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Background: KRAS mutation status is a well-established independent prognostic factor in advanced non-small cell lung cancer (NSCLC), yet its role in early-stage disease is unclear. Here, we investigate the prognostic value of combining survival data on KRAS mutation status and tumor size in stage I-II NSCLC., Methods: We studied the combined impact of KRAS mutational status and tumor size on overall survival (OS) in patients with stage I-II NSCLC. We performed a retrospective study including 310 diagnosed patients with early (stage I-II) NSCLCs. All molecularly assessed patients diagnosed with stage I-II NSCLC between 2016-2018 in the Västra Götaland Region of western Sweden were screened in this multi-center retrospective study. The primary study outcome was overall survival., Results: Out of 310 patients with stage I-II NSCLC, 37% harbored an activating mutation in the KRAS gene. Our study confirmed staging and tumor size as prognostic factors. However, KRAS mutational status was not found to impact OS and there was no difference in the risk of death when combining KRAS mutational status and primary tumor size., Conclusions: In our patient cohort, KRAS mutations in combination with primary tumor size did not impact prognosis in stage I-II NSCLC., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Eklund, Mourad, Wiel, Sayin, Fagman, Hallqvist and Sayin.)

Loss-of-function mutations in KEAP1 frequently occur in lung cancer and are associated with poor prognosis and resistance to standard of care treatment, highlighting the need for the development of targeted therapies. We previously showed that KEAP1 mutant tumors consume glutamine to support the metabolic rewiring associated with NRF2-dependent antioxidant production. Here, using preclinical patient-derived xenograft models and antigenic orthotopic lung cancer models, we show that the glutamine antagonist prodrug DRP-104 impairs the growth of KEAP1 mutant tumors. We find that DRP-104 suppresses KEAP1 mutant tumors by inhibiting glutamine-dependent nucleotide synthesis and promoting antitumor T cell responses. Using multimodal single-cell sequencing and ex vivo functional assays, we demonstrate that DRP-104 reverses T cell exhaustion, decreases T regs , and enhances the function of CD4 and CD8 T cells, culminating in an improved response to anti-PD1 therapy. Our preclinical findings provide compelling evidence that DRP-104, currently in clinical trials, offers a promising therapeutic approach for treating patients with KEAP1 mutant lung cancer.

Pulmonary adenocarcinoma (ADC) is a very diverse disease, both genetically and histologically, which displays extensive intratumor heterogeneity with numerous acquired mutations. ADC is the most common type of lung cancer and is believed to arise from adenocarcinoma in situ (AIS) which then progresses to minimally invasive adenocarcinoma (MIA). In patients of European ethnicity, we analyzed genetic mutations in AIS ( n = 10) and MIA ( n = 18) and compared the number of genetic mutations with advanced ADC ( n = 2419). Using next-generation sequencing, the number of different mutations detected in both AIS (87.5%) and MIA (94.5%) were higher ( p < 0.001) than in advanced ADC (53.7%). In contrast to the high number of mutations in Kirsten rat sarcoma virus gene ( KRAS ) in advanced ADC (34.6%), there was only one case of AIS with KRAS G12C mutation (3.5%; p < 0.001) and no cases of MIA with KRAS mutation ( p < 0.001). In contrast to the modest prevalence of epidermal growth factor receptor ( EGFR ) mutations in advanced ADC (15.0%), the fraction of EGFR mutant cases was higher in both in AIS (22.2%) and MIA (59.5%; p < 0.001). The EGFR exon 19 deletion mutation was more common in both MIA (50%; n = 6/12) and ADC (41%; n = 149/363), whereas p.L858R was more prevalent in AIS (75%; n = 3/4). In contrast to pulmonary advanced ADC, KRAS driver mutations are less common, whereas mutations in EGFR are more common, in detectable AIS and MIA.

Cellular redox homeostasis is an essential, dynamic process that ensures the balance between reducing and oxidizing reactions within cells and thus has implications across all areas of biology. Changes in levels of reactive oxygen species can disrupt redox homeostasis, leading to oxidative or reductive stress that contributes to the pathogenesis of many malignancies, including cancer. From transformation and tumor initiation to metastatic dissemination, increasing reactive oxygen species in cancer cells can paradoxically promote or suppress the tumorigenic process, depending on the extent of redox stress, its spatiotemporal characteristics and the tumor microenvironment. Here we review how redox regulation influences tumorigenesis, highlighting therapeutic opportunities enabled by redox-related alterations in cancer cells., (© 2024. Springer Nature America, Inc.)

