[Show abstract][Hide abstract] ABSTRACT: Localization of KLF6, KLF6-SV1, KLF6 NLS and the ZFs in 293T cells. Co-localization of KLF6, KLF6-SV1, KLF6 NLS or the ZFs EGFP constructs together with Cherry-H2A, which was used to show nuclear staining. Localization of the different constructs was observed by fluorescence microscopy.
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[Show abstract][Hide abstract] ABSTRACT: p21 promoter luciferase assays for KLF6, KLF6-SV1 and the NLS mutants as well as EGFP empty vector in 293T cells. Expression levels were calculated by normalizing each luciferase value to Renilla gene expression and representing the Relative Luciferase Units (RLU). All experiments were performed at least three times and in triplicate. Statistical significance was determined by two tailed, two-sample equal variance T-test (p<0.005).
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[Show abstract][Hide abstract] ABSTRACT: The tumor suppressor KLF6 and its oncogenic cytoplasmic splice variant KLF6-SV1 represent a paradigm in cancer biology in that their antagonistic cancer functions are encoded within the same gene. As a consequence of splicing, KLF6-SV1 loses both the C-terminus C2H2 three zinc finger (ZF) domain, which characterizes all KLF proteins, as well as the adjacent 5' basic region (5BR), a putative nuclear localization signal (NLS). It has been hypothesized that this NLS is a functional domain critical to direct the distinct subcellular localization of the tumor suppressor and its splice variant.
In this study, we demonstrate using EGFP fusion constructs that KLF6/KLF6-SV1 nucleo-cytoplasmic transport is not regulated by the 5' basic region but activated by a novel NLS encoded within the ZF domain, and a nuclear export signal (NES) located in the first 16 amino acids of the shared N-terminus sequence. We demonstrate KLF6 nuclear export to be Crm1-dependent. The dysregulation of nucleo-cytoplasmic transport when disrupting the KLF6 NLS using site-directed mutagenesis showed that its integrity is necessary for appropriate protein stability. Moreover, these mutations impaired transcriptional induction of two KLF6 well-characterized target genes, E-cadherin and p21, as shown by RT-PCR and luciferase promoter assays. The addition of the ZF domain to KLF6-SV1 results in its nuclear localization and a markedly decreased half-life similar to wild type KLF6.
We describe the domains that control KLF6 nucleo-cytoplasmic shuttling and how these domains play a role in KLF6 protein half-life and tumor suppressor function. The results begin to mechanistically explain, at least in part, the opposing functions of KLF6 and KLF6-SV1 in cancer.
[Show abstract][Hide abstract] ABSTRACT: Mutations in the N-terminus 16 amino acids results in increased KLF6 nuclear localization. Subcellular localization of the different NES mutants. Cherry-H2A construct was used to show nuclear staining. Localization of the different constructs was observed by fluorescence microscopy. Graphs with the percentage of cells with the different localization are shown on the right. N, Nuclear localization, C, Cytoplasmic localization, N = C, Nuclear and cytoplasmic distribution within the same cell is equal, N>C, Nuclear localization is more intense than cytoplasmic localization, N<C, Nuclear localization is less intense than cytoplasmic localization, and PN, perinuclear localization.
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[Show abstract][Hide abstract] ABSTRACT: Epithelial ovarian cancer is one of the most lethal gynecologic cancers and the fifth most frequent cause of female cancer deaths in the United States. Despite dramatic treatment successes in other cancers through the use of molecular agents targeted against genetically defined events driving cancer development and progression, very few insights into epithelial ovarian cancer have been translated from the laboratory to the clinic. If advances are to be made in the early diagnosis, prevention, and treatment of this disease, it will be critical to characterize the common and private (personalized) genetic defects underlying the development and spread of epithelial ovarian cancer. The tumor suppressor Kruppel-like factor 6 and its alternatively spliced, oncogenic isoform, Kruppel-like factor 6 splice variant 1, are members of the Kruppel-like zinc finger transcription factor family of proteins, which have diverse roles in cellular differentiation, development, proliferation, growth-related signal transduction, and apoptosis. Inactivation of Kruppel-like factor 6 and overexpression of Kruppel-like factor 6 splice variant 1 have been associated with the progression of a number of human cancers and even with patient survival. This article summarizes our recent findings demonstrating that a majority of epithelial ovarian cancer tumors have Kruppel-like factor 6 allelic loss and decreased expression coupled with increased expression of Kruppel-like factor 6 splice variant 1. The targeted reduction of Kruppel-like factor 6 in ovarian cancer cell lines results in marked increases in cell proliferation, invasion, tumor growth, angiogenesis, and intraperitoneal dissemination in vivo. In contrast, the inhibition of Kruppel-like factor 6 splice variant 1 decreases cellular proliferation, invasion, angiogenesis, and tumorigenicity; this provides the rationale for its potential therapeutic application. These results and our recent demonstration that the inhibition of Kruppel-like factor 6 splice variant 1 can dramatically prolong survival in a preclinical mouse model of ovarian cancer are reviewed and discussed.
