Up-regulation of prosaposin by the retinoid HPR and the effect on ceramide production and integrin receptors

N-(4-hydroxyphenyl)retinamide (HPR) is a synthetic retinoid of clinical use in breast cancer chemoprevention. Its in vitro effects on tumor cell lines include growth inhibition and apoptosis and are, to a large extent, independent of the activation of retinoic acid receptors. We have performed a cDNA differential display analysis to identify genes potentially relevant for understanding the mechanism of action of HPR. We report that prosaposin (PSAP), a gene encoding for the common precursor protein of saposins (cofactors involved in the metabolism of various sphingolipids), was significantly up-regulated, both at transcriptional and protein level, in MCF7 and T47D breast cancer cell lines after treatment with HPR, but not with all trans retinoic acid (ATRA). Remarkably, this response was also induced by oxidants, whereas it was abrogated by antioxidants in cells treated with HPR, which implies a direct role of free radicals generated by HPR in prosaposin up-regulation. Stable transfectants of T47D cells overexpressing prosaposin (T47D-PSAP) exhibited morphological changes, reduced proliferation, and impaired attachment to culture plates, which correlated with down-regulation of alpha6, beta1, and beta4 subunits of the integrin family of adhesion receptors. Concordant with a role of prosaposin in ceramide generation, increased amounts of this lipid were found in T47D-PSAP and in HPR-treated T47D cells. Moreover, treatment of T47D with C2-ceramide resulted in the up-regulation of prosaposin and down-regulation of integrin receptors. Altogether, these findings establish a link between prosaposin, ceramide, and integrin receptor regulation. In view of the association between integrin down-regulation and suppression of metastatic capacity, our data raise the possibility that the cancer chemopreventive activity of HPR may rely, at least in part, on its capacity to regulate integrin receptors through prosaposin.