Refractory and relapsed B cell lymphomas are often driven by the difficult-to-target oncogene MYC. Here, we report that high MYC expression stimulates proliferation and protects B lymphoma cells from apoptosis under normal oxidative stress levels and that compounds including N-acetylcysteine (NAC) and vitamin C (VitC) induce apoptosis by reducing oxidative stress. NAC and VitC injections effectively reduce tumor growth in lymphoma cells with high MYC expression but not in those with low MYC expression. MYC knockdown confers tumor resistance to NAC and VitC, while MYC activation renders B cells sensitive to these compounds. Mechanistically, NAC and VitC stimulate MYC binding to EGR1 through Cys117 of MYC, shifting its transcriptional output from cell cycle to apoptosis gene expression. These results identify a redox-controlled mechanism for MYC's role in maintaining proliferation and preventing apoptosis, offering a potential therapeutic rationale for evaluating NAC or VitC in patients with MYC-driven B cell lymphoma., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Selenium homeostasis depends on hepatic biosynthesis of selenoprotein P (SELENOP) and SELENOP-mediated transport from the liver to e.g. the brain. In addition, the liver maintains copper homeostasis. Selenium and copper metabolism are inversely regulated, as increasing copper and decreasing selenium levels are observed in blood during aging and inflammation. Here we show that copper treatment increased intracellular selenium and SELENOP in hepatocytes and decreased extracellular SELENOP levels. Hepatic accumulation of copper is a characteristic of Wilson's disease. Accordingly, SELENOP levels were low in serum of Wilson's disease patients and Wilson's rats. Mechanistically, drugs targeting protein transport in the Golgi complex mimicked some of the effects observed, indicating a disrupting effect of excessive copper on intracellular SELENOP transport resulting in its accumulation in the late Golgi. Our data suggest that hepatic copper levels determine SELENOP release from the liver and may affect selenium transport to peripheral organs such as the brain., (© 2023. The Author(s).)

Objectives: Immunotherapy by blocking programmed death protein-1 (PD-1) or programmed death protein-ligand1 (PD-L1) with antibodies (PD-1 blockade) has revolutionized treatment options for patients with non-small cell lung cancer (NSCLC). However, the benefit of immunotherapy is limited to a subset of patients. This study aimed to investigate the value of combining immune and genetic variables analyzed within 3-4 weeks after the start of PD-1 blockade therapy to predict long-term clinical response., Materials and Methodology: Blood collected from patients with NSCLC were analyzed for changes in the frequency and concentration of immune cells using a clinical flow cytometry assay. Next-generation sequencing (NGS) was performed on DNA extracted from archival tumor biopsies of the same patients. Patients were categorized as clinical responders or non-responders based on the 9 months' assessment after the start of therapy., Results: We report a significant increase in the post-treatment frequency of activated effector memory CD4 + and CD8 + T-cells compared with pre-treatment levels in the blood. Baseline frequencies of B cells but not NK cells, T cells, or regulatory T cells were associated with the clinical response to PD-1 blockade. NGS of tumor tissues identified pathogenic or likely pathogenic mutations in tumor protein P53, Kirsten rat sarcoma virus, Kelch-like ECH-associated protein 1, neurogenic locus notch homolog protein 1, and serine/threonine kinase 11, primarily in the responder group. Finally, multivariate analysis of combined immune and genetic factors but neither alone, could discriminate between responders and non-responders., Conclusion: Combined analyses of select immune cell subsets and genetic mutations could predict early clinical responses to immunotherapy in patients with NSCLC and after validation, can guide clinical precision medicine efforts., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Dutta, Rohlin, Eklund, Magnusson, Nilsson, Akyürek, Torstensson, Sayin, Lundgren, Hallqvist and Raghavan.)

