Computational modeling and synthesis of pyridine variants of benzoyl-phenoxy-acetamide with high glioblastoma cytotoxicity and brain tumor penetration.

Glioblastomas are highly aggressive brain tumors for which therapeutic options are very limited. In a quest for new anti-glioblastoma drugs, we focused on specific structural modifications to the benzoyl-phenoxy-acetamide (BPA) structure present in a common lipid-lowering drug, fenofibrate, and in our first prototype glioblastoma drug, PP1. Here, we propose extensive computational analyses to improve the selection of the most effective glioblastoma drug candidates. Initially, over 100 structural BPA variations were analyzed and their physicochemical properties, such as water solubility (- logS), calculated partition coefficient (ClogP), probability for BBB crossing (BBB_SCORE), probability for CNS penetration (CNS-MPO) and calculated cardiotoxicity (hERG), were evaluated. This integrated approach allowed us to select pyridine variants of BPA that show improved BBB penetration, water solubility, and low cardiotoxicity. Herein the top 24 compounds were synthesized and analyzed in cell culture. Six of them demonstrated glioblastoma toxicity with IC50 ranging from 0.59 to 3.24 µM. Importantly, one of the compounds, HR68, accumulated in the brain tumor tissue at 3.7 ± 0.5 µM, which exceeds its glioblastoma IC50 (1.17 µM) by over threefold.

Computational modeling and synthesis of Pyridine variants of Benzoyl-Phenoxy-Acetamide with high glioblastoma cytotoxicity and brain tumor penetration.

Glioblastomas are highly aggressive brain tumors for which therapeutic options are very limited. In a quest for new anti-glioblastoma drugs, we focused on specific structural modifications of benzoyl-phenoxy-acetamide (BPA) present in a common lipid-lowering drug, fenofibrate, and in our first prototype glioblastoma drug, PP1. Here, we propose extensive computational analyses to improve selection of the most effective glioblastoma drug candidates. Initially over 100 structural BPA variations were analyzed and their physicochemical properties such as water solubility (-logS), calculated partition coefficient (ClogP), probability for BBB crossing (BBB_SCORE), probability for CNS penetration (CNS-MPO) and calculated cardiotoxicity (hERG), were evaluated. This integrated approach allowed us to select pyridine variants of BPA that show improved BBB penetration, water solubility, and low cardiotoxicity. Herein the top 24 compounds were synthesized and analyzed in cell culture. Six of them demonstrated glioblastoma toxicity with IC50 ranging from 0.59 to 3.24mM. Importantly, one of the compounds, HR68, accumulated in the brain tumor tissue at 3.7+/-0.5mM, which exceeds its glioblastoma IC50 (1.17mM) by over 3-fold.

Serum microRNAs associated with concussion in football players.

Mild Traumatic Brain Injury (mild TBI)/concussion is a common sports injury, especially common in football players. Repeated concussions are thought to lead to long-term brain damage including chronic traumatic encephalopathy (CTE). With the worldwide growing interest in studying sport-related concussion the search for biomarkers for early diagnosis and progression of neuronal injury has also became priority. MicroRNAs are short, non-coding RNAs that regulate gene expression post-transcriptionally. Due to their high stability in biological fluids, microRNAs can serve as biomarkers in a variety of diseases including pathologies of the nervous system. In this exploratory study, we have evaluated changes in the expression of selected serum miRNAs in collegiate football players obtained during a full practice and game season. We found a miRNA signature that can distinguish with good specificity and sensitivity players with concussions from non-concussed players. Furthermore, we found miRNAs associated with the acute phase (let-7c-5p, miR-16-5p, miR-181c-5p, miR-146a-5p, miR-154-5p, miR-431-5p, miR-151a-5p, miR-181d-5p, miR-487b-3p, miR-377-3p, miR-17-5p, miR-22-3p, and miR-126-5p) and those whose changes persist up to 4 months after concussion (miR-17-5p and miR-22-3p).

Anti-glioblastoma effects of phenolic variants of benzoylphenoxyacetamide (BPA) with high potential for blood brain barrier penetration.

