Biology subjects

DeNicola, G. M.

Publications and source records attributed to DeNicola, G. M..

30 records · Page 2Linked to original sources

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↗

bioRxiv2022

NADK upregulation is an essential metabolic adaptation that enables breast cancer metastatic colonization

Metabolic reprogramming and metabolic plasticity allow cancer cells to fine-tune their metabolism to adapt to the ever-changing environments of the metastatic cascade, for which lipid metabolism and oxidative stress are of particular importance. Continuous production of NADPH, a cornerstone of both lipid and redox homeostasis, is essential for proliferation and cell survival suggesting that cancer cells may require larger pools of NADPH to efficiently metastasize. NADPH is recycled through reduction of NADP+ by several enzymatic systems in cells; however, de novo NADP+ is synthesized only through one known enzymatic reaction, catalysed by NAD+ kinase (NADK). Here, we show that NADK is upregulated in metastatic breast cancer cells enabling de novo production of NADP(H) and the expansion of the NADP(H) pools thereby increasing the ability of these cells to adapt to the oxidative challenges of the metastatic cascade and efficiently metastasize. Mechanistically, we found that metastatic signals lead to a histone H3.3 variant-mediated epigenetic regulation of the NADK promoter, resulting in increased NADK levels in cells with metastatic ability. Together, our work presents a previously uncharacterized role for NADK as an important contributor to breast cancer progression and suggests that NADK constitutes an important and much needed therapeutic target for metastatic breast cancers.

cancer biology↗

bioRxiv2022

A CRISPR screen identifies redox vulnerabilities for KEAP1/NRF2 mutant non-small cell lung cancer

The redox regulator NRF2 is hyperactivated in a large percentage of non-small cell lung cancer (NSCLC) cases, which is associated with chemotherapy and radiation resistance. To identify redox vulnerabilities for KEAP1/NRF2 mutant NSCLC, we conducted a CRISPR-Cas9-based negative selection screen for antioxidant enzyme genes whose loss sensitized cells to sub-lethal concentrations of the superoxide (O2*-)-generating drug {beta}-Lapachone. While our screen identified expected hits in the pentose phosphate pathway, the thioredoxin-dependent antioxidant system, and glutathione reductase, we also identified the mitochondrial superoxide dismutase 2 (SOD2) as one of the top hits. Surprisingly, {beta}-Lapachone did not generate mitochondrial O2*- but rather SOD2 loss enhanced the efficacy of {beta}-Lapachone due to loss of iron-sulfur protein function, loss of mitochondrial ATP maintenance and deficient NADPH production. Importantly, inhibition of mitochondrial electron transport activity sensitized to {beta}-Lapachone, demonstrating these effects may be translated to increase ROS sensitivity therapeutically.

cancer biology↗

bioRxiv2022

Unexpected growth of a classic yeast auxotroph

Organisms must either synthesize or assimilate essential organic compounds to survive. The homocysteine synthase Met15 has been considered essential for inorganic sulfur assimilation in yeast since its discovery in the 1970s. As a result, MET15 has served as a genetic marker for hundreds of experiments that play a foundational role in eukaryote genetics and systems biology. Nevertheless, we demonstrate here through structural and evolutionary modeling, in vitro kinetic assays, and genetic complementation, that an alternative homocysteine synthase encoded by the previously uncharacterized gene YLL058W enables cells lacking Met15 to assimilate enough inorganic sulfur for survival and proliferation. These cells however fail to grow in patches or liquid cultures unless provided with exogenous methionine or other organosulfurs. We show that this growth failure, which has historically justified the status of MET15 as a classic auxotrophic marker, is largely explained by toxic accumulation of the gas hydrogen sulfide due to a metabolic bottleneck. When patched or cultured with a hydrogen sulfide chelator, and when propagated as colony grids, cells without Met15 assimilate inorganic sulfur and grow, and cells with Met15 achieve even higher yields. Thus, Met15 is not essential for inorganic sulfur assimilation in yeast. Instead, MET15 is the first example of a yeast gene whose loss conditionally prevents growth in a manner that depends on local gas exchange. Our results have broad implications for investigations of sulfur metabolism, including studies of stress response, methionine restriction, and aging. More generally, our findings illustrate how unappreciated experimental variables can obfuscate biological discovery.

molecular 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↗

bioRxiv2021

The Actin Bundling Protein Fascin Promotes Lung Cancer Growth and Metastasis by Enhancing Glycolysis and PFKFB3 Expression

