EFFECTS OF 3-KETODESOGESTREL AND ALL-TRANS RETINOIC ACID ON PHOX2A AND PHOX2B EXPRESSION: A COMMON STRATEGY AS NEW THERAPEUTIC PERSPECTIVE IN CONGENITAL CENTRAL HYPOVENTILATION SYNDROME (CCHS) AND NEUROBLASTOMA (NB) TREATMENT.
Creators: name: BELPERIO, DEBORA
Publisher: name: Universit� degli Studi di Milano
Year: 2017

description: PHOX2B (Paired-like homeobox2B) and PHOX2A (Paired-like homeobox2A), are two paired like homeodomain proteins required for the correct autonomic nervous system development and for the specification of the noradrenergic phenotype. The neuronal development and specification driven by PHOX2 proteins is the result of a fine temporal and spatial control of the two-paralogue proteins. In particular, PHOX2B is considered a ?master regulatory gene? in the network leading to the cathecolaminergic phenotype specification, and regulate PHOX2A expression (Coppola et al., 2005). As PHOX2 proteins play crucial role in embryonic neuronal differentiation, their mutations or altered regulation are linked to congenital pathologies, such as Congenital Central Hypoventilation Syndrome (CCHS), a neurodevelopmental disorder characterized by a failure in the autonomic control of breathing, and neuroblastic tumours. The aim of this project was to disclose still unknown mechanisms involved in the pathogenetic mechanisms of PHOX2B-driven disease development, to improve the recognition of new sensitive therapeutic intracellular targets The results obtained during the three years of my PhD thesis are organized in three parts. Part 1) In the first part of the project, we better characterized PHOX2B homeodomain-mediated functions, as hetero-/homo-dimerization and nuclear import, also in the perspective of better analysing the possible effect of polyalanine expansion on these functions, as the expansion of the polyalanine tract in PHOX2B results in a protein with altered DNA-binding, reduced transcriptional activity and defect in nuclear localisation (Trochet et al., 2005; Bachetti et al., 2005; Di Lascio et al., 2013). Interestingly our data clearly underlined a strong interaction between PHOX2A and PHOX2B wild type/ mutants proteins, indicating a possible involvement of PHOX2A in the pathogenesis of CCHS. Part 2) Consistently with PHOX2B role as transcriptional regulator, it is reasonable to suppose that transcriptional dysregulation might be an important mechanism of CCHS pathogenesis and/or tumour development. Recently, studies conducted on NB cell lines have highlighted a correlation between drugs, used in NB treatment or clinical trials (e.g. GA, 17-AAG), and the modulation of PHOX2B gene expression. Moreover, NBs are very sensitive to retinoids, and they were introduced in NB treatment as ?additional drugs? in order to induce differentiation in residual cancer cells after NB surgical elimination. In the second part of the project, we showed that ?all trans retinoic acid? (ATRA), a retinoid drug that is known to suppress growth in cancer, differently regulates both PHOX2A and PHOX2B expression in SK-N-BE(2)C neuroblastoma cell line, thus suggesting that the molecular mechanism for retinoic-induced cellular differentiation, in NB treatment, is due to a direct regulation of PHOX2 protein expression. Part 3) An interesting strategy into treatments of CCHS, that is currently under investigation, regards the use of progestinic drugs for ameliorating respiratory symptoms in patients. This strategy stems from the fortuitous observation of Straus and colleagues, that progestin drug Desogestrel can relief some symptoms in CCHS female patients. The molecular mechanism of the unexpected Desogestrel pharmacological effect is at the moment completely unknown, but this observation gave us a useful indication in the perspective of pharmacological interventions in CCHS. As previously data collected in my laboratory, on T47D cell line, suggested us that 3-KDG can influence both the activity of PHOX2B and PHOX2A, and that this is mediated by the intracellular receptor PGR, we decided to deeply investigate the Desogestrel mechanism in a cellular background more properly associated with the physiological PHOX2B environment. To this purpose, we generated a neuroblastoma cell line (SK-N-BE(2)C) stably expressing nuclear progesterone receptor isoforms, and analysed the effect of 3-KDG treatment on endogenous protein amount and transcription levels by using western blot and quantitative PCR analyses. Our findings demonstrated that desogestrel treatment is able to alter PHOX2B and PHOX2A expression, as well as the expression of DBH and some other target genes of PHOX2 proteins, particularly reducing their expression. Taking together these results point out the existence of a specific cross-talk between ATRA and 3-KDG treatments and PHOX2 pathway, suggesting that the regulation of PHOX2 proteins expression could be a common strategy into the treatment of NB and CCHS.