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1.
Genome Res ; 31(10): 1867-1884, 2021 10.
Artículo en Inglés | MEDLINE | ID: mdl-34301623

RESUMEN

The metabolic heterogeneity and metabolic interplay between cells are known as significant contributors to disease treatment resistance. However, with the lack of a mature high-throughput single-cell metabolomics technology, we are yet to establish systematic understanding of the intra-tissue metabolic heterogeneity and cooperative mechanisms. To mitigate this knowledge gap, we developed a novel computational method, namely, single-cell flux estimation analysis (scFEA), to infer the cell-wise fluxome from single-cell RNA-sequencing (scRNA-seq) data. scFEA is empowered by a systematically reconstructed human metabolic map as a factor graph, a novel probabilistic model to leverage the flux balance constraints on scRNA-seq data, and a novel graph neural network-based optimization solver. The intricate information cascade from transcriptome to metabolome was captured using multilayer neural networks to capitulate the nonlinear dependency between enzymatic gene expressions and reaction rates. We experimentally validated scFEA by generating an scRNA-seq data set with matched metabolomics data on cells of perturbed oxygen and genetic conditions. Application of scFEA on this data set showed the consistency between predicted flux and the observed variation of metabolite abundance in the matched metabolomics data. We also applied scFEA on five publicly available scRNA-seq and spatial transcriptomics data sets and identified context- and cell group-specific metabolic variations. The cell-wise fluxome predicted by scFEA empowers a series of downstream analyses including identification of metabolic modules or cell groups that share common metabolic variations, sensitivity evaluation of enzymes with regards to their impact on the whole metabolic flux, and inference of cell-tissue and cell-cell metabolic communications.


Asunto(s)
Análisis de la Célula Individual , Transcriptoma , Perfilación de la Expresión Génica/métodos , Redes Neurales de la Computación , RNA-Seq , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , Secuenciación del Exoma
2.
Pharmacol Res ; 201: 107092, 2024 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-38311014

RESUMEN

AP endonuclease-1/Redox factor-1 (APE1/Ref-1 or Ref-1) is a multifunctional protein that is overexpressed in most aggressive cancers and impacts various cancer cell signaling pathways. Ref-1's redox activity plays a significant role in activating transcription factors (TFs) such as NFκB, HIF1α, STAT3 and AP-1, which are crucial contributors to the development of tumors and metastatic growth. Therefore, development of potent, selective inhibitors to target Ref-1 redox function is an appealing approach for therapeutic intervention. A first-generation compound, APX3330 successfully completed phase I clinical trial in adults with progressing solid tumors with favorable response rate, pharmacokinetics (PK), and minimal toxicity. These positive results prompted us to develop more potent analogs of APX3330 to effectively target Ref-1 in solid tumors. In this study, we present structure-activity relationship (SAR) identification and validation of lead compounds that exhibit a greater potency and a similar or better safety profile to APX3330. In order to triage and characterize the most potent and on-target second-generation Ref-1 redox inhibitors, we assayed for PK, mouse and human S9 fraction metabolic stability, in silico ADMET properties, ligand-based WaterLOGSY NMR measurements, pharmacodynamic markers, cell viability in multiple cancer cell types, and two distinct 3-dimensional (3D) cell killing assays (Tumor-Microenvironment on a Chip and 3D spheroid). To characterize the effects of Ref-1 inhibition in vivo, global proteomics was used following treatment with the top four analogs. This study identified and characterized more potent inhibitors of Ref-1 redox function (that outperformed APX3330 by 5-10-fold) with PK studies demonstrating efficacious doses for translation to clinic.


