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Pain Jan 2024The persistence of inflammatory and neuropathic pain is poorly understood. We investigated a novel therapeutic paradigm by targeting gene networks that sustain or...
The persistence of inflammatory and neuropathic pain is poorly understood. We investigated a novel therapeutic paradigm by targeting gene networks that sustain or reverse persistent pain states. Our prior observations found that Sp1-like transcription factors drive the expression of TRPV1, a pain receptor, that is blocked in vitro by mithramycin A (MTM), an inhibitor of Sp1-like factors. Here, we investigate the ability of MTM to reverse in vivo models of inflammatory and chemotherapy-induced peripheral neuropathy (CIPN) pain and explore MTM's underlying mechanisms. Mithramycin reversed inflammatory heat hyperalgesia induced by complete Freund adjuvant and cisplatin-induced heat and mechanical hypersensitivity. In addition, MTM reversed both short-term and long-term (1 month) oxaliplatin-induced mechanical and cold hypersensitivity, without the rescue of intraepidermal nerve fiber loss. Mithramycin reversed oxaliplatin-induced cold hypersensitivity and oxaliplatin-induced TRPM8 overexpression in dorsal root ganglion (DRG). Evidence across multiple transcriptomic profiling approaches suggest that MTM reverses inflammatory and neuropathic pain through broad transcriptional and alternative splicing regulatory actions. Mithramycin-dependent changes in gene expression following oxaliplatin treatment were largely opposite to and rarely overlapped with changes in gene expression induced by oxaliplatin alone. Notably, RNAseq analysis revealed MTM rescue of oxaliplatin-induced dysregulation of mitochondrial electron transport chain genes that correlated with in vivo reversal of excess reactive oxygen species in DRG neurons. This finding suggests that the mechanism(s) driving persistent pain states such as CIPN are not fixed but are sustained by ongoing modifiable transcription-dependent processes.
Topics: Humans; Plicamycin; Oxaliplatin; Antineoplastic Agents; Neuralgia; Hyperalgesia; Ganglia, Spinal
PubMed: 37366593
DOI: 10.1097/j.pain.0000000000002972 -
Journal of Experimental & Clinical... Apr 2023MiT-Renal Cell Carcinoma (RCC) is characterized by genomic translocations involving microphthalmia-associated transcription factor (MiT) family members TFE3, TFEB, or...
BACKGROUND
MiT-Renal Cell Carcinoma (RCC) is characterized by genomic translocations involving microphthalmia-associated transcription factor (MiT) family members TFE3, TFEB, or MITF. MiT-RCC represents a specific subtype of sporadic RCC that is predominantly seen in young patients and can present with heterogeneous histological features making diagnosis challenging. Moreover, the disease biology of this aggressive cancer is poorly understood and there is no accepted standard of care therapy for patients with advanced disease. Tumor-derived cell lines have been established from human TFE3-RCC providing useful models for preclinical studies.
METHODS
TFE3-RCC tumor derived cell lines and their tissues of origin were characterized by IHC and gene expression analyses. An unbiased high-throughput drug screen was performed to identify novel therapeutic agents for treatment of MiT-RCC. Potential therapeutic candidates were validated in in vitro and in vivo preclinical studies. Mechanistic assays were conducted to confirm the on-target effects of drugs.
RESULTS
The results of a high-throughput small molecule drug screen utilizing three TFE3-RCC tumor-derived cell lines identified five classes of agents with potential pharmacological efficacy, including inhibitors of phosphoinositide-3-kinase (PI3K) and mechanistic target of rapamycin (mTOR), and several additional agents, including the transcription inhibitor Mithramycin A. Upregulation of the cell surface marker GPNMB, a specific MiT transcriptional target, was confirmed in TFE3-RCC and evaluated as a therapeutic target using the GPNMB-targeted antibody-drug conjugate CDX-011. In vitro and in vivo preclinical studies demonstrated efficacy of the PI3K/mTOR inhibitor NVP-BGT226, Mithramycin A, and CDX-011 as potential therapeutic options for treating advanced MiT-RCC as single agents or in combination.
CONCLUSIONS
The results of the high-throughput drug screen and validation studies in TFE3-RCC tumor-derived cell lines have provided in vitro and in vivo preclinical data supporting the efficacy of the PI3K/mTOR inhibitor NVP-BGT226, the transcription inhibitor Mithramycin A, and GPNMB-targeted antibody-drug conjugate CDX-011 as potential therapeutic options for treating advanced MiT-RCC. The findings presented here should provide the basis for designing future clinical trials for patients with MiT-driven RCC.
