Lactate-induced MRP1 expression contributes to metabolism-based etoposide resistance in non-small cell lung cancer cells

Background Metabolic reprogramming contributes significantly to tumor development and is tightly linked to drug resistance. The chemotherapeutic agent etoposide (VP-16) has been used clinically in the treatment of lung cancer but possess different sensitivity and efficacy towards SCLC and NSCLC. Here, we assessed the impact of etoposide on glycolytic metabolism in SCLC and NSCLC cell lines and investigated the role of metabolic rewiring in mediating etoposide resistance. Methods glycolytic differences of drug-treated cancer cells were determined by extracellular acidification rate (ECAR), glucose consumption, lactate production and western blot. DNA damage was evaluated by the comet assay and western blot. Chemoresistant cancer cells were analyzed by viability, apoptosis and western blot. Chromatin immunoprecipitation (ChIP) was used for analysis of DNA-protein interaction. Results Here we showed that exposure to chemotherapeutic drug etoposide induces an exacerbation of ROS production which activates HIF-1α-mediated the metabolic reprogramming toward increased glycolysis and lactate production in non-small cell lung cancer (NSCLC). We identified lactic acidosis as the key that confers multidrug resistance through upregulation of multidrug resistance-associated protein 1 (MRP1, encoded by ABCC1), a member of ATP-binding cassette (ABC) transporter family. Mechanistically, lactic acid coordinates TGF-β1/Snail and TAZ/AP-1 pathway to induce formation of Snail/TAZ/AP-1 complex at the MRP1/ABCC1 promoter. Induction of MRP1 expression inhibits genotoxic and apoptotic effects of chemotherapeutic drugs by increasing drug efflux. Furthermore, titration of lactic acid with NaHCO3 was sufficient to overcome resistance. Conclusions The chemotherapeutic drug etoposide induces the shift toward aerobic glycolysis in the NSCLC rather than SCLC cell lines. The increased lactic acid in extracellular environment plays important role in etoposide resistance through upregulation of MRP expression. These data provide first evidence for the increased lactate production, upon drug treatment, contributes to adaptive resistance in NSCLC and reveal potential vulnerabilities of lactate metabolism and/or pathway suitable for therapeutic targeting. Graphical abstract

Effect of EGR1-Mediated LncRNA HOTAIR on MRP1 Gene Expression and MDR in Lung Cancer

Background: Multi-drug resistance-associated protein 1 (MRP1) plays critical roles in the multi-drug resistance (MDR) of cancer cells, and its expression is regulated by several transcription factors. However, the effects of EGR1 on MRP1 expression and MDR in lung cancer cells remain unknown.Methods: The expression of Transcription factors EGR1 and lncRNA HOTAIR in Non-small cell lung cancer were detected by using qRT-PCR, at the same time, Western Blot was used to detect the expression of transcription factor EGR1. MTT assay, Flow Cytometry and Observation with laser confocal microscope assay were used to explore the role of EGR1 and lncRNA HOTAIR in Non-small cell lung cancer cells. ChIP PCR assay and Luciferase reporter assays were used to demonstrate the molecular biological mechanism of EGR1 and lncRNA HOTAIR in Non-small cell lung cancer.Results: We found that EGR1 could bind to the MRP1 promoter at site -53/-42 bp and thereby regulate MRP1 expression. EGR1 knock-down reduced MRP1 expression, while EGR1 overexpression increased it. Knockdown of EGR1 increased the drug sensitivity to 5-Fluorouracil (5-FU), Toosendanin (TSN), and cisplatin (DDP), reduced the efflux ability of Rho-123, and induced apoptosis of drug-resistant lung cancer cells A549/DDP, while EGR1 overexpression had the opposite effects. Further, we demonstrated that lncRNA HOTAIR mediated the effects of EGR1 on MRP1 and MDR via sponging of miR-6807-3p. Moreover, we showed that miR-6807-3p exerts its function by targeting the EGR1 3'UTR. Conclusions: We revealed the role and molecular mechanisms of the novel HOTAIR/miR-6807-3p/EGR1 axis in the regulation of MRP1 expression and MDR in lung cancer cells. Our findings identify EGR1 as a potential biomarker and therapeutic target for MDR in human lung cancer.

Allyl isothiocyanate upregulates MRP1 expression through Notch1 signaling in human bronchial epithelial cells

Multidrug resistance associated protein-1 (MRP1) and Notch signaling are closely related and both play a critical role in chronic obstructive pulmonary disease (COPD) establishment and progression. The aim of our work was to test whether Notch1 is involved in allyl isothiocyanate (AITC) induced MRP1 expression. We used cigarette smoke extract (CSE) to simulate the smoking microenvironment in vitro. The results demonstrated that CSE led to apoptosis as well as reduced the expression of Notch1, Hes1, and MRP1, while AITC significantly reversed this downregulation. Transfected with Notch1 siRNA downregulated MRP1 expression and activity, aggravated the suppression effect by CSE, and abolished the AITC-induced Notch1, Hes1, and MRP1 expression. Validation of the correlation between Notch1 and MRP1 was implemented by gel-shift assays (electrophoretic mobility shift assay). The result revealed an interaction between a specific promoter region of MRP1 and the intracellular domain of Notch1. In conclusion, Notch1 signaling positively regulated MRP1 in 16HBE cells and AITC induced MRP1 expression and function may be attributed to Notch1 signaling. These findings show that Notch1 and MRP1 might have a potential protective effect in the COPD process and become a new therapeutic target for COPD or other lung diseases. It also provides a theoretical basis for the therapeutic effects of AITC.

lncRNA ROR Promotes Gastric Cancer Drug Resistance

Objective: Gastric cancer is one of the most common malignant tumors worldwide, and for resectable tumors, the most effective treatment is surgery with chemotherapy in neoadjuvant or adjuvant setting. However, the majority of patients fail to achieve the ideal initial response and/or develop resistance to chemotherapy. It was reported that long noncoding RNA regulator of reprogramming (ROR) is highly associated with the progression of gastric cancer. However, the role ROR in multidrug resistance (MDR) remains unclear. Methods: The messenger RNA levels of 63 specimens of patients with gastric cancer were determined by real-time polymerase chain reaction analysis and were correlated with drug resistance and survival of patients. To determine the cellular functions of ROR, we generated gastric cancer MDR cells. The effect of ROR depletion on multidrug resistance-associated protein 1 (MRP1) expression and cell apoptosis were examined by immunoblotting analyses, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and flow cytometry. Results: We found that ROR expression levels are positively associated with increased MDR and poor prognosis of patients with gastric cancer. Regulator of reprogramming expression is increased in gastric cancer cells resistant to adriamycin (ADR) and vincristine (VCR). Depletion of ROR reduced MRP1 expression and increased apoptosis of drug-resistant gastric cancer cells in response to ADR and VCR treatment. Conclusions: We demonstrated that ROR expression promotes MRP1 expression and MDR of gastric cancer cells and is correlated with increased MDR and poor prognosis of patients with gastric cancer. Our finding highlighted the potential of targeting ROR to improve the efficacy of chemotherapy.