Full-text · Article · Dec 2009 · Mount Sinai Journal of Medicine A Journal of Translational and Personalized Medicine
[Show abstract][Hide abstract] ABSTRACT: Cell death and differentiation is a monthly research journal focused on the exciting field of programmed cell death and apoptosis. It provides a single accessible source of information for both scientists and clinicians, keeping them up-to-date with advances in the field. It encompasses programmed cell death, cell death induced by toxic agents, differentiation and the interrelation of these with cell proliferation.
[Show abstract][Hide abstract] ABSTRACT: NK cell cytotoxicity requires the formation of an actin-rich immunological synapse (IS) with a target cell and the polarization of perforin-containing lytic granules toward the IS. Following the polarization of lytic granules, they traverse through the actin-rich IS to join the NK cell membrane in order for directed secretion of their contents to occur. We examined the role of myosin IIA as a candidate for facilitating this prefinal step in lytic NK cell IS function. Lytic granules in and derived from a human NK cell line, or ex vivo human NK cells, were constitutively associated with myosin IIA. When isolated using density gradients, myosin IIA-associated NK cell lytic granules directly bound to F-actin and the interaction was sensitive to the presence of ATP under conditions of flow. In NK cells from patients with a truncation mutation in myosin IIA, NK cell cytotoxicity, lytic granule penetration into F-actin at the IS, and interaction of isolated granules with F-actin were all decreased. Similarly, inhibition of myosin function also diminished the penetration of lytic granules into F-actin at the IS, as well as the final approach of lytic granules to and their dynamics at the IS. Thus, NK cell lytic granule-associated myosin IIA enables their interaction with actin and final transit through the actin-rich IS to the synaptic membrane, and can be defective in the context of naturally occurring human myosin IIA mutation.
Full-text · Article · Jul 2009 · The Journal of Immunology
[Show abstract][Hide abstract] ABSTRACT: Defects in apoptosis are not only a hallmark of cancer initiation and progression but can also underlie the development of chemoresistance. How the tightly regulated cascade of protein-protein interactions between members of three competing protein families regulating the apoptotic cascade is subverted in tumor cells is incompletely understood. Here, we show that KLF6-SV1, whose overexpression is associated with poor survival in several different cancers and is an alternatively spliced isoform of the Krüppel-like tumor suppressor KLF6, is a critical prosurvival/antiapoptotic protein. KLF6-SV1 binds the proapoptotic BH3-only protein NOXA, which results in their mutual HDM2-dependent degradation. In turn, this increases the intracellular concentration of the prosurvival binding partner of NOXA, Mcl-1, and effectively blocks apoptosis. In an ovarian cancer model, systemically delivered small interfering RNA against KLF6-SV1 induces spontaneous apoptosis of tumor cells, decreases tumor burden, and restores cisplatin sensitivity in vivo. Moreover, i.p. delivery of siKLF6-SV1 RNA halts ovarian tumor progression and improves median and overall survival (progression-free for >15 months; P < 0.0002) in mice in a dose-dependent manner. Thus, KLF6-SV1 represents a novel regulator of protein interactions in the apoptotic cascade and a therapeutically targetable control point.
[Show abstract][Hide abstract] ABSTRACT: Systemic hyalinosis is an autosomal recessive disease that encompasses two allelic syndromes, infantile systemic hyalinosis (ISH) and juvenile hyaline fibromatosis (JHF), which are caused by mutations in the CMG2 gene. Here we have analyzed the cellular consequences of five patient-derived point mutations in the extracellular von Willebrand domain or the transmembrane domain of the CMG2 protein. We found that four of the mutations led to retention of the protein in the endoplasmic reticulum (ER), albeit through different mechanisms. Analysis of recombinant CMG2 von Willebrand factor A (vWA) domains, to which three of the mutations map, indicated that the mutations did not prevent proper folding and ligand binding, suggesting that, in vivo, slow folding, rather than misfolding, is responsible for ER retention. Our work shows that systemic hyalinosis can be qualified as a conformational disease, at least for the mutations that have been mapped to the extracellular and transmembrane domains. The long ER half-life and the ligand binding ability of the mutated von Willebrand domains suggest that treatments based on chemical chaperones could be beneficial.