Topics: Humans; Carcinoma, Renal Cell; Kidney Neoplasms; MTOR Inhibitors; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Translocation, Genetic; Phosphatidylinositol 3-Kinase; Membrane Glycoproteins
PubMed: 37095531
DOI: 10.1186/s13046-023-02667-4 -
The Journal of Nutritional Biochemistry Jul 2023The abnormal iron metabolism in liver cancer leads to iron deficiency in tumor tissues. We previously found that iron deficiency promoted liver cancer metastasis, but...
The abnormal iron metabolism in liver cancer leads to iron deficiency in tumor tissues. We previously found that iron deficiency promoted liver cancer metastasis, but the mechanisms were not fully understood. In the present study, we identified that the angiogenesis-associated glutamyl aminopeptidase (ENPEP) was consistently decreased in iron-deficient liver tissues, iron-deficient liver tumors, and iron-deprived liver cancer cells. Interestingly, the lower expression of ENPEP was correlated with the poor prognosis of liver cancer patients, while the biomarkers of angiogenesis, CD31 and CD34, were increased in tumor tissues. In vivo imaging of liver-orthotopically implanted and tail vein-injected liver cancer cells showed that iron deficiency increased the pulmonary metastasis of liver cancer. The angiogenesis in iron-deficient tumors was enhanced, and the expression of ENPEP was decreased. Silencing ENPEP expression increased the migration of liver cancer cells and the proliferation of cocultured HUVECs. By sequence analysis, we found that the transcription factor SP1 possessed abundant binding sites in the ENPEP promoter region. Its combination with ENPEP promoters was verified by chromatin immunoprecipitation. The inhibition of SP1 by mithramycin A effectively restored the expression of ENPEP, which was decreased by iron deficiency. In conclusion, these results revealed that iron deficiency in liver tumors decreased the expression of ENPEP by SP1 and increased the angiogenesis and metastasis of liver tumors, which further explained the mechanism by which iron deficiency promoted liver cancer metastasis.
Topics: Humans; Cell Line; Plicamycin; Iron Deficiencies; Iron; Liver Neoplasms; Gene Expression Regulation, Neoplastic; Cell Line, Tumor
PubMed: 37085059
DOI: 10.1016/j.jnutbio.2023.109357 -
Molecular Metabolism Jun 2023A buildup of skeletal muscle plasma membrane (PM) cholesterol content in mice occurs within 1 week of a Western-style high-fat diet and causes insulin resistance. The...
OBJECTIVE
A buildup of skeletal muscle plasma membrane (PM) cholesterol content in mice occurs within 1 week of a Western-style high-fat diet and causes insulin resistance. The mechanism driving this cholesterol accumulation and insulin resistance is not known. Promising cell data implicate that the hexosamine biosynthesis pathway (HBP) triggers a cholesterolgenic response via increasing the transcriptional activity of Sp1. In this study we aimed to determine whether increased HBP/Sp1 activity represented a preventable cause of insulin resistance.
METHODS
C57BL/6NJ mice were fed either a low-fat (LF, 10% kcal) or high-fat (HF, 45% kcal) diet for 1 week. During this 1-week diet the mice were treated daily with either saline or mithramycin-A (MTM), a specific Sp1/DNA-binding inhibitor. A series of metabolic and tissue analyses were then performed on these mice, as well as on mice with targeted skeletal muscle overexpression of the rate-limiting HBP enzyme glutamine-fructose-6-phosphate-amidotransferase (GFAT) that were maintained on a regular chow diet.
RESULTS
Saline-treated mice fed this HF diet for 1 week did not have an increase in adiposity, lean mass, or body mass while displaying early insulin resistance. Consistent with an HBP/Sp1 cholesterolgenic response, Sp1 displayed increased O-GlcNAcylation and binding to the HMGCR promoter that increased HMGCR expression in skeletal muscle from saline-treated HF-fed mice. Skeletal muscle from these saline-treated HF-fed mice also showed a resultant elevation of PM cholesterol with an accompanying loss of cortical filamentous actin (F-actin) that is essential for insulin-stimulated glucose transport. Treating these mice daily with MTM during the 1-week HF diet fully prevented the diet-induced Sp1 cholesterolgenic response, loss of cortical F-actin, and development of insulin resistance. Similarly, increases in HMGCR expression and cholesterol were measured in muscle from GFAT transgenic mice compared to age- and weight-match wildtype littermate control mice. In the GFAT Tg mice we found that these increases were alleviated by MTM.
CONCLUSIONS
These data identify increased HBP/Sp1 activity as an early mechanism of diet-induced insulin resistance. Therapies targeting this mechanism may decelerate T2D development.
