Emerging role of Proline dehydrogenase in 2D and 3D growth of lung cancer cells resistant to tyrosine kinase inhibitors

Lung cancer is the leading cause of cancer death. Lung adenocarcinoma (LUAD), the prevalent subtype, is characterized by recurrent molecular alterations, such as EGFR mutations. These mutations make tumors susceptible to therapy with Tyrosine-Kinase Inhibitors (TKIs). However, resistance mechanisms occur and lead to cancer progression. Identification of prognostic and predictive biomarkers would be beneficial. Proline dehydrogenase (PRODH) is a mitochondrial flavoenzyme that catalyzes proline oxidation, electrons derived from the reaction can be used for ATP or ROS production. Thus, PRODH plays a critical role in cancer, affecting cellular processes such as survival, senescence, and apoptosis. We found higher PRODH expression in clinical LUAD samples, but not in LUAD cell lines. Interestingly, we observed increased PRODH expression in EGFR-mutant lung cell lines with in vitro acquired resistance to TKIs, compared to the TKI-sensitive parental lines. We hypothesize that PRODH may influence metastatic and survival capabilities during the development of resistance in lung cancer cells. The HCC827 cell line, harboring an EGFR exon 19 deletion, and its derivative, HCC827-GR5, with acquired resistance to Gefitinib, a second-generation TKI, were grown in both 2D and 3D to examine the role of PRODH in the TKI resistance. PRODH was either silenced or inhibited to evaluate its influence on cell survival, viability, and apoptosis. PRODH transcript and protein were increased in EGFR-mutant LUAD cell lines with in vitro acquired resistance to TKIs. Moreover, we observed that PRODH inhibition strongly impaired the survival and proliferation ability of HCC827-GR5 cells. In TKI-resistant cells, PRODH inhibition determined an increased expression of cell cycle arrest (p21 mRNA) and apoptotic (PUMA mRNA, cleaved caspase 8) markers, together with an increase in Annexin V positive cells, suggesting that PRODH inhibition may induce a combination of growth arrest and cell death. Furthermore, PRODH inhibition affected the 3D growth and viability of TKI-resistant, EGFR-mutated, lung cancer cells. Our results suggest that PRODH may be involved in the onset of TKI resistance. Specifically, PRODH inhibition or silencing impaired the growth of EGFR mutant, TKI-resistant cells in both 2D and 3D, while did not affect their TKI-sensitive parental cells. PRODH may potentially serve as a biomarker for predicting TKI-resistant LUAD therapy.