Hepatocellular carcinoma (HCC) is a lethal malignancy with high mortality and poor prognosis. Oncogenic transcription factor Late SV40 Factor (LSF) plays an important role in promoting HCC. A small molecule inhibitor of LSF, Factor Quinolinone Inhibitor 1 (FQI1), significantly inhibited human HCC xenografts in nude mice without harming normal cells. Here we evaluated the efficacy of FQI1 and another inhibitor, FQI2, in inhibiting endogenous hepatocarcinogenesis. HCC was induced in a transgenic mouse with hepatocyte-specific overexpression of c-myc (Alb/c-myc) by injecting N-nitrosodiethylamine (DEN) followed by FQI1 or FQI2 treatment after tumor development. LSF inhibitors markedly decreased tumor burden in Alb/c-myc mice with a corresponding decrease in proliferation and angiogenesis. Interestingly, in vitro treatment of human HCC cells with LSF inhibitors resulted in mitotic arrest with an accompanying increase in CyclinB1. Inhibition of CyclinB1 induction by Cycloheximide or CDK1 activity by Roscovitine significantly prevented FQI-induced mitotic arrest. A significant induction of apoptosis was also observed upon treatment with FQI. These effects of LSF inhibition, mitotic arrest and induction of apoptosis by FQI1s provide multiple avenues by which these inhibitors eliminate HCC cells. LSF inhibitors might be highly potent and effective therapeutics for HCC either alone or in combination with currently existing therapies.
LSF; HCC; FQI; mitotic arrest; apoptosis
Background and Aims
Understanding the molecular pathogenesis of hepatocellular carcinoma (HCC) would facilitate development of targeted and effective therapies for this fatal disease. We recently demonstrated that the cellular transcription factor Late SV40 Factor (LSF) is overexpressed in more than 90% of human HCC cases, compared to normal liver, and plays a seminal role in hepatocarcinogenesis. LSF transcriptionally upregulates osteopontin (OPN) that plays a significant role in mediating the oncogenic function of LSF. The present study aims at a better understanding of LSF function by analyzing the signaling pathway modulated by LSF.
Phospho-receptor tyrosine kinase (RTK) array was performed to identify which receptor tyrosine kinases are activated by LSF. Immunohistochemical analysis using tissue microarray was performed to establish correlation among LSF, OPN and phospho-c-Met levels in HCC patients. Co-immunoprecipitation analysis was performed to check OPN-induced CD44 and c-Met interaction. Inhibition studies using chemicals and siRNAs were performed in vitro and in vivo using nude mice xenograft models to establish the importance of c-Met activation in mediating LSF function.
Secreted OPN, induced by LSF, activates c-Met via a potential interaction between OPN and its cell surface receptor CD44. A significant correlation was observed among LSF, OPN and activated c-Met levels in HCC patients. Chemical or genetic inhibition of c-Met resulted in profound abrogation of LSF-mediated tumorigenesis and metastasis in nude mice xenograft studies.
The present findings elucidate a novel pathway of c-Met activation during hepatocarcinogenesis and support the rationale of using c-Met inhibitors as potential HCC therapeutics.
Late SV40 Factor; CD44; Hepatocellular carcinoma; c-Met; osteopontin
Late SV40 factor 3 (LSF), a transcription factor, contributes to human hepatocellular carcinoma (HCC). However, decreased expression level of LSF in skin melanoma compared to that in benign melanocytic tumors and nevi in mice and humans was found in this study. Anchorage-dependent and -independent growth of melanoma cells was suppressed by LSF overexpression through an increased percentage of G1 phase cells and an increased p21CIP1 expression level in vitro and in vivo. Anchorage-dependent growth in LSF-overexpressed melanoma cells was promoted by depletion of LSF in the LSF-overexpressed cells. Integrated results of our EMSA and chromatin immunoprecipitation assays showed binding of LSF within a 150-bp upstream region of the transcription start site of p21CIP1 in melanoma cells. Taken together, our results suggest potential roles of LSF as a growth regulator through control of the transcription of p21CIP1 in melanocytes and melanoma cells as well as a biomarker for nevus.
melanoma; transcription factor LSF; TFCP2; CDKN1A; cell cycle
Hepatocellular carcinoma (HCC) is a dreadful cancer and a major cause of death among patients with chronic liver disease and cirrhosis. The apparent alterations in a diversity of intracellular pathways found in HCC has set the rational for developing molecular-directed drugs that simultaneously inhibit multiple pathways, such as the multi-kinase inhibitor Sorafenib. However, recently this concept has been challenged by showing that HCC is heavily dependent on a single oncogene designated late SV-40 factor (LSF), a transcription factor that is over-expressed in liver cancer cells and that its expression is strongly correlated with tumor grade and aggressiveness. Furthermore, using an intensive screening for drugs that inhibit LSF activity, Grant et al have found a molecule designated factor quinolinone inhibitor 1 that can specifically block the ability of LSF to bind its target promoters, resulting in a massive death of HCC cells both in vitro and in vivo. The innovative findings of HCC representing “oncogene addiction” to LSF and the ability of a single molecule to block the activity of this oncogene resulting in tumor abolishment are encouraging and provide us with the hope that the “Achilles heel” of HCC has been found.