Topics: Mice; Animals; Insulin Resistance; Actins; Mice, Inbred C57BL; Cholesterol; Diet, High-Fat; Mice, Transgenic; Hexosamines
PubMed: 37019209
DOI: 10.1016/j.molmet.2023.101715 -
Biomedicine & Pharmacotherapy =... Jun 2023Osteosarcomas are frequently associated to a poor prognosis and a modest response to current treatments. EC-8042 is a well-tolerated mithramycin analog that has...
Osteosarcomas are frequently associated to a poor prognosis and a modest response to current treatments. EC-8042 is a well-tolerated mithramycin analog that has demonstrated an efficient ability to eliminate tumor cells, including cancer stem cell subpopulations (CSC), in sarcomas. In transcriptomic and protein expression analyses, we identified NOTCH1 signaling as one of the main pro-stemness pathways repressed by EC-8042 in osteosarcomas. Overexpression of NOTCH-1 resulted in a reduced anti-tumor effect of EC-8042 in CSC-enriched 3D tumorspheres cultures. On the other hand, the depletion of the NOTCH-1 downstream target HES-1 was able to enhance the action of EC-8042 on CSCs. Moreover, HES1 depleted cells failed to recover after treatment withdrawal and showed reduced tumor growth potential in vivo. In contrast, mice xenografted with NOTCH1-overexpressing cells responded worse than parental cells to EC-8042. Finally, we found that active NOTCH1 levels in sarcoma patients was associated to advanced disease and lower survival. Overall, these data highlight the relevant role that NOTCH1 signaling plays in mediating stemness in osteosarcoma. Moreover, we demonstrate that EC-8042 is powerful inhibitor of NOTCH signaling and that the anti-CSC activity of this mithramycin analog highly rely on its ability to repress this pathway.
Topics: Animals; Mice; Bone Neoplasms; Cell Line, Tumor; Neoplastic Stem Cells; Osteosarcoma; Plicamycin; Receptor, Notch1; Receptors, Notch
PubMed: 37018985
DOI: 10.1016/j.biopha.2023.114627 -
Clinical and Translational Medicine Mar 2023Atherosclerosis (AS), the main pathological basis of life-threatening cardiovascular disease, is essentially caused by chronic macrophage inflammation. Overexpression of...
BACKGROUND
Atherosclerosis (AS), the main pathological basis of life-threatening cardiovascular disease, is essentially caused by chronic macrophage inflammation. Overexpression of proline/serine-rich coiled-coil protein 1 (PSRC1) reduces macrophage inflammatory responses and delays AS development. However, the exact mechanism of PSRC1 is unclear.
METHODS
Proteins interacting with PSRC1 were screened by proteomics in RAW264.7 cells, followed by RT-qPCR, immunoprecipitation and immunofluorescence to explore the specific mechanistic pathways affecting inflammation. CRISPR-Cas9 constructs for PSRC1 ApoE (DKO) mice and high-fat diet-fed ApoE and DKO mice were used for AS models for in vivo experiments. Upstream transcription factors of PSRC1 were predicted by ATAC-seq, ChIP-seq and UCSC, and the regulatory mechanism was verified by ChIP-qPCR and dual luciferase assays. Peripheral blood serum and monocytes were collected from coronary artery disease (CAD) patients and non-CAD patients.
RESULTS
Increased binding of ANXA2 to PSRC1 in macrophages under oxidized low-density lipoprotein stimulation and decreased release of ANXA2 to the extracellular compartment were observed. Knockdown of ANXA2 in AS model mice delayed AS progression. Knockdown of ANXA2 in DKO mice reversed the AS-promoting effect of PSRC1 knockdown. Mechanistically, ANXA2 promotes STAT3 phosphorylation, which in turn promotes inflammatory responses. In addition, SP1 is a PSRC1 upstream repressive transcription factor, and the SP1 inhibitor mithramycin (Mith) elevated PSRC1 expression and exerted anti-AS effects in AS model mice. Patients with CAD had considerably greater serum levels of ANXA2 than those without CAD, and Mith reduced the secretion of ANXA2 in peripheral blood monocytes of CAD patients.
CONCLUSION
In macrophages, PSRC1 can interact with ANXA2 to inhibit its extracellular release and delay AS development. SP1 is an upstream transcription factor of PSRC1 and inhibits the transcription of PSRC1. The SP1 inhibitor Mith can elevate PSRC1 levels and slow AS progression while reducing ANXA2 release from monocytes in CAD patients. Mith is expected to be a new agent for AS treatment.
