Proline Dehydrogenase affects Lung Adenocarcinoma cell survival, proliferation, and 3D growth

Proline dehydrogenase (PRODH) is a mitochondrial flavoenzyme that catalyzes the oxidation of proline, generating two electrons that can be used to produce ATP or ROS. The latter can promote apoptosis, cellular senescence, and autophagy. We previously showed that PRODH is expressed in most lung adenocarcinomas (LUAD) at stages I-II, but not at higher stages, or in the other subtypes. We demonstrated that PRODH promotes cellular senescence in LUAD cell lines via ROS production, causing a reduction in proliferation and survival. This work aims to further analyze the role of PRODH in lung cancer development by investigating its effects on 3D growth and migration. We also aim to assess whether the growth retardation and the impaired cell survival we observed are due to the catalytic activity of PRODH. We used NCI-H1299 LUAD cell clones stably transfected with a PRODH expression construct or with empty vector (as control). To assess how PRODH expression affects 3D growth, we compared the size, morphology, and number of cells of spheroids obtained from PRODH-expressing or control clones. On the same clones, we compared the migration ability by wound healing assay. We also carried out survival and proliferation experiments with NCI-H1299 cell clones expressing a PRODH variant with reduced catalytic activity (p.L441P). The spheroids of PRODH-expressing clones are smaller, contain a lower number of cells than those of controls and appear visibly less compact and flatter. Wound healing assay did not show significant differences in the migration of cells from PRODH-expressing and control clones. We will confirm these preliminary results by transwell assays. In clonogenic assay, we observed that the reduced catalytic activity of PRODH-L441P is sufficient to decrease cell survival comparably to the wild-type enzyme. By growth curve analysis, we observed that clones expressing the mutated protein have an intermediate proliferation between controls and clones expressing the wild-type protein. This work has helped to better clarify the role of PRODH in LUAD. We have shown that catalytic activity of the enzyme affects proliferation more than cell survival in the tested conditions. We have also observed that PRODH expression affects the 3D growth of cells at both size and morphological levels. Our next studies will better define PRODH role in cell migration by transwell assays, analysis of epithelial-to-mesenchymal transition and stemness markers.