Infection with the hepatitis C virus (HCV) is a major cause of chronic liver diseases and hepatocellular carcinoma worldwide, and thus represents a significant public health problem. The type I interferon (IFN), IFN?, has been successful in treating HCV-infected patients, but current IFN-based treatment regimens for HCV have suboptimal efficacy, and relatively little is known about why IFN therapy eliminates the virus in some patients but not in others. Therefore, it is critical to understand the basic mechanisms that underlie the therapeutic resistance to IFN action in HCV-infected individuals, and there is an urgent need to identify those patients most likely to respond to IFN therapy for HCV. To characterize the response of HCV-infected patients to treatment with IFN?, the expression of an IFN-response gene signature comprised of IFN-stimulated genes and genes that play an important role in the innate immune response was examined in liver biopsies from HCV-infected patients enrolled in a clinical trial. In the present study we found that the expression of a subset of IFN-response genes was dysregulated in liver biopsy samples from nonresponsive hepatitis C patients as compared with virologic responders. Based on these findings, a statistical model was developed to help predict the response of patients to IFN therapy, and compared to results obtained to the IL28 mutation model, which is highly predictive of the response to IFN-based therapy in HCV-infected patients. We found that a model incorporating gene expression data can improve predictions of IFN responsiveness compared to IL28 mutation status alone.
In breast cancer, distinct expression profiles of miRNAs (microRNAs) have been associated with molecular subgroups and clinicopathological characteristics, implicating a diagnostic and prognostic role of miRNAs. However, the biological functions of deregulated miRNAs in tumor progression are not completely defined. In this study, we investigated the function of miR-18a (microRNA-18a) in regulating breast cancer metastasis through the HIF1A (hypoxia-inducible factor 1alpha) -dependent hypoxic response.
Despite advances in surgery, imaging, chemotherapy, and radiation, patients with glioblastoma multiforme (GBM), the most common histological subtype of glioma, have an especially dismal prognosis; >70% of GBM patients die within 2 years of diagnosis. In many human cancers, the microRNA miR-21 is overexpressed, and accumulating evidence indicates that it functions as an oncogene. Here, we report that miR-21 is overexpressed in human GBM cell lines and tumor tissue. Moreover, miR-21 expression in GBM patient samples is inversely correlated with patient survival. Knockdown of miR-21 in GBM cells inhibited cell proliferation in vitro and markedly inhibited tumor formation in vivo. A number of known miR-21 targets have been identified previously. By microarray analysis, we identified and validated insulin-like growth factor (IGF)-binding protein-3 (IGFBP3) as a novel miR-21 target gene. Overexpression of IGFBP3 in glioma cells inhibited cell proliferation in vitro and inhibited tumor formation of glioma xenografts in vivo. The critical role that IGFBP3 plays in miR-21-mediated actions was demonstrated by a rescue experiment, in which IGFBP3 knockdown in miR-21KD glioblastoma cells restored tumorigenesis. Examination of tumors from GBM patients showed that there was an inverse relationship between IGFBP3 and miR-21 expression and that increased IGFBP3 expression correlated with better patient survival. Our results identify IGFBP3 as a novel miR-21 target gene in glioblastoma and suggest that the oncogenic miRNA miR-21 down-regulates the expression of IGFBP3, which acts as a tumor suppressor in human glioblastoma.
Intact miRNAs can be isolated from the circulation in significant quantities despite the presence of extremely high levels of RNase activity. The remarkable stability of circulating miRNAs makes them excellent candidates for biomarkers in diagnostic applications as well as therapeutic targets in a variety of disease states including melanoma. Circulating RNA molecules are resistant to degradation by RNases because they are encapsulated in membrane-bound microvesicles. We describe a convenient method for the use of ExoQuick, a proprietary resin developed by Systems Biosciences (Mountain View, CA), whereby microvesicles can be purified under gentle conditions using readily available laboratory equipment. This protocol allows for isolation all microvesicles, regardless of their origin, and provides a convenient method for identifying potential cancer-specific biomarkers from biological fluids including serum and plasma.
