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    Molecular Mechanisms of Fas-Mediated Cell Death Induced by Tumor-Specific Ether Lysophospholipid Derivatives.

    Scientific Abstract:
    Molecular Mechanisms of Fas-Mediated Cell Death Induced by Tumor-Specific Ether Lysophospholipid Derivatives. Background: Tumor cells have a markedly reduced susceptibility to FasL-induced Fas-mediated apoptosis. In breast cancer, the aberrant loss of expression or function of Fas (CD95/APO1, a member of the TNF receptor superfamily) can lead to the development of cancer cells as well as the cytostatic drug resist- ance. Thus the understanding of molecular mechanisms of Fas-mediated apoptosis is vital to breast cancer treatment. Reports that Fas mediates apoptosis via rafts (membrane microdomains) offer a great promise as cytotoxic drugs may be used to enhance raft-dependent killing of tumor cells. Fas-mediated cell death has been related to the antitumor activity of ether lysophospholipid-derived drugs, namely alkyllysophospho- lipids (ALP), e.g. 1-O-Octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (Edelfosine) and alkylphospho- cholines (APC), e.g. hexadecylphosphocholine (Miltefosine). Edelfosine-induced cell death, although FasL -independent, requires Fas and drug internalization via rafts. While ALP and APC inhibit phosphatidylcho- line (PC) synthesis, the link between this action and raft-dependent Fas-mediated pathway is unclear. Also, as the ability to selectively kill cancer cells makes these drugs promising tools for chemotherapy, the side effects prevent usage at their full potential. Thus knowing the mechanisms by which they kill tumor cells will assist the design of new analogs with better therapeutic index. Objective/Hypothesis: our goal is to un- derstand how these drugs induce Fas-mediated cell death, especially relative to Fas localization to rafts. We hypothesize that their interference in phospholipid metabolism is responsible for raft-dependent Fas-med- iated cell death and that they trigger the death signal in the same manner as FasL. Specific aims: 1. To esta- blish whether ALP/APC-induced inhibition of CTP:phosphocholine citidyltransferase (CT), a key enzyme in PC synthesis, causes Fas aggregation in rafts, 2. to test whether ALP/APC affect Fas via ceramide (CER) accumulation, and 3. To determine whether ALP and APC induce cell death by mimicking FasL action. Study design: Aim 1: To test the requirement of CT inhibition by ALP/APC in Fas-mediated cell death, Fas distribution in rafts and cell death will be assessed in CT-expressing and CT-deficient breast cancer cells, with and without ALP/APC treatment. To link PC synthesis inhibition to ALP/APC-induced Fas-mediated cell death, parallel tests will be done in the presence of lysoPC, which enables PC synthesis via an alterna- tive pathway. Aim 2: On the basis that CER production via an increased acid sphingomyelinase (ASMase) activity upon Fas ligation is crucial for Fas-mediated cell death and that PC provides phosphocholine to CER to form sphingomyelin (SM), the effect of PC synthesis inhibition by ALP/APC (causing SM reduction and CER accumulation) will be tested. To determine the necessity of ASMase-dependent CER accumulation in ALP/APC-induced Fas-mediated cell death, Fas association to rafts and cell death will be assessed after ALP/APC treatment of cells lacking ASMase activity, e.g. breast cancer cells whose ASMase transcription is inhibited by siRNA. To show whether ALP/APC-induced cell death requires CER, ASMase-expressing cells will be treated with a CER synthesis inhibitor, and then with ALP/APC. Since exogenous CER can re- store death in cells resistant to FasL-induced Fas-mediated apoptosis, the effect of C2-CER and ALP/APC cotreatment on Fas localization to rafts and cell death will also be tested. Aim 3: Since ALP-induced cell death is FasL-independent and both FasL and ALP treatments involve Fas aggregation in rafts, the actions of ALP/APC and FasL may be similar. To validate the statement, breast cancer cells will be treated with ALP, APC, or FasL. The comparison will be in terms of Fas aggregation in rafts, death inducing signaling com- plex (DISC) formation (recruitment of the adaptor protein, FADD, and caspase-8 by Fas), as well as lipid and protein components in the rafts. Cells whose Fas cannot recruit FADD and cells defective in FADD and caspase-8 will be tested to confirm the importance of DISC formation in ALP/APC-induced cell death. Po- tential Outcomes and Benefits: From this research, an insight to how these drugs kill tumor cells will be a basis for better breast cancer therapeutic strategies and design of drugs with higher anticancer activity and reduced side effects, providing breast cancer patients with better treatments and thus a better quality of life.

