NRF3 suppresses squamous carcinogenesis, involving the unfolded protein response regulator HSPA5.

Epithelial skin cancers are extremely common, but the mechanisms underlying their malignant progression are still poorly defined. Here, we identify the NRF3 transcription factor as a tumor suppressor in the skin. NRF3 protein expression is strongly downregulated or even absent in invasively growing cancer cells of patients with basal and squamous cell carcinomas (BCC and SCC). NRF3 deficiency promoted malignant conversion of chemically induced skin tumors in immunocompetent mice, clonogenic growth and migration of human SCC cells, their invasiveness in 3D cultures, and xenograft tumor formation. Mechanistically, the tumor-suppressive effect of NRF3 involves HSPA5, a key regulator of the unfolded protein response, which we identified as a potential NRF3 interactor. HSPA5 levels increased in the absence of NRF3, thereby promoting cancer cell survival and migration. Pharmacological inhibition or knock-down of HSPA5 rescued the malignant features of NRF3-deficient SCC cells in vitro and in preclinical mouse models. Together with the strong expression of HSPA5 in NRF3-deficient cancer cells of SCC patients, these results suggest HSPA5 inhibition as a treatment strategy for these malignancies in stratified cancer patients.

Loss of NFE2L3 protects against inflammation-induced colorectal cancer through modulation of the tumor microenvironment.

We investigated the role of the NFE2L3 transcription factor in inflammation-induced colorectal cancer. Our studies revealed that Nfe2l3-/- mice exhibit significantly less inflammation in the colon, reduced tumor size and numbers, and skewed localization of tumors with a more pronounced decrease of tumors in the distal colon. CIBERSORT analysis of RNA-seq data from normal and tumor tissue predicted a reduction in mast cells in Nfe2l3-/- animals, which was confirmed by toluidine blue staining. Concomitantly, the transcript levels of Il33 and Rab27a, both important regulators of mast cells, were reduced and increased, respectively, in the colorectal tumors of Nfe2l3-/- mice. Furthermore, we validated NFE2L3 binding to the regulatory sequences of the IL33 and RAB27A loci in human colorectal carcinoma cells. Using digital spatial profiling, we found that Nfe2l3-/- mice presented elevated FOXP3 and immune checkpoint markers CTLA4, TIM3, and LAG3, suggesting an increase in Treg counts. Staining for CD3 and FOXP3 confirmed a significant increase in immunosuppressive Tregs in the colon of Nfe2l3-/- animals. Also, Human Microbiome Project (HMP2) data showed that NFE2L3 transcript levels are higher in the rectum of ulcerative colitis patients. The observed changes in the tumor microenvironment provide new insights into the molecular differences regarding colon cancer sidedness. This may be exploited for the treatment of early-onset colorectal cancer as this emerging subtype primarily displays distal/left-sided tumors.

Profound downregulation of neural transcription factor Npas4 and Nr4a family in fetal mice neurons infected with Zika virus.

Zika virus (ZIKV) infection of neurons leads to neurological complications and congenital malformations of the brain of neonates. To date, ZIKV mechanism of infection and pathogenesis is not entirely understood and different studies on gene regulation of ZIKV-infected cells have identified a dysregulation of inflammatory and stem cell maintenance pathways. MicroRNAs (miRNAs) are post-transcriptional regulators of cellular genes and they contribute to cell development in normal function and disease. Previous reports with integrative analyses of messenger RNAs (mRNAs) and miRNAs during ZIKV infection have not identified neurological pathway defects. We hypothesized that dysregulation of pathways involved in neurological functions will be identified by RNA profiling of ZIKV-infected fetal neurons. We therefore used microarrays to analyze gene expression levels following ZIKV infection of fetal murine neurons. We observed that the expression levels of transcription factors such as neural PAS domain protein 4 (Npas4) and of three members of the orphan nuclear receptor 4 (Nr4a) were severely decreased after viral infection. We confirmed that their downregulation was at both the mRNA level and at the protein level. The dysregulation of these transcription factors has been previously linked to aberrant neural functions and development. We next examined the miRNA expression profile in infected primary murine neurons by microarray and found that various miRNAs were dysregulated upon ZIKV infection. An integrative analysis of the differentially expressed miRNAs and mRNAs indicated that miR-7013-5p targets Nr4a3 gene. Using miRmimics, we corroborated that miR-7013-5p downregulates Nr4a3 mRNA and protein levels. Our data identify a profound dysregulation of neural transcription factors with an overexpression of miR-7013-5p that results in decreased Nr4a3 expression, likely a main contributor to ZIKV-induced neuronal dysfunction.

