FLI1 accelerates leukemogenesis through transcriptional regulation of pyruvate kinase-L/R and other glycolytic genes.

In cancer cells, multiple oncogenes and tumor suppressors control glycolysis to sustain rapid proliferation. The ETS-related transcription factor Fli1 plays a critical role in the induction and progression of leukemia, yet, the underlying mechanism of this oncogenic event is still not fully understood. In this study, RNAseq analysis of FLI1-depleted human leukemic cells revealed transcriptional suppression of the PKLR gene and activation of multiple glycolytic genes, such as PKM1/2. Pharmacological inhibition of glycolysis by PKM2 inhibitor, Shikonin, significantly suppressed leukemic cell proliferation. FLI1 directly binds to the PKLR promoter, leading to the suppression of this inhibitor of glycolysis. In accordance, shRNA-mediated depletion of PKLR in leukemic HEL cells expressing high levels of FLI1 accelerated leukemia proliferation, pointing for the first time to its tumor suppressor function. PKLR knockdown also led to downregulation of the erythroid markers EPOR, HBA1, and HBA2 and suppression of erythroid differentiation. Interestingly, silencing of PKLR in HEL cells significantly increased FLI1 expression, which was associated with faster proliferation in culture. In FLI1-expressing leukemic cells, lower PKLR expression was associated with higher expression of PKM1 and PKM2, which promote aerobic glycolysis. Finally, injection of pyruvate, a known inhibitor of glycolysis, into leukemia mice significantly suppressed leukemogenesis. These results demonstrate that FLI1 promotes leukemia in part by inducing glycolysis, implicates PKLR in erythroid differentiation, and suggests that targeting glycolysis may be an attractive therapeutic strategy for cancers driven by FLI1 overexpression.

A critical ETV4/Twist1/Vimentin axis in Ha-RAS-induced aggressive breast cancer.

RAS oncogenes are major drivers of diverse types of cancer. However, they are largely not druggable, and therefore targeting critical downstream pathways and dependencies is an attractive approach. We have isolated a tumorigenic cell line (FE1.2), which exhibits mesenchymal characteristics, after inoculating Ha-Ras-expressing retrovirus into mammary glands of rats, and subsequently isolated a non-aggressive revertant cell line (FC5). This revertant has lost the rat Ha-Ras driver and showed a more epithelial morphology, slower proliferation in culture, and reduced tumorigenicity in vivo. Re-expression of human Ha-RAS in these cells (FC5-RAS) reinduced mesenchymal morphology, higher proliferation rate, and tumorigenicity that was still significantly milder than parental FE1.2 cells. RNA-seq analysis of FC5-RAS vs FC5-Vector cells identified multiple genes whose expressions were regulated by Ha-RAS. This analysis also identified many genes including those controlling cell growth whose expression was altered by loss of HA-Ras in FC5 cells but remained unchanged upon reintroduction of Ha-RAS. These results suggest that targeting the Ha-Ras driver oncogene induces partial tumor regression, but it still denotes strong efficacy for cancer therapy. Among the RAS-responsive genes, we identified Twist1 as a critical mediator of epithelial-to-mesenchymal transition through the direct transcriptional regulation of vimentin. Mechanistically, we show that Twist1 is induced by the ETS gene, ETV4, downstream of Ha-RAS, and that inhibition of ETV4 suppressed the growth of breast cancer cells driven by the Ha-RAS pathway. Targeting the ETV4/Twist1/Vimentin axis may therefore offer a therapeutic modality for breast tumors driven by the Ha-RAS pathway.

An unusual indole-diterpenoid with C-17 norcassane skeleton from Euphorbia fischeriana induces HEL cell cycle arrest and apoptosis.

Two new polycyclic diterpenoids, euphkanoids H and I (1 and 2), along with 6 known analogues (2-8) were isolated from the roots of Euphorbia fischeriana, a traditional Chinese medicine. Their structures were identified by spectral methods, and the absolute configurations of 1 and 2 were determined by ECD calculation and single crystal X-ray diffraction, respectively. Compound 1 represents the first example of C-17 norcassane indole-diterpenes. All the isolates were screened for antiproliferative activity against a panel of human cancer cell lines using the MTT assay, and 1 showed significant cytotoxicity against HEL cells (IC50 = 3.2 µM). Simple mechanistic study revealed that 1 could induce cell cycle arrest at G0/G1 phase and apoptosis in HEL cells.

FLI1 regulates inflammation-associated genes to accelerate leukemogenesis.