Glioblastomas are the most aggressive brain tumors for which therapeutic options are limited. Current therapies against glioblastoma include surgical resection, followed by radiotherapy plus concomitant treatment and maintenance with temozolomide (TMZ), however, these standard therapies are often ineffective, and average survival time for glioblastoma patients is between 12 and 18 months. We have previously reported a strong anti-glioblastoma activity of several metabolic compounds, which were synthetized based compounds, which were synthetized based on the chemical structure of a common lipid-lowering drug, fenofibrate, and share a general molecular skeleton of benzoylphenoxyacetamide (BPA). Extensive computational analyses of phenol and naphthol moieties added to the BPA skeleton were performed in this study with the objective of selecting new BPA variants for subsequent compound preparation and anti-glioblastoma testing. Initially, 81 structural variations were considered and their physical properties such as solubility (logS), blood-brain partitioning (logBB), and probability of entering the CNS calculated by the Central Nervous System-Multiparameter Optimization (MPO-CNS) algorithm were evaluated. From this initial list, 18 compounds were further evaluated for anti-glioblastoma activity in vitro. Nine compounds demonstrated desirable glioblastoma cell toxicity in cell culture, and two of them, HR51, and HR59 demonstrated significantly improved capability of crossing the model blood-brain-barrier (BBB) composed of endothelial cells, astrocytes and pericytes.

Attenuated Negative Feedback in Monocyte-Derived Macrophages From Persons Living With HIV: A Role for IKAROS.

Persons living with HIV (PLWH) are at higher risk of developing secondary illnesses than their uninfected counterparts, suggestive of a dysfunctional immune system in these individuals. Upon exposure to pathogens, monocytes undergo epigenetic remodeling that results in either a trained or a tolerant phenotype, characterized by hyper-responsiveness or hypo-responsiveness to secondary stimuli, respectively. We utilized CD14+ monocytes from virally suppressed PLWH and healthy controls for in vitro analysis following polarization of these cells toward a pro-inflammatory monocyte-derived macrophage (MDM) phenotype. We found that in PLWH-derived MDMs, pro-inflammatory signals (TNFA, IL6, IL1B, miR-155-5p, and IDO1) dominate over negative feedback signals (NCOR2, GSN, MSC, BIN1, and miR-146a-5p), favoring an abnormally trained phenotype. The mechanism of this reduction in negative feedback involves the attenuated expression of IKZF1, a transcription factor required for de novo synthesis of RELA during LPS-induced inflammatory responses. Furthermore, restoring IKZF1 expression in PLWH-MDMs partially reinstated expression of negative regulators of inflammation and lowered the expression of pro-inflammatory cytokines. Overall, this mechanism may provide a link between dysfunctional immune responses and susceptibility to co-morbidities in PLWH with low or undetectable viral load.

Racial Disparities in Triple Negative Breast Cancer: A Review of the Role of Biologic and Non-biologic Factors.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer that lacks expression of the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor (HER2). TNBC constitutes about 15-30 percent of all diagnosed invasive breast cancer cases in the United States. African-American (AA) women have high prevalence of TNBC with worse clinical outcomes than European-American (EA) women. The contributing factors underlying racial disparities have been divided into two major categories based on whether they are related to lifestyle (non-biologic) or unrelated to lifestyle (biologic). Our objective in the present review article was to understand the potential interactions by which these risk factors intersect to drive the initiation and development of the disparities resulting in the aggressive TNBC subtypes in AA women more likely than in EA women. To reach our goal, we conducted literature searches using MEDLINE/PubMed to identify relevant articles published from 2005 to 2019 addressing breast cancer disparities primarily among AA and EA women in the United States. We found that disparities in TNBC may be attributed to racial differences in biological factors, such as tumor heterogeneity, population genetics, somatic genomic mutations, and increased expression of genes in AA breast tumors which have direct link to breast cancer. In addition, a large number of non-biologic factors, including socioeconomic deprivation adversities associated with poverty, social stress, unsafe neighborhoods, lack of healthcare access and pattern of reproductive factors, can promote comorbid diseases such as obesity and diabetes which may adversely contribute to the aggression of TNBC biology in AA women. Further, the biological risk factors directly linked to TNBC in AA women may potentially interact with non-biologic factors to promote a higher prevalence of TNBC, more aggressive biology, and poor survival. The relative contributions of the biologic and non-biologic factors and their potential interactions is essential to our understanding of disproportionately high burden and poor survival rates of AA women with TNBC.

