Cancer cell-autonomous cGAS-STING response confers drug resistance.

The cGAS-STING pathway has long been recognized as playing a crucial role in immune surveillance and tumor suppression. Here, we show that when the pathway is activated in a cancer-cell-autonomous response manner, it confers drug resistance. Targeted or conventional chemotherapy drugs promoted cytosolic DNA accumulation in cancer cells, activating the cGAS-STING pathway and downstream TBK1-IRF3/NF-κB signaling. This cancer cell-intrinsic response enabled the cells to counteract drug stress, allowing treatment resistance to be acquired and maintained. Blockade of stimulator of interferon genes (STING) signaling delayed and overcame resistance in models in vitro and in vivo. This finding uncovers an alternative face of cGAS-STING signaling other than the well-reported modulation of microenvironmental immune cells. It also implies a caution for the combination of STING agonist with targeted or conventional chemotherapy drug treatment, a strategy prevailing in current clinical trials.

Erratum: Aldehyde dehydrogenase 1A1 confers erlotinib resistance via facilitating the reactive oxygen species-reactive carbonyl species metabolic pathway in lung adenocarcinomas: Erratum.

[This corrects the article DOI: 10.7150/thno.35729.].

A phosphoglycerate mutase 1 allosteric inhibitor overcomes drug resistance to EGFR-targeted therapy via disrupting IL-6/JAK2/STAT3 signaling pathway in lung adenocarcinoma.

Resistance to epidermal growth factor receptor (EGFR) inhibitors, from the first-generation erlotinib to the third generation osimertinib, is a clinical challenge in the treatment of patients with EGFR-mutant lung adenocarcinoma. Our previous work found that a novel allosteric inhibitor of phosphoglycerate mutase 1 (PGAM1), HKB99, restrains erlotinib resistance in lung adenocarcinoma cells. However, the role of HKB99 in osimertinib resistance and its underlying molecular mechanism remains to be elucidated. Herein, we found that IL-6/JAK2/STAT3 signaling pathway is aberrantly activated in both erlotinib and osimertinib resistant cells. Importantly, HKB99 significantly blocks the interaction of PGAM1 with JAK2 and STAT3 via the allosteric sites of PGAM1, which leads to inactivation of JAK2/STAT3 and thereby disrupts IL-6/JAK2/STAT3 signaling pathway. Consequently, HKB99 remarkably restores EGFR inhibitor sensitivity and exerts synergistic tumoricidal effect. Additionally, HKB99 alone or in combination with osimertinib down-regulated the level of p-STAT3 in xenograft tumor models. Collectively, this study identifies PGAM1 as a key regulator in IL-6/JAK2/STAT3 axis in the development of resistance to EGFR inhibitors, which could serve as a therapeutic target in lung adenocarcinoma with acquired resistance to EGFR inhibitors.

Metabolic and Nonmetabolic Functions of PSAT1 Coordinate Signaling Cascades to Confer EGFR Inhibitor Resistance and Drive Progression in Lung Adenocarcinoma.

Emerging evidence demonstrates that the dysregulated metabolic enzymes can accelerate tumorigenesis and progression via both metabolic and nonmetabolic functions. Further elucidation of the role of metabolic enzymes in EGFR inhibitor resistance and metastasis, two of the leading causes of death in lung adenocarcinoma, could help improve patient outcomes. Here, we found that aberrant upregulation of phosphoserine aminotransferase 1 (PSAT1) confers erlotinib resistance and tumor metastasis in lung adenocarcinoma. Depletion of PSAT1 restored sensitivity to erlotinib and synergistically augmented the tumoricidal effect. Mechanistically, inhibition of PSAT1 activated the ROS-dependent JNK/c-Jun pathway to induce cell apoptosis. In addition, PSAT1 interacted with IQGAP1, subsequently activating STAT3-mediated cell migration independent of its metabolic activity. Clinical analyses showed that PSAT1 expression positively correlated with the progression of human lung adenocarcinoma. Collectively, these findings reveal the multifunctionality of PSAT1 in promoting tumor malignancy through its metabolic and nonmetabolic activities. SIGNIFICANCE: Metabolic and nonmetabolic functions of PSAT1 confer EGFR inhibitor resistance and promote metastasis in lung adenocarcinoma, suggesting therapeutic targeting of PSAT1 may attenuate the malignant features of lung cancer.

Targeting AKR1B1 inhibits glutathione de novo synthesis to overcome acquired resistance to EGFR-targeted therapy in lung cancer.

