Sphingolipid metabolic enzymes modulate anticancer drug resistance
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[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] One of the most challenging aspects of cancer therapy today is the problem of drug resistance. The aim of our research is to understand the molecular basis of anticancer drug resistance. By random insertional mutagenesis, previous research in our lab identified 6 novel genes, which are involved in cisplatin resistance in the cellular slime mold Dictyostelium discoideum. One of these genes encodes sphingosine-1- phosphate lyase, which is an enzyme that functions in the degradation of sphingosine-1- phosphate (S-1-P). The bioactive lipid S-1-P plays a central role in regulating multiple cellular functions, such as proliferation, motility, immunity and angiogenesis. Since disruption of S-1-P lyase gene results in increased resistance to cisplatin, we reasoned that this was due to increased S-1-P level in S-1-P lyase null mutant. To test this hypothesis, we generated additional S-1-P lyase and sphingosine kinase (functions in generating S-1-P) mutants. Our preliminary data in the model organism Dictyostelium suggested that increasing S-1-P level by overexpressing sphingosine kinase or knocking out S-1-P lyase causes the cells to be more resistant to platinum based anticancer drugs, while decreasing S-1-P level by overexpressing S-1-P lyase or knocking out sphingosine kinases causes the cells to be more sensitive to platinum based anticancer drugs. When extending our findings from Dictyostelium to human cell lines, we found that the human S-1-P lyase gene regulates cisplatin sensitivity in a p38 MAPK dependent manner. Our findings, for the first time, directly connected the sphingolipid metabolic pathway with the clinical problem of anticancer drug resistance and it provided a better understanding of the role of the bioactive lipid S-1-P in the development of cancer and drug resistance. Ceramide is bioactive sphingolipid (upstream of S-1-P) that plays a central role in triggering apoptosis following a variety of stimuli. The global hypothesis is that the balance between S-1-P and ceramide determines the cell's fate. It is well documented that ceramide formation plays an important role in cytokine- and chemotherapy-induced cancer cell death, while S-1-P is emerging as a key and novel regulator of carcinogenesis. But little is known about the role of intracellular ceramide in chemotherapeutic drug resistance. The recent identification and characterization of the LASS genes, which encode ceramide synthase for the de novo biosynthesis of ceramides, provided the opportunity to address the question. Cell lines stably overexpressing the LASS1, LASS4 or LASS5 genes were tested for sensitivity to a panel of chemotherapeutic drugs. Overexpression of LASS4 did not alter sensitivity to any of the tested drugs, while overexpression of LASS5 increased sensitivity to a subset of drugs known to be substrates for the multidrug resistance MDR1 protein. In contrast, LASS1 overexpression increased sensitivity to all drugs tested. Thus, it appears that ceramides with different acyl chains function by different mechanisms to control drug sensitivity. Further, we have shown that following drug treatment, there is a specific translocation and concentration of the LASS1 protein (but not LASS4 or LASS5) from the ER to the Golgi apparatus, accompanied with a concomitant reduction of LASS1 enzyme activity. The translocation of LASS1 to the Golgi apparatus is rapid, and occurs in response to chemotherapeutic drugs and UV-light. Moreover, treatment of cells with the general unfolded protein response inducer DTT also results in LASS1 translocation and establishes a role for the enzyme as a novel stress sensor. Understanding the mechanisms by which the ceramide synthase LASS1 senses and responds to stress stimuli, and traffics from the ER to Golgi will generate significant and unique insights about the role of ceramide synthase in sensitizing cells to stress stimuli. Taken together, our systematic studies defined the role of the sphingolipid metabolic enzymes in mediating the cellular response to anticancer drugs. We expected our results will lead to the identification of multiple novel strategies for cancer therapy as well as overcoming anticancer drug resistance.
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