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NRF2 is the master regulator for the cellular oxidative stress response and regulates γ-globin gene expression in human erythroid progenitors and sickle cell disease mice. To explore NRF2 function, we established a human β-globin locus yeast artificial chromosome transgenic/NRF2 knockout (β-YAC/NRF2−/−) mouse model. NRF2 loss reduced γ-globin gene expression during erythropoiesis and abolished the ability of dimethyl fumarate, an NRF2 activator, to enhance γ-globin transcription. We observed decreased H3K4Me1 and H3K4Me3 chromatin marks and association of TATA-binding protein and RNA polymerase II at the β-locus control region (LCR) and γ-globin gene promoters in β-YAC/NRF2−/− mice. As a result, long-range chromatin interaction between the LCR DNase I hypersensitive sites and γ-globin gene was decreased, while interaction with the β-globin was not affected. Further, NRF2 loss silenced the expression of DNA methylcytosine dioxygenases TET1, TET2, and TET3 and inhibited γ-globin gene DNA hydroxymethylation. Subsequently, protein-protein interaction between NRF2 and TET3 was demonstrated. These data support the ability of NRF2 to mediate γ-globin gene regulation through epigenetic DNA and histone modifications. Impact statement Sickle cell disease is an inherited hemoglobin disorder that affects over 100,000 people in the United States causing high morbidity and early mortality. Although new treatments were recently approved by the FDA, only one drug Hydroxyurea induces fetal hemoglobin expression to inhibit sickle hemoglobin polymerization in red blood cells. Our laboratory previously demonstrated the ability of the NRF2 activator, dimethyl fumarate to induce fetal hemoglobin in the sickle cell mouse model. In this study, we investigated molecular mechanisms of γ-globin gene activation by NRF2. We observed the ability of NRF2 to modulate chromatin structure in the human β-like globin gene locus of β-YAC transgenic mice during development. Furthermore, an NRF2/TET3 interaction regulates γ-globin gene DNA methylation. These findings provide potential new molecular targets for small molecule drug developed for treating sickle cell disease.

Background Circular RNAs (circRNAs) have been closely implicated in competing endogenous RNA (ceRNA) network among human cancers including non-small cell lung cancer (NSCLC). However, the role of most circRNAs in NSCLC remains to be determined. Here, we aimed to investigate the role of hsa_circ_0007385 (circ_0007385) in NSCLC cells. Methods Expression of hsa_circ_0007385 (circ_0007385), miRNA (miR)-519d-5p and high-mobility group box 1 (HMGB1) was measured by real-time quantitative PCR and western blotting. Functional experiments were evaluated by cell counting kit (CCK)-8, flow cytometry, fluorescein active caspase-3 staining kit, transwell assays, western blotting, and xenograft experiment. The relationship among circ_0007385,miR-519d-5p and HMGB1 was testified by dual-luciferase reporter assay. Kaplan-Meiersurvival curve identified overall survival in NSCLC patients. Results circ_0007385 expression was higher in NSCLC tissues and cell lines, and was associated with poor overall survival. Silencing circ_0007385 could suppress cell proliferation, migration and invasion in A549 and H1975 cells, as well as cisplatin (DDP) resistance. Moreover, circ_0007385 silence retarded tumor growth of A549 cells in vivo. Molecularly, there was a direct interaction between miR-519d-3p and either circ_0007385 or HMGB1; expression of miR-519d-3p was downregulated in NSCLC tumors in a circ_0007385-correlated manner, and circ_0007385 could indirectly regulate HMGB1 via miR-519d-3p. Functionally, both inhibiting miR-519d-3p and restoring HMGB1 could overturn the suppressive effect of circ_0007385 knockdown on cell proliferation, migration, invasion, and DDP resistance. Conclusions Collectively, circ_0007385 deletion could function anti-tumor role in NSCLC by suppressing malignant behaviors and DDP resistance in vitro and in vivo via circ_0007385/miR-519d-3p/HMGB1 axis. These outcomes might enhance our understanding of the molecular mechanisms underlying the malignant progression of NSCLC. Key points SIGNIFICANT FINDINGS OF THE STUDY: circ_0007385 was upregulated in NSCLC tissues and cells, and was associated with poor overall survival. Silenced circ_0007385 suppressed NSCLC cell proliferation, migration, invasion, and DDP resistance in vitro, and tumor growth in vivo. circ_0007385 was upregulated in NSCLC tissues and cells, and was associated with poor overall survival. What this study adds miR-519d-3p could directly interact with circ_0007385 and HMGB1 in NSCLC cells. A promising circ_0007385/miR-519d-3p/HMGB1 regulatory pathway was determined in NSCLC cells.