New Insights into CDK Regulators: Novel Opportunities for Cancer Therapy.

Cyclins and their catalytic partners, the cyclin-dependent kinases (CDKs), control the transition between different phases of the cell cycle. CDK/cyclin activity is regulated by CDK inhibitors (CKIs), currently comprising the CDK-interacting protein/kinase inhibitory protein (CIP/KIP) family and the inhibitor of kinase (INK) family. Recent studies have identified a third group of CKIs, called ribosomal protein-inhibiting CDKs (RPICs). RPICs were discovered in the context of cellular senescence, a stable cell cycle arrest with tumor-suppressing abilities. RPICs accumulate in the nonribosomal fraction of senescent cells due to a decrease in rRNA biogenesis. Accordingly, RPICs are often downregulated in human cancers together with other ribosomal proteins, the tumor-suppressor functions of which are still under study. In this review, we discuss unique therapies that have been developed to target CDK activity in the context of cancer treatment or senescence-associated pathologies, providing novel tools for precision medicine.

Correction to: Nrf3 promotes UV-induced keratinocyte apoptosis through suppression of cell adhesion.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

NFE2L3 Controls Colon Cancer Cell Growth through Regulation of DUX4, a CDK1 Inhibitor.

Constitutive nuclear factor κB (NF-κB) activation is a hallmark of colon tumor growth. Cyclin-dependent kinases (CDKs) are critical cell-cycle regulators, and inhibition of CDK activity has been used successfully as anticancer therapy. Here, we show that the NFE2L3 transcription factor functions as a key regulator in a pathway that links NF-κB signaling to the control of CDK1 activity, thereby driving colon cancer cell proliferation. We found that NFE2L3 expression is regulated by the RELA subunit of NF-κB and that NFE2L3 levels are elevated in patients with colon adenocarcinoma when compared with normal adjacent tissue. Silencing of NFE2L3 significantly decreases colon cancer cell proliferation in vitro and tumor growth in vivo. NFE2L3 knockdown results in increased levels of double homeobox factor 4 (DUX4), which functions as a direct inhibitor of CDK1. The discovered oncogenic pathway governing cell-cycle progression may open up unique avenues for precision cancer therapy.

Regulation of CXCL1 chemokine and CSF3 cytokine levels in myometrial cells by the MAFF transcription factor.

Cytokines play key roles in a variety of reproductive processes including normal parturition as well as preterm birth. Our previous data have shown that MAFF, a member of the MAF family of bZIP transcription factors, is rapidly induced by pro-inflammatory cytokines in PHM1-31 myometrial cells. We performed loss-of-function studies in PHM1-31 cells to identify MAFF dependent genes. We showed that knockdown of MAFF significantly decreased CXCL1 chemokine and CSF3 cytokine transcript and protein levels. Using chromatin immunoprecipitation analyzes, we confirmed CXCL1 and CSF3 genes as direct MAFF targets. We also demonstrated that MAFF function in PHM1-31 myometrial cells is able to control cytokine and matrix metalloproteinase gene expression in THP-1 monocytic cells in a paracrine fashion. Our studies provide valuable insights into the MAFF dependent transcriptional network governing myometrial cell function. The data suggest a role of MAFF in parturition and/or infection-induced preterm labour through modulation of inflammatory processes in the microenvironment.