The purpose of this study is to identify metastasis-associated genes/signaling pathways in basal-like breast tumors. Kaplan-Meier analysis of two public meta-datasets and functional classification was used to identify genes/signaling pathways significantly associated with distant metastasis free survival. Integrated analysis of expression correlation and interaction between mRNAs and miRNAs was used to identify miRNAs that potentially regulate the expression of metastasis-associated genes. The novel metastatic suppressive role of miR-17-5p was examined by in vitro and in vivo experiments. Over 4,000 genes previously linked to breast tumor progression were examined, leading to identification of 61 and 69 genes significantly associated with shorter and longer DMFS intervals of patients with basal-like tumors, respectively. Functional annotation linked most of the pro-metastatic genes to epithelial mesenchymal transition (EMT) process and three intertwining EMT-driving pathways (hypoxia, TGFB and Wnt), whereas most of the anti-metastatic genes to interferon signaling pathway. Members of three miRNA families (i.e., miR-17, miR-200 and miR-96) were identified as potential regulators of the pro-metastatic genes. The novel anti-metastatic function of miR-17-5p was confirmed by in vitro and in vivo experiments. We demonstrated that miR-17-5p inhibition in breast cancer cells enhanced expression of multiple pro-metastatic genes, rendered cells metastatic properties, and accelerated lung metastasis from orthotopic xenografts. In contrast, intratumoral administration of miR-17-5p mimic significantly reduced lung metastasis. These results provide evidence supporting that EMT activation and IFN pathway inactivation are markers of metastatic progression of basal-like tumors, and members of miR-17, miR-200, and miR-96 families play a role in suppressing EMT and metastasis. The metastasis-associated genes identified in this study have potential prognostic values and functional implications, thus, can be exploited as therapeutic targets to prevent metastasis of basal-like breast tumors.
Glioblastoma Multiforme (GBM) is a highly fatal disease and new chemotherapeutic agents are desperately needed to treat GBM patients. With the aim of identifying new antiglioma agents we screened the UC DDC library for compounds structurally related to the antiglioma lead molecule compound 1 (SP-6-27) and clinically used compound 2 (Azixa). We identified imidazoquinoline analog 3 (S-94403) as initial hit from the first screen which included the different heterocyclic set of 15 compounds. Based on the initial hit 3 (S-94403) second search was performed to explore the structure activity relationship (SAR) study on imidazoquinolines. Our SAR revealed that N-phenyl with EDGs/EWGs at C9 position, methyl group at C7 position and aryl or hetero-aryl groups at C2 position essential for the anticancer activity. These two consecutive screening have identified the compounds S-94403 (IC50 = 0.625 µM) and S-98950 (IC50 = 1.04 µM) as most potent imidazoquinoline-based antiglioma agents.
Cancer is a major devastating disease, and is a leading cause of death worldwide. Despite the progress in cancer treatment, cancer mortality rate remains high. Therefore, the discovery and development of improved anticancer drugs to treat cancer are needed. 4H-chromenes have strong cytotoxicity against a panel of human cancer cell lines involving pathways that include microtubule depolarization and tumor vasculature disruption. A chromene analog, Crolibulin™ (EPC2407) is currently in Phase I/II clinical trials for the treatment of advanced solid tumors. This article reviews the general synthesis, biological activities and structure-activity relatinships of different classes of chromenes.
MicroRNAs (miRNAs) regulate mRNA stability and translation through the action of the RNAi-induced silencing complex. In this study, we systematically identified endogenous miRNA target genes by using AGO2 immunoprecipitation (AGO2-IP) and microarray analyses in two breast cancer cell lines, MCF7 and MDA-MB-231, representing luminal and basal-like breast cancer, respectively. The expression levels of ?70% of the AGO2-IP mRNAs were increased by DROSHA or DICER1 knockdown. In addition, integrated analysis of miRNA expression profiles, mRNA-AGO2 interaction, and the 3-UTR of mRNAs revealed that >60% of the AGO2-IP mRNAs were putative targets of the 50 most abundantly expressed miRNAs. Together, these results suggested that the majority of the AGO2-associated mRNAs were bona fide miRNA targets. Functional enrichment analysis uncovered that the AGO2-IP mRNAs were involved in regulation of cell cycle, apoptosis, adhesion/migration/invasion, stress responses (e.g. DNA damage and endoplasmic reticulum stress and hypoxia), and cell-cell communication (e.g. Notch and Ephrin signaling pathways). A role of miRNAs in regulating cell migration/invasion and stress response was further defined by examining the impact of DROSHA knockdown on cell behaviors. We demonstrated that DROSHA knockdown enhanced cell migration and invasion, whereas it sensitized cells to cell death induced by suspension culture, glucose depletion, and unfolding protein stress. Data from an orthotopic xenograft model showed that DROSHA knockdown resulted in reduced growth of primary tumors but enhanced lung metastasis. Taken together, these results suggest that miRNAs collectively function to promote survival of tumor cells under stress but suppress cell migration/invasion in breast cancer cells.
