Biology subjects

Falzone, A.

Publications and source records attributed to Falzone, A..

7 recordsLinked to original sources

bioRxiv2023

Imaging the master regulator of the antioxidant response in non-small cell lung cancer with positron emission tomography

Mutations in the NRF2-KEAP1 pathway are common in non-small cell lung cancer (NSCLC) and confer broad-spectrum therapeutic resistance, leading to poor outcomes. The cystine/glutamate antiporter, system xc-, is one of the >200 cytoprotective proteins controlled by NRF2, which can be non-invasively imaged by (S)-4-(3-18F-fluoropropyl)--glutamate ([18F]FSPG) positron emission tomography (PET). Through genetic and pharmacologic manipulation, we show that [18F]FSPG provides a sensitive and specific marker of NRF2 activation in advanced preclinical models of NSCLC. We validate imaging readouts with metabolomic measurements of system xc- activity and their coupling to intracellular glutathione concentration. A redox gene signature was measured in patients from the TRACERx 421 cohort, suggesting an opportunity for patient stratification prior to imaging. Furthermore, we reveal that system xc- is a metabolic vulnerability that can be therapeutically targeted for sustained tumour growth suppression in aggressive NSCLC. Our results establish [18F]FSPG as predictive marker of therapy resistance in NSCLC and provide the basis for the clinical evaluation of both imaging and therapeutic agents that target this important antioxidant pathway.

cancer biology↗

bioRxiv2022

The Origin Of Cysteine and its Catabolism in Mammalian Tissues and Tumors

Cysteine plays critical roles in cellular biosynthesis, enzyme catalysis, and is an essential contributor to redox metabolism. While cultured cells are highly dependent on exogenous cystine for proliferation and survival, how diverse tissues obtain and use cysteine in vivo has not been characterized. We comprehensively interrogated cysteine metabolism in normal murine tissues and the cancer that arise from them using stable isotope 13C-serine and 13C-cystine tracing. We found that de novo cysteine synthesis was highest in normal liver and pancreas and absent in lung tissue. In tumors, cysteine synthesis was either inactive or downregulated during tumorigenesis. By contrast, cystine uptake and metabolism to downstream metabolites was a universal feature of normal tissues and tumors. Differences in cysteine catabolism were evident across tumor types, including glutathione synthesis. Thus, cystine is a major contributor to the cysteine pool in tumors and cysteine catabolic pathways are differentially active across tumor types.

cancer biology↗

bioRxiv2022

Distinct Nrf2 Signaling Thresholds Mediate Lung Tumor Initiation and Progression

Mutations in the KEAP1-NRF2 pathway occur in up to a third of non-small cell lung cancer (NSCLC) cases and often confer resistance to therapy and poor outcomes. Here, we developed murine alleles of the KEAP1 and NRF2 mutations found in human NSCLC and comprehensively interrogated their impact on tumor initiation and progression. Chronic Nrf2 stabilization by Keap1 or Nrf2 mutation was not sufficient to induce tumorigenesis, even in the absence of tumor suppressors p53 or Lkb1. When combined with KrasG12D/+, constitutive Nrf2 activation promoted lung tumor initiation and early progression of hyperplasia to low-grade tumors but impaired their progression to advanced-grade tumors, which was reversed by Nrf2 deletion. Finally, NRF2 overexpression in KEAP1 mutant NSCLC cell lines was detrimental to cell proliferation, viability, and anchorage-independent colony formation. Collectively, our results establish the context-dependence and activity threshold for NRF2 during the lung tumorigenic process. SignificanceThis study reports murine lung cancer models harboring mutations in the Keap1/Nrf2 pathway and highlights the context-dependent and diverse roles of Nrf2 during lung tumor initiation and progression.

cancer biology↗

bioRxiv2021

Genetic tools for the stable overexpression of circular RNAs

Circular RNAs (circRNAs) are a class of non-coding RNAs that feature a covalently closed ring structure formed through backsplicing. circRNAs are broadly expressed and contribute to biological processes through a variety of functions. Standard gain-of-function and loss-of-function approaches to study gene functions have significant limitations when studying circRNAs. Overexpression studies in particular suffer from the lack of efficient genetic tools. While mammalian expression plasmids enable transient overexpression of circRNAs in cultured cells, most cell biological studies require long-term ectopic expression. Here we report the development and characterization of genetic tools enabling stable circRNA overexpression in vitro and in vivo. We demonstrated that circRNA expression constructs can be delivered to cultured cells via transposons, whereas lentiviral vectors have limited utility for the delivery of circRNA constructs. We further showed that circRNA transposons can be supplied to mouse livers via hydrodynamic tail vein injection, resulting in ectopic circRNA expression in a hepatocellular carcinoma mouse model. Furthermore, we generated genetically engineered mice harboring circRNA expression constructs. We demonstrate that this approach enables constitutive, global circRNA overexpression as well as inducible circRNA expression directed specifically to melanocytes in a melanoma mouse model. Overall, these tools expand the genetic toolkit available for the functional characterization of circRNAs of interest.