Oncogene addiction; Hepatocellular carcinoma; Late SV40 factor; Transcription factor; Multi-kinase inhibit
Transcription factor LSF is required for progression from quiescence through the cell cycle, regulating thymidylate synthase (Tyms) expression at the G1/S boundary. Given the constant level of LSF protein from G0 through S, we investigated whether LSF is regulated by phosphorylation in G1. In vitro, LSF is phosphorylated by cyclin E/cyclin-dependent kinase 2 (CDK2), cyclin C/CDK2, and cyclin C/CDK3, predominantly on S309. Phosphorylation of LSF on S309 is maximal 1 to 2 h after mitogenic stimulation of quiescent mouse fibroblasts. This phosphorylation is mediated by cyclin C-dependent kinases, as shown by coimmunoprecipitation of LSF and cyclin C in early G1 and by abrogation of LSF S309 phosphorylation upon suppression of cyclin C with short interfering RNA. Although mouse fibroblasts lack functional CDK3 (the partner of cyclin C in early G1 in human cells), CDK2 compensates for this absence. By transient transfection assays, phosphorylation at S309, mediated by cyclin C overexpression, inhibits LSF transactivation. Moreover, overexpression of cyclin C and CDK3 inhibits induction of endogenous Tyms expression at the G1/S transition. These results identify LSF as only the second known target (in addition to pRb) of cyclin C/CDK activity during progression from quiescence to early G1. Unexpectedly, this phosphorylation prevents induction of LSF target genes until late G1.
The transcription factors of the LSF/Grainyhead (GRH) family are characterized by the possession of a distinctive DNA-binding domain that bears no clear relationship to other known DNA-binding domains, with the possible exception of the p53 core domain. In triploblastic animals, the LSF and GRH subfamilies have diverged extensively with respect to their biological roles, general expression patterns, and mechanism of DNA binding. For example, Grainyhead (GRH) homologs are expressed primarily in the epidermis, and they appear to play an ancient role in maintaining the epidermal barrier. By contrast, LSF homologs are more widely expressed, and they regulate general cellular functions such as cell cycle progression and survival in addition to cell-lineage specific gene expression.
To illuminate the early evolution of this family and reconstruct the functional divergence of LSF and GRH, we compared homologs from 18 phylogenetically diverse taxa, including four basal animals (Nematostella vectensis, Vallicula multiformis, Trichoplax adhaerens, and Amphimedon queenslandica), a choanoflagellate (Monosiga brevicollis) and several fungi. Phylogenetic and bioinformatic analyses of these sequences indicate that (1) the LSF/GRH gene family originated prior to the animal-fungal divergence, and (2) the functional diversification of the LSF and GRH subfamilies occurred prior to the divergence between sponges and eumetazoans. Aspects of the domain architecture of LSF/GRH proteins are well conserved between fungi, choanoflagellates, and metazoans, though within the Metazoa, the LSF and GRH families are clearly distinct. We failed to identify a convincing LSF/GRH homolog in the sequenced genomes of the algae Volvox carteri and Chlamydomonas reinhardtii or the amoebozoan Dictyostelium purpureum. Interestingly, the ancestral GRH locus has become split into two separate loci in the sea anemone Nematostella, with one locus encoding a DNA binding domain and the other locus encoding the dimerization domain.
In metazoans, LSF and GRH proteins play a number of roles that are essential to achieving and maintaining multicellularity. It is now clear that this protein family already existed in the unicellular ancestor of animals, choanoflagellates, and fungi. However, the diversification of distinct LSF and GRH subfamilies appears to be a metazoan invention. Given the conserved role of GRH in maintaining epithelial integrity in vertebrates, insects, and nematodes, it is noteworthy that the evolutionary origin of Grh appears roughly coincident with the evolutionary origin of the epithelium.