Malignant gliomas are locally aggressive, highly vascular tumors that have a dismal prognosis, and present therapies provide little improvement in the disease course and outcome. Many types of malignancies, including glioblastoma, originate from a population of cancer stem cells (CSCs) that are able to initiate and maintain tumors. Although CSCs only represent a small fraction of cells within a tumor, their high tumor-initiating capacity and therapeutic resistance drives tumorigenesis. Therefore, it is imperative to identify pathways associated with CSCs to devise strategies to selectively target them. In this study, we describe a novel relationship between glioblastoma CSCs and the Notch pathway, which involves the constitutive activation of STAT3 and NF-?B signaling. Glioma CSCs were isolated and maintained in vitro using an adherent culture system, and the biological properties were compared with the traditional cultures of CSCs grown as multicellular spheres under nonadherent culture conditions. Interestingly, both adherent and spheroid glioma CSCs show constitutive activation of the STAT3/NF-?B signaling pathway and up-regulation of STAT3- and NF-?B-dependent genes. Gene expression profiling also identified components of the Notch pathway as being deregulated in glioma CSCs, and the deregulated expression of these genes was sensitive to treatment with STAT3 and NF-?B inhibitors. This finding is particularly important because Notch signaling appears to play a key role in CSCs in a variety of cancers and controls cell fate determination, survival, proliferation, and the maintenance of stem cells. The constitutive activation of STAT3 and NF-?B signaling pathways that leads to the regulation of Notch pathway genes in glioma CSCs identifies novel therapeutic targets for the treatment of glioma.
Gliomas are considered the most malignant form of brain tumors, and ranked among the most aggressive human cancers. Despite advance standard therapy the prognosis for patients with gliomas remains poor. Chemotherapy has played an important role as an adjuvant in treating gliomas. The efficacy of the chemotherapeutic drug is limited due to poor drug delivery and the inherent chemo- and radio-resistance. Challenges of the brain cancer therapy in clinical settings are; i) to overcome the chemo- and radio-resistance, ii) to improve drug delivery to tumors and iii) the development of effective drug screening procedures.
Ionizing radiation (IR) is an essential component of therapy for alveolar rhabdomyosarcoma. Nuclear factor-kappaB (NF-??) transcription factors are upregulated by IR and have been implicated in radioresistance. We evaluated the ability of curcumin, a putative NF-?? inhibitor, and cells expressing genetic NF- ?? inhibitors (I?B? and p100 super-repressor constructs) to function as a radiosensitizer. Ionizing radiation induced NF-?? activity in the ARMS cells in vitro in a dose- and time-dependent manner, and upregulated expression of NF-?? target proteins. Pretreatment of the cells with curcumin inhibited radiation-induced NF-?? activity and target protein expression. In vivo, the combination of curcumin and IR had synergistic antitumor activity against Rh30 and Rh41 ARMS xenografts. The greatest effect occurred when tumor-bearing mice were treated with curcumin prior to IR. Immunohistochemistry revealed that combination therapy significantly decreased tumor cell proliferation and endothelial cell count, and increased tumor cell apoptosis. Stable expression of the super-repressor, SR-I?B?, that blocks the classical NF-?B pathway, increased sensitivity to IR, while expression of SR-p100, that blocks the alternative pathway, did not. Our results demonstrate that curcumin can potentiate the antitumor activity of IR in ARMS xenografts by suppressing a classical NF-?? activation pathway induced by ionizing radiation. These data support testing of curcumin as a radiosensitizer for the clinical treatment of alveolar rhabdomyosarcoma. IMPACT OF WORK: The NF-?? protein complex has been linked to radioresistance in several cancers. In this study, we have demonstrated that inhibiting radiation-induced NF-?? activity by either pharmacologic (curcumin) or genetic (SR-I?B?) means significantly enhanced the efficacy of radiation therapy in the treatment of alveolar rhabdomyosarcoma cells and xenografts. These data suggest that preventing the radiation-induced activation of the NF-?? pathway is a promising way to improve the antitumor efficacy of ionizing radiation and warrants clinical trials.