Molecular and Structural Traits of Insulin Receptor Substrate 1/LC3 Nuclear Structures and Their Role in Autophagy Control and Tumor Cell Survival.

Insulin receptor substrate 1 (IRS-1) is a common cytosolic adaptor molecule involved in signal transduction from insulin and insulin-like growth factor I (IGF-I) receptors. IRS-1 can also be found in the nucleus. We report here a new finding of unique IRS-1 nuclear structures, which we observed initially in glioblastoma biopsy specimens and glioblastoma xenografts. These nuclear structures can be reproduced in vitro by the ectopic expression of IRS-1 cDNA cloned in frame with the nuclear localization signal (NLS-IRS-1). In these structures, IRS-1 localizes at the periphery, while the center harbors a key autophagy protein, LC3. These new nuclear structures are highly dynamic, rapidly exchange IRS-1 molecules with the surrounding nucleoplasm, disassemble during mitosis, and require a growth stimulus for their reassembly and maintenance. In tumor cells engineered to express NLS-IRS-1, the IRS-1/LC3 nuclear structures repress autophagy induced by either amino acid starvation or rapamycin treatment. In this process, IRS-1 nuclear structures sequester LC3 inside the nucleus, possibly preventing its cytosolic translocation and the formation of new autophagosomes. This novel mechanism provides a quick and reversible way of inhibiting autophagy, which could counteract autophagy-induced cancer cell death under severe stress, including anticancer therapies.

A miRNA Signature for Cognitive Deficits and Alcohol Use Disorder in Persons Living with HIV/AIDS.

HIV-associated neurocognitive disorders (HAND) affects more than half of persons living with HIV-1/AIDS (PLWHA). Identification of biomarkers representing the cognitive status of PLWHA is a critical step for implementation of successful cognitive, behavioral and pharmacological strategies to prevent onset and progression of HAND. However, the presence of co-morbidity factors in PLWHA, the most common being substance abuse, can prevent the identification of such biomarkers. We have optimized a protocol to profile plasma miRNAs using quantitative RT-qPCR and found a miRNA signature with very good discriminatory ability to distinguish PLWHA with cognitive impairment from those without cognitive impairment. Here, we have evaluated this miRNA signature in PLWHA with alcohol use disorder (AUD) at LSU Health Sciences Center (LSUHSC). The results show that AUD is a potential confounding factor for the miRNAs associated with cognitive impairment in PLWHA. Furthermore, we have investigated the miRNA signature associated with cognitive impairment in an independent cohort of PLWHA using plasma samples from the CNS HIV Antiretroviral Therapy Effects Research (CHARTER) program. Despite differences between the two cohorts in socioeconomic status, AUD, and likely misuse of illicit or prescription drugs, we validated a miRNA signature for cognitive deficits found at LSUHSC in the CHARTER samples.

Anticancer potential of aminomethylidene-diazinanes I. Synthesis of arylaminomethylidene of diazinetriones and its cytotoxic effects tested in glioblastoma cells.

Diazinane and aryl moieties with vinylamine linkers were synthesized to investigate the importance of their structural variations as potential anti-glioblastoma agents. Structural variations incorporated on to the diazinane moiety included oxa and thio derivatives, each with a variety of nitrogen-bound substituents. The size and shape of the aromatic moiety was varied, with the final variation introducing two carbonyl groups, yielding a substituted anthraquinone. Readily available diazinanes and aryl amines were used asan advantageous foundation. Several parameters were calculated whilst engineering these compounds, including: ClogP, molecular polarizability, polar surface area, minimal molecular projected area, and pKa. In addition, a simple and efficient procedure was developed to synthesize these compounds. It was demonstrated that a vinylamine with 1,3-diazinane-2,4,6-trione and 1-anthraquinone moiety is the most promising drug candidate causing almost 70% of LN229 tumor cell death at 1µg/ml. In addition, its molecular polarizability, polar surface area and minimal molecular projected area indicate a possible potential of this molecule for crossing BBB.