Nrf3 promotes UV-induced keratinocyte apoptosis through suppression of cell adhesion.

The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) is a key regulator of the cellular stress response, but the biological functions of the related Nrf3 protein are largely unknown. Here we demonstrate a novel pro-apoptotic function of Nrf3 in mouse and human keratinocytes. In response to UV irradiation, Nrf3-deficient keratinocytes were protected from apoptosis in vitro and in vivo. The protective function was also seen under oxidative or hyperosmotic stress conditions, but not when apoptosis was induced by disruption of cell-matrix interactions. Mechanistically, we show that Nrf3-deficient keratinocytes exhibit stronger cell-cell and cell-matrix adhesion, which correlates with higher cell surface integrin levels and enhanced activation of focal adhesion kinase. Nrf3-deficient cells also formed more and larger focal adhesions and exhibited a higher motility. These results suggest that the strong expression of Nrf3 in basal keratinocytes promotes their elimination in response to DNA damage-inducing agents, thereby preventing accumulation of mutated stem and transit amplifying cells in the epidermis.

Intermittent hypoxia confers pro-metastatic gene expression selectively through NF-κB in inflammatory breast cancer cells.

Inflammatory breast cancer (IBC) is the most aggressive form of breast cancer. Treatment options are limited and the mechanisms underlying its aggressiveness are poorly understood. Intermittent hypoxia (IH) causes oxidative stress and is emerging as important regulator of tumor metastasis. Vessels in IBC tumors have been shown to be immature, which is a primary cause of IH. We therefore investigated the relevance of IH for the modulation of gene expression in IBC cells in order to assess IH as potential regulator of IBC aggressiveness. Gene array analysis of IBC cells following chronic IH (45-60 days) demonstrated increased expression of pro-metastatic genes of the extracellular matrix, such as tenascin-C (TNC; an essential factor of the metastatic niche) and matrix metalloproteinase 9 (MMP9), and of pro-inflammatory processes, such as cyclooxygenase-2 (COX-2). Investigating the oxidative stress-dependent regulation of TNC, we found a gradual sensitivity on mRNA and protein levels. Oxidative stress activated NF-E2-related factor 2 (Nrf2), c-Jun N-terminal kinase (JNK), c-Jun and nuclear factor κB (NF-κB), but TNC upregulation was only dependent on NF-κB activation. Pharmacological inhibition of inhibitor of NF-κB α (IκBα) phosphorylation as well as overexpression of IκBα prevented TNC, MMP9 and COX-2 induction, whereas the pro-inflammatory cytokine interleukin-1ß (IL-1ß) increased their expression levels. Analysis of the gene array data showed NF-κB binding sites for 64% of all upregulated genes, linking NF-κB with IH-dependent regulation of pro-metastatic gene expression in IBC cells. Our results provide a first link between intermittent hypoxia and pro-metastatic gene expression in IBC cells, revealing a putative novel mechanism for the high metastatic potential of IBC.

Stringent Control of NFE2L3 (Nuclear Factor, Erythroid 2-Like 3; NRF3) Protein Degradation by FBW7 (F-box/WD Repeat-containing Protein 7) and Glycogen Synthase Kinase 3 (GSK3).

The NFE2L3 transcription factor has been implicated in various cellular processes, including carcinogenesis, stress response, differentiation, and inflammation. Previously it has been shown that NFE2L3 has a rapid turnover and is stabilized by proteasomal inhibitors. The mechanisms regulating the degradation of this protein have not been investigated. Here we report ubiquitination of NFE2L3 and demonstrate that F-box/WD repeat-containing protein 7 (FBW7 or FBWX7), a component of Skp1, Cullin 1, F-box containing complex (SCF)-type E3 ligase, is the E3 ligase mediating the degradation of NFE2L3. We showed that FBW7 interacts with NFE2L3 and that dimerization of FBW7 is required for the degradation of the transcription factor. We also demonstrate that the kinase glycogen synthase kinase 3 (GSK3) mediates the FBW7-dependent ubiquitination of NFE2L3. We show phosphorylation of NFE2L3 by GSK3 and its significance in the regulation of NFE2L3 by the tumor suppressor FBW7. FBW7 abrogated NFE2L3-mediated repression of the NAD(P)H: quinone oxidoreductase 1 (NQO1) gene antioxidant response element (ARE). Our findings reveal FBW7 and GSK3 as novel regulators of the NFE2L3 transcription factor and a potential mechanism by which FBW7 might regulate detoxification and the cellular response to stress.