Transcriptional regulation in mammalian cells is driven by a complex interplay of multiple transcription factors that respond to signals from either external or internal stimuli. A single transcription factor can control expression of distinct sets of target genes, dependent on its state of post-translational modifications, interacting partner proteins, and the chromatin environment of the cellular genome. Furthermore, many transcription factors can act as either transcriptional repressors or activators, depending on promoter and cellular contexts (Alvarez, et al., 2003). Even in this light, the versatility of LSF (Late SV40 Factor) is remarkable. A hallmark of LSF is its unusual DNA binding domain, as evidenced both by lack of homology to any other established DNA-binding domains and by its DNA recognition sequence. Although a dimer in solution, LSF requires additional multimerization with itself or partner proteins in order to interact with DNA. Transcriptionally, LSF can function as an activator or a repressor. It is a direct target of an increasing number of signal transduction pathways. Biologically, LSF plays roles in cell cycle progression and cell survival, as well as in cell lineage-specific functions, shown most strikingly to date in hematopoietic lineages.
This review discusses how the unique aspects of LSF DNA-binding activity may make it particularly susceptible to regulation by signal transduction pathways and may relate to its distinct biological roles. We present current progress in elucidation of both tissue-specific and more universal cellular roles of LSF. Finally, we discuss suggestive data linking LSF to signaling by the amyloid precursor protein and to Alzheimer's disease, as well as to the regulation of latency of the human immunodeficiency virus (HIV).
GRH; DNA-binding; signal transduction; cell cycle progression; immune response; APP; HIV
Human immunodeficiency virus type 1 (HIV-1) establishes a persistent, nonproductive state within a small population of memory CD4+ cells. The transcription factor LSF binds to sequences within the HIV-1 long terminal repeat (LTR) initiation region and recruits a second factor, YY1, to the LTR. These factors then cooperatively recruit histone deacetylase 1 to the LTR, resulting in inhibition of transcription. This appears to be one mechanism contributing to HIV persistence within resting CD4+ T cells. We sought to further detail LSF binding to the HIV-1 LTR and factors that regulate LSF occupancy. We find that LSF binds the LTR as a tetramer and that binding is regulated by phosphorylation mediated by mitogen-activated protein kinases (MAPKs). In vitro, phosphorylation of LSF by Erk decreases binding to the LTR, while binding is increased by p38 phosphorylation. LSF occupancy at LTR chromatin is increased by the p38 agonist anisomycin and decreased by specific p38 inhibition. p38 inhibition also results in increased acetylation of histone H4 at the LTR nucleosome adjacent to the LSF binding site. p38 inhibition also blocked the ability of YY1 to inhibit activation of the integrated HIV promoter. Finally, HIV was recovered from the resting CD4+ T cells of aviremic, HIV-infected donors upon treatment of these cells with specific inhibitor of p38. These data suggest that the MAPK pathway regulates LSF binding to the LTR and thereby one aspect of the regulation of HIV expression. This mechanism could be exploited as a novel therapeutic target to disrupt latent HIV infection.
LSF is a mammalian transcription factor that is rapidly and quantitatively phosphorylated upon growth induction of resting, peripheral human T cells, as assayed by a reduction in its electrophoretic mobility. The DNA-binding activity of LSF in primary T cells is greatly increased after this phosphorylation event [Volker et al., 1997]. We demonstrate here that LSF is also rapidly and quantitatively phosphorylated upon growth induction in NIH 3T3 cells, although its DNA-binding activity is not significantly altered. Three lines of experimentation established that ERK is responsible for phosphorylating LSF upon growth induction in both cell types. First, phosphorylation of LSF by ERK is sufficient to cause the reduced electrophoretic mobility of LSF. Second, the amount of ERK activity correlates with the extent of LSF phosphorylation in both primary human T cells and NIH 3T3 cells. Finally, specific inhibitors of the Ras/Raf/MEK/ERK pathway inhibit LSF modification in vivo. This phosphorylation by ERK is not sufficient for activation of LSF DNA-binding activity, as evidenced both in vitro and in mouse fibroblasts. Nonetheless, activation of ERK is a prerequisite for the substantial increase in LSF DNA-binding activity upon activation of resting T cells, indicating that ERK phosphorylation is necessary but not sufficient for activation of LSF in this cell type.
ERK; LSF; T cells; fibroblasts; DNA-binding; phosphorylation
AIM: To investigate the relationship between late SV40 factor (LSF) and Notch signaling in the development and progress of hepatocellular carcinoma (HCC).
METHODS: Liver cancer tissue specimens from 25 patients were analyzed for Notch-1 and LSF expression by immunohistochemistry. The correlation between expression and the biological effects of Notch-1 and LSF were analyzed using genetic and pharmacological strategies in HCC cell lines and human normal cell lines, including hepatic stellate cells (HSC) and human embryonic kidney epithelial cells (HEK).