Acquired resistance represents a bottleneck to molecularly targeted therapies such as epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) treatment in lung cancer. A deeper understanding of resistance mechanisms can provide insights into this phenomenon and help to develop additional therapeutic strategies to overcome or delay resistance. Here, we identified a pharmacologically targetable metabolic mechanism that drives resistance to EGFR TKIs in lung cancer cell lines and patient-derived xenograft mice. We demonstrated that aldo-keto reductase family 1 member B1 (AKR1B1) interacts with and activates signal transducer and activator of transcription 3 (STAT3) to up-regulate the cystine transporter solute carrier family 7 member 11 (SLC7A11). This leads to enhanced cystine uptake and flux to glutathione de novo synthesis, reactive oxygen species (ROS) scavenging, protection from cell death, and EGFR TKI drug resistance in lung cancer cell lines and xenograft mouse models. Suppression of AKR1B1 with selective inhibitors, including the clinically approved antidiabetic drug epalrestat, restored the sensitivity of resistant cell lines to EGFR TKIs and delayed resistance in lung cancer patient-derived xenograft mice. Our findings suggest a metabolic mechanism for resistance to a molecularly targeted therapy and provide a potential therapeutic target for overcoming resistance to EGFR TKIs, including the third-generation inhibitor osimertinib.

Erratum: Overcoming erlotinib resistance in EGFR mutation-positive lung adenocarcinomas through repression of phosphoglycerate dehydrogenase: Erratum.

[This corrects the article DOI: 10.7150/thno.23177.].

NRF2-GPX4/SOD2 axis imparts resistance to EGFR-tyrosine kinase inhibitors in non-small-cell lung cancer cells.

Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) have achieved satisfactory clinical effects in the therapy of non-small cell lung cancer (NSCLC), but acquired resistance limits their clinical application. NRF2 has been shown to enhance the resistance to apoptosis induced by radiotherapy and some chemotherapy. In this study, we investigated the role of NRF2 in resistance to EGFR-TKIs. We showed that NRF2 protein levels were markedly increased in a panel of EGFR-TKI-resistant NSCLC cell lines due to slow degradation of NRF2 protein. NRF2 knockdown overcame the resistance to EGFR-TKIs in HCC827ER and HCC827GR cells. Furthermore, we demonstrated that NRF2 imparted EGFR-TKIs resistance in HCC827 cells via upregulation of GPX4 and SOD2, and suppression of GPX4 and SOD2 reversed resistance to EGFR-TKIs. Thus, we conclude that targeting NRF2-GPX4/SOD2 pathway is a potential strategy for overcoming resistance to EGFR-TKIs.

HKB99, an allosteric inhibitor of phosphoglycerate mutase 1, suppresses invasive pseudopodia formation and upregulates plasminogen activator inhibitor-2 in erlotinib-resistant non-small cell lung cancer cells.

Acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), such as erlotinib, remains a major challenge in the targeted therapy of non-small cell lung cancer (NSCLC). HKB99 is a novel allosteric inhibitor of phosphoglycerate mutase 1 (PGAM1) that preferentially suppresses cell proliferation and induces more apoptosis in acquired erlotinib-resistant HCC827ER cells compared with its parental HCC827 cells. In this study we identified the molecular biomarkers for HKB99 response in erlotinib-resistant HCC827ER cells. We showed that HCC827ER cells displayed enhanced invasive pseudopodia structures as well as downregulated plasminogen activator inhibitor-2 (PAI-2). Meanwhile, PAI-2 knockdown by siPAI-2 candidates decreased the sensitivity of HCC827 parental cells to erlotinib. Moreover, HKB99 (5 µM) preferentially inhibited the invasive pseudopodia formation and increased the level of PAI-2 in HCC827ER cells. Collectively, this study provides new insight into the role of PAI-2 in regulating the sensitivity of erlotinib resistant NSCLC cells to PGAM1 inhibitor. Furthermore, PAI-2 level might be considered as a potential biomarker for predicting the efficacy of the PGAM1 allosteric inhibitor on the erlotinib resistant NSCLC cells.

Development and validation of a rapid, robust and sensitive UPLC-QQQ-MS/MS method for simultaneous quantification of GSH metabolism in lung cancer cells.