Topics: Animals; Mice; Annexin A2; Atherosclerosis; Coronary Artery Disease; Inflammation; Macrophages; Proline; Serine; Transcription Factors; Phosphoproteins; Mice, Knockout, ApoE
PubMed: 36932468
DOI: 10.1002/ctm2.1220 -
International Journal of Radiation... 2023The oncogenic fusion protein EWS::FLI1 is an attractive therapeutic target in Ewing sarcoma (ES). Mithramycin A (MithA) is a potent and specific inhibitor of EWS::FLI1...
PURPOSE
The oncogenic fusion protein EWS::FLI1 is an attractive therapeutic target in Ewing sarcoma (ES). Mithramycin A (MithA) is a potent and specific inhibitor of EWS::FLI1 that can selectively radiosensitize ES cells through transcriptional inhibition of DNA double-strand break (DSB) repair. Here, we evaluate temporal changes in cell cycle progression and apoptosis in ES cells treated with MithA and/or ionizing radiation (RTx), testing the hypothesis that combining MithA with ionizing radiation would synergistically impair cell cycle progression and enhance apoptotic elimination to a greater extent than either agent alone.
MATERIALS AND METHODS
Four EWS::FLI1 ES cell lines TC-71, RD-ES, SK-ES-1, and A673, and one EWS::ERG cell line (CHLA-25) were exposed to 10nM MithA or vehicle and followed 24 h later by exposure to 2 Gy x-radiation or sham irradiation. Reactive oxygen species (ROS) activity was evaluated by cytometric assay, and assay of antioxidant gene expression by RT-qPCR. Cell cycle changes were evaluated by flow cytometry of nuclei stained with propidium iodide. Apoptosis was assessed by cytometric assessment of Caspase-3/7 activity and by immunoblotting of PARP-1 cleavage. Radiosensitization was evaluated by clonogenic survival assay. Proliferation (EdU) and apoptosis (TUNEL) were evaluated in SK-ES-1 xenograft tumors following pretreatment with 1 mg/kg MithA, followed 24 h later by a single 4 Gy fraction of x-radiation.
RESULTS
MithA-treated cells showed reduced levels of ROS, and were associated with increased expression of antioxidant genes , and . It nonetheless induced persistent G/G arrest and a progressive increase of the sub-G fraction, suggesting apoptotic degeneration. assays of Caspase-3/7 activity and immunoblotting of Caspase-3/7 dependent cleavage of PARP-1 indicated that apoptosis began as early as 24 h after MithA exposure, reducing clonogenic survival. Tumors from xenograft mice treated with either radiation alone, or in combination with MithA showed a significant reduction of tumor cell proliferation, while apoptosis was significantly increased in the group receiving the combination of MithA and RTx.
CONCLUSIONS
Taken together, our data show that the anti-proliferative and cytotoxic effects of MithA are the prominent components of radiosensitization of EWS::FLI1 ES, rather than the result of acutely enhanced ROS levels.
Topics: Humans; Mice; Animals; Sarcoma, Ewing; Poly(ADP-ribose) Polymerase Inhibitors; Reactive Oxygen Species; Antioxidants; Caspase 3; Cell Cycle Checkpoints; Apoptosis; Cell Line, Tumor; Gene Expression Regulation, Neoplastic
PubMed: 36913323
DOI: 10.1080/09553002.2023.2188930 -
Laboratory Investigation; a Journal of... Apr 2023Although linked to esophageal carcinogenesis, the mechanisms by which cigarette smoke mediates initiation and progression of esophageal adenocarcinomas (EAC) have not...
Although linked to esophageal carcinogenesis, the mechanisms by which cigarette smoke mediates initiation and progression of esophageal adenocarcinomas (EAC) have not been fully elucidated. In this study, immortalized esophageal epithelial cells and EAC cells (EACCs) were cultured with or without cigarette smoke condensate (CSC) under relevant exposure conditions. Endogenous levels of microRNA (miR)-145 and lysyl-likeoxidase 2 (LOXL2) were inversely correlated in EAC lines/tumors compared with that in immortalized cells/normal mucosa. The CSC repressed miR-145 and upregulated LOXL2 in immortalized esophageal epithelial cells and EACCs. Knockdown or constitutive overexpression of miR-145 activated or depleted LOXL2, respectively, which enhanced or reduced proliferation, invasion, and tumorigenicity of EACC, respectively. LOXL2 was identified as a novel target of miR-145 as well as a negative regulator of this miR in EAC lines/Barrett's epithelia. Mechanistically, CSC induced recruitment of SP1 to the LOXL2 promoter; LOXL2 upregulation coincided with LOXL2 enrichment and concomitant reduction of H3K4me3 levels within the promoter of miR143HG (host gene for miR-145). Mithramycin downregulated LOXL2 and restored miR-145 expression in EACC and abrogated LOXL2-mediated repression of miR-145 by CSC. These findings implicate cigarette smoke in the pathogenesis of EAC and demonstrate that oncogenic miR-145-LOXL2 axis dysregulation is potentially druggable for the treatment and possible prevention of these malignancies.
