Role Of Nuclear Respiratory Factor 1 Research Articles

Abstract 779: A NRF1-driven JRK-mediated β-catenin transcriptional activity contributes to the aggressive growth of ovarian cancer

Abstract Ovarian cancer is the most lethal gynecological cancer, accounting for thousands of cancer-related deaths in the United States every year. Though there has been progress towards understanding ovarian cancer pathogenesis, much is not yet understood about the molecular mechanisms that facilitate chemoresistance and metastasis. Our work has shown that nuclear respiratory factor 1 (NRF1) is highly active in several human cancers, though the role of NRF1 remains largely unexplored in ovarian carcinomas. Therefore, our aim was to examine the role of this transcription factor in ovarian cancer, since understanding its transcriptional landscape may reveal NRF1 or its target genes as possible therapeutic targets for this highly aggressive cancer. The Cancer Genome Atlas (TCGA) was utilized to collect RNA-Seq data from 379 serous cystadenocarcinoma samples, where we found that about 20% of ovarian cancer patients had altered NRF1 expression, showing either high expression or amplification of this transcription factor. Patients with high NRF1 expression were found to have decreased survival compared to patients with low NRF1 expression. NRF1 amplification was also correlated with the overamplification of one of its target genes - Jrk helix-turn-helix protein (JRK) in 26% of the samples. JRK regulates centrosome amplification, and through the production of mitotic spindle abnormalities, centrosome amplification contributes to genomic instability. Further investigation of the relationship between NRF1 and JRK uncovered that protein levels of JRK and β-catenin were significantly higher in NRF1-induced stem-like cells compared to the control group, and JRK is known to bind to β-catenin. Our NRF1 ChIP-Seq analyses showed that promoters of these two genes - JRK and CTNNB1 - were bound to NRF1. The Flow Assorted Cell Sorting (FACS), ChIP-Seq, RNA Seq and confocal microscopic studies suggest that through JRK, NRF1 regulates β-catenin transcriptional activity. Amplified gene expression is the one of the first steps towards oncogene activation, and our findings illustrate that altered NRF1 expression (i.e., overexpression or amplification) coupled with that of JRK influences the prognosis of patients diagnosed with ovarian cancer. The Wnt/β-catenin pathway is also highly active in the aggressive ovarian cancer. In addition to JRK, through the activation of β-catenin, NRF1may also affect the development of ovarian tumors. Taken together, our findings provide a novel insight into the molecular basis of the contribution of NRF1-driven JRK-mediated β-catenin transcriptional activity to the susceptibility of highly aggressive ovarian cancer. A better understanding of how ovarian neoplasm formation depends on NRF1-JRK-β-catenin pathway may open new avenues for therapeutic strategy against ovarian cancer. Citation Format: Ana Ruas, Quentin Felty, Jayanta K. Das, Alok Deoraj, Changwon Yoo, Deodutta Roy. A NRF1-driven JRK-mediated β-catenin transcriptional activity contributes to the aggressive growth of ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 779.

Nuclear Respiratory Factor 1 Acting as an Oncoprotein Drives Estrogen-Induced Breast Carcinogenesis.

We have previously shown nuclear respiratory factor 1 (NRF1)-mediated transcriptional programming of mitobiogenesis contributes to estrogen-induced breast cancer through modulating cell cycle progression. In this study, we report a new role of NRF1 that goes beyond that of programming mitobiogenesis. Specifically, we report a novel oncogenic function of NRF1 supporting its causative role in breast cancer development and progression. The gain of NRF1 and/or treatment with 17β-estradiol (E2) produced heterogeneous breast cancer stem cell (BCSC)-like subsets composed of more than 10 distinct cell sub-populations. Flow sorting combined with confocal imaging of markers for pluripotency, epithelial mesenchymal transition (EMT), and BCSCs phenotypically confirmed that the BCSC-like subset arise from cell re-programming. Thus, we determined the molecular actions of NRF1 on its target gene CXCR4 because of its known role in the acquisition of the BCSC-like subset through EMT. CXCR4 was activated by NRF1 in a redox-dependent manner during malignant transformation. An NRF1-induced BCSC-like subset was able to form xenograft tumors in vivo, while inhibiting transcription of CXCR4 prevented xenograft tumor growth. Consistent with our observation of NRF1-driven breast tumorigenesis in the experimental model, higher protein levels of NRF1 were also found in human breast cancer tissue specimens. This highly novel role of NRF1 in the stochastic acquisition of BCSC-like subsets and their progression to a malignant phenotype may open an entirely new research direction targeting NRF1 signaling in invasive breast cancer. Our discovery of targeting transcriptional activation of CXCR4 to inhibit NRF1-induced oncogenic transformation provides a mechanistic explanation for estrogen-dependent breast carcinogenesis and opens new avenues in strategic therapeutics to fight breast cancer.