Familial melanoma (FM) is a dominantly heritable cancer that is associated with mutations in the tumor suppressor CDKN2A/p16. In FM, a single inherited "hit" occurs in every somatic cell, enabling interrogation of cultured normal skin fibroblasts (SFs) from FM gene carriers as surrogates for the cell of tumor origin, namely the melanocyte. We compared the gene expression profile of SFs from FM individuals with two distinct CDKN2A/p16 mutations (V126D-p16 and R87P-p16) with the gene expression profile of SFs from age-matched individuals without p16 mutations and with no family history of melanoma. We show an altered transcriptome signature in normal SFs bearing a single-hit inherited mutation in the CDKN2A/p16 gene, wherein some of these abnormal alterations recapitulate changes observed in the corresponding cancer. Significantly, the extent of the alterations is mutation-site specific with the R87P-p16 mutation being more disruptive than the V126D-p16 mutation. We also examined changes in gene expression after exposure to ultraviolet (UV) radiation to define potential early biomarkers triggered by sun exposure. UV treatment of SFs from FM families induces distinct alterations in genes related to cell cycle regulation and DNA damage responses that are also reported to be dysregulated in melanoma. Importantly, these changes were diametrically opposed to UV-induced changes in SF from normal controls. We posit that changes identified in the transcriptome of SF from FM mutation carriers represent early events critical for melanoma development. As such, they may serve as specific biomarkers of increased risk as well as molecular targets for personalized prevention strategies in high-risk populations.
EF24 is a curcumin analog that has improved anticancer activity over curcumin, but its therapeutic potential and mechanism of action is unknown, which is important to address as curcumin targets multiple signaling pathways. EF24 inhibits the NF-?B but not the JAK-STAT signaling pathway in DU145 human prostate cancer cells and B16 murine melanoma cells. EF24 induces apoptosis in these cells apparently by inhibiting miR-21 expression, and also enhances the expression of several miR-21 target genes, PTEN and PDCD4. EF24 treatment significantly suppressed the growth of DU145 prostate cancer xenografts in immunocompromised mice and resulted in tumor regression. EF24 enhanced the expression of the miR-21 target PTEN in DU145 tumor tissue, but suppressed the expression of markers of proliferating cells (cyclin D1 and Ki67). In syngeneic mice injected with B16 cells, EF24 treatment inhibited the formation of lung metastasis, prolonged animal survival, inhibited miR-21 expression and increased the expression of miR-21 target genes. Expression profiling of miRNAs regulated by EF24 in vitro and in vivo showed that the antitumor activity of EF24 reflected the enhanced expression of potential tumor suppressor miRNAs as well as the suppressed expression of oncogenic miRNAs, including miR-21. Taken together, our data suggest that EF24 is a potent anticancer agent and selectively targets NF-?B signaling and miRNA expression, indicating that EF24 has significant potential as a therapeutic agent in various cancers.
MicroRNA-21 (miR-21) is overexpressed in many human tumors and has been linked to various cellular processes altered in cancer. miR-21 is also up-regulated by a number of inflammatory agents, including IFN, which is of particular interest considering the close relationship between inflammation and cancer. Because miR-21 appears to be overexpressed in human melanoma, we examined the role of miR-21 in cancer development and metastasis in B16 mouse melanoma cells. We found that miR-21 is a member of an IFN-induced miRNA subset that requires STAT3 activation. To characterize the role of miR-21 in melanoma behavior, we transduced B16 cells with lentivirus encoding a miR-21 antagomir and isolated miR-21 knockdown B16 cells. miR-21 knockdown or IFN treatment alone inhibited B16 cell proliferation and migration in vitro, and in combination they had an enhanced effect. Moreover, miR-21 knockdown sensitized B16 cells to IFN-induced apoptosis. In B16 cells miR-21 targeted tumor suppressor (PTEN and PDCD4) and antiproliferative (BTG2) proteins. To characterize the role of miR-21 in vivo, empty vector- and antagomiR-21-transduced B16 melanoma cells were injected via tail vein into syngeneic C57BL/6 mice. Although empty vector-transduced B16 cells produced large lung metastases, miR-21 knockdown cells only formed small lung lesions. Importantly, miR-21 knockdown tumor-bearing mice exhibited prolonged survival compared with empty vector tumor-bearing mice. Thus, miR-21 regulates the metastatic behavior of B16 melanoma cells by promoting cell proliferation, survival, and migration/invasion as well as by suppressing IFN action, providing important new insights into the role of miR-21 in melanoma.
