The tumor suppressor KLF6 is a member of the Krüppel-like family of transcription factors, which has been implicated in the pathogenesis of several human carcinomas. Uncovering the transcriptional targets relevant for its tumorigenic properties, including cellular proliferation and invasion, will be essential to understanding possible mechanisms by which KLF6 and its antagonistic splice form, KLF6-SV1, regulate this development. To begin defining possible metastatic-related pathways, we analysed the effect of KLF6 dysregulation on a recognized suppressor of cellular invasion, E-cadherin. Targeted KLF6 reduction in an ovarian cancer cell line, SKOV-3, resulted in a 50% reduction of E-cadherin expression (P<0.01) and conversely, KLF6-SV1 silencing upregulated E-cadherin approximately fivefold (P<0.0001). These changes resulted from KLF6 directly transactivating the E-cadherin promoter as demonstrated by luciferase promoter assay and chromatin immunoprecipitation (ChIP). KLF6-mediated changes in E-cadherin levels were accompanied by downstream changes in both the subcellular localization of β-catenin and c-myc expression levels. Moreover, and consistent with these experimental findings, patient-derived epithelial ovarian tumors with low KLF6 and high KLF6-SV1 expression ratios had significantly decreased E-cadherin expression (P<0.0001). These combined findings highlight the E-cadherin pathway as a novel and functionally important mediator by which changes in KLF6 and KLF6-SV1 can directly alter ovarian tumor invasion and metastasis.
The tumor suppressor KLF6 is a member of the Krüppel-like family of zinc-finger transcription factors, which regulate differentiation and development through their roles in growth-related signal transduction pathways, cell proliferation, apoptosis and angiogenesis (Bieker, 2001; Black et al., 2001). KLF6 inactivation was initially demonstrated in prostate cancer (Narla et al., 2001; Chen et al., 2003) and since then, additional studies have extended the range of human cancers associated with KLF6 inactivation including colorectal (Reeves et al., 2004), gastric (Cho et al., 2005), lung (Ito et al., 2004), hepatocellular (Tal-Kremer et al., 2004), nasopharyngeal (Chen et al., 2002), astrocytic glioma (Jeng and Hsu, 2003), as well as ovarian (DiFeo et al., in press). In addition, two converging lines of evidence suggest that KLF6 status can both define cancer susceptibility and long-term outcome. First, in a multi-institutional study of over 3400 men, we have recently shown that a specific germline KLF6 SNP, regardless of family history of disease, increases lifetime risk of prostrate cancer (Narla et al., 2005a). This single nucleotide polymorphism (SNP) increases KLF6 gene alternative splicing to yield the biologically active, growth-promoting isoform KLF6-SV1, which increases tumor cell proliferation, invasion and in vivo tumor growth (Narla et al., 2005a, 2005b). Second, multiple gene expression studies in different cancers have linked decreased KLF6 expression with tumor recurrence, aggressive behavior, poor clinical outcomes and chemotherapeutic resistance (Singh et al., 2002; Glinsky et al., 2004; Kettunen et al., 2004; MacLeod et al., 2005; Stanbrough et al., 2006).
One mechanism by which KLF6 suppresses cellular proliferation is through upregulation of p21 in a p53-independent manner in a number of cellular contexts (Narla et al., 2001). Tumor-derived KLF6 mutations can abrogate its growth-suppressive effects, at least in part, through a loss of ability to transactivate the p21 gene (Narla et al., 2001; Kremer-Tal et al., 2004; Reeves et al., 2004). Additional pathways by which KLF6 regulates cancer development and progression are emerging and include inhibition of key oncogenic signaling pathways, such as the cyclin-dependent kinase complex CDK4-cyclinD1 (Benzeno et al., 2004) and c-jun oncoprotein (Slavin et al., 2004) and through an uncharacterized, p53-independent induction of apoptosis (Ito et al., 2004). Identifying additional transcriptional and translational pathways regulated by KLF6 and KLF6-SV1 should therefore provide important insights into KLF6/KLF6-SV1-mediated tumorigenic mechanisms.