Abstract 1552: Integrative bioinformatics analyses of ChIP seq and RNA seq data reveals a complex regulatory landscape of NRF1 network involved in the pathogenesis of breast cancer

Abstract The expression of redox sensitive transcription factor -nuclear respiratory factor 1 (NRF1) expression significantly correlates with histological grades and prognosis of breast cancer. However, the molecular mechanism by which NRF1 may contribute in the development of breast cancer is not clear. In this study we examined whether NRF1 is a molecular risk factor of breast cancer. We also identified regulatory landscape of NRF1 network involved in the pathogenesis of breast cancer. Steps followed for the investigation included the selection of mRNA microarray dataset from TCGA and Metabric, selection and analysis of ChIP-Seq datasets from different breast cancer cell lines to identify NRF1 target genes possibly involved in breast cancer through Molecular Pathway, Gene Ontology and statistical analysis. Analysis of mRNA expression in breast cancer patients showed that NRF1 was significantly overexpressed in breast cancer tissues compared to normal tissues. ChIP-Seq analysis of two different breast cancer cell line - MCF7 and T7D showed that there are more than 8,000 genes, which contain NRF1 binding DNA motif(s). The NRF1 binding to ChIP DNA sequences of target gene is cell context dependent. High percentage of known breast cancer (~50%) susceptibility genes are transcriptional targets of NRF1. Network analysis revealed that NRF1 regulates genes, which are key regulators of epithelial-mesenchymal transition (EMT), stemness, cell apoptosis, cell cycle regulation, chromosomal integrity, and DNA damage and repair. Furthermore, we observed that NRF1 regulates target genes of both breast cancer and cancer KEGG pathways. Many of the MicroRNAs involved in cancer and genes of signaling pathways involved in cancer initiation and progression such as MAPK, PI3K/AKT and Notch signaling are also transcriptional targets of NRF1. In summary, this study reveals a complex regulatory landscape of NRF1 network involved in the pathogenesis of breast cancer. Dysregulation of NRF1 signaling pathways may contribute in the development of breast cancer. Clinical confirmation of our study will have significant impact on our understanding of the role of NRF1 as a valuable biomarker for breast cancer diagnosis and prognosis and will provide strong rationale for the future studies to further develop NRF1 for breast cancer therapeutic target. This work was in part supported by a VA MERIT Review (VA BX001463) grant to DR. Citation Format: Jairo Ramos, Deodutta Roy. Integrative bioinformatics analyses of ChIP seq and RNA seq data reveals a complex regulatory landscape of NRF1 network involved in the pathogenesis of breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1552. doi:10.1158/1538-7445.AM2017-1552

Abstract 803: Overexpression of NRF1 leads to the generation of cancer stem-like cells and resistance to anoikis _ pathways to anchorage-independent growth during estrogen-induced malignant transformation

Abstract The actual role nuclear respiratory factor-1 (NRF-1) plays in breast cancer remains the least studied of all transcription factors. A growth role for the NRF1 through regulation of cell cycle genes during estrogen-induced malignant transformation has recently been reported by us; however, the underlying mechanism of its contribution to estrogen-induced breast carcinogenesis is undefined. In this study we provide new mechanistic insight into the mode by which NRF1 may contribute in estrogen-induced neoplastic conversion of cells by suppressing anoikis; identified BNIP3 as a key transcriptional target of NRF1; and survival signals conferred by NRF1 seems to impinge on the hypoxia-inducible death factor BNIP3. ChIP, qPCR, mass spectrometry, redox Western blot, colony formation, cell proliferation, ROS assay, immunofluorescence microscopy were used to study the role of NRF-1. Our data showed that higher expresion of NRF-1 in MCF10A produced large sizes of spheroids with increased level of stem cell markers -CD44+CD24+CD49f+ compared to cells transfected with vector alone. E2-induced increase of spheroid formation, suppression of cell death and cell survival were amplified by expression of NRF1 and these effects were diminished by loss of NRF1 function through expression of dominant negative NRF1. Breast cancer cell lines - MCF7 and MDA-MB231 overexpressed both NRF1 and BNIP3, and formed larger tumor spheroids compared to the MCF10A or vector-transformed breast cancer cells. E2 treatment as well as overexpression of NRF1 increased mRNA and protein levels of BNIP3. In addition, we found the increased transcriptional interaction of NRF-1 transcription factor in the BNIP3 promoter region by ChIP-qPCR assay in cells treated with E2. We also found the increased functional activity of the BNIP3 promoter in cells treated with E2 and this was decreased with co-treatment of ROS scavenger-Ebselen. NRF1 spheroids have the ability to micro-invasion. In summary, overexpression of NRF1 altered the cell morphology towards mesenchymal stem-like shape and promoted cancer stem cell-like phenotype. Findings of this study unveiled the underlying mechanism of NRF1 action through the activation of BNIP3 which is regulated by NRF1 and is presumably responsible for the estrogen-induced malignant transformation of MCF10A cells, including anoikis resistance, anchorage-independent cell growth, and increased cell migration and invasion. This work was in part supported by a VA MERIT Review (VA BX001463) grant to DR. Citation Format: Jayanta Das, Deodutta Roy. Overexpression of NRF1 leads to the generation of cancer stem-like cells and resistance to anoikis _ pathways to anchorage-independent growth during estrogen-induced malignant transformation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 803. doi:10.1158/1538-7445.AM2015-803