Inflammation plays a critical role in cancer initiation and progression, and is induced by inflammatory factors that are direct target of oncogenes and tumor suppressors. The ETS related transcription factor Fli-1 is involved in the induction and progression of various cancers; yet its role in inflammation is not well-defined. Using RNAseq analysis, we herein demonstrate that FLI1 induces the inflammatory pathway in erythroleukemia cells. Majority of genes within the TNF signaling pathway including TNF and IL1B were identified as transcriptional targets of FLI1. TNF expression is indirectly regulated by FLI1 through upregulation of another ETS related oncogene, SPI1/PU.1. Pharmacological inhibition of TNF significantly inhibited leukemia cell proliferation in culture. In contrast, IL1B expression is directly regulated by FLI1 through promoter binding and transcriptional activation. The secreted factor IL1B binds its canonical receptors to accelerate cancer progression through changes in the surrounding tumor microenvironment, fostering cell survival, proliferation and migration. Through network analysis, we identified IL1B-interacting genes whose expression is also regulated by FLI1. Among these, IL1B-interacting proteins, FOS, JUN, JUNB and CASP1 are negatively regulated by FLI1. Treatment of leukemia cells with inhibitors of AP1 (TAN IIA) and CASP1 (765VX) significantly accelerated FLI1-dependent leukemia progression. These results emphasize the significance of FLI1 in regulating the inflammatory pathway. Targeting these inflammatory genes downstream of FLI1 offers a novel strategy to treat leukemic progression associated with overexpression of this oncogenic ETS transcription factor.

[Laser Induced Fluorescence Spectroscopic Analysis of Aromatics from One Ring to Four Rings].

In order to distinguish small aromatics preferably, a Nd : YAG Laser was used to supply an excitation laser, which was adjusted to 0.085 J x cm(-2) at 266 nm. Benzene, toluene, naphthalene, phenanthrene, anthracene, pyrene and chrysene were used as the representative of different rings aromatics. The fluorescence emission spectra were researched for each aromatic hydrocarbon and mixtures by Laser induced fluorescence (LIF). Results showed that the rings number determined the fluorescence emission spectra, and the structure with same rings number did not affect the emission fluorescence spectrum ranges. This was due to the fact that the absorption efficiency difference at 266 nm resulted in that the fluorescence intensities of each aromatic hydrocarbon with same rings number were different and the fluorescence intensities difference were more apparently with aromatic ring number increasing. When the absorption efficiency was similar at 266 nm and the concentrations of each aromatic hydrocarbon were same, the fluorescence intensities were increased with aromatic ring number increasing. With aromatic ring number increasing, the fluorescence spectrum and emission peak wavelength were all red-shifted from ultraviolet to visible and the fluorescence spectrum range was also wider as the absorption efficiency was similar. The fluorescence emission spectra from one to four rings could be discriminated in the following wavelengths, 275 to 320 nm, 320 to 375 nm, 375 to 425 nm, 425 to 556 nm, respectively. It can be used for distinguish the type of the polycyclic aromatic hydrocarbons (PAHs) as it exists in single type. As PAHs are usually exist in a variety of different rings number at the same time, the results for each aromatic hydrocarbon may not apply to the aromatic hydrocarbon mixtures. For the aromatic hydrocarbon mixtures, results showed that the one- or two-ring PAHs in mixtures could not be detected by fluorescence as three- or four-ring PAHs existed in mixture. This was caused by radiation energy transfer mechanism, in which the ultraviolet light was lost in mixtures but the fluorescence intensities were increased with the one- or two-ring PAHs adding. When the mixture only contained three- and four-ring PAHs, the fluorescence emission spectrum showed the both characteristics of three- and four-ring PAHs fluorescence. When three- and four-ring PAHs existed in mixtures at the same time, the fluorescence emission spectra were related to each concentration, so the rings number could be discriminated to a certain extent.

Divergent Sp1 protein levels may underlie differential expression of UDP-glucose dehydrogenase by fibroblasts: role in susceptibility to orbital Graves disease.

