Molecular Genetics

1) THE ROLE OF PROLINE DEHYDROGENASE IN TUMORIGENESIS. Proline dehydrogenase (PRODH) is a mitochondrial inner-membrane and stress-inducible 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. ROS generation by PRODH has been proposed as the mechanism by which this enzyme displays pro-apoptotic 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. Alterations in PRODH sequence and/or expression/activity are the cause of mendelian conditions or contribute to the onset of complex diseases. Indeed, a reduced PRODH activity (and accumulation of proline) are responsible for hyperprolinemia type 1 (HP1), an autosomal recessive disorder, but have also been proposed to play a role in some behavioural disorders, such as schizophrenia. Moreover, PRODH plays a role in tumorigenesis. In particular, our research group is characterizing PRODH expression, regulation and functions in lung carcinomas, one of the most frequent and deadly tumors in both sexes worldwide. Immunohistochemical characterization of PRODH expression in Non-Small Cell Lung Carcinoma (NSCLC), in collaboration with the Pathology laboratory of “Ospedale di Circolo” in Varese, showed that this protein is highly expressed in the majority of early stage adenocarcinomas, compared to squamocellular carcinomas, whereas its expression is absent in Small Cell Lung Carcinomas. Moreover, PRODH expression appears to correlate to a favourable prognosis. We are currently investigating which tumor growth features are influenced by PRODH expression in lung cancer cell line models. Preliminary data suggest that PRODH affects cancer cell survival and 3D growth, but also that the cellular genetic background can act as a modifier of PRODH effects. In parallel we are also investigating regulation of PRODH gene expression by transcription factors and signal transduction pathways.

2) QUANTIFICATION OF SERUM MICRORNAS AS BIOMARKERS FOR EARLY DIAGNOSIS OF NON-SMALL CELL LUNG CANCER (NSCLC) Lung cancer, composed predominantly by non-small cell lung cancer (NSCLC), is the leading cause of cancer-related deaths worldwide. NSCLC comprises adenocarcinomas (AD, 50% of cases) and squamous cell carcinomas (SCC, 40% of cases), both characterized by a high degree of heterogeneity due to an interplay of genetic and microenvironmental factors. Tobacco smoking and some pathological conditions, such as Chronic Obstructive Pulmonary Disease (COPD), are associated with the onset of lung cancer. The high mortality of lung cancer is contributed to by late diagnosis, that is in turn attributable to the lack of non-invasive screening methods. Recently, microRNAs (miRNAs) attracted the attention of scientists as possible lung cancer biomarkers since they are stable, retrievable and dysregulated in many types of cancers. miRNAs, which belong to the class of small RNAs, are endogenous 19-24 nucleotides long non-coding RNAs. They have an intracellular function in post-transcriptional regulation of target mRNAs, but they have also been shown to be secreted in the extracellular environment and reach bloodstream, potentially regulating gene expression in distant cells. Like standard genes, some microRNAs can act as oncogenes or oncosuppressors and their levels can be increased or reduced in cancer patients compared to controls. However, there is no consensus about which miRNAs are the best biomarkers for lung cancer, to refine risk assessment in CT screening. This is attributable to both clinico-pathological and methodological factors. To identify candidate miRNAs among those published as early stage lung cancer biomarkers, we performed a critical review of the literature. We are testing some miRNA candidates, including miR-223. In collaboration with University of Trento, we are currently comparing the performances of different technologies, such as “droplet digital PCR” and novel techniques that allow direct detection of miRNAs from biofluids and could make analysis more reliable and straightforward. The cohort studied, composed of samples from patients with stage I-II NSCLC and controls has been collected in collaboration with the Thoracic Surgery unit of Ospedale di Circolo in Varese and confirmed histopathologically by the Pathology laboratory of Ospedale di Circolo in Varese.