Tamoxifen regulation of sphingolipid metabolism--Therapeutic implications

Abstract

Tamoxifen, a triphenylethylene antiestrogen and one of the first-line endocrine therapies used to treat estrogen receptor-positive breast cancer, has a number of interesting, off-target effects, and among these is the inhibition of sphingolipid metabolism. More specifically, tamoxifen inhibits ceramide glycosylation, and enzymatic step that can adventitiously support the influential tumor-suppressor properties of ceramide, the aliphatic backbone of sphingolipids. Additionally, tamoxifen and metabolites N-desmethyltamoxifen and 4-hydroxytamoxifen, have been shown to inhibit ceramide hydrolysis by the enzyme acid ceramidase. This particular intervention slows ceramide destruction and thereby depresses formation of sphingosine 1-phosphate, a mitogenic sphingolipid with cancer growth-promoting properties. As ceramide-centric therapies are becoming appealing clinical interventions in the treatment of cancer, agents like tamoxifen that can retard the generation of mitogenic sphingolipids and buffer ceramide clearance via inhibition of glycosylation, take on new importance. In this review, we present an abridged, lay introduction to sphingolipid metabolism, briefly chronicle tamoxifen's history in the clinic, examine studies that demonstrate the impact of triphenylethylenes on sphingolipid metabolism in cancer cells, and canvass works relevant to the use of tamoxifen as adjuvant to drive ceramide-centric therapies in cancer treatment. The objective is to inform the readership of what could be a novel, off-label indication of tamoxifen and structurally-related triphenylethylenes, an indication divorced from estrogen receptor status and one with application in drug resistance.

Keywords: Acid ceramidase; Glucosylceramide synthase; Multidrug resistance; P-glycoprotein; Sphingolipid; Tamoxifen.

Fig. 1

Anabolic and catabolic routes of ceramide metabolism. Ceramide, shown in blue, the aliphatic backbone of sphingolipids, can be converted to sphingomyelin, ceramide 1-phosphate, and glucosylceramide. Ceramide, a tumor suppressor, induces apoptosis. Glycosylation (conversion to GC) is a prominent metabolic pathway in multidrug resistant cancer cells; glycosylation as well leads to ceramide resistance. If ceramide is hydrolyzed by ceramidase, sphingosine and free fatty acid are generated. The sphingosine can be metabolized to sphingosine 1-phosphate, a mitogenic entity, by sphingosine kinase. Various points in ceramide metabolism can be activated or inhibited, providing a useful strategy for studying ceramide-related events. GC, glucosylceramide. Note: arrows to designate several of the back-reactions are not included in this figure; for example, beta-glucocerebrosidase, also known as D-glucosyl-N-acylsphingosine glucohydrolase, which catalyzes cleavage by hydrolysis of the beta-glucosidic linkiage of glucosylceramide.

Fig. 2

De novo pathway enzymes for production of ceramide. Ceramide synthesis is initiated by serine palmitoyltransferase and culminates with addition of a 4,5-trans double bond in dihydroceramide, catalyzed by dihydroceramide desaturase. Ceramide synthases, which number 6, are the subject of engaged investigations due to their selectivity for an array of fatty acyl-CoAs, giving rise to a myriad of molecular species of ceramides with distinct biological properties. Enzymes of the de novo pathway are shown in italics.

Fig. 3

Chemical structure of triphenylethylenes and related SERM, raloxifene. Tamoxifen is composed of an aromatic triphenylethylene nucleus and an aminoethoxy side chain. Tamoxifen is metabolized in humans mainly to N-desmethyltamoxifen, 4-hydroxytamoxifen, 4-hydroxy-N-desmethyltamoxifen (endoxifen, structure shown with red hydroxyl group), and N-didesmethyltamoxifen (not shown). Metabolism is directed by the cytochrome P450 enzyme system. 4-hydroxytamoxifen is a potent anti-estrogen and is considered the most active metabolite; however, endoxifen is also a potent anti-estrogen. Raloxifene (Evista) belongs to the benzothiophene class of compounds and shares inhibition of ceramide glycosylation properties with tamoxifen. Toremifene is a chlorinated derivative of tamoxifen.

Fig. 4

Chemical structure of cyclosporin A and verapamil, prominent clinical agents employed as drug resistance modulators. Verapamil is a phenylalkylamine, L-type calcium channel blocker used in the treatment of hypertension, angina, and arrhythmia, and on the docket of the World Health Organization’s list of essential medicines. Cyclosporin A, a cyclic, 11-amino acid peptide, is an immunosuppressant used in organ transplantation to prevent rejection. Both verapamil and cyclosporin A inhibit ceramide glycosylation in cancer cells.

Fig. 5

Schematic illustrating effect of tamoxifen and classical P-gp antagonists on GC synthesis. Shown here using short-chain, C6-ceramide as starting substrate, conversion to GC is catalyzed by GCS (shown in blue) at the ER-Golgi interface. Resultant GC can be transported into the Golgi lumen by the flippase actions (shown in green) of P-gp where it is further metabolized to LC by LCS (shown in blue). Tamoxifen and other P-gp antagonists inhibit transit of newly synthesized GC into the Golgi lumen, interference that effectively halts ceramide glycosylation, resulting in ceramide buildup. P-gp, P-glycoprotein; GC, glucosylceramide; GCS, glucosylceramide synthase; ER, endoplasmic reticulum; LC, lactosylceramide; LCS, lactosylceramide synthase.

Fig. 6

A combinatorial approach to suppression of cancer growth that employs 4-HPR and tamoxifen. Using prostate cancer cells (PC-3) as an example, 4-HPR activates SPT. This step forms sphinganine that is used for synthesis of ceramides, a process resulting in ceramide production. The addition of tamoxifen blocks cellular conversion of ceramide to GC, resulting in ceramide buildup (up arrows) with concurrent magnification of ceramide cancer suppressor effects. Tamoxifen also inhibits AC, a vital enzymatic junction. This inhibition leads to diminished levels of sphingosine that in turn limit production of mitogenic S1P. Not depicted here: the de novo pathway intermediates of ceramide biosynthesis and the impact of 4-HPR on dihydroceramide desaturase. SPT, serine palmitoyltransferase; GC, glucosylceramide; AC, acid ceramidase; P-gp, P-glycoprotein; S1P, sphingosine 1-phosphate.