High expression of lysosomal cathepsin D inhibits neuroblastoma cell growth, migration and invasion, and confers better prognosis in neuroblastoma patients

Neuroblastoma (NB) is one of the most common extracranial solid tumors of childhood and it accounts for 15% of cancer-related deaths. NB is an embryonal malignancy arising during fetal or early postnatal life from neural crest-derived sympathetic cells. It is commonly found in the adrenal medulla or along the sympathetic chain. Neuroblastoma shows heterogeneous clinical phenotypes with different rate of aggressiveness and responsiveness to therapies. The broad spectrum of clinical manifestations ranges from spontaneous regression, maturation into a benign ganglioneuroma or, in the worst cases, into an aggressive and metastatic disease. Epidermal growth factor receptor (EGFR) is a protein involved in cellular growth and invasiveness, is found frequently overexpressed or aberrantly activated in human neuroblastoma cells, and its inhibition or decreased phosphorylation causes tumor growth suppression and apoptosis in neuroblastomas. Cathepsin D is a ubiquitous soluble aspartic endopeptidase found in acidic intracellular compartments. CD accomplishes bulk protein degradation and mediates the activation of hormones, as well as the inactivation of mature growth factors through extensive lysosomal degradation. In the first original article published, we found that CD reduced the growth of neuroblastoma cells by downregulating EGFR/MAPK signaling and confers better prognosis in neuroblastoma patients. In the second research article, we demonstrated a dual role of cathepsin D in 2D and in 3D growth of neuroblastoma cells in response to EGF. CD upregulation is necessary to guarantee the survival and proliferation of NB cells grown in suspension (as 3D neurospheres) while it is necessary to downregulate its expression for allowing adherence and anchorage-dependent growth of the tumor cells. To our knowledge, this is the first evidence for such a role of CD in neuroblastomas. Another important novelty of the present work is the use of mixed clones expressing CD at different level to mimic cancer heterogeneity arising during tumor evolution. The differential expression and role of CD in the two culture systems, let to hypothesize that cathepsin D could be regulated via epigenetic mechanisms. The bioinformatic analysis performed in this work, identified 38 miRNAs that downregulate CD in human neuroblastomas, and these miRNAs are associated with biological processes involved in cell migration, cell motility, cell growth and EGFR signaling, among others. Collectively, we have uncovered a novel role of CD in the metastatic spreading of tumors. This finding may have a translational relevance, and we propose CD as a possible biomarker for metastatic neuroblastomas and for the stratification of patients in a view of personalized medicine. Modulators of CD synthesis and activity could be a novel therapeutic strategy to ameliorate patients’ outcome. The third part of my work deals with the role of CD in the chaperone-mediated autophagy of annexin A2, which results in attenuation of the NB cell malignant phenotype.