MicroRNA-21 guide and passenger strand regulation of adenylosuccinate lyase-mediated purine metabolism promotes transition to an EGFR-TKI-tolerant persister state

Abstract

In EGFR-mutant lung cancer, drug-tolerant persister cells (DTPCs) show prolonged survival when receiving EGFR tyrosine kinase inhibitor (TKI) treatments. They are a likely source of drug resistance, but little is known about how these cells tolerate drugs. Ribonucleic acids (RNAs) molecules control cell growth and stress responses. Nucleic acid metabolism provides metabolites, such as purines, supporting RNA synthesis and downstream functions. Recently, noncoding RNAs (ncRNAs), such as microRNAs (miRNAs), have received attention due to their capacity to repress gene expression via inhibitory binding to downstream messenger RNAs (mRNAs). Here, our study links miRNA expression to purine metabolism and drug tolerance. MiR-21-5p (guide strand) is a commonly upregulated miRNA in disease states, including cancer and drug resistance. However, the expression and function of miR-21-3p (passenger strand) are not well understood. We found that upregulation of miR-21-5p and miR-21-3p tune purine metabolism leading to increased drug tolerance. Metabolomics data demonstrated that purine metabolism was the top pathway in the DTPCs compared with the parental cells. The changes in purine metabolites in the DTPCs were partially rescued by targeting miR-21. Analysis of protein levels in the DTPCs showed that reduced expression of adenylosuccinate lyase (ADSL) was reversed after the miR-21 knockdown. ADSL is an essential enzyme in the de novo purine biosynthesis pathway by converting succino-5-aminoimidazole-4-carboxamide riboside (succino-AICAR or SAICAR) to AICAR (or acadesine) as well as adenylosuccinate to adenosine monophosphate (AMP). In the DTPCs, miR-21-5p and miR-21-3p repress ADSL expression. The levels of top decreased metabolite in the DTPCs, AICAR was reversed when miR-21 was blocked. AICAR induced oxidative stress, evidenced by increased reactive oxygen species (ROS) and reduced expression of nuclear factor erythroid-2-related factor 2 (NRF2). Concurrently, miR-21 knockdown induced ROS generation. Therapeutically, a combination of AICAR and osimertinib increased ROS levels and decreased osimertinib-induced NRF2 expression. In a MIR21 knockout mouse model, MIR21 loss-of-function led to increased purine metabolites but reduced ROS scavenging capacity in lung tissues in physiological conditions. Our data has established a link between ncRNAs, purine metabolism, and the redox imbalance pathway. This discovery will increase knowledge of the complexity of the regulatory RNA network and potentially enable novel therapeutic options for drug-resistant patients.

An intrinsic purine metabolite AICAR blocks lung tumour growth by targeting oncoprotein mucin 1

“…These data might provide clues to recent discoveries that AICAR's anticancer roles are independent of AMPK, indicating that AICAR plays a prominent role in binding to other targets [18,19]. Targeting lung cancer cells with AICAR in vitro and in vivo has demonstrated that AICAR is an effective anticancer molecule by our and other labs [20,21]. It is still not well known how AICAR targets these lung cancer cells.…”

Efficient identification of new small molecules targeting succinate dehydrogenase in non- small cell lung cancer

“…RNA extraction was performed using mirVana miRNA Isolation Kit (Cat#AM1560, Invitrogen) as described previously 79 . A total of 500 ng RNA each sample was input for the Reverse Transcription reaction to acquire the cDNA.…”

MiRNA-Based Therapies for Lung Cancer: Opportunities and Challenges?

“…In addition, ectopic overexpression of miR-506-3p in erlotinibresistant cells targeted Sonic Hedgehog (SHH), increased E-cadherin expression, inhibited vimentin expression, and reversed EMT to a mesenchymal phenotype epithelial-like transformation, thereby counteracting EMT-mediated chemoresistance, increasing sensitivity to apoptosis, decreasing cell stemness, reducing proliferation, and enhancing sensitivity to erlotinib [118] (Figure 5). Numerous studies have shown that multiple miRNAs are extensively involved in EGFR-TKI regulation, suggesting that miRNAs may be new therapeutic molecules and biomarkers for anti-EGFR therapy and have a positive role in reversing drug resistance and improving sensitivity [119][120][121]. Alessandro et al collected plasma samples from 39 patients with advanced EGFR-mutated NSCLC treated with EGFR-TKIs, assessed the expression levels of miRNAs and found that miR-21 could be a useful indicator to monitor the effect of EGFR-TKI treatment [122].…”

“…Alessandro et al collected plasma samples from 39 patients with advanced EGFR-mutated NSCLC treated with EGFR-TKIs, assessed the expression levels of miRNAs and found that miR-21 could be a useful indicator to monitor the effect of EGFR-TKI treatment [122]. In addition, Zhang et al identified miR-608 and miR-4513 single nucleotide polymorphisms as independent candidate biomarkers for pre- Numerous studies have shown that multiple miRNAs are extensively involved in EGFR-TKI regulation, suggesting that miRNAs may be new therapeutic molecules and biomarkers for anti-EGFR therapy and have a positive role in reversing drug resistance and improving sensitivity [119][120][121]. Alessandro et al collected plasma samples from 39 patients with advanced EGFR-mutated NSCLC treated with EGFR-TKIs, assessed the expression levels of miRNAs and found that miR-21 could be a useful indicator to monitor the effect of EGFR-TKI treatment [122].…”