RESULTS: Immunohistochemistry showed that both Notch-1 and LSF were significantly upregulated in HCC samples (76%, 19/25, P < 0.0001 and 84%, 21/25, P < 0.0001, respectively) compared with non-cancer samples. Activation of Notch-1 by exogenous transfection of Notch1 intracellular domain increased LSF expression in HSC and HEK cells to levels similar to those seen in HepG2 cells. Furthermore, blocking Notch-1 activation with a γ-secretase inhibitor, DAPT, downregulated LSF expression in HepG2 cells. Additionally, a biological behavior assay showed that forced overexpression of LSF promoted HepG2 cell proliferation and invasion.
CONCLUSION: LSF is a key mediator of the Notch signaling pathway, suggesting that it might be a novel therapeutic target for the treatment of HCC.
Notch receptor; Late SV40 factor; Signal transduction; Hepatocellular carcinoma
The transcription factor LSF, identified as a HeLa protein that binds the simian virus 40 late promoter, recognizes direct repeats with a center-to-center spacing of 10 bp. The characterization of two human cDNAs, representing alternatively spliced mRNAs, provides insight into the unusual DNA-binding and oligomerization properties of LSF. The sequence of the full-length LSF is identical to that of the transcription factors alpha CP2 and LBP-1c and has similarity to the Drosophila transcription factor Elf-1/NTF-1. Using an epitope-counting method, we show that LSF binds DNA as a homodimer. LSF-ID, which is identical to LBP-1d, contains an in-frame internal deletion of 51 amino acids resulting from alternative mRNA splicing. Unlike LSF, LSF-ID did not bind LSF DNA-binding sites. Furthermore, LSF-ID did not affect the binding of LSF to DNA, suggesting that the two proteins do not interact. Of three short regions with a high degree of homology between LSF and Elf-1/NTF-1, LSF-ID lacks two, which are predicted to form beta-strands. Double amino acid substitutions in each of these regions eliminated specific DNA-binding activity, similarly to the LSF-ID deletion. The dimerization potential of these mutants was measured both by the ability to inhibit the binding of LSF to DNA and by direct protein-protein interaction studies. Mutations in one homology region, but not the other, functionally eliminated dimerization.
Colorectal cancer (CRC) is the third most common cancer worldwide and the fourth most common cause of cancer death. Therapy failure was the first cause of death. LSF is a transcription factor regulating gene expression of angiogenesis, tumor invasion and proliferation, and is identified as a chemoresistant gene. Real-time PCR and Western blot to analyze mRNA and protein expression of LSF in 23 paired CRC samples. Immunohistochemistry was used to detect protein expression of LSF in 166 paired CRC samples. Both LSF mRNA and protein were upregulated in CRC. High LSF expression in CRC correlated with large tumor size, advanced pN stage, advanced AJCC stage and high Ki-67 index (P < 0.001). High expression of LSF favored worse prognosis. 5-year survival rates of LSF high and low expression were 39.6% and 78.6%, respectively. The 5-year median OS were 34 months and 57 months, respectively. LSF is an important mediator in CRC tumorigenesis and progression, and LSF expression is an important index for and prognostic prediction.
LSF; colorectal cancer; prognosis
The killing of bacterial pathogens by macrophages occurs via the oxidative burst and bacteria have evolved to overcome this challenge and survive, using several virulence and defense strategies, including antioxidant mechanisms. We show here that the 1-Cys peroxiredoxin LsfA from the opportunistic pathogen Pseudomonas aeruginosa is endowed with thiol-dependent peroxidase activity that protects the bacteria from H2O2 and that this protein is implicated in pathogenicity. LsfA belongs to the poorly studied Prx6 subfamily of peroxiredoxins. The function of these peroxiredoxins has not been characterized in bacteria, and their contribution to host-pathogen interactions remains unknown. Infection of macrophages with the lsfA mutant strains resulted in higher levels of the cytokine TNF-α production due to the activation of the NF-kB and MAPK pathways, that are partially inhibited by the wild-type P. aeruginosa strain. A redox fluorescent probe was more oxidized in the lsfA mutant-infected macrophages than it was in the macrophages infected with the wild-type strain, suggesting that the oxidative burst was overstimulated in the absence of LsfA. Although no differences in the phagocytosis rates were observed when macrophages were infected with wild-type and mutant bacteria in a gentamicin exclusion assay, a higher number of wild-type bacterial cells was found in the supernatant. This difference was not observed when macrophages were pre-treated with a NADPH oxidase inhibitor, confirming the role of LsfA in the bacterial resistance to ROS generated via NADPH oxidase. In an acute pneumonia model, mice infected with the mutant strains presented higher cytokine release in the lungs and increased activated neutrophil recruitment, with reduced bacterial burden and improved survival rates compared to mice infected with the wild-type bacteria. LsfA is the first bacterial 1-Cys Prx shown to modulate host immune responses and its characterization will allow a better understanding of the role of redox signaling in host-pathogen interactions.