Topics: Cigarette Smoking; Adenocarcinoma; Esophageal Neoplasms; Nicotiana; MicroRNAs; Phenotype; Gene Expression Regulation, Neoplastic
PubMed: 36870293
DOI: 10.1016/j.labinv.2022.100014 -
Cancer Cell International Feb 2023Arginine auxotrophy constitutes a shortcoming for ~ 30% of glioblastoma multiforme (GBM). Indeed, arginine-depleting therapy using arginine deiminase from...
BACKGROUND
Arginine auxotrophy constitutes a shortcoming for ~ 30% of glioblastoma multiforme (GBM). Indeed, arginine-depleting therapy using arginine deiminase from Streptococcus pyogenes (SpyADI) has proven activity against GBM in preclinical studies. The good safety profile of SpyADI renders this agent an ideal combination partner for cytostatic therapy.
METHODS
In this study, we combined the antineoplastic antibiotic Mithramycin A (MitA) with SpyADI to boost single-agent activity and analyzed underlying response mechanisms in-depth.
RESULTS
MitA monotherapy induced a time- and dose-dependent cytotoxicity in eight patient-derived GBM cell lines and had a radiosensitizing effect in all but one cell line. Combination treatment boosted the effects of the monotherapy in 2D- and 3D models. The simultaneous approach was superior to the sequential application and significantly impaired colony formation after repetitive treatment. MitA monotherapy significantly inhibited GBM invasiveness. However, this effect was not enhanced in the combination. Functional analysis identified SpyADI-triggered senescence induction accompanied by increased mitochondrial membrane polarization upon mono- and combination therapy. In HROG63, induction of lysosomes was seen after both monotherapies, indicative of autophagy. These cells seemed swollen and had a more pronounced cortically formed cytoskeleton. Also, cytochrome C and endoplasmatic reticulum-stress-associated proteins ATF4 and Calnexin were enhanced in the combination, contributing to apoptosis. Notably, no significant increases in glioma-stemness marker were seen.
CONCLUSIONS
Therapeutic utilization of a metabolic defect in GBM along with cytostatic therapy provides a novel combination approach. Whether this SpyADI/MitA regimen will provide a safe alternative to combat GBM, will have to be addressed in subsequent (pre-)clinical trials.
PubMed: 36843002
DOI: 10.1186/s12935-023-02873-2 -
FASEB Journal : Official Publication of... Mar 2023The dysfunction of CRALBP, a key regulator of the visual cycle, is associated with retinitis punctata albescens characterized by night vision loss and retinal...
The dysfunction of CRALBP, a key regulator of the visual cycle, is associated with retinitis punctata albescens characterized by night vision loss and retinal degeneration. In this paper, we find that the expression of CRALBP is regulated by heat shock protein 90 (HSP90). Inhibition of HSP90α or HSP90β expression by using the CRISPR-Cas9 technology downregulates CRALBP's mRNA and protein expression in ARPE-19 cells by triggering the degradation of transcription factor SP1 in the ubiquitin-proteasome pathway. SP1 can bind to CRALBP's promoter, and inhibition of SP1 by its inhibitor plicamycin or siRNA downregulates CRALBP's mRNA expression. In the zebrafish, inhibition of HSP90 by the intraperitoneal injection of IPI504 reduces the thickness of the retinal outer nuclear layer and Rlbp1b mRNA expression. Interestingly, the expression of HSP90, SP1, and CRALBP is correlatedly downregulated in the senescent ARPE-19 and Pig primary RPE cells in vitro and in the aged zebrafish and mouse retinal tissues in vivo. The aged mice exhibit the low night adaption activity. Taken together, these data indicate that the HSP90-SP1 is a novel regulatory axis of CRALBP transcriptional expression in RPE cells. The age-mediated downregulation of the HSP90-SP1-CRALBP axis is a potential etiology for the night vision reduction in senior people.
Topics: Mice; Animals; Swine; Zebrafish; Down-Regulation; Vision, Ocular; Retina; Dark Adaptation; HSP90 Heat-Shock Proteins
PubMed: 36826429
DOI: 10.1096/fj.202201608RR