NFE2L2 Knockout Cell Line (A549)

It has been recently reported that CD38 expressed on tumor cells of multiple murine and human origins could be upregulated in response to PD-L1 antibody therapy, which led to dysfunction of tumor-infiltrating CD8 T immune cells due to increasing the production of adenosine. However, the role of tumor expressed-CD38 on neoplastic formation and progression remains elusive. In the present study, we aimed to delineate the molecular and biochemical function of the tumor-associated CD38 in lung adenocarcinoma progression. Our clinical data showed that the upregulation of tumor-originated CD38 was correlated with poor survival of lung cancer patients. Using multiple in vitro assays we found that the enzymatic activity of tumor expressed-CD38 facilitated lung cancer cell migration, proliferation, colony formation, and tumor development. Consistently, our in vivo results showed that inhibition of the enzymatic activity or antagonizing the enzymatic product of CD38 resulted in the similar inhibition of tumor proliferation and metastasis as CD38 gene knock-out or mutation. At biochemical level, we further identified that cADPR, the mainly hydrolytic product of CD38, was responsible for inducing the opening of TRPM2 iron channel leading to the influx of intracellular Ca and then led to increasing levels of NRF2 while decreasing expression of KEAP1 in lung cancer cells. These findings suggested that malignant lung cancer cells were capable of using cADPR catalyzed by CD38 to facilitate tumor progression, and blocking the enzymatic activity of CD38 could be represented as an important strategy for preventing tumor progression.

Non-small cell lung cancer (NSCLC) remains a lethal malignancy due to therapy resistance and recurrence. Ferroptosis, a regulated form of cell death, is a promising strategy to overcome cancer drug resistance, yet its mechanisms remain incompletely defined. Here, we report that Immediate Early Response 3 (IER3) is significantly upregulated in NSCLC tumors and linked to advanced stage and poor prognosis. Using IER3-overexpressing and knockout models in A549 and H1299 cells, we found that IER3 promotes NSCLC cell proliferation, migration, and invasion by suppressing ferroptosis. Conversely, IER3 knockout induced ferroptosis and reduced malignancy-effects reversed by the ferroptosis inhibitor Fer-1. Mechanistically, IER3 sustained AKT phosphorylation to inactivate GSK3β, both blocking GSK3β-dependent proteasomal degradation of NRF2 and enhancing its nuclear translocation, which collectively led to the transactivation of downstream ferroptosis-suppressive gene programs. This program maintained glutathione homeostasis, sequestered labile iron, scavenged ROS, and ultimately inhibited lipid peroxidation to counter ferroptosis. Rescue assays confirmed NRF2 overexpression or AKT/GSK3β activation reversed IER3 knockout-induced ferroptosis and viability loss. Additionally, low-IER3 NSCLC tumors were more sensitive to clinical/preclinical agents targeting survival/stress pathways. Collectively, our findings establish IER3 as an NSCLC oncogenic driver-suppressing ferroptosis via AKT/GSK3β/NRF2 to sustain malignancy-highlighting its potential as a prognostic biomarker and therapeutic target for improved NSCLC outcomes.

The nuclear factor erythroid 2-like 2 (NFE2L2; NRF2) signaling pathway is frequently deregulated in human cancers. The critical functions of NRF2, other than its transcriptional activation, in cancers remain largely unknown. Here, we uncovered a previously unrecognized role of NRF2 in the regulation of RNA splicing. Global splicing analysis revealed that NRF2 knockdown in non-small cell lung cancer (NSCLC) A549 cells altered 839 alternative splicing (AS) events in 485 genes. Mechanistic studies demonstrated that NRF2 transcriptionally regulated SMN mRNA expression by binding to two antioxidant response elements in the SMN1 promoter. Post-transcriptionally, NRF2 was physically associated with the SMN protein. The Neh2 domain of NRF2, as well as the YG box and the region encoded by exon 7 of SMN, were required for their interaction. NRF2 formed a complex with SMN and Gemin2 in nuclear gems and Cajal bodies. Furthermore, the NRF2-SMN interaction regulated RNA splicing by expressing SMN in NRF2-knockout HeLa cells, reverting some of the altered RNA splicing. Moreover, SMN overexpression was significantly associated with alterations in the NRF2 pathway in patients with lung squamous cell carcinoma from The Cancer Genome Atlas. Taken together, our findings suggest a novel therapeutic strategy for cancers involving an aberrant NRF2 pathway.