Acute Ethanol Increases IGF-I-Induced Phosphorylation of ERKs by Enhancing Recruitment of p52-Shc to the Grb2/Shc Complex.

Ethanol plays a detrimental role in the development of the brain. Multiple studies have shown that ethanol inhibits insulin-like growth factor I receptor (IGF-IR) function. Because the IGF-IR contributes to brain development by supporting neural growth, survival, and differentiation, we sought to determine the molecular mechanism(s) involved in ethanol's effects on this membrane-associated tyrosine kinase. Using multiple neuronal cell types, we performed Western blot, immunoprecipitation, and GST-pulldowns following acute (1-24 h) or chronic (3 weeks) treatment with ethanol. Surprisingly, exposure of multiple neuronal cell types to acute (up to 24 h) ethanol (50 mM) enhanced IGF-I-induced phosphorylation of extracellular regulated kinases (ERKs), without affecting IGF-IR tyrosine phosphorylation itself, or Akt phosphorylation. This acute increase in ERKs phosphorylation was followed by the expected inhibition of the IGF-IR signaling following 3-week ethanol exposure. We then expressed a GFP-tagged IGF-IR construct in PC12 cells and used them to perform fluorescence recovery after photobleaching (FRAP) analysis. Using these fluorescently labeled cells, we determined that 50 mM ethanol decreased the half-time of the IGF-IR-associated FRAP, which implied that cell membrane-associated signaling events could be affected. Indeed, co-immunoprecipitation and GST-pulldown studies demonstrated that the acute ethanol exposure increased the recruitment of p52-Shc to the Grb2-Shc complex, which is known to engage the Ras-Raf-ERKs pathway following IGF-1 stimulation. These experiments indicate that even a short and low-dose exposure to ethanol may dysregulate function of the receptor, which plays a critical role in brain development. J. Cell. Physiol. 232: 1275-1286, 2017. © 2016 Wiley Periodicals, Inc.

HIV-1-Tat Protein Inhibits SC35-mediated Tau Exon 10 Inclusion through Up-regulation of DYRK1A Kinase.

The HIV-1 transactivator protein Tat is implicated in the neuronal damage that contributes to neurocognitive impairment affecting people living with HIV/AIDS. Aberrant splicing of TAU exon 10 results in tauopathies characterized by alterations in the proportion of TAU isoforms containing three (3R) or four (4R) microtubule-binding repeats. The splicing factor SC35/SRSF2 binds to nuclear RNA and facilitates the incorporation of exon 10 in the TAU molecule. Here, we utilized clinical samples, an animal model, and neuronal cell cultures and found that Tat promotes TAU 3R up-regulation through increased levels of phosphorylated SC35, which is retained in nuclear speckles. This mechanism involved Tat-mediated increased expression of DYRK1A and was prevented by DYRK1A silencing. In addition, we found that Tat associates with TAU RNA, further demonstrating that Tat interferes with host RNA metabolism in the absence of viral infection. Altogether, our data unravel a novel mechanism of Tat-mediated neuronal toxicity through dysregulation of the SC35-dependent alternative splicing of TAU exon 10. Furthermore, the increased immunostaining of DYRK1A in HIV+ brains without pathology points at dysregulation of DYRK1A as an early event in the neuronal complications of HIV infection.

Anti-tumoral effects of miR-3189-3p in glioblastoma.

Glioblastoma is one of the most aggressive brain tumors. We have previously found up-regulation of growth differentiation factor 15 (GDF15) in glioblastoma cells treated with the anticancer agent fenofibrate. Sequence analysis of GDF15 revealed the presence of a microRNA, miR-3189, in the single intron. We then asked whether miR-3189 was expressed in clinical samples and whether it was functional in glioblastoma cells. We found that expression of miR-3189-3p was down-regulated in astrocytoma and glioblastoma clinical samples compared with control brain tissue. In vitro, the functionality of miR-3189-3p was tested by RNA-binding protein immunoprecipitation, and miR-3189-3p coimmunoprecipitated with Argonaute 2 together with two of its major predicted gene targets, the SF3B2 splicing factor and the guanine nucleotide exchange factor p63RhoGEF. Overexpression of miR-3189-3p resulted in a significant inhibition of cell proliferation and migration through direct targeting of SF3B2 and p63RhoGEF, respectively. Interestingly, miR-3189-3p levels were increased by treatment of glioblastoma cells with fenofibrate, a lipid-lowering drug with multiple anticancer activities. The attenuated expression of miR-3189-3p in clinical samples paralleled the elevated expression of SF3B2, which could contribute to the activation of SF3B2 growth-promoting pathways in these tumors. Finally, miR-3189-3p-mediated inhibition of tumor growth in vivo further supported the function of this microRNA as a tumor suppressor.