Downregulation of nuclear respiratory factor‐1 contributes to mitochondrial events induced by benzo(a)pyrene

Environmental carcinogen benzo(a)pyrene (BaP) has been shown to be a genotoxicant that affects both nuclear DNA (nDNA) and mitochondrial DNA (mtDNA). Nuclear respiratory factor 1 (NRF-1) is a transcriptional activator of nuclear genes that encode a range of mitochondrial proteins including mitochondrial transcription factor A (mtTFA). However, the role of NRF-1 in BaP-induced mitochondrial event is not clear. We investigated the change of NRF-1 and mtTFA in human bronchial epithelial cells (16HBE) elicited by BaP. The results indicated that BaP induced cell apoptosis, total mitochondrial enzymes activities and ATP levels decrease in dose- and time-dependent manners, respectively. The transcription and protein levels of NRF-1 and mtTFA decreased at 48 h after 16 μM BaP treatment. Our results indicated downregulation of NRF-1 and mtTFA is involved in BaP-induced mitochondrial events.

Abstract 2177: A novel protein interaction between Smad4 and NRF1 establishes a molecular switch on p15Ink4b expression

Abstract We have been investigating TGFβ signaling particularly concentrating on Smad4 as a key element in this biologically important pathway in cancer. In order to demonstrate the novel molecular ways in regulation of Smad4 in tumorigenesis, we previously have performed a baculovirus protoarray and identified nuclear respiratory factor 1 (NRF1) as one of the putative interacting protein partners of Smad4. NRF1 is the first mammalian transcriptional factor reported to act on nuclear respiratory genes. Other than mitochondrial respiratory function and biogenesis, NRF1 has also been associated with a subset of E2F-responsive genes, suggesting a role in DNA replication, mitosis and cytokinesis. In this study, we firstly demonstrated the novel interaction between NRF1 and Smad4 via using bimolecular fluorescence complementation (BiFC) assay. NRF1 amino-terminal domain, carrying a nuclear localization signal, was found to interact with Smad4 and direct the heterodimer into nucleus. Subsequently, to investigate further the role of NRF1 on Smad4 expression, a manual search of probable NRF1 binding sites on Smad4 promoter was carried out and putative sequences were identified. Upon ectopic expression of NRF1, luciferase reporter assays as well as western blot analysis resulted in decrements of both Smad4 transcriptional activity and protein expression. The core binding region of NRF1 was discovered within -186/-175 nucleotide region of Smad4 promoter via utilizing a site-directed mutagenesis approach. Thirdly, to further substantiate the role of Smad4 and NRF1's interaction in tumorigenesis, p15INK4b, a cell cycle regulator, was identified as a putative common target of both NRF1 and Smad4 through a comparison among previously reported subsets of target promoters. Indeed, the induction of p15INK4b promoter activity by Smad4 was found to be diminished to a basal level by overexpression of NRF1. Furthermore, the nature of this inhibition was shown to be both dose and time dependent in the presence of NRF1. Under cell synchronized conditions, both transcriptional factors were shown to bind p15INK4b promoter region in close proximity, whereas NRF1 was found to slightly inhibit Smad4's binding to its regulatory region when cells proceed in cell cycle. Finally, a computational analysis of gastric and breast cancer microarray expression public data on other putative common targets of NRF1 and Smad4 revealed a negative correlation between the effects of these two transcriptional factors on gene expressions. Thus, NRF1 appears to negatively interact with Smad4 and inhibit p15INK4b expression, through which it suggests a possible role in tumorigenesis. We will be representing our current findings in this research. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2177. doi:10.1158/1538-7445.AM2011-2177

Nuclear Respiratory Factor-1 is Involved in Mitochondrial Dysfunction Induced by Benzo(a)pyrene in Human Bronchial Epithelial Cells