molecular biology↗

bioRxiv2019

Inhibition of TXNRD or SOD1 overcomes NRF2-mediated resistance to β-lapachone

Alterations in the NRF2/KEAP1 pathway result in the constitutive activation of NRF2, leading to the aberrant induction of antioxidant and detoxification enzymes, including NQO1. The NQO1 bioactivatable agent {beta}-lapachone can target cells with high NQO1 expression but relies in the generation of reactive oxygen species (ROS), which are actively scavenged in cells with NRF2/KEAP1 mutations. However, whether NRF2/KEAP1 mutations influence the response to {beta}-lapachone treatment remains unknown. To address this question, we assessed the cytotoxicity of {beta}-lapachone in a panel of NSCLC cell lines bearing either wild-type or mutant KEAP1. We found that, despite overexpression of NQO1, KEAP1 mutant cells were resistant to {beta}-lapachone due to enhanced detoxification of ROS, which prevented DNA damage and cell death. To evaluate whether specific inhibition of the NRF2-regulated antioxidant enzymes could abrogate resistance to {beta}-lapachone, we systematically inhibited the four major antioxidant cellular systems using genetic and/or pharmacologic approaches. We demonstrated that inhibition of the thioredoxin-dependent system or copper-zinc superoxide dismutase (SOD1) could abrogate NRF2-mediated resistance to {beta}-lapachone, while depletion of catalase or glutathione was ineffective. Interestingly, inhibition of SOD1 selectively sensitized KEAP1 mutant cells to {beta}-lapachone exposure. Our results suggest that NRF2/KEAP1 mutational status might serve as a predictive biomarker for response to NQO1-bioactivatable quinones in patients. Further, our results suggest SOD1 inhibition may have potential utility in combination with other ROS inducers in patients with KEAP1/NRF2 mutations. HighlightsO_LIAberrant activation of NRF2 in non-small cell lung cancer promotes resistance to {beta}-lapachone via the antioxidant defense. C_LIO_LIInhibition of the thioredoxin-dependent system and superoxide dismutase 1 increase sensitivity to {beta}-lapachone treatment. C_LIO_LIMutations in the NRF2/KEAP1 pathway might serve as predictive biomarker for response to {beta}-lapachone in patients. C_LI

cancer biology↗

bioRxiv2019

PHGDH supports liver ceramide synthesis and sustains lipid homeostasis

BackgroundD-3-phosphoglycerate dehydrogenase (PHGDH), which encodes the first enzyme in serine biosynthesis, is overexpressed in human cancers and has been proposed as a drug target. However, whether PHGDH is critical for the proliferation or homeostasis of tissues following the postnatal period is unknown. MethodsTo study PHGDH inhibition in adult animals, we developed a knock-in mouse model harboring a PHGDH shRNA under the control of a doxycycline-inducible promoter. With this model, PHGDH depletion can be globally induced in adult animals, while sparing the brain due to poor doxycycline delivery. ResultsWe found that PHGDH depletion is well tolerated and no overt phenotypes were observed in multiple highly proliferative cell compartments. Further, despite detectable knockdown, liver and pancreatic function were normal. Interestingly, diminished PHGDH expression in the liver reduced serine and ceramide levels without increasing the levels of deoxysphingolipids. Further, liver triacylglycerol profiles were altered, with an accumulation of longer chain, polyunsaturated tails upon PHGDH knockdown. ConclusionsThese results suggest that dietary serine is adequate to support the function of healthy, adult murine tissues, but PHGDH-derived serine supports liver ceramide synthesis and sustains general lipid homeostasis.

biochemistry↗

bioRxiv2019

NNT Regulates Mitochondrial Metabolism in NSCLC Through Maintenance of Fe-S Protein Function

Human lung tumors exhibit robust and complex mitochondrial metabolism, likely precipitated by the highly oxygenated nature of pulmonary tissue. As ROS generation is a byproduct of this metabolism, reducing power in the form of nicotinamide adenine dinucleotide phosphate (NADPH) is required to mitigate oxidative stress in response to this heightened mitochondrial activity. Nicotinamide nucleotide transhydrogenase (NNT) is known to sustain mitochondrial antioxidant capacity through the generation of NADPH, however its function in non-small cell lung cancer (NSCLC) has not been established. We found that NNT expression significantly enhances tumor formation and aggressiveness in mouse models of lung tumor initiation and progression. We further show that NNT loss elicits mitochondrial dysfunction independent of substantial increases in oxidative stress, but rather marked by the diminished activities of proteins dependent on resident iron-sulfur clusters. These defects were associated with both NADPH availability and ROS accumulation, suggesting that NNT serves a specific role in mitigating the oxidation of these critical protein cofactors.

cancer biology↗