Oral cancer is arguably the most serious condition that dental providers may encounter in their practice. The relatively poor prognosis associated with oral cancer highlights the importance of the dental teams awareness of the disease. While many characteristics of oral cancer have endured over time, new research is revealing trends that are changing the way we approach its screening, diagnosis and treatment. In this report, we provide a translational overview of oral cancer, including risk factors, signs and symptoms, clinical management, as well as our recent findings on the role of chronic inflammation in the development of the disease. In addition, our recent genetic profiling approach in both cancer cell lines and in patients has identified potential biomarkers, molecular pathways and therapeutic drugs (Velcade and Aspirin) for oral squamous cell carcinomas. This comprehensive review should be of interest to all dental professionals.
The nuclear factor ?B (NF-?B) transcription factor regulates the expression of genes involved in cell survival and immune responses. We have identified a novel interferon (IFN)-activated signaling pathway that leads to NF-?B activation and demonstrate that a subset of IFN-stimulated genes and microRNAs that play key roles in cellular response to IFN is regulated by NF-?B. This review focuses on the IFN-induced NF-?B activation pathway and the role of NF-?B in the expression of IFN-induced coding and noncoding genes, antiviral activity and apoptosis, and the therapeutic application of IFN in cancer and infectious disease.
Matrix metalloproteinase-9 (MMP-9) is important in numerous normal and pathological processes, including the angiogenic switch during tumor development and tumor metastasis. Whereas TNF-? and other cytokines up-regulate MMP-9 expression, interferons (IFNs) inhibit MMP-9 expression. We found that IFN-? treatment or forced expression of the IFN-induced GTPase, mGBP-2, inhibit TNF-?-induced MMP-9 expression in NIH 3T3 fibroblasts, by inhibiting MMP-9 transcription. The NF-?B transcription factor is required for full induction of MMP-9 by TNF-?. Both IFN-? and mGBP-2 inhibit the transcription of a NF-?B-dependent reporter construct, suggesting that mGBP-2 inhibits MMP-9 induction via inhibition of NF-?B-mediated transcription. Interestingly, mGBP-2 does not inhibit TNF-?-induced degradation of I?B? or p65/RelA translocation into the nucleus. However, mGBP-2 inhibits p65 binding to a ?B oligonucleotide probe in gel shift assays and to the MMP-9 promoter in chromatin immunoprecipitation assays. In addition, TNF-? activation of NF-?B in NIH 3T3 cells is dependent on Rac activation, as evidenced by the inhibition of TNF-? induction of NF-?B-mediated transcription by a dominant inhibitory form of Rac1. A role for Rac in the inhibitory action of mGBP-2 on NF-?B is further shown by the findings that mGBP-2 inhibits TNF-? activation of endogenous Rac and constitutively activate Rac can restore NF-?B transcription in the presence of mGBP-2. This is a novel mechanism by which IFNs can inhibit the cytokine induction of MMP-9 expression.
High-grade glioblastomas have immature, leaky tumor blood vessels that impede the efficacy of adjuvant therapy. We assessed the ability of human interferon (hIFN)-? delivered locally via gene transfer to effect vascular stabilization in an orthotopic model of glioblastoma xenograft resection.
Constitutively activated STAT3 is found frequently in a wide variety of human tumors, including melanoma. Moreover, constitutive STAT3 activation actively participates in tumor formation and progression, making STAT3 an attractive target for cancer therapy. We report here that in murine B16 melanoma cells, which have been previously shown to express constitutively active STAT3, the expression of a mutant form of STAT3 with the canonical tyrosine phosphorylation site (residue 705) mutated to phenylanaine has dominant-negative properties (STAT3-DN). STAT3-DN inhibits STAT3 tyrosine phosphorylation and STAT3-dependent DNA binding activity. Most importantly, STAT3-DN expression in B16 cells inhibits their invasiveness, as well as their melanogenesis by down-regulation of tyrosinase mRNA and protein expression as well as tyrosinase activity. These results suggest that STAT3 signaling plays a critical role in regulating melanoma behavior, and may represent a druggable target for melanoma therapy.