1,8-Dinitropyrene (1,8-DNP) is a diesel exhaust constituent classified as a possible human carcinogen (Group 2B) by the International Agency for Research on Cancer. Its mutagenic properties can be attributed in part through the formation of covalent DNA adducts that result from mononitroreduction (e.g., -(deoxyguanosin-8-yl)-1-amino-8-nitropyrene). Recombinant aldo-keto reductases (AKRs) 1C1-1C3 catalyze the nitroreduction of 1,8-DNP, 1-nitropyrene, and 3-nitrobenzanthrone. Although are induced by nuclear factor erythroid 2-related factor 2 (NRF2), the contribution of NRF2 toward the nitroreduction of 1,8-DNP in human lung cells is currently unknown. We used highly sensitive and specific in-cell fluorescence assays to examine the ability of human lung A549 and HBEC3-KT cells to metabolize 1,8-DNP to yield 1-amino-8-nitropyrene (1,8-ANP) and 1,8-DNP to yield 1,8-diaminopyrene (1,8-DAP) via mono- and bis-nitroreduction, respectively. A549 cells generated both 1,8-ANP and 1,8-DAP from 1,8-DNP. By contrast, HBEC3-KT cells formed 1,8-ANP, but essentially no 1,8-DAP, from 1,8-DNP. We used genetic and pharmacological approaches to investigate the dependence of 1,8-DNP nitroreduction on AKR1C1-1C3 and NRF2. A549 cells with homozygous /NRF2 knockout did not exhibit decreased 1,8-ANP formation but showed decreased 1,8-DAP formation, indicating that the second but not the first nitroreduction step was NRF2-dependent. Treatment of HBEC3-KT cells with NRF2 activators (-sulforaphane (SFN) or 1-(2-cyano-3,12,28-trioxooleana-1,9(11)-dien-28-yl)-1-imidazole (CDDO-Im) did not increase the mononitroreduction of 1,8-DNP to 1,8-ANP but increased the conversion of 1,8-ANP to 1,8-DAP consistent with the second step requiring inducible NRF2. AKR1C isoform specific inhibitors showed that these enzymes accounted for the majority of 1,8-ANP and 1,8-DAP formation in both cell lines. The ability of A549 NRF2 knockout cells to still form 1,8-ANP coupled with their lack of AKR1C isoform expression indicated that a new nitroreductase was expressed as an adaptive response to NRF2 loss. We find that this nitroreductase is not NQO1, thioredoxin reductase, xanthine oxidase, or NADPH-P450 oxidoreductase.

1-Nitropyrene (1-NP) is a constituent of diesel exhaust and classified as a group 2A probable human carcinogen. The metabolic activation of 1-NP by nitroreduction generates electrophiles that can covalently bind DNA to form mutations to contribute to cancer causation. NADPH-dependent P450 oxidoreductase (POR), xanthine oxidase (XO), aldehyde oxidase (AOX), and NAD(P)H/quinone oxidoreductase 1 (NQO1) may catalyze 1-NP nitroreduction. We recently found that human recombinant aldo-keto reductases (AKRs) 1C1-1C3 catalyze 1-NP nitroreduction. and are genes induced by nuclear factor erythroid 2-related factor 2 (NRF2). Despite this knowledge, the relative importance of these enzymes and NRF2 to 1-NP nitroreduction is unknown. We used a combination of pharmacological and genetic approaches to assess the relative importance of these enzymes and NRF2 in the aerobic nitroreduction of 1-NP in human bronchial epithelial cells, A549 and HBEC3-KT. 1-NP nitroreduction was assessed by the measurement of 1-aminopyrene (1-AP), the six-electron reduced metabolite of 1-NP, based on its intrinsic fluorescence properties (λ and λ). We found that co-treatment of 1-NP with salicylic acid, an AKR1C1 inhibitor, or ursodeoxycholate, an AKR1C2 inhibitor, for 48 h decreased 1-AP production relative to 1-NP treatment alone (control) in both cell lines. -Sulforaphane or 1-(2-cyano-3,12,28-trioxooleana-1,9(11)-dien-28-yl)-1-imidazole (CDDO-Im), two NRF2 activators, each increased 1-AP production relative to control only in HBEC3-KT cells, which have inducible NRF2. Inhibitors of POR, NQO1, and XO failed to modify 1-AP production relative to control in both cell lines. Importantly, A549 wild-type cells with constitutively active NRF2 produced more 1-AP than A549 cells with heterozygous expression of /NRF2, which were able to produce more 1-AP than A549 cells with homozygous knockout of /NRF2. Together, these data show dependence of 1-NP metabolic activation on AKR1Cs and NRF2 in human lung cells. This is the second example whereby /NRF2 is implicated in the carcinogenicity of diesel exhaust constituents.

Acute respiratory distress syndrome (ARDS) is a deadly illness which presents with severe hypoxemia as well as diffuse alveolar damage. Jumonji domain‑containing 3 (JMJD3), which belongs to the UTX/UTY JmjC‑domain protein subfamily, is involved in infection, development, aging and immune disorders. However, the role of JMJD3 in acute lung injury (ALI) is still unclear. The present study explored the roles and potential mechanisms of JMJD3 in ALI. Alveolar epithelial cell‑specific knockout of JMJD3 mice and A549 alveolar epithelial cells were used to investigate the function of JMJD3 in ALI. Lipopolysaccharide (LPS) was used to establish an and ALI model. The expression of JMJD3 in murine lung tissue and alveolar epithelial cells was detected. Pathological injury of lung tissue and alveolar epithelial cells was also investigated following inhibition of JMJD3. The results showed that JMJD3 expression was significantly increased in murine lung tissues and in A549 cells following LPS stimulation. JMJD3‑deficient mice in alveolar epithelial cells exhibited alleviated lung pathological injury and ferroptosis following h stimulation. Mechanistically, it was found that JMJD3 knockout could increase the expression of nuclear factor erythroid‑2‑related factor‑2 (Nrf2) in lung tissues challenged with h. However, Nrf2 overexpression by adenovirus could further enhance the anti‑ferroptotic effect from JMJD3 silence in h‑treated A549 cells. Taken together, the present study revealed that JMJD3 deficiency may relieve LPS‑induced ALI by blocking alveolar epithelial ferroptosis in a Nrf2‑dependent manner, which may serve as a novel therapeutic target against ALI.