About

Stephen Safe is a professor associated with the Texas A&M University College of Veterinary Medicine & Biomedical Sciences (VMBS). The college is recognized as a leading institution in veterinary education and research, supporting collaborations that translate discoveries into solutions for animal, human, and environmental health. The college emphasizes innovative research, including basic and clinical studies, with a focus on a 'One Health' approach that recognizes the complex interactions between animal, human, and environmental health. While the specific research focus and key contributions of Stephen Safe are not detailed in the provided page text, his association with VMBS suggests involvement in a dynamic environment dedicated to advancing veterinary medicine through cutting-edge research, education, and outreach efforts.

Research topics

• Biology
• Biochemistry
• Pharmacology
• Chemistry
• Cancer research
• Computer Science
• Immunology
• Medicine
• Cell biology
• Environmental chemistry
• Organic chemistry
• Toxicology
• Computational biology
• Psychiatry
• Internal medicine
• Database
• Endocrinology
• Genetics
• Statistics
• Mathematics
• Bioinformatics

Selected publications

• Targeting Lineage-Specific Functions of NR4A1 for Cancer Immunotherapy

  International Journal of Molecular Sciences · 2025-05-30 · 7 citations

  reviewOpen access

  Orphan nuclear receptor 4A1 (NR4A1, Nur77) plays a crucial role in regulating immune cell metabolism and function within the tumor microenvironment (TME), thus influencing cancer progression and serving as a potential therapeutic target for cancer immunotherapy. A comprehensive review discussing the multifaceted roles of NR4A1 in immune cells and the exploitation of that knowledge for therapeutic development is lacking in the field. This review explores diverse functions of NR4A1 in tumor-associated immune cells, including T cells, monocytes, natural killer cells, B cells, dendritic cells, macrophages, and neutrophils. NR4A1 contributes to immune regulation by impacting cytokine production, cell differentiation, and immune cell exhaustion. We highlight how NR4A1 in immune cells within the TME may be either a positive (e.g., macrophages in colon cancer) or negative prognostic factor (e.g., T cells in melanoma), depending on the cancer and immune cell context. Additionally, this review also highlights potential therapeutic strategies targeting NR4A1, leading to its inhibition, activation, or degradation to restore immune cell function and enhance anti-tumor immunity. Such therapies could potentially improve patient outcomes by altering immune cell behaviors, blocking intrinsic tumor growth pathways, or via both mechanisms. However, the development of NR4A1-targeted therapies will be dependent on further research to better understand lineage-specific roles of NR4A1 and the underlying mechanisms across different cancer types and immune cells.

  PublisherOA PDFDOI

• Expression of Prooncogenic Nuclear Receptor 4A (NR4A)-Regulated Genes β1-Integrin and G9a Inhibited by Dual NR4A1/2 Ligands

  International Journal of Molecular Sciences · 2025-04-21 · 1 citations

  articleOpen accessSenior authorCorresponding

  Bis-indole-derived compounds including 1,1-bis(3'-indolyl)-1-(3,5-disubstitutedphenyl)methane (DIM-3,5) analogs bind both orphan nuclear receptors 4A1 (NR4A1) and NR4A2, and DIM-3,5 compounds act as dual receptor inverse agonists and inhibit both NR4A1- and NR4A2-regulated responses. Chromatin immunoprecipitation assays show that β1-integrin and the methyltransferase gene G9a are regulated by both NR4A1 and NR4A2 acting as cofactors for Sp1- and Sp4-dependent gene expression. DIM-3,5 treatment results in the loss of one or more of these nuclear factors from the β1-integrin and G9a promoters. Single-cell and RNAseq analyses show that both receptors regulate common (<10%) and unique genes in SW480 colon cancer cells; however, functional enrichment analysis of the differentially expressed genes converges to several common pathways and gene ontology terms.

  PublisherOA PDFDOI

• Abstract A047: Bis-indole derived dual NR4A1/2 inverse agonists synergistically enhance temozolomide-induced cytotoxicity in glioblastoma cells through downregulation of drug-resistance genes