The microRNA miR-21 is overexpressed in many human cancers, wherein accumulating evidence indicates that it functions as an oncogene. Here, we report that the cytokine IFN rapidly induces miR-21 expression in human and mouse cells. Signal transducer and activator of transcription 3 (STAT3) was implicated in this pathway based on the lack of IFN effect on miR-21 expression in prostate cancer cells with a deletion in the STAT3 gene. STAT3 ablation abrogated IFN induction of miR-21, confirming the important role of STAT3 in regulating miR-21. Chromatin immunoprecipitation analysis showed that STAT3 directly bound the miR-21 promoter in response to IFN. Experiments in mouse embryo fibroblasts with a genetic deletion of the p65 NF-?B subunit showed that IFN-induced miR-21 expression was also dependent on NF-?B. STAT3 silencing blocked both IFN-induced p65 binding to the miR-21 promoter and p65 nuclear translocation. Thus, IFN-induced miR-21 expression is coregulated by STAT3 and NF-?B at the level of the miR-21 promoter. Several cell death regulators were identified as downstream targets of miR-21, including PTEN and Akt. Functional experiments in prostate cancer cells directly showed that miR-21 plays a critical role in suppressing IFN-induced apoptosis. Our results identify miR-21 as a novel IFN target gene that functions as a key feedback regulator of IFN-induced apoptosis.
Oral squamous cell carcinoma (OSCC) is a major health problem worldwide, and patients have a particularly poor 5-year survival rate. Thus, identification of the molecular targets in OSCC and subsequent innovative therapies are greatly needed. Prolonged exposure to alcohol, tobacco, and pathogenic agents are known risk factors and have suggested that chronic inflammation may represent a potential common denominator in the development of OSCC. Microarray analysis of gene expression in OSCC cell lines with high basal NF-?B activity and OSCC patient samples identified dysregulation of many genes involved in inflammation, wound healing, angiogenesis, and growth regulation. In particular IL-8, CCL5, STAT1, and VEGF gene expression was up-regulated in OSCC. Moreover, IL-8 protein levels were significantly higher in OSCC cell lines as compared with normal human oral keratinocytes. Targeting IL-8 expression by siRNA significantly reduced the survival of OSCC cells, indicating that it plays an important role in OSCC development and/or progression. Inhibiting the inflammatory pathway by aspirin and the proteasome/NF-?B pathway by bortezomib resulted in marked reduction in cell viability in OSCC lines. Taken together our studies indicate a strong link between inflammation and OSCC development and reveal IL-8 as a potential mediator. Treatment based on prevention of general inflammation and/or the NF-?B pathway shows promise in OSCCs.
Physiological functions of mitochondria in contractile arterial myocytes are poorly understood. Mitochondria can uptake calcium (Ca(2+)), but intracellular Ca(2+) signals that regulate mitochondrial Ca(2+) concentration ([Ca(2+)](mito)) and physiological functions of changes in [Ca(2+)](mito) in arterial myocytes are unclear.
The zinc finger antiviral protein (ZAP) is an interferon-stimulated gene that restricts the replication of retroviruses, alphaviruses, and filoviruses. Relatively little is known, however, regarding the detailed mechanism of ZAP induction during viral infections. We show that, although being inducible by either interferon or virus, expression of ZAP is more efficiently activated by virus than are several other classical interferon-stimulated genes and that viral induction of ZAP occurs under the direct control of interferon regulatory factor 3 (IRF3) independent of interferon paracrine/autocrine signaling. ZAP was up-regulated in cells unresponsive to type I and III interferons upon engagement of TLR3, retinoic inducible gene I/melanoma differentiation-associated gene 5 pathways, or ectopic expression of a constitutively active IRF3 mutant. Conversely, induction of ZAP by virus or dsRNA was severely impaired in cells expressing a dominant-negative mutant IRF3 and completely abrogated in cells lacking IRF3. In contrast to IRF3, ZAP induction was independent of NF-kappaB activity. Mutational analysis of the human ZAP promoter revealed that multiple interferon-stimulated response elements far distal to the transcription start site serve redundantly to control IRF3-dependent induction of ZAP transcription. Chromatin immunoprecipitation assays demonstrated that IRF3 selectively binds the distal interferon-stimulated response elements in human ZAP promoter following viral infection. Collectively, these data suggest that ZAP is a direct target gene of IRF3 action in cellular antiviral responses.
