Human herpes virus-6 induced changes in the expression and activity of the E2F family transcription factors in human cells

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Étude du mécanisme d'autorégulation entre les facteurs de transcription E2F et le microARN miR-20a

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Human herpes virus-6 induced changes in the expression and activity of the E2F family transcription factors in human cells

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Cyclin-dependent kinase-mediated phosphorylation of RBP1 and pRb promotes their dissociation to mediate release of the SAP30·mSin3·HDAC transcriptional repressor complex

Eukaryotic cell cycle progression is mediated by phosphorylation of protein substrates by cyclin-dependent kinases (CDKs). A critical substrate of CDKs is the product of the retinoblastoma tumor suppressor gene, pRb, which inhibits G1-S phase cell cycle progression by binding and repressing E2F transcription factors. CDK-mediated phosphorylation of pRb alleviates this inhibitory effect to promote G1-S phase cell cycle progression. pRb represses transcription by binding to the E2F transactivation domain and recruiting the mSin3·histone deacetylase (HDAC) transcriptional repressor complex via the retinoblastoma-binding protein 1 (RBP1). RBP1 binds to the pocket region of pRb via an LXCXE motif and to the SAP30 subunit of the mSin3·HDAC complex and, thus, acts as a bridging protein in this multisubunit complex. In the present study we identified RBP1 as a novel CDK substrate. RBP1 is phosphorylated by CDK2 on serines 864 and 1007, which are N- and C-terminal to the LXCXE motif, respectively. CDK2-mediated phosphorylation of RBP1 or pRb destabilizes their interaction in vitro, with concurrent phosphorylation of both proteins leading to their dissociation. Consistent with these findings, RBP1 phosphorylation is increased during progression from G 1 into S-phase, with a concurrent decrease in its association with pRb in MCF-7 breast cancer cells. These studies provide new mechanistic insights into CDK-mediated regulation of the pRb tumor suppressor during cell cycle progression, demonstrating that CDK-mediated phosphorylation of both RBP1 and pRb induces their dissociation to mediate release of the mSin3·HDAC transcriptional repressor complex from pRb to alleviate transcriptional repression of E2F.

A transcription factor map as revealed by a genome-wide gene expression analysis of whole-blood mRNA transcriptome in multiple sclerosis

Background Several lines of evidence suggests that transcription factors are involved in the pathogenesis of Multiple Sclerosis (MS) but a complete mapping the whole network has been elusive. One of the reasons is that there are several clinical subtypes of MS and transcription factors which may be involved in one subtype may not be in others. We investigated the possibility that this network could be mapped using microarray technologies and modern bioinformatics methods on a dataset from whole blood in 99 untreated MS patients (36 Relapse Remitting MS, 43 Primary Progressive MS, and 20 Secondary Progressive MS) and 45 age-matched healthy controls, Methodology/Principal Findings We have used two different analytical methodologies: a differential expression analysis and a differential co-expression analysis, which have converged on a significant number of regulatory motifs that seem to be statistically overrepresented in genes which are either differentially expressed (or differentially co-expressed) in cases and controls (e.g. V$KROX_Q6, p-value < 3.31E-6; V$CREBP1_Q2, p-value < 9.93E-6, V$YY1_02, p-value < 1.65E-5). Conclusions/significance: Our analysis uncovered a network of transcription factors that potentially dysregulate several genes in MS or one or more of its disease subtypes. Analysing the published literature we have found that these transcription factors are involved in the early T-lymphocyte specification and commitment as well as in oligodendrocytes dedifferentiation and development. The most significant transcription factors motifs were for the Early Growth response EGR/KROX family, ATF2, YY1 (Yin and Yang 1), E2F-1/DP-1 and E2F-4/DP-2 heterodimers, SOX5, and CREB and ATF families.

Targeting amino acid transport in metastatic castration-resistant prostate cancer : effects on cell cycle, cell growth, and tumor development

