We investigated the role of the KLF6 tumor suppressor gene and its alternatively spliced isoform KLF6-SV1 in epithelial ovarian cancer (EOC).
We first analyzed tumors from 68 females with EOC for KLF6 gene inactivation using fluorescent loss of heterozygosity (LOH) analysis and direct DNA sequencing and then defined changes in KLF6 and KLF6-SV1 expression levels by quantitative real-time PCR. We then directly tested the effect of KLF6 and KLF6-SV1 inhibition in SKOV-3 stable cell lines on cellular invasion and proliferation in culture and tumor growth, i.p. dissemination, ascites production, and angiogenesis in vivo using BALB/c nu/nu mice. All statistical tests were two sided.
LOH was present in 59% of samples in a cell type-specific manner, highest in serous (72%) and endometrioid (75%) subtypes, but absent in clear cell tumors. LOH was significantly correlated with tumor stage and grade. In addition, KLF6 expression was decreased in tumors when compared with ovarian surface epithelial cells. In contrast, KLF6-SV1 expression was increased approximately 5-fold and was associated with increased tumor grade regardless of LOH status. Consistent with these findings, KLF6 silencing increased cellular and tumor growth, angiogenesis, and vascular endothelial growth factor expression, i.p. dissemination, and ascites production. Conversely, KLF6-SV1 down-regulation decreased cell proliferation and invasion and completely suppressed in vivo tumor formation.
Our results show that KLF6 and KLF6-SV1 are associated with key clinical features of EOC and suggest that their therapeutic targeting may alter ovarian cancer growth, progression, and dissemination.
"Immunoblotting of KLF6 in H23 cells transfected with pre-miR-141or pre-miR-200c showed that only miR-141 significantly reduced the KLF6 protein levels at 24 h (Figure 5A). Since KLF6 regulates the expression and secretion of VEGFA, a critical angiogenic factor , we examined the effect of increasing miR-141 expression levels on VEGFA levels. We overexpressed both miR-141 and miR-200c in the H23, A-549 and HCC-44 NSCLC cell lines and treated the cells with DFX to produce hypoxia. "
[Show abstract][Hide abstract] ABSTRACT: Background
Several treatments in non-small cell lung cancer (NSCLC) are histology-dependent, and the need for histology-related markers is increasing. MicroRNAs (miRNAs) are promising molecular markers in multiple cancers and show differences in expression depending on histological subtype. The miRNA family miR-200 has been associated with the regulation of epithelial-mesenchymal (EMT)/mesenchymal-epithelial transition (MET). EMT involves profound phenotypic changes that include the loss of cell-cell adhesion, the loss of cell polarity, and the acquisition of migratory and invasive properties that facilitates metastasis. A dual role for the miR-200 family in the prognosis of several tumors has been related to tumor cell origin. However, the prognostic role and function of miR-200 family in early-stage NSCLC adenocarcinoma and squamous cell carcinoma (SCC) have not been well established.
miRNA expression was determined using TaqMan assays in 155 tumors from resected NSCLC patients. Functional studies were conducted in three NSCLC cell lines: H23, A-549 and HCC-44.
High miR-200c expression was associated with shorter overall survival (OS) in the entire cohort (p = 0.024). High miR-200c (p = 0.0004) and miR-141 (p = 0.009) expression correlated with shorter OS in adenocarcinoma – but not in SCC. In the multivariate analysis, a risk score based on miR-141 and miR-200c expression emerged as an independent prognostic factor for OS in the entire cohort (OR, 2.787; p = 0.033) and in adenocarcinoma patients (OR, 10.649; p = 0.002). Functional analyses showed that miR-200c, was related to mesenchymal-epithelial transition (MET) and affected cell migration and E-cadherin levels, while overexpression of miR-141 reduced KLF6 protein levels and produced an increase of secretion of VEGFA in vitro (H23, p = 0.04; A-549, p = 0.03; HCC-44, p = 0.02) and was associated with higher blood microvessel density in patient tumor samples (p<0.001).
High miR-141 and miR-200c expression are associated with shorter OS in NSCLC patients with adenocarcinoma through MET and angiogenesis.
PLoS ONE 07/2014; 9(7):e101899. DOI:10.1371/journal.pone.0101899 · 3.23 Impact Factor
"The tumor suppressor function of KLF6 was further demonstrated in hepatic  and gastric cancer [46,47] and linked to a role in cellular differentiation. Several mechanisms leading to KLF6 loss have been described, including promoter hypermethylation , somatic mutations and loss of heterozygosity in prostate , gastric  and ovarian cancer . Interestingly, an alternative mechanism to downregulate KLF6 was found to involve the generation of three splice variants (SV1-3) with antagonistic effects on the wild type KLF6 tumor suppressor function . "
[Show abstract][Hide abstract] ABSTRACT: Krüppel-like factors (KLFs) comprise a highly conserved family of zinc finger transcription factors, that are involved in a plethora of cellular processes, ranging from proliferation and apoptosis to differentiation, migration and pluripotency. During the last few years, evidence on their role and deregulation in different human cancers has been emerging. This review will discuss current knowledge on Krüppel-like transcription in the epithelial-mesenchymal transition (EMT), invasion and metastasis, with a focus on epithelial cancer biology and the extensive interface with pluripotency. Furthermore, as KLFs are able to mediate different outcomes, important influences of the cellular and microenvironmental context will be highlighted. Finally, we attempt to integrate diverse findings on KLF functions in EMT and stem cell biology to fit in the current model of cellular plasticity as a tool for successful metastatic dissemination.
"This is the case of oncogenic splice variant 1 of KLF6 (KLF6-SV1), a tumor suppressor gene belonging to the Krüppel-like family of transcription factors, able to act as functional driver of the entire metastatic cascade through Twist induction [77, 78]. Notably, KLF6-SV1 antagonizes the tumor suppressive activity of the full-length KLF6 protein and sustains tumor growth and dissemination in ovarian and prostate cancer models [79–81]. Interestingly, increased expression of KLF6-SV1 occurs in many tumors and is associated with poor prognosis in prostate, lung, and ovarian cancers . "
[Show abstract][Hide abstract] ABSTRACT: Alterations in the abundance or activities of alternative splicing regulators generate alternatively spliced variants that contribute to multiple aspects of tumor establishment, progression and resistance to therapeutic treatments. Notably, many cancer-associated genes are regulated through alternative splicing suggesting a significant role of this post-transcriptional regulatory mechanism in the production of oncogenes and tumor suppressors. Thus, the study of alternative splicing in cancer might provide a better understanding of the malignant transformation and identify novel pathways that are uniquely relevant to tumorigenesis. Understanding the molecular underpinnings of cancer-associated alternative splicing isoforms will not only help to explain many fundamental hallmarks of cancer, but will also offer unprecedented opportunities to improve the efficacy of anti-cancer treatments.
International Journal of Cell Biology 10/2013; 2013(2):962038. DOI:10.1155/2013/962038
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