CAFs regulate HK2 through SKP2 driving NSCLC glycolysis to affect almonertinib resistance

Abstract Purpose Almonertinib, a third-generation EGFR-TKI, is becoming widely used in the clinic. In recent years, glycolysis and cancer-associated fibroblasts (CAFs) have emerged as hotspots for cancer therapy. The aim of this paper is to investigate the relationship among glycolysis, CAFs and almonertinib resistance. Methods In this study, we used cell count kit-8 assay, transcriptome sequencing, hierarchical clustering analysis, western blot, RT‒qPCR, detection of glucose consumption and lactate production assays to compare the differences in glycolytic capacity and hexokinase 2 (HK2) between constructed drug-resistant and drug-sensitive cells. Through KEGG pathway enrichment analysis, PPI network construction and modeling analysis, we selected SKP2 (S-phase kinase-associated protein 2) as the target to study the mechanism of HK2 regulation by CAFs. Two siRNAs were constructed to knock down HK2 and SKP2 by transfection. Finally, the effects on cell proliferation, migration, invasion and apoptosis were verified by colony formation, wound healing, Transwell invasion, and flow assays for apoptosis. Results First, the glycolytic capacity and HK2 content of H1975AR cells, which are resistant to almonertinib, were higher than those of H1975 cells. However, by knocking down HK2, cellular resistance was found to be reduced, and this result demonstrated that HK2 affected cellular resistance to almonertinib through glycolysis. Second, we obtained H1975/CAF-CM by coculturing with conditioned medium from extracted CAFs, which showed upregulation of drug resistance, glycolytic ability and HK2 content. This demonstrated that CAFs could make cells resistant to almonertinib through HK2-driven glycolysis. We knocked down SKP2 in H1975/CAF-CM cells and subsequently examined the protein expression and mRNA expression of HK2 and found that the amount of HK2 decreased with the reduction in SKP2. Finally, we verified in H1975/CAF-CM cells that the specific inhibitor of HK2 enhanced the ability of almonertinib to inhibit cell proliferation, migration, invasion and promote apoptosis. Conclusion Our study demonstrated that CAFs drive NSCLC glycolysis through SKP2 regulation of the glycolysis-associated kinase HK2, which affects almonertinib resistance.