[Show abstract][Hide abstract] ABSTRACT: Gastric cancer is the second most common cancer and a leading cause of cancer-related death worldwide. The Kruppel-like factor 6 (KLF6) tumour suppressor gene had been previously shown to be inactivated in a number of human cancers through loss of heterozygosity (LOH), somatic mutation, decreased expression and increased alternative splicing into a dominant negative oncogenic splice variant, KLF6-SV1. In the present study, 37 gastric cancer samples were analysed for the presence of loss of heterozygosity (LOH) of the KLF6 locus and somatic mutation. In total, 18 of 34 (53%) of the gastric cancer samples analysed demonstrated KLF6 locus specific loss. Four missense mutations, such as T179I, R198G, R71Q and S180L, were detected. Interestingly, two of these mutations R71Q and S180L have been identified independently by several groups in various malignancies including prostate, colorectal and gastric cancers. In addition, decreased wild-type KLF6 (wtKLF6) expression was associated with loss of the KLF6 locus and was present in 48% of primary gastric tumour samples analysed. Functional studies confirmed that wtKLF6 suppressed proliferation of gastric cancer cells via transcriptional regulation of the cyclin-dependent kinase inhibitor p21 and the oncogene c-myc. Functional characterisation of the common tumour-derived mutants demonstrated that the mutant proteins fail to suppress proliferation and function as dominant negative regulators of wtKLF6 function. Furthermore, stable overexpression of the R71Q and S180L tumour-derived mutants in the gastric cancer cell line, Hs746T, resulted in an increased tumourigenicity in vivo. Combined, these findings suggest an important role for the KLF6 tumour suppressor gene in gastric cancer development and progression and identify several highly cancer-relevant signalling pathways regulated by the KLF6 tumour suppressor gene.
Full-text · Article · Mar 2009 · European journal of cancer (Oxford, England: 1990)
[Show abstract][Hide abstract] ABSTRACT: The Krüppel-like zinc finger transcription factor (KLF6) gene encodes a family of proteins generated through alternative splicing involved in the regulation of cancer development and progression. Alternative splicing of the KLF6 gene results in the production of at least four alternatively spliced isoforms, two of which are extensively discussed in this review. The full length form of the KLF6 gene is a tumor suppressor gene that is frequently inactivated by loss of heterozygozity (LOH), somatic mutation, and/or decreased expression in human cancer. While the exact mechanisms underlying KLF6's tumor suppressor roles are not completely known, a number of highly relevant, overlapping pathways have been described: transactivation of p21 in a p53-independent manner, reduction of cyclin D1/cdk4 complexes via interaction with cyclin D1, inhibition of c-Jun proto-oncoprotein activities, decreased VEGF expression, and induction of apoptosis. Kruppel-like factor 6 splice variant 1 (KLF6-SV1) is an oncogenic splice variant of the KLF6 tumor suppressor gene that is specifically overexpressed in a number of human cancers. Increased KLF6-SV1 expression is associated with poor prognosis in prostate, lung, and ovarian cancer. 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. In addition, a common germline polymorphism in the KLF6 gene associated with increased prostate cancer risk in a large multi-institutional study of 3411 men results in increased expression of KLF6-SV1. Furthermore, recent studies have demonstrated that targeted reduction of KLF6-SV1 results in the induction of spontaneous apoptosis in cell culture, synergizes with chemotherapeutic agents like cisplatin, and results in significant tumor regression in vivo. Combined, these data make the KLF6 gene family a compelling therapeutic target for both the treatment of localized as well as metastatic cancer.
