Memet Fuat, Piraye ve Nazım

Taha Toros Arşivi, Dosya Adı: Nazım Hikmetİstanbul Kalkınma Ajansı (TR10/14/YEN/0033) İstanbul Development Agency (TR10/14/YEN/0033

HTLV-1 bZIP factor protein targets the Rb/E2F-1 pathway to promote proliferation and apoptosis of primary CD4+ T cells

Human T-cell leukemia virus type 1 (HTLV-1) is an oncogenic retrovirus that induces a fatal T-cell malignancy, adult T-cell leukemia (ATL). Among several regulatory/accessory genes in HTLV-1, HTLV-1 bZIP factor (HBZ) is the only viral gene constitutively expressed in infected cells. Our previous study showed that HBZ functions in two different molecular forms, HBZ protein and HBZ RNA. In this study, we show that HBZ protein targets retinoblastoma protein (Rb), which is a critical tumor suppressor in many types of cancers. HBZ protein interacts with the Rb/E2F-1 complex and activates the transcription of E2F-target genes associated with cell cycle progression and apoptosis. Mouse primary CD4+ T cells transduced with HBZ show accelerated G1/S transition and apoptosis, and importantly, T cells from HBZ transgenic (HBZ-Tg) mice also demonstrate enhanced cell proliferation and apoptosis. To evaluate the functions of HBZ protein alone in vivo, we generated a new transgenic mouse strain that expresses HBZ mRNA altered by silent mutations but encoding intact protein. In these mice, the numbers of effector/memory and Foxp3+ T cells were increased, and genes associated with proliferation and apoptosis were upregulated. This study shows that HBZ protein promotes cell proliferation and apoptosis in primary CD4+ T cells through activation of the Rb/E2F pathway, and that HBZ protein also confers onto CD4+ T-cell immunophenotype similar to those of ATL cells, suggesting that HBZ protein has important roles in dysregulation of CD4+ T cells infected with HTLV-1

Long Intergenic Noncoding RNA OIN1 Promotes Ovarian Cancer Growth by Modulating Apoptosis-Related Gene Expression

Patients with advanced ovarian cancer usually exhibit high mortality rates, thus more efficient therapeutic strategies are expected to be developed. Recent transcriptomic studies revealed that long intergenic noncoding RNAs (lincRNAs) can be a new class of molecular targets for cancer management, because lincRNAs likely exert tissue-specific activities compared with protein-coding genes or other noncoding RNAs. We here show that an unannotated lincRNA originated from chromosome 10q21 and designated as ovarian cancer long intergenic noncoding RNA 1 (OIN1), is often overexpressed in ovarian cancer tissues compared with normal ovaries as analyzed by RNA sequencing. OIN1 silencing by specific siRNAs significantly exerted proliferation inhibition and enhanced apoptosis in ovarian cancer cells. Notably, RNA sequencing showed that OIN1 expression was negatively correlated with the expression of apoptosis-related genes ras association domain family member 5 (RASSF5) and adenosine A1 receptor (ADORA1), which were upregulated by OIN1 knockdown in ovarian cancer cells. OIN1-specifc siRNA injection was effective to suppress in vivo tumor growth of ovarian cancer cells inoculated in immunodeficient mice. Taken together, OIN1 could function as a tumor-promoting lincRNA in ovarian cancer through modulating apoptosis and will be a potential molecular target for ovarian cancer management

Estrogen-Inducible LncRNA BNAT1 Functions as a Modulator for Estrogen Receptor Signaling in Endocrine-Resistant Breast Cancer Cells

Recent advances in RNA studies have revealed that functional long noncoding RNAs (lncRNAs) contribute to the biology of cancers. In breast cancer, estrogen receptor α (ERα) is an essential transcription factor that primarily promotes the growth of luminal-type cancer, although only a small number of lncRNAs are identified as direct ERα targets and modulators for ERα signaling. In this study, we performed RNA-sequencing for ER-positive breast cancer cells and identified a novel estrogen-inducible antisense RNA in the COL18A1 promoter region, named breast cancer natural antisense transcript 1 (BNAT1). In clinicopathological study, BNAT1 may have clinical relevance as a potential diagnostic factor for prognoses of ER-positive breast cancer patients based on an in situ hybridization study for breast cancer specimens. siRNA-mediated BNAT1 silencing significantly inhibited the in vitro and in vivo growth of tamoxifen-resistant ER-positive breast cancer cells. Notably, BNAT1 silencing repressed cell cycle progression whereas it promoted apoptosis. Microarray analysis revealed that BNAT1 silencing in estrogen-sensitive breast cancer cells repressed estrogen signaling. We showed that BNAT1 knockdown decreased ERα expression and repressed ERα transactivation. RNA immunoprecipitation showed that BNAT1 physically binds to ERα protein. In summary, BNAT1 would play a critical role in the biology of ER-positive breast cancer by modulating ERα-dependent transcription regulation. We consider that BNAT1 could be a potential molecular target for diagnostic and therapeutic options targeting luminal-type and endocrine-resistant breast cancer

Distinct gene expression signatures induced by viral transactivators of different HTLV-1 subgroups that confer a different risk of HAM/TSP

