Ruolo di Clusterina nella progressione del tumore prostatico

Clusterin (CLU) is a secreted glycoprotein with a broad distribution in human tissues and body fluids. It is involved in many biological processes, but its function is not completely understood. The human CLU gene encodes for three known mRNA variants currently listed in the NCBI database as NM_001831, NR_038335 and NR_045494, here named CLU 1, CLU 2 and CLU 3 respectively. All variants are transcribed as pre-mRNAs and comprise 9 exons and 8 introns. Exon 1 sequences are unique to each of the three mRNAs. Although in NCBI CLU 2 and CLU 3 are listed as non-coding mRNAs, bioinformatics analysis has predicted that all variants are translated in proteins with different length and cellular localization. Among all protein forms hypothesized, the only one isolated and sequenced is translated from the AUG present on exon 2. This translation produces a 449 amino acids citoplasmatic precursor (psCLU) which undergoes to post-translational modifications before its secretion as heterodimer (sCLU) in the extracellular environment, where it acts as an ATP-independent chaperone. In addition to the extracellular protein, it is possible to find a intracellular form with a not well-understood function. The aim of this thesis is to increase the knowledge about CLU 1 and CLU 2 transcripts and their regulation as well as the role of the intracellular protein in relationship with NF-kB signaling. We investigated CLU 1 and CLU 2 expression in a panel of different cell lines, with a special focus on prostate cancer cells. We found out that CLU 1 is shorter at its 5’ end than the NM_001831 sequence and the translation start site is on exon 2. Despite the information reported in NCBI, we demonstrated that CLU 2 mRNA is translated starting from the AUG on exon 2. The protein products of CLU 1 and CLU 2 are therefore identical and correspond to sCLU. The expression levels of CLU 1 and CLU 2 are different in the analyzed cell lines and, in particular, CLU 2 is expressed at low levels in prostate epithelial cells. Here, CLU 2 is epigenetically silenced and the administration of drugs able to relax the chromatin, such as trichostatin A and 5-aza-2'-deoxycytidine, increased CLU 2 expression. We demonstrated the presence of a new promoter region (P2) that directly drives the expression of CLU 2 by a bioinformatics analysis followed by in vitro assays in prostate epithelial cancer cell lines treated with epigenetic drugs. P2 is a TATA less, GC rich promoter, which remains silent in prostate cancer cells under basal conditions and becomes active after the administration of epigenetic drugs. The mechanism responsible of CLU 2 derepression is the dynamic remodeling of histone tails associate with P2. At last, we evaluated the role of intracellular CLU in relationship with the NF-kB pathway, using an experimental model of prostate cancer cell where CLU has been silenced or overexpressed. We observed that intracellular CLU inhibits the phosphorylation and activation of p65, the most representative member of NF-kB family. The consequence is a reduced transcription of some genes regulated by NF-kB and involved in the extracellular matrix remodeling like urokinase-type plasminogen activator, cathepsin B and matrix metallopeptidase 9. We demonstrated that CLU does not physically interact with p65, but its action is indirect.