The transcription factor BACH1 regulates the expression of a variety of genes including genes involved in oxidative stress responses, inflammation, cell motility, cancer cell invasion and cancer metabolism. Based on this, BACH1 has become a promising therapeutic target in cancer (as anti-metastatic target) and also in chronic conditions linked to oxidative stress and inflammation, where BACH1 inhibitors share a therapeutic space with activators of transcription factor NRF2. However, while there is a growing number of NRF2 activators, there are only a few described BACH1 inhibitors/degraders. The synthetic acetylenic tricyclic bis(cyanoenone),(±)-(4bS,8aR,10aS)-10a-ethynyl-4b,8,8-trimethyl-3,7-dioxo-3.4b,7,8,8a,9,10, 10a-octahydrophenanthrene-2,6-dicarbonitrile, TBE31 is a potent activator of NRF2 without any BACH1 activity. Herein we found that biotinylation of TBE31 greatly reduces its potency as NRF2 activator (50-75-fold less active) while acquiring a novel activity as a BACH1 degrader (100-200-fold more active). We demonstrate that TBE56, the biotinylated TBE31, interacts and promotes the degradation of BACH1 via a mechanism involving the E3 ligase FBXO22. TBE56 is a potent and sustained BACH1 degrader (50-fold more potent than hemin) and accordingly a powerful HMOX1 inducer. TBE56 degrades BACH1 in lung and breast cancer cells, impairing breast cancer cell migration and invasion in a BACH1-dependent manner, while TBE31 has no significant effect. Altogether, our study identifies that the biotinylation of TBE31 provides novel activities with potential therapeutic value, providing a rationale for further characterisation of this and related compounds., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Conditional mutation of protein geranylgeranyltransferase type I (GGTase-I) in macrophages (GLC) activates Rho-GTPases and causes arthritis in mice. Knocking out Rag1 in GLC mice alleviates arthritis which indicates that lymphocytes are required for arthritis development in those mice. To study GLC dependent changes in the adaptive immunity, we isolated CD4 + T cells from GLC mice (CD4 + GLCs). Spleen and joint draining lymph nodes (dLN) CD4 + GLCs exhibited high expression of Cdc42 and Rac1, which repressed the caudal HOXA proteins and activated the mechanosensory complex to facilitate migration. These CDC42/RAC1 rich CD4 + GLCs presented a complete signature of GARP + NRP1 + IKZF2 + FOXP3 + regulatory T cells (Tregs) of thymic origin. Activation of the β-catenin/Lef1 axis promoted a pro-inflammatory Th1 phenotype of Tregs, which was strongly associated with arthritis severity. Knockout of Cdc42 in macrophages of GLC mice affected CD4 + cell biology and triggered development of non-thymic Tregs. Knockout of Rac1 and RhoA had no such effects on CD4 + cells although it alleviated arthritis in GLC mice. Disrupting macrophage and T cell interaction with CTLA4 fusion protein reduced the Th1-driven inflammation and enrichment of thymic Tregs into dLNs. Antigen challenge reinforced the CD4 + GLC phenotype in non-arthritic heterozygote GLC mice and increased accumulation of Rho-GTPase expressing thymic Tregs in dLNs. Our study demonstrates an unexpected role of macrophages in stimulating the development of pro-inflammatory thymic Tregs and reveal activation of Rho-GTPases behind their arthritogenic phenotype., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The transcription factor BACH1 is a potential therapeutic target for a variety of chronic conditions linked to oxidative stress and inflammation, as well as cancer metastasis. However, only a few BACH1 degraders/inhibitors have been described. BACH1 is a transcriptional repressor of heme oxygenase 1 (HMOX1), which is positively regulated by transcription factor NRF2 and is highly inducible by derivatives of the synthetic oleanane triterpenoid 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO). Most of the therapeutic activities of these compounds are due to their anti-inflammatory and antioxidant properties, which are widely attributed to their ability to activate NRF2. However, with such a broad range of action, these compounds have other molecular targets that have not been fully identified and could also be of importance for their therapeutic profile. Herein we identified BACH1 as a target of two CDDO-derivatives (CDDO-Me and CDDO-TFEA), but not of CDDO. While both CDDO and CDDO-derivatives activate NRF2 similarly, only CDDO-Me and CDDO-TFEA inhibit BACH1, which explains the much higher potency of these CDDO-derivatives as HMOX1 inducers compared with unmodified CDDO. Notably, we demonstrate that CDDO-Me and CDDO-TFEA inhibit BACH1 via a novel mechanism that reduces BACH1 nuclear levels while accumulating its cytoplasmic form. In an in vitro model, both CDDO-derivatives impaired lung cancer cell invasion in a BACH1-dependent and NRF2-independent manner, while CDDO was inactive. Altogether, our study identifies CDDO-Me and CDDO-TFEA as dual KEAP1/BACH1 inhibitors, providing a rationale for further therapeutic uses of these drugs., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

There is an urgent need to identify new predictive biomarkers for treatment response to both platinum doublet chemotherapy (PT) and immune checkpoint blockade (ICB). Here, we evaluated whether treatment outcome could be affected by KRAS mutational status in patients with metastatic (Stage IV) non-small cell lung cancer (NSCLC). All consecutive patients molecularly assessed and diagnosed between 2016−2018 with Stage IV NSCLC in the region of West Sweden were included in this multi-center retrospective study. The primary study outcome was overall survival (OS). Out of 580 Stage IV NSCLC patients, 35.5% harbored an activating mutation in the KRAS gene (KRASMUT). Compared to KRAS wild-type (KRASWT), KRASMUT was a negative factor for OS (p = 0.014). On multivariate analysis, KRASMUT persisted as a negative factor for OS (HR 1.478, 95% CI 1.207−1.709, p < 0.001). When treated with first-line platinum doublet (n = 195), KRASMUT was a negative factor for survival (p = 0.018), with median OS of 9 months vs. KRASWT at 11 months. On multivariate analysis, KRASMUT persisted as a negative factor for OS (HR 1.564, 95% CI 1.124−2.177, p = 0.008). KRASMUT patients with high PD-L1 expression (PD-L1high) had better OS than PD-L1highKRASWT patients (p = 0.036). In response to first-line ICB, KRASMUT patients had a significantly (p = 0.006) better outcome than KRASWT patients, with a median OS of 23 vs. 6 months. On multivariable Cox analysis, KRASMUT status was an independent prognostic factor for better OS (HR 0.349, 95% CI 0.148−0.822, p = 0.016). kRAS mutations are associated with better response to treatment with immune checkpoint blockade and worse response to platinum doublet chemotherapy as well as shorter general OS in Stage IV NSCLC.

Cellular redox homeostasis is an essential and dynamic process that ensures the balance between reducing and oxidizing reactions within cells and regulates a plethora of biological responses and events. The study of these biochemical reactions has proven difficult over time, but recent technical and methodological developments have contributed to the rapid growth of the redox field and to our understanding of its importance in biology. The aim of this short review is to give the reader an overall understanding of redox regulation in the areas of cellular signaling, development, and disease, as well as to introduce some recent discoveries in those fields.