Pseudomonas aeruginosa is an important human pathogen that employs a vast arsenal of virulence factors and infects immunocompromised hosts, such as patients in intensive care units, causing pneumonia and other illnesses. Macrophages are cells in the first line of defense against pathogens in the lungs. After pathogen recognition, macrophages release pro-inflammatory cytokines to recruit other immune cells and employ a process known as oxidative burst to kill invading microbes. P. aeruginosa can counteract oxidative stress using antioxidant proteins, such as peroxiredoxins. We show here that LsfA, which belongs to the poorly characterized Prx6 subfamily of peroxiredoxins, is indeed endowed with a thiol-dependent activity that is required for full virulence. In vitro and in vivo infection models confirmed that LsfA peroxidase activity is required for the immunomodulation caused by P. aeruginosa and that its absence allows the host to overcome the infection. This study demonstrates for the first time the involvement of a bacterial Prx6 in virulence.
A subpopulation of stably infected CD4+ cells capable of producing virus upon stimulation has been identified in human immunodeficiency virus (HIV)-positive individuals (T.-W. Chun, D. Finzi, J. Margolick, K. Chadwick, D. Schwartz, and R. F. Siliciano, Nat. Med. 1:1284-1290, 1995). Few host factors that directly limit HIV-1 transcription and could support this state of nonproductive HIV-1 infection have been described. YY1, a widely distributed human transcription factor, is known to inhibit HIV-1 long terminal repeat (LTR) transcription and virus production. LSF (also known as LBP-1, UBP, and CP-2) has been shown to repress LTR transcription in vitro, but transient expression of LSF has no effect on LTR activity in vivo. We report that both YY1 and LSF participate in the formation of a complex that recognizes the initiation region of the HIV-1 LTR. Further, we have found that these factors cooperate in the repression of LTR expression and viral replication. This cooperative function may account for the divergent effects of LSF previously observed in vitro and in vivo. Thus, the cooperation of two general cellular transcription factors may allow for the selective downregulation of HIV transcription. Through this mechanism of gene regulation, YY1 and LSF could contribute to the establishment and maintenance of a population of cells stably but nonproductively infected with HIV-1.
Repression of human immunodeficiency virus type 1 (HIV-1) transcription may contribute to the establishment or maintenance of proviral quiescence in infected CD4+ cells. The host factors YY1 and LSF cooperatively recruit histone deacetylase 1 (HDAC1) to the HIV-1 long terminal repeat (LTR) and inhibit transcription. We demonstrate here regulation of occupancy of HDAC1 at a positioned nucleosome (nuc 1) near the transcription start site of integrated LTR. We find that expression of YY1 increases occupancy by HDAC1, decreases acetylation at nuc 1, and downregulates LTR expression. HDAC1 recruitment and histone hypoacetylation were also seen when Tat activation was inhibited by the overexpression of YY1. A YY1 mutant without an HDAC1 interaction domain and incompetent to inhibit LTR activation fails to recruit HDAC1 to LTR or decrease nuc 1 acetylation. Further, expression of a dominant-negative mutant of LSF (dnLSF), which inhibits LSF occupancy and LTR repression, results in acetylation and decreased HDAC1 occupancy at nuc 1. Conversely, exposure of cells to the histone deacetylase inhibitor trichostatin A or activation of LTR expression by HIV-1 Tat results in the displacement of HDAC1 from nuc 1, in association with increased acetylation of histone H4. Recruitment of HDAC1 to the LTR nuc 1 can counteract Tat activation and repress LTR expression. Significantly, when repression is overcome, LTR activation is associated with decreased HDAC1 occupancy. Since the persistence of integrated HIV-1 genomes despite potent suppression of viral replication is a major obstacle for current antiretroviral therapy, strategies to selectively disrupt the quiescence of chromosomal provirus may play a role in the future treatment of AIDS.