Regulation and function of the NFE2 transcription factor in hematopoietic and non-hematopoietic cells.

The NFE2 transcription factor was identified over 25 years ago. The NFE2 protein forms heterodimers with small MAF proteins, and the resulting complex binds to regulatory elements in a large number of target genes. In contrast to other CNC transcription family members including NFE2L1 (NRF1), NFE2L2 (NRF2) and NFE2L3 (NRF3), which are widely expressed, earlier studies had suggested that the major sites of NFE2 expression are hematopoietic cells. Based on cell culture studies it was proposed that this protein acts as a critical regulator of globin gene expression. However, the knockout mouse model displayed only mild erythroid abnormalities, while the major phenotype was a defect in megakaryocyte biogenesis. Indeed, absence of NFE2 led to severely impaired platelet production. A series of recent data, also summarized here, shed new light on the various functional roles of NFE2 and the regulation of its activity. NFE2 is part of a complex regulatory network, including transcription factors such as GATA1 and RUNX1, controlling megakaryocytic and/or erythroid cell function. Surprisingly, it was recently found that NFE2 also has a role in non-hematopoietic tissues, such as the trophoblast, in which it is also expressed, as well as the bone, opening the door to new research areas for this transcription factor. Additional data showed that NFE2 function is controlled by a series of posttranslational modifications. Important strides have been made with respect to the clinical significance of NFE2, linking this transcription factor to hematological disorders such as polycythemias.

Thioredoxin-interacting protein regulates the differentiation of murine erythroid precursors.

Thioredoxin-interacting protein (TXNIP) is involved in various cellular processes including redox control, metabolism, differentiation, growth, and apoptosis. With respect to hematopoiesis, TXNIP has been shown to play roles in natural killer cells, dendritic cells, and hematopoietic stem cells. Our study investigates the role of TXNIP in erythropoiesis. We observed a rapid and significant increase of TXNIP transcript and protein levels in mouse erythroleukemia cells treated with dimethyl sulfoxide or hexamethylene bisacetamide, inducers of erythroid differentiation. The upregulation of TXNIP was not abrogated by addition of the antioxidant N-acetylcysteine. The increase of TXNIP expression was confirmed in another model of erythroid differentiation, G1E-ER cells, which undergo differentiation upon activation of the GATA1 transcription factor. In addition, we showed that TXNIP levels are induced following inhibition of p38 or c-Jun N-terminal kinase (JNK) mitogen-activated protein kinases. We also observed an increase in iron uptake and a decrease in transferrin receptor protein upon TXNIP overexpression, suggesting a role in iron homeostasis. In vivo, flow cytometry analysis of cells from Txnip(-/-) mice revealed a new phenotype of impaired terminal erythropoiesis in the spleen, characterized by a partial block between basophilic and late basophilic/polychromatic erythroblasts. Based on our data, TXNIP emerges as a novel regulator of terminal erythroid differentiation.

The small MAF transcription factors MAFF, MAFG and MAFK: current knowledge and perspectives.