Full-text · Article · Feb 2009 · Drug resistance updates: reviews and commentaries in antimicrobial and anticancer chemotherapy
[Show abstract][Hide abstract] ABSTRACT: Multicentric osteolysis with nodulosis and arthropathy (MONA, NAO (OMIM no. 605156)) is an autosomal recessive member of the 'vanishing bone' syndromes and is notable for the extent of carpal and tarsal osteolysis and interphalangeal joint erosions, facial dysmorphia, and the presence of fibrocollagenous nodules. This rare disorder has been described previously in Saudi Arabian and Indian families. We now report on the first Turkish family with MONA, further confirming the panethnic nature of this disease. Strikingly, and in addition to the previously noted skeletal and joint features, affected members of this family also had congenital heart defects. Molecular analysis identified a novel MMP2 inactivating mutation that deletes the terminal hemopexin domains and thus confirmed the diagnosis of MONA. On the basis of these findings, we suggest that cardiac defects may also represent a component of this syndrome and thus a physiologically relevant target of MMP-2 activity.
Full-text · Article · Dec 2008 · European journal of human genetics: EJHG
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to correlate the status of the KLF6 tumour suppressor gene including loss of heterozygosity (LOH), mutation and alternative splicing in human pancreatic cancer with tumour grade and survival. Whereas neither KLF6 loss nor mutation was identified, expression of the KLF6 alternative splice forms was significantly increased in pancreatic tumour samples and cell lines. These cancers demonstrated marked cytoplasmic KLF6 expression, consistent with over-expression and accumulation of KLF6 splice form(s), which lack a nuclear localisation signal. In addition, KLF6 splicing correlated significantly with tumour stage and survival. In summary, pancreatic cancer displays a novel pattern of KLF6 dysregulation through selectively increased expression of KLF6 splice variants. Therefore, determination of KLF6 mRNA splicing levels may represent a novel biomarker predicting prognosis.
Full-text · Article · Oct 2008 · European journal of cancer (Oxford, England: 1990)
[Show abstract][Hide abstract] ABSTRACT: The advent of targeted therapies in hepatocellular carcinoma (HCC) has underscored the importance of pathway characterization to identify novel molecular targets for treatment. We evaluated mTOR signaling in human HCC, as well as the antitumoral effect of a dual-level blockade of the mTOR pathway.
The mTOR pathway was assessed using integrated data from mutation analysis (direct sequencing), DNA copy number changes (SNP-array), messenger RNA levels (quantitative reverse-transcription polymerase chain reaction and gene expression microarray), and protein activation (immunostaining) in 351 human samples [HCC (n = 314) and nontumoral tissue (n = 37)]. Effects of dual blockade of mTOR signaling using a rapamycin analogue (everolimus) and an epidermal/vascular endothelial growth factor receptor inhibitor (AEE788) were evaluated in liver cancer cell lines and in a xenograft model.
Aberrant mTOR signaling (p-RPS6) was present in half of the cases, associated with insulin-like growth factor pathway activation, epidermal growth factor up-regulation, and PTEN dysregulation. PTEN and PI3KCA-B mutations were rare events. Chromosomal gains in RICTOR (25% of patients) and positive p-RPS6 staining correlated with recurrence. RICTOR-specific siRNA down-regulation reduced tumor cell viability in vitro. Blockage of mTOR signaling with everolimus in vitro and in a xenograft model decelerated tumor growth and increased survival. This effect was enhanced in vivo after epidermal growth factor blockade.
MTOR signaling has a critical role in the pathogenesis of HCC, with evidence for the role of RICTOR in hepato-oncogenesis. MTOR blockade with everolimus is effective in vivo. These findings establish a rationale for targeting the mTOR pathway in clinical trials in HCC.
[Show abstract][Hide abstract] ABSTRACT: Metastatic prostate cancer (PCa) is one of the leading causes of death from cancer in men. The molecular mechanisms underlying the transition from localized tumor to hormone-refractory metastatic PCa remain largely unknown, and their identification is key for predicting prognosis and targeted therapy. Here we demonstrated that increased expression of a splice variant of the Kruppel-like factor 6 (KLF6) tumor suppressor gene, known as KLF6-SV1, in tumors from men after prostatectomy predicted markedly poorer survival and disease recurrence profiles. Analysis of tumor samples revealed that KLF6-SV1 levels were specifically upregulated in hormone-refractory metastatic PCa. In 2 complementary mouse models of metastatic PCa, KLF6-SV1-overexpressing PCa cells were shown by in vivo and ex vivo bioluminescent imaging to metastasize more rapidly and to disseminate to lymph nodes, bone, and brain more often. Interestingly, while KLF6-SV1 overexpression increased metastasis, it did not affect localized tumor growth. KLF6-SV1 inhibition using RNAi induced spontaneous apoptosis in cultured PCa cell lines and suppressed tumor growth in mice. Together, these findings demonstrate that KLF6-SV1 expression levels in PCa tumors at the time of diagnosis can predict the metastatic behavior of the tumor; thus, KLF-SV1 may represent a novel therapeutic target.
