NF-Y Recruits Both Transcription Activator and Repressor to Modulate Tissue- and Developmental Stage-Specific Expression of Human γ-Globin Gene

The human embryonic, fetal and adult β-like globin genes provide a paradigm for tissue- and developmental stage-specific gene regulation. The fetal γ-globin gene is expressed in fetal erythroid cells but is repressed in adult erythroid cells. The molecular mechanism underlying this transcriptional switch during erythroid development is not completely understood. Here, we used a combination of in vitro and in vivo assays to dissect the molecular assemblies of the active and the repressed proximal γ-globin promoter complexes in K562 human erythroleukemia cell line and primary human fetal and adult erythroid cells. We found that the proximal γ-globin promoter complex is assembled by a developmentally regulated, general transcription activator NF-Y bound strongly at the tandem CCAAT motifs near the TATA box. NF-Y recruits to neighboring DNA motifs the developmentally regulated, erythroid transcription activator GATA-2 and general repressor BCL11A, which in turn recruit erythroid repressor GATA-1 and general repressor COUP-TFII to form respectively the NF-Y/GATA-2 transcription activator hub and the BCL11A/COUP-TFII/GATA-1 transcription repressor hub. Both the activator and the repressor hubs are present in both the active and the repressed γ-globin promoter complexes in fetal and adult erythroid cells. Through changes in their levels and respective interactions with the co-activators and co-repressors during erythroid development, the activator and the repressor hubs modulate erythroid- and developmental stage-specific transcription of γ-globin gene.</p

IDO Immune Status after Chemoradiation May Predict Survival in Lung Cancer Patients

Host immunity influences the impact of radiotherapy (RT) in cancer, but mechanistic connections remain obscure. In this study, we investigated the relationship of indoleamine 2,3-dioxygenase (IDO) systemic activity on clinical outcomes in RT-treated non-small cell lung cancer (NSCLC). IDO-mediated production of kynurenine and the kynurenine:tryptophan ratio in patient blood serum were determined for stage III NSCLC patients at times before, during, and after RT administration and then correlated to overall survival (OS), progression-free survival, and disease progression rate in patients. We found the impact of RT on these serum IDO markers to be heterogeneous in patients. On average, kynurenine:tryptophan ratios were reduced during RT but restored after RT. Notably, both baseline levels of kynurenine:tryptophan and changes in the levels of kynurenine after RT were significantly associated with OS. When combined, favorable change and favorable baseline corresponded with very long-term OS (median OS was not reached after 57 months of median follow-up). Favorable change combined with unfavorable baseline still corresponded with a lack of distant metastases. Our results suggest that RT alters IDO-mediated immune status in NSCLC patients and that changes in this serum biomarker may be useful to predict outcomes and perhaps personalize RT dosage to improve survival.Significance: Radiotherapy appears to influence systemic IDO activity and to exert a significant impact on metastatic risk and overall survival, with possible implications for defining a biomarker to optimize radiation dose in patients to improve outcomes. Cancer Res; 78(3); 809-16. ©2017 AACR

Hydroxyurea differentially modulates activator and repressors of γ-globin gene in erythroblasts of responsive and non-responsive patients with sickle cell disease in correlation with Index of Hydroxyurea Responsiveness

Hydroxyurea (HU), the first of two drugs approved by the US Food and Drug Administration for treating patients with sickle cell disease (SCD), produces anti-sickling effect by re-activating fetal γ-globin gene to enhance production of fetal hemoglobin. However, approximately 30% of the patients do not respond to HU therapy. The molecular basis of non-responsiveness to HU is not clearly understood. To address this question, we examined HU-induced changes in the RNA and protein levels of transcription factors NF-Y, GATA-1, -2, BCL11A, TR4, MYB and NF-E4 that assemble the γ-globin promoter complex and regulate transcription of γ-globin gene. In erythroblasts cultured from peripheral blood CD34+ cells of patients with SCD, we found that HU-induced changes in the protein but not the RNA levels of activator GATA-2 and repressors GATA-1, BCL11A and TR4 correlated with HU-induced changes in fetal hemoglobin (HbF) levels in the peripheral blood of HU high and low responders. However, HU did not significantly induce changes in the protein or RNA levels of activators NF-Y and NF-E4. Based on HU-induced changes in the protein levels of GATA-2, -1 and BCL11A, we calculated an Index of Hydroxyurea Responsiveness (IndexHU-3). Compared to the HU-induced fold changes in the individual transcription factor protein levels, the numerical values of IndexHU-3 statistically correlated best with the HU-induced peripheral blood HbF levels of the patients. Thus, IndexHU-3 can serve as an appropriate indicator for inherent HU responsiveness of patients with SCD

Principal component analysis identifies patterns of cytokine expression in non-small cell lung cancer patients undergoing definitive radiation therapy

Radiation treatment (RT) stimulates the release of many immunohumoral factors, complicating the identification of clinically significant cytokine expression patterns. This study used principal component analysis (PCA) to analyze cytokines in non-small cell lung cancer (NSCLC) patients undergoing RT and explore differences in changes after hypofractionated stereotactic body radiation therapy (SBRT) and conventionally fractionated RT (CFRT) without or with chemotherapy

Oral Pathobiont Activates Anti-Apoptotic Pathway, Promoting both Immune Suppression and Oncogenic Cell Proliferation.