Background L-type amino acid transporters (LATs) uptake neutral amino acids including L-leucine into cells, stimulating mammalian target of rapamycin complex 1 signaling and protein synthesis. LAT1 and LAT3 are overexpressed at different stages of prostate cancer, and they are responsible for increasing nutrients and stimulating cell growth. Methods We examined LAT3 protein expression in human prostate cancer tissue microarrays. LAT function was inhibited using a leucine analog (BCH) in androgen-dependent and -independent environments, with gene expression analyzed by microarray. A PC-3 xenograft mouse model was used to study the effects of inhibiting LAT1 and LAT3 expression. Results were analyzed with the Mann-Whitney U or Fisher exact tests. All statistical tests were two-sided. Results LAT3 protein was expressed at all stages of prostate cancer, with a statistically significant decrease in expression after 4–7 months of neoadjuvant hormone therapy (4–7 month mean = 1.571; 95% confidence interval = 1.155 to 1.987 vs 0 month = 2.098; 95% confidence interval = 1.962 to 2.235; P = .0187). Inhibition of LAT function led to activating transcription factor 4–mediated upregulation of amino acid transporters including ASCT1, ASCT2, and 4F2hc, all of which were also regulated via the androgen receptor. LAT inhibition suppressed M-phase cell cycle genes regulated by E2F family transcription factors including critical castration-resistant prostate cancer regulatory genes UBE2C, CDC20, and CDK1. In silico analysis of BCH-downregulated genes showed that 90.9% are statistically significantly upregulated in metastatic castration-resistant prostate cancer. Finally, LAT1 or LAT3 knockdown in xenografts inhibited tumor growth, cell cycle progression, and spontaneous metastasis in vivo. Conclusion Inhibition of LAT transporters may provide a novel therapeutic target in metastatic castration-resistant prostate cancer, via suppression of mammalian target of rapamycin complex 1 activity and M-phase cell cycle genes.

Targeting ASCT2-mediated glutamine uptake blocks prostate cancer growth and tumour development

Glutamine is conditionally essential in cancer cells, being utilized as a carbon and nitrogen source for macromolecule production, as well as for anaplerotic reactions fuelling the tricarboxylic acid (TCA) cycle. In this study, we demonstrated that the glutamine transporter ASCT2 (SLC1A5) is highly expressed in prostate cancer patient samples. Using LNCaP and PC-3 prostate cancer cell lines, we showed that chemical or shRNA-mediated inhibition of ASCT2 function in vitro decreases glutamine uptake, cell cycle progression through E2F transcription factors, mTORC1 pathway activation and cell growth. Chemical inhibition also reduces basal oxygen consumption and fatty acid synthesis, showing that downstream metabolic function is reliant on ASCT2-mediated glutamine uptake. Furthermore, shRNA knockdown of ASCT2 in PC-3 cell xenografts significantly inhibits tumour growth and metastasis in vivo, associated with the down-regulation of E2F cell cycle pathway proteins. In conclusion, ASCT2-mediated glutamine uptake is essential for multiple pathways regulating the cell cycle and cell growth, and is therefore a putative therapeutic target in prostate cancer.

Mechanisms of E2F2-mediated transcriptional repression

In this work we wanted to study the mechanism of E2F2-mediated repression. Our hypothesis is that E2F2 activates the expression of one or more E2F members of the “repressor” subset of the family through the E2F motifs present in their promoters, and those repressor E2F(s) would subsequently repress the target promoters. To address this hypothesis, we focused on E2F7. E2F7 is a repressor that lacks the Rb binding domain, and associates with DNA through E2F binding sites (de Bruin et al., 2003). Furthermore, E2F7 itself is also regulated by E2F motifs on its own promoter, and it has been shown to repress DNA metabolism and replication genes in late S-phase (de Bruin et al., 2003; Westendorp et al., 2012). E2F7, together with E2F8 has been found to form heterodimers, being critical on cell proliferation and development, and both seem to have similar functions (Li et al., 2008). Preliminary results from Zubiaga’s group have indicated that E2F2 activates E2F7 transcription in U2OS cells, suggesting that E2F2’s repressor function could be mediated by E2F7. For this purpose, we focused on studying E2F7’s role on the target genes previously known to be repressed by E2F2: Chk1 and Mcm5. The specific aims for this work were the following: - Confirm that E2F2 induces E2F7 in HEK-293T cells - Assess whether E2F7 acts as a transcriptional repressor on E2F sites - Evaluate the role of E2F7 on E2F2-mediated transcriptional repression of Chk1 and Mcm5.

Papel de E2F1 y E2F2 en la instauración de esteatosis inducida por dieta rica en grasa

Los factores de transcripción E2F son cruciales en la transición G1/S del ciclo celular. El factor de transcripción E2F1 regula el metabolismo oxidativo y su falta genera resistencia a obesidad. Se desconoce si E2F1 y E2F2 están implicados en la regulación de la homeostasis metabólica hepática. Por ello, el objetivo fue investigar el papel de E2F1 y E2F2 en la modulación del metabolismo lipídico hepático y su repercusión a nivel orgánico. Se utilizaron ratones macho de 3 meses E2F1-/-, E2F2-/- y sus controles que serán alimentados con dieta control o rica en grasa (HFD) durante 10 semanas. Se analizaron parámetros séricos en estado de alimento y tras 13 horas de ayuno. Se investigaron in vivo los flujos metabólicos hepáticos implicados en la disponibilidad de fosfolípidos, diglicéridos y triglicéridos (TG) tras administración de sustratos radiactivos.

