Caratterizzazione molecolare e funzionale della lisil ossidasi (LOX) mediante silenziamento genico in colture cellulari primarie umane di cortex e carcinoma renale (RCC)

80% of sporadic clear cell renal cell carcinomas (ccRCCs) is characterized by biallelic inactivation of von Hippel-Lindau (VHL) tumor suppressor gene (VHL-/-) preventing the degradation of hypoxia-inducible factor 1 (HIF-1α). Therefore, constitutive expression of HIF-1α activates the transcription of hypoxia-inducible genes that code proteins involved in angiogenesis, cellular proliferation, migration and invasion. Among these HIF-1 target genes, the extracellular matrix enzyme Lysyl oxidase (LOX) shows multiple extra- and intracellular roles in the regulation of metastatic process and tumorigenesis. LOX enzyme is synthesized as pro-Lox (50kDa) and cleaved, by BMP-1 (bone morphogenetic protein-1) in the extracellular space, yielding the N-terminal propeptide (LOX-PP 18kDa) and the active enzyme (LOX 32kDa). Active LOX 32kDa has a role in cytoskeleton rearrangement and in regulation of cellular migration by involvement in Src/FAK and Rac signalling pathways, while LOX-PP 18kDa seems to inhibit tumor formation. Our collaborators have previously demonstrated, by transcryptomic analysis, that LOX transcript is overexpressed in RCC primary cultures. In this work we validated the results of transcryptomic analysis by performing expression studies through real time PCR and Western blot. We observed that LOX transcript, as well as pro-LOX and LOX protein, are more abundant in RCC respect to normal cortex primary cultures. LOX-PP, instead, is more expressed in normal cortex sample thus supporting its tumor suppressor role already described in literature. Moreover we elucidated the molecular and functional effects of LOX protein in the primary cultures by silencing experiments. We analyzed S100A4 and E-Cadherin expression in LOX silenced primary cultures in order to evaluate an hypothetical interaction between LOX and S100A4 and between LOX and E-Cadherin. S100A4 and E-Cadherin proteins are interesting for us since they are HIF targets and involved in tumour invasiveness and metastasis. In our model they are more abundant in normal cortex primary cultures. In the normal primary cultures, LOX silencing increases S100A4 transcript and protein levels that seem to be mediated by an effect of LOX on β-catenin expression. In LOX silenced RCC primary cultures, S100A4 and β-catenin levels decrease. Moreover, E-Cadherin is also affected by LOX regulation. We, indeed, saw that, in normal cortex primary cultures, LOX silencing induces an increase of E-Cadherin transcript and protein level. Instead, in RCC primary cultures LOX silencing induces a decrease of E-Cadherin expression. It has been recently described that LOX protein, that is a HIF target, may also promote the synthesis of HIF-1α. In our model of LOX silenced RCC primary cultures we observed a decrease in the protein level of HIF-1α as well as in the transcript level of three of HIF-1’s targets, such as Zeb-2, Snail, TGF-b3. In the last part of this work we further elucidated the role of LOX in cell motility/migration. We, indeed, observed that LOX down-regulation influences the cell-matrix adhesion that usually plays a key role in the cell motility. Adhesion assays showed that cell ability to adhere to the substrate is lower in LOX-silenced primary cultures than the control samples as well as migration activity, analyzed by boyden chamber system. Finally we performed an MTT assay to evaluate the cell viability that doesn’t change in LOX silenced cortex cultures. In RCC primary cultures, instead, LOX silencing induces a significant decrease of cell viability only in HIF-1α positive group. The role of LOX in the regulation of cellular motility and the regulatory loop between LOX and HIF1α described in this work might be the beginning of a future study about the use of LOX as an hypothetical target of RCC.