We recently demonstrated that inhibition of either KLF6 or KLF6-SV1 has dramatic and opposite effects on both ovarian cancer cell growth and in vivo tumor dissemination (DiFeo et al., manuscript in revision). Specifically, stably silencing KLF6 in SKOV-3 ovarian cancer cells resulted in rapid tumor growth, marked ascites and intraperitoneal dissemination in an in vivo mouse model. No viable tumors formed when KLF6-SV1 was silenced before tumor cell implantation. To explore possible underlying molecular mechanisms related to the invasive phenotypic changes, we analysed the effect of KLF6 dysregulation on a recognized suppressor of cellular invasion, E-cadherin. Reduced E-cadherin expression is a critical molecular event contributing to dysregulated cell adhesion, triggering cancer invasion and metastasis (Naora and Montell, 2005). Additionally, E-cadherin loss may result in β-catenin nuclear localization and subsequent upregulation of the transcriptional activities of TCF/LEF DNA-binding factors (Stockinger et al., 2001; Conacci-Sorrell et al., 2003). In turn, nuclear β-catenin upregulation activates growth-promoting genes, such as c-myc (Stockinger et al., 2001). Thus, resulting downstream changes in β-catenin subcellular localization could potentially affect two critical cancer-promoting processes, invasion and proliferation.
First, using SKOV-3 cells, which express low levels of both KLF6 and KLF6-SV1, we stably expressed siRNA to luciferase (si-luc), KLF6 (si-KLF6) and KLF6-SV1 (si-SV1) and examined the effects on E-cadherin expression by quantitative reverse transcription–polymerase chain reation (qtRT–PCR). Strikingly, E-cadherin expression and protein levels were reduced ∼50% in si-KLF6 cells (P<0.01) and upregulated approximately fivefold in si-SV1 cells (P<0.0001) (Figure 1). This effect on E-cadherin expression was not restricted to SKOV-3 cells, and in fact, was present in virtually all cell lines tested from multiple tissue origins including MCF10A (normal mammary epithelial), 293T (embryonic kidney), Hep3B (hepatoma), HCT116 (colon carcinoma), HuH7 (hepatoma), MDA-435 (breast carcinoma) and MDA-453 (breast carcinoma) (data not shown).
Therefore, we next sought to determine whether these differences were a result of direct transcriptional regulation by KLF6. The Kruppel-like family of transcription factors (KLFs) and the related Sp1-like proteins regulate a large number of genes through their direct binding of GC-box promoter elements (Kaczynski et al., 2003). Therefore, we generated a series of deletion constructs in the E-cadherin promoter sequentially eliminating five putative GC-box elements that were clustered within four upstream sites (Figure 2). Although the −1000 and −230 nt E-cadherin deletion constructs maintained strong transactivation of the reporter, this effect was lost in the -103 and -37 constructs. These findings strongly suggested that either the -150 and/or -117 GC-box elements, both contained within Site #3, were necessary and sufficient in vitro for full KLF6-mediated activation (P<0.0001). The specificity of these findings was reinforced by the finding that the -37 GC-rich region was insufficient for increasing basal transcription levels by KLF6, unlike previous results demonstrating WT1-mediated transactivation through this motif (Hosono et al., 2000) (Figure 2). As predicted, KLF6-SV1 failed to transactivate any of the constructs, as it lacks a DNA-binding domain and is localized to the cytoplasm (Narla et al., 2005a) (Figure 2). One explanation for KLF6-SV1-mediated changes in E-cadherin expression (Figure 1) may be owing to a dominant-negative interaction with endogenous KLF6, as suggested previously (Narla et al., 2005a, 2005b).
To establish a direct interaction between KLF6 and the native E-cadherin promoter in vivo, ChIP analysis was performed. In HeLa cells transfected with a mammalian KLF6 expression vector, KLF6 protein was shown to occupy the GC box elements contained within Sites 3 (−150 and −117 nt) and 4 (−53 nt) (Figure 3). Both these sites are contained within the region required for maximal transcriptional transactivation using the E-cadherin promoter–reporter constructs. (Figure 2). The two most upstream sites failed to demonstrate a detectable interaction; also consistent with the promoter–reporter assays. An additional confirmation of KLF6 binding selectivity was demonstrated by lysine acetylation. Chromatin immunoprecipitation analysis demonstrated that the level of lysine acetylation of histone H3 at lysine 9 (K9), an epigenetic modification directly associated with gene activation, clearly overlaps with KLF6 binding site occupation (Figure 3).