The basic leucine zipper transcription factor nuclear factor (erythroid-derived 2)-like 2 (NRF2) plays a critical role in the cellular antioxidant response under oxidative stress conditions. In this study, we investigated the role of NRF2 in fetal hemoglobin expression and the pathophysiology of sickle cell disease (SCD) in a NRF2 knockout (SCD/NRF2-/-) transgenic mouse model. NRF2 loss impaired survival of SCD pups during gestation and in the first 2 months of life. Furthermore, fetal hemoglobin expression was inhibited during erythropoiesis in embryonic day 13.5 and embryonic day 18.5 fetal liver and adult spleen and bone marrow cells, respectively. Examination of peripheral red blood cells revealed an increase of reactive oxygen species (ROS) and sickling under hypoxic conditions. Loss of NRF2 function in SCD/NRF2-/- mice produced greater splenomegaly with red pulp expansion and obscured architecture. In addition, NRF2 knockout reduced the expression of its target antioxidant proteins, leading to increased levels of ROS, proinflammatory cytokines, and adhesion molecules in SCD mice. Genetic knockout of NRF2 demonstrates its role in developmentally regulated γ-globin gene expression and the ability to control oxidative stress and the phenotypic severity of SCD.

Effective treatment of rheumatoid arthritis can be mediated by native chicken type II collagen (nCCII), recombinant peptide containing nCCII tolerogenic epitopes (CTEs), or a therapeutic DNA vaccine encoding the full-length CCOL2A1 cDNA. As recombinant CCII (rCCII) might avoid potential pathogenic virus contamination during nCCII preparation or chromosomal integration and oncogene activation associated with DNA vaccines, here we evaluated the importance of propeptide and telopeptide domains on rCCII triple helix molecular assembly. We constructed pC- and pN-procollagen (without N- or C-propeptides, respectively) as well as CTEs located in the triple helical domain lacking both propeptides and telopeptides, and expressed these in yeast Pichia pastoris host strain GS115 (his4, Mut+) simultaneously with recombinant chicken prolyl-4-hydroxylase α and β subunits. Both pC- and pN-procollagen monomers accumulated inside P. pastoris cells, whereas CTE was assembled into homotrimers with stable conformation and secreted into the supernatants, suggesting that the large molecular weight pC-or pN-procollagens were retained within the endoplasmic reticulum whereas the smaller CTEs proceeded through the secretory pathway. Furthermore, resulting recombinant chicken type II collagen pCα1(II) can induced collagen-induced arthritis (CIA) rat model, which seems to be as effective as the current standard nCCII. Notably, protease digestion assays showed that rCCII could assemble in the absence of C- and N-propeptides or telopeptides. These findings provide new insights into the minimal structural requirements for rCCII expression and folding.

Deregulated oncogenic signaling linked to PI3K/AKT and mTORC1 pathway activation is a hallmark of human T-cell acute leukemia (T-ALL) pathogenesis and contributes to leukemic cell resistance and adverse prognosis. Notably, although the multiagent chemotherapy of leukemia leads to a high rate of complete remission, options for salvage therapy for relapsed/refractory disease are limited due to the serious side effects of augmenting cytotoxic chemotherapy. We report that ablation of HSF1, a key transcriptional regulator of the chaperone response and cellular bioenergetics, from mouse T-ALL tumors driven by PTEN loss or human T-ALL cell lines, has significant therapeutic effects in reducing tumor burden and sensitizing malignant cell death. From a mechanistic perspective, the enhanced sensitivity of T-ALLs to HSF1 depletion resides in the reduced MAPK–ERK signaling and metabolic and ATP-producing capacity of malignant cells lacking HSF1 activity. Impaired mitochondrial ATP production and decreased intracellular amino acid content in HSF1-deficient T-ALL cells trigger an energy-saving adaptive response featured by attenuation of the mTORC1 activity, which is coregulated by ATP, and its downstream target proteins (p70S6K and 4E-BP). This leads to protein translation attenuation that diminishes oncogenic signals and malignant cell growth. Collectively, these metabolic alterations in the absence of HSF1 activity reveal cancer cell liabilities and have a profound negative impact on T-ALL progression. Implications: Targeting HSF1 and HSF1-dependent cancer-specific anabolic and protein homeostasis programs has a significant therapeutic potential for T-ALL and may prevent progression of relapsed/refractory disease.