Dietary antioxidants and supplements are widely used to protect against cancer, even though it is now clear that antioxidants can promote tumor progression by helping cancer cells to overcome barriers of oxidative stress. Although recent studies have, in great detail, explored the role of antioxidants in lung and skin tumors driven by RAS and RAF mutations, little is known about the impact of antioxidant supplementation on other cancers, including Wnt-driven tumors originating from the gut. Here, we show that supplementation with the antioxidants N-acetylcysteine (NAC) and vitamin E promotes intestinal tumor progression in the ApcMin mouse model for familial adenomatous polyposis, a hereditary form of colorectal cancer, driven by Wnt signaling. Both antioxidants increased tumor size in early neoplasias and tumor grades in more advanced lesions without any impact on tumor initiation. Importantly, NAC treatment accelerated tumor progression at plasma concentrations comparable to those obtained in human subjects after prescription doses of the drug. These results demonstrate that antioxidants play an important role in the progression of intestinal tumors, which may have implications for patients with or predisposed to colorectal cancer.

Cancer cells produce high levels of mitochondria-associated reactive oxygen species (ROS) that can damage macromolecules, but also promote cell signaling and proliferation. Therefore, mitochondria-targeted antioxidants have been suggested to be useful in anti-cancer therapy, but no studies have convincingly addressed this question. Here, we administered the mitochondria-targeted antioxidants MitoQ and MitoTEMPO to mice with BRAF-induced malignant melanoma and KRAS-induced lung cancer, and found that these compounds had no impact on the number of primary tumors and metastases; and did not influence mitochondrial and nuclear DNA damage levels. Moreover, MitoQ and MitoTEMPO did not influence proliferation of human melanoma and lung cancer cell lines. MitoQ and its control substance dTPP, but not MitoTEMPO, increased glycolytic rates and reduced respiration in melanoma cells; whereas only dTPP produced this effect in lung cancer cells. Our results do not support the use of mitochondria-targeted antioxidants for anti-cancer monotherapy, at least not in malignant melanoma and lung cancer.

Recent data suggest that the transcription factor Zfp148 represses activation of the tumor suppressor p53 in mice and that therapeutic targeting of the human orthologue ZNF148 could activate the p53 pathway without causing detrimental side effects. We have previously shown that Zfp148 deficiency promotes p53-dependent proliferation arrest of mouse embryonic fibroblasts (MEFs), but the underlying mechanism is not clear. Here, we showed that Zfp148 deficiency downregulated cell cycle genes in MEFs in a p53-dependent manner. Proliferation arrest of Zfp148-deficient cells required increased expression of ARF, a potent activator of the p53 pathway. Chromatin immunoprecipitation showed that Zfp148 bound to the ARF promoter, suggesting that Zfp148 represses ARF transcription. However, Zfp148 preferentially bound to promoters of other transcription factors, indicating that deletion of Zfp148 may have pleiotropic effects that activate ARF and p53 indirectly. In line with this, we found no evidence of genetic interaction between TP53 and ZNF148 in CRISPR and siRNA screen data from hundreds of human cancer cell lines. We conclude that Zfp148 deficiency, by increasing ARF transcription, downregulates cell cycle genes and cell proliferation in a p53-dependent manner. However, the lack of genetic interaction between ZNF148 and TP53 in human cancer cells suggests that therapeutic targeting of ZNF148 may not increase p53 activity in humans.

Rewiring of metabolic pathways is a hallmark of tumorigenesis as cancer cells acquire novel nutrient dependencies to support oncogenic growth. A major genetic subtype of lung adenocarcinoma with KEAP1/NRF2 mutations, which activates the endogenous oxidative stress response, undergoes significant metabolic rewiring to support enhanced antioxidant production. We demonstrate that cancers with high antioxidant capacity exhibit a general dependency on exogenous non-essential amino acids (NEAAs) that is driven by the Nrf2-dependent secretion of glutamate through system x c - (XCT), which limits intracellular glutamate pools that are required for NEAA synthesis. This dependency can be therapeutically targeted by dietary restriction or enzymatic depletion of individual NEAAs. Importantly, limiting endogenous glutamate levels by glutaminase inhibition can sensitize tumors without alterations in the Keap1/Nrf2 pathway to dietary restriction of NEAAs. Our findings identify a metabolic strategy to therapeutically target cancers with genetic or pharmacologic activation of the Nrf2 antioxidant response pathway by restricting exogenous sources of NEAAs., Competing Interests: Declaration of Interests T.P. reports grants and consulting fees outside of the submitted work from Dracen Pharmaceuticals, Agios, Bristol Meyers Squib, and Calithera Biosciences., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Rho family proteins are prenylated by geranylgeranyltransferase type I (GGTase-I), which normally target proteins to membranes for GTP-loading. However, conditional deletion of GGTase-I in mouse macrophages increases GTP-loading of Rho proteins, leading to enhanced inflammatory responses and severe rheumatoid arthritis. Here we show that heterozygous deletion of the Rho family gene Rac1, but not Rhoa and Cdc42, reverses inflammation and arthritis in GGTase-I-deficient mice. Non-prenylated Rac1 has a high affinity for the adaptor protein Ras GTPase-activating-like protein 1 (Iqgap1), which facilitates both GTP exchange and ubiquitination-mediated degradation of Rac1. Consistently, inactivating Iqgap1 normalizes Rac1 GTP-loading, and reduces inflammation and arthritis in GGTase-I-deficient mice, as well as prevents statins from increasing Rac1 GTP-loading and cytokine production in macrophages. We conclude that blocking prenylation stimulates Rac1 effector interactions and unleashes proinflammatory signaling. Our results thus suggest that prenylation normally restrains innate immune responses by preventing Rac1 effector interactions.