Asunto(s)
ADN-(Sitio Apurínico o Apirimidínico) Liasa , Neoplasias , Adulto , Humanos , Animales , Ratones , Inhibidores de la Angiogénesis , Apoptosis , Bioensayo , Neoplasias/tratamiento farmacológico , Microambiente Tumoral
3.
Br J Cancer ; 124(9): 1566-1580, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33658640

RESUMEN

BACKGROUND: MPNST is a rare soft-tissue sarcoma that can arise from patients with NF1. Existing chemotherapeutic and targeted agents have been unsuccessful in MPNST treatment, and recent findings implicate STAT3 and HIF1-α in driving MPNST. The DNA-binding and transcriptional activity of both STAT3 and HIF1-α is regulated by Redox factor-1 (Ref-1) redox function. A first-generation Ref-1 inhibitor, APX3330, is being tested in cancer clinical trials and could be applied to MPNST. METHODS: We characterised Ref-1 and p-STAT3 expression in various MPNST models. Tumour growth, as well as biomarkers of apoptosis and signalling pathways, were measured by qPCR and western blot following treatment with inhibitors of Ref-1 or STAT3. RESULTS: MPNSTs from Nf1-Arfflox/floxPostnCre mice exhibit significantly increased positivity of p-STAT3 and Ref-1 expression when malignant transformation occurs. Inhibition of Ref-1 or STAT3 impairs MPNST growth in vitro and in vivo and induces apoptosis. Genes highly expressed in MPNST patients are downregulated following inhibition of Ref-1 or STAT3. Several biomarkers downstream of Ref-1 or STAT3 were also downregulated following Ref-1 or STAT3 inhibition. CONCLUSIONS: Our findings implicate a unique therapeutic approach to target important MPNST signalling nodes in sarcomas using new first-in-class small molecules for potential translation to the clinic.


Asunto(s)
Biomarcadores de Tumor/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Regulación Neoplásica de la Expresión Génica , Neurofibrosarcoma/patología , Factor de Transcripción STAT3/metabolismo , Adolescente , Animales , Apoptosis , Biomarcadores de Tumor/genética , Proliferación Celular , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Neurofibrosarcoma/genética , Neurofibrosarcoma/metabolismo , Pronóstico , Factor de Transcripción STAT3/genética , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto
5.
Front Med (Lausanne) ; 10: 1146115, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37181357

RESUMEN

Pancreatic cancer or pancreatic ductal adenocarcinoma (PDAC) is characterized by a profound inflammatory tumor microenvironment (TME) with high heterogeneity, metastatic propensity, and extreme hypoxia. The integrated stress response (ISR) pathway features a family of protein kinases that phosphorylate eukaryotic initiation factor 2 (eIF2) and regulate translation in response to diverse stress conditions, including hypoxia. We previously demonstrated that eIF2 signaling pathways were profoundly affected in response to Redox factor-1 (Ref-1) knockdown in human PDAC cells. Ref-1 is a dual function enzyme with activities of DNA repair and redox signaling, responds to cellular stress, and regulates survival pathways. The redox function of Ref-1 directly regulates multiple transcription factors including HIF-1α, STAT3, and NF-κB, which are highly active in the PDAC TME. However, the mechanistic details of the crosstalk between Ref-1 redox signaling and activation of ISR pathways are unclear. Following Ref-1 knockdown, induction of ISR was observed under normoxic conditions, while hypoxic conditions were sufficient to activate ISR irrespective of Ref-1 levels. Inhibition of Ref-1 redox activity increased expression of p-eIF2 and ATF4 transcriptional activity in a concentration-dependent manner in multiple human PDAC cell lines, and the effect on eIF2 phosphorylation was PERK-dependent. Treatment with PERK inhibitor, AMG-44 at high concentrations resulted in activation of the alternative ISR kinase, GCN2 and induced levels of p-eIF2 and ATF4 in both tumor cells and cancer-associated fibroblasts (CAFs). Combination treatment with inhibitors of Ref-1 and PERK enhanced cell killing effects in both human pancreatic cancer lines and CAFs in 3D co-culture, but only at high doses of PERK inhibitors. This effect was completely abrogated when Ref-1 inhibitors were used in combination with GCN2 inhibitor, GCN2iB. We demonstrate that targeting of Ref-1 redox signaling activates the ISR in multiple PDAC lines and that this activation of ISR is critical for inhibition of the growth of co-culture spheroids. Combination effects were only observed in physiologically relevant 3D co-cultures, suggesting that the model system utilized can greatly affect the outcome of these targeted agents. Inhibition of Ref-1 signaling induces cell death through ISR signaling pathways, and combination of Ref-1 redox signaling blockade with ISR activation could be a novel therapeutic strategy for PDAC treatment.

