Novel selective estrogen mimics for the treatment of tamoxifen-resistant breast cancer.

Endocrine-resistant breast cancer is a major clinical obstacle. The use of 17ß-estradiol (E2) has reemerged as a potential treatment option following exhaustive use of tamoxifen or aromatase inhibitors, although side effects have hindered its clinical usage. Protein kinase C alpha (PKCα) expression was shown to be a predictor of disease outcome for patients receiving endocrine therapy and may predict a positive response to an estrogenic treatment. Here, we have investigated the use of novel benzothiophene selective estrogen mimics (SEM) as an alternative to E2 for the treatment of tamoxifen-resistant breast cancer. Following in vitro characterization of SEMs, a panel of clinically relevant PKCα-expressing, tamoxifen-resistant models were used to investigate the antitumor effects of these compounds. SEM treatment resulted in growth inhibition and apoptosis of tamoxifen-resistant cell lines in vitro. In vivo SEM treatment induced tumor regression of tamoxifen-resistant T47D:A18/PKCα and T47D:A18-TAM1 tumor models. T47D:A18/PKCα tumor regression was accompanied by translocation of estrogen receptor (ER) α to extranuclear sites, possibly defining a mechanism through which these SEMs initiate tumor regression. SEM treatment did not stimulate growth of E2-dependent T47D:A18/neo tumors. In addition, unlike E2 or tamoxifen, treatment with SEMs did not stimulate uterine weight gain. These findings suggest the further development of SEMs as a feasible therapeutic strategy for the treatment of endocrine-resistant breast cancer without the side effects associated with E2.

SERMs attenuate estrogen-induced malignant transformation of human mammary epithelial cells by upregulating detoxification of oxidative metabolites.

The risk of developing hormone-dependent cancers with long-term exposure to estrogens is attributed both to proliferative, hormonal actions at the estrogen receptor (ER) and to chemical carcinogenesis elicited by genotoxic, oxidative estrogen metabolites. Nontumorigenic MCF-10A human breast epithelial cells are classified as ER(-) and undergo estrogen-induced malignant transformation. Selective estrogen receptor modulators (SERM), in use for breast cancer chemoprevention and for postmenopausal osteoporosis, were observed to inhibit malignant transformation, as measured by anchorage-independent colony growth. This chemopreventive activity was observed to correlate with reduced levels of oxidative estrogen metabolites, cellular reactive oxygen species (ROS), and DNA oxidation. The ability of raloxifene, desmethylarzoxifene (DMA), and bazedoxifene to inhibit this chemical carcinogenesis pathway was not shared by 4-hydroxytamoxifen. Regulation of phase II rather than phase I metabolic enzymes was implicated mechanistically: raloxifene and DMA were observed to upregulate sulfotransferase (SULT 1E1) and glucuronidase (UGT 1A1). The results support upregulation of phase II metabolism in detoxification of catechol estrogen metabolites leading to attenuated ROS formation as a mechanism for inhibition of malignant transformation by a subset of clinically important SERMs.

Selective estrogen receptor modulator (SERM) lasofoxifene forms reactive quinones similar to estradiol.

The bioactivation of both endogenous and equine estrogens to electrophilic quinoid metabolites has been postulated as a contributing factor in carcinogenic initiation and/or promotion in hormone sensitive tissues. Bearing structural resemblance to estrogens, extensive studies have shown that many selective estrogen receptor modulators (SERMs) are subject to similar bioactivation pathways. Lasofoxifene (LAS), a third generation SERM which has completed phase III clinical trials for the prevention and treatment of osteoporosis, is currently approved in the European Union for this indication. Previously, Prakash et al. (Drug Metab. Dispos. (2008) 36, 1218-1226) reported that similar to estradiol, two catechol regioisomers of LAS are formed as primary oxidative metabolites, accounting for roughly half of the total LAS metabolism. However, the potential for further oxidation of these catechols to electrophilic o-quinones has not been reported. In the present study, LAS was synthesized and its oxidative metabolism investigated in vitro under various conditions. Incubation of LAS with tyrosinase, human liver microsomes, or rat liver microsomes in the presence of GSH as a trapping reagent resulted in the formation of two mono-GSH and two di-GSH catechol conjugates which were characterized by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Similar conjugates were also detected in incubations with P450 3A4, P450 2D6, and P450 1B1 supersomes. Interestingly, these conjugates were also detected as major metabolites when compared to competing detoxification pathways such as glucuronidation and methylation. The 7-hydroxylasofoxifene (7-OHLAS) catechol regioisomer was also synthesized and oxidized either chemically or enzymatically to an o-quinone that was shown to form depurinating adducts with DNA. Collectively, these data show that analogous to estrogens, LAS is oxidized to catechols and o-quinones which could potentially contribute to in vivo toxicity for this SERM.