Targeted Inhibition of the KLF6 Splice Variant, KLF6 SV1, Suppresses Prostate Cancer Cell Growth and Spread

Abstract

Prostate cancer is a leading cause of cancer death in men. Risk prognostication, treatment stratification, and the development of rational therapeutic strategies lag because the molecular mechanisms underlying the initiation and progression from primary to metastatic disease are unknown. Multiple lines of evidence now suggest that KLF6 is a key prostate cancer tumor suppressor gene including loss and/or mutation in prostate cancer tumors and cell lines and decreased KLF6 expression levels in recurrent prostate cancer samples. Most recently, we identified a common KLF6 germ line single nucleotide polymorphism that is associated with an increased relative risk of prostate cancer and the increased production of three alternatively spliced, dominant-negative KLF6 isoforms. Here we show that although wild-type KLF6 (wtKLF6) acts as a classic tumor suppressor, the single nucleotide polymorphismincreased splice isoform, KLF6 SV1, displays a markedly opposite effect on cell proliferation, colony formation, and invasion. In addition, whereas wtKLF6 knockdown increases tumor growth in nude mice >2-fold, short interfering RNAmediated KLF6 SV1 inhibition reduces growth by f50% and decreases the expression of a number of growth-and angiogenesis-related proteins. Together, these findings begin to highlight a dynamic and functional antagonism between wtKLF6 and its splice variant KLF6 SV1 in tumor growth and dissemination. (Cancer Res 2005; 65(13): 5761-8)

KLF4, p21 and context-dependent opposing forces in cancer

“…It is associated with increased production of three alternatively spliced, dominant-negative KLF6 isoforms. Whereas full-length KLF6 has a role in tumour suppression, the alternatively spliced KLF6 isoforms seem to have oncogenic functions, as specific disruption of these variants suppressed rather than enhanced tumorigenesis 68 . The dominantnegative properties of these splice variants are illustrated by their abilities to counteract induction of CDKN1A and proliferative arrest by full-length KLF6 (REF.…”

A small interfering RNA targeting the KLF6 splice variant, KLF6-SV1, as gene therapy for gastric cancer

“…To date, three alternatively spliced KLF6 isoforms, KLF6-SV1, -SV2 and -SV3, have been identified [21]. KLF6-SV1 is an oncogenic splice variant of KLF6, and increased KLF6-SV1 expression is associated with poor prognosis in prostate, lung, and ovarian cancers [10,13,[21][22][23]. Furthermore, KLF6-SV1 has been shown to be biologically active, antagonizing the tumor suppressor function of KLF6 and promoting tumor growth and dissemination in both ovarian and prostate cancer models [20,21,23].…”

“…KLF6-SV1 is an oncogenic splice variant of KLF6, and increased KLF6-SV1 expression is associated with poor prognosis in prostate, lung, and ovarian cancers [10,13,[21][22][23]. Furthermore, KLF6-SV1 has been shown to be biologically active, antagonizing the tumor suppressor function of KLF6 and promoting tumor growth and dissemination in both ovarian and prostate cancer models [20,21,23]. Evidence from many cancer types and different stages of tumorigenesis and progression has confirmed important roles for KLF6 and KLF6-SV1; in particular, that the overexpression of KLF6-SV1 is associated with metastatic potential and with decreased survival and that small interfering RNA (siRNA)-mediated downregulation of KLF6-SV1 decreases tumor growth in vitro [20,21] and in vivo [10,23].…”

“…Furthermore, KLF6-SV1 has been shown to be biologically active, antagonizing the tumor suppressor function of KLF6 and promoting tumor growth and dissemination in both ovarian and prostate cancer models [20,21,23]. Evidence from many cancer types and different stages of tumorigenesis and progression has confirmed important roles for KLF6 and KLF6-SV1; in particular, that the overexpression of KLF6-SV1 is associated with metastatic potential and with decreased survival and that small interfering RNA (siRNA)-mediated downregulation of KLF6-SV1 decreases tumor growth in vitro [20,21] and in vivo [10,23]. In addition, KLF6-SV1 has been identified as a novel anti-apoptotic protein, independent of a tumor cell's p53 status [10,24], which suggested its potential as a novel target for anti-cancer therapy.…”

E-cadherin is a novel transcriptional target of the KLF6 tumor suppressor

“…Polyclonal pools of each short interfering RNA (siRNA)-infected cell line were collected and the levels of KLF6 and KLF6-SV1 were determined by qRT-PCR and Western blot. RNA isolation, reverse transcription and quantification was performed as previously described (Narla et al, 2005b) using E-cadherin-forward: 5 0 -CAA AGT GGG CAC AGA TGG TGT G-3 0 and reverse: 5 0 -CTG CTT GGA TTC CAG AAA CGG-3 0 and GAPDH forward: 5 0 -CAA TGA CCC CTT CAT TGA CC-3 0 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) reverse: 5 0 -GAT CTC GCT CCT GGA AGA TG-3 0 primers. Expression levels were calculated by normalizing each cDNA to GAPDH and then using this normalized value to calculate fold change.…”