus (90) Google Scholar A better-characterized spectrum of passenger mutations of glioblastoma was used.16Parsons D.W. Jones S. Zhang X. Lin J.C. Leary R.J. Angenendt P. Mankoo P. Carter H. Siu I.M. Gallia G.L. Olivi A. McLendon R. Rasheed B.A. Keir S. Nikolskaya T. Nikolsky Y. Busam D.A. Tekleab H. Diaz Jr, L.A. Hartigan J. Smith D.R. Strausberg R.L. Marie S.K. Shinjo S.M. Yan H. Riggins G.J. Bigner D.D. Karchin R. Papadopoulos N. Parmigiani G. Vogelstein B. Velculescu V.E. Kinzler K.W. An integrated genomic analysis of human glioblastoma multiforme.Science. 2008; 321: 1807-1812Crossref PubMed Scopus (4616) Google Scholar The CHASM raw score is the fraction of trees that classified the mutations as a passenger; the null hypothesis (mutants are passengers) was tested, and P values were corrected by multiple testing (Benjamini-Hochberg false-discovery rate). PolyPhen-2 software version 2.2.2 with the HumVar data set was used to predict the impact of AA substitutions due to missense mutations of KLF6 exon 2 on the structure and function of KLF6 proteins.17Adzhubei I.A. Schmidt S. Peshkin L. Ramensky V.E. Gerasimova A. Bork P. Kondrashov A.S. Sunyaev S.R. A method and server for predicting damaging missense mutations.Nat Methods. 2010; 7: 248-249Crossref PubMed Scopus (9508) Google Scholar RNA was obtained from 74 frozen specimens, including 25 HN primary PCs (88% Gleason score 6 and 12% Gleason score 7), 21 primary hormone (androgen)–deprived PCs (HD-PCs) removed after 3 to 6 months of neoadjuvant hormone therapy (31% Gleason score 6, 44% Gleason score 7, and 25% Gleason scores 8 and 9), 6 CRPC mets, and 22 NPs. Representative areas of PCs, NP epithelia, and mets were obtained by LCM or microdissection. RNA, extracted as previously described, was reverse transcribed using random hexamers (Amersham-Pharmacia Biotech, St. Albans, UK) using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). The cDNA was amplified using the High Fidelity PCR Supermix (Invitrogen) with KLF6-specific primers (forward, 5′-GACATGGACGTGCTCCCCAT-3′; and reverse, 5′-ATGCCGCTTCTTACAGGA-3′) located at position 184 in exon 1 and 225 in exon 4, respectively. The 1030-bp amplicon was cloned, as previously described, and 12 clones per case were sequenced and analyzed, as previously described, except for comparison to the KLF6 GenBank mRNA sequence (NM001300). ScanProsite software (Swiss Institute of Bioinformatics) was used to predict the AA sequence and changes in functional ZF domains. The prevalence of the different KLF6 SVs was assessed by the number of clones with each SV in each specimen. HEK-293 and LNCaP cells (ATCC 2009, Manassas, VA) were grown in Eagle's minimum essential medium (Sigma-Aldrich, St. Louis, MO) and RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), respectively. For MTT assays, 4 × 103 LNCaP cells grown with 10% FBS or charcoal-stripped FBS were transfected with 0.2 μg of pFLAG-CMV-2 Expression Vector (Sigma-Aldrich) containing wt- or SV-KLF6 and analyzed 5 days after transfection in three independent experiments.18Liu X.M. Gomez-Pinillos A. Liu X.J. Johnson E.M. Ferrari A.C. Induction of bicalutamide sensitivity in prostate cancer cells by an epigenetic Pura-mediated decrease in androgen receptor levels.Prostate. 2010; 70: 179-189PubMed Google Scholar HEK-293, 5 × 104 cells per well, was transfected with DNA containing p21 reporter construct,19Wang L.G. Ossowski L. Ferrari A.C. Overexpressed androgen receptor linked to p21WAF1 silencing may be responsible for androgen independence and resistance to apoptosis of a prostate cancer cell line.Cancer Res. 2001; 61: 7544-7551PubMed Google Scholar pFLAG-CMV-2 vector containing wt-KLF6 or the test SV, and the pRL-SV40 Luciferase Vector (Promega, Madison, WI) to normalize transfections. At 48 hours, whole cell protein extracts were quantified by the dual-luciferase system (Promega), and 20 μg was used for 12% SDS-PAGE and transfer to a nitrocellulose membrane18Liu X.M. Gomez-Pinillos A. Liu X.J. Johnson E.M. Ferrari A.C. Induction of bicalutamide sensitivity in prostate cancer cells by an epigenetic Pura-mediated decrease in androgen receptor levels.Prostate. 