In this study, we investigated the role of nuclear respiratory factor-1(NRF-1) in benzo(a)pyrene (BaP)-induced mitochondrial events in human bronchial epithelial cells (16HBE). Cytotoxicity was determined with MTT assay, and apoptosis was measured by flow cytometry. The results showed that BaP inhibited cell proliferation in a dose-dependent manner and induced apoptosis in 16HBE cells. Time-dependent reactive oxygen species (ROS) generation induced by BaP was observed in 16HBE cells. The loss of mitochondrial membrane permeability transition (MPT) was obtained by a laser scanning confocal microscope, and the decreasing ATP level was detected by a Cell-Titer-Glo(®) Luminescent Cell Viability Assay. Results of western blotting assay revealed that both NRF-1 and mitochondrial transcription factor A (mtTFA) decreased in 12-μM BaP-treated cells at both 12 and 24 hr. The results of RT-PCR indicate that NRF-1 and mtTFA mRNA in 16HBE cells were not changed after BaP treatment 12 or 24 hr. Down-regulation of NRF-1 by shRNA further reduced the loss of MPT and increased ROS generation in response to BaP treatment. Therefore, our results demonstrate that NRF-1 is responsible for BaP-induced mitochondrial dysfunction in 16HBE cells and associated with the level of mtTFA protein, loss of MPT and ROS overproduction.

AMPK activation represses the human gene promoter of the cardiac isoform of acetyl-CoA carboxylase: Role of nuclear respiratory factor-1

The cardiac-enriched isoform of acetyl-CoA carboxylase (ACCβ) produces malonyl-CoA, a potent inhibitor of carnitine palmitoyltransferase-1. AMPK inhibits ACCβ activity, lowering malonyl-CoA levels and promoting mitochondrial fatty acid β-oxidation. Previously, AMPK increased promoter binding of nuclear respiratory factor-1 (NRF-1), a pivotal transcriptional modulator controlling gene expression of mitochondrial proteins. We therefore hypothesized that NRF-1 inhibits myocardial ACCβ promoter activity via AMPK activation. A human ACCβ promoter-luciferase construct was transiently transfected into neonatal cardiomyocytes ± a NRF-1 expression construct. NRF-1 overexpression decreased ACCβ gene promoter activity by 71 ± 4.6% ( p < 0.001 vs. control). Transfections with 5′-end serial promoter deletions revealed that NRF-1-mediated repression of ACCβ was abolished with a pPIIβ-18/+65-Luc deletion construct. AMPK activation dose-dependently reduced ACCβ promoter activity, while NRF-1 addition did not further decrease it. We also investigated NRF-1 inhibition in the presence of upstream stimulatory factor 1 (USF1), a known transactivator of the human ACCβ gene promoter. Here NRF-1 blunted USF1-dependent induction of ACCβ promoter activity by 58 ± 7.5% ( p < 0.001 vs. control), reversed with a dominant negative NRF-1 construct. NRF-1 also suppressed endogenous USF1 transcriptional activity by 55 ± 6.2% ( p < 0.001 vs. control). This study demonstrates that NRF-1 is a novel transcriptional inhibitor of the human ACCβ gene promoter in the mammalian heart. Our data extends AMPK regulation of ACCβ to the transcriptional level.

Activation of the Cytochrome c Gene by Electrical Stimulation in Neonatal Rat Cardiac Myocytes: ROLE OF NRF-1 AND c-Jun

Activation of cytochrome c (cyt c) transcription in electrically stimulated neonatal rat cardiac myocytes is preceded by transient expression of the activating protein-1 family of transcription factors, c-Fos, c-Jun, and JunB, as well as nuclear respiratory factor-1 (NRF-1). Mutations in either the NRF-1 or in the two cyclic AMP response elements on the cyt c promoter significantly reduce cyt c promoter activation produced either by electrical stimulation (Xia, Y., Buja, L. M., Scarpulla, R. C., and McMillin, J. B. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 11399-11404) or by transfection of c-jun into nonpaced cardiac myocytes. Electrical stimulation of cardiac myocytes activates the c-Jun N-terminal kinase (McDonough, P. M., Hanford, D. S., Sprenkle, A. B., Mellon, N. R., and Glembotski, C. C. (1997) J. Biol. Chem. 272, 24046-24053) so that the fold-activation of the cyt c promoter is increased by pacing when either c-jun or c-fos/c-jun were cotransfected. Physical association of NRF-1 protein with the NRF-1 enhancer element and of c-Jun with the cyclic AMP response element binding sites on the cyt c promoter was demonstrated by gel shift competition assays and by antibody super shifts. This is the first demonstration that induction of NRF-1 and c-Jun by pacing of cardiac myocytes directly mediates cyt c gene expression and mitochondrial proliferation in response to hypertrophic stimuli in the heart.