Changes in cellular metabolism accompany tumor therapeutic resistance. Metabolite concentrations specifically reflect the cellular state. Glutathione (GSH) metabolism maintains the redox homeostasis while also confers therapeutic resistance to cancer cells. However, analytical methods for studying GSH metabolism rely on high-resolution-based untargeted metabolomics. Since the aim of untargeted metabolomics studies is covering as much metabolites as possible, these methods lack sensitivity for simultaneous analysis of intracellular GSH-related metabolites with different polarities and structures. In this study, based on cultured lung cancer cells, we described a rapid, robust and sensitive ultra-performance liquid chromatography-triple quadrupole tandem mass spectrographic method (UPLC-QQQ-MS/MS) to simultaneously quantify a repertoire of GSH-related metabolites, including GSH, GSSG, glycine, cysteine, glutamine, glutamate, cystine, γ-glutamyl-cysteine and cysteinyl-glycine. This method avoided the use of derivatization and/or ion-pairing reagents and was validated according to United States Food and Drug Administration (US FDA) criteria. The lower limit of quantification was determined to be 0.5-100 ng/mL with lower limits of detection at 0.14-10.07 ng/mL. The intra- and inter-day precision values for all the analytes were <15% CV, and the accuracy ranged from 85.4% to 114% at three levels of quality control. This method combined simple preparation with rapid analytical procedure (8 min), allowed for high-throughput analysis of GSH metabolism in basic and therapeutic treatment conditions within cultured cells. Our data showed a significant alteration of GSH metabolism in two independent resistant cells compared to sensitive cells. This method monitored the impact of molecularly targeted drugs on GSH metabolism within lung cancer cells and therefore helped identifying potential metabolic vulnerability for the therapeutic resistance in lung cancer.

Aldehyde dehydrogenase 1A1 confers erlotinib resistance via facilitating the reactive oxygen species-reactive carbonyl species metabolic pathway in lung adenocarcinomas.

Background: Acquired resistance to epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) such as erlotinib is a major challenge to achieve an overall clinical benefit of the targeted therapy. Recently, aldehyde dehydrogenase 1 (ALDH1) induction has been found to render lung adenocarcinomas resistant to EGFR-TKIs, and targeting ALDH1A1 becomes a novel strategy to overcome resistance. However, the molecular mechanism underlying such effect remains poorly understood. Methods: Comprehensive assays were performed in a panel of lung adenocarcinoma cell lines and xenografts that acquired resistance to erlotinib. Cancer phenotype was evaluated by cell viability, apoptosis, migration, and epithelial-mesenchymal transition analysis in vitro, tumorsphere formation analysis ex vivo, and tumor growth and dissemination analysis in vivo. Reactive oxygen species (ROS) and reactive carbonyl species (RCS) were detected based on fluorescent oxidation indicator and liquid chromatography coupled to mass spectrometry, respectively. Protein target was suppressed by RNA interference and pharmacological inhibition or ecto-overexpressed by lentivirus-based cloning. Gene promoter activity was measured by dual-luciferase reporting assay. Results: Knockdown or pharmacological inhibition of ALDH1A1 overcame erlotinib resistance in vitro and in vivo. ALDH1A1 overexpression was sufficient to induce erlotinib resistance. Metabolomic analysis demonstrated lower ROS-RCS levels in ALDH1A1-addicted, erlotinib-resistant cells; in line with this, key enzymes for metabolizing ROS and RCS, SOD2 and GPX4, respectively, were upregulated in these cells. Knockdown of SOD2 or GPX4 re-sensitized the resistant cells to erlotinib and the effect was abrogated by ROS-RCS scavenging and mimicked by ROS-RCS induction. The ALDH1A1 overexpressed cells, though resisted erlotinib, were more sensitive to SOD2 or GPX4 knockdown. The ALDH1A1 effect on erlotinib resistance was abrogated by ROS-RCS induction and mimicked by ROS-RCS scavenging. Detection of GPX4 and SOD2 expression and analysis of promoter activities of GPX4 and SOD2 under the condition of suppression or overexpression of ALDH1A1 demonstrated that the RCS-ROS-metabolic pathway was controlled by the ALDH1A1-GPX4-SOD2 axis. The ROS-RCS metabolic dependence mechanism in ALDH1A1-induced resistance was confirmed in vivo. Analysis of public databases showed that in patients undergoing chemotherapy, those with high co-expression of ALDH1A1, GPX4, and SOD2 had a lower probability of survival. Conclusions: ALDH1A1 confers erlotinib resistance by facilitating the ROS-RCS metabolic pathway. ALDH1A1-induced upregulation of SOD2 and GPX4, as well as ALDH1A1 itself, mitigated erlotinib-induced oxidative and carbonyl stress, and imparted the TKI resistance. The elucidation of previously unrecognized metabolic mechanism underlying erlotinib resistance provides new insight into the biology of molecular targeted therapies and help to design improved pharmacological strategies to overcome the drug resistance.