Lung cancer remains one of the most genetically complex, aggressive, and lethal solid malignancies. Understanding how distinct lung cancer mutations give rise to altered nutrient requirements and promote immune evasion in the context of a heterogeneous lung tumor microenvironment is vital for the development of novel personalized therapeutic strategies., (Copyright © 2019 Elsevier Inc. All rights reserved.)

For tumors to progress efficiently, cancer cells must overcome barriers of oxidative stress. Although dietary antioxidant supplementation or activation of endogenous antioxidants by NRF2 reduces oxidative stress and promotes early lung tumor progression, little is known about its effect on lung cancer metastasis. Here, we show that long-term supplementation with the antioxidants N-acetylcysteine and vitamin E promotes KRAS-driven lung cancer metastasis. The antioxidants stimulate metastasis by reducing levels of free heme and stabilizing the transcription factor BACH1. BACH1 activates transcription of Hexokinase 2 and Gapdh and increases glucose uptake, glycolysis rates, and lactate secretion, thereby stimulating glycolysis-dependent metastasis of mouse and human lung cancer cells. Targeting BACH1 normalized glycolysis and prevented antioxidant-induced metastasis, while increasing endogenous BACH1 expression stimulated glycolysis and promoted metastasis, also in the absence of antioxidants. We conclude that BACH1 stimulates glycolysis-dependent lung cancer metastasis and that BACH1 is activated under conditions of reduced oxidative stress., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Approximately 30% of human lung cancers acquire mutations in either Keap1 or Nfe2l2, resulting in the stabilization of Nrf2, the Nfe2l2 gene product, which controls oxidative homeostasis. Here, we show that heme triggers the degradation of Bach1, a pro-metastatic transcription factor, by promoting its interaction with the ubiquitin ligase Fbxo22. Nrf2 accumulation in lung cancers causes the stabilization of Bach1 by inducing Ho1, the enzyme catabolizing heme. In mouse models of lung cancers, loss of Keap1 or Fbxo22 induces metastasis in a Bach1-dependent manner. Pharmacological inhibition of Ho1 suppresses metastasis in a Fbxo22-dependent manner. Human metastatic lung cancer display high levels of Ho1 and Bach1. Bach1 transcriptional signature is associated with poor survival and metastasis in lung cancer patients. We propose that Nrf2 activates a metastatic program by inhibiting the heme- and Fbxo22-mediated degradation of Bach1, and that Ho1 inhibitors represent an effective therapeutic strategy to prevent lung cancer metastasis., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Thioredoxin reductase-1 (TrxR1)-, glutathione reductase (Gsr)-, and Nrf2 transcription factor-driven antioxidant systems form an integrated network that combats potentially carcinogenic oxidative damage yet also protects cancer cells from oxidative death. Here we show that although unchallenged wild-type (WT), TrxR1-null, or Gsr-null mouse livers exhibited similarly low DNA damage indices, these were 100-fold higher in unchallenged TrxR1/Gsr-double-null livers. Notwithstanding, spontaneous cancer rates remained surprisingly low in TrxR1/Gsr-null livers. All genotypes, including TrxR1/Gsr-null, were susceptible to N -diethylnitrosamine (DEN)-induced liver cancer, indicating that loss of these antioxidant systems did not prevent cancer cell survival. Interestingly, however, following DEN treatment, TrxR1-null livers developed threefold fewer tumors compared with WT livers. Disruption of TrxR1 in a marked subset of DEN-initiated cancer cells had no effect on their subsequent contributions to tumors, suggesting that TrxR1-disruption does not affect cancer progression under normal care, but does decrease the frequency of DEN-induced cancer initiation. Consistent with this idea, TrxR1-null livers showed altered basal and DEN-exposed metabolomic profiles compared with WT livers. To examine how oxidative stress influenced cancer progression, we compared DEN-induced cancer malignancy under chronically low oxidative stress (TrxR1-null, standard care) vs. elevated oxidative stress (TrxR1/Gsr-null livers, standard care or phenobarbital-exposed TrxR1-null livers). In both cases, elevated oxidative stress was correlated with significantly increased malignancy. Finally, although TrxR1-null and TrxR1/Gsr-null livers showed strong Nrf2 activity in noncancerous hepatocytes, there was no correlation between malignancy and Nrf2 expression within tumors across genotypes. We conclude that TrxR1, Gsr, Nrf2, and oxidative stress are major determinants of liver cancer but in a complex, context-dependent manner., Competing Interests: The authors declare no conflict of interest.