  Molecular Cancer Therapeutics · 2025-10-22

  articleSenior author

  Abstract Backgroud and Purpose: Glioblastoma (GBM) is the most common form of adult brain cancer and the standard of care, temozolomide (TMZ) and radiation therapy, is subject to drug resistance. The orphan nuclear receptors 4A1 (NR4A1) and NR4A2 are highly expressed in GBM patients and their growth is inhibited by a series of bis-indole derived compounds (CDIMs) that bind both receptors and act as inverse agonists. It has been reported that CDIMs decrease expression of several NR4A-regulated genes such as G9a, several integrins and survivin in cancer cells, which have been shown to play a role in TMZ resistance in GBM. The objective of our study is to determine whether CDIMs synergistically enhance the cytotoxic effects of TMZ treatment through the downregulation of NR4A-regulated TMZ-resistance genes. Methods: The CellTiter-Blue assay kit from Promega was used as a measure for metabolic capacity and cell-viability. Small interfering RNAs (siRNAs) were used to transiently silence the expression of NR4A1/2. The Bliss-Independence model and SynergyFinder tool were used to quantify synergism in select combinations of CDIMs and TMZ. Western blot analysis was used to determine treatment-related effects on the NR4A-regulated TMZ-resistance genes. Results: Initial testing showed that several human-derived GBM cell-lines exhibited varied responsiveness to treatment with up to 1000 µM TMZ. The knockdown (KD) of NR4A1/2 by RNA interference (siRNAs) potentiates the cytotoxicity of TMZ in U87-MG GBM cells, which were the most resistant of the GBM cell lines tested. The increase in TMZ-induced cytotoxicity in N4RA1/2 deficient GBM cells was as high as 5-fold in combination. An array of combination doses of TMZ plus several CDIM analogs identified several treatments that exhibited a synergistic growth inhibitory effect in the GBM cell-lines, with some exhibiting inhibitory effects 25% greater or more than expected. When lysates of cells treated with these synergistic combinations were analyzed using western blot analysis and compared to controls, TMZ treatment alone, and CDIM treatment alone, there was a decreased expression of the TMZ-resistance genes: G9a, β1-integrin and surviving after cotreatment with CDIMs plus TMZ. Furthermore, NR4A1/2 KD treated cells showed decreased expression in the TMZ-resistance genes when compared to control and TMZ treatment alone. Conclusions: The CDIM-3,5 dual NR4A1/2 inverse agonists represent a novel mechanism-based approach for treating GBM by targeting the NR4A-mediated pro-oncogenic pathways. The CDIMs decrease expression of NR4A-mediated drug resistance genes and synergistically enhance the cytotoxicity of TMZ treatment. The battery of cell-lines are being used to investigate the synergistic responses and mechanisms. This will include studies on the effects of individual knockdown and pharmacologic inhibition of the NR4A-regulated resistance genes to determine their relative contributions to enhancing the potential therapeutic efficacy of TMZ and our ongoing in vivo study comparing the different treatments effects on survival. Citation Format: Evan Farkas, Ana Gutierrez, Andrei Mikheev, Robert Rostomily, Stephen Safe. Bis-indole derived dual NR4A1/2 inverse agonists synergistically enhance temozolomide-induced cytotoxicity in glioblastoma cells through downregulation of drug-resistance genes [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr A047.

  PublisherDOI

• Activation of Genes by Nuclear Receptor/Specificity Protein (Sp) Interactions in Cancer

  Cancers · 2025-01-17 · 5 citations

  reviewOpen access1st authorCorresponding

  The human nuclear receptor (NR) superfamily consists of 48 genes that are ligand-activated transcription factors that play a key role in maintaining cellular homeostasis and in pathophysiology. NRs are important drug targets for both cancer and non-cancer endpoints as ligands for these receptors can act as agonists, antagonists or inverse agonists to modulate gene expression. With two exceptions, the classical mechanism of action of NRs involves their interactions as monomers, dimers or heterodimers with their cognate response elements (cis-elements) in target gene promoters. Several studies showed that a number of NR-regulated genes did not directly bind their corresponding cis-elements and promoter analysis identified that NR-responsive gene promoters contained GC-rich sequences that bind specificity protein 1 (Sp1), Sp3 and Sp4 transcription factors (TFs). This review is focused on identifying an important sub-set of Sp-regulated genes that are indirectly coregulated through interactions with NRs. Subsequent studies showed that many NRs directly bind Sp1 (or Sp3 and Sp4), the NR/Sp complexes bind GC-rich sites to regulate gene expression and the NR acts as a ligand-modulated nuclear cofactor. In addition, several reports show that NR-responsive genes contain cis-elements that bind both Sp TFs and NRs, and mutation of either cis-element results in loss of NR-responsive (inducible and/or basal). Regulation of these genes involves interactions between DNA-bound Sp TFs with proximal or distal DNA-bound NRs, and, in some cases, other nuclear cofactors are required for gene expression. Thus, many NR-responsive genes are regulated by NR/Sp complexes, and these genes can be targeted by ligands that target NRs and also by drugs that induce degradation of Sp1, Sp3 and Sp4.