UDP-glucose dehydrogenase (UGDH) catalyzes the formation of UDP-glucuronate. Glucuronate represents an integral component of the glycosaminoglycan, hyaluronan, which accumulates in orbital Graves disease. Here we report that orbital fibroblasts express higher levels of UGDH than do those from skin. This is a consequence of greater UGDH gene promoter activity and more abundant steady-state UGDH mRNA. Six Sp1 sites located in the proximal 550 bp of the UGDH gene promoter appear to determine basal promoter activity, as does a previously unrecognized 49-bp sequence spanning -1436 nucleotides (nt) and -1388 nt that negatively affects activity. Nuclear Sp1 protein is more abundant in orbital fibroblasts, and its binding to specific sites on DNA is greater than that in dermal fibroblasts. Mutating each of these Sp1 sites in a UGDH gene promoter fragment, extending from -1387 to +71 nt and fused to a luciferase reporter, results in divergent activities when transfected in orbital and dermal fibroblasts. Reducing Sp1 attenuated UGDH gene promoter activity, lowered steady-state UGDH mRNA levels, and reduced UGDH enzyme activity. Targeting Sp1 and UGDH with specific siRNAs also lowered hyaluronan synthase-1 (HAS-1) and HAS-2 levels and reduced hyaluronan accumulation in orbital fibroblasts. These findings suggest that orbital fibroblasts express high levels of UGDH in an anatomic-specific manner, apparently the result of greater constitutive Sp1. These high UGDH levels may underlie susceptibility of the orbit to localized overproduction of hyaluronan in Graves disease.

Orbital fibroblasts from patients with thyroid-associated ophthalmopathy overexpress CD40: CD154 hyperinduces IL-6, IL-8, and MCP-1.

PURPOSE: Fibroblast diversity represents an emerging concept critical to our understanding of tissue inflammation, repair, and remodeling. Orbital fibroblasts heterogeneously display Thy-1 and exhibit unique phenotypic attributes that may explain the susceptibility of the human orbit to thyroid-associated ophthalmopathy (TAO). In the present study the authors investigated the role of CD40 ligation on macrophage chemoattractant protein-1 (MCP-1), IL-6, and IL-8 expression in fibroblasts from patients with TAO. METHODS: Human orbital fibroblasts were cultured from tissues obtained with informed consent from patients with TAO and from patients undergoing surgery for other noninflammatory conditions. The fibroblasts were then examined by flow cytometry, microscopy, and cytokine assays. RESULTS: The authors report that orbital fibroblasts from patients with TAO expressed elevated levels of CD40. Surface CD40 could be further upregulated by IFN-gamma in TAO and control fibroblasts. This upregulation was mediated through Jak2 and could be blocked by dexamethasone and AG490, a powerful and specific inhibitor of tyrosine kinase. Treatment with CD154, the ligand for CD40, upregulated the expression of IL-6, IL-8, and MCP-1 in TAO fibroblasts but failed to do so in control cultures. Thy-1(+) fibroblasts displayed higher CD40 levels than did their Thy-1(-) counterparts and were largely responsible for this cytokine production. IL-1beta also induced MCP-1, IL-6, and IL-8 more vigorously in TAO-derived fibroblasts. CONCLUSIONS: Characterization of orbital fibroblasts and their differential expression of cytokines and receptors should prove invaluable in understanding the site-specific nature of TAO and the development of specific therapies.

Unique attributes of orbital fibroblasts and global alterations in IGF-1 receptor signaling could explain thyroid-associated ophthalmopathy.

Tissue remodeling associated with thyroid-associated ophthalmopathy (TAO) involves the complex interplay between resident cells (endothelium, vascular smooth muscle, extraocular muscle, and fibroblasts) and those recruited to the orbit, including members of the "professional" immune system. Inflammation early in the disease can later culminate in fibrosis and diminished extraocular muscle motility. TAO remains a poorly understood process, in large part because access to tissues early in the disease is limited and because no robust and complete animal models of Graves' disease have yet been devised. Remaining uncertainty as to the identity of a pathogenic autoantigen(s) that underlies lymphocyte trafficking to the orbit complicates matters. These limitations in our understanding of extrathyroidal Graves' disease have resulted in poorly served patients with severe TAO. Therapies have targeted symptoms rather than the underlying disease processes. Our laboratory group has focused over the last several years on defining the peculiarities of the human orbital fibroblasts as a strategy for shedding more light on the pathologies occurring in TAO. We have reasoned that unique properties of these cells might ultimately prove the basis for why the manifestations of Graves' disease occur in an anatomically selective manner. In this brief review we attempt to survey our findings. We believe that they might provide a "roadmap" for further discovery into the pathogenesis of TAO. Clearly, more questions remain than those thus far answered.

Jak2 dampens the induction by IL-1beta of prostaglandin endoperoxide H synthase 2 expression in human orbital fibroblasts: evidence for divergent influence on the prostaglandin E2 biosynthetic pathway.

