Study of regulatory factors in Non Small Cell Lung Cancer: a role for Proline Dehydrogenase?

Proline dehydrogenase (PRODH) is a mitochondrial inner-membrane and stressinducible flavoenzyme catalysing the first step in the proline degradation pathway. Due to its distinctive chemical structure, in fact, proline is metabolized by a distinct set of enzymes, compared to the other aminoacids. Proline metabolizing enzymes constitute a “catalytic cycle”, transferring reducing potential into mitochondria and connecting proline to several metabolic pathways involved in basal metabolism, such as the tricarboxylic acids (TCA) cycle and the urea cycle. Thus, proline metabolism entails several regulatory pathways that are important in both redox regulation and bioenergetics. Electrons deriving from PRODH activity can be transferred from the flavin adenine dinucleotide (FAD) cofactor to the oxidative phosphorylation pathway, thus inducing the formation of Reactive Oxygen Species or ATP. ROS generation by PRODH has been proposed as the mechanism by which this enzyme displays proapoptotic effects; nevertheless, PRODH was also described to induce cell protective autophagy and to potentially promote survival during some types of stress by inducing ATP production. Therefore, PRODH seems capable of influencing the balance between survival and apoptosis, likely depending on the cell type and on the type and severity of stress acting on those cells. Mutations and alterations of PRODH activity are responsible for mendelian conditions or contribute to complex diseases. Indeed, absence or reduced PRODH activity (and proline accumulation) is responsible for “hyperprolinemia type 1” (HP1), an autosomal recessive disorder, but a role has also been proposed in behavioural disorders such as schizophrenia. Indeed, patients affected by HPI show increased susceptibility to schizoaffective disorders, further supporting the role of PRODH in these diseases. Noteworthy, at least 16 PRODH missense mutations have been identified with moderate to severe effect on PRODH activity. PRODH was also proposed to play a role in cancer. Indeed, its involvement in tumour development and progression has been supported by studies concerning its expression and biological functions in different types of tumour. In particular, immunohistochemistry experiments have shown that the levels of proline dehydrogenase are lower in tumour tissues than the corresponding healthy tissues in different types of cancer, such as kidney, bladder, and digestive tract tissues including colon, rectum, stomach, liver and pancreas. More recently, however, PRODH was shown to have a role in tumour promotion and progression in other types of tumours, favouring survival and invasion in breast and pancreatic cancer and in melanoma. In this PhD project, we focused on the characterization of PRODH expression, regulation and functions in lung cancer, the most frequent and one of the deadliest cancer types. Lung cancer is a highly heterogeneous disease, comprising Small Cell Lung Cancer (SCLC, comprising 15% of lung cancer cases) and Non Small Cell Lung Cancers (NSCLC, 85% of lung cancer cases), in turn comprising two subtypes, adenocarcinoma (ADC, ≈55% of cases) and squamous cell carcinoma (SCC, ≈ 45%). Immunohistochemical characterization of PRODH expression in NSCLC cases showed that this protein is strongly expressed in a high proportion of early stage lung ADCs compared to lung SCC, whereas no expression was detected in SCLC cases. Moreover, PRODH expression seemed to correlate with a favourable prognosis. Then, we aimed to investigate what cellular processes are influenced by PRODH in lung ADC. We tested the effect of modulation of PRODH expression in lung ADC tumor cell lines by performing a panel of phenotypic assays. We found that in 5 out of 7 lung adenocarcinoma cell lines tested in this work, PRODH overexpression led to a decrease in cell survival, as determined by clonogenic assays. However, in 2 cell lines, namely A549 and NCI-H1437 lung ADC cell lines, modulation of PRODH expression suggested that PRODH favoured cell survival; we hypothesized that the genetic background of these cell lines may influence the outcome. Moreover, in NCI-H1650 and NCI-H1299, PRODH overexpression led to an increase in cell motility and a decrease in the ability of these cells to form spheroids when grown in soft agar, indicating reduced anchorage independence, that represents a hallmark of tumorigenesis. In this project, we investigated if the PRODH gene is a target of TTF-1, also known as NKX2-1, a homeodomain containing transcriptional factor that regulates normal development and morphogenesis of the lung and adult lung physiology. This hypothesis was formulated based on observations and analogies between these two proteins and the dual function they can exert on lung cancer cell growth. Indeed, they are expressed in the same cell types in normal lung tissue and in the majority of lung ADC cases, and both can have either promoting or inhibiting effects on tumours. Transfection of a construct encoding TTF-1 in two adenocarcinoma cell lines (A549 and NCI-H1299) showed an increase in PRODH transcript compared to cells transfected with empty vector. Moreover, luciferase assays showed that one (RE1) of the four putative response elements (REs) for TTF-1 identified bioinformatically in the PRODH gene was able to increase luciferase activity and that mutagenesis of this RE abolished this induction, suggesting a direct binding of this sequence by TTF1. The results obtained support the hypothesis that TTF-1 may be a direct - albeit weak - transcriptional regulator of the PRODH gene and suggest that other cofactors may collaborate in PRODH transactivation. In conclusion, the data obtained in this PhD project suggest that PRODH can influence several aspects of cell behaviour in lung cancer cell lines and identify TTF1 as a novel regulator of PRODH gene expression in the lung. The results presented here encourage further work to elucidate PRODH roles and regulation during lung tumorigenesis, aiming at a possible application of this protein as a biomarker for prognosis and differential diagnosis of lung adenocarcinoma.