Unravelling the Role of Proline Dehydrogenase (PRODH) in Lung Adenocarcinoma: Implications for Cellular Senescence and the Tumor Microenvironment

Proline dehydrogenase (PRODH) is a mitochondrial flavoenzyme that catalyzes proline oxidation. The electrons generated from this reaction can be used to produce ATP or ROS, potentially affecting several cellular processes, such as survival, apoptosis, senescence, and inflammation. PRODH is frequently expressed in early-stage lung adenocarcinoma (LUAD), but little is known about its correlation with lung cancer development. Thus, we aim to investigate the effects of PRODH expression in LUAD cell lines. To investigate how PRODH affects the growth and proliferation of lung cancer cells, we stably transfected the NCI-H1299 LUAD cell line with expression constructs encoding wild-type PRODH or a variant with reduced catalytic activity (p.L441P), as well as the empty vector (control). We compared the survival and proliferation ability of the three types of clones by clonogenic and MTT assays. Furthermore, in wild-type PRODH-expressing clones and controls, ROS production was assayed by 2′,7′ dichlorofluorescin diacetate, and cellular senescence investigated by the senescence-associated-β galactosidase assay, analysis of the cell cycle inhibitor p21 and of senescence-associated secretory phenotype (SASP) performed by qPCR and secretome analysis. Wild-type and p.L441P PRODH-expressing clones had reduced cell viability and impaired proliferation, compared to control clones and the p.L441P variant displayed an intermediate phenotype. PRODH expressing clones produced higher levels of ROS and had more senescent cells, compared to controls. Moreover, PRODH expression led to higher levels of p21 and cytokines related to the SASP, such as MCP 1, IL-8, and TNF-α. Secretome analysis also revealed that PRODH expression increased the levels of soluble factors involved in monocyte recruitment (MCP-1, MCP-2, MCP-3), monocyte-to-macrophage differentiation (GM-CSF), cytokines involved in M2-like macrophage polarization (IL-10, IL-4, and IL-13) and angiogenesis (IL-8, FGF6, FGF7, and PIGF). These findings suggest that PRODH may influence the tumor microenvironment (TME). Our study shows that ROS production affects the induction of cellular senescence and LUAD cell proliferation. PRODH may also shape the TME towards an immune-regulatory phenotype. Based on these data, we speculate that, by inducing cellular senescence and cytokine production, PRODH may play a role in the development of lung cancer, in modulating the TME composition, and cancer cells’ response to therapy.