A study of the regulation of Trib family members expression in normal and malignant haematopoiesis

The Tribbles family of genes consist of three members; TRIB1, TRIB2 and TRIB3. Trib1 and Trib2 have been identified as oncogenes that can induce AML in mice. However little is known about how the expressions of the Tribbles family genes are controlled in the cell during haematopoiesis or leukaemogenesis. To investigate the Tribbles genes in leukaemia a bioinformatics approach was used. TRIB2 expression was found to be elevated in T-ALL and ALL with t(1;19). TRIB1 was found not to be significantly elevated in any leukaemic subtypes. Analyses of the TRIB1 and TRIB2 gene signatures in both leukaemic and normal haematopoietic cells identified pathways and transcription factors associated with these signatures. Pathways enriched for the TRIB1 signature included TLR signalling pathways and NF-κB pathways. Transcription factors enriched for this signature include C/EBP and SRF. Enriched for the TRIB2 signature includes T cell signalling pathways and Notch signalling pathways. Transcription factors enriched for this signature include E2F and ETS. Further investigation in vitro confirmed the finding that E2F1 was as a potential regulator of TRIB2 expression. E2F1 is able to directly bind to the TRIB2 promoter region and induce TRIB2 expression. C/EBPα p42 was found to inhibit E2F1 and the p30 isoform was found to cooperate with E2F1 induced activation of the TRIB2 promoter. Indicating the potential presence of a regulatory loop involved in the regulation of the TRIB2 gene. In conclusion we have investigated the Tribbles gene signatures in both normal haematopoietic and leukaemic cells. This has led to the identification of a number of pathways and transcription factors associated with these genes. We have also identified a family of transcription factors directly responsible for the regulation of TRIB2 expression. This regulatory pathway has the potential to be targeted in the treatment of leukaemia with a high TRIB2 signature.

Age-specific differences in oncogenic pathway deregulation seen in human breast tumors.

PURPOSE: To define the biology driving the aggressive nature of breast cancer arising in young women. EXPERIMENTAL DESIGN: Among 784 patients with early stage breast cancer, using prospectively-defined, age-specific cohorts (young or=65 years), 411 eligible patients (n = 200or=65 years) with clinically-annotated Affymetrix microarray data were identified. GSEA, signatures of oncogenic pathway deregulation and predictors of chemotherapy sensitivity were evaluated within the two age-defined cohorts. RESULTS: In comparing deregulation of oncogenic pathways between age groups, a higher probability of PI3K (p = 0.006) and Myc (p = 0.03) pathway deregulation was observed in breast tumors arising in younger women. When evaluating unique patterns of pathway deregulation, a low probability of Src and E2F deregulation in tumors of younger women, concurrent with a higher probability of PI3K, Myc, and beta-catenin, conferred a worse prognosis (HR = 4.15). In contrast, a higher probability of Src and E2F pathway activation in tumors of older women, with concurrent low probability of PI3K, Myc and beta-catenin deregulation, was associated with poorer outcome (HR = 2.7). In multivariate analyses, genomic clusters of pathway deregulation illustrate prognostic value. CONCLUSION: Results demonstrate that breast cancer arising in young women represents a distinct biologic entity characterized by unique patterns of deregulated signaling pathways that are prognostic, independent of currently available clinico-pathologic variables. These results should enable refinement of targeted treatment strategies in this clinically challenging situation.

An integrated approach to the prediction of chemotherapeutic response in patients with breast cancer.