Fascin is a pro-metastasis actin bundling protein upregulated in essentially all the metastatic carcinoma. It is believed that fascin promotes cancer cell migration and invasion by facilitating membrane protrusions such as filopodia and invadopodia. Aerobic glycolysis is a key feature of cancer metabolism that provides critical intermediate metabolites for tumor growth and cell proliferation. Here we report that fascin increase glycolysis in lung cancer to promote tumor growth and metastasis. Fascin promotes glycolytic flux by increasing the expression and activities of phosphofructose kinase 1 and 2 (PFK1 and 2). The glycolytic function of fascin depends on activation of YAP1 through its canonical actin bundling activity. Fascin promotes the binding of YAP1 to a TEAD1/4 binding motif located 30 bp upstream of the PFKFB3 transcription start site to activate its transcription. Our interrogation of the TCGA database suggest that the fascin-YAP1-PFKFB3 circuit is likely conserved across different types of cancer. We further showed that the glycolytic function of fascin is essential for promotion of lung cancer growth and metastasis. Importantly, pharmacological inhibitors of fascin could be used to suppress the YAP1-PFKFB3 signaling and inhibit glycolysis in cancer cell lines, organoid cultures and xenograft metastasis models. Taken together, our data reveal an important glycolytic role of fascin in lung cancer metabolism, and suggest that pharmacological inhibitors of fascin could be used to reprogram cancer metabolism in lung cancer and potentially other cancer with fascin upregulation.

cancer biology↗

bioRxiv2020

Establishing a Living Biobank of Patient-Derived Organoids of Intraductal Papillary Mucinous Neoplasms of the Pancreas

Pancreatic cancer (PaCa) is the third leading cause of cancer-related deaths in the United States. There is an unmet need to develop strategies to detect PaCa at an early, operable stage and prevent its progression. Intraductal papillary mucinous neoplasms (IPMNs) are cystic PaCa precursors that comprise nearly 50% of pancreatic cysts detected incidentally via cross-sectional imaging. Since IPMNs can progress from low- and moderate-grade dysplasia to high-grade dysplasia and invasion, the study of these lesions offers a prime opportunity to develop early detection and prevention strategies. Organoids are an ideal preclinical platform to study IPMNs, and the objective of the current investigation was to establish a living biobank of patient-derived organoids (PDO) from IPMNs. IPMN tumors and adjacent normal pancreatic tissues were successfully harvested from 15 patients with IPMNs undergoing pancreatic surgical resection at Moffitt Cancer Center & Research Institute (Tampa, FL) between May of 2017 and March of 2019. Organoid cultures were also generated from cryopreserved tissues. Organoid count and size were determined over time by both Image-Pro Premier 3D Version 9.1 digital platform and Matlab application of a Circular Hough Transform algorithm, and histologic and genomic characterization of a subset of the organoids was performed using immunohistochemistry and targeted sequencing, respectively. The success rates for organoid generation from IPMN tumor and adjacent normal pancreatic tissues were 81% and 87%, respectively. IPMN organoids derived from different epithelial subtypes showed different morphologies in vitro, and organoids recapitulated histologic and genomic characteristics of the parental IPMN tumor. In summary, this pre-clinical model has the potential to provide new opportunities to unveil mechanisms of IPMN progression to invasion and to shed insight into novel biomarkers for early detection and targets for chemoprevention.

cancer biology↗

bioRxiv2020

Non-canonical glutamate-cysteine ligase activity protects against ferroptosis

Cysteine is required for maintaining cellular redox homeostasis in both normal and transformed cells. Deprivation of cysteine induces the iron-dependent form of cell death known as ferroptosis; however, the metabolic consequences of cysteine starvation beyond impairment of glutathione synthesis are uncharacterized. Here, we find that cystine starvation promotes ferroptosis not only through the inhibition of glutathione (GSH) synthesis, but also through the accumulation of glutamate. Surprisingly, we find that glutamate-cysteine ligase catalytic subunit (GCLC) prevents glutamate accumulation through the generation of alternative {gamma}-glutamyl peptides. Further, inhibition of GCLC accelerates ferroptosis under cystine starvation in a GSH-independent manner. These results indicate that GCLC has an additional, non-canonical role in the protection against ferroptosis to maintain glutamate homeostasis under cystine starvation.

cancer 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↗

bioRxiv2018

Cysteine dioxygenase 1 is a metabolic liability for non-small cell lung cancer

NRF2 is emerging as a major regulator of cellular metabolism. However, most studies have been performed in cancer cells, where co-occurring mutations and tumor selective pressures complicate the influence of NRF2 on metabolism. Here we use genetically engineered, non-transformed primary cells to isolate the most immediate effects of NRF2 on cellular metabolism. We find that NRF2 promotes the accumulation of intracellular cysteine and engages the cysteine homeostatic control mechanism mediated by cysteine dioxygenase 1 (CDO1), which catalyzes the irreversible metabolism of cysteine to cysteine sulfinic acid (CSA). Notably, CDO1 is preferentially silenced by promoter methylation in non-small cell lung cancers (NSCLC) harboring mutations in KEAP1, the negative regulator of NRF2. CDO1 silencing promotes proliferation of NSCLC by limiting the futile metabolism of cysteine to the wasteful and toxic byproducts CSA and sulfite, and depletion of cellular NADPH. Thus, CDO1 is a metabolic liability for NSCLC cells with high intracellular cysteine, particularly NRF2/KEAP1 mutant cells.

cancer biology↗