Polycyclic aromatic hydrocarbons-induced ROS accumulation enhances mutagenic potential of T-antigen from human polyomavirus JC.

Polycyclic aromatic hydrocarbons (PAHs) are the products of incomplete combustion of organic materials, which are present in cigarette smoke, deep-fried food, and in natural crude oil. Since PAH-metabolites form DNA adducts and cause oxidative DNA damage, we asked if these environmental carcinogens could affect transforming potential of the human Polyomavirus JC oncoprotein, T-antigen (JCV T-antigen). We extracted DMSO soluble PAHs from Deepwater Horizon oil spill in the Gulf of Mexico (oil-PAHs), and detected several carcinogenic PAHs. The oil-PAHs were tested in exponentially growing cultures of normal mouse fibroblasts (R508), and in R508 stably expressing JCV T-antigen (R508/T). The oil-PAHs were cytotoxic only at relatively high doses (1:50-1:100 dilution), and at 1:500 dilution the growth and cell survival rates were practically unaffected. This non-toxic dose triggered however, a significant accumulation of reactive oxygen species (ROS), caused oxidative DNA damage and the formation of DNA double strand breaks (DSBs). Although oil-PAHs induced similar levels of DNA damage in R508 and R508/T cells, only T-antigen expressing cells demonstrated inhibition of high fidelity DNA repair by homologous recombination (HRR). In contrast, low-fidelity repair by non-homologous end joining (NHEJ) was unaffected. This potential mutagenic shift between DNA repair mechanisms was accompanied by a significant increase in clonal growth of R508/T cells chronically exposed to low doses of the oil-PAHs. Our results indicate for the first time carcinogenic synergy in which oil-PAHs trigger oxidative DNA damage and JCV T-antigen compromises DNA repair fidelity.

Differential Effects of MicroRNAs on Glioblastoma Growth and Migration.

Glioblastoma multiforme is characterized by rapid proliferation, aggressive metastatic potential, and resistance to radio- and chemotherapy. The matricellular protein CYR61 regulates cellular proliferation and migration and is highly expressed in Glioblastomas. MicroRNAs are 22-nucleotides long RNAs that regulate gene expression post-transcriptionally. Here, we utilized the LN229 glioblastoma cell line and found that CYR61 is a target of miR-136, miR-155, and miR-634. Over-expression of miR-136 and miR-634 miRNAs negatively affected proliferation, but not migration, while expression of miR-155 reduced migration but did not affect the proliferation of LN229 cells. Investigation of the molecular mechanisms affected by expression of miR-634 revealed an increased phosphorylation of p70S6 kinase, suggesting an induction of the mammalian target of rapamycin (mTOR) complex 1 pathway. Additionally, in miR-634 overexpressing cells, TSC2, a negative regulator of mTOR signaling, was found to be decreased. Altogether, our study provides insights on the differential roles of miRs-136, -155, and -634 in regulating glioblastoma cell growth and migration, and how microRNAs could be manipulated to decrease the aggressiveness and metastatic potential of tumor cells.

Nuclear IRS-1 and cancer.