Objective: In the liver, a contributing factor in the pathogenesis of non-alcoholic fatty liver disease (NASH) is oxidative stress, which leads to the accumulation of highly reactive electrophilic α/β unsaturated aldehydes. The objective of this study was to determine the impact of NASH on protein carbonylation and antioxidant responses in a murine model., Methods: Liver-specific phosphatase and tensin homolog (PTEN)-deletion mice (PTENLKO) or control littermates were fed a standard chow diet for 45-55 weeks followed by analysis for liver injury, oxidative stress and inflammation., Results: Histology and Picrosirius red-staining of collagen deposition within the extracellular matrix revealed extensive steatosis and fibrosis in the PTENLKO mice but no steatosis or fibrosis in controls. Increased steatosis and fibrosis corresponded with significant increases in inflammation. PTEN-deficient livers showed significantly increased cell-specific oxidative damage, as detected by 4-hydroxy-2-nonenal (4-HNE) and acrolein staining. Elevated staining correlated with an increase in nuclear DNA repair foci (γH2A.X) and cellular proliferation index (Ki67) within zones 1 and 3, indicating oxidative damage was zonally restricted and was associated with increased DNA damage and cell proliferation. Immunoblots showed that total levels of antioxidant response proteins induced by nuclear factor erythroid-2-like-2 (Nrf2), including GSTμ, GSTπ and CBR1/3, but not HO-1, were elevated in PTENLKO as compared to controls, and IHC showed this response also occurred only in zones 1 and 3. Furthermore, an analysis of autophagy markers revealed significant elevation of p62 and LC3II expression. Mass spectrometric (MS) analysis identified significantly more carbonylated proteins in whole cell extracts prepared from PTENLKO mice (966) as compared to controls (809). Pathway analyses of identified proteins did not uncover specific pathways that were preferentially carbonylated in PTENLKO livers but, did reveal specific strongly increased carbonylation of thioredoxin reductase and of glutathione-S-transferases (GST) M6, O1, and O2., Conclusions: Results show that disruption of PTEN resulted in steatohepatitis, fibrosis and caused hepatic induction of the Nrf2-dependent antioxidant system at least in part due to elevation of p62. This response was both cell-type and zone specific. However, these responses were insufficient to mitigate the accumulation of products of lipid peroxidation., Competing Interests: The authors have declared no competing interests exists.

Treating KRAS-mutant lung adenocarcinoma (LUAD) remains a major challenge in cancer treatment given the difficulties associated with directly inhibiting the KRAS oncoprotein. One approach to addressing this challenge is to define mutations that frequently co-occur with those in KRAS, which themselves may lead to therapeutic vulnerabilities in tumors. Approximately 20% of KRAS-mutant LUAD tumors carry loss-of-function mutations in the KEAP1 gene encoding Kelch-like ECH-associated protein 1 (refs. 2, 3, 4), a negative regulator of nuclear factor erythroid 2-like 2 (NFE2L2; hereafter NRF2), which is the master transcriptional regulator of the endogenous antioxidant response. The high frequency of mutations in KEAP1 suggests an important role for the oxidative stress response in lung tumorigenesis. Using a CRISPR-Cas9-based approach in a mouse model of KRAS-driven LUAD, we examined the effects of Keap1 loss in lung cancer progression. We show that loss of Keap1 hyperactivates NRF2 and promotes KRAS-driven LUAD in mice. Through a combination of CRISPR-Cas9-based genetic screening and metabolomic analyses, we show that Keap1- or Nrf2-mutant cancers are dependent on increased glutaminolysis, and this property can be therapeutically exploited through the pharmacological inhibition of glutaminase. Finally, we provide a rationale for stratification of human patients with lung cancer harboring KRAS/KEAP1- or KRAS/NRF2-mutant lung tumors as likely to respond to glutaminase inhibition.

During tumorigenesis, the high metabolic demand of cancer cells results in increased production of reactive oxygen species. To maintain oxidative homeostasis, tumor cells increase their antioxidant production through hyperactivation of the NRF2 pathway, which promotes tumor cell growth. Despite the extensive characterization of NRF2-driven metabolic rewiring, little is known about the metabolic liabilities generated by this reprogramming. Here, we show that activation of NRF2, in either mouse or human cancer cells, leads to increased dependency on exogenous glutamine through increased consumption of glutamate for glutathione synthesis and glutamate secretion by x c - antiporter system. Together, this limits glutamate availability for the tricarboxylic acid cycle and other biosynthetic reactions creating a metabolic bottleneck. Cancers with genetic or pharmacological activation of the NRF2 antioxidant pathway have a metabolic imbalance between supporting increased antioxidant capacity over central carbon metabolism, which can be therapeutically exploited.