As E-cadherin is a main binding partner of β-catenin and plays a pivotal role in its stabilization, localization and subsequent upregulation of the transcriptional activities of TCF/LEF DNA-binding factors (Stockinger et al., 2001; Conacci-Sorrell et al., 2003), we next investigated the effect of KLF6-mediated E-cadherin changes on β-catenin subcellular localization. Two complementary methods were used. First, cytosolic β-catenin protein levels were measured following stepwise -separation of cytosolic and nuclear fractions. KLF6 downregulation decreased cytosolic β-catenin fivefold (Figure 4a). Conversely, KLF6-SV1 silencing upregulated cytosolic β-catenin 2.5-fold and there was a concomitant upregulation of nuclear KLF6 (Figure 4a). Second, differences in β-catenin nuclear and cytosolic translocation were also measured by immunocytochemistry. Consistent with cell fractionation findings, si-KLF6 cells displayed twice as many cells with nuclear β-catenin expression (P<0.01) (Figure 4b), whereas it was half as frequent in si-SV1 stable cell lines (P<0.01) (Figure 4b).
We also assayed c-myc expression as a functional end point marker of β-catenin-LEF/TCF activity. In si-SV1 cells, c-myc was reduced ≈70% (Figure 4a), whereas in si-KLF6 cells, c-myc was not increased. This blunted response possibly reflects already highly increased c-myc expression in certain ovarian cancer cells (Berchuck and Charney, 1997).
Having established the role of KLF6 on E-cadherin expression in multiple cell lines, we next sought to further the physiologic relevance of these findings by examining patient-derived ovarian tumor samples. We recently demonstrated that changes in KLF6 and KLF6-SV1 expression levels are present in a majority of ovarian tumors and can affect tumor growth, invasion and dissemination (DiFeo et al., in press). Samples were binned according to their KLF6-SV1/KLF6 expression pattern. Samples with decreased KLF6 and increased KLF6-SV1 expression have a high KLF6-SV1/KLF6 ratio. As the expression of KLF6 increases or KLF6-SV1 decreases, the KLF6-SV1/KLF6 ratio decreases. In accordance with these findings, papillary serous tumors with a high KLF6-SV1/KLF6 ratio expression pattern (n=16) were associated with an approximately 80% reduction in E-cadherin expression compared to those samples with low levels (n=14) (P<0.0002) (Figure 5). As E-cadherin decreases are believed to contribute to EOC development and progression (Frixen et al., 1991; Vleminckx et al., 1991), it is particularly relevant that stage IV tumors with a high KLF6-SV1/KLF6 splicing ratio had minimal E-cadherin expression (P<0.0001). Similarly, stage III tumors with high KLF6-SV1/KLF6 splicing ratio had approximately 80% lower E-cadherin levels (P<0.0004) (Figure 5).
Taken together, our findings define E-cadherin as a novel KLF6 transcriptional target and suggest that KLF6-SV1/KLF6 regulation of this critical suppressor of cellular invasion is one mechanism by which KLF6-SV1/KLF6 contributes to ovarian cancer pathogenesis. KLF6 transcriptionally regulates a number of essential proliferation and differentiation genes, including p21(CIP1/Waf1) (Narla et al., 2001), transforming growth factor-β1, type I and II receptors (Kim et al., 1998) and the insulin-like growth factor I receptor (Rubinstein et al., 2004). Our combined findings, both in cultured cells and patient-derived ovarian cancer samples, now suggest that changes in tumor proliferation and invasion may, in part, be functionally linked through the action of KLF6 and KLF6-SV1 on E-cadherin. Indeed, loss of E-cadherin expression is proposed to be a central event in enhancing metastasis from ovarian tumors (Frixen et al., 1991; Vleminckx et al., 1991) and is associated with decreased patient survival (Gabbert et al., 1996). In turn, increased nuclear β-catenin may activate growth promoting genes as demonstrated by c-myc upregulation in cultured cells and si-KLF6 tumor xenografts (DiFeo et al., in press). As previously demonstrated in a mouse mammary epithelial cell system, β-catenin mislocalization ultimately affects cellular proliferation (Stockinger et al., 2001). Interestingly, changes in KLF6-SV1 levels which decrease cellular proliferation also upregulate E-cadherin expression and result in β-catenin's cytoplasmic sequestration. In summary, these findings define E-cadherin regulation by KLF6 and KLF6-SV1 as a potentially critical pathway linking together KLF6 and KLF6-SV1's dual effects on both proliferation and invasion in ovarian cancer.
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This research was financially supported by a Grant, JAM: DAMD17-03-10129.
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DiFeo, A., Narla, G., Camacho-Vanegas, O. et al. E-cadherin is a novel transcriptional target of the KLF6 tumor suppressor. Oncogene 25, 6026–6031 (2006). https://doi.org/10.1038/sj.onc.1209611
- alternative splicing
- ovarian cancer
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