  PublisherOA PDFDOI

• Orphan nuclear receptor transcription factors as drug targets

  Transcription · 2025-05-27 · 3 citations

  reviewOpen access1st authorCorresponding

  The nuclear receptor (NR) superfamily of ligand-activated receptors plays a key role in maintaining cellular homeostasis and in pathophysiology. NRs can be subdivided into functional activities structural similarity and the existence of endogenous ligands. Most NRs are classified as those that are adopted orphan or orphan receptors which have only possible ligands or no identified endogenous ligands, respectively. In this review, the activities of the complete orphan receptor sub-family of transcription factors have been reviewed with a focus on the effects of possible endogenous (biochemicals), natural product-derived and synthetic ligands. Despite their lack of a bona-fide ligand, the orphan receptors bind structurally diverse compounds that exhibit tissue-specific agonist, antagonist and inverse agonist activities with potential for future development as clinical therapeutics for the treatment of multiple diseases.

  PublisherOA PDFDOI

• Orphan nuclear receptor 4A1 (NR4A1) and NR4A2 are endogenous regulators of CD71 and their ligands induce ferroptosis in breast cancer

  Cell Death and Disease · 2025-11-03 · 2 citations

  articleOpen accessSenior author

  Ferroptosis is an iron-dependent cell death pathway that involves multiple genes, including the transferrin receptor (TFRC/CD71), glutathione peroxidase 4 (GPX4) and cystine-glutamate antiporter (SLC7A11). This study is based on the hypothesis that orphan nuclear receptor 4A1 (NR4A1) and NR4A2 maintain low levels of ferroptosis in triple negative breast cancer (TNBC) cells and bis-indole derived (CDIM) compounds act as NR4A1/2 ligands that induce ferroptosis by enhancing CD71 expression. 1,1-Bis(3'-indolyl)-1-(3,5-disubstitutedphenyl)methane (DIM-3,5) analogs were investigated for their cytotoxicity and effects on NR4A1 and NR4A2 regulated genes and induction of ferroptosis. Several assays also determined enhanced lipoperoxidation, reactive oxygen species and malondialdehyde formation in TNBC cells. Knockdown of NR4A1, NR4A2, Sp1 and Sp4 was carried out by RNA interference. Molecular mechanisms of NR4A1/2-mediated regulation of CD71 expression were determined using CD71-luciferase promoter constructs, overexpression of Sp1 and chromatin immunoprecipitation (ChIP) assays. Initial studies show that DIM-3,5 analogs act as an inverse NR4A1/NR4A2 agonists that downregulate the pro-oncogenic responses/gene products regulated by both receptors in TNBC cells. DIM-3,5 analogs also induced ROS, malondialdehyde and lipoperoxide formation in TNBC cells, and this was accompanied by decreased expression of GPX4 and SLC7A11 and induction of CD71. Induction of CD71, an important biomarker of ferroptosis was observed after treatment of TNBC cells with DIM-3,5 analogs, knockdown of NR4A1, NR4A2, Sp1 or Sp4 demonstrating that induction of CD71 was coregulated by both receptors. Moreover, both promoter and ChIP analysis indicated that NR4A1 and NR4A2 acted as ligand-dependent cofactors of Sp1/4-mediated expression of CD71 in TNBC cells. Thus, CD71, a key biomarker of ferroptosis is an NR4A1/2/Sp regulated gene that can be directly targeted by DIM-3,5 inverse NR4A1/2 agonists to induce ferroptosis in TNBC cells.

  PublisherOA PDFDOI

• Orphan Nuclear Receptor 4A1 (NR4A1) and NR4A2 are Endogenous Regulators of CD71 and Their Ligands Induce Ferroptosis in Breast Cancer

  Research Square · 2025-04-21

  preprintOpen access1st authorCorresponding
  PublisherOA PDFDOI

• Dual Targeting of Orphan Nuclear Receptors NR4A1 and NR4A2 for Nonhormonal Endometriosis Therapy