Prostaglandin endoperoxide H synthase 2 (PGHS-2) catalyzes the rate-limiting steps in the synthesis of PGE(2). It is substantially but transiently induced in human orbital fibroblasts treated with IL-1beta. In this study, we report that the induction of PGHS-2 by IL-1beta is dramatically enhanced and prolonged when Jak2 signaling is abrogated, either with the specific inhibitor AG490 or by transiently transfecting fibroblasts with a dominant negative mutant Jak2. Attenuating Jak2 increases PGHS-2 steady-state mRNA levels, a consequence of increased gene transcription and mRNA survival in IL-1beta-treated cultures. Surprisingly, interrupting Jak2 function also blocked the expected increase in PGE(2) synthesis usually provoked by IL-1beta. This resulted from the rapid loss of IL-1beta-dependent arachidonate release and by attenuation of group IIA secreted PLA(2) (sPLA(2)) gene induction. Supplying Jak2-compromised cultures with exogenous arachidonate failed to increase PGE(2) production in response to IL-1beta until cells were mechanically disrupted. However, transiently transfecting them with wild-type sPLA(2) fully restored prostanoid production to anticipated levels. sPLA(2) expression following transfection resulted in increased IL-1beta-dependent PGHS-2 and microsomal PGE(2) synthase levels. Thus, sPLA(2) plays important roles in PGE(2) synthesis in addition to its release of arachidonate. Our findings suggest that Jak2 ordinarily dampens and limits the duration of the PGHS-2 induction by IL-1beta. Moreover, it is required for IL-1beta-dependent signaling to sPLA(2), the expression and activity of which are necessary for up-regulating PGE(2) synthesis in orbital fibroblasts.

Interleukin-4 induces 15-lipoxygenase-1 expression in human orbital fibroblasts from patients with Graves disease. Evidence for anatomic site-selective actions of Th2 cytokines.

Orbital fibroblasts orchestrate tissue remodeling in Graves disease, at least in part, because they exhibit exaggerated responses to proinflammatory cytokines. A hallmark of late stage orbital disease is vision-threatening fibrosis, the molecular basis of which remains uncertain. We report here that the Th2 cytokines, interleukin (IL)-4 and IL-13, can induce in these cells the expression of 15-lipoxygenase-1 (15-LOX-1) and in so doing up-regulate the production of 15-hydroxyeicosatetraenoic acid. IL-4 increases 15-LOX-1 protein levels through pretranslational actions. The increased steady-state 15-LOX-1 mRNA is independent of ongoing protein synthesis and involves very modestly increased gene promoter activity. Importantly, IL-4 substantially enhances 15-LOX-1 transcript stability, activity that localizes to a 293-bp sequence of the 3'-untranslated region. IL-4 activates Jak2 in orbital fibroblasts. Interrupting signaling through that pathway, either with the specific chemical inhibitor, AG490, or by transiently transfecting the cells with a Jak2 dominant negative mutant kinase, attenuates the 15-LOX-1 induction. Interferongamma, a Th1 cytokine, could block this induction by attenuating IL-4-dependent mRNA stabilization. 15-LOX-1 protein and its mRNA were undetectable in IL-4-treated dermal fibroblasts, despite comparable levels of cell surface IL-4 receptor and phosphorylated Jak2 and STAT6. Our findings suggest that orbital connective tissues may represent a site of localized 15-hydroxyeicosatetraenoic acid generation resulting from cell type-specific 15-LOX-1 mRNA stabilization by IL-4. These results may have relevance to the pathogenesis of orbital Graves disease, an inflammatory autoimmune condition that gives way to extensive fibrosis associated with a Th2 response.

IL-1 beta induces IL-6 expression in human orbital fibroblasts: identification of an anatomic-site specific phenotypic attribute relevant to thyroid-associated ophthalmopathy.

Human orbital fibroblasts exhibit a unique inflammatory phenotype. In the present study, we report that these fibroblasts, when treated with IL-1beta, express high levels of IL-6, a cytokine involved in B cell activation and the regulation of adipocyte metabolism. The magnitude of this induction is considerably greater than that in dermal fibroblasts and involves up-regulation of IL-6 mRNA levels. IL-1beta activates both p38 and ERK 1/2 components of the MAPK pathways. Disrupting these could attenuate the IL-6 induction. The up-regulation involves enhanced IL-6 gene promoter activity and retardation of IL-6 mRNA decay by IL-1beta. Dexamethasone completely blocked the effect of IL-1beta on IL-6 expression. Orbital fibroblasts also express higher levels of IL-6R than do skin-derived cells. When treated with rIL-6 (10 ng/ml), STAT3 is transiently phosphorylated. Thus, the exaggerated capacity of orbital fibroblasts to express high levels of both IL-6 and its receptor in an anatomic site-selective manner could represent an important basis for immune responses localized to the orbit in Graves' disease.