Chronic periodontitis (CP) is a microbial dysbiotic disease linked to increased risk of oral squamous cell carcinomas (OSCCs). To address the underlying mechanisms, mouse and human cell infection models and human biopsy samples were employed. We show that the \u27keystone\u27 pathogen Porphyromonas gingivalis, disrupts immune surveillance by generating myeloid-derived dendritic suppressor cells (MDDSCs) from monocytes. MDDSCs inhibit CTLs and induce FOXP3 + 

Genetic Variations in the Transforming Growth Factor-β1 Pathway May Improve Predictive Power for Overall Survival in Non-small Cell Lung Cancer

Purpose: Transforming growth factor-β1 (TGF-β1), a known immune suppressor, plays an important role in tumor progression and overall survival (OS) in many types of cancers. We hypothesized that genetic variations of single nucleotide polymorphisms (SNPs) in the TGF-β1 pathway can predict survival in patients with non-small cell lung cancer (NSCLC) after radiation therapy. Materials and Methods: Fourteen functional SNPs in the TGF-β1 pathway were measured in 166 patients with NSCLC enrolled in a multi-center clinical trial. Clinical factors, including age, gender, ethnicity, smoking status, stage group, histology, Karnofsky Performance Status, equivalent dose at 2 Gy fractions (EQD2), and the use of chemotherapy, were first tested under the univariate Cox's proportional hazards model. All significant clinical predictors were combined as a group of predictors named "Clinical." The significant SNPs under the Cox proportional hazards model were combined as a group of predictors named "SNP." The predictive powers of models using Clinical and Clinical + SNP were compared with the cross-validation concordance index (C-index) of random forest models. Results: Age, gender, stage group, smoking, histology, and EQD2 were identified as significant clinical predictors: Clinical. Among 14 SNPs, BMP2:rs235756 (HR = 0.63; 95% CI:0.42-0.93; p = 0.022), SMAD9:rs7333607 (HR = 2.79; 95% CI 1.22-6.41; p = 0.015), SMAD3:rs12102171 (HR = 0.68; 95% CI: 0.46-1.00; p = 0.050), and SMAD4: rs12456284 (HR = 0.63; 95% CI: 0.43-0.92; p = 0.016) were identified as powerful predictors of SNP. After adding SNP, the C-index of the model increased from 84.1 to 87.6% at 24 months and from 79.4 to 84.4% at 36 months. Conclusion: Genetic variations in the TGF-β1 pathway have the potential to improve the prediction accuracy for OS in patients with NSCLC

A facilitated tracking and transcription mechanism of long-range enhancer function

In the human ε−globin gene locus, the HS2 enhancer in the Locus Control Region regulates transcription of the embryonic ε-globin gene located over 10 kb away. The mechanism of long-range HS2 enhancer function was not fully established. Here we show that the HS2 enhancer complex containing the enhancer DNA together with RNA polymerase II (pol II) and TBP tracks along the intervening DNA, synthesizing short, polyadenylated, intergenic RNAs to ultimately loop with the ε-globin promoter. Guided by this facilitated tracking and transcription mechanism, the HS2 enhancer delivers pol II and TBP to the cis-linked globin promoter to activate mRNA synthesis from the target gene. An insulator inserted in the intervening DNA between the enhancer and the promoter traps the enhancer DNA and the associated pol II and TBP at the insulator site, blocking mid-stream the facilitated tracking and transcription mechanism of the enhancer complex, thereby blocking long-range enhancer function

Augmentation of the anticancer activity of CYT997 in human prostate cancer by inhibiting Src activity

Abstract Background Abnormalities of tubulin polymerization and microtubule assembly are often seen in cancer, which make them very suitable targets for the development of therapeutic approach against rapidly dividing and aggressive cancer cells. CYT997 is a novel microtubule-disrupting agent with anticancer activity in multiple cancer types including prostate cancer. However, the molecular mechanisms of action of CYT997 in prostate cancer have not been well characterized. Methods Src knockdown cells were achieved by lentiviral-mediated interference. The drug effects on cell proliferation were measured by MTS. The drug effects on cell viability and death were determined by Cell Titer-Glo® Luminescent cell viability kit and flow cytometry with Zombie Aqua™ staining. The drug effects on apoptosis were assessed by Cell Death Detection Elisa kit and Western blot with a cleaved PARP antibody. The drug effects on cell invasion were examined by Matrigel-coated Boyden chambers. Oxidative stress was detected by DCFH-DA staining and electrochemical biosensor. Mouse models generated by subcutaneous or intracardiac injection were used to investigate the in vivo drug efficacy in tumor growth and metastasis. Results CYT997 effectively inhibited proliferation, survival, and invasion of prostate cancer cells via blocking multiple oncogenic signaling cascades but not the Src pathway. Inhibition of Src expression by small hairpin RNA or inactivation of Src by dasatinib increased the CYT997-induced cytotoxicity of in vitro. Moreover, the combination of dasatinib and CYT997 exhibited a superior inhibitory effect on tumor growth and metastasis compared with either of the drugs alone. Conclusion Our findings demonstrate that blockage of Src augments the anticancer effect of CYT997 on prostate cancer and suggest that co-treatment of dasatinib and CYT997 may represent an effective therapeutic regimen for limiting prostate cancer