e15191 Background: Inactivation of KLF6 by mutations and alternative splicing has been implicated in PC. However, there are significant discrepancies among human PC studies concerning the prevalence and significance of KLF6 mutations, and the prevalence of splice variants (SVs) has not been established. The objective of this study was to assess the prevalence and role of KLF6 splicing in human PC progression. Methods: Human specimens were obtained from the tissue banks of three institutions. Representative epithelium of 74 frozen PC specimens [25 primary hormone-naïve (HN) PCs, 21 primary hormone-deprived (HD) PCs, 6 castrate-resistant metastases] and 22 normal prostates (NPs) were microdissected. KLF6 cDNA was amplified by PCR, cloned and sequenced in both forward and reverse directions and compared to the KLF6 GenBank sequence. The transcriptional activity of KLF6 SVs was evaluated in co-transfection assays with a p21-reporter, and their effect on LNCaP cell growth was determined by MTT assays. Results: Sixteen different SVs were identified: 13 were novel; 14 affected the DNA binding domain (DBD) and were transcriptionally inactive. The incidence of SVs was increased in PC versus NP (75% vs 45%; p=0.01) and in HD-PC versus HN-PC (90% vs 64%; p=0.04) HD-PC were significantly enriched in SVs lacking the DBD. Forced expression of dysfunctional KLF6 SVs increased growth of LNCaP cells in androgen-depleted media. Conclusions: KLF6 SVs lacking p21-reporter transactivation activity occur in NP epithelium but are markedly increased in incidence, variety and level of expression in PC tissues that survive androgen-deprivation. This enrichment may enable resistance to hormone therapy and a proliferative advantage under castrate conditions.

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.