Interferon-alpha (IFNalpha) has shown promise in the treatment of various cancers. However, the development of IFN resistance is a significant drawback. Using conditions that mimic in vivo selection of IFN-resistant cells, the RST2 IFN-resistant cell line was isolated from the highly IFN-sensitive Daudi human Burkitt lymphoma cell line. The RST2 cell line was resistant to the antiviral, antiproliferative, and gene-induction actions of IFNalpha. Although STAT2 mRNA was present, STAT2 protein expression was deficient in RST2 cells. A variant STAT2 mRNA, which resulted from alternative splicing within the intron between exon 19 and 20, was expressed in several human cell lines but at relatively high levels in RST2 cells. Most importantly, the RST2 line showed an intrinsic resistance to apoptosis induced by a number of chemotherapeutic agents (camptothecin, staurosporine, and doxorubicin). Expression of STAT2 in RST2 cells not only rescued their sensitivity to the biological activities of IFNs but also restored sensitivity to apoptosis induced by these chemotherapeutic agents. The intrinsic resistance of the RST2 cells to IFN as well as chemotherapeutic agents adds a new dimension to our knowledge of the role of STAT2 as it relates to not only biological actions of IFN but also resistance to chemotherapy-induced apoptosis.
Chronic infection with hepatitis C virus (HCV) is a major global health problem. One way HCV may evade the host immune response is by inhibiting the production of type I interferon (IFN). In addition, the standard treatment for chronic HCV infection involves treatment with IFN-alpha (or its pegylated derivative), alone or in combination with ribavirin. Therefore, it is believed that an important reason that most HCV-infected individuals progress from acute to chronic infection is due to a defect in the host response. In this study, we examined the host response to HCV infection in a cohort of patients enrolled in the UTHSC Cooperative HCV Research Center by determining levels of biologically active IFN in the sera of patients. We found that 15 of 35 enrolled HCV-infected patients show serum levels of IFN (ranging from 2 to 40 IU/mL) before initiation of therapy. Uninfected individuals do not have circulating levels of IFN. Basal IFN levels do not correlate with the clinical response to therapy, nor do they reflect the age, sex, or race of patients. These results suggest that the differential response of patients most likely reflects a defect in the later stages of the host innate immune response, such as the cellular response to endogenous or exogenous IFN. In contrast, the early stage of the host immune response in vivo of many HCV-infected patients (approximately 40%) is intact as determined by IFN production.
Oral cancer is arguably the most serious condition that dental providers may encounter in their practice. The relatively poor prognosis associated with oral cancer highlights the importance of the dental teams awareness of the disease. While many characteristics of oral cancer have endured over time, new research is revealing trends that are changing the way we approach its screening, diagnosis and treatment. In this report, we provide a translational overview of oral cancer, including risk factors, signs and symptoms, clinical management, as well as our recent findings on the role of chronic inflammation in the development of the disease. In addition, our recent genetic profiling approach in both cancer cell lines and in patients has identified potential biomarkers, molecular pathways and therapeutic drugs for oral squamous cell carcinomas. This comprehensive review should be of interest to all dental professionals.
Epigenetic changes in chromatin through histone post-translational modifications are essential for altering gene transcription in response to environmental cues. How histone modifications are regulated by environmental stimuli remains poorly understood yet this process is critical for delineating how epigenetic pathways are influenced by the cellular environment. We have used the target of rapamycin (TOR) pathway, which transmits environmental nutrient signals to control cell growth, as a model to delineate mechanisms underlying this phenomenon. A chemical genomics screen using the TOR inhibitor rapamycin against a histone H3/H4 mutant library identified histone H3 lysine 56 acetylation (H3K56ac) as a chromatin modification regulated by TOR signaling. We demonstrate this acetylation pathway functions in TOR-dependent cell growth in part by contributing directly to ribosomal RNA (rRNA) biogenesis. Specifically, H3K56ac creates a chromatin environment permissive to RNA polymerase I transcription and nascent rRNA processing by regulating binding of the high mobility group protein Hmo1 and the small ribosomal subunit (SSU) processome complex. Overall, these studies identify a novel chromatin regulatory role for TOR signaling and support a specific function for H3K56ac in ribosomal DNA (rDNA) gene transcription and nascent rRNA processing essential for cell growth.