Cell cycle progression in mammalian cells from G1 into S phase requires sensing and integration of multiple inputs, in order to determine whether to continue to cellular DNA replication and subsequently, to cell division. Passage to S requires transition through the restriction point, which at a molecular level consists of a bistable switch involving E2Fs and pRb family members. At the G1/S boundary, a number of genes essential for DNA replication and cell cycle progression are upregulated, promoting entry into S phase. Although the activating E2Fs are the most extensively characterized transcription factors driving G1/S expression, LSF is also a transcription factor essential for stimulating G1/S gene expression. A critical LSF target gene at this stage, Tyms, encodes thymidylate synthetase. In investigating how LSF is activated in a cell cycle-dependent manner, we recently identified a novel time delay mechanism for regulating its activity during G1 progression, which is apparently independent of the E2F/pRb axis. This involves inhibition of LSF in early G1 by two major proliferative signaling pathways: ERK and cyclin C/CDK, followed by gradual dephosphorylation during mid- to late-G1. Whether LSF and E2F act independently or in concert to promote G1/S progression remains to be determined.
LSF; cyclin C/CDK; ERK; thymidylate synthetase; E2F; pRb; p53; G1 phase; S phase; restriction point
Our previous study demonstrated a decreased expression of tumor susceptibility gene 101 (TSG101) in cervical cancer cells. To identify the mechanism responsible for TSG101 downregulation during cervical cancer development, we analyzed the TSG101 promoter using cis-element cluster finder software. One of the transcription factors whose binding site was detected in the TSG101 promoter was late SV40 factor (LSF). The aim of this study was to analyze the TSG101 protein and LSF expression levels during cervical cancer development. Immunohistochemical analysis confirmed a previously observed decreased expression of TSG101, whereas quantitative polymerase chain reaction (qPCR) and immunohistochemistry analysis revealed high expression of LSF in cervical, precancer and cancer cells compared with human papillomavirus (HPV)-negative non-cancer samples. High expression of LSF in cervical cancer HPV-positive cells suggests that this protein may be important in the regulation of TSG101 expression, as well as in cervical carcinogenesis. The role of LSF as a mediator in cervical cancer development must be confirmed in future studies.
LSF transcription factor; TSG101 protein; cervical cancer
The LSF/Grainyhead transcription factor family is involved in many important biological processes, including cell cycle, cell growth and development. In order to investigate the evolutionary conservation of these biological roles, we have characterized two new family members in Caenorhabditis elegans and Xenopus laevis. The C.elegans member, Ce-GRH-1, groups with the Grainyhead subfamily, while the X.laevis member, Xl-LSF, groups with the LSF subfamily. Ce-GRH-1 binds DNA in a sequence-specific manner identical to that of Drosophila melanogaster Grainyhead. In addition, Ce-GRH-1 binds to sequences upstream of the C.elegans gene encoding aromatic l-amino-acid decarboxylase and genes involved in post-embryonic development, mab-5 and dbl-1. All three C.elegans genes are homologs of D.melanogaster Grainyhead-regulated genes. RNA-mediated interference of Ce-grh-1 results in embryonic lethality in worms, accompanied by soft, defective cuticles. These phenotypes are strikingly similar to those observed previously in D.melanogaster grainyhead mutants, suggesting conservation of the developmental role of these family members over the course of evolution. Our phylogenetic analysis of the expanded LSF/GRH family (including other previously unrecognized proteins/ESTs) suggests that the structural and functional dichotomy of this family dates back more than 700 million years, i.e. to the time when the first multicellular organisms are thought to have arisen.
Lisofylline (LSF), is the R-(−) enantiomer of the metabolite M1 of pentoxifylline, and is currently under development for the treatment of type 1 diabetes. The aim of the study was to develop a physiologically based pharmacokinetic (PBPK) model of LSF in mice and to perform simulations in order to predict LSF concentrations in human serum and tissues following intravenous and oral administration. The concentrations of LSF in serum, brain, liver, kidneys, lungs, muscle, and gut were determined at different time points over 60 min by a chiral HPLC method with UV detection following a single intravenous dose of LSF to male CD-1 mice. A PBPK model was developed to describe serum pharmacokinetics and tissue distribution of LSF using ADAPT II software. All pharmacokinetic profiles were fitted simultaneously to obtain model parameters. The developed model characterized well LSF disposition in mice. The estimated intrinsic hepatic clearance was 5.427 ml/min and hepatic clearance calculated using the well-stirred model was 1.22 ml/min. The renal clearance of LSF was equal to zero. On scaling the model to humans, a good agreement was found between the predicted by the model and presented in literature serum LSF concentration–time profiles following an intravenous dose of 3 mg/kg. The predicted LSF concentrations in human tissues following oral administration were considerably lower despite the twofold higher dose used and may not be sufficient to exert a pharmacological effect. In conclusion, the mouse is a good model to study LSF pharmacokinetics following intravenous administration. The developed PBPK model may be useful to design future preclinical and clinical studies of this compound.
