SPHINGOLIPID METABOLISM'S ALTERATIONS CORRELATED WITH TUMOR CELL INVASIVITY AND FENRETINIDE RESISTANCE IN A HUMAN OVARIAN CARCINOMA CELL LINE

Glycosphingolipids (GSL) modulate several signal transduction processes controlling cell proliferation, survival, differentiation and transformation. Alterations in the expression of carbohydrate epitopes associated with GSL are frequent in tumors, and it has been hypothesized that GSL could play important roles in modulating some of the properties of tumor cells. The contribution of transformation-associated changes in GSL composition to the tumor phenotype are very complex and not fully elucidated, and likely implies heterogeneous molecular mechanisms. However, at least two well established paradigms support this role: 1) gangliosides have been described as modulators of growth factor receptor function, associated tyrosine kinase activities and cellular compartmentalization. 2) GM3 and/or GM2 inhibit integrin-dependent tumor cell motility via the formation of a ganglioside/tetraspanin/integrin receptor complex (the “glycosynapse”) that is responsible for the negative regulation of c-Src tyrosine kinase activity [1]. In addition, several commonly used anticancer drugs exert their cytotoxic action at least in part by triggering the production of the sphingolipid ceramide, a mediator of apoptosis and an inhibitor of cell proliferation in a variety of tumor cell lines [2]. It has been demonstrated that chemoresistant tumor and tumor cell lines are frequently characterized by the increased glycosylation of ceramide with formation of glucosylceramide, due to an increased expression or activation of glucosylceramide synthase (GCS) [3]. Scavenging ceramide via its increased glycosylation would allow tumor cells to escape ceramide-induced apoptosis, thus contributing to the drug resistant phenotype [4-6]. However, it has been shown that GlcCer accumulation is not the only consequence of an altered sphingolipid metabolism in drug resistant cancer cells. In addition to its use as a precursor of complex sphingolipids, ceramide can be converted into sphingosine, which is then phosphorylated from sphingosine kinases to generate sphingosine 1-phosphate (S1P). Up-regulation of sphingosine kinase and concomitant decrease of the levels of sphingolipid mediators upstream to sphingosine kinase have been associated with resistance to camptothecin and docetaxel in prostate cancer cells [7, 8], to oxaliplatin in colon cancer cells [9] and to gemcitabine in pancreatic cancer cells [10]. During my PhD period, I have studied the alterations in sphingolipid metabolism and their functional relevance in different phenotypic variants of a human ovarian carcinoma cell line, A2780. A2780 cells are sensitive to a variety of antitumor drugs, including the synthetic retinoid N-(4-hydroxyphenyl)retinamide (fenretinide or HPR), that is currently it is under clinical trials as preventive chemotherapeutic drug for prostatic and ovarian cancer, neuroblastoma, lymphoma and leukemia. A2780/HPR is a HPR-resistant cell line obtained from the parental A2780 cell line by in vitro exposure to increasing sublethal HPR concentrations. The A2780/HPR cell line presents a multiple phenotypic differences compared to A2780 cells. Previous work from this lab showed that A2780/HPR cells express higher ganglioside levels respect to the parental cell line, due to the overexpression of GM3 synthase, a key enzyme in ganglioside biosynthetic pathway, and a higher sphingolipid degradation rate [11]. In addition to HPR resistance, A2780/HPR cells are characterized by a reduced in vitro motility respect to the parental cell line. Subsequent experiments were aimed at elucidating a possible link between alterations in sphingolipid metabolism and modulation of cell motility and/or the acquisition of resistance to HPR. At first, we examined the effect of the overexpression of sialyltransferase-1 (SAT-1, GM3 synthase) on A2780 cells. Our results indicate that 1) GM3 synthase overexpression led to a marked increase in the ganglioside; 2) this was associated with a strong reduction of in vitro cell motility without affecting the growth rate, nor the sensitivity to HPR; 3) the expression of the membrane adaptor protein caveolin-1 was markedly upregulated in SAT-1 transfected cells and in A2780/HPR cells, naturally expressing high GM3 synthase levels. 4) in SAT-1 overexpressing cells, GM3 directly interacts with caveolin-1, as demonstrated by photolabelling experiments, with the use of radioactive and photoactivable GM3; 4) caveolin-1 formed a multimolecular complex with gangliosides, integrin receptor subunits and c-Src, as demonstrated by differential solubilization and co-immunoprecipitation experiments; 5) In vitro motility of A2780 cells was strongly reduced in the presence of a selective Src inhibitor, and SAT-1 transfected A2780 cells were characterized by higher levels of the inactive c-Src p-Tyr-527 form, that was concentrated in a detergent-resistant membrane fraction, co-localizing with the caveolin-1/ganglioside complex. These data suggest that motility of A2780 cell can be negatively influenced by a caveolin-1/ganglioside complex controlling Src activity. Supporting this hypothesis, we observed that exogenous administration of gangliosides or a pharmacological treatment able to increase cellular ganglioside levels in A2780 cells were able to effectively reduce their in vitro motility of these cells, with a concomitant increased phosphorylation of caveolin-1. As mentioned above, GM3 synthase overexpression did not affect the sensitivity of A2780 cells to HPR. On the other hand, degradation of sphingolipids occurs at higher extent in drug-resistant A2780/HPR cells. Sphingolipid degradation pathway implies the conversion of ceramide in sphingosine, which afterwards can be phosphorylated to S1P by the action of sphingosine kinase (SK). We showed that the production of S1P is significantly higher in A2780/HPR vs. A2780 cells due to an increased SK activity and SK-1 mRNA and protein levels. Treatment of A2780 and A2780/HPR cells with a potent and highly selective pharmacological sphingosine kinase inhibitor effectively reduced S1P production and resulted in a marked reduction of cell proliferation. Moreover, A2780/HPR cells treated with a SK inhibitor were sensitized to the cytotoxic effect of HPR, due to an increased dihydroceramide production. On the other hand, the ectopic expression of SK-1 in A2780 cells was sufficient to induce HPR resistance in these cells. Challenge of A2780 and A2780/HPR cells with agonists and antagonists of S1P receptors had no effects on their sensitivity to the drug, suggesting that the role of SK in HPR resistance in these cells is not mediated by the S1P receptors. These data clearly demonstrate a role for SK in determining resistance to HPR in ovarian carcinoma cells, due to its effect in the regulation of intracellular ceramide/S1P ratio which is critical in the control of cell death and proliferation.