6.
Front Oncol ; 12: 826617, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35402225

RESUMEN

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers with a poor response to current treatment regimens. The multifunctional DNA repair-redox signaling protein Ref-1 has a redox signaling function that activates several transcriptional factors (TFs) including NF-κB (RelA), STAT3, AP-1. These have been implicated in signaling in PDAC and associated with cancer progression and therapy resistance. Numerous studies have shown a role for RelA in PDAC inflammatory responses and therapy resistance, little is known as to how these inflammatory responses are modulated through Ref-1 redox signaling pathways during pancreatic pathogenesis. RelA and STAT3 are two major targets of Ref-1 and are important in PDAC pathogenesis. To decipher the mechanistic role of RelA in response to Ref-1 inhibition, we used PDAC cells (KC3590) from a genetically engineered Kras G12D-driven mouse model that also is functionally deficient for RelA (Parent/Vector) or KC3590 cells with fully functional RelA added back (clone 13; C13). We demonstrated that RelA deficient cells are more resistant to Ref-1 redox inhibitors APX3330, APX2009, and APX2014, and their sensitivity is restored in the RelA proficient cells. Knockdown of STAT3 did not change cellular sensitivity to Ref-1 redox inhibitors in either cell type. Gene expression analysis demonstrated that Ref-1 inhibitors significantly decreased IL-8, FOSB, and c-Jun when functional RelA is present. We also demonstrated that PRDX1, a known Ref-1 redox modulator, contributes to Ref-1 inhibitor cellular response. Knockdown of PRDX1 when functional RelA is present resulted in dramatically increased PDAC killing in response to Ref-1 inhibitors. The enhanced cell killing was not due to increased intracellular ROS production. Although Ref-1 inhibition decreased the NADP/NADPH ratio in the cells, the addition of PRDX1 knockdown did not further this redox imbalance. This data suggests that the mechanism of cell killing following Ref-1 inhibition is at least partially mediated through RelA and not STAT3. Further imbalancing of the redox signaling through disruption of the PRDX1-Ref-1 interaction may have therapeutic implications. Our data further support a pivotal role of RelA in mediating Ref-1 redox signaling in PDAC cells with the Kras G12D genotype and provide novel therapeutic strategies to combat PDAC drug resistance.

7.
Cells ; 10(11)2021 11 12.
Artículo en Inglés | MEDLINE | ID: mdl-34831357

RESUMEN

Targeting the hedgehog (HH) pathway to treat aggressive cancers of the brain, breast, pancreas, and prostate has been ongoing for decades. Gli gene amplifications have been long discovered within malignant glioma patients, and since then, inhibitors against HH pathway-associated molecules have successfully reached the clinical stage where several of them have been approved by the FDA. Albeit this success rate implies suitable progress, clinically used HH pathway inhibitors fail to treat patients with metastatic or recurrent disease. This is mainly due to heterogeneous tumor cells that have acquired resistance to the inhibitors along with the obstacle of effectively targeting the tumor microenvironment (TME). Severe side effects such as hyponatremia, diarrhea, fatigue, amenorrhea, nausea, hair loss, abnormal taste, and weight loss have also been reported. Furthermore, HH signaling is known to be involved in the regulation of immune cell maturation, angiogenesis, inflammation, and polarization of macrophages and myeloid-derived suppressor cells. It is critical to determine key mechanisms that can be targeted at different levels of tumor development and progression to address various clinical issues. Hence current research focus encompasses understanding how HH controls TME to develop TME altering and combinatorial targeting strategies. In this review, we aim to discuss the pros and cons of targeting HH signaling molecules, understand the mechanism involved in treatment resistance, reveal the role of the HH pathway in anti-tumor immune response, and explore the development of potential combination treatment of immune checkpoint inhibitors with HH pathway inhibitors to target HH-driven cancers.