Full-text · Article · Aug 2008 · Journal of Clinical Investigation
[Show abstract][Hide abstract] ABSTRACT: Hepatocellular carcinoma (HCC) is the fifth most prevalent cancer worldwide and the third most lethal. Dysregulation of alternative splicing underlies a number of human diseases, yet its contribution to liver cancer has not been explored fully. The Krüppel-like factor 6 (KLF6) gene is a zinc finger transcription factor that inhibits cellular growth in part by transcriptional activation of p21. KLF6 function is abrogated in human cancers owing to increased alternative splicing that yields a dominant-negative isoform, KLF6 splice variant 1 (SV1), which antagonizes full-length KLF6-mediated growth suppression. The molecular basis for stimulation of KLF6 splicing is unknown.
In human HCC samples and cell lines, we functionally link oncogenic Ras signaling to increased alternative splicing of KLF6 through signaling by phosphatidylinositol-3 kinase and Akt, mediated by the splice regulatory protein ASF/SF2.
In 67 human HCCs, there is a significant correlation between activated Ras signaling and increased KLF6 alternative splicing. In cultured cells, Ras signaling increases the expression of KLF6 SV1, relative to full-length KLF6, thereby enhancing proliferation. Abrogation of oncogenic Ras signaling by small interfering RNA (siRNA) or a farnesyl-transferase inhibitor decreases KLF6 SV1 and suppresses growth. Growth inhibition by farnesyl-transferase inhibitor in transformed cell lines is overcome by ectopic expression of KLF6 SV1. Down-regulation of the splice factor ASF/SF2 by siRNA increases KLF6 SV1 messenger RNA levels. KLF6 alternative splicing is not coupled to its transcriptional regulation.
Our findings expand the role of Ras in human HCC by identifying a novel mechanism of tumor-suppressor inactivation through increased alternative splicing mediated by an oncogenic signaling cascade.
[Show abstract][Hide abstract] ABSTRACT: Liver transplantation usually cures hepatocellular carcinoma when the Milan selection criteria are applied, whereas there is substantial risk of posttransplant recurrence with tumors beyond these criteria. This study uses molecular data to identify a subgroup of patients who, despite having hepatocellular carcinoma beyond Milan criteria, have favorable outcomes.
Allelic imbalance of 18 microsatellites was analyzed in 70 consecutive patients (35 within Milan, 35 beyond Milan criteria) transplanted for hepatocellular carcinoma of whom 24 had recurrence and 46 survived at least 5 years recurrence-free. Fractional allelic imbalance (the fraction of significant microsatellites that demonstrated allelic imbalance) and relevant clinical/pathological variables were tested for correlation with time to recurrence.
Allelic imbalance in 9/18 microsatellites correlated with recurrence. Fractional allelic imbalance > 0.27 and macrovascular invasion were independent predictors of recurrence in patients with tumors beyond Milan criteria; the probability of recurrence at 5 years was 85% with fractional allelic imbalance > or = 0.27 vs. 10% when < 0.27 (p=0.0002). An algorithm including Milan criteria and fractional allelic imbalance status is 89% accurate in predicting tumor recurrence after transplantation.
Analysis of allelic imbalance of 9 microsatellites identifies a subgroup of patients who, despite having hepatocellular carcinoma beyond Milan criteria, have a low risk of posttransplant recurrence.
Full-text · Article · May 2008 · Journal of Hepatology
[Show abstract][Hide abstract] ABSTRACT: Kruppel-like factor 6 (KLF6) is a tumor suppressor gene that is functionally inactivated in human cancer by loss of heterozygosity, somatic mutation, decreased expression, and increased alternative splicing into an oncogenic splice variant, KLF6-SV1. Here we show that increased expression of KLF6-SV1 is associated with decreased survival in patients with lung adenocarcinoma. In addition, KLF6-SV1 is a novel antiapoptotic protein in lung cancer cell lines, and targeted reduction of KLF6-SV1 using siRNA induces apoptosis both alone and in combination with the chemotherapeutic drug cisplatin. Together, these findings highlight a critical role for KLF6-SV1 in lung cancer, and show a potential novel therapeutic strategy for the treatment of lung cancer.