The small MAFs, MAFF, MAFG and MAFK have emerged as crucial regulators of mammalian gene expression. Previous studies have linked small MAF function, by virtue of their heterodimerization with the Cap 'n' Collar (CNC) family of transcription factors, to the stress response and detoxification pathways. Recent analyses have revealed a complex regulatory network involving small MAF transcription factors and other cellular proteins. The expression and activity of small MAFs are tightly regulated at multiple levels. With regard to their clinical importance, small MAFs have been linked to various diseases, such as diabetes, neuronal disorders, thrombocytopenia and carcinogenesis. A better understanding of the molecular mechanisms governing the activity of small MAFs will provide novel insights into the control of mammalian transcription and may lead to the development of novel therapeutic strategies to treat common human disorders.

Abnormal differentiation of erythroid precursors in p45 NF-E2(-/-) mice.

The transcription factor p45 nuclear factor-erythroid-derived 2 (NF-E2) plays major roles in erythroid and megakaryocytic lineages. Here, we investigated the role of p45 NF-E2 in erythroid differentiation in vivo. Absence of p45 NF-E2 in mice leads to a twofold increase in serum erythropoietin levels. In the bone marrow of these animals, we found a different distribution of precursor populations compared to wild-type mice, suggesting abnormal differentiation. Loss of p45 NF-E2 was also associated with an increase in splenic erythropoiesis, as evidenced by an accumulation of early precursors, namely, late basophilic and polychromatic erythroblasts. These observations are consistent with a stress erythropoiesis phenotype and indicate that the spleen is likely compensating for ineffective erythropoiesis in the bone marrow. Analysis of bone marrow samples revealed increased GATA1 levels, as well as an increased proportion of erythroid cells arrested at the G(1) stage of cell cycle in p45 NF-E2-deficient mice. These results suggest that p45 NF-E2 is required for the differentiation of erythroid precursors.

NFE2L3 (NRF3): the Cinderella of the Cap'n'Collar transcription factors.

NFE2L3 [Nuclear factor (erythroid-derived 2)-like 3] or NRF3, a member of the Cap'n'Collar (CNC) family, is a basic-region leucine zipper (bZIP) transcription factor that was first identified over 10 years ago. Contrary to its extensively studied homolog NFE2L2 (NRF2), the regulation and function of the NFE2L3 protein have not yet attracted as much attention. Nevertheless, several recent reports have now shed light on the possible roles of NFE2L3. Structural and biochemical studies revealed a series of domains and modifications that are critical for its cellular regulation. The control of the subcellular localization of NFE2L3 appears to be essential for understanding its role in various cellular processes. Importantly, newer studies provide fascinating insights linking NFE2L3 to differentiation, inflammation, and carcinogenesis. Here, we present an overview of the current level of knowledge of NFE2L3 transcription factor biology in humans and mice. From being the Cinderella of the CNC transcription factors for many years, NFE2L3 may now rapidly come into its own.

Nfe2l3 (Nrf3) deficiency predisposes mice to T-cell lymphoblastic lymphoma.

We have previously generated mice deficient for Nfe213 (NF-E2 p45 related factor 3 or Nrf3), a member of the cap 'n' collar family of basic-leucine zipper transcription factors. To examine whether Nrf3 is involved in chemical-induced carcinogenesis, we exposed the mice to benzo[a]pyrene (B[a]P), a carcinogen found in cigarette smoke. Contrary to wild-type mice, Nrf3-null animals are highly susceptible to B[a]P, exhibiting significantly increased mortality. Pathology analysis of affected tissue sections revealed a high incidence of T-cell lymphoblastic lymphoma in B[a]P-treated Nrf3(-/-) mice. Lymphoblastic lymphoma occasionally metastasized into the lung as demonstrated by perivascular malignant lymphocytic infiltration. Together, our studies show that the absence of Nrf3 predisposes mice to lymphoma development, suggesting a protective role of this transcription factor in hematopoietic malignancies. Our data demonstrate the first in vivo function of Nrf3 and its link to tumor development. Nrf3-deficient mice may serve as a preclinical mouse model to study carcinogen-induced lymphomagenesis.

Terahertz white-light pulses from an air plasma photo-induced by incommensurate two-color optical fields.