Asunto(s)
Proteínas Hedgehog/antagonistas & inhibidores , Transducción de Señal , Microambiente Tumoral , Animales , Antineoplásicos/farmacología , Proteínas Hedgehog/metabolismo , Humanos , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Inmunidad/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/inmunología
8.
FASEB Bioadv ; 3(6): 459-469, 2021 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-34124601

RESUMEN

Vismodegib, a Smoothened antagonist, is clinically approved for treatment of human basal cell carcinoma (BCC), in the clinical trials of medulloblastoma (MB) and other cancers. However, a significant proportion of these tumors fail to respond to Vismodegib after a period of treatment. Here, we find that AMPK agonists, A769662, and Metformin, can inhibit GLI1 activity and synergize with Vismodegib to suppress MB cell growth in vitro and in vivo. Furthermore, combination of AMPK agonists with Vismodegib is effective in overcoming Vismodegib-resistant MB. This is the first report demonstrating that combining AMPK agonist (Metformin) and SHH pathway inhibitor (Vismodegib) confers synergy for MB treatment and provides an effective chemotherapeutic regimen that can be used to overcome resistance to Vismodegib in SHH-driven cancers.

9.
J Cell Signal ; 2(3): 151-161, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34557865

RESUMEN

In the realm of DNA repair, base excision repair (BER) protein, APE1/Ref-1 (Apurinic/Apyrimidinic Endonuclease 1/Redox Effector - 1, also called APE1) has been studied for decades. However, over the past decade, APE1 has been established as a key player in reduction-oxidation (redox) signaling. In the review by Caston et al. (The multifunctional APE1 DNA repair-redox signaling protein as a drug target in human disease), multiple roles of APE1 in cancer and other diseases are summarized. In this Review, we aim to expand on the contributions of APE1 to various diseases and its effect on disease progression. In the scope of cancer, more recent roles for APE1 have been identified in cancer cell metabolism, as well as chemotherapy-induced peripheral neuropathy (CIPN) and inflammation. Outside of cancer, APE1 signaling may be a critical factor in inflammatory bowel disease (IBD) and is also an emergent area of investigation in retinal ocular diseases. The ability of APE1 to regulate multiple transcription factors (TFs) and therefore multiple pathways that have implications outside of cancer, makes it a particularly unique and enticing target. We discuss APE1 redox inhibitors as a means of studying and potentially combating these diseases. Lastly, we examine the role of APE1 in RNA metabolism. Overall, this article builds on our previous review to elaborate on the roles and conceivable regulation of important pathways by APE1 in multiple diseases.

10.
Drug Discov Today ; 26(1): 218-228, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-33148489

RESUMEN

Apurinic/apyrimidinic (AP) endonuclease-reduction/oxidation factor 1 (APE1/Ref-1, also called APE1) is a multifunctional enzyme with crucial roles in DNA repair and reduction/oxidation (redox) signaling. APE1 was originally described as an endonuclease in the Base Excision Repair (BER) pathway. Further study revealed it to be a redox signaling hub regulating critical transcription factors (TFs). Although a significant amount of focus has been on the role of APE1 in cancer, recent findings support APE1 as a target in other indications, including ocular diseases [diabetic retinopathy (DR), diabetic macular edema (DME), and age-related macular degeneration (AMD)], inflammatory bowel disease (IBD) and others, where APE1 regulation of crucial TFs impacts important pathways in these diseases. The central responsibilities of APE1 in DNA repair and redox signaling make it an attractive therapeutic target for cancer and other diseases.