Lisofylline; PBPK model; Mice; Simulations; Human tissues
Enigmatic mechanisms restore the resting state in activated lymphocytes following human immunodeficiency virus type 1 (HIV-1) infection, rarely allowing persistent nonproductive infection. We detail a mechanism whereby cellular factors could establish virological latency. The transcription factors YY1 and LSF cooperate in repression of transcription from the HIV-1 long terminal repeat (LTR). LSF recruits YY1 to the LTR via the zinc fingers of YY1. The first two zinc fingers were observed to be sufficient for this interaction in vitro. A mutant of LSF incapable of binding DNA blocked repression. Like other transcriptional repressors, YY1 can function via recruitment of histone deacetylase (HDAC). We find that HDAC1 copurifies with the LTR-binding YY1-LSF repressor complex, the domain of YY1 that interacts with HDAC1 is required to repress the HIV-1 promoter, expression of HDAC1 augments repression of the LTR by YY1, and the deacetylase inhibitor trichostatin A blocks repression mediated by YY1. This novel link between HDAC recruitment and inhibition of HIV-1 expression by YY1 and LSF, in the natural context of a viral promoter integrated into chromosomal DNA, is the first demonstration of a molecular mechanism of repression of HIV-1. YY1 and LSF may establish transcriptional and virological latency of HIV, a state that has recently been recognized in vivo and has significant implications for the long-term treatment of AIDS.
Transcription factor CP2 (TFCP2) is overexpressed in hepatocellular carcinoma(HCC) and correlated with the progression of the disease. Here we report the use of an integrated systems biology approach to identify genome-wide scale map of TFCP2 targets as well as the molecular function and pathways regulated by TFCP2 in HCC.
We combined Chromatin immunoprecipitation (ChIP) on chip along with gene expression microarrays to study global transcriptional regulation of TFCP2 in HCC. The biological functions, molecular pathways, and networks associated with TFCP2 were identified using computational approaches. Validation of selected target gene expression and direct binding of TFCP2 to promoters were performed by ChIP -PCR and promoter reporter.
TFCP2 fostered a highly aggressive and metastatic phenotype in different HCC cells. Transcriptome analysis showed that alteration of TFCP2 in HCC cells led to change of genes in biological functions involved in cancer, cellular growth and proliferation, angiogenesis, cell movement and attachment. Pathways related to cell movement and cancer progression were also enriched. A quest for TFCP2-regulated factors contributing to metastasis, by integration of transcriptome and ChIP on chip assay, identified fibronectin 1 (FN1) and tight junction protein 1 (TJP1) as targets of TFCP2, and as key mediators of HCC metastasis. Promoter reporter identified the TFCP2-responsive region, and located the motifs of TFCP2-binding sites in the FN1 promoter, which then was confirmed by ChIP-PCR. We further showed that FN1 inhibition blocks the TFCP2-induced increase in HCC cell aggression, and that overexpression of TFCP2 can rescue the effects of FN1 inhibition. Knock down of TJP1 could also rescue, at least in part, the aggressive effect of TFCP2 knockdown in HCC cells.
The identification of global targets, molecular pathways and networks associated with TFCP2, together with the discovery of the effect of TFCP2 on FN1 and TJP1 that are involved in metastasis, adds to our understanding of the mechanisms that determine a highly aggressive and metastatic phenotype in hepatocarcinogenesis.
Electronic supplementary material
The online version of this article (doi:10.1186/s13046-015-0121-1) contains supplementary material, which is available to authorized users.
Hepatocellular carcinoma; Metastasis; Fibronectin 1; Tight junction protein 1; Transcription factor CP2
Hepatocellular carcinoma (HCC) is a highly virulent malignancy with diverse etiology. Identification of a common mediator of aggressive progression of HCC would be extremely beneficial not only for diagnostic/prognostic purposes but also for developing targeted therapies. AEG-1/MTDH/LYRIC gene is amplified in human HCC patients, and overexpression of AEG-1/MTDH/LYRIC has been identified in a high percentage of both hepatitis B virus and hepatitis C virus positive HCC cases, suggesting its key role in regulating hepatocarcinogenesis. Important insights into the molecular mechanisms mediating oncogenic properties of AEG-1/MTDH/LYRIC, especially regulating chemoresistance, angiogenesis, and metastasis, have been obtained from studies using HCC model. Additionally, analysis of HCC model has facilitated the identification of AEG-1/MTDH/LYRIC downstream genes and interacting proteins, thereby unraveling novel players regulating HCC development and progression leading to the development of novel interventional strategies. Characterization of a hepatocyte-specific AEG-1/MTDH/LYRIC transgenic mouse (Alb/AEG-1) has revealed novel aspects of AEG-1/MTDH/LYRIC function in in vivo contexts. Combination of AEG-1/MTDH/LYRIC inhibition and chemotherapy has documented significant efficacy in abrogating human HCC xenografts in nude mice indicating the need for developing effective AEG-1/MTDH/LYRIC inhibition strategies to obtain objective response and survival benefits in terminal HCC patients.