  Endocrinology · 2025-09-26 · 1 citations

  articleOpen accessSenior author

  Previous studies show that orphan nuclear receptor 4A1 (NR4A1) regulates endometriotic cell growth, survival, estrogen receptor β (ERβ), mechanistic target of rapamycin signaling and fibrosis. NR4A2 is also expressed in epithelial and stromal derived endometriotic cells, and in this study the effects of 1,1-bis(3'-indolyl)-(3,5-disubstitutedphenyl)methane (DIM-3,5) dual NR4A1/nuclear receptor 4A2 (NR4A2) ligands and knockdown of NR4A1 and NR4A2 were investigated. The dual NR4A1/2 DIM-3,5 analogs inhibited previously identified proendometriotic pathways and gene products, and they also inhibited TWIST1 and multiple markers associated with epithelial-to-mesenchymal transition (EMT). The results show that both NR4A1 and NR4A2 regulate the same pathways, including endometriotic cell growth, survival, and migration and also some of the same genes in endometriotic epithelial and stromal cells. For example, DIM-3,5 compounds downregulate ERβ in stromal but not epithelial endometriotic cells, and this response is NR4A1- and not NR4A2-dependent. Among the EMT-related markers, claudin-1 is induced by DIM-3,5 ligands and after knockdown of NR4A1 or NR4A2 in both epithelial and stromal cells. Most of the EMT markers are downregulated by DIM-3,5 ligands and are coregulated by NR4A1 and NR4A2. In vivo studies showed that DIM-3,5-Cl2 significantly reduced the growth of endometriotic lesions in a mouse model without inducing cytotoxicity during treatment. Thus, DIM-3,5 derivatives simultaneously suppress NR4A1- and NR4A2-dependent endometriosis progression effectively and represent a promising nonhormonal therapeutic strategy to replace current hormone-based treatments that can be associated with adverse effects.

  PublisherOA PDFDOI

• Perfluorooctane Sulfonate (PFOS) and Related Compounds Induce Nuclear Receptor 4A1 (NR4A1)-Dependent Carcinogenesis

  Chemical Research in Toxicology · 2025-03-11 · 2 citations

  articleOpen accessSenior authorCorresponding

  Polyfluoroalkyl substances (PFAS) are widely used industrial compounds that have been identified as contaminants in almost every component of the global ecosystem, and in human studies, higher levels of PFAS have been correlated with increased incidence of multiple diseases. Based on the results of human and laboratory animal studies, we hypothesize that the orphan nuclear receptor 4A1 (NR4A1) may be a critical target for some PFAS such as the legacy linear polyfluorooctanesulfonate (PFOS) and other sulfonates. We show that PFOS and related compounds bound the ligand binding domain (LBD) of NR4A1 and induced the growth of several cancer cell lines and enhanced tumor growth in an athymic nude mouse model. Using NR4A1-responsive rhabdomyosarcoma Rh30 cells as a model, PFOS induced NR4A1-dependent cell proliferation and Rh30 cell migration and invasion. Moreover, in Rh30 cells, PFOS also induces several NR4A1-regulated genes including the PAX3-FOXO1 oncogene and downstream gene products, and in a chromatin immunoprecipitation assay, PFOS does not decrease NR4A1 binding to the promoter. These results demonstrate that PFOS is an NR4A1 ligand and enhances tumorigenesis through the activation of this receptor.

  PublisherOA PDFDOI

• A Chalcone Synthase-like Bacterial Protein Catalyzes Heterocyclic C-Ring Cleavage of Naringenin to Alter Bioactivity Against Nuclear Receptors in Colonic Epithelial Cells

  Metabolites · 2025-02-21 · 1 citations

  articleOpen access

  Gut microbial metabolism of dietary flavonoids leads to a diverse array of bioactive products that are closely associated with human health. Combining enzyme promiscuity prediction, metabolomics, and in vitro model systems, we identified a chalcone-synthase-like bacterial polyketide synthase that can initiate the metabolism of naringenin by catalyzing the C-ring cleavage. This was validated using a mutant strain of the model organism Bacillus subtilis (ATCC 23857). Our prediction–validation methodology could be used to systematically characterize the products of gut bacterial flavonoid metabolism and identify the responsible enzymes and species. In vitro experiments with Caco-2 cells revealed that naringenin and its bacterial metabolites differentially engage the aryl hydrocarbon receptor (AhR) and orphan nuclear receptor 4A (NR4A). These results suggest that metabolism by gut bacterial species could directly impact the profile of bioactive flavonoids and influence inflammatory responses in the intestine. These results are significant for understanding gut-microbiota-dependent physiological effects of dietary flavonoids.

  PublisherOA PDFDOI

Recent grants

• NIH Grant R01CA104116

  NIH · $682k · 2009

• NIH Grant R01CA076636

  NIH · $543k · 2003

• NIH Grant R01CA142697

  NIH · $1.3M · 2017

• NIH Grant R01CA136571

  NIH · $1.2M · 2015

• NIH Grant R01ES004176

  NIH · $2.7M · 2007

Frequent coauthors

• Maen Abdelrahim

  Houston Methodist

  252 shared

• Syng‐Ook Lee

  232 shared

• Sudhakar Chintharlapalli

  221 shared

• Kumaravel Mohankumar

  131 shared

• Kyounghyun Kim

  University of Arkansas for Medical Sciences

  129 shared

• Kyungsil Yoon

  127 shared

• Sabitha Papineni

  122 shared

• Erik Hedrick

  97 shared