Cancer is a multistep process that arises from a series of genetic and epigenetic events. With recent technological advances there has been a burst in genome sequencing and epigenetic studies revealing a plethora of alterations that may contribute to cancer. However, the great challenge for the cancer research community is the systematic functional characterization of these genetic and epigenetic events to assess their role in cancer initiation and progression. Recent advances in genome engineering using CRISPR/Cas9, an ancient bacterial immune-like system, have revolutionized cancer genetics. Here we highlight the breakthroughs in the effective use of these novel genome-editing techniques, and we discuss the challenges and potential applications of these tools for cancer biology., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Thousands of long non-coding RNAs (lncRNAs) lie interspersed with coding genes across the genome, and a small subset has been implicated as downstream effectors in oncogenic pathways. Here we make use of transcriptome and exome sequencing data from thousands of tumours across 19 cancer types, to identify lncRNAs that are induced or repressed in relation to somatic mutations in key oncogenic driver genes. Our screen confirms known coding and non-coding effectors and also associates many new lncRNAs to relevant pathways. The associations are often highly reproducible across cancer types, and while many lncRNAs are co-expressed with their protein-coding hosts or neighbours, some are intergenic and independent. We highlight lncRNAs with possible functions downstream of the tumour suppressor TP53 and the master antioxidant transcription factor NFE2L2. Our study provides a comprehensive overview of lncRNA transcriptional alterations in relation to key driver mutational events in human cancers.

The transcription factor Zinc finger protein 148 (Zfp148, ZBP-89, BFCOL, BERF1, htβ) interacts physically with the tumor suppressor p53, but the significance of this interaction is not known. We recently showed that knockout of Zfp148 in mice leads to ectopic activation of p53 in some tissues and cultured fibroblasts, suggesting that Zfp148 represses p53 activity. Here we hypothesize that targeting Zfp148 would unleash p53 activity and protect against cancer development, and test this idea in the APCMin/+ mouse model of intestinal adenomas. Loss of one copy of Zfp148 markedly reduced tumor numbers and tumor-associated intestinal bleedings, and improved survival. Furthermore, after activation of β-catenin-the initiating event in colorectal cancer-Zfp148 deficiency activated p53 and induced apoptosis in intestinal explants of APCMin/+ mice. The anti-tumor effect of targeting Zfp148 depended on p53, as Zfp148 deficiency did not affect tumor numbers in APCMin/+ mice lacking one or both copies of Trp53. The results suggest that Zfp148 controls the fate of newly transformed intestinal tumor cells by repressing p53 and that targeting Zfp148 might be useful in the treatment of colorectal cancer., Competing Interests: The authors have no conflict of Interest to declare.

KRAS and BRAF are among the most commonly mutated oncogenes in human cancer that contribute to tumorigenesis in both distinct and overlapping tissues. However, KRAS and BRAF mutations are mutually exclusive; they never occur in the same tumor cell. The reason for the mutual exclusivity is unknown, but there are several possibilities. The two mutations could be functionally redundant and not create a selective advantage to tumor cells. Alternatively, they could be deleterious for the tumor cell and induce apoptosis or senescence. To distinguish between these possibilities, we activated the expression of BRAF(V600E) and KRAS(G12D) from their endogenous promoters in mouse lungs. Although the tumor-forming ability of BRAF(V600E) was higher than KRAS(G12D), KRAS(G12D) tumors were larger and more advanced. Coactivation of BRAF(V600E) and KRAS(G12D) markedly reduced lung tumor numbers and overall tumor burden compared with activation of BRAF(V600E) alone. Moreover, several tumors expressed only one oncogene, suggesting negative selection against expression of both. Similarly, expression of both oncogenes in mouse embryonic fibroblasts essentially stopped proliferation. The expression of both oncogenes hyperactivated the MEK-ERK-cyclin D pathway but reduced proliferation by increasing the production of p15, p16 and p19 proteins encoded by the Ink4/Arf locus and thereby increased senescence-associated β-galactosidase-positive cells. The data suggest that coexpression of BRAF(V600E) and KRAS(G12D) in early tumorigenesis leads to negative selection due to oncogene-induced senescence.

Antioxidants in the diet and supplements are widely used to protect against cancer, but clinical trials with antioxidants do not support this concept. Some trials show that antioxidants actually increase cancer risk and a study in mice showed that antioxidants accelerate the progression of primary lung tumors. However, little is known about the impact of antioxidant supplementation on the progression of other types of cancer, including malignant melanoma. We show that administration of N-acetylcysteine (NAC) increases lymph node metastases in an endogenous mouse model of malignant melanoma but has no impact on the number and size of primary tumors. Similarly, NAC and the soluble vitamin E analog Trolox markedly increased the migration and invasive properties of human malignant melanoma cells but did not affect their proliferation. Both antioxidants increased the ratio between reduced and oxidized glutathione in melanoma cells and in lymph node metastases, and the increased migration depended on new glutathione synthesis. Furthermore, both NAC and Trolox increased the activation of the small guanosine triphosphatase (GTPase) RHOA, and blocking downstream RHOA signaling abolished antioxidant-induced migration. These results demonstrate that antioxidants and the glutathione system play a previously unappreciated role in malignant melanoma progression., (Copyright © 2015, American Association for the Advancement of Science.)