A Novel Allosteric Inhibitor of Phosphoglycerate Mutase 1 Suppresses Growth and Metastasis of Non-Small-Cell Lung Cancer.

Phosphoglycerate mutase 1 (PGAM1) plays a pivotal role in cancer metabolism and tumor progression via its metabolic activity and interaction with other proteins like α-smooth muscle actin (ACTA2). Allosteric regulation is considered to be an innovative strategy to discover a highly selective and potent inhibitor targeting PGAM1. Here, we identified a novel PGAM1 allosteric inhibitor, HKB99, via structure-based optimization. HKB99 acted to allosterically block conformational change of PGAM1 during catalytic process and PGAM1-ACTA2 interaction. HKB99 suppressed tumor growth and metastasis and overcame erlotinib resistance in non-small-cell lung cancer (NSCLC). Mechanistically, HKB99 enhanced the oxidative stress and altered multiple signaling pathways including the activation of JNK/c-Jun and suppression of AKT and ERK. Collectively, the study highlights the potential of PGAM1 as a therapeutic target in NSCLC and reveals a distinct mechanism by which HKB99 inhibits both metabolic activity and nonmetabolic function of PGAM1 by allosteric regulation.

UPLC-MS-based metabolomics reveals metabolic dysregulation in ALDH1A1-overexpressed lung adenocarcinoma cells.

INTRODUCTION: Specific oncogenotypes can produce distinct metabolic changes in cancer. Recently it is considered that metabolic reprograming contributes heavily to drug resistance. Aldehyde dehydrogenase 1A1 (ALDH1A1), is overexpressed in drug resistant lung adenocarcinomas and may be the cause of acquired drug resistance. However, how ALDH1A1 affects metabolic profiling in lung adenocarcinoma cells remains elusive. OBJECTIVE: We sought to investigate metabolic alterations induced by ALDH1A1 in lung adenocarcinoma in order to better understand the reprogramming and metabolic mechanism of resistance induced by ALDH1A1. METHODS: Metabolic alterations in lung adenocarcinoma HCC827-ALDH1A1 cells were analyzed by ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS). HCC827-ALDH1A1 metabolic signatures were extracted by univariate and multivariate statistical analysis. Furthermore, metabolite enrichment analysis and pathway analysis were performed using MetaboAnalyst 4.0 software. RESULTS: Twenty-two metabolites were positively identified using authentic standards, including uridine monophosphate (UMP), uridine diphosphate (UDP), adenosine diphosphate (ADP), malic acid, malonyl-coenzyme A, nicotinamide adenine dinucleotide (NAD), coenzyme A and so on. Furthermore, metabolic pathway analysis revealed several dysregulated pathways in HCC827-ALDH1A1 cells, including nucleotide metabolism, urea cycle, tricarboxylic acid (TCA) cycle, and glycerol phospholipid metabolism etc. CONCLUSION: Lung cancer is the most frequent cause of cancer-related deaths worldwide. Nearly all patients eventually undergo disease progression due to acquired resistance. Mechanisms of biological acquired resistance need to be identified. Our study identified altered metabolites in HCC827-ALDH1A1 cells, enhancing our knowledge of lung adenocarcinoma metabolic alterations induced by ALDH1A1, creating a novel therapeutic pathway. These metabolic signatures of ALDH1A1 overexpression may shed light on molecular mechanisms in drug-resistant tumors, and on candidate drug targets. Furthermore, new molecular targets may provide the foundation for potential anticancer strategies for lung cancer therapy.

Overcoming erlotinib resistance in EGFR mutation-positive lung adenocarcinomas through repression of phosphoglycerate dehydrogenase.