    Lay Abstract:
    Molecular Mechanism of Tumor-Specific Fas-Mediated Cell Death Induced by Ether Lysophospholipid-Derived Antitumor Drugs. Alterations in the control of cell death/survival contribute to the pathogenesis of many human diseases. Breast cancer has been linked to the suppression of programmed cell death, also known as apoptosis. To successfully develop anticancer therapeutic strategies, the understanding of molecular mechanisms involved in apoptosis is necessary. One of the best characterized apoptotic signaling pathways by far is triggered by a protein called Fas, located on the cell surface. In breast cancer, the abnormal regulation and function of Fas can lead to the development of cancer cells as well as the resistance of cancer cells to anticancer drugs. Recent findings that Fas molecules activate the cell death signal from small, specialized sites on the cell surface called ‘rafts’ present a great promise for anticancer treatment since cytotoxic drugs may be used in chemotherapy to enhance raft-dependent killing of tumor cells. Notably, actions of some cytotoxic drugs derived from ether phospholipids have been related to Fas-mediated cell death. This class of drugs includes alkyl-lysophospholipids (ALP), e.g. Edelfosine, which has been frequently used to purge autologous bone marrow during transplantation, and alkyl-phosphocholines (APC), e.g. Miltefosine, which has been successfully used as a topical treatment for cutaneous metastases in breast cancer patients. These drugs interfere with the metabolism of a class of lipids called phospholipids and induce apoptosis. Although the ability of these drugs to selectively kill cancer cells while sparing the healthy cells makes them promising tools for chemotherapy, their side effects prevent applications at their full potential. Recently it has been reported that in order for Edelfosine to induce cell death, it must be internalized into the cells via ‘rafts’ and Fas protein must be present on the cell surface. Although ALP and APC are known to interfere with synthesis of phospholipids, particularly phosphatidylcholine (PC), the link between this action and raft-dependent Fas-mediated cell death pathway remains unclear. We propose to determine how ALP and APC trigger Fas-mediated cell death, particularly in relation to Fas localization to ‘rafts’. We will demonstrate whether the interference in phospholipid metabolism by ALP and APC is the cause of raft-dependent Fas-mediated cell death and that the mechanism by which these drugs induce the death signal is similar to the natural mechanism used by a protein called FasL, which works specifically to induce cell death by binding to Fas. The first aim of this study is to determine whether the inhibition of CTP:phosphocholine citidyltransferase (CT), a key enzyme in PC synthesis, by these drugs causes Fas aggregation in rafts. The results from this aim will tell us whether the inhibition of PC synthesis is important to the action of the drugs on the Fas-mediate cell death and whether it is the PC synthesis inhibition at the step requiring CT that is particularly essential. The second aim is to determine whether these drugs affect Fas-mediated cell death via the accumulation of a lipid called ceramide. This accumulation is one of the results of PC synthesis inhibition by the drugs. This will give us more information on the sequential steps following the PC synthesis inhibition that lead to cell death. The third aim is to determine whether the drugs induce cell death by mimicking the action of the natural substance, FasL, on Fas. The effects of these drugs on Fas localization and the subsequent Fas-mediated events leading to cell death will be compared to the effects caused by FasL. These results will provide us with a better understanding of the tumor-killing mechanism of these drugs. The molecular mechanism and targets elucidated will be a basis of better breast cancer therapeutic strategies including the combined therapies (e.g. radiotherapy and chemotherapy) as well as the design of new drugs with superior anticancer activity and reduced damaging side effects, which will provide breast cancer patients with better treatments and thus a better quality of life.