The family of insulin receptor substrates (IRS) consists of four proteins (IRS-1-IRS-4), which were initially characterized as typical cytosolic adaptor proteins involved in insulin receptor (IR) and insulin-like growth factor I receptor (IGF-IR) signaling. The first cloned and characterized member of the IRS family, IRS-1, has a predicted molecular weight of 132 kDa, however, as a result of its extensive serine phosphorylation it separates on a SDS gel as a band of approximately 160-185 kDa. In addition to its metabolic and growth-promoting functions, IRS-1 is also suspected to play a role in malignant transformation. The mechanism by which IRS-1 supports tumor growth is not fully understood, and the argument that IRS-1 merely amplifies the signal from the IGF-1R and/or IR requires further investigation. Almost a decade ago, we reported the presence of nuclear IRS-1 in medulloblastoma clinical samples, which express viral oncoprotein, large T-antigen of human polyomavirus JC (JCV T-antigen). This first demonstration of nuclear IRS-1 was confirmed by several other laboratories. Nuclear IRS-1 was also detected by cells expressing the SV40 T-antigen, v-Src, in immortalized fibroblasts stimulated with IGF-I, in hepatocytes, 32D cells, and in an osteosarcoma cell line. More recently, nuclear IRS-1 was detected in breast cancer cells in association with estrogen receptor alpha (ERα), and in JC virus negative medulloblastoma cells expressing estrogen receptor beta (ERß), further implicating nuclear IRS-1 in cellular transformation. Here, we discuss how nuclear IRS-1 acting on DNA repair fidelity, transcriptional activity, and cell growth can support tumor development and progression.

Estrogen receptor beta-mediated nuclear interaction between IRS-1 and Rad51 inhibits homologous recombination directed DNA repair in medulloblastoma.

In medulloblastomas, which are highly malignant cerebellar tumors of the childhood genotoxic treatments such as cisplatin or gamma-irradiation are frequently associated with DNA damage, which often associates with unfaithful DNA repair, selection of new adaptations and possibly tumor recurrences. Therefore, better understanding of molecular mechanisms which control DNA repair fidelity upon DNA damage is a critical task. Here we demonstrate for the first time that estrogen receptor beta (ERbeta) can contribute to the development of genomic instability in medulloblastomas. Specifically, ERbeta was found highly expressed and active in mouse and human medulloblastoma cell lines. Nuclear ERbeta was also present in human medulloblastoma clinical samples. Expression of ERbeta coincided with nuclear translocation of insulin receptor substrate 1 (IRS-1), which was previously reported to interfere with the faithful component of DNA repair when translocated to the nucleus. We demonstrated that ERbeta and IRS-1 bind each other, and the interaction involves C-terminal domain of IRS-1 (aa 931-1233). Following cisplatin-induced DNA damage, nuclear IRS-1 localized at the sites of damaged DNA, and interacted with Rad51--an enzymatic component of homologous recombination directed DNA repair (HRR). In medulloblastoma cells, engineered to express HRR-DNA reporter plasmid, ER antagonist, ICI 182,780, or IRS mutant (931-1233) significantly increased DNA repair fidelity. These data strongly suggest that both molecular and pharmacological interventions are capable of preventing ERbeta-mediated IRS-1 nuclear translocation, which in turn improves DNA repair fidelity and possibly counteracts accumulation of malignant mutations in actively growing medulloblastomas.

Tumour necrosis factor-alpha stimulates expression of TNF-alpha converting enzyme in endothelial cells.

Tumor necrosis factor-alpha converting enzyme (ADAM17) is a major metalloproteinase involved in the shedding of several membrane-bound cytokines and cytokine receptors. Interplay of cytokines and their soluble receptors might be an important regulatory element in the network of interactions responsible for maintaining homeostasis in the immune system. ADAM17 thus has the potential to participate in a broad range of immune reactions. We studied the mechanisms of ADAM17 activation in endothelial cells and found that pro-inflammatory cytokines (tumor necrosis factor-alpha, interleukin-1beta, interferon-gamma) and growth factors (epidermal growth factor, vascular endothelial growth factor) are able to upregulate transcription of ADAM17 and expression of ADAM17 protein. This process might constitute an important mechanism of regulation of ADAM17 activity. Stimulation of transcription, rather than increased ADAM17 mRNA stability, was responsible for increased levels of ADAM17 mRNA. Importantly, the increase in ADAM17 was accompanied by increased shedding of TNF-Receptor I (p55) in tumor necrosis factor-alpha-stimulated endothelial cells. Therefore, ADAM17-dependent depletion of membrane-bound tumor necrosis factor receptors from endothelial cells might constitute a mechanism of self-protection in states of prolonged immunostimulation.

Role of the insulin-like growth factor I/insulin receptor substrate 1 axis in Rad51 trafficking and DNA repair by homologous recombination.