How to improve the efficacy and reverse the resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), such as erlotinib, remains a major challenge in the targeted therapy of lung adenocarcinoma with EGFR-activating mutation. Phosphoglycerate dehydrogenase (PHGDH) is the key enzyme of de novo serine biosynthesis over-expressed in various types of cancer including lung cancer. Elevated PHGDH expression is correlated with a worse overall survival in clinical lung adenocarcinoma patients. Here we investigated the role of PHGDH in lung adenocarcinoma with the acquisition of resistance to erlotinib. Methods: The necessary genes required for the acquired erlotinib resistance in lung adenocarcinoma cells were screened out by RNA-Seq analysis. Then the protein and mRNA levels of PHGDH were confirmed by immunoblotting and qRT-PCR in the erlotinib resistant cells. The effects of PHGDH inhibition or overexpression on erlotinib resistance were examined using cell culture and tumor xenograft mouse models respectively. To explore mechanism, the ROS level and DNA damage marker, γH2AX, were tested by DCFH-DA staining and immunofluorescence after PHGDH inhibition. Results: We found that PHGDH level was significantly increased in the lung adenocarcinoma PC9ER4 and HCC827ER9 cells that acquired resistance to erlotinib. Perturbation of PHGDH by siPHGDH transfection or NCT-503, a small molecular PHGDH inhibitor, synergistically augmented the tumoricidal effect and restored sensitivity to erlotinib in cell lines and xenografts. Over-expression of PHGDH caused xenografts resistant to erlotinib. Furthermore, multiple DNA damage repair pathways related genes were changed by PHGDH depletion specifically in erlotinib resistant cells. ROS stress and DNA damage marker γH2AX were enhanced by siPHGDH and NCT-503, which was reversed by NAC. Conclusion: Our study indicated that PHGDH inhibition has potential therapeutic value in lung adenocarcinoma with the acquired resistance to EGFR-TKIs.

Interleukin-6 identified as an important factor in hypoxia- and aldehyde dehydrogenase-based gefitinib adaptive resistance in non-small cell lung cancer cells.

Gefitinib resistance and relapse of the disease were the greatest challenges facing clinical therapy of non-small-cell lung cancer (NSCLC). Of note, regarding the hypoxia condition in solid tumor tissues in vivo, roles of hypoxia in gefitinib adaptive resistance and its association with lung cancer stem cells (LCSCs) have not been fully elucidated. In the present study, the role of hypoxia in gefitinib adaptive resistance and its association with aldehyde dehydrogenase (ALDH)-based LCSC gefitinib resistance were comparatively studied using RNA-sequencing (RNA-seq) technology. Co-treatment of PC9 cells with gefitinib and hypoxia (1% O2) significantly enhanced adaptive resistance compared with gefitinib or hypoxia treatment alone. An ALDEFLUOR assay demonstrated that there was a significant increase of ALDH expression level in hypoxia and gefitinib co-treated PC9 cells, in addition to a higher ratio of G0/G1 quiescent cell enrichment and acquisition of epithelial-mesenchymal transition. RNA-seq analysis revealed that interleukin-6 (IL-6) is an important common factor in hypoxia and ALDH-based gefitinib resistance, supported by inflammation-associated tumor necrosis factor, nuclear factor-κB and Janus kinase-signal transducer and activator of transcription signaling pathway enrichment. Furthermore, exposure of PC9 and HCC827 cells to IL-6 increased gefitinib adaptive resistance. Consequently, IL-6 expression level was a poor prognostic marker for patients with NSCLC and adenocarcinoma. Thus, targeting IL-6 combined with tyrosine kinase inhibitor treatment may be promising in NSCLC clinical therapy in the future.

Autocrine glutamatergic transmission for the regulation of embryonal carcinoma stem cells.

Glutamate behaves as the principal excitatory neurotransmitter in the vertebrate central nervous system and recently demonstrates intercellular signaling activities in periphery cancer cells. How the glutamatergic transmission is organized and operated in cancer stem cells remains undefined. We have identified a glutamatergic transmission circuit in embryonal carcinoma stem cells. The circuit is organized and operated in an autocrine mechanism and suppresses the cell proliferation and motility. Biological analyses determined a repertoire of glutamatergic transmission components, glutaminase, vesicular glutamate transporter, glutamate NMDA receptor, and cell membrane excitatory amino-acid transporter, for glutamate biosynthesis, package for secretion, reaction, and reuptake in mouse and human embryonal carcinoma stem cells. The glutamatergic components were also identified in mouse transplanted teratocarcinoma and in human primary teratocarcinoma tissues. Released glutamate acting as the signal was directly quantified by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Genetic and pharmacological abolishment of the endogenously released glutamate-induced tonic activation of the NMDA receptors increased the cell proliferation and motility. The finding suggests that embryonal carcinoma stem cells can be actively regulated by establishing a glutamatergic autocrine/paracrine niche via releasing and responding to the transmitter.