2010; 70: 179-189PubMed Google Scholar to characterize wt-KLF6 and SVs by anti-KLF6 (sc-134374; Santa Cruz), anti-FLAG (F3165; Sigma-Aldrich), and secondary antibodies. Continuous variables were described by the mean and SD and compared by the Student's t-test. Categorical variables were described by counts and percentages and were compared with the χ2 or Fisher's statistical tests. All tests were two sided; P < 0.05 indicated statistical significance (IBM SPSS Statistics 19, Chicago, IL). KLF6 mutation analysis was focused on exon 2, because >80% of previously identified mutations were located there.6Narla G. Heath K.E. Reeves H.L. Li D. Giono L.E. Kimmelman A.C. Glucksman M.J. Narla J. Eng F.J. Chan A.M. Ferrari A.C. Martignetti J.A. Friedman S.L. KLF6, a candidate tumor suppressor gene mutated in prostate cancer.Science. 2001; 294: 2563-2566Crossref PubMed Scopus (381) Google Scholar, 7Chen C. Hyytinen E.R. Sun X. Helin H.J. Koivisto P.A. Frierson Jr, H.F. Vessella R.L. Dong J.T. Deletion, mutation, and loss of expression of KLF6 in human prostate cancer.Am J Pathol. 2003; 162: 1349-1354Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar We identified 26 BCs in 20 of 81 PC specimens, including 18 (26%) of 70 primary PCs and 2 (29%) of 7 HN-LN mets; 0 BCs were detected in 7 NPs (Table 1). All BC-positive PC specimens were FFPE, whereas all BC-negative specimens, including four CRPC mets and seven NPs, were fresh frozen, raising the question of whether the detection of BC could be related to FFPE processing. However, BCs were only present in 1 (6%) of 18 NECs derived from the same FFPE specimens as primary PCs (P = 0.11).Table 1Distribution of PC Cases with BCs in Two or More ClonesCase no.⁎The case numbers that are duplicated mean for example, that patient number 2 has the T3118C mutation and the A3149G and the C3556T. The table is organized according to the sequential location of the BC in the KLF6 gene.BC in KLF6 geneExon 2 positionAA changeNS or SCHASM scoreP valueBHFDRPolyPhen-2No. of clones†Clones that showed the specific BC in both DNA strands.Tumor groupGleason score1T3089C40C48RNS0.4860.0460.15Benign2Primary62T3118C69F57FS2Primary103T3147C98I67TNS0.6420.1180.15Benign2Primary64T3147C98I67TNS0.6420.1180.15Benign2Primary62A3149G100I68VNS0.540.0650.15Benign2Primary105G3354T305S136INS0.5320.0630.15Benign2Primary106C3390T341P148SNS0.3720.0250.15Probably damaging2Primary64T3400C351S151SS2Primary67G3401A352P152PS2Primary68C3407T358L154LS2Primary69A3410G361S155GNS0.660.1340.15Benign2Primary610C3428T379L161LS2Primary64C3428T379L161LS2NEC11G3432A383W162stopNSDamaging2Met12G3434A385G163SNS0.8380.4960.5Benign2Primary613A3468G419K174RNS0.2980.0140.15Possibly damaging4Met14A3468G419K174RNS0.2980.0140.15Possibly damaging2Primary615C3478T429S177SS2Primary816A3482G433T179ANS0.5680.0770.15Benign2Primary817G3487A438S180SS7Primary613G3490A441G181GS4Met13A3504G455K186ENS0.550.0680.15Benign2Met18A3533T484R196WNS0.610.0980.15Probably damaging2Primary619G3550A501R201RS9Primary82C3556T507H203HS2Primary1020T3577C528V210VS2Primary6Distribution of cases with BCs in two or more clones and effect on AA sequence of KLF6 exon 2. CHASM raw score, P value, and BHFDR values for the prediction of driver/passenger NS missense mutations. PolyPhen-2 prediction of the effect of NS missense
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