Abstract Background Among human T cell leukemia virus type 1 (HTLV-1)-infected individuals, there is an association between HTLV-1 tax subgroups (subgroup-A or subgroup-B) and the risk of HAM/TSP in the Japanese population. To investigate the role of HTLV-1 subgroups in viral pathogenesis, we studied the functional difference in the subgroup-specific viral transcriptional regulators Tax and HBZ using microarray analysis, reporter gene assays, and evaluation of viral-host protein–protein interaction. Results (1) Transcriptional changes in Jurkat Tet-On human T-cells that express each subgroup of Tax or HBZ protein under the control of an inducible promoter revealed different target gene profiles; (2) the number of differentially regulated genes induced by HBZ was 2–3 times higher than that induced by Tax; (3) Tax and HBZ induced the expression of different classes of non-coding RNAs (ncRNAs); (4) the chemokine CXCL10, which has been proposed as a prognostic biomarker for HAM/TSP, was more efficiently induced by subgroup-A Tax (Tax-A) than subgroup-B Tax (Tax-B), in vitro as well as in unmanipulated (ex vivo) PBMCs obtained from HAM/TSP patients; (5) reporter gene assays indicated that although transient Tax expression in an HTLV-1-negative human T-cell line activated the CXCL10 gene promoter through the NF-κB pathway, there was no difference in the ability of each subgroup of Tax to activate the CXCL10 promoter; however, (6) chromatin immunoprecipitation assays showed that the ternary complex containing Tax-A is more efficiently recruited onto the promoter region of CXCL10, which contains two NF-κB binding sites, than that containing Tax-B. Conclusions Our results indicate that different HTLV-1 subgroups are characterized by different patterns of host gene expression. Differential expression of pathogenesis-related genes by subgroup-specific Tax or HBZ may be associated with the onset of HAM/TSP

HBZ upregulates TIGIT expression.

<p>(A) <i>TIGIT</i> mRNA was increased in CD4⁺ T cells of HBZ-Tg mice (n = 7) compared with those of non-Tg mice (n = 7). (B) TIGIT expression was enhanced on resting and activated CD4⁺ T cells from HBZ-Tg mice by flow cytometry (FCM). The representative histogram of two independent experiments is shown. (C) A retrovirus expressing HBZ or control retrovirus was transduced into mouse primary CD4⁺ T cells. <i>TIGIT</i> transcripts were measured by realtime PCR. Results are the mean ± SD in triplicate. Three independent experiments were performed and the representative results are shown. (D) Expression of TIGIT on HBZ transduced mouse primary CD4⁺ T cells was measured by FCM. The representative result was shown for two independent experiments. (E) Expression of TIGIT on CD3^lowCD4⁺ T cells from ATL patients (n = 5) and CD4⁺CADM1⁺ T cells from HAM/TSP patients (n = 6) and healthy donors (HD, n = 6) were measured by FCM. (F) Expression of TIGIT on HBZ-transduced murine primary CD4⁺ cells and control cells in the presence or absence of TGF-β was measured by FCM. Two independent experiments were performed and the representative results are shown. *<i>P</i> < 0.05, **<i>P</i> < 0.01.</p

TIGIT transcription is regulated by HBZ.

<p>(A) Volcano plots of p-value (y-axis) against log₂ fold change (x-axis: HBZ expressing murine primary T cells vs. control cells) for RNA-seq data. (B) RNA-seq and ChIP-seq results of three Treg related genes. RNA-seq was performed twice and ChIP-seq was performed once for each. (C) The enrichment of the <i>TIGIT</i> promoter region by the indicated histone marks or isotype control was measured by ChIP-qPCR. The % input of HBZ expressing murine primary CD4⁺ T cells or control cells is shown. (D) The enrichment of the TIGIT promoter regions by anti-FLAG was analyzed by ChIP-seq in HBZ-FLAG expressing cells. The detected peak in the TIGIT promoter region was shown as the dotted box. The enrichment of the TIGIT promoter regions by anti-FLAG or isotype control was measured by ChIP-qPCR using the primers indicated. Results shown are the mean ± SD in triplicate (C, D).</p

HBZ suppresses Fgl2 and CD226 expression.

<p>(A) <i>Fgl2</i> mRNA was quantified in HBZ-expressing murine primary CD4⁺ T cells and control cells by realtime PCR. This experiment was carried out in triplicate, and the mean values ± SD are shown. Three independent experiments were performed and the representative results are shown. (B) The levels of <i>Fgl2</i> mRNA were measured in CD4⁺ T cells from non-Tg (n = 4) and TIGIT⁺CD4⁺ and TIGIT^-CD4⁺ T cells from HBZ-Tg (n = 3) mice. (C) Jurkat cells were cotransfected with 500 ng of FGL2-promoter-Luc, pME18Sneo-sHBZ, 500 ng of pcDNA-C/EBPα and pGL4-TK. Luciferase activity was measured after 24 hours. Luciferase assay was carried out in triplicate, and the mean values ± SD are shown. Three independent experiments were performed and the representative results were shown. (D) The <i>CD226</i> gene transcript was quantified in HBZ-expressing murine primary CD4⁺ T cells and control cells. This assay was carried out in triplicate and the mean values ± SD are shown. Three independent experiments were performed and the representative results were shown. (E) The <i>CD226</i> gene expression was measured in CD4⁺ T cells from HBZ-Tg (n = 7) and non-Tg (n = 7) mice by realtime PCR. *<i>P</i> < 0.05, **<i>P</i> < 0.01.</p