The receptor for insulin-like growth factor I (IGF-IR) controls normal and pathological growth of cells. DNA repair pathways represent an unexplored target through which the IGF-IR signaling system might support pathological growth leading to cellular transformation. However, this study demonstrates that IGF-I stimulation supports homologous recombination-directed DNA repair (HRR). This effect involves an interaction between Rad51 and the major IGF-IR signaling molecule, insulin receptor substrate 1 (IRS-1). The binding occurs within the cytoplasm, engages the N-terminal domain of IRS-1, and is attenuated by IGF-I-mediated IRS-1 tyrosine phosphorylation. In the absence of IGF-I stimulation, or if mutated IGF-IR fails to phosphorylate IRS-1, localization of Rad51 to the sites of damaged DNA is diminished. These results point to a direct role of IRS-1 in HRR and suggest a novel role for the IGF-IR/IRS-1 axis in supporting the stability of the genome.

T-antigen of human polyomavirus JC cooperates withIGF-IR signaling system in cerebellar tumors of the childhood-medulloblastomas.

Polyomaviruses are implicated in a number of cancers, and the transforming activity of their early protein, large T-antigen, has been documented in a variety of cell types and in experimental animals (1). Although the pathways by which T-antigen induces uncontrolled cell growth are not fully defined, T-antigen mediated inactivation of tumor suppressors, p53 and pRB, is well-documented in some malignancies (2). Here we postulate that functional interaction between the insulin-like growth factor (IGF-IR) and the T-antigen of human polyomavirus JC (JCV T-antigen) may contribute to the process of malignant transformation in medulloblastomas: (i) the IGF-IR signaling system is strongly activated in medulloblastoma cell lines and medulloblastoma biopsies; (ii) the cytoplasmic protein, insulin receptor substrate 1 (IRS-1), is translocated to the nucleus in the presence of JCV T-antigen; (iii) molecular characterization of the interaction between IRS-1 and JCV T-antigen indicates that the binding involves the N-terminal portion of IRS-1 (PH/PTB domain) and the C-terminal region of JCV T-antigen (aa 411-628); and finally (iv) competition for the IRS-1-JCV T-antigen binding attenuates anchorage-independent growth of T-antigen positive medulloblastoma cells in culture. Based on these findings, we propose a novel role for IRS-1 in JCV T-antigen-mediated deregulation of cellular equilibrium, which may involve uncoupling of IRS-1 from the surface receptor and translocation of its function to the nuclear compartment of the cell.

Members of the AP-1 family, c-Jun and c-Fos, functionally interact with JC virus early regulatory protein large T antigen.

The activating protein 1 (AP-1) family of regulatory proteins is characterized as immediate-early inducible transcription factors which were shown to be activated by a variety of stress-related stimuli and to be involved in numerous biological processes, including cellular and viral gene expression, cell proliferation, differentiation, and tumorigenesis. We have recently demonstrated the involvement of the AP-1 family members c-Jun and c-Fos in transcriptional regulation of the human polyomavirus, JC virus (JCV), genome. Here, we further examined their role in JCV gene regulation and replication through their physical and functional interaction with JCV early regulatory protein large T antigen (T-Ag). Transfection and replication studies indicated that c-Jun and c-Fos can significantly diminish T-Ag-mediated JCV gene transcription and replication. Affinity chromatography and coimmunoprecipitation assays demonstrated that c-Jun and T-Ag physically interact with each other. Results from band shift assays showed that the binding efficiency of c-Jun to the AP-1 site was reduced in the presence of T-Ag. In addition, we have mapped, through the use of a series of deletion mutants, the regions of these proteins which are important for their interaction. While the c-Jun interaction domain of T-Ag is localized to the middle portion of the protein, the T-Ag interacting domain of c-Jun maps to its basic-DNA binding region. Results of transient-transfection assays with various c-Jun mutants and T-Ag expression constructs further confirm the specificity of the functional interaction between c-Jun and T-Ag. Taken together, these data demonstrate that immediate-early inducible transcription factors c-Jun and c-Fos physically and functionally interact with JCV major early regulatory protein large T-Ag and that this interaction modulates JCV transcription and replication in glial cells.