NF-?B activation induced by genotoxic treatment in cancer cells has been associated with therapeutic resistance in multiple human malignancies. Therapeutic resistance also correlates with high metastatic potential in human cancers, including breast cancer. Whether genotoxic treatment-activated NF-?B also contributes to cancer metastasis following radiation and chemotherapy is unclear. Here, we show that chemotherapeutic drug-induced NF-?B activation promotes breast cancer cell migration and invasion. The increased metastatic potential is dependent on IL-6 induction mediated by genotoxic NF-?B activation. Moreover, genotoxic treatment also up-regulates oncogenic microRNA-21 (miR-21) expression through eliciting NF-?B recruitment to the miR-21 promoter region, where it cooperates with signal transducer and activator of transcription 3 (STAT3) to activate miR-21 transcription. DNA damage-induced histone H3 phosphorylation via activated MSK1 creates an open chromatin structure for NF-?B/STAT3-driven transactivation of miR-21. NF-?B-dependent IL-6 up-regulation is responsible for STAT3 activation and recruitment to the miR-21 promoter upon genotoxic stress. Induction of miR-21 may enable cancer cells to elude DNA damage-induced apoptosis and enhance the metastatic potential of breast cancer cells through repressing expression of PTEN and PDCD4. Our data support a critical role of DNA damage-induced NF-?B activation in promoting cancer metastasis following genotoxic treatment, and NF-?B-dependent miR-21 induction may contribute to both therapeutic resistance and metastasis in breast cancer.
DiGeorge Critical Region 8 (DGCR8) is a double-stranded RNA-binding protein that interacts with Drosha and facilitates microRNA (miRNA) maturation. However, the role of DGCR8 in vascular smooth muscle cells (VSMCs) is not well understood. To investigate whether DGCR8 contributes to miRNA maturation in VSMCs, we generated DGCR8 conditional knockout (cKO) mice by crossing VSMC-specific Cre mice (SM22-Cre) with DGCR8(loxp/loxp) mice. We found that loss of DGCR8 in VSMCs resulted in extensive liver hemorrhage and embryonic mortality between embryonic days (E) 12.5 and E13.5. DGCR8 cKO embryos displayed dilated blood vessels and disarrayed vascular architecture. Blood vessels were absent in the yolk sac of DGCR8 KOs after E12.5. Disruption of DGCR8 in VSMCs reduced VSMC proliferation and promoted apoptosis in vitro and in vivo. In DGCR8 cKO embryos and knockout VSMCs, differentiation marker genes, including ?SMA, SM22, and CNN1, were significantly down-regulated, and the survival pathways of ERK1/2 mitogen-activated protein kinase and the phosphatidylinositol 3-kinase/AKT were attenuated. Knockout of DGCR8 in VSMCs has led to down-regulation of the miR-17/92 and miR-143/145 clusters. We further demonstrated that the miR-17/92 cluster promotes VSMC proliferation and enhances VSMC marker gene expression, which may contribute to the defects of DGCR8 cKO mutants. Our results indicate that the DGCR8 gene is required for vascular development through the regulation of VSMC proliferation, apoptosis, and differentiation.
The nuclear factor-kappa B (NF?B) signal transduction pathway plays an important role in immunity, inflammation, cell growth, and survival. Since dysregulation of this pathway results in high, constitutive NF?B activation in various cancers and immune disorders, the development of specific drugs to target this pathway has become a focus for treating these diseases. NF?B regulates various aspects of the cellular response to interferon (IFN). However, the role of the upstream regulator of the NF?B signaling pathway, the inhibitor of ?B kinase (IKK) complex, on IFN function has not been examined. In the present study, we examined the effects of 2 IKK inhibitors, N-(1,8-Dimethylimidazo[1,2-a]quinoxalin-4-yl)-1,2-ethanediamine hydrochloride (BMS-345541) and 2-[(aminocarbonyl)amino]-5-(4-fluorophenyl)-3-thiophenecarboxamide (TPCA-1), on IFN action in several human glioma cell lines. IKK inhibitors inhibit glioma cell proliferation, as well as TNF-induced RelA (p65) nuclear translocation and NF?B-dependent IL8 gene expression. Importantly, BMS-345541 and TPCA-1 differentially inhibit IFN-induced gene expression, completely suppressing MX1 and GBP1 gene expression, while having only a minor effect on ISG15 expression. Furthermore, these IKK inhibitors displayed marked differences in blocking IFN-induced antiviral action against cytopathic effects and replication of vesicular stomatitis virus (VSV) and encephalomyocarditis virus (EMCV). Our results show that the IKK complex plays an important function in IFN-induced gene expression and antiviral activity. Since VSV and EMCV are oncolytic viruses used in cancer therapy, our results indicate the potential synergy in combining IKK inhibitors with oncolytic viruses.
Nuclear factor-?B (NF-?B) has been implicated in tumor cell proliferation and survival and in tumor angiogenesis. We sought to evaluate the effects of curcumin, an inhibitor of NF-?B, on a xenograft model of disseminated neuroblastoma.
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