BACKGROUND: A major challenge in oncology is the selection of the most effective chemotherapeutic agents for individual patients, while the administration of ineffective chemotherapy increases mortality and decreases quality of life in cancer patients. This emphasizes the need to evaluate every patient's probability of responding to each chemotherapeutic agent and limiting the agents used to those most likely to be effective. METHODS AND RESULTS: Using gene expression data on the NCI-60 and corresponding drug sensitivity, mRNA and microRNA profiles were developed representing sensitivity to individual chemotherapeutic agents. The mRNA signatures were tested in an independent cohort of 133 breast cancer patients treated with the TFAC (paclitaxel, 5-fluorouracil, adriamycin, and cyclophosphamide) chemotherapy regimen. To further dissect the biology of resistance, we applied signatures of oncogenic pathway activation and performed hierarchical clustering. We then used mRNA signatures of chemotherapy sensitivity to identify alternative therapeutics for patients resistant to TFAC. Profiles from mRNA and microRNA expression data represent distinct biologic mechanisms of resistance to common cytotoxic agents. The individual mRNA signatures were validated in an independent dataset of breast tumors (P = 0.002, NPV = 82%). When the accuracy of the signatures was analyzed based on molecular variables, the predictive ability was found to be greater in basal-like than non basal-like patients (P = 0.03 and P = 0.06). Samples from patients with co-activated Myc and E2F represented the cohort with the lowest percentage (8%) of responders. Using mRNA signatures of sensitivity to other cytotoxic agents, we predict that TFAC non-responders are more likely to be sensitive to docetaxel (P = 0.04), representing a viable alternative therapy. CONCLUSIONS: Our results suggest that the optimal strategy for chemotherapy sensitivity prediction integrates molecular variables such as ER and HER2 status with corresponding microRNA and mRNA expression profiles. Importantly, we also present evidence to support the concept that analysis of molecular variables can present a rational strategy to identifying alternative therapeutic opportunities.

Evolution of networks and sequences in eukaryotic cell cycle control.

The molecular networks regulating the G1-S transition in budding yeast and mammals are strikingly similar in network structure. However, many of the individual proteins performing similar network roles appear to have unrelated amino acid sequences, suggesting either extremely rapid sequence evolution, or true polyphyly of proteins carrying out identical network roles. A yeast/mammal comparison suggests that network topology, and its associated dynamic properties, rather than regulatory proteins themselves may be the most important elements conserved through evolution. However, recent deep phylogenetic studies show that fungal and animal lineages are relatively closely related in the opisthokont branch of eukaryotes. The presence in plants of cell cycle regulators such as Rb, E2F and cyclins A and D, that appear lost in yeast, suggests cell cycle control in the last common ancestor of the eukaryotes was implemented with this set of regulatory proteins. Forward genetics in non-opisthokonts, such as plants or their green algal relatives, will provide direct information on cell cycle control in these organisms, and may elucidate the potentially more complex cell cycle control network of the last common eukaryotic ancestor.

Punctuated evolution and transitional hybrid network in an ancestral cell cycle of fungi.

Although cell cycle control is an ancient, conserved, and essential process, some core animal and fungal cell cycle regulators share no more sequence identity than non-homologous proteins. Here, we show that evolution along the fungal lineage was punctuated by the early acquisition and entrainment of the SBF transcription factor through horizontal gene transfer. Cell cycle evolution in the fungal ancestor then proceeded through a hybrid network containing both SBF and its ancestral animal counterpart E2F, which is still maintained in many basal fungi. We hypothesize that a virally-derived SBF may have initially hijacked cell cycle control by activating transcription via the cis-regulatory elements targeted by the ancestral cell cycle regulator E2F, much like extant viral oncogenes. Consistent with this hypothesis, we show that SBF can regulate promoters with E2F binding sites in budding yeast.

The NRF-1/α-PAL transcription factor regulates human E2F6 promoter activity

E2F6 is widely expressed in human tissues and cell lines. Recent studies have demonstrated its involvement in developmental patterning and in the regulation of various genes implicated in chromatin remodelling. Despite a growing number of studies, nothing is really known concerning the E2F6 expression regulation. To understand how cells control E2F6 expression, we analysed the activity of the previously cloned promoter region of the human E2F6 gene. DNase I footprinting, gel electrophoretic-mobility shift, transient transfection and site-directed mutagenesis experiments allowed the identification of two functional NRF-1/α-PAL (nuclear respiratory factor-1/α-palindrome-binding protein)-binding sites within the human E2F6 core promoter region, which are conserved in the mouse and rat E2F6 promoter region. Moreover, ChIP (chromatin immunoprecipitation) analysis demonstrated that overexpressed NRF-1/α-PAL is associated in vivo with the E2F6 promoter. Furthermore, overexpression of full-length NRF-1/α-PAL enhanced E2F6 promoter activity, whereas expression of its dominant-negative form reduced the promoter activity. Our results indicate that NRF-1/α-PAL is implicated in the regulation of basal E2F6 gene expression.