We demonstrate plasma-THz generation in ambient air with two-color (ω-2ω) optical excitation using input pulses with a bandwidth supporting sub-20-fs duration, yielding a continuous bandwidth exceeding 100 THz with no significant roll-off and a pulse energy of 360 nJ at a 1-kHz repetition rate. The key aspect in achieving this performance is an optimized geometry of the second-harmonic generation (SHG) crystal, producing a 2ω-field detuned from the second-harmonic of the ω-field, which promotes both a high polarization bandwidth and optimal ω-2ω temporal overlap in the plasma, as supported by theoretical results.

Antagonistic roles of the ERK and p38 MAPK signalling pathways in globin expression, haem biosynthesis and iron uptake.

Late-stage erythroid cells synthesize large quantities of haemoglobin, a process requiring the co-ordinated regulation of globin and haem synthesis as well as iron uptake. In the present study, we investigated the role of the ERK (extracellular-signal-regulated kinase) and p38 MAPK (mitogen-activated protein kinase) signalling pathways in MEL (mouse erythroleukaemia) cell differentiation. We found that treatment of HMBA (hexamethylene bisacetamide)-induced MEL cells with the ERK pathway inhibitor UO126 results in an increase in intracellular haem and haemoglobin levels. The transcript levels of the genes coding for ß(major)-globin, the haem biosynthesis enzyme 5-aminolevulinate synthase 2 and the mitochondrial iron transporter mitoferrin 1 are up-regulated. We also showed enhanced expression of globin and transferrin receptor 1 proteins upon UO126 treatment. With respect to iron uptake, we found that ERK inhibitor treatment led to an increase in both haem-bound and total iron. In contrast, treatment of MEL cells with the p38 MAPK pathway inhibitor SB202190 had the opposite effect, resulting in decreased globin expression, haem synthesis and iron uptake. Reporter assays showed that globin promoter and HS2 enhancer-mediated transcription was under the control of MAPKs, as inhibition of the ERK and p38 MAPK pathways led to increased and decreased gene activity respectively. Our present results suggest that the ERK1/2 and p38α/ß MAPKs play antagonistic roles in HMBA-induced globin gene expression and erythroid differentiation. These results provide a novel link between MAPK signalling and the regulation of haem biosynthesis and iron uptake in erythroid cells.

Nrf3-deficient mice are not protected against acute lung and adipose tissue damages induced by butylated hydroxytoluene.

We found that both wild type and Nrf3 (NF-E2-related factor 3) deficient mice are sensitive to BHT single administration exhibiting respiratory distress and considerably lose body weight following treatment. At time of sacrifice, the BHT-treated Nrf3-/- mice had lost significantly more body weight than their WT counterparts. In the lung, transcript levels of the transcription factors Nrf1, Nrf2 and Nrf3 were differentially regulated by BHT treatment. In addition, genes implicated in adipogenesis were repressed following BHT exposure in the white adipose tissue. Together, our data provide the first evidence that BHT exposure not only affects lung function but also leads to impaired adipogenesis in adipocytes.

Characterizing large-area electro-optic crystals toward two-dimensional real-time terahertz imaging.

We have characterized the homogeneity of large-area (>10 mm x 10 mm) CdTe(110) and ZnTe(110) crystals using a raster electro-optic scanning method to assess their usability in two-dimensional electro-optic terahertz (THz) imaging with parallel read out. The spatial variation in the detected THz signal (at 0.2 and 0.645 THz, respectively) is due to nonuniform residual birefringence and scattering. For CdTe, this depends critically on the growth method, and has an important contribution from slip planes in the crystals, as is evident in the scanned images. For the highest-quality CdTe(110) crystals investigated, the rms signal variations are less than 15%, comparable to those for ZnTe(110). For electro-optic scanning, we introduce a hybrid measurement system based on a fs Nd:glass laser and a continuous-wave electronic THz source.