ErbB2/Neu oncogene is overexpressed in 25% of invasive/metastatic breast cancers. We have found that deletion of heat shock factor Hsf1 in mice overexpressing ErbB2/Neu significantly reduces mammary tumorigenesis and metastasis. Hsf1(+/-)ErbB2/Neu(+) tumors exhibit reduced cellular proliferative and invasive properties associated with reduced activated ERK1/2 and reduced epithelial-mesenchymal transition (EMT). Hsf1(+/+)Neu(+) mammary epithelial cells exposed to TGFβ show high levels of ERK1/2 activity and EMT; this is associated with reduced expression of E-cadherin and increased expression of Slug and vimentin, a mesenchymal marker. In contrast, Hsf1(-/-)Neu(+) or Hsf1(+/+)Neu(+) cells do not exhibit activated ERK1/2 and show reduced EMT in the presence of TGFβ. The ineffective activation of the RAS/RAF/MEK/ERK1/2 signaling pathway in cells with reduced levels of HSF1 is due to the low levels of HSP90 in complex with RAF1 that are required for RAF1 stability and maturation. These results indicate a powerful inhibitory effect conferred by HSF1 downstream target genes in the inhibition of ErbB2-induced breast cancers in the absence of the Hsf1 gene.

Summary The inhibitory effects of cancer on T cell metabolism have been well established, but the metabolic impact of immunotherapy on tumor cells is poorly understood. Here, we developed a CD4+ T cell-based adoptive immunotherapy protocol that was curative for mice with implanted colorectal tumors. By conducting metabolic profiling on tumors, we show that adoptive immunotherapy profoundly altered tumor metabolism, resulting in glutathione depletion and accumulation of reactive oxygen species (ROS) in tumor cells. We further demonstrate that T cell-derived tumor necrosis factor alpha (TNF-α) can synergize with chemotherapy to intensify oxidative stress and tumor cell death in an NADPH (nicotinamide adenine dinucleotide phosphate hydrogen) oxidase-dependent manner. Reduction of oxidative stress, by preventing TNF-α-signaling in tumor cells or scavenging ROS, antagonized the therapeutic effects of adoptive immunotherapy. Conversely, provision of pro-oxidants after chemotherapy can partially recapitulate the antitumor effects of T cell transfer. These findings imply that reinforcing tumor oxidative stress represents an important mechanism underlying the efficacy of adoptive immunotherapy.

Immunotherapy of rheumatoid arthritis (RA) using oral-dosed native chicken or bovine type II collagen (nCII) to induce specific immune tolerance is an attractive strategy. However, the majority of clinical trials of oral tolerance in human diseases including RA in recent years have been disappointing. Here, we describe a novel recombinant peptide rcCTE1-2 which contains only two tolerogenic epitopes (CTE1 and CTE2) of chicken type II collagen (cCII). These are the critical T-cell determinants for suppression of RA that were first developed and used to compare its suppressive effects with ncCII on the collagen-induced arthritis (CIA) model. The rcCTE1-2 was produced using the prokaryotic pET expression system and purified by Ni-NTA His affinity chromatography. Strikingly, our results showed clearly that rcCTE1-2 was as efficacious as ncCII at the dose of 50 microg/kg/d. This dose significantly reduced footpad swelling, arthritic incidence and scores, and deferred the onset of disease. Furthermore, rcCTE1-2 of 50 microg/kg/d could lower the level of anti-nCII antibody in the serum of CIA animals, decrease Th1-cytokine INF-gamma level, and increase Th3-cytokine TGF-beta(1) produced level by spleen cells from CIA mice after in vivo stimulation with ncCII. Importantly, rcCTE1-2 was even more potent than native cCII, which was used in the clinic for RA. Equally importantly, the findings that the major T-cell determinants of cCII that are also recognized by H-2(b) MHC-restricted T cells have not previously been reported. Taken together, these results suggest that we have successfully developed a novel recombinant peptide rcCTE1-2 that can induce a potent tolerogenic response in CIA.

Chicken type II procollagen (ccol2a1) has become as an important oral tolerance protein for effective treatment of rheumatoid arthritis. However, its molecular identity remains unclear. Here, we reported the full-length cDNA and nearly complete genomic DNA encoding ccol2a1. We have determined the structural organization, evolutional characters, developmental expression and chromosomal mapping of the gene. The full-length cDNA sequence spans 4837 bp containing all the coding region of the ccol2a1 including 3' and 5' untranslation region. The deduced peptide of ccol2a1, composed of 1420 amino acids, can be divided into signal peptide, N-propeptide, N-telopeptide, triple helix, C-telopeptide and C-propeptide. The ccol2a1 genomic DNA sequence was determined to be 12,523 bp long containing 54 exons interrupted by 53 introns. Comparison of the ccol2a1 with its counterparts in human, mouse, canine, horse, rat, frog and newt revealed highly conserved sequence in the triple helix domain. Chromosomal mapping of ccol2a1 locates it on 4P2. While the ccol2a1 mRNA was expressed in multiple tissues, the protein was only detected in chondrogenic cartilage, vitreous body and cornea. The ccol2a1 was found to contain two isoforms detected by RT-PCR. The distribution of the ccol2a1 lacking exon 2wasfrequently detected in chondrogenic tissues, whereas the exon 2-containing isoform was more abundant in non-chondrogenic tissues. These results provide useful information for preparing recombinant chicken type II collagen and for a better understanding of normal cartilage development.