Hepatocellular carcinoma (HCC) is a highly aggressive vascular cancer characterized by diverse etiology, activation of multiple signal transduction pathways, and various gene mutations. Here, we have determined a specific role for astrocyte elevated gene-1 (AEG1) in HCC pathogenesis. Expression of AEG1 was extremely low in human hepatocytes, but its levels were significantly increased in human HCC. Stable overexpression of AEG1 converted nontumorigenic human HCC cells into highly aggressive vascular tumors, and inhibition of AEG1 abrogated tumorigenesis by aggressive HCC cells in a xenograft model of nude mice. In human HCC, AEG1 overexpression was associated with elevated copy numbers. Microarray analysis revealed that AEG1 modulated the expression of genes associated with invasion, metastasis, chemoresistance, angiogenesis, and senescence. AEG1 also was found to activate Wnt/β-catenin signaling via ERK42/44 activation and upregulated lymphoid-enhancing factor 1/T cell factor 1 (LEF1/TCF1), the ultimate executor of the Wnt pathway, important for HCC progression. Inhibition studies further demonstrated that activation of Wnt signaling played a key role in mediating AEG1 function. AEG1 also activated the NF-κB pathway, which may play a role in the chronic inflammatory changes preceding HCC development. These data indicate that AEG1 plays a central role in regulating diverse aspects of HCC pathogenesis. Targeted inhibition of AEG1 might lead to the shutdown of key elemental characteristics of HCC and could lead to an effective therapeutic strategy for HCC.
Maintaining reactive oxygen species (ROS) homeostasis plays a central role in plants, and is also critical for plant root development. Threshold levels of ROS act as signals for elongation and differentiation of root cells. The protein phosphatase LIKE SEX FOUR2 (LSF2) has been reported to regulate starch metabolism in Arabidopsis, but little is known about the mechanism how LSF2 affect ROS homeostasis. Here, we identified that LSF2 function as a component modulating ROS homeostasis in response to oxidative stress and, thus regulate root development. Compared with wild type Arabidopsis, lsf2-1 mutant exhibited reduced rates of superoxide generation and higher levels of hydrogen peroxide upon oxidative stress treatments. The activities of several antioxidant enzymes, including superoxide dismutase, catalase, and ascorbate peroxidase, were also affected in lsf2-1 mutant under these oxidative stress conditions. Consequently, lsf2-1 mutant exhibited the reduced root growth but less inhibition of root hair formation compared to wild type Arabidopsis plants. Importantly, protein phosphatase LSF2 interacted with mitogen-activated protein kinase 8 (MPK8), a known component of ROS homeostasis pathways in the cytoplasm. These findings indicated the novel function of LSF2 that controls ROS homeostasis to regulate root development.
Upon stimulation of mature B cells, class switch recombination (CSR) can alter the specific immunoglobulin heavy chain constant region that is expressed. In a tissue culture cell line, we previously demonstrated that inhibition of late SV40 factor (LSF) family members enhanced IgM to IgA CSR. Here, isotype specificity of CSR regulation by LSF family members is addressed in primary mouse splenic B cells. First, we demonstrate that LBP-1a is the prevalent family member in B lymphocytes. Second, we demonstrate by ChIP that LBP-1a binds genomic sequences around mouse switch regions (S) in an isotype-specific manner, in accordance with computational predictions: binding is observed to Sμ and Sα, but not to the tested Sγ1, regions. Importantly, binding of LBP-1a is tightly regulated, with occupancy at genomic S regions dramatically decreasing following LPS stimulation. Finally, the consequence of DNA-binding by LBP-1a is determined using bone marrow chimeric mice in which LSF/LBP-1 activity is inhibited in hematopoietic lineages. Upon in vitro stimulation of such primary B-cells, CSR occurs with a higher efficiency to IgA, but not to IgG1. These results are supportive of a model whereby LBP-1a represses CSR in an isotype-specific manner via direct interaction with switch regions involved in the recombination.
B cells; Immunoglobulins; Molecular Biology; Recombinant Viral Vectors