Rationale: Cell proliferation and cell cycle control mechanisms are thought to play central roles in the pathogenesis of atherosclerosis. The transcription factor Zinc finger protein 148 (Zfp148) was shown recently to maintain cell proliferation under oxidative conditions by suppressing p53, a checkpoint protein that arrests proliferation in response to various stressors. It is established that inactivation of p53 accelerates atherosclerosis, but whether increased p53 activation confers protection against the disease remains to be determined., Objective: We aimed to test the hypothesis that Zfp148 deficiency reduces atherosclerosis by unleashing p53 activity., Methods and Results: Mice harboring a gene-trap mutation in the Zfp148 locus (Zfp148(gt/+)) were bred onto the apolipoprotein E (Apoe)(-/-) genetic background and fed a high-fat or chow diet. Loss of 1 copy of Zfp148 markedly reduced atherosclerosis without affecting lipid metabolism. Bone marrow transplantation experiments revealed that the effector cell is of hematopoietic origin. Peritoneal macrophages and atherosclerotic lesions from Zfp148(gt/+)Apoe(-/-) mice showed increased levels of phosphorylated p53 compared with controls, and atherosclerotic lesions contained fewer proliferating macrophages. Zfp148(gt/+)Apoe(-/-) mice were further crossed with p53-null mice (Trp53(-/-) [the gene encoding p53]). There was no difference in atherosclerosis between Zfp148(gt/+)Apoe(-/-) mice and controls on a Trp53(+/-) genetic background, and there was no difference in levels of phosphorylated p53 or cell proliferation., Conclusions: Zfp148 deficiency increases p53 activity and protects against atherosclerosis by causing proliferation arrest of lesional macrophages, suggesting that drugs targeting macrophage proliferation may be useful in the treatment of atherosclerosis., (© 2014 American Heart Association, Inc.)

Antioxidants are widely used to protect cells from damage induced by reactive oxygen species (ROS). The concept that antioxidants can help fight cancer is deeply rooted in the general population, promoted by the food supplement industry, and supported by some scientific studies. However, clinical trials have reported inconsistent results. We show that supplementing the diet with the antioxidants N-acetylcysteine (NAC) and vitamin E markedly increases tumor progression and reduces survival in mouse models of B-RAF- and K-RAS-induced lung cancer. RNA sequencing revealed that NAC and vitamin E, which are structurally unrelated, produce highly coordinated changes in tumor transcriptome profiles, dominated by reduced expression of endogenous antioxidant genes. NAC and vitamin E increase tumor cell proliferation by reducing ROS, DNA damage, and p53 expression in mouse and human lung tumor cells. Inactivation of p53 increases tumor growth to a similar degree as antioxidants and abolishes the antioxidant effect. Thus, antioxidants accelerate tumor growth by disrupting the ROS-p53 axis. Because somatic mutations in p53 occur late in tumor progression, antioxidants may accelerate the growth of early tumors or precancerous lesions in high-risk populations such as smokers and patients with chronic obstructive pulmonary disease who receive NAC to relieve mucus production.

Hematopoiesis is regulated by transcription factors that induce cell fate and differentiation in hematopoietic stem cells into fully differentiated hematopoietic cell types. The transcription factor zinc finger protein 148 (Zfp148) interacts with the hematopoietic transcription factor Gata1 and has been implicated to play an important role in primitive and definitive hematopoiesis in zebra fish and mouse chimeras. We have recently created a gene-trap knockout mouse model deficient for Zfp148, opening up for analyses of hematopoiesis in a conventional loss-of-function model in vivo. Here, we show that Zfp148-deficient neonatal and adult mice have normal or slightly increased levels of hemoglobin, hematocrit, platelets and white blood cells, compared to wild type controls. Hematopoietic lineages in bone marrow, thymus and spleen from Zfp148 (gt/gt) mice were further investigated by flow cytometry. There were no differences in T-cells (CD4 and CD8 single positive cells, CD4 and CD8 double negative/positive cells) in either organ. However, the fraction of CD69- and B220-positive cells among lymphocytes in spleen was slightly lower at postnatal day 14 in Zfp148 (gt/gt) mice compared to wild type mice. Our results demonstrate that Zfp148-deficient mice generate normal mature hematopoietic populations thus challenging earlier studies indicating that Zfp148 plays a critical role during hematopoietic development.

The transcription factor Zfp148 (Zbp-89, BFCOL, BERF1, htβ) interacts physically with the tumor suppressor p53 and is implicated in cell cycle control, but the physiological role of Zfp148 remains unknown. Here we show that Zfp148 deficiency leads to respiratory distress and lethality in newborn mice. Zfp148 deficiency prevented structural maturation of the prenatal lung without affecting type II cell differentiation or surfactant production. BrdU analyses revealed that Zfp148 deficiency caused proliferation arrest of pulmonary cells at E18.5-19.5. Similarly, Zfp148-deficient fibroblasts exhibited proliferative arrest that was dependent on p53, raising the possibility that cell stress is part of the underlying mechanism. Indeed, Zfp148 deficiency lowered the threshold for activation of p53 under oxidative conditions. Moreover, both in vivo and cellular phenotypes were rescued on Trp53(+/-) or Trp53(-/-) backgrounds and by antioxidant treatment. Thus, Zfp148 prevents respiratory distress and lethality in newborn mice by attenuating oxidative stress-dependent p53-activity during the saccular stage of lung development. Our results establish Zfp148 as a novel player in mammalian lung maturation and demonstrate that Zfp148 is critical for cell cycle progression in vivo.