Asunto(s)
Reparación del ADN , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Descubrimiento de Drogas , Humanos , Terapia Molecular Dirigida/métodos , Oxidación-Reducción/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Factores de Transcripción/metabolismo
11.
J Exp Clin Cancer Res ; 40(1): 251, 2021 Aug 10.
Artículo en Inglés | MEDLINE | ID: mdl-34376225

RESUMEN

BACKGROUND: Pancreatic cancer is a complex disease with a desmoplastic stroma, extreme hypoxia, and inherent resistance to therapy. Understanding the signaling and adaptive response of such an aggressive cancer is key to making advances in therapeutic efficacy. Redox factor-1 (Ref-1), a redox signaling protein, regulates the conversion of several transcription factors (TFs), including HIF-1α, STAT3 and NFκB from an oxidized to reduced state leading to enhancement of their DNA binding. In our previously published work, knockdown of Ref-1 under normoxia resulted in altered gene expression patterns on pathways including EIF2, protein kinase A, and mTOR. In this study, single cell RNA sequencing (scRNA-seq) and proteomics were used to explore the effects of Ref-1 on metabolic pathways under hypoxia. METHODS: scRNA-seq comparing pancreatic cancer cells expressing less than 20% of the Ref-1 protein was analyzed using left truncated mixture Gaussian model and validated using proteomics and qRT-PCR. The identified Ref-1's role in mitochondrial function was confirmed using mitochondrial function assays, qRT-PCR, western blotting and NADP assay. Further, the effect of Ref-1 redox function inhibition against pancreatic cancer metabolism was assayed using 3D co-culture in vitro and xenograft studies in vivo. RESULTS: Distinct transcriptional variation in central metabolism, cell cycle, apoptosis, immune response, and genes downstream of a series of signaling pathways and transcriptional regulatory factors were identified in Ref-1 knockdown vs Scrambled control from the scRNA-seq data. Mitochondrial DEG subsets downregulated with Ref-1 knockdown were significantly reduced following Ref-1 redox inhibition and more dramatically in combination with Devimistat in vitro. Mitochondrial function assays demonstrated that Ref-1 knockdown and Ref-1 redox signaling inhibition decreased utilization of TCA cycle substrates and slowed the growth of pancreatic cancer co-culture spheroids. In Ref-1 knockdown cells, a higher flux rate of NADP + consuming reactions was observed suggesting the less availability of NADP + and a higher level of oxidative stress in these cells. In vivo xenograft studies demonstrated that tumor reduction was potent with Ref-1 redox inhibitor similar to Devimistat. CONCLUSION: Ref-1 redox signaling inhibition conclusively alters cancer cell metabolism by causing TCA cycle dysfunction while also reducing the pancreatic tumor growth in vitro as well as in vivo.


Asunto(s)
ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Neoplasias Pancreáticas/genética , Animales , Humanos , Ratones , Neoplasias Pancreáticas/patología , Transfección
12.
Cell Metab ; 29(4): 993-1002.e6, 2019 04 02.
Artículo en Inglés | MEDLINE | ID: mdl-30527740

RESUMEN

Mitochondria are dynamic organelles that have been linked to stem cell homeostasis. However, the mechanisms involved in mitochondrial regulation of stem cell fate determination remain elusive. Here we discover that epithelial-mesenchymal transition (EMT), a key process in cancer progression, induces mitochondrial fusion through regulation of the miR200c-PGC1α-MFN1 pathway. EMT-activated MFN1 forms a complex with PKCζ and is required for PKCζ-mediated NUMB phosphorylation and dissociation from the cortical membrane to direct asymmetric division of mammary stem cells, where fused mitochondria are tethered by MFN1-PKCζ to the cortical membrane and asymmetrically segregated to the stem cell-like progeny with enhanced glutathione synthesis and reactive oxygen species scavenging capacities, allowing sustaining of a self-renewing stem cell pool. Suppression of MFN1 expression leads to equal distribution of the fragmented mitochondria in both progenies that undergo symmetric luminal cell differentiation. Together, this study elucidates an essential role of mitofusin in stem cell fate determination to mediate EMT-associated stemness.


Asunto(s)
Polaridad Celular , Transición Epitelial-Mesenquimal , GTP Fosfohidrolasas/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Células Madre/citología , Células Madre/metabolismo , Animales , Línea Celular , Femenino , Humanos , Ratones , Ratones Noqueados , MicroARNs/metabolismo , Mitocondrias/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo
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